Hematology Lectures

1. Platelet Function Testing
By:
Ahmed Makboul Ahmed
Assistant Lecturer of Clinical Pathology, SECI, Assiut University

2. Outlines:
- Platelet structure.
- Functions of Platelets.
- Overview of normal platelet function.
- Platelet function tests:
o Overview.
o Light transmission platelet aggregometry
(LTA).
o Other platelet function tests.

3. INTRODUCTION:
Stages of Hemostasis:
1. Primary Hemostasis:
- Platelets and the injured vessels interact to form the primary hemostatic plug,
which is a clump of platelets.
- It only temporarily stops the bleeding and is very fragile.
2. Secondary Hemostasis:
- The fibrin-platelet plug is then formed by a series of interactions involving the
coagulation factors (coagulation cascade).
- The fibrin stabilizes the clot covering the hole in the vessel.
3. Fibrinolysis:
- The process of removing the clot once the wound has been healed.

4. PLATELET STRUCTURE:
Normal human platelets are:
Size: Small in size (0.5 x 3.0 μm)
Shape:
- Discoid in shape have a mean volume of 7–11 fL.
- Anucleated.
Number: They circulate in relatively high numbers (between 150
and 400 x 109 /L).
Life span: Their lifespan is approximately 10 days (9 – 12 days).

5. Platelet Ultrastructure
1
2
3
4

6. 1

7. 1. Peripheral Zone:
Function:
- Adhesion and aggregation.
- Responsible for cell's negative charge.
a). Glycocalyx (exterior coat):
It contains receptors:
- GPIb-IX-V: It is the major platelet receptor for VWF.
- GPIa/-Ia and GPVI: receptors for collagen.
- GPIIb-IIIa: receptor for vWF.
- Thromboxane A2 (TXA2) receptor.
- ADP receptors: P2Y12 and P2Y1.
b). Membrane:
- Has a number of phospholipids and proteins
embedded within it.
- Responsible for the release of fatty acid derivatives:
PGs, prostacyclins.

8. 2

9. 2. Structural Zone:
- Organized network of protein filaments.
- Maintain shape of resting platelet—discoid.
- 3 principle types of filaments:
a). Microtubules:
- Located beneath the cell membrane of resting
platelet.
- Maintain discoid shape.
b). Microfilaments:
- Mediate contractile events.
c). Intermediate filaments.

10. 3

11. 3. Membranous Zone:
Function:
- Structure and support.
a). Open Canalicular System (OCS):
- Responsible for the secretion of granule
contents.
- Provides a route for entry and secretion.
b). Dense Tubular System (DTS):
- Responsible for the storage of calcium.
- Major site of prostaglandin and
thromboxane synthesis

12. 4

13. 4. Organelle Zone:
Function:
- Secretion and storage.
a). Mitochondria.
b). Glycogen particles: support metabolic activities
c). Granules:
dispersed within the cytoplasm, which serve as storage
sites for proteins and other substances necessary for
platelet function:
i. Dense granules: contain platelet agonists and
signaling molecules that amplify platelet activation
(approximately 3-8 dense granules per platelet):
- Agonists:
ADP.
ATP.
- Serotonin.
- Calcium.

14. ii. Alpha granules: contain proteins that enhance adhesion, growth factors, and coagulation
factors (approximately 80 alpha granules per platelet).
- Adhesive proteins:
 Fibronectin.
 vWF.
 Thrombospondin.
 Vitronectin.
- Growth modulators:
 PDGF.
 TGF-β.
 Platelet factor 4 (PF4).

15. - Coagulation factors:
 Factor V.
 High-molecular-weight kininogen (HMWK).
 C1 inhibitor.
 Fibrinogen.
 Factor XI.
 Protein S.
 Plasminogen activator inhibitor-1 (PAI-1).
- Membranes of platelet alpha granules contain P-selectin.

16. FUNCTIONS OF PLATELETS:
Role of Platelets in the Circulation
1. Surveillance of blood vessel continuity:
- Checks endothelial lining for gaps and
breaks.
- Fill-in small gaps caused by separation of
endothelial cells.
2. Formation of primary hemostatic plug.
3. Surface for coagulation factors to make
secondary hemostatic plug.
4. Aid in healing injured tissue.
Platelet Function
1. Adhesion.
2. Activation and shape change.
3. Secretion or release.
4. Aggregation.

17. OVERVIEW OF NORMAL PLATELET FUNCTION:
• Endothelial cells produce a number of potent antiplatelet substances
(e.g., nitric oxide, prostacyclin, and CD39) that normally inhibit vessel
wall–platelet interactions.
• Vessel wall damage exposes highly adhesive substrates (e.g., P selectin,
von Willebrand factor (VWF), collagen, and many other extracellular
matrix components), which overcome these inhibitory factors and result in
a sequence of stepwise events resulting in the formation of a hemostatic
plug:

18. 1. Platelet Adhesion:
o Platelets are marginated along the vessel wall
where they are well-positioned to monitor the
integrity of the endothelium.
o When vessel wall injury occurs and there is
endothelial damage, the initial response of
platelets is that of adhesion to collagen fibers in
the exposed subendothelium.
o Initial binding of platelets is considered to occur
via GPIa-IIa receptor, which allows for further
binding to collagen via the GPVI receptor,
initiating transmembrane and, subsequently,
intracellular signalling.
o Adhesion of platelets to the exposed
subendothelium is influenced by shear rates. At
high shear, GPIa-IIa and GPVI are not sufficient to
initiate binding to collagen, and binding of the
GPIb-IX-V receptor to vWF that is immobilized on
collagen, becomes essential in platelet adhesion.

19. 2. Platelet Activation and Granule
Release:
Platelet adhesion at the site of vessel wall
damage initiates activation events that result in
aggregation:
1. Adherent platelets undergo a dramatic shape
change to an irregular sphere with multiple
pseudopodia spreading on the subendothelium
increasing their area of surface contact.
2. Adherent platelets also secrete or release the
contents of their storage granules -- the alpha-
and dense granules -- by an exocytic process.
This provides a high local concentration of
effector molecules essential for platelet plug
formation at the site of vascular injury.

20. 2. Platelet Activation and Granule
Release:
3. Platelet activation stimulates the
formation of another aggregating
agent, thromboxane A2 via the
arachidonic acid cascade.
4. ADP, thromboxane A2 and thrombin
bind to specific platelet membrane
receptors and stimulate aggregation on
and around the platelets adherent to
the subendothelium via receptor-
mediated signal transduction events.

21. 3. Platelet Aggregation:
- Aggregation is an active metabolic process:
binding of any of the agonists to their
respective membrane receptors initiates
signalling pathways that ultimately convert
GPIIb-IIIa - from a low affinity resting state
to a high-affinity activated state for binding
extracellular soluble ligands such as plasma
fibrinogen and vWF.
- Divalent fibrinogen and multivalent von
Willebrand factor function as bridges
between GPIIb-IIIa receptors on adjacent
activated platelets, thus allowing platelet
aggregation to proceed to form a platelet
plug that arrests blood loss from a vascular
injury.

22. PLATELET FUNCTION TESTING:
Overview:
- Platelet function testing is indicated to determine if bleeding is due to defective
platelet function:
oAdhesion
oActivation
oGranule release
oAggregation
- Defective platelet function suspected when platelet count is normal in setting of
bleeding that is characteristic of disorder of primary hemostasis.
- Light transmission platelet aggregometry (LTA) is considered gold standard for
platelet function testing.
- Platelet function is evaluated by observing response of platelets to agonists that
bind to platelets via their receptors leading to activation and aggregation.

23. Light transmission platelet aggregometry (LTA):
CLINICAL APPLICATIONS:
1. Diagnosis of Bleeding Disorder:
- Pattern of response to agonists can help in determining if there is platelet
function defect and can help categorize type of defect.
2. Monitoring Antiplatelet Therapy:
- Effects of antiplatelet medications can be seen with LTA.
- LTA used for monitoring antiplatelet therapy typically in research setting.
- At present, clinical guidelines advise that there is no indication for routine
laboratory monitoring of antiplatelet therapy.

24. PRINCIPLE:
- PRP has high optical density and transmits
light poorly because platelets prevent passage
of light.
PPP has been depleted of platelets and is able
to transmit light well (low optical density).
- When substance that promotes platelet
activation and aggregation (agonist) is added
to PRP, normal platelets are activated and
aggregate.
- Platelet aggregometer measures change in
optical density (or light transmittance) over
time of stirred PRP in cuvettes at 37°C after
addition of agonists.
Aggregation of platelets in PRP results in
decrease in optical density (increased light
transmittance) as aggregated platelets fall out
of path of light.

26. METHOD:
1. SPECIMEN:
1.1. Collection:
- Patients should not take medications known to affect platelet function for 10-14
days before specimen collection.
- 3.2% sodium citrate:
 1:9 citrate-to-whole blood ratio.
1.2. Transport:
- After collection, samples should be transported to laboratory at room temperature.
- Tubes should not be subjected to vibration, shaking, vortexing, continuous mixing,
or agitation.
- Samples should be tested between 30 min and no more than 4 hr from blood
collection.

27. 2. PROCESSING:
2.1. Citrated PRP:
- Prepared by centrifugation at 800 – 1000
rpm for 15–20 minutes.
- Platelet Count of PRP:
• Platelet count of PRP is performed and
should be 200-400 x 10⁹/L.
• If platelet count is > 400 x 10⁹/L,
concentration can be adjusted with
autologous PPP.
- PRP is added to a cuvette at 37C and
preincubated for up to 5 minutes.
- The PRP is stirred at a recommended speed
(e.g., 1000–1200 rpm using a magnetic stir
bar) to allow platelets to come in contact
with each other.

28. 2.2. Instrument calibration:
Aggregometers are calibrated for light transmission using
autologous PPP and PRP:
o 100% transmission is set with autologous platelet-poor plasma
(PPP) (prepared by centrifugation at 3000 rpm for 10 minutes).
o 0% transmission is set with PRP and a stable baseline
established before addition of agonist.

29. 2.3. Addition of agonist:
Agonist is added at maximum of one-tenth volume of agonist to PRP to
initiate aggregation (10% of total volume).
Baseline panel of agonists includes:
Agonist Dose Comment
ADP 2.5–20 μmol/L
Collagen 1–5 μg/mL Usually mediates a steep aggregation curve but
after a characteristic lag phase.
Epinephrine 3–30 μmol/L
Arachidonic acid (AA) 0.5–1.5 mmol/L
Ristocetin 0.5–2 mg/mL Not strictly an activating agonist but stimulates
platelet agglutination through binding of plasma
vWF to GPIb.

30. INTERPRETATION OF RESULTS:
- LTA measures change in optical density
(or light transmittance) of stirred PRP
that occurs after addition of agonists.
- Aggregation tracing presents change in
light transmission (y-axis) against time in
minutes (x-axis).
- Several parameters are evaluated for
interpretation of these studies:
1. Lag phase (time between addition of
agonist and start of aggregation).
2. Platelet shape change.
3. Primary aggregation slope.
4. Secondary aggregation.
5. Disaggregation.
6. Maximum amplitude.
7. Percent aggregation.

31. Normal LTA tracing with ADP, epinephrine, collagen, and ristocetin agonists

32. INTERPRETATION OF ABNORMAL LTA RESULTS:
1. Defective platelet adhesion:
a). Abnormal aggregation response only to ristocetin agonist may be noted if there
is:
o defect in GPIb receptor (Bernard Soulier Syndrome)
o decreased/abnormal vWF (vWD(.
- Examination of peripheral blood smear can be helpful in distinguishing vWD and
BSS:
o BSS is usually accompanied by macrothrombocytopenia (giant platelets).
- Addition of cryoprecipitate (source of vWF) corrects abnormal response to
ristocetin that may be seen in vWD, but not in BSS.
- vWF antigen and activity studies and factor VIII activity assay can be performed if
vWD is suspected.

34. In vWD, there is increased response to low-dose ristocetin. This occurs in:
Type 2B vWD
o Increased affinity of vWF for platelet
GPIb-V-IX.
o Mutations causing type 2B vWD lead
to gain-of-function mutations in GPIb
binding site on vWF, leading to
spontaneous binding of vWF to
platelets.
o Spontaneous binding of vWF to
platelets leads to formation of platelet
aggregates. These aggregates are
removed from circulation leading to
thrombocytopenia.
Platelet type (pseudo) vWD
o Caused by mutations in GPIba gene
(vWF receptor on platelets).
o This mutation results in abnormal
GPIb receptor with increased binding
(gain-of-function) affinity for normal
vWF

35. These 2 disorders can be differentiated by:
1. Modification of low dose Ristocetin-induced platelet aggregation (RIPA)
test:
- Patient platelets mixed with normal plasma.
- Patient plasma mixed with normal platelets.
Increased low-dose RIPA with patient platelets mixed with normal plasma is
consistent with platelet-type vWD
Increased low-dose RIPA with patient plasma mixed with normal platelets is
consistent with type 2B vWD
2. Genetic studies: can also be used to distinguish type 2B vWD from platelet-
type vWD.

36. b). Abnormal aggregation response only to collagen agonist may be
noted if there is:
o defect in collagen receptors (GPIa-IIa or GPVI).
- Flow cytometry can be used to evaluate for low levels of GPIa-IIa or
GPVI.

37. 2. Defective platelet activation and granule release:
a). Secretion defects:
- Defect in platelet-dense granules is due to either:
o Contents of granules (storage pool disorder).
o Inability to release granules (release defect).
- Manifests as lack of secondary aggregation on LTA in response to ADP,
epinephrine, collagen.

38. Additional studies can be performed to distinguish between storage pool
disorder and release defect:
1. Electron microscopy can be used to examine contents of platelet-dense
granules to investigate storage pool disorder.
2. Measurement of total platelet content of ADP and ATP and release of
ATP in response to agonists can help to distinguish between storage pool
disorder and release defect:
o Storage pool defects have decrease in amount of stored and released
ADP with increased ratio of ATP to ADP.
o Release defects have normal stored ADP levels, normal ATP:ADP ratio,
but decreased ADP release.

39. b). Thromboxane pathway defects:
In these defects, there are:
o Absence/ severe reduction of aggregation in response to arachidonic acid
(for cyclooxygenase defects, as in case of administration of aspirin), or
absence of aggregation response to both arachidonic acid and
thromboxane mimetics (for thromboxane receptor defects).
c). P2Y12 inhibitors (e.g. clopidogrel):
o Reduced or absent aggregation to ADP with notable deaggregation even
at high concentrations of agonist.

40. 3. Defective platelet aggregation:
- Lack of platelet aggregation in response to all agonists except for ristocetin
indicates defect in platelet aggregation.
This aggregation pattern can be seen in Glanzmann Thrombasthenia (GT) in
which patients lack or have defect of GPIIb-IIIa receptor.
- Similar aggregation pattern may be seen in severe hypofibrinogenemia or
afibrinogenemia:
o Defects in aggregation can be remedied by addition of cryoprecipitate
(source of fibrinogen) to PRP in the case of hypofibrinogenemia or
afibrinogenemia.
o GT diagnosis can be confirmed by performing flow cytometry to examine
number of GPIIb-IIIa receptors on platelets.

42. Disorders Aggregation Response Comment
ADP Collagen AA Ristocetin
Primary
wave
Secondary
wave
Defective adhesion:
Bernard Soulier syndrome N N N N A - Platelet
morphology.
- vWD assay.
vWD N N N N A
Defective activation and secretion:
Storage pool disease N A A N N - ADP/ATP levels
Secretion defect N A A N N
P2Y12 inhibitor (Clopidogrel) R/A R/A N N N
COX deficiency N A/N R A N
TX Synthase deficiency N A/N R A N
Aspirin ingestion N A/N R A N
Defective aggregation:
Glanzmann Thrombasthenia A A A A N GPIIb/IIIa level by
flow cytometry
N: Normal, A: Absent, R: Reduced

43. From: Dacie and Lewis Practical Hematology, 12th Edition, 2017.

44. Other Platelet Function Tests:
1. Whole-Blood (Impedance) Aggregometry:
- Uses electric impedance to evaluate platelet aggregation in whole blood.
- Citrated whole blood sample is stirred at 37°C.
- Electric current is passed between 2 electrodes immersed in sample.
oImpedance is measured between electrodes.
- Platelet aggregation occurs on surface of electrodes and increases impedance.
- Addition of agonist to sample increases aggregation of platelets on surface of electrodes.
oThis is detected as increase in impedance measured in ohms.
- WBA uses same agonists as LTA:
o ADP.
o Epinephrine.
o Collagen.
o Ristocetin.
oAA.
- WBA requires one-quarter of blood needed for LTA.

45. 2. Platelet Function Analyzer (PFA-100®):
- PFA-100® uses whole blood to measure platelet function under
high shear flow conditions.
- Sample collected in 3.2% sodium citrate.
- Whole blood passes through opening cut into coated membrane.
o Membrane coated with collagen and epinephrine or collagen
and ADP as agonists.
- These agonists promote platelet adhesion, activation, and
aggregation leading to occlusion of aperture and cessation of blood
flow.
- Time to occlusion of opening by platelet plug is measured:
o Prolonged occlusion or “closure” times suggest impaired
platelet function.

46. 3. Flow cytometry:
- Whole-blood flow cytometry offers a
very attractive and reliable test for the
diagnosis of various platelet receptor,
granular, and other defects.
- Platelet responses to various agonists
can also be studied by measuring
various activation markers. This is
potentially useful for measuring
platelet function in thrombocytopenia,
as flow cytometry responses are
independent of platelet count.

47. Applications of flow cytometry platelet function tests:
i. Diagnosis of platelet function disorders:
- Mainly used clinically for quantification of surface GP receptor density:
o Glanzmann thrombasthenia:
 Flow cytometry shows lack of surface GPIIb-IIIa.
o Bernard-Soulier syndrome:
 Flow cytometry shows lack of surface GPIb.
ii. Platelet activation markers:
- Degranulation markers: CD62p, CD63, and CD40L.
iii. Measuring platelet production.
iv. Accurate platelet counting: platelet to RBCs ratio.
v. Blood bank tests:
- Quality control of concentrates.
- Leukocyte contamination.
vi. Antiplatelet drugs monitoring.

48. 4. Measurement of Platelet Nucleotides:
- ATP and ADP are released from platelet granules after platelet activation.
- Evaluation of released adenine nucleotides (ATP and ADP) can be used as
independent measure of granule release.
oRelease of ATP from dense granules is quantitated using luciferin/luciferase
system.
oATP release occurs during secondary aggregation phase in LTA.
oMeasurement of ATP release alone does not distinguish between storage pool
and release defects.
- Measurement of total platelet content of ADP and ATP and release of ATP in
response to agonists can help to distinguish between a storage pool disorder and a
release defect:
oStorage pool defects have decrease in amount of stored and released ADP with
increased ATP-to-ADP ratio.
oRelease defects have normal ADP levels, normal ATP:ADP ratio, but decreased
ADP release.

49. 5. The VerifyNow®:
The VerifyNow® device is a fully automated and near patient testing
aggregation system, is available solely for the monitoring of the
three major classes of antiplatelet drugs (e.g., GPIIb/IIIa inhibitors,
aspirin, and P2Y12 receptor inhibitors/ antagonists).
6. Plateletworks®:
Plateletworks® is a standardized platelet counting ratio technique,
based upon comparing platelet counts within a control EDTA tube
and after aggregation with platelet agonists within citrated tubes,
could theoretically also be applied to the diagnosis of platelet
disorders, although experience with this assay is limited.