Rh Molecular Testing

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October 31 HLA: Transfusion and Transplant

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Continuing Education

• PACE, Florida and California DHS• 1.0 Contact Hours• Each attendee must register to receive CE at:

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• Registration deadline is October 13, 2017

• Certificates will be sent via email only to those who have registered by October 27, 2017

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• Course content is for information and illustration purposes only. Immucor makes no representation or warranties about the accuracy or reliability of the information presented, and this information is not to be used for clinical or maintenance evaluations.

• The opinions contained in this presentation are those of the presenter and do not necessarily reflect those of Immucor.

RH Molecular TestingAn Immucor Webinar

JOANN MOULDS PHD, MT(ASCP)SBBIMMUNOHEMATOLOGY CONSULTANT

*Levine P, Stetson RE. An Unusual Case of Intra-Group Agglutination. JAMA 1939;113(2):126-27. 1

• 25 y.o. group O woman delivered a stillborn fetus* • Transfused with 500 ml of blood from her husband• Hemolytic reaction: chills, pain in legs and back• Antibody reacted with 83/104 (80%) group O donors

Different from the ABO, MN, P blood groups

History of RH - Act I

*Landsteiner K, Wiener AS. An agglutinable factor in human blood recognized by immune sera for rhesus blood Proc. Soc. Exp. Biol. and Med. 1940;43:223.

Macaca mulatta (Rhesus monkey) red blood cells injected to rabbit

History of RH- Act II

Rabbit produced an antibody named anti-Rhesus

Anti-Rhesus agglutinates RBCs of 85% random individuals

• Antibody referred to as anti-Rh (Rh for Rhesus)*• Apparently identical to Levine & Stetson’s case of HDN• 1941: Levine published a full account of the etiology of their case

History of Rh- Act III

• Newborn RBC typing:Human anti-Rh: can differentiate Rh+ and Rh-Rabbit anti-Rh: both Rh+ and Rh- typed positive

• Human & rabbit anti-Rh react against 2 different antigensHuman antibody named “anti-Rh”, defining “Rh” blood groupRabbit antibody named “anti-LW”, defining “LW” (or “Rhesus”)

• Antibody described by Levine & Stetson later identified as anti-Rh(D)

Nomenclature: Who is Right, Who is Wrong?

Wiener Fisher Rosenfield

R1 DCe RH 1,2,-3,-4,5

r dce RH -1,-2,-3,4,5

R2 DcE RH 1,-2,3,4,-5

R0 Dce RH 1,-2,-3,4,5

r’’ dcE RH -1,-2,3,4,-5

r’ dCe RH -1,2,-3,-4,5

Rz DCE RH 1,2,3,-4,-5

Ry dCE RH -1,2,3,-4,-5

• Wiener: Rh-Hr terminologyOne locus, multiple allelic forms

• Fisher-Race: CDE terminologyThree closely linked loci producing antithetical antigens: D or d; C or c; E or e

• Rosenfield: Numerical terminologyRepresent serological data “free of bias and divorced from speculative implication”

• Rh genes at 1p36.11

• RHD and RHCE originated by ancestral duplication

Two DNA segments “Rh box” border RHD

• Closely linked genes:Each gene has10 exons~94% homology

They Were All Wrong….

RHD RHCE

1 2 3 4 5 6 7 8 9 10 10 9 8 7 6 5 4 3 2 1

13,500 >5,000 13,500>5,000ATG STOP ATGSTOP

RHD RHCE

5’3’5’ 3’

Rh box

Rh box

Rh null: No D, Cc or Ee antigensDue to mutations in RHAG gene (regulator type) or deleted RHD paired with silent RHCE allele (amorph)

D– –: Lacks Cc, Ee; has enhanced DDue to mutation in RHCE gene

Rh negative: Lacks D antigenDeletion of RHD gene most common in CaucasiansIn African descent: RHD pseudogene & hybridsIn Asians: Del

www.uni-ulm.de/~wflegel/RH/

D Negative Polymorphism

Caucasian: Frequency ~15%

Due to misalignment of the two chromosomes in trans during meiosis

Molecular assays for RHD zygosity:

Hybrid Rhesus box PCRPst I RFLP

RhD Negative Genotypes in Blacks

PercentSouth Africa Tunisia

Brazil (S.P.)

Brazil (Camp.)

USA(Shv)

Deletion 18 98.6 94 87 83

Pseudogene 66 <1 4 11 7

Hybrid 16 1 2 2 5

Hybrid/Ψ 0 0 0 0 5

# Studied 82 443 188 206 41

Negative on direct agglutination testing (IS); positive only at IAT phase: initially named DU

• Mutations result in aa substitution in intramembranous portion (>90 SNPs)

• Folding & integration of the Rh protein into membrane is affected

• Reduction of D antigen >10 fold• Expresses all D epitopes• Weak D types 1, 2, 3 don’t form

ALLO anti-D

Weak D Polymorphism

NH2COOH

Type 1 (809T>G)

SNPs causing Weak D and position of aa. change in Rh protein

Type 22 (1224G>C)

Type 6 (29G>A)

Type 12 (830G>A)

Type 23 (634G>T)

Type 34 (809T>A)

Type 37 (399G>T)

Type 43 (605T>C)Type 51

(594A>T)(602C>G)

Type 52 (92T>C)

Type 53 (740T>G)

Type 71 (29G>C)

Type 72 (1212C>A)

Type 73 (1241C>T)

• D antigen is composed of >30 epitopes on the extracellular portion of the RBC membrane

• Normal D expression dependent on linear & 3-D conformational arrangements

Due to single aa changes or hybrids Partial D carriers CAN form allo anti-D

Partial D PolymorphismEvans+DIVb Type 2

KOU, FKDw+DV Type 1

667T>G 697G>C

DHK, DYODw-DV Type 5

697G>A

BARC+DVI Type 2

Rh32+DBT Type 2

Weak Partial D 15

845 G>A

RHD mutations causing Partial D

Clinical Considerations for Partial D

Donors: have stimulated allo anti-D but rarePatients:• Partial D has less risk of forming anti-D than Rh

negative individuals who have no RhD protein • Those missing a single Rh epitope can make

antibody to the missing piece• Partial Ds are not necessarily compatible with

each other

RhD Genotypes In SCD Patients*188

136 17 25“Normal D” D neg Homozygous variant

or heter. & silenced D

2 anti-D 8 anti-D 8 anti-D(5 deletion) (includes r’s)

*Moulds, JM. LifeShare Blood Centers unpublished data

• ~20% of D- develop anti-D after exposure to small volume of D+ blood• Rh antibodies cause severe hemolytic transfusion reactions (HTR) and

hemolytic disease of the fetus and newborn (HDFN)

• Accurate Rh phenotype identification remains problematic despite advances in serological methods and reagents

Clinical Relevance of Rh

HTR:o Donor: Typed negative when in fact

weak or partial antigen present –induce antibody

o Recipient: Typed positive when in fact weak or partial antigen present – produce antibody

HTR:o Donor: Typed negative when in fact

weak or partial antigen present –induce antibody

o Recipient: Typed positive when in fact weak or partial antigen present – produce antibody

HDFN (Mother)o Typed positive when in fact weak or

partial antigen present – produce antibody

o Typed negative when in fact weak (do not produce antibody) –unnecessary interventions

HDFN (Mother)o Typed positive when in fact weak or

partial antigen present – produce antibody

o Typed negative when in fact weak (do not produce antibody) –unnecessary interventions

Comprehensive Transfusion Medicine Survey by CAPSandler SG, et al. Arch Pathol Lab Med 2014:138;620-25

QUESTION 1999 2012

Routinely perform weak D-AHG on patients

58.2% 19.8%

Weak D reported as RhDpositive

50.7% 46.9%

Transfusion of RhDnegative to weak D recipient

43.5% 49.2%

Recommend RhIg to weak D transfused with RhD+

19.9% 15.7%

Rh type performed on all pregnancies at delivery

39.8% (32.3%) 48% (21.6%)

Rh Working Group

Representatives from:• AABB• CAP• ABC• ACOG• ARC• US Armed Services• Scientific consultants

• Patient Neg/Donor Pos is confusing to everyone including physicians

• 95% of women with weak D don’t make anti-DWaste of RhIgUnnecessary exposure to blood product

• 95% of weak D transfusion recipients do not need Rh negative blood

Rh Discrepant/InconclusiveRh typing ≤ 2+ @ RT

Rh PosRh Neg

RHD Genotype for weak D

Not RhIG candidate RhD+ for transfusionNot RhIG candidate

RhD+ for transfusionRhIG candidate

RhD neg. for transfusion

Weak D type 1,2,3 Not RhIG candidate RhD+ for transfusion

Weak D type 1,2,3 Not RhIG candidate RhD+ for transfusion

No weak D type 1,2,3 RhIG candidateRhD negative for

transfusion

Problems With The 2+ Rule

• Differences in typing reagentspolyclonal vs monoclonal vs blends

• Method usedautomated, eg. PK 7200manual: gel, tube, etc.

• Presence of C or E in trans

Sandler SG, et. al. Transfusion 2015;55:680-9

485,836

4,8585,830

46% minorities

Kacker et al., Transfusion 2015;55:2095Financial Implications

• Modeled a dynamic US population, tracking pregnancy-related costs, and contemplating the diversity of races

• It compared the expected financial impact of two strategies: Weak D treated as D–, at risk of HDFN and received RhIGWeak D genotyped for weak D type 1, 2, or 3 managed as D+

• Average costs of tests + RhIg administrations per pregnancy over 10 yrs.

Conservative strategy: $34.75 Alternative strategy: $34.73• Over 10 years RHD genotyping is cost-saving if <$256/test

• Availability of labs doing RH genotyping

• Cost of testing - depends on test; $100-300

• FDA approved tests - none

• TAT - 2 to 5 days

• Convincing OBs - is giving RhIg safer?

• Full RHD gene sequencing (10 exons)

• Sequencing exons encoding weak D 1,2,3

• Microarray RHD assays

• Allele specific PCR or RFLP

• Full RHD gene sequencing (10 exons)will detect all mutations in coding regions

• Sequencing exons encoding weak D 1,2,3will detect relevant weak D mutationswill detect other mutations in exons 6, 9, 1

101011 22 33 44 6655 77 9988 101 2 3 4 65 7 98RHDGene

• Microarray RHD assaysvary by manufacturerall detect weak D 1, 2, 3 some detect partial RHD

RhD BeadChip Assay

• RhD negative: RhD deletion, DΨ, 5 hybrids, 16X, 269X

• Del: 1227A, IVS3+1G>A

• Weak D: 1, 1.1, 2, 3, 5, 14, 17, 29, 34, 40, 41, 47, 51

• Partial D: DBS0,1,2; DAR, DAR-E, DAU 1,2,3,4,5; DOL 1,2,3; DBT 1,2; DIII a,b,c; DIII 4,6,7; DIVa,b; DIVa-2, DIV 3,4,5; DV 1,2,4,5,6,7,8,9; DVI, DCS 1,2; DFR1,2,3,4; DHMi, DNB, DUC2, ceHAR, DFV, weak D 4.0,4.1,4.3,11,15

Policies regarding RH will continue to evolve but will most certainly involve RH genotyping

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All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.

Continuing Education

• PACE, Florida and California DHS• 1.0 Contact Hours• Each attendee must register to receive CE at:

https://www.surveymonkey.com/r/RhDMouldsImmucor

• Registration deadline is October 13, 2017

• Certificates will be sent via email only to those who have registered by October 27, 2017

All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.

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