Lab Medicine Conference
Leucocytes, Erythrocytes, Platelets,
and Clotting Studies
Lab Medicine Conference : Hematology :
Topics Covered
ƒ Methodology & indications for :
–White blood cell count
–Differential cell count
–Peripheral blood smears
–Hemoglobin, hematocrit
–Platelet count
–Prothrombin Time
–Partial Thromboplastin Time
–Thrombin Time, Bleeding Time
Components of the Complete
Blood Count (CBC)
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Hemoglobin
Hematocrit
Red blood cell count
Red blood cell indices
White blood cell count
Platelet estimate
White Blood Cell (Leucocyte)
Counts : Background
ƒ One of most commonly ordered lab tests in E.D.
ƒ Classic example of a lab test of variable or low
sensitivity & specificity
ƒ Consisit of separate cell lines :
–Neutrophil
–Lymphocyte
–Basophil
–Eosinophil
–Monocyte - tissue macrophage
Categories of Neutrophils
ƒ Two categories in bone marrow :
–Mitotic pool (precursors still capable of mitotic
division)
ƒ Myeloblast
promyelocyte myelocyte
–Postmitotic pool
ƒ Metamyelocytes
bands mature neutrophils
(polymorphonuclear leucocytes or PMN's)
ƒ Is the storage pool for neutrophils & represents 15
to 20 times the circulating population
Body Distribution of
Neutrophils
ƒ Maturation sequense from myeloblast to PMN
takes 5 to 7 days
ƒ Mature PMN's released from marrow into
circulation
ƒ About 1/2 circulate freely (circulating PMN pool) &
1/2 adhere to blood vessel walls (marginal PMN
pool)
ƒ PMN's carry out their phagocytic functions in
extravascular sites (in tissue) & then do not
return to circulation
Body Distribution of
Lymphocytes
ƒ Peripheral lymphocytes represent only
5 % of total body pool
ƒ Cells mature in bone marrow, thymus,
spleen, lymph nodes, & other lymphoid
collections
ƒ Cells can freely enter & leave
circulation
ƒ Consist of three cell subtypes : T cells,
B cells, & null cells
Factors Affecting the
Peripheral Leucocyte Count
ƒ Lymphoid, marrow, circulating, marginal, & tissue
pools of leucocytes are in dynamic flux
ƒ Rate at which new cells enter vascular system
normally equals the loss into tissues
ƒ 3 determinants of peripheral leucocyte count :
–Relative rates of marrow & lymphoid production
–Cell margination
–Tissue consumption
Methods of Lab Measurement
of Total Leucocyte Counts
ƒ Older manual methods had variance of 20 %
ƒ Modern automated methods have variance of 4
%
ƒ Automated counting methods :
–Electrical impedance
–Darkfield optical
Electrical Impedance Method
Used by the Coulter Counter
ƒ WBC's suspended in electrically conductive media
which also lyses the erythrocytes
ƒ This suspension flows thru aperture located in
insulated strip separating two electrodes
ƒ Aperture allows only single cell to pass at a time
ƒ Each cell causes reduction in electrical flow
proportional to the cell's volume
ƒ Changes in the electrical flow then quantitate the
number of leucocytes
Darkfield Optical Technique for
Measuring Leucocyte Counts
ƒ Diffraction of a light beam is caused by a
thin column of suspended cells passing at
the microscopic focal point
ƒ Diffracted impulse is used to determine
the cell count
ƒ Knowing concentration of the diluent,
result then expressed as cells per
microliter
Stains for Determination of
Leucocyte Differential Count
ƒ Manual exam under microscope
ƒ The most time consuming part of heme exam
ƒ Uses either Wright's or May-Gruenwald- Giemsa
stain
–Both contain methylene blue & eosin
–Acid components of cells stain with methylene
azures & basic components take up eosin
–Different stain uptake by cell granules allows cell
type differentiation
Problems with "Manual" Method for
Leucocyte Differential Counts
ƒ Time consuming
ƒ Poor precision
–> 15 % variation in same sample checked by
same person at different times
–Only 100 to 500 cells counted
–Cell type distribution varies from edge of smear
to middle
–At a pathologists' convention, 50 % called a test
cell a band & 50 % called it a poly
Continuous Flow Cytochemistry Automated
Method for Leucocyte Differential
Measurement
ƒ Cells flow thru 3 channels where RBC's are lysed & WBC's
stained supravitally
ƒ Stains evaluate presence of peroxidase, esterase, &
basophilic granules
ƒ Cell size & staining uptake identified by light absorption &
light scattering properties of cell
ƒ Counts 10,000 cells per sample so precision is improved
over manual methods (which count 100 to 500 cells)
ƒ False positive cell identification rate is about 10 % & false
negative rate is 0.8 to 10 %
Coupling of Coulter Channelizer
to Standard Coulter Counter
ƒ Allows rapid differential counts independent
of operator supervision
ƒ Cells are sorted by electrical impedance &
grouped according to cell volume
ƒ Bimodal cell volume distribution results
–Heavier cells are granulocytes (neutrophils,
eosinophils, & monocytes)
–Second peak represents lymphocytes
ƒ Can be done at rate of 100 runs per hour
Quantitative Analysis of Buffy Coat
to Determine Differential Count
ƒ Capillary tube containing a plastic cylinder
is centrifuged (the plastic cylinder is needed to
augment height of the buffy coat)
ƒ Buffy layer then separated into
lymphocytic & granulocytic elements by
staining with acridine orange
ƒ Multiplication of column heights by
predetermined constants yields neutrophil
& lymphocyte percentages
Effects of Age on Normal
Leucocyte Counts
ƒ Total leucocyte count peaks in first 12 hours of
life, then generally declines till adult level
reached at age 21 years
ƒ Absolute neutrophil counts (total WBC count multiplied
by % of neutrophils) :
–10,000 /mm3 at birth
–3500 /mm3 at age 2 years
–4400 /mm3 age 3 to adult
ƒ Lymphocytes peak at 7000/mm3 at age 1 year
Gender and Race Effects on
Leucocyte Counts
ƒ Premenopausal female levels average 500
per mm3 more than males
ƒ 1000 /mm3 increase in total count with
pregnancy
ƒ Black women & children ages 1 to 5 have
total counts lower by 1000 cells /mm3
ƒ Smokers may have chronic elevation of 1000
to 1800 cells /mm3
Lab Problems Causing Errors
in Leucocyte Counts
ƒ Uneven distribution of WBC's in
peripheral portions of slide smear
ƒ Incomplete RBC lysis causes false
high count
ƒ WBC lysis causes false low count
ƒ Clumping of platelets causes false high
count
ƒ Improper dilution causes 2 cells to be
counted as single cell
Average Total Leucocyte and Differential
Counts for U.S. Adults Ages 25 to 74
Para meter
Total
Leucocytes
Segmented
Neutrophils
Band
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
# of
7.6
Cells in +/- 0.06
Billions
per Liter
4.51
+/- 0.05
0.02
2.69
+/- 0.04
0.22
+/- 0.02
0.15
+/- 0.01
0.01
Percent
of 100
Cells
58.9
+/- 0.36
0.2
35.9
+/- 0.41
2.9
+/- 0.19
2.0
+/- 0.06
0.1
Mean and Ranges of Normal
Leucocyte Counts at Different Ages
TOTAL
WBC'S
AGE
BIRTH
1 Week
1 Month
1 Year
6 Years
21 Years
MEAN
(RANGE)
18.1
(9 to 30)
12.2
(5 to 21)
10.8
(5 to 20)
11.4
(6 to 18)
8.5
(5 to 15)
7.4
(4 to 11)
NEUTROPHILS
MEAN
(RANGE)
11.0
(6 to 26)
5.5
(2 to 10)
3.8
(1 to 9)
3.5
(2 to 9)
4.3
(2 to 8)
4.4
(2 to 8)
Percent
61
45
35
31
51
59
MONOCYTES
EOSINOPHILS
Percent
31
MEAN
(%)
1.1 (6)
MEAN
(%)
0.4 (2)
41
1.1 (9 )
0.5 (4)
56
0.7 (7 )
0.3 (3)
61
0.6 (5 )
0.3 (3)
42
0.4 (5 )
0.2 (3)
34
0.3 (4)
0.2 (3)
LYMPHOCYTES
MEAN
(RANGE)
5.5
(2 to 11)
5.0
(2 to 17)
6.0
(2 to 17)
7.0
(4 to 11)
3.5
(2 to 7)
2.5
(1 to 5)
Methods of Blood Smear
Preparation
ƒ Coverslip method
–2nd coverslip placed on drop of blood on first slip &
rotated 45 degrees ; slips then pulled apart horizontally ;
usually produces even blood film
ƒ Slide method
–Edge of slide pushed away from drop of blood on base
slide ; disadvantage is uneven distribution of WBC's in
tapered smear
ƒ Spinner method
–Centrifuge spins slide briefly at 5000 rpm ; can be used
with automated differential analyzers
Choices for Blood Stains
ƒ Rowmanowsky types contain basic (thiazine)
and acidic (eosin) components
–Wright's stain : most common
ƒ Has methyl alcohol, eosin, thiazines (azure B,
etc.)
–Giemsa : useful for malaria
–Leishman
–May-Grunwald
–Jenner
–MacNeal
Appearance of Blood Components
On a Properly Done Wright's Stain
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Overall pink color
Erythrocytes : pink
Leucocyte nuclei : blue to purple
Neutrophil granules : violet
Eosin granules : red / orange
Basophil granules : blue to purple
Lymphocyte cytoplasm : light blue
Platelets : purple granules
ƒ Reticulocytes (immature RBC's) have RNA that stains
blue as granules or reticulum within the cells
Neutrophil Appearance on
Blood Smears
ƒ Mature cells have 2 to 4 nuclear lobes
ƒ Causes of hypersegmented (> 5 lobes) :
–Megaloblastic anemia
–Severe sepsis
–Uremia
–Myeloproliferative disorders
–Metastatic malignancy
–Heat stroke
ƒ Immature cells ("band" forms) : show increased % in response
to acute inflammatory conditions, especially bacterial
infections
Wright stain showing an S.L.E. cell (a polymorphonuclear
leucocyte with phagocytized nuclei)
Characteristics of Atypical
Lymphocytes
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Seen in infectious mononucleosis
Large (12 to 16 microns in length)
Abundant finely granular cytoplasm
Nucleus is more variable in shape
Presence of small cytoplasmic
vacuolations is the most characteristic
feature
Atypical lymphocytes
Physical Characteristics of
Erythrocytes (RBC's)
ƒ Normal adult blood contains 5 million RBC's per
microliter
ƒ 26 trillion RBC's = circulating red cell mass
ƒ Are anucleate biconcave discs
ƒ Each 6.7 to 7.7 microns in diameter
ƒ Internal cell volume is 80 to 100 femtoliters (10
L)
ƒ Each cell contains 250 million hemoglobin (Hgb)
molecules
ƒ Hgb constitutes about 1/3 of total cell content
-15
“Sickled” erythrocytes of Sickle Cell Disease
Fragmentation hemolysis from a prosthetic aortic valve
Hereditary elliptocytosis
Erythrocyte Turnover
ƒ Produced mainly in marrow of ribs,
vertebrae, skull, clavicles, & pelvis
ƒ Low O2 tension & reduced O2 carrying
capacity stimulate erythropoetin
ƒ Cannot produce own proteins after
maturation
ƒ 1 % of cells removed daily, mainly by
spleen
ƒ Average cell lifespan 100 to 120 days
Variations in Hemogram Measurements from
Different Blood Drawing Techniques
ƒ First capillary sample gives lower cell counts than
subsequent samples
ƒ Ear lobe Hgb can be 15 % > fingerstick Hgb
ƒ If skin cold or squeezed, can cause 6 % higher values
ƒ In neonates, capillary Hgb is 3.5 g/dl > venous Hgb
ƒ Hemoconcentration results from tourniquet times > 60
seconds
ƒ Hemolysis can result from small needle draw or
improper technique
Choices of Anticoagulants for
Hemogram Measurements
ƒ EDTA (lavender top tube) :
–Prevents platelet clumping
–Agent of choice for blood cell counts & morphologic studies
–Causes changes in RBC indices if analysis delayed > 6 hours
ƒ Trisodium citrate (blue top tube) :
–Needs ratio of one part 3.8 % solution to 9 parts sample
blood for effective anticoagulation
–This is why tube must be fully filled for accurate result
ƒ Sodium heparin (green top tube) :
–Best for hemolysis prevention & osmotic fragility tests
–Causes cell clumping so is not good for cell counts
–Causes blue background on Wright's stain
Methods for Red Cell Count
Measurement
ƒ Manual
–Blood sample diluted 1 to 200 with isotonic Hayem's Solution,
them micropipetted into counting chamber (hemocytometer)
–Number of RBC's in 1/5 square mm area counted & multiplied
by 10,000 to yield number of cells per mm3
–Range of error for this method : +/- 20 %
ƒ Automated - most are multichannel (also calculate Hct) :
–Electrical impedance (Coulter Counter, etc.)
ƒ Measure voltage change when cells displace conductive
fluid
–Light Scattering systems (Hemalog, Ortho, etc.) :
ƒ Measure voltage pulses that occur when cells interrupt a
laser beam & scatter light onto a photodetector
Measurement Methods for
Hemoglobin
ƒ Colorimetric methods have superceded specific gravity,
gasometric, & chemical methods
ƒ Colorimetric methods measure photoelectric absorption
of pigment derived from Hgb
–Cyanomethemoglobin method is standard
ƒ Sample treted with KCN & absorption at 540 nm
measured
ƒ Anything causing incresed sample turbidity (lipemia,
etc.) can cause measurement errors
ƒ Accuracy is +/- 2 %
ƒ Expressed as grams per deciliter
Manual Measurement Techniques for
Hematocrit (Hct)
ƒ Hct expressed as ml per ml, or %
ƒ Represents % of blood volume occupied by RBC's
ƒ Macrohematocrit method :
–Large tube centrifuged for 30 minutes ; Hct then read by scale on
tube
–Allows measurement of sed rate & WBC counts on same sample
ƒ Microhematocrit method :
–7 cm length capillary tube (1 ml volume) used
–End of tube sealed with clay after sample entered
–Centrifuged for 4 minutes ; Hct then read by scale next to tube
–Accuracy is +/- 2 %
–Sometimes false elevation from plasma trapping
MCV : Mean Corpuscular
Volume
ƒ Is average volume of single RBC
ƒ Calculated by : measure Hct & divide
by RBC count
ƒ MCV < 80 femtoliters = microcytic
ƒ MCV > 100 femtoliters = macrocytic
MCH : Mean Corpuscular
Hemoglobin
ƒ Is average weight or content of Hgb in single
RBC
ƒ Determined by dividing measured Hgb by RBC
count
ƒ Value expressed in picograms
ƒ MCH < 27 = microcytic or hypochromic
normocytic
ƒ MCH > 31 = macrocytic
ƒ Helps to classify anemias as hypochromic or
normochromic
MCHC : Mean Corpuscular
Hemoglobin Concentration
ƒ Is average Hgb concentration in a given volume of
packed RBC's
ƒ Determined by dividing Hgb content by Hct
ƒ Result is weight per 100 ml of packed RBC's,
expressed as "%"
ƒ Has less independent correlation with RBC
appearance on smear than does MCV or MCH
ƒ If < 30 % suggests severe iron deficiency
ƒ If > 38 % suggests hereditary spherocytosis
ƒ If > 40 % suggests error in RBC measurements
Hemogram Values for Different Ages
(at sea level)
Hemoglobin
(gm / dL)
Hematocrit
(%)
RBC Count
(100,000 / uL)
17.1 +/- 1.8
52 +/- 5
4.64 +/- 0.5
MCV
(cubic
microns)
113 +/- 6
1 Day
19.4 +/- 2.1
58 +/- 7
5.30 +/- 0.5
1 Month
14.1 +/- 1.9
45 +/- 7
3 Months
11.2 +/- 0.8
5 Years
Birth
(cord blood)
MCH
(picograms)
MCHC
(gm / dL)
37 +/- 2
33 +/- 1
110 +/- 6
37 +/- 2
33 +/- 1
4.35 +/- 0.6
104 +/- 11
32 +/- 3
31 +/- 3
37 +/- 3
3.88 +/- 0.4
95 +/- 9
29 +/- 3
30 +/- 2
12.7 +/- 1.0
37 +/- 3
4.65 +/- 0.5
80 +/- 4
27 +/- 2
34 +/- 1
Adult Men
15.5 +/- 1.1
46 +/- 3
5.11 +/- 0.4
90 +/- 5
30 +/- 2
34 +/- 1
Adult Women
13.7 +/- 1.0
41 +/- 3
4.51 +/- 0.4
90 +/- 5
30 +/-2
34 +/- 1
Reticulocyte Counts
ƒ Normally comprise 0.5 to 1.5 % of all erythrocytes
ƒ Reflects release of young RBC's from marrow
ƒ Marrow failure is reflected by low retic count :
–Iron deficiency
–Thalassemia
–Aplastic anemia
ƒ Calculation of reticulocyte index (RI) allows correction of %
for decreased hematocrit :
–RI = reticulocyte % X Hct / Normal Hct
ƒ ( Normal Hct entered as 45 to 48 %)
ƒ Normoblasts = nucleated RBC's (pre-retic precursor)
–normally not seen on peripheral smear
Additional Lab Tests Useful in
Diagnosis of Hemolytic Anemias
ƒ Unconjugated bilirubin : elevated
ƒ Haptoglobin levels decreased
–Alpha-2 macroglobulin normally at high levels in plasma
–Binds to free Hgb ; complex actively cleared by
monocytes
–Once haptoglobin binding capacity exceeded, free Hgb
levels increase & Hgb is filtered into urine
–Filtered Hgb is catalyzed into hemosiderin causing
hemosiderinuria
– Hemoglobinuria then results if hemolysis is more severe
exceeding capacity to make hemosiderin
Diagnostic Interpretations of
Hemogram Measurements
ƒ MCHC > 38 % without spherocytes present suggests
inaccuracy of all CBC values due to cold agglutinins,
lipemia, or rouleaux formation
ƒ Hct < 25 implies not anemia of chronic disease alone
ƒ MCV < 80 indicates chronic iron deficiency or
thalassemia minor
ƒ Hypochromic normocytic anemia implies plumbism
(lead toxicity)
ƒ MCV > 120 implies liver disease or megaloblastic
anemia
Red Cell Distribution Width
(RDW)
ƒ Reported by newer automatic RBC counter
machines
ƒ Provides distribution frequency of red cell volume
ƒ RDW = standard deviation of measured RBC size X
100 / MCV
ƒ RDW = coefficient of variation (CV %) of RBC size
ƒ Normal RDW = 11.5 to 14.6 % (can be high but not
low)
ƒ Error is +/- 0.5 %
Diagnostic Use of RDW
ƒ Nutritional deficiency (iron, folate, or B12)
results in dimorphic subpopulation of
RBC's
–RDW then is high (even if MCV & Hgb are
normal)
ƒ RDW is normal with hypoproliferative
anemias and reticulocytosis
ƒ Usually is normal with hemorrhage
Polycythemias
ƒ Defined as increase in number of circulating RBC's
per unit volume of blood
ƒ Classed as :
–Absolute increase in red cell mass :
ƒ Primary (Polycythemia vera)
ƒ Secondary (chronic hypoxia, etc.)
–No increase in total red cell mass :
ƒ Relative (hemoconcentration from loss of plasma)
ƒ Stress
ƒ Pseudopolycythemia
Polycythemia Vera
Diagnostic Criteria
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Normal O2 saturation
Thrombocytosis : platelets > 600,000 / mm3
Leucocytosis : WBC > 12,000 / mm3
Leucocyte alkaline phosphatase > 100
Elevated B12 levels
Usually splenomegaly present
Symptoms related to increased blood
volume & increased blood viscosity
Morphologic Variants of Erythrocytes
Red Cell Variant
Acanthocyte
Basophilic stippling
Major Clinical Associations
Abetalipoproteinemia
Alcoholic cirrhosis with hemolysis
Lead poisoning, thalassemia
Hemolytic states
Blister cells
DIC
Sickle cell disease
Burr cells (echinocytes)
Uremia
Renal disease, Pyruvate kinase deficiency
Elliptocytes
Hereditary elliptocytosis
( a few may be seen on normal smears)
Heinz bodies (denatured Hgb)
Drug - induced oxidative hemolysis
Unstable hemoglobinopathies
Howell-Jolly bodies (nuclear fragments)
Hemolytic anemias, Megaloblastic anemia
Hyposplenism, Thalassemia
Macroovalocytes
Megaloblastic anemia
Myeloproliferative disease
Nucleated RBC's
Increased erythropoesis
( blood loss or hemolysis)
Morphologic Variants of RBC's (cont.)
Red Cell Variant
Poikilocytes (teardrop cells)
Pappenheimer Bodies
Parasites
Rouleaux of RBC's
Major Clinical Associations
Myelophthistic states
Sideroblastic anemias, Lead poisoning
Thalassemia
Malaria
Bartonella
Increased plasma proteins
( multiple myeloma)
Schistocytes (schizocytes)
DIC, Mechanical cardiac valves,
Severe burns, Uremia
Sickle cells (drepanocytes)
Sickle cell disease & variants
(Hb - SC, etc.)
Spherocytes
Stomatocytes
Target cells
Hereditary spherocytosis
Immune & other hemolytic anemias
Hereditary stomatocytosis
Alcoholism
Thalassemias, Other hemoglobinopathies
Iron deficiency, liver disease
Effects of Disease Processes on
Erythrocyte Morphologies
Process
Abnormal erythropoesis
Abnormal hemoglobin
formation
Damage to red cells after
leaving the bone marrow
Increased erythropoiesis to
compensate for anemia
Effect
Increased variation in size
(anisocytosis) & shape
(poikilo-cytosis)
Reduced or unequal Hgb (hypochromasia or anisochromasia)
Spherocytosis, irregular
contraction or fragmentation
(schistocytosis)
Signs of immaturity (polychromasia, punctate basophilia,
and nucleated RBC's)
Causes of Poikilocytosis
(Alterations in RBC Shape)
ƒ Megaloblastic anemia
–Helmet-, pear-, tear-, or oval- shaped RBC's
ƒ Acanthocytes (spherical RBC's with irregular spicules) :
–Indicate permanent RBC damage
–Seen in abetalipoproteinemia, anorexsia nervosa,
postsplenectomy, renal disease, alcoholic liver disease
ƒ Burr cells (echinocytes) : contracted, spurred RBC's :
–Seen in hyperosmolar states
ƒ Elliptocytes
–If > 90 % indicate hereditary elliptocytosis
Causes of Poikilocytosis (cont.)
ƒ Blister cells : RBC's with single or multiple vacuoles
–Rupture of blisters results in distorted & fragmented cells
(schizocytes) : helmet cells, triangle cells, keratocytes
ƒ Burns, prosthetic heart valves, DIC
ƒ If seen in sickle cell anemia, implies pulmonary embolus
ƒ Sickle cells (drepanocytes)
–Seen in sickle cell disease ; % of sickle cells does not
correlate with crisis
ƒ Spherocytes : have increased osmotic fragility
ƒ Stomatocytes (cup - shaped RBC's)
–Seen in alcoholism, infectious mononucleosis, lead
poisoning, thalassemia minor, malignancies
Alterations in RBC Hemoglobin
Content
ƒ Hypochromasia (decreased RBC hemoglobin concentration
–Iron deficiency anemia
–Thalassemias (deficient globin production)
–Sideroblastic anemias
ƒ Intramitochondrial defect in heme synthesis
ƒ Cytoplasmic ferretin granules (stain with Prussian blue)
ƒ Alcoholism, malignancies, rheumatoid, drugs, lead
ƒ Anisochromasia (increased variation in Hgb content in RBC's) :
–Usually combo of hypo- & normo- chromic cells on same
smear
–Seen in Sideroblastic anemias, after Rx with iron, and after
transfusion
Platelets
ƒ Key component in normal clotting
ƒ Cytoplasmic fragments released from bone marrow
megakaryocytes
ƒ Normally are round, oval, 2 to 4 microns in diameter (1/4 to 1/2
size of RBC's)
ƒ Normally one per 10 to 30 RBC's on smear
ƒ Contain fine purple-staining granules which fill cytoplasm
ƒ In thrombocytopenia, usually few or none are seen on several
microscope fields
ƒ Upon leaving marrow, 2/3 enter systemic circulation & 1/3
enter splenic pool
ƒ Turn over at 35,000 per microliter per day
ƒ Life span 7 to 10 days
Platelet Counts
ƒ Determined from lavender top (EDTA) tube
ƒ Normal range 150,000 to 400,000 per microliter
ƒ High risk of spontaneous bleeding below
20,000
ƒ 3 measurement methods :
–Estimation from blood peripheral smear
–Manual chamber counts
–Automated
Manual Methods for Platelet
Counts
ƒ Rees and Ecker method :
–Error range 15 to 25 %
–Uses light microscope, hemocytometer, & diluting
fluid containing cresyl blue stain
ƒ Becker - Cronkite method :
–Blood diluted & hemolyzed with ammonium oxalate
–Platelets counted in phase hemocytometer & phase
microscope
–8 to 10 % error rate
–Most precise method for severe thrombocytopenia
Automated Platelet Counts
ƒ Precision : 2 to 3 % variance
ƒ Electrical or optical methods used
ƒ Can be done on whole blood
(uncentrifuged)
ƒ Still require comparison check with
stained smear (to rule out count errors)
Sources of Error in
Automated Platelet Counts
ƒ If machine has pre-fixed upper & lower size
thresholds, it can underestimate count of nonstandard size platelets
ƒ Can report false high counts from cytoplasmic
fragments of RBC's or WBC's or malarial
parasites
ƒ High IgM levels can alter light scattering
ƒ Platelet aggregation (sometimes from sampling
problems) causes count errors but most
machines "flag" for this
Thrombocytopenia
ƒ Defined as count below 100,000 per mm3
ƒ If count < 50,000, hemocytometer manual
counting with phase microscopy recommended
ƒ General causes :
–Lab artifact (pseudothrombocytopenia)
–Decreased or defective production
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Congenital, drugs, infections, marrow problems
–Increased destruction (as in TTP, DIC, or ITP)
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Immunologic, mechanical, or toxic causes
–Loss, dilution, or sequestration
Thrombocytosis
ƒ Defined as counts > 400,000 to 600,000 / mm3
ƒ Can be associated with bleeding or thrombosis
ƒ Classed as :
–Reactive : reaction to hemorrhage, inflammation,
malignancy, or necrosis
–Autonomous : due to increased megakaryocytes
ƒ Essential thrombocythemia
ƒ Myeloproliferative disorders
ƒ Sideroblastic anemias
Test Choices for Evaluation of
Bleeding or Clotting Problems
ƒ Initial standard tests :
–Platelet count
–Prothrombin time (PT)
–Activated Partial Thromboplastin Time (aPTT)
–Peripheral blood smear exam
ƒ Secondary tests :
–Bleeding time
–Thrombin time
–Fibrinogen level
–Fibrin split products
–Specific clotting factor assays, Urea clot solubility for XIII
–Mix patient plasma with normal plasma
Coagulation Factor Nomenclature
ƒ I : fibrinogen
ƒ II : Prothrombin
ƒ III : Tissue thromboplastin
ƒ IV : Calcium
ƒ V : Labile factor
ƒ VII : Stable factor
ƒ VIII : Antihemophilic A factor
ƒ IX : Antihemophilic B factor
ƒ X : Stuart factor
ƒ XI : Plasma thromboplastin antecedent
ƒ XII : Hageman factor
ƒ XIII : Fibrin stabilizing factor
Note : # VI not
assigned
Coagulation Pathway / Clotting Cascade
CONTACT
XII
INTRINSIC
EXTRINSIC
Tissue
XIIA
XIA
XI
IX
Thromboplastin
VII
Calcium
Calcium
IXA
VA
VIII
Calcium
Prekallikrein
V
Platelet lipid
Kallikrein
Kinins
X
COMMON
Inflammation
XA
II
Insoluble fibrin
XIII
IIA
I
IA
XIIIA
XII
XII A
TF + VII
Another diagram of the coagulation cascade
Comments on Coagulation
Pathway
ƒ All coag factors synthesized in liver
ƒ Factors II, VII, IX, & X are dependent on vitamin K for hepatic
synthesis of active forms
ƒ Intrinsic & extrinsic pathways activated independently
ƒ Intrinsic pathway operates within the circulation
–Activated when blood contacts a non-epithelial surface
ƒ Extrinsic pathway activated by blood exposure to
phospholipoprotein surface of disrupted tissue cells
–Forms thrombin & fibrin more rapidly than intrinsic
pathway
ƒ Common pathway involves X, II, XIII, and I
Fibrinolytic System
ƒ Responsible for dissolution of clot &
occurs by lysis of fibrin polymers
ƒ Plasminogen is absorbed from plasma
onto fibrin polymers & converted to active
enzyme plasmin
–Plasmin hydrolyzes fibrin polymers &
also fibrinogen, factor V, & factor VIII
ƒ Plasmin inhibitors in circulation serve to
prevent systemic fibrinolysis
Measurement of Prothrombin
Time
ƒ "Quick" one stage test
–Measures clotting time of plasma after adding
calcium & thromboplastin
–Measures coagulant activity of the extrinsic
pathway, including the common pathway
–Measures effect of factors I, II, V, VII, & X
Methodology of Prothrombin
Time (PT) Measurement
ƒ Citrate - anticoagulated sample is
centrifuged to obtain plasma
ƒ Calcium chloride & tissue thromboplastin
(from human or rabbit brain) added to
plasma
ƒ PT then is the time from addition of
calcium & thromboplastin to visual
appearance of first fibrin strands
ƒ PT value is unaffected by platelet or
intrinsic factor defects
Prothrombin Time
Measurement Reporting
ƒ Sample PT is compared to control from
pooled fresh frozen plasma
ƒ Normal range is 11 to 15 seconds
ƒ For normal result, requires adequate
levels of factors X, VII, V, prothrombin, &
at least 100 mg/dL fibrinogen
ƒ Usually one factor must fall to less than
30 % of normal amount to alter the PT
Partial Thromboplastin Time
(PTT)
ƒ Measures intrinsic & common coagulation pathways
ƒ Does not test coag factors VII & XIII
ƒ Centrifuged platelet-poor anticoagulated plasma mixed with
phospholipid emulsion (the emulsion only activates the
intrinsic path)
ƒ Calcium chloride added to plasma, & time to appearance of
fibrin threads in the tube is recorded as tube is slowly tilted
back & forth
ƒ Results in variable amount of time of exposure of sample to
glass surface, sometimes causing premature intrinsic
activation
ƒ Normal range 60 to 85 seconds
Activated Partial
Thromboplastin Time (aPTT)
ƒ Contact activating agent (kaolin, Celite, or
ellagic acid) added to sample at test onset
ƒ Provides more rapid & standardized
activation of contact factors
ƒ Normal range 22 to 38 seconds ; must be
reported against a control sample
ƒ aPTT lengthens when relevant factor is <
30 % of normal level
Use of International Normalized Ratio
(INR) in Reporting Prothrombin Times
ƒ Allows more accurate measuring of intensity of anticoagulant
effect (esp. warfarin)
ƒ Allows correction for degree of sensitivity of the thromboplastin
used by the lab in PT measurement (different labs use
thromboplastins from different sources & with different
sensitivities)
ƒ Utilizes International Sensitivity Index (ISI) : a measure of the
responsiveness of a given thromboplastin to vitamin K dependent coag factors
–Standard W.H.O. reference thromboplastin has ISI of 1.0
(commercially available ones in U.S. have ISI from 1.2 to 2.8)
ƒ INR value = sample PT / lab control PT ratio raised to the power
of the ISI number
Clinical Use of INR
ƒ Patient should be dosed with warfarin & monitored to
achieve specific INR
–For conventional anticoagulation ( S/P pulmonary
embolus, prosthetic heart valve, recurrent emboli, etc.) :
ƒ Goal is INR 2.5 to 3.5
–For less intense anticogulation (chronic stroke
prophylaxis, etc.) :
ƒ Goal is INR 2.0 to 3.0
–In acute anticoagulation, can D/C heparin when INR from
concurrent warfarin reaches 2.0
–If lab changes to new thromboplastin reagent with
different ISI, recalculation of INR should not change
Causes of Lab Error in PT & PTT
Values
ƒ Incomplete blue top tube filling ( false high value)
ƒ Tissue thromboplastins from needlestick (enter first
blood tube drawn)
ƒ Sample is partially clotted or hemolyzed
ƒ Relative deficiency of coagulant in a sample with
severe anemia (falsely low result)
ƒ Relative excess of coagulant in a sample with
polycythemia (falsely high result)
ƒ Lipemic or icteric plasma interferes with optical
methods
Differential Diagnosis of
Prolonged PTT
ƒ Factor XII deficiency
ƒ Factor XI deficiency
ƒ Prekallikrein deficiency
ƒ Kininogen deficiency
ƒ Factor IX deficiency
–(Hemophilia A)
ƒ Factor VIII deficiency
–(Hemophilia B)
ƒ Lupus - like
anticoagulants
ƒ Circulating antibodies to
factors VIII or IX
ƒ Heparin effect
ƒ Factor II deficiency
ƒ Factor V deficiency
ƒ Factor X deficiency
ƒ Hypofibrinogenemia
ƒ Dysfibrinogenemia
ƒ D.I.C.
ƒ Hepatocellular disease
Differential Diagnosis of
Prolonged PT
ƒ Vitamin K deficiency
ƒ Fat malabsorption
ƒ Liver failure, biliary obstruction
ƒ Warfarin effect
ƒ Factor II deficiency
ƒ Factor V deficienncy
ƒ Factor VII deficiency
ƒ Factor X deficiency
ƒ Hypofibrinogenemia
ƒ Dysfibrinogenemia
ƒ D.I.C.
Thrombin Time Measurement
ƒ Used to assess quantitative & functional
deficiencies of fibrinogen
ƒ Measures defects in common pathway
ƒ Done by adding exogenous thrombin to sample
citrated plasma
–Time to clot formation then measured
ƒ Normal is 10 to 15 seconds
ƒ Prolonged if fibrinogen is < 100 mg/dL, functionally
abnormal, or inhibitors like heparin present
ƒ Not affected by isolated deficiencies in intrinsic or
extrinsic pathways
Bleeding Time Measurement
ƒ Measures efficiency of vascular & platelet phases of hemostasis
ƒ Acts as screening test for platelet quantity & / or quality
ƒ Requires no reagents
ƒ Is the time for bleeding to stop from a standard forearm incision
–Ivy method : 3 small incisions
–Mielke (template) method : spring loaded device
–Incisions are 1 mm deep & 5 mm long
–Oozing blood absorbed into filter paper at 30 second intervals till
bleeding stops
ƒ Normal values : Duke (without BP cuff) : 1 to 3.5 minutes; Ivy : 2 to
7 minutes ; Template 2.5 to 7.5 minutes
ƒ Aspirin use prolongs bleeding time for 5 to 8 days
Causes of Abnormal Bleeding Time
(BT)
ƒ BT starts to increase if platelets < 100,000
ƒ BT increases linearly for platelet counts between
100,000 & 10,000
ƒ Use corrected bleeding time to assess for platelet
dysfunction (separate effect from count) :
–Expected BT = 30 minus platelet count / mm3
divided by 4000
ƒ Causes of increased BT include drug effects,
vonWillebrand's disease, uremia, collagen disorders,
amyloidosis
Charges at H.M.C. for
Hematology Tests (in $)
Test
Hgb
Hct
RBC count
WBC count
CBC
Differential
Eosinophil count
Retic count
Routine
15
15
13
16
27
32
16
19
Stat
19
19
16
19
32
39
Charges at H.M.C. for
Hematology Tests (in $)
TEST
PT
PTT
Platelet count
Thrombin time
DIC Screen (FSP + platelets)
Sickle cell screen
Fibrinogen
Clotting inhibitor screen
Malaria smear
Factor assays
Haptoglobin
Plasmin
Plasminogen
Bleeding times
ROUTINE
21 (15)
21 (15)
20 (11)
43
58
8
32
68
37
56 to 156
21
40
48
24 to 39
STAT
32
32
30
64
87
12
48
Lab Medicine : Hematology
Studies Summary
ƒ Familiarize yourself with your lab's
hematology methods
ƒ Decide what first line tests are clinically
needed
ƒ Decide if secondary tests are needed
based on abnormal values of first line
tests
ƒ Decide if abnormal values may be
artifactual
ƒ Consider costs of tests when ordering