[Application of free flow preparative methods for the separation of blood cells]

Human blood cells can be separated using an apparatus described by Hanning and co-workers (Model FF4 Desaga Heidelberg). The method of free flow preparative electrophoresis uses a specific property of blood cells when subjected to an electric field: their electrophoretic mobility. Three regions of migration can be defined: --a high mobility region (HMR); --a low mobility region (LMR) and --an intermediate mobility region (IMR). After the electrophoretic migration, the various cell fractions are identified. If human leucocytes are subjected to an electric field (40 volts/cm) it is possible to isolate several populations which differ by their membranes electric charge. The repartition of the neutrophiles granulocytes is twofold: --one fraction of high mobility (HMR) and --one fraction of low mobility (LMR). The meaning of these two populations is being discussed. Between these teo fractions is located the lymphocyte fraction which is represented by a single peak of intermediate mobility (IMR). Eosinophiles and monocytes are concentrated in low mobility fraction (LMR). With appropriate migration parameters (80 volts/cm, various component buffers) it is possible to obtain from lymphocytes, purified by density gradient, an electrophoretic separation of T and B lymphocytes population. Histograms obtained from quantitative measures (on collected cell fractions) show an unimodal distribution. When separated cells are characterized by three membranes markers (E. Rosettes, EAC Rosettes and surface immunoglobulins) the electrophoretic heterogeneity of B and T cells is then demonstrated. With E Rosettes (T cells) the majority of T lymphocytes is distributed in HMR and IMR. With EAC Rosettes and membrane immunofluorescence (B cells markers) the majority of B lymphocytes is found in the LMR. Thus an important enrichment of T and B sub-population is obtained, but contamination from one another still remains too important. More selective methods should lead to an improvement of this cell separation.