RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. / Name of Candidate
and
Address
(in block letters) / Dr.PARISMRITA BORAH,
#15, Sibasthan bylane, Sibamandir Path,
Six mile, Khanapara,
Guwahati, Assam
2. / Name of Institution / JJM Medical College,
Davangere,
Karnataka. PIN-577004
3. / Course of study and subject / Post-Graduate,
MD in Pathology
4. / Date of admission to course / 30.07.2013
5. / Title of the Topic:
“SCREENING AND QUANTIFICATION OF INHIBITORS IN HEMOPHILIA”
6. / Brief resume of the intended work
6.1 Need for the study
Some hemophilia patients develop antibodies or inhibitors to the deficient coagulation factor when they are infused with that factor. The antibodies inactivate the pro coagulant activity of the infused factor and inhibits patients response to replacement therapy. The accurate diagnosis of these inhibitors is plagued with many unresolved problems. Furthermore, even when diagnosed, the management of these patients is very challenging.
Factor inhibitors increase the morbidity and mortality associated with hemophilia.1 Many different risk factors have been implicated in the development of inhibitors, but there is no conclusive data yet, especially in India.
In view of the availability of various blood products for the treatment of these patients, this study is mainly aimed to document the presence or absence of inhibitors and the various factors influencing the development of inhibitors.
6.2 Review of Literature
Hemophilia is a hereditary disorder characterized by deficiency of factor VIII or factor IX coagulant activity.2 Hemophilia is clinically classified into three groups: severe, with less than 1% F VIII activity; moderate, with 1% to 5% F VIII activity; and mild, with 6% to 40% F VIII activity.3 The incidence of hemophilia A is 1 case per 10,000 population. In its severe form, the incidence is 1 in 16,000 population. It is therefore likely that there are over 50,000 people with severe hemophilia in India.4 It has been calculated that there are around 3,50,000 people with severe or moderately severe Hemophilia A worldwide and 70,000 people with severe or moderately severe Hemophilia B.5
Severely affected persons with hemophilia tend to bleed spontaneously into their joints.6 There is currently no cure for hemophilia but the bleeding tendency can be effectively corrected by intravenous substitution of clotting factors. Currently the mainstay of treatment of hemophilia A and B is lifelong replacement of coagulation factors. A major challenge in the treatment of people with hemophilia is the development of neutralizing anti factor VIII and factor IX antibodies that compromise the activity of the administered factor.7 The development of inhibitors in hemophilia patients is a major obstacle affecting the quality of life of treated individuals.3 Inhibitors inactivate the pro coagulant activity of infused factors and patients with high titre in particular do not respond to factor therapy even at high doses.8 Between 10-20% of people with hemophilia A and 2-3% of those with hemophilia B develop inhibitors against the deficient factor.4 Prospective studies of recombinant F VIII concentrates in previously untreated patients with severe hemophilia A demonstrated that, although inhibitors may arise at any time in the patient’s life, the majority develop early, after a median of 10 exposure days.9
Early diagnosis of factor inhibitors is essential. Although the presence of inhibitor can be suspected on clinical grounds, as when a patient does not respond to conventional doses of factors, laboratory diagnosis is required for confirmation. Many hemophilia A patients plasma do not display correction of APTT (Activated Partial Thromboplastin Time) on mixing studies with Normal Pooled Plasma (NPP) which indicates the presence of an inhibitor.10 The most common screening test for inhibitors is an APTT on a mixture of patient and normal plasma incubated together for one to two hours at 37C.In the presence of an inhibitor, the APTT after incubation is prolonged compared to controls without inhibitors. Inhibitors are quantified by the Bethesda test in which normal pooled plasma (as a source of factor VIII) is incubated with undiluted patient plasma for two hours at 37C and then assayed for residual factor VIII. One inhibitor unit (Bethesda unit, BU) is defined as the amount that destroys half the factor VIII in that mixture.11 Inhibitors are classified into low or high responding inhibitors. The International Society on Thrombosis and Hemostasis Scientific and Standardisation committee has recommended that an inhibitor titre of 5 BU differentiates low from high responding titres.12
6.3 Objectives of the study
1.  To document the presence / absence of inhibitors among patients with hemophilia.
2.  To quantify inhibitors by modified Bethesda assay & stratify patients for appropriate therapeutic decisions.
7. / Materials and methods
7.1 Source of data
Prospective study will be undertaken in the Department of Pathology, J.J.M.Medical college, Davangere for a period of two years from August 2013 to July 2015.
7.2 Method of collection of data (including sampling procedure, if any)
Detailed clinical history including previous treatment and with informed consent, blood will be collected in 3.2% sodium citrate in the ratio of 9:1 under aseptic condition. Screening of inhibitors will be done by APTT mixing studies in which normal plasma and test plasma is incubated at 37 ̊C for 60 minutes both separately and as 50:50 mixture. The APTT is then determined on the normal plasma, test plasma, incubated mixture and on a mixture prepared from equal volumes of test and normal plasma after separate incubation (immediate mix) The degree of correction of the APTT of each mixture is compared. Quantification of inhibitors in positive cases will be done by nijemegen modification of Bethesda Assay in which equal parts of pooled normal plasma is added to each of doubling dilutions (1/2,1/4,1/8,1/16 etc) of the test plasma prepared using imidazole buffer as diluent and also to an immunodeficient factor VIII deficient plasma which is taken as standard. Factor VIII assay is then performed on all the mixtures after incubation at 37 ̊C for 2 hours. The inhibitor concentration is calculated from a graph of residual factor VIII activity versus inhibitor units.
Statistical analysis: Results will be subjected for appropriate statistical analysis.
Sample size: 100
Inclusion criteria:
·  Patients with congenital Hemophilia due to factor VIII and factor IX deficiency who received treatment with blood and blood products.
Exclusion criteria:
·  Other hereditary bleeding disorders
7.3 Does the study require any investigations or interventions to be conducted on patients or other humans or animals? If so, please describe briefly.
Yes,
Venous blood sample for the necessary investigations.
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
Yes.
List of references
1.  Shapiro A, Inhibitors in Hemophilia: Current Perspectives and Future Directions. New York: National Hemophilia Foundation; 2001,p.4
2.  Srivastava A, Hemophilia in Developing countries- The challenge of Detection and Diagnosis. In: Sohail M.T, Heijnen, editor. Comprehensive Hemophilia Care in Developing Countries. Lahore: Ferozsons; 2001, p.17-25
3.  Amblo JK, Seren BP, Butenas S, Kathleen E, Ziedins B, Gomperts ED et al, Quantification of anti-factor VIII antibodies in human plasma. Blood 2009;113(11):2587-93
4.  Srivastava A, Guidelines for Management of Hemophilia in India. New Delhi: Hemophilia Federation; 2006. p.5-6
5.  Jones P, Guidelines for the Development of a national programme for hemophilia. Geneva: World Health Organisation; 1996. p.1-76
6.  Iorio A, Puccetti P and Makris M, Clotting factor concentrate switching and inhibitor development in hemophilia A. Blood 2012;120(4):720-27
7.  Astermark J, Altisent C, Botorova A, Diniz MJ, Gringeri A, Holme PA et al, Non genetic risk factors and the development of inhibitors in hemophilia: a comprehensive review and consensus report. Hemophilia 2010;16:747-66
8.  Jarres RK, Inhibitors : our greatest challenge. Can we minimize the incidence. Hemophilia 2013;19(suppl.1):2-7
9.  Hay CRM, Brown S, Collins PW, Keeling DM and Liesner R, The diagnosis and management of factor VIII and IX inhibitors: a guideline from the United Kingdom Hemophilia Centre Doctors Organisation. British Journal of Haematology 2006;133:591-605
10.  Pinto P, Ghosh K, Shetty S, Inhibitors in Hemophilia, Advances in diagnosis and treatment. In: Rathi SA, Agarwal M.B, editor. Haematology Today 2013. India: 2003. p.145-52
11.  Kasper CK, Diagnosis and Management of inhibitors to factors VIII and IX. Canada: World Federation of Hemophilia; 2004. p.1-12
12. Witmer C, Young G, Factor VIII inhibitors in hemophilia A: rationale and latest evidence. Therapeutic Advances in Hematology 2013;4(1):59-72
9. / Signature of Candidate
10. / Remarks of the Guide / As there are limited studies on this complication in hemophilia, this study will enable us to understand the factors influencing the development of inhibitors in Indian hemophilia population & review with the western literatures.
11. / Name and Designation of the Guide (in block letters).
11.1 Guide
11.2 Signature
11.3 Co-Guide (if any)
11.4 Signature
11.5 Head Of the Department
11.6 Signature / DR. HANAGAVADI SURESH, MD.
PROFESSOR,
DEPARTMENT OF PATHOLOGY,
JJM MEDICAL COLLEGE.
DAVANGERE-577004
_
DR. S.S.HIREMATH, MD.
PROFESSOR AND HOD,
DEPARTMENT OF PATHOLOGY,
JJM MEDICAL COLLEGE.
DAVANGERE-577004.
12. / 12.1 Remarks of the Chairman & Principal
12.2 Signature

8