Bob Ashcroft replies to Jill Martin's question about analyzing WBC markers in unlysed whole blood as follows: >Basically, you need to run at high flow rates, of >50,000 cells/s and then >you need to threshold out most RBCs (say 90%, when 0.1% are WBCs), but the >remainder comprise WBCs at or below 1/1000 cells. Every white cell has 1, 2 >, 3, 4,.. RBCs coincident except for a MoFlo MLS system with 5 microsecond >dead-times, where you get mainly none, one or two coincident RBCs. > >My patent protocols generally use a pulse width and a DNA dye to add an >exclusion gate for non-nucleated cells, but then you must resolve the issue >of RBC coincidences and how they affect gates for the WBC subsets and the >dispersion in fluorescences of positives and negatives! > >If you don't have a MoFlo, then buy one; else forget it! > Bob may be correct about needing a MoFlo to sort white cells uncontaminated by red cells; however, analysis of WBC's in unlysed whole blood can be done on more conventional machines. The field dates back to the 1960's, when a group working with Leitz in Germany (Hallermann L, Thom R, Gerhartz H: Elektronische Differentialzählung von Granulocyten und Lymphocyten nach intravitaler Fluochromierung mit Acridinorange. Verh Deutsch Ges Inn Med 70:217, 1964) considered adding a fluorescence channel to the then current scatter-based blood cell counter. They showed that using acridine orange as a dye would not only allow WBC counting in unfixed blood using fluorescence as a trigger signal, but that different levels of red AO fluorescence could discriminate mononuclear and granulocytic cells. Adams and Kamentsky (Adams LR, Kamentsky LA: Machine characterization of human leukocytes by acridine orange fluorescence. Acta Cytol 15:289, 1971; Adams LR, Kamentsky LA: Fluorimetric characterization of six classes of human leukocytes. Acta Cytol 18:389, 1974 590) showed that differential leukocyte counting in unlysed whole blood could be accomplished with AO; the Los Alamos group confirmed cell identifications by sorting (Steinkamp JA, Romero A, Van Dilla MA: Multiparameter cell sorting: Identification of human leukocytes by acridine orange fluorescence. Acta Cytol 17:113, 1973). The prototype "Cytomat" hematology counters built at Block Engineering in the 1970's analyzed whole blood at 50,000 cells/second, using a nuclear DNA stain to identify and make 6-parameter, 3-beam measurements on WBC's while counting RBC's and platelets and measuring hemoglobin on a cell by cell basis (Curbelo R, Schildkraut ER, Hirschfeld T et al: A generalized machine for automated flow cytology system design. J Histochem Cytochem 24:388, 1976; Kleinerman M: Differential counting of leukocytes and other cells. U S Patent 3,916,205, 1975; Shapiro HM, Schildkraut ER, Curbelo R et al: Combined blood cell counting and classification with fluorochrome stains and flow instrumentation. J Histochem Cytochem 24:396, 1976; Shapiro HM, Schildkraut ER, Curbelo R et al: Cytomat-R: a computer-controlled multiple laser source multiparameter flow cytophotometer system. J Histochem Cytochem 25:836, 1977; Shapiro HM: Fluorescent dyes for differential counts by flow cytometry: does histochemistry tell us much more than cell geometry? J Histochem Cytochem 25:976, 1977). Since then, B-D has obtained patents on counting and measuring surface markers on lymphocytes using immunofluorescence as a trigger signal instead of a nucleic acid stain; Terstappen and Loken (Terstappen LWMM, Loken MR: Five-dimensional flow cytometry as a new approach to blood and bone marrow differentials. Cytometry 9:548-56, 1988), while at B-D, also described staining procedures using a combination of antibodies and the nucleic acid stains thiazole orange and LDS-751. This wasn't commercialized because, while B-D holds patents on thiazole orange, Abbott holds patents on LDS-751. However, the use of cyanine dyes (in which class thiazole orange falls) and styryl dyes (in which class LDS-751 falls) as nucleic acid stains was described in the literature as far back as the 1930's. Thus, there's a great deal of prior art in this area which might have some bearing on current (and should have had some bearing on past) patent applications in this area. I've done T-cell subset analysis in whole blood using nucleic acid stains and/or CD3 immunofluorescence for triggering. The use of a signal other than scatter is critical, as the signals from RBC's will choke the scatter channel of most instruments at rates higher than a few thousand cells/second. With a fluorescence trigger, not only don't you need a MoFlo, you can almost certainly do the job on a FACScan or EPICS XL, and I've done it myself on Cytomutts and the Ortho Cytoron Absolute as well as on the Block instruments. -Howard
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