HEMOPHILIA THE ROYAL DISEASE

Understanding of hemophilia increased over years, better understanding now lead us to better comprehensive care for such unfortunate patients. this presentation is derived from the text of world federation of hemophilia and indian academy of pediatrics.

1. HEMOPHILIA DR AKSHAYA TOMAR DEPT OF IMMUNOHEMATOLOGY AND BLOOD TRANSFUSION AFMC PUNE

2. INTRODUCTION Hemophilia is an X-linked congenital bleeding disorder caused by a deficiency of coagulation factor VIII (FVIII) (in hemophilia A) or factor IX (FIX) (in hemophilia B).

3. Hemophilia has been called a "royal disease". This is because the hemophilia gene was passed from Queen Victoria, who became Queen of England in 1837, to the ruling families of Russia, Spain, and Germany. It was caused by spontaneous mutation.

4. ROYAL DISEASE

5. FACTOR VIII Factor VIII serves as a cofactor for the serine proteinase factor IXa in the conversion factor X to factor Xa during the propagation phase of blood coagulation Glycoprotein synthesized mainly in hepatocytes , but also in kidneys, endothelial cells and lymphatic tissue. The gene for factor VIII is located on the X chromosome (Xq28). Human factor VIII is a single chain of about 300 kDa consisting of domains described as A1-A2-B-A3-C1- C2.

6. FACTOR VIII Present in the bloodstream in association with von Willebrand factor ( vWF ) in a non-covalent complex The vWF protects factor VIII from premature proteolysis and transfers it to sites of endothelial injury. The function of FVIIIa in the coagulation cascade is to accelerate FX activation in the presence of FIXa , phospholipids and calcium ions Half life is 12 hrs

7. STRUCTURE OF FACTOR VIII It consists of 2332 AA In the blood, under the influence of proteolytic processes ( furin protease), this protein is divided into two chains: a heavy chain of 200 kDa (A1-A2-B) and a light chain of 80 kDa (A3-C1-C2).

8. STRUCTURE OF FACTOR VIII A DOMAIN B DOMAIN C DOMAIN Homologous to factor V a domain It comprises 40% of FVIII mass 2 in number – C1 and C2 3 in number – A1,A2,A3 Function not fully understood C2 responsible for phospholipid linkage to FVIII Main Ab epitope is present in A2 Not affect FVIII activity in coagulation C2 also contains binding sites for thrombin and FXa A1 and A2 offers binding sites for FIXa and protein C Highly glycosylated,FVIII conc which are B domain deleted shows comparable or high activity C1 – no specific role but strengthen the interaction between C2 and vWF

9. ACTIVATION OF FACTOR VIII Proteolytically activated by thrombin Activation results from cleavage of the heavy chain in: Arg372 (A1 — A2 domain linkage) and Arg740 (A2 — B domain linkage) cleavage of the light chain in amino acid site Arg1689 (B — A3 domain linkage) Active form of coagulation factor VIII, FVIIIa is a trimer consisting of A1 (amino acids 1–372), A2 (amino acids 373–740) and linked A3 — C1–C2 (amino acids 1690–2332) domains .

10. IMPORTANCE OF FVIII

11. STRUCTURE OF FACTOR VIII

12. The major types of this condition are: Hemophilia A (classic hemophilia or factor VIII deficiency) Hemophilia B (Christmas disease or factor IX deficiency). Hemophilia C (Rosenthal syndrome) results from deficiency in factor XI).

13. HEMOPHILIA A

14. INTRODUCTION X-linked recessive bleeding disorder Males are affected and females are asymptomatic or mildly affected carriers The prevalence is around 1 in 5000 male births for hemophilia A and 1 in 30,000 male births for hemophilia B

15. PROBLEM STATEMENT World Federation of Hemophilia (WFH) report on the annual global survey 2015, which covered 91% of world population, identified nearly 190,000 Hemophilia patients (nearly 150,000 (80%) with hemophilia A) There were around 17,500 hemophilia patients (83% hemophilia A) identified from India in this survey Most probably its an underestimate

16. GENETIC BASIS Most common mutation in patients with a severe form of hemophilia A (about 45% cases ) is large inversion with translocation of exons 1–22 consequence of homologous recombination between the F8A gene in intron 22 and one of the F8A copies present outside the coagulation factor VIII gene Other mutations causing hemophilia are point mutations

17. HEMOPHILIA INHERITENCE

18. DISEASE CLASSIFICATION AND SEVERITY OF BLEEDING

19. CLINICAL FEATURES FVIII do not cross the placenta; therefore, bleeding may occur in utero , although this is rare 50% of severe hemophilia bleed during circumcision (neonates) 90% of severe hemophilia present in 1 st year of life (when they crawl or walk) Excessive bruising and intramuscular hematomas are common, but bleeding into a joint space, or hemarthrosis , is the hallmark of hemophilia

20. JOINTS PREDOMINANTLY AFFECTED

21. CLINICAL FEATURES Older children describe a burning or tingling sensation preceding other physical stigmata of hemarthrosis (warmth, major swelling, pain, or limited range of motion) Recurrent hemorrhage into a particular joint or “target joint” often develops.

22. LIFE THREATENING BLEEDING

23. LAB DIAGNOSIS A correct diagnosis is essential to ensure that a patient gets the appropriate treatment.

24. PRINCIPLES OF DIAGNOSIS Understanding the clinical features of hemophilia and the appropriateness of the clinical diagnosis. Using screening tests to identify the potential cause of bleeding (platelet counts,function,PT,aPTT ) Confirmation of diagnosis by factor assays and other appropriate specific investigations.

25. TECHNICAL ASPECTS Patient preparation Fasting not necessary, fatty meal should be avoided Aspirin/NSAIDs : interferes with plt function(screening test) Avoid strenuous exercise/smoking Sample collection Collected as close to the lab as possible Should be tested within 4 hrs Ideal temp range 20 – 25°C Torniquet application should not be >1 min

26. TECHNICAL ASPECTS Needle should be 19-21G for adults/23-25G for children Sample should not be taken from indwelling catheters Sample to be collected in sodium citrate (3.2% aqueous trisodium citrate dihydrate ), minimum sample reqmt is 80% of the total quantity If the sample cannot be processed within four hours of collection, the platelet poor plasma can be frozen at -30°C and stored for a few weeks, or up to six months if stored at -70°C . Storage at -20°C is usually inadequate.(WFH 2016 recommendation)

27. TECHNICAL ASPECTS End point detection For manual testing, the tube should be tilted three times every five seconds through an angle of approximately 90° during observation. Semi or fully automated coagulation analysers are preferred

28. SCREENING TESTS TESTS RESULTS PT NORMAL APTT PROLONGED PLATELET COUNT NORMAL MIXING STUDY PNP CORRECTION IN PROLONGED APTT Correction or mixing studies using pooled normal plasma (PNP) will help to define whether prolonged coagulation times are due to factor deficiency or circulating anticoagulants of inhibitors.

29. FACTOR ASSAYS Factor assays are required to To confirm diagnosis To monitor treatment To evaluate presence of factor inhibitor To assess quality of cryoprecipitate (if no factor conc available) One-stage assays based on APTT are the most commonly used techniques

30. FACTOR VIII ASSAY aPTT based assay/Clot based assay Requirements: Factor VIII deficient plasma (STA – Deficient VIII) Patient/Test Plasma Owren Koller / Imidazole buffer (pH 7.4) Clot detection method/platform Reference / calibration curve interpretation

31. Most commercially available substrate plasmas (lyophilized or frozen) are prepared by selective immunadsorption of FVIII from human pooled normal plasma 0.1 M imidazole buffer pH 7.4 Rationale is to prolong clotting times in order to create a larger discriminatory window for interpretation of results

32. The result is compared with a standard curve generated from samples containing known FVIII activities (e.g., serial dilutions of a standard reference plasma).

33. The first stage of the assay generates FXa and includes a reaction mixture with excess phospholipid , calcium, and FV (from bovine serum). -The amount of functional FVIII present is the rate-determining step in generation of FXa . The second stage of the assay adds prothrombin and fibrinogen in normal pooled plasma. -Clotting time is measured as a proxy for the amount of FVIII present and is read against a standard curve - Two-stage assay has less reagent variation, but is more complicated to perform and automate than the one-stage assay

34. Based on a similar principle to the two-stage assay When the reaction is stopped, FXa production is assumed to be proportional to the amount of functional FVIII present in the sample The second assay stage measures FXa through cleavage of an FXa specific peptide nitroanilide substrate P- nitroaniline is produced, giving a color that can be measured photometrically by absorbance at 405 nm. The color produced is directly proportional to the amountof functional FVIII present in the sample based on a standard curve

35. CHROMOGENIC ASSAYS

36. CALIBRATION CURVE Slope is calculated by the spread in seconds between serial dilutions The greater the change in y in relation to x, the steeper the slope and the better the assay precision

37. INTERPRETATION

38. INHIBITOR TESTING The presence of some form of inhibitor is suspected when there is a prolonged APTT that is not fully corrected by mixing patient plasma with PNP Most frequently encountered functional inhibitors of hemostasis are lupus anticoagulants (LA) (non specific, must be excluded) Confirmation that an inhibitor is directed against a specific clotting factor requires a specific inhibitor assay.

39. BETHESDA ASSAY The Bethesda assay is the most widely used method for detection of FVIII: C inhibitors FVIII: C inactivation in both a test mixture (normal plasma/patient plasma) and a control mixture (normal plasma/0.1 M imidazole buffer pH 7.4) is measured and expressed in inhibitor activity After 2 h of incubation at 37° C, the relative percentage FVIII: C activity of the test mixture compared to the control mixture (residual FVIII: C activity) was determined.

40. BETHESDA ASSAY The inhibitor activity was read in BU/ml from a semilogarithmic plot representing the correlation between residual FVIII :C activity (logarithmic) and inhibitor activity (linear) High titer inhibitors have ≥5 BU/ mL and low titers have 0.6 to <5 BU/ mL. The Nijmegen modification of the FVIII inhibitor assay offers improved specificity and sensitivity over the original Bethesda assay

41. NIJMEGEN MODIFICATION – THE ISTH GOLD STANDARD FOR INHIBITOR TESTING TO REMOVE FACTOR CONCENTRATE RECENTLY ADMINISTERED If testing of the undiluted patient plasma gives 25%-100% RA, then the original result is used.

42. INHIBITOR ASSAYS Methods developed to detect antibodies to FVIII and FIX include the enzyme-linked Imunosorbent assay (ELISA), Immunoprecipitation assay (IP) Fluorescence immunoassay (FLI)

43. GENETIC DIAGNOSIS Genetic analysis is recommended, wherever feasible, not only for the affected male, but also for all the at-risk female family members to identify carriers. It helps in genetic counseling for the family and provides prenatal diagnosis. Performed using CVS at 11-14 wks POG

44. GENETIC DIAGNOSIS In patients with severe hemophilia A, inversions of intron 22 (reported in 40–45%) and intron 1 (reported in 1–6%) of factor VIII gene are the most common mutations In Hemophilia B, majority are point mutations (commonly missense mutations).

45. MANAGEMENT OF HEMOPHILIA MAJ AKSHAYA TOMAR

46. PRINCIPLE OF MANAGEMENT PRIMARY AIM To prevent and treat bleeding with the deficient clotting factor SECONDARY AIM Preventing and minimizing long-term morbidity Specific treatment should be initiated in acute bleeds as soon as possible

47. TREATMENT OF ACUTE BLEEDING Provide RICE (Rest , Ice , Compression , Elevation) Factor replacement should be started immediately (as soon as possible) Joint movements should be initiated as soon as pain and swelling start to subside and gradually increased

48. CALCULATION OF DOSE Calculation of dose of factors: Calculation of the doses in individual episodes for different patients to achieve a desired factor level is based on the formulae given below: Factor VIII (IU per dose) = U/ dL desired rise (%) × Body wt (kg) × 0.5 (1 U/kg of factor VIII increases the body level by 2%; half-life of factor VIII is 8-12 hours)

49. CALCULATION OF DOSE Factor IX (IU per dose) = U/ dL desired rise (%) × Body wt (kg) (1 U/kg of factor IX increases the body level by 1%; half life of factor VIII is 18-24 hours). Because recovery of recombinant factor IX activity is less than that of therapeutic plasma-derived factor IX, 1.2 to 1.5 times the dose should be administered if using recombinant factor IX. The frequency of doses should be 12-hourly in hemophilia A and 24-hourly in hemophilia B.

50. Continuous infusion is preferable in life-threatening bleeds. It avoids peaks and troughs. It may lead to reduction in total factors consumed (cost-effective). Factor levels should be monitored and doses adjusted accordingly.

51. CHOICE OF FACTOR CONCENTRATE In the 1950s and 1960s, bleeding episodes were treated with fresh frozen plasma (FFP) Modern treatment started in 1965 with identification of the cryoprecipitate fraction Subsequently, plasma-derived factor VIII and IX concentrates were introduced. The recombinant factor VIII and recombinant factor IX were introduced in 1992 and 1997 respectively

52. CHOICE OF FACTOR CONCENTRATE SIPPET trial was one such prospective trial which reported that early replacement therapy with plasma-derived factor VIII (23.2%) was associated with a lower incidence of inhibitor development than with recombinant factor VIII (37.3%) IAP and WFH recommend the use of factor concentrates (either plasma derived or recombinant) in preference to cryoprecipitate or FFP due to concerns about their quality and safety.

53. ADJUNCTIVE TREATMENT DESMOPRESSIN ACETATE: In mild to moderate forms of hemophilia A and in carriers, the synthetic vasopressin analogue Desmopressin acetate ( given IV, SC or intranasal) can be used to increase plasma concentrations of factor VIII and VWF. DOSE : (0.3 μg /kg IV infusion/SC) increases factor VIII levels by 3-to 6-folds IV Desmopressin is available in 40 μg /10 mL vials The intranasal desmopressin is given at a dose of 150 μg for children weighing <50 kg and 300 μg for those weighing >50 kg. Repeated doses may result in tachyphylaxis,water retention and hyponatremia

54. ADJUNCTIVE TREATMENT ANTIFIBRINOLYTICS: Tranexemic acid (25 mg/kg oral or 10 mg/kg IV every 6-8h) is useful, especially for bleeds in areas rich in fibrinolytic activity Contraindicated in hematuria and those receiving activated prothrombin complex concentrates ( aPCC ) due to risk of thrombosis

55. ADJUNCTIVE TREATMENT Pain management: Pain is a common symptom in patients with hemophilia. The cause of pain may vary from acute pain caused by venipuncture or bleed to chronic arthropathy . Paracetamol is the preferred analgesic. In cases of severe chronic arthropathy , COX-2 inhibitors or opioids might be required.

56. PAIN MANAGEMENT

57. LIFESTYLE MEASURES Regular physical activity (non contact sports) Elastic, neoprene, splints and arch supports may be used to support a joint and some adjacent muscles. Activities should be withheld during acute bleeds and restarted gradually to avoid re-bleeds. Aspirin /NSAIDs avoided , paracetamol is preferred Oral hygiene to avoid gum bleed, using soft bristles toothbrush Hemophiliacs should carry their identity card all the time Veins should be handled with care

58. PROPHYLAXIS As compared to severe hemophilia, moderate hemophilia (>1% factor activity) patients seldom experience spontaneous bleeding with preserved joint function. Prophylaxis aims to maintain nadir factor activity >1% in severe hemophilia by regular factor VIII injections. The objective of prophylaxis is to prevent bleeding and joint destruction, thereby preserving normal musculoskeletal function

59. PROPHYLAXIS

60. PROPHYLAXIS

61. PROPHYLAXIS (DOSAGE) Malmo protocol (25-40 IU/kg/dose) Utrecht protocol (15-30 IU/kg/dose) IAP recommends 10-20 IU/kg/dose , twice to thrice/week in severe hemophilia There is now data from resource-poor settings to demonstrate that lower doses of Factor VIII used for prophylaxis would also limit the number of acute bleeds, and hence the long-term joint morbidity However, episodic prophylaxis has not been found to be useful.

62. PROPHYLAXIS 10-20 IU/kg/dose should be the starting dose Further modifications made as per intercurrent /breakthrough bleeding frequency Preferable time for administering prophylaxis doses is in morning and/or just before undertaking activities at-risk for trauma (to effectively cover these activities) Home-based treatment is feasible for mild to moderate bleeds, if caretakers are trained properly.

63. FACTOR CONCENTRATES WFH strongly recommends the use of viral inactivated plasma-derived or recombinant concentrates in preference to Cryo or FFP for the treatment of hemophilia and other inherited bleeding disorders Factor concentrates are selected on the basis of : Purity (refers to the percentage of desired component in relation with other ingredients) Viral inactivation

64. FVIII CONCENTRATE

65. DOSAGE AND ADMINISTRATION Vials available from 250 to 3000 IU In the absence of an inhibitor, 1U FVIII /Kg body weight  raises plasma FVIII by 2IU/dl Half life 8-12 hrs FVIII should be infused by slow IV injection not to exceed 3 ml/min in adults - 100 U /min in young children

66. FACTOR IX CONCENTRATE 2 CLASSES Pure FIX Prothrombin complex concentrate (II,VII,IX, X) Whenever possible, the use of pure FIX concentrates is preferable for the treatment of hemophilia B as opposed to PCC In absence of an ihibitor 1U/Kg of FIX  raises Plasma FIX by 1 U/dl Plasma half life is 12-24 hrs

67. OTHER PLASMA PRODUCTS Factor concentrates are preferred over FFP/ Cryo except in resource constraint situations CRYO  preferred in Hemophilia A FFP  preferrred in Hemophilia B Other steps to reduce viral/disease transmission Quarantining plasma until the donor has been tested or even retested Nucleic acid testing (NAT) to detect viruses Preferably viral inactivated product

68. DOSAGE AND ADMINISTRATION FFP 1 ml of FFP  1U of factor activity Generally difficult to achieve FVIII levels higher than 30 IU/dl with FFP alone FIX levels above 25 IU/dl are difficult to achieve Acceptable starting dose : 10-20ml/kg CRYO 1 ml of CRYO  3-5 ml of FVIII, no FIX Starting dose : 1 U/ 10kg Body weight

69. VACCINATION IN HEMOPHILIACS Subcutaneous (SC) administration of vaccines is preferred over intramuscular (IM) or intradermal There is no compromise in immunogenicity Use a thin needle (25-27 G) and apply prolonged pressure for 5 minutes. Avoid factor replacement close to vaccinations, as vaccination induces an inflammatory reaction and thus may increase the chance of developing inhibitors,

70. LONG TERM COMPLICATION

71. CHRONIC ARTHROPATHY Recurrent hemorrhages lead to target joints which progress to chronic arthropathy with functional impairment and pain. Synovitis : Compression bandages , Splints , Analgesics , Factor conc Restricted ROM : superficial thermotherapy , joint traction , active and passive exercises Unresolved Synovitis : open or arthroscopic synovectomy Permanent joint damage : tissue release , osteotomy , joint replacement Surgery and joint replacement is not needed in those who have been adequately managed and do not have inhibitors (on continuous prophylaxis)

72. FACTOR INHIBITORS Neutralizing Ab directed against FVIII and FIX Incidence : Severe Hemophilia A : 33% Mild to Moderate Hemophilia A : 13% Hemophilia B : 3 – 4% Risk Factors : Early age of exposure Common inversion mutation Large deletion of FVIII gene Sibling with hemophilia and inhibitor

73. FACTOR INHIBITOR Majority of inhibitors develop early in the treatment trajectory after a median exposure of 10 exposure days and less common after 150 exposure days Inhibitor Screen Once in every 5 exposure days until 20 days of exposure Every 10 day from 21 to 50 days 2 times a year until 150 days and beyond that annually

74. FACTOR INHIBITOR Later, inhibitor should be screened in the following situations: Prior to and after any surgery or invasive procedure Before and after a switch of products Whenever clinically indicated (bleeding on prophylaxis, response to factor therapy is sub-optimal) In hemophilia A, factor VIII inhibitors are characteristically time-dependent. Immediate mixing results show correction of aPTT , which is lost when the same 1:1 mix is incubated for 2 hours at 37°C. A difference of >5 seconds between immediate mix and incubated mix indicates factor VIII inhibitors

75. FACTOR INHIBITOR BYPASS ACTIVITY In patients with high titer inhibitors, bypassing agents (Activated Prothrombin Complex Concentrates (FEIBA) or recombinant Factor VIIa ) should be used for treatment of bleeding

76. ERADICATING INHIBITORS Immune Tolerance Induction(ITI) - mainstay of treatment to eradicate the inhibitors Regular, frequent, and prolonged exposure of the patient to specific factor concentrates thereby inducing peripheral tolerance Bleeding episodes need treatment with bypassing agents for patients on ITI (especially with titers >10 BU) Overall, ITI is successful in 70% of hemophilia A and in 30% of hemophilia B patients with inhibitors

77. TRANSFUSION TRANSMITTED INFECTIONS Use of plasma-derived product (with efficient viral inactivation) and recombinant factors have significantly decreased TTIs in developed countries. IAP however recommend vaccination against hepatitis B in all children diagnosed with hemophilia. Screening of HIV, hepatitis B and HCV should be performed in all hemophilia patients

78. COMPREHENSIVE CARE ■■ Prevention of bleeding and joint damage ■■ Prompt management of bleeding ■■ Management of complications including: Joint and muscle damage and other sequelae of bleeding Inhibitor development Viral infection(s) transmitted through blood products ■■ Attention to psychosocial health

79. BIBLIOGRAPHY Consensus Statement of the Indian Academy of Pediatrics in Diagnosis and Management of Hemophilia, July 2018 Laboratory testing for factor VIII and IX inhibitors in haemophilia : A review , Hemophilia , March 2018 WFH guidelines for hemophilia, 2012 CLOT – ED by Marlies Ledford, 2017 Chromogenic factor VIII activity assay, AJH , 2014 The factor VIII protein and its function, ABP , 2016

80. T hanks !