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
= 10
3
plt/uL
= 10
9
plt / L
*15 = constant
1. Platelet Size and Platelet Shape

2. Manual platelet count
-venous blood + EDTA : prevent plt clumping
-in capillary blood : low plt count –due to plt adhesion to the wound
-Dilution : 1:100 or 1:200 (Neubauer Counting Chamber)

a. Ress-Ecker / Tocantin’s 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
-K
2
EDTA
-Ammonium oxalate
-based on hemolysis of rbc and complete blockage of plt activity by chelating Ca and Mg
-Dilution: 1:50
-0.22% K
3
EDTA
-0.44% NH
4
Oxalate
-Stain : Crystal violet (visualization)
-Total capacity: 25uL
-Normal Range: 145-375 x 10
9
/L

c. Brecker-Cronkite Method
-2 syringe/tube technique
-phase-contrast microscopy
-Diluting fluid :1% NH
4
Oxalate
-Counting Chamber :Spencer-Briteline #1475 -25 squares
-NV: 140-440 x 10
9
/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

*Fonio’s Method
-indirect platelet count
-anticoagulant: 14% MgSO
4

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





















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 mmHg – sphygmomanometer
-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










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 up – “Petechiae”
-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









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 β-THROMBOGLOBULIN
-heparin binding proteins
-found in platelet α-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