Capillary electrophoresis for chimerism monitoring by PCR amplification of microsatellite markers after allogeneic hematopoietic cell transplantation.
BACKGROUND
Hematopoietic chimerism has been demonstrated to be relevant for donor cell engraftment and detection of minimal residual disease after allogeneic hematopoietic cell transplantation (aHCT). In the light of increasing numbers of non-myeloablative aHCT as a treatment modality sensitive, rapid, and accurate chimerism monitoring techniques acquire novel relevance.


METHODS
We evaluated the informativeness of five microsatellite markers in 376 donor/recipient pairs and evaluated the ability of capillary electrophoresis to detect mixed chimerism after aHCT. The sensitivity for capillary electrophoresis with respect to different markers was determined by limiting dilution assays with mixed chimerism samples containing defined amounts of cells or DNA. Furthermore, capillary electrophoresis was applied in 17 retrospectively selected patients with a mixed chimerism detected previously by gel electrophoresis, having undergone aHCT for different hematologic diseases and initially achieving a complete donor chimerism.


RESULTS
In 163 of 165 (98%) of all related and 210 of 211 (99%) unrelated transplants the microsatellites identified informative alleles. The sensitivity and accuracy was higher with capillary electrophoresis when compared with gel electrophoresis with three of five microsatellites. Potential pitfalls with the application of capillary electrophoresis was preferential amplification and the occurrence of stutter peaks in the representative area. Investigation of the selected patient samples demonstrated that detection of a mixed chimerism was earlier with capillary electrophoresis when compared with gel electrophoresis. The detected recipient genotype by capillary electrophoresis examination, despite a negative gel electrophoresis result, ranged from 0.7 to 7.1%.


CONCLUSIONS
We conclude that chimerism assessment with our five microsatellites identified informative alleles in 99% of all donor/recipient pairs and may therefore be of use when establishing an institutional chimerism testing procedure. Capillary electrophoresis displayed a high sensitivity and accuracy for detecting a mixed chimerism in vitro and in vivo.
Spyridonidis
Alexandros
A
Department of Hematology and Oncology, Albert Ludwigs University Medical Center, Freiburg, Germany.
Zeiser
Robert
R
Wäsch
Ralph
R
Bertz
Hartmut
H
Finke
Jürgen
J
D046528:Chimerism; D019075:Electrophoresis, Capillary / Q000379:methods*; D006402:Hematologic Diseases / Q000628:therapy; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D066298:In Vitro Techniques; D008206:Lymphatic Diseases / Q000628:therapy; D018895:Microsatellite Repeats / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014184:Transplantation, Homologous