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1. Classification
of thrombocyte
disorders using
platelet
aggregation
assays
JAVIER, Christine Joyce M.

2. Thrombocyte disorders
• Thrombocyte disorders can either show
thrombocytosis or thrombocytopenia.

3. Platelet function tests
• Platelet function tests are designed to
detect qualitative platelet abnormalities.
• Platelet count → platelet morphology →
platelet function tests
• Platelet counts > 50 x 103/uL & presence of
bleeding symptoms
• Bleeding time and platelet aggregometry

4. Platelet aggregometry
• Platelet aggregometry is an in vitro test
which allows the assessment of platelet
adhesion, aggregation, and secretion.
• Normal adhesion
• Normal aggregation
• Normal secretion

5. Platelet aggregometry
• Aggregometer is an instrument designed to
measure platelet aggregation.
• Two types:
• Platelet aggregometry (conventional)
• Light-transmittance
• Electrical impedance
• Lumiaggregometry

6. Platelet aggregometry –
light- transmittance
• Sample required: platelet-rich plasma
• Sample preparation:
• Centrifuge blue-capped tubes for 30 minutes.
• Transfer supernatant to a plastic tube.
• Stand at room temperature for 30 minutes.

7. Platelet aggregometry –
light- transmittance
• Process:
• Transfer PRP to a cuvette.
• Add stir bar.
• Place in incubation wells.
• Transfer the tube to reaction wells.
• Start the stirring device.

8. Platelet aggregometry –
light- transmittance

9. Platelet aggregometry –
light- transmittance

10. Platelet aggregometry –
electrical impedance
• Sample required: sodium citrate whole blood
• Sample preparation:
• Hold blue-capped tubes for at room
temperature until testing.

11. Platelet aggregometry –
electrical impedance
• Process:
• Transfer whole blood sample to a cuvette.
• Add equal volume of physiologic saline.
• Add stir bar.
• Place in incubation wells.
• Transfer the tube to reaction wells.

12. Platelet aggregometry –
electrical impedance

13. Platelet aggregometry –
electrical impedance

14. lumiaggregometry
• Sample required: sodium citrate whole blood
or platelet-rich plasma.
• Sample preparation:
• Platelet-rich plasma – same with light
transmittance aggregometry
• Whole blood – same with electrical
impedance aggregometry

15. lumiaggregometry
• Process:
• Transfer sample to a cuvette.
• Add stir bar.
• Place in incubation wells.
• Transfer the tube to reaction wells.
• Add agonist, then luciferin-luciferase.

16. lumiaggregometry

17. types of platelet agonists
• Thrombin or TRAP
• ADP
• Collagen
• Epinephrine
• Ristocetin
• Arachidonic acid

18. Classification of
thrombocyte disorders
• Disorders of platelet adhesion
• Disorders of aggregation
• Disorders of secretion
• Disorders of thromboxane synthesis
• Acquired disorders due to drugs (aspirin,
nonsteroidal anti-inflammatory drugs or
NSAIDs and clopidogrel)

19. Case study
• A 20 year old woman has an average platelet
count of 60, 000/uL to 80, 000/uL with
significant bleeding symptoms requiring
frequent interventions and has no response to
platelet raising therapies.
• Splenectomy
• Tonsillar bleed from eating a potato chip

20. Case study
• Questions
1. Is qualitative platelet abnormality
suspected? If yes, which of the patient’s
clinical features supports your claim?
2. What further laboratory tests should be
performed?
3. What is the clinical diagnosis?

21. Case study
• Answer to question #1: Yes, qualitative
platelet abnormality is suspected. It was
mentioned earlier that qualitative platelet
abnormalities are only suspected if platelet
counts exceed 50, 000/uL and bleeding
symptoms are present.

22. Case study
• Answer to question #2: Review morphology
of platelets in a stained smear. Also, perform
a platelet aggregation assay.

23. Case study

24. Case study
PATIENT
NORMAL

25. Case study
• Answer to question #3: Clinical diagnosis is
Bernard-Soulier syndrome. Bernard-Soulier
syndrome is a qualitative platelet disorder
caused by the absence of GP Ib. This disease
is characterized by prolonged bleeding,
thrombocytopenia, giant platelets, and
diminished aggregation with ristocetin in a
platelet aggregation assay.

26. Current advances
• Replacement of platelet aggregometry by flow
cytometry in assessment of platelet function
• Flow cytometry – performed regardless of
platelet count
• Light transmittance – 200,000/uL-300,000/uL
• Electrical impedance > 100,000/uL
• Development of point-of-care testing machines
to immediately asses platelet function

27. La fin.

28. Slide notes!
SLIDE 2: Thrombocyte disorders can either show an increase or decrease in platelet count. Diseases that exhibit
decreased platelet count can be divided into quantitative and qualitative. Qualitative abnormalities are evaluated in
platelet function tests.
SLIDE 3: Platelet function tests are designed to detect qualitative or functional platelet abnormalities in patients who
are experiencing the symptoms of mucocutaneous bleeding.
Platelet count is performed first, the morphology of platelets are reviewed in a stained smear before platelet function
tests are began.
Qualitative platelet abnormalities are suspected if only the platelet counts exceed 50,000/uL and there is a presence
of bleeding symptoms. Platelet counts below 50,000/uL are considered quantitative platelet disorders.
Two tests used to determine platelet function are bleeding time and platelet aggregometry. Bleeding time was the
original test used yet was largely replaced by platelet aggregometry.
SLIDE 4: Platelet aggregometry is an in vitro test which allows the assessment of platelet adhesion, aggregation, and
secretion.
Functional platelets adhere to subendothelial collagen, aggregate with one another, and secrete the contents of their
alpha granules and dense granules.
Adhesion is considered normal if the platelet membrane is intact and the plasma von Willebrand factor is functional.
Aggregation is considered normal if the platelet membrane and platelet activation pathways are intact, and if there is
a normal concentration of fibrinogen.
Secretion is considered normal if platelet granules are able to release its contents.

29. Slide notes!
SLIDE 5: Aggregometer is an instrument designed to measure platelet aggregation.
There are two types as of today. The conventional platelet aggregometry allows the measurement of platelet
aggregation only, while lumiaggregometry allows the measurement of both platelet aggregation and secretion.
Conventional platelet aggregometry uses an aggregometer which either follows the principle of light-transmittance,
or the principle of electrical impedance.
Allow me to discuss each one by one.
SLIDE 6: Conventional platelet aggregometry following the light-transmittance principle requires the use of patient’s
platelet-rich plasma. The sample is prepared by centrifuging blue-capped tubes for 30 minutes. After that, the
supernatant is transferred to a plastic tube and must stand at room temperature undisturbed for 30 minutes. This
allows the platelet to regain their responsiveness after the spin.
SLIDE 7: PRP or platelet-rich plasma is transferred to a cuvette. A magnetic stir bar is added for each cuvette. These
cuvettes are placed in incubation wells which allows it to be incubated at 37 degrees Celsius for 5 minutes. After
incubation, the cuvette is placed to reaction well. Stirring device is started which turns the stir bar at 800-1200 rpm
to keep the platelets in suspension.
SLIDE 8: A focused light is directed onto a sample cuvette. Photodetector at the end detects the light transmittance.

30. Slide notes!
SLIDE 9: There is a zero percent light transmittance when resting platelets are in suspension, It also allows the
recording of baseline data which is zero percent aggregation. After the addition of an agonist, for example, ADP, there
is a platelet shape change from discoid to spherical. Primary-wave aggregation happens after, which is the formation
of small clumps. There is a secretion of granules. Lastly, second-wave aggregation occurs, which is the formation of
large clumps.
As more platelet aggregates form, percent light transmittance increases and is directly proportional to percent
aggregation.
Allow me to show a video on how a light-transmittance platelet aggregometry is performed.
SLIDE 10: Conventional platelet aggregometry following the electrical impedance principle requires the use of
sodium citrate whole blood. There is no special procedure for sample preparation. The tubes must be held at room
temperature until testing.
SLIDE 11: Sodium citrate whole blood is transferred to a cuvette. Equal volume of normal saline solution is added to
the cuvette to achieve the 1 is to 1 ratio of whole blood saline suspension that can be either 300 microliter or 500
microliter in total volume. Stir bar is added and these cuvettes are placed in incubation wells which allows it to be
incubated at 37 degrees Celsius for 5 minutes. After incubation, the cuvette is placed to reaction well.
SLIDE 12: An agonist is directly added to the cuvette, followed by the suspension of a pair of low-voltage cartridge-
mounted disposable direct current electrodes. Platelet aggregates adhere to the electrodes in suspension, which
impedes the current.

31. Slide notes!
SLIDE 13: There is zero impedance of current or resistance when resting platelets are in suspension, It also allows the
recording of baseline data which is zero ohm of resistance. After the addition of an agonist, for example, ADP, there is
a platelet shape change from discoid to spherical. Primary-wave aggregation happens after, which is the formation of
small clumps. There is a secretion of granules. Lastly, second-wave aggregation occurs, which is the formation of large
clumps.
As more platelet aggregates form, impedance in current or resistance increases and is directly proportional to
percent aggregation.
SLIDE 14: Let’s go to lumiaggregometry.
Samples required for lumiaggregometry can either be sodium citrate whole blood or platelet-rich plasma.
Preparations of these samples are same with how PRP for light-transmittance aggregometry is prepared, and with
how whole blood for electrical impedance aggregometry is prepared.
SLIDE 15: The procedure of lumiaggregometry differs a little from the conventional platelet aggregometry. Sample is
transferred to a cuvette. Stir bar is added and these cuvettes are placed in incubation wells which allows it to be
incubated at 37 degrees Celsius for 5 minutes. After incubation, the cuvette is placed to reaction well.
ATP standard is added to the first sample cuvette, followed by the addition of luciferin-luciferase to test for full
luminescence. For the second sample cuvette, an agonist is added followed by the luciferin-luciferase.
Usually, thrombin is the first agonist used for it is able to induce full secretion, yet other agonists are also be tested.
The luminescence induced by thrombin is measured, recorded, and used for comparison with the luminescence
produced by other agonists.

32. Slide notes!
SLIDE 16: When platelet granules are able to secrete, molecules like ATP is released. It can oxidize a firefly-derived
luciferin-luciferase reagent and is able to generate chemiluminescence directly proportional to the concentration of
analyte measured. A photodetector amplifies the luminescence, which is recorded as a second tracing on the
aggregation.
SLIDE 17: Types of Platelet Agonists used in platelet aggregation assays
Thrombin can be used or the synthetic thrombin receptor activating peptide to asses ATP release.
ADP, collagen and epinephrine are used to evaluate abnormalities in Glycophorin 2b 3a.
Ristocetin is used to evaluate abnormalities in plasma von Willebrand Factor and Glycophorin 1b.
Arachidonic acid is used in assessing eicosanoid synthesis deficiencies.
SLIDE 18: Classification of Thrombocyte Disorders
Disorders of platelet adhesion include the von Willebrand disease and Bernard-Soulier syndrome. Platelets do not
aggregate with ristocetin.
Disorders of aggregation include the Glanzmann thrombasthenia. Platelets do not aggregate with ADP, collagen and
epinephrine.
Disorders of secretion include alpha granule deficiency called gray platelet syndrome, and dense granule deficiency
called delta storage pool defect. Secretion and aggregation do not happen in the presence of ADP, arachidonic acid
and collagen.
Disorders of thromboxane synthesis include diseases that inhibit cyclooxygenase pathway. Together with the acquired
disorders due to drugs, storage and secretion with these category diminish with arachidonic acid and collagen.

33. Slide notes!
SLIDE 19: The case study presents a 20 year old woman who has an average platelet count of 60000 per microliter to
80000 per microliter with significant bleeding symptoms requiring frequent interventions and has no response to
platelet raising therapies. Bleeding episodes include splenectomy, tonsillar bleed from eating a potato chip and
menorrhagia resulting to iron-deficiency anemia. She has always had bruising, petechiae and oral bleeding. Her
parents are healthy and so is her 15 year old brother.
SLIDE 23: Stained smear showed giant platelets.
SLIDE 24: Platelet aggregation assay showed normal aggregation with agonist ADP, epinephrine, collagen and
arachidonic acid. Aggregation diminished with ristocetin.
SLIDE 26: Flow cytometry is now slowly being favored over the light transmittance and electrical impedance
aggregometry for it only requires small volume of whole blood and is independent of platelet count. Platelet-rich
plasma for aggregometry requires a platelet count of about 200000 to 300000 per microliter, while whole blood
samples require a platelet count greater than 100000 per microliter.
Platelet function tests are increasingly proposed as one of the tests to be assessed prior to surgery as an aid in
prediction of bleeding.