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LABORATORY EVALUATION OF PLATELETS

A. QUANTITATION

1. Estimates

-from peripheral blood film

-counting (area: rbc barely touching

-140,000/150,000 to 400,000/440,000 plt / cumm

- 10-40 rbc = 1 plt

- 1 field (OIO)

- 3-10 plts/100 rbc

- 5-20 plts/200 rbc

NB:

1.

Hct of ptx should be NORMAL

2.

No plt clumping (Microclots) on the field

*EDTA

-Advantage: detecting causes of artifactually low counts secondary to platelet clumping caused

by anticoagulant-dependent platelet agglutinins or clot from poorly selected specimen

(plt count in 1 field) x 15 = plt / cumm

= 103 plt/uL= 109plt / L

*15 = constant

1.

Platelet Size and Platelet Shape

2.

Manual platelet count

-venous blood + EDTA : prevent plt clumping

-in capillary blood : low plt countdue to plt adhesion to the wound

-Dilution : 1:100 or 1:200 (Neubauer Counting Chamber)

a. Ress-Ecker / Tocantins Method

- Light microscopy method-Diluent : Rees-Ecker Diluent : citrate-formaldehyde buffer with brilliant cresyl blue

: fixes and preserves rbc and plt to prevent disintegration

Composition:

Citrate -anticoagulant

Formaldehyde -preservative

BCB -stain for light microscopy (Blue)

-Counting Chamber : Neubauer (4 large squares)

b. Unopette Method

-K2EDTA

-Ammonium oxalate

-based on hemolysis of rbc and complete blockage of plt activity by chelating Ca and Mg

-Dilution: 1:50

-0.22% K3EDTA

-0.44% NH4Oxalate

-Stain : Crystal violet (visualization)

-Total capacity: 25uL

-Normal Range: 145-375 x 109/L

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c. Brecker-Cronkite Method

-2 syringe/tube technique

-phase-contrast microscopy

-Diluting fluid :1% NH4Oxalate

-Counting Chamber :Spencer-Briteline #1475 -25 squares

-NV: 140-440 x 109/L

2. Automated Methods

1. Optical Method

-measurement of degree of light scattering

2. Electrical Method

-change in electrical resistance/capacitance that will be detected on the tubing

-precise

-Limitations:

False INCREASE:

-false increase result -> reagent contaminated (particles, bacteria)

-carry over of samples with patient with high plt count-particles/cellular debris is read as plt

False DECREASE:

-platelet satellitism (plts stick to neutrophil)

-large plts are not read by the machine

*Fonios Method

-indirect platelet count

-anticoagulant: 14% MgSO4

-Procedure: capillary blood -> 1 drop Anticoagulant, puncture, smear

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PLATELET FUNCTION TEST

A. PLATELET ADHESION-adhere to other surfaces

1. BLEEDING TIME

-measure time for a standard wound to stop bleeding

-observe blood flow

-comprehensive in vivo test for platelet action or function test

-sensitive to abnormalities of

-platelet number and function

-plasma VIII: vWF deficiencies

-vessel wall composition that interfere with plt fxn

a. IVY METHOD

-40 mmHgsphygmomanometer

-3 incision wound on the volar surface of the forearm

-2 mins -> blot on filter paper

-Normal Reference: 2-8 mins-no standardization of wound

b. DUKES METHOD

-performed on children and toddlers

-ear lobe

-glass slide

-back of ear

-puncture

-Normal Reference: 1-3 mins

-no standardization of wound

c. MIELKE/TEMPLATE METHOD

-modification of IVY method

-use a template for incision wound

-has standardization of incision

-Normal Reference: 1-8 mins

d. SIMPLATE METHOD

-use of Simplate Bleeding device

-uniformed incision wound

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2. CAPILLARY FRAGILITY / TOURNIQUET TEST

-relationship of blood vessel and platelets

-normal pressure / trauma

-GAPS formed between endothelial cells

If NORMAL plt fxn: gaps are filled up

If ABNORMAL plt fxn: gaps are not filled upPetechiae

-100 mmHg for 5 mins ->release -> observe for the presence of Petechiae after 2 mins

3. GLASS BEAD RETENTION TEST

-plt adhesion test

-glass bead column

-normal platelet that have access to normal vWF will adhere and aggregate to the beads

-effluent from column will have a much lower plt ct than the starting sample

4. PLATELET ADHESIVENESS IN VIVO

-serial platelet counts on blood exuding from forearm incision

-normally, plt ct is decreased due to plt adhesion to the wound- plt cts are compared with a venous blood plt ct as a control

-calculate % platelet adhesiveness

-Normal Range: 15-45%

B. PLATELET AGGREGATION TEST

-use Platelet Aggregometer

-diagnose acquire or hereditary plt disorders

(1) activated platelet rich plasma (PRP) -> stirred into aggregometer, light will pass thru

-Stimuli:-ADP

-Epinephrine

-Collagen

-Thrombin

-Ristocetin

-Arachidonic acid

(2) Platelet shape change

-discoid to spherical

-initial decrease of light transmittance

(3) Platelet Aggregation

-if there is COMPLETE aggregation: HIGH light transmittance

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TESTS FOR PLATELET FACTOR

1. PF3 ASSAY / AVAILABILITY

-coagualation factor derived from platelets that would act to thromboplastin

-convert: Prothrombin to Thrombin

Kaolin + Epinephrine -> stimulated to provide PF3 Activity

2. CLOTTING TIME

-against a control

-37-51 seconds

3. PF3 and -THROMBOGLOBULIN

-heparin binding proteins

-found in platelet -granules

-indicate platelet activity in

-MI

-DM-venous thrombosis

-other myeloproliferative disorders

4. vWF ASSAY

- agglutination of fixed plts in response to ristocetin depends on the presence of vWF in plasma

-rate/percentage of agglutination is proportional to the amount of vWF