Rajendra Chaudhary, MD, DNB SGPGI, Lucknow The Elusive D Antigen Rajendra Chaudhary, MD, DNB SGPGI, Lucknow

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 It received its name in 1940, when Landsteiner and Wiener immunized rabbits with red blood cells from rhesus monkeys and found that the rabbit antirhesus antibody agglutinated approximately 85 percent of human red blood cells tested. They gave the name "Rh" to this determinant present on all rhesus monkey cells and apparently present on 85 percent of human red blood cells.

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.

Rh Antigen Frequency Antigen Caucasians Indians D 85 95 d 15 5 C 70 c 80 E 30 e 98

Structure of Rh D Gene

Structure of Rh Antigen Rh antigen in the red cell membrane. The Rh proteins form twelve transmembrane helices and six extracellular loops. Only extracellular loops 3, 4, and 6 contain amino acids that differ between C and D. Amino acid positions differing between RhD and the ce allele of RhCE are dispersed throughout the protein (whaite circles). Three amino acid positions do not differ between the C allele of RhCE and RhD (yellow circles).

Rh Designations D positive 95% D Negative 5%

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

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

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

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

Weak D Expression

Frequency of Weak D Expression Country Year Frequency Scotland 1967 0.5% France 1974 0.6 USA 2004 0.4 Germany 2006 India 2011 0.9 SGPGI data 2009 0.5

Variants of D Antigen Quantitative variants Qualitative 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

Weak D, Partial D Normal D Partial D Weak D Partial Weak D DVI The circles represent the RBC membrane, the rectangles represent an RhD antigen and the numbers above each RhD Ag are representation of different D epitopes on the Ag. The D epitopes are arbitrarily numbered 1, 2, and 3. In this example, 8 D antigens on the RBC surface are schematically shown as normal, and each D antigen has 3 D epitopes. In reality, the number of D antigens ranges from 10 000 to 25 000 and more than 30 D epitopes are expressed on the D antigen. The weak D RBC features D antigens with the full complement of D epitopes, but the number of D antigens is reduced compared to normal. The partial D RBC demonstrates the normal number of RhD antigens but each Ag is lacking at least 1 D epitope. The partial D type DVI demonstrates both weak D and partial D features. DVI

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)

Molecular Basis of Weak D This figure depicts missense mutations in the RHD gene associated with weak D phenotypes. The locations of these mutations are depicted as checkered ovals; the D-specific amino acids are shown as open ovals. Most of the mutations are located within transmembrane spans (gray) and cytoplasmic regions. Regions of conserved Rh protein family sequence are indicated as black rectangles.

D Antigen Copy Member

Some Weak D Types

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 NO weak D

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

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

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

Epitopes in Different Partial D Categories Epitopes present Epitopes absent II 1, 2, 3, 5, 6 / 7, 8 4, 9 III 1, 2, 3, 4, 5, 6 / 7, 8, 9 Must be others missing IVa 4, 5, 6 / 7, 8 1, 2, 3, 9 IVb 5, 6 / 7, 8 1, 2, 3, 4, 9 Va 2, 3, 4, 6 / 7, 8, 9 1, 5 VI 3, 4, 9 1, 2, 5, 6 / 7, 8 VII 1, 2, 3, 4, 5, 6 / 7, 9 8 DFR 1, 3, 4, 9 2, 5, 6 / 7, 8 DBT 6 /7, 8

Molecular Basis of Partial / 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

Anti-D Antisera Monoclonal anti D Polyclonal 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.

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 Blend of MAb IgM and polyclonal (human) IgG can be used for IAT to identify weak D antigen.

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

Interpretation of Aberrant Results Immediate Spin IAT Interpretation Anti-D Rh Control Blood Donor -- D Negative + D Positive (weak / partial) WK+ Blood Recipient D Negative (weak / partial)

Confusion Over Weak Expression of D Individual Rh status Donor Rh + Recipient Rh - Prenatal RhIg? Newborn Postpartum RhIg? Autologous donor @#!&%*~?

Clinical Significance D phenotype Changes in AA D antigen express Test to detect D Recipient Donor Can make anti-D Component Transfusion RhIg Can produce anti-D in D neg D pos None Normal IS No D pos / D neg Yes Partial D Extra cellular Altered IS + IAT D neg Partial Weak D Unlikely Weak D Transmemb / cytoplasm Normal but weak IAT No? D Neg RhD absent Absent

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 Klauher Bettke tests. Today's blood donor can be recipient tomorrow

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

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? 34

Clinical Considerations What is the risk of developing an anti D Should the patient be given RhIg What is the risk of HDN Classically, 80%; more recently 32% 35

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

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% Incidence varies with different serological & molecular techniques used & the population studied

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

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

Strategy for Identification of D Variant in Indians Rh discrepancy Test for “C” antigen If “C” positive, test for D antigen using cell line LHM 70/45 Negative (D Variant) Further characterization using panel of epitope specific monoclonal antisera and molecular study

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

Strategy in France 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

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 Limited specificity means not able to detect DVI or common D variants 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

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

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, 8 cases of weak D and 123 normal Rh phenotypes were used in the study.

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

Rh D Typing Strategy & Selection of Anti-D Reagents Subjects D variant RhD status Anti-D reagents Blood donors Cord blood Husband of Rh neg women Partial D D pos Identification of weak D antigen important Broad spectrum anti-D reagent which is a blend of many clinically significant epitopes ANC Recipients of blood D neg Common D variants are non reactive by IS and reported as negative Anti- D reagent with limited specificity

RhD Typing Strategy Used In Western Countries 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 IS phase, incubate at 37 C RHD genotyping to identify D variants in individuals who demonstrate weak agglutination at IS phase of testing. Blood donors and cord blood Use broad specificity anti-D reagents (mix of IgM and IgG oligoclonal 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.

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 Ig prophylaxis.

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

Does knowledge of partial D and weak D status serve a clinically useful purpose? Carriers of most partial D and some weak D types can be anti-D immunized D typing should avoid their being transfused with Rh positive blood Carriers of most weak D types cannot be anti-D immunized transfuse with Rh positive blood avoid common practice of wasting Rh neg. blood. Superior sensitivity uncover many weak D in the “Rh negative“ donor pool

Tying ourselves in knots!!!