Evaluation and automation of hematopoietic chimerism analysis based on real-time quantitative polymerase chain reaction.
Abstract Chimerism analysis is an essential tool in the follow-up of patients after allogeneic stem cell transplantation. High-resolution methods for chimerism analysis based on real-time quantitative polymerase chain reaction (RQ-PCR) with a detection limit of 0.1% marker-specific cells are especially valuable in the detection of patient-derived subpopulations for the monitoring of minimal residual disease. Using artificial chimeric mixtures of genotypically different cells, we optimized and evaluated the intrasample variation, accuracy, and detection limit of chimerism analysis based on RQ-PCR of short insertion and deletion polymorphisms. Furthermore, automated setup by robot was evaluated. The results were accurate, with acceptable intrasample variation at and above 0.1% marker-specific cells. The sensitivity was mainly limited by background values. Chimerism results based on RQ-PCR were similar to results based on PCR of short tandem repeats when samples from recipients of transplants with nonmyeloablative conditioning were analyzed. Furthermore, automated setup was feasible in a time-, labor-, and reagent-conserving manner.
Masmas
Tania N
TN
Lymphocyte Research Laboratory, Department of Hematology, Rigshospitalet, Copenhagen, Denmark. masmas@dadlnet.dk
Madsen
Hans O
HO
Petersen
Søren L
SL
Ryder
Lars P
LP
Svejgaard
Arne
A
Alizadeh
Mehdi
M
Vindeløv
Lars L
LL
D005069:Evaluation Studies as Topic; D006410:Hematopoiesis / Q000235:genetics; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D018365:Neoplasm, Residual / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012680:Sensitivity and Specificity; D018183:Transplantation Chimera / Q000235:genetics*