The document provides an overview of platelet function, including their role in hemostasis, structure, and types of platelet function tests. It describes various tests to assess platelet adhesion, aggregation, and secretion, including techniques such as the PFA-100 and aggregometry, which measure the ability of platelets to aggregate and their functionality. Additionally, it outlines the advantages and disadvantages of these tests and highlights the significance of flow cytometry in diagnosing platelet defects.

1. PLATELET FUNCTION TESTS

2. INTRODUCTION
• Definition: Small, colourless,anucleated cellular
fragment formed from megakaryocyte
• Platelets are 2nd
major component of the hemostatic
system
• There role is first established by Bizzozero in 1892

3. PLATELET FORMATION
• From megakaryocytes in the bone marrow
• To generate 2000 – 3000 platelets per megakaryocyte

4. PLATELET STRUCTURE
• Platelets are small, anucleate cells
• Discoid in shape
• Diameter : 1.5-3.5 µm
• Normal count : 150 – 440 x 109
/L
• Storage granules : α, dense, lysosomes
• Primary function : rapid formation of a vascular plug
following vessel injury

5. Peripheral zone
Structural zone
Organelle zone
Membrane systems

6. α Granules
Group 1- hemostatic proteins
• Fibrinogen
• Factor v
• Von willebrand factor
• Plasminogen
• Plasminogen activator
inhibitor(PAI-1)
• α2antiplasmin
Group II- non hemostatic proteins
• Platelet specific
 Β thromboglobulin
 Platelet factor 4
 Platelet derived growth factor
• Non platelet specific
 Albumin
 Thrombospondin
 Fibronectin

7. PLATELET FUNCTIONS
• Formation of primary haemostatic plug
• Surface for coagulation factor to make secondary
haemostatic plug
• Aid in healing of injured tissue
1. Adhesion
2. Shape change
3. Aggregation
4. Secretion

8. PLATELET FUNCTION TEST
• Group of test that measure the ability of
platelet to aggregate and promote clotting in
sample.
• OR it measures any dysfunction in platelet.

9. PLATELET FUNCTION TESTS
• ADHESION TESTS
 Retention in a glass-bead column
 Baumgartner’s technique
 PFA-100
• AGGREGATION TESTS
 Turbidometric technique using
 ADP
 Collagen
 Ristocetin
 Adrenaline (epinephrine)
 Thrombin
 Arachidonic acid
 Endoperoxide analogues
 Calcium ionophore

10. • INVESTIGATION OF GRANULE CONTENT AND
RELEASE
 Dense bodies
 Electron microscopy
 ADP and ATP content (bioluminescence)
 Serotonin release
• GRANULES
 b-thromboglobulin
 Platelet factor 4
 VWF
 Fluorescence by flow cytometry

11. • PROSTAGLANDIN PATHWAYS
 TXA2 radioimmunoassay
• PLATELET COAGULANT ACTIVITY
 Prothrombin consumption index
• FLOW CYTOMETRY
 Glycoprotein surface expression
 Activation
 P selectin (CD62) surface expression
• FIBRINOGEN BINDING
 Annexin binding (to phosphatidyl serine)
 Conformational changes in Gp IIbIIIa

12. PLATELET FUNCTION ANALYSER
• New generation of rapid whole-blood analyzers
• Screen for platelet function defect
• Whole blood is passed through a hole in a membrane
coated with collagen and either ADP or epinephrine
• Combined influence of high shear rates and VWF,
platelets encountering this membrane spontaneously
adhere to collagen

13. PFA-100 [Platelet Function Analyser]

14. • And aggregate because of stimulation by ADP or
epinephrine
• Measures closure time (CT), the amount of time
required for a platelet plug to occlude the aperture
and stop flow

15. CT is prolonged in
• Severe congenital and acquired platelet dysfunction
• Defects in platelet glycoprotein receptors GPIIb/IIIa
(Glanzmann thrombasthenia) and GPIb (Bernard-
Soulier syndrome)
• Monitoring of patients taking aspirin – aspirin
resistance

16. ADVANTAGES
• Whole blood test
• Small blood volumes
• Simple
• Rapid
DISADVANTAGES
• Inflexible
• vWf dependent

17. OTHER ADHESION TESTS :
• Retention in a glass bead column
• Baumgartners technique

18. Retention in a glass-bead column

19. AGGREGATION TESTS
• Measures the ability of various agonists of platelets to
induce in vitro activation and platelet-to-platelet
aggregation
• Instrument: Aggregometer

20. • 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.

22. • Platelet aggregometer measures change in
optical density for 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.

23. SEMINAR
STRONG AGONISTS
• Directly induce platelet
aggregation, TxA2
synthesis and platelet
granule secretion
• Eg : Collagen
Thrombin
TxA2
WEAK AGONISTS
• Induce platelet
aggregation without
inducing secretion
• Eg : ADP
Epinephrine
20/08/2018

24. SECRETION ASSAYS
• Measured by 2 different ways
 Measuring ATP secretion from platelets
 14
C-labelled serotonin

25.  ATP secretion from platelets
• PRP or whole blood is incubated with firefly
luciferase and its substrate luciferin
• Addition of platelet agonists causes release of ATP, a
cofactor in a light-producing luciferin-luciferase
assay
• Resulting light can be measured with a photodetector
system
• Often performed simultaneously with a platelet
aggregation assay

26. FLOW CYTOMETRY
• Measure multiple properties of single cells in
suspension
• Specific monoclonal antibodies are used to detect
antigens or proteins in these cells
• Antibodies are bind with fluorescent dyes that are
activated as the cells pass in a single-file stream
through the flow cytometer

27. • Light emitted from each fluorochrome can be
individually detected and its intensity measured for each
cell
Diagnosis of:
• inherited defects in platelet surface glycoproteins
• Decreased expression or absence of GPIb - Bernard-
Soulier syndrome
• GPIIb/IIIa - Glanzmann thrombasthenia
• Multiple antibodies against different GPIIb/IIIa epitopes
- acquired Glanzmann thrombasthenia

29. other tests
• Test of platelet secretion or release reaction
• Tests for detection of abnormalities in arachidonic
acid metabolism
• Test for platelet procoagulant activity( prothrombin
consumption test)
• clot retraction test
• Platelet micro RNA analysis
• Impact cone and plate analyser
• Thromboelastography

30. REFERENCES
• 1.Hoffbrand A V, Catovsky D, Tuddenham E G D, editors.
Postgraduate Hematology.5th
ed. Massachusetts:Blackwell
Publishing Ltd; 2005.P.808-24.
• 2. Seegmiller A, Sarode R. Laboratory Evaluation of Platelet
Function. Hematol Oncol Clin N Am 2007;21:731–42.
• 3. Quinn M, Fitzgerald D, Platelet function, assesment,
diagnosis and treatment. New Jersy:Humana press;2005:P.202-
328.
• 4. Platelet Function Testing: Light Transmission Aggregometry
[LTA], 29-4-2012, Available from http://www.platelet-
research.org/3/aggregometry.htm.
• 5.Laffan M A, Manning R , Investigation of hemostasis, Bain
B.J, Bates I, Laffan M.A, Lewis S.M, Dacie and Lewis Practical
Haematology. 11th
ed. London: 2012. P.424-39.

31. Thank you!!!