A Rapid Automated Method for the Sequential Isolation of CD19, CD3 and Myeloid Cells from One Tube of Whole Blood.

Abstract Immune ablation followed by allogeneic hematopoietic cell transplantation in humans necessitates hematopoietic cell reconstitution and immune re-education. All blood cell lineages are affected and post-transplantation immune restoration depends upon the graft’s ability to generate both lymphoid and myeloid lineage cells. Decisions regarding immunomodulation treatment post-transplantation are often made on the basis of chimerism testing. Chimerism analysis is typically performed on small blood samples, especially with pediatric patients. Since lymphoid and myeloid engraftment is asynchronous the determination of lineage-specific chimerism is needed. Analysis of purified cell subsets requires techniques which can isolate >1 cell type from a single small starting sample. This avoids dividing the sample. Performing flow cytometry as well as isolation of DNA from the purified subsets means that high cell recovery is essential. Preparation of the sample using a ficoll-based method often results in cell loss of 50% while certain lysis and wash steps can affect granulocyte content. We describe a method of sequential selections to isolate B cells, then T cells and finally myeloid lineages (CD33+ and/or CD66b+) using a fully automated pipetting robot called RoboSep®. RoboSep® can process sample sizes that range from 0.5 to 4.25 ml of human whole blood. CD19 (B cell) positive and CD3 (T cell) positive and myeloid cell fractions are isolated using immunomagnetic, column-free positive selection (EasySep®). Briefly, cells are first labeled with antibody targeting CD19 positive cells. These are then coupled to magnetic nanoparticles and the sample is placed in a magnet. The supernatant with unlabeled cells is removed to a new tube, leaving isolated CD19 positive cells in the magnet. The supernatant is then labeled with anti-CD3 antibody, magnetic nanoparticles, placed in a magnet and the supernatant is removed to a new tube leaving isolated CD3 positive cells. Finally, a cocktail of antibodies (anti-CD33, anti-CD66b) is used to label and select the myeloid cells from the supernatant. The resultant positive cells are collected in the magnet. Assessment by flow cytometry yields average purities over 90% for all cell types. Cell isolation using this method produces highly purified cells in quantities sufficient to generate genomic DNA for chimerism testing, even from very small amounts of starting sample. For example, 2.0 ml of whole blood yields on average 1.3ug of B cell genomic DNA, 10.2ug of T cell genomic DNA and 6.1ug of myeloid cell genomic DNA. In conclusion, we have developed a rapid, fully automated RoboSep® method to sequentially isolate highly purified B cells, then T cells and finally myeloid cells from a single starting sample of whole blood. The number of cells (x106) and amount of total genomic DNA (range) obtained from 2.0 ml of whole blood starting sample (n=3). No. Enriched Cells Total DNA (ug) CD19+ 0.12 – 0.34 1.1 – 1.6 CD3+ 1.8 – 3.2 7.9 – 11.9 Myeloid 2.2 – 2.9 4 – 7.2

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