laboratory hematology practice (kottke-marchant/laboratory hematology practice) || cellular...

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Laboratory Hematology Practice, First Edition. Edited by Kandice Kottke-Marchant, Bruce H. Davis.

© 2012 Blackwell Publishing Ltd. Published 2012 by Blackwell Publishing Ltd.

Introduction

The history of qualitative cellular analysis originates from the 14th century in Italy with the introduction of spectacles to correct vision. In the late 16th and early 17th centuries opticians invented the compound microscope, most likely as a result of accidentally inverting a telescope. Because of its intrinsic absorption and refraction of visible light, the com-pound microscope allowed the visualization of small objects, although the magnifi cations possible were only × 20 –30. In 1665 Robert Hooke, the English Father of Microscopy, pub-lished Micrographia. In examining the microscopic structure of cork, he analogized its organization to monks ’ cells in a monastery. The word cell to describe the basic structure of biologic organisms remains to this day. Better known as the Father of Microscopy was the Delft scientist Antonie van Leeuwenhoek. He crafted microscopes that used double -convex lenses of high quality glass that he ground himself, which produced an optical magnifi cation of slightly greater than ×200. With these he observed bacteria, muscle fi bers, spermatozoa, lymphatics, and individual blood cells. van Leeuwenhoek fi rst described red blood cells in 1674 and estimated their size as 1.1 times the value accepted as accu-rate today (approximately 7.5 μ m).

The Prussian physician Rudolph Ludwig Karl Virchow is known as the Father of Pathology by virtue of his emphasis on the central role of the cell in biology. He stated in DieCellularpathologie in 1858 that “every cell originates from another existing cell like it. ” And he was perhaps the fi rst physician to recognize leukemia, which became easier to describe with the advent of synthetic dyes that allowed reproducible staining of both microorganisms and blood cells.

Synthetic aniline dyes were the discovery of W. H. Perkin in 1853. In 1879 Paul R. Ehrlich, a German physician, expanded the use of these colorful dyes, classifying them as basic, acidic, or neutral. It was while trying to synthesize quinine that Ehrlich inadvertently made his discovery, which enabled reproducible staining of blood cells including the granules in white blood cells [1,2]. His acidic/basic dye combinations for blood cells became the basis for the Romanowsky [3], Giemsa [4], Wright [5], and May–Grünwald modifi cations [6]. Dimitri Leonidovich Romanowsky modifi ed Ehrlich ’s technique using an aqueous mixture of eosin Y and oxidized methylene blue [3]. Because the aqueous dye solutions were unstable, James Homer Wright introduced methanol as a solvent and advocated using methanol as a fi xative prior to staining [5]. Gustav Giemsa standardized the dye solutions and added glycerol to increase solubility and stability [4]. Richard May and Ludwig Gr ünwald used saturated solutions to increase the intensity of the staining of the individual blood cells [6]. For general screening and surveillance purposes, Wright –Giemsaor May –Grünwald–Giemsa-stained fi lms “display unrivaled beauty and discrimination of form and color ” [7].

Every day throughout the world hematology laboratories use these stains to examine blood smears by light micros-copy. Qualitative and quantitative descriptions of cellular changes are observed and recorded, and reports are duly issued. The percentage of complete blood count (CBC) speci-mens followed by a manual review, scan, or differential count ranges from less than 10% to more than 50% with an average of nearly 27% in laboratories the United States [8] . Is this labor -intensive, expensive activity really neces-sary? With the advent of optical and electronic cell counters for enumeration, immunophenotyping for proteomic char-acterization, immunocytochemistry for localization (nuclear,

2 Cellular Morphologic Analysis of Peripheral Blood

Powers Peterson 1, Sheila McNeill 2, and Gene Gulati 3 1 Quest Diagnostics Nichols Institute, Valencia, CA, USA 2 Sentara Norfolk General Hospital, Norfolk, VA, USA 3 Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA, USA

CHAPTER 2 Morphologic Analysis of Peripheral Blood

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site and secured for no more than 1 minute. The selected site should be cleaned with 70% isopropyl alcohol and allowed to air dry. The puncture should be made with the needle bevel -up at approximately a 30 ° angle. Following the puncture, a tube is applied to the needle inside the needle holder. The order of draw is prescribed and the fi rst draw should be for blood cultures if required, in which case the skin cleansing should be performed with betadine. This is followed by: sodium citrate, plain, gel separator, heparin, ethylene diamine tetraacetic acid (EDTA), acid citrate dex-trose, and oxalate or fl uoride [19]. Each tube is allowed to fi ll, and as it is pulled from the needle holder, it must be mixed by inversion according to the manufacturer ’s recom-mendations. Tubes must be fi lled to within 10% of the recommended volume. When collection is accomplished and the needle is removed from the draw site, pressure is applied to the site. All tubes should then be labeled imme-diately with the appropriate patient information, date and time of draw, and the identifi cation of the person obtaining the specimen(s). The needle and its holder should be dis-posed of in designated sharps biohazard containers only.

Blood collection with a syringe differs from that of a veni-puncture with needle holder in two aspects. First, the pres-sure applied to the barrel provides the vacuum to fi ll the syringe with blood. Second, a blood transfer device that resembles a needle holder with an attachment to connect to a syringe is then attached to the syringe to aliquot the blood, which is done by attaching tubes to the needle inside of the holder. This is the currently approved method in the United States for blood transfer from syringe to laboratory tubes.

A winged infusion set has a smaller needle connected to tubing that can then be connected to a needle holder. Because the tubing contains air, a sodium citrate tube must not be drawn fi rst. If a sodium citrate tube is the only draw, then a blank tube must be utilized fi rst to remove the air from the collection set.

Skin punctures are performed primarily on newborns and pediatric or geriatric patients. If indicated, capillary speci-mens can also be collected directly into Unopettes or lavender -capped microcontainers for cell counting. Direct blood smears may be made from the microcontainer or from the puncture site after the fi rst drop of blood is wiped away. When a skin puncture is performed, the fi rst tube fi lled is the EDTA or other anticoagulant -containing microcollection tube. This will ensure more accurate hematologic results.

The vacuum tubes for blood collection and microcollection tubes have colored caps that follow a universal coding system among manufacturers. For hematology specimens EDTA is the recommended anticoagulant [19–21]. Dipotassium (K 2)EDTA powder is sprayed onto the wall of plastic lavender -capped tubes. The use of plastic tubes mitigates many of the issues regarding the safety of glass tubes and the potential risks associated with their breakage. Wherever possible, glass tubes are being phased out of use [22].

cytoplasmic, or membrane), and advanced techniques for detection and identifi cation of molecular abnormalities, is light microscopic evaluation of the blood smear a clinically useful activity? The answer is unequivocally yes, because the blood smear remains a crucial diagnostic aid [9].

Atlases and articles abound with intimate details of the colors, shapes, and sizes of the cells that inhabit the periph-eral blood, both in their normal forms, their variations, and in abnormal forms [10–16]. This chapter on cellular mor-phologic analysis will not recapitulate these extensive bodies of work. Instead the authors will concentrate on the periph-eral blood smear as a unique diagnostic tool in the labora-tory hematology armamentarium. This discussion will address technical topics as they relate most specifi cally to the blood smear: sample collection, pre -analytic variables that affect the quality of the blood smear, and staining artifacts. This will be followed by further discussions of blood cell examination, fi rst with respect to the results generated by automated analyzers, the CBC, and then in terms of the examination of the peripheral blood smear. Subsequent dis-cussion will focus on the diagnostic potential of the blood smear. There are limitations, but in the right clinical setting there is unquestionably clinical utility. What diagnoses are possible? Can a defi nitive diagnosis be rendered? Lastly, the authors have constructed fi gures to illustrate those condi-tions, clinical and artifactual, in which defi nitive fi ndings are present on peripheral blood smears.

Sample collection

A signifi cant variety of clinical information can be obtained from a well -made, well -stained, and thoughtfully analyzed blood smear [9,17,18]. According to Jandl more information can be gained from examining the blood smear than from any other single hematologic procedure [7]. Proper sample collection, processing, and staining allow the laboratory professional and/or clinical physician to identify spurious results, reach a diagnosis, and suggest further testing if warranted.

Blood sample collection must follow standard precautions. Proper patient identifi cation is the critical fi rst step in the collection process. The method of blood collection is based on the patient ’s age, physical condition, and the volume of blood needed. Venipuncture utilizing an evacuated tube system is the most frequent sampling technique. Use of a syringe or winged infusion set is generally reserved for small or fragile veins. Skin punctures are performed primarily on newborns and pediatric or geriatric patients.

For a venipuncture, the needle must be engineered with sharps injury protection in mind and the needle holder is for single use only. The preferred venipuncture site is a vein in the antecubital fossa, wrist, or hand. A tourniquet, usually a latex band, is applied 7 –10cm (3 –4 inches) above the draw

PART I Cellular Analysis

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of one of the authors serves as an example: a patient pre-sented with a WBC of 10 × 109/L, which was within the reference range. However, this patient ’s normal WBC was 3 × 109/L and the patient in fact had an acute infection that required antibiotics.

Peripheral blood smears

Wedge pull smears are made after the tube has been mixed either by inversion at least 20 times or by placing on a tube rocker. A small drop of blood is placed at one end of a clean glass slide with a microhematocrit tube or a specifi c transfer device. The spreader slide touches the slide at a 30 ° angle and is placed slightly in front of the drop of blood. It is pulled back until contact with the blood is made and then pushed forward in one continuous motion. This results in a smear with one edge that is rectangular and no visible blood droplet. The rounded end is feathered and the smear appears

Pre -analytic variables

Transport of specimens to the laboratory should occur as soon as possible. They should remain at room temperature, with the one exception of specimens obtained from patients with severe cryoglobulinemia or cold agglutinins. These specimens may need to be transported at as near as possible to body temperature. Analysis by an automated hematology instrument should ideally be performed within 6 hours for EDTA -evacuated tubes and within 4 hours for microcollec-tion tubes [20,23]. The integrity of a specimen, which includes proper labeling and correct fi ll volume, should be verifi ed at analysis. Any specimen with visible clots must be rejected. Adequate mixing ensures accurate cell counts on a hematology analyzer. As delineated in Table 2.1, there are also patient conditions that can affect a single CBC. Results of an isolated CBC may not be a true indicator of patient status without the patient ’s clinical history. An experience

Table 2.1 Variables that may affect complete blood count ( CBC) results.

Cause Corrective action

Patient identifi cation Specimen drawn from wrong patient Follow appropriate procedure step -by-step

Collection Inadequate or excess fi ll volume Collect within acceptable volume range

Inadequate mixing Follow manufacturer ’s instructions

Improper or incorrect labeling Follow appropriate procedure step -by-step

Clot(s) in the specimen Collect new specimen

Transport time Prolonged Perform test(s) as soon as possible, certainly

within 24 hours, otherwise report partial CBC

(Hemoglobin, WBC, RBC, and platelet count)

Temperature Extremes of temperature (high or low) during

transport

Use cooler with temperature packs for transport

to distant laboratory or off -site facility

Specimen settling Failure to or inadequate mixing prior to cell

counting

Follow manufacturer ’s instructions or mix by

tube inversion 20 times

Instrument Inaccurate/improper calibration Follow manufacturer ’s instructions

Quality control failure Follow appropriate procedure(s)

Performance interferences Increased lipids Perform plasma replacement

Increased bilirubin Perform plasma replacement

Cryoproteins Incubate at 37 °C for 10 –15 minutes

Cold agglutinins Incubate at 37 °C for 10 –15 minutes

Cytoplasmic fragments Report estimated platelet count from smear, if

appropriate

Hemolysis ( in vitro) Perform plasma replacement or obtain new

specimen

Platelet clumps Vortex or collect citrated blood (blue -top tube)

White cell clumps Report estimated WBC from smear. If

appropriate, report with a comment

Physiologic and environmental factors Age, sex, ethnic origin; pregnancy; smoking;

diurnal variation; high altitude; exercise; stress;

chemotherapy

Be familiar with expected variations

RBC, red blood cell count; WBC, white blood cell count.


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smooth with no streaks or ridges. All slides are labeled with patient information.

After being air -dried, the slides can be stained manually or on an automated slide stainer [23]. For manual staining, the slides are placed on a staining rack over a sink, and stain, buffer, and rinse are applied in a timed fashion. There are two types of automated stainer. The fi rst is a dip stainer that holds the slides vertically either in a basket or in individual cassettes and robotically moves them through the stain, buffer, and rinse processes. The second has two conveyer spirals and each slide is pushed along a platen and individu-ally stained. With the robotic systems common in high -volume laboratories, a tube of blood may be directed to a robotic slide maker/stainer. There the blood is mixed, then a wedge pull slide is made, which is labeled and stained.

Artifacts on peripheral blood smears

Regardless of whether a blood smear is manually or auto-matically generated, artifacts may be present [24]. The fi rst step to correcting the problem of an artifact is to recognize it as such, whether it is due to an abnormality in the patient, for example an elevated hemoglobin level, or to a technical problem, for example stain precipitation. Table 2.2 lists the most common reasons for artifactual changes on a blood smear and makes recommendations for correction of the problem. Some artifacts cannot be corrected except by obtaining a new specimen. Specimens that have been exposed to high temperatures or that have sat around for more than 6 hours show irreversible changes [25].

Table 2.2 Artifacts that can be present on peripheral blood smears.

Artifact Cause Corrective action

Smudge cells Nuclear remnants of lymphocytes

on peripheral blood smear

One drop 22% albumin added to fi ve drops blood prior to making

blood smear

Vacuoles in neutrophils and/or

monocytes

Increase with age of specimen in

EDTA

Prepare blood smear as soon as possible, preferably within 4 hours

Increased necrobiotic cells Increase with age of specimen Prepare blood smear as soon as possible, preferably within 4 hours

Refractile red blood cells Excess humidity Make a new slide, dry well; check stain for excess water

Water in stain Check stain/methanol for excess water

Stain precipitate Excess stain Clean stain tubing/lines and platen with methanol

Pale white blood cells with

increased numbers of white cells

Too many cells for staining

procedure

Stain slide a second time

Red blood cells very pink or blue Altered buffer volume or pH Make new slide, adjust buffer volume and/or pH

No area for proper red cell

morphology

Smear too thick or drop of blood

too large

Make new blood smear with more pressure on pusher slide and

more rapid pushing motion

Elevated hemoglobin Make new slide; decrease angle of pusher slide (manual slide

preparation)

Excess larger white blood cells at

periphery

Too much pressure on pusher

slide

Make new slide and decrease pressure on pusher slide (manual slide

preparation)

Blood cell examination: the completeblood count

Blood cell examination is usually performed in a sequential manner, beginning with analysis by an automated instru-ment. Samples are selected for further analysis if quantita-tive or qualitative abnormalities are found [8,26–34].Quantitative abnormalities include aberrant values for cell counts or cell size for the instrument -generated leukocyte differential. Qualitative abnormalities include alert fl ags that may indicate the possibility of inaccurate results or the pres-ence of abnormal cell types. Qualitative abnormalities vary in clinical importance. Some refl ect expected variations in clinical circumstances; others indicate conditions that warrant attention.

A related issue is that of false -positive and false -negativeresults from automated analyzers [7,27–31,35,36]. Falsely abnormal results suggested by automated analyzers can be identifi ed with careful observation of the blood smear. Table 2.1 lists the potentially overlooked but unequivocally impor-tant variables that can affect results generated by an auto-mated analyzer. Equally important, a normal result from an automated analyzer does not exclude the possibility of an inherited or acquired hematologic or other disorder, exam-ples of which are listed in Table 2.3. Examination of the blood smear may clarify whether the numerical result from the analyzer is spurious (Figure 2.1) or real (Figures 2.2 and 2.3). An example of a condition that can cause a spurious result is shown in Figure 2.4. (Note: Figures 2.4–2.13 are all images from blood smears stained with either Wright or Wright –Giemsa stains.)


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Figure 2.1 Causes of spurious complete blood count (CBC) results from automated analyzers. These conditions can be diagnosed on examination of a

peripheral smear. Some conditions causing erroneous (spurious) results are artifactual; others refl ect biologic variations or abnormalities. Some clinical

conditions, such as hyperlipidemic states and extreme leukocytoses, may give spurious results on particular analyzers.

* instrument dependent; NRBC, nucleated red blood cell; WBC, white blood cell count.

SPURIOUS CBC RESULTS

Platelets

Decreased Increased

Plateletagglutination in

EDTA

Lipids*

Decreased Increased

White blood cell

clumping

Cryoproteins

White blood cells

Insufficiently Iysed red blood cells

Abnormalhemoglobin

Newborn

Platelet aggregates

Decreased Increased

Red blood cells

Coldagglutinins

Very small red blood cells

In vitro hemolysis

WBC >100 × 109/L*

Plateletsatellitism

Microorganisms

Cryoproteins

Bacteria

Fungi

Fragmentedred cells

Giantplatelets

Increased NRBCs*

Fungi

Giant platelets

Fibrin strands

*Pappenheimerbodies

Disease/clinical condition Findings on blood fi lm

Compensated immune hemolytic anemia Spherocytosis, red cell agglutination

Hereditary spherocytosis Spherocytosis, polychromatophilia

Hemoglobin C disease Target cells, hemoglobin C crystals

Post-splenectomy state Howell–Jolly bodies, target cells, acanthocytes

Hereditary elliptocytosis Elliptocytes

Hereditary acanthocytosis Acanthocytes

Lead poisoning Basophilic stippling (insensitive indicator)

Macroglobulinemia, myeloma Rouleaux formation

Malaria Intra-erythrocytic parasites

Babesiosis Intra- and extra -erythrocytic parasites

Anaplasmosis (ehrlichiosis) Intraleukocytic morulae

Coccemia Intra- and extraleukocytic cocci

Fungemia Intra- and extraleukocytic fungal organisms

Disseminated intravascular coagulation Schistocytes (insensitive indicator)

Hemolysis due to physical injury to red cells Schistocytes

Infectious mononucleosis Reactive (atypical) lymphocytes

Hairy cell leukemia Hairy cells

Myelodysplastic syndrome Dysplastic granulocytes and nucleated red cells

Modifi ed from Ryan DH [44], with permission from McGraw -Hill.

Table 2.3 Conditions in

which the complete blood

count ( CBC) may be

unremarkable but examination

of the blood fi lm will suggest

or confi rm a disorder.


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Figure 2.2 Medical disorders or conditions that cause increased or decreased cell counts from automated analyzers. These abnormalities can be

diagnosed on examination of the peripheral blood smear.

ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CML, chronic myelogenous leukemia; CMML, chronic myelomonocytic leukemia;

CTCL, cutaneous T -cell lymphoma; ET, essential thrombocythemia; G6PD, glucose -6-phosphate dehydrogenase; G -CSF, granulocyte colony stimulating

factor; GM -CSF, granulocyte -macrophage colony stimulating factor; Hb, hemoglobin; LGL, large granular lymphocytosis; MPN, myeloproliferative

neoplasm; PV, polycythemia vera.

QUANTITATIVE CBC ABNORMALITIES

Platelets

Decreased Increased

Gray plateletsyndrome

May–Hegglin, MYH9-related

disorders

Neoplasm

EDTAclumping

Red cellfragments

Decreased Increased

May–Hegglin

Chediak–Higashi

Alder Reilly

Neoplasm

White blood cells

Infection

Bordetella pertussis

Inf. mononucleosis

G-CSF, GM-CSF

Infection

Hairy cell leukemia

Plasma cell leukemia

Leukemic phase,follicular lymphoma

Fungi

Bacteria

Decreased Increased

Red blood cells

Thalassemiaminor

G6PDdeficiency

Hereditaryspherocytosis

Nutritionaldeficiency

Hb SS, SC

Hb CC

β0-thalassemia

Hereditaryelliptocytosis

AML, M7

MPD/MPN

P Vera

ET

Iron

Folate

Vitamin B12

Plateletsatellitism

CML

CMML

CTCL (Sezary)

AML, M3

ALL, L3Burkitt lymphoma/

leukemia

Chronic & prolymphocytic

Leukemias

Fungi(Candida)

LGL leukemia

Paris-Trousseausyndrome

HbEE

AML,M6

All laboratories should have a protocol for the examina-tion of a laboratory -initiated blood smear [9,38]. A compre-hensive set of guidelines for laboratories worldwide has been developed and validated by an international consensus group for hematology review and published by the International Society for Laboratory Hematology (ISLH) [ 26;www.islh.org ]. The suggested criteria, or rules, as they relate to generating a slide for review are shown in Table 2.4.Ideally, a laboratory ’s protocols take into account clinical data such as the age and sex of the patient, analyzer -generated results, and laboratory organization and resources. Hierarchical protocols vary among testing sites and are struc-tured to refl ect the level of training and experience of testing personnel, the sophistication of the automated analyzers, and the incidence of variations or abnormalities in the popu-lation being tested [39]. For each laboratory, the written procedures delineate which quantitative and/or qualitative abnormalities mandate a microscopic review of the blood

smear and by whom this should be performed [26,40–43].When abnormalities are unfamiliar, rare, or potentially sig-nifi cant for diagnostic and/or therapeutic reasons, a physi-cian or laboratory specialist may need to review the blood smear. Optimally, integration of clinical information with the numerical and/or morphologic abnormalities improves the quality of laboratory results and enhances patient care [37,44].

Blood cell examination

An initial scan of a stained blood smear at low power will allow assessment of the quality of the slide, distribution of the cells, and quality of the stain [10,18,23,37,45,46].A poorly made slide with ridges in the smear will yield inac-curate results and a new slide must be made. Stain precipi-tate deposited on the slide indicates the need to troubleshoot


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Figure 2.3 Medical disorders or conditions that cause morphologic cellular abnormalities that can be diagnosed on a peripheral blood smear. These

disorders may be genetic (hereditary) or acquired.

*extra-erythrocytic organisms; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; B. recurrentis, Borrelia recurrentis; CLL, chronic

lymphocytic leukemia; CML, chronic myelogenous leukemia; CMML, chronic myelomonocytic leukemia; CTCL, cutaneous T -cell lymphoma; ET, essential

thrombocythemia; G6PD, glucose -6-phosphate dehydrogenase; G -CSF, granulocyte colony stimulating factor; GM -CSF, granulocyte -macrophage colony

stimulating factor; Hb, hemoglobin; HPPK, hereditary pyropoikilocytosis; LGL, large granular lymphocyte; MDS, myelodysplastic syndrome; MPN,

myeloproliferative neoplasm; PMF, primary myelofi brosis; PV, polycythemia vera.

QUALITATIVE ABNORMALITIES

Platelets

Genetic Acquired

Gray plateletsyndrome

Paris-Trousseausyndrome

Neoplasm May–Hegglin, other MYH9

Chediak–Higashi

Alder–Reilly

Pelger–Huet

Acquired

Red blood cells

AML, M7

MPN

PV

ET

Genetic Acquired

White blood cells

Infection

Anaplasma, Ehrlichia

Bacteria

G-CSF, GM-CSF

Tay–Sachs

Neoplasm

CML

CMMLCTCL (Sezary)

AML, M3

Mast cellleukemia

CLL

Plasma cell leukemia

Leukemic phase,follicular lymphoma

Fungi

Genetic

G6PD deficiency

Hereditaryspherocytosis

Nutritionaldeficiency

Hb SS, SC

Hb CC

Hb S-β0

thalassemia

Hereditaryelliptocytosis

Iron

Folate

Vit.B12

AML, M6

Infections

Drugs/Toxins

LeadArsenic

*Filarialspecies

*Trypano-soma

species

Malaria

*B. recurrentis

Babesia

Hairy cell leukemia

LGL leukemia

May–Hegglin, other MYH9-

related disorders

MDS

IMF

Myelofibrosis

HPPK**

Prolymphocyticleukemia

Figure 2.4 Platelet satellitosis.

the staining process. The stain tube lines will need to be cleaned with methanol and the rinse lines checked for fl uid before a new slide is stained. A large number of smudge cells with nuclear remnants of lymphocytes on a smear may indicate the presence of fragile cells. These can be reduced by adding one drop of 22% albumin to fi ve drops of blood and making a new blood smear from this mixture. The pres-ence of red blood cells that appear too pink or blue on an initial scan indicates the need to verify the pH of the stain and/or the quantity of the buffer. Increased proteins can also cause the slide to have a bluish -purple appearance.

Review of the blood smear can result in a more rapid and accurate diagnosis of a variety of hematologic and other disorders [7,44,47–49]. These include malignancy as well as infectious, congenital, and acquired disorders. Specifi c fi nd-


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Table 2.4 Reasons for slide review based on results from an automated analyzer.

a Automated CBC.

Parameter Result In combination with:

Neonate First sample —

WBC <4.0 or >30.0/μL First time

WBC <4.0 or >30.0 Delta check failure (within 3 days)

Platelet count <100 or >1 × 109/L First time

Platelet count Any value Delta check failure

Hemoglobin <7g/dL or >2g/dL above

reference range for age, sex

First time

MCV <75fL or >105fL (adult) First time (if specimen is <24 hours old)

RDW >22 First time

b White cell differential and reticulocytes.

Parameter Result ( ×109/L) In combination with:

No differential or incomplete differential Any —

Neutrophil # <1.0 or >20.0 First time

Lymphocyte # >5.0 (adult) or <7.0 ( <12 years old) First time

Monocyte # >1.5 (adult) or <3.0 ( <12 years old) First time

Eosinophil # >2.0 First time

Basophil # >0.5 First time

NRBC # Any value First time

Absolute reticulocyte # >0.100 First time

c Suspect fl ags.

Parameter Result In combination with:

Red cell fragments Flag + —

Dimorphic red cells Flag + First time

Platelet fl ags Platelet & MPV fl ags —

Immature granulocyte fl ag Flag + First time

Immature granulocyte fl ag Flag + Previous confi rmed result and delta failure for WBC

Atypical/variant lymphocytes Flag + First time

Atypical/variant lymphocytes Flag + Previous confi rmed result and delta failure for WBC

Blast fl ag Flag + First time

Blast fl ag Flag + Previous confi rmed result and delta failure for WBC

NRBC fl ag Flag + —

Extensively modifi ed, but consistent with International Society for Laboratory Hematology (ISLH) -endorsed international consensus recommendations

[26].

MCV, mean cell volume; NRBC, nucleated red blood cell; RDW, red cell distribution width; WBC, white blood cell count.

#, absolute number.


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Figure 2.5 Wuchereria bancrofti. Figure 2.6 β-thalassemia major ( β0).

ings in the peripheral blood smear can tailor or truncate further testing. For example, the detection of red cell agglu-tination in a patient with a previously undiagnosed anemia might suggest that further studies should be done for an autoimmune hemolytic process. If the red blood cell fi nding were rouleaux instead, further studies for a plasma cell dys-crasia, such as myeloma, would be indicated. The fi nding of fragmented red blood cells could imply a microangiopathic process, a severe megaloblastic anemia, or a more benign disorder.

Peripheral blood smear fi ndings may yield an unequivocal diagnosis that dictates the therapeutic option. For example, the classifi cation of a parasitemia as Plasmodium infection instead of Babesia infection, or Wuchereria bancrofti (Figure 2.5) instead of Loa loa or Mansonella perstans infection would permit appropriate therapy to be initiated. In other cases, a diagnosis can be suggested but additional confi rmatory studies are warranted. For example, the presence of nucle-ated red blood cells (NRBCs) in an adult could indicate any of the following: recent signifi cant blood loss; a hemolytic process, either congenital such as thalassemia (Figure 2.6)or acquired such as severe thermal injury; myelodysplasia; or acute erythroid leukemia. Basophilic stippling of red cells indicates disordered erythropoiesis, which could also indi-cate any of the medical conditions listed for NRBCs. In a child, however, the possibility of lead poisoning should also be considered. Table 2.5 summarizes the possible diagnostic utilities of review of the blood smear.

Limitations of the blood smear

If Table 2.5 represents the reasons to review the blood smear, then what are the limitations of peripheral blood smear review? The major limitation is that identifying an abnormal-ity is not necessarily equivalent to making a diagnosis. Table 2.6 elucidates on this concept. For example, two of the cat-

Table 2.5 Reasons for review of a blood smear.

QuantitativeAssess accuracy of platelet count

Enumerate or confi rm leukocyte populations if analyzer -generated

differentials are unavailable or invalid

Verify accuracy of analyzer results if spurious results are suspected (fl ags)

QualitativeDiagnose hematologic malignancy: acute or chronic leukemia

Diagnose hematologic stem cell disorder

Chronic myeloproliferative neoplasm

Myelodysplasia, primary or secondary (therapy -related)

Diagnose hereditary leukocyte disorder (e.g. Pelger –Huet anomaly;

May–Hegglin anomaly)

Both qualitative and quantitativeEvaluate cytopenia(s)

Acquired anemia (e.g. hemolysis, liver disease, and combined anemias)

Acquired thrombocytopenia (e.g. schistocytes in DIC or TTP)

Plasma cell dyscrasia

Inherited platelet disorder (e.g. gray platelet syndrome)

Differential diagnosis of macrocytic anemias

Evaluate hereditary hemolytic disordersHemoglobinopathies, thalassemias

Enzyme defects (e.g. oxidative stress hemolysis in G6PD defi ciency)

Membrane defects (e.g. hereditary elliptocytosis and hereditary

spherocytosis)

Evaluate presence of infectious agents (e.g. malaria, Babesia,fungi)

Classify lymphoproliferative disorders (infectious versus neoplastic)

Modifi ed from Peterson P et al. [51], with permission from Carden

Jennings Publishing.

DIC, disseminated intravascular coagulation; G6PD, glucose -6-

phosphate dehydrogenase; TTP, thrombotic thrombocytopenic

purpura.


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Figure 2.7 Glucose-6-phosphate defi ciency —hemolysis with

characteristic bite cells.

Table 2.6 Limitations of peripheral smear review.

Quantitative—may not determine the:Etiology of a decreased or increased platelet count

Etiology of a decreased or increased RBC or Hb

Etiology of a decreased or increased WBC

Qualitative—may not classify a:Leukemia (e.g. myeloid versus lymphoid)

Hematologic stem cell disorder

Chronic myeloproliferative neoplasm (e.g. polycythemia vera versus

essential thrombocythemia)

Myelodysplasia, primary versus secondary (therapy -related)

Both qualitative and quantitative —may not establish the:Etiology of some acquired anemias

Etiology of rouleaux or background protein in a plasma cell dyscrasia

Etiology of dyspoiesis of any cell line

Pathogenesis of a granulocytosis (reactive versus neoplastic)

Pathogenesis of a lymphocytosis (reactive versus neoplastic)

Evaluate hereditary hemolytic disorders —may not be able to categorize:

A hemoglobinopathy or a thalassemia

An enzyme defect

A membrane defect

Evaluate presence of infectious agents —may not be able to:Speciate malaria with certainty

RBC, red blood cell count; WBC, white blood cell count.

egories of hematologic malignancies listed as qualitative (leukemias and stem cell disorders) both require additional and far more sophisticated laboratory studies for an une-quivocal and accurate diagnosis. It could be hazardous to classify an acute leukemia on the basis of a peripheral blood smear alone. In the category of both qualitative and quan-titative abnormalities, the limitation of evaluating cytopenias is identical to that stated above for malignancies and stem cell disorders (myeloma, paroxysmal nocturnal hemoglob-inuria). The same applies to the hereditary hemolytic disor-ders. Although there are hemoglobinopathies in which the peripheral blood smear fi ndings are nearly diagnostic, such as hemoglobin SS [50] and hemoglobin CC, any such fi nding necessitates further confi rmatory laboratory study and family studies. A similar logic follows for potential red cell enzyme (Figure 2.7) and membrane defects detected on smear review.

Clinical utility of the blood smear

With these limitations in mind, review of the blood smear is an essential medical step in suggesting or even making a diag-nosis. Table 2.7 presents a medically, results -oriented way of

summarizing the rules listed in Table 2.4. An intelligent and thorough examination of the blood smear will be suffi cient to correctly diagnose some disorders. Inherited leukocyte and platelet disorders largely fall into this category.

The relevant leukocyte disorders include the Pelger –Huet[51] and Alder –Reilly anomalies and other mucopolysac-charidoses [52], and the Chediak –Higashi anomaly (Figure 2.8) [53]. The platelet disorders include the gray platelet (α-storage pool disease) [54] and Paris -Trousseau (del 11q23) syndromes [55,56], and the May –Hegglin family of 22q11 disorders [57]. Paris -Trousseau syndrome and its vari-ants (Jacobsen syndrome) are dysmegakaryopoietic throm-bocytopenias characterized by fused α−granules, which in the peripheral blood smear appear as enlarged platelets that contain giant red α-granules. The May –Hegglin anomaly is characterized by macrothrombocytopenia and inclusions in the leukocytes. The leukocyte defect consists of the presence of 2 –5-μm cytoplasmic inclusions (D öhle bodies). May –Hegglin and three other disorders that display the same genetic abnormality, a mutation that involves the gene encoding the nonmuscle myosin heavy chain IIA ( MYH9),together comprise the MYH9 -related disorders. The other disorders are the Sebastian and Fechtner syndromes, and the Epstein syndrome, which is characterized by macrothrom-bocytopenia without leukocyte inclusions.

Hematologic malignancies are often initially diagnosed on the blood smear. These disorders generally require further testing, which includes bone marrow aspiration and biopsy, immunophenotyping, cytogenetic studies, fl uorescent in situhybridization, and proteomics. Even with the advent of digital microscopy and computerized image analysis [58],some of these leukemias have such unique morphologic features that they are instantly recognizable on the blood smear. These include the mature B -cell neoplasms chronic lymphocytic leukemia (CLL) [59], prolymphocytic leukemia (PLL) [60,61], hairy cell leukemia [62], and plasma cell leukemia (PCL) [63]. In addition, acute lymphoblastic leuke-


20

Figure 2.8 Chediak–Higashi syndrome.

Figure 2.9 AML, M3 —example of a faggot cell.

mia (FAB ALL L3) is morphologically identical to Burkitt leukemia/lymphoma [64]. The mature T -cell neoplasms that are morphologically distinct include large granular lym-phocytic leukemia [65,66], adult T -cell leukemia/lymphoma [67,68], S ézary syndrome [69], and the leukemic phase of follicular lymphoma [70,71]. The myeloid malignancies include acute promyelocytic leukemia and its variants (Figure 2.9) [72–74]; some cases of acute erythroid [72,75]and acute megakaryoblastic leukemia [76,77]; and some of the myeloproliferative neoplasms, which include poly-cythemia vera (PV), essential thrombocythemia (ET), and typical chronic myelogenous leukemia (CML) in chronic phase [78–82]. In addition, the myelodysplastic disorder typical chronic myelomonocytic leukemia (CMML) can sometimes be included here [78,79].

Similar diagnostic certainty can accompany some mor-phologic changes in red blood cells. The constellation of abundant and small target cells and ovoid -to-rectangular, polyhedral intra -erythrocytic crystals is unlikely to be present in anything other than homozygous hemoglobin C disease [83,84]. Similarly, the peculiar boat -shaped cells and clam-shaped intra -erythrocytic crystals observed in hemo-globin SC disease are not observed in any other hemoglob-inopathy or other red blood cell disorder [50,83]. The common nutritional defi ciencies of iron, folate, and vitamin B12 each display characteristic morphologic changes (Figure 2.10). The combined fi nding of pencil cells, prekeratocytes, and target cells is indicative of iron defi ciency anemia [11,85]. Heavy -metal poisoning may be detected on the peripheral blood smear because of changes in the red blood cells. Arsenic poisoning causes a hemolytic anemia that results in dehemoglobinization of red cells, leaving the cell membranes visible as ghosts [86]. Lead poisoning character-istically results in coarse basophilic stippling [14,87].

One of the clearest examples of the utility of peripheral blood smears is the diagnosis and quantitation of para-

Table 2.7 Medical indications for peripheral smear review.

Finding Example(s) of condition

First occurrence or observationAbnormal cells Blast; abnormal lymphoid cells;

lymphocytosis

Abnormal red cell indices/

morphology

Markedly decreased/increased

MCV

Increased RDW in an outpatient

NRBC; rouleaux; schistocytes;

spherocytes; teardrop cells

Suspicion of microorganisms

on blood smear

Malaria; Babesia; Borrelia;

Anaplasma(Ehrlichia);

Trypanosoma; Candida

Atypical or dysplastic cells

of any lineage

Pelger–Huet cells; giant

platelets

Inclusions in red blood cells Hemoglobin C or SC crystals;

Howell–Jolly bodies;

Pappenheimer bodies

Inclusions in platelets Paris-Trousseau syndrome

Intranuclear/intracytoplasmic

inclusions in leukocytes

Auer rod; Chediak –Higashi

syndrome; HIV; other

systemic infection; May –

Hegglin anomaly

Pancytopenia or marked

cytopenia

Acute leukemia; hairy cell

leukemia; Gray platelet

syndrome

Subsequent occurrence or observationPersistence of abnormal cells in

a patient under treatment for a

known hematologic/stem cell

malignancy

Residual acute leukemia

Persistence of infectious organisms

in a patient under treatment

Document level of parasitemia

Modifi ed from Peterson P et al. [51], with permission from Carden

Jennings Publishing.

MCV, mean cell volume; NRBC, nucleated red blood cell; RDW, red

cell distribution width.


21

Figure 2.10 Megaloblastic anemia —examples of a six -lobed

polymorphonuclear leukocyte and a Howell –Jolly body.

sitemias. Although newer serologic and polymerase chain reaction (PCR) tests are available for malaria, identifi cation of the parasite and its speciation depend on recognizing the intra-erythrocytic organisms and noting their morphologic hallmarks. Not only is this critical for speciation, but it is also important to distinguish Plasmodium from Babesia species (Figure 2.11) [88,89]. Other blood parasitemias, which include fi lariasis ( Wuchereria species [Figure 2.5], Loa loa,and Mansonella species) [90–92] and trypanosomiasis ( T. cruzi, T. brucei rhodiense, and T. gambiense) [93,94], can also be diagnosed on peripheral blood smears. Additionally, the diagnosis of relapsing fever depends on microscopic demon-stration of the spirochetes in the blood; Borrelia species are the only pathogenic human spirochetes that stain with Wright and Giemsa stains [95,96]. Similarly, the fi nding of membrane-bound clusters of bluish -purple bacteria within either granulocytes or monocytes is diagnostic of anaplas-mosis, which was formerly known as ehrlichiosis. These two

Figure 2.11 Babesia microti—examples of multiply infected red blood

cells, including one with a tetrad form.

Figure 2.12 Histoplasma capsulatum infection.

tick-borne diseases caused by Anaplasma phagocytophilum and Ehrlichia chafeensis are generally classifi ed as Rickettsiaceae[97,98], although this classifi cation is in fl ux. Lastly, in over-whelming septic states bacteria or fungi (Figure 2.12) may be detected on the blood smear [32,33,99,100] although reportedly the level of fungemia must be high [101].

Defi nitive diagnoses for devotees of the blood smear

Figure 2.2 represents those disorders that result in abnormal numerical results from an automated analyzer and can potentially be diagnosed on a peripheral blood smear (Figures 2.4, 2.6, 2.7, 2.10, 2.12, 2.13). Similarly, Figure 2.3shows the disorders resulting from a genetic or acquired abnormality that can also potentially be diagnosed on a blood smear (Figures 2.5, 2.7–2.13).Of course, the caveat is: not in every case. Figure 2.2 includes disorders that can be initially detected by increases or decreases in the cell count results—thrombocytopenia or thrombocytosis, leukopenia or leukocytosis, anemia or erythrocytosis. Some of the dis-orders listed in these fi gures have been discussed previously because they may cause spurious results (Figure 2.1).

Disseminated infection can lead to thrombocytosis, leuko-cytosis, or leukopenia. Some infectious agents that cause septicemia can lead to a thrombocytosis that is spurious: bacteria and fungi, especially Candida species, can mimic platelets in size [32,33,99,100]; the platelet histograms, however, are usually abnormal. When present in high con-centrations in the blood, some Candida species, including C.albicans, can cause marked spurious leukocytosis. A leuko-penia due to bacteremia or fungemia is likely to be a patho-logic fi nding that indicates overwhelming life -threateninginfection. Some of the infections that cause lymphopenia may be morphologically unique; these include retroviral


22

CHAPTER 2 Morphologic Analysis of peripheral Bood

Acknowledgments

The authors gratefully acknowledge the contribution of Ms. Eman Al -Hadi to Figures 2.1–2.3.

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Despite the advent of optical and electronic cell counters for enumeration; immunophenotyping for characterization; immunocytochemistry for localization; computerized image analysis; and advanced techniques for the detection and identifi cation of molecular abnormalities, the peripheral blood smear remains a unique diagnostic tool in clinical laboratory hematology. As numerous experts have stated, the blood smear remains a crucial diagnostic aid [9,10,17,39].An accurate and insightful analysis of the blood fi lm is dependent on having technically superior, well -stained slides available for review. This occurs when sample collection is correct and timely, when pre -analytic variables that affect the quality of the blood smear are controlled for or elimi-nated, and when staining artifacts are absent. The results of an automated analysis, the CBC, often determine whether a peripheral blood smear is examined. Although not every blood smear examined will result in an unequivocal diagno-sis, the reason for a spurious result may become obvious. Despite limitations, in the right setting the blood smear has unquestionable clinical utility. An observant, knowledgeable microscopist will often be able to eliminate some diagnoses and suggest more likely diagnoses. At other times a skilled microscopist can render a defi nitive diagnosis.

Figure 2.13 Bordetella pertussis infection.


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