the elusive d antigen rajendra chaudhary, md, dnb sgpgi, lucknow
TRANSCRIPT
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The Elusive D Antigen
Rajendra Chaudhary, MD, DNB
SGPGI, Lucknow
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• 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
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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.
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Antigen Caucasians Indians
D 85 95
d 15 5
C 70 70
c 80 85
E 30 15
e 98 98
Rh Antigen Frequency
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Structure of Rh D Gene
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Structure of Rh Antigen
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Rh Designations
D positive 95%
D Negative 5%
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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
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Rh D Negative - Deletion
• Locus 1 deletion of RHD therefore, no D antigen.• Common in Caucasian population
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Rh D Negative - Insertion
• Locus 1 – 37 bp insertion & several mutations in RHD results in no product
• 66% of African Americans have RHDψ
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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
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Weak D Expression
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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
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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
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Weak D, Partial D
Normal D Partial D
Weak D Partial Weak D
DVI
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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)
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Molecular Basis of Weak D
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D Antigen Copy Member
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Some Weak D Types
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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
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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
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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
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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
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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
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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
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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.
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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.
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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
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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)
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Confusion Over Weak Expression of D
Individual Rh status
Donor Rh +
Recipient Rh -
Prenatal RhIg?
Newborn Postpartum RhIg?
Autologous
donor
@#!&%*~?
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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
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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
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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
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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?
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Clinical Considerations
What is the risk of developing an anti D
Should the patient be given RhIg
What is the risk of HDN
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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
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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%
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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
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• 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
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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
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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
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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
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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
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• 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
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• 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.
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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
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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
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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
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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
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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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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
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Tying ourselves in knots!!!