Method development of magnetic cell isolation and DNA extraction of small cell populations from Ficoll-separated hematopoietic cells

Sammanfattning: Clonal haematopoiesis of indeterminate potential, or CHIP are a family of mutations present in the general population. CHIP-mutations are prevalent in the haematopoietic stem cells and in the more mature cell populations, T-lymphocytes, B-lymphocytes and myeloid cells (CD3+, CD19+ and CD33+ cells) in blood. By separating these cell populations using magnetic isolation, extracting DNA from the cell populations, and detecting the same mutation in all cell populations, one can prove the presence of CHIP-mutations in a hematopoietic stem cell. At least 50 ng good quality DNA is needed for the gene analysis to detect CHIP-mutations. The magnet separated cell population may be very small, so the DNA extraction method must be optimized to achieve enough DNA yield. The main purpose of the method development was to compare two storage methods before DNA-extractions, and then three different DNA-quantification methods after the DNA-extractions. After the best storage and quantification methods were identified, five samples of cryo-preserved viable cells were used to isolate cell populations using magnetic beads covered in specific antibodies and a magnetic field, and then quantified. Results of the study showed that the best way was to store the cells in ATL-buffer and Proteinase K. To quantify DNA, qPCR was the most accurate method, since the other methods showed incorrect results because of the low DNA concentrations. Magnet cell separation was partly successful. All except one of the DNA yields from the cell separation protocols reached the critical amount of DNA, but some yields were not pure yields of the sought-after cell population. In general, the method must be worked on more with further research.