Download 1 NAME OF THE CANDIDATE JASIM VP ADDRESS DEPARTMENT

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