atlas of-clinical-hematology-6th-edition

H. Loffler � J. Rastetter � T. Haferlach

Atlas of Clinical Hematology

H. Loffler � J. Rastetter � T. Haferlach

Atlas ofClinicalHematologyInitiated by L. Heilmeyerand H. Begemann

Sixth Revised Edition

With 199 Figures, in 1056 separate Illustrations,Mostly in Color, and 17 Tables

Professor Dr. med. Helmut LofflerEhem. Direktor der II. Medizinischen Klinik und Poliklinikder Universitat Kiel im Stadtischen KrankenhausSeelgutweg 7, 79271 St. Peter, Germany

Professor Dr. med. Johann RastetterEhem. Leiter der Abteilung fur Hamatologie und Onkologie1. Medizinische Klinik und PoliklinikKlinikum rechts der Isar der Technischen Universitat MunchenWestpreußenstraße 71, 81927 Munchen, Germany

Professor Dr. med. Dr. phil. T. HaferlachLabor fur Leukamie-DiagnostikMedizinische Klinik IIILudwig-Maximilians-Universitat GroßhadernMarchioninistraße 1581377 Munchen

English editionsª Springer-Verlag Berlin Heidelberg1st ed. 19552nd ed. 19723rd ed. 19794th ed. 19895th ed. 2000

German editionsAtlas der klinischen Hamatologieª Springer-Verlag Berlin Heidelberg1st ed. 19552nd ed. 19723rd ed. 19784th ed. 19875th ed. 1999

Japanese editionRinsho Ketsuekigaku Atlasª Springer-Verlag Tokyo, 1989

Translated by: Terry C. Telger, Fort Worth, Texas, USA

ISBN 3-540-21013-X Springer Berlin Heidelberg New York

ISBN 3-540-65085-1 5th Edition Springer Berlin Heidelberg New York

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Spanish editionpublished byEditorial Cientifico-MedicaBarcelona, 1973

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Preface to the Sixth Edition

Soon after the 5th edition of this volume appeared, the WHO published de-tails on the pathology and genetics of the hematopoietic and lymphatic tis-sues. Work in progress found in short journal articles had already been in-tegrated into the last edition. Now it was possible to incorporate the newproposals for classification and diagnosis and to include figures of new typesof leukemia and lymphoma. These include leukemias of dendritic cells, in-travascular large B-cell lymphoma, the liver-spleen T-cell lymphoma as wellas persistent polyclonal B-cell lymphocytosis, which is placed between be-nign and malignant.

The present volume completes and extends the cytogenetic and molecu-lar-genetic characterization of the different diseases and incorporates newfigures. At this point we would like to thank PD Dr. Claudia Schoch, Munich,for her valuable help and for graciously providing new zytogenetic and FISHfigures. In addition, several figures and tables were replaced, and a schematicdrawing of the topography of lymphoma infiltration in bone marrow (cour-tesy of Prof. Dr. H.E. Schaefer, Freiburg) was added to the lymphoma chap-ter.

Even in 2004, diagnosis in hematology and lymphomas starts, as a rule,with the morphological examination of blood, bone marrow or lymphatictissues. It can direct the subsequent use of immunophenotyping, cytoge-netics and molecular genetics, in this way demonstrating ways of savingmoney and avoiding unnecessary investigations.

Gene expression profiling and, in the future, proteomics still representvery expensive methods that must find their place in diagnosis and prognos-tic evaluation. Gene profiling studies have already confirmed morphologicalsubtypes in AML, e.g., M3 and M3V, which cannot be distinguished intostrictly separate groups by cytogenetic and molecular-genetic methods.New therapeutic measures (especially immunotherapy) have brought inter-esting progress into the MDS group. For example, the biological entity 5qminus syndrome, which is well defined by morphology and cytogenetics, re-sponds very well to treatment with the thalidomide derivative CC 5013. Thefusion gene BCR-ABL, which was originally detected by cytogenesis and istoday routinely detected by FISH or PCR in CML, was the first example of aspecifically tailored molecular therapy in a tumor; certainly other exampleswill follow. Cases of ALL involving t(9;22), t(4;11) and t(8;14) have also beenestablished as separate prognostic groups with special therapeutic problems.

All of these examples demonstrate that a comprehensive arsenal of diag-nostic methods has to be used today for diagnostic and prognostic decisionsand individualized therapeutic planning.

We are again grateful to Prof. Dr. R. Disko of Munich who agreed to reviseand update the chapter on the principal causative agents of tropical diseases.Finally we wish to thank Mrs. Stephanie Benko and the entire staff of Spring-er-Verlag in Heidelberg as well as Ms. Marina Litterer at ProEdit GmbH fortheir thoughtful and effective support.

V

Preface to the Fifth Edition

The first edition of the Atlas of Clinical Hematology was published over 40years ago. The first four editions were coauthored by Herbert Begemann,who died unexpectedly in April of 1994. We wish to dedicate the fifth editionas a memorial to this dedicated physician and hematologist.

Since the fourth edition was published in 1987, hematology has undergoneprofound changes. New methods such as cytochemistry and immunopheno-typing have been joined by cytogenetics and, more recently, molecular ge-netic techniques, which have assumed amajor role in routine diagnostic pro-cedures. This has been due in part to significant advances in methodologyand new tools in molecular biology. When used in standardized protocols,these tools can furnish swift results that are relevant to patient care. Since theadvent of cytogenetics and molecular genetics, we have formulated new de-finitions for clinical and biological entities. An example is promyelocytic leu-kemia with its two variants (M3 and M3v), the (15;17) translocation, and thePML/RARA fusion gene, which has been successfully treated for the first timewith differentiation therapy. Another example is acute myelomonocytic leu-kemia with abnormal eosinophiles (M4Eo), inversion 16, and the MYH/11/CBFB fusion gene, which has a very good prognosis. The transmission ofmorphologic findings by electronic data transfer is also gaining importancein hematology, as it permits the immediate review of difficult findings byspecialists. Several colleagues seated at their own desks and microscopescan communicate with one another instantaneously by computer monitor.These advances do not alter the fact that hematologists must still have asound grasp of morphologic principles. Diagnostic problems often arisewhen modern counting devices and cell sorters, with their impressive cap-abilities, are used without regard for cellular morphology. There is no ques-tion that classical morphology has gained much from its competition andcomparison with the new techniques, leading to significant diagnosticand prognostic advances.

While retaining the basic concept of the previous editions, we found itnecessary to eliminate several chapters. Now that many hematologic centersand laboratories are equipped with fluorescence-activated cell sorters(FACS) for immunotyping, and given the availability of reliable commercialkits and precise staining instructions for immunocytochemistry, the chapterby B. R. Kranz has been omitted from the present edition. We have alsodropped the methodology section andmost of the electronmicrographs sup-plied by Prof. D. Huhn. Both colleagues merit our sincere thanks. Ever sincethe first edition, Prof. W. Mohr of Hamburg has authored the chapter onblood parasites as the principal causative agents of tropical diseases, andwe gratefully acknowledge his contribution. Following the death of Prof.Mohr, we have chosen to include this chapter owing to the special impor-tance of tropical diseases in the modern world. We are grateful to Prof. R.Disko of Munich, who agreed to revise and update the chapter.

The chapters on chronic myeloproliferative diseases, and especially thosedealing with myelodysplasias, acute leukemias, malignant lymphomas, andmalignant mastocytoses, had to be extensively revised or rewritten. We haveadded new sections and illustrations on therapy-induced bone marrowchanges, cytologic changes in the cerebrospinal fluid due to leukemic or lym-

VII

VIII

phomatous meningeal involvement, and NK cell neoplasias. We have alsoendeavored to give due attention to issues in pediatric hematology.

In compiling this revised fifth edition, in which over 90 % of the illustra-tions are new, we benefited greatly from our two decades of central morpho-logical diagnostics for the ALL and AML studies in adults and the morpho-logical consulting of the BFM treatment study on AML in children (H. L.).We thank the directors of these studies, Professors D. Hoelzer, T. Buchner, U.Creutzig, and J. Ritter, for their consistently fine cooperation. We also thankthe Institute of Pathology of the University of Kiel, headed by Prof. Karl Len-nert, and the current head of the Department of Hematologic Pathology,Prof. Reza Parwaresch, for preparing histologic sections of the tissue coresthat we submitted.

Acknowledgements

We are indebted to Prof. Brigitte Schlegelberger, Prof. Werner Grote (direc-tor of the Institute of Human Genetics, University of Kiel), Dr. Harder, andMr. Blohm for providing the cytogenetic findings and schematic drawings.We limited our attention to important findings that have bearing on the dia-gnosis or confirmation of a particular entity.

A work of this magnitude cannot be completed without assistance. Mysecretary of many years, Mrs. Ute Rosburg, often freed me from distractingtasks so that I could gain essential time. Mrs. Margot Ulrich efficiently or-ganized the processing of the photographic materials, while Mrs. Ramm-Pe-tersen, Mrs. Meder, and Mrs. Tetzlaff were meticulous in their performanceof cytologic, cytochemical, and immunocytochemical methodologies. My se-nior staff members in Kiel, Prof. Winfried Gassmann and Dr. Torsten Ha-ferlach, helped with the examination and evaluation of many of the speci-mens pictured in the Atlas. My colleague Dr. Haferlach collaborated with thestudy group of Prof. Schlegelberger to introduce the FISH technique intoroutine clinical use. Finally, we thank Mrs. Monika Schrimpf and the entirestaff at Springer-Verlag in Heidelberg as well as Ms. Judith Diemer at PROEDIT GmbH for their thoughtful and effective support.

St. Peter and Munich Helmut Loffler · Johann RastetterSummer 1999

VIII Preface to the Fifth Edition

Preface to the First Edition

So far the diagnostic advances of smear cytology have found only limitedapplications in medical practice. This is due largely to the fact that availableillustrative materials have been too stylized to give the novice a realistic in-troduction to the field. In the present atlas we attempt to correct this situa-tion by portraying the great morphologic variety that can exist in individualcells and in pathologic conditions. In so doing, we rely mainly on artist’sdepictions rather than photographs. On the one hand the “objectivity” ofcolor photos, though much praised, is inherently questionable and is furtherdegraded by the process of chemographic reproduction. An even greaterdrawback of photomicrographs is their inability to depict more than oneplane of section in sharp detail. By contrast, a person looking through a mi-croscope will tend to make continual fine adjustments to focus through mul-tiple planes and thus gain an impression of depth. A drawing can recreatethis impression much better than a photograph and so more closely approx-imates the subjective observation. We have avoided depicting cells in blackand white; while there is merit in the recommendation of histologists thatstudents’ attention be directed toward structure rather than color, this israrely practicable in the cytologic examination of smears. The staining meth-ods adopted from hematology still form the basis for staining in smear cy-tology. For this reason most of the preparations shown in this atlas werestained with Pappenheim’s panoptic stain. Where necessary, various specialstains were additionally used. For clarity we have placed positional drawingsalongside plates that illustrate many different cell types, and we have usedarrows to point out particular cells in films that are more cytologically uni-form.

We were most fortunate to have our color plates drawn by an artist, HansDettelbacher, in whom the faculties of scientific observation, technical pre-cision, and artistic grasp are combined in brilliant fashion. We express ourthanks to him and to his equally talented daughter Thea, who assisted herfather in his work. Without their contribution it is doubtful that the atlascould have been created.

We are also grateful to a number of researchers for providing scientifichelp and specimens, especially Prof. Dr. Henning and Dr. Witte of Erlangen,Dr. Langreder of Mainz, Prof. Dr. Mohr of the Tropical Institute of Hamburg,Dr. Moeschlin of Zurich, Dr. Undritz of Basel, and Dr. Kuhn of our FreiburgClinic. We also thank our translators, specifically Dr. Henry Wilde of ourFreiburg Clinic for the English text, Dr. Rene Prevot of Mulhouse for theFrench text, and Dr. Eva Felner-Kraus of Santiago de Chile for the Spanishtext. We must not fail to acknowledge the help provided by the scientific andtechnical colleagues at our hematology laboratory, especially Mrs. HildegardTrappe andMrs. WaltraudWolf-Loffler. Finally, we express our appreciationto Springer Verlag, who first proposed that this atlas be created and took thesteps necessary to ensure its technical excellence.

Freiburg, Spring 1955 Ludwig Heilmayer · Herbert Begemann

IX

Contents

Methodology

I Techniques of Specimen Collection and Preparation 3

Blood Smear 4

Bone Marrow 4

Fine-Needle Aspiration of Lymph Nodes and Tumors 5

Splenic Aspiration 6

Concentrating Leukocytes from Peripheral Blood in Leukocytopenia 6

Demonstration of Sickle Cells 6

II Light Microscopic Procedures 7

1 Staining Methods for the Morphologic and CytochemicalDifferentiation of Cells 8

1.1 Pappenheim’s Stain (Panoptic Stain) 8

1.2 Undritz Toluidine Blue Stain for Basophils 8

1.3 Mayer’s Acid Hemalum Nuclear Stain 8

1.4 Heilmeyer’s Reticulocyte Stain 8

1.5 Heinz Body Test of Beutler 8

1.6 Nile Blue Sulfate Stain 9

1.7 Kleihauer-Betke Stain for Demonstrating Fetal Hemoglobinin Red Blood Cells 9

1.8 Kleihauer-Betke Stain for Demonstrating Methemoglobin-Containing Cells in Blood Smears 10

1.9 Berlin Blue Iron Stain 10

1.10 Cytochemical Determination of Glycogen in Blood Cellsby the Periodic Acid Schiff Reaction and Diastase Test(PAS Reaction) 11

1.11 Sudan Black B Stain 13

1.12 Cytochemical Determination of Peroxidase 13

1.13 Hydrolases 13

1.14 Appendix 16

XI

XII

2 Immunocytochemical Detection of Cell-Surface and IntracellularAntigens 18

3 Staining Methods for the Detection of Blood Parasites 19

3.1 “Thick Smear” Method 19

3.2 Bartonellosis 19

3.3 Detection of Blood Parasites in Bone Marrow Smears 19

3.4 Toxoplasmosis 19

3.5 Microfiliariasis 19

3.6 Mycobacterium Species (M. tuberculosis, M. leprae) 19

Illustrations

III Overview of Cells in the Blood, Bone Marrow,and Lymph Nodes 23

IV Blood and Bone Marrow 27

4 Individual Cells 28

4.1 Light Microscopic Morphology and Cytochemistry 28

5 Bone Marrow 67

5.1 Composition of Normal Bone Marrow 69

5.2 Disturbances of Erythropoiesis 80

5.3 Reactive Blood and Bone Marrow Changes 107

5.4 Bone Marrow Aplasias (Panmyelopathies) 118

5.5 Storage Diseases 122

5.6 Hemophagocytic Syndromes 129

5.7 Histiocytosis X 132

5.8 Chronic Myeloproliferative Disorders (CMPD) 134

5.9 Myelodysplastic Syndromes (MDS) 158

5.10 Acute Leukemias 170

5.11 Neoplasias of Tissue Mast Cells (Malignant Mastocytoses) 286

V Lymph Nodes and Spleen 293

6. Cytology of Lymph Node and Splenic Aspirates 294

6.1 Reactive Lymph Node Hyperplasia 295

6.2 Infectious Mononucleosis 304

6.3 Persistent Polyclonal B Lymphocytosis 307

6.4 Malignant Non-Hodgkin Lymphomasand Hodgkin Lymphoma 308

XII Contents

VI Tumor Aspirates from Bone Marrow Involvedby Metastatic Disease 385

VII Blood Parasites and Other Principal Causative Organismsof Tropical Diseases 399

7 Blood Parasites 400

7.1 Malaria 400

7.2 African Trypanosomiasis (Sleeping Sickness) 410

7.3 American Trypanosomiasis (Chagas Disease) 411

7.4 Kala Azar or Visceral Leishmaniasis 414

7.5 Cutaneous Leishmaniasis (Oriental Sore) 416


7.7 Loa Loa 417

7.8 Wuchereria bancrofti and Brugia malayi 417

7.9 Mansonella (Dipetalonema) Perstans 420

8 Further Important Causative Organismsof Tropical Diseases 421

8.1 Relapsing Fever 421

8.2 Bartonellosis (Oroya Fever) 421

8.3 Leprosy 423

Subject Index 425

XIIIContents

Methodology

I Techniques of Specimen Collectionand Preparation 3

II Light Microscopic Procedures 7

I3

Techniques of Specimen Collectionand Preparation

Blood Smear 4

Bone Marrow 4

Fine-Needle Aspiration of Lymph Nodes and Tumors 5

Splenic Aspiration 6

Concentrating Leukocytes from Peripheral Blood in Leukocytopenia 6

Demonstration of Sickle Cells 6

IBlood Smear

Differentiation of the peripheral blood is still animportant procedure in the diagnosis of hemato-logic disorders. The requisite blood smears areusually prepared from venous blood anticoagu-lated with EDTA (several brands of collectingtube containing EDTA are available commer-cially). However, many special tests require thatthe blood be drawn from the fingertip or earlobeand smeared directly onto a glass slide with nochemicals added. The slide must be absolutelyclean to avoid introducing artifacts. Slides arecleaned most effectively by degreasing in alcoholfor 24 h, drying with a lint-free cloth, and finalwiping with a chamois cloth (as a shortcut, theslide may be scrubbed with 96 % alcohol andwiped dry).

Preparation of the Smear. The first drop of bloodis wiped away, and the next drop is picked up onone end of a clean glass slide, which is held by theedges. (When EDTA-anticoagulated venousblood is used, a drop of the specimen is trans-ferred to the slide with a small glass rod.) Nextthe slide is placed on a flat surface, and a cleancoverslip with smooth edges held at about a 45tilt is used to spread out the drop to create a uni-form film. We do this by drawing the coverslipslowly to the right to make contact with the blooddrop and allowing the blood to spread along theedge of the coverslip. Then the spreader, held atthe same angle, is moved over the specimen slidefrom right to left (or from left to right if the op-erator is left-handed), taking care that no portionof the smear touches the edge of the slide. Thelarger the angle between the coverslip and slide,the thicker the smear; a smaller angle results in athinner smear.

Once prepared, the blood smear should bedried as quickly as possible. This is done mostsimply by waving the slide briefly in the air (hold-ing it by the edges and avoiding artificial warm-ing). The predried slide may be set down in aslanted position on its narrow edge with thefilm side down. For storage, we slant the slidewith the film side up, placing it inside a drawerto protect it from dust and insects.

The best staining results are achieved when thesmear is completely air-dried before the stain isapplied (usually 4–5 h or preferably 12–24 h afterpreparation of the smear). In urgent cases thesmear may be stained immediately after air dry-ing.

Bone Marrow

Percutaneous aspiration of the posterior iliacspine is the current method of choice for obtain-ing a bone marrow sample. It is a relatively safeprocedure, and with some practice it can be donemore easily and with less pain than sternal aspira-tion. Marrow aspirate and a core sample can beobtained in one sitting with a single biopsy needle(e.g., a Yamshidi needle). When proper techniqueis used, the procedure is not contraindicated byweakened host defenses or thrombocytopenia.However, there is a significant risk of postproce-dural hemorrhage in patients with severe plas-matic coagulation disorders (e.g., hemophilia),in patients on platelet aggregation inhibitors,and in some pronounced cases of thrombocyto-sis. In all cases the biopsy site should be com-pressed immediately after the needle is with-drawn, and the patient should be observed. Theprocedure should be taught by hands-on trainingin the clinical setting.

Aspiration is usually performed after a corebiopsy has been obtained. The needle is intro-duced through the same skin incision and shouldenter the bone approximately 1 cm from thebiopsy site. A sternal aspiration needle may beused with the guard removed, or a Yamshidi nee-dle can be used after removal of the stylet.

The operator rechecks the position of the spineand positions the middle and index fingers of theleft hand on either side of the spine. The sternalaspiration needle, with adjustable guard re-moved, is then inserted until bony resistance isfelt and the needle tip has entered the periosteum.This is confirmed by noting that the tip can nolonger be moved from side to side. The needleshould be positioned at the center of the spineand should be perpendicular to the plane ofthe bone surface. At this point a steady, graduallyincreasing pressure is applied to the needle, per-haps combined with a slight rotary motion, to ad-vance the needle through the bone cortex. Thismay require considerable pressure in some pa-tients. A definite give will be felt as the needle pe-netrates the cortex and enters the marrow cavity.The needle is attached to a 20-mL glass syringe,the aspiration is performed, and specimens areprepared from the aspirated material.

After the needle is withdrawn, the site is cov-ered with an adhesive bandage and the patient in-structed to avoid tub bathing for 24 h.

The usual practice in infants is to aspirate bonemarrow from the tibia, which is still active hema-topoietically.

We prefer to use the needle described by Klimaand Rosegger, although various other designs aresuitable (Rohr, Henning, Korte, etc.). Basically it

4 Chapter I · Techniques of Specimen Collection and Preparation

I

does not matter what type of needle is used, aslong as it has a bore diameter no greater than2–3 mm, a well-fitting stylet, and an adjustabledepth guard. All bone marrow aspirations canbe performed in the ambulatory setting.

Sternal aspiration is reserved for special indi-cations (prior radiation to the pelvic region, se-vere obesity). It should be practiced only by ex-perienced hematologists. It is usually performedon the sternal midline at approximately the levelof the second or third intercostal space. The skinaround the puncture site is aseptically prepared,and the skin and underlying periosteum are de-sensitized with several milliliters of 1 % mepiva-caine or other anesthetic solution. After the anes-thetic has taken effect, a marrow aspiration nee-dle with stylet and guard is inserted vertically atthe designated site. When the needle is in contactwith the periosteum, the guard is set to a depth ofabout 4–5 mm, and the needle is pushed throughthe cortex with a slight rotating motion. A definitegive or pop will be felt as the needle enters themarrow cavity. Considerable force may have tobe exerted if the cortex is thick or hard. Whenthe needle has entered the marrow cavity, the sty-let is removed, and a 10- or 20-mL syringe is at-tached. The connectionmust be airtight so that aneffective aspiration can be performed. The plun-ger is withdrawn until 0.5 to 1 mL of marrow isobtained. Most patients will experience painwhen the suction is applied; this is unavoidablebut fortunately is of very brief duration. If nomarrow is obtained, a small amount of physiolo-gic saline may be injected into the marrow cavityand the aspiration reattempted. If necessary, theneedle may be advanced slightly deeper into themarrow cavity. The procedure is safe when per-formed carefully and with proper technique.Complications are rare and result mainly fromthe use of needles without guards or from carelesstechnique. The procedure should be used withcaution in patients with plasmacytoma, osteo-porosis, or other processes that are associatedwith bone destruction (e.g., metastases, thalasse-mia major). Bone marrow aspirations can be per-formed in the outpatient setting.

For preparation of the smears, we expel a smalldrop of the aspirated marrow onto each of severalglass slides (previously cleaned as described on p.3) and spread it out with a coverslip as describedfor the peripheral blood. We also place some ofthe aspirate into a watch glass and mix it with sev-eral drops of 3.6 % sodium citrate. This enables usto obtain marrow particles and prepare smears ina leisurely fashion following the aspiration. If theaspirate is not left in the citrate solution for toolong, the anticoagulant will not introduce cellchanges that could interfere with standard inves-

tigations. We vary our smear preparation techni-que according to the nature of the inquiry and thedesired tests. Spreading the marrow particlesonto the slide in a meandering pattern will causeindividual cells to separate from the marrowwhile leaving the more firmly adherent cells,especially stromal cells, at the end of the track.In every bone marrow aspiration an attemptshould be made to incorporate solid marrowparticles into the smear in addition to marrowfluid in order to avoid errors caused by theadmixture of peripheral blood. We see no advan-tage in the two-coverslip method of smear pre-paration that some authors recommend. Wefind that simple squeeze preparations often yieldexcellent results: Several marrow particles or adrop of marrow fluid are expelled from the syr-inge directly onto a clean glass slide. A secondslide is placed over the sample, the slides arepressed gently together, and then they are pulledapart in opposite directions. This technique per-mits a quantitative estimation of cell content. Allmarrow smears are air dried and stained as in theprocedure for blood smears. Thicker smears willrequire a somewhat longer staining time withGiemsa solution. Various special stains mayalso be used, depending on the nature of thestudy.

If cytologic examination does not provide suf-ficient information, the histologic examination ofa marrow biopsy specimen is indicated. This isespecially useful for the differentiation of pro-cesses that obliterate the bone marrow, includingosteomyelosclerosis or -fibrosis in neoplastic dis-eases and abnormalities of osteogenesis, theblood vessels, and the marrow reticulum. In re-cent years the Yamshidi needle has become in-creasingly popular for bone marrow biopsies.

Fine-Needle Aspiration of Lymph Nodesand Tumors

The fine-needle aspiration of lymph nodes andtumors is easily performed in the outpatient set-ting. The diagnostic value of the aspirate varies indifferent pathologic conditions. An accurate his-tologic classification is usually essential for soundtreatment planning and prognostic evaluation,and so the histologic examination has becomea standard tool in primary diagnosis. The unques-tioned value of the cytologic examination of aspi-rates is based on the capacity for rapid orientationand frequent follow-ups, adding an extra dimen-sion to the static impression furnished by histo-logic sections.

The technique of lymph node aspiration is verysimple: Using a 1 or 2 gauge (or smaller) hypoder-

5

Chapter I · Techniques of Specimen Collection and Preparation

Imic needle with a 10- or 20-mL syringe attached,we fixate the lymph node between two fingers ofthe free hand, insert the needle into the node, andapply forceful suction to aspirate a small amountof material. A thinner needle should be used fortissues that contain much blood, and someauthors routinely use needles of gauge 12, 14, or16 (outer diameter 0.6–0.9 mm). Special equip-ment is available that permits one-handed aspira-tion (e.g., the Cameco pistol grip syringe holder)and even the use of one-way syringes.

The tissue fragments on the needle tip and in-side the needle are carefully expelled onto a glassslide, and a smear is prepared. It is rare for tissueto be drawn into the syringe, but if this material ispresent it may be utilized for bacteriologic study.The smears are stained like a blood film, and spe-cial stains may be used as needed. The aspirationis almost painless and does not require anesthe-sia. If the lymph node is hard or if histologic ex-amination of the aspirate is desired, we use asomewhat larger gauge needle (approximately1–2 mm in diameter) that has a stylet and a sharpfront edge. The stylet is withdrawn before thenode is punctured. Of course, the use of a largerneedle requires preliminary anesthesia of the skinand lymph node capsule. All tumors that are ac-cessible to a percutaneous needle can be aspiratedin similar fashion.

Splenic Aspiration

Splenic aspiration is rarely practiced nowadaysand is always performed under some form ofradiologic guidance. Today it is indicated onlyin certain forms of hypersplenism or unexplainedsplenic enlargement. We consider the Moeschlintechnique to be the safest. Splenic aspiration iscontraindicated in patients with hemorrhagic dia-thesis, septic splenomegaly, splenic cysts, or pain-ful splenomegaly due to excessive capsular ten-sion or infarction. The procedure should beused with caution in patients with hypertensionof the portal or splenic vein (Banti syndrome,splenic vein thrombosis, splenomegalic cirrho-sis). It should be withheld from dazed patientswho are unable to cooperate. Moeschlin recom-mends that splenic aspiration be performed

only when definite splenic enlargement is notedand only under stringent aseptic conditions. Theprocedure is safest when performed under ultra-sound guidance, as this will demonstrate not onlythe size and position of the spleen but also anypathologic changes (e.g., splenic cysts) that wouldcontraindicate the procedure.

Concentrating Leukocytes from PeripheralBlood in Leukocytopenia

Principle. White blood cells are centrifuged aftersedimentation of the erythrocytes to concentratethe nucleated cells and make it easier to detectabnormal cell forms.

Reagents

1. Gelatin, 3 %, in 0.9 % NaCI (or plasma gel in-fusion solution; B. Braun, Melsungen)

2. Heparin (cresol-free)

Method. Place 3–5 mL of venous blood or EDTA-treated blood into a narrow tube, add 1/4 volumegel to the sample and carefully mix by tilting. Letstand at 37 for 14 min, 7 min at a 45 slant, and7 min upright. Pipet off the leukocyte-rich layerand centrifuge lightly at 2000 rpm. Decant thesupernatant, gently shake out the sediment,and prepare the smears.

Demonstration of Sickle Cells

Method. Place 1 drop of blood onto a slide andcover with a coverslip.

Place 1 drop of 2 % sodium thiosulfate(Na2S2O4) along one edge of the coverslip andhold a blotter against the opposite edge, the objectbeing to draw the Na thiosulfate beneath the cov-erslip so that it mixes with the blood. (If this isunsuccessful, it may be necessary to raise the cov-erslip slightly or even add the Na thiosulfate di-rectly to the blood before covering. However, it isbest to mix the thiosulfate and blood in the ab-sence of air, as described above!)

Create an airtight seal around the coverslipwith paraffin, and let stand for 30 min at roomtemperature. Examine the unstained slide underthe microscope.

6 Chapter I · Techniques of Specimen Collection and Preparation

Light Microscopic Procedures

1 Staining Methods for the Morphologic and Cytochemical Differentiation of Cells 8

1.1 Pappenheim’s Stain (Panoptic Stain) 8

1.2 Undritz Toluidine Blue Stain for Basophils 8

1.3 Mayer’s Acid Hemalum Nuclear Stain 8

1.4 Heilmeyer’s Reticulocyte Stain 8

1.5 Heinz Body Test of Beutler 8

1.6 Nile Blue Sulfate Stain 9

1.7 Kleihauer-Betke Stain for Demonstrating Fetal Hemoglobin in Red Blood Cells 9

1.8 Kleihauer-Betke Stain for Demonstrating Methemoglobin-Containing Cellsin Blood Smears 10

1.9 Berlin Blue Iron Stain 10

1.10 Cytochemical Determination of Glycogen in Blood Cells by the Periodic Acid SchiffReaction and Diastase Test (PAS Reaction) 11

1.11 Sudan Black B Stain 13

1.12 Cytochemical Determination of Peroxidase 13

1.13 Hydrolases 13

1.13.1 Cytochemical Determination of Leukocyte Alkaline Phosphatase (LAP)in Blood Smears 13

1.13.2 Cytochemical Determination of Acid Phosphatase 141.13.3 Detection of Esterases with Naphthyl Acetate or Naphthyl Butyrate

(”Neutral Esterases”) 14, Acid Esterase (ANAE) 151.13.4 Naphthol AS-D Chloroacetate Esterase (CE) 15

1.14 Appendix 16

2 Immunocytochemical Detection of Cell-Surface and Intracellular Antigens 18

3 Staining Methods for the Detection of Blood Parasites 19

3.1 “Thick Smear” Method 19

3.2 Bartonellosis 19

3.3 Detection of Blood Parasites in Bone Marrow Smears 19


3.5 Microfiliariasis 19

3.6 Mycobacterium Species (M. tuberculosis, M. leprae) 19

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1. Staining Methods for theMorphologic and CytochemicalDifferentiation of Cells

1.1 Pappenheim’s Stain (Panoptic Stain)

The hematologic stain that we use most fre-quently, and which was used in most of the platespictured in this atlas, is Pappenheim’s panopticstain. It is based on a combination of the Jen-ner-May-Grunwald stain and Giemsa stain.

Method. Place the air-dried slide with the film sideup in prepared May-Grunwald eosin-methyleneblue solution for 3 min. Dilute with water or buf-fer solution (phosphate buffer pH 7.3, see below)for an additional 3 min. Pour off this solution andapply Giemsa stain immediately, without inter-mediate rinsing. The stock Giemsa stain is dilutedwith neutral distilled water by adding 10 mLwaterper 10 drops of Giemsa solution. Stain the speci-men for 15 to 20 min. The dilution ratio andGiemsa staining time should be individually ad-justed to allow for inevitable variations in thecomposition of the solution. After Giemsa stain-ing, wash the slide with neutral water and tilt toair-dry. Fixation is effected by the methyl alcoholalready contained in the May-Grunwald solution.The quality of the stain depends greatly on the pHof the water that is used. The smear will be too redif the water is too acidic and too blue if the wateris too alkaline. Standard pH strips can be used totest the water for proper acidity. Water left stand-ing in the laboratory can easily become too acidicthrough exposure to acid fumes, especially fromcarbon dioxide. The latter problem is solved bypreboiling. A more accurate way to ensure correctacidity for staining is to use a pH 7.3 buffer solu-tion (22.3 mL of 1/15 mol/L KH2PO4 + 77.7 mL of 1/15 mol/L Na2HPO4) instead of water.

1.2 Undritz Toluidine Blue Stain for Basophils

Reagent. Saturated toluidine blue-methanol: dis-solve 1 g toluidine blue in 100 mL methanol. Thesolution will keep indefinitely.

Method. Fix and stain the air-dried smears on thestaining rack by covering with the toluidine blue-methanol for 5 min. Wash in tap water, air dry.

Interpretation. The granulations in basophils andmast cells stain a red-violet color owing to thestrong metachromatic effect of the sulfate presentin the heparin. As a result, these cells are easilyidentified even at moderate magnification. Bycontrast, azurophilic granules (even in severe

“toxic” granulation) and the coarse granules inleukocytes affected by Adler anomaly showvery little violet transformation of their blue col-or.

1.3 Mayer’s Acid Hemalum Nuclear Stain

This is used for the blue contrast staining of nu-clei in assays of cytoplasmic cell constituents (gly-cogen, enzymes; pp. 9 ff.) and in immunocyto-chemistry.

Reagents. Dissolve 1 g hematoxylin (Merck) in 1 Ldistilled water and add 0.2 g sodium iodate(NaIO3) and 50 g aluminum potassium sulfate(KAl(SO4)2 · 12H2O). After these salts are dis-solved, add 50 g chloral hydrate and 1 g crystal-lized citric acid. The hemalum will keep for atleast 6 months at 20 8C with no change in stainingproperties. The solution can also be purchased inready-to-use form.

Method. The necessary staining time in the hema-lum bath varies with the method of specimen pre-paration and must be determined by progressivestaining. After staining, wash the slide for at least15 min in several changes of tap water (acid resi-dues may reduce the intensity of the stain).

1.4 Heilmeyer’s Reticulocyte Stain

Draw a 1 % brilliant cresyl blue solution in phy-siologic saline to the 0.5 mark of a white cellcounting pipet, and draw up the blood to the1.0 mark. Expel the mixture carefully, withoutforming air bubbles, into a paraffinated watch-glass dish, mix carefully with a paraffinated glassrod, and place in a moist chamber for 15–20 min.Then remix carefully with a paraffinated glassrod. With the rod, transfer 1 or 2 drops of the mix-ture to a microscope slide and smear in standardfashion using a ground coverslip. Examine theair-dried slides under oil-immersion magnifica-tion, and count the number of reticulocytes per1000 red cells at multiple sites in the smear.Very high-quality films can be obtained by Giem-sa counterstaining.

1.5 Heinz Body Test of Beutler1

This test is used to detect defects of red cell me-tabolism that do not allow glutathione to be

1 After Huber H, Loffler H, Faber V (1994) Methoden der diag-nostischen Hamatologie. Springer, Berlin Heidelberg NewYork Tokyo.

8 Chapter II · Light Microscopic Procedures

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maintained in a reduced state. The defect may re-sult from a glucose-6-phosphate dehydrogenasedeficiency, a glutathione reductase deficiency,diseases with “unstable hemoglobin,” or an “idio-pathic” Heinz body anemia. The test involves theoxidative denaturation of hemoglobin to intra-erythrocytic “Heinz bodies” following incubationof the red cells with acetylphenylhydrazine.

Reagents

1. Sorensen phosphate buffer, pH 7.6, 0.67 M:1/15 M KH2PO4 13 parts.1/15 M Na2HPO4 87 parts.

2. Glucose phosphate buffer: dissolve 0.2 g glu-cose in 100 mL phosphate buffer. The solutionmay be stored frozen or at 4 8C (watch forclouding!).

3. Acetylphenylhydrazine solution: dissolve20 mg acetylphenylhydrazine in 20 mL glucosephosphate buffer at room temperature. Thissolution is prepared fresh and should beused within 1 h.

4. (a) Dissolve saturated alcohol solution of bril-liant cresyl blue; or (b) 0.5 g methyl violet in100 mL of 0.9 % NaCI, and filter; blood: hepar-inized, defibrinated, or treated with EDTA.

Method. Centrifuge the blood lightly for 5 min. Pi-pet 0.05 mL of test erythrocytes into 2 mL of theacetylphenylhydrazine solution. Suspend normalerythrocytes in an identical solution to serve as acontrol. Aerate the suspensions by drawing themup into the pipet and carefully blowing them outwith a small quantity of air; repeat several times.Incubate for 2 h at 37 8C, aerate again, and incu-bate 2 h more.

To stain with brilliant cresyl blue: spread asmall drop of stain solution 4(a) onto a clean, de-greased slide and dry the thin stain film rapidly inair. Place a small drop of the incubated erythro-cyte suspension on a coverglass and invert theglass onto the stain; examine with the micro-scope.

To stain with methyl violet: mix a small drop ofthe erythrocyte suspension with 2 or 3 drops ofstain solution 4b on the slide and cover with acoverslip. Let the mixture stand for 5– 10 minand examine with the microscope.

Interpretation. The percentage of erythrocytesthat contain more than four Heinz bodies is de-termined. Normal values range from 0 % to 30 %.The number of Heinz bodies is elevated in the dis-eases listed above.

1.6 Nile Blue Sulfate Stain

This stain is used for the visualization of Heinzinclusion bodies. A 0.5 % Nile blue sulfate solu-tion in absolute alcohol is transferred to theend of a slide with a glass rod until about 1/3of the slide is covered. The slide is dried by blow-ing on it, and the stain film is spread out evenlywith a cotton swab. Slides prepared in this way areplaced face-to-face and wrapped in paper for sto-rage. Staining is performed by dropping 2 or 3large drops of blood onto the prepared part ofthe slide and covering with the prepared partof the second slide. The slides, held by their un-stained outer ends, are separated and placed backtogether several times to thoroughly mix theblood with the stain. Finally the slides are left to-gether for 3 to 5 min, separated, and a groundcoverslip is used to collect the blood from eachslide and smear it onto another slide, which is al-lowed to dry. The Heinz bodies appear as small,dark blue bodies situated at the margin of the yel-low to bluish erythrocytes.

1.7 Kleihauer-Betke Stainfor Demonstrating Fetal Hemoglobinin Red Blood Cells

Principle.Normal adult hemoglobin (HbA) is dis-solved out of the red cells by incubating air-driedand fixed blood smears in citric acid phosphatebuffer (of McIlvain), pH 3.3, at 37 8C. Fetal hemo-globin (HbF) is left undissolved in the red cellsand can be made visible by staining.

Reagents

– Ethyl alcohol, 80 %– McIlvaine citric acid-phosphate buffer, pH 3.3– Stock solution A:

Sorensen citric acid, 21.008 g in 1 L water¼ 0.1 M

– Stock solution B:Disodium hydrogen phosphate Na2HPO4

2H2O, 27.602 g in 1 L water ¼ 0.2 M– For pH 3.3:

266 mL of solution B + 734 mL of solution A,Ehrlich hematoxylin, 0.1 % erythrosin solution

Method. Prepare thin blood smears, air dry, andfix in 80 % ethyl alcohol for 5 min. Wash in waterand dry. If further processing is delayed, the slidesmay be stored in a refrigerator for 4–5 days. Forelution, place the slides upright in a beaker con-taining the buffer in a 37 8C water bath for 3 min,moving the slides up and down after 1 and 2 minto keep the buffer mixed. Then wash in runningwater.

9

1 · Staining Methods for the Morphologic and Cytochemical Differentiation of Cells

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Staining. Stain in Ehrlich hematoxylin for 3 min,then poststain in 0.1 % aqueous erythrosin solu-tion for 3 min. Examine at 40� using dry or oil-immersion magnification.

Interpretation. Erythrocytes that contain HbA ap-pear as unstained “shadows,” while cells that con-tain HbF will stain a bright red color.

The method can be used for the diagnosis ofthalassemia major and for the detection of fetalerythrocytes that have entered the maternal circu-lation.

1.8 Kleihauer-Betke Stainfor Demonstrating Methemoglobin-Containing Cells in Blood Smears

Principle.Methemoglobin combines with KCN toform cyanmethemoglobin, while oxyhemoglobindoes not react with cyanides. Oxyhemoglobinfunctions as a peroxidase, whereas cyanmethe-moglobin has very low perixodase activity.

Method. Add 1/50 vol of a 0.4 M KCN solution toblood anticoagulated with heparin or sodium ci-trate. Prepare thin smears from this mixture, dry,and immerse in the following mixture at roomtemperature: 80 mL of 96 % ethyl alcohol + 16mL of 0.2 M citric acid + 5 mL of 30 % H2O2.Move the smears rapidly in the solution for about1 min, then leave them in the solution for 2 min.Wash the smears first in methyl alcohol, then indistilled water, and stain with hematoxylin anderythrosin (see stain for HbF). Examine at 40�using dry or oil-immersion magnification.

Interpretation. Oxy-Hb-containing cells stain abright red. Cells that contain met-Hb (convertedto cyanmet-Hb) are eluted and appear as sha-dows.

The same staining procedure can be used todifferentiate erythrocytes with a glucose-6-phos-phate dehydrogenase (G-6-PDH) deficiency bycombining it with the Brewer test (method ofBetke, Kleihauer and Knotek). This test is basedon the principle that hemoglobin converted tomet-Hb by the addition of nitrite reduces tooxy-Hb in the presence of methylene blue andglucose. Red blood cells with a G-6-PDH deficitcannot undergo this reduction. Even after severalhours, when all the methemoglobin in normalerythrocytes has converted back to oxy-Hb, cellswith a G-6-PDH deficiency retain all or most oftheir met-Hb. This causes the deficient cells to ap-pear “blank” with appropriate staining (see top ofthis section).

1.9 Berlin Blue Iron Stain

Principle

The Berlin blue reaction is used for the histo-chemical demonstration of trivalent iron. Ironin protein compounds can also be demonstratedby the addition of dilute hydrochloric acid. Ironin hemoglobin is not detected.

Reagents

� Methanol� Potassium ferrocyanide (potassium hexacya-

noferrate), 2 %� HCl, 37 %� Pararosaniline solution inmethanol, 1 % (alter-

native: nuclear red stain)

Method

� Fix the air-dried smears in formalin vapor for30 min (alternative: fix in methanol for 10–15min).

� Wash in distilled water for 2min and air dry.� Place the specimens in a cuvet that contains

equal parts of a 2 % solution of potassium fer-rocyanide and a dilute HCl solution (1 part37 % HCl mixed with 50 parts distilled water)for 1 h.

� Wash in distilled water.� Nuclear stain in pararosaniline solution: 300

lL of 1 % pararosaniline solution in methanoldiluted with 50 mL distilled water.Alternative. Stain nuclei with nuclear true redsolution (which yields a fainter nuclear stain).

All materials should be iron-free, and metal for-ceps should not be introduced into the solution.Pappenheim- or Giemsa-stained smears can sub-sequently be used for iron staining. They are firstprepared by destaining them for 12–24 h in puremethanol. These smears do not need to be fixedprior to staining.

Interpretation

Iron is stained blue, appearing either as diffuselyscattered granules or as clumps in the cytoplasm.There are two applications for iron staining in he-matology:(a) demonstrating sideroblasts and siderocytes,

and(b) demonstrating iron stored in macrophages

and endothelial cells.

Regarding (a): sideroblasts and siderocytes are,respectively, erythroblasts and erythrocytes thatcontain cytochemically detectable iron. Thisiron can be demonstrated in the form of smallgranules that may be irregularly scattered


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throughout the cytoplasm or may encircle the nu-cleus of erythroblasts like a ring. Normally thegranules are very fine and can be identified in ery-throblasts only by closely examining the smearswith oil-immersion microscopy in a darkenedroom. Generally 1 to 4 fine granules will beseen, rarely more. When iron deficiency is pre-sent, the percentage of sideroblasts is reducedto less than 15 %. Sideroblasts containing coarseiron granules that form a partial or complete ringaround the nucleus (ringed sideroblasts) are de-finitely abnormal. The detection of siderocyteshas little practical relevance: they are increasedin the same diseases as sideroblasts, and theyare elevated in the peripheral blood followingsplenectomy, as the spleen normally removesiron from intact red blood cells.

Regarding (b): the content of stored iron is as-sessed by examining bone marrow fragments insmears or sections. Iron stored in macrophagesmay occur in a diffusely scattered form, a finelygranular form, or in the form of larger granules orclumps that may cover part of the nucleus. Ironcan also be demonstrated in plasma cells as a re-sult of alcohol poisoning or sideroblastic anemiaand hemochromatosis.

The differential diagnosis afforded by ironstain is summarized in Table 1.

1.10 Cytochemical Determinationof Glycogen in Blood Cells by the PeriodicAcid Schiff Reaction and Diastase Test(PAS Reaction)

Principle

This method is based on the oxidation of a-gly-cols in carbohydrates and carbohydrate-contain-ing compounds. The resulting polyaldehydes aredemonstrated with the Schiff reagent (leukofuch-sin).

Reagents

� Formalin.� Periodic acid solution, 1 %, in distilled water.� Sulfite water: add tap water to 10 mL of a 10 %

sodium metabilsulfite solution (Na2S2O5) and10 mL of 1mol/L HCL to make a volume of200 mL. The stock solutions can be stored inthe refrigerator; the mixture should alwaysbe freshly prepared.

� Prepare Schiff reagent (commercially available)as follows: completely dissolve 0.5 g pararosa-niline in 15 mL of 1mol/L HCl by shaking(no heating) and add a solution of 0.5 g potas-sium metabisulfite (K2S2O5) in 85 mL distilled

water. The clear, bright red solution will gradu-ally lighten to a yellowish color. After 24 h,shake with 300mg activated charcoal (pow-dered) for 2min and then filter. The colorlessfiltrate is ready to use and, when stored in adark stoppered bottle in a cool place, willkeep for several months. Schiff reagent thathas turned red should no longer be used!

Method

� Fix the smears for 10min in a mixture of 10 mL40 % formalin and 90 mL ethanol (alternative:fix for 5min in formalin vapor).

� Wash for 5min in several changes of tap water.� Place the smears in 1 % periodic acid for 10min

(prepared fresh for each use).� Wash in at least two changes of distilled water

and dry.� Place in Schiff reagent for 30min (in the dark at

room temperature).� Rinse in sulfite water (changed once) for 2–

3min.� Wash in several changes of distilled water for

5min.� Nuclear stain with hemalum for approx.

10min, then blue in tap water for approx.15–20min, and air dry.

Even older slides that have been stained withGiemsa or Pappenheim can be reused for thePAS reaction. Specimens that have been treatedseveral times with oil or xylene should not beused for PAS staining. The smears can be placedunfixed in periodic acid after washing in distilledwater (Step 3) to remove the color.

Interpretation

PAS-positive material in the cytoplasm may pro-duce a diffuse red stain or may appear as pink toburgundy-red granules, flakes, or clumps of vary-ing size that may occupy large areas of the cyto-plasm. The distribution of PAS-positive materialin normal leukocytes is summarized in the Table.Some plasma cells, macrophages, and osteoblastsmay also show a positive PAS reaction, and mega-karyocytes are strongly positive.

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Table 1. Differential diagnosis by iron stain in the bone marrow

Sideroblasts Iron-storingreticulum cells,sideromacrophages

Special features

Normal bone marrow � 20–60 %finely granular,1 –4 granules

Isolated,mostly finelygranular deposits

Siderocytes inperipheral blood0–0.3 ‰

Hypochromic anemias– Iron deficiency < 15 % finely granular None Serum FeQ

– Infection, tumor < 15 % finely granular Increased finelygranular or (rarely)coarsely granulardeposits

Serum FeQ

– Sideroachrestic anemias(RARS)

> 90 % coarselygranular;ringedsideroblasts(> 15 %)

Greatly increased,many diffuse orcoarsely granulardeposits

Serum Feq,siderocytesmay be increased

– Lead poisoning > 90 % coarselygranular;ringed sidero-blasts



– Thalassemia > 90 % coarselygranular;ringed sidero-blasts



Hemolytic anemias � 80 % finely granular Increased finelygranular or (rarely)coarsely granulardeposits

Secondary sideroachrestic anemias � 80 % finely granular Increased finelygranular or (rarely)coarsely granulardeposits

Vitamin B6 deficiency � 80 % finely granular Increased finelygranular or (rarely)coarsely granulardeposits

Megaloblastic anemias � 80 % finely granular Increased finelygranular or (rarely)coarsely granulardeposits

Aplastic anemias � 80 % finely granular Increased finelygranular or (rarely)coarsely granulardeposits

Myeloproliferative disorders � 80 % finely granular Increased finelygranular or (rarely)coarsely granulardeposits

Hemochromatosis � 80 % finely granular IncreasedPlasma cells containiron

Bone marrow isnot useful fordiagnosis exceptpositive plasma cells

Postsplenectomy state � 80 % finely granular Somewhat increased Siderocytesgreatly increased


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PAS reaction in normal leukocytes

Cell type PAS reaction

Myeloblast ˘Promyelocyte (+)

Myelocyte +

Metamyelocyte ++

Band andsegmented cells

+++

Eosinophils + (intergranular reaction)

Basophils + (granular!)

Monocytes (+) to +Lymphocytes ˘ to + (granular)

Reaction: ˘ ¼ negative; (+) ¼ weakly positive;+ ¼ positive; ++ ¼ markedly positive; +++ ¼ stronglypositive

1.11 Sudan Black B Stain

Principle

Sudan black B is a fat-soluble dye that becomeshighly concentrated in lipids. The sudanophilia,which occurs even after degreasing, is based onan oxidative coupling of Sudan black derivativeswith phenols. It is peroxidase-dependent and thuscorresponds to the peroxidase reaction. It ishardly used anymore.

1.12 Cytochemical Determinationof Peroxidase

Principle

Benzidine or diaminobenzidine (more oftenused) is converted, in the presence of peroxide,from the leuko form into a high-polymer formthat is detectable by cytochemical staining.

Reagents.

� Fixative: methanol + 37 % formalin (10 : 1).� DAB solution: 5mg diaminobenzidine tetrahy-

drochloride in 20 mL of 0.05mol/L tris-HClbuffer (pH 7.6) with 50 lL of 1 % H2O2 added

� Tris-HCl: 50 mL of solution A (121.14 g trishy-droxymethylaminomethane dissolved in 1 Ldistilled water) + 40 mL of solution B (1mol/L HCl) + 960 mL distilled water

� Mayer’s hemalum:

Method

� Fix the air-dried smears for 15 s at 4 8C (30 s forthicker bone marrow smears).

� Wash 3 times in tap water.

� Air dry.� Incubate in DAB solution for 10min.� Wash briefly in tap water.� Incubate in Mayer’s hemalum for 3min.� Wash in tap water for 3min.� Air dry.

Interpretation

From the promyelocytic stage on, neutrophils andeosinophilic granulocytes show a yellowish greento brownish granular stain. Monocytes may showa positive reaction, which is weaker than that ofgranulocytes.

1.13 Hydrolases

Principle

The principle is the same for all hydrolases andmay be summarized as follows: Today only theazo dye method is still in routine clinical use.It is based on the hydrolytic splitting of an arylester by the enzyme and the immediate couplingof the liberated phenol derivative to a dye sub-stance, usually a diazonium salt or hexazotizedpararosaniline.

1.13.1 Cytochemical Determinationof Leukocyte Alkaline Phosphatase (LAP)in Blood Smears

Reagents

� Fixative: 10 % formalin in absolute methanol(one part 37 % formalin, 9 parts 100 % metha-nol)

� Staining solution: dissolve 35mg sodium-a-naphthyl phosphate in 70 mL of 2 % veronalsodium solution, pH 9.4; add 70mg concen-trated variamine blue salt B, and stir. Immedi-ate filter the solution and use.

� Mayer’s hemalum.

Method

� Fix the air-dried smears at 4 8C for 30 s.� Wash 3 times thoroughly in tap water.� Incubate in refrigerator at 4–7 8C for 2 h.� Wash thoroughly in tap water.� Nuclear stain inMayer’s hemalum for 5–8min.� Air dry the smears and mount in glycerine ge-

latin or Aquatex.

Interpretation

Neutrophilic granulocytes (a few band cells,mostly segmented forms) are the only types ofblood cell that show enzymatic activity. The in-tensity of the phosphatase reaction is usuallyscored on a four-point scale. The activity score,

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or index, is based on groups of 100 cells and iscalculated from the sum of the cells assigned tothe different reaction grades, which is multipliedby a corresponding factor (1 –4). The indexranges from 0 to 400. Cells in the bone marrowthat have phosphatase activity are neutrophilicgranulocytes, vascular endothelial cells, and os-teoblasts. The location of structures in bone mar-row smears, lymph node touch preparations, andsections can be determined more accurately byusing methods that employ the substratesnaphthol-AS-BI phosphate or -MX phosphate.

1.13.2 Cytochemical Determinationof Acid Phosphatase

Reagents

� Fixative: see Appendix� Staining solution: mix together 0.8 mL hexazo-

tized pararosaniline (mix equal parts 4 % so-dium nitrite and 4 % pararosaniline in HCl,see Appendix) + 30 mL Michaelis buffer pH7.4 (58 mL of 0.1mol/L sodium barbital +41.9 mL of 0.1mol/L HCl) + 10mg naphthol-AS-BI phosphate, dissolved in 1 mL dimethyl-formamide. Adjust the solution to pH 4.9–5.1and filter before use.

� Mayer’s hemalum

Method

� Fix the air-dried smears at 4 8C for 30 s.� Wash 3 times in tap water.� Air dry.� Incubate in stain solution for 3 h at room tem-

perature.� Wash briefly in tap water.� Place in Mayer’s hemalum for 3min.� Blue in tap water for 3min.� Air dry.

Interpretation

A bright red homogeneous or granular precipitateforms in the cytoplasm of cells with acid phospha-tase activity. In the case of plasmacytomas, theabnormal plasma cells tend to show stronger ac-tivity than normal plasma cells or plasma cells af-fected by reactive changes. A dotlike staining pat-tern is seen in T-lymphocytes, while the blasts ofT-ALL usually show a circumscribed (focal) para-nuclear acid phosphatase reaction.

Acid Phosphatase Reactionwith Inhibition by Tartrate

Method

Add 60mg of L-tartaric acid to 30 mL of the stain-ing solution, then analyze as described for acid

phosphatase. Fast garnet GBC can be used as acoupling salt instead of the pararosaniline solu-tion. This requires the following modificationsin the staining solution: Dissolve 10mgnaphthol-AS-BI phosphate in 0.5 mL dimethylfor-mamide, andadd0.1mol/L acetate buffer pH5.0 tomake 10mL. Dissolve 10– 15mg of fast garnet GBCin 20 mL of 0.1mol/L acetate buffer solution. Mixboth solutions well. Filtering is not required. In-cubate the smears at 37 8C for 60–90min.

Interpretation

Most of the cells of hairy cell leukemia are posi-tive even after tartrate inhibition, and macro-phages and osteoclasts do not show significant in-hibition. Today immunophenotyping, especiallywith CD 103, is more important.

1.13.3 Detection of Esterases with NaphthylAcetate or Naphthyl Butyrate(”Neutral Esterases”)

Reagents

� Solution a: mix 1 drop (0.05 mL) sodium nitritesolution (4 %) + 1 drop (0.05 mL) pararosani-line solution (4 % in 2mol/L HCl) for about1min (yields a pale yellow solution), then dis-solve in 5 mL of 0.2mol/L phosphate buffer, pH7.0–7.1 (250 mL Na2HPO4+130 mL NaH2 PO4).

� Solution b: dissolve 10mg a-naphthyl acetate in0.2–0.3 mL chemically pure acetone; add 20mL of 0.2mol/L phosphate buffer pH 7.0–7.1while stirring vigorously.

� Mix solutions a and b and filter into small cu-vets.

Method

� Fix the thin, air-dried smears (will keep up to 3days when sheltered from dust, longer at 4–8 8C) in formalin vapor for 4min or in the fixa-tive solution for 30 s (see Appendix).

� Wash in tap water.� Incubate for 60min.� Wash in tap water.� Stain in Mayer’s hemalum for approx. 8min.� Blue in tap water for approx. 15min.� Mount smears with glycerine gelatin or Aqua-

tex (Merck).� Air-dried smears may be mounted with Eukitt.

Interpretation

Positive cells stain with a brown to reddish-browndiffuse or granular pattern. The a-naphthyl buty-rate stain yields a dark red color. The result is verysimilar to the a-naphthyl acetate stain, so theslightly different method used with a-naphthylbutyrate will not be described in detail.


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Monocytes in the peripheral blood are stronglypositive for a-naphthyl acetate stain, while neu-trophilic and eosinophilic granulocytes are nega-tive. Some lymphocytes stain with a circum-scribed, dotlike pattern. The strongest activityin bone marrow cells is found in monocytes,macrophages, and megakaryocytes.

Acid a-Naphthyl Acetate Esterase (ANAE)

Reagents

� Fixative: see Appendix.� Staining solution: dissolve 50mg a-naphthyl

acetate in 2.5 mL ethyleneglycolmonomethylether + 44.5 mL of 0.1mol/L phosphate bufferpH 7.6 þ 3.0 mL hexazotized pararosaniline(1.5 mL 4 % pararosaniline in 2mol/L HCl +1.5 mL 4 % sodium nitrite solution). Adjustthe solution to pH 6.1–6.3 with 1mol/L HCland filter before use. The solution must beclear.

� Mayer’s hemalum.

Method

� Fix air-dried smears in fixative solution at 4 8Cfor 30 s.

� Wash 3 times in tap water.� Air dry for 10–30min.� Incubate in staining solution at room tempera-

ture for 45min.� Rinse briefly in tap water.� Place in Mayer’s hemalum for 3min.� Blue in tap water for 3min.� Air dry.

Interpretation

The reaction product appears as a reddish-brownhomogeneous or granular precipitate. Acid ester-ase is used to identify T-lymphocytes. The meth-od is reliable only for more mature forms, how-ever, and inconsistent results are obtained inacute lymphocytic leukemias with T characteris-tics.

1.13.4 Naphthol AS-D ChloroacetateEsterase (CE)

Reagents

� Methanol-formalin solution, 9 : 1 (v/v).� 0.1mmol/L Michaelis buffer, pH 7.0.� Naphthol AS-D chloroacetate.� Dimethylformamide.� Sodium nitrite solution, 4 %.� Pararosaniline solution, 4 %, in 2mol/L HCl.� Staining solution A: mix 0.1 mL sodium nitrite

solution and 0.1 mL pararosaniline solutionwith 30 mL Michaelis buffer.

� Staining solution B: dissolve 10mg naphtholAS-D chloroacetate in 1 mL dimethylforma-mide.

� Staining solution C: mix solutions A) and B),adjust to pH 6.3 with 2mol/L HCl, and filterinto a cuvet. Use immediately.

Method

� Fix smears in methanol-formalin for 30 s atroom temperature, wash thoroughly in tapwater without delay.

� Place smears in staining solution for 60min,then wash thoroughly in tap water.

� Nuclear stain with hemalum for 5– 10min,wash thoroughly with tap water, and blue forapprox. 10min.

� After air drying, the smears may be directly ex-amined or mounted with Eukitt.

Interpretation

A bright red reaction product forms at sites of en-zymatic activity in the cytoplasm. Neutrophilicgranulocytes normally display a positive reactionfrom the promyelocytic stage on, the late promye-locyte to myelocyte stages showing the strongestreaction. A slightly weaker reaction is seen inband and segmented forms. Monocytes mayalso show a weak chloroacetate esterase reaction.Besides neutrophils, tissue mast cells display verystrong activity. In acute myelomonocytic leuke-mia, which is associated with an anomaly of chro-mosome 16, some of the abnormal eosinophilsshow a positive chloroacetate esterase reaction.Normal eosinophils are negative.

15


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1.14 Appendix

Fixation (Suitable for Esterase,Acid Phosphatase, DAP IV)

The fixative solution is composed of:� 30 mL buffer solution (20mg disodium hydro-

gen phosphate · 12H2O and 100mg potassiumdihydrogen phosphate dissolved in 30 mL dis-tilled water; pH should be 6.6)

� +45 mL analytical grade acetone� +25 mL formalin (37 %)

Fix air-dried smears in this solution for 30 s at 4–10 8C, wash in three changes of distilled water, anddry at room temperature for 10–30min.

Schaefer universal fixative. Mix 0.5 mL of 25 %glutardialdehyde solution and 60 mL analyticalgrade acetone in distilled water to make 100mL. Air-dried smears are incubated in this fixa-tive solution at room temperature: 1min for per-oxidase, 10min for chloroacetate esterase, 5minfor detecting esterase with naphthyl acetate ornaphthyl butyrate, 1min for acid phosphatase,1min for alkaline phosphatase, 10min for detect-ing iron, and 10min for the PAS reaction.

Sodium Nitrite Solution 4 %

Dissolve 4 g sodium nitrite in distilled water tomake 100 mL.

Pararosaniline Solution 4 %

Dissolve 2 g Graumann pararosaniline (Merck) in50 mL of 2mol/L HCl by gentle heating. Cool andfilter the solution.

The sodium nitrite and pararosaniline solu-tions will keep for several months when storedin a dark bottle under refrigeration. Most ofthe reagents and even commercial staining kitscan be ordered from pharmaceutical houses(Merck, Serva, Sigma, etc.). Before kits are usedfor routine tests, they should be compared againstsolutions prepared by the methods indicated.

The cytochemical features of blood cells andbone marrow cells are reviewed in Table 2.


II

Table 2. Cytochemistry of blood and bone marrow cells

Per-oxidase

PAS Esterase

a-Naphthyl-acetate-,Naphthol-AS-acetate-

Naphthol-AS-D-chloracetate-

Phosphatases

alkaline acid

Remarks

Reticulum cells ˘ ˘ � + ++ ˘ ˘ (1) ++ (1) vascularendothelia +++

Plasma cells ˘ ˘ (+) ˘ ˘ + Acid phospha-tasc is stronglypositive inmultiplemyeloma

Myeloblast ˘ ˘ � (+) ˘ � (+) ˘ ˘ ˘Promyelocyte ++ (+) ˘ � (+) +++ ˘ +

Myelocyte ++ + ˘ � (+) +++ ˘ +

Metamyelocyte ++ ++ ˘ � (+) +++ ˘ � (+) (+)

Band form ++ +++ ˘ � (+) +++ ˘ � (+) (+)

Segmented form +++ +++ ˘ � (+) +++ ˘ � +++ (+)

Eosinophils ++ + ˘ � (+) ˘ ˘ (+) � +

BasophilsBlood ˘ � + + ˘ � (+) ˘ ˘ ˘Tissue ++ ++

Monocytes ˘ � + (+) � + +++ (+) ˘ ˘Lymphocytes ˘ ˘ � + + ˘ ˘ ˘ Hairy cells are

acid-phospha-tase positive

Erythroblasts ˘ ˘ ++ ˘ ˘ ˘ Positive PASreaction inerythremiasand erythroleu-kemias andsome MDS

Erythrocytes ˘ ˘ (+) ˘ ˘ ˘Megakaryocytesand platelets

˘ + +++ ˘ ˘ ++ PAS reactionmay bedecreased inWerlhof’sdisease

Osteoblasts ˘ ˘ + ˘ +++ +Osteoclasts ˘ ˘ � (+) ++ ˘ ˘ +++

Reaction: ˘ ¼ negative; (+) ¼ weakly positive; + ¼ positive; ++ ¼ markedly positive; +++ ¼ strongly positive

17


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2. ImmunocytochemicalDetection of Cell-Surfaceand Intracellular Antigens

Today the immunologic characterization of cellsis based on the use of monoclonal antibodies.This may involve the immunocytologic stainingof smears or analysis by flow cytometry, in which

a number of different fluorochrome-labeled anti-bodies are used for studies of cell suspensions.We refer the reader to commercial kits, whichcome with detailed instructions, and to the infor-mation that has become available in recent text-books on immunocytology and diagnostic hema-tology. (Hrusak O, Porwit-MacDonald A (2002)Antigen expression patterns reflecting genotypeof acute leukemias. Leukemia 16: 1233– 1258)


II

3. Staining Methods for theDetection of Blood Parasites1

3.1 “Thick Smear” Method

One drop of blood is placed on a slide and spreadwith the edge of a second slide to cover an area thesize of a dime. The film should not be too thick, orit will flake off during drying or displace duringstaining (it should be thin enough that printedtext can still be read through it). The film is airdried and may be stained after it is completelydry.

The film is stained without preliminary fixa-tion. Owing to the concentrating effect of thethick smear method, a parasitic infection canbe detected even when the organisms are presentin small numbers. Staining without preliminaryfixation induces a massive hemolysis that dis-lodges the parasites from the erythrocytes sothat they can be identified.

The staining solution is prepared fresh for eachuse and consists of 1 drop of stock Giemsa staindistilled to 1.0 mL and buffered water (pH 7.2).This solution hemolyzes and stains simulta-neously.

The stain is applied for 20–30min, then theslide is carefully washed by dipping it in tapwater. It is dried in an upright position.

Besides the thick smear preparation, a thinblood smear (fixed in methanol for 5min) shouldalso be prepared so that the parasites can be ac-curately identified if doubt exists. Often this is dif-ficult to accomplish in thick smears.

Thick smear preparations for trypanosomes(T. gambiense, T. rhodesiense, T. cruzi) arestained in the same way as for malaria parasites.This method is also used to examine for Borreliarecurrentis.

3.2 Bartonellosis

Bartonella organisms are most readily detectedby the examination of Pappenheim-, or Giem-sa-stained blood smears.

3.3 Detection of Blood Parasitesin Bone Marrow Smears

Blood parasites are best demonstrated in marrowsmears by Giemsa staining (17mm) after fixationin methanol (5min) (see p. 7).

3.4 Toxoplasmosis

Giemsa staining of the touch preparation or othersample is also recommended for the detection oftoxoplasmosis. Direct immunofluorescence andthe peroxidase reaction can detect the organismwith high sensitivity.

3.5 Microfiliariasis

1. Wet preparation (thick smear method): Exam-ine a drop of fresh (anticoagulated) blood under acoverslip on a microscope slide (bearing in mindthe periodicity in microfilarial activity, see p.403). The highly motile organisms are clearly visi-ble even at low magnification (250�).

2. Concentrating the sample: To 3–5mL of drawnvenous blood, add 10–15 mL of a mixture of 95mL formalin (5 %), 5 mL acetic acid, and 2 mLof an alcoholic gentian violet solution (4 g per100 mL 96 % alcohol). Centrifuge the mixture,and examine the sediment for stained microfilar-iae. (Membrane filtration methods provide a par-ticularly good yield.)

3. Examination of a skin snip for microfilariae(Onchocerca volvulus). Place a large drop of phy-siologic saline solution onto a slide. Immerse inthe saline a pinhead-size piece of skin excisedwith a Walser dermatome (if that is not available,use a razor blade). Cover with a coverslip, letstand 20min, then examine with the microscopeat low power (�300�). The organisms will passfrom the skin into the saline medium and willmove vigorously in the fluid.

3.6 Mycobacterium Species(M. tuberculosis, M. leprae)

One or two of the following reactions are used toexamine a suspicious sample. The Kinyoun andauramine stains are usually combined and havelargely replaced the Ziehl-Neelsen stain. The my-cobacteria stain red with both the Kinyoun andZiehl-Neelsen stains.

a. Kinyoun cold stain(alternative to Ziehl-Neelsen):

1. Fix the specimen (with heat or methanol).2. Immerse in Kinyoun solution for 3min.3. Wash with water for 30 s.4. Place in Gabett solution for 2min.5. Wash and dry.

1 Revised by Prof. Dr. R. Disko, Munchen.

19


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b. Auramine stain:1. Fix the specimen with heat.2. Stain with Auramin solution for 3min.3. Decolorize with acid alcohol for 1min.4. Wash off acid alcohol with water.5. Restain with blue-black ink

solution for 1min.6. Rinse off ink solution with water and dry.

c. Ziehl-Neelsen stain:1. Fix the specimen with heat.2. Cover with 10 % carbolfuchsin and heat to

steaming 3 times; stain for 3min.3. Decolorize in 3 changes of acid

alcohol for 3min.4. Wash with water.5. Counterstain with dilute methylene

blue solution for 3min.6. Wash with water and dry between sheets of

blotting paper.


Illustrations

III Overview of Cells in the Blood, Bone Marrow,and Lymph Nodes 23

IV Blood and Bone Marrow 27

V Lymph Nodes and Spleen 293

VI Tumor Aspirates from Bone Marrow Involvedby Metastatic Disease 385

VII Blood Parasites and Other Principal CausativeOrganisms of Tropical Disease 399

23

Overview of Cells in the Blood,Bone Marrow, and Lymph Nodes

III

III

Figure 1 presents an overview of the various cellsof hematopoiesis. The figure does not attempt toanswer unresolved questions of cell origins and isintended only as an introductory scheme to helpthe beginner find some order in the bewilderingvariety of cells. The cells of hematopoiesis devel-op from CD 34–positive stem cells, which resem-ble large lymphocytes or small, undifferentiatedblasts (Fig. 2).When cultured, these cells form co-lonies that can sometimes be identified by theirintrinsic color (Fig. 3).

Red and white cell precursors account for mostof the cells found in normal bone marrow. In ad-dition there are variable numbers of reticulumcells, vascular and sinus endothelial cells, mega-karyocytes, tissue mast cells, lymphocytic ele-ments, plasma cells and, very rarely, osteoblastsand osteoclasts (more common in children).The earliest precursors of the red and white bloodcells have a basophilic cytoplasm and are very si-milar to one another. As hemoglobin synthesis in-creases, the erythroblasts lose their basophilic cy-toplasm while their nuclei undergo a characteris-tic structural change. After losing their nuclei, theyoung erythrocytes still contain remnants of theirformer cytoplasmic organelles as evidence oftheir immaturity. They are reticulocytes and arereleased as such into the peripheral blood. Thereticulocytes can be demonstrated by supravitalstaining (see p. 8).

The myeloblasts, which are the precursors ofneutrophilic granulocytes and monocytes, devel-op into neutrophilic promyelocytes and promo-nocytes. The eosinophilic and basophilic granulo-cytes pursue their own lines of development andtherefore have their own promyelocytes with spe-cific granules.

Platelets (thrombocytes) develop from the cy-toplasm of the megakaryocytes.

The common progenitor cell from whichmonocytes and neutrophilic granulocytes origi-nate might be termed the myelomonoblast(CFU-GM).

The reticulum cells described and counted incytologic preparations from bone marrow, lymphnodes, and spleen form a heterogeneous group. Alarge portion belong to the macrophage systemand are derived from blood monocytes. Theyalso include segregated vascular and sinus

endothelial cells in addition to dendritic cellsbelonging to the stroma. The reticulum cells ofthe bone marrow constitute the reticular orspongy tissue of the bone marrow in whichthe actual hematopoietic cells reside. Apparentlythey perform important tasks relating to nutri-tion and differentiation of the blood cellprecursors.

Two different types of reticulum cell areknown to occur in the lymph nodes and spleen:the “dendritic reticulum cell,” which occurs ex-clusively in germinal centers, primary follicles,and occasionally in the peripheral zones of folli-cles, and the “interdigitating reticulum cell,”which is specific to the thymus-dependent regionof the lymph node (see Fig. 132 for details).

The “fibroblastic reticulum cell” described byLennert and Muller-Hermelink can occur in allregions of the lymph node as well as in bone mar-row, but as yet it has not been positively identifiedby light microscopy. The cells formerly describedas small “lymphoid reticulum cells” are probablytissue lymphocytes.

In the lymphatic system, a basic distinction isdrawn between B lymphocytes and T lympho-cytes based on the development, differentiation,and function of the cells. Unfortunately, the dif-ferentiation of these two cell types cannot be ac-complished with traditional staining methodsand must rely on immunocytologic or flow cyto-metric analysis. Both lymphatic cell lines appearto arise from a common, committed stem cell thatprobably resides in the bone marrow. Thereafterthe primary differentiation of the T cell line takesplace in the thymus, while that of the B cells (inhumans) takes place in the bone marrow, whichtoday is viewed as the equivalent of the fabricianbursa in birds. Further development and prolif-eration of both cell lines take place in the lymphnodes.

The final maturation stage of B lymphocytes isthe plasma cell, whose function is to produce im-munoglobulins. Plasma cells occur ubiquitously.Apparently they can develop anywhere in thebody but are most plentiful in lymph nodes,spleen, and bone marrow. A positive correlationexists between the amount of immunoglobulinspresent in the serum and the size of the plasmacell population.

24 Chapter III · Overview of Cells in the Blood, Bone Marrow, and Lymph Nodes

III

Fig. 1. The various cell lines of hematopoiesis

25

Chapter III · Overview of Cells in the Blood, Bone Marrow, and Lymph Nodes

III

Fig. 2. CD 34–positive stem cells

Fig. 3. Colonies of CD34-positivestem cells in cultures

26 Chapter III · Overview of Cells in the Blood, Bone Marrow, and Lymph Nodes

27

Blood and Bone Marrow

4 Individual Cells 28

4.1 Light Microscopic Morphology and Cytochemistry 284.1.1 Cells of Erythropoiesis (Fig. 4 a– f) 284.1.2 Granulocytopoiesis and Mast Cells (Tissue Basophils) 384.1.3 Degenerate Forms, Toxic Changes and Artifacts (Fig. 9 a–d) 444.1.4 Congenital Anomalies of Granulocytopoiesis (Fig. 10 a– f) 464.1.5 Cells of the Monocyte-Macrophage System (Fig. 13 a–h) 544.1.6 Megakaryocytes (Fig. 15 a–e) 604.1.7 Osteoblasts and Osteoclasts (Fig. 16 a– f) 634.1.8 Lymphocytes and Plasma Cells (Fig. 17 a–g) 66

5 Bone Marrow 69

5.1 Composition of Normal Bone Marrow 69

5.2 Disturbances of Erythropoiesis 805.2.1 Hypochromic Anemias (Fig. 23a–d) 805.2.2 Hemolytic Anemias 825.2.3 Megaloblastic Anemias 905.2.4 Toxic Disturbances of Erythropoiesis 965.2.5 Acute Erythroblastopenia 985.2.6 Chronic Erythroblastopenia (Pure Red Cell Aplasia) 1015.2.7 Congenital Dyserythropoietic Anemias 1015.2.8 Synartesis 105

5.3 Reactive Blood and Bone Marrow Changes 1075.3.1 Agranulocytosis 1145.3.2 Kostmann Syndrome 1175.3.3 Thrombocytopenias and Thrombocytopathies 1175.3.4 Pseudothrombopenia 117

5.4 Bone Marrow Aplasias (Panmyelopathies) 119

5.5 Storage Diseases 1225.5.1 Gaucher Disease 1225.5.2 Niemann-Pick Disease 1255.5.3 Glycogen Storage Disease Type II (Acid Maltase Deficiency, Pompe Disease) 127

5.6 Hemophagocytic Syndromes 129

5.7 Histiocytosis X 132

5.8 Chronic Myeloproliferative Disorders (CMPD) 1345.8.1 Myeloid Leukemia and Transient Abnormal Myelopoiesis (TAM)

of Down Syndrome (DS) 1405.8.2 Special Variants of Megakaryocyte Proliferation 1405.8.3 Familial Erythrocytosis (Fig. 48 a–c) Cytochemical Detection of Alkaline Phosphatase 1435.8.4 Chronic Myeloid (Granulocytic) Leukemia 1445.8.5 Chronic Neutrophilic Leukemia (CNL) 1575.8.6 Chronic Eosinophilic Leukemia (CEL) and the Hypereosinophilic Syndrome (HES) 157

5.9 Myelodysplastic Syndromes (MDS) 158

5.10 Acute Leukemias 1805.10.1 Acute Myeloid Leukemia (AML) 1835.10.2 Acute Lymphoblastic Leukemia (ALL) (Fig. 115 a–d) 265

5.11 Neoplasias of Tissue Mast Cells (Malignant Mastocytoses) 286

IV

IV

4. Individual Cells

4.1 Light MicroscopicMorphologyand Cytochemistry

4.1.1 Cells of Erythropoiesis (Fig. 4 a– f)

The proerythroblasts, called also pronormo-blasts or rubriblasts, are the earliest precursorsof erythropoiesis. They range from 15 to 22 lmin size and do not yet contain hemoglobin.They typically have a darkly basophilic, oftenshadowy cytoplasm that sometimes shows pseu-dopodia. The nucleus has a dense, finely honey-combed chromatin structure (Fig. 4 a–c). Mostproerythroblasts have several (at most five) indis-tinct pale blue nucleoli, which disappear as thecell matures. Like all erythropoietic cells, proery-throblasts tend to produce multinucleated forms.Typically there is a perinuclear clear zone, whichis found to contain minute granules on phasecontrast examination. Hemoglobin first appearsadjacent to the nucleus and produces a flaringof the perinuclear clear zone, later expandingto occupy the whole cell and heralding a transi-tion to the polychromatic forms. Meanwhile thenucleus undergoes a characteristic structuralchange: the nucleoli disappear while the chroma-tin becomes coarser and acquires typical erythro-blastic features.

A continuum exists from the proerythroblaststo the basophilic erythroblasts (macroblasts)(Fig. 4d). These cells tend to be smaller thanproerythroblasts (8– 15 lm in diameter). The nu-clear-cytoplasmic ratio is shifted in favor of thecytoplasm. The polychromatic erythroblasts

show a coexistence of basophilic material witha greater abundance of hemoglobin. The nucleusappears coarse and smudgy, and there is partialclumping of the nuclear chromatin.

As development progresses, the cell loses moreof its basophilic cytoplasm and further di-minishes in size (7– 10 lm in diameter), graduallyentering the stage of the orthochromatic normo-blast (Fig. 4e). The nuclear- cytoplasmic ratio isfurther shifted in favor of the cytoplasm, whichacquires an increasingly red tinge ultimatelymatching that of the mature erythrocyte.Supravital staining of the youngest erythrocytesreveals a network of strands (see p. 8) calledthe “substantia reticulofilamentosa” of the reticu-locytes. Staining with brilliant cresyl blue causesthe aggregation or precipitation of ribonucleo-proteins. It takes four days for the cells to passthrough the four maturation stages. The clump-like erythroblastic nucleus then condenses to astreaklike, featureless, homogeneous mass.Some authors subdivide the normoblasts into ba-sophilic, polychromatic, and orthochromaticforms according to their degree of maturity, whileothers use the terms rubricyte (basophilic normo-blast) and metarubricyte (orthochromatic nor-moblast). Such fine distinctions are unnecessaryfor the routine evaluation of marrow smears,however. Normoblasts are incapable of dividing.The nucleus is expelled through the cell mem-brane.

Particularly when erythropoiesis is increased,examination of the smear will reveal nests or is-lands of erythroblasts with central reticulum cellswhose cytoplasm is in close contact (metabolicexchange) with the surrounding erythroblasts(Fig. 4 f).

28 Chapter IV · Blood and Bone Marrow

IV

Fig. 4 a–d

a

b

c

d

29

4 · Individual Cells

IV

Fig. 4 e– f

e

f


IV

Erythrocytes (Figs. 5 a–h, 6 a– i)

The morphologic evaluation of erythrocytes isbased on the following criteria:– Size– Shape– Hemoglobin: concentration, distribution– Stainability– Distribution in the smear– Inclusions

Normal erythrocytes (Fig. 5 a) (diam. 7–8 lm).

Hypochromic erythrocytes (Fig. 5 b) in iron defi-ciency anemia. The cells, which have normal dia-meters, are conspicuous for their paucity of he-moglobin, which may form only a thin peripheralrim (anulocytes).

Poikilocytes (Fig. 5 c) are dysmorphic erythro-cytes of variable shape that occur in the settingof severe anemias. Their presence indicates a se-vere insult to the bone marrow. Teardrops andpear shapes are particularly common and arenot specific for osteomyelosclerosis or -fibrosis.

Microspherocytes (Fig. 5 d) are smaller than nor-mal erythrocytes (diam. 3–7 lm) but arecrammed with hemoglobin and have a greaterthickness, giving them an approximately spheri-cal shape. They are typical of congenital hemoly-tic jaundice (spherocytic anemia) but also occurin acquired hemolytic anemias.

Elliptocytes (ovalocytes) (Fig. 5 e) result from aninherited anomaly of erythrocyte shape that isusually innocuous but may be linked to a propen-sity for hemolytic anemia (elliptocytic anemia).

Basophilic stippling (Fig. 5 f) of erythrocytes is asign of increased but abnormal regeneration. It isparticularly common in lead poisoning. The nor-mal prevalence of basophilic stippling is 0–4 er-ythrocytes per 10,000

Polychromatic erythrocytes (Fig. 5 g) (diam. 7–8 lm), Cabot rings. Polychromasia occurs whenmature erythrocytes show increased stainingwith basic dyes (violet stain) in addition to hemo-globin staining. It is usually associated with reti-culocytosis. Polychromasia occurs in red cellsthat still have a relatively high RNA contentand in which hemoglobin synthesis is not yetcomplete. It is especially common in chronic he-molytic anemias. The variable staining of the er-ythrocytes is also termed anisochromia. Cabotrings are remnants of spindle fibers and are a pro-duct of abnormal regeneration (see also Fig. 46c).

Megalocytes (Fig. 5 h) are very large, mostly ovalerythrocytes that are packed with hemoglobin(> 8lm in diameter). They occur predominantlyin megaloblastic anemias (see sect. 5.2.3)

31


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Fig. 5 a–d

a

b

c

d


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Fig. 5 e–h

e

f

g

h

33


IV

Erythrocytes (Fig. 6 a) containing nuclear rem-nants in the form of Howell-Jolly bodies, whichare observed after splenectomy and in cases ofsplenic atrophy. Chromatin dust, like the Ho-well-Jolly bodies, consist of nuclear remnants.

Target cells (Mexican hat cells) (Fig. 6 b) are dis-tinguished from anulocytes by the deeper stainingof their central zone and peripheral rim. They areparticularly common in hemoglobin abnormal-ities, occurring also in other hemolytic anemias,severe iron deficiency, and after splenectomy.

Acanthocytes or “burr cells” (Fig. 6 c) are distin-guished by their jagged surface, which usually isdeeply clefted. Acanthocytes are seen in a rarehereditary anomaly, A-b-lipoproteinemia. Theyare also a feature of uremia and hepatic coma,where large numbers of these cells are considereda poor prognostic sign. Acanthocyte formationhas also been linked to the use of alcohol and cer-tain drugs.

Sickle cells (drepanocytes) (Fig. 6 d). Sickle-shaped erythrocytes occasionally form sponta-neously, but sickling is consistently induced byoxygen withdrawal in the sickle cell test (see p.5). It signifies a common hemoglobinopathy,HbS disease (sickle cell anemia), which affectsblacks almost exclusively. Red cell sickling alsooccurs in the less common HbC disease.

Knizocytes (triconcave erythrocytes) (Fig. 6 e)occur mainly in hemolytic anemias. The affectederythrocyte appears to have a “handle.”

Stomatocytes (Fig. 6 f) have a slitlike central lu-cency. They are found in the very rare hereditarystomatocytosis and in other anemias.

Schizocytes (fragmentocytes) (Fig. 6 g) resultfrom the fragmentation of erythrocytes, consist-ing either of a fragmented red cell or a fragmentdetached from such a cell. They resemble bits ofbroken egg shell. They may be caused by in-creased mechanical hemolysis (turbulence fromartificial heart valves) or by increased intravascu-lar coagulation (e.g., in hemolytic uremic syn-drome) as fast-flowing red cells are sliced apartby fibrin filaments.

Siderocytes (Fig. 6 h) are erythrocytes that con-tain iron granules detectable with iron staining.They are a common feature of severe hemolyticanemias, lead poisoning, and pernicious anemia.Siderocytes containing coarse iron granules,which may encircle the nucleus (see Fig. 60),are pathognomonic for sideroachrestic anemias.Normal blood contains 0.5– l siderocyte per1000 red cells.

Left: At the center is a siderocyte containing sev-eral large iron granules and two sideroblasts alsocontaining coarse iron granules, which normallyare very small and difficult to see.

Right: At the center are three erythrocytes withnumerous gray-violet granules that containiron (Pappenheimer bodies). This is a clear-cutpathologic finding that is rarely observed.

Reticulocytes (Fig. 6 i) in various stages of matur-ity. The more filamentous reticula are character-istic of younger cells (brilliant cresyl blue stain,see p. 8).


IV

Fig. 6 a–d

a

b

c

d

35


IV

Fig. 6 e–h

e

f

g

h


IV

Fig. 6 i

i

37


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4.1.2 Granulocytopoiesis and Mast Cells(Tissue Basophils)

Myeloblasts and Promyelocytes (Fig. 7 a–h)

Myeloblasts are the earliest precursors of granu-locytopoiesis that can be identified by light mi-croscopy. Today it is believed that they also func-tion as precursors of monocytes, i.e., as myelomo-noblasts. They range from 12 to 20 lm in diameter(Fig. 7 a–c). The cytoplasmic rim is basophilicbut may show a range of hues from soft paleblue to dark blue. The cytoplasm is agranularon ordinary panoptic staining, although oldercells frequently show incipient granulation sig-nifying transition to the promyelocyte stage(Fig. 7 d). The peroxidase reaction is usually ne-gative, but there is no question that agranular,peroxidase-positive myeloblasts exist. The nu-cleus shows a very fine, dense chromatin struc-ture with as many as six nucleoli, which generallyare distinct and pale blue.

Promyelocytes evolve directly from the myelo-blasts by incorporating azurophilic granules intotheir cytoplasm in a concentric pattern surround-ing the clear zone in the nuclear indentation (Gol-gi zone), which itself is devoid of granules. Initi-ally there are few granules, but these subsequentlythicken and spread to fill the cytoplasm. At firstthe cytoplasm is basophilic but gradually lightensuntil it acquires the typical myelocytic tinge. Thevariable staining properties of the cytoplasm haveled some authors to subdivide the promyelocytesinto mature and immature forms, groups I and II,etc. As in myeloblasts, the promyelocyte nucleusis finely structured and contains up to six nucleo-li. The cells are peroxidase-positive. First to ap-pear are the primary or azurophilic granule,which contain peroxidase; they are joined laterby the specific secondary granules (peroxidase-negative), which increase as maturation proceeds.The cells are 20–25 lm in diameter, making pro-myelocytes the largest cell of the granulocyto-poietic and erythropoietic lines. In absoluteterms, mitoses are more frequent than in themye-loblasts (Fig. 7 d–h).


IV

Fig. 7 a–d

a

b

c

d

39


IV

Fig. 7 e–h

e

f

g

h


IV

Neutrophilic Myelocytes, Metamyelocytes, Bandand Segmented Forms, also Eosinophilic andBasophilic Granulocytes and Tissue Basophils(Fig. 8 a–h)

The offspring of the promyelocytes are the mye-locytes (Fig. 8 a–e). Generally these cells aresomewhat smaller than their immediate precur-sors, with diameters ranging from 14 to 20 lm.As the coarse promyelocytic granules becomemore sparse, the typical fine neutrophilic granu-larity becomes predominant. The basophilic cyto-plasm lightens from the nucleus outward, becom-ing acidophilic, while the nuclear chromatin ac-quires a coarser structure. Nucleoli are rarely visi-ble. The myelocyte is the most plentiful granulo-cytopoietic cell type found in the bone marrow.

As maturation proceeds, the nuclear chroma-tin becomes even coarser and more dense. Thenucleus becomes indented or horseshoe-shaped,while the cytoplasm and granules remain essen-tially the same as in mature myelocytes. The cellsat this stage are calledmetamyelocytes or juvenileforms (Fig. 8 d). A few may be found in the per-ipheral blood. Metamyelocytes are no longer cap-able of division.

The band neutrophil (Fig. 8 e) is distinguishedfrom the metamyelocyte by its smaller and morecoarsely structured nucleus. Its cytoplasm andgranules are like those of metamyelocytes. Con-strictions begin to appear in the nucleus, butthe cell is not classified as a segmented form untilthe bridge between two nuclear lobes is filiform orless than one-third the width of the adjacent lobes.

The nuclear lobes of the segmented neutrophil(Fig. 8 b, c, g) present a coarse, clumped chroma-tin structure. Most of these cells contain 3–5 nu-clear lobes usually joined by thin, short chroma-tin filaments. A cell containing more than 5 lobesis said to be hypersegmented. This is especiallycommon in pernicious anemia but is not pathog-nomonic for that condition. The band and seg-mented forms range from 10 to 15 lm in size.

Eosinophilic granulocytes (Fig. 8 b, e, f) developin much the same way as neutrophils, but the twocell types are always distinguishable from the pro-myelocyte stage onward. The nuclei have struc-

tures similar to the corresponding maturationstages. The typical large eosinophilic granules al-most completely obscure the cytoplasm. Con-spicuously large granules, sometimes of a deepblue color, may be found among these mature eo-sinophilic granules in the early stages of promye-locytes and myelocytes (Fig. 8 b, e) but are nolonger present in the mature stages. Another ty-pical feature of eosinophils is their nuclear seg-mentation, with most cells possessing two nuclearlobes and a smaller number having three. Mitosesof eosinophils are also occasionally found in bonemarrow. All eosinophils are peroxidase-positive.

Basophilic granulocytes (Fig. 8 g) also have a de-velopmental pathway similar to neutrophils. Ty-pically they contain large basophilic granules thatobscure the cytoplasm and even cover the nu-cleus. In segmented basophils the nucleus con-sists of multiple lobes and often presents a clover-leaf shape. The cells tend to be somewhat smallerthan neutrophils and eosinophils. They are usual-ly peroxidase-negative when standard techniqueis used.

Mast cells (tissue basophils, Fig. 8 h) are classi-fied into two types based on nuclear size andshape and granule density. Mastoblasts and pro-mastocytes have a relatively large nucleus with anindistinct structure and sparse granules. Masto-cytes are large cells (diam. 15–30 lm) with around, compact nucleus showing structural simi-larities to the lymphocyte or plasma cell. Acidmucopolysaccharides are responsible for the typi-cal metachromasia of the granular stain patternand can be demonstrated cytochemically with to-luidine blue stain (for method, see p. 7). A strongnaphthol AS-D chloroacetate esterase reaction isalso typical (Fig. 8 h, right). Mast cells produceheparin and histamine in a ratio of 3 : 1. Isolatedmast cells may be found in fragments of normalbone marrow. The mast cell population in bonemarrow is increased in severe inflammatory dis-orders, panmyelophthisis, non-Hodgkin lympho-ma, and especially in Waldenstrom disease (lym-phoplasmocytoid immunocytoma) and hairy cellleukemia. Atypical mast cells may be found inmastocytoses (Figs. 128, 129).

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Fig. 8 a–d

a

b

c

d


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Fig. 8 e–h

e

f

g

h

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4.1.3 Degenerate Forms, Toxic Changesand Artifacts (Fig. 9 a–d)

Rarely, degenerate leukocyte forms (Fig. 9 a) maybe found in the peripheral blood of patients ex-posed to certain irritants. They are more com-monly seen in smears prepared from long-storedblood previously treated with EDTA or citrate so-lution. Most of these cells have the same diameteras segmented forms, but many are considerablysmaller (4–8 lm). Usually their cytoplasm isslightly more basophilic than in segmented forms,and their granules are coarser and oftensmudged. Pronounced nuclear pyknosis is typi-cal, and 3–5 solid, featureless nuclear remnantsmay be found scattered like droplets in the cyto-plasm. There are few if any filaments intercon-necting the nuclear remnants.

In cases of severe infection or bone marrowinjury, it is common to find large purple granulesin the myelocytes and in more mature stages upto the segmented forms. Often they are similar tothe granules seen in promyelocytes, and manyauthors consider them to be identical. This “toxicgranulation” (Fig. 9 b) is variable in its intensity.In very pronounced cases the neutrophils maycome to resemble basophilic granulocytes.

Cytoplasmic vacuoles in leukocytes, like toxicgranulations, can develop in response to varioustoxic insults. They are often observed duringlong-term chloramphenicol therapy, phenylketo-nuria, diabetes mellitus, and in the setting of se-vere bacterial and viral infections. The vacuolesreflect a metabolic disturbance in the affectedcells.

Heparin artifact. Adding heparin to peripheralblood or especially bone marrow before prepar-ing smears leads to artifacts when a panoptic stainis used (Giemsa or Pappenheim): the cells showscant or atypical staining, and a purple, crumblyprecipitate forms on the background, making itdifficult or impossible to identify the cells(Fig. 9 c).

Necrotic bone marrow. Bone marrow that hasbeen aspirated and stained may be found to con-tain unstructured purple material or faint,shadowy cells with indistinct outlines. The causeis necrotic bonemarrow at the aspiration site. Thenecrosis may be quite extensive and is occasion-ally seen in acute leukemias and other diseases(Fig. 9 d).


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Fig. 9 a–d

a

b

c

d

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4.1.4 Congenital Anomaliesof Granulocytopoiesis (Fig. 10 a– f)

Pelger-Huet Anomaly (Fig. 10 a–c)

This is an inherited anomaly involving the nucleiof granulocytes. The heterozygous form predomi-nates in man while the homozygous form, char-acterized by small round or oval nuclei (Fig. 10 c),is extremely rare. The nucleus of neutrophils isindented and resembles the band form, givingrise to a “pseudoregenerative” white blood pic-ture. When nuclear segmentation (lobulation) oc-curs, the neutrophils acquire two nuclear lobesand rarely three. These lobes are exceptionallyshort, thick, and chromatin-rich. Pelger myelo-cytes and band forms also have very coarse,clumped nuclei rich in chromatin. The patientis classified as a full carrier if all neutrophilsare affected by the anomaly and a partial carrierif normal band and segmented forms are also pre-

sent. The Pelger-Huet anomaly is harmless in itseffect on leukocyte function. Severe infectionsand particularly myelodysplasias, acute myeloidleukemia, and advanced chronic myeloid leuke-mia can produce transient, qualitatively similarchanges in white cell nuclei, creating what isknown as “pseudo Pelger-Huet forms.”

Alder-Reilly Anomaly (Fig. 10 d– f)

Here the granulocytes contain large, bluish gran-ules that often resemble those of promyelocytes;monocytes have large granules, too. The abnor-mal granulation is especially marked in eosino-phils, which appear basophilic rather than eosino-philic (Fig. 10 e, left). The lymphocytes also con-tain particularly large azurophilic granules(Fig. 10 f). Carriers of this anomaly frequentlyhave associated bone and joint deformities (gar-goylism). The anomaly is known to occur in mu-copolysaccharidosis VI and VII.


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Fig. 10 a–d

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b

c

d

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Fig. 10 e– f

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Steinbrinck-Chediak-Higashi Anomaly (GranularGigantism of Leukocytes) (Fig. 11 a– f)

This condition affects virtually all leukocytes. Theneutrophils contain irregular, grayish-blue cyto-plasmic inclusions 1–3 lm in diameter. Thesebodies are sharply demarcated and contain per-oxidase and also CE in some cases, identifyingthem as primary granules (Fig. 11 a, b, e, f).The granules of eosinophilic leukocytes are alsoenlarged to 2–3 times the size of normal eosino-philic granules. They are round to oval in shapeand variable in size. Most lymphocytes andmono-cytes also contain intensely red-staining granules1–2 lm in diameter. The inclusions in the mono-cytes are 5 lm in diameter and stain pink(Fig. 11 d). In the bone marrow, red-violet bodies

1– 3lm in diameter can be demonstrated in semi-mature and mature cells starting with the pro-myelocytes. In addition, the myeloblasts andmyelocytes frequently contain large vacuoles inwhich a large, round inclusion is often found(Fig. 11 c– f). Phase contrast and electron micro-scopy reveal coarse cytoplasmic inclusions of apleomorphic structure in neutrophils and eosino-phils, lymphocytes, and even in erythroblasts.There is further evidence indicating that thedisease is based on a defect in the lysosomalmembrane. This pathogenic disturbance, whichexerts its major biochemical effect on glycolipids,affects not only the blood cells but other organsas well and thus cannot be considered innocu-ous. Affected individuals usually die at an earlyage.

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Fig. 11 a–d

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b

c

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Fig. 11 e– f

e

f

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May-Hegglin Anomaly (Fig. 12 a–d)

This disorder has an autosomal dominant modeof inheritance and is associated with mild leuko-penia and thrombocytopenia. The neutrophilicgranulocytes contain predominantly rod-shapedinclusions of a pale- to dirty-blue color, approxi-mately 2–5lm in diameter, which are found onelectron microscopy to consist of dense RNA fi-brils and are distinguishable from the Dohle

bodies that occur in severe infections. The inclu-sions also occur in monocytes and eosinophils,but they are very difficult to detect in these cells.They can be selectively demonstrated with methylgreen-pyronine stain (red) (Fig. 12 a, b). Giantplatelets are also detected (Fig. 12 c). There isone reported case (H.L.) in which bone marrowexamination revealed a coarse, nonhomogeneousclumping of granules in the cytoplasm of themegakaryocytes (Fig. 12 d).


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Fig. 12 a–d

a

b

c

d

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4.1.5 Cells of the Monocyte-MacrophageSystem (Fig. 13 a–h)

Typical monocytes with pleomorphic nucleus,pale blue cytoplasm, and fine, barely perceptibleazurophilic granules (Fig. 13 a, b).

Monocyte, with a metamyelocyte at upper left(Fig. 13 c).

Monocyte with a small nucleus and, below it, asegmented neutrophil (Fig. 13 d).

Two promonocytes in a bone marrow smear(Fig. 13 e).

Two promonocytes with nucleoli, and a mono-cyte at upper left (Fig. 13 f).

Monocyte with phagocytized nuclear residue(Fig. 13 g).

Monocyte with phagocytized material in the cyto-plasm (macrophage) (Fig. 13 h).

Monocytes. The monocyte is an exceptionallypleomorphic blood cell ranging from 12 to20 lm in size. Its cytoplasm often has irregularborders and stains a characteristic grayish blue.Some monocytes contain azurophilic granulesmuch finer than those seen in lymphocytes.The nucleus is seldom round; usually it is deeplyindented and lobulated (often bean-shaped). Itsloose, delicate chromatin pattern is uniqueamong the blood cells. Clumps of chromatinmay be found scattered among larger, chroma-tin-poor areas. Nucleoli are rarely present.


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Fig. 13 a–d

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b

c

d

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Fig. 13 e–h

e

f

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Macrophages in the Bone Marrow (Fig. 14 a–h)

Macrophage with nuclei, erythrocytes and plate-lets in the cytoplasm (nucleus at lower right)(Fig. 14 a).

Two macrophages with cellular residues(Fig. 14 b).

Macrophage showing high acid phosphatase ac-tivity (red) and nuclear residues (bluish gray)(Fig. 14 c).

Binucleated macrophage with predominantlyfine-grained hemosiderin in the cytoplasm (yel-low-gold) (Fig. 14 d).

Abundant hemosiderin, some coarsely granular,in the cytoplasm (Fig. 14 e).

Clumped and diffusely scattered iron in the cyto-plasm of macrophages (Berlin blue reaction)(Fig. 14 f).

Peculiar blue pigment in the cytoplasm (Fig. 14 g).

Two lipophages containing droplets of stored fat(Fig. 14 h).

The bone marrow cells formerly known as reticu-lum cells in cytologic practice are actually quitediverse. A great many cells that cannot be fitted

into standard classification schemes have beengrouped under the heading of “reticulum cells.”Current concepts regarding the origin and classi-fication of the cell forms of interest here will bediscussed below. A large portion of the “reticulumstorage cells,” but probably not all, are derivedfrom the monocyte-macrophage system. The re-ticulum cells of the bone marrow in the strictsense form the reticular or spongy tissue that con-stitutes the matrix of all hematopoiesis. Othertypes are vascular endothelial cells and sinus en-dothelial cells, which occasionally are found inclusters. Arterial endothelial cells are distinguish-able from other “reticulum cells” by their positivealkaline phosphatase reaction. When spread ontoa slide, the cells lose part of their cytoplasm andpresent as “naked nuclei.” The cells formerlycalled small lymphoid reticulum cells probably re-present tissue-bound lymphocytes. The shapeand appearance of reticulum cells vary greatlywith the purpose and functional status of thecell. The typical nucleus presents a loose “reticu-lar” structure with one or more small nucleolithat are fairly indistinct. Usually there is a broadrim of cytoplasm that is poorly demarcated fromits surroundings. The cytoplasm usually appearsclear or pale blue on staining. Vacuoles are com-mon.

Dendritic and interdigitating reticulum cells,which carry out important immunologic func-tions, will be described later.

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Fig. 14 a–d

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b

c

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Fig. 14 e–h

e

f

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4.1.6 Megakaryocytes (Fig. 15 a–e)

These largest of the bone marrow cells show ex-treme morphologic diversity. The young precur-sors are called megakaryoblasts, of which thesmallest diploid forms are only slightly largerthan myeloblasts, which they resemble (Fig.15 a). The diploid forms develop into polyploidforms, which have two or four small nuclei orone large nucleus depending on whether the nu-cleus undergoes true division or simple redupli-

cation (endomitosis). A polyploid promegakaryo-cyte with only one nucleus is shown in Fig. 15 b.

Cell c is somewhat more mature, as we seefrom its polychromatic cytoplasm in which acid-ophilic elements coexist with the basophilicground substance (Fig. 15 c).

Cell d is a mature megakaryocyte with an in-dented nucleus, blurred chromatin pattern, and ared-violet, shadowy cytoplasm (Fig. 15 d).

Cell e conveys the impression of platelet for-mation at lower left and upper right (Fig. 15 e).


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Fig. 15 a–d

a

b

c

d

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Fig. 15 e

e


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4.1.7 Osteoblasts and Osteoclasts(Fig. 16 a–f)

Group of osteoblasts in a bone marrow smear.The clear area in the cytoplasm lies distantfrom the nucleus (Golgi region) (Fig. 16 a).

Cluster of osteoblasts at high magnification. Theclear area in the cytoplasm is plainly visible andlies farther from the nucleus than in plasma cells(Fig. 16 b).

Extremely high alkaline phosphatase activity inosteoblasts. Besides mature granulocytes and vas-cular endothelial cells, these are the only cells inthe bone marrow that display high alkaline phos-phatase activity (Fig. 16 c).

Multinucleated osteoclast (separate round nuclei)in the bone marrow (Fig. 16 d).

Elongated portion of an osteoclast, showing a ser-ies of adjacent nuclei (Fig. 16 e).

Twisted osteoclast with coarse inclusions (re-sorbed bone) (Fig. 16 f).

A familiarity with osteoblasts is important so thatthey can be distinguished from plasma cells or,when clustered, from tumor cells. Most osteo-blasts are oblong with a maximum diameter ofapproximately 30 lm. As a rule the nucleus is ec-centrically placed, has a reticular chromatin pat-tern, and usually contains several deep-blue nu-cleoli. The cytoplasm is dark blue and contains aclear zone, the archoplasm, situated some dis-tance from the nucleus. Osteoblasts have high al-kaline phosphatase activity.

Osteoclasts are a special type of foreign-bodygiant cell derived from monocytes. They containseveral or numerous round or oval nuclei, usuallywith one small nucleolus. They display high acidphosphatase activity. The cytoplasm is reddish-violet and finely granular. It may contain coarseinclusions believed to be remnants of resorbedbone. Exceptionally large osteoclasts with asmany as 100 nuclei (polykaryocytes) may befound in the bone marrow of children and espe-cially in patients with osteitis fibrosa generalisata.Osteoclasts are always multinucleated giant cellsin contrast to megakaryocytes with one large,sometimes polymorpheous nucleus.

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Fig. 16 a–d

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b

c

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Fig. 16 e– f

e

f

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4.1.8 Lymphocytes and Plasma Cells(Fig. 17 a–g)

Small lymphocyte with narrow rim of pale tomoderate blue cytoplasm (Fig. 17 a).

Small lymphocyte at top, compared with a mono-cyte below. Note the difference in nuclear struc-ture (Fig. 17 b).

Coarsely granular lymphocyte with numerous, re-latively coarse azurophilic granules in a broad,pale cytoplasmic rim. Cellular morphology is con-sistent with a T cell or NK cell (Fig. 17 c).

Plasma cell from peripheral blood (“blood plasmacell”) with highly basophilic cytoplasm and aperinuclear halo (Fig. 17 d).

Typical mature plasma cell in the bone marrowwith an eccentric nucleus and perinuclear halo(Fig. 17 e).

Group of mature plasma cells with a spoked-wheel nuclear structure, which usually is apparentonly in tissue sections (Fig. 17 f).

Binucleated mature plasma cell (not uncommonin reactive states) (Fig. 17 g).

The dominant forms in healthy adults are smalllymphocytes with a compact nucleus. Occasion-ally they are accompanied by larger cells thatmay contain azurophilic granules in their cyto-plasm.

Plasma cells in the bone marrow are character-ized by a basophilic cytoplasm and an eccentricnuclear position. The cells range from 14 to20 lm in size. The cytoplasm of mature formsis darkly basophilic (due to a high content of er-gastoplasm), often shadowy, and always agranu-lar. Small to moderately large cytoplasmic va-cuoles also are commonly seen. The nucleus ofimmature cells is centered, shows a delicate chro-matin pattern, and contains 1– 3 nucleoli. The nu-cleus of mature plasma cells is always eccentricand shows a coarse chromatin pattern similarto that in mature lymphocytes. Nucleoli cannotbe demonstrated in the mature forms with ordin-ary stains. Binucleated or even multinucleatedforms are not uncommon. The individual chro-mosomes in mitotic figures are relatively coarse.


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Fig. 17 a–d

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b

c

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Fig. 17 e–g

e

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5. Bone Marrow

5.1 Composition of Normal Bone Marrow

As stated on p. 23, normal bone marrow containsthe precursors of erythrocytes, granulocytes, pla-telets, stromal cells, tissue mast cells, B lympho-cytes, and occasional osteoblasts. The abundanceof stromal cells varies with the smear techniquethat is used. Reticulum cells are sparse in purebone marrow fluid but are more plentiful inthe smear from a bone marrow fragment. Maturehematopoietic cells develop within a few daysafter bone marrow transplantation and evenmore rapidly following peripheral stem cell trans-plantation (Fig. 20a–c).

Various techniques can be used to perform adifferential cell count in bone marrow aspirates.Formerly we determined the cell counts per 100leukocytes and their precursors. In the countingof marrow fragments, which should be free ofblood if possible, a maximum of 10 lymphocytesare found per 100 leukocytes. We also find ap-proximately 30 or at most 40 erythrocyte precur-sors per 100 leukocytes, 2 to 6 plasma cells, andapproximately 10 reticulum cells, the exact countvarying, as noted, with the smear technique. It isvery difficult to state figures for megakaryocytes,but this is not essential for routine clinical tests. Abetter way to obtain comparable absolute figures,however, is by counting all bonemarrow cells (ex-cluding megakaryocytes) sequentially, continu-ing until at least 200 cells have been counted. Ta-ble 3 shows the distribution that Bain obtained by

counting 500 nucleated cells in the bone marrowsmears from 50 healthy subjects.

Table 4 shows the changes in differential cellcounts that are associated with certain commondiseases. Cytochemical procedures have gainedmajor importance in the diagnosis of hematologicdisorders in recent years. Cytochemical stainingmethods are described on pp. 7– 16, and the stain-ing characteristics of the various blood cells andtheir precursors are reviewed in Table 2 and inFigs. 18 and 19 for the most common stainingmethods.

Morphologic, cytochemical, and immunologictechniques must be used for the accurate differ-entiation of leukemias and lymphomas.

Figure 21 a–d illustrates the different degreesof cellularity that may be found in bone marrowsmears in various pathologic conditions. Thesevariations underscore the importance of examin-ing the smear at low magnification before pro-ceeding with a more detailed evaluation.Figure 22 f, g shows the appearance of a normalbone marrow smear viewed at high magnifica-tion. Figure 22 a, b illustrates the range of varia-tion in the cellular and fat distribution that maybe found in normal bone marrow (histologic sec-tions). Figure 22 c shows how the naphthol AS-Dchloroacetate esterase method can be used to lo-calize early neutrophilic granulocytopoiesis closeto bony trabeculae. Figure 22 d, e illustrates smallintact vessels in a bone marrow smear demon-strated by the alkaline phosphatase method. De-tached endothelial cells appear as “reticulumcells” when this method is used.

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Fig. 18. a–d

a Peroxidase reaction(POX of Graham-Knoll)

b POX in bone marrow. Mature granu-locytes are positive, and erythroblasts arenegative. Nuclei stained with hemalum

c POX in bone marrow. At center is anegative blast; next to it is a positive,isomorphic cell (promyelocyte)

d Leukocyte alkaline phosphatase (LAP)with the substrate a-naphthyl phosphateand variamin blue salt B. Grades 1–4 inthe peripheral blood


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Fig. 18. e–h

e LAP (Sigma method). Very high activitylevels (grades 3 and 4) are observed afterG-CSF therapy

f LAP in bone marrow. Only maturegranulocytes are positive (red)

g Nonspecific esterase reaction(a-naphthyl acetate, hexazonium-para-rosaniline) in bone marrow. Monocytesare strongly positive, and a weak reactionis seen in the cytoplasm of erythroblasts

h Very strong nonspecific esterasereaction in macrophages and monocytes.A dotlike reaction pattern is seen in twolymphocytes

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Fig. 19 a–d

a Naphthol AS-D chloroacetate esterase(CE) in the bone marrow. Neutrophilicgranulocytes are strongly positive (red),and two eosinophils are negative

b CE: a promyelocyte with a positivereaction (red) in the nuclear indentationis visible. Below it is a positive myelocyte

c CE in a lymph node smear shows twotissue mast cells with strongly positivegranules, also negative lymphocytes

d PAS reaction: strong diffuse staining ina neutrophil, weak intergranular stainingin an eosinophil (left), and fine positivegranules in a monocyte (right). Neutro-phils show an increasing reaction as theymature. Some lymphocytes contain finePAS-positive granules


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Fig. 19 e–h

e Acid phosphatase (Sigma method) in amacrophage

f Berlin blue reaction for detecting iron:a “reticulum cell” at the center of anerythroblast cluster shows strong, diffusecytoplasmic staining

g Iron detection: coarse granular irondeposits in a macrophage

h Iron detection: fine granularcytoplasmic reaction in an endothelialcell (may be seen after the intravenousadministration of iron)

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Fig. 20 a–c. Mature cells of hemato-poiesis following peripheral stem celltransplantation

a

b

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Fig. 21 a–d. Variations in the cellular-ity of bone marrow smears

a Normal bone marrow

b Hypocellular bone marrow

c Hypercellular bone marrow

d Hypercellular bone marrow withnumerous megakaryocytes

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Fig. 22 a–d

a, b Range of variation in the cellular andfat distribution of normal bone marrow

b

c Localization of early neutrophilicgranulocytopoiesis (naphthol AS-Dchloroacetate esterase method)

d, e Small intact vessels in the bonemarrow smear (e demonstrated byalkaline phosphatase method)


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Fig. 22 e–g e

f, g Normal marrow smear viewed athigh magnification

g

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Table 3. Percentage of bone marrow cells found in smears from 50 healthy subjects (slightly modified fromBain BJ 1996) Br J Haematol 14:206–209

Observed range 95 % range (Average)

Blasts .0–3.2 .0–3.0 (1.4)

Promyelocytes 3.6– 13.2 3.2– 12.4 (7.8)

Myelocytes 4.0–21.4 3.7– 10.0 (7.6)

Metamyelocytes 1.0–7.0 2.3 –5.9 (4.1)

Band and segmented forms

Men 21.0–45.6** 21.9–42.3 (32.1)

Women 29.6–46.6** 28.8–45.9 (37.4)

Eosinophils 0.9–7.4 0.7–6.3 (3.5)

Basophils .0–0.8 .0–0.4 (0.1)

Erythroblasts

Men 18.0–39.4** 16.2–40.1 (28.1)

Women 14.0–31.8** 13.0–32.0 (22.5)

Lymphocytesa 4.6–22.6 6.0–20.0 (13.1)

Plasma cells .0– 1.4 .0– 1.2 (0.6)

Monocytes .0–3.2 .0–2.6 (1.3)

Macrophages .0– 1.8 .0– 1.3 (0.4)

Myeloid:erythroid ratio

Men 1.1 –4.0* 1.1 –4.1 (2.1)Women 1.6–5.4* 1.6–5.2 (2.8)

Significance of the difference between men and women: *p = 0.01, **p = 0.001.a The percentage of lymphocytes in small children may reach approximately 35 %, and isolated lymphatic blasts(“hematogones”) are found which express CD 19.


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Table 4. Changes in differential counts of bone marrow in various diseases

Normal Iron deficiencyanemias

Hemolyticanemias

Megaloblasticanemias

Infection Chronic myeloidleukemia

Chronic lympho-cytic leukemia

Agranulocytosis

20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80

Reticulum cells

Plasma cells

Megaloblasts

Basophilic

proerythroblasts

Polychromatic

erythroblasts

Orthochromatic

normoblasts

Myeloblasts

Neutrophilic

promyelocytes

Neutrophilic

myelocytes

Neutrophilic

metamyelocytes

Band neutrophils

Segmentedneutrophils

Immatureeosinophils

Mature eosinophils

Basophils

Monocytes

Lymphoblasts

Lymphocytes

Megakaryocytes þ þ�þþ þ (þ) þ þþ (+) +

(+) = weakly positive; + = positive; ++ = markedly positive

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5.2 Disturbances of Erythropoiesis

5.2.1 Hypochromic Anemias (Fig. 23a–d)

Hypochromic anemias are the morphologic pro-totype of all anemias that arise from a disturbanceof hemoglobin synthesis in erythrocytes. In casesof severe iron deficiency, the erythrocytes aresmall, flat, and feature a large area of central pal-lor (anulocytes) (Fig. 23a). Typically there is a“left shift” of erythropoiesis, meaning that thereis a predominance of younger basophilic forms.Nuclear-cytoplasmic dissociation is also present,i.e., the nucleus is relatively mature while the cy-toplasm still appears strongly basophilic and maybe poorly marginated (Fig. 23b, c).

The quantitative changes in erythropoiesis canbe quite diverse. Iron deficiency due to an acute orchronic blood loss is usually associated with amarked increase of erythropoiesis, with a shiftin the balance of erythropoiesis and granulocyto-poiesis in favor of red cell production. Addition-ally, the megakaryocytes are usually increased innumber.

By contrast, there is often an absolute reduc-tion of erythropoiesis in toxic-infectious processesand neoplastic diseases (“chronic disease ane-mias”), although there are no hard and fast rules.

The bone marrow changes found in iron utili-zation disorders (sideroachrestic anemia, “irondeficiency without iron deficiency”) are similarto those observed in iron deficiency. They canbe differentiated by iron staining (see p. 9).

In the iron deficiency anemias (Fig. 23a–d) itis rare to find siderocytes and sideroblasts, andiron deposits are never detected in macrophages(Fig. 23d). On the other hand, sideroachrestic an-emias are characterized by numerous sideroblastswith coarse granular iron deposits (ringed sidero-blasts, Fig. 61) and massive iron storage in themacrophages [see myelodysplastic syndromes,refractory anemia with ringed sideroblasts(RARS)]. Iron-storing cells can also be found ininfectious and neoplastic anemias. The variousforms of iron deficiency and their pathogenesisare reviewed in Scheme 1.

Scheme 1. Etiologic factors in iron deficiency and its symptoms.[From Begemann H, Begemann M (1989) Praktische Hamatologie, 9th ed. Thieme, Stuttgart]


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Fig. 23 a–d

a Erythrocytes in severe iron deficiency.The large area of central pallor (anulo-cytes) is typical. The erythrocytes are flat,small, and appear pale

b Group of bonemarrow erythroblasts iniron deficiency. The basophilic cytoplasmcontrasts with the relatively mature nu-clei (nuclear-cytoplasmic dissociation)

c In severe iron deficiency, even the cy-toplasm of some mature erythroblasts isstill basophilic and has indistinct margins

d Iron stain reveals absence of ironstores in bone marrow fragments due tosevere iron deficiency

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5.2.2 Hemolytic Anemias

Hemolytic anemias (HA) are characterized by ashortening of the erythrocyte life span, whichnormally is about 120 days. Anemia will develop,however, only if the bone marrow is unable to in-crease red cell production sufficiently to compen-sate for the increased rate of destruction. If theerythropoietic response is adequate, “compen-sated hemolytic disease” is present. “Decompen-sated hemolytic disease” exists when there is a dis-proportion between the destruction and produc-tion of erythrocytes. The best way to detect shor-tened erythrocyte survival is by chromium radi-olabeling of the cells (51Cr). This technique canalso identify the preferential site of erythrocytedestruction (e.g., the spleen).

When functioning normally, the bone mar-row will respond to an increase in hemolysiswith erythroid hyperplasia, which is manifestedby a predominance of mature, nucleated red cellprecursors (normoblasts). Usually these precur-sor cells do not show significant qualitative ab-normalities. But if the hemolysis is of long dura-tion, megaloblastic changes can develop mainlyas a result of folic acid deficiency, which can bedetected in the serum.Granulocytopoiesis is qua-litatively and quantitatively normal in mostcases. It is common to find increased numbersof phagocytized red cells (erythrophagocytosis)and iron deposits in the macrophages (seeFig. 14a). Examination of the peripheral bloodmay show an increased reticulocyte count (usual-ly by several hundred per thousand), basophilicstippling of red cells, occasional normoblasts(Fig. 24a), especially in acute hemolysis, and leu-kocytosis, depending on the rate of red cell de-struction and the level of bone marrow activity.Besides these nonspecific changes, there arefindings considered pathognomonic for specificentities [spherocytes (Fig. 24b), ovalocytes(Fig. 24c), and sickle cells (Fig. 25d, e)]. In addi-tion, Heinz body formation is characteristic ofa number of enzymopenic HA, and methemoglo-bin is increased in toxic HA.

Several groups of hemolytic anemias are re-cognized on the basis of their pathogenetic me-chanisms, as shown in Scheme 2.

Hemolytic anemias may also be classifiedclinically as acute (acute hemolytic crisis) orchronic. It is common for the chronic course tobe punctuated by episodes of acute disease.

The absolute increase of erythropoiesis thatoccurs during the course of regenerative hemoly-tic anemias is illustrated in Fig. 24e, f.

The most common corpuscular HA in CentralEurope is spherocytic anemia ormicrospherocyto-sis, which is easily recognized by the typical mor-

phology of the red blood cells (Fig. 24b) (see alsoPrice-Jones curves, Scheme 3).

The principal hematologic features of thalasse-mia are anisocytosis, hypochromic erythrocytes,poikilocytosis, schistocytes, and especially targetcells (see Fig. 24g). The marked elevation of HbFin thalassemia major can be demonstrated bystaining (see Fig. 24h; for method, see p. 9). Ex-amination of the bone marrow in thalassemiashows, in addition to increased erythropoiesis,iron-storing macrophages along with scatteredpseudo-Gaucher cells (Fig. 25a, b). Some matureerythroblasts contain PAS-positive granules, andsome macrophages show a bright red PAS reac-tion (Fig. 25c, left and right).

Sickle cells are most easily detected by the di-rect examination of a deoxygenated blood sample(see Fig. 25d, e; for method, see p. 5). CO hemo-globin also can be visualized by staining.

One class of toxic HA is characterized by ery-throcytes that contain deep-blue, rounded, ofteneccentrically placed inclusion bodies after specialstaining that were first described by Heinz. TheseHeinz bodies display a special affinity for vitalstains (Nile blue sulfate, brilliant cresyl blue)(see p. 8 and Fig. 24d). They occur almost exclu-sively in mature erythrocytes and are very rarelyfound in normoblasts and reticulocytes. Heinzbody formation results from the oxidative dena-turation of hemoglobin and is particularly com-mon in glucose-6–phosphate dehydrogenase de-ficiency.

However, this phenomenon occurs only afterthe ingestion or administration of substancesthat are harmless in persons with a normal ery-throcyte metabolism, such as antimalarial drugs,anticonvulsants, analgesics, sulfonamides, nitro-furan, sulfones, certain vegetables, fava beans,and a number of other drugs and chemicals.

Heinz bodies can also occur in the absence ofprimary erythrocyte metabolic defects followingintoxication with phenols, aniline, phenacetin,salicylazosulfapyridine, and many other sub-stances. Again, this probably results from thedose-dependent blocking of various intraerythro-cytic enzymes by the offending compound.

Very rarely, Heinz body formation is seen incongenital hemolytic anemias following splenec-tomy (hereditary Heinz body anemia). Since thepresence of an instable hemoglobin with a patho-logic thermal stability has been demonstrated inthis anemia, the disease has been classified as ahemoglobinopathy.

The principal serogenic HA caused by isoanti-bodies is hemolytic disease of the newborn (HDN),a consequence of fetomaternal Rh incompatibil-ity. Examination of the infant’s blood usually re-veals large numbers of erythroblasts. These cells


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Scheme 2. Classification of hemolytic anemias (HA)

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probably originate from extramedullary hemato-poietic foci, which can be quite extensive in new-borns. The example in Fig. 25f shows a number ofnormoblasts.

Erythrocyte-storing macrophages (erythro-phagocytosis) are a very common finding in auto-immune hemolytic anemia caused by warm-reac-tive, cold-reactive and bithermal antibodies (seeFig. 25i, left). In cold agglutinin disease, the ag-glutination of erythrocytes is observed on acold microscope slide but is inhibited on awarm slide (Fig. 25i, right).

In acute alcoholic HA with associated lipide-mia (Zieve syndrome), examination of the bone

marrow reveals abundant fat cells in additionto increased erythropoiesis.

Hemolytic anemias due to mechanical causesare marked by the presence of characteristic ery-throcyte fragments (fragmentocytes, schizocytes).Erythroblasts are also found if hemolysis is severe(Fig. 25g).

Finally, reference should be made to the Hchains (b-chain tetramers) that can be demon-strated by supravital staining. When these chainsare present, densely stippled erythrocytes arefound (Fig. 25h, center).


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Fig. 24 a–d

a Blood smear in autoimmune hemolyticanemia (AIHA) with three normoblastsand polychromatic erythrocytes (reticu-locytes)

b Blood smear in spherocytic anemiashows small, round erythrocytes packedwith hemoglobin (microspherocytes).These cells are characteristic but notspecific, as they also occur in autoim-mune hemolytic anemias

c Elliptocytes: the narrow elliptical form,as shown here, is specific for hereditaryelliptocytosis

d Heinz bodies demonstrated by Nileblue sulfate staining. These bodies occurmainly in association with enzymopenichemolytic anemias or hemoglobin in-stability

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Fig. 24 e–h

e Greatly increased, predominantly nor-moblastic erythropoiesis in hemolyticanemia

f Predominantly mature, morphologi-cally normal erythroblasts in hemolyticanemia

g Blood smear in b-thalassemia withmarked anisocytosis, poikilocytosis, andseveral typical target cells

h Detection of HbF in the peripheralblood. Erythrocytes that contain HbF arestained red


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Fig. 25 a–d

a Bone marrow smear in b-thalassemia.Increased erythropoiesis is accompaniedby hemosiderin-containing macrophages

b Storage cell in the bone marrow inb-thalassemia

c Left: two normoblasts in the bonemarrow with a granular PAS reaction inthalassemia. Right: macrophage in whichbright red-staining material is inter-spersed with yellow-gold hemosiderin(PAS reaction)

d Sickle cells in the peripheral blood insickle cell anemia

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Fig. 25 e–h

e Sickling test with sodium metabisulfitein Hb S disease

f Normoblasts in a blood smear in fetalerythroblastosis

g Fragmentocytes and a freshly expellederythroblast nucleus (still adherent) inthrombotic thrombocytopenic purpura(TTP)

h Reticulocytes and large Heinz bodiesadjoined at center by a finely stipplederythrocyte with H chains


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Fig. 25 i Cold agglutinin disease,peripheral blood. Left: smear on a coldslide; right: smear on a warm slide

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5.2.3 Megaloblastic Anemias

This term is applied to a class of anemias whosemajor representative in Europe is the cryptogenicpernicious anemia. They are characterized mor-phologically by the appearance of megaloblastsin the bone marrow – erythropoietic cells thatdiffer from normal erythroblasts in their sizeand especially in their nuclear structure. Butthe disease process does not affect erythropoiesisalone; the granulocytes and their precursors aswell as the megakaryocytes also display typicalchanges.

These disturbances of hematopoiesis aremanifested by anemia (usually hyperchromic)and by a reduction of leukocytes and plateletsin the peripheral blood. Examination of the bloodsmear shows marked anisocytosis and poikilocy-tosis with large, usually oval erythrocytes wellfilled with hemoglobin. These megalocytes(Figs. 26, 27), as they are called, result in abroad-based Price-Jones curve whose peak isshifted to the right (see Scheme 4). Nucleatedred cell precursors that may show basophilic stip-pling are also occasionally found in the peripheralblood. Leukocytopenia results from a decreasednumber of granulocytes, some showing hyperseg-mentation.

Scheme 3. Schematic diagram of the major pathogenic factors in megaloblastic anemias and their clinicalsymptoms. [Slightly modified from Begemann H, Begemann M (1989) Praktische Hamatologie, 9th ed. Thieme,Stuttgart]


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Besides their hematologic manifestations,megaloblastic anemias affect various organ sys-tems. Gastrointestinal changes are well knownand consist mainly of Hunter’s glossitis andatrophic gastritis. Central nervous system involve-ment is present in a high percentage of cases,usually in the form of degenerative spinal corddisease with its associated symptoms. The extentand severity of organ involvement and the var-ious hematologic manifestations depend on thenature, duration, and degree of the underlyingavitaminosis as well as on individual, possibly ge-netic factors.

In the last four decades we have learned muchabout the pathogenesis of megaloblastic anemias.The great majority of these diseases are based ona deficiency of either vitamin B12 or folic acid.Both vitamins play a crucial role in the nucleicacid metabolism of the cell, and each comple-ments but cannot replace the other. A deficiencyof either vitamin (in the absence of adequatestores) will lead to a disturbance of DNA synth-esis and to a megaloblastic anemia. Disease in a

different organ systemmay even precede and pre-cipitate the anemia. Once a megaloblastic anemiahas been diagnosed, it is imperative that its causebe identified. Two large etiologic groups are re-cognized: anemias caused by a vitamin B12 defi-ciency and anemias caused by a folic acid deficit(see Scheme 3). In most cases the cause of the un-derlying vitamin deficiency can be determined. In“cryptogenic” pernicious anemia, the type mostcommon in Europe, the gastric juice lacks an in-trinsic factor necessary for the absorption of in-gested vitamin B12 (extrinsic factor) in the smallbowel. The gastric lesion responsible for the fail-ure of intrinsic factor formation is also mani-fested in a “histamine-refractory” anacidity,which is a typical symptom of the disease. The ab-sent or deficient absorption of orally adminis-tered vitamin B12 can be accurately detected inthe Schilling urinary excretion test. Today ithas become routine practice to determine the ser-um vitamin B12 level or measure the folic acidcontent of the erythrocytes.

5.00

Nu

mb

er o

f re

d c

ells

5

10

15

20

25

30

6.0 7.0 8.0 9.0 10.0 11.0µ

Hemolyticjaundice

NormalPerniciousanemia

%

Scheme 4. Price-Jones curves in hemolyticjaundice (spherocytic anemia), in health, and inpernicious anemia (megalocytic anemia)

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Fig. 26 a–h. Megaloblastic anemias

a Blood smears in pernicious anemia: atleft, severe anisocytosis, poikilocytosis, avery large megalocyte, and a normoblastwith an extra Howell-Jolly body. At rightare three megalocytes with Howell-Jollybodies

b Mitotic megaloblast with a chromo-some fragment that will develop into aHowell-Jolly body

c Very cellular bone marrow in megalo-blastic anemia, here showing a predo-minance of immature megaloblasts andthe typical fine, loose chromatin struc-ture. Incipient hemoglobin formation inthe cytoplasm signals a decrease in ba-sophilia

d Group of promegaloblasts showing atypical nuclear structure. The appearanceof the cells reflects the disturbance inDNA synthesis


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Fig. 26 e–h

e Megaloblasts at various stages of ma-turity, also metamyelocytes and seg-mented forms showing a loose chroma-tin structure

f Very pronounced nuclear abnormal-ities in megaloblasts

g Megaloblasts showing incipientapoptosis. At lower right is a giant me-tamyelocyte

h Very large megaloblast with unusuallybroad cytoplasm already showing partialhemoglobination. Below it and to theright are giant forms in the granulocy-topoiesis series

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Fig. 27 a–d

a Two giant metamyelocytes in perni-cious anemia

b Hypersegmented neutrophil in ablood smear in pernicious anemia

c Iron stain demonstrates two sidero-megaloblasts and one sideromegalocytecontaining coarse iron granules

d Hypersegmented megakaryocyte inmegaloblastic anemia


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Fig. 27 e–h

e Hypersegmented megakaryocyte witha bizarre-shaped nucleus in megaloblas-tic anemia

f, g Nonspecific esterase (a-naphthylacetate, pH 7.2): megaloblasts showstrong esterase activity that is mostpronounced in the perinuclear area

h Subtle megaloblastic change(transitional form) like that seen in mildmegaloblastic anemia or shortly after theinstitution of vitamin B12 therapy

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5.2.4 Toxic Disturbances of Erythropoiesis

In cases of chronic alcohol abuse, examination ofthe bone marrow may show vacuolation of boththe red and white cell precursors (Fig. 28c, d).Chloramphenicol is among the drugs that can im-pair erythropoiesis. Once widely used as an anti-

biotic, this drug leads to the increased formationof abnormal sideroblasts and to vacuolation ofthe cytoplasm in erythroblasts (Fig. 28a, b).Rare cases of irreversible aplastic anemia (pan-myelophthisis) have been reported as a fatalside effect.


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Fig. 28 a–d

a, b Conspicuous vacuolation in thecytoplasm of early proerythroblastsfollowing treatment with chlorampheni-col

c Vacuolation in the cytoplasm ofproerythroblasts due to alcohol abuse

d Iron in the cytoplasm of a plasma cell,demonstrated by iron staining. At upperleft is a vacuolated proerythroblast(alcohol abuse)

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5.2.5 Acute Erythroblastopenia

This severe reduction of erythropoiesis in thebone marrow occurs mainly in children butmay also develop in hemolytic anemias (aplasticcrisis). Parvovirus B19 infection has been identi-fied as the causal agent for the decreased erythro-poiesis and consequent reticulocytopenia. The di-

agnosis is established by the presence of giantproerythroblasts in the bone marrow, whichreach the size of megakaryocytes (Fig. 29a–h).Most cases resolve spontaneously in 1 to 2 weeks.

Transient erythroblastopenia in children mayoccur in the absence of parvovirus B19 infection,but these cases do not present with giant erythro-blasts in the bone marrow.


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Fig. 29 a–d

a Bone marrow smear in acute erythro-blastopenia. At the center of the field is agiant proerythroblast with intenselybasophilic cytoplasm, a loose nuclearchromatin structure, and very largenucleoli. This cell is several times largerthan a normal erythroblast and is roughlythe size of a megakaryocyte

b Another giant proerythroblast

c Giant proerythroblast next to a maturemegakaryocyte

d Overview with a group of giantproerythroblasts

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Fig. 29 e–h

e Histologic bone marrow section inacute erythroblastopenia. Two giantproerythroblasts, each with a palenucleus and very large nucleolus, appearat upper center and lower right of center.Hematoxylin-eosin

f Bone marrow section. Three giantproerythroblasts with large nucleoli andvery pale chromatin are visible to the leftand right of center. Below them are twomature megakaryocytes and granulocy-topoietic cells. Hematoxylin-eosin

g Bone marrow section. At left center is agiant proerythroblast with a pale nucleusand very large nucleolus. To the right of itis a megakaryocyte with a round nucleus,and above that is a mature segmentedmegakaryocyte. CE stain

h Bone marrow section. A pair of giantproerythroblasts are visible in the upperand lower central part of the field.Granulocytopoiesis with red cytoplasmicstain. CE reaction


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5.2.6 Chronic Erythroblastopenia(Pure Red Cell Aplasia)

This is an “aplastic anemia in the strict sense” in-volving a profound disturbance of erythropoiesis.It is characterized by an absence or severe reduc-tion of red cell precursors in the bone marrow.Granulocytopoiesis and thrombocytopoiesis areessentially normal. Reticulocytes are either absentfrom the peripheral blood or present in very smallnumbers. The result is a severe anemic state thatdominates the clinical picture. Giant proerythro-blasts are absent.

5.2.7 Congenital DyserythropoieticAnemias

These rare disorders are characterized by a severedisturbance of erythropoiesis, which leads to con-spicuous morphologic changes. Type I congenitaldyserythropoietic anemia (CDA) shows a hazynuclear structure with fine chromatin strands in-terconnecting the nuclei of separate erythroblasts(Fig. 30a, ultrastructure Fig. 30b). Multinucleatederythroblasts characterize the type II form of CDA(Fig. 30c–e). Approximately 15 %–20 % of all redcell precursors contain 2–4 nuclei, found mainlyin the more mature forms, and there are bizarreaberrations of nuclear division (karyorrhexis).The blood film shows aniso- and poikilocytosis,basophilic stippling, and Cabot rings. In typeIII CDA, bone marrow examination reveals ery-throid hyperplasia with a multinucleation of ery-throblasts affecting all maturation stages(Fig. 30f–h). Giant cells with 10– 12 nuclei are ob-served.

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Fig. 30 a–b. Dyserythropoietic anemia

a Type I bone marrow, containing erythroblasts withan indistinct chromatin structure. Right: chromatinstrands interconnecting the erythroblast nuclei.Bottom: two neutrophils nuclei showing a positivePAS reaction

b Normoblast in Type I dyserythropoietic anemia.Note the typical morphologic features of this rare dis-ease: cordlike condensations of chromatin with manysmall, rounded clear zones; invagination of cytoplasminto the nucleus (X) and disruptions in the nuclearmembrane; tiny iron deposits in the mitochondria;and cytoplasmic inclusions (E) that probably representphagolysosomes

Fig. 30 a

Fig. 30 b


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Fig. 30 c– f

c Type II dyserythropoietic anemia,characterized by mature erythroblastswith very small nuclei, often duplicated,or with bizarre nuclear shapes (karyor-rhexis)

d Binucleated erythroblast and “daisyforms”

e Erythroblasts with very small orduplicated nuclei

f Proerythroblast with six nuclei in typeIII dyserythropoietic anemia

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Fig. 30 g–h

g Erythroblasts with two, three, and sixnuclei in type III dyserythropoietic ane-mia

h Multinucleated erythroblast withmature cytoplasm and a bizarre-shapednucleus


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5.2.8 Synartesis

Figure 31 illustrates the phenomenon of synartesisin a case showing marked dyserythropoietic dis-turbances of erythropoiesis. This phenomenoninvolves a syncytium-like aggregation of erythro-blasts, which form pale cytoplasmic bridges atpoints of contact between the individual cells.On electron microscopy it is characterized bythe presence of septate-like membrane junctionsbetween erythroblasts which are caused by amonoclonal immunoglobulin which is directedagainst an antigen of erythroblast membrane.An isolated severe anaemia with reticulocytope-nia results, which is reversible after therapywith corticosteroids.

E.M. Cramer, I. Garcia, J.-M. Masse, J.-M. Zini,P. Lambin, E. Oksenhendler, F. Souni, M. Smith,G. Flandrin, J. Breton-Gorius, G. Tobelem andN. Casadevall (1999) Erythroblastic Synartesis:An Auto-immune Dyserythropoiesis. Blood 94:3683–3693

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Fig. 31 a–d. Dyserythropoieticchanges in synartesis.

a At upper left is a binucleated erythro-blast, and at center is a karyorrhecticfigure. Below there is another binu-cleated erythroblast with deformednuclei

b Five erythroblasts in close contact withclear zones at the connections

c At center are three erythroblasts,apparently clustered and featuring verylarge perinuclear halos

d A cluster of four proerythroblasts atthe center of the field appear to be closelyconnected and contain large perinuclearclear zones


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5.3 Reactive Blood and Bone MarrowChanges

The response of the bone marrow to infectionconsists of an increase in granulocytopoiesisand a relative decrease in erythropoiesis(Fig. 32a). If an abnormality of iron distributioncoexists with the infection, as is frequently thecase, some increase in erythropoiesis will alsobe seen. Usually the immature precursors ofgranulocytopoiesis are markedly increased aswell and may lead to a predominance of promye-locytes (see Fig. 32a). Eosinophilia is also com-mon. Qualitative changes in erythropoiesis andgranulocytopoiesis are manifested by nuclear-cy-toplasmic dissociation, persistent cytoplasmic ba-sophilia, toxic granulations, and nuclear abnorm-alities. The plasma cells also are increased innumber and in extreme cases can mimic a plas-macytoma (Fig. 32b; see Table 6). This is mostcommon in chronic inflammatory processes in-volving the liver (chronic hepatitis, cirrhosis)and biliary tract (cholangitis, cholecystitis, etc.).Whether the individual picture is dominated bya left shift in granulocytopoiesis or a proliferationof plasma cells depends on the virulence and toxi-city of the causative infectious organism. HIV in-fection can lead to very severe “toxic bone mar-row changes” that may be associated with signif-icant plasmacytosis and even dysplastic changesas in MDS (Figs. 32c, d and 67d, e).

The bone marrow responds to neoplastic dis-eases in much the same way as to infection andvarious toxic insults. The most commonmorpho-

logic findings in the blood and bone marrow ofpatients with infections and neoplastic diseaseare summarized in Table 5. The anemia thatdevelops in these conditions has been referredto as “anemia of chronic disease.” In cases wherethe serum iron level is low but there is a normal orelevated ferritin and adequate bone marrowstores, an abnormality of iron distribution existsrather than a true iron deficiency. The reactivechanges consist of plasma cell alterations (Russellbodies), monocytosis, and small granulomasformed by monocytes or epitheloid cells as wellas circumscribed lymphocytic foci (polyclonal)in chronic inflammatory or immunologic dis-eases (Fig. 33a–d).

Occasionally the response of the eosinophils isso pronounced that they come to dominate theblood picture. This pattern can be difficult to in-terpret etiologically. It is most commonly seen inallergic reactions, certain infectious diseases, in-sect bites, skin diseases, parasitic infections (hel-minths!), collagen diseases (hypereosinophilicsyndrome), blood diseases, and endocrine disor-ders. Eosinophils tend to be increased in the pre-sence of carcinoma, especially if the tumor hasalready metastasized. Eosinophilia is also seenin patients on hemodialysis. If the eosinophilsare sufficiently numerous to produce a more orless pronounced leukocytosis, hypereosinophiliais said to be present (Fig. 34a–h).

Eosinophilia with splenomegaly and eosinophi-lia persistans, in which splenomegaly usually co-exists with diffuse enlargement of the lymphnodes are special disorders. Apparently these

Table 5. Reactive bone marrow changes (findings in the blood and bone marrow)

Peripheral blood Bone marrow

Anemia (aplastic, hemolytic)Abnormal erythrocytesBasophilic stippling oferythrocytes

Erythropoiesis: Maturation defectsCytoplasmic vacuolesMegaloblastoid formsReduction RErythroblastophthisisHyperplasia

Pancytopenia — GranulocytopeniaAgranulocytosisToxic granulationsCytoplasmic vacuolesDoehle bodies

Panmyelopathy-Granulocytopoiesis: Maturation defectsCytoplasmic vacuolesLeft shiftEosinophiliaReductionRAgranulocytosisMonocytesGranuloma formation

ThrombocytopeniaAbnormal platelets

Thrombocytopoiesis: Maturation defectsReductionRAbsenceof megakaryocytes

Monocytopoiesis

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conditions represent severe allergic disorders ofvarying etiology. It can be difficult to differentiatethe hypereosinophilias (Fig. 34a–h) from the veryrare eosinophilic leukemias, in which the periph-eral blood is flooded with large numbers ofmature eosinophils (see Fig. 112). Often this dis-tinction can be accomplished only by observingthe clinical course, but eosinophilic leukemiascan be confirmed by the detection of chromo-somal abnormalities.

Hypereosinophilic syndrome (HES) – inwhich recently anomalies of the PDGF-receptorgene have been found – is often difficult to dis-tinguish from the eosinophilic leukemias andother hypereosinophilias. Diagnostic criteria in-clude the persistence of eosinophilia for at least6 months with leukocyte counts of 10,000 to30,000/lL and a 30 % to 70 % proportion of eo-sinophils. Most patients manifest signs and symp-toms of organ involvement such as hepato-splenomegaly, congestive cardiomyopathy, pul-monary fibrosis, etc. Frequently the eosinophilsshow morphologic abnormalities (enlarged cellswith a decreased number of weakly staining gran-ules, vacuolation, multisegmentation). Immatureforms are often present in the peripheral blood.The bone marrow is dominated by eosinophils(25 %–75 %) with a shift to the left and qualitativechanges.

“Tropical eosinophilia” is associated withlymph node enlargement, splenomegaly, pul-monary infiltration, and severe asthmatic com-plaints. B. malayi or W. bancrofti microfilariae(see Figs. 198, 199d) have been identified in theenlarged lymph nodes of most patients. Nonehave been detected in the blood, although ahigh titer against filariae has been demonstratedin the serum by complement fixation. The term“tropical eosinophilia” should no longer beused to designate a separate disease entity. Severeeosinophilia diagnosed in persons returning fromthe tropics is usually the result of a helminthiasis.

Administration of the hematopoietic growthfactors G-CSF and GM-CSF stimulates the prolif-eration and differentiation of progenitor cells.While GM-CSF also leads to the stimulation ofmonocytes and eosinophils, the administrationof G-CSF leads only to an increase in neutrophils.This is accompanied by a left shift and in somecases by mild atypias that do not adversely affectcellular function. Neutrophilic alkaline phospha-tase is maximally activated. Bone marrow exam-ination following chemotherapy shows a more ra-pid regeneration of neutrophilic granulocytopoi-esis (Fig. 35a–d). The more rapid and increasedrecruitment of stem cells has proven to be a veryuseful phenomenon in the practice of bone mar-row transplantation and especially in peripheralstem cell transplantation.

Table 6. Differential diagnosis of a plasma cell increase in the bone marrow

Number Morphology Remarks

Normal bone marrow < 5 % Almost exclusively small,mature plasma cells

Reactive bone marrowchanges (toxic-infectiousor neoplastic process)

5 %-10 % Predominantly small,mature plasma cells

Very strong increase after HIV infection,in chron. inflammatory processes of theliver, biliary tract, etc., frequent bonemarrow eosinophilia

Multiple myeloma > 10 % Marked pleomorphism ofplasma cells (“immatureforms,” atypical nucleoli)

Strong acid phosphatase activity in themyeloma cells

Lymphoplasmacytoidimmunocytoma (Walden-stroms disease, Waldenstromsmacroglobulinemia)

> 10 % Marked pleomorphism Marked lymphatic infiltration, mast cells

Monoclonal gammopathyof undetermined signifance(MGUS)

> 10 % Slight pleomorphism Transition to multiple myclomais possible

Concomitantparaproteinemia

> 10 % Slight pleomorphism Especially in systemic lymphatic disease,carcinoma


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Fig. 32 a–d. Reactive blood and bonemarrow changes

a Greatly increased granulocytopoiesiswith a left shift and markedly reducederythropoiesis due to an acute infection

b Marked proliferation of mature plasmacells with eosinophilia, scattered baso-phils, and a slight increase in lympho-cytes

c Bone marrow in HIV infection, showingsignificant “toxic” abnormalities

d Marked plasmacytosis in the bonemarrow of an HIV-infected patient

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Fig. 33 a–d Reactive bone marrowchanges

a Plasma cell with numerous Russellbodies (reactive change)

b At center is a granuloma-like aggre-gation of monocytes, and below that is acell containing phagocytized material inrheumatoid arthritis

c High-power view of a monocyticgranuloma. One cell contains a phago-cytized erythrocyte

d Lymphocytic focus in the bone mar-row. Note the relatively sharp demarca-tion of the focus from the hematopoieticbackground (lower right and side of field)


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Fig. 34 a–d. Reactive eosinophilia andhypereosinophilic syndrome

a, b Pronounced eosinophilia in theperipheral blood, with typical eosinophilsthat generally contain bilobed andoccasionally trilobed nuclei

c Pronounced eosinophilia in the bonemarrow, with immature precursors con-taining small numbers of purple-stainedgranules

d At center is an eosinophilic promyelo-cyte with immature purple granulesalong with scattered mature granules.The cytoplasm is basophilic

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Fig. 34 e–h

e Naphthol AS-D chloroacetate esterasein the bone marrow in reactive eosino-philia. Only the neutrophilic granulocytesare positive; the eosinophils are negative

f Various maturation stages in reactiveeosinophilia. The cell at center left is abasophilic granulocyte

g Extreme eosinophilia of the bonemarrow in hypereosinophilic syndrome.The eosinophils are negative for naphtholAS-D chloroacetate esterase, and fourneutrophils in the field are positive

h Macrophage containing Charcot-Ley-den crystals in eosinophilia. The crystalsdevelop from the eosinophilic granules


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Fig. 35 a–d. Effect of hematopoieticgrowth factors

a Peripheral blood smear after the ad-ministration of G-CSF

b Left shift following G-CSF

c Maximum leukocyte alkaline phos-phatase activity following G-CSF

d Bonemarrow following chemotherapyand the administration of G-CSF de-monstrates promyelocytes and myelo-cytes, some showing mild atypia

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5.3.1 Agranulocytosis

Agranulocytosis is a collective term applied todiseases associated with an extreme reductionor complete absence of granulocytes in the pe-ripheral blood. A more accurate and descriptiveterm is granulocytopenia, which in fact hasbeen adopted for most diseases in this category.Agranulocytosis in the strict sense is reserved fordiseases that are based on a specific, individualhypersensitivity to exogenous or (rarely) endo-genous agents and usually have an acute onset.(This does not include “cyclic agranulocytosis,”whose pathogenesis is not yet fully understood.)Potential exogenous offenders include almost allchemicals and especially drugs, the most notor-ious being pyrazolone. The causal agent incitesan immune response in which antigen-antibodiescomplexes are formed and bring about the de-struction of granulocytes. The cytolytic processaffects not only granulocytes in the peripheralblood but also precursors in the bone marrow, in-cluding promyelocytes in some cases. The patho-genetic mechanism underlying the agranulocyto-

sis is unrelated to the formation of antibodies di-rected against endogenous granulocytes. Thus, astrict distinction must be drawn between allergicagranulocytosis and the true autoimmune granu-locytopenias.

The morphologic substrate of the antibody-mediated agranulocytosis depends on the timingof the examination. If bone marrow is sampledearly in the course, granulocytopoiesis may be al-most completely absent; if later, the characteristicpromyelocytic marrow is found (Fig. 36e). Exam-ination during the remission phase shows only anincrease in granulocytopoiesis. Erythropoiesis isnot impaired in uncomplicated agranulocytoses,and often it is relatively increased due to the over-all reduction in granulocytopoiesis. Septic com-plications can also lead to changes in red cell pre-cursors and megakaryocytes. If there is an arrestof normal granulocytopoiesis, the bone marrowfindings may closely resemble those in aplasticanemias and acute leukemias, with a potentialfor confusion. In particular, it may be possibleto distinguish hypoplastic acute leukemias onlyby observing their course.


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Fig. 36 a–h. Bone marrow inagranulocytosis

a Overall decrease in cellularity withscattered early promyelocytes and rela-tively numerous lymphocytes

b Bone marrow in agranulocytosis in avery early regenerative stage. Smallnumbers of early transitional forms be-tween blasts and promyelocytes are in-termingled with lymphocytes. An acutehypoplastic leukemia should also beconsidered at this stage. The diagnosis isestablished by serial examinations

c Bonemarrow in agranulocytosis. In thisearly regenerative phase, some neutro-phils are already peroxidase-positive. Aneutrophil mitotic figure is seen at upperright

d Early regenerative phase with “pro-myelocytic marrow,” clearly recognizedhere by its granularity

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Fig. 36 e–h

e Overview of the regenerative stage:promyelocytes are predominant but arealready accompanied by small numbersof mature forms

f Promyelocytic marrow in agranulocy-tosis. At center is a mitotic figure. Incontrast to promyelocytic leukemia, thecells are quite regular and have uniformlydistributed granules with an absence ofAuer rods

g Lymphocytic focus in the marrow inagranulocytosis. Usually these foci areclearly demarcated from their surround-ings

h Severe granulocytopenia followingchemotherapy, virtually indistinguishablefrom agranulocytosis. This specimencontains relatively large numbers ofeosinophilic granulocytes


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5.3.2 Kostmann Syndrome

Kostmann syndrome (severe congenital neutro-penia) is inherited as an autosomal recessive trait.Affected children are born with an extreme pau-city or absence of granulocytes. Granulocytopoi-esis in the bone marrow becomes arrested at thepromyelocyte stage, rarely progressing to themyelocyte stage; more mature forms are absent.

Shwachman syndrome, which has the samemode of inheritance, is characterized by neutro-penia in addition to various malformations.Granulocytopoiesis in the bone marrow is dimin-ished.

5.3.3 Thrombocytopeniasand Thrombocytopathies

Hemorrhagic disorders rarely present obviousmorphologic clues that permit a diagnosis, doingso only when they are associated with quantita-tive or qualitative changes in the blood platelets.Twomajor disease groups can be identified: thosein which the platelets are reduced in number, andthose in which the platelets are present in normalnumbers but do not function normally. Diseasesof the first group are called thrombocytopenias;those of the second, thrombocytopathies. Bothgroups have similar effects on blood coagulation,and both lead to similar clinical manifestations.

The most important of the diseases in whichplatelet counts are reduced is idiopathic thrombo-cytopenic purpura (Werlhof disease, Werlhof syn-drome). It is associated with a thrombocytopenichemorrhagic diathesis. The pathogenesis is basedon an acceleration of platelet destruction due toan autoimmune response. No characteristicchanges are found in the bone marrow(Fig. 37a). The megakaryocytes may be normalbut usually are markedly increased. There arelarge numbers of young basophilic forms witha round or minimally lobulated nucleus. Eosino-philia may be present in the bonemarrow. A com-pensatory increase in erythrocytopoiesis is usual-ly present due to prior blood losses. The abnorm-alities of platelet morphology seen in this diseaseresult from the acceleration of thrombocytopoi-esis. Giant platelets with a dense granulomereare a frequent finding and may approach thesize of leukocytes. Marked platelet anisocytosisis also common.

Deficient platelet production is causative ofthrombocytopenia in cases where bone marrowfunction has become severely compromised inthe setting of a lymphoma, plasmacytoma, leuke-mia, or by elements extrinsic to the marrow (e.g.,bone marrow metastases from solid tumors). Oc-casionally, however, essential thrombocytopeniasare encountered that must result from a depopu-lation of the megakaryocyte compartment of thebone marrow (amegakaryocytic thrombocytope-nia).

One way to determine whether a thrombocy-topenia is caused by deficient platelet productionor increased platelet turnover is by the use ofradiolabeled platelets. In hemorrhagic diathesesdue to platelet dysfunction, there is rarely a char-acteristic morphologic correlate that can be de-monstrated by panoptic staining. While these dis-eases are usually diagnosed at an early age, theydo not always present overt hemorrhagic symp-toms. The most important of these disordersare the May-Hegglin anomaly (with an autosomaldominant mode of inheritance), congenitalthrombocytic dystrophy (Bernard-Soulier syn-drome) (Fig. 37b), and Glanzmann-Naegelithrombasthenia.

Glanzmann-Naegeli thrombasthenia is not as-sociated with abnormalities of platelet count ormorphology. By contrast, giant platelets are char-acteristic of Bernard-Soulier syndrome (Fig. 37b)and May-Hegglin syndrome. Bernard-Souliersyndrome is also associated with concomitantplatelet dysfunction (autosomal recessive inheri-tance). The giant platelets and thrombocytopeniain the May-Hegglin anomaly are accompanied byspecific pale dirty-blue leukocyte inclusions,which are seen most clearly in neutrophilic gran-ulocytes but are difficult to detect in eosinophilsand monocytes. They are distinguishable fromthe “Doehle bodies” of infectious conditions byelectron microscopy (see Fig. 12a–d).

5.3.4 Pseudothrombopenia

A reduced platelet count in EDTA-treated blood(pseudothrombopenia) may result from plateletaggregation or from platelet adhesion to leuko-cytes. This laboratory phenomenon can be iden-tified as such by the microscopic examination ofsmears (Fig. 37c).

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Fig. 37a–c. Thrombocytopenias andthrombocytopathies

a Accumulation of megakaryocytes inthe bone marrow in idiopathic throm-bocytopenia. The increase is not alwaysdramatic, and a large proportion of im-mature megakaryocytes is sometimesfound

b Giant platelet in Bernard-Souliersyndrome

c EDTA-induced pseudothrombocyto-penia may result from the autoagguti-nation of platelets (platelet aggregate)left or from the adherence of platelets toneutrophils right or monocytes (satellit-ism). It is a laboratory phenomenon thatdoes not occur when other anticoagu-lants are used (citrate, heparin) because itrequires a calcium-poor medium


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5.4 Bone Marrow Aplasias(Panmyelopathies)

Bone marrow aplasias are conditions in which thebone marrow is acellular or severely hypoplastic,resulting in peripheral cytopenia. Hyperplasticbone marrow with peripheral cytopenia shouldraise suspicion of a myelodysplasia and excludesbone marrow aplasia. The old term “panmyelopa-thy” covers such a range of conditions involvingbone marrow injury that it should no longer beused.

Bone marrow aplasia can result from varioushematopoietic disturbances, which fall into thefollowing groups (see also Table 7):1. Aplastic anemia, primary or secondary2. Bone marrow aplasia secondary to cytostatic

therapy or irradiation3. “Displacement” of normal hematopoietic tis-

sue by hematologic or nonhematologic tumorcells or by myelofibrosis or myelosclerosis

Regarding group 1, aplastic anemia, which isequivalent to a panmyelophthisis when fully de-veloped, may be congenital (very rare in children)or acquired. Congenital Fanconi anemia and Dia-mond-Blackfan syndrome are known to occur inchildren along with a few extremely rare condi-tions that involve other congenital anomaliesor metabolic disorders. Aplastic anemia is classi-fied into three grades of severity on the basis ofhematologic findings:

Granulo-cytes

Platelets Reticulo-cytes

Aplasticanemia (AA)

< 1 � 109 / l < 50 � 109 / l < 40 � 109 / l

Severe aplasticanemia (SAA)

< 0,5 � 109 / l < 20 � 109 / l < 20 � 109 / l

Very severeaplastic anemia(VSAA)

< 0,2 � 109 / l < 20 � 109 / l < 20 � 109 / l

A diagnosis of aplastic anemia requires at leasttwo of the above criteria in the peripheral bloodin addition to hypoplastic or aplastic bone mar-row.

Groups 2 and 3 above must be excluded beforeaplastic anemia is diagnosed. Whenever bonemarrow aspiration yields insufficient materialfor a positive diagnosis or if the aspiration is un-successful (dry tap), the diagnosis should be es-tablished by biopsy and histologic examination.

Besides the idiopathic form of aplastic anemia,the secondary acquired forms are most frequentlycaused by drugs, particularly analgesic and anti-rheumatic agents. Viral hepatitis (< 1 % of pa-tients) and exposure to chemicals are rarely im-plicated. Finally, it should be noted that aplasticphases may occur during the course or even at theonset of paroxysmal nocturnal hemoglobinuria(PNH). Today diagnosis is made by flowcyto-metric detection of an absence or reduction ofGPI-bound antigens. Acute leukemia (particu-larly ALL) may also run through a preliminaryaplastic stage.

Table 7. Classification of panmyelopathies.[After Begemann and Rastetter (eds.) (1986) KlinischeHamatologie, 3rd ed., Thieme, Stuttgart]

I. Idiopathic or primary formsII. Symptomatic or secondary forms

a) Injurious physical or chemical agents in suffi-cient doses to produce sustained hypo- or aplasiaof the bone marrow1. Ionizing radiation (X-rays, radium, radiopho-sphorus, radiogold, etc.)

2. Cytostatic drugs3. Benzene4. Other toxic substances (e.g., arsenic com-pounds, inorganic gold preparations)

b) Agents that occasionally cause hypo- or aplasia ofthe bone marrow1. Chemical agents (e.g., chloramphenicol, an-ticonvulsants, antithyroid drugs, analgesics,insecticides, sulfonamides, etc.)

2. Infectious insults (hepatitis, etc.)c) Infiltrative neoplastic processes in the marrow

spaces (medullary carcinomatoses and sarcomasincluding hemoblastoses and malignant lym-phomas).

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Fig. 38 a–h. Bone marrow aplasia

a Complete absence of hematopoiesis inaplastic anemia (AA). The bone marrowcontains only fat cells embedded in aproteinaceous fluid (right edge of field)

b Another case of AA, also showingpanmyelophthisis. Isolated plasma cellsand lymphocytes are still interspersedamong the fat cells

c– f Smears and histologic sections froma patient with aplastic anemia. All sam-ples were taken at the time of diagnosis

c Predominance of fat cells in a bonemarrow smear. An erythropoietic area isseen at lower left

d Higher-power view of an erythro-poietic area shows interspersed lym-phocytes and two tissuemast cells (centerand lower right). The incidental aspirationof an erythropoietic area may causemisinterpretation


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Fig. 38 e–h

e Typical features of panmyelophthisis ina histologic section

f In this section, a nest of proerythro-blasts is visible at upper center

g, h Bone marrow at different stages ofparoxysmal nocturnal hemoglobinuria(PNH)

g Initial sample shows typical picture ofAA, consisting almost entirely of lym-phocytes, plasma cells, two tissue mastcells, and increased numbers of fat cells

h Bone marrow smear 3 years latershows a very cellular marrow with in-creased erythropoiesis. One megakaryo-cyte is visible in the field. This pattern issuspicious for hemolytic anemia

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5.5 Storage Diseases

5.5.1 Gaucher Disease

Gaucher disease is a cerebroside storage diseasecaused by a deficiency of b-glucocerebrosidase.It is a familial disorder that is usually diagnosedin children after 1 year of age (juvenile form), butinfantile and adult forms are also known. It pre-sents clinically with splenomegaly and occasionalhepatomegaly. A yellowish brown skin discolora-tion is typical in adults. Leukopenia is frequent,and a moderate normochronic anemia may beseen in later stages. Today the disease can be di-agnosed by detecting the enzyme deficiency in theblood. If there is no known family history, thepresumptive diagnosis can usually be confirmedby bone marrow aspiration. Gaucher cells canalso be demonstrated in splenic and liver aspi-rates or biopsies. Gaucher cells are very large sto-rage cells (up to 60 lm in diameter) with small

central or eccentric nuclei that show a round orirregular outline and little if any internal struc-ture. The abundant cytoplasm stains a pale gray-ish blue with panoptic stain and has a character-istic wispy or fibrillary appearance (like crumpledpaper). Rarely the cells may contain two or morenuclei. Gaucher cells have extremely high tar-trate-resistant acid phosphatase activity, theygive a diffusely positive PAS stain, and someshow a diffuse iron reaction.

Isolated pseudo-Gaucher cells may be found inbone marrow samples from patients with chronicmyeloid leukemia, where they have no particulardiagnostic or clinical significance. They are dou-bly refractive under polarized light, a feature thatdistinguishes them from true Gaucher cells. Pseu-do-Gaucher cells are also found in thalassemias(strongly PAS positive).

Figure 39a–h illustrates the appearance ofGaucher cells in bone marrow.


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Fig. 39 a–h. Gaucher disease

a Bone marrow smear in Gaucher dis-ease. Numerous typical storage cellsshow distinctive crumpled, fibrillary cy-toplasm with a bluish-gray tinge. Thenuclei have a loose, sometimes moth-eaten appearance

b High-power view of Gaucher cells

c Gaucher cells still containing erythro-cyte remnants are seen above and belowthe center of the field

d Prominent fibrillary cytoplasmic struc-ture in a Gaucher cell

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Fig. 39 e–h

e Very intense acid phosphatase reac-tion in Gaucher cells

f Acid phosphatase. The fibrillary struc-ture of the cytoplasm is apparent in theslightly crushed cells

g Strong diffuse PAS reaction

h Iron stain produces marked diffusestaining of the cytoplasm


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5.5.2 Niemann-Pick Disease

Niemann-Pick disease is a sphingomyelin storagedisease (sphingolipoidosis) that is based on a de-ficiency of sphingomyelinase. It is inherited as anautosomal recessive trait and produces clinicalmanifestations during childhood. Five differentbiochemical subtypes have been identified. Char-acteristic foam cells are found in the bone mar-row, liver, spleen, and lymph nodes.

Another variant is type C (NPC 1-protein de-fect) with a defect of cholesterol transport.Here you find vacuoles of different size in the cy-toplasm, sometimes blue granules. An infantileand a juvenile-adult course can be distinguished.

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Fig. 40 a–d. Niemann-Pick disease

a, b Storage cells with very small nucleiand fine, closely spaced, partially con-fluent pale bluish-gray inclusions, someof which are dislodged during stainingand appear as vacuoles (foamy cyto-plasm)

c Relatively weak PAS reaction

d The inclusions may show marked ba-sophilic staining like the storage cells insea-blue histiocytic disease, considered avariant of Niemann-Pick disease. These“sea-blue histiocytes” may also occur asstorage cells when cellular breakdown isincreased (as in this case)


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5.5.3 Glycogen Storage Disease Type II(Acid Maltase Deficiency, Pompe Disease)

In our examination of an adult with severe mus-cular dystrophy, we noted severe vacuolation inthe plasma cells of the bone marrow (Fig. 41a–d). The PAS reaction demonstrated coarse posi-tive inclusions. Electron microscopy of semithinsections and cytochemical analysis revealed thepresence of a polysaccharide- and protein-con-taining material in the “vacuoles.” 1

1 Pralle H, Schroder R, Loffler H (1975) New kind of cytoplas-mic inclusions of plasma cells in acidmaltase deficiency. ActaHaematol 53 : 109–117

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Fig. 41 a–d. Type II glycogen storagedisease (acid maltase deficiency,Pompe disease)

a, b Plasma cells contain closely spacedvacuoles of varying size, found on elec-tron microscopy and cytochemical ana-lysis to contain glycopeptide

b

c Coarse PAS-positive inclusions in theplasma cells

d The “vacuoles” are strongly positive foracid phosphatase


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5.6 Hemophagocytic Syndromes

The phagocytosis of blood cells by macrophagesmay occur in the setting of inflammatory pro-cesses, immune responses, or malignant diseases.An hereditary form, familial hemophagocyticlymphohistiocytosis, predominantly affects in-fants, with 80 % of cases occurring before thesecond year of life. Marked phagocytic stateswith greatly increased numbers of macrophageswere formerly described as malignant histiocy-

toses or histiocytic medullary reticuloses. Manyof these states may be caused by viruses (e.g., cy-tomegalovirus) and other infectious organisms.They are most common in immunosuppressedpatients but also occur in the setting of malignantdiseases. The “malignant histiocytoses” probablyconsist mainly of different forms of monocyticleukemia, and some may represent misidentifiedforms of large-cell malignant lymphoma. Trueneoplasias with a macrophagic phenotype areprobably quite rare.

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Fig. 42 a–h. Hemophagocyticsyndrome

a Low-power view of bone marrowshows several macrophages that havephagocytized platelets and erythrocytes.The cause in this case is unknown

b Macrophages with erythrocytes,platelets, and (at top right) small nuclei inthe cytoplasm

c Bone marrow from the same patientshows a phagocytized neutrophil atupper right

d Phagocytized erythrocytes andplatelets have displaced the macrophagenucleus to the edge of the cell


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Fig. 42 e–h

e Macrophage with phagocytizednormoblasts

f Phagocytosis of two rod neutrophilsand a nuclear remnant. Macrophagenucleus is at lower right

g Macrophages preserved in air-driedsmears for 15 months still show strongacid phosphatase activity

h Sample from the same patient (freshsmear) shows strong esterase activity inthe macrophages

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5.7 Histiocytosis X

Histiocytosis X (Langerhans cell histiocytosis,Fig. 43) is characterized by large cells with abun-dant grayish-blue cytoplasm and round to ovalnuclei. CD11c, CD1, and S-100 protein serve asmarkers. The Birbeck granules that are specificfor Langerhans cells can be demonstrated by elec-tron microscopy. Multinucleated giant cells arecharacteristic.


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Fig. 43 a–d

a, b Bone marrow involvement byhistiocytosis X (Langerhans cell histiocy-tosis). Note the large cells with broad,bluish-gray cytoplasm and round to ovalnuclei

b

c Nonspecific esterase reaction (ANAE)demonstrates fine positive granules inthe cytoplasm

d Demonstration of acid phosphatase inthe cytoplasm of malignant cells. Thereaction is weaker than in the macro-phages

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5.8 Chronic Myeloproliferative Disorders(CMPD)

Dameshek introduced the “myeloproliferativesyndrome” as a collective term encompassing es-sential thrombocythemia, polycythemia vera, os-teomyelosclerosis, and chronic myeloid leuke-mia. Since the detection of the Philadelphia chro-mosome (Ph) by Nowell and Hungerford in 1960and later the underlying BCR/ABL translocationby Bartram et al., a sharp distinction must bedrawn between chronic myeloid (granulocytic)leukemia and the other chronic myeloprolifera-tive disorders. The concept of CMPD is justifiedby certain similarities in the course of these dis-eases. Several apparent transitions between thedifferent forms have been elucidated using mole-cular genetic techniques and have been classifiedas various manifestations of chronic myeloid leu-kemia. Many questions remain unanswered, how-ever, and it is necessary to provide an accuratedescription of individual cases.

The diagnosis of essential thrombocythemia isbased on a consistently elevated platelet count(higher than 6� 109/l), the exclusion of a differentcause (including chronic inflammatory disease),and an increase of megakaryocytes in the bonemarrow, which often show only subtle abnormal-ities (hypersegmented nuclei) and are grouped inclusters. The peripheral blood film may show amild leukocytosis with slight basophilia and eosi-nophilia in addition to thrombocytosis. These

cases require a chromosomal and/or moleculargenetic evaluation to exclude chronic myeloidleukemia. Polycythemia vera can be diagnosedonly when findings meet the criteria defined bythe Polycythemia Vera Study Group. The cellular-ity of the bonemarrow is markedly increased, andfat cells are completely absent in fully establishedcases. There is a significant increase in megakar-yocytes, which show an extreme diversity of sizes.Erythropoiesis and usually granulocytopoiesisare markedly increased, and iron stores are ab-sent from the marrow. A slight increase of baso-phils is observed in the blood and bone marrow.Histologic examination is necessary for an accu-rate quantitative evaluation of bone marrowstructures. An increase in leukocyte alkalinephosphatase activity is detected in blood smears.Osteomyelosclerosis or myelofibrosis is character-ized by an increase in reticular fibers and/or can-cellous bone ranging to the complete obliterationof the bonemarrow and by extramedullary hema-topoiesis. The differential blood count may bevery similar to that in chronic myeloid leukemia,but there are significant erythrocyte abnormal-ities that include teardrop-shaped cells and thepresence of erythroblasts in the blood smear. Leu-kocyte alkaline phosphatase is usually elevated ornormal. L. Pahl et al. (Blood 100, 2441 (2002)) de-scribed a membrane receptor PRV-1, which isoverexpressed in polycythemia vera, partly in es-sential thrombocythemia and myelofibrosis.


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Fig. 44 a–d. Essential thrombocythe-mia (ET)

a Blood smear reveals anisocytosis and agreatly increased number of platelets

b Bone marrow smear in ET shows largemasses of platelets and scatteredmegakaryocytes

c Three mature megakaryocytes andlarge platelet aggregations

d Histologic section in ET shows asubstantial increase in moderately pleo-morphic megakaryocytes, some ar-ranged in clusters. There is a normalproportion of fat cells. Giemsa stain

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Fig. 45 a–e. Polycythemia vera

a Bone marrow smear shows markedhypercellularity with a significant in-crease in megakaryocytes, which varymarkedly in size and maturation

b High-power view shows the sizevariation of the megakaryocytes

c Bone marrow area with increasederythropoiesis and granulocytopoiesis. Atleft is a basophil

d Histologic section shows residual fatcells, a typical increase in megakaryo-cytes of varying size, and increased ery-thropoiesis. Giemsa stain


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Fig. 45 e–h

e Blood smear shows a substantialincrease in leukocyte alkaline phospha-tase activity (red)

f Bone marrow in myelofibrosis. Notethe clustering of the pleomorphicmegakaryocytes. Hematoxylin-eosinstain

g Bone marrow section in myelofibro-sis. Silver stain demonstrates heavy fiberproliferation. At lower left is a cluster ofmegakaryocytes

h Bone marrow in osteomyelosclerosis(OMS). The marrow cavity is almostcompletely obliterated by collagen andincreased cancellous trabeculae. Hema-toxylin-eosin stain

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Fig. 46 a–d

a Blood smear in osteomyelosclerosis(OMS). Monocyte and segmented cell atleft, erythroblast at center, and promye-locyte at right

b Blood smear in OMS shows significantpoikilocytosis with teardrop-shapederythrocytes. At top is a normoblast

c Blood smear in OMS shows heavybasophilic stippling and two erythrocyteswith Cabot rings. At center is anerythrocyte with Howell-Jolly bodies anda nuclear remnant

d Increased leukocyte alkaline phos-phatase (LAP) in OMS. There is one grade1 and one grade 4 neutrophil, a normo-blast, and a negative blast at upper left


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Fig. 46 e–h

e Blood smear in chronic myeloproli-ferative disease (CMPD) shows fiveerythroblasts and, at the center of thefield, basophilic stippling. Such caseswere once termed “chronic erythremia.”Erythroblastosis can occur in variousforms of CMPD

f Blood smear in chronic myeloid leu-kemia (CML) during the acceleratedphase after splenectomy. At center is amegakaryocyte nucleus, at right are twomegakaryoblasts, and at left is a myelo-blast

g Histologic section from an iliac crestbiopsy in “pure” megakaryocytic myelosisconsists almost entirely of megakaryo-cytes at various stages of maturity.Giemsa stain

h Silver-stained specimen from the samepatient clearly shows the darkly stainednuclei of the megakaryocytes and fiberproliferation

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5.8.1 Myeloid Leukemia and TransientAbnormal Myelopoiesis (TAM)of Down Syndrome (DS)

Acute myeloid leukemia of DS is immunologi-cally characterized by blast cells with featuresof megakaryoblasts. The blasts have a basophiliccytoplasm which might remind of proerythro-blasts. The disease responds quite well to theusual treatment. There are no biological differ-ences between MDS and AML in Down syn-drome. AML in older children with DS (3 yearsand older) behave more like AML in childrenwithout DS and has a poorer prognosis.

Transient abnormal myelopoiesis (TAM) ortransient myeloproliferation may show a clinicaland morphological picture indistinguishablefrom AML.

Spontaneous remission appears in the major-ity within 3 months. AML develops 1–3 years laterin about one quarter of the children.

5.8.2 Special Variants of MegakaryocyteProliferation

The pure malignant proliferation of megakaryo-cytes (Fig. 46g and h) is as rare as tumorousmegakaryoblastoma (Fig. 104d and e)

In one case with an exceptional increase inmegakaryoblasts and promegakaryocytes and avery high proportion of mitoses, we were ableto classify the disease as promegakaryocytic-megakaryoblastic leukemia (megakaryocyte pre-cursor cell leukemia). The cells and mitoses couldbe positively identified by the immunocytochem-ical detection of themegakaryocyte markers CD41and CD61. This case is more characteristic of aCMPD than an acute leukemia (Fig. 47a–h; jointobservation with D. Muller, Hof).

Figure 48a–c shows an example of familialpolyglobulia with positive erythrocyte alkalinephosphatase. Cytochemical and biochemical testsin four family members (three generations)showed that some of the erythrocytes and100 % of the erythroblasts contained alkalinephosphatase, which differs from the phosphatasein neutrophils. It is likely that the increasedbreakdown of 2,3–diphosphoglycerate plays arole in the pathogenesis of the erythrocytosis.

Fig. 46 i. Three blast cells in the periph-eral blood in TAM. All three cells haveintensely basophilic cytoplasm, which ishardly visible in the two cells below.There is no morphological difference toAML of DS


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Fig. 47 a–h. Promegakaryocytic-mega-karyoblastic leukemia (after Loffler andMuller, unpublished)

a Six megakaryocytic mitoses andnumerous small megakaryoblasts

b Higher-power view of megakaryo-blasts and four mitoses

c Four mitoses, blasts, and a promega-karyocyte in the lower half of the field

d Megakaryoblasts, a mitosis, and apromegakaryocyte

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Fig. 47 e–h

e Immunocytochemical detection ofCD41. A large proportion of the mega-karyoblasts and promegakaryocytes arepositive

f CD41: three positive mitoses are seenat upper left and lower right. Other fea-tures are the same as in e

g CD41: besides blasts and promega-karyocytes, a positive mature megakar-yocyte is visible at right

h CD61: the result is the same as withCD41


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5.8.3 Familial Erythrocytosis(Fig. 48 a–c)Cytochemical Detectionof Alkaline Phosphatase

a Blood smear. Erythrocytes show weakdiffuse reaction with fine positive gran-ules

b Erythroblast cluster in bone marrowsmear with marked cytoplasmic reaction(substrate a-naphthyl phosphate)

c Marked reaction (red) in erythroblastswith the substrate naphthol-AS-Bi phos-phate

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5.8.4 Chronic Myeloid (Granulocytic)Leukemia

The blood picture in chronic myeloid leukemia(CML) often contributes more to the diagnosisthan the bone marrow. Besides the high leukocytecount and a pathologic left shift with the appear-ance of immature granulocytopoietic forms at allstages (including promyelocytes and myelo-blasts), eosinophilia and basophilia are presentin the peripheral blood and corroborate the diag-nosis of CML. In addition, individual granulo-cytes show qualitative changes such as anisocyto-sis, nuclear-cytoplasmic asynchrony, and hypo-segmentation (“pseudo-Pelger forms”). Thesechanges are largely absent during the earlychronic phase. The same changes may be foundin severe reactive leukocytoses. The bone marrowis very cellular. Erythropoiesis is greatly sup-pressed in favor of granulocytopoiesis, whichdominates the cell picture. The granulocytopoie-tic line include a great many immature forms,producing a marked shift to the left. Marrow ba-sophils and eosinophils are usually increased. De-spite these findings, it can be difficult to distin-guish the bone marrow changes from those asso-ciated with severe reactive leukocytoses. Beforethe discovery of the Philadelphia chromosomeand the BCR-ABL translocation, the demonstra-tion of low or even negative leukocyte alkalinephosphatase (LAP, see p. 13) was of key impor-tance. Today the diagnosis is established by detec-tion of the Philadelphia chromosome (Ph). It re-presents a reciprocal translocation between thelong arms of chromosomes 9 and 22 [i.e.,t(9;22)], resulting in a translocation of the BCRand ABL genes. This creates a new fusion genecalled the BCR-ABL gene. The correspondingproteins, which have molecular weights of210 (p210) and 190 (p190), and very rarely 230(p230) can be detected in very low concentrationby PCR. Thus, molecular biology and its combi-nation with cytogenetic analysis in the FISH tech-nique provide highly sensitive detection methodsthat complement morphology and cytogenetics inthe diagnosis and especially the follow-up of CMLafter intensive therapy.

The differentiation of CML from chronic mye-lomonocytic leukemia (CMML), can be difficultto accomplish by morphology alone, since thereare “intermediate forms” that the FAB grouphas classified as atypical CML. The most reliabledifferentiating method is the cytogenetic detec-tion of the (9;22) translocation or the moleculargenetic detection of the BCR-ABL translocation.The absence of these changes precludes a diagno-sis of CML (CGL).

Almost all chronic myeloid leukemias progressto an acute phase (acute blast phase, blast crisis)during the course of the disease. This acute phasemay arise by transformation from the chronicphase, or an accelerated phase may precede it.The accelerated phase can be diagnosed by itsclinical manifestations (fever, bone pain, spleno-megaly) and an increasing left shift of the gran-ulocytopoiesis. There may also be an increasein basophilic granulocytes, which are already nu-merous in this disease. An increase in leukocytealkaline phosphatase activity is occasionally de-tected during the blast phase. Sometimes theacute phase has its onset in a particular organsuch as the spleen or lymph nodes.

The blasts consist predominantly of cyto-chemically and immunologically identifiablemyeloblasts and less commonly (20 %–30 %)of lymphoblasts, which may be PAS-positiveand display the immunologic features of commonALL. Megakaryoblast or erythroblast transforma-tion is less frequent, but mixed blast phases aresomewhat more common. Besides the t (9;22)translocation, the accelerated phase or blast phaseis often characterized by other cytogeneticchanges that mainly consist of an extra Ph chro-mosome, an isochromosome 17, trisomy 8, or acombination of these.

Because the BCR-ABL translocation occurs inearly stem cells, CML affects a portion of the Tlymphocytes and may affect all hematopoieticcells, although the involvement need not be com-plete. It is not surprising, therefore, when a highpercentage of eosinophils or basophils are discov-ered in variants of CML. As long as the typicalcytogenetic or molecular genetic abnormality ispresent, there is no need to classify the leukemiaas “eosinophilic” or “basophilic.” True eosinophi-lic and basophilic leukemias do exist, but they aremore aptly classified as acute leukemias and arediscussed under that heading.


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Table 8. Stages of CML

a) Chronic phase

Blood smear Bone marrow

GP: Pathologic left shift GP: Very hyperplasticIncreased eosinophils Shift to the leftIncreased basophils Increased eosinophils

Increased basophils

EP: Scattered normoblastsAnisocytosis, polychromatophilia

EP: Decreased (absolute or relative)

ThP: Platelets usually increasedAnisocytosis, giant plateletsScattered megakaryocyte nuclei

ThP: Megakaryocytes usually increased, some ab-normal forms (microkaryocytes)

b) Accelerated phase


GP: Pathologic left shift, pseudo-Pelger formsIncreased numbers of blasts,< 20 %Basophils may be markedly increased,< 30 %

GP: Pathologic left shiftIncreased numbers of N.C. or “blasts,”20 %Basophils may be markedly increased

EP: Scattered normoblasts anisocytosis,polychromatophilia

EP: Decreased

ThP: Platelets normal or decreasedAnisocytosis, scattered megakaryocytenuclei

ThP: Normal or decreased

c) Acute phase (blast crisis)


GP: Practically all cells are blasts GP: Practically all cells are blasts > 30 %

EP: Pronounced anisocytosisPolychromatophilia, normoblasts

EP: Greatly decreased

ThP: Platelets absent or greatly decreasedAnisocytosis, megakaryocyte nuclei

ThP: Greatly decreased

GP, granulocytopoiesis;EP, erythropoiesis;ThP, thrombocytopoiesis, megakaryocytopoiesis

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Fig. 49 a–h. Chronic myeloid leukemia(CML)

a Blood smear shows a preponderanceof mature neutrophilic granulocytes.Myelocyte at center, basophil at upperright

b Promyelocyte (center) in a blood smear

c Blood smear showing a greater leftshift. At center is a myeloblast

d Two basophils in blood smear


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Fig. 49 e–h

e Two blasts in blood smear – an unusualfinding during the chronic phase

f Increased granulocytopoiesis in a bonemarrow smear. Neutrophilic granulo-cytes, two basophils, and four eosinophils

g Bone marrow smear in CML showsgreatly increased numbers of megakar-yocytes, including many with round nu-clei and mature cytoplasm

h Bone marrow smear. Despite the pre-sence of a large megakaryocyte cluster,the patient had typical CML with BCR/ABL translocation

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Fig. 50 a–d. Chronic myeloid leukemia(CML)

a Megakaryocytes are greatly increasedand interspersed with (red) neutrophilicgranulocytes. Histologic section,naphthol AS-D chloroacetate esterasereaction

b Blood smear in untreated CML. All cellsare negative for leukocyte alkalinephosphatase (index 0)

c Histologic section from an iliac crestcore biopsy (Giemsa stain) illustrates theextremely high cellular density andpaucity of fat cells

d Histologic section during chronicphase of CML. Naphthol AS-D chloro-acetate esterase reaction shows an ex-treme increase in neutrophilic granulo-cytopoiesis


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Fig. 51 a– f. Chronic myeloid leukemia(CML)

a Bone marrow smear shows two sto-rage cells with abundant cytoplasm(pseudo-Gaucher cells) against a back-ground of increased granulocytopoiesis

b Pseudo-Gaucher cell, virtually indis-tinguishable from a true Gaucher cell inits cytologic features

c Several pseudo-Gaucher cells with aslightly bluer cytoplasmic tinge. Thedouble refraction in the cytoplasm of thepseudo-Gaucher cells under polarizedlight distinguishes them from true Gau-cher cells

d Blasts, promyelocytes, and atypicallymaturing forms with nuclear atypia; alsoseveral small basophils in the acceleratedphase of CML

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Fig. 51 e– f

e Higher-power view in acceleratedphase of CML shows atypical forms andbasophils

f Blood smear in accelerated CML showsa marked increase in basophilic granu-locytes. Toluidine blue stain

Fig. 52. Schematic diagram and partial karyotype of a Philadelphia translocation t(9;22)(q34;q11) andinterphase FISH with a BCR-ABL probe. By reciprocal translocation, a splitting of the signals of both probes(red and green signal) results in the following signal constellation in a Philadelphia positive cell: a red and agreen signal on the unchanged chromosome 9 and 22 as well as a red-green “colocalization signal” on thederivative chromosomes 9 and 22


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Fig. 53. BCR/ABL (Philadelphia) trans-location in a megakaryocyte, demon-strated by fluorescence in situ hybridi-zation (FISH) combined with fluores-cent immunophenotyping (FICTION).The Philadelphia translocations in thecell are identified by the co-location of agreen (BCR) and red (ABL) signal. Theblue cytoplasmic stain indicates CD41positivity. (Figure in cooperation withT. Haferlach et al.)

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Fig. 54 a–g. Blast phases in CML

a Blood smear in myeloid blast phase ofCML shows myeloblasts and promyelo-cytes with a rod form and basophil at thecenter of the field

b Myeloid blast phase. Blasts andbasophils

c Myeloid blast phase. Undifferentiatedblasts

d Blood smear from the same patientshows two peroxidase-positive precur-sors at upper center


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e Same smear series as in c and d,immunoperoxidase reaction. All theblasts are positive. If findings areequivocal, other myeloid markers such asCD13 and CD33 can also be used

f Histologic section in blast phase. A nestof blasts (paler nuclei) is visible at center(HE stain)

g Bone marrow smear in myeloblastphase shows predominant basophils andscattered eosinophilic granulocytes

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Fig. 55 a–d. Lymphoblast phase inCML. Immunophenotype of a c-ALL

a Blasts with numerous large vacuoles inthe cytoplasm

b Coarse PAS-positive clumps andgranules in the vacuoles

c Immunocytochemical detection ofCD19. All blasts are positive

d Immunocytochemical detection ofCD10. All blasts are positive (red)


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Fig. 56 a–h. Megakaryocytic-megakar-yoblastic and erythroblastic phases inCML

a Megakaryocytic-megakaryoblastictransformation

b Bone marrow from the same patient.Predominantly mature megakaryocytesare strongly esterase-positive

c Megakaryoblastic transformation in adifferent patient

d Blood smear in megakaryoblast phasedemonstrates three blasts

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Fig. 56 e–h

e Three blasts in megakaryoblast phase

f Bone marrow smear from the patient ine. Immunocytochemical detection ofCD61. The blasts are positive. Note thepositive platelets

g Bone marrow from a patient in theerythroblast phase of CML shows veryimmature, partially megaloblastoiderythroblasts

h Smear from the patient in g shows avery intense PAS reaction in coarsegranules and clumps


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5.8.5 Chronic Neutrophilic Leukemia (CNL)

The disease is extremely rare. It is characterizedby sustained peripheral blood neutrophilia; thebone marrow is hypercellular due to neutrophilgranulocyte proliferation. There is no Philadel-phia chromosome or BCR-ABL fusion gene de-tectable. The peripheral blood smear shows seg-mented and band neutrophils, in almost all casesfewer than 5 % of myeloid precursors; blast cellsare almost never observed. The neutrophils maybe toxic or normal, dysplasia is not present, redcells and platelets usually appear normal, alkalineleukocyte phosphatase is increased, cytogeneti-cally nearly 90 % of the patients are normal,and specific abnormal karyotypes have notbeen found. This badly defined leukemia occursin adults and adolescents; diagnosis is one of ex-clusion of all causes of reactive neutrophilia andof all other myeloproliferative diseases.

5.8.6 Chronic Eosinophilic Leukemia (CEL)and the Hypereosinophilic Syndrome (HES)

Distinguishing the two diseases is difficult be-cause clinical data, hematological and other la-boratory parameters do not allow a clear-cut de-finition. In both cases you find a persistent eosi-nophilia of � 1.5 � 109/L, there are fewer than20 % of blasts in the blood or bone marrow. InCEL there should be evidence of clonality ofthe eosinophils or an increase in blasts (lessthan 20 %) in the blood or bone marrow. If thesecriteria are missing and no underlying cause foreosinophilia can be found, the diagnosis is idio-pathic HES. Recently, changes of the PDGF-recep-tor in HES have been found (s. p. 111 – 112, Fig. 34,and pp 259–261, Fig. 112).

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5.9 Myelodysplastic Syndromes (MDS)

The diagnostic criteria for myelodysplastic syn-dromes (MDS) and a classification of these syn-dromes were presented by the FAB group in 1982.MDS are a class of subacute to chronic diseasesthat are associated with quantitative and qualita-tive blood and bone marrow changes.1 The syn-dromes are seen predominantly in older patients,with some cases showing transformation to acuteleukemia. An essential criterion is the number ofblasts that can be detected in the bone marrowand/or the peripheral blood. Cytopenia is invari-ably present (anemia, granulocytopenia, orthrombocytopenia) despite normocellular or hy-percellular bone marrow (“ineffectual hemato-poiesis”). Disturbances of erythropoiesis (dysery-thropoiesis) are reflected in quantitative and qua-litative abnormalities of nucleated precursors inthe bone marrow and of erythrocytes in the per-ipheral blood (see Table 9). The proportion of er-ythropoiesis in the bone marrow may be in-creased (> 50 %) or reduced (< 5 %). Disturb-ances of granulocytopoiesis (dysgranulocyto-poiesis) are manifested in the bonemarrow by ab-normalities in granules or nuclei (pseudo-Pelgerforms, ring forms, etc.). Occasionally the cyto-plasmic basophilia is irregularly distributed,and a heavy basophilic rim is present at thecell periphery. Neutrophils in the peripheralblood are frequently agranular or hypogranular,and the mature forms sometimes show a basophi-lic cytoplasm. Disturbances of megakaryocyto-poiesis lead to the appearance of abnormal cells(micromegakaryocytes, large mononuclearforms, hypersegmented forms, many single nu-clei). Abnormal platelets (giant forms, conspicu-ous anisocytosis) are found in the peripheralblood (Table 9).

Two types of blast are observed in MDS (seeFig. 68):

Type I: The cells of type I range from forms indis-tinguishable from myeloblasts to cells of variablesize that cannot be classified. The cytoplasm isagranular, and the nucleus shows distinct nucleoliand a loose chromatin pattern.

Type II: The cells contain few primary (azurophi-lic) granules, the nuclear-cytoplasmic ratio issmaller, and the nucleus is centrally located.

Five forms of MDS were recognized by the FABgroup (Table 10 b). WHO distinguishes betweenseven subtypes with two variants of RAEB(Table 10), CMML is included in a special group(s. below), and pediatric hematologists would liketo retain RAEB-t [Leukemia 17, 277 (2003)]. Thesubtype with dysplastic changes in two or morecell lines is called refractory cytopenia with multi-lineage dysplasia (RCMD) (s. Table 10 b):1. Refractory anemia (RA). The cardinal symp-

tom is anemia. Reticulocytes are diminishedin the peripheral blood, and blasts are absentor do not exceed 1 %. The bone marrow is nor-mo- or hyperplastic and usually shows a pre-dominance of more or less dysplastic erythro-poiesis, though there are cases in which ery-thropoiesis is reduced. Granulocytopoiesisand megakaryocytopoiesis may show evidenceof dysplasia. The proportion of blasts is lessthan 5 %.

2. Refractory anemia with ringed sideroblasts(RARS). In contrast to RA, the proportion ofringed sideroblasts exceeds 15 %. The prog-nosis is most favorable in cases where granu-locytopoiesis and megakaryocytes are unaf-fected. This form (“pure” sideroblastic ane-mia) should be distinguished from RARS,therefore. The variant with dysplastic changesin two or more cell lines is called RCMD-RS.

3. Refractory anemia with excess blasts (RAEB).Cytopenia is usually present, and all three celllines in the peripheral blood display abnorm-alities. Small numbers of blasts (< 5 %) may befound in the peripheral blood. The bone mar-row is hypercellular with variable hyperplasiaof erythropoiesis or granulocytopoiesis. Dyser-ythropoiesis, dysgranulocytopoiesis, and/or

Table 9. Signs of dysplasia

ErythropoiesisMultinuclearity, megaloblastic nuclear changes,abnormal mitoses, karyorrhexis, ringed sideroblasts,coarse iron granules, cytoplasmic vacuoles

GranulocytopoiesisPseudo-Pelger forms, hypersegmentation, giant forms,ring nuclei, hypogranulation, abnormal basophilia,peroxidase defect, atypical positive esterase inneutrophils and eosinophils

MegakaryopoiesisMicro(mega)karyocytes (small round nucleus, maturecytoplasm), large mononucleate forms, many smallnuclei, hypersegmented nuclei, nuclear-cytoplasmicasynchrony, anisocytosis of platelets, giant platelets

1 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA,Gralnick HR, Sultan C (1982) Proposals for the classificationof the myelodysplastic syndromes. Brit J Haematol 51 : 189–199


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Table 10a. FAB criteria for the subtypes of MDS (key findings are in boxes)

Bone marrow Blood

Diagnosis Abbreviation Blastsa Ringedsideroblastsb

Blasts Monocytes(� 109/ l)

Refractory anemia RA < 5% < 15% � 1% < 1

Refractory anemia with ringedsideroblasts

RARS < 5% � 15% � 1% < 1

Refractory anemia with excessblasts

RAEB 5–20% Variable < 5% < 1

RAEB in transformation RAEB-t > 20c < 30 Variable �5% < 1Chronic myelomonocyticleukemia

CMML < 20% Variable < 5% � 1

a Percentage of all nucleated cells.b Percentage of erythroblasts.c 20 % is sufficient if peripheral blasts exceed 5 % or Auer rods are present

Tabelle 10b. WHO classification of myelodysplastic syndromes (MDS)

Disease Blood findings Bone marrow findings

Refractory anemia (RA) AnemiaNo or rare blasts

Erythroid dysplasia only< 5% blasts< 15% ringed sideroblasts

Refractory anemia with ringedsideroblasts (RARS)

AnemiaNo blasts

� 5% ringed sideroblastsErythroid dysplasia only< 5% blasts

Refractory cytopenia withmultilineage dysplasia (RCMD)

Cytopenias (bicytopenia orpancytopenia)No or rare blastsNo Auer rods< 1 � 109/L monocytes

Dysplasia in � 10 % of the cellsor two or more myeloid cell lines< 5% blasts in marrowNo Auer rods< 15% ringed sideroblasts

Refractory cytopenia withmultilineage dysplasia and ringedsideroblasts (RCMD-RS)

Cytopenias (bicytopenia orpancytopenia)No or rare blastsNo Auer rods< 1 � 109/L monocytes

Dysplasia in � 10 % of the cellsin two or more myeloid cell lines< 5% blastsNo Auer rods

Refractory anemia withexcess blasts � 1 (RAEB-1)

Cytopenias< 5 % blastsNo Auer rods< 1 � 109/L monocytes

Unilineage or multilineage dysplasia< 5 � 9% blastsNo Auer rods

Refractory anemia withexcess blasts � 2 (RAEB-2)

Cytopenias5 � 19% blastsAuer rods �< 1 � 109/L monocytes

Unilineage or multilineage dysplasia10 % � 19 % blastsAuer rods �

Myelodysplastic syndrome �unclassified (MDS-U)

CytopeniasNo or rare blastsNo Auer rods

Unilineage dysplasia:one myeloid cell line< 5% blastsNo Auer rods

MDS associated withisolated del(5q)

AnemiaUsually normal or increasedplatelet count< 5% blasts

Normal to increased megakaryocyteswith hypolobated nuclei< 5% blastsIsolated del(5q) cytogeneticabnormalityNo Auer rods

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4. Chronic myelomonocytic leukemia (CMML)has been grouped together with atypicalCML, juvenile myelomonocytic leukemia andmyelodysplastic/myeloproliferative disease asunclassifiable in a new category. The dominantfeature is monocytosis, which must exceed1� 109/L in the peripheral blood. Frequentlythis is associated with an increase in maturegranulocytes, with or without dysgranulocyto-poiesis (such as hypogranular or Pelgerforms). Monocytes are best demonstrated byesterase staining. Atypical CML is Philadel-phia- and BCR-ABL-negative. The diagnosticcriteria of CMML are the same as in theFAB classification. According to the blast per-centage, a subtype 1 with < 5 % blasts in per-ipheral blood and < 10 % blasts in bone mar-row is separated from subtype 2 with 5 %–19 % blasts in peripheral blood and 10 %–19 % in bone marrow. If Auer rods are found,the case falls into subtype 2, provided the per-centage of blasts in peripheral blood and mar-row is less than 20 %; if the percentage is 20 %or more, the case falls into the AML group. Afurther variant is CMML with eosinophilia ifthe criteria for CMML are fulfilled and thereare, in addition, � 1.5 � 109/l eosinophils inperipheral blood. In this variant, which mightbe a special entity, the translocationt(5;12)(q31;p12) with the abnormal fusiongene TEL/PDGR has been recognized. In spiteof being a rare finding (less than 1 %–2 % ofall patients with CMML), the favorable res-ponse to imatinib therapy is significant.The findings in JMML are:1. Monocytosis in peripheral blood of � 1 �

109/l2. Blasts < 20 % of the leukocytes in p. b. and

< 20 % of all nucleated cells in b.m.

3. No Philadelphia or BCR-ABL rearrange-ment

4. Two or more of the following criteria:HbF increased for ageMyeloid precursors in p.b.Leukocytes � 10 � 10 /lClonal chromosome anomalies (e.g.,monosomy 7)Hypersensitivity of the myeloid precursorsagainst GM-CSF in vitro

5. RAEB in transformation. The hematologic fea-tures resemble those of RAEB, except that thepercentage of blasts in the peripheral blood ex-ceeds 5 %, the marrow contains 20 % to 30 %blasts (types I and II), and conspicuous Auerrods are sometimes found in granulocytopoie-tic precursors. RAEB in transformation usuallyruns a brief course with a relatively swift pro-gression to AML.

A separate biologic entity is the 5q-minus syn-drome, which was formerly included in the RAor RARS subgroups. Occurring mainly in olderwomen, it is characterized by a macrocytic ane-mia with normal or increased numbers of plate-lets. The bone marrow contains abundant mega-karyocytes that mostly display round nuclei, nu-clear hypolobulation, and a granular cytoplasm.Cytogenetic demonstration of the deletion onthe long arm of chromosome 5 (5q-) establishesthe diagnosis.

Another entity, which is characterized cyto-genetically by a deletion or translocation of theshort arm of chromosome 17 – del (17p) – andmorphologically by pronounced dysgranulocyto-poiesis with almost complete (“homozygotic”)pseudo-Pelger transformation of the granulo-cytes, is usually associated with additional chro-mosome abnormalities. A very prominent clump-ing of the chromatin is often seen. Mutations ofthe p53 gene have been postulated as the cause.

Some cases of MDS do not fit into any of thesubtype categories noted above. These cases pre-sent with bone marrow hypoplasia or fibrosis,which usually requires a histologic diagnosis, orwith an increase in tissue or blood basophils.Many of these cases are secondary to treatmentwith cytostatic drugs.

Tabelle 10c. WHO classification of myelodysplastic /myeloproliferative diseases

Chronic myelomonocytic leukemia (CMML)

Atypical chronic myeloid leukemia (ACML)

Juvenile myelomonocytic leukemia (JMML)

Myelodysplastic /myeloproliferative disease, unclassifiable


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Fig. 57 a– f. Myelodysplastic syn-dromes (MDS)

a Erythroblasts with megaloblasticchanges. Binucleated form at top center.Refractory anemia (RA)

b Abnormal erythroblast mitoses withclumped and fragmented chromosomes

c Chromatin strands linking erythroblastnuclei in RA. Indistinguishable from thechanges in type I form of CDA

d Trinucleated proerythroblast at top,atypical mitosis at lower right, agranularpseudo-Pelger form at center, hypergra-nular pseudo-Pelger form at bottom,myeloblast at lower left. RAEB-t

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Fig. 57 e– f

e Pseudo-Pelger formwithout granules atleft, vacuolated cytoplasm at center,vacuolation and unevenly distributedgranules at right – typical of anomaliesinvolving the short arm of chromosome17 (17p)

f Two pseudo-Pelger forms, at left with“toxic” granules


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Fig. 58 a–d Dysgranulocytopoiesis

a Bone marrow smear in RAEB-t showshypogranular cells of granulocytopoiesis.Twomyeloblasts appear at the center andright edge of the field

b Polyploid giant segmented form atcenter, below it a normal-appearingsegmented neutrophil

c Peroxidase reaction. One cell is positiveat top, three neutrophils below it arenegative: partial peroxidase defect

d Abnormal nonspecific esterase reac-tion (a-naphthyl acetate). Most of theneutrophils in the field show weak tomoderate esterase activity; normally theyare negative

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Fig. 59 a–d. Signs of megakaryocyticdysplasia in MDS

a Micro(mega)karyocyte with a typicalsmall round nucleus and largely maturecytoplasm. RAEB

b Binucleated microkaryocyte at center.RAEB

c Five abnormal megakaryocytes, eachwith multiple nuclei and an abnormalcytoplasmic stain. Cluster of myeloblastsat right. Transition from RAEB-t to AML

d Cluster of multinucleated megakaryo-cytes in CMML


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Fig. 60–67. Subtypes of MDSFig. 60 a–h. RARS(subtype RA, s. Fig. 57a–c)

a Bone marrow smear in RARS. Pap-penheim stain. The cytoplasm is stillmostly basophilic, shows some indistinctboundaries, and contains normal-shapednuclei. Similar to findings in severe irondeficiency

b Bone marrow smear from the samepatient. Iron stain. Most erythroblastscontain coarse iron granules (blue) thatencircle the nucleus like a ring. At left is a“normal” erythroblast with a prominentiron granule. The other erythroblasts aretypical ringed sideroblasts

c Five, more mature ringed sideroblastsand one proerythroblast, also with irongranule rings. This finding is unusual,since iron granules normally are not de-tected in the proerythroblast stage

d Ringed sideroblasts with unusuallylarge iron granules

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Fig. 60 e–h

e Another case of RARS. Erythropoiesis ismostly very immature and shows somemegaloblastic changes

f Higher-power view more clearlydemonstrates the megaloblastoidchanges

g Erythroblasts with vacuolatedcytoplasm in RARS

h Iron stain of same bone marrow as in gshows a ringed sideroblast (at top) withvacuolated cytoplasm


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Fig. 61 a, b

a Electron microscopic view of iron deposition in themitochondria, which encircle the nucleus (ringedsideroblast)

b Higher-power view of the iron-laden mitochondria.(Courtesy Prof. Dr. H. E. Schafer, Freiburg)

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Fig. 61 b

Fig. 61 a


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Fig. 62 a–d

a Massive iron deposition, includingcoarse clumps, in a bone marrowfragment in MDS (partly transfusion-related)

b Pappenheim stain shows hemosiderindeposition in a plasma cell (left). At right isiron-stained bone marrow from the samepatient. Note the coarse granular irondeposition in the cytoplasm of a plasmacell in RARS. Nucleus is at lower right.

c– f The 5q-minus syndrome

c Two megakaryocytes with round nu-clei and different stages of cytoplasmicmaturity – a typical finding in thissyndrome

d Three typical megakaryocytes withround nuclei and relatively maturecytoplasm

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Fig. 62 e– f

e Large megakaryocyte with a nonseg-mented nucleus. The cytoplasm is stillpartly basophilic. Below the megakaryo-cyte is an erythroblast in mitosis and amyeloblast

f Erythropoiesis showing subtle mega-loblastic features in 5q-minus syndrome

Fig. 63. Schematic diagram and partial karyotype ofthe deletion on the long arm of chromosome 5del(5)(q13q31), the so-called 5q-minus syndromewhich, in addition, is found in the setting of complexaberrant clones in AML


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Fig. 64 a–h. RAEB and RAEB-ta

a Bone marrow smear in RAEB. At left is amyeloblast, and below it is a lymphocyte.Most granulocytes in the myelocyticstage have no detectable granules. Notethe absence of monocytes in the field!

b Bone marrow from the same patient.Nonspecific esterase reaction shows ab-normally high enzyme activity in granu-lopoietic cells as further evidence ofdysplasia. No monocytes!

c Cluster of myeloblasts next to severalmaturing granulopoietic cells in RAEB-ta

d Transition from RAEB-ta to AML. Theproportion of blasts is just below 30 %.Cluster of myeloblasts and one normal-appearing eosinophilic myelocyte.

ta Subtype RAEB-t was not removed sinceit was retained in a recent classificationfor MDS in children

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Fig. 64 e–h

e Myeloblasts and atypical erythroblastsin RAEB-t

f Blasts and atypical binucleatederythroblast in the bone marrow inRAEB-t

g Cluster of micro(mega)karyocytes inbone marrow from the same patient

h Dysplastic erythropoiesis and blastincrease in RAEB-t


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Fig. 65 a–h. Chronic myelomonocyticleukemia (CMML)

a Blood smear shows a substantialincrease in monocytes. To the right ofcenter is an immature myelocyte

b Smear from the same patient.Nonspecific esterase. Most of themonocytes are strongly positive (brownstain)

c Blood smear. At left is a large promo-nocyte

d Bone marrow smear shows increasedmonocytes and some mild dysplasticchanges in neutrophilic granulocytopoi-esis

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Fig. 65 e–h

e Bone marrow from the patient ind. Nonspecific esterase reaction clearlydifferentiates the monocytes (oftendifficult with Pappenheim stain)

f Monocytes and dysplastic precursors ofgranulocytopoiesis in a bone marrowsmear; granules are extremely sparse

g Exceptionally large, strongly polyploidmonoblasts in the blast phase of CMML.At bottom is a mitosis

h Bone marrow from the same patient.Nonspecific esterase reaction. The verylarge blasts and promonocytes are asstrongly positive as the normal-sizedmonocytes


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Fig. 66 a–h. Myelodysplasias withpseudo-Pelger changes

a Bone marrow smear. Nuclei ofneutrophilic granulocytopoiesis show acoarse, clumped, dissociated chromatinpattern

b Bone marrow from the same patientshows dysplastic megakaryocytes anderythroblasts. The round granulopoieticcells correspond to pseudo-Pelger formsof the homozygotic type

c Unusually strong chromatin fragmen-tation (center). Note the granules in thecytoplasm

d Homozygotic type of pseudo-Pelgerforms in a different patient

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Fig. 66 e–h

e Bone marrow from the patient ind. Nonspecific esterase reaction shows alarge proportion of strongly positivemonocytes, most with round nuclei. Thepositive cells correspond to the round-nuclear forms in d with grayish-bluecytoplasm

f Blood smear from a different patientshows abundant pseudo-Pelger forms ofthe homozygotic type

g Bone marrow from the patient inf. Obvious chromatin clumping is limitedto the more mature forms

h Peroxidase reaction. Most of thehomozygotic-type pseudo-Pelger formsare strongly peroxidase-positive


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Fig. 66 i Partial karyotype of a dicentricchromosome 5;17 that leads to asimultaneous loss of the long arm ofchromosome 5 and the short arm ofchromosome 17. The loss of the shortarm of chromosome 17 (17p-) causesthe loss of an allele of p53, which islocalized there in the band 17p13.Structural changes of 17p are detectedin MDS and AML with pseudo-pelgeranomalies

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Fig. 67 a–d

a Bone marrow from a patient withatypical MDS shows a marked increase intissue mast cells, some containing sparsegranules

b Bone marrow from the same patient.Higher-power view of the atypical tissuemast cells

c Bone marrow from the same patient.Toluidine blue stain shows typical me-tachromasia of the tissue mast cellgranules

d Bone marrow in HIV infection. At thebottom of the field is a dysplasticerythroblast with four nuclei


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Fig. 67 e Higher-power view of dysplas-tic erythroblasts in the same patient.Panels d and e illustrate that HIV maypresent with significant dysplasticchanges that are morphologicallyindistinguishable from MDS

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5.10 Acute Leukemias

Acute leukemias are clonal diseases of hemato-poietic precursors with molecular genetic ab-normalities. All hematopoietic cell lines may beaffected. Proliferation of the leukemic cell clonereplaces normal hematopoiesis in varying de-grees. In acute myeloid leukemia (AML), it ismost common for granulocytopoiesis and mono-cytopoiesis to be affected. Erythropoiesis is lessfrequently affected, and megakaryopoiesis rarelyso. The distribution of the subtypes varies accord-ing to age. Acute lymphocytic leukemia (ALL) oc-curs predominantly in children, while AML hasits peak in adults. The involvement of severalmyeloid cell lines is relatively common, but thesimultaneous involvement of myeloid and lym-phoid cell lines is very rare (hybrid and bilinearacute leukemias). WHO has recently proposed

that the percentage of blasts in the bone marrowmust be approximately 20 % to justify a diagnosisof acute leukemia. Examination of the peripheralblood is not essential for diagnosis but can pro-vide important additional information. The diag-nosis and classification (subtype assignment) al-ways rely on the bone marrow. The most widelyaccepted system at present for the classification ofAML is based on the criteria of the French-Amer-ican-British (FAB) Cooperative Group (Table 11)and of the WHO (Table 11a, b). Because the quan-tity and quality of the blasts is of central impor-tance in this classification, they should be definedas accurately as possible. The leukemic blasts in-clude myeloblasts, monoblasts, and megakaryo-blasts. The cells of erythropoiesis are generallycounted separately, although in acute erythroleu-kemia (M6 subtype) the erythroblasts account fora preponderance of the leukemic cells. Blasts are

Table 11a. FAB classification of akute myeloid leukemia (AML), modified


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traditionally defined as immature cells that donot show signs of differentiation. But sinceFrench hematologists have always accepted blastswith granules, and cells having the nuclear andcytoplasmic features of classic blasts but contain-ing granules are known to occur in leukemias, adistinction is now drawn between type I blasts,which are devoid of granules, and type II blasts,which contain scattered granules or Auer rodsand do not show perinuclear pallor (Fig. 68). Oc-casional reference is made to “type III blasts,” butwe consider this a needless distinction except forthe abnormal cells of promyelocytic leukemia(M3), which may be heavily granular but are stillclassified as blasts even though they do notstrictly meet the criteria. Besides bone marrowand blood smears, histologic sections are re-quired in cases where aspiration yields insuffi-cient material. The standard stains (Pappenheim,Giemsa) are useful for primary evaluation, but amore accurate classification of the cells requirescytochemical analysis and immunophenotyping(flow cytometry and/or immunochemistry). Inorder to make a prognosis or define biological en-tities (Table 12), it is further necessary to performcytogenetic and molecular genetic studies, fluor-

escence in situ hybridization (FISH), and com-bine FISH technology with immunocytochemis-try.

As mentioned, the classification of AML essen-tially follows the recommendations of the FABand the WHO. However, the experience of recentyears in cooperative and prospective studiesusing modern methods supports the notion ofa future-oriented, prognostic classification thattakes into account the results of therapies and pa-tient follow-ups. This will allow for a more indi-vidually tailored approach. At present this biolo-gical classification, or subdivision into biologicalentities, does not cover all the subtypes of AML orALL, but in the future these gaps will be filledthrough the application of more sensitive meth-ods.

Acute lymphocytic leukemias (ALL) are cur-rently classified according to immunologic crite-ria (Table 13). The classification into three sub-types (L1–L3) originally proposed by the FABno longer has clinical importance today exceptfor the L3 subtype, which is virtually identicalto the immunologic B4 subtype (mature B-ALL). Morphology and cytochemistry form thebasis of diagnosis in ALL. The immunologic clas-sification of ALL is based on two principal groups– the B-cell line and T-cell line with their sub-types. Cytogenetic and/or molecular genetic stu-dies can also be used to define prognostic entities.These include c-ALL with t(9;22) and pre-B-cellALL with t(4;11), which has a characteristic immu-nophenotype.

Besides morphologic analysis, the peroxidasetechnique is the most important basic method be-cause lymphoblasts are always peroxidase-nega-tive, regardless of their immunophenotype.

CSF Involvement in Acute Leukemiasand Malignant Non-Hodgkin Lymphomas

Involvement of the cerebrospinal fluid (CSF) isusually associated with clinical symptoms. Thequestion of whether CSF involvement is presenthas major implications for further treatment, in-asmuch as curative therapy in children with acutelymphoid leukemias has been possible only sincethe introduction of prophylacis against menin-geal involvement. In this area as in others, thefull arsenal of available analytic methods hasbeen brought the bear. Decision-making can beparticularly difficult in patients with mild CSF in-volvement, because cells in the CSF are subject tosubstantial changes. It is important, therefore, al-ways to cerrelate the CSF findings with the find-ings in the blood or bone marrow (see Fig. 127).

Table 11b. WHO classification of acute myeloidleukemias

Acute myeloid leukemia with recurrent geneticabnormalities– Acute myeloid leukemia with t(8;21)(q22;q22);(AML 1(CBFa)/ETO)– Acute myeloid leukemia with abnormal bone mar-row eosinophils inv(16)(p13q22) or t(16;16)(p13;q22);(CBFb/MYH11)– Acute promyelocytic leukemia (AML witht(15;17)(q22;q12) (PML/RARa) and variants– Acute myeloid leukemia with 11q23 (MLL)abnormalities

Acute myeloid leukemia with multilineage dsyplasia

Acute myeloid leukemia and myelodysplasticsyndromes, therapy-related

Acute myeloid leukemia not otherwise categorizedAcute myeloid leukemia minimally differentiatedAcute myeloid leukemia without maturationAcute myeloid leukemia with maturationAcute myelomonocytic leukemiaAcute monoblastic and monocytic leukemiaAcute erythroid leukemiasAcute megakaryoblastic leukemiaAcute basophilic leukemiaAcute panmyelosis with myelofibrosisMyeloid sarcoma

Acute leukemia of ambiguous lineageUndifferentiated acute leukemiaBilineal acute leukemiaBiphenotypic acute leukemia

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Table 12. Biological entities in acute myeloid leukemia

Morphology FABclassification

Cytogenetics Molecular genetics Frequency (%)

Promyelocytic leukemiaHypergranular M3 t(15;17) PML/RARa � 5Microgranular M3v

Myeloblastic leukemia M1 /M2 t(8;21) AML1 /ETO � 10

Myelomonocytic leukemiawith abnormal Eo

M4Eo inv(16)t(16;16)

MYH11 /CBFb � 5

Myelomonocytic/monocytic-monoblastic leukemia

M4/5 11q23 various > 5; morefrequent inchildren

Myeloblastic leukemia etc.,often with increased basophils

M1 / 2 MDS t(6;9) DEK/CAN < 1

special “myelo-monoblast”with ery phagocytosis

M4 /5 (5 ep) t(8;16) CBP-MOZ < 0,1

Myeloblastic leukemia withdysmegakaryo-cytopoiesis

M1 etc. 3q21 and/or 3q26 e. g., EVI 1 /EAP/MDS1

� 1

AML and MDS with pseudo-Pelger-Huet anomalies

AML u. MDS 17p with complexaberration

p53 alterations 3–5

Megakaryoblastic leukemia M7 t(1;22) ? � 1(small children)

}

Table 13. Immunologic classification of acute lymphoblastic leukemias (ALL) [after Bene, MC et al (1995) Pro-posals for the immunological classification of acute leukemias. European Group for the Immunological Char-acterization of Leukemias (EGIL). Leukemia 9: 1783–1786]

ALL Classification

1) B lineage-Alla (CD19 + ) and/or CD 79a+ and/or CD22+

a) B1 (pro-B) (no other differentiation antigens)

b) B2 (common) (CD10+)

c) B3 (pre-B) Cytoplasmic IgM+

d) B4 (mature B) Cytoplasmic surface kappa or lambda +

2) T lineage-ALLb (cytoplasmic/membrane CD3+)

a) T1 (pro-T) CD7+

b) T2 (pre-T) CD2+ and/or CD5+ and/or CD8+

c) T3 (cortical T) CD1a+

d) T4 (mature T) Membrane CD3+, CD1a-

T-a/b and T-c/d anti-TCR a/b+, anti-TCR c/d+

All with myeloid antigen expression (My + ALL)a At least two of three markers must be positive. Most cases are TdT positive and HLA-DR positive, except subtypeB IV, which is often TdT negativeb Most cases are TdT positive, HLA-DR- and CD34 negative; these markers are not considered for diagnosis andclassification


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5.10.1 Acute Myeloid Leukemia(AML)

Fig. 68 a–d. Acute myeloid leukemia(AML). Definition of blasts

a Two type I blasts. The cytoplasm isdevoid of granules

b Cytoplasmic granules distinguish thistype II blast from type I. At left is agranulocyte in mitosis, above it is apromyelocyte

c At left are two type I blasts, at right isone type II blast with distinct granules.Below is a promyelocyte with very coarseprimary granules. Between blasts I and IIis an eosinophil

d At left is a type I blast. At center is anatypical promyelocyte with an eccentricnucleus and numerous granules occu-pying an area where a clear Golgi zonewould be expected to occur

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Fig. 69 a–h. AML, M0 subtype

a Morphologically undifferentiatedblasts with distinct nucleoli are perox-idase-negative and do not show theesterase reaction typical of monocytes

b Bone marrow smear from the samepatient. Immunocytochemical detectionof CD13. A large proportion of the blastsare positive (red). APAAP technique

c Bone marrow smear from a differentpatient with the M0 subtype of AML. Theblasts are difficult to distinguishmorphologically from ALL

d Smear from patient in c. Demonstra-tion of CD13


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Fig. 69 e–h

e Same patient as in c and d. Esterasereaction shows a patchy positive reactionthat is not sufficiently intense formonoblasts

f Morphologically undifferentiated blastsin a smear from a different patient

g Peroxidase reaction in the samepatient. Blasts are negative, and amongthem is a positive eosinophil

h Same patient as in f and g. Patchyesterase reaction is not sufficientlyintense for monoblasts

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a The pale blue areas in the nuclearindentation are caused by overlyingcytoplasm (three lower blasts at left ofcenter). This is a typical finding in AML-M1and should not be confused with nucleoli

b Smear from the same patient. Perox-idase reaction. Positive Auer rods arevisible in the nuclear indentation of theuppermost cell

c Pseudonucleoli in blasts from adifferent patient

d Same patient as in c. Positive perox-idase reaction


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Fig. 70 e–h

e Small blasts appear morphologicallyundifferentiated

f Same patient. Very strong peroxidasereaction

g Blasts appear undifferentiated exceptfor purple inclusion

h Same patient. Peroxidase reaction.Numerous Auer rods, which often arevery small (Phi bodies)

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Fig. 71 a–h. AML, M1 and M2 subtypes

a Most of these blasts show no signs ofdifferentiation, although the cytoplasmin some contains long, thin Auer rods

b High-power view of the same speci-men. Long, thin Auer rods appear in thecytoplasm at upper left and right andlower right

c Peroxidase reaction in the same pa-tient shows an accentuated perinuclearreaction with conspicuous Auer rods. The(8;21) translocation was detected in thispatient

d Bone marrow from a different patientshows a long, thin Auer rod and pro-nounced maturation (more than 10 %).Chromosome findings were normal


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Fig. 71 e–h

e Blood smear in AML. Undifferentiatedblasts with scant cytoplasm

f Peroxidase reaction in the same pa-tient. All blasts in the field are stronglypositive

g Bone marrow from the same patientshows pronounced maturation (morethan 10 %)

h Very strong peroxidase reaction in thesame patient. This case demonstratesthat bone marrow examination is ne-cessary for an accurate classification

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Fig. 72 a–g. AML, variants of M2subtype

a Blasts with markedly basophiliccytoplasm and characteristic perinuclearpallor (Golgi zone). Cell at lower left showsatypical maturation. The FAB classifiesthis morphologic variant as M2. Thet(8;21) translocation is also present

b Dysplastic maturation in M2. Severaleosinophils are visible

c M2 with eosinophilia. Three eosino-philic precursors contain scattered blueto violet immature granules and palematuring granules. They differ from theeosinophils in inv(16)

d Peroxidase reaction in the samepatient. The eosinophils are distin-guished by their characteristic granules.The blasts are also positive


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Fig. 72 e–g

e CE reaction. Enzyme activity (red) isdetected only in neutrophils. All theeosinophils are negative, in contrast toinv(16)

f Different case of M2 with eosinophilia.To the right of center is a blast with Auerrods. The t(8;21) is also found in thismorphologic variant

g M2 with eosinophilia. Peroxidase re-action. Auer rods appear above the nu-cleus of the cell at center (eosinophil?)

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Fig. 73 a–h. Cytoplasmic inclusions inAML

a Secondary AML following chemother-apy for NHL with t(10;11). Unusually largereddish inclusions (pseudo-Chediakanomaly)

b Same patient. Large vacuoles withgranular contents

c CE reaction. At center are vacuoles withCE-positive inclusions in the cytoplasm

d Pink to red, round or angular inclusionsin a different patient


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Fig. 73 e–h

e Same patient as in d. At center is a pale,oblong inclusion over a binucleated cell

f Rounded and rod-shaped inclusions inthe same patient. At bottom is an extra-cellular rod-shaped inclusion

g PAS reaction. Positive rod-shapedinclusion at center, positive roundedinclusion above it

h CE reaction. Rod-shaped inclusion atcenter are strongly positive, like Auerrods. Cytogenetic analysis in this patientshowed only a hyperdiploid clonewithout specific aberrations(Prof. Fonatsch, then in Lubeck)

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Fig. 74. Schematic diagram and partial karyotype of thetranslocation t(8;21)(q22;q22), found predominantly in theM2 subtype of AML


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Fig. 75 a–h. AML, M2 subtype, charac-terized by increased numbers of tissuemast cells or basophilic granulocytes

a Bone marrow in M2 with t(8;21). Thecytoplasm of two blasts (left of center andupper right) contains long, thin Auer rods

b Three tissue mast cells in the samepatient. At right is a younger form withrelatively few granules

c Cytopenic bonemarrow from the samepatient following chemotherapy. Theblasts have disappeared, and the marrowfragment contains abundant tissue mastcells

d Higher-power view of a group of tissuemast cells following chemotherapy. In-creased tissue mast cells are found in asmall percentage of patients with AML-M2 and t(8;21)

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Fig. 75 e–h

e AML-M2with t(6;9). Blasts and atypical-appearing basophilic granulocytes

f High-power view in the same patient.The atypical basophils with predomi-nantly fine granules are metachromaticafter staining with toluidine blue

g Very strong increase of basophils in adifferent patient. At upper left is a baso-phil in mitosis. A (6;9) translocation couldnot be demonstrated

h Toluidine blue stain in same patientshows metachromatic reaction of baso-philic granulocytes


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Fig. 75 i Schematic diagram and partial karyotype ofthe t(3;12)(q26;p13), which is found in AML and MDS.Changes in the short arm of chromosome 12 (12p), liket(6;9) [below t(3;12)], may be associated with increasedbasophilic granulocytes in the bone marrow. A similarincrease is occasionally found in the M4 subtype in theabsence of detectable anomalies

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Fig. 76 a–g. AML, M2 subtype, with“Golgi esterase.” In some cases of M2with t(8;21), circumscribed nonspecificesterase activity in the perinuclear zonemay cause the cells to be mistaken formonocytes. All the criteria of AML-M2are present, however, including amarked peroxidase reaction.

a M2 with t(8;21). Peroxidase reaction.The blasts are peroxidase-positive, and apositive Auer rod is visible at center abovethe nucleus

b Nonspecific esterase in the samepatient. Strong “Golgi esterase”

c Different case of M2. Pappenheim stain

d Same as c. Nonspecific esterase.Typical “Golgi esterase”


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Fig. 76 e–g

e Different case of M2 with t(8;21).Peroxidase reaction. At top center is aneedle-shaped Auer rod above the nu-cleus

f Sudan black B reaction in the same caseis identical to peroxidase

g Same as e. Typical “Golgi esterase”

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Fig. 77 a–h. Other atypias in AML

a CE reaction. Abundant Auer rods intwo blasts in the M2 subtype of AML. Thisshould not be confused with AML-M3

b Numerous Auer rods in a rod cell fromthe peripheral blood of a patient withAML-M4

c Dysplastic cytoplasmic maturationwith a peripheral basophilic zone in thetwo cells at top. AML-M2

d Basophilic poles of cytoplasm at topand center, mature granulocytes devoidof granules. AML-M2


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Fig. 77 e–h

e–g Polyploid giant forms of granulo-cytopoiesis, reaching the size of mega-karyocytes. e At upper left is a normal-sized atypical promyelocyte next to agiant form

f Normal-sized blast to the left of a giantpromyelocyte

g Peroxidase reaction demonstratesthree positive giant forms and, at top,three negative blasts

h Giant segmented form with centralvacuolation of cytoplasm

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Fig. 78 a–c. Bone marrow smears inAML M1 and inv(3)(q21;q26) illustratethe morphologic changes that are as-sociated with inversion or translocationanomalies of the long arm of chromo-some 3

a At center is a cluster of micro(mega)-karyocytes

b Peroxidase reaction in the samepatient. At top is a positive cell. At centerand below is a group of negativemicrokaryocytes

c Schematic diagram and partialkaryotype of the inversioninv(3)(q21;q26), which is observed inAML with megakaryocytic abnormalities


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a Bone marrow smear with heavilygranulated atypical promyelocytesand cells whose cytoplasm is packedfull of Auer rods (faggots)

b Same patient. High-power view of theheavily granulated cells and cells withnumerous Auer rods. The granules andAuer rods may coexist, or Auer rods mayoccur without other granules

c At left of center is a cell with Auer rodsand pale, agranular cytoplasm

d Three atypical promyelocytes withAuer rods of various shapes

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Fig. 79 e–h

e “Hyperbasophilic” variant withstrongly basophilic ground substanceand coarse granules

f Atypical promyelocytes with dispersedviolet granules of exceptional size(like basophilic promyelocytes)

g Very dark violet granules in a differentcase

h Toluidine blue stain in the same pa-tient shows a metachromatic reaction insome of the abnormal granules. Panels gand h illustrate the basophilic variant ofM3


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Fig. 80 a–d. AML, M3V subtype

a Typical preponderance of bilobed nu-clei with an apparently agranularcytoplasm, but a more extensive searchof the bone marrow reveals heavilygranulated cells or cells with Auer rods

b Peroxidase reaction in the samepatient demonstrates a marked granularreaction in all cells

c Peripheral blood in AML-M3V withtypical bilobed or constricted nuclei andbasophilic-appearing cytoplasm, foundon electron microscopy to containminute granules

d Histologic section in M3 V. HE stain.The monocytoid, frequently bilobedshape of the nuclei may cause confusionwith monocytic leukemia. In all the casesshown in Figs. 79–84, the diagnosis hasbeen established cytogenetically by de-tecting the t(15;17) translocation or bythe molecular genetic demonstration ofPML/RARA

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Fig. 81 a–d. AML, M3V subtype withincrease of basophilic granulocytes

a At center is an abnormal promyelocytewith Auer rods. Above it and to the rightare two basophilic granulocytes

b Toluidine blue stain in the same caseshows three basophilic granulocyteslocated along a diagonal at center

c CE reaction in the same patient. Atcenter is a faggot cell with numerouspositive Auer rods. Above it are twoblasts with monocytoid nuclei showing afaint diffuse reaction

d Markedly increased basophilic granu-locytes in a different case of M3 V. Twocells with Auer rods at top and center.Scattered basophilic granulocytes, somecontaining indistinct granules


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Fig. 82 a–e. Cytochemical findingsin M3

a Intense peroxidase reaction. It iscommon to find superimposition of theAuer rods

b CE reaction gives particularly clearvisualization of the Auer rods

c Intensely CE-positive Auer rods and“Auer bodies.”

d Histologic section in untreated M3. CEreaction again shows coarse positivegranules and scattered Auer rods.(Courtesy of Prof. R. Parwaresch, Kiel)

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Fig. 82 e PAS reaction. Some positiveAuer rods are found in M3, visible here inthe middle cells and to the left


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Fig. 83 a–g. AML, M3 subtype,response to treatment with all-transretinoic acid (ATRA)

a Bone marrow smear in promyelocyticleukemia shows relatively mild atypiasprior to therapy

b Smear from same patient after 6weeks’ therapy shows definite matura-tion and “normalization” of the cells. Thepromyelocytes and myelocytes appearpredominantly normal, and a few corre-spond to later maturation stages

c After a total of 8 weeks’ therapy,conspicuous bone marrow abnormalitieshave disappeared, and only subtlechanges are noted in several matureforms

d Different case prior to therapy

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Fig. 83 e–g

e Case in d after 4 week’s therapy. Notethat the granules generally are somewhatfiner and the nuclei appear slightly moremature

f After another 3 week’s therapy, there ismarked progression of maturation withsegmented granulocytes

g After a total of 8 weeks’ therapy themarrow appears morphologically normal,signifying a complete remission


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Fig. 84 a–g. Other changes in responseto ATRA therapy

a Another case of M3 prior to therapy. Atcenter is a cell with Auer rods. At left is abasophilic granulocyte, and two othersappear at upper right and lower left

b After 4 week’s therapy, definite signsof maturation are already seen. At centeris a cell containing remnants of Auer rods

c Morphologic findings are normal aftera total of 6 weeks’ therapy. Extensiveerythropoiesis and mature granulocytesare observed

d Different case of M3 after just 3 weeks’treatment with ATRA. Segmented formsare already present, some still containingAuer rods (at bottom and lower right)

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Fig. 84 e–g

e Same case as d shows macrophagewith phagocytized cellular material,including Auer rods

f Blood smear in complete remissionfollowing ATRA therapy. The neutrophilicgranulocytes appear essentially normal

g Hypersegmented neutrophil in thesame patient


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Fig. 85 a, b. Electron microscopic view of atypicalpromyelocytes in the M3 and M3V subtypes ofpromyelocytic leukemia (Courtesy Prof. Dr. H. K.Muller-Hermelink, Wurzburg)

a Atypical promyelocyte in M3. Note the large,irregular primary granules

b M3V subtype. At center is a typical bilobed nucleuswith a thin chromatin strand linking the two nuclearsegments. At right is a large nucleolus. The cytoplasmcontains small, sparse granules

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Fig. 85 a

Fig. 85 b


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Fig. 86. Schematic diagram and partial karyotype ofthe translocation t(15;17)(q22;q12), which is foundin both the M3 and M3V subtypes of promyelocyticleukemia. In the rare cases where cytogeneticanalysis is unrewarding – the so-called kryptic PML-RARA rearrangement – diagnosis can be ensured bymeans of FISH or PCR. The interphase FISH-figuredemonstrates a normal cell with two red and twogreen signals as well as a cell with the t(15;17)/PML-RARA-rearrangement in which a red and a greensignal for the uninvolved chromosomes and a red-green colocalization signal are seen.In addition to the t(15;17) there exist the extremelyrare variants with the karyotypes t(11;17)(q13;q21)and the fusion transcript NuMA-RARA, thet(11;17)(q23;q21), PLZF-RARA, and thet(5;17)(q32;q12), NPM-RARA. Morphologically thesevariants do not exactly correspond in most cases tothe classical subtypes M3 or M3V

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Fig. 87 a–g. Acute myelomonocyticleukemia, M4 subtype

a Bone marrow smear with undifferen-tiated blasts and precursors of thegranulocytic and monocytic lines

b Peroxidase reaction in the samepatient shows peroxidase-positive blastsand later granulocytic precursors.Monocytic line is negative

c Nonspecific esterase in the samepatient shows strong, diffuse activity(brown) in four monocytes

d Different patient. Blasts with grayish-blue cytoplasm (monocytic line). Severalblasts contain coarse granules (granulo-cytic line)


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Fig. 87 e–g

e CE reaction in the same case (d) showsstrong activity in some cells (granulocyticline) and no activity in others

f Nonspecific esterase reaction in thesame case shows strong, diffuse activity(grade 4) in most of the cells

g Blood smear in M4 subtype demon-strates a large proportion of promono-cytes and monocytes. There was just over20 % granulocytopoiesis in the bonemarrow, confirming the diagnosis of theM4 subtype

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Fig. 88 a–g. Acute myelomonocyticleukemia, M4 subtype

a Pappenheim stain in bone marrowsmear. Blasts and early precursors ofgranulocytopoiesis and monocytopoiesisare difficult to distinguish morphologi-cally

b Bone marrow from the same patient.Peroxidase reaction. Almost 100 % of thecells are peroxidase-positive

c Nonspecific esterase reaction in thesame patient. Approximately 85 % of thecells are strongly and diffusely positive,confirming the diagnosis of M4. A largepercentage of the leukemic cells haveboth monocytic and granulocytic prop-erties

d Bone marrow from a different case ofM4


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Fig. 88 e–g

e Blood smear from the patient in d. Atcenter is a segmented neutrophil withAuer rods. Below it are two monocyteswith atypical nuclei

f Bone marrow histology in M4. Mega-karyocytes are increased, and some aredysplastic. HE stain

g Blood smear from the same patientshows greatly increased platelets andtwo monocytes

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Fig. 89 a–d. Variants of the M4 sub-type

a Severely dysplastic erythropoiesis. Atcenter are three erythroblasts, two ofwhich are very immature forms. The cy-toplasm of the upper erythroblast is va-cuolated

b PAS reaction in bone marrow from thesame patient shows a strong, coarselygranular reaction in four atypicalproerythroblasts and a diffuse reaction inan erythroblast at left. Three precursors ofthe white cell line show weak diffusestaining. There was less than 50 %erythropoiesis, excluding a diagnosis oferythroleukemia (M6 subtype)

c Bone marrow in M4 shows increasednumbers of basophilic granulocytes.Three basophils surround the center ofthe field

d Increased basophilic granulocytes inthe bone marrow from a different patientwith M4. At top and right are two baso-phils with unusually fine granules. To-luidine blue stain confirms the meta-chromic reaction of the granules


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Fig. 90 a–h. AML with abnormal eosi-nophils. M4Eo subtype

a This bone marrow smear containsprecursors of granulocytopoiesis, mono-cytopoiesis, and blasts along with a highpercentage of eosinophils, some of which(left and right of center) contain abnormaldark purple-violet granules. There arealso mature eosinophils, some with verysmall granules

b Different case shows a striking abun-dance of abnormal eosinophils. Thesecells are easily mistaken for basophilswith Pappenheim stain, but they do notshow a metachromatic reaction withtoluidine blue stain

c Different case with many abnormaleosinophils. Note the pleomorphism ofthe abnormal granules

d Different case with two dysplasticeosinophils at the center of the field

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Fig. 90 e–h

e Severely dysplastic granules at center.The presence of this cell is sufficient tomake a diagnosis

f Severely abnormal, immature eosino-phil and an eosinophil containing veryfine granules

g Abnormal eosinophil (bottom) next toa group of monocytes

h Blood smear in M4Eo. At top are threevery small monocytes and one monocytewith an abnormal nuclear structure.Below it is a pseudo-Pelger neutrophilform. Abnormal eosinophils are rarelyfound in blood films, and bone marrowexamination is necessary to confirm thediagnosis


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Fig. 91 a– f. Cytochemical findings inM4Eo

a The principal finding is the detection ofCE in the granules of at least some of theabnormal eosinophils. This differentiatesthe abnormal eosinophils of M4Eo fromreactive states and from eosinophils inother leukemias (e.g., M2 subtype ofAML) in which eosinophils are increased.An exception is “pure” acute eosinophilicleukemia (see p. 253). This panel showsthat a varying percentage of abnormaleosinophilic granules are CE-positive. Atupper left is an eosinophilic myelocytewith large, 100 % positive granules

b From top to bottom center are threeabnormal eosinophils that are CE-positive

c Nonspecific esterase reaction in thebone marrow in M4Eo. The monocytesshow the usual strong diffuse reaction. Itis not uncommon, however, to find casesof M4Eo in which esterase activity is lowcompared with M4 and M5, withoutabnormal eosinophilia

d PAS reaction. The abnormal granulesmay be PAS-positive. This finding ischaracteristic but not specific

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Fig. 91 e– f

e, f Adam’s reaction for the detection oftryptophan. In the blood and bonemarrow, this reaction is positive only ineosinophilic granules and so is the besttechnique for identifying eosinophils.Note that the abnormal granules andsome Charcot-Leyden crystals, whichform from eosinophilic granules (91f), arepositive


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Fig. 92 a–g. Other findings in M4Eo

a, b In bone marrow with a highpercentage of eosinophils, it is not unu-sual to find macrophages containingCharcot-Leyden crystals that havedeveloped from eosinophilic granules.a Bone marrow smear in M4Eo. Twomacrophages are laden with Charcot-Leyden crystals, which appear bright bluein this sample

b Several pale Charcot-Leyden crystals ina slightly crushed macrophage, whichalso contains bluish material (phagocy-tized granules?)

c Histologic section in M4Eo. Giemsastain. The immature (bluish-violet) andmature (reddish) granules in the eosino-phils are easily identified

d Blood smear. Even in M4Eo, Auer rodsare sometimes found in bone marrowcells and rarely in the peripheral blood

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Fig. 92 e–g

e Meningeal involvement may occur inM4Eo. In this case eosinophils were foundin the CSF during a recurrence

f Electron micrograph of an abnormaleosinophil. Predominantly eosinophilicprogranules are visible in the cytoplasm.At upper left is a condensed vacuole,which develops into eosinophilic pro-granules. The crystalloid inclusions thatare a typical feature of mature eosino-philic granules are poorly visualized. Thesmall dark granules in the intergranularcytoplasm and in the pale rim of thecondensed vacuole at upper left consist ofglycogen

g Normal eosinophil for comparison. Thegranules contain typical crystalloidinclusions !


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Fig. 93 a, b

a Schematic diagram and partial karyo-type of the inversion inv(16)(p13;q22),which is found in acute myeloid leukemiaof the M4Eo subtype. The arrows indicatethe breakpoints

b FISH technique for detecting theinv(16) in interphase cells. Note the brightred eosinophilic granules in the cyto-plasm and, above the nucleus, a signal(blue-green dot at lower right) from thenormal chromosome 16 and a two-partsignal (two groups of small dots at left)from the inverted chromosome 16. (FromHaferlach et al. [1996] Blood. 87 : 2459–2463)

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Fig. 94 a–h. Monocytic leukemia, M5bsubtype. According to FAB criteria, atleast 80 % of the nonerythroblasticbone marrow cells must be of mono-cytic lineage, and fewer than 80 % ofthese must be monoblasts

a Blood smear shows five somewhatatypical monocytes. At top is a neutrophilwith a rod-shaped nucleus

b Blood smear from the same patient.ANAE shows strong diffuse activity (grade4) in all monocytic cells

c Blood smear from a different patientwith severe leukocytosis. Besides amonoblast at top center, the cells consistof monocytes and promonocytes

d Bonemarrow from the patient in c. Theproportion of monoblasts is larger butstill less than 80 %


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Fig. 94 e–h

e Blood smear from the same patient.ANAE demonstrates very strong diffusereactivity (grade 4)

f Peroxidase reaction in the same patientshows a positive segmented neutrophilwith negative monocytes and promo-nocytes

g Monocytes and promonocytes in theperipheral blood from a different patient

h Bone marrow smear from the patientin g shows a higher proportion of pro-monocytes and monoblasts than in theblood smear

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Fig. 95 a–d. Monocytic leukemia, M5bsubtype

a Bone marrow smear from a differentpatient shows a large proportion ofmonoblasts. Borderline form with M5a

b ANAE in the same case shows strongdiffuse activity in four cells

c, d Pronounced erythrophagocytosis ina case of M5b. The t(8;16) translocationcould not be detected

d


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Fig. 96 a–h. Monoblastic leukemia,M5a subtype. Diagnostic criteria: atleast 80 % of nonerythropoietic bonemarrow cells must be of monocyticlineage, and at least 80 % of these mustbe monoblasts

a Bone marrow smear consists exclu-sively of monoblasts with round, fre-quently eccentric nuclei, distinct nucleoli,and abundant gray-blue cytoplasm

b Peripheral blood from the samepatient also shows 100 % monoblasts,which appear slightly smaller androunder

c Blood smear from the same patient.ANAE shows strong diffuse esteraseactivity in the cytoplasm of the blasts

d Peroxidase reaction in a bone marrowsmear from the same patient. Oneresidual granulocyte is positive, and allmonoblasts are negative

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Fig. 96 e–h

e Bone marrow smear in a different caseshows very immature-appearing mono-blasts, which are sometimes interpretedas nonhematopoietic tumor cells

f Extremely strong ANAE activity in thebone marrow of the patient in e

g Bone marrow smear from a differentpatient with M5a

h Bone marrow smear from the patientin g. PAS reaction. A segmented neu-trophil at lower right shows typical strong,diffuse reactivity. The monoblasts have afaint diffuse or finely granular reaction,which is condensed at the periphery ofthe cytoplasm to form coarse granules.This is a typical finding in the monocyticline


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Fig. 97 a–h. Monoblastic leukemia,M5a subtype

a, b Different regions in the same bonemarrow smear. In panel a, groups ofmonoblasts are seen to the left of centerand on the right, along with abundantnormal hematopoiesis. Panel b shows adifferent region with 100 % infiltration ofthe bone marrow by monoblasts

b

c–e Different regions of a histologicbone marrow section.

c Largely normal-appearing bonemarrowwith abundant megakaryocytes, ery-thropoiesis, and granulocytopoiesis

d Another site in the same specimen.A line from top center to lower leftseparates the monoblastic infiltrate atlower right from the area of normalhematopoiesis at upper left

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Fig. 97 e–h

e Another site showing 100 % mono-blastic infiltration

f ANAE reaction in a bone marrow smearfrom the patient in c–e shows extremelystrong, diffuse reactivity in the mono-blasts

g Very immature monoblasts in anotherpatient with M5a

h Peroxidase reaction in a bone marrowsmear from the patient in g. All themonoblasts are negative; only onenormal residual granulocyte is positive


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Fig. 98a. Karyotype of AML, FAB type M5a, with the translocation t(9;11)(p22;q23). Myelo-monocytic andmonocytic or monoblastic leukemias (FAB subtypes M4, M5a, M5b) are mainly associated with translo-cations involving band q23 of chromosome 11, on which theMLL gene is localized. Besides t(9;11), the mostimportant of these translocations are t(11;19), t(10;11) and t(6;11). The translocation t(9;11) leads to thefusion gene AF9/MLL, which, as the others, can be identified by molecular genetic analysis

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Fig. 98b, c. Bone marrow smears oftwo patients with the translocationt(8;16)

b Side by side heavy and only scantygranulated blasts. Below the middle,erythrophagocytosis

c High-power view. In the center, oneblast with erythrophagocytosis


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Fig. 98d. A special type of cell is found in AML with the rare translocation t(8;16)(p11;p13). Mostlymonoblasts/monocytic or myelomonocytic leukemias (FAB M 4 or 5) have been associated with thistranslocation. According to our studies of seven cases, we find a type of cell which lies between monoblastand promyelocyte and in most cases shows as well strong diffuse esterase (naphthyl acetate) as peroxidasereaction. Generally, there is erythrophagocytosis in a few, rarely in many blast cells. Phagocytosis ofleukocytes and platelets is very rare. The translocation t(8:16) leads to a CBP-MOZ fusion transcript. It isrelatively often found in therapy-associated AML

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Fig. 99 a–h. Acute erythroleukemiasubtype AML-M6. To meet thedefinition, erythroid progenitors mustconstitute at least 50 % of the marrownucleated cells, and blasts must con-stitute at least 30 % of the nonery-throblastic cells (NEC). There are also“pure” erythroblastic leukemias (acuteerythremia) with marked atypias of redcell precursors but very few myelo-blasts. The proportion of red cell pre-cursors may decline during the courseof an erythroleukemia (e.g., after bloodtransfusion), resulting in an M1 or M2type of pattern

a Megaloblastoid erythropoiesis. At rightis a binucleated cell

b At the bottom of the field is an ery-throblast with four nuclei, and above it tothe left is a myeloblast with an Auer rod

c At left is an erythroblast with sevennuclei and mature cytoplasm. At right is atrinucleated erythroblast with immaturecytoplasm

d CE reaction. At top is a multinucleatederythroblast with mature cytoplasm. Arelatively large number of maturegranulocytes are still seen


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Fig. 99 e–h

e Marked megaloblastoid erythropoiesisin a case of M6

f Intense granular and diffuse PASreaction in the bone marrow of the samepatient. Such a strong PAS reaction is notseen in megaloblastic anemias caused bya vitamin B12 or folic acid deficiency

g Bone marrow from a different patientwith M6 and mature erythropoiesis. Theerythrocytes and some of the erythro-blasts have cytoplasmic inclusionsconsisting of Pappenheimer bodies

h Smear from the patient in g after ironstaining. The inclusions in the erythro-cytes (center of field) are positive. Ringedsideroblasts may be numerous in M6

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Fig. 100 a–h. Acute erythroleukemia,subtype M6b

a Bone marrow from a patient with moremature but dysplastic erythropoiesis.Myeloblasts are visible above and belowthe center of the field

b PAS reaction in a bone marrow smearfrom the same patient demonstrates avery strong, predominantly diffuse reac-tion in the cytoplasm of the erythroblasts.Generally the reaction is clumped andgranular in earlier precursors and diffusein more mature forms

c Group of myeloblasts in another caseof M6b

d Peroxidase-positive myeloblast in asmear from the patient in c


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Fig. 100 e–h

e Focal acid phosphatase reaction in theerythroblasts of the same patient. Thisfinding should not be mistaken for T-cellALL

f PAS reaction in the bone marrow of thesame patient

g Extremely immature form of M6a.Some of the cells show vacuolatedcytoplasm, and there is one abnormalmitosis

h PAS reaction in a smear from the samepatient

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Fig. 101 a–h. Acute erythroleukemia,M6a subtype

a Extremely immature cells can beidentified as erythroblasts only by thestrong basophilia of their cytoplasm

b PAS reaction as an additional criterionin the same patient. This is a case of“pure” erythremia, corresponding to theM6 a subtype

c Another case shows a markedly diffusePAS reaction in an erythrocyte (targetcell)

d Severely dysplastic erythropoiesis inanother case of M6b. Myeloblasts arevisible at above left


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Fig. 101 e–h

e Bone marrow smear from the patientin d. ANAE reaction shows strong, diffuseactivity in the cytoplasm of the blasts andmore mature forms. This case is an M6subtype involving the monocytic ratherthan granulocytic line

f Pappenheim stain in the same caseshows a blast with a short Auer rod and,next to it, elliptical erythrocytes (acquiredelliptocytosis)

g, h Giant erythrocytes in another caseof M6. Note the basophilic stippling andchromatin particles in h

h

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Fig. 102 a–g. Megakaryoblastic leuke-mia (M7). This last FAB-defined subtypeof AML is more common in childrenthan adults, with more than 15 % ofcases diagnosed before 2 years of age.Only infants with the M7 subtype arefound to have the t(1;22) translocationas a specific chromosome abnormality.The definition requires 30 % megakar-yoblasts in the bone marrow smear.Often these cells cannot be identifiedmorphologically, and the diagnosismust be established by immunophe-notyping (expression of the antigensCD41 and CD61). Bone marrow fibrosisoften results in a dry tap; these casesrequire core biopsy and histologicevaluation

a Undifferentiated blasts with cytoplas-mic budding (pseudopods)

b Smear from the same patient. Peroxi-dase reaction. The blasts are negative

c Immunocytochemical detection ofCD41. APAAP technique. Blasts arepositive (red)

d Undifferentiated blasts and cytoplasmfragments in bone marrow from a child.CD41 positive


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Fig. 102 e–g

e ANAE reaction in the same patientshows a circumscribed, stippled patternof activity

f, g Bone marrow smears from a differ-ent patient. The megakaryoblasts showrelatively large nuclei, dense chromatin,and irregular cytoplasmic margins withdetached flecks of cytoplasm. Cytoplas-mic budding is particularly marked in g

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Fig. 103 a–c. Megakaryoblasticleukemia (M7)

a Different case showing large,pleomorphic blasts

b Minimal cytoplasmic budding

c APAAP technique. CD41 positive


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Fig. 104 a–c. Transition from mega-karyoblastic leukemia to a mixedmegakaryoblastic-basophilic leukemia

a The blasts appear very immature, and ahigh percentage express CD34 in addi-tion to CD41 and CD61

b Following a poor response to che-motherapy, the blasts contain dark-pur-ple cytoplasmic granules that show me-tachromatic reactivity

c Metachromatic reaction of the gran-ules after toluidine blue staining. Therehas been a transformation from themegakaryoblast phenotype to basophilicprecursors

d, e Samples aspirated from the iliaccrest in the area of a localized process

d Group of relatively large blasts showdense, homogenous-appearing nuclearchromatin and a wide, moderatelybasophilic cytoplasmic rim. CD61 andCD41 positive

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Fig. 104 e– f

e ANAE reaction. Cytoplasm shows in-tense enzymatic activity. Panels d and eillustrate a megakaryoblastoma

f Bone marrow smear from a small childwith Down syndrome shows almostexclusive proliferation of megakaryo-blasts and promegakaryocytes


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Fig. 105. Partial karyotype of a t(1;22)translocation from the bone marrow ofan infant with megakaryoblastic leu-kemia. (Courtesy Prof. O. Haas, Vienna)

Fig. 106. Electron microscopic detec-tion of platelet peroxidase in a mega-karyoblast. The reaction is detectable inthe nuclear membrane (arrow) andendoplasmic reticulum (open arrow)(upper panel). Typically the Golgicomplex (open arrows) is peroxidase-negative while the nuclear membrane(arrow) shows definite peroxidasestaining (lower panel). Mitochondriashow nonspecific peroxidase staining.(Courtesy Dr. Heil, Hannover)

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Therapy-Induced Changes in AcuteLeukemias (Figs. 107, 108; see alsoFigs. 83, 84)

Fig. 107 a–d.

a AML, M2 subtype, prior to treatment

b Severe cytopenia following two cyclesof chemotherapy

c Higher-power view of a small focus ofgranulocytopoietic regeneration withyoung promyelocytes. It can be difficultto distinguish between normal andleukemic precursors at this stage

d Complete remission after an additionalfour weeks’ therapy


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Fig. 107 e–h

e Hypocellular bone marrow from a dif-ferent patient following induction che-motherapy. Stromal cells, fat cells, plasmacells, and lymphocytes are found. Twomature granulocytes are seen at the top.This state may evolve toward remissionor recurrence

f Bone marrow findings in a differentpatient with AML-M5b, 18 days afterinduction chemotherapy with TAD(thioguainine, cytosine-arabinoside,daunorubicin). There is incipient regen-eration of neutrophilic granulocytopoi-esis

g Higher magnification of the samebone marrow demonstrates promyelo-cytes and myelocytes

h Bone marrow smear with fat cellsfollowing chemotherapy

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Fig. 108 a–d

a Bone marrow smear in ALL followingcombination chemotherapy thatincluded vincristine demonstratesmegaloblastic erythropoiesis andabnormal mitosis

b Atypical chromosomes in a mitosisfollowing cytosine-arabinoside therapy

c Bone marrow smear in recurrentmonoblastic leukemia (M5a) followingvincristine therapy, with abundantarrested mitoses in erythroid precursors

d ANAE reaction in the same patient. Atcenter is a monoblast in mitosis (diffuseesterase activity) engulfed by the ester-ase-positive cytoplasm of a macrophage.At left are two arrested erythroblasticmitoses


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Secondary Acute Myeloid Leukemias(Fig. 109 a–h)

Secondary AML may develop followingchemotherapy with alkylating cytostaticdrugs or with topoisomerase II inhibitors(particularly etoposide)

Fig. 109 a–d.

a Primary bone marrow findings inimmunologically confirmed T-cell ALL.Complete remission was achieved initi-ally

b Findings 12 months later suggested a“recurrence,” which proved to be asecondary monocytic leukemia (M5b).Promonocytes and monocytes are foundin the bone marrow

c Higher-power view of b

d Bone marrow smear in c, ANAEreaction

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Fig. 109 e–h

e Primary bone marrow findings in apatient with a long history of mantle celllymphoma (MCL) in stage IV, which hadbeen treated several times with che-motherapy including etoposide

f Bonemarrow findings on diagnosis of asecondary monoblastic leukemia (M5a).This field shows almost 100 % infiltrationby monoblasts

g ANAE reaction shows marked activityin the monoblasts

h View of a different site shows mono-blasts interspersed with abundant lym-phatic cells from the preexisting MCL.Concurrent cytogenetic studies identifieda (11;16) translocation with an 11q23breakpoint (characteristic of monocyticleukemia as well as secondary AML aftertopoisomerase II inhibitors) and also at(11;14) with an 11q13 breakpoint (typicalof MCL), confirming the coexistence ofresidual MCL cells and monoblasticleukemia


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Fig. 110 a– f. Other secondary acutemyeloid leukemias

a Monoblastic leukemia (M5a) with de-tection of the 11q23 aberration followingchemotherapy for maxillary sinus carci-noma

b, c Primary bone marrow findings inpromyelocytic leukemia (M3). Faggot celland hypergranular promyelocytes.Complete remission was achieved withcombined ATRA and chemotherapy

d An apparent recurrence diagnosed2 1/2 years later proved to be monocyticleukemia (M5b). The 11q23 aberrationwas present, but the primary t(15;17)translocation had disappeared

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Fig. 110 e– f

e Higher-power view of d

f ANAE reaction in the same smear


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Fig. 111 a–e. Recurrence of AML withdeficient granulocyte maturation

a Bone marrow at onset of therecurrence shows increased blasts

b Mature granulocytes are still relativelyabundant but are accompanied bymarkedly dysplastic, polypoid forms

c, d Peroxidase reaction differentiatesgranulocytes that are still normal fromthe predominantly dysplastic granulo-cytes, which are peroxidase-negative

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Fig. 111 e CE reaction shows the samepattern as peroxidase


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Rare Acute Myeloid Leukemias(Figs. 112– 114)

Fig. 112 a–h. Acute eosinophilicleukemia

a Blasts with reddish to purple granulesand some large vacuoles that containcoarse inclusions

b Some of the granules are dark purpleand highly variable in appearance

c Large vacuoles containing orange-colored granules

d, e Peroxidase reaction. Two neutro-philic precursors are seen at top andcenter. All other cells contain variablegranules, some quite large and somewith central pallor. These granules cor-respond to eosinophilic granules in pre-cursors

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Fig. 112 e–h

f, g CE reaction. The granules (red) areCE-positive and correspond in size andshape to those with peroxidase activity

g

h The cells were identified as immatureeosinophils by the positive Adam reac-tion of the granules (here grayish-blue)and by staining with luxol fast blue andthe peroxidase modification for eosino-phils. A specific chromosome aberrationcould not be identified in this patient,and molecular genetic analysis did notdetect inversion 16 (CBFb/MYH11)


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Fig. 112 i, j Two additional cases ofeosinophilic leukemia with a higherdegree of maturation but also withatypical eosinophilic granulocytescontaining very fine granules. Bothpatients were men, 20 and 63 years ofage

i Clumped purple granules and pleo-morphic nuclei in the cells of the 20-year-old man. As in the case above, thegranules gave a positive Adam reaction.Cytogenetic analysis identified at(10;11)(q11;p13–14) translocation. TheWilms tumor gene (wt1) was expressed, incontrast to hypereosinophilic syndrome

j The 63-year-old man had immaturecells, also with Adam-positive granules.Some of the granules were CE- andperoxidase-positive. No chromosomeabnormalities were found. The greaterthan 30 % proportion of blasts clearlyidentifies the disease as an acute leuke-mia

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Acute basophilic leukemia(Fig. 113 a–e)

These cases require differentiation froma basophilic phase of CML and fromtissue mast cells leukemia

Fig. 113 a–d.

a, b Acute basophilic leukemia with alarge proportion of blasts. Some cellscontain abundant basophilic granules,and scattered inclusions like Auer rodsare seen. Pappenheim stain (a), toluidineblue stain (b)

c Higher degree of maturation in adifferent patient

d Toluidine blue stain


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Fig. 113 e

e Distinct, in part large granules in adifferent patient

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Hypoplastic AML (Fig. 114 a–d)

Sample evaluation is feasible in thesecases only if sufficient bone marrowfragments can be aspirated andmounted as smears. Otherwise the caseshould be evaluated by core biopsy andhistologic examination. It can be verydifficult to distinguish this variant ofAML from aplastic anemias. Even his-tologic examination of the bone mar-row can be misleading unless blasts arepositively identified. The diagnosis re-lies on demonstrating a frequently cir-cumscribed collection of blasts. Spora-dic cases of acute lymphocytic leukemiamay also start with an aplastic preli-minary stage, and frequent follow-upsare necessary to establish a clear di-agnosis

Fig. 114 a–d.

a Hypocellular bone marrow withincreased fat cells

b Higher-power view of a more cellularsite shows myeloblasts and erythroblasts(left and bottom)

c Hypocellular bone marrow in adifferent patient

d Myeloblasts and isolated lymphocytesin an isolated hypercellular area of thesame sample


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5.10.2 Acute LymphoblasticLeukemia (ALL) (Fig. 115 a–d)

Fig. 115 a–d.

a Small blasts. These may closelyresemble lymphocytes but are distin-guished by their finer chromatin struc-ture and the occasional presence of nu-cleoli

b Different case showing blasts ofvarying sizes, some with pleomorphicnuclei. Panels a and b illustrate B-lineageALL

c Peroxidase reaction. All lymphoblastsare negative and are interspersed withresidual cells of granulocytopoiesis,whose proportion is more clearlydemonstrated by the peroxidase reaction

d Terminal deoxynucleotidyl transferase(TDT), detected by the immunoperoxi-dase reaction in the nuclei. TDT is notspecific for lymphoblasts but is useful fordifferentiating forms of ALL from mature-cell lymphatic neoplasias and large-celllymphomas. Today this reaction is usuallyperformed in a fluorescence-activatedcell sorter

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B-lineage ALL (Fig. 116 a–g)

Fig. 116 a–d.

a The pan-B marker CD19 is very usefulfor the immunocytochemical detectionof B-cell lineage. 100 % of the blasts inthis sample are positive (red)

b Contrast with the T-lineage markerCD3: the blasts are negative, andinterspersed mature T lymphocytes arepositive (same case as a)

c Different case with lymphoblasts ofvarying size

d Same case as c, demonstration ofCD 19


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Fig. 116 e–g

e Same case as c and d, demonstration ofCD10. The detection of CD19 and CD10correspond to c-ALL (BII)

f PAS stain yields a granular reaction in avariable percentage of lymphoblasts,which show a coarse granular or floccu-lent reaction pattern

g Coarse granular and globular PAS re-action in a different case. The reactionpattern seen in f and g, when combinedwith a pale cytoplasmic background andnegative peroxidase and ANAE, providesmorphologic and cytochemical evidenceof ALL

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Fig. 117 a–g B-lineage ALL

a A range of morphologic features maybe found in B-lineage ALL. This sampleshows relatively large blasts with anintensely basophilic cytoplasm

b Large blasts with marked cytoplasmicbudding, at first suggesting megakaryo-blastic leukemia. Both a and b representB-lineage ALL

c Vacuolation is not uncommon in ALL.Relatively large vacuoles are seen in thiscase

d Vacuoles in a different case. The coarsegranular PAS reaction in the cytoplasm isnot associated with the vacuoles


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Fig. 117 e–g

e Large, partially confluent vacuoles in adifferent patient

f PAS reaction in the same case (e) showsthat the vacuoles are filled with glycogen

g Peroxidase reaction in the same caseclearly demonstrates the vacuoles in theperoxidase-negative blasts

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Fig. 118 a–d. Mature B-ALL (B IV) cor-responding to the FAB subtype L3. Thissubtype of ALL is rare, but its recogni-tion is important because the prognosiscan significantly improve with specifictherapy. Morphologic examinationshows round, relatively uniform blastsof moderate size with intensely baso-philic cytoplasm and sharply defined(fat-containing) vacuoles

a–c Various examples of this subtype ofALL

b

c

d Bone marrow smear in whichinfiltration is confined to the upper partof the field


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Fig. 119 a–h. ALL with cytoplasmicgranules. One morphologic subtype ofALL violates the dogma that cytoplas-mic granules are suggestive of AML.This case is a B-lineage type of ALL withcytoplasmic granules that are peroxi-dase-negative and behave as lyso-somes

a Blasts above the center of the fieldcontain coarse purple granules. Severalfiner granules are visible below

b PAS reaction. The abnormal granulesstain pink, contrasting with the burgun-dy-red staining of the other glycogengranules

c ANAE reaction. The distinct, intenselyreacting granules in the cytoplasmcorrespond to those seen in a and b

d Another case with very prominentdark-purple granules. They might bemistaken for basophilic granules but aredistinguished by an absence ofmetachromasia

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Fig. 119 e–h

e Relatively large, intensely staininggranules in a different case

f Acid phosphatase reaction. Some ofthe granules are markedly positive

g, h Electron micrographs reveal large,membrane-bound inclusions in thecytoplasm with finely granular contentscorresponding to the granules describedabove. (Courtesy of Prof. Dr. Muller-Her-melink, Wurzburg)

h


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Fig. 120 a, b. Chromosome abnormal-ities in ALL. The (9;22) translocation inALL indicates a particularly unfavorableprognosis. This finding correspondscytogenetically to that described inCML. In terms of molecular genetics, theminor breakpoint cluster region (m-bcr)is predominantly affected in ALL. a The(4;11) translocation, which also impliesa poor prognosis. b The translocationt(8;14) (q24;q32), connected to Burkittlymphoma and mature B-ALL (B IV)

a

b

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T-lineage ALL (Fig. 121 a–e)

Usually the B-cell and T-cell forms of ALLare morphologically indistinguishable.The pronounced irregularity of the nu-clear contour (“convoluted nucleus”) andhigh rate of mitoses are more consistentwith T-ALL

Fig. 121 a–c.

a Pappenheim stain in T-ALL

b The focal or paranuclear acid phos-phatase reaction pattern shown here istypical of T-lineage ALL

c Acid phosphatase reaction using adifferent technique (Sigma)


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Fig. 121 d, e

d A case of T-ALL with consistentlyround nuclei

e Another case with some irregularnuclear contours

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Fig. 122 a–d. T-lineage ALL

a Vacuolation of the cytoplasm may alsobe observed in T-ALL

b Acid phosphatase reaction at a typicallocation in the same case

c Detection of CD3 in the same case

d Basophilic cytoplasm and vacuoles in adifferent case


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Other Morphologic Variants of ALL(Fig. 123 a–h)

Fig. 123 a–d.

a “Hand-mirror” form with a handle-likeextension of the cytoplasm. Thismorphologic subtype of ALL has nospecial significance but is shown to avoidconfusion with monoblasts

b Different case with fine granules and ahand-mirror configuration of thecytoplasm

c Detection of CD19 in smears from thesame patient

d Detection of CD10 in a smear from thesame patient. Findings are consistentwith c-ALL

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Fig. 123 e–g

e Very rarely, pseudo-Gaucher cells arealso found in ALL. This case involves aT-ALL

f Acid phosphatase reaction in the samecase shows strong activity in the cyto-plasm of a pseudo-Gaucher cell and afocal reaction in lymphoblasts

g Bone marrow smear of a patient withc-ALL and eosinophilia and the raretranslocation t(5;14). In the center, oneblast and the nucleus of a blast, aboveand below one eosinophil each


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Acute Leukemias with MixedPhenotypes

Fig. 124 a–g. Acute leukemias invol-ving the lymphatic and granulocyticcell lines (hybrid, biphenotypic, bi-linear, mixed lineage). The EuropeanGroup for the Immunological Charac-terization of Leukemias has proposed apoint system to assist in classificationwhich has been taken over byWHO [seeBene, MC et al (1995) Proposals for theimmunological classification of acuteleukemias. European Group for theImmunological Characterization ofLeukemias (EGIL). Leukemia. 9: 1783–1786]. These types of leukemia mostcommonly occur in early T-ALL withmyeloid markers. They also occur intrue bilinear forms and occasionally inPhiladelphia-positive forms of ALL,which also have a myeloid component

a Lymphatic blasts are interspersed withscattered mature lymphatic cells andlarge blasts with somewhat pleomorphicnuclei and lighter cytoplasm

b Higher-power view shows reddishgranules in the cytoplasm of the largerblasts (lower right of center)

c A number of the large blasts are po-sitive for peroxidase stain, confirmingtheir granulocytic lineage

d The peripheral blood contains verysmall numbers of mature granulocyteswith Auer rods

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Fig. 124 e–g

e Almost all the blasts express CD13 onimmunocytochemical analysis

f More than 60 % of the small blasts areCD3–positive. Panels a through f illus-trate the coexistence of T-ALL and AML

g In another case previously classified asT-ALL, we found myeloid precursors withatypias (center right, upper left), againdemonstrating the involvement ofgranulocytopoiesis


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Fig. 125 a– f. Originally diagnosed asAML, this case was identified by im-munocytochemical analysis as bilinearAL with concomitant involvement ofthe T-ALL and myeloid lines.

a Peripheral blood reveals a blast with around nucleus and mature granulocyteswith Auer rods

b Higher-power view of a granulocytewith multiple Auer rods

c Low-power view of bone marrowshows a predominance of small blastswith round nuclei interspersed with largeblasts showing pseudo-Chediak anomalyand splinter-like inclusions (arranged di-agonally from upper left to lower right)

d Numerous fine Auer rods are seen athigher magnification

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Fig. 125 e– f

e Low-power view of CE reaction. TheAuer rods and positive inclusions areclearly demonstrated (red)

f Higher magnification demonstratesAuer rods and, below, inclusions withAuer rods


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Fig. 126 a, b. In a case of Ph-positiveALL, severely dysplastic granulocytesare interspersed among the lympho-blasts, again confirming the involve-ment of granulocytopoiesis. The pa-tient did not have prior therapy. Ph-positive cases of ALL are commonlyassociated with the coexpression ofmyeloid markers without detectablemorphologic changes in granulocyto-poiesis

a, b Marked dysplasia is evident in thematuration of granulocytopoieticprecursors

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CSF Cytology in Acute Leukemias andMalignant Lymphomas (Fig. 127 a– f)

Samples were cytocentrifuged andPappenheim-stained

Fig. 127 a– f.

a, b Inflammatory CSF findings forcomparison

a CSF in purulent meningitis with neu-trophilic leukocytes. Amonocyte is visibleat upper right

b CSF in viral meningitis. Lymphocytechanges in the CSF are similar to thoseseen when EDTA-treated blood is storeda long time before smears are prepared

c CSF. Numerous typical lymphoblasts inleukemic meningitis

d CSF findings during lymphatic blastcrisis in chronic myeloid leukemia


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Fig. 127 e– f

e CSF findings inmeningeal involvementby follicular lymphoma (cb/cc). Note thehighly pleomorphic lymphatic cells andnumerous mitoses

f CSF findings in meningeal involvementby monoblastic leukemia

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5.11 Neoplasias of Tissue Mast Cells(Malignant Mastocytoses)

We can distinguish mature-cell systemic masto-cytoses with or without cutaneous involvement(urticaria pigmentosa) from immature-cell andleukemic forms (mast cell leukemias). As withnormal mast cell, the tissue mast cells in ma-ture-cell mastocytoses are identified by toluidineblue staining (to detect metachromasia) and bythe CE reaction of the granules. As atypias be-come more pronounced, however, the metachro-masia persists while the CE reaction weakens orbecomes negative. By contrast, a positive tryptasereaction can be demonstrated both in mature-cell

mastocytoses and in atypical immature-type tis-sue mast cells using immunocytochemical meth-ods1.

WHO proposed the following classification ofmast cell diseases:– Cutaneous mastocytosis– Indolent systemic mastocytosis (ISM)– Systemic mastocytosis with associated clonal,

hematological non-mast cell lineage disease(SM-AHNMD)

– Aggressive systemic mastocytosis (ASM)– Mast cell leukemia (MCL)– Mast cell sarcoma (MCS)– Extracutaneous mastocytoma

1 Baghestanian M, Bankl Hc, Sillaber C, Beil WJ, RadaszkiewiczT, FurederW, Preiser J, Vesely M, Schernthaner G, Lechner K,Valent P (1996) A case of malignant mastocytosis with circu-lating mast cell precursors: biologic and phenotypic charac-terization of the malignant clone. Leukemia 10 : 159–166


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Fig. 128 a–d. Neoplasias of tissue mastcells (malignant mastocytoses). Tissuemast cells are positively identified bydetecting a metachromatic reaction inthe granules. Even with high grades ofcellular atypia, it is usually possible todetect at least isolated metachromaticgranules. Naphthol AS-D chloroacetateesterase (CE) is present in normal andreactive mast cells and in the mast cellsof mature-cell (systemic) mastocytoses.As atypias increase, the enzyme disap-pears, and often it is no longer de-tectable when atypias are severe.Tryptase, however, can be detectedeven in immature malignant mastocy-toses. Mature-cell systemic mastocy-toses, which may or may not have cu-taneous manifestations (urticaria pig-mentosa), are distinguishable fromimmature-cell malignant mastocytosesand leukemic forms (tissue mast cellleukemia)

a Indolent systemic mastocytosis in abone marrow smear from a child.Pappenheim stain

b Toluidine blue stain in the same caseshows pronounced metachromasia ofthe granules

c CE reaction demonstrates very highactivity in the tissue mast cells

d Dense infiltration of a bone marrowfragment in themarrow smear of an adultwith mature-cell systemic mastocytosis.Toluidine blue stain

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Fig. 129 a–h Malignant mastocytosis

a Aleukemic mast cell leukemia with verycoarse granules

b Same case shows loosely arrangedchromatin pattern in the enlarged andirregularly shaped nuclei. Granules areloosely distributed and markedly larger,and some are contained in vacuoles

c View of a different site in the samesample

d Toluidine blue stain demonstratesmasses of confluent granules


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e Toluidine blue stain, higher-powerview

f Different case of malignant mastocy-tosis (aleukemic mast cell leukemia) withvery sparse granules, some localized tothe perinuclear area

g Loosely scattered granules in the samecase

h Granules are visible only in some cellsand appear indistinct

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Fig. 130 a–e. Malignant mastocytosiswith erythrophagocytosis

a Bone marrow smear shows very im-mature blasts that contain only a fewmetachromatic granules mostly localizedto the perinuclear zone (center)

b Different site in the same bonemarrowsmear

c Same case, showing a high percentageof “mastoblasts” with erythrophagocy-tosis

d A mastoblast in the peripheral blood


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Fig. 130 e Toluidine blue stain of a bonemarrow smear from the patient in a–d

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Lymph Nodes and Spleen

6. Cytology of Lymph Node and Splenic Aspirates 294

6.1 Reactive Lymph Node Hyperplasia 295

6.2 Infectious Mononucleosis 304

6.3 Persistent Polyclonal B Lymphocytosis 307

6.4 Malignant Non-Hodgkin Lymphomas and Hodgkin Lymphoma 308

6.4.1 Some Mature B-cell Lymphomas (Table 14) 3116.4.2 T-cell Lymphomas 3356.4.3 Plasma Cell Myeloma (Multiple Myeloma, Kahler Disease) 3556.4.4 Natural Killer (NK)-Cell Neoplasias 3726.4.5 Leukemia of Dentric Cells (CD4 + CD56 + Neoplasia) 3776.4.6 Hodgkin Lymphoma (Lymphogranulomatosis) 379

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An accurate diagnosis requires considerable ex-perience in the interpretation of lymph node as-pirates or touch preparations. Above all, the ex-aminer must be familiar with the great morpho-logic diversity of reactive lymph node changes,and so particular attention is given to thesechanges in the present chapter. Four main typesof cytologic pattern may be seen on the examina-tion of smears prepared from lymph node aspi-rates (after Theml 1986):1. A reactive cellular composition without speci-

fic cellular elements. Infections are usuallycausative, and reactive hyperplasia should besuspected.

2. A reactive cellular composition with specificelements, such as a preponderance of epithe-loid cells (consistent with sarcoidosis) or Lang-hans cells (consistent with tuberculous lym-phadenitis). Hodgkin cells and Sternberg cellsshould raise suspicion of Hodgkin disease.

3. A monotonic picture composed of lymphaticcellular elements. This should raise suspicionof non-Hodgkin lymphoma.

4. Cells extrinsic to the lymph nodes, consistentwith a metastatic tumor.

Splenic aspiration has been used as a diagnosticprocedure for more than half a century. Its im-portance has grown in recent decades owing togeneral improvements in cytologic methodsand their expanded use. The procedure is bestperformed under direct laparoscopic vision orunder ultrasound guidance when a percutaneousneedle is used (see p. 5). In all cases it is preferableto use the Menghini needle or other thin-gaugeneedle in order to obtain material for both cyto-logic and histologic evaluation. The criteria forassessing the benignancy or malignancy of a cy-tologic smear are the same as in lymph node as-pirates. Unfortunately, smear cytology is of littlevalue in a number of conditions that are asso-ciated with splenomegaly (e.g., fibroadenia).The counting of splenograms is usually unre-warding.

In all diseases that are associated with lymphnode enlargement, a serious effort should bemade to supplement the cytologic examinationwith a histologic diagnosis.

6. Cytology of Lymph Nodeand Splenic Aspirates

The great majority of cells in aspirates or touchpreparations of normal lymphatic tissue are smallto medium-sized mature lymphocytes. These arelargely identical to blood lymphocytes and are ac-companied by smaller lymphatic cells or earlierprecursors of lymphocytopoiesis, which areknown collectively as large lymphoid cells. Usual-ly the prevalence of these cells is 10 % or less.They consist of lymphoblasts, immunoblasts,and centroblasts. Small numbers of immatureand mature plasma cells are also present. Tissuemast cells, granulocytes, histiocytes, and epithe-loid cells are less commonly found. More com-mon are heterogeneous groups called “reticulumcells.” Below we shall describe cell types that dis-play characteristic quantitative or qualitativechanges during the course of reactive or autono-mous lymph node disorders and are thereforehelpful in making a diagnosis. Immunocytologicmethods are particularly useful in differentiatingnormal cells of lymphocytopoiesis and their ma-lignant equivalents.

The larger basophilic cells provide an impor-tant clue to the function and proliferative beha-vior of the lymphatic tissue, and they are readilydistinguished from the mass of mature lympho-cytes in the low-power microscopic field. How-ever, they may show a range of morphologiesthat make it difficult to differentiate them accord-ing to type. The following three cell types are ofprimary interest:

The immunoblast (Fig. 131 a, left) can rangefrom 25 to 30 lm in diameter according to itsfunctional state. Normally its cytoplasmic rimis moderately wide and darkly basophilic, appear-ing broader and lighter when the cell is function-ally stimulated. Often several vacuoles are pre-sent. The nucleus is usually centered and isround, oval, or slightly indented. It shows afine reticular chromatin pattern that at times ap-pears almost homogeneous. Usually there arewell-defined, irregularly shaped nucleoli of apale to dark blue color.

The second cell type is somewhat smaller thanthe immunoblast (approximately 20 lm in di-ameter). Its cytoplasmic rim is narrow and darklybasophilic. The centrally placed nucleus contrastssharply with the cytoplasm owing to its pale color,which serves to distinguish the cell from the im-munoblast, plasmablast, and lymphoblast. Thenuclear chromatin pattern is relatively coarseand meshlike. One or more nucleoli may be visi-

294 Chapter V · Lymph Nodes and Spleen

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ble and tend to be close to the nuclear membraneand of a pale color, especially in histologic sec-tions. These cells (Fig. 131 a, center) are identicalto the “centroblasts” described by Lennert.

Plasmablasts (Fig. 131 a, right) are usuallysomewhat smaller than immunoblasts. Theirabundant cytoplasm stains a deep, dark blue.Perinuclear pallor is often found in the more ma-ture forms. One or more cytoplasmic vacuoles areoccasionally seen. The nucleus, which is concen-trically placed in the younger “proplasmablasts”(¼ B immunoblast?), migrates closer to the cyto-plasmic border as the cell matures, much as inmature plasma cells. The chromatin has a coarsetrabecular structure and often appears stippled.Usually the nucleoli are poorly defined by panop-tic staining. Plasma cells are described on p. 65,66, tissue mast cells on p. 41, and epitheloid cellsin Figs. 136 a, d, 137 b, and 138.

The lymphoblast (see Fig. 131 b, c) is 15 to20 lm in diameter and has a relatively narrowand lightly basophilic cytoplasmic rim. The nu-cleus is centered, coarsely reticular, and containsa pale, medium-sized nucleolus.

The histiocyte is a relatively large cell (15–25 lm) whose morphology is frequently ill-de-fined. It is probably related to the monocyte.Its cytoplasmic rim is pale gray-blue to violet, de-pending on its functional state, and often con-tains fine reddish granules. The nuclear structureusually has a coarse, stringy appearance but maybe relatively loose. One or more small, deep bluenucleoli are usually present. The cell is rich in en-zymes, especially nonspecific esterase. Unlike the“lymphatic” reticulum cells, the histiocyte is per-oxidase-positive.

Among the reticulum cells of the lymphatic tis-sue, the interdigitating and dendritic forms ap-pear to play a special role. The interdigitating re-ticulum cell (see Fig. 132) is a specific cell of thethymus-dependent region of the lymph node(paracortical and interfollicular regions of thenodes and periarteriolar areas of the spleen).These cells have an irregularly shaped, often bi-zarre nucleus with a relatively loose, delicatechromatin pattern and small, pale, indistinct nu-cleoli. The cytoplasm stains very weakly andtends to blend with its surroundings. These cellsshow an extremely weak acid phosphatase reac-tion and nonspecific esterase reaction on cyto-chemical staining (Fig. 132 c, d).

Dendritic reticulum cells occur exclusively ingerminal centers, primary follicles, and occasion-ally in the outer follicular zone. Their cytoplasmstains very weakly with panoptic stain and con-trasts poorly with its surroundings. The nucleusis oblong and sharply marginated with loose,coarsely meshed chromatin and a pale blue nu-

cleolus of moderate size. Cytochemical staining(Fig 133 a) elicits a weakly positive nonspecific es-terase reaction and an apparently negative acidphosphatase reaction.

The remaining reticulum cells are at leastpartly derived from histiocytes and macrophages.It is by no means certain, however, that all storagecells are descendants of the monocyte-macro-phage system, although this question is not criti-cal to clinical evaluation.

The “starry sky macrophages” (Fig. 134 a–c)are unique among the macrophages in their mor-phologic and diagnostic importance. They arethought to be the result of an “ineffective germ-inal center proliferation” whose production ismediated by the effects of antigens, antibodies,or antigen-antibody complexes. These cells areeasily recognized by their size and by the coarseparticles (phagocytized nuclear debris) that theycontain. They are strongly positive for acid phos-phatase and nonspecific esterase (Fig. 134 b, c).

Splenic aspirates additionally contain cellforms that are not present in lymph node smears.Serosa cells are shed from the single layer of peri-toneum that covers the spleen. Besides splenic as-pirates, these cells are commonly found in the se-diment of aspirated ascitic fluid. They are 20 to40 lm in diameter and are often grouped in smallclusters. Their borders are irregular, and their cy-toplasm stains gray-blue to deep blue. The nu-cleus is often eccentrically positioned. The chro-matin is relatively dense and coarse, and two orthree bluish nucleoli are often present. Multinu-cleated forms sometimes occur.

6.1 Reactive Lymph Node Hyperplasia

This term may be applied collectively to all mor-phologic changes that affect lymph nodes, such asthe nodes draining an area of infection or nodesresponding to other kinds of immunologic chal-lenge. While the morphologies of these changesare extremely varied, they are characterizedvery generally by an increase in the large basophi-lic cell forms described on p. 286. These cells alsomanifest significant qualitative changes. Typicallythey increase in size, their cytoplasmic rim widensand often becomes lighter and vacuolated, andthe nuclear-cytoplasmic ratio increases. The nu-cleus enlarges to some degree, and its chromatinpattern appears less dense. Marked enlargementof the nucleoli is often a prominent feature. Thesechanges can result in cells that resemble mono-nuclear Hodgkin cells (“Hodgkin-like cells,” seep. 297. As a rule, storage cells are also increasedin size and number. Macrophages dotted with nu-clear debris (“starry sky macrophages,” see pp.

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292–294) are frequently seen as evidence of a fol-licular lymphatic hyperplasia. Besides the lym-phatic cells, increased numbers of tissue mast cellsand granulocytes are found in hyperplastic lymphnodes. The eosinophils are often very prominent,although their increase cannot be attributed toany specific pathogenic factors. A lymph node hy-perplasia with a particularly marked increase ineosinophils is described as hyperergic. Thisterm is appropriate in that the diffuse lymphnode enlargement seen in these conditions ismerely symptomatic of a severe disorder (prob-ably allergic) that is associated with hypereosino-philia in the peripheral blood (see p. 105). Thesedisorders probably belong to the large class of“undefined, abnormal immune reactions,” whosebest known representative is “immunoblasticlymphadenopathy.”1

There are few diseases for which smear cytol-ogy can establish an etiology. The most important

are tuberculosis (Langhans giant cells, epitheloidcells, liquefactions Fig. 137 a, b), sarcoidosis(Boeck disease, epitheloid cells, isolated Lan-ghans giant cells Fig. 138 a, b), and toxoplasmosis(epitheloid-cell lymphadenitis of Piringer-Ku-chinka, see Fig. 136 a–d).

In the examination of cervical lymph node as-pirates, an enlarged branchiogenic cyst should beconsidered in the differential diagnosis. The mor-phologic features of these cysts are highly charac-teristic. They consist mainly of large epithelialcells with a small, dense, central nucleus andhomogeneous cytoplasm. Abundant storage cellsand necrotic material are usually seen as well.

1 Lukes RJ, Tindle BH (1971) Immunoblastic lymphadeno-pathy. A hyperimmune entity resembling Hodgkin’s disease.N Engl J Med 292 : 1–8.


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Fig. 131 a–c. Lymph node cytology

a Immunoblast at left, two centroblastsat center, and a plasmablast at right

b Immunoblast at center, lymphoblast attop center

c Plasmablast at upper left, lymphoblastat center

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Fig. 132 a–d. Lymph node cytology,interdigitating reticulum cell

a, b Panoptic stain shows typical closerelationship of lymphocytes to reticulumcells and their cytoplasmic extensions

b

c Acid phosphatase reaction

d Nonspecific esterase reaction.(Fig. 132 courtesy of Prof. E. Kaiserling,Tubingen)


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Fig. 133. Lymph node cytology,dendritic reticulum cell

Panoptic stain

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Fig. 134 a–d. Lymph node cytology,storage cells

a Starry sky macrophage (macrophagecontaining nuclear debris), panoptic stain

b Starry sky macrophage, acidphosphatase reaction

c Two starry sky macrophages, nonspe-cific esterase reaction. (Fig. 134 a–ccourtesy of Prof. E. Kaiserling, Tubingen)

d Two storage cells containing anthra-cotic material (mediastinal lymph node).Immunoblast at right, above it twoplasmablasts


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Fig. 135 a–d. Lymph node hyperplasia

a Two immunoblasts and an eosinophilin lower half of field

b Plasmablast at top center, severalplasma cells at bottom, tissue basophilat left

c Starry sky macrophage at center,several immunoblasts at right

d Immunoblast at upper left, above it acentroblast, at right a reticulum cell

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Fig. 136 a–d. Lymph node hyperplasiain toxoplasmosis

a Epitheloid cell lymphadenitis (Piringer-Kuchinka type). Epitheloid cell cluster atleft, an immunoblast at center, to its righta binucleated plasma cell

b Starry sky macrophage at center

c Starry sky macrophage. Typical speci-men with many coarse granular inclu-sions and cytoplasmic vacuoles

d Epitheloid cell shows typical fine butdense chromatin pattern with one or twoblue, sharply defined nucleoli


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Fig. 137 a, b. Tuberculous lymphade-nitis

a Langhans giant cell

b Syncytium of epitheloid cells

Fig. 138 a, b. Sarcoidosis(Boeck disease), lymph node

a Numerous epitheloid cells resemblinga school of fish

b Epitheloid cells in a “footprint”configuration

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6.2 Infectious Mononucleosis

Infectious mononucleosis is caused by the Ep-stein-Barr virus. Cytologically it is the prototypeof lymph node hyperplasias of viral etiology. Theclinical picture may be dominated by lymph nodeenlargement and splenomegaly or by a pseudo-membranous, lacunar, or ulcerative sore throat.Fever may reach 39 8C and persists for severaldays or as long as 2 to 3 weeks. Often the feverprecedes the onset of glandular swelling. Thered blood count is usually normal, and mild an-emia is rare. Generally there is a leukocytosis of10,000 to 15,000/lL, which in rare cases exceeds50,000/lL. Sporadic cases present with leukocy-topenia; this finding plus the pharyngitis can mi-mic agranulocytosis. Thrombocytopenia is occa-sionally seen and may be so pronounced that ahemorrhagic diathesis results. The leukocyte dis-tribution is characteristic. Examination of theblood smear shows a predominance of pleo-morphic mononucleated cells (60 %–90 %),which give the disease its name. Morphologicallythese cells may resemble young lymphocytes orlymphoblasts. In the early phase of the diseaseone finds granulated lymphocytes or lympho-cytes with small vacuoles at the circumferenceof the cytoplasm. Some appear as large blast-like cells with strongly basophilic cytoplasm. Asignificant increase in monocytes is not observed.

Because similar cells can occur in other viral dis-eases, they have also been referred to as “viro-cytes” or lymphoid cells. The nuclei are pleo-morphic, often kidney-shaped or irregular, andthe nuclear chromatin forms a coarse meshworkof loosely arranged strands. One or more nucleoliare usually present. Azurophilic granules arefound in some cells, which represent transformedT-lymphocytes. Bone marrow findings are gener-ally nonspecific, and thus the changes are typicalof infection. The mononuclear elements of thebone marrow may be increased in a few cases,but never to a high degree.

By contrast, large numbers of typical cells arefound in lymph node aspirates (Fig. 139). The pic-ture is dominated by mononuclear cells very si-milar or identical to those in the peripheral blood.Particularly striking are the large basophilic cellswhose “transformed” state is evidenced by theirenlarged, blue-tinged nucleoli. The cytologicchanges can even cause confusion with Hodgkincells (“Hodgkin-like cells”). Isolated epitheloidcells are also found.

The diagnosis of infectious mononucleosis isestablished by detecting antibodies against theEpstein-Barr virus (EBV). While the rapid testsgive a general impression as to whether infectiousmononucleosis is present, their capabilities areotherwise limited.


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Fig. 139 a–d. Infectious mononucleo-sis, lymph node

a Marked increase of immunoblasts,macrophage at lower right

b At center an immunoblast, severalplasma cells

c Various immunoblasts, with severalpleomorphic lymphoid cells in lower halfof field

d Two strongly stimulated immuno-blasts, Hodgkin-like cells

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Fig. 140 a–d. Blood smears ininfectious mononucleosis

a–c Typical pleomorphic lymphaticcells. Often these cells have irregularnuclear contours and basophilic cyto-plasm, and some resemble blasts

b

c

d Detection of CD3, APAAPmethod. CD3is detectable in the typical cells of in-fectious mononucleosis (red). These cellsrepresent transformed T lymphocytes


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6.3 Persistent Polyclonal B Lymphocytosis

This is a lymphocytosis stable for years, and bi-nucleated lymphocytes are detected in peripheralblood smears (about 3 %). These changes arealmost only found in cigarette-smoking womenunder 50 years of age. In most cases there is apolyclonal increase of IgM; an association withHLA-DR 7 seems to exist. Of 25 women withthis anomaly, 77 % had an isochromosome3q(+i(3q)) (Fig. 140 e).

Fig. 140 e. Lympho-cytes with two nucleiin persistent polyclo-nal B lymphocytosis

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6.4 Maligne Non-Hodgkin-Lymphomeund Hodgkin-Lymphom

Wahrend das Hodgkin-Lymphom ein eigenesKrankheitsbild (mit großer Variationsbreite inSymptomatik und Prognose) darstellt, bildetdie Gruppe der malignen Non-Hodgkin-Lym-phome eine hinsichtlich Erscheinungsbild, Prog-nose und Therapie recht heterogener Krankheits-gruppe, deren Ubersichtlichkeit zudem durch diewechselnden Klassifizierungsvorschlage noch zu-satzlich erschwert wird. Tabelle 14 und 15 gebeneinen Uberblick uber die Non-Hodgkin-Lym-phome. In Tab. 16 sind typische Immunmar-ker-Profile zusammengestellt.

Nach der Herkunft der erkrankten Zellen kon-nen B- und T-Zell-Lymphome unterschieden wer-den. B-Zell-Lymphome sind in Europa und Ame-rika haufiger, wahrend die T-Zell-Lymphome inAsien uberwiegen. Unter Einbeziehung von mor-phologischen und zytochemischen Befunden, Im-munphanotyp, Zytogenetik und Molekulargene-tik sowie klinischen Daten hat die WHO-Arbeits-gruppe nach den Prinzipien der Revised Euro-pean-American Classification of Lymphoid Neo-plasms (REAL) die neue Einteilung der malignenNon-Hodgkin-Lymphome und verwandter Er-krankungen konzipiert. Nach wie vor stutztsich der Primarbefund auf die Morphologieund manche Erkrankungen sind alleine morpho-logisch definiert, haufig ist aber die Definitioneiner Entitat ohne Immunphanotyp oder gene-tisch-molekulargenetisches Profil nicht moglich.B-, T- und NK-Zell-Neoplasien werden weiterin Vorstufen-(Precursor) und reife (mature) Er-krankungen unterteilt. Auf die Einteilung nachprognostischen Kriterien (niedrig-maligne – in-dolent, intermediar – aggressiv, hoch-maligne –sehr aggressiv) wurde verzichtet.

Es ist nicht immer moglich, allein mit Hilfe derAusstrichzytologie eine eindeutige Klassifizie-rung der verschiedenen Lymphome vorzuneh-men. Trotzdem gibt es eine Reihe von zytomor-phologischen Kriterien, die in den meisten Fallenzumindest eine Verdachtsdiagnose gestatten. Siesollen in den folgenden Abbildungen dargestelltwerden.

Die aktuelle Diagnostik und Klassifizierungder malignen NHL stutzt sich auf die histologi-sche und immunologische Untersuchung von ex-stirpierten Lymphknoten oder anderen befalle-nen Geweben. Wie bei den akuten Leukamiensind zytogenetische oder molekulargenetischeBefunde wichtige Erganzungen oder sogar ent-scheidend fur die genaue Einordnung. Lymph-knotenpunktate spielen in der Primardiagnostikeine untergeordnete Rolle, es sei denn, periphere

Lymphknoten konnen aus technischen Grundennicht exstirpiert werden. Andererseits ist diePunktatdiagnostik eine wichtige Erganzung,wenn Organe im Abdomen, Thorax oder intraab-dominell gelegene Lymphknoten unter sonogra-phischer oder computertomographischer Kon-trolle gezielt punktiert werden konnen. Auchzur schnelleren Orientierung oder Verlaufskon-trolle sind sie hilfreich. In unterschiedlichem Gra-de ist auch das Knochenmark befallen, und /oderes kommt zur leukamischen Ausschwemmung indas periphere Blut. Wir werden in diesem Ab-schnitt auf die Beteiligung von Knochenmarkund Blut bei NHL eingehen, im ubrigen verweisenwir auf die Publikation zur WHO-Klassifizierung.Beim Hodgkin-Lymphom ist der Knochenmark-befall am ehesten nach histologischer Untersu-chung von Biopsien, selten auch im Ausstrichvon Aspiraten nachzuweisen. Das periphereBlut liefert nur indirekte Hinweise, wenn eineLymphopenie oder selten eine Eosinophilie be-stehen. Wir richten uns bei der Nomenklaturnach der WHO-Klassifizierung.

Bei der histologischen Beurteilung von Kno-chenmark-Schnittpraparaten spielt die Art desInfiltrationsmusters speziell in den fruhen Sta-dien der Erkrankung eine wichtige Rolle, da esteilweise recht spezifisch ist.

In der Schemazeichnung 1 ist die Topographievon Lymphominfiltraten im Knochenmark sche-matisch dargestellt (Schaefer, H. E.).

308 Kapitel V · Lymphknoten und Milz

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Paratrabekular: Splenic marginal zone lympho-ma, mantle cell lymphoma

Paratrabekular osteoclastic: Plasma cell myelo-ma, adult T-cell leukemia/lymphoma

Random interstitial: Advanced hairy cell leuke-mia, large B-cell lymphoma

Random tumorforming: Large B-cell lymphoma,Hodgkin-lymphoma, advanced hairy cell leuke-mia

Courtesy Prof. Dr. H. E. Schaefer, Freiburg. Upuntil now, he has only presented his schematicdrawings publicly once previously, during a Japa-nese-Korean workshop in 1995.

Suggested Further Reading

World Health Organisation (2001) Classificationof Tumours: Pathology and Genetics. IARC Press,Lyon (Tumours of Haematopoietic and Lym-phoid Tissues. Ed.: E. S. Jaffe, N. L. Harris, H.Stein, J. W. Vardiman)

Table 14. WHO classification of mature B-cell neoplasms

B-cell neoplasmsPrecursor B-cell neoplasmPrecursor B lymphoblastic leukemia lymphoma

Mature B-cell neoplasmsChronic lymphocytic leukemia / small lymphocytic lymphomaB-cell prolymphocytic leukemiaLymphoplasmacytic lymphomaSplenic marginal zone lymphomaHairy cell leukemiaPlasma cell myelomaMonoclonal gammopathy of undetermined significance (MGUS)Solitary plasmacytoma of boneExtraosseous plasmacytomaPrimary amyloidosisHeavy chain diseasesExtranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT-lymphoma)Nodal marginal zone B-cell lymphomaFollicular lymphomaMantle cell lymphomaDiffuse large B-cell lymphomaMediastinal (thymic) large B-cell lymphomaIntravascular large B-cell lymphomaPrimary effusion lymphomaBurkitt lymphoma/leukemia

B-cell proliferations of uncertain malignant potentialLymphomatoid granulomatosisPost-transplant lymphoproliferative disorder, polymorphic

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Table 15. Immune markers in leukemic forms of non-Hodgkin lymphoma

Marker B-CLL B-PLL HCL FL MCL SLVL T-CLL /LGL SS T-PLL ATLL

S Ig (þ) þþ þþ þþ þþ þþ � � � � �CD2 � � � � � � þ þ þ þ þCD3 � � � � � � þ � þ þ þCD4 � � � � � � � � þ þ /� þCD5 þþ � � � þ � � � þ þ þCD7 � � � � � � � � þ /� þ � /þCD8 � � � � � � þ þ /� þ /� þ /� þ /�CD19 /20 /24 þþ þþ þþ þ þ þþ � � � � �CD22 þ /� þþ þþ þ þ þþ � � � � �CD10 � � � þ /� � � � � � � �CD25 � � þþ � � þ /� � � � � þþCD56 � � � � � � � þ � � �CD103 � � þþ � � � � � � � �HL-DR þþ þþ þþ þþ þþ þþ � � � � �� ¼ negative; (+) ¼ weakly positive; + ¼ positive; ++ ¼ markedly positive; +++ ¼ strongly positive;+/� ¼ majority of cases positive; � /+ ¼ majority of cases negative. CLL, chronic lymphocytic leukemia;PLL, prolymphocytic leukemia; HCL, hairy cell leukemia; FL, follicular lymphoma; MCL, mantle cell lymphoma;SLVL, splenic lymphoma with villous lymphocytes; LGL, large granulated lymphocyte leukemia; SS, Sezarysyndrome; ATLL, adult T-cell leukemia/lymphoma


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6.4.1 Some Mature B-cell Lymphomas(Table 14)

Chronic Lymphocytic Leukemia(Chronic Lymphadenosis, CLL)

CLL is a chronic disease characterized by hema-tologic changes, multiple or generalized lymphnode enlargement, splenomegaly, and frequenthepatomegaly. It predominantly affects older in-dividuals. Peripheral blood examination usuallyreveals a leukocytosis with a strong preponder-ance of lymphocytes. Most of the cells appearmorphologically as small, mature lymphocyteswith a dense, coarse nuclear structure. Nucleoliare found only in a few “immature” cells. The cy-toplasmic rim is narrow with a medium blue col-oration. Gumprecht shadows or “smudge cells”(Fig. 141 a–c) is the term applied to crushed cellsthat, while often present in B-CLL, are not specificfor that disease. A large percentage of the lympho-cytes show marked, usually fine to moderatePAS granulations on cytochemical staining(Fig. 141 f). Small lymphocytes also predominatein the bone marrow (Fig. 141 d), lymph nodes,and spleen. Granulocytopoiesis, erythropoiesis,and thrombocytopoiesis are quantitatively de-pressed in the bone marrow, resulting in a pro-gressive anemia, absolute granulocytopenia,and thrombocytopenia with their associated ef-fects (which could also result from autoantibo-dies). The depletion of normal B-lineage cellsfrom the bone marrow and lymph nodes almostalways produces an increasing hypogammaglo-bulinemia that may culminate in an antibody de-ficiency syndrome.

The lymphocytes of CLL have B-cell properties(B-CLL) in the majority of cases seen in Europeand America. The existence of a T-CLL is contro-versial. The mature-cell type of T-cell leukemia orNK-cell leukemia is referred to as LGL (largegranulated lymphocyte) leukemia or T-cell lym-phocytosis. It is characterized by neutropeniaand a chronic course. The abnormal cells havefine to medium-sized azurophilic granules intheir cytoplasm and should be further identifiedby immunologic or molecular genetic criteria. T-cell leukemias (except for T-ALL) are classifiedmainly as T-cell prolymphocytic leukemias. Thecells are frequently small and thus may be mista-ken for the cells of B-CLL in improperly preparedsmears. Most have nucleoli, however, and somehave an irregular nuclear contour. A variantwith a broader cytoplasmic rim and sharply de-fined vesicular nucleoli resembles B-cell prolym-phocytic leukemia. Immunophenotyping is es-sential. The other leukemic forms of NHL are de-scribed in the figure legends.

Either of two staging systems may be used forevaluating the course of CLL and planning ther-apy:

1. Staging system of Rai et al.:Stage 0: Blood lymphocytosis < 15,000/lL,

bone marrow infiltrationStage I: Stage 0 plus enlarged lymph nodesStage II: 0 with or without I, plus hepatome-

galy and/or splenomegalyStage III: 0 with or without I and II, plus ane-

mia (Hb < 11g/dl)Stage IV: 0 with or without I– III, plus throm-

bocytopenia (<100,000/lL)

2. Staging system of Binet:A. Lymphocytosis > 4000/lL,

bone marrow infiltration > 40 %,hemoglobin > 10 g/dL,platelets > 100,000/lL,two lymph node regions involved (enlarged)

B. Same as stage A,plus enlargement of at least threelymph node regions

C. Same as A,plus anemia (Hb<10 g/dL) and/orthrombocytopenia <100,000/lL,with involvement of any numberof lymph node regions

Since the introduction of fluorescence in-situ hy-bridization (FISH) it has been possible to detectprognostic important cytogenetic aberrations inB-CLL. Today the 13q(-) aberration is thoughtto be favorable, the 17p(-) aberration (by muta-tion or loss of p53) is unfavorable, the 11q deletionis unfavorable (but less so), and the trisomy 12 hasa median position, with a median survival of 114months [Dohner, H. et al. (2000) N Engl T Med343: 1910].

Immunocytoma (IC)

In the Kiel classification, this group of low-gradenon-Hodgkin lymphomas includes lymphoplas-mocytoid and lymphoplasmacytic immunocyto-ma. Cases that are CD5–positive on immunologictesting are presently classified as a form of B-CLL,whereas CD5-negative cases, often associatedwith an IgM type of monoclonal gammopathyand featuring lymphatic cells transitional withplasma cells, are classified as Waldenstrom dis-ease. In up to 50 % of the patients, the transloca-tion t(9;14)(p13;q32) and rearrangements of thePAX-5 gene are found, as in other lymphomas,with plasmacytoid differentiation. The abnormalcell forms range from small lymphocytes to plas-

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ma cells. Bone marrow examination sometimesshows an increase in tissue mast cells(Fig. 142 d, h).

Prolymphocytic Leukemias

Prolymphocytic leukemias can be classified phe-notypically as having a B-cell or T-cell lineage. B-cell prolymphocytic leukemia (B-PLL) is diag-nosed if at least 55 % prolymphocytes are foundon examination of the peripheral blood. Thesecells are larger than lymphocytes, have a broadercytoplasmic rim, and a round nucleus with a well-defined, vesicular nucleolus that is usually cen-tered within the nucleus. Generally the leukocytecount is greatly elevated. Splenomegaly is presentbut there is little or no lymph node enlargement.Immunophenotypic analysis shows strong ex-pression of the surface immunoglobulin (SJg).The B-cell markers CD19, CD20, CD24, andCD22 are markedly positive, FMC7 is positive,and CD5 is negative. No specific cytogenetic aber-rations are known (Fig. 150).

T-cell prolymphocytic leukemia (T-PLL) isalso associated with an elevated leukocyte countand splenomegaly. In contrast to B-PLL, however,lymph node enlargement is usually present and issometimes accompanied by cutaneous infiltratesand effusions. The cells resemble those of B-PLLin approximately 50 % of cases but usually showan irregular nuclear contour. In other cases thecells are smaller and have a narrower and mark-edly basophilic cytoplasmic rim. In approxi-mately one-third of cases the nucleoli are difficultto detect with light microscopy but are clearly de-fined by electron microscopy. At one time suchcases were frequently diagnosed as T-CLL, and in-deed there are sporadic cases that do not displaythe features of LGL leukemia and must be classi-fied as T-CLL. The T-cell markers CD2, CD3, CD5,and CD7 are positive on immunophenotypic ana-lysis. CD4 and the T-cell receptor are positive inmore than 50 % of cases, and CD8 is rarely detect-able.

Cytogenetic analysis frequently demonstratesan inv(14) or aberrations of chromosome 8(Figs. 157, 158).

Mantle Cell Lymphoma (MCL)

This lymphomatous disease was previously re-cognized by the Kiel group as a separate entity(“centrocytic lymphoma”) because of its poorprognosis, but the entity was accepted interna-tionally only after the cytogenetic detection ofthe (11;14) translocation (Fig. 151 f). The abnormal

cells are derived from the mantle zone of the lym-phoid follicle and require differentiation fromtrue follicular cells (from the center of the folli-cle). They may equal or greatly surpass lympho-cytes in size. The smaller cells usually have an ir-regular nuclear contour but are sometimes diffi-cult to distinguish from the cells of CLL; even theimmunologic detection of CD5 and standard B-cell markers cannot provide reliable differentia-tion. These cases are diagnosed by using cytoge-netic analysis or the FISH technique to detect the(11;14) translocation. The variant with larger andsometimes anaplastic cells is identified by a con-spicuous pleomorphism of nuclear contours anda very coarse (“rocky”) chromatin pattern(Fig. 151).

Follicular Lymphoma

The cells are derived from the center of the folli-cle. Mature lymphatic forms predominate, andblasts are rarely observed in blood and bone mar-row smears. Deep nuclear clefts are typicallyfound in the more mature forms and may almostcompletely divide the nucleus (“cleaved cell,”Fig. 152 e). Additionally there are cells with roundnuclei and isolated blasts. Depending on the pro-portion of centroblasts in the histological sectionper high-power microscopic field, three gradesare distinguished. This method cannot be appliedto smears. Cytogenetic analysis reveals a t(14;18)in approximately 70 % of cases, and molecularanalysis shows the hyperexpression of bcl-2.The t(14;18) can be detected with high sensitivityby FISH and PCR and therefore makes a usefuldifferentiating criterion. On immunophenotyp-ing, the cells of low-grade follicular lymphomamay express CD10 in addition to standard B-cell markers. CD5 is negative.

Hairy Cell Leukemia (HCL)

Hairy cell leukemia is always associated with sple-nomegaly, which tends to progress slowly duringthe course of the disease. Lymph node enlarge-ment is usually absent. Most cases present hema-tologically with leukopenia, neutropenia, and ty-pically with monocytopenia. Lymphocytes andonly small numbers of hairy cells are found inblood films (Fig. 144 a). The hairy cells areroughly the size of lymphocytes or slightly larger.The grayish-blue cytoplasm appears finely honey-combed and has irregular borders with typicalhairlike projections that give this disease itsname. Other cells may present denser, pseudo-pod-like cytoplasmic extensions. Azurophilic


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granules are occasionally present in the cyto-plasm. The nucleus is usually oval, and its chro-matin is finer than in lymphocytes (Figs. 144–146). A single, small nucleolus is rarely present.Cytochemical staining demonstrates a strongacid phosphatase activity that is not inhibitedby tartrate (see method on p. 14). This is dueto the presence of isoenzyme 5 in the hairy cells(Fig. 147 a–d). When these cells are seen in thebone marrow, which may be difficult or impossi-ble to aspirate (dry tap), they are frequently ar-ranged in clusters. If a dry tap is obtained, acore biopsy is necessary for a definitive diagnosis(Fig. 146 c–e). The hairy cells are a special variantof the B-lymphocyte line that have a typical im-munophenotype. They express the standard B-cell markers in addition to CD11c and CD103. Be-sides typical hairy cell leukemia, there is a variant(HCL-V) characterized by elevated white cellcounts in the peripheral blood. In contrast to ty-pical HCL, a decrease in monocytes is usually notobserved. Most of the cells have a broader andmore basophilic cytoplasm than the typical hairycells. Their denser nuclear chromatin and distinctnucleoli create a picture that resembles the nucleiin B-PLL. Tartrate-resistance acid phosphatase isusually absent (Fig. 148).

Splenic Marginal Zone Lymphoma (SMZL)

Formerly known as “splenic lymphoma with vil-lous lymphocytes (SLVL)”, this is rare disease thataccounts for less than 1 % of the lymphatic neo-plasms. Most patients are over 50 years of age.Spleen, hilary lymph nodes of the spleen, bonemarrow and, in most cases, peripheral bloodare involved, liver rarely, and peripheral lymphnodes are usually not infiltrated. In peripheralblood one finds lymphocytes with polar villi, nor-mal appearing and plasmocytoid lymphatic cells.On immunphenotyping the cells have surface IgMand IgD, they are CD20 and CD79a positive, andCD5, CD10, and CD23 negative, which makes iteasier to distinguish from CLL and HCL. In upto 40 % of the patients there is an allelic loss at7q21-32; trisomy 3 and the t(11;18), which can bedemonstrated in extranodal marginal zone lym-phoma, are not found.

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Figs. 141–152. Mature B-cell lympho-mas

Fig. 141 a–h. Chronic lymphocyticleukemia of B lineage (B-CLL)

a–c Peripheral blood smears show pre-dominantly mature lymphocytes, somewith a clumped chromatin pattern (c).Occasional crushed nuclei (Gumprechtnuclear shadows) are seen

b

c

d Bone marrow smear in B-CLL, withdiffuse infiltration of the bone marrow


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Fig. 141 e–h

e Bone marrow smear from the samepatient. Immunocytochemical detectionof CD5. All the lymphocytes are positive(red) – a characteristic feature of B-CLL.Standard B-lineage markers are alsofound

f Peripheral blood, PAS reaction. Nu-merous cells show a strong, finely gran-ular PAS reaction

g Schematic diagram and interphaseFISH of a deletion 13q. In cases of CLLdeletions in the chromosome band13q14 are often observed. They can bedetected with FISH on interphase nuclei.While a normal cell shows two signals, acell with deletion 13q has only one signal.

h Interphase FISH of a trisomy 12. It canbe detected by FISH on interphase nuclei.While a normal cell shows two signals, acell with a trisomy 12 has three signals.

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Fig. 142 a–g

a Cellular pleomorphism is greater thanin Fig. 141, and there are scatteredyounger forms with well-defined nu-cleoli. This is an atypical form of CLL(formerly classified as immunocytoma)

b Different case with vacuoles in thecytoplasm

c Different case with rod-shapedcrystalline inclusions in the cytoplasm

d Two mature tissue mast cells sur-rounded by lymphocytes, which oftenhave a somewhat broader and morebasophilic cytoplasm. Similar findings areseen in Waldenstrom disease


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Fig. 142 e–g

e, f Filiform, spaghetti-like inclusions inthe cytoplasm

f

g Bone marrow smear showing granu-loma-like infiltration and five tissue mastcells. Sample from a patient with Wal-denstrom disease

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Fig. 143 a–d. Transitional form of CLL/PLL or atypical CLL

a Besides small lymphocytes, there aremore than 10 % prolymphocytes withdistinct nucleoli

b Larger cells with broader, basophiliccytoplasm

c Different case with small lymphocytesand larger lymphocytes containing adistinct nucleolus (prolymphocytes)

d Different site in smear c. Here all thecells have nucleoli, showing that thedistribution of cells in a smear can be veryheterogeneous


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Fig. 144 a–g. Hairy cell leukemia (HCL)

a Typical hairy cell, shown with a normallymphocyte for comparison

b Hairy cell with abundant, agranular,very “hairy” cytoplasm

c More hairy cells with abundant cyto-plasm, which has a shredded appearance

d Exceptionally cellular bone marrowsmear in HCL. The nuclear indentation atcenter is not unusual. The cytoplasmshows a fine honeycomb structure

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Fig. 144 e–g

e Large-cell type of HCL with abundantcytoplasm

f Very conspicuous “hairs” are seen in theleukocyte concentrate from a differentcase

g Different case with a broad, smoothcytoplasmic rim


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Fig. 145 a, b. Hairy cell leukemia. Thecytoplasm contains pale, streaklike,relatively well-defined structurescorresponding to the ribosome-lamel-lar complex (RLC) seen on electronmicroscopy (upper left in b)

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Fig. 145 c, d. Electron microscopic ap-pearance of the RLC, whose light mi-croscopic equivalent is shown in a andb.

c RLC in cross section

d RLC in longitudinal section. The pa-rallel structures are each composed offive lines. (Fig. 145 c, d courtesy of Prof.F. Gudat, Institute of Pathology, Basel)


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Fig. 146 a–d. Hairy cell leukemia

a Unusual nuclear form (“flower cell”) notnormally seen in HCL. Typical hairy cellswere found elsewhere in the sample

b Bone marrow smear in HCL showsloosely arranged nuclei of hairy cells andinterspersed tissue mast cells (violet“blotches”)

c Bone marrow section, Giemsa stain.The loose arrangement of the hairy cellscontrasts with the dense arrangementseen in B-CLL

d Higher-power view of an isolated hairycell at the center of the field. Below it is atissue mast cell

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Fig. 146 e. Appearance of reticular fi-bers in histologic section. Note how thehairy cells are trapped in the reticularfiber meshwork. This explains thegenerally poor aspirability of the bonemarrow (dry tap)


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Fig. 147 a–g. Cytochemistry andimmunocytochemistry of HCL

a Tartrate-resistant acid phosphatase(TRAP) activity in three hairy cells

b Different smear shows varying degreesof positive TRAP staining

c TRAP in bone marrow from a differentpatient. Neutrophils and lymphocytes arenegative

d TRAP demonstrated by a differenttechnique (Sigma)

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Fig. 147 e–g.

e Esterase reaction (pH 7.4) showsweakly diffuse and polar-cap patterns ofactivity

f Immunocytochemical detection ofCD11c

g Immunocytochemical detection ofCD103. APAAP technique


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Fig. 148a–d. Variant of hairy cell leu-kemia (HCL-V). This is another B-lineageform, usually associated with markedleukocytosis. The nuclear structure re-sembles that of prolymphocytes withnucleoli, but the cytoplasm is some-what less “hairy” than in typical HCL

a, b Typical cells of HCL-V with small,distinct nucleoli

c A lymphocyte with azure granulesappears among the abnormal cells

d Different case of HCL-V

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Fig. 149 a, b. Splenic marginal zonelymphoma (SMZL), formerly SLVL. Thecells in the blood smear are more var-iegated than in HCL. The nuclei usuallyhave a distinct nucleolus and some-times resemble the nuclei of B-cellprolymphocytic leukemia. The cyto-plasm shows variable basophilia.Cytoplasmic irregularities (“hairs”) atone pole of the cell are characteristic.

Panels a and b show typical samples ofperipheral blood


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Fig. 150 a–e. B-cell prolymphocyticleukemia. The cells are larger thanlymphocytes and have round nucleiwith distinct, usually sharply circum-scribed nucleoli

a Typical prolymphocytes withwell-defined nucleoli

b Peripheral blood in a different case

c Different case showing two typicalprolymphocytes and one lymphocyte

d PAS reaction shows relatively coarsegranules in the cytoplasm, similar tothose in ALL. Therefore this reaction isnot useful for distinguishing B-PLL fromALL

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Fig. 150 e. Extremely unusual case withcoarse azurophilic granules in thecytoplasm. Definite B-cell markers couldbe detected immunologically


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Fig. 151 a–e. Mantle cell lymphoma.Called centrocytic lymphoma in the Kielclassification, this disease has a poorprognosis. It differs from the follicularlymphomas and other low-grade lym-phomas by the (11;14) translocationand can also be identified by moleculargenetic testing. CD5 is detected onimmunocytochemical analysis; this canmake mantle cell lymphoma difficult todistinguish from B-CLL, especially whenthe cells are very small. Morphologi-cally, the cells often have somewhatirregular nuclei, dense chromatin, andscant cytoplasm. Less mature formswith distinct nucleoli are sometimesdifficult to distinguish from blasts.There are also highly pleomorphicforms with clefted, “rocky”-appearingnuclear chromatin

a Very small cell type with an irregularnuclear contour

b Blood smear in a different case withlarger cells clefted on the nuclear surface

c Peripheral blood from another caseshows multiple nuclear indentations

d Bonemarrow from another case showsa high degree of nuclear pleomorphism

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Fig. 151 e– f

e Higher-power view of the case in dshows relatively large cells with a cleftedchromatin pattern

f Schematic diagram and partial karyo-type of a translocation t(11;14)(q13;q32)and interphase FISH with an IGH/CCND1probe. From the reciprocal translocation,a splitting of the signals of both probesresults (red: CCND1, green: IGH-locus),such that the following signal constella-tion develops in a cell with t(11;14)/IGH-CCND1 rearrangement: a red and a greensignal on the respective unchangedchromosomes 11 and 14 as well as red-green “colocalization signal” on the de-rivative chromosomes 11 and 14. Thet(11;14) is found in mantle cell lymphomaand in plasma cell myeloma


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Fig. 152 a–e. Follicular lymphoma (FL)

a–c Typical cases with singly notchednuclei and small cells

b

c

d A centroblast. Centroblasts are anextremely rare finding in blood smears

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Fig. 152 e– f

e Deeply clefted nuclei in follicularlymphoma

f Schematic diagram and partial karyo-type of the translocationt(14;18)(q32;q21), which is foundin follicular lymphoma. Arrows indicatethe breakpoints


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6.4.2 T-cell Lymphomas

Sezary Syndrome

Sezary syndrome is a cutaneous T-cell lymphomathat is closely related to mycosis fungoides. It pre-sents clinically with diffuse erythroderma, and ty-pical histologic skin changes are observed. Highlycharacteristic T lymphocytes called Sezary cellsare found in the peripheral blood. These cellsrange from the size of lymphocytes to the approx-imate size of monocytes. They have scant, lightlybasophilic cytoplasm, and the typically oval nu-cleus shows a pathognomonic morphology.When seen in blood smears, the nucleus appearspermeated by fine furrows caused by highly con-

voluted nuclear folding, which creates a “cerebri-form” pattern on electron microscopy. Usuallythere are no detectable nucleoli. The nuclear con-tour may be smooth or slightly irregular, anddeep indentations are rare. On immunopheno-typing, the cells predominantly express CD4along with CD3, and they rarely express CD8. Cy-tochemical stains are relatively nonspecific, de-monstrating a dotlike esterase reaction andweak acid phosphatase activity.

A rare variant, which some also consider a var-iant of T-cell prolymphocytic leukemia, is Sezarycell leukemia. Peripheral blood examinationshows an increased cell count with an overwhelm-ing predominance of relatively small Sezary cells(Table 16).

Table 16. WHO classification of mature T-cell and NK-cell neoplasms

Leukemic / disseminatedT-cell prolymphocytic leukemiaT-cell large granular lymphocytic leukemiaAggressive NK-cell leukemiaAdult T-cell leukemia/lymphoma

CutaneousMycosis fungoidesSezary syndromePrimary cutaneous anaplastic large cell lymphomaLymphomatoid papulosis1

Other extranodalExtranodal NK/T cell lymphoma, nasal typeEnteropathy-type T-cell lymphomaHepatosplenic T-cell lymphomaSubcutaneous panniculitis-like T-cell lymphoma

NodalAngioimmunoblastic T-cell lymphomaPeripheral T-cell lymphoma, unspecifiedAnaplastic large cell lymphoma

Neoplasm of uncertain lineage and stage of differentiationBlastic NK-cell lymphoma

1 Clinically not considered a neoplastic disorder.

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Figs. 153–158. Mature lymphomas ofT-cell lineage

Fig. 153 a– f. Sezary syndrome. Lym-phatic cells in the peripheral bloodhave distinctively furrowed, convolutednuclei, which sometimes are indented.Electron microscopy shows cerebriformnuclear folding (s. Fig. 153 f)

a Very deep nuclear indentation inSezary syndrome

b, c Two different cases with nuclearfurrowing

d Pleomorphic nucleus at left, largeSezary cell at right


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Fig. 153 e– f

e Leukocyte concentrate demonstratesmany small Sezary cells and one largercell

f Electron micrograph of a Sezary cellshows the typical bizarre, cerebriformfolding of the nucleus

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Fig. 154 a– f. Sezary cell leukemia.These patients generally do not haveerythroderma as in Sezary syndrome,but there is pronounced leukocytosis. Ithas been postulated that Sezary cellleukemia may be a variant of T-cellprolymphocytic leukemia

a–d Peripheral blood examinationshows leukocytosis with many smallSezary cell displaying a typical nuclearstructure

b

c

d


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Fig. 154 e– f

e Immunocytochemical detection ofCD3

f Immunocytochemical detection ofCD4. The cells express typical T-cellmarkers, and some have the properties ofT-helper cells

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Large Granular Lymphocytic(LGL) Leukemia

Another form of mature-cell T- or NK-cell leuke-mia is LGL leukemia (large granulated lympho-cyte leukemia), which is very rare. Two distinctimmunologic forms have been identified. Thefirst is the T-cell type, characterized by an indo-lent course, increased T cells in the peripheralblood, and neutropenia. Immunologic studies de-monstrate CD2, CD3, CD8, and CD16. CD 57 may

be positive or negative, and the T-cell receptor a/b is positive. The second is the NK-cell type,which takes an aggressive clinical course. CD2is positive, CD3 is negative, CD16 is positive,and CD56 and CD57 may be positive or negative.The T-cell receptor a/b is negative. Peripheralblood examination in both variants shows maturelymphatic cells with azurophilic granules, some ofwhich are very fine and difficult to identify(Fig. 155).


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Fig. 155 a–d. LGL leukemia a

b–d These cells contain distinct azuro-philic granules, some of which are veryfine. If possible, a molecular genetic studyshould be performed to establish thediagnosis

c

d

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Adult T-Cell Leukemia/Lymphoma (ATLL)

Adult T-cell leukemia/lymphoma (ATLL) occursin an endemic form in southwestern Japan andthe Caribbean. Its etiology is based on infectionwith the human T-cell leukemia virus(HTLV-1). Sporadic cases have also been reportedin Europe and other regions (Fig. 156 a, b).


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Fig. 156 a–d

a, b Adult T-cell leukemia/lymphoma(ATLL). The pleomorphic, flower-likenuclei (“flower cells”) are characteristic.(Courtesy Prof. D. Catovsky, London)

b

kemia, which was observed in Germany.There was no evidence of infection withHTLV-I

c, d Peripheral blood, Pappenheim stain

d

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c–d Morphologically similar T-cell leu-

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Fig. 157 a–g. T-cell prolymphocyticleukemia (T-PLL). In contrast to B-PLL,the nuclei are usually irregular and thenucleoli are not as distinct. Cytochem-ical staining shows a pronounced acidesterase reaction (ANAE)

a Pleomorphic nuclei with indistinctnucleoli

b Characteristic dotlike ANAE stainingpattern

c Acid phosphatase reaction similar tothat found in T-ALL


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Fig. 157 d–g. T-PLL

d Immunocytochemical detection ofCD3

e Different case of T-PLL with distinctnucleoli and irregular nuclear contours

f Two prolymphocytes in the peripheralblood

g Very strong ANAE activity in the samecase

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Fig. 158 a– f. T-prolymphocyticleukemia (T-PLL)

a Round nuclei and distinct nucleoli inT-PLL

b Different case, apt to be mistaken forB-PLL

c Same case shows a typical dotlikereaction pattern of ANAE

d Immunocytochemical detection ofCD3


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Fig. 158 e– f

e, f Large-cell T-PLL with stronglybasophilic cytoplasm

f Dotlike ANAE staining pattern

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Large Cell Lymphomas (Fig. 159 a– j)

Fig. 159 a– j. Large cell non-Hodgkinlymphomas (NHL)

a Lymph node touch preparation inBurkitt-type B-cell lymphoma with “starrysky macrophages” among the lympho-blasts

b–g Centroblastic variant (large-cell,diffuse) with a fine chromatin pattern andpale nucleoli. The cells may also showgreater pleomorphism

c

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Fig. 159 e–g e

f

g Detection of CD10. This marker, orig-inally used in diagnosing c-ALL, is alsoexpressed in the centroblastic variant ofthe diffuse large B-cell lymphoma

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Fig. 159 h– j

h–k Large-cell form of anaplastic T-celllymphoma

i These cells have large nuclei, usuallyindented and occasionally lobulated, anda strongly basophilic cytoplasm

j Extremely strong acid phosphataseactivity localized to the paranuclear area


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Fig. 160 a– f. Diffuse large B-cell lym-phoma

a Large-cell high-grade B-cell lymphomawith intensely basophilic cytoplasm anddistinct nucleoli

b Immunocytochemical detection of theB-cell marker CD19 in the same case

c Diffuse large B-cell lymphoma withlarge nucleoli (immunoblastic variant)

d Different large-cell lymphoma withexpression of CD30 (Ki-1 lymphoma)

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Fig. 160 e– l

e Same patient as in d

f Immunocytochemical detection ofCD30

g, h Richter syndromeCharacterized by the presence of largeblasts with large nucleoli in a patient withpreexisting B-CLL. In some cases theblasts belong to the same clone as thepreexisting CLL. In bone marrow smears,large blasts with copious cytoplasm anddistinct nucleoli are interspersed amongthe small lymphocytes

h


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Rare Lymphomas

Primary Effusion Lymphoma (PEL). The PEL is alymphoma of large blast like B-cells with baso-philic, sometimes vacuolated cytoplasm; the nu-clei are round or irregular and contain prominentnucleoli. The cells remind one of plasmoblasts,sometimes of Hodgkin cells. In all cases the cellsare positive for the herpes virus 8 (HHV 8/Kaposisarcoma herpes virus), and the majority of pa-tients are HIV positive. Lymphoma cells usuallyexpress leukocyte common antigen (CD45), butare usually negative for pan B-cell and T-cellmarkers. Activation and plasma cell-related mar-kers (CD30, CD38, CD138) are usually demon-strable.

Intravascular Large B-cell Lymphoma. Lympho-ma cells are only present in the lumina of smallvessels, particularly capillaries. The lymphoma is

usually disseminated in extranodal sites, e.g.,CNS, skin, lung, kidney, adrenals or bone mar-row. Since in smears the cells are in close contactthey may be mistaken for non-hematological tu-mor cells. The demonstration of B-cell associatedmarkers (CD19, 20, 22, 79a) protects against mis-diagnosis (Fig. 160 i, j).

Hepatosplenic T-cell Lymphoma. This extranodaland systemic disseminated lymphoma is derivedfrom cytotoxic T-cells usually of cdT-cell recep-tor type demonstrating marked sinusoidal infil-tration of spleen, liver and bone marrow. Onsmears the cells are medium in size, irregular,usually oval. Since they are grouped in close con-tact, there is the danger of mistaking them fornon-hematological tumor cells. On immunphe-notyping they express CD3 and usually the T-cell receptor cd; a minority appears to be of abtype (Fig. 160 k, l).

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Fig. 160 i– l.

i Intravascular large B-cell lymphoma.Bone marrow section, Giemsa stain. Onesees cone-like the vessel lumina fillingcells with large clear nuclei and distinctnucleoli

j In the bone marrow smear the cells liein complexes, so they may be mistakenfor cells of a carcinoma or sarcoma. Theycan be distinguished by B-cell markers

k Cluster of cells in bone marrow smearin a case of hepatosplenic cdT-cell lym-phoma. The large polymorphic cells maybe mistaken for cells of a carcinoma orsarcoma. By immunphenotyping the T-cell nature can be demonstrated

l High power view. The homogeneouschromatin structure and the clearnucleoli can be seen

(The histologic section and the smearfor the Fig. 160i, j courtesy of Prof. Dr.H. E. Schaefer, Freiburg, the smearsfor the Fig. 160k, l courtesy of Dr.M.-T. Daniel, Paris)


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6.4.3 Plasma Cell Myeloma(Multiple Myeloma, Kahler Disease)

Plasma cell myeloma is characterized by a prolif-eration of abnormal plasma cells. It may take theform of a solitary tumor or multiple tumors (mul-tiple myeloma), or the changes may permeate thebone marrow diffusely. The most common form ismultiple myeloma, characterized by multiple,sharply circumscribed osteolytic foci that are con-spicuous on radiographs. The diffuse form of thedisease presents radiographically as a generalizeddecalcification and rarefaction of the bony struc-ture, resembling osteoporosis. Solitary plasmacy-tomas of bone, extraosseous plasmacytomas maybecome generalized in the course of the disease,just as initially diffuse lesions can in time producemultiple plasma cell tumors. Since plasma cells aredescendants of B lymphocytes, which are ubiqui-tous, plasmacytomas are not confined to the bonemarrow (extramedullary plasmacytoma). It is re-latively uncommon for large numbers of plasmacells to enter the peripheral blood (� 20 %).This condition is called plasma cell leukemia. Pa-tients with smoldering and indolent myeloma areasymptomatic and have no signs of reduction oftheir bone marrow or renal function; calcium isnormal. The patients with indolent myelomahave up to three lytic bone lesions without pain(according to WHO).

Because plasma cells manufacture immuno-globulins, qualitative and quantitative protein ab-normalities are a consistent feature of plasmacy-tomas. Generally there is a pronounced increasein immunoglobulins, whose homogeneity on im-munoelectrophoresis (“monoclonal paraprotei-nemia,” “monoclonal gammopathy”) reflectsthe monoclonality of the malignant plasma cellpopulation, demonstrable by immunocytologicanalysis. Several immunologic classes of plasma-cytoma are currently recognized: IgA, IgG, IgD,and IgE. IgM paraproteinemia, by contrast, isseen chiefly in lymphoplasmacytic lymphoma(Waldenstrom’s macroglobulinemia). If thestructure of the immunoglobulins in the plasmacells is rudimentary, a Bence Jones myeloma(light-chain plasmacytoma) is said to be present,and the patient will manifest a characteristicBence Jones proteinuria. Non-secretory myelo-

mas are very rare (1 %). The different variantsof plasmacytomas cannot be distinguished mor-phologically. The clinical picture of plasmacyto-ma is marked by the proliferation of plasma cellsand by the diverse effects of abnormal proteinformation.

Themorphologic features of plasmacytoma arecharacteristic and are easily recognized in fullydeveloped cases. There are sheets or islands ofplasma cells that usually show the characteristicsof mature plasma cells (“mature-cell” or “plasma-cytic” myeloma) butmay show considerable pleo-morphism (Figs. 161– 169) or may even lose theirplasma cell characteristics (“immature-cell” or“plasmablastic” myeloma; see Fig. 162; the refer-ence to plasma cell precursors may be a misno-mer, as it is more reasonable to assume a highergrade of malignant transformation based on im-munologic findings). The cytoplasm of the mye-loma cells may contain reddish-violet inclusions,called Russell bodies (dilated, immunoglobulin-filled ergastoplasm sacs, see Fig. 163 f, g), or va-cuoles (Russell bodies dislodged by the stainingprocess), which may culminate in the formationof morula cells. Some plasma cells take a reddish-violet cytoplasmic stain, which has earned themthe name “flaming plasma cells” (see Fig. 161 f)and is seen in IgA myelomas.

Cytochemical staining typically demonstratesvery strong acid phosphatase activity and oftenshows nonspecific esterase activity (seeFig. 164 d–g), but this is rarely necessary to es-tablish a diagnosis. Differential diagnosis mayprove difficult in early stages when the plasmacell increase does not yet permit a definitive di-agnosis to be made. The picture is further com-plicated by the substantial plasma cell increasesthat can arise in the course of severe, prolongedinfections and neoplastic diseases (see also Ta-ble 7). When doubt exists, the immunocytologicdifferentiation of monoclonal and polyclonalplasma cell populations can be helpful. The neo-plastic plasma cells usually do not express CD19,but the majority express CD38, CD138 and CD56.

As the plasma cell population expands, thecells of normal hematopoiesis become less andless prominent, and the blood picture may ulti-mately become pancytopenic. The rare plasmacell leukemias have a particularly poor prognosis.

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Fig. 161 a–g. Plasma cell myeloma(plasmacytoma)

a, b Rouleaux formation of erythrocytesin the blood smear, caused by the in-creased amount of abnormal immuno-globulin in the plasma. A small plasmacell is seen in b

b

c Moderately pleomorphic plasma cellswith nucleoli, and one binucleated form

d These plasma cells appear fairly ma-ture but contain distinct nucleoli


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Fig. 161 e–g

e Mature-cell myeloma in a differentcase

f “Flaming” myeloma cells with red-staining, homogeneous-appearing cyto-plasm. This type of myeloma cell is seenmainly in IgA gammopathy

g Giant vacuoles in myeloma cells

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Fig. 162 a– f. Plasma cell myeloma(plasmacytoma)

a Plasmablastic type of multiplemyeloma

b Distinct nucleoli

c The large nucleoli are clearly demon-strated by specific nucleolar staining

d Extremely immature myeloma


V

Fig. 162 e– f

e Many multinucleated forms inimmature myeloma

f Giant plasma cells (multinucleated)

359


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Fig. 163 a–g. Plasma cell myeloma

a, b Immature plasma cells with pleo-morphic nuclei in multiple myeloma

b

c–e Myelomawith granular plasma cells.Some of the plasma cells contain abun-dant purple granules that are peroxidase-negative but express the myeloid markerCD33 (e)

d


V

Fig. 163 e–g

f Binucleated myeloma cell with smallRussell bodies in the cytoplasm

g Large Russell bodies in the cytoplasmof myeloma cells. Some extracellularbodies are also seen

361


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Fig. 164 a–g. Plasmacell myeloma

a Erythrophagocytosis in myeloma cells

b, c Crystalline inclusions in myelomacells

c

Fig. 164 d–g. Cytochemistry ofmyeloma

d, e Very strong acid phosphataseactivity in myeloma cells


V

Fig. 164 e–g

f, g Strong ANAE activity in myelomacells

g

363


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Fig. 165 a–c. Immunocytochemicalfindings in myeloma

a CD38-positive myeloma cells

b CD56-positive myeloma cells

c As is usually the case, the myelomacells are CD19-negative

d, e Different case of myeloma beforeand after therapy

d Bone marrow sample prior to therapy


V

Fig. 165 e. Bone marrow from the samepatient 6 months after chemotherapy.Complete remission

365


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Fig. 166 a–d. Histologic section ofplasma cell myeloma

a Giemsa stain. Myeloma cells withintensely basophilic cytoplasm

b Immunohistochemical detection ofmonoclonal kappa chains

c Higher-power view of the same sample

d Section from the patient in a–c,k-chain detection. The myeloma cells arenegative, and there are very few inter-spersed (normal) plasma cells that arek-chain-positive (red)


V

Fig. 167 a–e. Coexistence of lympho-cytic lymphoma and plasma cellmyeloma

a Lymphocytes intermingled with largeplasma cells with foamy cytoplasm

b Different site in the same sampleconsists almost entirely of foamy plasmacells

c Acid phosphatase reaction of plasmacells in the same case

d, e Two different monoclonal proteins –IgA and IgM – in a different patient

d Bone marrow region showing purelymphatic infiltration

367


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e Different site in the same sample,showing an area of plasma cell infiltration


V

Fig. 168 a–d. Plasma cell leukemia

a Small plasma cells in the peripheralblood

b Small plasma cells in a different case ofplasma cell leukemia

c Blood smear from the case in b showspeculiar, partially confluent proteinglobules

d Blood smear from the same patient,PAS reaction. The protein masses stainpink, indicating a carbohydrate compo-nent in the protein

369


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Fig. 169 a–d. Plasma cell leukemia

a, b Peripheral blood in a different caseof plasma cell leukemia

b

c ANAE in myeloma cells that haveentered the peripheral blood

d Bone marrow from the case in b and c


V

Fig. 169 e, f. Hypoplastic plasma cellmyeloma

e Bone marrow smear from a patientwith hypoplastic myeloma shows fattymarrow fragments along with a group ofplasma cells

f Higher-power view of the same casewith CD56–positive myeloma cells

g Blood smear of a case of plasma cellleukemia with CD138 positive cells

371


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6.4.4 Natural Killer (NK)-Cell Neoplasias

These diseases, which occur as extranodal tumorsor leukemias are grouped together by WHO withT-cell lymphomas and leukemias, including largegranular leukemia (see p. 340).


V

Fig. 170 a–e. Extranodal NK/T-celllymphoma, nasal type (midline granu-loma, angiocentric T-cell lymphoma)with bone marrow involvement

a, b Relatively large blasts with abun-dant cytoplasm containing distinct red-dish granules

b

c Intense perinuclear acid phosphatasereaction in a bone marrow smear fromthe patient in a, b

d The blasts are CD56-positive

373


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Fig. 170 e–h

e Immunocytochemical analysis alsodemonstrates CD7 in the blasts. This caseis an acute NK-cell leukemia that is verysimilar morphologically to AML

f –h Different case of acute NK-cellleukemia

f –h Similar findings as in a–e, with asomewhat narrower cytoplasmic rim.The blasts are also CD56-positive

g

h


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Fig. 171 a– f. NK-cell neoplasia.Sometimes only isolated granules arefound in the abnormal NK cells, and someblasts are devoid of granules

a–c Bone marrow smears in which onlyisolated blasts with granules are seen

b

c

d Peripheral blood smear

375


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Fig. 171 e– f

e Immunocytochemical detection ofCD56

f Detection of acid phosphatase activity


V

6.4.5 Leukemia of Dendritic Cells(CD4 + CD56 + Neoplasia)

The malignant cells are type 2 dendritic cells orplasmacytoid dendritic cells. They may presentas a malignant lymphoma or as leukemia. TheseCD4 and CD56 coexpressing neoplasias are rare.They have no B, T or myeloid markers. A bonemarrow infiltration is found in 90 % of the pa-tients, and often there are cutaneous nodules,lymphadenopathy or spleen enlargement or

both. To date, prognosis has been poor. Thesize of the pathologic cells varies; in most casesthey are of medium size, and they may remindone of ALL blasts. Their moderately broad,weakly basophilic cytoplasm contains small va-cuoles, no granules, and it forms pseudopodia-like projections. Peroxidase and esterase are ne-gative. There is no common cytogenetic aberra-tion, but more than 60 % have complex aberra-tions (Fig. 171 g, h).

377


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Fig. 171 g–h Bone marrow smear in acase of leukemia of dendritic cells. Pre-vailing round cells with partly cleftednuclei, small nucleoli and narrow cyto-plasm. In other cases broader cytoplasmwith vacuoles and pseudopodia-likeextensions has been observed

h High-power view. Coarse structure ofnuclear chromatin. The cells remind oneof lymphoblasts. (The smears for the Fig.171 g, h courtesy of Dr. M.-T. Daniel, Paris)


V

6.4.6 Hodgkin Lymphoma(Lymphogranulomatosis)

At present the diagnosis of Hodgkin disease can beestablished only by histologic or cytologic exami-nation. All other clinical data, including the bloodpicture and radiographic findings, provide usefulinformation but cannot verify the diagnosis.

The cytologic features of the disease, seen mostclearly in lymph node aspirate or touch prepara-tions, are highly diverse but easily recognized. Itsdistinctive features are the mononuclear granulo-ma cells orHodgkin cells (Figs. 172– 174) andmul-tinucleated Reed-Sternberg cells (Figs. 172– 174).Both cell types are characterized by a coarse re-ticular chromatin pattern and especially byvery large, often irregularly shaped nucleoli,which usually stain a deep blue. The cytoplasmmay be well marginated, but usually it is notclearly demarcated from its surroundings. Itstains a pale to dark blue. Mitoses are rare in thesecells. In addition, it is usual to find increasednumbers of plasma cells, neutrophils, and even eo-sinophils. The latter have been described as highlycharacteristic but in fact do not afford proof ofthe diagnosis, any more than their absence wouldexclude Hodgkin disease.

Four subtypes of Hodgkin disease were recog-nized based on the criteria established by the RyeConference. They can be accurately classifiedonly by the histologic examination of a biopsiedlymph node.

1. Lymphocyte predominance type (LP)a) Nodular paragranulomab) Diffuse paragranuloma

2. Nodular sclerosing type (NS)3. Mixed cellular type (MC)4. Lymphocyte depletion type (LD)Recently the WHO recommended the followingclassification along with the renaming of Hodgkindisease as Hodgkin lymphoma:1

Nodular lymphocyte predominanceHodgkin lymphomaClassical Hodgkin lymphoma

Nodular sclerosis (grades I and II)Mixed cellularityLymphocyte richLymphocyte depleted

The histologic subtype of Hodgkin lymphoma isnot considered to have a bearing on prognosis.The histologic subtypes cannot be identifiedwith reasonable accuracy in cytologic smears.Especially nodular sclerosis and mixed cellularityrequire histologic differentiation, especially sincethe “lacunar cells” thought to characterize nodu-lar sclerosis can be recognized only in histologicsections.

1 Jaffe ES, Harris NL, Diebold J, Muller-Hermelink HK (1998):World Health Organization classification of lymphomas: awork in progress. Ann Oncol 9 [Suppl 5]: 25–30

379


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Fig. 172 a–e. Bone marrow infiltrationin Hodgkin lymphoma

a–c Mononucleated Hodgkin cells withcharacteristic giant nucleoli

b

c

d Constricted, elongated nucleus of aHodgkin cell with a large nucleolus


V

Fig. 172 e. Multinucleated Sternberg-Reed cell

381


V

Fig. 173 a, b. Hodgkin lymphoma(lymphogranulomatosis)

a Lymph node. At center is a multinu-cleated Sternberg-Reed giant cell. Thelarge, dark nucleoli are typical

b Spleen. At bottom center are a mono-nuclear Hodgkin cell and several stimu-lated immunoblasts. At left a plasmablastand numerous neutrophils


V

Fig. 174 a–d. Hodgkin lymphoma(lymphogranulomatosis)

a Two adjacent mononuclear Hodgkincells

b Multinucleated Sternberg-Reed giantcell

c Multinucleated Sternberg-Reed giantcell

d Multinucleated Sternberg-Reed giantcell. Above it is an eosinophilic granulo-cyte

383


385

Tumor Aspirates from Bone Marrow Involvedby Metastatic Disease

VI

VI

A number of nonhematologic malignancies canmetastasize to the bone and bone marrow. Forthe staging of these cases, it is important to deter-mine whether bone marrow involvement is pre-sent. Not infrequently, however, bone marrow in-vasion by tumor cells is detected incidentally inpatients who are examined for unexplainedsymptoms or suspicion of leukemia. It can bevery difficult to distinguish morphologically un-differentiated tumor cells from the cells of acuteleukemia or malignant lymphoma based on mor-phologic criteria alone.

An accurate classification requires the use ofimmunologic methods, which also help to assignthe neoplastic cells to a specific primary tumor.These methods employ antibodies against com-ponents of the cytoskeleton (cytokeratin, vimen-tin, desmin, etc.), a number of tumor markers,and organ-specific antibodies. Since new antibo-

dies are constantly being offered by various firms,we refer the reader to the monograph by Hastka1

as well as the overview by G. Rabenhorst.2

In table 17 several possibilities for marking fre-quent carcinomas with cytokeratines are shown(G. Rabenhorst). For the identification of sarco-mas the following antibodies may be used: tu-mors of muscle tissue – actine and desmine, neu-rogenic tumors – S 100 and neuronspecific eno-lase, tumors of the blood vessels – factor VIII,tumors of connective tissue – vimentine, tumorsof stroma – CD 34.

Table 17.

Organ Antibody

CK5/6 CK7 CK8 CK20 NSE SpecificAntibodies

Small cell bronchus – – + – + TTFI

Bronchus, epidermoid + – + – – TTF1

Bronchus, unclassified – + + – – TTF1

Gastrointestinal tract – – + + (+) CEA

Bilary tract – + + (+) – (CEA)

Breast – + + – – Hormonrec

Urinary tract + + + – – (34bE12)

Endometrium – (+) + (+) – Vimentin

1 Hastka J (1997) Immunocytology. Schattauer, Stuttgart2 Rabenhorst G (2004) Histo- und zytomorphologische Me-thoden zur Diagnose maligner Tumoren. In: Bruhn HD,Folsch UR, Kneba M, Loffler H, Schattauer, Stuttgart

386 Chapter VI · Tumor Aspirates from Bone Marrow Involved by Metastatic Disease

VI

Fig. 175 a–d. Tumor cells from a small-cell bronchial carcinoma in the bonemarrow

a Cluster of tumor cells in a bonemarrowsmear

b Same smear at high magnification

c Two tumor cells in a blood smear fromthe same patient. (It is unusual to findtumor cells in the peripheral blood unlessthe sample has been specially concen-trated.)

d Neuron-specific enolase (NSE) is auseful immunologic marker for small-cellbronchial carcinoma. Positive detectionin a bone marrow smear

387

Chapter VI · Tumor Aspirates from Bone Marrow Involved by Metastatic Disease

VI

Fig. 176 a–e. Cells from breastcarcinoma in the bone marrow

a Pappenheim stain

b Immunocytochemical detectionof cytokeratin

c Histologic examination of a corebiopsy in breast carcinoma. HE stain

d Histologic detection of cytokeratin in abone marrow smear with metastaticbreast carcinoma


VI

Fig. 176 e. Detection of cytokeratin in abone marrow smear in breast carcinoma

389


VI

Fig. 177 a–e. Detection of tumor cellsin the bone marrow

a Cells of metastatic gastric carcinoma ina bone marrow smear

b Detection of cytokeratin in the tumorcells of the same case

c Osteoclasts along with tumor cellsfrom prostatic carcinoma in a bonemarrow smear

d Tumor cell infiltration in a bonemarrow smear


VI

Fig. 177 e. Leukoerythroblastic reactionin peripheral blood from the samepatient. A normoblast, myelocyte and ametamyelocyte are seen

391


VI

Fig. 178 a–c. Detection of tumor cellsin the bone marrow

a Nest of tumor cells in a patient withprostatic carcinoma

b Histologic examination of a corebiopsy in prostatic carcinoma. Adenoidstructures can be seen

c Vessel-like mass of angiosarcoma cellsin a bone marrow smear


VI

Fig. 179 a–e. Detection of neurologictumor cells in pediatric patients

a Bone marrow involvement by medul-loblastoma. Small, round cells with palechromatin

b Higher-power view of the same case

c Cells of neuroblastoma, very similar inappearance to a and b

d NSE reaction in a bone marrow smearfrom the case in c

393


VI

e “Rosette” arrangement of tumor cellsin sympathicoblastoma


VI

Fig. 180 a–g. Metastatic rhabdomyo-sarcoma in the bone marrow. Rhabdo-myosarcoma can metastasize to thebone marrow in children and in youngadults. Not infrequently, the cells aremistaken for those of an acute leuke-mia. In about 70 % of alveolar rhab-domyosarcoma the translocationt(2;13)(q35;q14) is found as cytogeneticaberration

a Rhabdomyosarcoma cells in a bonemarrow smear. These cells are easilyconfused with the cells of acute leukemia

b Higher-power view of the same case

c PAS reaction in the same case. Thedegree of the PAS reaction is highlyvariable and sometimes is very intense

d Metastatic rhabdomyosarcoma in ahistologic marrow section. Note therelatively pale nuclei

395


VI

Fig. 180 e–g

e Different case of rhabdomyosarcomawith bone marrow involvement

f PAS reaction in the same case

g Same case, showing the detection ofdesmin in the tumor cells


VI

Fig. 181 a, b. Detection of tumor cellsin lymph nodes

a Renal cell carcinoma. Cluster of tumorcells

b Renal cell carcinoma. PAS reaction

397


VII399

Blood Parasites and Other Principal CausativeOrganisms of Tropical Diseases1

7 Blood Parasites 400

7.1 Malaria 400

7.1.1 Tertian Malaria (Plasmodium vivax and Plasmodium ovale) (Fig. 182) 4017.1.2 Quartan Malaria (Plasmodium malariae) (Fig. 183) 4017.1.3 Malignant Tertian Malaria (Plasmodium falciparum) (Figs. 184, 186) 401

7.2 African Trypanosomiasis (Sleeping Sickness) 410

7.3 American Trypanosomiasis (Chagas Disease) 411

7.4 Kala Azar or Visceral Leishmaniasis 414

7.5 Cutaneous Leishmaniasis (Oriental Sore) 416

7.6 Toxoplasmosis (Fig. 190) 416

7.7 Loa Loa 417

7.8 Wuchereria bancrofti and Brugia malayi 417

7.9 Mansonella (Dipetalonema) Perstans 420

8 Further Important Causative Organisms of Tropical Diseases 421

8.1 Relapsing Fever 421

8.2 Bartonellosis (Oroya Fever) 421

8.3 Leprosy 423

1 Revised by Prof. R. Disko, Munich.

VII

7 Blood Parasites

7.1 Malaria

The causative Plasmodium organisms are trans-mitted to man as sporozoites in the saliva ofthe feeding anopheles mosquito. The organismstravel in the bloodstream from the capillariesto the liver, where they undergo further develop-ment. They enter the liver cells (primary tissueforms) in the “pre-erythrocytic phase” of theirlife cycle, and after a variable period they are re-leased into the peripheral blood as merozoites.There they invade the erythrocytes, inciting a par-oxysm of fever. Inside the red cells the plasmodia(merozoites) mature through several stages, cul-minating in the discharge of merozoites from theremains of the erythrocyte; these then proceed toinfect fresh erythrocytes. This process takes about48 h for the parasite of tertian malaria (Plasmo-dium vivax and Plasmodium ovale) and malig-nant tertian malaria (Plasmodium falciparum)and 72 h for the parasite of quartan malaria (Plas-modiummalariae). The different stages can be re-cognized in the blood, depending on the time atwhich the sample is drawn (see Figs. 182– 186).Besides asexual reproduction through division,the organisms are capable of sexual reproductionin their male and female gametocyte forms. Thegametocytes unite only in the mosquito, formingoocysts. Sporozoites soon emerge from the cystsand make their way to the insect’s salivary glands,to enter the capillaries and bloodstream of thenext person on whom the mosquito feeds. Game-tocytes that do not enter a mosquito can surviveno more than about 40 days in the human body,after which time they can no longer harm theirhuman host.

Cycle in the Tissue

Infected mosquito bites a host#

Inoculation of sporozoites#

1 Parenchymal cells of the liver (tissue schizont)#

2 Strong asexual proliferation (hepatic phase)#

3 After several generations, release of mero-zoites from the schizonts

#4 Entry into the blood (erythrocytic phase)

Entry of the Parasite into the Erythrocyte

1. Merozoite recognizes the host erythrocyte.2. Attaches to the surface of the cell.3. Must reorient so that its apical end touches the

surface of the cell.4. Invagination develops at the attachment site,

and the parasite enters the cell.5. Erythrocyte reseals, enclosing the merozoite

within a vacuole.

Development in the Erythrocyte

1. Ring form with a peripheral nucleus and cen-tral vacuole.

2. Trophozoites.3. Repeated nuclear division.4. Erythrocyte becomes completely filled with

merozoites (Blood schizonts).5. Erythrocyte rupture, discharging the mero-

zoites (8– 12 P. malariae, 18–24 P. vivax).6. Invasion of fresh erythrocytes.

400 Chapter VII · Blood Parasites and Other Principal Causative Organisms of Tropical Diseases

VII

7.1.1 Tertian Malaria (Plasmodium vivaxand Plasmodium ovale (Fig. 182))

The clinical pattern of paroxysmal fever, spleno-megaly, and hepatomegaly is characteristic. Thefever usually begins with chills, followed 8–10 h later by sweating and defervescence. The di-agnosis is established by examination of theblood, i.e., by detecting the parasites in a thickor thin smear. Serologic tests using indirect im-munofluorescence are not positive until 14– 18days after the parasites gain access to the blood.The course of infection with Plasmodium ovale islargely identical to that of Plasmodium vivaxma-laria. Both forms can incite a double tertian feverpattern with daily attacks caused by two nonsyn-chronous plasmodium populations, each ofwhich has a schizogenic peak on the followingday. A maximum of 2 % of erythrocytes are para-sitized in both infections. P. vivax and P. ovale arethe only malaria organisms that cause enlarge-ment of the affected erythrocytes with the appear-ance of “Schuffner dots” in the cells.

7.1.2 Quartan Malaria (Plasmodiummalariae) (Fig. 183)

The fever attacks of quartan malaria occur at 72-hintervals, or every fourth day. In some cases adouble quartan pattern is observed. Enlargementof the spleen and liver is not so pronounced as intertian malaria. Because the parasite burden isonly 1 % at maximum, more time is requiredfor anemia to develop. The development of aquartan malaria duplicata is possible. Late recru-descence of quartan malaria is known to occurdecades after the primary infection has subsided.Relapse is confirmed by identifying the parasitesin the blood.

7.1.3 Malignant Tertian Malaria(Plasmodium falciparum) (Figs. 184, 186)

Malignant tertian malaria (falciparum malaria) isthe most severe, life-threatening form of the dis-ease. The fever pattern is, due to an incompletesynchronization of the parasite population, non-specific and may resemble that of tertian malariaor may consist of daily attacks mimicking a dou-ble tertian pattern. In other cases the fever may beconstant or may occur in irregular episodes. Sple-nomegaly and hepatomegaly are slowly progres-sive over the course of the disease. The parasiteburden is virtually endless due to the “chain re-action” mode of proliferation. Because all parasi-tized cells are destroyed, the erythrocyte countfalls precipitously along with the hemoglobinand serum iron. Untreated, this disease can ter-minate fatally. Permanent disability in nonim-mune individuals is rare.

The gametes of malignant tertian malaria arecrescent-shaped. Some rupture the erythrocytemembrane and consequently are found outsidethe red cells in smears.

Erythrocytes infected by Plasmodium falcipar-um frequently contain “Maurer spots,” which arecoarser than Schuffner dots. Erythrocytes in ma-lignant tertian malaria are not enlarged (impor-tant sign for differential diagnosis!).

Relapse can occur in any type of malarial in-fection, but a true recurrence is seen only in ter-tian malaria (P. vivax and P. ovale). Recurrencesdevelop from the persistence of sporozoites in theliver cells (called hypnozoites). These dormantstages may develop into tissue schizonts over aperiod of time ranging from a few months tofive years, and the schizonts provide a source ofnew merozoites that can reinvade the red cells.The hypnozoites are also responsible for thelong incubation periods (up to 1 year) of tertianmalaria.

P. falciparum and P. malariae do not formhypnozoites, but they may recrudesce (for up to1 year in the case of P. falciparum, decades inthe case of P. malariae). This recrudesence alwaysarises from a nonimmunologic or pharmacologi-cally cured involvement of the blood, and it mayoccur in tertian malaria as well.

Some cases manifest a long latent period inwhich an infection contracted in the fall, for ex-ample, does not produce initial symptoms untilthe following spring, 7–9 months later. This phe-nomenon is seen mainly with vivax malaria and isless common in the ovale form.

401

7 · Blood Parasites

VII

Fig. 182 a–d. Tertian malaria (Plasmodium vivax), Giemsa stain.

Thick smear

a Left: erythrocytes are destroyed, and all that re-main of the leukocytes (l) are nuclei. Parasites (p)with a red nucleus (k) and blue protoplasm. Ty-pical ring forms (r) are scarce in the thick smear

b Left: older ring form (r) with increased proto-plasm and initial deposition of pigment (pi)

c Left: semiadult schizonts (h) with divided nu-clei, more abundant protoplasm, and pigment de-posits

d Left: macrogametocyte (w, female gametocyte)with round nucleus, larger than an erythrocyte.Microgametocyte (m, male gametocyte) with abandlike nucleus, roughly the size of an erythro-cyte. Pigment is distributed throughout the para-site. Young schizont (t), mature schizont (t1) with24–26 merozoites (me)

Thin smear

a Right: young ring forms (r) with a red nucleusand blue protoplasm surrounding the vacuole.Note that the erythrocytes are not yet enlarged

b Right: older rings (r). Parasite (r1) with in-creased amounts of protoplasm and pigment. En-largement of erythrocytes, bizarre parasite forms

c Right: semiadults (h) with brownish pigmentspots in erythrocytes, now enlarged and speckledwith Schuffner dots (s)

d Right: macrogametocyte (w), microgametocyte(m), young schizont (t), mature schizont (t1), alsomorula or mulberry form with 18–24 merozoites.In infections with Plasmodium ovale, the affectederythrocytes are slightly enlarged and are dis-torted into oval shapes. The schizonts containonly 8– 12 merozoites (Fig. 185)


VII

Abb. 182 a–d

403


VII

Fig. 183 a–d. Quartan malaria (Plasmodium malariae), Giemsa stain.

Thick smear

a. Left: ring forms (r) are more compact than fal-ciparum rings, have red nuclei and blue proto-plasm. Leukocyte nucleus (l)

b Left: band forms (b) (a semiadult) and olderring forms (r), more compact than in the vivaxforms

c Left: schizonts (t) usually contain 8– 12 mero-zoites (me); 16 merozoites are rare

d Left: macrogametocyte (w, female gametocyte)completely occupies the normal-size erythrocyte;the microgametocyte (m, male gametocyte) issmaller than the erythrocyte. Both are smallerthan the vivax gametocyte

Thin smear

a Right: ring form (r) in an unenlarged erythro-cyte

b Right: band form (b) and young schizont (t)with pigment and a divided nucleus. No Schuffnerdots

c Right: schizont (t) with 8 merozoites (me) andcentral pigment in an unenlarged erythrocyte

d Right: macrogametocyte (w) and microgame-tocyte (m). The parasite burden in quartan malar-ia is the lowest of all the malarias (only up to 1 %of affected erythrocytes)


VII

Abb. 183 a–d

405


VII

Fig. 184 a–d. Malignant tertian malaria (Plasmodium falciparum or P. immaculatum), Giemsa stain.

Thick smear

a Left: numerous ring forms (r) with a red nu-cleus and an adjacent spot of blue protoplasm.The band and segmented neutrophils (l) havebeen distorted by the staining process.

b Left: many larger falciparum rings (r), somewith two nuclei. The parasite burden is highestin malignant tertian malaria (may equal morethan 50 % of affected erythrocytes). Remnantsof two segmented neutrophils (l) are visible inthe field.

c Left: schizont (t) with 8–24 merozoites (me),platelets (thr), segmented neutrophil (l), and lym-phocyte remnant (ly).

d Left: macrogametocyte (w) and microgameto-cyte (m), also called crescents, segmented neutro-phil (l), lymphocyte (ly), and cellular debris.

Thin smear

a Right: ring forms (r) in unenlarged erythro-cytes. Infection of one erythrocyte by 2–4 para-sites (r2) is common. These rings are indistin-guishable from young vivax forms!

b Right: larger falciparum rings (r) in unenlargederythrocytes, no Schuffner dots.

c Right: young schizont (t1) and mature schizont(t2) with 24 merozoites.

d Right: microgametocyte (w) and macrogame-tocyte (m). Remnants of the erythrocyte are stillvisible around the macrogametocyte, which tendsto be longer than the host cell.

The erythrocytes are not enlarged in falciparummalaria, whose early stage is indistinguishablefrom the three other types of malaria. Thereforethe blood examination should be repeated every12 to 24 h. Falciparum affects all erythrocytestages, not just the early ones as in vivax malaria


VII

Fig. 184 a–d

407


VII

Fig. 185 a–d. Malaria

a Left: Plasmodium vivax: ring form in aslightly enlarged erythrocyte stippledwith Schuffner dots

Right: schizont of P. vivax in an enlargederythrocyte

b Left: tertian malaria (P. vivax): thicksmear with large ring forms, segmentedneutrophil at upper left

Right: young vivax schizonts and severalring forms in a thick smear

c Left: Plasmodium ovale; ring form inenlarged erythrocytes, which show el-liptical distortion and Schuffner stippling

Center and right: young schizonts inenlarged, elliptically distorted erythro-cytes with conspicuous Schuffner dots

d Left: quartan malaria (Plasmodiummalariae); ring forms in a thick smear(erythrocytes are clumped, not disinte-grated!)

Right: thin smear with ring forms andyoung schizonts in unenlarged erythro-cytes


VII

Fig. 186 a–d. Malignant tertian malar-ia (Plasmodium falciparum)

a Thin smear with numerous rings; someerythrocytes contain 2, 3, or 4 parasites

b Young schizont and ring form inunenlarged erythrocytes

c Left: thick smear in falciparum malariawith an extremely heavy parasite burden.Beside the lymphocyte at upper left, thefield consists entirely of falciparumparasites

Right: thick smear in falciparum malaria(erythrocytes are not completely hemo-lyzed)

d Left: gametocyte in a thin smear

Right: gametocytes in a thick smear

409


VII

Fig. 187 a–b. Sleeping sickness (Trypanosomagambiense), Giemsa stain

7.2 African Trypanosomiasis(Sleeping Sickness)

This is a trypanosomiasis caused by two subspe-cies of the same species (Trypanosoma brucei),which differ in their virulence: Trypanosomagambiense, which occurs chiefly in western Africaand causes protracted illness, and Trypanosomarhodesiense, which is prevalent in eastern Africaand takes a more fulminating course.

The organism is introduced through the bite ofthe tsetse fly. Often a primary focus (trypanoso-mal chancre) develops at the site of inoculation(e.g., on the shoulder, upper arm, or neck), pro-ducing central swelling, erythema and often whitescaling over an area up to several inches in dia-meter. The trypanosomes can be detected in as-pirates from this area and from the associated re-gional lymph node.

The second, lymphoglandular phase of the dis-ease is characterized by fever, severe headache,and slight enlargement of the liver and spleen.

At this stage the parasites can be detected in thickand thin blood smears.

In the final, meningo-encephalitic stage of thedisease, the trypanosomes invade the central ner-vous system and can be detected in the cerebro-spinal fluid.

A Thick smear

Trypanosomes (tr) with a red nucleus, whichusually is centered red kinetoplasts, and blue pro-toplasm. The posterior end is usually roundedand contains the micronucleus, which gives riseto the red, whiplike flagellum. The trypanosomesare approximately 13–42 lm in length, averaging20–29 lm. They are longer than the erythrocytes.Their shape is pleomorphic, and dividing formsare occasionally seen

B Thin smear

Nucleus (k), kinetoplast (bl), flagellum (g), undu-lating membrane (u), protoplasmic body (pr),dark stained granular inclusions (gr). The kineto-plast is usually rounded or oval


VII

7.3 American Trypanosomiasis(Chagas Disease)

A primary lesion called a chagoma forms at thesite of inoculation by the reduviid bug (triatome).The parasite, Trypanosoma cruzi, is introducednot through the bite of the bug but through itsinfected feces, which are rubbed into the conjunc-tiva or a break in the skin. The infection spreadsrapidly through lymphatic pathways, causing en-largement of the regional nodes. This oculogland-ular syndrome (Romana’s sign) becomes evidentfrom 4 days to 3 weeks after the infection is ac-quired.

The disease is particularly life-threatening ininfants and small children, as it can progress toa meningoencephalitic form.

The flagellated parasites (Trypanosoma cruzi)can be detected in the blood stream only duringthe acute febrile phase. Later they are detectableonly by animal inoculation and xenodiagnosis(using laboratory-cultivated triatomes). The or-ganisms multiply in an intracellular, nonflagel-lated form (amastigotic or “leishmanial” form)within the tissue (e.g., the myocardium).

Today serologic methods are also available forthe diagnosis of late cases and chronic stages andfor identifying Chagas cardiopathy. Severe myo-cardial damage and organ enlargement are latesequelae whose etiology can be established onlyby serologic procedures.

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Fig. 188. Chagas disease(Trypanosoma cruzi)

Blood smear showing T. cruzi in the per-ipheral blood (Giemsa stain). The bodyof the parasite is stubbier than that of T.gambiense, but otherwise its dimensionsare much the same: length approximately20 lm, width 2–3 lm. Typical features arethe large nucleus, the large terminal kine-toplast, and the long, usually C-shaped fla-gellum


VII

Fig. 189 a, b. Trypanosomes

a Trypanosoma rhodiense in a thin bloodsmear displays a dark red nucleus, blueprotoplasm containing darker granules,an undulating membrane, and a flagel-lum

b Thick smear with two parasites atcenter. The general form of the parasite isrecognizable, but the fine features of thetrypanosome body are not appreciatedas clearly as in the thin smear

Fig. 190. Toxoplasmosis

Thin smear from the peritoneal exudateof a mouse infected intraperitoneallywith Toxoplasma gondii shows numerousfree organisms shaped like crescents ororange slices with a dark blue nucleusand bluish cytoplasm

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VII

Fig. 191. Kala azar (Leishmania donovani).

Splenic aspirate contains numerous leishmaniae, mostintracellular, with a dark nucleus and rod-shaped ki-netoplasts (picture of “two nuclei”) in a pale bluish-redcytoplasm. Some of the organisms are pear-shaped(diameter 4lm).

7.4 Kala Azar or Visceral Leishmaniasis

Causative Organism: Leishmania donovaniAll types of leishmania are macrophage para-

sites.The disease is transmitted to humans by

blood-sucking sandflies of the genus Phleboto-mus, with dogs and humans serving as the prin-cipal reservoir. The incubation period is highlyvariable, ranging from days to many months oreven years. A nonspecific prodromal stagemarked by lethargy and body ache is followedby episodes of fever, often accompanied by chills.In the acute stage, measurements taken at 1– to 2-h intervals will very often show two fever spikes ina 24-h period.

A very large, hard spleen, a large firm liver, leu-kocytopenia, thrombocytopenia, and anemia arecharacteristic. There may also be cardiovascular

abnormalities and, without prompt initiation oftreatment, permanent myocardial injury. Thereis reversal of the serum protein ratio with hyper-gammaglobulinemia, hypoalbuminemia. The bestmethod for the direct demonstration of the para-site is the bone marrow biopsy. The best methodfor serological detection is the indirect immuno-fluoroscence test (IIF). The serological analysisshould always precede the detection of the para-site; in case of negative serology parasite detec-tion is nearly impossible.


VII

Fig. 192 a–d. Leishmaniae

a Leishmania donovani in a sternal mar-row smear. The numerous parasites areall intracellular

b Sternal marrow smear with extracel-lular leishmaniae, expelled from an in-fected macrophage that ruptured whenthe smear was prepared

c L. donovani in a splenic touch pre-paration (golden hamster). Macrophageshave engulfed massive numbers of theparasites

d Free forms in a splenic touchpreparation (golden hamster)

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7.5 Cutaneous Leishmaniasis (Oriental Sore)

Causative Organism: Leishmania tropica, Leish-mania maior

The disease is transmitted by the bite of thephlebotomus sandfly on unprotected skin. Theprincipal reservoirs are humans (L. tropica)and small rodents (L. maior). The incubation per-iod ranges from several days to weeks or evenmonths.

Small, red, pruritic papules appear at the site ofinoculation and develop into firm nodules whichulcerate within months in the case of L. tropica,already after a few weeks after L. maior inocula-tion. The lesions are most commonly seen on ex-posed areas of the face (nose, ocular canthi,cheeks, ears) and on unclothed portions of thetrunk and limbs. Ulcerating lesions may coexistwith nonulcerative lesions that spread to involveadjacent skin areas (L. recidivans).

In Central and South America, the causativeorganisms of cutaneous leishmaniasis are trans-mitted by flies of the genus Lutzomyia. A distinc-tion is drawn between the L. mexicana and L. bra-ziliensis complexes. Espundia (mucocutaneousleishmaniasis), a malignant form caused by L.braziliensis, causes destruction of skin, mucousmembranes, and cartilage in the nasopharyngealregion. “Uta,” caused by L. peruviana, is benignbut leads to scarring, especially in the face.

For the detection of the parasite of cutaneousleishmaniasis, PCR has growing importance.However, the result should if possible be ascer-tained by the microscopical detection of the para-site in the biopsy.

7.6 Toxoplasmosis (Fig. 190)

Causative organism: Toxoplasma gondiiInfection is usually contracted orally by the in-

gestion or handling of contaminated food, such asraw meat, or by touching the feces of acutely in-fected cats, which shed oocytes in the stool. Theseoocytes are highly resistant and can remain infec-tive for long periods in amoist environment, suchas wet soil. The Sabin-Feldman dye test (SFT), in-direct immunofluorescence (IIF), and indirecthemagglutination (IHA) test become positivewithin 8 days of infection, and the complementfixation text (CFT) becomes positive within 2–3 weeks. Recently the enzyme immune test or ELI-SA assay has been employed to detect toxoplasmainfection.

While the CFT becomes negative after a time,the SFT, IHA, and IIF usually remain positivewith low titers for the rest of the patient’s life, sig-nifying that the infection has entered its latentstage. Recently, the so-called test of avidityproved to be of help in calculating the durationof infection (acute or chronic).

Infection must be massive before there is sig-nificant lymphadenitis with clinical manifesta-tions such as headache, low-grade fever, and sin-gle or multiple lymph node enlargement, particu-larly in the neck. Involvement of the liver (hepa-titis) is occasionally reported.

Severe, generalized forms of the disease areuncommon. Generalized cases with central neu-rologic involvement and a fatal outcome havebeen observed in patients with AIDS.

The parasite can be detected in the acute stageby animal inoculation or PCR, but the diagnosis isbest established by serologic examination of theblood. The histologic examination of an excisedlymph node or lymph node aspirate also maybe considered.


VII

7.7 Loa Loa

Transmission: By flies of the genus Chrysops. Aperiod ranging from weeks to several monthspasses after inoculation before microfilariae aredetectable in the peripheral blood.

Occurrence: Exclusively in the rain forest regionsof western and central Africa.

Clinically, severe itching is an early sign and isoften accompanied by Calabar swellings: tense,circumscribed edematous swellings 1– 10 cm indiameter with erythema, heat, and moderatepain. The swellings appear on the forearm,face, and occasionally on the trunk and legs. Itch-ing is caused by the migration of the filiariae be-neath the skin. Migrating filariae can be seen be-neath the conjunctiva, where they cause lacrima-tion and conjunctival irritation. Psychoneuroticsequelae and severe encephalitic syndromeswith microfilariae in the CSF are occasionally ob-served.

Early diagnosis: The serologic reactions can de-tect many cases 12– 18 months before microfilar-iae appear in the peripheral blood.

Fig. 193. Loa loa, hematoxylin stain.

The Loa loamicrofilaria is 250–300 lm long, lar-ger and thicker than Mansonella perstans, withwhich it often coexists in the blood. Its delicatesheath (h) extends past the extremities of thehead and tail. The nuclei of the somatic cellsare densely arranged and extend to the tip ofthe tail. This microfilaria occurs in the peripheralblood only in the daytime (hence the old name,Microfilaria diurna)

7.8 Wuchereria bancrofti and Brugia malayi

Wuchereria bancrofti and Brugia malayi microfi-lariae are nocturnally active, reaching a peak inthe peripheral blood around midnight. A Pacificisland subspecies of W. bancrofti (var. pacifica)does not show this nocturnal periodicity.

Geographic distribution of W. bancrofti:Western, central, and eastern Africa, Egypt, Ma-dagascar, Comoro Islands, Central and SouthAmerica, India, Southeast Asia, Sri Lanka, China,Korea, New Guinea, and the South Sea Islands. B.malayi is found in India, Sri Lanka, and SoutheastAsia. Both parasites have many features in com-mon. They are found in the lymphatic vessels andin the sinuses of the lymph nodes.

Transmission: By Anopheles, Culux and Aedesspecies of mosquitoes.

Initial symptoms appear 3 to 16 months afterthe infection is acquired and include deafness,limb weakness, and itching in the axilla and groin.Periodic pain in the extremities and testes also oc-cur. Pea- to walnut-sized lymph nodes develop atthe base of the thigh, in the axilla, and at the el-bow, and lymphangitis develops in the genital re-gion and extremities. A syndromic condition mayarise that has been described as “tropical eosino-philia,” i.e., a pulmonary filariasis with no detect-able microfilariae in the peripheral blood. Thisform occurs predominantly in Asia.

Late Sequelae: Lymph scrotum, chyluria, and ele-phantiasis

Very often, serologic reactions become posi-tive approximately 4 to 8 weeks after the infectionis acquired. Considerable time passes before mi-crofilariae can be detected in the peripheralblood.

Fig. 194. Wuchereria bancrofti, hematoxylin stain.

The microfilaria of this species is 224–296 lmlong and sheathed (h). The nuclei (k) of the so-matic cells are loosely arranged and do not extendto the tip of the tail (s). The parasites tend to formlarge, smooth coils in thick smears.

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VII

Abb. 193

Abb. 194


VII

Fig. 195 a–d. Filariasis

a Microfilaria of Mansonella perstans, seealso Fig. 196

b High-power view of a Loa loa micro-filaria with granules in the cell body andconspicuous protoplasm (see alsoFig. 193)

c Low-power view of numerous Loa loamicrofilariae in a thick smear

d Wuchereria bancrofti, see also Fig. 194

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VII

7.9 Mansonella (Dipetalonema) Perstans

Transmission: By biting flies of the genusCulicoides.

Distribution: Western and central Africa andCentral America.

This nematode commonly occurs in associa-tion with Loa loa (Figs. 193, 199 b, c) andWucher-eria bancrofti (Figs. 194, 199 d). The infection isusually innocuous, causing nomore than pruritusand transient skin swelling.

Fig. 196. Mansonella perstans, hematoxylin stain.

Thick smears are best for the detection and dif-ferential diagnosis of the various microfilariaethat occur in the blood (see method on p 19).M. perstans microfilariae are 165–216 lm long(shorter than the others), slender, and un-sheathed. The rows of nuclei extend to the tipof the relatively blunt tail

Abb. 196


VII

8 Further Important CausativeOrganisms of Tropical Diseases

8.1 Relapsing Fever

Causative Organisms: Borrelia recurrentis(Louse-Borne Relapsing Fever) and Borrelia dut-toni (Sporadic Tick-Borne Relapsing Fever)

The disease is transmitted by lice (Borrelia re-currentis, epidemic in Ethiopia central and SouthAmerica and other countries) or by ticks of thegenus Ornithodorus, for example (endemic inAfrica). Following an incubation period of 5 to8 days, the disease begins abruptly with feverup to 40 8C, often accompanied by chills. The fe-ver lasts 3–7 days and is followed by an afebrileperiod of 2–20 days. The number of relapses isvariable. The spleen and liver are enlarged, andthere is a propensity for bleeding from the skinandmucosae. The ESR and transaminases are ele-vated, mild anemia is present, and the leukocytecount is normal.

Fig. 197 a, b. Relapsing fever (Borrelia recurrentis),Giemsa stain.

A Thick smear: Borrelia spirochetes (s), leuko-cyte nucleus (l), cellular debris (z). The spiro-chetes are appreciably longer than the red cells.Length 15–30 lm with 6–8 turns and pointedends

B Borrelia spirochetes (s) in a thin smear. Thesizes of the red cells and spirochetes are easierto compare in this type of preparation

8.2 Bartonellosis (Oroya Fever)

Causative Organism: Bartonella bacilliformisThis disease is transmitted by female flies of

the genus Lutzomyia. An animal reservoir hasnot yet been identified. The flies pick up B. bacil-liformis when they bite an infected individual andtransmit them when they feed again. Bartonellainfections occur in the high mountain valleysof the Andes (Ecuador, Peru, and Bolivia). Theincubation period is from 15 to 100 days.

A clinically nonspecific prodromal stage withremitting or intermittent fever is followed bythe development of a severe hypochromic,macrocytic, hemolytic anemia. During this acutestage, which lasts 2–3 weeks, organisms can bedetected in the erythrocytes of the peripheralblood. Within several weeks granulomatousskin lesions appear, mainly in exposed areas (ver-ruga peruana). Very massive infections terminatefatally within 10 days.

Fig. 198. Oroya fever (Bartonella bacilliformis),Giemsa stain

Thin blood smear: the bacteria are pleomorphicand stain a bright red in the unenlarged erythro-cytes. Most of the organisms are spherical, a feware rod-shaped, and some unite to form smallchains. It is typical for several bartonellae to infectone red cell

421

8 · Further Important Causative Organisms of Tropical Diseases

VII

Abb. 197 a, b

Abb. 198


VII

8.3 Leprosy

Causative Organism: Mycobacterium leprae(Hansen’s Bacillus)

The infection is transmitted from person toperson and requires prolonged, intimate contactdue to the low infectivity of the bacilli. The incu-bation period is very long (months to years).

The onset of leprosy is insidious. Several formsare recognized:

I. The tuberculoid form develops when resis-tance to infection is intact. The lepromintest is positive and few bacteria are found.

II. The lepromatous form develops when resis-tance to infection is impaired (anergicphase). The lepromin test is negative, andbacteria are abundant.

III. The indeterminate form can develop in eitherdirection, i.e., the lepromin test and bacterialcount are variable.

IV. The borderline form, in which the lepromintest is usually negative and the bacterialcount is usually high.

The natural history of leprosy is marked by per-iods of activation (“reactions”) that commencewith chills, fever, and severe malaise. In thewake of this “reaction,” new lesions appear dueto the dissemination of leprosy bacilli via theblood stream or lymphatic pathways.

Fig. 199. Leprosy (Mycobacterium leprae),Ziehl-Neelsen stain

The leprosy bacillus can be demonstrated in nasalsmears or skin biopsies (e.g., from the auricle).Because M. leprae is an acid-fast organism, itstains red with Ziehl-Neelsen stain. The bacillimay occur singly or may be grouped into heapsor cigar-shaped clusters. The organisms are 3–4 lm long and 0.4 lm thick.

Abb. 199

423

8 · Further Important Causative Organisms of Tropical Diseases

Subject Index

A

aberration– 13q(-) 311– 17p(-) 311abnormal– eosinophils 221– erythroblast mitose 161– nonspecific esterase reaction 163acanthocytes 34acid– a-naphthyl acetate esterase

(ANAE) 15– phosphatase 14,0 73, 124– – cytochemical determination

14acute basophilic leukemia 262– eosinophilic leukemia 259– erythroblastopenia 98, 99– erythroleukemia– – subtype AML-M6 238– – subtype M6a 242– – subtype M6b 240– leukemia 180– – CSF cytology 284– – CSF involvement 181– – therapy-induced changes 250– – with mixed phenotypes 279– NK-cell leukemia 374acute lymphocytic leukemia (ALL)

180, 265– B4 subtype 181– B-cell line 181– B-lineage 265, 266– c-ALL with t(4;11) 181– chromosome abnormalities 273– immunologic classification 182– immunologic criteria 181– L3 subtype 181– Ph-positive 283– pre-B-cell ALL with t(4;11) 181– T-cell line 181– T-lineage 274– with cytoplasmic granules 271acute myeloid leukemia (AML) 180,

183, 253– abnormal eosinophils 221– biological classification 181– biological entities 182– coexistence with T-ALL 280– hyperbasophilic variant 204– karyotype 235– M0 subtype 184– M1 subtype 186, 188– M2 subtype 188, 195– – variants 190– – with eosinophilia 190– – with t(6;9) 196

– M3 subtype 203– M3 V subtype 205, 206– – cytochemical findings 207– – response to treatment 209– prognostic classification 181– rare forms 259– type I/II blasts 183– WHO classification 181– with deficient granulocyte matura-

tion 257acute myelomonocytic leukemia– M4 subtype 216– – cytochemical findings 223– – meningeal involvement 226– – variants 220Adam’s reaction 224adult T-cell leukemia 342, 343African trypanosomiasis 410agranulocytosis 114alcohol abuse 96Alder-Reilly anomaly 46aleukemic mast cell leukemia 288alkaline phosphatase activity 63, 113allergic agranulocytosis 114amegakaryocytic thrombocytopenia

117American trypanosomiasis 411anaplastic T-cell lymphoma 350anemia of chronic disease 107angiosarcoma 392anisochromia 31antibodies against components of the

cytoskeleton 386aplastic– anemia 96, 119– – severe type 119– crisis 98apoptosis 93atypical MDS 178Auer rod 160autoimmune– granulocytopenia 114– hemolytic anemia 84, 85azurophilic granule 38, 66

B

B marker CD19 266band neutrophil 41Bartonella bacilliformis 421bartonellosis 19, 421basophilic– erythroblast 28– granulocytes 41– stipling 31, 138B-cell– lymphoma 308

– prolymphocytic leukemia 311, 312,329

bcl-2 312B-CLL 311– cytogenetic aberration 311BCR/ABL translocation 134, 144Bence-Jones myeloma 355Berlin blue– iron stain 10– reaction 73Bernard-Soulier syndrome 117, 118bilinear acute leukemia 180bilobed nucleus 205binucleated– erythroblast 103– lymphocyte 307– microkaryocyte 164Birbeck granule 132blast-like cell 304blood– cytochemistry 17– reactive changes 107– smear 4– – differentiation 4– – preparation 4Boeck disease 303bone marrow 4, 69– aplasia 119, 120– aspirate differential cell count 69– cell cytochemistry 17– cellular distribution 76– change in differential counts 79– fat distribution 76– in agranulocytosis 115– in HIV infection 109– lymphocytic focus 110– paroxysmal nocturnal hemoglobi-

nuria (PNH) 121– reactive changes 107– smear 123– – cellularity 75– toxic changes 107Borrelia– duttoni 421– recurrentis 421branchiogenic cyst 296breast carcinoma 388Brugia malayi 417

C

Cabot ring 31, 138c-ALL eosinophilia 278CBP-MOZ fusion transcript 237CD10 267CD103 313, 326CD11c 313, 326

425 A–C

CD34-positive stem cell 24CD41 244CD61 244cell-surface antigen– immunocytochemical detection

18centroblast 294, 295, 297, 312, 333centroblastic variant 348Chagas disease 411, 412Charcot-Leyden crystals 112, 224, 225chloramphenicol 96chromatin fragmentation 175chromosome 12 197chronic– disease anemia 80– erythroblastopenia 101– lympadenosis 311chronic eosinophilic leukemia

(CEL) 157chronic lymphocytic leukemia

(CLL) 311– atypical form 316, 318– of B-lineage 314chronic myeloid leukemia 134, 144,

146– accelerated phase 150– blast phases 152– erythroblastic phase 155, 156– lymphoblast phase 154– megakaryocytic-megakaryoblastic

phase 155– myeloid blast phase 152– stages 144, 145chronic myelomonocytic leukemia

(CMML) 158, 173– with eosinophilia 160chronic myeloproliferative disorders

(CMPD) 134chronic myeolomonocytic leukemia

(CMML) 160chronic neutrophilic leukemia

(CNL) 157cleaved cell 312clustering 137cold agglutinini disease 89compensated hemolytic disease 82congenital– dyserythropoietic anemia 101– – type I–III 101– Fanconi anemia 119core sample 4cutaneous leishmaniasis 416cyclic agranulocytosis 114cytochemical procedures 69cytokeratine 386cytopenia 158cytoplasmic vacuoles 44

D

daisy form 103decompensated hemolytic disease

82deletion– 11q 311– 13q 315dentritic reticulum cell 57, 295, 299

desmin 386Diamond-Blackfan syndrome 119dicentric chromosome 177diffuse large B-cell lymphoma 351– immunoblastic variant 351Doehle body 117dotlike ANAE 345Down syndrome 140– acute myeloid leukemia 140dry tap 119, 313dyserythropoiesis 158dyserythropoietic anemia 102– type I 102– type II/III 103dysgranulocytopoiesis 158, 163dysmegakaryocytopoiesis 160dysplasia 158dysplastic eosinophils 221

E

early neutrophilic granulocytopoi-esis 76

EDTA 4– EDTA-induced pseudothrombocy-

topenia 118elliptocyte 31, 85eosinophilia 107eosinophilic– granulocytes 41– leukemia 108– promyelocyte 111epitheloid cell 302, 303Epstein-Barr virus 304erythroblast islands 28erythrocyte 31– fragments 84– in severe iron deficiency 81– Rouleaux formation 356erythroid hyperplasia 82erythrophagocytosis 82, 230erythropoiesis– disturbances 80– left shift 80– toxic disturbances 96essential thrombocythemia (ET) 135extra Ph chromosome 144extramedullary plasmacytoma 355extranodal NK/T-cell lymphoma 373

F

FAB 180– subtype L3 270– type M5a 235faggot 203familial– erythrocytosis 143– hemophagocytic lymphohistiocy-

tosis 129– polyglobulia 140fetal– erythroblastosis 88– hemoglobin 9filariasis 419fine-needle aspiration 5

FISH technique 144, 228, 311fixation 16flaming– myeloma cell 357– plasma cell 355flower cell 344foamy plasma cell 367focal acid phosphatase reaction 274follicular lymphoma 312, 333foreign body giant cell 63fragmentocyte 34, 84, 88fusion gene 144, 160– AF9/MLL 235

G

gametocyte 402, 409gastric carcinoma 390Gaucher– cell 122, 123– disease 122, 123G-CSF 108– left shift 113giant– metamyelocyte 93, 94– platelet 52, 117, 118– proerythroblast 98, 99Glanzmann-Naegeli thrombasthe-

nia 117glucose-6-phosphate dehydrogenase

deficiency 82glycogen 268– storage disease type I 127– cytochemical determination 11GM-CSF 108Golgi esterase 198granular lymphocyte 66granule 38granulocytopenia 114granulocytopoiesis 109– giant forms 201– left shift 109Gumprecht shadow 311

H

H chains 84, 88hairy cell leukemia (HCL) 312, 319– cytochemistry 325– immunocytochemistry 325– variant 327Hb S disease 88Hbf 86Heinz body 82, 85– test 8hematopoiesis 25hemolytic– anemia 82– – classification 83– disease of the newborn 82hemophagoctic syndrome 129hemosiderin 169heparin artifact 44hepatosplenic– cdT-cell lymphoma 354– T-cell lymphoma 353

426 Subject Index

hereditary Heinz body anemia 82histiocyte 295histiocytosis X 132histologic examination 5HIV infection 178Hodgkin– cell 379– lymphoma 308, 379– – bone marrow infiltration 380Hodgkin-like cell 304, 305Howell-Jolly body 34, 92hybrid acute leukemia 180hydrolases 13hypereosinophilic syndrome (HES)

107, 108, 111, 157hypersegmented– megakaryocyt 94– neutrophil 94hypochromic erythrocytes 31hypoplastic– acute myeloid leukemia (AML)

264– plasma cell myeloma 371

I

idiopathic thombocytopenic pur-pura 117, 118

immunoblast 294, 297immunoblastic lymphadenopathy

296immunocytoma (IC) 311indolent– myeloma 355– systemic mastocytosis 287ineffectual hematopoiesis 158infectious mononucleosis 304interdigitating reticulum cell 295,

298interphase cell 227intracellular antigen– immunocytochemical detection

18intravascular large B-cell lymphoma

353, 354intrinsic factor 91inversion– inv(14) 312– inv(16)(p13;q22) 228– inv(3)(q21;q26) 202iron 97– deficiency 80– stain– – differential diagnosis 11– utilization disorder 80iron-laden mitochondria 167isochromosome– 17 144– 3q 307

J

juvenile myelomonocatic leukemia(JMML) 160

K

Kala azar 414karyorrhexis 101Ki-1 lymphoma 351Kleihauer-Betke stain 9, 10– demonstrating methemoglobin

10knizocytes 34Kostmann syndrome 117kryptic PML-RARA rearrangement

215

L

Langerhans– cell histiocytosis 132– giant cell 303large cell non-Hodgkin lymphoma

348large granulated lymphocyte (LGL)

leukemia 311, 340, 341Leishmania 415– donovani 414leprosy 423leukemia of dentritic cell 377, 378leukemic blast 180leukocyte– alkaline phosphatase (LAP) 70– – cytochemical determination 13– concentration 6– degenerate forms 44leukocytopenia 90leukoerythroblastic reaction 391light-chain plasmacytoma 355lipophages 57Loa loa 417, 419lymph node cytology 294lymphadenitis of Piringer-Kuchinka

296lymphatic tissue 294lymphoblast 294, 295lymphocyte 66– with polar villi 313lymphocytic focus 116lymphocytopoiesis 294lymphogranulomatosis 379lymphoid cell 304lymphoma infiltration in bone mar-

row 308

M

macroblast 28macrogametocyte 402macrophage 57, 130malaria 400malignant– histiocytosis 129– lymphoma 284– mastocytosis 286, 290– – with erythrophagocytosis 290– non-Hodgkin lymphoma 181, 308– tertian malaria 400, 401, 406, 409Mansonella perstans 419, 420mantle cell lymphoma 312, 331

marrow– aspirate 4– erythroblasts in iron deficiency 81mast cell 41– disease 286mastoblast 41, 290mature– B-ALL 181, 270– B-cell neoplasm– – WHO classification 309– T-cell neoplasm 309– – WHO classification 310Mayer’s nuclear stain 8May-Hegglin anomaly 52, 117medulloblastoma 393megakaryoblast 60, 180megakaryoblastic leukemia (M7)

244, 246megakaryocyte 60– marker 140megakaryocytic dysplasia in MDSmegaloblast 93megaloblastic anemia 90– pathogenic factors 90megaloblastoid changes 166megalocyte 31, 92membrane receptor PRV-1 134merozoites 402metachromasia 41metamyelocytes 41microfiliariasis 19microgametocyte 402micromegakaryocyte 158, 164microspherocytosis 31, 82midline granuloma 373mixed megakaryoblastic-basophilic

leukemia 247MLL gene 235monoblast 180monoblastic leukemia– M5b subtype 231monoclonal gammopathy 355monocyte 54, 110– granuloma-like aggregation 110monocytic– granuloma 110– leukemia– – M5b subtype 228mucopolysaccharidosis 46multinucleated erythroblast 101multiple myeloma 355– plasmablastic type 358Mycobacterium– leprae 423– species 19myeloblast 38, 180myelocyte 41myelodysplasia with pseudo-Pelger

changes 175myelodysplastic– disease 160– syndrome 158– – diagnostic criteria 158myelofibrosis 137myeloid leukemia 140myeloma– cytochemistry 362– immunocytochemistry 364

427 C–M

myelodysplastic syndrome 159myeloproliferative syndrome 134,

160

N

naphthol AS-D chloroacetate ester-ase 15, 72

naphthyl– acetate 14– butyrate 14natural killer cell neoplasia 372necrotic bone marrow 44neoplasia of tissue mast cells 286neuroblastoma 393neuron-specific enolase (NSE) 387neutral esterase 14Niemann-Pick disease 125Nile blue sulfate stain 9NK-cell neoplasm 310Non-Hodgkin lymphoma 335– immune markers 335non-secretory myeloma 355nonspecific esterase reaction 71NPM-RARA 215nuclear cleft 312

O

organ-specific antibodies 386Oriental sore 416Oroya fever 421orthochromatic normoblast 29osteoblast 63osteoclast 63, 390osteomyelosclerosis 134, 137

P

p53 177, 311panmyelopathy 119panmyelophtisis 119Pappenheim– stain 8pappenheimer body 34paranuclear acid phosphatase reac-

tion 274paroxysmal nocturnal hemoglobi-

nuria (PNH) 119, 121partial peroxidase defect 163PAS reaction 11, 72– in normal leukocytes 12PAX-5 gene rearrangements 311PDGF receptor 108Pelger-Huet anomaly 46periodic acid Schiff reaction (PAS

reaction) 11peripheral stem cell transplantation

74pernicious anemia 90peroxidase– cytochemical determination 13– reaction 70persistent polyclonal B lymphocyto-

sis 307

Philadelphia chromosome 134, 144plasma cell 66, 97, 128, 294– increase 108– leukemia 355, 369– myeloma 355, 356plasmablast 295, 297plasmablastic myeloma 355plasmacytic myeloma 355plasmacytosis 107– in HIV infection 109Plasmodium– falciparum 400, 401, 409– malariae 400, 401– ovale 400–402, 408– vivax 400, 401, 408PLZF-RARA 215PML/RARA 205poikilocytes 31polychromatic erythrocytes 31polycythemia vera 134, 135polyploid monoblast 174Pompe disease 127posterior iliac spine aspiration 4Price-Jones curve 90primary– effusion lymphoma (PEL) 353– granule 38proerythroblast 28prolymphocyte 312prolymphocytic leukemia 312promastocytes 41promegakaryocytes 60promegakaryocytic-megakaryocytic

leukemia 141promonocyte 54promyelocyte 38promyelolytic marrow 15, 114prostatic carcinoma 390pseudo-Chediak anomaly 192pseudo-Gaucher cell 82, 122, 149pseudo-Pelger 158, 162pseudothrombopenia 117pure– megakaryocytic myelosis 139– red cell aplasia 101

Q

5q-minus syndrome 160, 169quartan malaria 400, 401, 403, 408

R

RAEB 158, 171– in transformation 160– RAEB-1 160– RAEB-t 171RARS 165reactive eosinophilia 111Reed-Sternberg cell 379refractory anemia (RA) 158, 161– with excess blasts (RAEB) 158– with ringed sideroblasts (RARS)

158refractory cytopenia with multilineage

dysplasia (RCMD) 159

relapsing fever 421renal cell carcinoma 397reticulocyte 34, 88– count 82– stain 8reticulum 295rhabdomyosarcoma 395ribosome-lamellar complex 321Richter syndrome 352ring form 402, 406ringed sideroblast 80, 165Russell body 107, 110, 355, 361

S

sarcoidosis 303sarcoma 386schizocytes 34, 84schizont 402, 408sea-blue histiocyte 126segmented neutrophil 41serosa cell 295Sezary– cell leukemia 335, 338– syndrome 335, 336Shwachman syndrome 117sickle cell 6, 34, 87sickling test 88sideroachrestic anemia 80sideroblast 80siderocytes 34sideromegaloblasts 94sleeping sickness 410small-cell bronchial carcinoma 387smoldering 355smudge cell 311solitary plasmacytoma 355spherocytic anemia 85splenic aspiration 6– cytology 294splenic marginal zone lymphoma

(SMZL) 313, 328staging system– of Binet 311– of Rai 311staining method 8starry sky macrophages 295, 300Steinbrinck-Chediak-Higashi anom-

aly 49stem cell– in culture 26– transplantation 69sternal aspiration 4Sternberg-Reed cell 381stomatocyte 34storage disease 122strong acid phosphatase activity

313Sudan black B stain 13sympathicoblastoma 394synartesis 105

T

T-ALL, coexistence with AML 280target cell 34, 82

428 Subject Index

tartrate-resistant acid phosphatase313, 325

T-cell– leukemia 311– lymphocytosis 311– lymphoma 308, 335– prolymphocytic leukemia

(T-PLL) 311, 312, 344T-CLL 311terminal deoxynucleotidyl transferase

(TDT) 265tertian malaria 400–402, 408thalassemia 82– b-thalassemia 86thick smear 19, 403thrombocythemia 134thrombocytopathy 117thrombocytopenia 117thrombotic thrombocytopenic pur-

pura (TTP) 88tissue– basophil 41– mast cell 286T-lineage marker CD3 266toluidine blue stain basophil 8toxic granulation 44toxoplasmosis 19, 296, 302, 413,

416transient– abnormal myelopoiesis (TAM)

140– erythroblastopenie 98– myeloproliferation 140translocation

– (11;14) 312– 17p 177– 5 del(5)(q13q31) 170– involving band q23 of chromosome

11 235– t(1;22) 244, 249– t(10;11) 192– t(11;14) 331– t(11;14)(q13;132) 332– t(11;17)(q13;q21) 215– t(11;17)(q23;q21) 215– t(14;18)(q32q21) 334– t(15;17) 205– t(15;17)(q22;q12) 215– t(2;13)(q35;q14) 395– t(3;12)(q26;p13) 197– t(4;11) 273– t(5;12)(q31;p12) 160– t(5;14) 278– t(5;17)(q32;q21) 215– t(8;14)(q24;q32) 273– t(8;16) 236– t(8;16)(p11;p13) 237– t(8;21) 191– t(8;21)(q22;q22) 194– t(9;11)(p22;q23) 235– t(9;14)(p13;q32) 311– t(9;22) 273trisomy– 8 144– 12 311, 315tropical eosinophilia 108Trypanosoma– cruzi 412

– gambiense 410– rhodiense 413trypanosome 413tuberculosis 296tuberculous lymphadenitis 303tumor marker 386tumorous megakaryoblastoma

140

V

vacuolation 96, 268vimentin 386virocyte 304vitamin B12 91

W

Waldenstrom– disease 311, 317– macroglobulinemia 355Wucheria bancrofti 417, 419

Y

Yamshidi needle 4

Z

Zieve syndrome 84

429 M–Z