Transplant Webinar Series: Ep. 7 The Role of NGS in the … Program Handouts/OUS... ·...

Transplant Webinar Series: Ep. 7The Role of NGS in the Transplant Setting

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The Role of MICA testing in Solid Organ Transplantation

Featuring

Dr Mohit ChowdhryIndraprastha Apollo Hospitals, New Delhi, India

19 July 2018


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NGS for a Solid Organ only Transplant Laboratory

Peter Jindra

Outline

Decision process to advocate for NGS Lab planning Test Validation Implementation into Clinical PracticeReporting out NGS data Areas where NGS can help an HLA lab in

solid organ tranplantation

Baylor College of Medicine

Independent HLA in the Department of Surgery CHI-SL Medical Center (Kidney, Heart, Lung, Liver)

Texas Children’s Hospital (Kidney, Heart, Lung, Liver)

Veteran’s Affairs Michael E. DeBakey Hospital (Kidney)

Houston Area Donor Typing Center for LifeGiftOPO

Current HLA typing techniques for Solid Organ Transplant Serology

Confirmation of Expression on the cell surface SSP

Confirmation of Various Loci, Mainly DPB1

SSO (Primary Method) Recipients, Living Donors, Confirmatory Donor Typing

Real-Time PCR (Primary Method) Deceased Donor Typing

All 11 loci A, B, C, DRB1, DRB345, DQA1, DQB1, DPA1, DPB1

HLA Typing by RT-PCR

Pros Donor typing on-call, done in 3 hrs.

1 Tray = 1 Donor Minimize Sample Switch

Easy for new technologists to run and interpret results

Resolution is at SAB Level

Cons Not easy to batch

Some common alleles still are not excluded

High cost per test

HLA typing by SSO

Pros Batching is possible

Good Resolution with some ambiguities

Relatively good price per HLA typing all 11 Loci

Able to run single loci.

Cons Cannot batch more than 8.

Concern about sample switch

Technologists need to learn proper technique for washing

Running the test is longer and analysis takes time

HLA typing volume

Needed to consider HLA typing volume

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Jan Feb Mar Apr May Jun Jul Aug Sep Oct

TAT Totals 2017

HLA Typing

The Population of Houston

http://www.city-data.com/city/Houston

Talking with the Administration

BCM Mission statement Baylor College of Medicine is a health sciences university that

creates knowledge and applies science and discoveries to further education, healthcare and community service locally and globally.

Clinical volumes continue to increase at all centers Greater Understanding of HLA alleles Research opportunities Open new business ventures

Bone Marrow Disease Association

What equipment/space would we really need?

How many samples are we going to run at a time?

Run our New Patients, Living Donors, and any Confirmatory Typings

Various sizes are available to run 8,16, 25, 48, 96 depending on the flow cell using 300-cycles 2 X 150 output Nano Flow Cell 1 M reads 300 Mb (17 hrs) Micro Flow Cell 4 M reads 1.25 GB (19 hrs) v.2 Flow Cell 15 M reads 4.5 GB (24 hrs)

Depth of coverage was best running v.2 Flow Cell with 25 samples.Although made for 96 samples the extra reads increase our

call confidence

Lab Work Flow and TAT

Wednesday

Thursday

Friday

What if the HLA typing can not be resolved by NGS?

Real-Time PCROnly if the complete typing failed and

needed results STAT.

SSO Run the specific loci which failed and

have results on Tuesday. Use to confirm homozygosity

Personnel Required

With automation it is feasible for one technologist trained in DSA methods to perform the test. Ideally two technologist in the DNA section

with a third person rotating in.

Initial training DNA section head and Supervisor perform the

procedure. Understand the timing involved and analysis

of the data. Decrease the learning curve

Validation

Test a series of samples capable of helping to guide the QA/QC process Homozygousity (confirm by SSO)

Rare alleles (we see commonly in our population)

Null alleles (we still have serology trays)

Runs with the same sample run twice on the flow cell and all other runs.

Reproducibility between technologists in the section

Seek out blind samples to ensure accuracy and performance

General CAP Validation

Validation Summary Molecular Pathology Checklist Validation Studies

Use adequate number and representative distribution of samples

40 samples Whole Blood/Buccal Swab

Accuracy Blinded samples and samples previously typed by SSO.

Precision/Reproducibility Samples run by different technologists

Same sample run on multiple experiments

Reportable Range

Reference Interval (ensure each run has a good sampling of the population)

Lower Limit of Detection

CAP Molecular Pathology Checklist NGS FOCUS

NGS Analytical Wet Bench Procedure / Validation / Quality Management Program

Controls, Metrics, QC parameters

Exception Log/Record

NGS Analytical Bioinformatics Procedure / Validation / QM

NGS Data Transfer Confidentiality and Storage

Monitoring for Upgrades and Track Versions

Sequence Variants Interpretation and Reporting

At what level of resolution will we report?

Currently report at the allele group or serological level

With high res we will transition to the specific HLA protein

Gene variants with differences Silent Mutations Non-Coding

Regions

Example: HLA-A Typing A*24:23

What do the Solid Organ Clinicians / Coordinators need?

Listing patients for transplant in UNet.

How do should they proceed with A*24:23?

Post Transplant Monitoring for DSA

How do we call a donor specific antibody if we don’t have a bead specific to the high res donor type or α/β pair?

We use an * to indicate specific bead not available on single antigen panel.

We report the MFI from the bead that is most similar and aligned on the panel

Is NGS too accurate to call a donor specific HLA antibody?

Areas where NGS can help a solid organ lab.

Epitope Mapping High Resolution Typing of Donor and Recipients

Exact amino acid sequence to define the HLA epitopes present

Define the number of eplet mismatches (eplet load) between the donor and recipient

An eplet mismatch threshold >11 predicted dnDSA development for HLA-DR or -DQ with sensitivity >90%.

J Am Soc Nephrol. 2017 Nov;28(11):3353-3362

Guide Clinical Decision Making

For clinicians seeking to minimize tacrolimus in the setting of infection or to limit serious adverse effects HLA-DR/DQ eplet mismatch provides a precise and individualized assessment of

alloimmune risk to guide decision making

Choose between various potential living donors in Kidney Living Donor Program Selection based on eplet mismatched

National Kidney Registry (NKR) Large national pool of paired exchange

Find matches with less risk

Additional Areas of Interest due to Typing HLA by NGS

Studies address HLA gene polymorphism and its effect of HLA cell surface expression and signaling.

Find novel gene variants among the population Anthropology

Graft vs Host Disease Analysis

GvHD in solid organ transplantation

A 55-yr-old Korean woman underwent liver transplantation owing to decompensated hepatitis C virus-related cirrhosis.

Donor was a 40-yr-old woman.

received immunosuppressive therapy consisting of prednisone, one injection of basiliximab, and low-dose tacrolimus, which was discontinued when GVHD symptoms appeared.

The patient developed fever of 39 23 days after the transplant, with five days of severe leucopenia (0.38×109 white blood cells [WBC]/L), accompanied by anemia (6.4g of Hb/dL) and thrombocytopenia (81×109 platelets/L) with symptoms suggesting GVHD.

Pancytopenia persisted despite administration of granulocyte-colony-stimulating factor. Bone marrow was aspirated to evaluate the etiology of hematologic disorder and to diagnose GVHD. Hypocellular marrow was observed Ann Lab Med. 2016 Jan; 36(1)

Using NGS for Chimerism Testing

Within solid organ transplant we get calls to run chimerism testing to confirm graft vs host disease

Liver Lung

Use STR-PCR to amplify 11 polymorphic STR alleles 8 of 11 markers discriminated donor vs recipient 41 days post liver txp markers

NGS Chimerism 41 days post liver txp

NGS Chimerism

32 of 90 SNPs discriminated between donor and recipient.

Average percent of donor DNA was 95.1% in the bone marrow aspirate

Depth of coverage Recipient 674 reads

Donor 1609 reads

Post transplant 1080 reads

Comparison of STR and NGS

% of Recipient DNA in mixed chimerism from bone marrow aspriate5.6% (4.1-7.7%) by STR-PCR

4.9% (2.5-6.7%) by NGS

Overall Conclusion

NGS provides a powerful tool for a solid organ only HLA laboratory

The numerous positives out weight the learning curve for implementing this new method

Reaching out to the HLA community and a reliable vendor are key to the successful implementation

AcknowledgementsBaylor Immune Evaluation lab: Dr. Kerman Dr. Cusick Thuy Tu Lauren Clark Noriel Acorda Nicholas Woolley Luis Rodriguez Stacie Gray Derrick Giang Brittany Howell Trang Dao Katherine Foye


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Continuing Education

• ABHI, ASCLS/P.A.C.E., Florida and California Credits • 1.0 Contact Hour or 0.15 continuing education credits

(CECs) awarded• Each attendee must register to receive CE credits at:

https://www.surveymonkey.co.uk/r/ImmucorTransplantEp7

• Registration deadline is 13 July 2018• Certificates will be sent via email only to those who

have registered by 27 July 2018


Future Webinars

Link to register: https://immucor.webinato.com/register

The Role of MICA testing in Solid Organ Transplantation

Featuring

Dr Mohit ChowdhryIndraprastha Apollo Hospitals, New Delhi, India

19 July 2018

Transplant Webinar Series: Ep. 7 The Role of NGS in the … Program Handouts/OUS... ·...

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