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The Myth and Mystery of RhD

Christine Lomas-Francis MSc, FIBMS

Technical Director

Immunohematology and Genomics

New York Blood Center

Quotient Biodiagnostics Industry Workshop

October 24, 2011

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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

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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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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

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Rh positive

RHD RHCE

D antigen Cc and Ee antigens

RhD

5’ 3’ ce, Ce, cE, or CE 5’ 3’

Adapted from: Westhoff CM., Semin.Hematol. 2007;44:42-50

RHD and RHCE encode RhD and RhCE proteins

RhCE

RhD and RhCE differ by 32 to 35 amino acids

Genes

Proteins C/c Ser103Pro

E/e Pro226Ala

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Rh positive

RHD RHCE

D antigen Cc and Ee antigens

RhD

5’ 3’ ce, Ce, cE, or CE 5’ 3’

Adapted from: Westhoff CM., Semin.Hematol. 2007;44:42-50

RHD and RHCE encode RhD and RhCE proteins

RhCE

RhD and RhCE differ by 32 to 35 amino acids

Genes

Proteins C/c Ser103Pro

E/e Pro226Ala

D epitopes

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Rh positive

RHD RHCE

D antigen Cc and Ee antigens

ce Rh negative Deleted X X

RhCE

C/c Ser103Pro

E/e Pro226Ala

5’ 3’ ce, Ce, cE, or CE 5’ 3’

Adapted from: Westhoff CM., Semin.Hematol. 2007;44:42-50

5’ 3’

RhD: D Phenotype and Immunogenicity Genes

Protein

No RhD protein

All D epitopes missing

RHD gene deletion: most common in populations of European ancestry

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D Antigen Expression

• 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

RhD D is composed of many epitopes

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Partial D Phenotype: Qualitative Variant of D

RhD Changes predicted to be in

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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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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Weak D Phenotype: Quantitative Variant of D

•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%)

Changes predicted to be in

the transmembrane or

cytoplasmic regions

RhD

V270G

Type 1

G385A

Type 2

S3C

Type 3

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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

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•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

The Del (DEL) Phenotype

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D and D-like Epitopes Expressed on RhCE

Flegel et al. The RHCE allele ceCF: the molecular basis of Crawford (RH43). Transfusion, 2006; 46:1334-1342

1 2 3 4 5 6 7 8 9 10

W16C

RHD*DHARRh33+, FPTT+

Q233E L245V

RHce*CFVS+, Crawford+

•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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Partial D and Weak D: Comparison

All epitopes

present

Make anti-D Patient

considered

Location of

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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Case Study

Anti-D reagents

IgM + IgG

Reaction with

Patient RBCs

# 1 2+

# 2 3+

# 3 3+

# 4 2+

Results obtained in direct testing

Patient:

African American

Delivered her 3rd baby

Anti-D, 3+ by PEG IAT in her

plasma at delivery

Autocontrol negative

D typing results indicate her

RBCs are D+ with (slightly)

weakened expression

RBCs also C E c+ e+

DNA analysis predicts:

Presence of weak D type 4

Associated with 2 amino acid

changes: T201R and F223V

that are predicted to be in the

internal portion of RhD

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What is a True Weak D Phenotype?

All epitopes

present

Make anti-D Patient

considered

Location of

changes

Weak D Yes/no No/yes/don’t

know

D+?? Internal

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?

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

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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

S Africa

DV: 1 in 30,000

DFR: 1 in 60,000 DIIIa: 4 in 100 in

African Americans

Weak D type 15: 1

in 15,000

DHar: 1 in 60,000 DIV: 1 in 10,000

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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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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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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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FDA-licensed Anti-D: Reactions with Selected D variants

Anti-D IgM IgG DVI

IS/IAT

DBT

IS/IAT

DHAR

IS/IAT

Crawf

IS/IAT

ceRT ceSL

G-clone GAMA4

01

F8D8 neg/pos pos pos pos

IC Ser 4 MS201 MS26 neg/pos pos pos neg weak neg

IC Ser 5 Th28 MS26 neg/pos pos pos neg weak neg

O tube MAD2 Poly neg/pos neg/pos neg/neg neg

O gel MS201 neg pos pos neg weak neg

Biot. RH1 BS226 neg pos neg

Biot. RH1

blend

BS221 BS232

H41

11B7

neg/pos pos/neg neg

Quot. alpha LDM1 neg pos neg

Quot. Beta LDM3 neg pos neg

Quot. Delta LDM1

ESD-M

pos pos neg

Quot. blend LDM3 EDS1 neg/pos pos neg

Adapted from Chou & Westhoff; AABB Technical Manual; 17th ed, page 399

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Regulatory Aspects: Donors

AABB Standards, 27th ed

5.8.2 Determination of Rh Type

for All Collections

The Rh type shall be

determined for each

collection with anti-D

reagent. If the initial test with

anti-D is negative, the blood

shall be tested using a

method designed to detect

weak D.

When either test is positive,

the label shall read “Rh

POSITIVE”

UK BTS Guidelines for the Blood

Transfusion Services

• The D blood group must be determined

on each donation of blood.

• … for first time donors use two anti-D

blood grouping reagents, capable of

detecting between them DIV, DV and

DVI. If two monoclonal anti-Ds are used,

they should be from different clones.

• If the results … are discordant or

equivocal, the tests should be repeated.

Where the D group is in doubt it is safer

to classify such donors as D positive.

• For known (repeat) donors one anti-D

reagent, or blended reagent, that detects

weak D, DIV, DV and DVI can be used.

USA UK (Europe)

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Regulatory Aspects: Patients

AABB Standards, 27th ed.

5.13.2 Rh Type

The Rh type shall be

determined with anti-D

reagent. The test for weak

D is unnecessary when

testing the patient.

UK BTS Guidelines for the Blood

Transfusion Services

• 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.

• Reagents used for D grouping

patients should not detect

category DVI.

USA UK (Europe)

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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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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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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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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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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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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