Tabares, MA©

Specimen collection

 Specimen of choice
o 9:1 venous bld with 3.2% sodium citrate (0.105-0.109 mol/L) – preserves Factors V and VIII better.
o EDTA: inhibits fibrinogen-thrombin rxn ad Factor V is unstable in its presence
o No fasting requirement
o Extend time for observing venipuncture site
 Consider drugs that may affect the tests
 Instruct px to discontinue those w/c may interfere with the tests
1. Aspirin
o suppresses most platelet fxn
2. Warfarin
o reduces activities of several coagulation factors and prolongs prothrombin time
 If a specimen is part of a series of tubes to be filled, it must be collected first or immediately after a non-additive tube
 Clotted specimens are useless
 Hemolyzed samples are unreliable
 Traumatic venipuncture can falsely shorten tests results
 Tourniquet must be removed w/in 1 minute to avoid venous stasis
 Centrifugation
o 2,000 x g for10 mins (2,500 x g for 15mins)
o Platelet-poor-plasma (<10x10^9/L plts)
 Release heparin-neutralizing substances
 Calculation of anticoagulant if Hct is <55%
o C= (1.85x10.3) (100-H) V
 C= volume of sodium citrate
 H= hematocrit
 V=volume of whole blood

Platelet Function Tests

 Detect qualitative (functional) platelet abnormalities

o Platelet count vs Platelet function tests
 Number of platelets in the pxs blood
o Calculated number of platelets in a volume of blood platelets per cubic millimeter (cu.mm) of whole blood
 Thrombocyte count
 Why are platelets difficult to count?
1. They easily disintegrate
2. Small, colorless, refractile bodies
3. Difficult to distinguish from debris
4. Unevenly distributed in the blood
5. Have a tendency to attach to foreign surface
6. Have a tendency to clump with each other

Methods for Platelet counting

1. Indirect method
o Study of blood smears
2. Direct method
o hemocytometry
3. Automated method
o Automation

Indirect method
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 Platelets are counted in relation to 100 RBC
1. Dameshek method
2. Fonio’s method
3. Olef’s method
4. Cramer and Bannerman Method
5. Modified Indirect Platelet count
1. Dameshek method
a. Diluting fluid is placed over the puncture site
b. Mixure is placed on a coverslip and inverted on a slide
c. Platelets are counted in 1000RBCs
2. Fonio’s method
o Similar to Dameshek but mixture is smeared on a slide, dried ad stained
o Computation for 1 and 2:
Plt/cu.mm = plt ct x RBC ct
1000
3. Modified indirect platelet count
a. Prepare a good quality smear
b. Stain with Wright’s stain
c. Using the battlement method of counting platelets in 10 consecutive OIF (moderately thin portion of smear)

Plts ctd X 2000 =platelets/cu.mm

Direct method

o Most accurate way of platelet count

o RBC pipette and diluting fluids
1. Brecher-Cronkite
2. Rees-Ecker
3. Guy’s and Leake
4. Unopette method
5. Tocantin’s method
1. Brecher-Cronkite method
o Recommended method for manual platelet count
o Principle
 Diluent contains ammonium oxalate w/c completely lyses RBC
 Phase contrast microscope
 Enhance the refractileness of the platelets making identification easier
2. Rees-Ecker method
 Rees-Ecker diluting fluid
 Brilliant cresyl blue
 Platelets as light blue
 Count using HPO ; 25 squares of the central large square

Precautions to be taken in doing platelet counts

1. Glasswares must be scrupulously clean

2. Diluting fluid must be filtered before use to remove particles
3. Blood shoud be rapidly diluted to prevent clumping
4. Blood should be thoroughly mixed
5. Charged chamber should be kept for 15mins under petri dish to prevent evaporation and for cells to settle down
6. Finger should not be squeezed excessively to collect blood

Automated platelet counting

1. Coulter counter
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2. Technicon HEmolab
3. Fisher Autocytometer
4. MK-4 Platelet counter

Platelet Function Tests

 Bleeding Time Test

o Measures the time reqd for bleeding to stop
o Reflects both platelet number and platelet functional integrity
o If standardized, considered the best test
 Platelet Aggregometry
o Assesses platelet adhesion ,aggregation and secretion
o Using platelet-rich plasma
o Performed using a light transmittance aggregometer
 Diminished or absent aggregation
 40% aggregation as the lower limit of normal
 Tabares, MA©

 Whole blood platelet aggregometry

o Aggregation is measured by electrical impedance
o Rise in impedance is directly proportional to platelet aggregation
 Platelet Lumiaggregometry
o May be performed using whole blood or PRP
o Luminescence induced by thrombin is measured
o Measures platelet aggregation and secretion of ATP from activated platelet granules

Testing for Heparin Induced Thrombocytopenia with Thrombosis (HIT)

 Aggregation-based tests for HIT include:

1. Light-transmittance aggregometry
2. Washed-platelet lumiaggregometry
3. Whole blood lumiaggregometry
4. Washed platelet carbon 14 serotonin release assay

Clot retraction time (CRT)

o Determines the length of time required for firm clot formation by platelets and fibrinogen
o Clot retraction decreases clot size during formation of homeostatic plug
o Results indicate platelet and fibrinogen quantity and function
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 Clot retraction is influenced by:

1. Number and activity of platelets
2. Concentration of fibrinogen
3. Packed cell volume
 Methods for CRT:
1. Hirschbeeck method
2. Stefanini-Dameshek method
3. MacFarlane method
4. Tocantin’s method

Capillary Fragility Test

1. Tourniquet Test
2. Rumpel-Leede Test

o Evaluates the stability of capillaries under increased hypoxia and hydrostatic pressure
o Positive test indicates weakness of the capillary walls

Platelet adhesiveness test

o Measures the ability of platelets to adhere to glass surface

 Ca, RBC, fibrinogen, vWF
a. a standard incision is made in the forearm and blood is collected at intervals for platelet count
b. blood is allowed to pass through a column of glass beads

Test for Platelet Factor 3

a. 1:1 (PRP: control PPP) + caolin = to activate platelets to release PF3

b. CaCl2, is added and clotting time is recorded.
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Quantitative Measurement of Platelet Markers

 Immunoassay for the antiplatelet Factor-IV (HIT) Antibody

o Detects antibodies early in the development of HIT
 Assays for platelet activation markers
o Enzyme immunoassays for PF4 and B-thromboglobulin