Standardisation of multiplex fluorescent short tandem repeat analysis for chimerism testing.

Area of Science:

• Biochemistry
• Genetics
• Immunology

Background:

• Assessing cell origin post-allogeneic hematopoietic stem cell transplantation (HSCT) is critical for patient outcomes.
• Multiplex fluorescent short tandem repeat (STR) analysis is a key technology for chimerism analysis.

Purpose of the Study:

• To optimize and evaluate two commercial STR analysis systems for determining cell origin after HSCT.
• To establish a standardized procedure for reproducible chimerism analysis.

Main Methods:

• Evaluation of AmpFlSTR Profiler Plus and GenePrint Powerplex-16 systems using multiplex fluorescent STR analysis.
• Development of a standard interpretation procedure for electropherographs, including classification of allelic distribution types (TYPE-I, TYPE-II, TYPE-III) and exclusion of stutter peaks.
• Intralaboratory and multicentre validation of the AmpFlSTR Profiler Plus system.

Main Results:

• A standard interpretation procedure was essential for reproducible results.
• Mixed blood samples were determined with <2% absolute deviation using the AmpFlSTR Profiler Plus system.
• Sensitivity thresholds were set at 5% for TYPE-I and 10% for TYPE-II markers.
• No significant difference in chimerism values was observed between shared STR markers across systems.
• The method's applicability was demonstrated in 26 allogeneic peripheral blood stem cell transplants.

Conclusions:

• Optimized STR analysis provides a reliable method for evaluating cell origin after allogeneic HSCT.
• The validated AmpFlSTR Profiler Plus system offers accurate and reproducible chimerism quantification.
• Standardized protocols and defined sensitivity thresholds are crucial for robust chimerism monitoring in clinical practice.