17 laboratory hematology

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Chapter 17

Laboratory hematologyGeorgette A. Dent, Jay H. Herman and Jamie E. Siegel

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is 1 of 2 test modules. To decide which test module is right for you or tochapters, each dedicated to a specific area of hematology. Also included

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A publication of the American Society of Hematology

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General concepts

Laboratory tests are ordered and interpreted within thecontext of a specic patient as routine screening during aperiodic examination, in the setting of an illness for diagno-sis or follow-up, or for preoperative certication of normal.Clinical judgment is applied in both the selection of tests andin their interpretation, and unexpected results should beconsidered within the framework of the patient and the dis-ease. Some unexpected results may require conrmation,particularly if the integrity of the specimen cannot be insured(eg, heparin contamination or delayed processing). Also, anylaboratory result can be incorrect for a variety of reasons,from mislabeling to technical problems with an assay.

Terminology

Sensitivity is the ability of a test to detect a true abnormality;as the sensitivity of a test is increased, the risk of a false-positive result increases (increasing sensitivity comes atthe cost of decreasing specicity). Very sensitive tests arehelpful in ruling out a disease when the test is negative.

Specicity is the ability of a test to detect a normal result if theabnormality is not present; maximum specicity may leadto too many false-negative results. As a rule, specic testsare useful for ruling in a disease when the test is positive.

Precision is reproducibility of a value during repeated testingof a sample.

Accuracy is the ability of a test to obtain the assigned value of anexternal standard (run as though it were a clinical sample).

Predictive value is the likelihood that an abnormal test indi-cates a patient with the abnormality ( positive predictivevalue), or the likelihood that a normal test indicates apatient without the abnormality ( negative predictive value).Positive and negative predictive values depend on the

frequency of the abnormality being sought in the popula-

tion as well as the factors that produce false-positiveresults. The tests for rare abnormalities require higher sen-sitivity and specicity to have high predictive values thando the tests for common abnormalities.

Reference ranges represent collected statistical data from awell population. Reference ranges usually represent datafor 90% or 95% of the well population.

Specic laboratory testing

Automated cell counting

Automated cell counting provides the best means of countinglarge numbers of cells while minimizing statistical error. The2 major types of cell counters are the aperture-impedancecounters and the optical method counters.

Aperture-impedance counters

Cells are counted and their size estimated by measuring changein electrical resistance as cells in solution ow through a nar-row aperture across which a direct current (DC) is maintained.Because the diluent in which cells are suspended is moreelectrically conductive than are the cells, when the cells passthrough the orice there is a decrease in electrical conduc-tance. The drop in voltage is proportional to cell size, allowingthe average cell size to be determined. Different blood ele-ments can be counted by using size limitation windows.

The accuracy of the aperture-impedance counters isdependent on even cell suspensions; distortion occurs whencells do not pass through the center of the aperture or whenmultiple cells pass at the same time.

Coulter (Hialeah, FL), Sysmex (Baxter Diagnostics,Waukegan, IL), and some Cell-Dyn instruments (AbbottDiagnostics, Santa Clara, CA) use this technology.

Laboratory hematologyGeorgette A. Dent, Jay H. Herman, and Jamie E. Siegel

General concepts, 444 Terminology, 444

Specic laboratory testing, 444 Bibliography, 464

CHAPTER

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Specic laboratory testing | 44

evaluation of anemia. The RDW is more frequently elevatedin iron deciency anemia than in thalassemia or anemia ofchronic disease.

Leukocyte analysisThe automated 5-part differential (neutrophils, monocytes,eosinophils, basophils, lymphocytes) can be obtained usingseveral methods. Technicon instruments use a combina-tion of light scatter and peroxidase staining. Coulter instru-ments use electrical impedance, light scatter, and electricalconductivity. Sysmex uses differential resistance to lysis ofeosinophils and basophils in specic detergents at differenttemperatures. Cell-Dyn uses patterns of light scatter at4 different angles.

Specimens are frequently agged by the instruments for

morphologic review. Morphologic review is indicated in cer-tain clinical circumstances regardless of instrument agging;for example, circulating lymphoma cells and atypical lym-phocytes are occasionally not reported or detected by theinstruments.

Platelet analysis

Automated counters provide both a platelet count and amean platelet volume (MPV). The MPV has an inverse rela-tionship with platelet number because platelets tend to belarger in patients with destructive thrombocytopenia. The

MPV tends to be increased in hyperthyroidism and myelo-proliferative disorders and decreased with megakaryocytichypoplasia.

Erythrocyte fragments can falsely increase the plateletcount. The presence of platelet clumping secondary to ethylene-diaminetetraacetic acid (EDTA)-related platelet antibodiescan falsely decrease the platelet count. If EDTA platelet anti-bodies are suspected as the cause of thrombocytopenia, thediagnosis can be conrmed by showing a normal plateletcount on a citrated sample. In patients with EDTA anti bodieswho are inaccurately reported to have thrombocytopenia,the blood smear obtained from the EDTA-anticoagulatedblood may show thrombocytopenia, though platelet clumpsare typically also seen. The smear made directly from a n-gerstick should be normal.

Examination of the blood smear for platelet numbers,morphology, and size variation can be very helpful in con-rming the automated count and directing further testing;for example, patients with BernardSoulier syndrome usu-ally have more uniformly large platelet population thanpatients with immune thrombocytopenic purpura (ITP).Phase microscopy can also be used to conrm plateletnumbers.

Optical method counters

This method takes advantage of the light scattering proper-ties of blood cells. A ow cytometer is used to produce a nestream of cells in suspension; the cells are then run in singlele across the path of a unifocal laser. The amount of lightscattered at a low angle from the incident light path dependson cell size. The amount of light scattered at a wide angledepends on factors such as cytoplasmic granules and nuclearshape. The pattern of light scattering is used to determinecell size, volume, shape, and complexity.

Technicon (Bayer Diagnostics, Kent, WA) and someCell-Dyn instruments (Abbott Diagnostics, Santa Clara, CA)use this technology.

Erythrocyte analysis

Automated instruments measure the number of cells perunit volume, cell size, and hemoglobin concentration; allother parameters are calculated. Hemoglobin concentrationis expressed in grams per deciliter (g/dL). Hemoglobin andits other forms, oxyhemoglobin and carboxyhemoglobin, areconverted by potassium ferrocyanide to cyanmethemoglobinto determine the amount of total hemoglobin. The absor-bance of the product, cyanmethemoglobin, is measured by aspectrophotometer at 540 nm. An increase in the measuredhemoglobin concentration from increased sample turbiditycan be an artifact of improperly lysed red cells, leukocytosis,paraproteinemia, and hyperlipidemia. Automated hemato-

crit is a calculated value that is obtained by multiplying thered cell volume by the red cell number (hematocrit red cellvolume red cell number).

The mean corpuscular volume (MCV) is expressed infemtoliters (10 15 L) and is measured directly by the auto-mated instrument. Agglutination of red cells, resulting inmeasurement of more than one cell at a time, and hyper-glycemia, causing osmotic swelling of the red cell, can arti-factually elevate the MCV.

The mean corpuscular hemoglobin (MCH) is a calculatedvalue; it is expressed in picograms (10 12 g). The MCH isobtained by dividing the hemoglobin concentration bythe red cell count (MCH hemoglobin [g/L]/red cell count[ 1012/L]). An elevated MCH can be an artifact of increasedplasma turbidity. The mean corpuscular hemoglobin con-centration (MCHC) is a calculated value; it is expressed ingrams of hemoglobin per deciliter of packed red blood cells.The MCHC is obtained by dividing the hemoglobin concen-tration by the hematocrit (MCHC hemoglobin [g/dL]/hematocrit L/L). Any artifact impacting hematocrit or hemo-globin determinations can alter the MCHC.

The red cell distribution width (RDW) is the coefcient ofvariation of red cell size distribution. The RDW is used in the

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| Laboratory hematology446

Reticulocyte analysis

New methylene blue is used to stain residual RNA andaggregates the RNA, making it easily visible for manual reti-culocyte counting. Automated reticulocyte counting useseither methylene blue or uorescent RNA-binding dyes.Leukocytes, large platelets, or platelet aggregates need to beexcluded because these cells will also contain RNA.

Peripheral blood morphology

The best quality peripheral blood smears are obtained fromngerstick procedures. Blood smears are stained with eitherthe Wright or the MayGrunwaldGiemsa stains.

With the advent of automatic cell counters performing5-part differentials, peripheral blood morphology needs tobe performed in only about 30% of specimens in a general

hospital or clinic population.The optimal area to examine the peripheral blood smear isthe transitional area between the thick part of the smear andthe feathered edge (Table 17-1). In the transitional area, thereare only a few overlapping red cells, and central pallor of

normal red cells is evident. Abnormal distribution of largercells should be excluded by examination of the edges of thesmear.

Supravital stains are used to detect red cell inclusions.Crystal violet detects denatured hemoglobin inclusions

(Heinz bodies); brilliant cresyl blue is used to precipitate anddetect unstable hemoglobins.

Erythrocyte sedimentation rate

The erythrocyte sedimentation rate (ESR) is measured byallowing red blood cells in a capillary tube to settle over time.Time to sedimentation is measured in millimeters per hourby either the Westergren or Wintrobe methods; both meth-ods are affected by the red cell count of the specimen. ESRincreases with age and tends to be more elevated in women

than in men. Increases in brinogen, immunoglobulins,acute phase proteins, and anemia will result in more rapidsettlement of the red blood cells and will elevate the ESR.Sickle cells are unable to rouleaux and will not settle rapidly;this will decrease the ESR.

Table 17-1 Red cell abnormalities.*

Abnormality Description Cause Disease association

Acanthocytes (spur cells) Irregularly spiculated red cell Altered membrane lipids Liver disease, abetalipoproteinemia,postsplenectomy

Basophilic stippling Punctate basophilic inclusions Precipitated ribosomes Lead toxicity, thalassemiasBite cells (degmacyte) Smooth semicircle taken from

one edgeHeinz body pitting by spleen G6PD deciency, drug-induced oxidant

hemolysisBurr cells (echinocytes) Short, evenly spaced spicules May be related to abnormal

membrane lipidsUsually artifactual, uremia, bleeding

ulcers, gastric carcinomaCabot ring Circular, blue, threadlike inclusion

with dotsNuclear remnant Postsplenectomy, hemolytic anemia,

megaloblastic anemiaHowellJolly bodies Small, discrete basophilic dense

inclusions; usually singleNuclear remnant Postsplenectomy, hemolytic anemia,

megaloblastic anemiaPappenheimer bodies Small dense basophilic granules Iron-containing siderosomes or

mitochondrial remnantSideroblastic anemia, postsplenectomy

Schistocytes (helmet cells) Distorted, fragmented cell, with 23pointed ends

Mechanical distortion in themicrovasculature by brinstrands; damage by mechanicalheart valves

Microangiopathic hemolytic anemia,prosthetic heart valves, severe burns

Spherocytes Spherical cell with dense appearanceand absent central pallor; usuallydecreased diameter

Decreased membrane redundancy Hereditary spherocytosis,immunohemolytic anemia

Stomatocytes Mouth- or cuplike deformity Membrane defect with abnormalcation permeability

Hereditary stomatocytosis,immunohemolytic anemia

Target cell (codocyte) Targetlike appearance, oftenhypochromic

Increased redundancy of cellmembrane

Liver disease, postsplenectomy,thalassemia, HbC

Teardrop cell (dacryocyte) Distorted, drop-shaped cell Myelobrosis, myelophthisic anemia

*Blood smear abnormalities can be artifacts of poor slide preparation or viewing the wrong part of the smear.Modied from Kjedsberg C et al. Practical Diagnosis of Hematologic Disorders. 2nd ed. Chicago: ASCP Press; 1995.G6PD glucose-6-phosphate dehydrogenase; HbC hemoglobin C.



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ESR is not an appropriate screening test in asymptomaticpatients; it is used to follow the course of disease in patientswith lymphomas and collagen vascular diseases.

Leukocyte alkaline phosphatase score

The leukocyte alkaline phosphatase (LAP) enzyme is in thegranules of mature neutrophils. This enzyme reacts with anadded substrate, resulting in a colored precipitate. The scor-ing of the stain intensity and granulation is called the LAPscore and is determined by counting the staining activity ona scale of 04 in 100 neutrophils. Normal LAP scores rangefrom 15 to 130. LAP scores should be performed on freshcapillary blood or samples anticoagulated with heparinbecause there is rapid loss of alkaline phosphatase activity insamples drawn in EDTA.

The LAP score is decreased in chronic myeloid leukemia,paroxysmal nocturnal hemoglobinuria, and, in some patients,myelodysplasia. The LAP score is increased by infection,inammation, polycythemia vera, agnogenic myeloid meta-plasia, pregnancy, estrogens, oral contraceptives, granulocytecolony-stimulating factor (G-CSF), granulocytemacro-phage-colony stimulating factor (GM-CSF), lithium, andcorticosteroids.

Bone marrow aspirate and biopsy

Bone marrow aspirate and biopsy are commonly performedin adults by collecting specimens from the anterior or poste-rior iliac crests. The posterior crest is usually assessed in chil-dren. Bone marrow aspirates can also be obtained from thesternum. In newborn and young infants, marrow aspirates

are often obtained from the anterior tibia. Good qualitysmears require adequate spicule harvesting because peri-spicular areas are the most representative areas to examine.

Hemophilia and other coagulation factor deciencies oranticoagulation are contraindications to marrow aspirations

and biopsies; thrombocytopenia is not a contraindication.The bone marrow aspirate and touch prep are stained with

either the Wright or MayGrunwaldGiemsa stains; unstainedsmears should be retained and kept frozen in a dry environ-ment for possible special stains. The bone marrow aspirate isused for cytologic examination of the bone marrow cells andfor performing the marrow differential. Bone marrow aspi-rate clot and core biopsies are xed in formalin or in a coagu-lative xative, and the biopsy specimen is decalcied andembedded in parafn; 34- m sections are then cut andstained with hematoxylin and eosin or Giemsa stains.

Plastic embedding does not require decalcication, andenzymatic activity is better preserved, thereby allowing cyto-chemical staining of the biopsy; thin cuts of 2 m can beobtained. Because of the technical expertise required andprolonged processing time, plastic embedding is less fre-quently performed.

When examining pediatric marrows, it is understood thaterythroid hyperplasia is present at birth because of high lev-els of erythropoietin. Lymphocytes may compose 40% of themarrow cellularity in children 4 years of age, and eosino-phils are present in higher numbers than in adults.

Perls or Prussian blue reactions are used to identify ferritinand hemosiderin in nucleated red cells (sideroblastic iron) andhistiocytes (reticuloendothelial iron). Siderocytes containingone or more blue-staining granules account for 2050% of thecells. (See Table 17-2 for other cytochemical stains.)

Table 17-2 Cytochemical stains.

Cytochemical stain Substrate and staining cells

Myeloperoxidase Primary granules of neutrophils and secondary granules of eosinophils. Monocytic lysosomalgranules stain faintly

Sudan black B Stains intracellular phospholipids and other lipids. Pattern of staining is similar tomyeloperoxidase.

Naphthol AS-D chloroacetate esterase(specic esterase)

Granulocytes stain; monocytes do not stain. Can be used in biopsies to stain granulocytes andmast cells

-Naphthyl butyrate (nonspecic esterase) Stains monocytes, macrophages, and histiocytes. Does not stain neutrophils-Naphthyl acetate (nonspecic esterase) Megakaryocytes stain with -naphthyl acetate but not -naphthyl butyrate

Terminal deoxynucleotidyl transferase (TDT) Intranuclear enzyme. Stains thymocytes and lymphoblasts. Some myeloblasts stain positively Tartrate-resistant acid phosphatase (TRAP) Stains an acid phosphatase isoenzyme. Positive staining in hairy cell leukemia, Gaucher cells,

activated T lymphocytesPeriodic acidSchiff (PAS) Detects intracellular glycogen and neutral mucosubstances. Positive staining in acute

lymphoblastic leukemia, acute myeloid leukemia, erythroleukemia, and Gaucher cellsToluidine blue Detects acid mucopolysaccharides. Positive in mast cells and basophilsTryptase Positive in mast cells, negative in basophils. Mast cells in systemic mast cell disease frequently

have a spindled shapeReticulin Reticulin bers stain black



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Ringed sideroblasts are dened as nucleated red cells withblue-staining iron granules surrounding at least two thirdsof the nucleus. These iron granules are present in mitochon-dria surrounding the nuclear membrane.

Iron staining of the biopsy can underestimate the marrow

iron stores because of the loss of iron during decalcication.

Immunocytochemical and immunohistochemical stains

A large array of specic antibodies detected by enzymaticformation of a colored product linked to the antigen antibody complex are now available for use on blood smears,marrow aspirates, and bone marrow biopsies or other tis-sues. Many cytochemical stains such as terminal deoxynucle-otidyl transferase (TDT), tartrate-resistant acid phosphatase(TRAP), and myeloperoxidase have been converted intoimmunocytochemical and immunohistochemical stains.

Immunocytochemical stains are used on marrow aspiratesand blood smears when ow cytometry specimens have notbeen collected or when ow cytometry results are confusing.The advantage of immunocytochemistry is the ability to cor-relate morphology with phenotype. Immunohistochemistrycan be used to phenotype undifferentiated tumors, lympho-proliferative disorders, and atypical lymphoid aggregates. Inpatients whose marrow cannot be aspirated (dry tap),immunohistochemistry can be performed on the biopsy sec-tion. Immunohistochemistry can also be used on sections oflymph nodes or other tissues when there is concern aboutlymphoma or some other neoplastic disease.

In addition to determining cell lineagefor example, dif-ferentiating hematopoietic neoplasms from nonhemato-poietic neoplasms or lymphoid processes from myeloiddisordersimmunohistochemistry can also be used to helpdetermine prognosis. Immunohistochemical stains can beused to delineate prognostic groups of diffuse large B-celllymphoma rst detected by gene microarray technology (seeChapter 1). Gene microarray technology has been used tosubdivide diffuse large B-cell lymphomas into germinal cen-ter B-cell (GCB) and nongerminal center B-cell (NGCB)types. Patients with the GCB phenotype have a better event-

free survival and overall survival than patients with the NGCBphenotype. Gene microarray technology is not currentlypractical for most clinical laboratories, but studies have shownthat differentiation of diffuse large B-cell lymphomas into theGCB and NGCB phenotypes can be done by immunohisto-chemistry using antibodies to CD10, BCL-6, and MUM1.

Preparation of bone marrow samples forancillary studies

Bone marrow collected in EDTA is adequate for both owcytometry and molecular analysis. Sodium heparin is suitable

for ow cytometry but may contaminate DNA preparationsand interfere with endonuclease digestion or the polymerasechain reaction (PCR). Bone marrow collected for cytogeneticstudies should be collected in a sterile tube containing tissueculture medium such as RPMI (containing fetal bovine

serum, L-glutamine, antibiotics) and anticoagulant.Bone marrow samples are stable at room temperature for

24 hours for ow cytometry, molecular analysis, and cyto-genetics. For cytogenetic studies, shorter storage periods arebetter. Marrow samples can be frozen in liquid nitrogen forow cytometry and molecular analysis. However, becauseow cytometry requires viable cells, freezing and thawing ofsamples may yield less than optimal results. Parafn- embeddedtissue can be used for PCR of genomic DNA sequences.Reverse transcription PCR (RT-PCR) assays require that RNApreparations be performed as early as possible to preventdigestion by ubiquitous nucleases; alternatively, the RNA canbe isolated from cell aliquots stored in frozen nitrogen.

Ancillary testingFlow cytometry

The most common applications of ow cytometry in hema-tology include the detection of cellular proteins usinguorescent-labeled monoclonal antibodies or assessing DNAcontent using DNA-binding dyes.

Flow cytometry is used for phenotyping populations ofcells, enumerating early progenitors for stem cell transplants,detecting minimal residual disease, detecting targets forimmunotherapy, and assessing the presence of prognosticmarkers. See Table 17-3 for a summary of clinical uses of owcytometry in ancillary studies. When immunophenotyping acellular population, the panel of antibodies used depends onthe cells being analyzed and the question being asked.

Gating is necessary to identify cells of interest in a mixedpopulation of cells. Three major leukocyte populations(lympho cytes, monocytes, and neutrophils) can be denedusing light scatter. Forward angle scatter (low angle; FS)measures cell size, and side light scatter (SS) measures inter-nal cellular granularity. Lymphocytes have the lowest FS and

SS signals, monocytes have intermediate FS and SS signals,and neutrophils have high SS and slightly lower FS signals.The most common method for gating different cell popu-

lations is by plotting right angle SS against CD45. Cells canbe separated based on the intensity of staining they displaywith the conjugated antibody that is classied as either brightor dim. Lymphocytes are bright CD45 and have a low SS sig-nal, neutrophils are dim to moderately bright CD45 and havea high SS signal, and monocytes are bright CD45 and have anintermediate SS.

Flow cytometry can also be used to detect populations ofnatural killer (NK) cells. NK cells express CD2, CD7, CD16,



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and CD56 and show variable expression of CD57 and CD8.NK cells do not express CD3, and the absence of CD3 expres-sion can be used to differentiate NK cells from T cells.

In addition to determining cell lineage, ow cytometrycan also be used to detect prognostic markers. For example,ow cytometric analysis of the tyrosine kinase, ZAP-70, canbe used to subdivide chronic lymphocytic leukemia (CLL)into prognostic groups. Positivity for ZAP-70 is highly cor-related with unmutated DNA, a feature of CLL arising frompregerminal center cells, and patients with pregerminal cen-ter CLL have a decreased overall survival when comparedwith patients with CLL arising from postgerminal centercells. Positivity for CD38 by ow cytometric analysis is alsocorrelated with unmutated DNA, but the correlation is notas strong as it is with ZAP-70.

Normal hematopoiesis

Uncommitted hematopoietic progenitors are CD34 and

CD38; expression of CD38 is evidence of lineage commit-ment. In myeloid differentiation, CD33 is one of the earliest

antigens to appear. CD33 is followed by CD13, which is thenfollowed by CD15 and CD11b. CD16 and CD10 are seen inlate maturation.

Appearance of CD71, loss of CD34 and CD33, anddecreased expression of CD45 characterizes erythroidmaturation. With further differentiation, CD71 expressiondecreases, glycophorin expression increases, and CD45disappears.

Megakaryocytic differentiation is indicated by the expres-sion of glycoprotein (GP) IIb (CD41). GPIIb-IIIa (CD61)

expression increases as CD34 expression decreases. GPIb(CD42b) is expressed at the promegakaryocyte stage.GPV (CD42d) expression increases with megakaryocytedifferentiation.

B- and T-cell precursors express TDT, HLA-DR, andCD34. B-cell differentiation is indicated by the expression ofCD19 followed by CD10. Surface immunoglobulin is com-posed of and light chains. A predominance of one type isknown as light chain restriction and is indicative of a mono-clonal process. CD34 and CD10 expression cease by the timeimmunoglobulin M (IgM) is expressed at the cell surface.Expression of surface IgM is associated with the expressionof mature B-lymphocyte markers (CD20, CD21, CD22,CD79b).

T-cell precursors express TDT, HLA-DR, and CD34. Dif-ferentiation is indicated by the expression of cytoplasmicCD3 and CD7, followed by the expression of CD2 and CD5.The common thymocyte also expresses CD1, CD4, and CD8.The mature helper/inducer lymphocyte expresses CD2, CD3,

CD4, and CD5 and may express CD7. The mature suppres-sor/cytotoxic T lymphocyte expresses CD2, CD3, CD4, CD5,and CD8 and may express CD7. (See Tables 17-4 through17-10 for useful CD markers.)

Cytogenetics

Cytogenetic analysis can be performed from cultured (indi-rect) and uncultured (direct) preparations. In the indirectassay, cells are grown so that mitotic forms can be visualizedand distinct chromosomal banding patterns can be assessed(conventional cytogenetics). Growing or culturing the cells

Table 17-3 Specimen allocation for ancillary studies.

Clinical problem Ancillary techniques

Pancytopenia Flow cytometry (LGL, hairy cell leukemia, PNH clone, AML)Cytogenetics (AML, MDS)Molecular genetics

Myeloid leukemia Flow cytometry (phenotyping)Cytogenetics and FISHMolecular genetics ( BCR-ABL, PML/RARA, AML1/ETO)

Lymphoproliferative disorder Flow cytometry (phenotyping, prognostic markers such as ZAP-70)Cytogenetics: t(1;19) in pre-B cell ALL, t(14;18) in follicular lymphomas, etcFISHMolecular genetics (clonality, specic markers such as BCL2, BCL6 , etc)Immunohistochemistry (phenotyping, prognostic markers)

Myeloproliferative disorders CytogeneticsFISH (BCR-ABL)Molecular genetics ( BCR-ABL, JAK2)

Plasmaproliferative disorders Flow cytometry (phenotyping, labeling index)

Cytogenetics

ALL acute lymphoblastic leukemia; AML acute myelogenous leukemia; CLL chronic lymphocytic leukemia; FISH uorescencein situ hybridization; LGL large granular lymphocyte leukemia; MDS myelodysplasia; PNH paroxysmal nocturnal hemoglobinuria.



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increases the mitotic rate and improves chromosome mor-phology. Mitogens may be useful in improving the yield ofkaryotyping abnormal cells and are particularly useful whenanalyzing mature B- or T-cell processes.

Cytogenetically, a minimum of 2 mitotic cells with gain ofthe same chromosome or with the same structural abnor-mality or 3 mitotic cells with loss of the same chromosome

dene a clone.

Constitutional chromosome abnormalities, associatedwith either congenital genetic syndromes or normal variants,are determined on peripheral blood T lymphocytes grown inculture with phytohemagglutinin (PHA), a T-cell mitogen.

Fluorescence in situ hybridization (FISH) employs specicuorescently labeled DNA probes to identify each chromo-somal segment. FISH can be performed using either cultured

or uncultured preparations. In the uncultured technique,

Table 17-4 Clinically useful CD markers.

Marker Lineage association

Progenitor cellsCD34 Progenitor cells, endotheliumCD38 Myeloid progenitors, T, B, NK cells, plasma cells, monocytes, CLL subset

B-cell markersCD10 Pre-B lymphocytes, germinal center cells, neutrophilsCD19 B cells (not plasma cells or follicular dendritic cells)CD20 B cells (not plasma cells)CD21 Mature B cells, follicular dendritic cells, subset of thymocytesCD22 Mature B cells, mantle zone cells (not germinal center cells)CD23 B cells, CLLCD79b B cells (not typical CLL)CD103 Intraepithelial lymphocytes, hairy cell leukemia, T cells in enteropathic T-cell lymphomaFMC7 B cells (not typical CLL), hairy cell leukemia

T-cell markersCD2 Pro- and pre-T cells, T cells, thymocytes, NK cells, some lymphocytes in CLL and B-ALLCD3 Thymocytes, mature T cells, cytoplasm of immature T cellsCD5 Thymocytes, T cells, B cells in CLL, B cells in mantle cell lymphomaCD4 Helper T cells, monocytes, dendritic cells, activated eosinophils, thymocytesCD7 Pro- and pre-T cells, T cells, thymocytes, NK cells, some myeloblastsCD8 Suppressor T cells, NK cells, thymocytesCD25 Activated T and B cells, adult T-cell leukemia/lymphoma

NK/cytotoxic T-cell markersCD16 NK cells, monocytes, macrophages, neutrophilsCD56 NK cells, myeloma cellsCD57 NK cells, T-cell subset

Myeloid and monocytic markersCD13 Monocytes, neutrophils, eosinophils, and basophilsCD14 Monocytes, macrophages, subset of granulocytes

CD33 Myeloid lineage cells and monocytesCD117 Immature myeloid cells, AML

MonocytesCD11c Monocytes, macrophages, granulocytes, activated B and T cells, NK cells, hairy cell leukemiaCD15 Myeloid lineage cells and monocytesCD64 Monocytes, immature myeloid cells, activated neutrophils

Megakaryocytic markersCD41 Platelets and megakaryocytes (GPIIb)CD42 Platelets and megakaryocytes (CD42a: GPI; CD42b: GPIb)CD61 Platelets, megakaryocytes, endothelial cells (GPIIb-IIIa)

Erythroid markersCD71 Transferrin receptor is up-regulated upon cell activationCD235a Glycophorin A

AML acute myelogenous leukemia; B-ALL B-lineage acute lymphoblastic leukemia; CLL chronic lymphocytic leukemia;GP glycoprotein; NK natural killer.



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Table 17-5 Acute myeloid leukemia phenotyping.

HLA-DR CD34 CD33 CD13 CD11c CD14 CD41 CD235a

M0 / / M1 / / M2 / / / /

M3 / M4 / M5 M6 / / M7 / / /

Table 17-6 B-lineage acute lymphoblastic leukemia phenotyping.

TDT CD19 CD10 CD20 Cyto- Surface Ig

Pro-B Pre-Pre-B (Common ALL) Pre-B /

Early B (Burkitt)

Cyto- ; cytoplasmic ; Ig immunoglobulin; TDT terminal deoxynucleotidyl transferase.

Table 17-7 T-lineage acute lymphoblastic leukemia phenotyping.

Surface TDT CD7 CD2 CD5 CD1 CD3 Cy CD3 CD4/CD8

Prothymocyte /Immature thymocyte /Common thymocyte / /Mature thymocyte CD4 or CD8Mature T cell CD4 or CD8

Cy CD3 cytoplasmic CD3; TDT terminal deoxynucleotidyl transferase.

Table 17-8 Common B-cell neoplasms.

CD20 CD5 CD10 CD23 CD43 CIg SIg Cyclin D1 Other

CLL/SLL 5% LPL / PLL / HCL / CD11c , CD25 , CD103MCL MZL / / FCL 60% BCL2LCL 10% 2550% / / / BCL2 in 3040%

Burkitt Myeloma Occ 15 20% CD56 , CD38 , CD138

CIg cytoplasmic immunoglobulin; CLL chronic lymphocytic leukemia; FCL follicular center cell lymphoma; HCL hairy cell leukemia;LCL large cell lymphoma; LPL lymphoplasmacytic lymphoma; MCL mantle cell lymphoma; MZL marginal zone lymphoma;PLL B-cell prolymphocytic leukemia; SIg surface immunoglobulin; SLL small lymphocytic lymphoma.

the assay is performed using nuclear DNA from interphasecells that are afxed to a microscope slide. FISH can beaccomplished with bone marrow or peripheral blood smears,or xed and sectioned tissues.

Hybridization of centromere-specic probes is used to

detect monosomy, trisomy, and other aneuploidies.

Chromosome-specic libraries, which paint the chromo-somes, are useful in identifying marker chromosomes orstructural rearrangements, such as translocations. Transloca-tions and deletions can also be identied in interphase ormetaphase by using genomic probes that are derived from

the breakpoints or recurring translocations or within the



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deleted segment. Multiplex FISH (spectral karyotyping)consists of simultaneously painting all chromosomes in a cellusing different colors for each chromosome.

Cytogenetics is particularly useful in the subclassicationof acute myeloid leukemias and in conrming the diagnosisand prognosis of B-cell neoplasias. Chronic lymphocytic leu-kemia, acute leukemias, B-cell lymphomas, and multiplemyeloma all have cytogenetic abnormalities that can bedetected using either conventional cytogenetics or FISH.

Molecular diagnosticsSouthern blotting

Southern blot analysis begins with the extraction of high-molecular-weight (HMW) DNA, followed by its digestion byrestriction enzymes. The digested DNA fragments are elec-trophoresed through a gel to separate the fragments by size.The separated fragments are then blotted onto a piece of l-ter paper, and the lter is soaked in a solution containinglabeled single-stranded DNA probes. The probe hybridizesto the spot on the paper where its complementary strand isfound. Unbound DNA is washed away, the lter is exposed tophotographic lm, and the lm reveals the position and sizeof the electrophoresed fragments.

This technique is slow and has been largely replaced byPCR. Southern blot analysis is used when the genomic frag-ments being investigated are large and the precise location ofthe marker sequence is unknown.

Southern blotting has been used on peripheral blood andbone marrow samples to detect immunoglobulin gene rear-rangements, T-cell receptor gene rearrangements, and chro-mosome translocations ( BCR-ABL and BCL2).

Polymerase chain reaction

The method is designed to permit selective amplication ofa specic target DNA sequence within total genomic DNA ora complex complementary DNA (cDNA) population. PartialDNA sequence information from the target sequences isrequired. Two oligonucleotide primers, which are specic forthe target sequence, are used. The primers are added to dena-tured single-stranded DNA. A heat-stable DNA polymeraseand the 4 deoxynucleotide triphosphates are used to initiatethe synthesis of new DNA strands. The newly synthesizedDNA strands are used as templates for further cycles of

amplication. The amplied DNA sequence can be detectedby electrophoresis on an agarose gel, and visualization can beaccomplished by the use of a DNA dye; alternatively, theamplied DNA can be directly sequenced in an automaticsequencer.

Quantitative real-time PCR is based on detection of auorescent signal produced proportionally during the ampli-cation of a PCR product. Forward and reverse primers areextended with Taq polymerase as in a traditional PCR reac-tion. A probe is designed to anneal to the target sequencebetween the forward and reverse primers. The probe is labeledat the 5 end with a reporter and a quencher uorochrome.

Table 17-10 Immunohistochemical diagnosis of Hodgkin disease.

CD45 CD30 CD15 CD20 CD3

Hodgkin (R-S cells) LPHD( ) B lymphoma / T lymphoma / /

LPHD lymphocyte-predominant Hodgkin disease; R-S ReedSternberg.

Table 17-9 Common mature T-cell and NK-cell neoplasms.

CD3S CD3C CD5 CD7 CD4 CD8 CD30 CD16 CD56 EBV

T-PLL 4 8 4 8 T-LGL NK-leukemia / /

EN-NK/T / HS- lym / Ent-T lym / / SC pannic T lym / PTCL-NOS / / / / / /AILD / /ALCL / / / /

AILD angioimmunoblastic lymphadenopathy; ALCL anaplastic large cell lymphoma; CD3C cytoplasmic CD3; CD3S surface CD3;EBV EpsteinBarr virus; Ent-T lym enteropathic T-cell lymphoma; EN-NK/T extranodal natural killer/T-cell lymphoma;HS- lym hepatosplenic lymphoma; NK-leukemia natural killer cell leukemia, PTCL-NOS peripheral T-cell lymphoma, nototherwise specied; SC pannic T lym subcutaneous panniculitis T-cell lymphoma; T-LGL T-large granular lymphocyte leukemia;T-PLL T-prolymphocytic leukemia.



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As long as both uorochromes are on the probe, the quenchermolecule stops all uorescence from the reporter. As Taq polymerase extends the primer, the 5 to 3 nuclease activityof Taq degrades the probe, releasing the reporter uoro-chrome. The amount of uorescence released during ampli-

cation is proportional to the amount of product generated ineach cycle.

Uses of PCR in clinical laboratories include detection ofthe BCR-ABL translocation in chronic myeloid leukemia anddetection of the JAK2 mutation in polycythemia vera, essen-tial thrombocythemia, and chronic idiopathic myelobrosis.

Hemostasis and thrombosis

A patients hemostatic status is usually evaluated only whenthe clinical setting calls for it, such as during episodes of clin-ical bleeding or prior to a planned invasive challenge, if thereis a personal or family history of excessive bleeding or bruis-ing. Testing for thrombophilia is usually performed after apatient has had a symptomatic thrombosis or recent history.The decision to test a patient for a predisposition to throm-bosis depends on the patients age, current medical status,past history of thrombosis, family history of thrombosis, andplanned use of this information in the future management ofthe patient. A personal and family history of bleeding orthrombosis and a list of current medications are an impor-tant part of the evaluation, and appropriate laboratoryscreening is usually directed by this history.

Generally, laboratory screening prior to surgery includesa prothrombin time (PT), activated partial thromboplastin

time (aPTT), and a quantitative assessment of platelets. Ifthere is a history of excessive bleeding, bruising, or menor-rhagia in women, then specic testing for von Willebranddisease (vWD) is indicated. However, false-normal resultsoccur during acute stress. Laboratory testing for inherited

predisposition to thrombosis should be done after thepatient has completed treatment of a thrombotic episode.The levels of normal circulating inhibitory factors can bedecreased during the acute phase of thrombosis. Protein Cand S levels are affected by anticoagulation with warfarin.The antithrombin level will decrease with the use of unfrac-tionated heparin. Lupus anticoagulant testing should beordered at the time of presentation before anticoagulationis begun, followed by serologic assays (anticardiolipinantibody and antibody to 2-glycoprotein I can be per-formed at any time). Molecular diagnostic testing can beordered at any time and is not affected by clinical status ormedications.

Coagulation testing

An outline of the traditional extrinsic and intrinsic pathwaysis presented in Figure 17-1. Our current understanding indi-cates that most coagulation reactions are initiated by expo-sure of tissue factor and that important interactions occurbetween the extrinsic and intrinsic pathways. Although thedivision into 2 separate pathways, as shown in Figure 17-1,does not reect these extrinsicintrinsic pathway interac-tions, it does provide a useful way to select coagulation testswhen evaluating a clinical problem.

Contact factors: XII, XI,prekallikrein, HMW kininogen

aPTTProthrombintime

ExtrinsicIntrinsic

XII

XI

IX

VIII

VII

"Tenase"Ca2+, PL

V, Ca 2+, PL

Prothrombin Thrombin

Tissue factor

"Prothrombinase"

Thrombintime

Fibrin clot

XIII

Fibrinogen

XFigure 17-1 Simplied coagulation cascadeindicating the intrinsic pathway measured bythe activated partial thromboplastin time(aPTT), the extrinsic pathway measured bythe prothrombin time (PT), and theconversion of brinogen to brin measuredby the thrombin time. Prekallikrein andhigh-molecular-weight (HMW) kininogenare not considered coagulation factors, butthey can prolong the aPTT if they aredecient.



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Overview of key points

Mixing studiesSpecic factor assaysScreening studies

PTFactor VII deciency

International normalized ratio (INR)aPTT

Deciencies of factors VIII, IX, XI, XII, prekallikrein,HMW kininogen

Lupus anticoagulant testingInhibitors to factor VIII and other intrinsic pathway

factorsHeparin and monitoring heparin

Combined abnormalities of PT and aPTTThrombin time

Fibrinogen assaysvon Willebrand factor (vWF) assaysActivity and antigen assaysRistocetin-induced platelet aggregation (RIPA)vWF multimers

Disseminated intravascular coagulation (DIC)FibrinolysisEvaluation of platelets

QuantitationPlatelet aggregationPlatelet function analyzersBleeding time

Platelet antibodiesAssays for heparin-induced thrombocytopenia (HIT)Assays for thrombotic thrombocytopenic purpura (TTP)

Evaluation of bleeding patients with normal screening testsThrombophilia

Protein C, protein S, antithrombinFactor V Leiden and activated protein C resistance (APCR)Prothrombin 20210 mutation Dimerized plasmin fragment D (D-dimer) and deepvenous thrombosis (DVT) evaluationLupus anticoagulants and thrombosis

1:1 mixing study

Traditionally, when a screening test is prolonged and the causeis not apparent, the next study performed is a 1:1 mixingstudy with patient plasma and pooled normal plasma (PNP).The clotting test is usually repeated within 5 minutes of themix (immediate time point). For a prolonged aPTT, themixing assay should be performed immediately after the mixand again after a 1-hour or, optimally, 2-hour incubation at37 C to detect a progressive inhibitor (usually progressiveinhibition indicates a factor VIII inhibitor).

A lack of correction or only partial correction in the aPTTmixing study should be determined by the laboratory per-forming the testing. Criteria used to determine lack of cor-rection with the mixing study may be dened as 5 secondsof the PNP or as outside the reference range of the test in the

laboratory performing the test. These criteria have not beenstandardized. Nevertheless, this then leads to testing for alupus anticoagulant and to specic intrinsic pathway factorassays to determine if a specic factor is inhibited. A correc-tion to the normal range generally indicates that the patienthas a deciency of a factor, but a low afnity or low titerinhibitor may give a correction in a mixing study, and testsfor a lupus anticoagulant and for specic factors are usuallystill necessary.

aPTT s that are only slightly prolonged (24 seconds)often may correct, and clinical judgement is necessary indeciding how much laboratory evaluation is necessary. Theclinical history and the clinical setting helps to determinehow to proceed (eg, a patient who is to undergo brain sur-gery has need for more extensive evaluation than one who isto have an accessible lymph node excised).

Specic factor assays

Specic factor assays are performed using substrate plasmasthat are decient in the factor being studied. These are thenmixed with the patient plasma to see if abnormalities in thePT or the aPTT correct. Most normal adults have levels ofeach factor that range between 50% and 150%, but the mini-mal hemostatic level of most factors is lower than this refer-ence range (usually below 30% activity). Many factor assaysare based on detection of clot formation, and they have beenautomated; the instruments use either a mechanical endpoint or a change in the optical density to detect the clot.Chromogenic assays are available for some factors such asfactor VIII, but they are not readily available. The activatedclotting factor cleaves a small chromogenic substrate that isadded to the reaction; the chromogenic substrate generates acolor change that is used for detection. Chromogenic assayscan be more precise than clotting assays, but they may not

detect some defects in a factor that disrupt the binding of thefactor to its natural (larger) substrate.

Expected ranges in pediatric patients

Many coagulation factors are abnormally low in the newborncompared with adult normal ranges. Of the screening tests, theaPTT is prolonged, whereas the PT, brinogen, and thrombintime are normal (the latter should be measured with calciumin the reagents to prevent prolongation by fetal brinogen).The brinogen rises during the rst week after birth and thenreturns to the normal range. The vitamin K-dependent



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proteins, including factors II, VII, IX, X, protein C, and proteinS, are decreased (often in the 30% range) and gradually rise tothe normal adult range by 6 months, with the exception of pro-tein C, which remains below the adult normal range until atleast 5 years of age. Vitamin K is administered on a routine basis

shortly after birth to prevent vitamin K deciency during therst week(s) of life. Factor VIII levels are normal in the newborn,and vWF levels are increased; there are also unusually HMWvWF multimers present in the circulation of 65% of neonates.The contact factors (factors XI and XII, prekallikrein, and high-molecular-weight kininogen [HMWK]) are low at birth (andmarkedly so in premature infants) and rise to the adult range at6 months. Antithrombin and plasminogen levels are also low atbirth and reach normal levels by 6 months of age.

Prothrombin time

The PT measures the extrinsic pathway and nal commonpathway and assesses 3 of the 4 vitamin K-dependent factors(factors II, VII, and X; factor IX is measured in the aPTT).The PT is performed by adding a commercial thromboplastinreagent (tissue factor, a lipoprotein normally present inmembranes of perivascular broblasts and activated endo-thelial cells) and calcium chloride to citrated plasma andmeasuring the time to clot formation.

Prolongation of the PT most often reects a deciency of 1or more of the vitamin K-dependent factors due to poornutrition, inadequate absorption of vitamin K, or decreasedhepatic synthesis of coagulation factors from liver disease;congenital deciencies of factors II and X are very rare (1 in12 million people). Congenital deciency of VII has an esti-mated incidence of 1 in 300,000 people. Coumarin anticoag-ulants also cause a prolonged PT due to their inhibition of thepostsynthetic carboxylation of the vitamin K-dependentfactors, which renders the protein less effective. Dysbrino-genemia occasionally causes a prolongation of the PT with-out a prolongation of the aPTT. Specic causes for prolongedPTs include the following (see Figure 17-2):

Deciency of factor VII causes a prolonged PT in the pres-ence of a normal aPTT. Congenital deciency is rare, but low

factor VII due to decreased hepatic synthesis accompanyingliver disease is not uncommon because of the short half-lifeof factor VII (t 1/2 is 46 hours). A specic assay using factorVII-decient substrate plasma and a PT method is available.Inhibitors of factor VII are extremely rare.

Argatroban and lepirudin are monitored by the aPTT butwill cause a long PT because of their direct effect on throm-bin inhibition, with argatroban having a much more pro-nounced effect than lepirudin. Both drugs are characterizedby a short half-life but may be prolonged in the acutely illpatient. Continued effect on the coagulation testing resultsmay occur after the drug has been discontinued.

Coumarin anticoagulants cause prolonged PTs (and, vari-ably, prolonged aPTTs depending on II, X, and IX levels).The PT/INR is used to monitor warfarin anticoagulation.Thromboplastins used in testing have different sensitivities

to the effects of warfarin on the vitamin K-dependent pro-teins; the INR is used to standardize these differences andmake the comparability of the assay results better betweenlaboratories. The INR is a calculation that represents theratio of the patients PT to the laboratorys mean normal PTas though the test had been performed with an internationalreference preparation (IRP) of thromboplastin. The com-mercial supplier calibrates the reagent to the IRP and assignsan International Sensitivity Index (ISI) to it. The laboratoryuses this value to calculate the INR.

INR PTof patient

Mean Normal PTof lab

ISI=

Normal subjects have an INR of 1.0 0.12. The INR isdesigned to follow patients who have been stabilized on adose of coumadin and to make it possible for the patient tohave PTs measured in different laboratories and still main-tain correct dosing. Instrument variables are also importantand can add imprecision to the ISI; this problem can be min-imized by calibration between specic reagents and instru-ments by the suppliers.

The INR is a mathematical conversion based on effects ofwarfarin and is not intended for assessing factor deciencies

Mixing study

Not corrected Corrected

Lupus anticoagulanttesting

Deficiency of factor:perform specific

assays for factor VII(also for X, V, II if

APTT is prolonged)

Positive Negative

Lupusanticoagulantpresent

Inhibitor of clotting factor:perform factor VII assay;if low perform inhibitor assay

Evaluation of prolongedprothrombin time

Figure 17-2 Algorithm for evaluation of an isolated prolongedprothrombin time (PT).



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due to congenital deciency or liver disease because the ISI isnot tested for nonvitamin K-dependent proteins, particu-larly factor V and brinogen. Thus, it should not be used forthis purpose because its sensitivity cannot be predicted.

Lupus anticoagulants are a common cause of a prolongedaPTT and are not infrequent in both hospitalized patientsand ambulatory patients. When a 1:1 mixing study for a pro-longed aPTT does not shorten to the normal range, a test fora lupus anticoagulant should be performed (a lupus anti-coagulant should also be suspected in some cases when theaPTT mix is corrected due to a low titer or an atypical lupusanticoagulant). Testing is also ordered based on clinicalpresentation.

Several types of tests are available; they all have in com-mon the use of a limiting concentration of phospholipid in arst step and the provision of excess phospholipid in a sec-ond step that corrects the clotting end point in a patient witha lupus anticoagulant, conrming the diagnosis. Becauseplatelets provide a source of phospholipid, the plasma sam-

ple must be prepared carefully to eliminate platelet frag-ments; this is usually accomplished by ltering the plasma orperforming a hard spin (5000g) before testing and before freezing if the test is to be done later.

Lupus anticoagulants are a family of antibodies directedagainst different antigens, and they are not positive in allassays. Included in the strict denition of a lupus anticoagu-lant, as opposed to an antiphospholipid antibody, is thedemonstration of a prolonged clotting assay that is correctedby provision of excess phospholipid. Many laboratories useseveral different assays to avoid missing the antibody. Testsfor the lupus anticoagulant include the dilute Russell viper

Prolonged PT:Poor nutrition ([darrow] vitamin K)Malabsorption of vitamin KLiver disease ( synthesis of vitamin K-dependent factors)CoumarinsRare congenital deciency or inhibitor (factor VII)

Key points

Activated partial thromboplastin time

The aPTT measures the intrinsic pathway factors (contactsystem factors, factors XII, XI, IX, and VIII) and the nalcommon pathway factors. It is performed by adding a com-mercial preparation of phospholipid (the partial thrombo-plastin), an activator such as kaolin or celite (in somesystems ellagic acid is used), and calcium chloride to citratedplasma and measuring the time to clot formation. The acti-vator and the phospholipid are incubated with the plasmafor approximately 4 minutes, during which time factors XIIaand XIa are formed; calcium chloride is then added, whichpermits activation of factor IX and the remaining reactionsto proceed to form a brin clot. Causes for a prolonged aPTT

include the following (see Figure 17-3):

Deciency of factors VIII, IX, XI, or XIIor deciency of otherproteins in the contact activation pathway, prekallikrein andHMWK , prolong the aPTT. The 2 latter proteins, along withfactor XII, do not cause a bleeding disorder but do lengthenthe aPTT. Depending on the aPTT method used, in order fora specic factor deciency to prolong the aPTT, its level isusually decreased to 3040%. Factor assays using specicdecient substrate plasmas and based on an aPTT methodare applicable for measuring these factors; a chromogenicassay for factor VIII is also available.

Factors VIII and IX decienciesor hemophilia A and B areX-linked inherited disorders that are often diagnosed early inlife but may not be identied until adulthood if there is amild deciency (540%).

vWD may manifest with a slightly prolonged aPTT if the fac-tor VIII level is at the sensitivity level of the laboratorysaPTT reagent.

Deciency of factor XIshould be suspected with a prolongedaPTT in a person of Jewish background. It can be associatedwith bleeding complications.

Mixing study

Not corrected Corrected


Deficiency of factor:perform long incubation APTT specific assays forfactor VIII, IX, XI, XII

Positive Negative

Lupusanticoagulant

present

Inhibitor of clotting factor. Perform: specific factor assay for VIII, IX, XI, XII inhibitor assay for factor that is decreased

Evaluation ofprolonged APTT

Figure 17-3 Algorithm for evaluation of an isolated prolongedactivated partial thromboplastin time (aPTT).



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patients as well as those with impaired renal function orthose with increased body mass ( 144 kg).

The aPTT is used to monitor standard heparin therapy. Atherapeutic heparin level for standard unfractionated hepa-rin is determined by the clinical laboratory using the anti-Xa

assay to correlate the aPTT results to a level of 0.30.7 anti-Xa IU/mL. The anti-Xa assay can also be used to assess theheparin level and is particularly helpful if there appears to beheparin resistance when excessively large doses of heparinare needed to prolong the aPTT to the therapeutic range or ifthe patient has a lupus anticoagulant that prolongs the base-line aPTT. The therapeutic range for LMW heparin wasbased on specimens obtained approximately 4 hours afterthe therapeutic dose of LMW heparin has been administeredand is often considered to be 0.61.0 anti-Xa IU/mL. TheaPTT is also used to measure the direct thrombin inhibitors,lepirudin and argatroban, and is considered to be therapeu-tic when it is 1.52.5 times the baseline levels.

Combined abnormalities of PT and aPTT

Deciency of a factor in the nal common pathway (factors X,V, prothrombin, and brinogen) or dysbrinogenemias cancause an isolated PT prolongation or a combined prolonga-tion of the PT and the aPTT. Except for dysbrinogenemia,specic factor assays are available to measure them. Decreasedhepatic synthesis from advanced liver disease will cause de-ciency of all coagulation factors except for factor VIII. Thiswill often manifest rst with a prolonged PT. Dysbrinogen-emia may also be acquired in hepatic disease and is suggestedby nding a low level of brinogen in a functional assay com-bined with a normal or disproportionately high level ofimmunologic brinogen (see discussion later in the chapter).See Figure 17-4 for evaluation of a prolonged PT and aPTT.

Inhibitors to factor V , which rarely arise spontaneously, canbe seen after exposure to bovine thrombin (it also contains

bovine factor V), which is part of brin glue; this productis used relatively frequently for hemostasis in orthopedic,cardiac, and urologic surgery, as well as neurosurgery. Theantibody may cross-react with human factor V and can causelife-threatening bleeding. A low level of factor V and aninhibitor of factor V will be found in specic tests. Productsthat contain human thrombin have become available, andthey do not usually cause antibodies to factor V.

Inhibitors to prothrombin can be associated with lupusanticoagulants and may lead to bleeding if prothrombin lev-els are sufciently low. These antibodies usually cannot be

Prolonged aPTT:Lupus anticoagulantFactors VIII or IX deciency or inhibitor vWD (if factor VIII is decreased)Factors XI or XII deciency, prekallikrein, or HMW kininogen

deciency or inhibitor Heparin contamination of sample

Key points

Figure 17-4 Algorithm for evaluationof a prolonged prothrombin time (PT) andactivated partial thromboplastin time(aPTT).

No clinical bleeding Clinical bleeding

If thrombin time longcheck protamine SO 4

neutralization

Notnormalized byprotamine SO 4

Mixing studiesusing both PT

and APTT assays

Fibrinogen level,FDP and D-dimer

platelet count

Normalization ofthrombin time =

heparincontamination

Notcorrected

Corrected fibrinogen+FDP and D-dimer platelet count

DIC. Follow plateletcount and fibrinogen(may perform factorassays for status: V,

VIII)

Deficiency of factor:perform assays for

factor X, V, II(possibly VIII, IX, XI)


Positive Negative

Lupusanticoagulant

present

Possible inhibitor of clotting factor. Perform: specific factor assays for factor X, V, II inhibitor assay for factor that is decreased



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detected in in vitro mixing studies because they are notdirected against the active site of the molecule. Rather, theyform a complex with other antigenic sites on prothrombinand are cleared from the circulation as immune complexes,lowering the prothrombin level.

tumors and after suramin administration. They prolong thethrombin time by interacting with antithrombin in a man-ner similar to heparin. The reptilase time will be normal inthese patients.

Prolonged PT and aPTT:Factors X and V or prothrombin deciencyHypo- or dysbrinogenemiaLiver disease ( hepatic synthesis of factors)Coumarins and vitamin K deciencyInhibitor to factor V or prothrombin

Key points

Thrombin time

The thrombin time measures the time of conversion ofbrinogen to brin and requires sufcient amounts of nor-mal brinogen and the absence of an inhibitor to thrombin.The thrombin reagent is commercial bovine or humanthrombin, and the normal reference range in each laboratoryis determined by the quantity of thrombin added in the assay.Citrated plasma is used as the test sample.

Unfractionated heparin, LMH heparin, lepirudin, and arg-atroban prolong the thrombin time. As noted previously,blood drawn from a central venous line can be contaminatedwith heparin, and very small quantities, less than the amountrequired to prolong the aPTT, will prolong the thrombintime.

Dysbrinogenemias usually prolong the thrombin time andare suspected if the functional test (clottable brinogen) isdisproportionately low compared with an immunologicmeasurement of brinogen.

Hypobrinogenemia usually prolongs the thrombin timewhen levels of brinogen are below approximately 5070mg/dL. Therapeutic doses of L-asparaginase can cause signi-cant hypobrinogenemia by inhibiting synthesis.

Inhibitors to thrombin occur uncommonly, but antibodiesto bovine thrombin are seen after the use of brin glue or

other products that use bovine thrombin for hemostasis (eg,in neurosurgery and orthopedic, urologic, and cardiacsurgery). The antibody to bovine thrombin usually does notcross-react with human thrombin. The thrombin time willbe prolonged when the assay reagent is bovine thrombin,and it will be normal when human thrombin is substitutedin the assay. These antibodies are of no clinical consequenceand usually disappear after several weeks to months.

Fibrin degradation products (FDPs) in very high levels caninhibit the thrombin time.

Heparin-like anticoagulants (heparan sulfates ) haveoccurred in patients with multiple myeloma and other

Prolonged thrombin time:Heparin, lepirudin, or argatroban in plasma sampleHypo- or dysbrinogenemiaInhibitor of thrombinHeparin-like anticoagulants FDPs

Key points

Fibrinogen assays

The functional brinogen assay is the most common brin-ogen assay used, and it is performed using a modiedthrombin time where the brinogen rather than the throm-bin is limiting. Fibrinogen can also be measured in immu-nologic tests (radial immunodiffusion) or as protein presentin the clot that results from the addition of thrombin toplasma.

Reptilase time

Reptilase is a snake venom that cleaves brinopeptide Adirectly from brinogen and results in brin clot formation.

This assay is prolonged in the presence of dysbrinogen-emias and is usually a sensitive assay for this condition.

von Willebrand factor assays

The majority of patients with vWD have mild disease thatcan be difcult to diagnose because of the overlap of valuesin normal subjects and patients. The variable levels of vWFin patients with different blood groups and during physio-logic alterations associated with acute phase reactions or themenstrual cycle can also make the diagnosis problematic,and patients may require repeat testing after an interval of

several weeks. Because of the lower vWF levels present innormal subjects with blood group O (approximately 25%lower as compared to normal subjects with types A, B, andAB), some laboratories prepare a separate reference range forpatients with blood group O.

Although the bleeding time has been used as a screeningtest for vWD, it is too insensitive and nonspecic, and it is nolonger widely used for the diagnosis of vWD. The factor VIIIlevel, which is decreased in moderate and severe vWD due tolack of protection for circulating factor VIII by vWF, is alsovariable and is helpful in the diagnosis of vWD only when itis abnormal (see Table 17-11 for vWF assays).



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Ristocetin cofactor and vWF antigen.

There are two specic tests for the initial diagnosis of vWD:ristocetin cofactor and vWF antigen.

VWF activity Ristocetin cofactor measures the activity of vWF by testing

the ability of vWF to bind to its platelet receptor, GPIb, andcause platelet agglutination in the presence of ristocetin (ris-tocetin alters vWF to allow binding to platelet membraneGPIb). It is performed by mixing different dilutions of patientplasma (the source of vWF) with washed normal platelets orplatelet membranes that are devoid of vWF, adding ristocetinat a nal concentration of approximately 1.2 mg/mL, andobserving the time to agglutination or aggregation. A stan-dard curve is executed by using dilutions of a normal plasmapool in the same manner, and the normal range, dened ineach laboratory, is usually near 45150%. The exceptions areindividuals with blood type O who may range as low asapproximately 35%; in these cases, it is prudent to repeat thetest and review the personal and family bleeding history care-fully before making a positive diagnosis of vWD.

Collagen binding assays measure a different activity of vWF,

assessing how well plasma vWF binds to collagen coated onwells of an enzyme-linked immunoadsorbent assay (ELISA)plate. The bound vWF is measured by specic antibodies tovWF.

vWF antigen measures the quantity of plasma vWF byimmunologic means such as an ELISA assay. Because indi-viduals with blood type O have levels that can be decreased,some laboratories have established separate reference rangesfor these individuals.

There are 3 types of vWD: type I, a quantitative decreasein vWF activity and antigen; type II, qualitative defects caus-ing dysfunction of vWF; and type III, lack of production of

vWF, leading to severe vWD. Additional tests are necessary toclassify the vWD type once a diagnosis has been establishedusing the vWF activity and antigen assays (see Table 17-12).These tests are also performed if the suspicion for vWD isstill high in a patient with borderline results. These include RIPA and vWF multimers.

RIPA. This assay is often confused with the ristocetin cofac-tor assay described previously, but the RIPA is used to testwhether the patient might have a qualitative defect in vWFthat causes the abnormal vWF to bind to platelets

spontaneously or at very low concentrations of ristocetin(a gain-of-function mutation). Such a nding is characteris-tic of type IIB vWD. The RIPA uses the patients platelet-richplasma (PRP) as the source of both vWF and platelets; risto-cetin is added separately in progressively lower concentra-tions (to 0.4 mg/mL) to different tubes containing aliqoutsof the patients PRP, and the presence or absence of aggrega-tion is noted (all or none). Most normal PRP will not aggre-gate at concentrations of ristocetin below 0.8 mg/mL, whereaspatients with type IIB vWD will aggregate at 0.40.6 mg/mL.Pseudo-vWD or platelet-type vWD, an abnormality in theplatelet GPIb receptor for normal vWF, also shows increased

Table 17-11 von Willebrand factor assays.

Name Function Assay

vWF activity Activity of vWF that causes binding of vWF to plateletGPIb in the presence of ristocetin with consequentaggregation

Ristocetin cofactor activity: quantitates platelet agglutinationafter addition of ristocetin and vWF

Ability of vWF to bind to collagen Collagen binding activity: quantitates binding of vWF tocollagen-coated plates

vWF antigen vWF protein as measured by protein assays; does notmeasure functional ability

Immunologic assays such as ELISA, RIA, and Laurellelectroimmunoassay

vWF multimers Size distribution of vWF multimers as assessed by agarosegel electrophoresis

vWF multimer assay: electrophoresis of plasma in low- concentration agarose gel and visualization by monospecic

antibody to vWFRIPA Measures the ability of patient vWF to bind to platelet

receptor GPIb in the presence of variable concentrationsof r istocetin

RIPA: aggregation of patient platelet-rich plasma with decreasingconcentrations of ristocetin

ELISA enzyme-linked immunoadsorbent assay; RIA radioimmunoassay; RIPA ristocetin-induced platelet aggregation;vWD von Willebrand disease; vWF von Willebrand factor.

Table 17-12 Assays for von Willebrand disease classication.

vWD type Activity Antigen RIPA Multimer pattern

Type I Uniform

Type IIA Large and intermediateType IIB LargeType IIM Uniform

Type IIN Normal Normal Normal NormalType III Undetectable

RIPA ristocetin-induced platelet aggregation;vWD von Willebrand disease.



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binding in the RIPA at low ristocetin concentrations and isphenotypically indistinguishable from type IIB vWD. Mix-ing studies using normal washed platelets plus patientplasma, or vice versa, can be used to distinguish whether thepatients vWF or platelet receptor is abnormal.

vWF multimers. Plasma vWF is composed of different-sizedmultimers that can be visualized in low concentration aga-rose gels after electrophoresis and detected using an antibodyto vWF. The absence of larger multimers signies a qualita-tive defect and aids in classifying the type of vWD in thepatient. This test can also detect larger than normal multim-ers that occur in 65% of normal neonates, in another vWDsubtype (Vicenza), and in some patients with TTP.

It is particularly important to evaluate carefully a femalewho presents with a factor VIII level of 1015% accompa-nied by normal or borderline levels of vWF activity and anti-

gen. This nding may be due to type IIN vWD, in which thepatients vWF has an abnormal binding site for factor VIII,leading to the rapid clearance and decreased plasma level offactor VIII. Type IIN vWD is evaluated functionally using abinding study that tests the patients vWF for its ability tobind normal factor VIII. The binding test is available in sev-eral laboratories that study vWF, and assays for genetic muta-tions that cause type IIN are available. Low levels of factorVIII can also be found (rarely) in carriers of hemophilia Awho have skewed lyonization of their X chromosome and infemales with XO phenotypes.

The diagnosis of vWD can be very problematic during the

second and especially the third trimester of pregnancy, whenthe vWF level normally increases 2 to 3 times above its base-line level. In these cases, a careful personal and family historyand family testing can be helpful. However, a denitive diag-nosis cannot usually be made with condence until 46weeks after delivery.

The diagnosis of acquired vWD, often associated withautoimmune or lymphoproliferative diseases, is suggested bya history of recent onset mucous membrane or skin bleedingwithout a past or family history of bleeding and by a pro-longed aPTT on screening studies. The latter is due to adecreased factor VIII associated with low levels of vWF. Theristocetin cofactor is usually abnormal, and multimer studiesusually show a decrease in the HMW multimers (a type IIAor B vWD pattern). Mixing studies with patient and normalplasma do not consistently demonstrate an inhibitor, andfurther specialized testing may need to be carried out to doc-ument the diagnosis.

Disseminated intravascular coagulation

Coagulation assay results vary during the course of DIC.Although thrombocytopenia is present, the coagulationscreening tests (PT, aPTT, thrombin time, and brinogen)

may be normal early in the course of acute DIC or duringchronic DIC. With hemorrhagic DIC when substantial con-sumption has occurred, the PT, aPTT, thrombin time, andbrinogen are usually all abnormal, and factor levels aredecreased. Fibrin/brinogen degradation products (FDP/

Fdps) and D-dimer (a cross-linked degradation product ofbrin) are elevated. Advanced liver disease has a similarcoagulation prole to that seen in DIC, and the 2 disorderscan be difcult to distinguish without clinical correlation.

Fibrinolysis

The activity of the brinolytic system does not have readilyavailable screening assays for assessment; the euglobulin clotlysis is a global test that was once used but is no longer readilyavailable in detecting activation of brinolysis. Other testsinclude FDPs, D-dimer tests, and specic assays for plasmin-

ogen, tissue plasminogen activator, plasminogen activatorinhibitor 1 (PAI-1), and plasminantiplasmin complex. Plas-minogen and related proteins (discussed previously) can bemeasured in functional or antigenic assays.

2-Antiplasmin is an important inhibitor of plasmin, andcongenital deciency of this protein can lead to seriousbleeding. Symptoms typically occur without warning duringinvasive procedures because deciency of 2-antiplasmindoes not cause abnormalities of the preoperative screeningcoagulation tests. 2-Antiplasmin can be measured in a func-tional assay.

Primary brinolysis, activation of plasminogen to plasminwith subsequent cleavage of brinogen, is unusual as a pri-mary disorder. In most circumstances, activation of brino-lysis is secondary to coexisting activation of coagulation (asseen in DIC). However, circulating plasminogen activatorshave been described in malignant diseases such as amyloido-sis and with envenomization from several species of snakes.In primary brinolysis, the FDPs are elevated without a sig-nicant increase in the D-dimer, and the platelet count isnormal (unless the underlying disease causes a decrease inplatelets).

ELISA assays are sensitive assays of hypercoagulable statesthat can detect subclinical activation of coagulation, such as foractivation peptides of coagulation factors and products of plate-let or endothelial cell activation. They are used only for clinicalresearch studies rather than as an aid in clinical management;examples are ELISA assays for circulating prothrombin frag-ment 1.2, thrombinantithrombin complex, brinopeptide Aand B, platelet factor 4, and thrombomodulin.

Quantitation of platelets

Platelet numbers are quantitated accurately by automated cellcounters that depend on electrical impedance or light scatter.



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Other methods for quantitating platelets and potential artifactsthat lead to falsely low platelet counts are described previously.

Evaluation of platelet function

Platelet aggregationPlatelet aggregation, a very sensitive and time-consumingtest, has been the primary method available for evaluation ofplatelet function until recently. In this test, either citratedwhole blood or PRP is prepared, and agonists such as adenos-ine diphosphate (ADP), epinephrine, collagen, arachidonicacid, or thrombin are added. In the PRP system, the agonistsare added to PRP and stirred to initiate platelet aggregation;the formation of platelet aggregates causes an increase inlight transmission through the PRP as the aggregates fall tothe bottom of the tube. The change in light transmission isrecorded by a mechanized recording instrument. The plateletrelease reaction can be assessed in a lumi-aggregometer dur-ing the assay by the addition of a reagent that requires ade-nosine triphosphate (ATP) for signal production (the plateletrelease reaction is the only source of ATP in this assay). In thewhole blood system, a change in light impedance is recordedas the aggregation end point. Although measurement ofplatelet aggregation can be very helpful, its time-consumingnature, the need for a fresh patient sample, the technical skilldemanded in its performance, and the lack of quantitativeparameters for its interpretation have decreased its use.

Platelet function analyzers

Platelet function analyzers have been developed as less com-plicated methods to measure platelet plug formation in vitro.One instrument, the PFA-100, has been developed to be usedas a screening instrument for both platelet function and forvWF concentration and function. Citrated blood is exposedto epinephrine or ADP as it is aspirated at arterial shear ratesthrough a collagen-coated membrane that contains an aper-ture. The platelets are activated, aggregate, and form a plugthat occludes the aperture in the membrane; the end point ismeasured as the time required for a drop in pressure within

the system caused by the occlusion of the aperture. Theinstrument is sensitive to intrinsic platelet function as well asto vWF levels. Though not as sensitive or specic as plateletaggregation and release studies (eg, for the diagnosis of stor-age pool disease), the PFA-100 is technically easy to use, hasa quantitative end point, is reproducible, and is much lesstime consuming.

The template bleeding time

The template bleeding time has been used for the diagnosisof intrinsic platelet abnormalities. It may also be abnormal

in moderate and severe vWD. The test is performed by

making a standard incision in the forearm using controlledconditions and measuring the time for the bleeding to stop. Itis operator dependent, reects the integrity of the microvascu-lature as well as the platelets, and is not particularly sensitive.Most importantly, it does not reliably predict the individuals

hemostatic capacity and should not be used as a general pre-operative screening study to predict the risk of bleeding.

Assays for platelet antibodies

Assays for platelet antibodies may be performed as part of anevaluation for immunologic causes of thrombocytopenia,but they have limited usefulness in clinical settings due totheir low sensitivity and, with some assays, due to their lowspecicity. Flow cytometry has been used to measure theimmunoglobulin on the surface of platelets, but this test doesnot reect specic platelet antibodies and has not correlatedwell with the clinical status of patients with immune throm-bocytopenias. More specic tests, using specic monoclonalantibodies to platelet antigens such as glycoproteins Ib andIIb/IIIa, can be performed in microtiter plate assays using amonoclonal antibody immobilization of platelet antigen(MAIPA) format. These can be difcult tests because theyrequire sufcient numbers of patient platelets to provide theplatelet-bound antibody, and adequate numbers of plateletsare not always available from patients with ITP. The tests arepositive in only 5070% of patients with clinically diagnosedITP. A technically similar indirect test using normal plateletsexposed to patient serum is positive in only 3050% ofpatients.

Assays for heparin-induced thrombocytopenia

Assays for HIT can be performed by

17 laboratory hematology

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