1. COAGULATION DISORDERS

2. Hemostasis Hemostasis depends on the integrity of –Blood vessels –Platelets –Coagulation factors –Anticoagulation factors

3. Steps in Hemostasis Formation of primary platelet plug due to adhesion of platelets to collagen and traces of thrombin. Conversion into permanent plug supported by fibrin clot. Lysis of fibrin and confinement of clot to the site of injury.

5. XIIXIIa XIXIa IXIXa XXa ProthrombinThrombin VIIVIIa TF FibrinogenFibrin VIIIa Va Intrinsic Pathway HMWK Prekallikerin Surface Extrinsic Pathway Cross linked fibrin XIIIa

6. XIXIa IXIXa XXa ProthrombinThrombin VII TF VIIa FibrinogenFibrin VIIIa Va Cross linked fibrin XIIIa VIII V XIII Coagulation Cascade Tissue injury

7. Coagulation Cascade Initiation of coagulation is not dependent on the contact factors (factor XII, prekellikerin or HMWK). Coagulation is initiated by exposure of blood to TF. TF-VIIa complex activates factors X and IX. Factor IXa accelerates factor X acivation by more than 50 folds.

8. Prothrombin Time(PT) XII XIIa XIXIa IXIXa XXa ProthrombinThrombin VIIVIIa TF FibrinogenFibrin VIIIa Va HMWK Prekallikerin Surface Cross linked fibrin XIIIa

9. Partial Thromboplastin Time XII XIIa XIXIa IXIXa XXa ProthrombinThrombin VII VIIa TF FibrinogenFibrin VIIIa Va HMWK Prekallikerin Surface Cross linked fibrin XIIIa

10. Thrombin Time XII XIIa XIXIa IXIXa XXa ProthrombinThrombin VIIVIIa TF FibrinogenFibrin VIIIa Va HMWK Prekallikerin Surface Cross linked fibrin XIIIa

11. Initial Laboratory Tests For Bleeding Abnormalities Platelet count. Bleeding time. Partial thromboplastin time (PTT). Prothrombin time (PT). Thrombin time(TT).

12. PTT and APTT Purpose: – Screen for deficiencies of the coagulation factors of the intrinsic and common pathways. All factors except VII and XIII. – Detect circulating anticoagulants. Principle of test: Phospholipid is incubated with platelet poor plasma; Then Ca++ is added and time for clot formation is measured. In APTT: the plasma is incubated with activating agent such as kaolin.

13. PTT and APTT Interpretation Prolongation Deficiencies in one or more of factors XII, XI, X, IX, VIII, V and II and Fibrinogen. Inhibitors to one of the above factors. High concentrations of fibrinogen and fibrin split products, heparin and protamine sulfate. Shortening Poor venipuncture. If plasma contains platelets. High factor VIII. DIC.

14. Prothrombin Time Purpose: – Screen for deficiencies of the coagulation factors of the exrinsic and common pathways. Factors Prothrombin (Factor II), V, VII, X and fibrinogen are measured. – Monitor oral anticoagulant therapy. Principle: Ca++ and tissue extract such as brain are added to plasma leading to activation of VII Prolongation: – Deficiency of Factors II, V, VII and X. – Fibrinogen level below 100mg/dl. – Heparin and fibrin split products

15. Thrombin Time Purpose of test: screen for reduction of fibrinogen concentration and presence of fibrin split products. Principle: thrombin is added to plasma. Time needed to clot is measured as TT. Interpretation: prolongation is seen in: – Low fibrinogen level below 100 mg/dl. – Qualitative change in fibrinogen. – Heparin therapy. – Fibrin or fibrinogen split products.

16. Bleeding Time Definition: time taken for bleeding from a standardized skin wound to stop. Principle of test: measurement of platelet function. Purpose of test: screening test for disorders of platelets (congenital or acquired), and for von Willebrand disease. Normal range: 2.5 to 7.5 min. Aspirin and other anti- inflammatory drugs prolong bleeding time.

17. Initial Laboratory Tests For Bleeding Abnormalities Platelet count. Bleeding time: prolongation is seen in platelet diseases or in patients receiving drugs interfering with platelet function. Partial thromboplastin time (PTT). – Prolongation is seen in hemophiliacs and in patients with lupus anti-coagulant. Prothrombin time (PT). – Prolongation is seen in factor VII deficiency. – Factors V, X, prothrombin and fibrinogen affect APTT and PT. Thrombin time.

18. Causes of Abnormal Bleeding Vascular disorders. Thrombocytopenia. Platelet function defects. Defective coagulation. Vascular disorders. Thrombocytopenia. Platelet function defects. Defective coagulation.

19. Vascular Bleeding Disorders Easy bruising and bleeding from small vessels. Caused by abnormal vessels or abnormal perivascular connective tissue. Sites of bleeding: skin and mucous membranes. Bleeding time and other screening tests are normal. Congenital or acquired.

20. Acquired Vascular Defects Benign disorders in women of child bearing age. Senile purpura due to atrophy of perivascular connective tissue. Henoch-Schoenlein syndrome. Steroid purpura (defective CT). Scurvy. Infections. – measles – dengue – meningococcemia – ricketsial infections Hereditary hemorrhagic telangiectasia Ehlers-Danlos syndrome.

21. Coagulation disorders

22. Deficiency Fibrinogen Prothrombin Factor V Factor VII Factor VIII Factor IX Factor X Factor XI Factor XIII Incidence in Population 1:1 million 1:2 million 1:1 million 1:500,000 1:10,000 1:600,000 1:1 million 1:2 million Gene on Chromosome 4 11 1 13 X 13 4 6 & 1 Mode of Inheritance AR XLR AR Inherited Deficiencies of Coagulation Factors

23. Hemophilia A Incidence: 1 in 10,000 (common). Sex-linked. 30% of the cases have no family history (recent mutation or generations of silent carriers). Caused by absolute reduction of factor VIII or normal amount but defective factor VIII. Severity of disease depends on factor VIII level – Normal level100 U/dl – Severe cases level <2 U/dl – Moderate cases level2-5 U/dl – Mild cases level5-25 U/dl

24. Hemophilia A Affects male Less commonly excessive bleeding occurs in heterozygous females…preferential inactivation of the X chromosome carrying the normal factor VIII gene ( unfavorable lyonization) C/O: -Easy bruising and massive hemorrhage after trauma or operative procedure. -Petechia are characteristically absent.

25. Hemophilia A Sites of bleeding: – Large joints and soft tissue – Urinary tract and GI tract – Brain – Nose Laboratory tests: – Prolonged PTT (corrected by mixing study). Normal PT and TT. – Low factor VIII assay. Treatment: replacement therapy (factor VIII concentrate or recombinant VIII). Inhibitor antibodies develop in 18-50% of patients

26. Nomenclature of Factor VIII Factor VIII Factor VIII c Factor VIII Ag Protein lacking or aberrant in hemophilia A Functional property of factor VIII missing in hemophilia A, measured by coagulation assays Antigenic property of factor VIII, measured by immunoassays

27. Factor VIII (FVIII) and the FVIII Gene The gene is located at the tip of the X chromosome. Huge gene composed of 26 exons. FVIII mRNA is ~9kb, and encodes a 300kD protein. FVIII is a cofactor for F IX in the proteolytic activation of F X. Bound to vWF in plasma. Markedly unstable in the absence of vWF.

28. Molecular Genetics of (Hemophilia A) Point mutation in exons or splice junctions (~50%) Inversion of intron 22 (~45%) Deletions or less commonly insertions (~5%)

29. Hemophilia in Females Exceedingly rare, seen in: – Mating between a carrier mother and affected father – Carriers with abnormalities of X-chromosome Extreme lyonization X mosaicism or deletion Newly mutant gene

30. Hemophilia B Incidence: 1 in 60,000. Sex-linked. Severity of disease depends on factor IX level –Normal level100 U/dl –Severe cases level<2 U/dl –Moderate cases level2-5 U/dl –Mild cases level5-25 U/dl Bleeding sites: similar to hemophilia A. Laboratory tests: –Prolonged PTT. Normal PT and TT. –normal factor VIII assay.

31. von Willebrand Factor (vWF) Coded by a gene located on the short arm of chromosome 12 The primary protein product is a 250,000 Da dimer. Transformed by multiple disulfide bonds to form a series of multimers ranging in weight from 10,000,000 to 20,000,000 Da

32. vWF Adhesive protein, bridges collagen to platelets receptor GPIb Carrier protein to factor VIII Ristocetin induces platelets agglutination in the presence of vWF (RIPA) Stored in Weibel-Palade bodies of endothelial cells and  granules of platelets

33. Willebrand von Willebrand Disease * *The most common hereditary bleeding disorder (prevalence 0.1-1%). Mild bleeding problems – Mucous membrane bleeding – Easy bruising – menorrhagia – Post-operative bleeding Most cases are inherited as Autosomal dominant disorder. ? When to suspect: *Both sexes are affected, and presented with prolonged bleeding times (BT) despite normal platelet counts.

34. Willebrand von Willebrand Disease *vWD differs from classic Hemophilia A in 3 cardinal manifestations: 1.Autosomal inheritance rather than sex linked 2.Consistently prolonged bleeding time (BT) 3.Mucocutaneous bleeding rather than hemarthroses and deep muscle hemorrhage.

35. Willebrand von Willebrand Disease * *vWD : quantitative or qualitative defects of plasma vWF. Either Dominant or Recessive. Divided into 3 main type: 1,2, and 3. Type 2 vWD is further subdivided into 4 subtypes, designated as 2A,2B, 2M, and 2N. Additional disease related to platelet vWF receptor, platelet type/pseudo-vWD (similar presentation of vWD, but doesn’t involve a mutation of the vWF gene).

36. Willebrand von Willebrand Disease * *vWD : Severe forms generally present with A normal PT A prolonged PTT that corrects with mixing study Normal platelet count An abnormal BT *Lab. Assessment should include assays for:. F VIII:C activity, vWF: Ag, RIPA, and vWF multimeric analysis.

38. Willebrand von Willebrand Disease type 1 The most common type (70-80% VWD cases). Autosomal dominant disorder with variable penetrance (60%) Partial deletion in gene…partial quantitative defect…reduced circulating vWF and factor VIII…mild symptoms. All sizes of vWF multimers are present. Slight prolongation of APTT/BT Platelets fail to agglutinate by ristocetin (decreased RIPA) Rx: DDAVP before dental work, surgery and after bleeding.

39. Willebrand von Willebrand Disease type 2 * Exhibits qualitative defect of vWF molecule with abnormalities in the multimeric distribution. *Type 2A: Most common of type 2 Dysfunctional defect of vWF Disproportionately lower vWF activity that antigenic concentration. Symptoms: moderate to severe Lab: BT, RIPA, vWF activity markedly abnormal. Factor VIII level may be normal or decreased. Rx: vWF concentrate. ? DDAVP

40. Willebrand von Willebrand Disease type 2 *Type 2B: Qualitative defect Increased affinity for platelet GP 1b Absence of only HMW multimers. * smaller ristocetin doses cause aggregation of platelets in type IIb (LD- RIPA enhanced aggregation). Lab: prolonged BT, vWF activity normal or decreased. Rx: vWF concentrate. ? DDAVP??? C/I

41. Willebrand von Willebrand Disease type 2 *Type 2M (multimer): Very rare Qualitative defect of vWF Decreased or absent binding of vWF to GP1b As in Type 2A, However, the multimer distribution is normal. * RIPA response is decreased. Lab: prolonged BT, vWF activity decreased. Rx: vWF concentrate. ? DDAVP ineffective

42. Willebrand von Willebrand Disease type 2 *Type 2N (Normandy): AR Qualitative defect of vWF Decreased affinity of vWF to F VIII: C *Level of F VIII: C is disproportionately lower than levels of vWF ? Misdiagnosed as Hemophilia. *The platelet-dependent function of vWF is normal…so…RIPA response is normal. Lab: BT is normal. Rx: vWF concentrate. ? DDAVP ineffective

43. Willebrand von Willebrand Disease type 3 * The most severe form: AR Quantitative defect of vWF Complete absence of vWF in the plasma, platelet, and endothelial cells. ? Misdiagnosed as Hemophilia A. * Lab: BT and RIPA are severly abnormal. Rx: Replace vWF and F VIII with concentrate.

45. *Platelet type/pseudo- VWD Is similar to type 2B, but GPIb is defective (no genetic mutation in in the vWF gene). Rx: Platelet concentrate.

46. Platelet disorders

47. Relationship Between Platelet Count and Bleeding Normal range 150-450X10 3 per µl. Levels above 60x10 3 /µl will not cause bleeding under normal conditions. Levels below 20x10 3 /µl will cause: Petechiae, mucosal bleeding. Post-operative bleeding, CNS bleeding. Levels around 5x10 3 /µl can lead to fatal CNS or GI hemorrhage. Levels between 20 and 60x10 3 /µl may cause bleeding (depending on platelets functional status).

48. Classification of Thrombocytopenia 1-Failure of production ( aplastic anemia, radiation, chemo.Rx ) 2-Increased platelet destruction (ITP) 3-Abnormal distribution ( Splenic sequestration )

49. (1)Thrombocytopenia Due to Failure of Production Decreased BM megakaryocytes – Infiltrative diseases of BM – Aplastic BM – Amegakaryocytic thrombocytopenia – HIV infection Increased BM megakaryocytes – Megaloblastic anemia – Myelodysplasia – Alcohol induced

50. (2)Thrombocytopenia Due to Increased Platelets Destruction ITP Secondary immune thrombocytopenia as in SLE Drug related immune thrombocytopenia as in quinidine and heparin. Post transfusion thrombocytopenia Neonatal thrombocytopenia either due to autoantibodies or alloantibodies DIC and microangiopathic hemolytic anemia

51. ITP Primary (idiopathic) or secondary Primary (idiopathic) or secondary Acute or chronic. Acute or chronic.

52. Acute ITP (Idiopathic/Childhood) Affects children. Develops acutely with 1-2 week duration. Preceded by infection or vaccination in 75% of cases. Initial Plt.count <20,000 Self limited, Spontaneous remission in >90% of cases. Severe cases benefit from steroids or IV immunoglobulins.

53. Chronic Immune Thrombocytopenic Purpura (ITP) High incidence in women of child bearing age (20-50). Mostly idiopathic, secondary causes include SLE, HIV, CLL, Hodgkin’s disease, drugs (uncommon). Autoantibodies against GP IIb/IIIa, or Ib/IX. Platelets lifespan reduced to hours. Megakaryocytes increased. Petechial bleeding, easy bruising, menorrhagia.

54. ITP Diagnosis Decreased platelet count (10-50x10 9 /l). Hb. and WBCs are normal. Increased Megakaryocytes numbers in BM. Antiplatelet antibodies

55. ITP Treatment Steroids. Splenectomy. High dose IV immunoglobulins. Immunosuppressive therapy.

56. Microangiopathic Thrombocytopenia TTP/HUS

57. Thrombotic thrombocytopenic Purpura (TTP) Usually affects adult Pathogenic mechanism: Inherited and Sporadic: Deficiency of metalloprotease (ADAMTS 13) needed for cleaving very HMW_vWF (multimers). More common, non-familial acquired: autoantibody against ADAMTS 13 Platelet microaggregrate (Hyaline microthrombi) formation. Acute Thrombocytopenia, fever, microangiopathic hemolytic anemia, neurologic abnormalities, renal dysfunction.

58. Laboratory Results: Microangiopathic hemolytic anemia picture: Schistocytes Reticulocytosis NRBC’s in PB Signs of hemolysis: Increase LDH Increase indirect bilirubin Decrease Haptoglobin Normal PT, PTT, D-Dimer but elevated BT. Rx: Plasma exchange.

59. Hemolytic Uremic Syndrome (HUS) NONIMMUNE THROMBOCYTOPENIA More commonly seen in pediatric population. E. coli O157:H7 (toxin induced endothelial damage) Bloody diarrhea followed by acute renal failure. Platelet microaggregrate (Hyaline microthrombi) formation, usually limited to the glomerular capillaries. Acute Thrombocytopenia, Microangiopathic hemolytic anemia, Renal failure. Normal PT, PTT, D-Dimer but elevated BT. Rx: Conservative (dialysis, antihypertensive,…).

60. Platelet Membrane Glycoproteins GP Ia-IIa: adhesion to collagen. GP Ic-IIa: laminin receptor. GP IIb-IIIa: binding to fibrinogen. GP Ib-IX: adhesion to subendothelial tissue via vWF.

61. Qualitative disorders of platelets 1.Bernard Soulier syndrome – Adhesion Defect – Autosomal recessive. – Deficiency of GpIb/IX (CD42) which serves as the receptor for vWF (adhesion of unactivated platelet). – Lab: Giant platelets. Platelet aggregation in response to ADP, collagen, and epinephrine is normal Absent platelet aggregation in response to vWF and ristocetin

62. Qualitative disorders of platelets 2. Glanzman’s thrombasthenia – Aggregation defect Failure to aggregate in response to ADP, collagen, epinephrine and thrombin – Autosomal recessive – Deficiency of GpIIb/IIIa (CD41/CD61) which serves as the receptor for fibrinogen and mediates the the binding of fibrinogen, vWF, and fibronectin to activated platelets. – Lab: N. PLT. count & morphology Prolonged BT. 3. Storage pool diseases – Initial aggregation normal, but secretion of thromboxane, ADP, and other granules contents impaired

63. DIC

64. DIC (Consumptive Coagulopathy) Thrombosis, fibrinolysis, bleeding. Obstetric complications, infections, cancers (panc, lung, prost, stomach, APML), massive tissue injury (trauma, burn), others (shock, liver, heat stroke, vasculitis, intravascular hemolysis). Lab: Low plat, fibrinogen high PT. PTT and FDP