Immucor Transplant Ep 7 Webinar 21 June 2018.pptx [Read-Only] Program Handouts/OUS Trans… ·...
Transcript of Immucor Transplant Ep 7 Webinar 21 June 2018.pptx [Read-Only] Program Handouts/OUS Trans… ·...
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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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Handouts
http://www.immucor.com/en-us/Pages/Educational-Program-Handouts.aspx
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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
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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.
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NGS for a Solid Organ only Transplant Laboratory
Peter Jindra
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Outline
Decision process to advocate for NGSLab 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
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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
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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?
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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
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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
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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
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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.
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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
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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
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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
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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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Questions?
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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
All Content © 2015 Immucor, Inc.
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
19/06/2018
16
All Content © 2015 Immucor, Inc.
Thank you!