Chimerism Analysis in the Pediatric Setting: Direct PCR from Bone Marrow, Whole Blood, and Cell Fractions

Abstract

Certain blood components and anticoagulants interfere with the PCR process and subsequent analysis. Here we demonstrate that reliable test results can be obtained for chimerism analysis despite omitting a DNA-extraction step and performing PCR and fragment analysis directly on bone marrow, whole blood, and individual cell fractions. For chimerism analysis, direct-tissue PCR is possible with the use of a robust, commercially available PCR mix containing a DNA polymerase capable of DNA amplification directly from the sample without the need for pretreatment. A total of 178 chimerism samples were processed directly, and results were compared to those obtained from the corresponding DNA sample. No differences were observed between the two sets of results. For the cell fraction-purity assessment, commercially available PCR kitswere used directly on T and B cells without the use of any additional lysing agent. A total of 53 purity samples and their corresponding DNA samples were analyzed and showed a correlation similar to thatobtained for the chimerism samples. The results show that chimerism testing and associated cell fraction-purity assessment can be performed reliably without the need for prior DNA extraction andthatthis method can easily be integrated into existing routine laboratory procedures.