Week 3: Platelet Function Testing

platelet closure times

time required to obtain full occlusion of the aperture

what types of conditions can be detected using the PFA?

1. von willebrand disorder

2. glanzmann thrombasthenia

3. aspirin

4. anti-platelet drugs

PFA stimulates the process of primary hemostasis _________

in vitro

the PFA measures the ability of platelets in __________ to occlude a cut in a membrane coated with collagen and epinephrine and collagen and ADP

whole blood

______ is the primary screener with PFA

Col/EPI

________ is used to indicate platelet dysfunction due to aspirin with PFA

Col/ADP

PFA sample requirements

1. whole blood in 3.8% or 3.2% sodium citrate

2. room temp

3. run within 4 hours

the specimens used for PFA cannot be what?

centrifuged or hemolyzed

the verify now system is an ________ detection system that measures platelet induced aggregation by microbead agglutination

optical

what does the verify now system cartridge contain?

lyophilized fibrinogen coated beads and a platelet agonist specific for the test

what type of specimen is needed for the verify now system?

whole blood

what is the agonist used for aspirin screening?

arachidonic acid

what is the agonist for P212 inhibitor or Clopidogrel screening?

ADP

what is the agonist for GP IIb/IIIa inhibitor, abciximab, or tirofiban screening?

thrombin

The phenomenon of platelet aggregation can be induced by adding aggregating agents to:

• platelet rich plasma

• whole blood

list of aggregation agents

• ADP

• epinephrine

• collagen

• ristocetin

• thrombin

• arachidonic acid

normal PLT count range

200,000 - 400,000 per uL

what type of specimen is used with optical aggregation?

platelet rich plasma PRP

principle of optical aggregation

1. infrared light thru 2 cuvettes — PRP sample + PPP reference

2. stimulus added → change in light transmission

what methodology is this?

optical aggregation

what is the % light transmission at 0% aggregation?

0

what are the 4 stages of platelet function?

1. shape change

2. primary aggregation

3. ATP release

4. second wave aggregation

what causes 1?

slight increase in light transmission from the addition of the aggregating agent (like ADP)

what causes 2?

slight decrease in light transmission due to shape change of platelets

what causes 3?

primary wave of aggregation

what causes 4?

release reaction

what causes 5?

completion of secondary wave of aggregation

with ___________ addition, there is a lag phase then single wave

collagen

with _______________ there is a single curve

arachidonic acid

what are the limitations of optical aggregation?

1. lack of other blood cells

2. excessive sample prep

3. loss of large platelets

4. lipemia and thrombocytopenia

5. pediatric patients

what is the specimen for impedance aggregation?

anticoagulated whole blood

with impedance aggregation, the entire ____________________ is present

PLT population

impedance method is non-__________

optical

principle of impedance aggregation methodology

1. electrode probe with 2 wires are placed in specimen

2. AC current applied

3. electrical resistance between the wires is measured

with impedance, a ______________ of platelets form on the wires with a baseline of 0 ohms

monolayer

with impedance methods, the aggregating agent is added to stimulate the platelets to adhere to the _____________

monolayer

the increase in impedance is directly proportional to the ______________

aggregation

how is whole blood diluted for impedance aggregation?

0.9% saline, 1:1 ratio

what does luminescence measure?

ATP release

principle of luminescence method

1. ATP is measured using firefly luciferin-luciferase reagent

2. photomultiplier = detection

firefly reaction

chrono-lume reagent + ATP → light

chrono-lume reagent components

1. luciferin

2. luciferase

3. magnesium sulfate

4. stabilizers and buffer

most common platelet defects

1. drugs (aspirin)

2. von willebrand disease (1-3% of population)

3. storage pool and secretion defects

conditions resulting in abnormal platelet release

1. prostaglandin synthetase deficiency

2. storage pool disease

3. release defect

prostaglandin synthetase deficiency

1. cyclooxygenase

2. also called aspirin-like defect

storage pool disease

dense granules are absent or deficient

release defect

a normal amount of stored nucleotides are present, but it requires increased concentrations of aggregating agent to induce release

what can these disorders be described as?

• von willebrand disease

• bernard soulier

• glanzmann

• storage pool

qualitative platelet disorders

what causes von willebrand disease?

a defect in the vWf on the platelet membrane which binds with Ib

what causes bernard soulier?

missing Ib which is the receptor for vWf

what causes glanzmann?

lack of IIb/IIIa for the attachment of fibrinogen

what platelet disorder is indicated?

• ADP, Collagen, Epinephrine — Absent or severely impaired aggregation (flat curve)

• ristocetin — normal

glanzmann thrombasthenia

what platelet disorder is indicated?

• Ristocetin — Abnormal/absent agglutination that does not correct with the addition of normal plasma

• ADP, Collagen, Epinephrine — Normal

bernard-soulier

what platelet disorder is indicated?

• Ristocetin — Abnormal/absent agglutination that corrects when normal plasma (containing vWF) is added

• ADP, Collagen, Epinephrine: Normal

von willebrand disease

what platelet disorder is indicated?

• ADP, Epinephrine, Collagen — Biphasic or abnormal secondary wave

• ristocetin — normal

storage pool disease

what platelet disorder is indicated?

• arachidonic acid — absent

• ADP and EPI — impaired secondary aggregation

• ristocetin and high-dose collagen — usually normal

aspirin/COX defect