An understanding of the subtle immune interactions of mother and fetus has been hindered by an inability to measure and correlate the frequency and type of fetal cells which have crossed the placenta into the mother's blood with information about the mother's immune system. Such information has been obtained only in a crude fashion and before monoclonal antibodies against B- and T- cell subsets were available. While it has been shown to be theoretically possible to isolate humman fetal cells directly from mother's blood by multiparameter cell sorting techniques, the currently available cell sorting technology is very slow (less than 5000 cells/sec) and unreliable for detecting and isolating rare (less than 0.1 percent) cell subpopulations. Consequently, many hours of cell sorting and/or manual counting of sorter enriched subpopulations are required to isolate these cell subpopulations. Current cytogenetic techniques are geared for relatively large numbers of cells and consequently not very suitable for small subpopulations of sorted cells for which expansion in tissue culture is either undesirable or impossible (e.g., fetal blood cells). Many of the above problems are addressed in this grant. We have developed new high-speed cell analysis and sorting technology capable of accurately and reproducibly analyzing live cells at rates in excess of 100,000 cells/sec and high-speed two-step sorting capable of isolating rare cell populations. Analysis experiments which currently require 5-6 hours of difficult and frequently unreliable work on a conventional cell sorter can be performed in our laboratory in 15 minutes with both ease and reliability using unique hardware and software on a modified commercial cell sorter. Cell sorting can be performed by new high-speed two-step sorting that will yield in less than an hour the same number and purity of cells that it would take a traditional cell sorter more than 8-9 hours to isolate. In addition we demonstrate the feasibility of obtaining karyotypes on small numbers of rare-cell subpopulations which cannot be expanded in tissue culture.
