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

22. 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 1st 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

23. JOINTS PREDOMINANTLY AFFECTED

24. 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.

25. LIFE THREATENING BLEEDING
INTRACRANIAL
•Most common cause of
death in hemophiliacs
•Spontaneous or trivial
trauma
•Any hemophiliac with
persistent headache should
be managed immediately
ILIOPSOAS
•Hematoma can be
spontaneous or trauma
related
•Expand into retroperitoneum
and cause compression or
hypovolumic shock
•Patients usually presents
with vague groin pain
RETROPHARYNGEAL
•Hematoma
•Compress or occlude the
airway

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

27. 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.

28. 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

29. 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)

30. 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

31. 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.

32. 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

33. 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

34. 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

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

37. -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

38. • 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

39. CHROMOGENIC ASSAYS

40. 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

41. INTERPRETATION

42. 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.

43. 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.

44. 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

45. 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.

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

47. 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

48. 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).

49. MANAGEMENT OF HEMOPHILIA
MAJ AKSHAYA TOMAR

50. 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

51. 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

52. 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)

53. 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.

54. • 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.

55. 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

56. 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.

57. 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

58. 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

59. 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.

60. PAIN MANAGEMENT

61. 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

62. 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

63. PROPHYLAXIS
Prophylaxis
Episodic
Factor replacement
only during evident
bleeding
Continuous
Factor replacement
for >85% of the year

64. PROPHYLAXIS
Primary (before 2nd episode of joint bleed)
Secondary (after 2 or more joint bleeds and before
developing any joint disease)
Tertiary (after the onset of joint disease)

66. 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.

67. 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.

68. 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

69. FVIII CONCENTRATE

70. 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

71. 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

76. 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

77. 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

78. 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,

79. LONG TERM COMPLICATION

80. 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)

81. 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

82. 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

83. 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

84. 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

85. 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

86. 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

87. 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

88. 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