The Next Generation of Post-Transplant Chimerism Monitoring

Why NGS Outperforms Traditional STR Analysis

Intro

Haematopoietic stem cell transplantation (HSCT) is the predominant curative treatment for more than 70 malignant and non-malignant diseases, with accurate post-transplantation monitoring being essential in promoting successful outcomes. Detection and tracking of donor-recipient cell chimerism over time helps identify potential complications such as graft failure or disease relapse.

The term ‘chimerism’ describes the presence of both donor and recipient cells in the recipient’s body post-transplant. ‘Complete chimerism’ refers to 100% donor cells in a post-transplant sample, indicating complete haematopoietic replacement, while ‘mixed chimerism’ (donor cells comprising 5% to 95% of the population) indicates incomplete replacement with some level of recipient cells remaining. Additionally, ‘split chimerism’ describes varying donor-recipient percentages in different cell lineages; many labs choose to sort and track chimerism within specific populations such as CD3+ and CD19+ cells.  Finally, ‘microchimerism’ describes situations where less than 1% of recipient cells are present: a sensitivity level used for the earliest detection of disease relapse.

Historically, Short Tandem Repeat (STR) analysis has been the gold standard for chimerism monitoring. However, this method has limitations in sensitivity and resolution which can impact the timing and precision of clinical decisions.

In contrast, Next-Generation Sequencing (NGS)-based methods like the One Lambda Devyser Chimerism NGS assay provide significant benefits over older methods through enhanced sensitivity and precision; these raise the bar for post-transplant monitoring, providing clinicians and scientists with clearer, faster and more actionable insights. The efficiency and accuracy of NGS chimerism monitoring can also reduce the need for follow-up/repeat testing and avoid complications by allowing earlier intervention.

NGS-based assays incorporate several advanced molecular techniques and processes designed to deliver high sensitivity, specificity and accuracy. Here is a closer look at how this works:

Methodology

1. Targeted DNA Amplification

Multiplex polymerase chain reaction (PCR) is used to amplify specific regions of the genome.  Unlike STR analysis which uses short tandem repeats which can sometimes lead to ambiguous results, NGS assays amplify either target single nucleotide polymorphisms (SNPs) or insertion-deletion polymorphisms (indels); these serve as genetic markers to distinguish donor and recipient DNA.

• A SNP is a variation at a single position in the DNA sequence where one nucleotide (A, T, C, or G) is replaced by another; these are the most common type of genetic variation in humans and occur frequently across the genome. However, these have limitations in chimerism monitoring since sequence variation of a single base change can be difficult to detect in low-frequency variants affecting the sensitivity of the assay.
• An indel refers to the addition or removal of one or more nucleotides in a DNA sequence; these either lengthen or shorten the DNA sequence at a specific location. Due to their structural difference, indels provide a more distinct marker than SNPs so are less prone to miscalling errors in sequencing and reduce the chance of misinterpretation. This is crucial in NGS-based chimerism monitoring where accurately distinguishing donor from recipient DNA, even at low levels, is essential.

While SNPs and indels are both abundant and stable markers allowing for the differentiation of donor and recipient, indels have the edge for producing the highest sensitivity. The One Lambda Devyser Chimerism NGS assay uses indels to provide the highest sensitivity of any NGS chimerism assay (limit of detection 0.05%).

2. Library Preparation and Indexing

After PCR amplification, the targeted DNA fragments undergo library preparation before sequencing. Adapters (short DNA sequences) are attached to the amplified fragments so they can bind to the sequencing platform. At this step, each sample is assigned a unique index/barcode after which all samples are pooled into a single tube. Pooling indexed samples saves time and cost by enabling multiple samples to be run in parallel, increasing throughput without compromising accuracy. The unique barcodes also prevent sample cross-contamination and ensure data is accurately attributed to its source since each sample can be readily distinguished.

3. Next-Generation Sequencing

Once the DNA samples have been prepared and indexed, the pooled library can be loaded onto a NGS platform where sequencing begins. The One Lambda Devyser Chimerism NGS assay is validated to run on a variety of Illumina-based sequencers for flexibility. Each of these sequencers provides a high depth of coverage by analysing hundreds of thousands of short DNA reads for each sample. This deep sequencing capability allows for precise quantification of donor and recipient DNA, detecting even small amounts of residual recipient cells, while also minimising the impact of sequencing errors. This is crucial for low-level chimerism monitoring as minor populations can be detected with high confidence.

4. Data Analysis and Bioinformatics

After sequencing, the generated data must be processed and interpreted. The One Lambda Devyser Chimerism NGS assay includes the associated Advyser software, a bioinformatics package which automatically aligns sequencing reads to reference genomes, analyses the specific indels/markers, and calculates the proportion of donor-recipient DNA.

Traditionally STR analysis has required multiple processing steps, making it relatively slow and delaying clinical decisions. In contrast, the Advyser software analysis is automated, takes seconds per sample to facilitate throughput, and includes validation checks and quality control metrics to ensure reliable results. While Advyser automatically picks informative markers based on indels that differ between recipient/donor, the user can also make manual selections based on individual laboratory preference. When reporting, easy-to-interpret tables and graphs highlight any longitudinal shifts in chimerism ratios; if running cell-sorted samples, the graph can also include these different cell lineages for clarity. This user-friendly interface simplifies the analysis process to support timely and informed clinical interventions where appropriate.

5. Reporting

Advances in library preparation, streamlined workflows and integrated bioinformatics ensure that NGS-based chimerism methods, such as the One Lambda Devyser Chimerism NGS assay, easily meet established standards for timely reporting of chimerism results (i.e. that routine chimerism reports must be generated within 5 days and urgent cases within 3 days from sample receipt). Due to its streamlined workflow, the One Lambda Devyser Chimerism NGS kit has the shortest amount of hands-on setup of any NGS-based chimerism assay (45 minutes) with next-day results without compromising accuracy or sensitivity. As a result, NGS chimerism monitoring fits seamlessly within the time-sensitive requirements of clinical settings, providing clinicians with high-quality and timely data to inform patient management decisions.

Why choose One Lambda Devyser Chimerism for NGS?

NGS-based chimerism monitoring has many advantages over traditional STR analysis including greater sensitivity and precision, improved quantitative accuracy, enhanced data interpretation and bioinformatics, reduced turnaround time and long-term cost-effectiveness. With its innovative design, the One Lambda Devyser Chimerism NGS assay offers a reliable, streamlined workflow designed specifically for chimerism monitoring. Compared to competitor NGS alternatives, this assay has the highest sensitivity (0.05%), lowest hands-on time (45 minutes), and is the only one to offer flexibility to the lab; the Chimerism library can be pooled with other One Lambda Devyser NGS workflows including HLA typing (AllType FASTplex) or donor-derived cell-free DNA monitoring (Accept cfDNA) on the same sequencing run.

Changing the game

In an era where precision medicine is centre stage, the shift from STR to NGS represents a significant leap forward for chimerism monitoring. Offering improved sensitivity, NGS enables the delivery of better post-transplant care for patients as clinicians can detect, diagnose and respond to changes in chimerism more effectively than ever. As technology evolves, there is a need for the gold standard in transplant monitoring to also keep pace. As such, the adoption of NGS-based chimerism monitoring not only represents the latest in medical technology but also a powerful tool for improving the lives of transplant patients.

Want to find out more?

Get in touch with your transplant diagnostic colleagues at VH Bio to discuss how to bring NGS-based chimerism into your testing repertoire.