Quantitative analysis of chimerism using a short tandem repeat method on a fluorescent automated DNA sequencer.
Stem cell transplantation (SCT) is the treatment of choice for a number of malignant and nonmalignant diseases. Monitoring of SC engraftment or microchimerism (MC) is important for diagnosis of relapse, rejection or graft vs. host disease (GVHD). The goal of this study was to develop a sensitive and relatively simple method for MC lineage analysis using the Visible Genetics fluorescence automated sequencer. Sensitivity of the method was studied by polymerase chain reaction (PCR) amplification of informative short tandem repeats (STR) using donor/recipient DNA mixtures as the templates and DNA extracted from donor and recipient CD3+, CD19+ and CD15+ cells mixed at various ratios. Semi-quantitative analysis was performed using the Visible Genetics software and percent of donor specific signal was calculated. The sensitivity of this method varied from 0.8% to 6.2% for both DNA and cellular MC in CD3+, CD19+ and CD15+ subsets. Regression analysis revealed linearity (r = 0.94) between the number of donor cells in the mixture and intensity of MC fluorescent signal. These data indicate that the Visible Genetics polyacrylamide gel sequencer can be successfully used for MC analysis in SC recipients providing a relatively high level of sensitivity.
Lobashevsky
A L
AL
Immunology Histocompatibility Laboratory, James Whitcomb Riley Hospital for Children, Indianapolis, IN, 46033, USA. alobashe@iupui.edu
Senkbeil
R W
RW
Townsend
J E
JE
Mink
C A
CA
Thomas
J M
JM
D015703:Antigens, CD / Q000097:blood; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods; D033581:Stem Cell Transplantation; D020080:Tandem Repeat Sequences / Q000235:genetics*; D018183:Transplantation Chimera / Q000097:blood / Q000235:genetics*
D015703:Antigens, CD