LABORATORY EVALUATION OF PLATELETS

- Laboratory investigation of a disorder of hemostasis may center on:

o Platelet function
o Platelet number
- These two aspects are interrelated in that abnormalities in either or both result in hemostatic defect but
test of platelet function is indicated only if platelet numbers are adequate

ENUMERATION OF PLATELETS
- Counting of platelets is an important and logical starting point in evaluating bleeding problems
- Done by estimating the number of platelets on PBS or by manual or automated techniques

1. Estimates from Peripheral Blood Films

- Guidelines:
a. Approximate reference range for platelets is 140 to 440 x 109/L or 150 to 400 x 109/L
b. There are approximately 10 to 40 red cells per platelet in the normal peripheral blood. (An oil-
immersion field containing 100 red cell should have 3-10 platelets, whereas a field with 200 red
cells should have 5-20 platelets
c. Platelet estimates in the presence of abnormal hematocrit values tend to be inaccurate
d. Accurate estimates are possible only when there are no platelet clumps. Larger platelet clumps
cause inaccurate estimates. If clumps are noted, the original blood specimen should be reexamined
for clots. If clots are not found, platelet clumping in the presence of EDTA should be suspected
and noted in the report
e. Abnormally sized platelets should also be noted

2. Manual Platelet Counts

 1:100 or 1:200 dilution of blood applied to the Neubauer hemocytometer chamber
 REES-ECKER METHOD/TOCANTINS METHOD
- Uses Rees-Ecker diluent uses a citrate-formaldehyde buffer with brilliant cresyl blue as platelet
stain for light microscopy
- This diluent fixes and preserves red blood cells and platelets to prevent disintegration
- Counted using 4 W sqaures
 UNOPETTE METHOD
- K3EDTA and ammonium oxalate
- Based on the hemolysis of red cells by hypotonicity and complete blockage of platelet activity by
chelating Mg and Ca with EDTA and ammonium oxalate
- 1:50 dilution of blood in a diluent with 0.44% ammonium oxalate and 0.22% K3EDTA gives a
uniform suspension of platelets and leukocytes causing complete hemolysis of red cells
- 0.75% Crystal violet (3uL) is added to the diluent after hemolysis is complete
- Reservoir contains 1.225 mL of the diluent
- Unopette has a 25 uL capacity
- DF is 50
- VCF is 50
- Counting is made using the 5 R squares
- NV: 145, 000 to 375, 000/cumm
  BRECKER-CRONKITE METHOD/PHASE CONTRAST MICROSCOPY METHOD
- More popular method
- Uses 1% ammonium oxalate diluting fluid
- Involves the 2-syringe method of collection
- Sample drawn up to the 1 mark of RBC pipette; DF up to the 101 mark
- Counting chamber is Spencer-Briteline No. 1475 using 25 squares
- NV: 140,000 – 440,000 platelets/cumm

 FONIO’S METHOD
- Indirect method that uses 14% MgSO4 as anticoagulant

 SPECIMEN REQUIREMENTS
- Venous blood collected with EDTA
- Capillary puncture can yield low platelet counts secondary to platelet adhesion to the wound
o Capillary puncture should be performed with a larger blade so that a deep wound with a free
flowing blood is obtained

3. Automated Platelet Counts

- Optical method is employed (detection of degree of light scattering) or electrical method (change
in electrical resistance or capacitance across a circuit) to detect particles as they stream through an
aperture tube or cell
- Advantage: Precision
- Falsely increased:
o Contamination of reagents with particles or bacteria
o Carry over from a prior sample with a high platelet count
o Particles that should not be counted but are registered as platelets (fragments of red or white blood
cells, insufficiently lysed red cells or red cells with inclusion bodies)
- Falsely decreased:
o Platelet agglutinins or platelet satellitism
o Exceptionally large platelets may not be counted if upper or lower thresholds are used

PLATELET FUNCTION
o ADHESION
 Tests:
1. Bleeding Time Test
- Time it takes for a standard wound to stop bleeding
- Comprehensive test of platelet action in vivo
- Sensitive to abnormalities of platelet numbers and function to plasma VIII:vWF deficiencies
- Sensitive to abnormalities of vessel wall composition that interfere with platelet function
- Methods:
a. Duke Method
- Small cut in the earlobe is made and the length of time required for the bleeding to stop
is recorded
- NV: 1-3 minutes
b. Ivy Method
- Introduced standardization into the procedure and the time required to stop bleeding is
recorded
-
 c. Mielke/Template BT
- Modified Ivy method
- Uses a template to produce a standardized slit in place of the disposable lancets

d. Simplate Method
- Uses a Simplate bleeding time device
- Uniform incision is made on the forearm and the length of time required for bleeding to
stop is recorded

2. Capillary Fragility Test (Tourniquet test/Rumple-Leede test)

- Temporarily increased intracapillary pressure may lead to rhexis bleeding of capillaries
- The capillary fragility test is a crude assay to assess platelet-vessel wall interactions. Increasing
the intravascular pressure in the arm causes “gaps” to appear between endothelial cells.
However the extravasation of red blood cells does not normally occur, since these gaps are
normally occluded by platelets. However, petechiae form if platelets are decreased or
dysfunctional

3. Glass Bead Retention Test

- Blood is passed through a glass bead column, normal platelets that have normal access to
normal vWF will adhere and aggregate to the beads so that the effluent from the column will
have a much lower platelet count than the starting sample
- The percentage of platelets retained in the glass bead column should be approximately 70%
or greater

4. Platelet Adhesiveness In Vivo

- Involves serial platelet counts on blood exuding from a forearm incision
- Normally, the platelet counts decrease because of platelet adhesion to the wound
- These count are compared with venous blood platelet count as a control to calculate platelet
adhesiveness
- Percentage range of platelet adhesiveness is 15% to 45%

o AGGREGATION
 Performed to assist in the diagnosis of hereditary and acquired platelet disorders
 Tests
1. Platelet Aggregation
- Measured with a platelet aggregometer
- Basic principle: Citrated, plasma-rich plasma is stirred in the aggregometer while a light beam
is passed through the suspension
- Chemical stimulus is added
 ADP
 Epinephrine
 Collagen
 Thrombin (retains aggregating properties but lacks clotting ability)
 Ristocetin
 Arachidonic acid
- The shape change from discoid to spheroid is monitored as an initial decrease in light
transmittance
- Subsequent aggregation allows an increase of light to pass through the suspension to the
photodetector and to be recorded as an increase in light transmittance
o PLATELET FACTOR ASSAYS
1. Platelet Factor 3 Availability
- Platelet factor 3 is a blood coagulation factor derived from platelets that act with certain plasma
thromboplastin factors to convert prothrombin to thrombin
- When platelets are incubated with kaolin and epinephrine, they are stimulated to provide PF3
activity
- The test compares the clotting time of the patient’s PRP with that obtained for a group of normal
individuals
- NV: 37 to 51 seconds for PRP
- Decreased in: Acquired or congenital defects of platelet secretion or release; impaired function
of factors V and Xa

2. Assays of PF4 and Beta-Thromboglobulin

- PF4 and Beta-thromboglobulin are heparin-binding proteins found in platelet alpha granules
- Levels in vivo are indicator of the presence of ongoing platelet activation in diseases like M.I.,
venous thrombosis, diabetes, inflammatory states and myeloproliferative disorders
- Release of these proteins from aggregating platelets in PRP can be examined for patients
suspected of having storage pool disease or a release defect

o VON WILLEBRAND FACTOR ASSAY

- vWF circulates in plasma as a noncovalent complex with factor VIII coagulant
- Agglutination of fixed platelets in response to ristocetin depends on the presence of vWF in plasma
- The rate or percentage of agglutination is proportional to the amount of von Willebrand present