PLATELET FUNCTION TESTS Aggregometer

• Platelet function tests are designed to detect is an instrument designed to measure platelet aggregation in
qualitative (functional) platelet abnormalities in a suspension of citrated whole blood or PRP.
patients who are experiencing the symptoms of Platelet Aggregometry and Lumiaggregometry
mucocutaneous bleeding. 1. Specimens must be maintained at ambient
Qualitative Platelet Abnormalities temperature (18° C to 24° C) until testing begins.
• Excessive bruising, superficial (mucocutaneous) 2. Specimens must be tested within 4 hours of
bleeding, and a prolonged bleeding time in a patient collection to avoid spontaneous in vitro platelet
whose platelet count is normal suggest an acquired activation and loss of normal activity.
or a congenital disorder of platelet function. 3. 3. Specimens for PRP-based light-transmittance
• Congenital disorders have been described that result aggregometry must stand undisturbed for 30
from abnormalities of each of the major phases of minutes after centrifugation while the platelets
platelet function: adhesion, aggregation, and regain their responsiveness
secretion. 4. 4. Specimens for impedance whole blood
aggregometry are diluted 1:1 with normal saline and
tested immediately.

PLATELET AGGREGOMETRY: Using Platelet-Rich Plasma

• PRP aggregometry is performed using a specialized
photometer called a light-transmittance
aggregometer (PAP-8E Platelet Aggregation Profiler;
Bio/Data Corp., Horsham, PA).
Procedure
1. Calibrate instrument as per manufacturer’s
instructions.
2. Operator pipettes the PRP to instrument compatible
cuvettes, usually 500 uL and drops one clean
plasticized stir bar per sample.
3. Operator places places the cuvettes in incubation
wells; and allows the samples to warm to 37° C for 5
minutes.
4. The operator then transfers the first cuvette,
containing specimen and stir bar, to the instrument’s
reaction well and starts the stirring device and the
recording computer. The stirring device turns the stir
bar at 800 to 1200 rpm, a gentle speed that keeps
the platelets in suspension.
5. The instrument directs focused light through the
sample cuvette to a photodetector
6. As the PRP is stirred, the recorder tracing stabilizes to
generate a baseline, near 0% light transmittance.
After a few seconds, the operator pipettes an agonist
directly into the sample to trigger aggregation.
7. In a normal specimen, after the agonist is added, the
shape of the suspended platelets changes from
discoid to spherical followed by pseudopod
extension.
• A platelet count is performed, and the blood film is
8. The intensity of light transmittance initially (and
reviewed before platelet function tests are begun, because
briefly) decreases, then increases in proportion to
thrombocytopenia is a common cause of hemorrhage.
the degree of shape change.
• Qualitative platelet abnormalities are suspected only
Platelet Agonists (Activating Agents)
when bleeding symptoms are present and the platelet
Used in Aggregometry
count exceeds 50,000 /uL.
The optical PRP-based aggregation method is employed most
PLATELET FUNCTION TESTS
frequently in clinical practice, and the agonists used are
1. Platelet Aggregometry
thrombin or synthetic thrombin receptor-activating peptide
A. Using Platelet-Rich Plasma
(TRAP), adenosine diphosphate (ADP), epinephrine, collagen,
B. Using Whole Blood
arachidonic acid, and ristocetin.
C. Platelet Lumiaggregometry
2. Bleeding Time test
Platelet Agonists
1. Thrombin or synthetic TRAP
2. Adenosine diphosphate (ADP): the most
commonly used agonist, particularly in aggregometry PLATELET AGGREGOMETRY: Using Whole blood
systems that measure only aggregation and not In whole-blood platelet aggregometry, platelet aggregation is
luminescence. measured by electrical impedance using a 1:1 saline–whole
3. Epinephrine: does not work in whole-blood blood suspension (Model 700 Whole Blood/Optical Lumi-
aggregometry. Platelets in suspension Aggregometer; Chrono-log Corp.,
4. Collagen Havertown, PA)
5. Arachidonic acid Procedure
6. Ristocetin 1. Operator pipettes aliquots of properly mixed whole blood
Continuation to cuvettes and adds equal volumes of physiologic
9. Percent light transmittance is monitored saline. Suspension volume may be 300 to 500 uL.
continuously and recorded. 2. The operator drops in one stir bar per cuvette and places
10. As platelet aggregates form, more light passes the cuvettes in 37° C incubation wells for 5 minutes.
through the PRP, and the tracing begins to move 3. The operator transfers the first cuvette to a reaction well,
toward 100% light transmittance. pipettes an agonist directly into the specimen, and
11. Platelet function deficiencies are reflected in suspends a pair of low-voltage cartridge-mounted
diminished or absent aggregation. disposable direct current electrodes in the mixture
12. Many laboratory directors choose 40% aggregation 4. As aggregation occurs, platelets adhere to the electrodes
as the lower limit of normal. and one another, impeding the current.
5. The rise in impedance, which is directly proportional to
platelet aggregation, is amplified and recorded by
instrument circuitry.
6. A whole-blood aggregometry tracing closely resembles a
PRP-based light transmittance aggregometry tracing.
Platelet Lumiaggregometry
• The Chrono-Log Model 700 Whole Blood/Optical
LumiAggregometer is used for the simultaneous measurement
of platelet aggregation and the secretion of adenosine
triphosphate (ATP) from activated platelet dense granules.
• The procedure for lumiaggregometry differs little from that
for light transmittance or whole blood aggregometry, but
because of its detection properties lumiaggregometry
simplifies the diagnosis of platelet dysfunction
• As ATP is released, it oxidizes a firefly derived luciferin-
luciferase reagent (Chrono-Lume from Chrono-Log) to generate
cold chemiluminescence proportional to the ATP
concentration.
• A photodetector amplifies the luminescence, which is
recorded as a second tracing on the aggregation report.
 Platelet Lumiaggregometry
Lumiaggregometry may be performed using whole blood or
PRP.
Procedure
1. The operator adds an ATP standard to the first
sample and then adds luciferin-luciferase and tests
for full luminescence.
2. The operator then adds luciferin-luciferase and an
agonist to the second sample; the instrument
monitors for aggregation and secretion
simultaneously.
*Thrombin (or thrombin receptor-activating peptide, TRAP) is
typically the first agonist used because thrombin induces full
secretion.
3. The operator ordinarily begins with 1 unit/mL of
thrombin to induce the release of 1 to 2 nM of ATP,
detected by luminescence of the firefly luciferin
luciferase.
4. The luminescence induced by thrombin is measured,
recorded, and used for comparison with the
luminescence produced by the additional agonists.
5. Normal secretion induced by agonists other than
thrombin produces luminescence at a level of about
50% of that resulting from thrombin.
6. Thrombin-induced secretion may be diminished to
less than 1 nM in storage pool deficiencies, but it is
relatively unaffected by membrane disorders or
pathway enzyme deficiencies.