Continuous measurement and analysis of staining kinetics by flow cytometry

Abstract

The measurement of color development with time in cells following the start of a staining reaction is of interest in a number of biological systems. These include the subsets of peripheral white blood cells after acridine orange staining, the uptake by cells and nuclei of fluorescent agents, especially antitumor drugs, and measurement of intracellular enzyme kinetics using fluorogenic o r absorbing substrates. The present work describes a simple computer program for analyzing flow cytometric (FCM) data versus time, including both the population kinetics of color development and the variability of staining speed within one population of cells.A single-channel absorption measurement in flow (Technicon Hemalog D) was used to record peroxidase kinetics in peripheral blood cells. Every 5 s, a 64-channel absorption histogram was recorded, up to a maximum of 64 histograms. The data were then analyzed by a computer program which searched for the peak channel of each histogram. A leastsquares fit was computed for these maxima. The asymmetries of the 64 absorption histograms were compared to see if there was more than one population present with different time constants.Although developed for enzyme kinetic measurements, this program may have wider usefulness in any measurements of time-dependent phenomena by FCM.Key terms: Blood cells, granulocytes, enzymes, enzyme kinetics Flow cytometry (FCM) is used generally for static (endpoint) measurements with quantification of the amount of absorbing or fluorescent dye in a cell at a stable endpoint equilibrium. Occasionally, multiple time points of a dynamic dye-uptake process are sampled by manual collection of FCM data from time-defined subpopulations (6,11,17). Martin and Swartzendruber (13) described a time-base generator which allows time to be used as a true parameter in FCM multi-parameter analysis and so allows FCM analysis of the dynamics of cell staining. Data collected using this time parameter provide an embarrassment of riches, since for any given time interval a population of cells may provide one, two, or more parameters of light absorption, scattering, or emission data as well as electronic cell volume data.The present study reports one approach to dealing with time data in a biologically meaningful way, using time and enzyme product (leukocyte myeloperoxidase) as two parameters. Our approach is similar in principle to that previously reported using a static image processing system (12). MATERIALS AND METHODSFive milliliters whole blood was collected from clinically normal blood donors in ethylenediaminetetraacetate (EDTA) in plastic tubes. For FCM analysis of whole blood, 250 pl blood was mixed with 145 p1 sample diluent and 517 pl fixative a t 4°C (both standard proprietary reagents for the Hemalog D, Technicon Inc. Tarrytown, NY, USA, as are the other reagents described below). The sample was then incubated 5 min at 55°C. After a further 5 min at 4"C, 1.92 ml peroxidase acid (an acetic acid formulation) at 4°C and 0.739 ml dye (4 chloro, 1 naphthol) at room t...

Measurement of myeloid maturation by flow cytochemistry in HL‐60 leukemia: Esterase is inducible, myeloperoxidase is not

“…This overstaining may be masking information about the differences within the neutrophil population. Further studies are needed that use altered staining reactions with less substrate concentration or reduced temperature (1).…”