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Session 1 - Clinical Significance of RhD
1. The Myth and Mystery of RhD
Quotient Biodiagnostics Industry Workshop
October 24, 2011
Christine Lomas-Francis MSc, FIBMS
Technical Director
Immunohematology and Genomics
New York Blood Center
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2. The Importance of RhD Typing
• The RhD antigen is the most
immunogenic of the Rh antigens
• Second only to ABO in clinical
significance
• Determine the RhD type of patients and
donors to prevent sensitization to RhD
and thus transfusion reactions and
hemolytic disease of the fetus and
newborn (HDFN) due to anti-D
2
3. Establishing the “correct” D Type
• Fundamental to safe transfusion practice
• Potent monoclonal anti-D are used and yet………….
• Interpretation of the D type of some patients and
donors is a challenge because some people have:
– qualitative variation in D antigen expression, referred
to as partial D
– quantitative reduction in D antigen expression,
referred to as weak D
• Careful reagent selection, an understanding of the
reagent characteristics and of the nature of the D
antigen is valuable when interpreting D typing
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4. Objectives
• Discuss D antigen expression
• Review weak and partial D phenotypes
• Review the regulatory requirements and
reagent use when typing patients and
donors for D
• Explain the clinical relevance of
distinguishing between weak and partial
D phenotypes in patient and donor
testing
4
5. RHD and RHCE encode RhD and RhCE proteins
Genes RHCE
RHD
Rh positive 5’ 3’ 3’ ce, Ce, cE, or CE 5’
D antigen Cc and Ee antigens
Proteins C/c E/e
Ser103Pro Pro226Ala
RhD RhCE
RhD and RhCE differ by 32 to 35 amino acids
Adapted from: Westhoff CM., Semin.Hematol. 2007;44:42-50
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6. RHD and RHCE encode RhD and RhCE proteins
Genes RHCE
RHD
Rh positive 5’ 3’ 3’ ce, Ce, cE, or CE 5’
D antigen Cc and Ee antigens
D epitopes
Proteins
C/c E/e
Ser103Pro Pro226Ala
RhD RhCE
RhD and RhCE differ by 32 to 35 amino acids
Adapted from: Westhoff CM., Semin.Hematol. 2007;44:42-50
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7. RhD: D Phenotype and Immunogenicity
Genes RHCE
RHD
Rh positive 5’ 3’ 3’ ce, Ce, cE, or CE 5’
D antigen Cc and Ee antigens
X Deleted X
Rh negative 3’ ce 5’
Protein C/c E/e
Ser103Pro Pro226Ala
No RhD protein
All D epitopes missing RhCE
RHD gene deletion: most common in populations of European ancestry
Adapted from: Westhoff CM., Semin.Hematol. 2007;44:42-50
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8. D Antigen Expression
RhD D is composed of many epitopes
• Continuum of strength of expression
• “Conventional” D+ (expresses all D epitopes)
• ~ 200 different RHD alleles encode proteins with amino acid
changes that cause variation in antigen expression
• Partial D (D categories, D mosaics); more prevalent in Blacks
• Weak D (formerly DU); 0.2 to 1% of Whites; prevalence can
depend on anti-D reagent
• Del (DEL); lowest antigen density
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9. Partial D Phenotype: Qualitative Variant of D
Changes predicted to be in
RhD the external loops of RhD
•Discovered as some D+ people made alloanti-D or because
RBCs reacted with some but not all anti-D
•Most partial D due to hybrid genes: parts of RHD replaced by
parts of RHCE; some are due to single nucleotide changes
•RhD protein with missing D epitopes
•RBCs may type as D-positive, but reagent dependant
•Alloanti-D can be made against missing epitopes
•Some partial D express novel low prevalence antigens: eg.
Goa on DIVa; DW on DVa; Tar on DVII
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10. Partial D Phenotypes
• Originally serologically divided into
categories DII to DVII (based on reaction
with anti-D made by D+ people)
• Later by use of monoclonal anti-D
• Further sub-division of categories by
molecular studies: e.g. 6 types of DIV and 4
types of DVI
• ~ 80 alleles that encode partial D; not all can
be serologically distinguished
• Usually given names; often 3 or 4 letters
such as DBT, DAR, DNB, DHAR….
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11. Weak D Phenotype: Quantitative Variant of D
G385A
Type 2
RhD Changes predicted to be in
the transmembrane or
S3C
Type 3 V270G
cytoplasmic regions
Type 1
•Reduced amount of D
•All D epitopes present but weakly expressed
•May require indirect antiglobulin test (IAT) for detection
•Not (usually) associated with alloanti-D production
•Now 80+ different weak D types (Types -1,-2,-3 = ~ 90%)
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12. Weak D Phenotypes
• Weak D phenotypes given numbers
– Weak D type 1, type 2, type 3……up to 76 with
some sub-types (4.1, 4.2 etc)
• Weak D classification is tricky
– Can be reagent and method dependent
– Sample can be 2+ in tube at IS, stronger in gel and
negative in solid phase
– Very ‘fluid’ statistics for prevalence of weak D
based on serology
• Weak D types usually cannot be distinguished
serologically; requires DNA analysis
12
13. The Del (DEL) Phenotype
•RBCs type as D negative (including at IAT)
•RBCs express very low level of D antigen (20
antigen sites/RBC); reduced amount of RhD
protein in membrane
•Detected only by adsorption and elution
•Del most often found in Asian populations (10
to 30% of D– Asians; 0.027% in European D– )
•Most Del RBCs express C, a few express E
•More than 20 molecular bases
13
14. D and D-like Epitopes Expressed on RhCE
1 2 3 4 5 6 7 8 9 10
RHce*CF
VS+, Crawford+
W16C Q233E L245V
Flegel et al. The RHCE allele ceCF: the molecular basis of Crawford (RH43). Transfusion, 2006; 46:1334-1342
RHD*DHAR
Rh33+, FPTT+
•Several Rhce proteins have a few D-specific amino acids
•Yet they react (strongly) with some anti-D reagents
•Patient typed D at one hospital, D+ at another, different reagents used for
typing, transfused D+ RBCs and made anti-D
Patient returned as a donor; caused donor D typing discrepancy
•DHar found in people with German ancestry
•Crawford phenotype found in people with African ancestry
•Also ceRT and ceSL variants; more likely to be an issue in Europe
because of cell lines in reagents
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15. Partial D and Weak D: Comparison
All epitopes Make anti-D Patient Location of
present considered changes
Partial D No Yes D– External
Weak D Yes No D+ Internal
In a clinical setting all we need to do is to determine if
the patient is D+, or has a partial or weak D phenotype!
We’ll come to donors later…………..
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16. Case Study
Patient: D typing results indicate her
African American RBCs are D+ with (slightly)
Delivered her 3rd baby weakened expression
Anti-D, 3+ by PEG IAT in her RBCs also C E c+ e+
plasma at delivery
Autocontrol negative
Results obtained in direct testing
Anti-D reagents Reaction with DNA analysis predicts:
IgM + IgG Patient RBCs Presence of weak D type 4
#1 2+ Associated with 2 amino acid
#2 3+
changes: T201R and F223V
that are predicted to be in the
#3 3+ internal portion of RhD
#4 2+
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17. What is a True Weak D Phenotype?
Comment added to patient report:
“This patient has a RHD allele first reported to encode a weak D
but now known to encode a partial D phenotype associated with
the production of alloanti-D.”
This should be considered a weak partial D phenotype
All epitopes Make anti-D Patient Location of
present considered changes
Weak D Yes/no No/yes/don’t D+?? Internal
know
In real life it’s a different story!
Some weak D types do make alloanti-D, e.g.:
Weak D type 4.0, 4.2, 11, 15, 33
Yet changes in the RhD protein appear to be internal
Does the terminology add to our confusion?
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18. Prevalence of Phenotypes with Altered D
• Limited statistics; more studies in last few years at DNA level
• Overall ~ 2% of people express altered D
• ~ 1% of Europeans express a weak D phenotype
• DVI most “common” partial D in Caucasian populations
– 0.02% to 0.05% in Caucasians (~ 0.02% in Germany; 0.04% in UK;
2.9% in Palestinians)
• DNB also “common”; highest in Swiss (1 in 292)
• Partial D phenotype more “common” in populations of African
ancestry, especially DIII and DAR
DVII: 1 in 900 DAR: 5 in 100 in DV: 1 in 30,000
S Africa
DFR: 1 in 60,000 DIIIa: 4 in 100 in Weak D type 15: 1
African Americans in 15,000
DHar: 1 in 60,000 DIV: 1 in 10,000
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19. RhD Variants in Multiethnic Prenatal Population
• Recent study** from Boston University Medical Center
• Screened 501 patients with 4 anti-D (2 in tubes, 2 in
solid phase) and referred discrepant results for DNA
analysis
• 11 discrepancies (2.2%)
– One tube reagent reacted with all 11 samples (1+ to 3+)
– Another tube reagent reacted with 7 of 11 samples (1+ to 2+)
– Solid phase: 4 of 11 reacted with one reagent (1+ to 4+); 2 of
11 reacted with another reagent (3+)
• DNA analysis found: weak D type 4 (n=4); weak D type
3 (n=1); DAR (n=3); DV (n=2); unknown (1)
**Wand D, et al. Am J Clin Pathol; 2010: 134: 438-442
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20. Monoclonal Anti-D Reagents: Background
• Potent and specific; because they are monoclonal each clone
recognizes a single D epitope
• Antibody to single epitope does not react with all partial (and weak) D
therefore “blended” reagents:
– Blend of monoclonal (IgM) and polyclonal (IgG) antibodies
– Blend of two or more monoclonal antibodies, each from a different cell line:
IgG or IgM, or a combination of IgG + IgM
– Limited number of stable IgM-secreting cell lines available
• Clones for anti-D reagents selected based on:
– Detection, or not, of partial DVI, most prevalent partial D in Caucasians
– DVI strategy: D-positive as donors; D-negative as recipients/RhIG candidates
– -IgM antibody does not react with DVI – (Initial Spin=negative)
– -IgG antibody reacts with DVI in weak D test = positive
• Similar criteria in USA and Europe
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21. D Typing and Result Interpretation can be Problematic!
Different anti-D reagents:
– Contain different clones
– Can react differently with weak or partial D
phenotypes
– FDA: only reactivity with DIV, DVa, & DVI need be
specified
• Multiple methods:
– Hospitals: tube tests, gel, solid phase, may or may
not proceed to AHG test for weak D
– Donor centers: automated analyzers, tube tests
• Variability in expression of D
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23. Regulatory Aspects: Donors
USA UK (Europe)
UK BTS Guidelines for the Blood
AABB Standards, 27th ed
Transfusion Services
5.8.2 Determination of Rh Type
for All Collections • The D blood group must be determined
on each donation of blood.
The Rh type shall be
determined for each • … for first time donors use two anti-D
collection with anti-D blood grouping reagents, capable of
reagent. If the initial test with detecting between them DIV, DV and
anti-D is negative, the blood DVI. If two monoclonal anti-Ds are used,
shall be tested using a they should be from different clones.
method designed to detect • If the results … are discordant or
weak D. equivocal, the tests should be repeated.
When either test is positive, Where the D group is in doubt it is safer
the label shall read “Rh to classify such donors as D positive.
POSITIVE” • For known (repeat) donors one anti-D
reagent, or blended reagent, that detects
weak D, DIV, DV and DVI can be used.
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24. Regulatory Aspects: Patients
USA UK (Europe)
UK BTS Guidelines for the Blood
AABB Standards, 27th ed. Transfusion Services
5.13.2 Rh Type • Patients should not be classified
The Rh type shall be as D positive on the basis of a
determined with anti-D weak reaction with a single anti-
reagent. The test for weak D reagent. If clear positive
D is unnecessary when results are not obtained with
testing the patient. two monoclonal anti-D reagents
it is safer to classify the patient
as D negative.
• Reagents used for D grouping
patients should not detect
category DVI.
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25. UK Guidelines for Patient Testing
More detailed than those in the USA
Weak D and Partial D
• …” reagents vary widely in their ability to detect both partial D
and weak D”
• …” when two different reagents are used it is helpful to use
those of a similar reactivity with partial D and weak D red cells,
to reduce the number of discrepancies”
• …” if a discrepancy occurs the patient should be treated as D
negative until the D status is resolved”
• ….”patients should not be classified as D positive on the basis
of a weak reaction with a single anti-D reagent. If clear positive
results are not obtained with two monoclonal anti-D reagents
it is safer to classify the patient as D negative”
• …”It is useful when investigating patients with suspected
weak D or partial D to test the patients' cells against an
identification kit containing monoclonal antibodies directed
against the different epitopes of the D antigen”
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26. Defining Weak D and Partial D status
Is it clinically useful?
• Patient setting:
– Carriers of partial D and some “weak D” phenotypes can be
immunized to make anti-D by transfusion and pregnancy;
detect those at risk and make informed decision
– Avoid transfusion of D+ blood and provide Rh immune
globulin
– Ideal method for identification? Requires special reagents
(monoclonal anti-D kits) and/or DNA analysis to do so
– Carriers of true weak D phenotypes cannot be immunized to
make anti-D
• D+ blood can be transfused
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27. Give RhIG to Women with Weak or Discrepant D types?
• No definitive answer; range of expert opinions
• Flegel et al: if reactions with anti-D at immediate
spin are less then 2+ consider as D– and give RhIG;
if DNA analysis performed, weak D type 1, 2, 3, 4.0,
4.1 do not need RhIG
• Noizat-Pirenne et al: weak D type 1, 2, 3 do not need
RhIG; but beware of weak D in Dce haplotype as
this is often a partial D
• Excellent summary of current dilemma in:
Questions & Answers; AABB News (April 2011) Vol
13 # 4: page 6 (Glenn Ramsey, MD)
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28. Strategies for D testing
Donors
• Goal: label donor RBCs with any amount of
D as “Rh positive”
• Potential Problem: Weak D; some are
missed; even with IAT testing :
– those with low antigen expression (type 2, 5, 9,
10,12,15,17,18)
– Del; all are typed as D negative (prevalent in
Asians)
• Less immunogenic, but appear to be able to
stimulate anti-D in D– patients; weak D types
1, 2, 26, Del , have stimulated anti-D
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29. Strategies for D testing (cont’d)
Donor:
• Select reagents to detect as many D
variants as possible
• Test for weak D
• Understand the differences in the
reagents and know how to manage
“conflicts”
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30. Important!
• Be aware of the ethnicity of the patient and
donor population being tested
• Prevalence of the various partial and weak D
phenotypes is not the same in all ethnic
groups
• Be familiar with the reaction profile of the
anti-D clones used in a particular reagent
• Be aware that formulation of a reagent can
affect the reactivity of a monoclonal anti-D
• Accept that a small number of samples will
be challenging to classify
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