Evaluation and Automation of Hematopoietic Chimerism Analysis Based on Real-Time Quantitative Polymerase Chain Reaction

• Published in: Biology of blood and marrow transplantation 2005-07, Vol.11 (7), p.558-566
• Main Authors: Masmas, Tania N., Madsen, Hans O., Petersen, Søren L., Ryder, Lars P., Svejgaard, Arne, Alizadeh, Mehdi, Vindeløv, Lars L.
• Format: Article
• Language: English
• Subjects: Evaluation Studies as Topic; Hematopoiesis - genetics; Hematopoietic Stem Cell Transplantation; Humans; Minimal residual disease; Neoplasm, Residual - genetics; Polymerase chain reaction; Polymerase Chain Reaction - methods; Polymorphism; Polymorphism, Genetic; Sensitivity and Specificity; Transplantation chimera; Transplantation Chimera - genetics
• ISSN: 1083-8791; 1523-6536
• DOI: 10.1016/j.bbmt.2005.04.004

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.