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Seeing Beyond Limits

2020 Webinar Schedule

Link to register: https://app.livestorm.co/immucor

Seeing Beyond Limits

2020 “Mini” WebinarsShort,10-minute educational videos with an industry leader.

Link to register: https://app.livestorm.co/immucor


learn.immucor.com


Continuing Education

• PACE, Florida and California DHS

• 1.0 Contact Hours

• Each attendee must register to receive CE at:

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

• Registration deadline is August 28, 2020

• Certificates will be sent via email only to those who have registered September 4, 2020


The Rh System

Regina Castor BS MT(ASCP)SBB cm

Area Technical Consultant – East Region


Objectives

• Review characteristics of the Rh antigens

• Discuss the genetics of the Rh system as it relates to antigen expression

• Describe the difficulties experienced with testing for Rh antigens

• Explore some of the reasons for typing discrepancies

• Present the proposed typing algorithm introduced by AABB/CAP for RHD genotyping


Rh System

• Composed of 2 genes• RHD and RHCE

• Responsible for 50+ antigens

• Both code for 417 amino acids

• Closely linked

• 97% sequence identity is shared

• Several nomenclatures• Fisher-Race (ex: DCe)

• Weiner (ex: R1)

• Large molecular weight proteins


Rh Genetics Source: “Discrepancies in RhD Typing: Picking up the Rhesus Pieces” Sue Johnson


Rh Antigens

• Found only on red blood cells

• Well developed at birth

• HDFN

• Enhanced with enzymes

• Ammonium channel transporter

• Important for RBC structural integrity

• Highly immunogenic• HTR

• Combination antigens possible

• Wide amount of genetic variation


Rh Antigens

“Rh-positive” or “Rh-negative”

D+ or D –

85% 15%

Caucasians

African

Americans Asians

D: 85% 92% 99%

C: 68% 27% 93%

E: 29% 22% 39%

c: 80% 96% 47%

e: 98% 98% 96%


Genetic Variation

• >500 RHD alleles reported involving amino acid changes

• Estimated 1% have an allele that codes for altered D expression (European ethnicity)

• Higher rates in African ethnicity


Genetic Variation


Genetic Variation

• Altered D categories • Weak D

• Quantitative difference in the amount of D antigens present on the cell

• Partial D*• Qualitative difference in the expressed antigen• “mosaic”

• Del*• Extremely low levels of antigen present cannot be detected

by routine serologic testing methods

• Nonfunctional RHD*• Gene does not encode full protein and are non-functional• Aka D null

*Capable of making Anti-D


Genetic Variation

• Altered D categories • Weak D

• Quantitative difference in the amount of D antigens present on the cell

• Partial D*• Qualitative difference in the expressed antigen• “mosaic”

• Del*• Extremely low levels of antigen present cannot be detected

by routine serologic testing methods

• Nonfunctional RHD*• Gene does not encode full protein and are non-functional• Aka D null

*Capable of making Anti-D

Sometimes the term “Weak D” is used as a generic term to describe serologic results as opposed to a specific group of RhD variants


“Rh-positive”

D+

D+, Partial D, Partial weak D, Weak D, D –

“Rh-negative”

D –WEAK D+

22

Typing and Testing Discrepancies


Reasons for Typing Discrepancies

• Genetic variants (as previously discussed)• Decreased quantity of Rh antigens

• Missing epitopes

• Both qualitative and quantitative differences

• Reagent differences• Polyclonal vs. Monoclonal

• Different manufacturers

• Different clones detect different epitopes

• May detect at different phases of testing


Typing Discrepancies


Reasons for Typing Discrepancies

• Differences in testing methods• Serological vs. Molecular

• Serologic methods cannot distinguish between Weak D and Partial D types (those who can make Anti-D and those who cannot)

• Hemagglutination, Gel, Solid Phase

• Manual vs. Automation

• Why the testing is being performed vs. interpretation• Donor testing

• Transfusion recipient

• Neonatal typing/RhIG Administration

• Age of the patient


Testing Algorithms Lack of standard practice


Testing Algorithms

• 2012 CAP survey: • Number of transfusion services performing a

serological weak D test on patients from 58.2% to 19.8% as a strategy to manage those with a weak D as Rh negative

• 2014 CAP survey:• How do you report the patient Rh type when it is

weak or variable?• RhD positive (47 %)

• RhD negative (11 %)

• If female or OB, report as RhD negative (some)

• Weak D positive (30 %)


Transfusion Volume 55, Issue 3, pages 680-689, 1 DEC 2014 DOI: 10.1111/trf.12941 http://onlinelibrary.wiley.com/doi/10.1111/trf.12941/full#trf12941-fig-0002

http://onlinelibrary.wiley.com/doi/10.1111/trf.v55.3/issuetoc

http://onlinelibrary.wiley.com/doi/10.1111/trf.12941/full#trf12941-fig-0002


AABB/CAP issue joint statement on phasing-in RHD Genotyping for pregnant women and other females of childbearing potential with a serologic weak D


Guidance Updated in 2020

Type 4.0 and 4.1 added to those that

can be treated as Rh +

“Use of two different serologic methods or Anti-D reagents have been shown to be an

effective practice for identifying those patients who

will benefit from RHDgenotyping”


• 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.

Identification and Management of patients

with Weak D and Partial D phenotype

A risk-based approach in a daily practice

NYP Lower Manhattan Hospital, New York

3 years experience

Daniel Alvarado MLS(ASCP)CMSBBCM, CQA(ASQ)Lisa Huynh MLS(ASCP)

Ljiljana Vasovic MD

Serologic weak D phenotype• Strength of reaction weaker than expected • Inconclusive Result • Discrepant Result vs. Historical or vs. other institution

Send for RHD genotyping• Result as per tube testing (or INV)• Manage as Rh Negative

genotyping result: Weak D type 1, 2, or 3 • Not at risk for anti-D • Not candidate for RhIG• Result as D positive

genotyping result: Weak D type 1, 2, or 3 Not detected • May be at risk for anti-D • Give Rh(D) negative blood • Candidate for RhIG• Result as D negative

@ LMH

Rh D Discrepancy Analysis / Partial D Analysis

• 6mL EDTA whole blood (lavender or pink top)

Rh D Genotyping

Manual Tube Weak D Cells Testing

Solid Phase ECHO or Manual Tube D Testing

Immucor Anti-D (Series 4) (Monoclonal Blend) Blood Grouping Reagent is prepared by blending IgM monoclonal anti-D

secreted by a human/murine heterohybridoma (MS201) with IgG anti-D heterohybridoma (MS26)

Immucor Anti-D (Series 5) (Monoclonal Blend) Blood Grouping Reagent is prepared by blending IgM monoclonal anti-D

secreted by a human/murine heterohybridoma (TH28) with IgG anti-D of another heterohybridoma (MS26).

the director Connie Westhoff, SBB, PhD

63 2+ ? 3+/3+ 3+/3+ hemizygote RHD*DFL

Solid Phase ECHO TUBE TESTING IS/AHG GENOTYPING

Anti-D

(series4)

Anti-D

(series5)

Anti-D

(series4)

Anti-D

(series5)

RHD alleles PREDICTED RHD PHENOTYPE 2nd alleleN

25 y.o. white female

LIS Comments :Day 0: Please Give O Neg RBCS till results from NYBC for D typing molecular are received.

Sample sent to the Molecular lab.Day +10 : Patient must receive RhIg (Rhogam).

Molecular Results for D Antigen: RHD*weak D type 1 Val207Gly

Predicted RHD Phenotype: D+ (weak partial)The patient has a RHD allele first reported as a weak D but now known to also encode a partial phenotype, hence the terminology "weak partial D phenotype". The RBCs may have weaker than expected D antigen expression depending on the reagent and method used.

Allo anti-D has been observed in patients with this RHD genotype who are on chronic transfusion therapy.

The risk for Anti-D in pregnancy is unknown and for transfusion appears to be far less than for other partial D phenotypes. Females of child bearing potential with a partial D phenotype are better served as Rh negative for transfusion and candidates for Rh immune globulin (if they have not produced Anti-D).

27 y.o. female delivered a healthy male child,Rh testing: serologic weak D phenotype

Solid Phase ECHO TUBE TESTING IS/AHG

Anti-D (series4) Anti-D (series5) Anti-D (series4) Anti-D (series5)

1+ 0 1+/3+ 1+/3+

RHD alleles

RHD genotype: RHD*DAR encodes amino acid changes 201Arg, 223Val, and 342Thr. Inactive RHD Ψ

D phenotype: D+ (partial)

COMMENTS: The patient has a partial D phenotype associated with risk for anti-D, the RBCs may have weaker than expected D antigen expression depending on the reagent and method used. Females of child bearing potential with a partial D phenotype are better served as Rh negative for transfusion and candidates for Rh immune globulin (if they have not produced active anti-D). (Transfusion 2015:55:680-89)


Anti-D

(series4)

Anti-D

(series5)

Anti-D

(series4)

Anti-D

(series5)

RHD alleles PREDICTED RHD PHENOTYPE 2nd alleleN

61 1+ ? 2+/2+ 2+/2+ RHD alleles RHD*DAR Inactive RHD Ψ

22 y.o. black female

Specimen sent to NYBC Molecular Lab for D Variant Testing on 03/14/2018.

RHD with no changes associated with common or partial D or weak D.PREDICTED RHD Phenotype: D+

Gene sequencing did not identify any changes from conventional. The patient is predicted to have two RHD alleles, and to have normal expression of D antigen and to not be at risk for anti-D, The results are consistent with massive transfusion with D- donor units.

50 y.o. Asian female Hx of A Pos

MHX: Beta thalassemia. consistent with massive transfusion with D- donor units at other institution

Solid Phase ECHO TUBE TESTING IS/AHG

Anti-D (series4) Anti-D (series5) Anti-D (series4) Anti-D (series5)

0 0 0/w+ 0/w+

PREDICTED RHD PHENOTYPE Designation n %D+ RHD 4 6.0%

D+ (partial) RHD*DAR 12 17.9%

RHD*DAU4 3 4.5%

RHD*DCS1 2 3.0%

RHD*DFL 1 1.5%

RHD*DOL 1 1.5%

D+ (weak partial ) RHD*weak partial D type 4.0 25 37.3%

D+ (weak) RHD*weak D type 1 6 9.0%

RHD*weak D type 122 1 1.5%



RHD*weak D type 841c 1 1.5%

GENDER

PREDICTED RHD PHENOTYPE Female Male Grand Total

D+ 2 2 4

D+ (partial) 17 2 19

D+ (weak partial ) 16 9 25

D+ (weak) 12 7 19

Grand Total 47 20 67

PREDICTED RHD PHENOTYPE Female Male Grand Total

D+ 3% 3% 6%

D+ (partial) 25% 3% 28%

D+ (weak partial ) 24% 13% 37%

D+ (weak) 18% 10% 28%

Grand Total 70% 30% 100%

At LMH GENDER Female Male

PREGNANCY STATUS No Yes N/A

PREDICTED RHD PHENOTYPE

D+ 2 2

D+ (partial) 9 8 2

D+ (weak partial ) 9 7 9

D+ (weak) 3 9 7

Grand Total 23 24 20

GENDER Female Male


PREDICTED RHD PHENOTYPE

D+ 3% 0% 3%

D+ (partial) 13% 12% 3%

D+ (weak partial ) 13% 10% 13%

D+ (weak) 4% 13% 10%

Grand Total 34% 36% 30%

Average of AGE Female Male Grand Total


D+ 70.0 45.5 57.75

D+ (partial) 43.1 30.1 49.5 38.31578947

D+ (weak partial ) 46.7 28.9 59.7 46.36

D+ (weak) 36.3 34.9 51.1 41.10526316

Grand Total 46.0 31.5 54.3 43.26865672

PREDICTED RHD PHENOTYPE White Black Asian

Pacific

Islander Other Refused U/K

Grand

Total

D+ 2 1 1 4

D+ (partial) 3 11 3 1 1 19

D+ (weak partial ) 2 14 1 5 1 2 25

D+ (weak) 12 1 1 3 1 1 19

Grand Total 17 27 2 2 12 3 4 67

PREDICTED RHD PHENOTYPE White Black Asian

Pacific

Islander Other Refused U/K

Grand

Total

D+ 0% 3% 1% 0% 1% 0% 0% 6%

D+ (partial) 4% 16% 0% 0% 4% 1% 1% 28%

D+ (weak partial ) 3% 21% 0% 1% 7% 1% 3% 37%

D+ (weak) 18% 0% 1% 1% 4% 1% 1% 28%

Grand Total 25% 40% 3% 3% 18% 4% 6% 100%


Anti-D

(series4)

Anti-D

(series5)

Anti-D

(series4)

Anti-D

(series5)

RHD alleles PREDICTED RHD PHENOTYPE Amino Acid Change 2nd alleles

1+ 0 1+/3+ 1+/3+N Anti-D Anti-D Anti-D Anti-D RHD alleles PREDICTED RHD PHENOTYPE Amino Acid Change 2nd alleles 1 0 0 0/3+ 0/3+ hemizygote RHD*weak D type 1 Val207Gly

2 0 0 1+/2+ 0/1+ hemizygote RHD*weak D type 1 Val207Gly

3 ? 0 0/3 0/2+ hemizygote RHD*weak D type 1 Val207Gly

4 ? 0 0/2+ 0/W+ hemizygote RHD*weak D type 1 Val207Gly

5 ? 0 1+/3+ 0/3+ hemizygote RHD*weak D type 1 Val207Gly

6 1+ 0 2+/3+ 2+/3+ hemizygote RHD*weak D type 1 Val207Gly

7 0 0 0/2+ 0/1+ hemizygote RHD*weak D type 2 Gly385Ala

8 0 0 0/2+ 0/2+ hemizygote RHD*weak D type 2 Gly385Ala

9 0 0 2+/2+ 2+/2+ hemizygote RHD*weak D type 2 Gly385Ala

10 0 0 3+/4+ 3+/4+ hemizygote RHD*weak D type 3 Ser 3 Cys

11 ? 0 2+/3+ 2+/3+ hemizygote RHD*weak D type 3 Ser 3 Cys

12 ? ? 1+/4+ 1+/4+ hemizygote RHD*weak D type 3 Ser 3 Cys

13 1+ ? 2+/2+ 2+/2+ hemizygote RHD*weak D type 3 Ser 3 Cys




17 1+ ? 2+/3+ 0/3+ hemizygote RHD*weak D type 3 Ser 3 Cys

18 1+ ? 2+/2+ 2+/2+ hemizygote RHD*weak D type 841c Val281Leu

19 0 ? 2+/2+ 2+/2+ hemizygote RHD*weak D type 122 Arg70Trp

N


Anti-D

(series4)

Anti-D

(series5)

Anti-D

(series4)

Anti-D

(series5)


20 0 0 2+/3+ 1+/3+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val

21 0 0 3+/3+ 2+/3+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val

22 ? ? 3+/4+ 2+/4+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val

23 1+ 0 1+/3+ 1+/3+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val


25 1+ 0 2+/3+ 2+/3+ RHD alleles RHD*weak partial D type 4.0 201Arg and 223ValHYBRID RHD*DIIIa-

CE(4-7)-D




29 1+ ? 0/2+ 0/1+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val

30 1+ ? 0/4+ 0/3+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val

31 1+ ? 1+/4+ 1+/4+ RHD alleles RHD*weak partial D type 4.0 201Arg and 223Val RHD*330delGT

32 1+ ? 2+/2+ 0/2+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val

33 1+ ? 2+/3+ 1+/2+ hemizygote RHD*weak partial D type 4.0 201Arg and 223Val






39 1+ ? 2+/4+ 2+/4+ RHD alleles RHD*weak partial D type 4.0 201Arg and 223Val Inactive RHD Ψ



42 1+ ? 3+/4+ 3+/4+ RHD alleles RHD*weak partial D type 4.0 201Arg and 223Val Inactive RHD Ψ


44 1+ ? 2+/2+ 2+/2+ RHD alleles RHD*weak partial D type 4.0 201Arg and 223ValHybrid MID*D111a-

CE(4- 7) -D

N


Anti-D

(series4)

Anti-D

(series5)

Anti-D

(series4)

Anti-D

(series5)


45 0 0 0/2+ 0/2+ RHD alleles RHD*DAR 201Arg,223Val and 342Thr Inactive RHD Ψ

46 0 1+ 1+/3+ 2+/4+ hemizygote RHD*DAU4 233Lys and 379Met



49 ? 0 0/4+ 0/3+ hemizygote RHD*DAR 201Arg,223Val and 342Thr

50 ? 0 1+/2+ 1+/2+ hemizygote RHD*DAR 201Arg,223Val and 342Thr

51 ? 0 2+/3+ 0/3+ hemizygote RHD*DCS1 223Val and 226Pro

52 ? 0 3+/4+ 2+/3+ hemizygote RHD*DAR 201Arg,223Val and 342Thr

53 1+ 0 w+/4+ w+/3+ hemizygote RHD*DAR 201Arg,223Val and 342Thr

54 1+ 0 2+/4+ 1+/3+ RHD alleles RHD*DOL 170Thr and 223Val partial, DAK+

55 1+ 0 2+/3+ 2+/3+ hemizygote RHD*DAR 201Arg,223Val and 342Thr



58 1+ 0 2+/3+ w+/2+ hemizygote RHD*DCS1 223Val and 226Pro

59 1+ ? w+/4+ w+/4+ hemizygote RHD*DAR 201Arg,223Val and 342Thr

60 1+ ? 2+/3+ 1+/3+ hemizygote RHD*DAR 201Arg,223Val and 342Thr

61 1+ ? 2+/2+ 2+/2+ RHD alleles RHD*DAR 201Arg,223Val and 342Thr Inactive RHD Ψ

62 1+ ? 2+/2+ 2+/2+ hemizygote RHD*DAR 201Arg,223Val and 342Thr

63 2+ ? 3+/3+ 3+/3+ hemizygote RHD*DFL Tyr165Cys

N


Anti-D

(series4)

Anti-D

(series5)

Anti-D

(series4)

Anti-D

(series5)

RHD alleles PREDICTED RHD PHENOTYPE 2nd allele

1+ 0 1+/3+ 1+/3+N Anti-D Anti-D Anti-D Anti-D RHD alleles PREDICTED RHD PHENOTYPE 2nd alleles note Hx

64 0 0 0/w+ 0/w+ homozygote RHD

weak D performed due

to Hx Rh Pos

ex: Tx Rh neg Beta thalassemia. consistent with

massive transfusion with D- donor units

65 1+ 0 0/3+ 0/3+ homozygote RHD

weak D performed due

to Hx Rh Pos fall rib fracture

66 1+ 1+ N/A N/A homozygote RHD RHD

Anti-D African American male; sickle cell disease.

Transfused. Anti-D identified at another facility.

67 3+ 4+ N/A N/A homozygote RHD RHD

Anti-D African American male; sickle cell disease.

Possible auto or allo anti-D.

N note Hx

Thank you Questions?

www.aabb.org 20

Daniel Alvarado MLS(ASCP)CMSBBCM, Lisa Huynh MLS(ASCP)Meta Morrison MLS(ASCP)


Continuing Education

• PACE, Florida and California DHS

• 1.0 Contact Hours

• Each attendee must register to receive CE at:


• Registration deadline is August 28, 2020

• Certificates will be sent via email only to those who have registered September 4, 2020



Thank you!

all content © immucor program...type 4.0 and 4.1 added to those that can be treated as rh + “use...

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