High-throughput cell analysis and sorting technologies for clinical diagnostics and therapeutics

A number of theoretical and practical limits of high-speed flow cytometry/cell sorting are important for clinical diagnostics and therapeutics. Three applications include: (1) stem cell isolation with tumor purging for minimal residual disease (MRD) monitoring and treatment, (2) identification and isolation of human fetal cells from maternal blood for prenatal diagnostics (PD) and in-vitro therapeutics, and (3) high-speed library screening for recombinant vaccine production (RVP) against unknown pathogens. For clinical diagnostics it is important to obtain highly purified cell subpopulations for subsequent DNA sequencing or gene expression microarray analyses. Using single-cell DNA PCR and TA cloning techniques it is possible to detect a single base pair mutation in a single gene of a tumor cell for MRD or PD. Practical gene expression microarray analysis requires analyses of relatively large numbers of purified cell subpopulations isolated from heterogeneous populations unless sequences can be reliably pre-amplified. Post-separation molecular characterization technologies such as single-cell PCR and single cell sequencing and gene expression microarray analyses depend on the delivery of purified cells or cell subpopulations. Otherwise these molecular characterization technologies superimpose the DNA sequence or gene expression profile patterns of the different cell types present. In particular, gene expression profiles from microarray analyses, performed on mixtures of cell types, cell cycle and cell differentiation stages, activated and non-activated cells are at best compromised in value and at worst of little value. Clinical therapeutics requires safe and sterile isolation of cell subpopulations for subsequent manipulation (e.g. genetic-engineering) prior to re-infusion into the patient in MRD therapeutics. Contaminating cell types, especially those dangerous to the patient (e.g. tumor cells within autologous transplants), can compromise patient therapy. Some measure of quality assurance is needed to give a measure of the purity of the cell separation. RVP against unknown pathogens requires very high-speed screening of large libraries of bacteria containing expressed human immunoglobulin sequences. Theoretical and practical implications of high-speed library screening are discussed in this context.