PFA-200 :: Platelet Function Analyser

An automated analyser assesses primary haemostasis under shear stress.

Two cartridges:

• CEPI: collagen and epinephrine (adrenaline).
• CADP: collagen and ADP

Blood sample is aspirated through the cartridge membrane – leading to platelet activation and aggregation.  The time to close the membrane aperture (closure time) is measured.

Aggregation depends on adequate platelet numbers, and platelet functional ability (vWF, platelets GP Ib, GP IIb/IIIa and fibrinogen).

Variables affecting results:

• Collection Time: Assay within 4 hours of collection.
• Haematocrit: Anaemia increases closure time (Hct <20%).
• Platelet Count: Closure times increase as plate count<100.
• Blood group/vWF levels
• Drugs: Aspirin, NSAID, clopidogrel (variable)
• Acquired platelet function defects: cardio-pulmonary bypass, liver disease, renal failure.

PFA-200 patterns

Disorder	Col/Epi	Col/ADP
Aspirin/NSAID	↑	N
Clopidogrel	N/↑	N/↑
vWD	↑	↑
Glanzmann’s;  Bernard Soulier	↑	↑
Storage Pool Defect	↑	N (or ↑)
Macrothrombocytopathy	↑	N
Renal Failure, MPD, CABG	↑	↑

Platelet aggregation studies

Tests the ability of certain agonists (stimulants) to produce platelet clumping or aggregation.

Patterns of abnormality can help identify the type of platelet defect.

Common defect patterns:

Disorder	Defect	Plat Count	Blood Film	ADP 2uM	ADP 5-10uM	Collagen 1ug/mL	AA 100uM	Adrenaline 10UM	Risocetin 1.2mg/mL
Aspirin/NSAID	Inhibit COX	N	N	1Y wave no 2y wave	1y no 2y	↓absent	Absent	↓	N
ADP antagonists (clopidogrel)	Inhib ADP receptor	N	N	Absent	Absent	N	N	N	N
Glanzmanns’s Thrombasthenia	GpIIb/IIIa receptor	N	N	Absent	Absent	Absent	Absent	Absent	N
Bernard Soulier Syndrome	GpIb-V-IX receptor	↓	Giant plats ê plat	N	N	N	N	N	Absent
vWD		N/↓	N	N	N	N	N	N	↓
Gray Platelet Syndrome	α-granule defic	↓	Gray plats	N/↓	N/↓	N/↓	N	N	N
Dense Granule Deficiency	δ-granules	N	N	N	Absent	↓	N	No 2y wave	? 1Y only
VWD Type IIb	VWF protein	↓		N	N	N	N	N	N   ↑ 0.5 risto
Platelet type vWD	Mutn GpIb receptor	↓		N	N	N	N	N	N   ↑ 0.5 risto
Renal Failure	Acquired SPD	N/↓		N	↓	N/↓	N/↓	↓	N/↓
Liver Disease		N/↓		1Y only	1Y only	↓	?	1Y only	N/↓
Afibrinogenaemia				Absent	Absent	Absent		Absent

Strong agonists: collagen, thrombin, TXA2 – directly induce plat agg, TX synthesis and plat gran secretion.

Weak agonists: ADP, Adren: Induce platn agg without secretion. (biphasic curve)

Agonists:

Agonist	Conc	Comment
ADP	LD:1–5 uM HD 10uM	ADP binds ADP receptor on plat. Shape change – 1y agg. 2y wave = ADP from plat storage grans. LD –> 1y only. 2y wave inhib by aspirin and NSAIDs. Clopidogrel inhibs P2Y1 receptor
Collagen	1, 4ug/mL	Collagen binds GpVI and GpIa/IIa inducing granule release, TX and GPIIb/IIIa activn. Lag phase.
Ristocetin	LD: 0.5mg/mL HD: 1.5-5mg/mL	Ristocetin (not LD) cuases plats agglutination (not agg) via vWF abd GP1b-IX-V complex.
Adrenaline	5,10uM	Adrenaline blinds a2 recepotor on plat — Ca release. Similar to ADP agg. 1y , 2y wave with ADP released from grans. 2y wave inhib by aspirin,. NSAIDs.  Variation in a receptor density – reduced agg.
Arachadonic Acid	500ug/mL	AA is precursor to TXA2. Via cyclo-oxygenase and thromboxane synthase.  TX is potent plat activator