Chimerism monitoring after transplantation is now easier, more sensitive, and more precise
Chimerism monitoring after transplantation is now easier, more sensitive, and more precise
Improving chimerism measurement in post-transplant patient monitoring
Chimerism monitoring following allogenic hematopoietic stem cell transplantation
Hematopoietic cell transplantation is the predominant curative treatment for many malignant and non-malignant haematological diseases. Each year, over 50,000 HCTs are performed world-wide. Better pre-transplantation matching methods, treatment and follow-up has led to increased patient survival, with nearly one million patients worldwide today living with donated Hematopoietic cells. Better post-transplant follow-up can further improve the quality of life for these patients, as well as reduce health care costs. Early detection of graft rejection and disease relapse following HCT improves patient outcomes by allowing treatment to be initiated as quickly as possible after onset of relapse. In order to evaluate the level of donor engraftment, mixed chimerism levels must be carefully monitored after transplantation.
While early detection makes a real difference in post-transplant patient management, the methods commonly used today are associated with challenges. These include:
- Real-time PCR methods are sensitive but have a low accuracy and precision at elevated levels of mixed chimerism
- STR fragment analysis methods are less sensitive and require frequent monitoring to allow early enough detection of relapse to increase the chances for successful intervention
- A combination of real-time PCR and STR fragment analysis methods enables effective monitoring but takes time and requires multiple protocols
- Data analysis and interpretation can be a challenge, especially when using a combination of test protocols
Devyser Chimerism for NGS
Devyser Chimerism for NGS provides labs with one simple protocol for fast and reliable chimerism measurement and monitoring in transplanted patients. Complete relapse monitoring is possible with high sensitivity, accurate measurement and a custom software that makes it easy to follow chimerism trends in patients.
Chimerism analysis using NGS enables high sensitivity and accurate measurement throughout the dynamic range. This is achieved by combining the sensitive analysis of real-time PCR with the accurate measurements at high chimerism levels of STR fragment analysis, as illustrated in these two diagrams.
The first diagram shows high accuracy and correlation between Devyser Chimerism for NGS and STR fragment analysis at high chimerism levels (more than >5% of minority fraction). It is also clear that real-time PCR measurements are generally not accurate at chimerism levels above 20% of minority fraction.
The second diagram, a magnified view of the low end of the range, shows that STR fragment analysis has its limitations in correctly measuring minority fraction below 3-5%. However, Devyser Chimerism for NGS and real-time PCR show high accuracy and correlation at low level minority fraction.
Kit features and benefits of Devyser Chimerism for NGS
High sensitivity allows very early detection of relapse
- Detects down to 0.1% fraction of chimerism
- Accurate and precise measurement over the entire dynamic range from 0.2 – 100% chimerism using a single method
- Allows earlier detection of graft rejection and
disease relapse. - One measurement method regardless of the level of mixed chimerism
Fast and effective workflow from patient sample to report
- A single workflow for both screening and monitoring
- Single-tube NGS method
- Eliminates the need for sample-specific primers and patient-specific marker tracking
- One universal reagent mix for all patients helps reduce reagent wastage and the need for keeping multiple reagents in stock
Easy to use
- User-friendly, designed-for-purpose software with automatic marker identification and chimerism calculation
- Screening samples are sequenced only once
- Speeds up results analysis and eliminates manual data input
- Saves time and increases lab productivity
Learn more about how our single-tube NGS assay can help your team get the results they need.
How this is achieved
24 highly discriminative indel markers
Screening and monitoring is performed using one reagent mix for all genetic markers. All 24 indel markers have strong discriminative power with low bias from ethnic parameters, and ability to analyse HLA identical siblings. Markers were further selected to allow sensitive detection combined with accurate and precise quantification of mixed chimerism.
- Markers were carefully selected using data mining from human genome databases, followed by empirical studies to verify their discriminative power
- Genetic markers with population independent discriminative power distributed across 17 chromosomes
- Enables efficient screening and monitoring of patients with both matched related and unrelated donors
- Allows highly sensitive detection of low level mixed chimerism as well as accurate and precise quantification throughout the dynamic range (0,2-100%)
- Cost efficient
PCA for 48 random markers:
- These markers capture the variation between populations
- Samples from the same populations (i.e. related samples) cannot be distinguished.
Parameter results:
- High FST-index and LD
PCA for Devyser Chimerism markers:
- No clear population structure meaning that the
markers are able to distinguish persons from the same
population/family
Parameter results:
- Low FST-index and LD
Robust performance with highly variable amounts of input DNA
Supports highly variable amounts of input DNA
- Achieve high levels of accuracy and precision throughout a wide dynamic range (0,2 – 100%)
- Suitable for various sample types, including DNA extracted from whole blood, bone marrow and
selected cell populations - Achieve high accuracy even when only a limited amount of DNA is available
Workflow - In short
One streamlined workflow allows your lab to perform marker screening and patient monitoring in parallel, for multple samples, in one simple workflow.
Fast and reliable results
- Significantly reduces assay complexity and
hands-on time - Significantly reduces risk of sample contamination and unnecessary lead time
- Less than 5 hours total assay
- Minimal hands-on time (<45 minutes)
suitable for processing of a large number
of samples - From DNA to results in less than 24 hours
Efficient use of resources
- One-tube solution
- One kit for all patients
- Streamlined, simple and robust NGS workflow uses just one multiplex PCR reaction per patient sample
- Screening samples sequenced only once
- Eliminates the need for sample replicates
NGS library workflow
Devyser’s NGS kits offer a single tube library protocol with indexes that are delivered pre-dispensed in strip or plate format. All patient samples are pooled to a single tube before clean-up. This workflow minimises hands-on time and significantly reduces the risk for sample mix up and contamination.
From DNA to sequencing in under 5 hours with less than 45 minutes hands-on time.
Target amplification
All target markers are amplified using one single tube per patient sample. Recipient/donor pair screening and chimerism monitoring can be performed simultaneously."
Index addition
Patient specific molecular indexes are added to all samples.
Pooling and cleanup
Combine and purify up to 96 samples simultaneously in a single tube.
NGS
Patient samples are sequenced using Next Generation Sequencing.
Data analysis
Data analysis and results reporting using the tailored software ADVYSER for Chimerism.
Data Analysis
Simple and flexible data analysis
The ADVYSER for Chimerism software is used for both marker screening and patient monitoring. In the marker screening process, the software will suggest suitable marker pairs to use for post-transplant monitoring. The software allows analysis and visualization of an unlimited number of monitoring samples and cell types to be followeed for every patient.
- User-friendly, designed-for-purpose software perfectly complements testing kit
- One easy-to-use software for simplified marker selection and monitoring
- Eliminates time-consuming, manual and
complex results analysis and calculations - Simplify challenging monitoring situations
- Reducing the risk for potential sample mix-up
Marker Screening
- Automated comparison and identification of up to 24 informative markers in a recipient/donor pair
- Customizable marker selection or approval of the automated selection
Post-transplant monitoring
- Automated calculation of mixed chimerism
- Visualization of results enables patient monitoring over time
- Visualizations can include an unlimited number of cell types for each patient.
Reporting
- Generate custom reports
- Export patient data for use in other systems