Single-laser Flow Cytometer Research Articles

A single laser flow cytometer: simplifying multi-color immunophenotyping. (P3303)

Abstract Flow cytometry has been an indispensable tool for immunologists, clinicians and cell biologists for almost 40 years. The power of flow cytometry is in its ability to simultaneously measure multiple features (parameters) per cell and this unique aspect has formed the basis for most cell analysis applications to date. Initially cytometers were only able to distinguish 2 fluorescent dyes (or colors) but the technology has since advanced to multi-laser systems capable of up to 18 color detection. However, these instruments can be considered complex and costly for simple immunophenotyping experiments. The BD Accuri C6 was able to address this need as a simple to use instrument that could perform 4 color immunophenotyping on an individual’s lab bench as opposed to relying on core facilities. We have taken this concept a step further leveraging the exceptionally bright polymeric Sirigen dyes. These dyes, excited off the violet laser, span the visible to near IR spectrum giving one the opportunity to perform up to four color immunophentyping with minimal compensation while maintaining good signal resolution. In support of this, we will show data for simple T cell, B cell and NK cell immunophenotyping using a customized single violet laser Accuri. Details on optimizing filter sets will be given as well as details on how to optimize the reagent panels to be used with this laser/filter set up.

Effect of gene polymorphisms and ethanol consumption on micronucleus frequency in human reticulocytes: a preliminary study

Results from previous studies suggest that alcohol consumption can be genotoxic on peripheral lymphocytes. The aim of our study was to examine the association of alcohol consumption and its genotoxic effect on hematopoietic stem cells in vivo. We investigated 156 healthy Japanese males in a cross-sectional study. Lifestyles, including alcohol drinking behavior and cigarette smoking status, were investigated by means of a self-completed questionnaire. Polymorphisms of ADH1B and ALDH2 were identified by PCR-restriction fragment length polymorphism (RFLP) analysis. The presence of micronuclei in transferrin-positive reticulocytes (MN-RET) was detected with a single-laser flow cytometer. Associations between the genetic polymorphisms, lifestyle factors, and MN-RET frequency were statistically analyzed. We found a significant difference in the mean frequencies of MN-RET between habitual drinkers and non-habitual drinkers (P=0.043), and between the ALDH2*1/*1 and ALDH2*2/*2 genotype (P=0.015). The ADH1B*2 and ALDH2*2 haplotype was estimated to have a significantly higher influence on MN-RET frequency than the ADH1B*2 and ALDH2*1 haplotype (P=0.00035), and the frequency of alcohol consumption played a significant role in MN-RET frequency on the background of the ADH1B*2 and ALDH2*1 haplotype (P=0.012). The results of our study suggest a possible association between the ADH1B and ALDH2 polymorphism and the genotoxic effects of alcohol drinking on hematopoietic stem cells.

Concentrate levels and Saccharomyces cerevisiae affect rumen fluid-associated bacteria numbers in dairy heifers

Total viable rumen bacteria counts through the use of colony-unit forming assays lack accuracy because they only include culturable bacteria capable of initiating cell division. Thus, bacterial counts can be underestimated. The use of fluorescent characteristics of cell membranes allows flow cytometry to enumerate and distinguish dead from live bacteria cells. The objective of this experiment was to investigate the viable and total ruminal bacteria counts when 3 levels of forage:concentrate in diets were fed at restricted levels with the addition of Saccharomyces cerevisiae (YC). Three cannulated post-pubertal Holstein heifers (age 18 ± 1.0 months) were fed corn silage (CS)-based diets in a 3-period (35 d) Latin square design. Heifers were fed the diets for 21 d with no yeast addition, followed by 14 d where yeast culture (YC) was added (1 g/kg as-fed basis); (Yea-Sacc 1026, Alltech, Inc., Nicholasville, KY) . A low concentrate (LC) TMR (80% CS, 20% concentrate; 12.4% CP, 35% NDF), a medium concentrate (MC) TMR (60% CS, 40% concentrate; 12.3% CP. 28% NDF), and a high concentrate (HC) TMR (40% CS, 60% concentrate; 12.6% CP, 25% NDF), were fed once per day. Rumen fluid was sampled − 2, 0, 2, 4, 6, 8, 10, 12 h after feeding. Samples were immediately stained with fluorescent dyes using the BacLight kit (Molecular Probes Inc., Eugene, OR) and analyzed with a Coulter XL-MCL single laser flow cytometer. Mean rumen viable bacteria counts linearly increased among treatments (4.96, 4.78, 6.73 × 10 11 ± 0.53 × 10 11 cells/ml; P = 0.02) for LC, MC and HC respectively, and YC addition increased number of viable bacteria cells ( P < 0.01). Total and viable bacteria counts decreased for the first 2 h after feeding then increased 4 h post-feeding. Dietary concentrate level and YC can alter rumen bacteria counts as measured by this method.

Cross‐Species Genetic Toxicity Assessment Accomplished by Flow Cytometric Analysis of Blood

The formation of micronuclei in blood cells has been an established indicator of genotoxicity for decades. Standard microscopy methods are time-consuming and lack the objectivity that fully automated methods can provide. The ability of flow cytometric technology to rapidly and objectively survey thousands of cells for micronuclei can significantly improve the value of this endpoint. In addition, since many more cells can be scored, and specific populations can be targeted, species that historically have been difficult to obtain micronucleus data from, such as humans, can now be readily evaluated using this methodology. This unit describes a procedure for fixation, staining, and analysis of blood samples using materials supplied in MicroFlow kits (Litron Laboratories) and a single-laser flow cytometer. This methodology provides a reliable, robust assessment of chromosome damage that is used in basic science research as well as drug-safety screening programs at large pharmaceutical and chemical companies.

Analysis of Micronuclei in the Transferrin-receptor Positive Reticulocytes from Peripheral Blood of Nasopharyngeal Cancer Patients Undergoing Radiotherapy by a Single-laser Flow Cytometer

The automated micronucleus test is now accepted as a simple, objective, and accurate method for evaluating potential mutagenic effects caused by physical, chemical or biotic factors. This paper describes a single-laser flow cytometry, based on an immunomagnetic isolation technique in combination with acridine orange staining, to detect frequencies of micronucleated transferrin-receptor positive reticulocytes from human peripheral blood. Using this flow cytometric system, we detected the frequencies of micronucleated transferrin-receptor positive reticulocytes from 10 nasopharyngeal cancer patients undergoing radiotherapy and the baseline of the frequencies of micronucleated transferrin-receptor positive reticulocytes from 7 healthy donors. The results showed that the mean frequency of micronucleated transferrin-receptor positive reticulocytes from healthy donors was 0.236% and that from nasopharyngeal cancer patients before radiotherapy was 0.297%. After radiotherapy it was significantly elevated. When the cumulative dose of radiotherapy was about 20Gy, it reached a maximum of 6.905%, and then, as the cumulative dose of radiotherapy continued to increase to 30Gy, 40Gy and 50Gy, the frequency decreased to 6.258%, 5.119% and 5.007% respectively. Our results indicated that the single-laser flow cytometric system was quick, reasonable and acceptable for detecting the frequency of micronucleated transferrin-receptor positive reticulocytes from human peripheral blood.

Early detection of cytogenetic damage from radiation or chemical agents by flow-cytometric analysis of micronucleated cd71-positive reticulocytes in human peripheral blood

Early detection of cytogenetic damage from radiation or chemical agents by flow-cytometric analysis of micronucleated cd71-positive reticulocytes in human peripheral blood

Micronucleated CD71-positive reticulocytes: a blood-based endpoint of cytogenetic damage in humans

The frequency of micronuclei (also known as Howell–Jolly bodies) in peripheral blood erythrocytes of humans is extremely low due to the efficiency with which the spleen sequesters and destroys these aberrant cells. In the past, this has precluded erythrocyte-based analyses from effectively measuring chromosome damage. In this report, we describe a high-throughput, single-laser flow cytometric system for scoring the incidence of micronucleated reticulocytes (MN-RET) in human blood. Differential staining of these cells was accomplished by combining the immunochemical reagent anti-CD71-FITC with a nucleic acid dye (propidium iodide plus RNase). The immunochemical reagent anti-CD42b-PE was also incorporated into the procedure in order to exclude platelets which can interfere with analysis. This analytical system was evaluated with blood samples from ten healthy volunteers, one splenectomized subject, as well as samples collected from nine cancer patients before and over the course of radio- or chemotherapy. The mean frequency of MN-RET observed for the healthy subjects was 0.09%. This value is nearly two orders of magnitude higher than frequencies observed in mature erythrocytes, and is approximately half the MN-RET frequency observed for the splenectomized subject (0.20%). This suggests that the spleen’s effect on micronucleated cell incidence can be minimized by restricting analyses to the youngest (CD71-positive) fraction of reticulocytes. Furthermore, MN-RET frequencies were significantly elevated in patients undergoing cancer therapy. Collectively, these data establish that micronuclei can be quantified in human peripheral blood reticulocytes with a single-laser flow cytometer, and that these measurements reflect the level of chromosome damage which has occurred in red marrow space.

Enumeration of micronucleated CD71-positive human reticulocytes with a single-laser flow cytometer

The extreme rarity of micronucleated reticulocytes (RETs) in the peripheral blood of non-splenectomized humans has precluded facile enumeration of these cells, as well as evaluation of this endpoint as an index of cytogenetic damage. In this report, we describe a high-throughput, single-laser flow cytometric system for scoring the incidence of micronuclei (MN) in newly formed human RETs. The procedure is based on an immunochemical reagent that differentially labels the most immature fraction of RETs from mature erythrocytes based on the expression level of the transferrin receptor (also known as CD71). The resolution of four erythrocyte populations (young RETs and mature erythrocytes, with and without MN) was achieved for human blood cells treated with phycoerythrin-conjugated anti-CD71, RNase, and either SYTOX Green or SYBR Green I nucleic acid dyes. Anti-glycophorin A labeling of erythroid cells (CyChrome conjugate) was also incorporated into the staining procedure to ensure that debris or other potential artifacts did not adversely impact the analyses. Instrument calibration procedures utilizing malaria-infected rodent erythrocytes were also developed, and are described. Using this analytical system, blood samples from 10 healthy non-splenectomized human volunteers were analyzed for micronucleus frequencies with a single-laser flow cytometer. Average micronucleus frequencies in the mature and most immature fraction of RETs were 0.016 and 0.19%, respectively. Blood samples from three healthy splenectomized volunteers were also evaluated. As expected, these samples exhibited higher micronucleus frequencies in the mature subset of erythrocytes (range 0.03–0.18%). The resulting data suggest that MN can be quantified in human erythrocyte populations with a single-laser flow cytometer, and that the frequency of MN cells in the youngest reticulocyte population approaches values expected in the absence of splenic selection against MN-erythrocytes. This high throughput system is potentially important for evaluating the value of the micronucleated reticulocyte endpoint as an index of chromosome breakage and/or chromosome segregational abnormalities in human populations.

Analysis of single and double-stained alveolar macrophages by flow cytometry

The quantification of cell surface antigens on human alveolar macrophages using flow cytometry is complicated by strong autofluorescence which varies with cell size and granularity. We report here a new method for overcoming the analytical problems caused by autofluorescence. After positioning the unstained cells along the 0, 0; 10 4, 10 4 diagonal on all three fluorescence dot-plots (FL1 vs. FL2; FL2 vs. FL3; FL1 vs. FL3) of a single-laser flow cytometer (excitation wavelength at 488 nm) and adjusting compensation so that the reference FL3 channel profile is not changed by PE-staining, the cell population on the FL3 histogram is arbitrarily classified into subpopulations having similar autofluorescence intensity. These are subsequently back-gated onto the FL1 vs. FL2 dot-plot and separately analyzed. The percentage of stained cells in the whole population is then calculated on the basis of absolute numbers or as the weighted mean of all the subpopulations. This approach permits the analysis not only of single-stained but also double-stained human alveolar macrophages.

A simplified method for Ca2+ flux measurement on isolated human B cells that uses flow cytometry.

A method for Ca2+ flux measurement on isolated human peripheral B cells that uses flow cytometry is described. B cells were isolated by anti-CD19 magnetic bead sorting, and Ca2+ flux was measured with the fluo-3 reagent on a standard single-laser flow cytometer. The response of B-cell stimulation by anti-immunoglobulin B (anti-IgM), anti-IgD, protein A, concanavalin A, and ionomycin was determined. Percentage of responder B cells, the level of Ca2+, and the time of peak stimulation were measured. Bound anti-CD19 monoclonal antibody coupled with small paramagnetic particles did not affect Ca2+ flux. All the isolated B cells responded maximally at 10s with stimulation by 8 microg of ionomycin. The average isolated preparation contains 70% IgM+ and 85% IgD+ cells, all of which showed peak stimulation with 10 microg of anti-IgM and anti-IgD per ml, respectively, at 30s. Only at high concentrations of 80 microg/ml, concanavalin A produced a slower response, peaking at 90 s after stimulation. Stimulation with 20 microg of protein A per ml resulted in Ca2+ flux in only 40 to 60% of cells that had a rapid response and maximal stimulation resembling the pattern of activation of ionomycin. B cells from three patients with mixed cryoglobulinemia with high concentrations of monoclonal rheumatoid factors showed stimulation with aggregated IgG, whereas those from healthy control subjects did not, demonstrating the applicability of the methodology to detection of specific antigen stimulation of B cells. This methodology may be useful in testing the functional capacity of B cells in a variety of diseases. The methodology may also prove useful in studying antigen-specific B-cell responses when they involve a significant percentage of B cells.

Simple and reliable enumeration of micronucleated reticulocytes with a single-laser flow cytometer

A flow cytometric procedure for scoring micronuclei in mouse peripherral blood erythrocytes, especially reticulocytes, is described. The methods reported herein were developed in an effort to simplify the techniques and to reduce the equipment requirements associated with automated micronucleus analyses. With this procedure, fluorescein-conjugated monoclonal antibodies which bind to the CD71-defined antigen (the transferrin receptor) are used to label reticulocytes. The nucleic acid dye propidium iodide is used to identify cells with micronuclei. Given 488 nm excitation, four populations of erythrocytes are clearly resolved: normochromatic erythrocytes with and without micronuclei, and reticulocytes with and without micronuclei. Since the method is capable of simultaneously providing the incidence of micronuclei in both mature and immature erythrocyte populations, it is compatible with either chronic or acute treatment regimens. To demonstrate cell handling and flow cytometric procedures for quantitatively analyzing peripheral blood micronuclei, an experiment with the model clastogen methyl methanesulfonate is described. Additionally, a reconstruction experiment was performed whereby three mouse blood samples were spiked with successively greater volumes of blood from a clastogen-treated animal so each preparation differed slightly, but definitely, in micronucleus content. Each sample was scored six times by conventional microscopy and by flow cytometry so that the two methods could be directly compared. Collectively, the results from the methyl methanesulfonate experiment and the reconstruction study demonstrate the accuracy and reliability of the flow cytometric method. Furthermore, advantages associated with objective, high throughput scoring methodology are clearly indicated.

Establishing a pure lymphocyte gate for subset analysis by flow cytometry

Development of a more cost-effective and efficient method of performing lymphocyte subset analysis is of continuing importance in clinical flow cytometry laboratories. Current two-color methods utilize forward and right angle light scatter and multiple tubes per sample and are thereby liable to gate contamination. Methods using three-color analysis with CD45 vs. right angle light scatter (RALS) gating cannot always exclude a contaminating nonlymphoid population. We have established a two tube approach to directly measure total T cells, T suppressor, and T helper subsets, total B cells and total natural killer cells. The technique involves staining of whole blood with a mixture of five monoclonal antibodies conjugated to three fluorochromes: CD4+CD19 fluorescein isothiocyanate (FITC), CD3+CD33 phycoerythrin (PE), CD45 peridin chlorophyll alpha protein (PerCP), CD8+CD16 FITC, CD3+CD33 PE, and CD45 PerCP. Analysis is performed using a single laser flow cytometer. This method has equivalent recovery to and improved purity of the lymphocyte gate when compared to well-established methods. These antibody combinations additionally allow clear separation of lymphocytes from other leukocytes and debris as well as separation of the T cell helper and suppressor subsets, natural killer cells and B lymphocytes. We additionally provide preliminary data that an accurate lymphocyte subset analysis can be performed on a single tube containing five antibodies (CD4+CD19 FITC, CD3+CD33 PE, and CD45 PerCP), although some measurements are performed deductively.

Use of fluorescence threshold triggering and high-speed flow cytometry for rare event detection.

A simple rare event detection method utilizing dual-parameter flow cytometry is described, which allows quantitation of specific cellular events at the level of two cells in 10(7) total cells. Using a standard unmodified single laser flow cytometer sampling at a rate of 25,000 events/sec and a fluorescence discriminator, 10(7) total cells are processed in 7 min. The assay involves precise characterization of instrument flow rates to calculate total events processed by the cytometer rather than accumulate total events in computer memory. This method of detecting rare events is demonstrated by using a model system of breast cancer cells labeled with a metabolically activated dye and serially diluted into normal peripheral blood. Potential applications include validation of methods to detect minimum residual disease following myeloablative therapy, detection of any remaining tumor cells following purging methods, and validation of methods to detect circulating fetal cells in maternal blood.

Triple immunofluorescence flow cytometry, using whole blood, of CD4 + and CD8 + lymphocytes expressing CD45RO and CD45RA

New fluorescent monoclonal antibody-dye conjugates permit three-color immunofluorescence analysis of leukocytes in whole blood using a single laser flow cytometer. The fluorochrome used in this study is a tandem conjugate of phycoerythrin (PE) and Cyan-5, which is excitable at 488 nm with a maximum in the emission spectrum at > 650 nm and it can be used together with PE and fluorescin isothiocyanate (FITC). The directly labelled monoclonal antibodies are incubated with unseparated anticoagulated blood and subsequently erythrocytes are lysed by a standardized automated procedure. The resulting leukocyte suspension can then be analyzed for three different surface markers in an individual sample of 100 μl blood. When compared simultaneously with single-color analysis triple-color immunofluorescence yielded identical quantitative and qualitative results on various lymphocyte subpopulations. The efficacy of this method was evaluated by analyzing leukocytes of 42 healthy donors for the following markers: CD3, CD4, CD8, CD14, CD16, CD19, CD25, CD38, CD45RO, CD45RA, CD56, CD57, TCR-γ/δ and HLA-DR. Of special interest was the finding that CD45RA and CD45RO are differently expressed in CD4 and CD8 cells. The reliability and convenience of this three-color analysis will make it possible to do more sophisticated examinations of subpopulations and their relevance in the monitoring of autoimmune diseases, immunodeficiency syndromes including AIDS and malignant disorders such as leukemias.

Quantification of effector/target conjugation involving natural killer (NK) or lymphokine activated killer (LAK) cells by two-color flow cytometry

Precise estimates of the frequency of NK- and LAK-target conjugates were obtained by two-color flow cytometry using hydroethidine and calcein as intracellular labels for target cells and effector cells, respectively. These two dyes can easily be used with a standard single-laser flow cytometer with excellent signal separation and dye retention. Hydroethidine labeling did not alter target susceptibility, and calcein labeling did not significantly alter NK function. Excellent agreement was obtained between this flow cytometric method and cisual estimation of the frequency of fresh or IL-2-activated human lymphocytes that from conjugates with K-562 target cells. The percentage of cloned NK or LAK cells that form conjugates with K-562 target cells was dependent on the E : T ratio, with extrapolated maximum conjugate frequencies ( α max) of 40–50%. However, the frequency of lymphocytes forming conjugates with K-562 cells did not closely correlate with the cytolytic activity of a given lymphocyte population. This two color flow cytometric method employing a pair of fluorochromes that do not modify cell membranes or alter cell function in cytotoxicity assays should facilitate further studies of mechanisms involved in the initial stages of target cell recognition by NK and LAK cells.

Flow cytometric multiparameter analysis of proliferating cell nuclear antigen/cyclin and Ki-67 antigen: A new view of the cell cycle

Flow cytometric multiparameter analysis of two proliferation-associated nuclear antigens (proliferating cell nuclear antigen (PCNA)/cyclin and Ki-67) was performed on seven human hematopoietic cell lines. PCNA/cyclin, an S phase-related antigen, was detected using an autoantibody and a fluorescein isothiocyanate-labeled anti-human antibody. The Ki-67 antigen, which in cycling cells is expressed with increasing levels during the S phase with a maximum in the M phase, was detected using a monoclonal antibody and a phycoerythrin-conjugated anti-mouse antibody. In some experiments the PCNA/Ki-67 staining was combined with a DNA stain, 7-amino actinomycin D, and simultaneous detection of the three stains was performed by a single laser flow cytometer. Using this technique four distinct cell populations, representing G 1, S, G 2, and M, respectively, could be demonstrated in cycling cells on the basis of their PCNA/cyclin and Ki-67 levels. The cell cycle phase specificity could be verified using metaphase (vinblastine, colcemide) and G 2 phase (mitoxantrone) blocking agents, as well as by stainings with a mitosis-specific antibody (MPM-2). Also, G 0 cells could be discriminated from G 1 cells in analysis of a mixture of resting peripheral mononuclear blood cells and a proliferating cell line. This technique can be valuable in detailed cell cycle analysis, since all cell cycle phases can be visualized and calculated using a simple double staining procedure.

Single laser flow cytometric detection of lymphocytes binding three antibodies labelled with fluorescein, phycoerythrin and a novel tandem fluorochrome conjugate

In order to implement three-color surface immunofluorescence on a single laser flow cytometer, we combined a new fluorescent tandem conjugate (TC, R-phycoerythrin plus Texas red, available as DuoCHROME) with fluorescein (FITC)- and phycoerythrin (PE)-labelled monoclonal antibodies for the simultaneous detection of three antigens on individual lymphoid cells. Considerable amounts of fluorescence leaked from the PE component of the TC complex into the PE detector, which necessitated compensation of the PE signal. Single and double stainings of peripheral lymphocytes with FITC-, PE-and/or TC-labelled antibodies revealed that the individual labels all identified populations of similar size, and that subsets of T cells could be properly and accurately detected with TC as one label. Triple staining was used to study phenotypically activated HLA-DR + cells within functionally important subsets of CD4 + T cells, i.e., CD45R + CD4 + (naive, ‘suppressor inducer’ T) and UCHL1 + CD4 + (memory, ‘helper’ T) cells. In the blood of healthy subjects as well as patients with primary biliary cirrhosis, CD4 + UCHL1 + (i.e., memory) T cells were significantly more activated than CD4 + UCHL1 − cells. The reciprocal CD45R + CD4 + (naive) T subset expressed HLA-DR to a much lesser extent. In patient samples, this difference in CD4 + T subset activation was more pronounced than among normal lymphocytes. The described triple immunofluorescence staining technique facilitates true multicolor analysis of individual cells on a single laser flow cytometer. Such multiparameter investigations may prove to be important for the further understanding of the phenotypic and functional diversity of immunocompetent cells.

Simultaneous measurement of DNA content and cell‐surface immunofluorescence of human bone marrow cells using a single laser flow cytometer

This paper describes the measurement of S phase DNA content in human bone marrow subpopulations using a single laser method for bivariate analysis of DNA content and cell-surface immunofluorescence (s-IF). Low density (less than 1.077 g/ml) bone marrow cells were labeled with a panel of unconjugated monoclonal antibodies (MoAb) for the lymphoid (CD2 + CD19), T-lymphoid (CD2), B-lymphoid (CD19), erythroid (anti-glycophorin-A), myelomonocytic (CD13, CD33; single and as cocktail) and monocytic (CD14) lineages. A fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse label was used as second step. Unfixed, MoAb-labeled cells were incubated for 24 h with a hypotonic propidium iodide solution for DNA staining. Cells were analysed on a single-laser flow cytometer, operating at 488 nm. The effect of the combined staining protocol upon both s-IF and DNA stainability was evaluated. Only a slight decrease (mean: 29.0%) in s-IF intensity was observed after DNA staining. The percentages of immunofluorescent cells in the bone marrow samples of 10 normal individuals before and after DNA staining were essentially unchanged for all the MoAbs used. The DNA histograms of the immunophenotypically defined subpopulations were of excellent quality with a mean coefficient or variation of 1.8%. This procedure allows the assessment of very low levels of S-phase DNA content, as measured in normal low density blood cells of 8 healthy volunteers (mean 0.07%).(ABSTRACT TRUNCATED AT 250 WORDS)

Resolving leukocytes using axial light loss.

Axial light loss (ALL) is the measurement of the total light lost from the laser beam at 0 degrees when a particle passes through the beam. Used in combination with the monoclonal antibody CD45, ALL can effectively resolve lymphocytes, monocytes, granulocytes, and dead cells in viable or fixed preparations of human peripheral blood. A bivariate display of ALL vs. CD45 clearly resolves all granulocytes from lymphocytes; although degranulated granulocytes cannot be resolved with forward-angle and right-angle light scatter, they are clearly resolved in right-angle scatter vs. CD45. A blood differential can be performed, with a single laser flow cytometer and three colors of fluorescence, when ALL is combined with fluoresceinated CD45 to resolve leukocytes, phycoerythrin-labeled NKH1 to resolve natural killer cells, and biotinylated CD3 in combination with DuoCHROME, the phycoerythrin/Texas red conjugate fluorochrome from Becton Dickinson, to resolve T-cells. B-cells are the only cells negative for both phycoerythrin and Texas red. When PE CD4 is included, the CD3+ CD4+ T-cell subset is resolved from the CD3+ CD4- subset comprising mainly the CD3+ CD8+ T-cell subset. The addition of propidium iodide is unnecessary since ALL clearly resolves dead cells in a viable preparation of human peripheral blood. Furthermore, since ALL resolves these cells even after fixation in paraformaldehyde, all samples can be fixed prior to analysis, thereby minimizing the potential biohazard risk.

A Venn diagram model that allows triple-label immunophenotypic analysis of cells based upon double-label measurements.

Using Venn diagrams derived from set theory, a mathematic model is presented that allows triple-label immunophenotypic analysis of cells based upon double-label measurements. Practical applications of this method are illustrated and discussed. Using this model, triple-label data concerning Leu-8 and Leu-9 (CD7) antigen co-expression by Leu-4+ (CD3+) leukocytes were derived from double-label measurements obtained with a single laser flow cytometer. Mean values were as follows: Leu-4+8+9+ (62%), Leu-4+8-9+ (36%), Leu-4+8+9- (0%), and Leu-4+8-9- (0%). Examples are given of additional models based upon similar principles.