WELCOME Immucor MIH User Group Meeting Program Handouts/MIH User Gro… · Validation of Automated...

1 1

WELCOME

Immucor MIH User Group Meeting

January 31, 2017

2 All Content © 2016 Immucor, Inc. 2 All Content © 2016 Immucor, Inc.

Agenda

Review of Proficiency Testing Results Monica Kalvelage, MT(ASCP)MB, SBB LifeShare Blood Centers Review of AABB 2016 Abstract: Validation of Automated DNA Extraction from Buccal Swab Samples Katrina Billingsley, MT(ASCP)SBBCM LifeShare Blood Centers Buccal Swab DNA Isolation for Patients with a Mixed Chimerism Jules Zinni, MLS(ASCP)CMMBCM Northwestern Memorial Hospital RHCE Genotyping: Investigating Discrepancies Between Molecular and Serologic Results Trina Horn, MS MLT(ASCP) SBBCM

Jessica Keller, MS MB(ASCP)CM

American Red Cross National Molecular Laboratory


Handouts

• User Group Meeting Handouts:

http://www.immucor.com/en-us/Pages/User-

Group-Meeting-Handouts-and-Slides.aspx

http://www.immucor.com/en-us/Pages/User-Group-Meeting-Handouts-and-Slides.aspx













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

Handouts


CE

• PACE, California DHS

– 437-302-17

• California and Florida CA

– 20-585936

• 1.0 Contact Hours


Continuing Education

• Each attendee must register to receive CE at:

– https://www.surveymonkey.com/r/MIHUGM

• Registration deadline is February 17, 2017

• Certificates will be sent via email only to those

who have registered by March 3, 2017

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



Other

• Session will be recorded and posted.

– Access information will be sent to each

registrant when the recording becomes

available

• No CE issued for participating in recording


Q&A

• You are all muted

• To ask a question….


• 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 these case studies

are those of the presenter and do not

necessarily reflect those of Immucor.

All Content © 2014 Immucor, Inc. 10

Proficiency Samples

2016 - Set 2, overview

Monica Kalvelage

LifeShare Blood Centers

All Content © 2014 Immucor, Inc. 11 All Content © 2014 Immucor, Inc. 11

Proficiency Samples – 2016 - Set 2, overview

• 60 gDNA sets mailed to participants

• 53 responses were submitted (88% response rate)

• 100% concordance of all results among all respondents

• 21 labs used the (0)* reporting style for Fyb predicted phenotype, all

others used 0

• 6 labs made specific comment about the homozygous FY-GATA mutation

in sample 81410

• No other reporting differences noted

Validation of Automated DNA Extraction Using Buccal Swab

Samples

Katrina Billingsley, MT(ASCP)SBBCM

Supervisor, Scientific Support Services LifeShare Blood Centers

Why Buccal Swabs?

• Extraction most commonly performed on whole blood, EDTA

• Occasions when whole blood is not an option

– Aplastic anemia

– Neonatal patients

– Bone Marrow Transplant patients

• What is the degree of engraftment?

Automated Extraction Challenges

• Buccal swabs often yield very low DNA concentrations and poor purity ratios

• Most samples would not meet standard acceptance criteria

– Concentration ≥10ng/μl

– Purity Absorbance A260/A280 ratios of 1.6 – 2.2

• PreciseType HEA criteria

– Concentration: ≥15ng/μl

– Purity: not defined

• Qiagen tech support

Sample Collection

• No smoking, food or drink within 30 minutes of collection

• Firmly brush swab up & down inside of each check about 6x per side

• Allow to air dry 24 hrs

QIAcube Protocol

• 25 samples processed using the QIAamp DNA Blood Mini - Buccal swab protocol

• 2 step protocol

– Buccal Swab

• Lyses cheek epithelial cells

– Buccal Swab Lysate

• Purify DNA

Concentration ng/µl

0

5

10

15

20

25

30

35

40

0 5 10 15 20 25 30

Targ

et

>10

ng/µ

l

n = 25

19 of 25 samples <10 ng/µl

A260/280 Absorbance

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25 30

Targ

et

1.6

- 2

.2

n = 25

12 of 25 samples A260/280 – 0.55-1.5

Would the DNA amplify?

• Buccal swab samples amplified with HGH specific primers

• Run on 2% agarose gel stained with EtBr

What about HEA?

• 3 of 25 samples run on HEA v1.2

– 2 donors, 1 patient

– Concordant with previous HEA results

– No IC, LS

Conclusion

• Using the QIAcube protocol for buccal swab extraction yields DNA of sufficient quality for amplification despite obtaining low purity and quantity readings.

• This validation demonstrates that conventional sample acceptance criteria may be too strict for buccal swab extraction.

Thank You!

Katrina Billingsley, MT(ASCP)SBBCM

Supervisor, Scientific Support Services LifeShare Blood Centers 318-673-1463 [email protected]

mailto:[email protected]

Buccal Swab DNA Isolation Hematopoietic Stem Cell Patient with a Mixed Chimerism

Presented to: Immucor Molecular User Group

Presented on: January 31st, 2017

Presented by: Jules Zinni, MLS(ASCP)CMMBCM

Background

• 64y Male

• Second clinical remission

• FMS-like tyrosine kinase -3 (FLT3) AML

• Reduced intensity conditioning (fludarabine & busulfan)

• Matched unrelated donor (10/10)

• ABO matched transplant

Recipient Rh(D) negative (no history of anti-D)

Donor Rh(D) positive

24

Transfusion Timeline

Day 49 70 74 71-113 115-122

126-131

136 137 140-142

143

Patient Rh(D) Typing

4+ Tube

- - - - - Positive (Ref lab)

- 4+mf Rh(D) neg

Ab ID - - - 95: Anti-E, -Jka, WAA, CAA, ERACT

- - Anti-D in alloadsorbed plasma (ID at ref lab day 138)

- DAT: 4+ IgG 4+ C3

RBC Transfused

- 1st Rh+

- 13 Rh+ ~2/wk span

2 Rh+ 2 Rh+ 1Rh+ @1455

2 Rh+ ~1200-1600

5 Rh- -

Hgb (g/dL) - - 11.3 ~8.9 10.4 -

5.5 @2100

~5.0 @1152 ~5.0 @2237

~8.0 -

ABUS: Antibody of Unknown Specificity, nonspecific WAA: Warm Autoantibody

CAA: Cold Autoantibody ERACT: Eluate Reacts with All Cells Tested 3

Laboratory Values Final Admission d136 - 144

0

50

100

150

200

250

0

1

2

3

4

5

6

7

8

9

13

6.0

0

13

6.5

4

13

6.5

9

13

6.6

1

13

6.8

7

13

7.0

5

13

8.0

0

13

8.1

5

13

8.4

8

13

8.6

3

13

9.1

8

14

0.0

0

14

0.2

5

14

0.5

9

14

0.7

3

14

0.9

3

14

1.0

0

14

1.1

8

14

1.3

4

14

2.0

0

14

2.1

7

14

2.2

9

14

2.3

0

14

2.3

1

14

2.4

6

14

2.7

6

14

2.8

5

14

3.0

1

14

3.1

7

14

3.3

3

14

3.4

3

14

3.5

1

14

3.6

7

14

3.8

5

14

4.0

0

14

4.0

1

14

4.1

4

nR

BC

s/1

00

WB

Cs

Days post-HSCT

nRBCs Hgb (g/dL) Total Bilirubin (mg/dL)

Potassium (mEq/L) RBC Trx (unit) PLT Trx (unit)

pHEA Testing of Sample with Antibody Detected

DNA Extracted from Day 95, Whole Blood

• Rh(D) positive serologically

• Anti-E identified

• Possible anti-Jka

• Warm autoantibody

• Cold autoantibody

• Nonspecific reaction

• DAT: IgG & C3 positive

• Eluate reacts with all cells tested

27

pHEA Testing of Sample Prior to Antibody Detection

DNA Extracted from Day 70, Whole Blood

• Rh(D) positive serologically

• Negative antibody screen

• C positive

• Jka positive

• Jkb positive

• Indeterminate Call (IC) on both E and N antigen predictions

28

Antigen Result IC(2)

C +

E IC

Jka +

Jkb +

N IC

One Patient, Two Samples, Two Phenotypes

HEA Sample Comparison

• Difficult to tell which phenotype is donor and which is recipient

Genetic material tested

• Probably not allo-anti-Jka

• IC can be due to a dual cell population- “contamination”

29

Antigen Day 95 Day 70

C 0 +

E 0 IC

Jka + +

Jkb 0 +

N + IC

Baseline Recipient and Donor Comparison

MUD

Rh+

30

Buccal Swab on Recipient & DNA of Donor from HLA Lab Recipient Buccal Swab

Rh -

Antibodies: Anti-E, -D, Nonspecific, WAA, CAA, ERACT

Chimerisms Leukemic Relapse

31

76.2 93.6 86

23.8 6.4 14

67 81 124

Days of Engraftment

Bone Marrow

Recipient (%) Donor (%)

39.5

15.5 14.2 31.2

52

94.3

60.5

84.5 85.8 68.8

48

5.7

25 39 53 74 80 108

Days of Engraftment

Peripheral Blood

Recipient (%) Donor (%)

Anti-E

95

Anti-E

95

O+

62

O+

62

O+ mf

140

O+ mf

140

143

O=

Exp

146 Exp

146

O=

143

136

Anti-D

136

Anti-D

1st HEA Sample

1st HEA Sample

70

2st HEA Sample

70

2st HEA Sample

144

Buccal HEA

144

Buccal HEA

C E Jka Jkb N

Day 70 + IC + + IC

Day 95 0 0 + 0 +

Buccal 0 0 + 0 +

MUD + + + + 0

Summary

• Indeterminate call may indicate a dual cellular population, not necessarily external contamination

• Buccal swab is beneficial for a baseline pHEA

• If antibodies are difficult to identify, providing antigen negative units that correspond with both the donor and recipient may be useful in patients with a mixed chimerism

Thank You

• Karyn Hartman, CHS(ABHI), MT(ASCP)SBB

• Glenn Ramsey, MD

• Paul Lindholm, MD

Contributing Authors

Plate with a depiction of a Chimera in ancient Greek and

Roman mythology. In the Illiad, Homer described it as, “a thing of immortal make,

not human, lion-fronted and snake behind, a goat in the

middle, and snorting out the breath of the terrible flame of

bright fire”.

The need is constant.

The gratification is instant.

Give blood. TM

Novel RHCE alleles containing c.307T cause C typing discrepancies Horn T1, Keller, J1, Pucillo, T2, Iqbal, J3, Mansfield, P4, Keller MA1

Transfusion 56(S4):159A, 2016.

1 National Molecular Laboratory, American Red Cross, Philadelphia PA; 2 Immunohematology Reference

Laboratory, Greater Chesapeake and Potomac Region, Baltimore, MD; 3 VA Medical Center, Los Angeles, CA; 4

Immunohematology Reference Laboratory, Penn Jersey Region, Philadelphia, PA

Trina Horn, MS MLT(ASCP) SBBCM

American Red Cross National Molecular Laboratory

Philadelphia, PA

Red blood cell (RBC) genotyping of donors and patients is becoming

the standard of care for certain patient groups, such as those with

sickle cell disease, where it is common to match for C, c, E, e and K

antigens to avoid alloimmunization.

With an increase in RBC genotyping comes the identification of

samples in which the genotype-predicted phenotype is found to be

discordant with the serologic type.

Here we report several cases of C typing discrepancies where RBC

genotyping predicted an individual to type C negative, while the

sample serologically typed C positive.

Investigation using higher resolution RHCE genotyping identified 2

novel RHCE*ce alleles that share Ser103, an amino acid substitution

involved in RhC antigen expression.

BACKGROUND

RESULTS

Case Patient/Donor

History

Genotype-

predicted

Phenotype

Serologic

phenotype

RHCE array

result

1 62 yo AA

male C negative C+

c.307C/T

c.733C/G

2 36 yo AA female

donor C negative

C+ (2-3+)

Immucor c.307C/T

c.733G C+ (4+) BioRad

3 17 yo AA male

donor C negative

C+ (2+)

Immucor c.307C/T

C+ (3+) BioRad

http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/

Conventional RHCE*Ce

48G>C 178C>A 203A>G 307C>T

Case 1 733C/G 307C/T

733G 307C/T Case 2

307C/T Case 3

These cases highlight the need for investigation of discrepancies between

a genotype-predicted phenotype and serologic type for the C antigen

While the RhC antigen is encoded by a RHCE allele containing c.48G>C

(Trp16Cys), c.178C>A (Leu60Ile), c.203A>G (Asn68Ser) and c.307C>T

(Pro103Ser), these cases illustrate that anti-C reactivity does not require all

of these residues

Case #1 carries c.307C/T and c.733C/G

— cDNA analysis would be needed to resolve the phase of these variants.

Case #2 carries RHCE*ce with c.307T and c.733G

Case #3 carries RHCE*ce with 307T

Taken together, these cases show that the serine at residue 103 is

responsible for reactivity with anti-C reagents

It is not clear if these alleles encode partial C antigens

DISCUSSION

These cases highlight the need for investigation of discrepancies

between genotype-predicted phenotype and serologic type.

Use of multiple genotyping methods, including Sanger sequencing

of cDNA transcripts can help resolve cases where allele dropout is

suspected.

These variants aid in deciphering the residues responsible for anti-C

reactivity.

CONCLUSIONS

1. Blood Group Antigen FactsBook, 3rd ed. ME Reid and C Lomas-

Francis 2012

2. http://www.isbtweb.org/working-parties/red-cell-immunogenetics-

and-blood-group-terminology/blood-group-terminology/blood-group-

allele-terminology/

REFERENCES

E typing discrepancy due to

a variant RHCE allele

Jessica Keller, MS MB(ASCP)CM

National Molecular Laboratory American Red Cross Philadelphia, PA Transfusion 56(S4):160A, 2016.

Introduction • Array platforms are useful tools to detect the

presence or absence of an allele associated with blood group antigen expression – PreciseTypeTM HEA , RHCE and RHD BeadChip kits are

routinely utilized at the Red Cross to define genotypes of patient and donors.

• Typing discrepancies arise from variation in a gene that could result in altered protein expression. – Discordant serologic typings or molecular vs serology – Sometimes, alternative methods are needed in

conjunction with BeadChip testing to resolve such typing discrepancies.

Case Study • The RHCE gene encodes C, E, c and e antigens

– Variations in the RHCE gene can cause discrepancies between serology and molecular methods.

• Caucasian 64yo male blood donor

– The donor historically phenotyped E negative.

– The current donation was E negative with

standard tube agglutination using Biotest

antisera.

– The hospital phenotyped the donor weakly E

positive using Gel.

Case Study – Molecular Methods

• PreciseTypeTM HEA BeadChip predicts an E+e+ phenotype (c.676G/C)

• Investigation involved additional testing:

– RHCE BeadChip is concordant with HEA (c.676G/C)

• No variant markers detected

– Genomic sequencing of RHCE exons 1, 3, 4, 6

• no changes from reference sequence.

– RHCE-cDNA analysis revealed a novel RHCE*cE allele

• nucleotides in exon 3: c.361T, c.380T, c.383A and c.455A.

RHCE*cE allele with RHD Exon 3

455C>A

Thr152Asn

383G>A

Gly128Asp

380C>T

Ala127Val 361A>T

Met121Leu

…

…

RHCE Exon1 2 3 4 5 6 7 8 9 10

Case Study Conclusions • Novel RHCE*CE-D(3)-cE allele identified

– Most likely the cause of the variable serologic E typings

– Most similar to the RHCE*D(1-3)-cE hybrid allele (aka RHCE*cEKK)

• Partial E+ expression

– RHCE*cE BA and RHCE*cE JU

• Weakened E phenotype

• Of Note: RHCE BeadChip & gDNA Seq of RHCE exon 3 showed no changes – Exon 3 allele dropout of the hybrid allele observed

– likely due to RHD-specific nucleotides in IVS2 and/or IVS3 at location of RHCE-specific primer binding regions

Summary

• This case highlights the need for investigation of discrepancies. – Between serologic tests as well as between genotype-predicted

phenotype and serologic type.

• Use of multiple genotyping methods can be useful when allele dropout is suspected.

• Known Limitations of the RHCE BeadChip to consider: – “For a particular sample, unanticipated point mutations in the probe or

primer binding region may impact the accuracy of the result for that sample.”

– “The RHCE assay is unable to detect RHCE-RHD hybrids.”

• High-resolution methods including Sanger sequencing of cDNA transcripts can be helpful to resolve such cases.

Thank You

• Questions?


Questions


Continuing Education

• Each attendee must register at

– https://www.surveymonkey.com/r/MIHUGM

Registration deadline is

February 17, 2017

Certificates will be sent via email by

March 3, 2017

Thank you!



WELCOME Immucor MIH User Group Meeting Program Handouts/MIH User Gro… · Validation of Automated...

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