PowerPoint Presentation

Platelet dysfunction disorder by

Dr.saima mansoor bugviHaematologist CH & ICH lahore

A patient with recurrent episodes of gum bleed since childhood is referred to you Platelet counts and coagulation profile is normal. suspecting an inherited disorder.What will be your diagnostic approach? Explain the rationale.

Give under lying defects of two major inherited platelet function disorders

What is the principle of platelet function testHow will you interpret its different wave forms Pitfalls of the technique

Diagnostic approachDetailed history of the patient bleeding from any other site besides gum bleeding pts with platelet dysfunction may have history of epistaxis and if female menorrhagiaExcessive bleeding in response to major nicks is suggestive of platelet dysfunction and VWDAsk about fatique, tiredness the bleeders usually suffer from anaemia due to chronic blood loss

Assess the extent of haemorrhages against the background of traumaPast history of bleeding during hemostatic challenge tooth extractionsurgery, trauma, child birth

Obtaining objective confirmation of the subjectivei nformation conveyed in the bleeding history is valuable.Objective data include Previous hospital or physician visits for bleeding symptoms,Results of previous laboratory evaluations,Previous transfusions of blood products for bleeding episodes, and A history of anemia and/or previous treatment with iron.

Drug historyesp non- prescription drugsHerbal medicines poses particular problemspatients do not readily shareactive ingredient difficult to determineGinkgo biloba and ginseng can cause platelet dysfunctionAsk about dietary supplements

A detailed family history draw pedigree for at least two generations, inherited platelet disorders are autosomal recessive in inheritanceHistory of consanguineous marriage, consanguineous marriages are more chances of expressing autosomal recessive disorders

ExaminationLook for the pallor Look for bruises and petechiae

Laboratory evaluationFirst line screening testsSecond line specific test

Laboratory tests for platelet disorders include

Assessing platelet number and size [MPV] Assessing platelet morphology blood filmScreening tests of platelet function Bleeding Time [BT] and PFA-100 Light Transmission Aggregometry e.g. classical Born aggregometry Flow cytometry e.g. to quantitate the presence or absence of platelet membrane glycoproteinsmeasurement of platelet nucleotides [ADP, ATP and the ADP:ATP ratio]Electron microscopy to look for the presence or absence of platelet granulesFlow analysis to look for platelet granules [mepacrine binding]Lumiaggregometry to look at platelet granule release.

Bleeding time Screening test for the presence of platelet function defectPatients with functional defects of platelets can show BTs in excess of 20 minutesWith platelet counts greater than 100,000/ul the BT should be less than 08 minutes

Complete blood count and blood filmA complete blood count (CBC) with examination of the blood film can also be helpfulPlatelet morphology can help in diagnosing disorders as bernard soulier syndrome gray platelet syndrome

Measurements of platelet activation/aggregationDirect 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 in response to agonists such as ADP, epinephrine or collagen and the agglutination response to ristocetinSpecific functional defects respond differently to these agonists.

Give under lying defects of two major inherited platelet function disorders

Platelets have a complex ultrastructure comprising a multitude of molecules and the malfunctioning of any of these may give rise to a specific disease .

Platelets participate in haemostasis by adhering to exposed elements of the subendothelial matrix. They then spread onto the subendothelial surface, become activated, release the contents of their storage organelles, and aggregate to each other. Abnormalities in any of these stages adhesion, activation, secretion and aggregation may give rise to congenital disorders of platelets. Patientssuffering from any of these diseases usually show a bleeding diathesis with a prolonged bleeding time and a normal platelet count.

GLANZMANS THROMASTHENIASTORAGE POOL DEFECT

Under lying defect of Glanzmans Thrombasthenia The platelets contain defective or low levels of glycoprotein IIb/IIIa (GpIIb/IIIa), which is a receptor for fibrinogen. As a result, no fibrinogen bridging of platelets to other platelets can occur, (GpIIb/IIIa), also known as IIb3, which is an integrin aggregation receptor on platelets. This receptor is activated when the platelet is stimulated by ADP, epinephrine, collagen, or thrombin. GpIIb/IIIa is essential to blood coagulation since the activated receptor has the ability to bind fibrinogen (as well as von Willebrand factor, fibronectin, and vitronectin), which is required for fibrinogen-dependent platelet-platelet interaction (aggregation)

Disorders of platelet aggregationPlatelet aggregation may be defined as the interaction of activated platelets with one another and occurs after adhesion of platelets to the wall of the injured blood vessel. A series of factors are capable of inducing platelet aggregation and may be classified as primary and secondary platelet - aggregating agents.

Primary aggregating agents are those factors, such as ADP, adrenaline and thrombin, able to directly induce platelet aggregation independently of their ability to release intraplatelet ADP or to induce the production of prostaglandins.

Mutations within the genes that code for IIb 3 subunits have been described in GT patients.

Type I GT is characterized by the lack of surface -detectable I Ib 3 complex and a profound defect in platelet aggregation and clot retraction Platelets of patients suffering from type II GT have detectable, but mark-edly reduced, amounts of the I Ib 3 receptor on their surface, usually 10 20% of normal values a series of patients with a variant form have been described who present near - normal levels of the I Ib 3 complex, which is dysfunctional in that platelets, when activated, can neither aggregate nor bind fibrinogen

Storage pool defectWith a Storage Pool Disease (SPD), there may not be enough of a certain type of granule, the granule may be abnormal, or there may not be enough of the chemicals it is supposed to hold. In Delta Storage Pool Disease, the delta granules (also called dense granules) are affected. In Alpha Storage Pool Disease, it is the alpha granules. Alpha SPD is also called Gray Platelet Syndrome. This is because the platelets of someone with Alpha SPD look gray when viewed under a microscope. It is possible to have Alpha/Delta SPD in which both types of granules are affected.When platelets are not able to store chemicals or secrete them when needed, they cant let other platelets know to come and help form a plug. It takes longer for a clot to form. SPD usually results in mild to moderate bleeding symptoms.

Defects of secondary a ggregationSecondary aggregation disorders are more frequent thanprimary aggregation disorders and the most common in thiscategory are the storage pool defi ciency (SPD) syndromes. SPDsyndromes may be classifi ed in a system that takes into accountthe content of both dense and granules

The disorder is heterogeneous and the termSPD includes a group of disorders having as their commonfeature a diminution in secretable substances stored in plateletgranules.A storage pool diseasePatients with SPD or - SPD usually have absent ADP - and adrenaline- inducedsecondary aggregation waves, although the primary waves arePresent Collagen - induced aggregation is absent ormarkedly reduced, whereas ristocetin - induced agglutination isnormal.

What is the principle of platelet function test

The light absorbance of PRP decreases as platelets aggregate.The amount and the rate of fall are dependent on platelet reactivity to the added agonist provided that other variables, such as temperature, platelet count and mixing speed, are controlled. The absorbance changes are monitored on a chart recorder.

Light transmission aggregometry (LTA) is regarded as the gold standard of platelet function testing and is still the most used test for the identification and diagnosis of platelet function defects. Platelet rich plasma (PRP) is stirred within a cuvette located between a light source and a detector. After addition of a various panel of agonists, such as collagen, ADP, thrombin, ristocetin, epinephrine, and arachidonic acid, the platelets aggregate and light transmission increases.

The platelet aggregation pattern is thought as a primary response to an exogenous agonist, followed by a secondary response to the release of dense granule contents. This biphasic response can be masked if high concentrations of agonists are added. Parameters measured include the rate or slope of aggregation (%/min) and the maximal amplitude (%) or percentage of aggregation after a fixed period of time, usually 610min

Platelet aggregation is studied by means of a platelet aggregometer, Used Principle: Photo-optical Method luminescence technology (Platelet Lumiaggregometry) Electrical Impedance Method

AGGREGATING AGENTS

Arachidonic acid: used to assess the viability of the thromboxane pathway.Thrombin: reacts with several membrane sites to induce full aggregation and secretion of organelle contents independent of the prostaglandin or ADP pathways.ADP: binds to a specific platelet membrane receptor and causes platelet activation and release of dense granule stored ADP. Shows biphasic aggregation.Epinephrine: binds to specific receptor and causes ADP secretion, but does not cause aggregation in storage pool disorder or release defects.Collagen: Shows no primary wave of aggregation and depends on intact membrane receptors, membrane phospholipase pathway integrity and normal cyclooxygenase and thromboxane pathway function.Ristocetin: requires vWF and intact surface membrane including a functional vWF receptor site (GPIb).

How will you interpret its different wave forms

Wave form of platelet aggregationPrimary Response Primary response is the reversible aggregation of platelets by the aggregating agent. The appearance of a biphasic reaction, showing both primary and secondary response, can occur for some agonists at low concentrations

Secondary response is the result of enhancement of the initial aggregation process caused by the release of endogenous ADP and the formation of thromboxane A2. the secondary response Parameters measured include the rate or slope of aggregation (%/min) and the maximal amplitude (%) or percentage of aggregation after a fixed period of time, usually 610min is irreversible.

The most obvious abnormalities in this series of aggregation traces is a lack of aggregation to all agonists except ristocetinThere are two possible explanations: 1. Glanzmann's Thrombasthenia [GTT] in which there is a defect in the GpIIb/IIIa receptorby lack of or reduction in platelet aggregation to all agonists because fibrinogen cannot bind to produce a platelet aggregate. Platelet responses to ristocetin and vWF are normal, and platelet secretion to collagen and thrombin remains existent, although at a reduced level because there is no additional burst in secretion resulting from platelet aggregation.

2. Afibrinogenaemia.

These aggregation traces are essentially the inverse of those seen in Q1. That is aggregation to all agonists except ristocetin. This suggests that the problem lies with the GpIb receptor. Remember the GpIb receptor is involved in the binding of Von Willebrand factor and therefore this pattern of traces would suggest either Bernard Soulier Syndrome [BSS] or VWD [probably severe Type 1 or Type 3.] The binding of platelets to VWF via the GpIb receptor is critical for the binding of binding of platelets to the damaged vascular endothelium.

The obvious abnormalities in these series of aggregation traces are the lack of second wave aggregation with ADP and adrenaline [remember these are weak agonists] but in addition the aggregation with collagen and ristocetin is abnormal [reduced.]This suggests either a platelet storage pool disorder or an abnormality of platelet granule release.

Platelet

Aggregation

Aggregation StudiesADPreversible 1o waveif ADP is released, then 2o waveabnormal with aggregation and release problemsEpinephrinsimilar to ADPCollagendirect release so only one wave of aggregationRistocetinantibioticaggregation only with vWF and GP-Ib

Pitfalls of the techniquePlatelet function tests are still labor intensive and time-consuming and require special equipment and experts of specialized laboratories.A review of recent and regular medications is also required

The main disadvantage of platelet function studies is the use of PRP instead of the whole blood under relatively low shear conditions, and, in the absence of red and white cells, it does not accurately simulate primary hemostasis. It also requires large sample volume and is time-consuming and there are many preanalytical and analytical variables that affect the LTA results. The LTA technique is not standardized, despite the fact that guidelines have been published.

There are many preanalytical and analytical variables that affect the LTA resultsSample quality is criticalFibrinogen levels are importantAgonists must be prepared fresh dailyThrombocytopenia makes result interpretation difficultComplete patient history is essential