Session 1 - Clinical Significance of RhD

The Myth and Mystery of RhDQuotient Biodiagnostics Industry Workshop October 24, 2011 Christine Lomas-Francis MSc, FIBMS Technical Director Immunohematology and Genomics New York Blood Center 1

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

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 3

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

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 antigensProteins C/c E/e Ser103Pro Pro226AlaRhD RhCE RhD and RhCE differ by 32 to 35 amino acidsAdapted from: Westhoff CM., Semin.Hematol. 2007;44:42-50 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 D epitopesProteins C/c E/e Ser103Pro Pro226AlaRhD RhCE RhD and RhCE differ by 32 to 35 amino acidsAdapted from: Westhoff CM., Semin.Hematol. 2007;44:42-50 6

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 RhCERHD gene deletion: most common in populations of European ancestryAdapted from: Westhoff CM., Semin.Hematol. 2007;44:42-50 7

D Antigen ExpressionRhD 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 8

Partial D Phenotype: Qualitative Variant of D Changes predicted to be inRhD the external loops of RhD•Discovered as some D+ people made alloanti-D or becauseRBCs reacted with some but not all anti-D•Most partial D due to hybrid genes: parts of RHD replaced byparts 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 9

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…. 10

Weak D Phenotype: Quantitative Variant of D G385A Type 2RhD Changes predicted to be in the transmembrane orS3CType 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%) 11

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

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

D and D-like Epitopes Expressed on RhCE 1 2 3 4 5 6 7 8 9 10 RHce*CF VS+, Crawford+ W16C Q233E L245VFlegel 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 fortyping, transfused D+ RBCs and made anti-DPatient 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 Europebecause of cell lines in reagents 14

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+ InternalIn a clinical setting all we need to do is to determine ifthe patient is D+, or has a partial or weak D phenotype!We’ll come to donors later………….. 15

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+ 16

What is a True Weak D Phenotype?Comment added to patient report:“This patient has a RHD allele first reported to encode a weak Dbut now known to encode a partial D phenotype associated withthe 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? 17

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 18

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 19

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 20

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 21

FDA-licensed Anti-D: Reactions with Selected D variants Anti-D IgM IgG DVI DBT DHAR Crawf ceRT ceSL IS/IAT IS/IAT IS/IAT IS/IAT G-clone GAMA4 F8D8 neg/pos pos pos pos 01 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 BS221 BS232 neg/pos pos/neg neg blend H41 11B7 Quot. alpha LDM1 neg pos neg Quot. Beta LDM3 neg pos neg Quot. Delta LDM1 pos pos neg ESD-M Quot. blend LDM3 EDS1 neg/pos pos negAdapted from Chou & Westhoff; AABB Technical Manual; 17th ed, page 399 22

Regulatory Aspects: Donors USA UK (Europe) UK BTS Guidelines for the BloodAABB Standards, 27th ed Transfusion Services5.8.2 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. 23

Regulatory Aspects: PatientsUSA UK (Europe) UK BTS Guidelines for the BloodAABB Standards, 27th ed. Transfusion Services5.13.2 Rh Type • Patients should not be classifiedThe 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. 24

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” 25

Defining Weak D and Partial D statusIs 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 26

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) 27

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 28

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” 29

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 30

Session 1 - Clinical Significance of RhD

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Session 1 - Clinical Significance of RhD — Presentation Transcript