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Platelet function disorders, assessment and testing.pptx
- 1. Platelet function disorders, assessment and testing
- 2. Platelet ontogeny • Originates from the common megakaryocyte erythrocyte precursor • Promegakaryocyte, megakaryocyte, proplatelet, platelet
- 4. Normal platelet function • Megakaryopoietic agents • stem cell factor, GM-CSF, IL3, IL6 • Thrombopoietin, c-mpl – hepatocytes, bone marrow stromal cells • Endomitosis – upto 128n • Produce 2000-3000 platelet per megakaryocyte • Maturation - surface GPIb, GPII, von Willebrand factor • Reside in bone marrow adjacent to sinusoids with pseudopods projecting into the sinuses
- 7. Endomitosis
- 8. Platelet physiology • Discoid shape – normal volume 7-11fL – margination • Rigid structure – Microtubules, actin filaments linked to GPIb • Lifespan – 10 days • 4 types of granules – • Dense granules – ADP, ATP, phosphates,serotonis, Ca++ • Alpha granules – fibrinogen, VWF, fV, protein S • Lysosomes • Peroxisomes
- 9. Platelet function • Platelet capture and stable adhesion • Platelet spreading • Granule secretion and TxA2 formation • Aggregation • Thrombus stabilization • Procoagulant activity
- 10. Platelet capture and stable adhesion • Low flow/ shear rates – directly adhere to exposed subendothelial connective tissue • High flow/ shear rates – mediated by the action of VWF with GPIb-IX-V Spreading • Fillopodia formation • Fried egg appearance
- 11. Granule sectretion/ TxA2 • ADP release from dense granules • De novo formation of TxA2 • From arachidonic acid by the action of phospholipase A2 • Alpha granules – P selectin – Binds leucocytes Aggregation • specific term used for the cross- linking of activated platelets through binding of bivalent or multivalent ligands to integrin αIIbβ3 • Fibrinogen is considered to be the main ligand (higher concentration in blood)
- 12. Thrombus stabilization • Remodeling of actin cytoskeleton • Clot retraction Procoagulant activity • Provides a negatively charged phospholipid surface for coagulation pathway
- 13. VWF – what, where and how • Multimer varying in size from 500 – 20,000kDa • Ultra-large VWF is stored in endothelial cells and released (Weibel- palade bodies) • Broken down into smaller multimers by ADAMTS-13 • VWF stabilizes factor VIII • Globular form and extended forms (activated) • Binds to GPIb-V-IX
- 14. Important molecules summarized • GPIb–IX–V • GPVI and integrin 𝛂2𝛃1 – Collagen receptor • Integrin 𝛂IIb𝛃3 (GPIIb/IIIa) • P2Y1 and P2Y12 ADP receptors – Adenosine receptor • TxA2 receptor • PAR-1 and PAR-4 – Thrombin receptors • Ca++
- 16. Congenital platelet function disorders • Disorders of adhesion • BSS • Collagen receptor defects • Activation and aggregation • Defects in receptors for ADP, collagen, and thromboxane A2 • Glanzmann thrombasthenia • Storage pool defects • Grey platelet syndrome – alpha granule • Hermansky-Pudlak syndrome - Dense (or delta) granule deficiency • Combined alpha delta deficiency • Quebec platelet disorder • Membrane changes – Promote coagulation • Scott syndrome – altered phospholipid distribution
- 17. Indication of platelet function testing • Bleeding manifestations with unrevealing initial evaluation for common bleeding disorders • Positive family history for bleeding disorders • Genetic test that point to platelet function disorder • Usually not done for abnormal platelet morphology
- 18. Not useful for • Monitoring antiplatelet therapy • Predicting bleeding risk in people with thrombocytopenia
- 19. Adult with bleeding disorder- Actively bleeding individual • Search for anatomic and surgical lesions • PT, aPTT, platelet count, fibrinogen levels, peripheral smear • PFA-100, vWf testing • Intervention – • Platelet transfusion if thrombocytopenia • Fibrinogen or Vit K in prolonged clotting time • Clotting factors • Antifibrinolytic drugs
- 20. Adult with bleeding disorder- Not actively bleeding • History • Underlying conditions • Cancer • Excess alcohol use • Liver disease • Kidney disease • Connective tissue disorders • Hypothyroidism • Response to bleeding challenges • Prior bleeding history • During infancy/ stump bleeding • Adolescence/ menstrual bleeding • Severe bleeds requiring intervention • History of iron deficiency • Pregnancy history • Bruising history • Family history
- 22. BAT • Low BAT score predicts absence of a bleeding disorder and can limit excess testing • Elevated BAT score predicts risk of subsequent bleeding • Validated for • Inherited platelet disorders • VWD • Heavy menstrual bleeding • General bleeding evaluation
- 23. Bleeding assessment tool (BAT)
- 24. Pre testing evaluation • Testing required prior to platelet function testing:- • Thorough history, Bleeding assessment tool (BAT) • Complete blood counts and peripheral smear study • PT, aPTT • Screening for von Willebrand disease (optional)
- 25. What are the available tests for assessing platelet function • Platelet aggregometry is gold standard • PFA-100 – screening test • Genetic testing • Flow cytometry • Bleeding time is not recommended
- 26. Pretesting considerations • Medications: antiplatelet agents, NSAIDs, SSRIs • Discontinue for at least 10 days prior to testing • Thrombocytopenia: usually not recommended below a count of 80,000. Some cases require flow cytometric testing • Preferably overnight fasting before collection of sample - chylomicrons
- 27. Collection and transportation • 20ml blood to be collected • Minimal venous occlusion • 1/10 volume of trisodium citrate • Should not be chilled • Testing done 30mins – 3hrs post centrifuging
- 28. Aggregometry techniques Platelet rich plasma – light transmission • Centrifuge at 200g for 10-15 mins • Agonist is added to PRP • Light transmission through the sample is measured • Transmission increases as platelet aggregates are formed and separated Whole blood • Measured using electrical impedance • Platelets aggregate onto the electrode causing change in impedance
- 29. Platelet aggregometry – First panel • Aggregometry measures platelet response (aggregation) to a panel of agonists: 1. Collagen---------------------------------- Physiologic platelet activator 2. Adenosine diphosphate (ADP)------ Physiologic platelet activator 3. Epinephrine------------------------------ Physiologic platelet activator 4. Ristocetin -------------------------------- Antibiotic with platelet activation 5. Arachidonic acid------------------------ Physiologic platelet activator • Agonists added at a ratio of 1:10 by volume
- 30. Second line of agonists that may be used • 46619 (a thromboxane receptor agonist) • Gamma thrombin • TRAP (thrombin receptor activating peptides) that stimulate PAR-1 (peptide sequence SFLRRN) or PAR-4 (peptide sequence AYPGKF) • Collagen Related Peptide (CRP) and Convulxin (a rattlesnake toxin), which stimulate platelet GpVI • Calcium ionophore A23187 • Phorbol 12-myristate-13-acetate
- 31. Interpretation of aggregometry • A- shape change • B- primary wave aggregation • X- angle of initial aggregation • Y – height of aggregation trace • D – lag phase
- 32. Caveats with aggregometry and technical consideration • May be inaccurate in individuals with thrombocytopenia (1- 6Lakhs/uL) • Is not well standardized • Not sensitive to all granule storage and release defects – Lumi aggregometry
- 34. Expected aggregometry panel for thrombasthenias
- 36. PFA - 100 Attempts to reproduce under high shear rates VWF binding, platelet attachment, activation and aggregation, which slowly build a stable platelet plug at the aperture Can be used to exclude a diagnosis of function defect
- 37. Platelet Lumiaggregometry • ATP secreted by dense granules is measured luminescence technique • Luminescence measurement of ATP secretion provides unequivocal evidence of normal or impaired dense granule release
- 38. Flow cytometry • Assay platelet surface glycoprotein deficiencies seen in heritable platelet disorders • Assay for activation markers after exposure to platelet agonists. • Assay for dense granule deficiency and storage pool disease using a granule binding dye
- 39. References
- 40. References • ISTH/SSC bleeding assessment tool: a standardized questionnaire and a proposal for a new bleeding score for inherited bleeding disorders. Rodeghiero F, Tosetto A, Abshire T, Arnold DM. 2010;8(9):2063. • https://www.uptodate.com/contents/approach-to-the-adult-with-a- suspected-bleeding- disorder?search=Platelet%20disorders&source=search_result&select edTitle=2~150&usage_type=default&display_rank=2#H2100446941 • https://www.uptodate.com/contents/inherited-platelet-function- disorders- ipfds?search=Platelet%20disorders&source=search_result&selectedTi tle=1~150&usage_type=default&display_rank=1
Editor's Notes
- Proplatelets are released into circulation
- Identifying megakaryocyte Localization of megakaryocyte Sinus vs fat spaces
- Multinucleated???
- Endomitosis – normal finding
- Questionable function
- FG- fibrinogen CRP – collagen related peptide THR - thrombin
- P selectin binds ??microvesicles
- A complex of FIXa and FVIIIa on the negatively charged lipid surface converts FX to FXa (tenase complex), which in turn forms a complex with FVa on the same surface to efficiently convert prothrombin to thrombin (prothrombinase complex). Scott syndrome
- TTP – lack of ADAMTS13 – large multimers – thrombosis Mutations in GPIbα, GPIbβ and GPIX give rise to the rare bleeding disorder Bernard–Soulier syndrome, which is characterized by macrothrombocytopenia
- Clopidogrel, prasugrel,,,, ticargrelor Aspirin Abciximab
- History of easy bruising, which is commonly reported, lacks sensitivity and specificity and may be a sign of trauma or skin fragility rather than a true bleeding disorder. Excessive menstrual bleeding has many causes (eg, an anatomic lesion, hormonal factors, or impaired hemostasis) Platelet and vascular disorders are generally characterized by mucosal bleeding Coagulation factor disorders are generally characterized by bleeding into muscles and joints and delayed bleeding after a bleeding challenge Inherited disorders tend to present in childhood if severe but frequently present in adulthood or only after a bleeding challenge if mild. Some inherited disorders have additional syndromic features Some acquired disorders are associated with an underlying condition such as pregnancy, cancer, or connective tissue diseases.
- Difficulty - Vague symptomatology Different presentation for identical disease processes Environmental effect – different exposure to bleeding challenges – trauma, surgeries, physical work
- Vwd - cutoff score of ≥4 points had a sensitivity of 100 percent, specificity of 87 percent, positive predictive value (PPV) of 0.20, and negative predictive value (NPV) of 1 for the diagnosis of VWD IPFD – median score approx. 9 Heavy menstrual bleeding – In a 2012 study involving 30 females with heavy menstrual bleeding for whom anatomic, hormonal, and medication-related causes had been excluded, use of the score with a cutoff of 3.5 points had a sensitivity of 85 percent, specificity of 90 percent, PPV of 0.89, and NPV of 0.86
- Distinction between 0 and 1 is of critical importance Normal range is <4 in adult males, <6 in adult females and <3 in children (3). https://bleedingscore.certe.nl
- Control sample will be platelet poor plasma 2000g for 20 mins
- ADP - aggregation testing, prepare 100, 50, 25, 10 and 5 mmol/l Collagen- 1 and 4 mg/ml Ristocetin sulphate – 12.5mg/ml Epinephrine – 20-200umol/L Arachidonic acid – 5, 10 mmol/l solution