Laboratory Evaluation of Platelets

by: Tom Anthony A. Tonguia, RMT

Objectives
• To know the purpose and importance
of evaluation of platelets.
• Discuss the following techniques in
evaluating your platelets.
Enumeration of Platelets
Estimates from Peripheral Blood Films

• A general reference range for platelets is 140 to 440 x

10^9/L, meaning there are approximately 10 to 40 red
cells per platelet in normal peripheral blood.
• Review of peripheral films is advantageous in detecting
causes of artifactually low counts secondary to platelet
clumping caused by anticoagulant-dependent platelet
agglutinins or clots from poorly collected specimens.
Manual platelet counts

• The two most commonly employed methods of manual platelet

counts utilize a 1:100 or 1:200 dilution of blood applied to a
Neubauer hemocytometer chamber.
• The Tocantins method which uses the Rees-Ecker diluent which fixes
and preserves RBCs as well as platelets to prevent disintegration.
• More popular is the phase-contrast microscopy which uses a phase
micros and ammonium oxalate which lyses red cells and allows
platelets to form pseudopods.
• Reference range is 140 to 440 x 10^9/L (Steininger)
Phase Microscope Platelets under Phase microscopy
Automated Platelet Counts

• Automated counters employ either optical method or electrical

methods to detect particles as they stream through an aperture tube
or flow cell.
• Automatic platelet counters may sample whole blood and
discriminate platelets from red cells on the basis of their size; sample
of whole blood and lyse red cells.
• Discriminate between platelets and leukocytes on the basis of their
size; or sample PRP and calculate platelet counts om the basis of
sample dilution, volume counted, and hematocrit of original sample.
Sources of Error

• Falsely high platelet counts

– contamination of reagents with particles or
bacteria
• Falsely low platelet counts
– platelet agglutinins or platelet satellitism
Automated Cell Counter
Platelet Adhesion tests
Bleeding time

• it is the time it takes for a standard wound to stop bleeding.

• it is a comprehensive test of platelet action in vivo.
• sensitive to abnormalities of platelet numbers and function to
plasma VIII:vWF deficiencies, and to abnormalities of vessel
wall composition that interfere with platelet funtion.
• Two methods is used, Duke and Ivy bleeding time.
• Reference range- 2 to 9 minutes (Ivy method), 1 to 3 minutes
(Duke method).
 Duke bleeding time
Ivy bleeding time
Glass Bead Retention Test

• When blood is passed through a glass bead column, normal

platelets that have access to normal vWF will adhere and
aggregate to the beads such that the effluent from the column
will have much lower platelet count than the starting sample.
• Reference range- the percentage of platelets retained in the
glass bead column should be approximately 70% or greater.
• Each laboratory should establish its own reference range with
the equipment used.
Platelet adhesiveness in vivo

• the in vivo adhesiveness test involves serial platelet

counts on blood excluding from a forearm incision.
• the literature range for the percentage of platelet
adhesiveness is 15% to 45%.
• Each laboratory should establish its own reference
range.
Clot retraction test

• The principle of the test is within 1 hour after whole blood

is allowed to clot in a clean glass tube at 37ᵒC, the clot will
begin to shrink and retract from the walls of the tube.
• Clot retraction is abnormal with thrombocytopenia, low or
abnormal fibrinogens, paraproteinemias that interfere with
fibrin formation, and Glanzmann's thrombasthenia in which
platelet is incapable of interacting with fibrin.
Platelet Factor assays
Platelet Factor 3 availability

• 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.
• Specimen requirements: blood collected in 3.2% sodium
citrate
Reagents and Equipment

• 0.030 M CaCl2
• Kaolin, 1.5% suspension
• Epinephrine (109 uM)
• Commercially available normal control plasma or
pooled PPP.
Assays of PF4 and Beta-Thromboglobulin

• PF4 and beta-thromboglobulin are heparin binding proteins

found in platelet alpha-granules.
• Their levels in vivo are an indicator of the presence of ongoing
platelet activition in a variety of disease such as myocardial
infarction, venous thrombosis, diabetes, inflammatiry states
and myeloproliferative disorders.
• This test is done in patients with suspected of having storage
pool disease or release defect.
• At present used as research procedures.
Procedure

• RIA kits are available for PF4 and beta-

thromboglobulin measurement in plasma or from
alpha-granule release.
Platelet Aggregation tests
Platelet Aggregometry

• Aggregating agents added to a stirred suspension of PRP

induce a shape change and aggregation of platelets.
• This measure and records a change in light transmission.
• Preferably, the patient and the normal conrtol should be
fasting, and should not be taking aspirin or nonsteroidal anti-
inflammatory drugs.
• Specimen requirements: Venous blood from a plastic syringe.
Aggregating agents

• ADP (125 uM)

• Epinephrine (109 uM)
• Collagen
• Thrombin (human)
• Ristocetin
• Arachidonic acid
Platelet aggregometry
Von Willebrand Factor Assay

• 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 vWF present.
• Blood is collected in 3.2% sodium citrate.
Reagent and Equipment used:
• Platelet aggregometer
• Ristocetin
• Phosphate-buffered salin (PBS)
• Bovine serum albumin (BSA)
• Pooled normal PPP or commercial lyophilized control
plasma assayed for vWF
• Commercial formalin fixed platelet suspension adjusted to
400 to 500x10^9/L
• Plastic tubes and pipets