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The Elusive D Antigen

The Elusive D Antigen. Rajendra Chaudhary , MD, DNB SGPGI, Lucknow. Rhesus System. The 2 nd most important after ABO Major cause of HDN T he most complex system, with over 45 antigens The complexity of the Rh blood group Ags is due to the highly polymorphic genes that encode them .

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The Elusive D Antigen

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  1. The Elusive D Antigen RajendraChaudhary, MD, DNB SGPGI, Lucknow

  2. Rhesus System • The 2nd most important after ABO • Major cause of HDN • The most complex system, with over 45 antigens • The complexity of the Rh blood group Ags is due to the highly polymorphic genes that encode them. • Multiple gene conversions & mutations • Discovered in 1940 after work on Rhesus monkeys

  3. Clinical Significance of D Antigen • D antigen, after A and B, is the most important RBC antigen in transfusion practice. • Individuals who lack D antigen DO NOT have anti-D. • Antibody produced through exposure to D antigen through transfusion or pregnancy. • Immunogenicity of D greater than that of all other RBC antigens studied. • 80%> of D neg individuals who receive single unit of D pos blood can be expected to develop immune anti-D. • Testing for D is routinely performed so D neg will be transfused with D neg.

  4. Rh Antigen Frequency

  5. Structure of RhD Gene

  6. Structure of Rh Antigen

  7. Rh Designations D positive 95% D Negative 5%

  8. Genetics of RhD Negative Phenotype • Molecular mechanism producing D negative phenotype differs in various ethnic population • Deletion: • RHD gene is deleted in majority of D negative Caucasians, 30% Japanese, 10-23% South Africans • Insertion: • In Africans, Pseudogene (37 bp insertion) major cause of D negative • Hybrid allele: • In African Americans, RHCE inserted in RHD results in no D antigen . Hybrid RHD-CE-D

  9. Rh D Negative - Deletion • Locus 1 deletion of RHD therefore, no D antigen. • Common in Caucasian population

  10. Rh D Negative - Insertion • Locus 1 – 37 bp insertion & several mutations in RHD results in no product • 66% of African Americans have RHDψ

  11. Rh D Negative – Hybrid RHD-CE-D • Locus 1 – RHCE inserted in RHD results in no D antigen • hybrid RHD-CE-D - common in Africans

  12. Weak D Expression

  13. Frequency of Weak D Expression

  14. Variants of D Antigen • Quantitative variants • Weak D (Genetically transmissible) • Position effect • Del variant • Qualitative variants • Partial D – missing one or more epitopes of D antigen • Partial Weak D – less number of D sites and missing epitopes

  15. Weak D, Partial D Normal D Partial D Weak D Partial Weak D DVI

  16. Quantitative D Variants Weak D (Genetically Transmissible) • RHD gene codes for weak expression of D antigen • D antigen is complete (all epitopes of D antigen are present), there are just fewer D Ag sites on RBC. • Normal D sites – 15,000 – 33,000 D sites/cell • Weak D – 70- 5200 D sites/cell RBC with normal amounts of D antigen Weak D (Du)

  17. Molecular Basis of Weak D

  18. D Antigen Copy Member

  19. Some Weak D Types

  20. Position Effect (Gene Interaction Effect) • C allele in trans position to D allele • Example : Dce/dCe , DcE/dCE • D antigen is normal , C antigen appears to be crowding the D antigen (steric hindrance) D c e / d C e C in trans position to D Weak D C in cis position to D D C e / d c e NOweak D

  21. Del Phenotype • Weakest D variants • Appears D negative at IS and Du test • Low D antigenic sites, only detectable by adsorption – elution and flowcytometry • Deletion of exon 9 in Asians • 16-30% of D negative in China, Japan, Korea are DEL phenotype • Reported in literature to make anti-D • 3 cases of Del in 500 D negative at SGPGI

  22. Serological Test for Del D negative red cells +Anti-D Incubate at 37 X 1 hr Perform Elution Test Eluate with D pos red cells If positive - Del

  23. Qualitative D Variant (Partial D) • The difference between A and B is a single epitope of the D antigen. • Patient B can make an antibody to donor A , even though both appear to have the entire D antigen present on their red blood cell’s A B • Multiple epitopes make up D antigen. • Each color represents a different epitope of the D antigen

  24. Epitopes in Different Partial D Categories

  25. Molecular Basis of Partial / Weak D Weak D Partial D • Partial D – characterized by AA changes in extracellular portions of D polypeptide • 60 known partial D variants • Weak D- characterized by single or few AA changes primarily in trans membrane or cytoplasmic part of D protein • 50 different mutations in weak D

  26. Anti-D Antisera • Monoclonal anti D • Antibody directed against a single epitope of the D antigen • Produced in vitro from a cell line (recombinant) expressing a particular immunoglobulin gene sequence • Several monoclonals may be “blended” • Polyclonal anti D • A group of anti D antibodies directed against a variety of epitopes on the protein; • naturally occurring following an immune response to D immunization.

  27. Requirements for Rh D Typing in India DGHS, DCGI, requirements for reliable Rh(D) typing: • Use two distinct anti–Rh(D) reagents of two different manufacturers or • Use of two distinct anti–Rh(D) reagents of two different batches of same manufacturer. • Blend of IgM and IgG monoclonal anti–D or • Blend of MAbIgM and polyclonal (human) IgG can be used for IAT to identify weak D antigen.

  28. When to Suspect D Variant The possibility of D variants must be considered • Weak reaction (< +2) with anti-D reagents • Significant discrepancy in the strength of reaction obtained with different anti-D reagents • Discrepancy between the current test and historical test result • If anti-D is detected in an individual who is serologically typed as RhD positive

  29. Interpretation of Aberrant Results

  30. Confusion Over Weak Expression of D

  31. Clinical Significance

  32. Reasons to Resolve Weak Expression • Conserve Rh-negative blood for D-negative recipients (high risk of making anti-D). • Avoid giving RhIG to women who do not need it (Rh status is confirmed for historical discrepancies) • Resolve early in pregnancy to eliminate false-positive KlauherBettke tests. • Today's blood donor can be recipient tomorrow

  33. Variable D Results • Perinatal results differ from hospital results • Previously positive; new reagent or method, now negative • Previously negative; new reagent or method, now positive • Doctors confused • Lab credibility suffers a blow

  34. Controversies Abound! • Should 1+ be considered positive or negative? • And the reaction strength is method specific • What about type of reagent used? • Should technical staff be expected to record or enter clear positive results as negative? • Will the LIS allow blood group interpretation if weak reactions are present and the interpretation doesn’t match?

  35. Clinical Considerations What is the risk of developing an anti D Should the patient be given RhIg What is the risk of HDN

  36. Variables Affecting D Typing Results • Rh antigen expression • RHD and RHCE gene mutations • Anti-D reagent • Monoclonal Vs polyclonal • Monoclonal IgM / IgG / blend • Testing platform • Slide / tube / gel / solid phase • Individual being Rh typed • Donor / Recipient / Cord blood / ANC

  37. Incidence of D Variants • Frequency of Du variants in Caucasians – 0.1- 1% • U.S (2010) 501 prenatal patients screened by 3 commercially available serologic method – discrepant results in 2.2% • Mezoka et al 2009 – D variant alleles in African – American blood donors – 35/400 (8.8%) • Central Europe – screening by molecular techniques – 5.23%

  38. We are not uninitiated • Kulkarni et al – Study from IIH • to identify D variants amongst antenatal women labeled as RhD negative • Of the 700 apparently Rh negative ANC, 24 (3.43%) were identified as D variants • One third (34%) of apparently Rh D negative women with positive ‘C’ antigen are D variants • Typing for the presence of ‘C’ antigen is helpful in identifying D variants in apparently D negative antenatal women

  39. D variants in RhD discrepant cases - IIH Study • Total 60 samples studied at IIH • 97% of D variants showed presence of “C”

  40. Strategy for Identification of D Variant in Indians

  41. Commonly Used D Testing Protocol Rh D Testing Blend of IgG +IgM > +2 Positive 0 - < +2 Weak D / Negative Incubate > +2 Positive 0 - < +2 Weak D / Negative IAT Positive Weak D Negative D negative

  42. Routine typing with 2 anti-D Genotype with C, c, E, e reagent Strategy in France ddCcee ddccEe Du test DwCcee DwccEe Molecular typing for weak D 1, 2, 3 Weak D 1, 2, 3 Other Weak D or Partial D Test with 3 IgM anti-D that do not detect DVI Positive D Pos as Donor & Patient Negative D Pos as Donor & D neg as Patient

  43. D typing strategy in Germany for recipients Recipient’s RBC + limited specificity anti-D reagent Perform immediate spin 0 - < 2+ agg Strong agg > 2+ Extended Incubation Recipient D positive Should receive D pos Blood/ no need of RhIg prophylaxis Strong agg >2+ 0 - <2+ agg no Is genetic evaluation of RHD gene accessible Recipient as D negative Rh prophylaxis required yes Assignment of individual D type Depends on the underlying RHD allele

  44. The aim of the study was to screen Indian population for detection of partial D by serology and classify them by multiplexPCR. • 10 000 RhD-positive individuals from West India • 15 cases of partial D detected (0.15%) • DFR was the commonest type of partial D

  45. The aim of this study was to estimate D antigen on RBC in weak D and partial D variants in Indian population by using flow cytometry. • 42 cases of partial D, 8cases of weak D and 123 normal Rh phenotypes were used in the study.

  46. Problems encountered in recognizing D variants • Partial D individuals may type as D pos or D negative with an anti-D reagent depending on the epitopes against which it has been raised • Monoclonal anti-D may give strong positive reaction with weak D phenotypes without performing IAT • Different commercial monoclonal anti-D of different manufacturer show variation in reactivity with weak D • Difference in reactivity with method used for RhD typing using same commercial monoclonal anti-D • At Blood bank it is difficult to differentiate between partial D and weak D

  47. Rh D Typing Strategy & Selection of Anti-D Reagents

  48. RhD Typing Strategy Used In Western Countries Blood donors and cord blood • Use broad specificity anti-D reagents (mix of IgM and IgGoligoclonal anti-D). • Weak D test must on blood donors and on cord blood samples. • RHD genotyping to identify D variants in individuals who appear D negative using the weak D test. Recipients and pregnant women: • Use limited specificity anti-D reagent (contains a single IgM monoclonal anti-D). • Do not perform the weak D test • If negative or weak at ISphase, incubate at 37 C • RHD genotyping to identify D variants in individuals who demonstrate weak agglutination at IS phase of testing.

  49. Transfusion 2008: 48: 473 Samples that were positive by automated Gel technology but negative by test tube were studied by multiplex PCR for RhD variants To limit anti-D alloimmunization, it is recommended that samples with immediate-spin tube test score of not more than 5 (i.e., 1+ agglutination) or a score of not more than 8 (i.e., 2+ hemagglutination) by gel technology be considered D– for transfusion and Rh Igprophylaxis.

  50. We are not uninitiated Conclusions from IIH studies • Anti-D obtained from Cell lines LHM 70/45, • negative with most discrepant samples • useful for patient typing • Anti-D obtained from LHM 76/59, 76/55, 77/ 64 • positive with most discrepant samples • useful for donor typing

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