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

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 Antigen Caucasians Indians D 85 95 d 15 5 C 70 c80 E 30 e 98

5. Structure of Rh D Gene

6. 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).

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, % 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
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

14. 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

15. 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 to 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

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 – D sites/cell
RBC with normal amounts of D antigen
Weak D (Du)

17. 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.

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
NO weak 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
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

25. 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

26. 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.

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

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

31. 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

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 Klauher Bettke 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? 34

35. 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

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 – %
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

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
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

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. 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

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
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

44. The aim of the study was to screen Indian population for detection of partial D by serology and classify them by multiplex PCR.
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, 8 cases 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
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

48. 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.

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 Ig prophylaxis.

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

51. 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

52. Tying ourselves in knots!!!