PS1539 A COMPARISON OF POST ALLOGENEIC STEM CELL TRANSPLANT CHIMERISM ANALYSIS METHODS: HIGH THROUGHPUT SNP GENOTYPING ANALYSED BY MALDI‐TOF MASS SPECTROMETRY VS. SHORT TANDEM REPEAT (STR) MARKER ANALYSIS
Authors: Wall K. , Skowronska A. , Eltawil N. , Mason J. , Griffiths M. , Larkins S. , Nakorchevsky A. 
Published: June 2019

Background:

Monitoring of donor chimerism levels is required in the post allogeneic stem cell transplant (SCT) setting to assess engraftment status. Chimerism levels can be assessed by a variety of methods including XY FISH (sex‐mismatch), microsatellite short tandem repeat (STR) analysis or single nucleotide polymorphism (SNP) genotyping by Q‐PCR. MALDI‐TOF Mass spectrometry (MS) is an alternative technique which can be used for high‐throughput SNP genotyping analysis for the purposes of post‐SCT monitoring. MALDI‐TOF MS offers rapid automated analysis and report generation compared to our existing STR methodology which requires expert analysis. We have evaluated MALDI‐TOF MS SNP genotyping as an alternative to STR analysis for chimerism status.

Aims:

1. To validate the use of the MALDI‐TOF MS SNP assay for clinical post‐SCT chimerism analysis across a range of transplant types (sibling, matched unrelated and double cord transplants). 2. To assess the accuracy and reproducibility of the assay across a range of defined recipient percentages using specifically prepared chimeric DNA samples. 3. To define the limits of sensitivity for detection of mixed chimerism. 4. To define the lowest DNA input concentration which permits detection of mixed chimerism.

Methods:

MALDI‐TOF MS SNP genotyping covers a panel of 92 SNPs. Locus specific multiplex PCR is followed by single base extension into the SNP site and detection with MALDI‐TOF MS. Mixed chimerism is assessed by analyzing the cumulative skew in genotyping data across informative SNPs. STR analysis comprises fluorescently tagged PCR of a panel of 16 highly polymorphic microsatellites. Relative peak height analysis of donor and recipient peaks is performed post‐PCR to give chimerism levels.

114 post‐SCT clinical samples from 38 single donor and two double cord donor transplants were analyzed by both methods to assess the concordance of results obtained.

Synthetic chimeras representing known % levels of donor and recipient DNA (1, 2, 5, 10, 30 and 50% recipient) were prepared and analyzed by both methods to assess accuracy and reproducibility across a range of recipient percentages and also to determine the limit of sensitivity for detection of mixed chimerism.

DNA dilutions of the synthetic 2% recipient chimera were prepared in order to define the minimum acceptable DNA template concentration for detection of mixed chimerism in both assays.

Results:

Good concordance between methodologies was seen across all clinical samples tested, with greatest variation seen between 40–60% chimerism levels. Analysis of synthetic chimera samples also showed good concordance, with greatest variation seen at the expected 50% chimerism level.

Analysis of the synthetic chimeras defined the sensitivity of detection for recipient DNA at 2% by both systems. The minimum input template DNA concentration for detection of mixed chimerism was shown to be 1ng/μl for MALDI‐TOF MS and 2ng/μl for STR analysis.

Summary/Conclusion:

The MALDI‐TOF MS SNP Assay gave highly comparable results to STR analysis for determination of post‐SCT chimerism levels and offered identical sensitivity to STR analysis for detection of mixed chimerism. The assay could reliably detect mixed chimerism at DNA concentrations as low as 1ng/μl. Results analysis and report generation is fully automated, taking less than 20 seconds per sample compared to STR analysis which takes 5–10 minutes of expert analysis per sample. We conclude that MALDI‐TOF MS SNP analysis offers a viable alternative to STR chimerism analysis with comparable accuracy and sensitivity and significantly increased analytical speed.