1. Platelets Platelets are: Disc shaped anuclear fragments Shed from megakaryocytes in the bone marrow Reference range 150 – 450 x 10 9 /L Involved in various haemostatic mechanisms Normal platelet function - primary haemostasis Dysfunctional platelets - haemorrhage Excessive platelet activation - thrombosis

3. Platelet Maturation 1. Megakaryoblast, a giant cell with 8 to 32 nuclei as a result of division of nuclei without cell division. 2. Promegakaryocyte 3. Megakaryocyte 4. Thrombocyte (Mature Platelet). Megakaryocyte maturation influenced by thrombopoietin Platelets are produced in the bone marrow It takes 5 days to mature from the megakaryoblast

4. Platelet Circulation 2/3 released into the bloodstream (peripheral blood) 1/3 are trapped in the spleen Average lifespan is 7-10 days

5. Platelets structure and function:

6. The ultrastructure of the platelet is observed in the fig. The glycoprotein of the surface coat are important in the platelet reaction of adhesion and aggregation which are the initial events leading to platelet plug formation during haemostasis. Specific glycoprotein receptors react with aggregating agents, inhibitors and aggregation factors: Adhesion to collagen is facilitated by glycoprotein (GP) Ia. Glycoprotein Ib,IIb and IIIa are important in the attachment of platelet to VWF and hence to vascular subendothelium. The binding site for IIb-IIIa is also the receptor for fibrinogen which is important in platelet aggregation.

8. The plasma membrane invaginates into the platelet anterior to form an open membrane (canalicular) system which provides a large reactive surface to which the plasma coagulation proteins may be selectively absorbed. The membrane phospholipids (platelet factor 3)are of particular important in the conversion of coagulation factor X to Xa and prothrombin to thrombin.

9. The platelet contain two types of granules: The electron-dense granules contain the calcium, ADP, ATP, and serotonin. Specific α-granules contain a heparin antagonist (platelet factor 4), β- thrombomodulin, fibronectin, VWF and other clotting factors.

10. During the release reaction, the contents of these granules are discharged into the canalicular system. The dense tubular system of platelets contains substantial quantities of calcium and may be the site of synthesis of Prostaglandins and thromboxane A2 Collagen exposure and thrombin promote platelet aggregation and the platelet release reaction whereby platelets release their granule contents.

11. Adeno­sine diphosphate (ADP) promotes platelet aggregation to form a primary haemostatic plug. Platelet prostaglandin synthesis is activated to form thromboxane A2. which potentiates the platelet release reaction, promotes platelet aggre­gation and also has vasoconstrictor activity.

12. Fibrin, pro­duced by blood coagulation binds to vWF and enmeshes the platelets to form a stable haemostatic plug. Activated platelets promote coagulation, as they have exposed phos­pholipid binding sites (the prothromhinase complex) which are involved in activation of factor X and of prothrombin to thrombin in the coagulation cascade. Platelet interactions in coagulation are initiated by adhesion to areas of vascular injury.

13. Platelets provide surfaces for the assembly of coagulation factors (e.g.,VIIIa/Ca2+/IXa and Va/Ca2+/Xa complexes). The platelets aggregate and increase the mass of the hemostatic plug. They also mediate blood vessel constriction (by releasing serotonin) and neutralize heparin.

14. Platelet Function –The main function of the platelet is to Arrest bleeding from small blood vessel injuries by forming mechanical plug during normal haemostatic response to vascular injury –Role in fibrin clot formation in larger injuries –Promote tissue repair following injury

15. Central of this function (function No. 1) are the platelets reactions of adhesion, secretion, aggregation and fusion as well as their pro-coagulant activity. Platelets undergo changes when activated Changes are due to the structure of the platelet External openings on platelets resemble “sponge”

16. Primary Hemostatic Mechanism (Platelet Phase) This mechanism leads to the formation of a reversible aggregate of platelets: a temporary hemostatic plug. To fulfill this function the platelet should go through three steps, they are: 1- Platelet adhesion: Following blood vessel injury, platelets adhere to the exposed subendothelial connective tissues. Sub-endothelial microfibrils bind the larger multimers of VWF and through these react with platelets membrane GPIb.

17. Platelet adhesion induces a series of metabolic reactions which initiate the platelets release reaction, shape change and aggregation. Platelet adhesion induces a series of metabolic reactions which initiate the platelets release reaction, shape change and aggregation. Following adhesion, platelets become more spherical and extrude long pseudo pods which enhance interaction between adjacent platelets. Following adhesion, platelets become more spherical and extrude long pseudo pods which enhance interaction between adjacent platelets. Von Willebrand factor VWF is involved in the platelet aggregation to subendothelial tissues and to the platelets. It also carries factor VIII. Von Willebrand factor VWF is involved in the platelet aggregation to subendothelial tissues and to the platelets. It also carries factor VIII.

18. 2- Release reaction: Collagen exposure or thrombin action result in the secretion of platelets granules contents which includes ADP, serotonin, fibrinogen, ß Thrombomodulin and heparin neutralizing factor (platelets factor 4).

19. 3- Platelet aggregation: Released ADP and thromboxine A2 cause additional platelet aggregation at the site of the injury. ADP causes platelet to swell and encourages platelets to adhere to each other, leading finally to secondary platelete aggregation. This will lead to formation of a platelet mass large enough to plug the area of the endothelial injury

21. Assays for primary Haemostasis Bleeding time Von Willebrand factor: Antigen determination, Activity Platelet count Platelet aggregation Activation markers Specialised tests for platelet function

22. Laboratory investigation of platelets and platelet function: A number of simple tests are employed to assess the platelet dysfunction and thrombocytopenia: Blood count and blood film examination: Peripheral blood platelet count and skin bleeding time are the first-line tests for platelet function.

23. If the results of these two tests are within the normal limits, it is unlikely that a clinically important platelet defect is responsible for the bleeding tendency. Additional information may be obtained by inspecting a fresh blood film which may show abnormalities of platelet size or morphology which can be diagnostic importance.

24. If the screening tests suggest a disorder of platelet function, further investigations are organized. Drugs and certain foods may affect the platelet function tests, for that the patient must be asked to refrain from taking such substances for at least seven days before the test. Platelet function tests can be divided into five main groups: 1.Adhesion tests 2.Aggregation tests 3.Assessment for the granular contents and the release reaction 4.Investigation of the prostaglandin pathway 5.Test for platelet coagulant activity.

25. Substances which commonly affect platelet function: Aspirin, Corticosteroids. Some anti-inflammatory drugs. Antihistamines. Local Anaesthetics. Penicillin, Cephalosporins.Heparin Ethanol, Garlic.

26. Blood count & film Normal platelet countLow platelet count 1- Bleeding time 1- Bone marrow examination 2- Platelet aggregation studies with ADP, adrenalin, collagen and restocetin. 2- Platelet antibodies 3- Other special platelet tests, e.g. Adhesion studies, pool measurement. 3- Screening test for DIC 4- Factor VIII clotting assay. vWF assay. vWF antibody assay.

27. Screening tests: As thrombocytopenia is a common cause of bleeding abnormality, patients with suspected bleeding disorders should be initially have a blood count including platelet count and blood film examination (in addition to establishing the presence of thrombocytopenia, the cause may be obvious, e.g. acute leukaemia).

28. Bleeding time test (BT): When blood count, platelet count and blood film examination are normal the bleeding time should be done to detect abnormal platelet function (dysfunction).

29. Principle: A standard incision is made on the volar surface of the forearm and the time the incision bled is measured. Cessation of bleeding indicates the formation of haemostatic plugs which are in turn dependent on the adequate number of platelet and the ability of the platelet to adhere to the subendothelium and form aggregation.

30. Methods for BT: 1- STANDARDIZED TEMPLATE METHOD: Materials: Sphygmomanometer Clearing swaps. Template bleeding time device. Filter paper. Stop watch.

31. Method:

32. –Place the Sphygmomanometer cuff around the patient's arm above the elbow, initiate to 40 mm Hg and keep it at this pressure through the test. –Clean the volar surface using the swap, chose an area with is devoid of visible superficial veins. –Press a sterile metal template with a linear slit 7-8 mm long firmly against the skin and use a scalpel blade with a guard so arranged that the tip of the blade protrudes 1 mm through the template slit. –In this way make an incision 6mm long and 1mm deep. Start the stop watch. –Blot off gently but completely with filter paper at 15s intervals the blood exuding from the cut. –When bleeding has cease, stop the watch and carefully oppose the edges of the incision and apply an adhesive strip to lessen the risk of keloid formation. –Normal range: –2.5-9.5 mins

33. B. IVY'S METHOD: The test is similar to the template method, but instead of a standardized incision two separate punctures, few cm apart are made in quick succession using a disposable lancet ( ay lancet cutting depth of 2.5 mm and width of just over 1 mm is suitable. A source of inaccuracy of ivy's method is depth is not be standardized. Normal range: 2-7 min.

34. C. DUKE METHOD: Old method, based on madding a small cut using a lancet in the earlobe. Blood flows from the puncture; the time it takes for the bleeding to stop is measured. Materials: Sterile blood lancet. Ether 1 slide Filter paper. Stopwatch

35. Method: Gently clean the lobe of the ear with cotton dipped in ether. Do not rub. Allow to dry Puncture the earlobe deeply. Start the stop watch. The blood should flow freely, with out need to squeeze the earlobe. After 30 seconds, collect the first drop of the blood on the corner of the filter paper. Do not touch the skin with the paper. Wait 30 seconds, collect the second drop of the blood in the same way. Continue to collect blood by the same way. When no more blood appears, top the watch. Normal range: 1-5 minutes.

36. PLATELTE COUNTS USING BLOOD FILM: BLOOD FILM SPREADIGN AND STAINING. Materials: Dust free clean glass microscope slide. Spreader which should be a microscope slide and one corner removed at each end.

37. Method: Place a small drop of well mixed blood on the slide about 1-2 Cm from one end. Posses the spreader at an angle of 45o about one cm in front of the drop of the blood. Move the spreader back to make contact with the drop of blood, and then move it quickly and smoothly along the line of contact of the spreader with the slide. Allow film to dry completely in the air. Note: The edge of the spreader should be very smooth, and narrow than that of the slide. The spreader must be cleaned and dried if it had been used for spreading more than five films.

38. Staining procedures: Place dried film on the staining rack with film facing upwards. Dip the well dried film in reagent one for 5 seconds. Dip the film in reagent two for 5 seconds Dip the film in reagent three for 5 seconds Clean under side of the slide and leave to dry upright. Use the 40X lens to choose a proper area. Use the oil immersion lens to investigate the morphology. Count the mean number of platelets in 10 fields at least, multiply by 20,000 The result is expressed in No./cmm