17 Laboratory Hematology

Chapter 17
Laboratory hematology
Georgette A. Dent, Jay H. Herman and Jamie E. Siegel
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444
General concepts
Laboratory tests are ordered and interpreted within the
context of a specic patient as routine screening during a
periodic 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 and
in their interpretation, and unexpected results should be
considered 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, any
laboratory 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 at
the cost of decreasing specicity). Very sensitive tests are
helpful in ruling out a disease when the test is negative.
Specicity is the ability of a test to detect a normal result if the
abnormality is not present; maximum specicity may lead
to too many false-negative results. As a rule, specic tests
are useful for ruling in a disease when the test is positive.
Precision is reproducibility of a value during repeated testing
of a sample.
Accuracy is the ability of a test to obtain the assigned value of an
external 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 predictive
value), or the likelihood that a normal test indicates a
patient 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-positive
results. The tests for rare abnormalities require higher sen-
sitivity and specicity to have high predictive values than
do the tests for common abnormalities.
Reference ranges represent collected statistical data from a
well population. Reference ranges usually represent data
for 90% or 95% of the well population.
Specic laboratory testing
Automated cell counting
Automated cell counting provides the best means of counting
large numbers of cells while minimizing statistical error. The
2 major types of cell counters are the aperture-impedance
counters and the optical method counters.
Aperture-impedance counters
Cells are counted and their size estimated by measuring change
in 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 more
electrically conductive than are the cells, when the cells pass
through the orice there is a decrease in electrical conduc-
tance. The drop in voltage is proportional to cell size, allowing
the 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 is
dependent on even cell suspensions; distortion occurs when
cells do not pass through the center of the aperture or when
multiple cells pass at the same time.
Coulter (Hialeah, FL), Sysmex (Baxter Diagnostics,
Waukegan, IL), and some Cell-Dyn instruments (Abbott
Diagnostics, Santa Clara, CA) use this technology.
Laboratory hematology
Georgette A. Dent, Jay H. Herman, and Jamie E. Siegel
General concepts, 444
Terminology, 444
Specic laboratory testing, 444 Bibliography, 464
CHAPTER
17
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evaluation of anemia. The RDW is more frequently elevated
in iron deciency anemia than in thalassemia or anemia of
chronic disease.
Leukocyte analysis
The automated 5-part differential (neutrophils, monocytes,
eosinophils, basophils, lymphocytes) can be obtained using
several methods. Technicon instruments use a combina-
tion of light scatter and peroxidase staining. Coulter instru-
ments use electrical impedance, light scatter, and electrical
con ductivity. Sysmex uses differential resistance to lysis of
eosinophils and basophils in specic detergents at different
temperatures. Cell-Dyn uses patterns of light scatter at
4 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 the
instruments.
Platelet analysis
Automated counters provide both a platelet count and a
mean platelet volume (MPV). The MPV has an inverse rela-
tionship with platelet number because platelets tend to be
larger in patients with destructive thrombocytopenia. The
MPV tends to be increased in hyperthyroidism and myelo-
proliferative disorders and decreased with megakaryocytic
hypoplasia.
Erythrocyte fragments can falsely increase the platelet
count. The presence of platelet clumping secondary to ethylene-
diaminetetraacetic acid (EDTA)-related platelet antibodies
can falsely decrease the platelet count. If EDTA platelet anti-
bodies are suspected as the cause of thrombocytopenia, the
diagnosis can be conrmed by showing a normal platelet
count on a citrated sample. In patients with EDTA anti bodies
who are inaccurately reported to have thrombocytopenia,
the blood smear obtained from the EDTA-anticoagulated
blood may show thrombocytopenia, though platelet clumps
are 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 than
patients with immune thrombocytopenic purpura (ITP).
Phase microscopy can also be used to conrm platelet
numbers.
Optical method counters
This method takes advantage of the light scattering proper-
ties of blood cells. A ow cytometer is used to produce a ne
stream of cells in suspension; the cells are then run in single
le across the path of a unifocal laser. The amount of light
scattered at a low angle from the incident light path depends
on cell size. The amount of light scattered at a wide angle
depends on factors such as cytoplasmic granules and nuclear
shape. The pattern of light scattering is used to determine
cell size, volume, shape, and complexity.
Technicon (Bayer Diagnostics, Kent, WA) and some
Cell-Dyn instruments (Abbott Diagnostics, Santa Clara, CA)
use this technology.
Erythrocyte analysis
Automated instruments measure the number of cells per
unit volume, cell size, and hemoglobin concentration; all
other parameters are calculated. Hemoglobin concentration
is expressed in grams per deciliter (g/dL). Hemoglobin and
its other forms, oxyhemoglobin and carboxyhemoglobin, are
converted by potassium ferrocyanide to cyanmethemoglobin
to determine the amount of total hemoglobin. The absor-
bance of the product, cyanmethemoglobin, is measured by a
spectrophotometer at 540 nm. An increase in the measured
hemoglobin concentration from increased sample turbidity
can be an artifact of improperly lysed red cells, leukocytosis,
paraproteinemia, and hyperlipidemia. Automated hemato-
crit is a calculated value that is obtained by multiplying the
red cell volume by the red cell number (hematocrit red cell
volume red cell number).
The mean corpuscular volume (MCV) is expressed in
femtoliters (10
15
L) and is measured directly by the auto-
mated instrument. Agglutination of red cells, resulting in
measurement 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 calculated
value; it is expressed in picograms (10
12
g). The MCH is
obtained by dividing the hemoglobin concentration by
the red cell count (MCH hemoglobin [g/L]/red cell count
[ 10
12
/L]). An elevated MCH can be an artifact of increased
plasma turbidity. The mean corpuscular hemoglobin con-
centration (MCHC) is a calculated value; it is expressed in
grams 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 of
variation of red cell size distribution. The RDW is used in the
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Laboratory hematology 446
Reticulocyte analysis
New methylene blue is used to stain residual RNA and
aggregates the RNA, making it easily visible for manual reti-
culocyte counting. Automated reticulocyte counting uses
either methylene blue or uorescent RNA-binding dyes.
Leukocytes, large platelets, or platelet aggregates need to be
excluded because these cells will also contain RNA.
Peripheral blood morphology
The best quality peripheral blood smears are obtained from
ngerstick procedures. Blood smears are stained with either
the Wright or the MayGrunwaldGiemsa stains.
With the advent of automatic cell counters performing
5-part differentials, peripheral blood morphology needs to
be performed in only about 30% of specimens in a general
hospital or clinic population.
The optimal area to examine the peripheral blood smear is
the transitional area between the thick part of the smear and
the feathered edge (Table 17-1). In the transitional area, there
are only a few overlapping red cells, and central pallor of
normal red cells is evident. Abnormal distribution of larger
cells should be excluded by examination of the edges of the
smear.
Supravital stains are used to detect red cell inclusions.
Crystal violet detects denatured hemoglobin inclusions
(Heinz bodies); brilliant cresyl blue is used to precipitate and
detect unstable hemoglobins.
Erythrocyte sedimentation rate
The erythrocyte sedimentation rate (ESR) is measured by
allowing red blood cells in a capillary tube to settle over time.
Time to sedimentation is measured in millimeters per hour
by either the Westergren or Wintrobe methods; both meth-
ods are affected by the red cell count of the specimen. ESR
increases 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 rapid
settlement 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, thalassemias
Bite cells (degmacyte) Smooth semicircle taken from
one edge
Heinz body pitting by spleen G6PD deciency, drug-induced oxidant
hemolysis
Burr cells (echinocytes) Short, evenly spaced spicules May be related to abnormal
membrane lipids
Usually artifactual, uremia, bleeding
ulcers, gastric carcinoma
Cabot ring Circular, blue, threadlike inclusion
with dots
Nuclear remnant Postsplenectomy, hemolytic anemia,
megaloblastic anemia
HowellJolly bodies Small, discrete basophilic dense
inclusions; usually single
Nuclear remnant Postsplenectomy, hemolytic anemia,
megaloblastic anemia
Pappenheimer bodies Small dense basophilic granules Iron-containing siderosomes or
mitochondrial remnant
Sideroblastic anemia, postsplenectomy
Schistocytes (helmet cells) Distorted, fragmented cell, with 23
pointed ends
Mechanical distortion in the
microvasculature by brin
strands; damage by mechanical
heart valves
Microangiopathic hemolytic anemia,
prosthetic heart valves, severe burns
Spherocytes Spherical cell with dense appearance
and absent central pallor; usually
decreased diameter
Decreased membrane redundancy Hereditary spherocytosis,
immunohemolytic anemia
Stomatocytes Mouth- or cuplike deformity Membrane defect with abnormal
cation permeability
Hereditary stomatocytosis,
immunohemolytic anemia
Target cell (codocyte) Targetlike appearance, often
hypochromic
Increased redundancy of cell
membrane
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 asymptomatic
patients; it is used to follow the course of disease in patients
with lymphomas and collagen vascular diseases.
Leukocyte alkaline phosphatase score
The leukocyte alkaline phosphatase (LAP) enzyme is in the
granules of mature neutrophils. This enzyme reacts with an
added substrate, resulting in a colored precipitate. The scor-
ing of the stain intensity and granulation is called the LAP
score and is determined by counting the staining activity on
a scale of 04 in 100 neutrophils. Normal LAP scores range
from 15 to 130. LAP scores should be performed on fresh
capillary blood or samples anticoagulated with heparin
because there is rapid loss of alkaline phosphatase activity in
samples 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, granulocyte
colony-stimulating factor (G-CSF), granulocytemacro-
phage-colony stimulating factor (GM-CSF), lithium, and
corticosteroids.
Bone marrow aspirate and biopsy
Bone marrow aspirate and biopsy are commonly performed
in 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 the
sternum. In newborn and young infants, marrow aspirates
are often obtained from the anterior tibia. Good quality
smears require adequate spicule harvesting because peri-
spicular areas are the most representative areas to examine.
Hemophilia and other coagulation factor deciencies or
anticoagulation 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; unstained
smears should be retained and kept frozen in a dry environ-
ment for possible special stains. The bone marrow aspirate is
used for cytologic examination of the bone marrow cells and
for 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 and
embedded in parafn; 34-m sections are then cut and
stained with hematoxylin and eosin or Giemsa stains.
Plastic embedding does not require decalcication, and
enzymatic activity is better preserved, thereby allowing cyto-
chemical staining of the biopsy; thin cuts of 2 m can be
obtained. Because of the technical expertise required and
prolonged processing time, plastic embedding is less fre-
quently performed.
When examining pediatric marrows, it is understood that
erythroid hyperplasia is present at birth because of high lev-
els of erythropoietin. Lymphocytes may compose 40% of the
marrow 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 ferritin
and hemosiderin in nucleated red cells (sideroblastic iron) and
histiocytes (reticuloendothelial iron). Siderocytes containing
one or more blue-staining granules account for 2050% of the
cells. (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 lysosomal
granules stain faintly
Sudan black B Stains intracellular phospholipids and other lipids. Pattern of staining is similar to
myeloperoxidase.
Naphthol AS-D chloroacetate esterase
(specic esterase)
Granulocytes stain; monocytes do not stain. Can be used in biopsies to stain granulocytes and
mast 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 lymphocytes
Periodic acidSchiff (PAS) Detects intracellular glycogen and neutral mucosubstances. Positive staining in acute
lymphoblastic leukemia, acute myeloid leukemia, erythroleukemia, and Gaucher cells
Toluidine blue Detects acid mucopolysaccharides. Positive in mast cells and basophils
Tryptase Positive in mast cells, negative in basophils. Mast cells in systemic mast cell disease frequently
have a spindled shape
Reticulin Reticulin bers stain black
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Ringed sideroblasts are dened as nucleated red cells with
blue-staining iron granules surrounding at least two thirds
of 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 enzymatic
formation 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 into
immunocytochemical and immunohistochemical stains.
Immunocytochemical stains are used on marrow aspirates
and blood smears when ow cytometry specimens have not
been collected or when ow cytometry results are confusing.
The advantage of immunocytochemistry is the ability to cor-
relate morphology with phenotype. Immunohistochemistry
can be used to phenotype undifferentiated tumors, lympho-
proliferative disorders, and atypical lymphoid aggregates. In
patients whose marrow cannot be aspirated (dry tap),
immunohistochemistry can be performed on the biopsy sec-
tion. Immunohistochemistry can also be used on sections of
lymph nodes or other tissues when there is concern about
lymphoma or some other neoplastic disease.
In addition to determining cell lineagefor example, dif-
ferentiating hematopoietic neoplasms from nonhemato-
poietic neoplasms or lymphoid processes from myeloid
disordersimmunohistochemistry can also be used to help
determine prognosis. Immunohistochemical stains can be
used to delineate prognostic groups of diffuse large B-cell
lymphoma rst detected by gene microarray technology (see
Chapter 1). Gene microarray technology has been used to
subdivide 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 NGCB
phenotype. Gene microarray technology is not currently
practical for most clinical laboratories, but studies have shown
that differentiation of diffuse large B-cell lymphomas into the
GCB and NGCB phenotypes can be done by immunohisto-
chemistry using antibodies to CD10, BCL-6, and MUM1.
Preparation of bone marrow samples for
ancillary studies
Bone marrow collected in EDTA is adequate for both ow
cytometry and molecular analysis. Sodium heparin is suitable
for ow cytometry but may contaminate DNA preparations
and interfere with endonuclease digestion or the polymerase
chain reaction (PCR). Bone marrow collected for cytogenetic
studies should be collected in a sterile tube containing tissue
culture 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 are
better. Marrow samples can be frozen in liquid nitrogen for
ow cytometry and molecular analysis. However, because
ow cytometry requires viable cells, freezing and thawing of
samples may yield less than optimal results. Parafn- embedded
tissue can be used for PCR of genomic DNA sequences.
Reverse transcription PCR (RT-PCR) assays require that RNA
preparations be performed as early as possible to prevent
digestion by ubiquitous nucleases; alternatively, the RNA can
be isolated from cell aliquots stored in frozen nitrogen.
Ancillary testing
Flow cytometry
The most common applications of ow cytometry in hema-
tology include the detection of cellular proteins using
uorescent-labeled monoclonal antibodies or assessing DNA
content using DNA-binding dyes.
Flow cytometry is used for phenotyping populations of
cells, enumerating early progenitors for stem cell transplants,
detecting minimal residual disease, detecting targets for
immunotherapy, and assessing the presence of prognostic
markers. See Table 17-3 for a summary of clinical uses of ow
cytometry in ancillary studies. When immunophenotyping a
cellular population, the panel of antibodies used depends on
the cells being analyzed and the question being asked.
Gating is necessary to identify cells of interest in a mixed
population of cells. Three major leukocyte populations
(lympho cytes, monocytes, and neutrophils) can be dened
using 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 can
be separated based on the intensity of staining they display
with the conjugated antibody that is classied as either bright
or dim. Lymphocytes are bright CD45 and have a low SS sig-
nal, neutrophils are dim to moderately bright CD45 and have
a high SS signal, and monocytes are bright CD45 and have an
intermediate SS.
Flow cytometry can also be used to detect populations of
natural 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 cytometry
can also be used to detect prognostic markers. For example,
ow cytometric analysis of the tyrosine kinase, ZAP-70, can
be 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 from
pregerminal center cells, and patients with pregerminal cen-
ter CLL have a decreased overall survival when compared
with patients with CLL arising from postgerminal center
cells. Positivity for CD38 by ow cytometric analysis is also
correlated with unmutated DNA, but the correlation is not
as 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 then
followed by CD15 and CD11b. CD16 and CD10 are seen in
late maturation.
Appearance of CD71, loss of CD34 and CD33, and
decreased expression of CD45 characterizes erythroid
maturation. With further differentiation, CD71 expression
decreases, glycophorin expression increases, and CD45
disappears.
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 megakaryocyte
differentiation.
B- and T-cell precursors express TDT, HLA-DR, and
CD34. B-cell differentiation is indicated by the expression of
CD19 followed by CD10. Surface immunoglobulin is com-
posed of and light chains. A predominance of one type is
known as light chain restriction and is indicative of a mono-
clonal process. CD34 and CD10 expression cease by the time
immunoglobulin M (IgM) is expressed at the cell surface.
Expression of surface IgM is associated with the expression
of 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 cytoplasmic
CD3 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 through
17-10 for useful CD markers.)
Cytogenetics
Cytogenetic analysis can be performed from cultured (indi-
rect) and uncultured (direct) preparations. In the indirect
assay, cells are grown so that mitotic forms can be visualized
and 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 FISH
Molecular 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, etc
FISH
Molecular genetics (clonality, specic markers such as BCL2, BCL6, etc)
Immunohistochemistry (phenotyping, prognostic markers)
Myeloproliferative disorders Cytogenetics
FISH (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 uorescence
in 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 of
karyotyping abnormal cells and are particularly useful when
analyzing mature B- or T-cell processes.
Cytogenetically, a minimum of 2 mitotic cells with gain of
the 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, associated
with either congenital genetic syndromes or normal variants,
are determined on peripheral blood T lymphocytes grown in
culture with phytohemagglutinin (PHA), a T-cell mitogen.
Fluorescence in situ hybridization (FISH) employs specic
uorescently 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 cells
CD34 Progenitor cells, endothelium
CD38 Myeloid progenitors, T, B, NK cells, plasma cells, monocytes, CLL subset
B-cell markers
CD10 Pre-B lymphocytes, germinal center cells, neutrophils
CD19 B cells (not plasma cells or follicular dendritic cells)
CD20 B cells (not plasma cells)
CD21 Mature B cells, follicular dendritic cells, subset of thymocytes
CD22 Mature B cells, mantle zone cells (not germinal center cells)
CD23 B cells, CLL
CD79b B cells (not typical CLL)
CD103 Intraepithelial lymphocytes, hairy cell leukemia, T cells in enteropathic T-cell lymphoma
FMC7 B cells (not typical CLL), hairy cell leukemia
T-cell markers
CD2 Pro- and pre-T cells, T cells, thymocytes, NK cells, some lymphocytes in CLL and B-ALL
CD3 Thymocytes, mature T cells, cytoplasm of immature T cells
CD5 Thymocytes, T cells, B cells in CLL, B cells in mantle cell lymphoma
CD4 Helper T cells, monocytes, dendritic cells, activated eosinophils, thymocytes
CD7 Pro- and pre-T cells, T cells, thymocytes, NK cells, some myeloblasts
CD8 Suppressor T cells, NK cells, thymocytes
CD25 Activated T and B cells, adult T-cell leukemia/lymphoma
NK/cytotoxic T-cell markers
CD16 NK cells, monocytes, macrophages, neutrophils
CD56 NK cells, myeloma cells
CD57 NK cells, T-cell subset
Myeloid and monocytic markers
CD13 Monocytes, neutrophils, eosinophils, and basophils
CD14 Monocytes, macrophages, subset of granulocytes
CD33 Myeloid lineage cells and monocytes
CD117 Immature myeloid cells, AML
Monocytes
CD11c Monocytes, macrophages, granulocytes, activated B and T cells, NK cells, hairy cell leukemia
CD15 Myeloid lineage cells and monocytes
CD64 Monocytes, immature myeloid cells, activated neutrophils
Megakaryocytic markers
CD41 Platelets and megakaryocytes (GPIIb)
CD42 Platelets and megakaryocytes (CD42a: GPI; CD42b: GPIb)
CD61 Platelets, megakaryocytes, endothelial cells (GPIIb-IIIa)
Erythroid markers
CD71 Transferrin receptor is up-regulated upon cell activation
CD235a 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 CD8
Mature 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
, CD103
MCL
MZL / /
FCL 60% BCL2
LCL 10% 2550% / / / BCL2

in

3040%
Burkitt
Myeloma Occ 1520% 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 interphase
cells that are afxed to a microscope slide. FISH can be
accomplished 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 or
structural rearrangements, such as translocations. Transloca-
tions and deletions can also be identied in interphase or
metaphase by using genomic probes that are derived from
the breakpoints or recurring translocations or within the
|
deleted segment. Multiplex FISH (spectral karyotyping)
consists of simultaneously painting all chromosomes in a cell
using different colors for each chromosome.
Cytogenetics is particularly useful in the subclassication
of acute myeloid leukemias and in conrming the diagnosis
and prognosis of B-cell neoplasias. Chronic lymphocytic leu-
kemia, acute leukemias, B-cell lymphomas, and multiple
myeloma all have cytogenetic abnormalities that can be
detected using either conventional cytogenetics or FISH.
Molecular diagnostics
Southern blotting
Southern blot analysis begins with the extraction of high-
molecular-weight (HMW) DNA, followed by its digestion by
restriction 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 containing
labeled single-stranded DNA probes. The probe hybridizes
to the spot on the paper where its complementary strand is
found. Unbound DNA is washed away, the lter is exposed to
photographic lm, and the lm reveals the position and size
of the electrophoresed fragments.
This technique is slow and has been largely replaced by
PCR. Southern blot analysis is used when the genomic frag-
ments being investigated are large and the precise location of
the marker sequence is unknown.
Southern blotting has been used on peripheral blood and
bone 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 of
a specic target DNA sequence within total genomic DNA or
a complex complementary DNA (cDNA) population. Partial
DNA sequence information from the target sequences is
required. Two oligonucleotide primers, which are specic for
the target sequence, are used. The primers are added to dena-
tured single-stranded DNA. A heat-stable DNA polymerase
and the 4 deoxynucleotide triphosphates are used to initiate
the synthesis of new DNA strands. The newly synthesized
DNA strands are used as templates for further cycles of
amplication. The amplied DNA sequence can be detected
by electrophoresis on an agarose gel, and visualization can be
accomplished by the use of a DNA dye; alternatively, the
amplied DNA can be directly sequenced in an automatic
sequencer.
Quantitative real-time PCR is based on detection of a
uorescent signal produced proportionally during the ampli-
cation of a PCR product. Forward and reverse primers are
extended with Taq polymerase as in a traditional PCR reac-
tion. A probe is designed to anneal to the target sequence
between the forward and reverse primers. The probe is labeled
at 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 Reed
Sternberg.
Table 17-9 Common mature T-cell and NK-cell neoplasms.
CD3S CD3C CD5 CD7 CD4 CD8 CD30 CD16 CD56 EBV
T-PLL 48 48
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, not
otherwise specied; SC pannic T lym subcutaneous panniculitis T-cell lymphoma; T-LGL T-large granular lymphocyte leukemia;
T-PLL T-prolymphocytic leukemia.
| 453
As long as both uorochromes are on the probe, the quencher
molecule stops all uorescence from the reporter. As Taq
polymerase extends the primer, the 5 to 3 nuclease activity
of 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 in
each cycle.
Uses of PCR in clinical laboratories include detection of
the BCR-ABL translocation in chronic myeloid leukemia and
detection 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 when
the clinical setting calls for it, such as during episodes of clin-
ical bleeding or prior to a planned invasive challenge, if there
is a personal or family history of excessive bleeding or bruis-
ing. Testing for thrombophilia is usually performed after a
patient 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, and
planned use of this information in the future management of
the patient. A personal and family history of bleeding or
thrombosis and a list of current medications are an impor-
tant part of the evaluation, and appropriate laboratory
screening is usually directed by this history.
Generally, laboratory screening prior to surgery includes
a prothrombin time (PT), activated partial thromboplastin
time (aPTT), and a quantitative assessment of platelets. If
there is a history of excessive bleeding, bruising, or menor-
rhagia in women, then specic testing for von Willebrand
disease (vWD) is indicated. However, false-normal results
occur during acute stress. Laboratory testing for inherited
predisposition to thrombosis should be done after the
patient has completed treatment of a thrombotic episode.
The levels of normal circulating inhibitory factors can be
decreased during the acute phase of thrombosis. Protein C
and S levels are affected by anticoagulation with warfarin.
The antithrombin level will decrease with the use of unfrac-
tionated heparin. Lupus anticoagulant testing should be
ordered at the time of presentation before anticoagulation
is begun, followed by serologic assays (anticardiolipin
antibody and antibody to
2
-glycoprotein I can be per-
formed at any time). Molecular diagnostic testing can be
ordered at any time and is not affected by clinical status or
medications.
Coagulation testing
An outline of the traditional extrinsic and intrinsic pathways
is 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 occur
between the extrinsic and intrinsic pathways. Although the
division 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 tests
when evaluating a clinical problem.
Contact factors: XII, XI,
prekallikrein, HMW kininogen
aPTT
Prothrombin
time
Extrinsic Intrinsic
XII
XI
IX
VIII
VII
"Tenase"
Ca
2+
, PL
V, Ca
2+
, PL
Prothrombin Thrombin
Tissue factor
"Prothrombinase"
Thrombin
time
Fibrin clot
XIII
Fibrinogen
X
Figure 17-1 Simplied coagulation cascade
indicating the intrinsic pathway measured by
the activated partial thromboplastin time
(aPTT), the extrinsic pathway measured by
the prothrombin time (PT), and the
conversion of brinogen to brin measured
by the thrombin time. Prekallikrein and
high-molecular-weight (HMW) kininogen
are not considered coagulation factors, but
they can prolong the aPTT if they are
decient.
|
Overview of key points
Mixing studies
Specic factor assays
Screening studies
PT
Factor VII deciency
International normalized ratio (INR)
aPTT
Deciencies of factors VIII, IX, XI, XII, prekallikrein,
HMW kininogen
Lupus anticoagulant testing
Inhibitors to factor VIII and other intrinsic pathway
factors
Heparin and monitoring heparin
Combined abnormalities of PT and aPTT
Thrombin time
Fibrinogen assays
von Willebrand factor (vWF) assays
Activity and antigen assays
Ristocetin-induced platelet aggregation (RIPA)
vWF multimers
Disseminated intravascular coagulation (DIC)
Fibrinolysis
Evaluation of platelets
Quantitation
Platelet aggregation
Platelet function analyzers
Bleeding time
Platelet antibodies
Assays for heparin-induced thrombocytopenia (HIT)
Assays for thrombotic thrombocytopenic purpura (TTP)
Evaluation of bleeding patients with normal screening tests
Thrombophilia
Protein C, protein S, antithrombin
Factor V Leiden and activated protein C resistance (APCR)
Prothrombin 20210 mutation
Dimerized plasmin fragment D (D-dimer) and deep
venous thrombosis (DVT) evaluation
Lupus anticoagulants and thrombosis
1:1 mixing study
Traditionally, when a screening test is prolonged and the cause
is not apparent, the next study performed is a 1:1 mixing
study with patient plasma and pooled normal plasma (PNP).
The clotting test is usually repeated within 5 minutes of the
mix (immediate time point). For a prolonged aPTT, the
mixing assay should be performed immediately after the mix
and again after a 1-hour or, optimally, 2-hour incubation at
37C to detect a progressive inhibitor (usually progressive
inhibition indicates a factor VIII inhibitor).
A lack of correction or only partial correction in the aPTT

mixing 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 seconds
of the PNP or as outside the reference range of the test in the
laboratory performing the test. These criteria have not been
standardized. Nevertheless, this then leads to testing for a
lupus anticoagulant and to specic intrinsic pathway factor
assays to determine if a specic factor is inhibited. A correc-
tion to the normal range generally indicates that the patient
has a deciency of a factor, but a low afnity or low titer
inhibitor may give a correction in a mixing study, and tests
for a lupus anticoagulant and for specic factors are usually
still necessary.
aPTT s that are only slightly prolonged (24 seconds)
often may correct, and clinical judgement is necessary in
deciding how much laboratory evaluation is necessary. The
clinical history and the clinical setting helps to determine
how to proceed (eg, a patient who is to undergo brain sur-
gery has need for more extensive evaluation than one who is
to have an accessible lymph node excised).
Specic factor assays
Specic factor assays are performed using substrate plasmas
that are decient in the factor being studied. These are then
mixed with the patient plasma to see if abnormalities in the
PT or the aPTT correct. Most normal adults have levels of
each 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 assays
are based on detection of clot formation, and they have been
automated; the instruments use either a mechanical end
point or a change in the optical density to detect the clot.
Chromogenic assays are available for some factors such as
factor VIII, but they are not readily available. The activated
clotting factor cleaves a small chromogenic substrate that is
added to the reaction; the chromogenic substrate generates a
color change that is used for detection. Chromogenic assays
can be more precise than clotting assays, but they may not
detect some defects in a factor that disrupt the binding of the
factor to its natural (larger) substrate.
Expected ranges in pediatric patients
Many coagulation factors are abnormally low in the newborn
compared with adult normal ranges. Of the screening tests, the
aPTT is prolonged, whereas the PT, brinogen, and thrombin
time are normal (the latter should be measured with calcium
in the reagents to prevent prolongation by fetal brinogen).
The brinogen rises during the rst week after birth and then
returns to the normal range. The vitamin K-dependent
| 455
proteins, including factors II, VII, IX, X, protein C, and protein
S, are decreased (often in the 30% range) and gradually rise to
the normal adult range by 6 months, with the exception of pro-
tein C, which remains below the adult normal range until at
least 5 years of age. Vitamin K is administered on a routine basis
shortly after birth to prevent vitamin K deciency during the
rst week(s) of life. Factor VIII levels are normal in the newborn,
and vWF levels are increased; there are also unusually HMW
vWF 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 (and
markedly so in premature infants) and rise to the adult range at
6 months. Antithrombin and plasminogen levels are also low at
birth and reach normal levels by 6 months of age.
Prothrombin time
The PT measures the extrinsic pathway and nal common
pathway 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 thromboplastin
reagent (tissue factor, a lipoprotein normally present in
membranes of perivascular broblasts and activated endo-
thelial cells) and calcium chloride to citrated plasma and
measuring the time to clot formation.
Prolongation of the PT most often reects a deciency of 1
or more of the vitamin K-dependent factors due to poor
nutrition, inadequate absorption of vitamin K, or decreased
hepatic synthesis of coagulation factors from liver disease;
congenital deciencies of factors II and X are very rare (1 in
12 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 the
postsynthetic carboxylation of the vitamin K-dependent
factors, 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 prolonged
PTs 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 accompanying
liver disease is not uncommon because of the short half-life
of factor VII (t
1/2
is 46 hours). A specic assay using factor
VII-decient substrate plasma and a PT method is available.
Inhibitors of factor VII are extremely rare.
Argatroban and lepirudin are monitored by the aPTT but
will 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 characterized
by a short half-life but may be prolonged in the acutely ill
patient. Continued effect on the coagulation testing results
may 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 and
make the comparability of the assay results better between
laboratories. The INR is a calculation that represents the
ratio of the patients PT to the laboratorys mean normal PT
as though the test had been performed with an international
reference preparation (IRP) of thromboplastin. The com-
mercial supplier calibrates the reagent to the IRP and assigns
an International Sensitivity Index (ISI) to it. The laboratory
uses 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 is

designed to follow patients who have been stabilized on a
dose of coumadin and to make it possible for the patient to
have PTs measured in different laboratories and still main-
tain correct dosing. Instrument variables are also important
and 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 of
warfarin and is not intended for assessing factor deciencies
Mixing study
Not corrected Corrected
Lupus anticoagulant
testing
Deficiency of factor:
perform specific
assays for factor VII
(also for X, V, II if
APTT is prolonged)
Positive Negative
Lupus
anticoagulant
present
Inhibitor of clotting factor:
perform factor VII assay;
if low perform inhibitor assay
Evaluation of prolonged
prothrombin time
Figure 17-2 Algorithm for evaluation of an isolated prolonged
prothrombin time (PT).
|
due to congenital deciency or liver disease because the ISI is
not tested for nonvitamin K-dependent proteins, particu-
larly factor V and brinogen. Thus, it should not be used for
this purpose because its sensitivity cannot be predicted.
Lupus anticoagulants are a common cause of a prolonged
aPTT and are not infrequent in both hospitalized patients
and ambulatory patients. When a 1:1 mixing study for a pro-
longed aPTT does not shorten to the normal range, a test for
a lupus anticoagulant should be performed (a lupus anti-
coagulant should also be suspected in some cases when the
aPTT mix is corrected due to a low titer or an atypical lupus
anticoagulant). Testing is also ordered based on clinical
presentation.
Several types of tests are available; they all have in com-
mon the use of a limiting concentration of phospholipid in a
rst step and the provision of excess phospholipid in a sec-
ond step that corrects the clotting end point in a patient with
a lupus anticoagulant, conrming the diagnosis. Because
platelets 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 or
performing a hard spin (5000g) before testing and before
freezing if the test is to be done later.
Lupus anticoagulants are a family of antibodies directed
against different antigens, and they are not positive in all
assays. Included in the strict denition of a lupus anticoagu-
lant, as opposed to an antiphospholipid antibody, is the
demonstration of a prolonged clotting assay that is corrected
by provision of excess phospholipid. Many laboratories use
several different assays to avoid missing the antibody. Tests
for the lupus anticoagulant include the dilute Russell viper
Prolonged PT:
Poor nutrition ([darrow] vitamin K)
Malabsorption of vitamin K
Liver disease ( synthesis of vitamin K-dependent factors)
Coumarins
Rare congenital deciency or inhibitor (factor VII)
Key points
Activated partial thromboplastin time
The aPTT measures the intrinsic pathway factors (contact
system factors, factors XII, XI, IX, and VIII) and the nal
common 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 some
systems ellagic acid is used), and calcium chloride to citrated
plasma and measuring the time to clot formation. The acti-
vator and the phospholipid are incubated with the plasma
for approximately 4 minutes, during which time factors XIIa
and XIa are formed; calcium chloride is then added, which
permits activation of factor IX and the remaining reactions
to 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 XII or deciency of other
proteins in the contact activation pathway, prekallikrein and
HMWK, prolong the aPTT. The 2 latter proteins, along with
factor XII, do not cause a bleeding disorder but do lengthen
the aPTT. Depending on the aPTT method used, in order for
a specic factor deciency to prolong the aPTT, its level is
usually decreased to 3040%. Factor assays using specic
decient substrate plasmas and based on an aPTT method
are applicable for measuring these factors; a chromogenic
assay for factor VIII is also available.
Factors VIII and IX deciencies or hemophilia A and B are
X-linked inherited disorders that are often diagnosed early in
life but may not be identied until adulthood if there is a
mild deciency (540%).
vWD may manifest with a slightly prolonged aPTT if the fac-
tor VIII level is at the sensitivity level of the laboratorys
aPTT reagent.
Deciency of factor XI should be suspected with a prolonged
aPTT in a person of Jewish background. It can be associated
with bleeding complications.
Mixing study
Not corrected Corrected
Lupus anticoagulant
testing
perform
long incubation APTT
specific assays for
factor VIII, IX, XI, XII
Positive Negative
Lupus
anticoagulant
present
Inhibitor of clotting factor. Perform:
specific factor assay for VIII, IX, XI, XII
inhibitor assay for factor that is
decreased
Evaluation of
prolonged APTT
Figure 17-3 Algorithm for evaluation of an isolated prolonged
activated partial thromboplastin time (aPTT).
| 457
venom time (dRVVT), the aPTT, the kaolin clotting time, the
dilute PT, and others. The dRVVT, also called the Stypven
(snake venom) time, is a phospholipid-based reagent that in
the dilute form is particularly sensitive to the lupus antico-
agulant. Kaolin is another reagent that has been found, in
some laboratories, to be sensitive to the lupus anticoagulant.
In these assays, the phospholipid component that is required
for clot formation is added initially in limiting amounts; it is
bound by the patients lupus anticoagulant antibody in the
assay, resulting in a prolonged time needed to form a clot.
Step 2 is then done with added excess phospholipid to over-
whelm the lupus anticoagulant, leading to a correction of
the prolonged clotting time. One must also be certain that a
factor deciency is not the cause for a prolonged test.
As an example, if a prolonged dRVVT is found, a mixing
study is performed. If the 1:1 mix does not correct, it suggests
the presence of an inhibitor. The conrmatory step is then
done on the 1:1 mix; increased phospholipid is added to a
repeat assay so the phospholipid is no longer limiting the clot
formation. If a lupus anticoagulant is present, the added
phospholipid will result in a shortening of the dRVVT to the
normal range.
In the situation where a mixing study (1:1 with normal
plasma) corrects without excess phospholipid, it suggests a
factor deciency or deciencies. Inherited deciency of fac-
tors X, V, or brinogen (and rarely factor IX), warfarin ther-
apy, and liver disease, for example, can result in a prolonged
dRVVT that corrects with a 1:1 mix but does not correct with
added phospholipid alone. Heparin will also prolong the
dRVVT, but it will not correct with either excess phospho-
lipid or mixing with normal plasma. Deciencies of other
intrinsic factors can prolong the lupus anticoagulant tests
that are based on the aPTT; these, also, will not correct with
excess phospholipid.
Lupus anticoagulants may prolong the clot-based test used
for specic factors assays and can give a false result suggest-
ing deciency of specic factors. However, if one measures a
minimum of 3 dilutions of the patient plasma in the specic
factor assays (eg, 1/10, 1/20, 1/40), one usually sees that each
dilution yields a higher level of the factor due to a diluting
out of the lupus anticoagulant.
Lupus anticoagulants may also prolong the PT by 12
seconds, but if the PT is more prolonged, evaluation for fac-
tor deciency may be necessary. Antiprothrombin anti bodies
coexist with lupus anticoagulants and can result in serious
bleeding. The baseline prolongation of the PT in patients
with lupus anticoagulants may also cause problems with the
correct dosing of coumarin anticoagulants in those patients
requiring them. A chromogenic factor X assay has been used
as a measure of coumadin effects on the vitamin K- dependent
clotting factors to select the dose of coumadin in these
patients, but it is not performed routinely in most labs.
An alternative is to measure factors II, VII, IX, and X in a

PT system that is not sensitive to the effects of the lupus
anticoagulant.
Inhibitors to factor VIII are detected in 2530% of patients
with severe hemophilia A. The patient develops alloanti-
bodies to the foreign factor VIII. Acquired hemophilia caused
by autoantibodies to factor VIII is the most common acquired
inhibitor to a specic coagulation protein (lupus anticoagu-
lants are the most common inhibitor causing prolonged
clotting times). A factor VIII antibody is suspected in the
patient who has a prolonged aPTT that fails to correct with
1:1 mixing and/or prolongs 45 seconds after a further
12-hour incubation of the mixture at 37C. A low factor
VIII level is also detected by the specic factor assay. The test
for an inhibitor to factor VIII, based on the factor VIII assay,
is then performed. The antibody titer is measured in Bethesda
units (BU); 1 BU is the amount of antibody that inhibits
50% of the factor VIII present in a normal plasma sample
after incubation with an equal volume of the patients plasma
for 2 hours at 37C (or, the reciprocal of the highest dilution
of patient plasma that causes the 50% inhibition). The inhib-
itor may be apparent only after prolonged incubation because
it may be directed against a protected site in factor VIII.
Inhibitors to other factors in the intrinsic pathway may also
develop in patients with deciencies of factors IX or XI who
are receiving replacement therapy with the needed factor
because these patients are at risk for inhibitor development.
Inhibitors occur to a lesser extent in patients with hemophilia
B as compared with hemophilia A. Inhibitors have also been
reported to occur 33% of the time in patients with factor XI
levels of 1%, but they rarely occur spontaneously in patients
without a deciency and factor replacement therapy.
Heparin causes a prolonged aPTT and can be present in
the central lines used for blood sampling. Even after discard-
ing the initial blood drawn through a central line, there is
commonly sufcient heparin present to prolong the aPTT. A
direct peripheral venipuncture is required in these cases to
exclude heparin as a cause of the prolonged aPTT. Heparin
used in prophylactic doses may also cause a prolonged aPTT.
Heparin can be detected by noting a concomitant prolonga-
tion of the thrombin time. Heparin-absorbing laboratory
reagents can differentiate heparin contamination as a cause
of prolonged aPTT but may not be readily available. The rep-
tilase time, also done in limited places, is normal in the pres-
ence of heparin (see discussion later in the chapter) and is
another way to appraise this problem.
LMW heparin will also prolong the aPTT, but it is not a
linear relationship and therefore cannot be used for moni-
toring. LMW heparin was developed to be given in a weight-
based dosing regimen that will not require monitoring.
However, measuring the anti-Xa activity (chromogenic
assay) of the drug is recommended in pediatric and pregnant
|
patients as well as those with impaired renal function or
those with increased body mass (144 kg).
The aPTT is used to monitor standard heparin therapy. A
therapeutic 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 the
heparin level and is particularly helpful if there appears to be
heparin resistance when excessively large doses of heparin
are needed to prolong the aPTT to the therapeutic range or if
the patient has a lupus anticoagulant that prolongs the base-
line aPTT. The therapeutic range for LMW heparin was
based on specimens obtained approximately 4 hours after
the therapeutic dose of LMW heparin has been administered
and is often considered to be 0.61.0 anti-Xa IU/mL. The
aPTT 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 can
cause 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. Decreased
hepatic synthesis from advanced liver disease will cause de-
ciency of all coagulation factors except for factor VIII. This
will often manifest rst with a prolonged PT. Dysbrinogen-
emia may also be acquired in hepatic disease and is suggested
by nding a low level of brinogen in a functional assay com-
bined with a normal or disproportionately high level of
immunologic 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, can
be seen after exposure to bovine thrombin (it also contains
bovine factor V), which is part of brin glue; this product
is used relatively frequently for hemostasis in orthopedic,
cardiac, and urologic surgery, as well as neurosurgery. The
antibody may cross-react with human factor V and can cause
life-threatening bleeding. A low level of factor V and an
inhibitor of factor V will be found in specic tests. Products
that contain human thrombin have become available, and
they do not usually cause antibodies to factor V.
Inhibitors to prothrombin can be associated with lupus
anticoagulants and may lead to bleeding if prothrombin lev-
els are sufciently low. These antibodies usually cannot be
Prolonged aPTT:
Lupus anticoagulant
Factors 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 evaluation
of a prolonged prothrombin time (PT) and
activated partial thromboplastin time
(aPTT).
No clinical bleeding Clinical bleeding
If thrombin time long
check protamine SO
4
neutralization
Not
normalized by
protamine SO
4
Mixing studies
using both PT
and APTT assays
Fibrinogen level,
FDP and D-dimer
platelet count
Normalization of
thrombin time =
heparin
contamination
Not
corrected
Corrected fibrinogen
+FDP and D-dimer
platelet count
DIC. Follow platelet
count and fibrinogen
(may perform factor
assays for status: V,
VIII)
perform assays for
factor X, V, II
(possibly VIII, IX, XI)
Lupus anticoagulant
testing
Positive Negative
Lupus
anticoagulant
present
Possible inhibitor of clotting factor. Perform:
specific factor assays for factor X, V, II
inhibitor assay for factor that is decreased
| 459
detected in in vitro mixing studies because they are not
directed against the active site of the molecule. Rather, they
form a complex with other antigenic sites on prothrombin
and are cleared from the circulation as immune complexes,
lowering the prothrombin level.
tumors and after suramin administration. They prolong the
thrombin time by interacting with antithrombin in a man-
ner similar to heparin. The reptilase time will be normal in
these patients.
Prolonged PT and aPTT:
Factors X and V or prothrombin deciency
Hypo- or dysbrinogenemia
Liver disease ( hepatic synthesis of factors)
Coumarins and vitamin K deciency
Inhibitor to factor V or prothrombin
Key points
Thrombin time
The thrombin time measures the time of conversion of
brinogen 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 human
thrombin, and the normal reference range in each laboratory
is 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 contaminated
with heparin, and very small quantities, less than the amount
required to prolong the aPTT, will prolong the thrombin
time.
Dysbrinogenemias usually prolong the thrombin time and
are suspected if the functional test (clottable brinogen) is
disproportionately low compared with an immunologic
measurement of brinogen.
Hypobrinogenemia usually prolongs the thrombin time
when levels of brinogen are below approximately 5070
mg/dL. Therapeutic doses of L-asparaginase can cause signi-
cant hypobrinogenemia by inhibiting synthesis.
Inhibitors to thrombin occur uncommonly, but antibodies
to 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 cardiac
surgery). The antibody to bovine thrombin usually does not
cross-react with human thrombin. The thrombin time will
be prolonged when the assay reagent is bovine thrombin,
and it will be normal when human thrombin is substituted
in the assay. These antibodies are of no clinical consequence
and usually disappear after several weeks to months.
Fibrin degradation products (FDPs) in very high levels can
inhibit the thrombin time.
Heparin-like anticoagulants (heparan sulfates) have
occurred in patients with multiple myeloma and other
Prolonged thrombin time:

Heparin, lepirudin, or argatroban in plasma sample
Hypo- or dysbrinogenemia
Inhibitor of thrombin
Heparin-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 modied
thrombin 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 present
in the clot that results from the addition of thrombin to
plasma.
Reptilase time
Reptilase is a snake venom that cleaves brinopeptide A
directly 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 that
can be difcult to diagnose because of the overlap of values
in normal subjects and patients. The variable levels of vWF
in patients with different blood groups and during physio-
logic alterations associated with acute phase reactions or the
menstrual 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 in
normal subjects with blood group O (approximately 25%
lower as compared to normal subjects with types A, B, and
AB), some laboratories prepare a separate reference range for
patients with blood group O.
Although the bleeding time has been used as a screening
test for vWD, it is too insensitive and nonspecic, and it is no
longer widely used for the diagnosis of vWD. The factor VIII
level, which is decreased in moderate and severe vWD due to
lack of protection for circulating factor VIII by vWF, is also
variable and is helpful in the diagnosis of vWD only when it
is abnormal (see Table 17-11 for vWF assays).
|
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, and
cause platelet agglutination in the presence of ristocetin (ris-
tocetin alters vWF to allow binding to platelet membrane
GPIb). It is performed by mixing different dilutions of patient
plasma (the source of vWF) with washed normal platelets or
platelet membranes that are devoid of vWF, adding ristocetin
at a nal concentration of approximately 1.2 mg/mL, and
observing the time to agglutination or aggregation. A stan-
dard curve is executed by using dilutions of a normal plasma
pool in the same manner, and the normal range, dened in
each laboratory, is usually near 45150%. The exceptions are
individuals with blood type O who may range as low as
approximately 35%; in these cases, it is prudent to repeat the
test 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 on
wells of an enzyme-linked immunoadsorbent assay (ELISA)
plate. The bound vWF is measured by specic antibodies to
vWF.
vWF antigen measures the quantity of plasma vWF by
immunologic 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 ranges
for these individuals.
There are 3 types of vWD: type I, a quantitative decrease
in 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 to

classify the vWD type once a diagnosis has been established
using the vWF activity and antigen assays (see Table 17-12).
These tests are also performed if the suspicion for vWD is
still 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 test
whether the patient might have a qualitative defect in vWF
that 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-rich
plasma (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 aliqouts
of 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, whereas
patients with type IIB vWD will aggregate at 0.40.6 mg/mL.
Pseudo-vWD or platelet-type vWD, an abnormality in the
platelet 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 platelet
GPIb in the presence of ristocetin with consequent
aggregation
Ristocetin cofactor activity: quantitates platelet agglutination
after addition of ristocetin and vWF
Ability of vWF to bind to collagen Collagen binding activity: quantitates binding of vWF to
collagen-coated plates
vWF antigen vWF protein as measured by protein assays; does not
measure functional ability
Immunologic assays such as ELISA, RIA, and Laurell
electroimmunoassay
vWF multimers Size distribution of vWF multimers as assessed by agarose
gel electrophoresis
vWF multimer assay: electrophoresis of plasma in low-
concentration agarose gel and visualization by monospecic
antibody to vWF
RIPA Measures the ability of patient vWF to bind to platelet
receptor GPIb in the presence of variable concentrations
of ristocetin
RIPA: aggregation of patient platelet-rich plasma with decreasing
concentrations 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 intermediate
Type IIB Large
Type IIM Uniform
Type IIN Normal Normal Normal Normal
Type III Undetectable
RIPA ristocetin-induced platelet aggregation;
vWD von Willebrand disease.
| 461
binding in the RIPA at low ristocetin concentrations and is
phenotypically indistinguishable from type IIB vWD. Mix-
ing studies using normal washed platelets plus patient
plasma, or vice versa, can be used to distinguish whether the
patients vWF or platelet receptor is abnormal.
vWF multimers. Plasma vWF is composed of different-sized
multimers that can be visualized in low concentration aga-
rose gels after electrophoresis and detected using an antibody
to vWF. The absence of larger multimers signies a qualita-
tive defect and aids in classifying the type of vWD in the
patient. This test can also detect larger than normal multim-
ers that occur in 65% of normal neonates, in another vWD
subtype (Vicenza), and in some patients with TTP.
It is particularly important to evaluate carefully a female
who 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 the
patients vWF has an abnormal binding site for factor VIII,
leading to the rapid clearance and decreased plasma level of
factor VIII. Type IIN vWD is evaluated functionally using a
binding study that tests the patients vWF for its ability to
bind 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 factor
VIII can also be found (rarely) in carriers of hemophilia A
who have skewed lyonization of their X chromosome and in
females with XO phenotypes.
The diagnosis of vWD can be very problematic during the
second and especially the third trimester of pregnancy, when
the vWF level normally increases 2 to 3 times above its base-
line level. In these cases, a careful personal and family history
and family testing can be helpful. However, a denitive diag-
nosis cannot usually be made with condence until 46
weeks after delivery.
The diagnosis of acquired vWD, often associated with
autoimmune or lymphoproliferative diseases, is suggested by
a history of recent onset mucous membrane or skin bleeding
without a past or family history of bleeding and by a pro-
longed aPTT on screening studies. The latter is due to a
decreased factor VIII associated with low levels of vWF. The
ristocetin cofactor is usually abnormal, and multimer studies
usually show a decrease in the HMW multimers (a type IIA
or B vWD pattern). Mixing studies with patient and normal
plasma do not consistently demonstrate an inhibitor, and
further 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 coagulation
screening tests (PT, aPTT, thrombin time, and brinogen)
may be normal early in the course of acute DIC or during
chronic DIC. With hemorrhagic DIC when substantial con-
sumption has occurred, the PT, aPTT, thrombin time, and
brinogen are usually all abnormal, and factor levels are
decreased. Fibrin/brinogen degradation products (FDP/
Fdps) and D-dimer (a cross-linked degradation product of
brin) are elevated. Advanced liver disease has a similar
coagulation prole to that seen in DIC, and the 2 disorders
can be difcult to distinguish without clinical correlation.
Fibrinolysis
The activity of the brinolytic system does not have readily
available screening assays for assessment; the euglobulin clot
lysis is a global test that was once used but is no longer readily
available in detecting activation of brinolysis. Other tests
include FDPs, D-dimer tests, and specic assays for plasmin-
ogen, tissue plasminogen activator, plasminogen activator
inhibitor 1 (PAI-1), and plasminantiplasmin complex. Plas-
minogen and related proteins (discussed previously) can be
measured in functional or antigenic assays.
2
-Antiplasmin is an important inhibitor of plasmin, and
congenital deciency of this protein can lead to serious
bleeding. Symptoms typically occur without warning during
invasive procedures because deciency of
2
-antiplasmin
does not cause abnormalities of the preoperative screening
coagulation tests.
2
-Antiplasmin can be measured in a func-
tional assay.
Primary brinolysis, activation of plasminogen to plasmin
with 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 (as
seen in DIC). However, circulating plasminogen activators
have 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 is
normal (unless the underlying disease causes a decrease in
platelets).
ELISA assays are sensitive assays of hypercoagulable states
that can detect subclinical activation of coagulation, such as for
activation peptides of coagulation factors and products of plate-
let or endothelial cell activation. They are used only for clinical
research studies rather than as an aid in clinical management;
examples are ELISA assays for circulating prothrombin frag-
ment 1.2, thrombinantithrombin complex, brinopeptide A
and B, platelet factor 4, and thrombomodulin.
Quantitation of platelets
Platelet numbers are quantitated accurately by automated cell
counters that depend on electrical impedance or light scatter.
|
Other methods for quantitating platelets and potential artifacts
that lead to falsely low platelet counts are described previously.
Evaluation of platelet function
Platelet aggregation
Platelet aggregation, a very sensitive and time-consuming
test, has been the primary method available for evaluation of
platelet function until recently. In this test, either citrated
whole blood or PRP is prepared, and agonists such as adenos-
ine diphosphate (ADP), epinephrine, collagen, arachidonic
acid, or thrombin are added. In the PRP system, the agonists
are added to PRP and stirred to initiate platelet aggregation;
the formation of platelet aggregates causes an increase in
light transmission through the PRP as the aggregates fall to
the bottom of the tube. The change in light transmission is
recorded by a mechanized recording instrument. The platelet
release 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 platelet
release reaction is the only source of ATP in this assay). In the
whole blood system, a change in light impedance is recorded
as the aggregation end point. Although measurement of
platelet aggregation can be very helpful, its time-consuming
nature, the need for a fresh patient sample, the technical skill
demanded in its performance, and the lack of quantitative
parameters 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 used
as a screening instrument for both platelet function and for
vWF concentration and function. Citrated blood is exposed
to epinephrine or ADP as it is aspirated at arterial shear rates
through a collagen-coated membrane that contains an aper-
ture. The platelets are activated, aggregate, and form a plug
that occludes the aperture in the membrane; the end point is
measured as the time required for a drop in pressure within
the system caused by the occlusion of the aperture. The
instrument is sensitive to intrinsic platelet function as well as
to vWF levels. Though not as sensitive or specic as platelet
aggregation and release studies (eg, for the diagnosis of stor-
age pool disease), the PFA-100 is technically easy to use, has
a quantitative end point, is reproducible, and is much less
time consuming.
The template bleeding time
The template bleeding time has been used for the diagnosis
of 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 controlled
conditions and measuring the time for the bleeding to stop. It
is 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 an
evaluation for immunologic causes of thrombocytopenia,
but they have limited usefulness in clinical settings due to
their low sensitivity and, with some assays, due to their low
specicity. Flow cytometry has been used to measure the
immunoglobulin on the surface of platelets, but this test does
not reect specic platelet antibodies and has not correlated
well with the clinical status of patients with immune throm-
bocytopenias. More specic tests, using specic monoclonal
antibodies to platelet antigens such as glycoproteins Ib and
IIb/IIIa, can be performed in microtiter plate assays using a
monoclonal antibody immobilization of platelet antigen
(MAIPA) format. These can be difcult tests because they
require sufcient numbers of patient platelets to provide the
platelet-bound antibody, and adequate numbers of platelets
are not always available from patients with ITP. The tests are
positive in only 5070% of patients with clinically diagnosed
ITP. A technically similar indirect test using normal platelets
exposed to patient serum is positive in only 3050% of
patients.
Assays for heparin-induced thrombocytopenia
Assays for HIT can be performed by several different
methods. One of the most widely used tests is an ELISA assay
that measures antibodies against platelet factor 4heparin
complexes. These are sensitive assays that detect antibodies
that may not be clinically signicant as well as antibodies
that cause thrombocytopenia and thrombosis. They must be
interpreted within the clinical context. A functional assay,
deemed the gold standard by many hematologists, is the
radiolabeled serotonin-release assay. Donor platelets are
incubated with radiolabeled serotonin. The platelets inter-
nalize the serotonin and are subsequently exposed to the
patients serum and heparin at a therapeutic concentration.
The platelets undergo a release reaction if antibodies to the
platelet factor 4heparin complex are present, and the
released radiolabeled serotonin is measured. A control incu-
bation is performed using a high concentration of heparin
(10.0 U/mL) to ensure specicity (the high concentration
of heparin prevents the correct stoichiometric complex of
platelet factor 4 and heparin, and no release reaction occurs).
The serotonin release assay is not available in many centers
| 463
because of the requirement for radioactive materials in the
assay, and another functional test that does not require radio-
activity measures the release of ATP in an assay similar to the
serotonin-release assay. This test is available commercially,
but it requires that platelet aggregation and release techniques
(ie, lumi-aggregometry) be available in the laboratory. Clini-
cally, it is important to discontinue heparin immediately and
institute another anticoagulant if heparin is thought to be a
possible cause for thrombocytopenia or thrombosis in a
patient; there can be both false-positive and false-negative
tests for HIT. It may be possible to restart heparin if a differ-
ent cause for the thrombocytopenia is established and tests
for HIT antibodies are negative.
Assays for thrombotic thrombocytopenic purpura and the
von Willebrand factor cleaving protease (ADAMTS13)
In sporadic cases of TTP, an autoantibody directed against
the enzyme that normally cleaves the procoagulant ultra-
HMW multimers of vWF is causal. The ultra-large forms of
vWF initiate the formation of platelet aggregates and lead to
the formation of thrombi and thrombocytopenia. In these
cases, the activity of the vWF cleaving protease, ADAMTS13,
is decreased in the patient, and an inhibitor can be identi-
ed in many of them. In hereditary forms of TTP, there are
mutations in the gene encoding the enzyme, and the activ-
ity of ADAMTS13 is decreased; however, no inhibitor is
present. There is currently debate about the value of mea-
suring the level of the ADAMTS13 activity in TTP patients
because other pathologic conditions such as inammation
and renal disease can cause a decrease in the level of the
enzyme.
Many of the assays for ADAMTS13 measure the decrease
in HMW vWF multimers that occurs under conditions that
promote enzyme activity in vitro (low ionic strength and
conditions that partially denature vWF). Several end points
have been used, including direct visualization of the multim-
ers or vWF cleavage products in gel electrophoresis systems
or ELISA format, collagen binding activity, and ristocetin
cofactor activity. Inhibitors are measured by mixing studies
that use a 1:1 mixture of the patient plasma and normal
plasma in the test system. The tests have varying sensitivity
and cannot be directly compared.
The decision to initiate treatment of TTP should be based
on the clinical syndrome, not on the assay for ADAMTS13
activity. However, the assay can be helpful for following the
effectiveness of plasma exchange and immunosuppressive
therapy. Conrmation of the clinical diagnosis can be secured
if an antibody to ADAMTS13 is demonstrated. Cases of TTP
that are secondary to bone marrow transplantation, cancer
chemotherapy, or medications such as cyclosporine appear
to have a different pathogenesis because ADAMTS13 activity
is normal in these diseases.
Bleeding in the absence of abnormal coagulation tests
and platelet abnormalities
Several recognized conditions can lead to a bleeding diathe-
sis despite normal screening coagulation tests and platelet
studies noted previously. Bleeding most often occurs in
these patients after invasive surgery or trauma, though some
patients have spontaneous bleeding and are discovered early
in life. These include factor XIII deciency,
2
-antiplasmin
deciency, and PAI-1 deciency; assays are available for each
of these factors. The most commonly used assay for factor
XIII (brin stabilizing factor, covalent bonds formed at the
D-domain) is a screening test that is abnormal when levels
are 2%, the minimal hemostatic level. A recently intro-
duced test that is more quantitative is based on the ability of
factor XIII to incorporate a biotinylated substrate into
brinogen coated on microtiter plates. Vessel wall defects,
such as collagen diseases (eg, EhlersDanlos and Marfan
syndromes), can also lead to serious bleeding; genetic test-
ing is becoming more readily available for some of these
syndromes.
Bleeding disorders with normal screening tests:
Factor XIII deciency
2
-Antiplasmin deciency
PAI-1 deciency
Vessel wall defects (eg, inherited collagen disorders)
Key points
Evaluation of thrombophilia
Deciency of one or more of the identied natural inhibi-
tors of coagulation has been associated with venous and
rarely arterial thrombosis, and functional and immunologic
assays are available to measure them. Their use is generally
restricted to patients who present with spontaneous throm-
bosis (not postoperative, after trauma, or with vascular
obstruction, etc) at a young age (45 years), those with
recurrent or unusual (eg, mesenteric) thrombosis, or those
with a family history of thrombosis. Except for genetic tests,
evaluation for these factors is best carried out several weeks
after completion of treatment to allow the patient to return
to baseline after a thrombotic episode; studies of family
members are also appropriate in some cases, although pro-
phylactic treatment with anticoagulation is not generally
indicated for asymptomatic carriers unless they are in high-
risk settings.
Protein C, protein S, and antithrombin are measured in
both functional (clot-based and chromogenic) and antigenic
assays. Because 60% of protein S is normally inactive due to
its binding to C4b-binding protein in plasma, interpretation
of the antigen assay for protein S needs to take into account
|
whether the test measures total or free protein S. In general,
the functional assays are most helpful for clinical evaluation,
especially for protein S, because the functional assay better
reects the free protein S. Functional deciency of these fac-
tors can be due to an absolute decrease in the factor (type I)
or a qualitatively abnormal protein (type II). Coumarin anti-
coagulants lower the levels of protein C and protein S and
should be discontinued for at least 2 weeks before measuring
these factors. The factors may also be decreased during a
thrombotic episode, and assessment should be delayed until
after treatment is completed.
D-dimer assays are used to help rule out acute thrombo-
embolic disease in patients with suspected spontaneous DVT.
ELISA assays for D-dimer, as opposed to latex agglutination
assays, should be used for this purpose because they have
been shown to have a higher sensitivity (approximately 95%).
For patients with clinically suspected thromboembolic dis-
ease, a normal value is strong evidence against the diagnosis;
for patients with an abnormally high value, the test is of no
utility because its specicity is low.
Genetic defects that predispose to thrombosis and are
measured by molecular tests include the factor V Leiden
mutation that leads to protein C resistance (due to a muta-
tion in factor V) and the prothrombin 20210 mutation that
is associated with increased levels of prothrombin and
thrombosis. A genetic test for a mutation in the 510 methy-
lene tetrahydrofolate reductase gene is also available. Patients
homozygous for the C677T mutation may have high levels of
circulating homocysteine.
The functional test for the factor V leiden mutation based
on a clotting end point called APCR is also available. This test
can be performed using a format based on the aPTT assay.
The test is rst done with APC added to patient plasma
(yielding a longer clotting time); a second test is performed
with buffer added in place of APC. The ratio of the aPTT
clotting times is decreased if the patient has factor V Leiden
(90% of patients with APC resistance) or another yet
undened cause for resistance to APC. It can also be abnor-
mal when lupus anticoagulants are present. Another APCR
test based on a tissue factor-dependent factor V assay is help-
ful in assessing patients who are on warfarin or who have a
lupus anticoagulant.
Circulating factors that predispose to thrombosis include
high levels of homocysteine and lipoprotein(a). Also, very
high levels of individual factors (factors VIII, IX, XI, and
brinogen) have been associated in epideminologic studies
with venous thrombosis. Abnormal brinogens and plas-
minogens are also unusual causes for thrombosis, but no
screening assays are available to detect them.
Lupus anticoagulants are an important cause of both arte-
rial and venous thromboses; assays for these antibodies are
discussed previously.
Thrombophilic states:
Deciency of protein C, protein S, antithrombin
Abnormal factors: factor V Leiden, prothrombin 20210
mutation
High levels of factors VIII, IX, and XI and brinogen
Lupus anticoagulant
Key points
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Chapter 17

; expression of CD38 is evidence of lineage commit-

Mature T cell CD4 or CD8

Cy CD3 cytoplasmic CD3; TDT terminal deoxynucleotidyl transferase.

LCL 10% 2550% / / / BCL2

A lack of correction or only partial correction in the aPTT

Coumarin anticoagulants cause prolonged PTs (and, vari-

Normal subjects have an INR of 1.0 0.12. The INR is

An alternative is to measure factors II, VII, IX, and X in a

Prolonged thrombin time:

vWF, leading to severe vWD. Additional tests are necessary to

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