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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE-41 PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION 1 NAME OF THE CANDIDATE JASIM VP 1. ADDRESS DEPARTMENT OF CLINICAL PATHOLOGY, ST.JOHN’S MEDICAL COLLEGE, BANGALORE-34 2 NAME OF THE INSTITUTION ST.JOHN’S NATIONAL ACADEMY OF HEALTH SCIENCES 3 COURSE OF STUDY &SUBJECT MSc.MLT HEMATOLOGY & TRANFUSION REACTION 4 DATE OF ADMISSION TO COURSE 25-8-2010 5 TITLE PREVALENCE OF Rh PHENOTYPE AND SIGNIFICANCE OF DU TESTING 6.0 BRIEF RESUME OF THE INTENTED WORK: 6.1NEED FOR STUDY The Rh blood-group system is clinically important because antibodies against Rh antigens are involved in hemolytic disease of the newborn, hemolytic transfusion reactions, and autoimmune hemolytic anemia.1,2 Individuals are classified as Rh-positive and Rh-negative according to the presence or absence of the D antigen on the surface of their red cells.1 The RhD blood group antigen has been shown to be subject to many phenotypic variations3 and the frequency of weak D phenotype varies with the method used, the reagent used, and the racial mix tested (Division of Medical Laboratory Science, 2001). Weak D phenotypic expression is known to arise from three mechanisms.3 In one of these mechanisms, referred to as gene interaction, there is a suppressive effect of the C gene when in trans to the D gene (e.g., Dce/Ce). The second is when part of the D antigen is missing (partial D).Thirdly, the presence of an aberrant form of D (e.g., at the molecular level) would result in weak phenotypic expression. The term’ Weak D' actually refers to red cells with the aberrant RhD protein, expressing reduced membrane surface D antigens. The partial Ds, sometimes give weak phenotypic reactions in serologic procedures. Because serologic distinction is currently not possible, many laboratories would detect some partial D's as weak D. It is known that many partial D phenotypes present as normal D types so they are often not classified until they make anti-D. In the laboratory, serologic typing techniques employing the use of anti-D sera are mainly used to detect the presence of the Rh D antigens on the red blood cells of individuals. The number of samples classified as weak D depends on the characteristics of the typing reagent.4 Weak D individuals (who are actually Rh -positive) were commonly mistyped as Rh- negative through the use of polyclonal antibodies with grave consequences.5 However, with the advent of monoclonal anti-D reagents, most weak D individuals are now typed as Rh-positive. It is now becoming evident that there are some immunogenic weak D samples that would not be detected by direct agglutination. Antihuman globulin test may therefore need to be performed to detect weak D red cells in individuals who initially type as Rh-negative. The objective of this study therefore, is to determine the prevalence of Rh phenotype and weak D (Du) among Rh-negative blood donors. 6.2 REVIEW OF LITERATURE With the discovery of the Rh antigen and its relationship to hemolytic disease of the newborn in 1939 and 1940, it soon became a recognized standard of care to make every attempt to avoid transfusing Rh(D)-positive red blood cells to Rh(D)-negative individuals.6,7 This was particularly true for Rh(D)-negative women of childbearing age. As early as 1943 and 1944, a weakly reacting form of the Rh(D) antigen, which gave ‘‘intermediate’’ reactions with anti-D typing sera, was recognized and described by Wiener.8 In 1946, Stratton coined the term Du for the phenotypic weak expression of the Rh(D) antigen. More recently, the term weak D has been proposed as a more appropriate characterization for the quantitative or qualitative differences observed for weakened expression of the Rh(D) antigen.9 The complexity of the Rh blood group antigens begins with the highly polymorphic genes that encode them. There are two genes, RHD and RHCE that are closely linked. Numerous genetic rearrangements between them have produced hybrid Rh genes that encode a myriad of distinct Rh antigens. To date, 49 Rh antigens are known. The significance of the Rh blood group is related to the fact that the Rh antigens are highly immunogenic. In the case of the D antigen, individuals who do not produce the D antigen will produce anti-D if they encounter the D antigen on transfused RBCs (causing a hemolytic transfusion reaction, HTR) or on fetal RBCs (causing HDN). For this reason, the Rh status is routinely determined in blood donors, transfusion recipients, and in mothers-to-be. Despite the importance of the Rh antigens in blood transfusion and HDN, we can only speculate about the physiological function of the proteins, which may involve transporting ammonium across the RBC membrane and maintaining the integrity of the RBC membrane The number of D antigen sites on the Rh(D)-positive red blood cell is normally in the range of 9900 to 33 000.10,11 The weak D phenotype appears to be a quantitative variation in the number of D antigen sites on the red blood cell (i.e, 110 to, 9000 per red blood cell).10,11 More than a half century has passed since weak expression of the Rh(D) antigen was initially observed. Confusion has continued to plague issues surrounding weak D terminology and its clinical significance. A suggestion was made in 1984 to abandon the term Du and to use the more appropriate weak D designation.12 A further proposal was made in 1992 to standardize terms for weak D.9 Practice guidelines related to testing for the weak D antigen and recommendations related to the administration of Rh immune globulin have also been recently published.13,14 Antigens of the Rh blood group Number of Antigens 49: D, C, E, c, and e are among the most significant Antigen Protein Specificity The sequence of amino acids determines the specificity of most of the Rh antigens. Antigen Proteins with unknown function Carrying The RhD and RhCE proteins are both transmembrane, multipass molecules proteins that are integral to the RBC membrane. The RhCE protein encodes the C/c antigen (in the 2nd extracellular loop) and the E/e antigen (in the 4th extracellular loop), plus many other Rh antigens e.g., Cw, Cx. Unlike most cell surface molecules, the Rh proteins are not glycosylated (they do not contain oligosaccharides) but they are closely associated with a RBC membrane glycoprotein called Rh-associated glycoprotein (RhAG). The function of the Rh-RhAG complex might involve transporting ammonium or carbon dioxide. The RhD protein encodes the D antigen. Molecular Basis Two genes, RHD and RHCE, encode the Rh antigens. The Rh genes are 97% identical, and they are located next to each other on chromosome 1. The D/d polymorphism most commonly arises from a deletion of the entire RHD gene. The C/c polymorphism arises from four SNPs that cause four amino acid changes, one of which (S103P) determines the C or c antigen specificity. The E/e polymorphism arises from a single SNP (676G→C) that causes a single amino acid change (A226P). Frequency of Rh antigens in Asians D: 99% C: 93% E: 39% c: 47% e: 96%15 Frequency of Rh phenotypes in Asians Rh haplotype DCe: 70% Rh D-negative phenotype: 1%15 Antibodies produced against Rh antigens Antibody type Mainly IgG, some IgM The majority of Rh antibodies are of the IgG type Antibody reactivity Capable of hemolysis Rh antibodies rarely activate complement. They bind to RBCs and mark them up for destruction in the spleen (extra vascular hemolysis). Transfusion reaction Typically delayed hemolytic transfusion reactions Anti-D, anti-C, anti-e, and anti-c can cause severe hemolytic transfusion reactions. Hemolysis is typically extra vascular15 Hemolytic disease of The most common cause of HDN. The newborn The D antigen accounts for 50% of maternal alloimmunization16 Anti-D and anti-c can cause severe disease. Anti-C, anti-E, and anti-e can cause mild to moderate disease. Common Rh phenotypes The most common Rh haplotype in Caucasians, Asians, and Native Americans is DCe. In Blacks, the Dce haplotype is slightly more common.15 In Caucasians, the Rh D-negative phenotype results from a deletion of the RHD gene. About 15% of Caucasians are Rh D-negative. Uncommon Rh phenotypes The D antigen contains over 30 epitopes. Variations of the D phenotype arise when these epitopes are only weakly expressed ("weak D phenotype") or when some are missing ("partial D phenotype"). Partial D: some D antigen epitopes are missing In contrast, people who have been identified as having the "partial D" phenotype should not receive Rh D-positive blood but in practice, people with partial D are difficult to identify. This phenotype is usually caused by the creation of a hybrid RhD and RhCE protein. The hybrid protein is similar enough to RhD to be correctly inserted in the RBC membrane, but it lacks several epitopes found on the complete RhD protein. If a person with the partial D phenotype encounters the complete D antigen on transfused RBCs, they may form anti-D and suffer from a transfusion reaction. Expression of Rh antigens The Rh antigens are expressed as part of a protein complex in the RBC membrane. This complex is only expressed in cells of the erythroid line, and therefore Rh antigens are only expressed in RBCs. The composition of the complex is unknown, but it is thought to be a tetramer, consisting of two molecules of Rh-associated glycoprotein (RhAG) and two molecules of Rh proteins. Function of Rh proteins The Rh antigens are thought to play a role in maintaining the integrity of the RBC membrane- RBCs which lack Rh antigens have an abnormal shape. Individuals with the rare Rhnull phenotype caused by the deletion of RHAG have RBCs that do not express any of the Rh antigens because they cannot be targeted to the RBC membrane. The absence of the Rh complex alters the RBC shape, increases its osmotic fragility, and shortens its lifespan, resulting in a hemolytic anemia that is usually mild in nature. These patients are at risk of adverse transfusion reactions because they may produce antibodies against several of the Rh antigens. Rh antigens may also be involved in the transport of ammonium across the RBC membrane. Interestingly, the first member of a family of water channels (aquaporins) and the first member of a family of urea transporters were both found in blood group proteins (the Colton blood group and Kidd blood group, respectively). Clinical significance of Rh antibodies The Rh antigens are highly immunogenic, and most of the Rh antibodies should be considered as potential causes of hemolytic transfusion reactions and HDN. Whereas most blood types are determined by red cell antigens that differ by one or two amino acids, the Rh blood group contains the D antigen which differs from the C/c and E/e antigens by 35 amino acids. This large difference in amino acids is the reason why the Rh antigens are potent at stimulating an immune response.4 The majority of antibodies formed against the Rh antigens are of the IgG type. They are capable of causing significant HTR and HDN. Rh antibodies rarely, if ever, bind complement, and therefore RBC destruction is mediated almost exclusively via macrophages in the spleen (extravascular hemolysis). There are a few examples of Rh alloantibodies that are naturally occurring and are of the IgM type, but they are in the minority. Transfusion reactions Anti-D, anti-C, anti-E, and anti-e have all been involved in hemolytic transfusion reactions; particularly delayed reactions.17 Routine blood typing for Rh D status in both blood donors and transfusion recipients has reduced the incidence of transfusion reactions caused by anti-D. But sensitization to other Rh antigens can be a problem in transfusion medicine, particularly in patients with sickle cell anemia (SCA). SCA is more common in Blacks, and the treatment of SCA involves blood transfusions. Blacks are also more likely to express variants of the Rh e antigen, and therefore produce anti-e, along with other Rh alloantibodies, this increases the difficulty in finding Rh-compatible blood donors. Hemolytic disease of the newborn Anti-D causes the most severe form of HDN and it used to be a major cause of fetal death. Since the introduction of anti-D immunoglobulin along with careful monitoring of at-risk pregnancies, the prevalence of HDN because of Rh D incompatibility has decreased dramatically. However, all cases cannot be prevented, and RhD alloimmunization remains a major cause of disease.18 Other Rh alloantibodies that are capable of causing severe HDN include anti-c19, 20 which clinically is the most important Rh antigen after the D antigen. Moderate disease can be caused by anti-Cw 21 and anti-Cx 22 . Rh alloantibodies that are typically associated with mild HDN include anti-C (relatively common) 23, anti-E24 and anti-e25. 6.3 OBJECTIVE OF THE STUDY: The objective of this study therefore, is to determine the prevalence of Rh phenotype and weak D (Du) among Rh-negative blood donors in St. John’s medical college hospital blood bank, Bangalore. • To study the prevalence of Rh phenotype • To evaluate the significance of Du testing DESIGN OF THE STUDY The study will include both prospective and retrospective data analysis All Rh negative tests would be tested further for weak D 7 MATERIALS AND METHODS 7.1 SOURCE OF DATA St. John’s medical college hospital blood bank, Bangalore 7.2 METHODS Retrospective data: Donors from January 2005 to December 2010. Prospective study: Blood samples of donors from January 2011 to December 2011 are tested for RhD phenotype and negative samples are further tested for Du or weak D testing. 8.0 References 1. Issitt, P.D. The Rh blood group system, 1988: eight new antigens in nine years and some observations on the biochemistry and genetics of the system. Transfuse Med Rev1989; 3: 1-12. 2. Issitt PD, Telen MJ. D, weak D (Du), and partial D: the molecular story unfolds [editorial]. Transfusion. 1996; 36:97–100. 3. Jones, J, Scott, M.L., Voak, D. Monoclonal anti-D specificity and RhD structure: criteria for selection of monoclonal anti-D reagents for routine typing of patients and donors. Transfuse Med.1995; 5: 171-184. 4. Westhoff CM. The Rh blood group system in review: a new face for the next decade. Transfusion 2004; 44:1663-73. 5. L, Petz, L.D. (1995). Erythrocyte antigens and antibodies. In: Beutler, E, Litchman, M.A, Coller, B.S. and Kipps, T.J.(Eds). 6. Potter EL. Rh: Its Relation to Congenital Hemolytic Disease and to Intragroup Transfusion Reactions. Chicago, Ill: The Year Book Publishers Inc; 1947: 8–11. 7. Domen RE. Discovery of the Rh red blood cell antigen. Arch Pathol Lab Med. 1986; 110:162– 164. 8. Wiener AS. The Rh series of allelic genes. Science. 1944;100:595–597. 9. Stratton F. A new Rh allelomorph. Nature. 1946;158:25–26. 10. Agre PC, Davies DM, Issitt PD, et al. A proposal to standardize terminology for weak D antigen. Transfusion. 1992;32:86–87. 11. Mollison PL, Engelfriet CP, Contreras M. Blood Transfusion in Clinical Medicine. 10th ed. Oxford, England: Blackwell Scientific Ltd; 1997:154–158, 415. 12. Moore BPL. Does knowledge of Du status serve a useful purpose? Vox Sang. 1984;46(suppl 1):95–97. 13. Snyder EL, Lipton KS. Prevention of Hemolytic Disease of the Newborn Due To Anti-D. Bethesda, Md: American Association of Blood Banks; February 16, 1998. Association Bulletin 98–2. 14. American College of Obstetricians and Gynecologists. Prevention of Rh D Alloimmunization. Washington, DC: The American College of Obstetricians and Gynecologists; May 1999. ACOG Practice Bulletin 4. 15. Reid ME and Lomas-Francis C. The Blood Group Antigen Facts Book. Second ed. 2004, New York: Elsevier Academic Press. 16. Avent ND, Reid ME. The Rh blood group system: a review. Blood 2000; 95:375-87. 17. Daniels GL. Human Blood Groups. 2nd ed. 2002: Blackwell Science. 18. Urbaniak SJ, Greiss MA. RhD haemolytic disease of the fetus and the newborn. Blood Rev 2000; 14:44-61. 19. Hackney DN, Knudtson EJ, Rossi KQ, Krugh D, O'Shaughnessy RW. Management of pregnancies complicated by anti-c isoimmunization. Obstet Gynecol 2004; 103:24-30. 20. Appelman Z, Lurie S, Juster A, Borenstein R. Severe hemolytic disease of the newborn due to anti-c. Int J Gynaecol Obstet 1990; 33:73-5. 21. Bowman JM, Pollock J. Maternal CW alloimmunization. Vox Sang 1993; 64:226-30. 22. Finney RD, Blue AM, Willoughby ML. Haemolytic disease of the newborn caused by the rare Rhesus antibody anti-CX. Vox Sang 1973; 25:39-42. 23. Bowman JM, Pollock JM, Manning FA, Harman CR. Severe anti-C hemolytic disease of the newborn. Am J Obstet Gynecol 1992; 166:1239-43. 24. Joy SD, Rossi KQ, Krugh D, O'Shaughnessy RW. Management of pregnancies complicated by anti-E alloimmunization. Obstet Gynecol 2005; 105:24-8. 25. Chapman J, Waters AH. Haemolytic disease of the newborn due to Rhesus anti-e antibody. Vox Sang 1981; 41:45-7. 09 SIGNATURE OF CANDIDATE 10 REMARKS OF THE GUIDE 11 NAME & DESIGNATION OF 11.1 GUIDE Dr. SITALAKSHMI S. DCP, DNB, PhD. PROFESSSOR, DEPT.CLINICAL PATHOLOGY. ST.JOHNS MEDICAL COLLEGE HOSPITAL. BANGALORE. 11.2 SIGNATURE OF THE GUIDE 11.3 HEAD OF DEPARTMENT Dr. KARUNA RAMESH KUMAR MD,DCP,PhD PROFESSSOR AND HEAD, DEPT.CLINICAL PATHOLOGY. ST.JOHNS MEDICAL COLLEGE HOSPITAL. BANGALORE. 11.4 REMARKS OF THE HOD 11.5 SIGNATURE OF THE HOD 11.6 SIGNATURE OF DEAN