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DigitalTRACE™: bringing microchimerism to transplant diagnostics

23 March 2022

Haematopoietic stem cell transplantation (HSCT) is the treatment of choice for a variety of malignant and non-malignant haematological disorders. However, HSCT remains a high-risk procedure so post-transplant monitoring of donor-recipient chimerism has become an important tool to monitor graft health. Chimerism analysis detects the relative amount of living donor cells and residual recipient cells, but what is clinically useful is the longitudinal monitoring over time which can show changes in the percentages of donor and recipient cells between different time points. Early detection of engraftment failure is key so that appropriate interventions can be provided.

From the laboratory perspective, a careful balance must be struck between three competing factors when choosing a technique: cost, turnaround time, and sensitivity. Samples received from post-transplant patients must be reported with minimal delay given the clinical urgency, and techniques used must be able to detect low levels of donor-recipient chimerism to have utility. However, consideration must be given to the best use of the laboratory’s budget as a highly sensitive technique may not represent value for money.

JETA DigitalTRACETM

DigitalTRACETM by JETA Molecular is a combined software and reagent package for post-transplant monitoring of patients following HSCT. This includes a wide panel of bi-allelic INDEL (insertion/deletion) markers which ensure individuals can be uniquely identified; this enables the software to calculate the percentage of circulating patient/donor cells. The monitoring assay utilises digital PCR (dPCR) technology to provide a highly sensitive, reproducible microchimerism technique that can detect disease relapse earlier than qPCR and STR methods. Another key point is that DigitalTRACETM has been validated to run on a variety of dPCR instruments, providing laboratories with greater flexibility when choosing to implement dPCR monitoring.

Figure 1. JETA Genotyping kit and Monitoring (INDEL marker) tubes.

DigitalTRACETM contains two main workstreams:

Genotyping

Informative markers are initially identified by genotyping patient and donor(s) using either qPCR or dPCR against the DigitalTRACETM panel; informative markers are present in either the donor or recipient and absent in the other. This technique is suitable for multiple donor transplantation as the panel consists of over 75 markers, ensuring the patient and each donor can be distinguished.

Post-Transplant Monitoring

Post-transplant monitoring samples from the patient can then be assayed against several informative INDEL markers, with different post-transplant timepoints and INDEL reactions in separate wells. Using dPCR each individual well is partitioned into thousands of discrete reactions; each of which, with or without the target, is amplified and evaluated separately. Wells are assigned as either positive (1) or negative (0) for the target enabling quantitative measurement of the amount of target present. The need for duplicates is also minimised as each partition is treated as a separate reaction; results also show high precision using different INDEL markers for the same time point.

Figure 2. Depending on the dPCR instrument/reagents used, each well can be separated into 8,500 to 26,000 partitions. These images depict how partitioned wells are graded positive (green) or negative (grey) for the target, with increased target concentration from left to right.

The results from both workstreams are checked using the instrument software then imported into the TRACE Analysis software. This has a wide range of functionality, including creating lab protocols for each run, performing virtual typing between patients and donors, longitudinal sample tracking, and producing automated reports.

Available Methodologies

Although PCR-STR has been considered the de rigueur technique for post-transplant monitoring in the UK, it is unlikely to continue unchallenged given the implementation of routine next-generation sequencing (NGS) which reduces the reliance on genetic analysers. There is also an appetite for microchimerism, where small populations of <1% can be detected, following several studies (such as Ahci 2017, Sellman 2018, Valero-Garcia 2019) which highlight the clinical utility of this level of sensitivity. Microchimerism provides strong evidence to exclude relapse when appropriate and additionally can predict relapses more than a month (up to 58 days) before clinical diagnosis. Unfortunately, STR lacks the sensitivity to assess <1% cell populations so the current alternative methods offering microchimerism sensitivity fall into three categories: qPCR, NGS, and dPCR (DigitalTRACETM).

Figure 3. Comparison between four different techniques for chimerism monitoring. Note that the STR limit of detection is insufficient for microchimerism analysis.

qPCR uses relative quantification so large volumes of pre-transplant DNA (for the minor population; either patient or donor) are needed to create reference curves to interpret each run; there is also reduced accuracy for this method when measuring higher concentrations (>10%).

NGS is the most sensitive technique, reporting the actual nucleotide differences present. However, the method is time-consuming for both practical and analysis work and has shown reduced accuracy when multiple donors are involved. It is also considerably more expensive than all other techniques available and could potentially slow routine laboratory workflow as it requires the use of a sequencing instrument.

DigitalTRACETM is ideally placed in between the alternatives of qPCR and NGS methods, sharing many benefits while avoiding many disadvantages. Unlike qPCR, DigitalTRACETM does not need large volumes of pre-transplant DNA as the method provides absolute quantification, and measurement accuracy is consistent across all concentrations. Unlike NGS, the DigitalTRACETM technique is streamlined for minimal amounts of practical and analysis time and is effective in situations where multiple donors have been transplanted (e.g., cord transplantation/HSCT re-transplant). It is also significantly cheaper than NGS and relies on different instrumentation therefore would not affect routine NGS workflow.

 

VH Bio Ltd. has partnered with JETA Molecular to bring dPCR microchimerism to the UK and Eire market.  Contact VH Bio Ltd. to discuss your post-transplant monitoring needs and arrange a DigitalTRACETM demo!

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