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 N.B. WITH
;BERNARDSOULIER
the
abnormal RISTO
is not corrected
by the addition
of vWF

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 Laboratory Studies
MEASUREMENTS OF PLATELET ACTIVATION/AGGREGATION .3
Direct measurements of platelet activation/aggregation are •
.possible using an aggregometer or flow cytometer
The aggregometer provides a graphic display of the wave of •
,platelet aggregation seen in response to agonists such as ADP
epinephrine, or collagen, and the agglutination response to
ristocetin. Specific functional defects respond differently to
.these agonists
For example, patients with vWD specifically show decreased •
or absent agglutination with ristocetin [ristocetin-induced
platelet agglutination assay (RIPA)], whereas other disorders
such as storage pool disease demonstrate poor responses to
.ADP, epinephrine, and collagen

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1) for the identification of patients
with bleeding disorders; 2) for
monitoring the response to
antiplatelet treatment; 3) in the
evaluation of perioperative
hemostasis; and 4) in transfusion
medicine. Because there are diverse
employment fields in which platelet
function is studied and the platelets
present a plethora of functions,
different methodologies have been
developed.

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 The PFA test is a rapid, accurate screening test of platelet function.
Common clinical applications
:include the following
Preoperative evaluation of platelet function •
Evaluation of women with menorrhagia •
Determining the presence of drug-induced platelet dysfunction •
Determining patient compliance with aspirin and other antiplatelet drugs •
Determining platelet functionality in high-risk pregnancy •
Evaluation of patients with suspected inherited or acquired platelet •
disorders, such as
von Willebrand disease (vWD)
Evaluation of the bleeding patient •
Monitoring DDAVP treatment in patients with Type I vWD •

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 ?How are the results interpreted .5
The PFA test result is dependent on platelet function, plasma von Willebrand Factor level, platelet
.number, and (to some extent) the hematocrit
The PFA test is initially performed with the Col/Epi membrane. A normal Col/Epi closure time
.excludes the presence of a significant platelet function defect )seconds 183<(
If the Col/Epi closure time is prolonged (>183 sec onds), the Col/ADP test is automatically
performed. If the Col/ADP result is normal (<122 seconds), aspirin-induced platelet dysfunction is
.most likely
Prolongation of both test results (Col/Epi >183 seconds, Col/ADP >122 seconds) may indicate
;the follow ing
Anemia (hematocrit <0.28) •
Thrombocytopenia (platelet count < 100 x 109/L) •
A significant platelet function defect other than aspirin •
Once anemia and thrombocytopenia have been excluded, further evaluation to exclude von
Willebrand disease and inherited/acquired platelet dysfunction such as renal failure storage pool
disease, release defect, Bernard-Soulier disease, and Glanzmann thromboasthenia should be
.considered. Long-term aspirin therapy may modestly prolong the Col/ADP

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 Haemostasis & Thrombosis
Adhesion
Activation
Spreading
Secretion
Aggregation
Procoagulant activity
Clot retraction
Tissue Repair

Vascular Tone
Uptake of serotonin when restingMaintenance/Regulation
restingMaintenance/Regulation of
Release of serotonin, thromboxane, prostaglandins
upon activation
Host Defence
Phagocytosis/Internalisation of
Viruses and Bacteria
Killing of Bacteria
Release of Platelet microbicidal
proteins
Superoxide production
Inflammation
Atheroslerosis
Allergic Asthma
Renal Disease
Chemotaxis
Platelet-Leukocyte interactions
Tumour Biology
Tumour Growth
Tumour killing
Tumour Metastasis

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Increased Platelet
Destruction
• The hallmark of increased platelet
destruction is increased
marrow megakaryocytes or, when available,
high reticulated
platelet count. Platelet destruction results
from various
immune conditions, including the following:
• Immune thrombocytopenic purpura (ITP)
• Thrombotic microangiopathies
• Post-transfusion purpura (PTP)
• Heparin-induced thrombocytopenia (HIT)
• Disseminated intravascular coagulation (DIC)

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Platelets Function Test

Dr. Saifeldein M.A.Elimam

Consultant Hematopathology
Laboratory & Blood Bank Department
MCH – N
 Platelet Function

Platelet must be adequate in number and function to

participate optimally in homeostasis.

The functions of platelets in homeostasis include;

Maintenance of vascular integrity

Initial arrest of bleeding by platelet plug formation
Stabilization of hemostatic plug by contributing to fibrin
formation.
 Platelet Adhesion, Activation and
Aggregation Mediators
vWF
INJURY ADP-receptor
THROMBUS
Shear Forces

Adhesion Activation Aggregation

• vWF • Membrane changes • GPIIb/IIIa-mediated

• Thrombin • Granule secretion • Fibrinogen
• Collagen • GPIIb/IIIa expression • vWF
• Fibronectin • Multiple agonists
• Feedback loops
 Platelet Adhesion and Activation

Platelets adhering to Aggregation

Normal platelets damaged endothelium of platelets into a
in flowing blood and undergoing activation thrombus

Platelet
thrombus
Platelets Platelets adhering
to subendothelial
space

Endothelial cells
Subendothelial space
 Platelet Function (Adhesion)

Following blood vessel injury, platelets adhere to the

exposed subendothelial connective tissues.

Subendothelial bind to the larger multimers of vWF and

through these react with Platelet membrane GPIb .

large number of adhesion proteins are involved in

platelet—vessel wall and platelet- platelet interaction.
Adhesion to collagen is facilitated by GPIa.
 Platelet Function

Platelet adhesion induces a series of metabolic reactions

which initiate the platelet release reactions, shape
change and aggregation.

Following adhesion, platelets become more spherical and

extrude long pseudopods which enhance interaction
between adjacent platelets.

von Willebrand factor (VWF) is involved in platelet

adhesion to the vessel wall and to other platelets
(aggregation).
 Platelet Function

vWF is encoded by a gene on chromosome 12 and is

synthesized by endothelial cells and Megakaryocytes.

Release of vWF from endothelial cells occurs under the

influence of several hormones.
 Platelet Aggregation

Flowing Rolling Hemisphere-shaped Spreading

disc-shaped ball-shaped platelet platelet
platelet platelet

FIRM, BUT REVERSIBLE IRREVERSIBLE

ADHESION ADHESION

Scanning electron micrograph Activated, aggregating platelets

of discoid, dormant platelets illustrating fibrin strands

.
 Platelet Function (Release Reaction)

Collagen exposure or thrombin action results in the

secretion of platelet granule contents which include ADP,
serotonin, fibrinogen, lysosomal enzymes, β-
thromboglobulin.
Collagen and thrombin activate platelet prostaglandin
synthesis.
Released ADP and thromboxane A2 cause additional
platelets to aggregate at the site of vascular injury.
ADP causes platelets to swell and encourages the platelet
membranes of adjacent platelets to adhere to each other.
 Platelet Function (Platelet aggregation)

As they do so further release reactions occur liberating

more ADP and thromboxane A2 causing secondary
platelet aggregation.

This positive feedback process results in the formation of

a platelet mass large enough to plug the area of
endothelial injury.
 Platelet Function Test

Bleeding Time
 Principle: Bleeding time is defined as the time taken for a
standardized skin wound to stop bleeding.

 Platelet aggregation

 Platelets function in primary hemeostasis by forming an

initial platelet plug at the site of vascular injury. The
phenomenon occurs partly through the ability of
platelets to adhere to one another, a process known as
platelet aggregation.
 Platelet Function Test

Substances that can induce platelet aggregation include;

Collagen, ADP, epinephrine, thrombin, serotonin,

arachidonic acid, restocetin, snake venoms.

Platelet aggregation is an essential part of the

investigation of any patient with a suspected platelet
dysfunction.

Platelet aggregation is studied by means of a platelet

aggregometer.
 Platelet Function Test

A photo-optical instrument connected to a chart

recorder.

Platelet rich plasma (PRP) which is turbid in appearance,

is placed in a cuvette warmed to 37°C in the heat block of
the instrument, and stirred by a small magnetic bar.

Light transmittance through the PRP is recorded.

 Platelet Function Test

The addition of aggregating agent cause the formation of

larger platelet aggregates with a corresponding increase
in light transmittance, owing to a clearing in the PRP.

The change in the light transmittance is converted to

electric signal and recorded as a tracing by the chart
recorder.
 Platelet Function Test

There are some basic requirements for platelet

aggregation as in vitro means of evaluating platelet
functions.

1- A clean venipuncture is crucial. Hemolyzed samples

should not be studied because RBCs contained ADP.

2- Plasma from fasting patients is preferred for testing.

Lipemic samples may obscure change in optical density
owing to platelet aggregation.
 Platelet Function Test

3- Sodium citrate is the anticoagulant used in

aggregation studies. In vitro aggregation is dependent on
the presence of calcium ions.

4- Fibrinogen must be present in the test sample for

aggregation to occur.

5- Aggregation studies should be performed at 37°C at a

pH of 6.5 to 8.5. To help maintain platelet values, all
samples, once collected, should be capped to prevent Co2
loss.
 Platelet Function Test

6- Test samples, should be maintained at room temp

during processing. Cooling inhibits the platelet
aggregation response.

7- Stirring is necessary to bring the platelets in close

contact with one another to allow aggregation to occur.

8- It is essential that the patient refrain from taking any

anti-inflammatory drugs, one week before the test. The
drugs inhibit the platelet’s release reaction.

9- Aggregating reagents should be prepared fresh daily

and brought to room temp before use. They must have
known potency and be added in small volume.
 Thank You
?Any Question
 Haemostasis & Thrombosis
Adhesion
Activation
Spreading
Secretion
Aggregation
Procoagulant activity
Clot retraction
Tissue Repair
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 Maintenance/Regulation of
Vascular Tone
Uptake of serotonin when resting
Release of serotonin, thromboxane,
prostaglandins
upon activation

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 Inflammation
Atheroslerosis
Allergic Asthma
Renal Disease
Chemotaxis
Platelet-Leukocyte interactions

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 Host Defence
Phagocytosis/Internalisation of
Viruses and Bacteria
Killing of Bacteria
Release of Platelet microbicidal
proteins
Superoxide production

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 Tumour Biology
Tumour Growth
Tumour killing
Tumour Metastasis

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