The Elusive D Antigen

Rajendra Chaudhary, MD, DNB

SGPGI, Lucknow

• 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

Rhesus System

Clinical Significance of D Antigen D antigen, after A and B, is the most important RBC

antigen in transfusion practice.o Individuals who lack D antigen DO NOT have anti-D.o Antibody produced through exposure to D antigen through

transfusion or pregnancy.o 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.

Antigen Caucasians Indians

D 85 95

d 15 5

C 70 70

c 80 85

E 30 15

e 98 98

Rh Antigen Frequency

Structure of Rh D Gene

Structure of Rh Antigen

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 0.4

India 2011 0.9

SGPGI data 2009 0.5

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

Weak D, Partial D

Normal D Partial D

Weak D Partial Weak D

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

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

D C e / d c e

Weak D

NO weak D

C in trans position to D

C in cis position to 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

Partial D 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 1, 2, 3, 4, 5, 6 / 7, 9

Molecular Basis of Partial / Weak 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

Weak D Partial D

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.

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 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 ResultsImmediate Spin IAT Interpretation

Anti-D Rh Control Anti-D Rh Control

Blood Donor

-- -- -- -- D Negative

-- -- + -- D Positive (weak / partial)

WK+ -- + -- D Positive (weak / partial)

Blood Recipient

-- -- -- -- D Negative

-- -- + -- D Negative (weak / partial)

WK+ -- + -- 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 SignificanceD

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

No Yes

Partial D Extra cellular

Altered IS + IAT Yes D neg Yes Yes

Partial Weak D

Extra cellular

Altered IS + IAT Yes D neg Yes Unlikely

Weak D Transmemb / cytoplasm

Normal but weak

IAT No? D neg No? Unlikely

D Neg RhD absent

Absent IAT Yes D neg Yes No

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?

Clinical Considerations

What is the risk of developing an anti D

Should the patient be given RhIg

What is the risk of HDN

Variables Affecting D Typing Results

Rh antigen expressiono RHD and RHCE gene mutations

Anti-D reagentoMonoclonal Vs polyclonaloMonoclonal IgM / IgG / blend

Testing platformo Slide / tube / gel / solid phase

Individual being Rh typedo 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%

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

• Total 60 samples studied at IIH

• 97% of D variants showed presence of “C”

DFR37%

DOL23%

DAR5%

DCS3%

DVI3%

DV5%

DMH5%

weak D12%

not classified7%

D variants in RhD discrepant cases - IIH Study

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 TestingBlend of IgG +IgM

> +2Positive

0 - < +2Weak D / Negative

> +2Positive

0 - < +2Weak D / Negative

PositiveWeak D

NegativeD negative

Incubate

IAT

Routine typing with 2 anti-D

Genotype with C, c, E, e reagent

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

PositiveD Pos as Donor

& Patient

NegativeD Pos as Donor &D neg as Patient

Strategy in

France

D typing strategy in Germany for recipientsRecipient’s RBC + limited specificity anti-D reagent Perform immediate spin

Recipient D positiveShould receive D posBlood/ no need of RhIgprophylaxis

Extended Incubation

0 - < 2+ aggStrong agg > 2+

Strong agg >2+ 0 - <2+ agg

Is genetic evaluation of RHD gene accessible

Recipient as D negativeRh prophylaxis required

no

Assignment of individual D typeDepends on the underlyingRHD allele

yes

• 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

Partial D D neg • Common D variants are non reactive by IS and reported as negative

• Anti- D reagent with limited specificity

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.

RhD Typing Strategy Used In Western Countries

Transfusion 2008: 48: 473

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.

Samples that were positive by automated Gel technology but negative by test tube were studied by multiplex PCR for RhD variants

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 statusserve a clinically useful purpose?

Carriers of most partial D and some weak D types can be anti-D immunizedo D typing should avoid their being transfused with Rh

positive blood Carriers of most weak D types cannot be anti-D

immunizedo transfuse with Rh positive bloodo avoid common practice of wasting Rh neg. blood.

Superior sensitivityo uncover many weak D in the “Rh negative“ donor pool

Tying ourselves in knots!!!