Viable and formaldehyde fixed rat bone marrow or spleen cells and rat or human blood leukocytes were incubated with fluorescein (fluorescein-diacetate, -dibutyrate and -1aurate) or umbelliferone (4-methylumbelliferyl acetate and -phosphate) fluorogenic substrates for subsequent flow cytometric determination of cell fluorescence and cell volume. Formaldehyde-fixed cells preserved between 14 and 20% of the enzyme activity of the unfixed cells and the number of cell clusters for fixed and unfixed cells was the same. The esterase substrates revealed one cell cluster for spleen cells, two for bone marrow cells and four for peripheral blood leukocytes. Phosphatase activity was only associated with a cell cluster of large cells. The time course of substrate cleavage was linear during the first 10 min of incubation. Later on a plateau was reached. Specific enzyme activities were calculated on a single cell level from the simultaneous cell volume and cell fluorescence measurement. Two enzyme activities could be measured simultaneously by using a substrate mixture of umbelliferone acetate and fluorescein acetate.Key terms: Flow cytometry, enzyme kinetics, esterase, phosphatase, hematopoietic cells Enzymatic activities of cells or tissues can be determined by biochemical or by histochemical methods. For biochemical determination the cells or tissue are usually homogenized, the enzymatic activities can then be precisely quantified. Such measurements are, however, bulk measurements, therefore the results are difficult to interpret on a cellular level if the tissue or the cell suspension contain different cell populations. Furthermore the homogenization destroys the cells as functional units which may influence the enzymatic activities. The histochemical approach avoids these problems because enzymatic activities become morphologically apparent on a single cell level. The quantitation of histochemical stains is, however, difficult and time consuming. Since the f i s t cytochemical demonstration of esterase activity in blood cells by GomoriPresented at Automated Cytology VII, Asilomar, California, November 25-30, 1979. 1953 (5), many enzyme staining techniques mostly based on light absorption have been developed (12). The methods are applicable in investigative as well as in diagnostic pathology and play an important role in the identification of hematopoietic cells (15), the investigation and diagnosis of enzyme abnormalities (7), the recognition of cell maturation processes (11) and the classification of leukemias (2, 4). Flow cytometric measurements combine the advantages of biochemical and histologic enzyme determination. They are single cell measurements at high speed (1000-2000 cells/second) and the enzyme activity can be precisely quantified. Flow cytometers have furthermore the advantage to be capable of evaluating simultaneously other cell parameters as cell volume, DNA content or cell membrane receptors. Due to these possibilities flow cytometers can be used for automated differential white cell counting (1 The ai...

The relative surface binding of 11 lectins to human peripheral blood T‐ and B‐lymphocytes, to Molt‐4 and JM T‐cell lines, and to 6410 and NC37 B‐cell lines was determined by flow cytometry. The lectins from Lens culinaris (LCA), Ricinus communis (RCA), Arachis hypogaea (PNA), Abrus precatorius (APA), Ulex europaeus (UEA‐F), Sarothamnus scoparius (SAS‐F), Helix pomatia (HPA), Phaseolus coccineus (L‐PHA), Glycine max (SBA), and Triticum vulgare (WGA) were fluoresceinated and incubated with living, formaldehyde‐fixed, or neuraminidase‐treated cells. Except LCA, which preferentially bound to the two B‐cell lines tested in this study, none of the other lectins exhibited selective binding to the undifferentiated cells of the cell lines. The T‐cell lines and, in part, the peripheral blood T‐cells bound less WGA, APA, LCA, and L‐PHA than the B‐cell lines and the peripheral blood B‐cells. Binding of PNA was found only after neuraminidase treatment of the cells; the binding of PNA, HPA, and UEA‐F after neuraminidase treatment was higher for the T‐cells than the B‐cells from peripheral blood. No significant differences were detected between both cell types for RCA, ConA, SBA, and SAS‐F.

Flow cytometry, usually applied to cells which have time independent features, can also be used for kinetic experiments where the change of cell populations with time is investigated. Dedicated time sequencing programs written in Assembler and incorporated in the CYTOMIC 12 analyzer (4) are described. A sequence of 64 one parameter histograms can be automatically acquired and immediately displayed as a pseudo-twoparameter histogram. The acquisition time for each of the subsequent histograms can be selected between 1 and 32 seconds. Kinet- ~_ _~Flow cytometric techniques have been used to study time dependent changes of biological cells (1, 6, 10, 11). For fast kinetic experiments in the time range of seconds, special transducers are required where the cells are directly introduced from the reaction vessel into the sensing zone of the flow cytometer ( 5 ) . At the same time, the requirements for the data acquisition and processing equipment differ from the requirements for conventional systems in which the cells are considered to have features that are stable with time. The data system must be able to measure, store, and display automatically a large number of data which are coordinated to the different time phases of the cell kinetics under consideration. By composing suitably the data for the different time phases, the course of the cell kinetics can be reconstructed.In general two methods can be applied in flow cytometric time kinetics:First method: With the histogram method, cell features are measured and classified in histograms over distinct time intervals which are normally equal in length. The absolute length of the intervals depends on the rate of change of the cells being investigated. For example, cell change which lasts 60 sec may be observed over 60 time intervals of 1 sec each, and with a pulse rate of 2000 pulses/sec, an average of 2000 cells would be investigated and stored in the form of a histogram during each time interval.ics lasting up to 34 minutes are resolved into 64 time intervals. Two parameter kinetics can be resolved into 12 32 X 32 channel, two parameter histograms which are displayed and evaluated immediately on the analyzer screen in groups of 4 without using complicated list mode procedures. The standard CYTOMIC 12 software can be applied for processing and printing of the sequence distribution curves.