Improved flow cytometric method to enumerate residual cells: minimal linear detection limits for platelets,erythrocytes, and leukocytes

Increasing demand for quality control of blood products requires more sensitive methods to enumerate residual cells. Presently, the reported threshold (in cells per microliter) is 400 for red blood cells, 30-500 for platelets, and 1 for leukocytes. To examine precision and linearity in enumerating residual platelets and red blood cells, EDTA-anticoagulated blood from healthy donors was serially diluted with serum, stained in TruCount tubes using a no-lyse/no-wash procedure and a monoclonal antibody cocktail against the CD42a (FL1) and glycophorin-A (FL2) epitopes, and analyzed by flow cytometry. Leukocyte counts were determined in separate tubes. Cell preparation and analysis were performed once for 20 blood samples each and 20 times using the same specimen. Acquisition from the same tube was performed separately for platelets (threshold on FL1) and red blood cells (threshold on FL2). Multiparameter analysis was used for data evaluation. Linear results were obtained for platelets per microliter between 3,410 and 5 and for red blood cells per microliter between 54,000 and 3. For the lower cell concentrations, the coefficient of variation was 16.7% for platelets and 10.9% for red blood cells. The presented method allows the distinction between physiologically intact and ghost red blood cells. The method represents a reliable, sensitive, and accurate approach to quantify platelets and red blood cells in diluted blood. It can be applied to enumerate residual cells in plasma products and meets the increasing demand for quality control in blood components.     

Improved quantification of cell survival on stromal monolayers by flow cytometric analyses

BACKGROUND: The ex vivo survival of leukemic cells maintained on bone marrow stroma is an important tool for the investigation of cell survival and leukemogenesis. Currently, ex vivo survival of leukemic cell survival is measured by coculture on stromal cell monolayers. In these assays, we postulated that two important sources of error might be introduced through either variations in flow volume or in donor stromal cells. METHODS: A previously reported coculture assay that maintains leukemic cells on bone marrow stromal cells was employed. RESULTS: We identified two means of optimizing the coculture assay. First, biologically inert beads having well-characterized fluorescent properties were added to each sample to mathematically adjust for flow-based variations in volume acquisition. The inclusion of fluorescent beads to the basic stromal cell assay showed a significantly lower coefficient of variation as compared to samples analyzed without beads or manually counted using a hemacytometer. Second, in order to minimize variability in bone marrow hematopoietic function between donors, an adherent stromal cell line known to support hematopoiesis (HS-5) was used. When normal human donor stromal cells were used, variability in the survival of leukemic cells was observed on stromal cells derived from different donors. In contrast, statistically significant variability in survival of leukemic cells was not seen on HS-5 monolayers. Finally, we demonstrate that patient-derived leukemic samples may be examined for cell survival using these modifications. CONCLUSIONS: The novel use of fluorescent beads and a hematopoietic-supportive stromal cell line together makes the quantification of stroma-supported cell survival more reproducible, accurate, and amenable to patient-derived samples. These improvements in flow cytometry-based cell quantification are an important step in establishing a role for stromal cell assays in the study of leukemia biology and therapy.     

Mathematical evaluation of two parameter flow cytometric histograms

A general procedure was developed to evaluate two-parameter flow cytometric data. These data usually show overlapping distributions representing different subpopulations. To calculate the fractions of cells in the various subpopulations, the maximum likelihood method is used, assuming an unknown number of Gaussian distributions with constant coefficient of variation in the histogram. A test on simulated data shows that Gaussians can be separated clearly by the procedure if the distance between their mean values is larger than twice the maximum standard deviation. The analysis of different measurements of double-stained (DNA/protein) cultured cells out of the same pool yield the same fractions in the phases of the cell cycle with acceptable error rates. To simulate samples with different fractions in the phases of the cell cycle, defined mixtures of proliferating and resting cells of the same type were prepared and measured for DNA and cell size. The calculated fractions are consistent with the expected variation of G1, S and G2 + M fractions depending on the mixture ratio.     

Improved flow cytometric analysis of the budding yeast cell cycle

The budding yeast, Saccharomyces cerevisiae has been a remarkably useful model system for the study of eukaryotic cell cycle regulation. Flow cytometric analysis of DNA content in budding yeast has become a standard tool for the analysis of cell cycle progression. However, popular protocols utilizing the DNA binding dye, propidium iodide, suffer from a number of drawbacks that confound accurate analysis by flow cytometry. Here we show the utility of the DNA binding dye, SYTOX Green, in the cell cycle analysis of yeast. Samples analyzed using SYTOX Green exhibited better coefficients of variation, improved linearity between DNA content and fluorescence, and decreased peak drift associated with changes in dye concentration, growth conditions or cell size.     

Improved stability of 2,3-bisphosphoglycerate during storage of hexokinase-overloaded erythrocytes

Human red blood cells were overloaded with homogeneous human hexokinase using a procedure of encapsulation based on hypotonic hemolysis and isotonic resealing and reannealing to achieve a final activity that was 15 times higher than that in control cells. Storage for 5 weeks at 4 degrees C of hexokinase-overloaded erythrocytes shows that these cells undergo small K+ leakage and mean cell volume increase compared with control cells. Furthermore, after these 5 weeks of storage the 2,3-bisphosphoglycerate content was normal while the ATP concentration was slightly reduced. These results and other properties suggest that encapsulation of key glycolytic enzymes in erythrocytes can provide a new way to maintain in vitro functionally active red blood cells for at least 5 weeks.     

Multiparameter flow cytometric approach for simultaneous evaluation of T lymphocyte-endothelial cell interactions.

Since vascular endothelium is now recognized as an immunologically active tissue, a better understanding of the relationship between endothelial cells and T lymphocytes is critical to the field of solid organ transplantation. Investigations of endothelial cell-T cell interactions have been limited by methodology. We developed a flow cytometric method allowing for concurrent investigation of multiple cell populations within the same culture that can be applied to these complex interactions. Allogeneic CD8+ or CD4+ T cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) were added to a murine endothelial cell monolayer, in which endothelial proliferation was not inhibited by irradiation or addition of a cell cycle-blocking agent. At specific time points, the coculture was analyzed by flow cytometry. T-cell proliferation could be detected by gating on the T-cell subset and evaluating the CFSE fluorescence peaks. By directly analyzing cellular division, we minimized erroneous interpretation of the data encountered by previous studies, which utilized (3)H-thymidine incorporation as sole measure of proliferation. Further subgating on cells that divided facilitated the study of CD8+ lymphocyte activation, differentiation, and acquisition of effector function. By gating on the endothelial cell population, phenotypic changes such as upregulation of surface MHC molecules or immune-mediated apoptosis could be detected. In conclusion, we present a flow cytometric approach that could have important applications for clinical immunological monitoring in allogeneic or xenogeneic transplantation, and might provide the requisite information to better tailor immunotherapy to prevent chronic rejection.     

Bivalent response to long-term storage in liquid-preserved boar semen: a flow cytometric analysis

The fertility of liquid-preserved boar semen declines during storage at 17°C, insemination trials even indicating early losses in fertilizing ability within the first 24-48 h of storage. Standard semen parameters barely reflect these changes in semen quality, and new approaches for assessment of functional changes in stored spermatozoa are needed. Capacitation, the essential prefertilization step for spermatozoa in the female genital tract, is specifically induced in vitro by bicarbonate. Therefore, we have investigated changes in responsiveness of boar spermatozoa to bicarbonate during storage. Ejaculates of 14 boars were diluted in Beltsville thawing solution, cooled to 17°C and stored for 12, 24, 72, 120, and 168 h before investigation. At each time, basic semen quality was characterized by sperm motility and viability. Subsequently, washed subsamples were incubated in variants of an in vitro fertilization (IVF) medium and assessed for kinetic changes of viability (plasma membrane integrity) and intracellular calcium concentration using flow cytometry in combination with propidium iodide and Fluo-3. By this means, it was possible to determine specific effects of bicarbonate and calcium on sperm subpopulations over incubation time. During storage, standard semen parameters remained on a high level. However, flow cytometric analysis of sperm responses to capacitating and control media revealed two opposing effects of storage. There was a loss of response to bicarbonate in part of the live sperm population but an increasing degree of instability in the rest. Assessment of response to capacitating media by flow cytometry appears a markedly more sensitive way of monitoring sperm functionality during storage than the standard semen parameters of motility and viability.     

Improved method for computing potential doubling time from flow cytometric data

Relative movement methods use the timed progression of the mean fluorescence of cells which have been labeled with monoclonal antibodies against bromodeoxyuridine and displayed with bivariate flow cytometry according to DNA and label content to compute duration of DNA synthesis, TS. The relative movement is the difference of the mean DNA fluorescence of the labeled undivided cells from the G1 channel relative to the difference between the G1 and G2M channels. In this communication, we show how to extend this method to compute the potential doubling time, Tpot, the time required for a population of cells to double, given quiescent cells but no cell loss. A quantity v is introduced that is a function of the fraction of labeled divided cells and the fraction of labeled undivided cells. We show that v is independent of time and is equal to ln(2)Ts/Tpot so that Tpot (equal to ln(2)Ts/v) can be directly found from the information available in computing the relative movement. The method is applied to Chinese hamster ovary cells to demonstrate its utility.     

Multiparametric evaluation of flow cytometric synthesis phase fraction determination in dual-labelled breast carcinomas.

Multiparametric, two-color DNA and cell cycle analyses were performed on 112 consecutive mechanically dissociated, ethanol-fixed breast carcinomas using a dual-label method with monoclonal antibodies (CAM 5.2) to cytokeratin (CK) and leukocyte common antigen (LCA) with propidium iodide (PI) staining. There was marked intertumoral variation of CK-positive (range, 3-87%; mean, 40%) and LCA-positive (range, 1-28%; mean, 6.5%) events in DNA histograms. Approximately 70% of DNA aneuploid cells were CK positive. CAM 5.2-stained (avidin-biotin technique) Cytospin preparations correlated with flow cytometric (FCM) detection of CK-positive cells in 15/21 (71%) cases. In each discrepant case, FCM detected greater numbers of CK-positive cells. Cytospin controls of tumor suspensions revealed that cytoplasmic loss was the major cause of decreased CK staining. Synthesis phase fraction (SPF) calculation from CK-gated histograms resulted in kinetic indices (mean ungated, 12.3%, vs. mean CK-gated, 16.8%; P less than .01) with improved statistical correlations with tumor grade and estrogen receptor (ER) status. Differences between ungated vs. CK-gated SPF were greatest in cases having less than 20% CK-positive events (P less than .05). Cases with lower CK staining events generally had higher SPF and were more often high grade (below median CK staining, 61% high grade, vs. above median CK staining, 31% high grade) and ER-negative (below median CK staining, 55% ER negative, vs. above median CK staining, 12% ER negative).(ABSTRACT TRUNCATED AT 250 WORDS)     

Realtime acquisition, storage, and display of correlated three-parameter flow cytometric data

A microprocessor-based system which performs realtime correlated acquisition, storage and display of multiparameter (3-parameter) data from a flow cytometer (FACS-III) is presented. List-mode techniques are not employed. The 3-parameter data is collected and correlated, then displayed along with cell-frequency as a realtime 3-parameter colour scattergram, while the experiment is in progress; in addition, correlated and uncorrelated higher-resolution projections of the 3-parameter data are collected and stored. The data projections may also be displayed: as 1-parameter histograms, or as 2-parameter colour or grey-scale scattergrams. Examples of 2- and 3-parameter colour scattergrams are presented. The speed and some characteristics of the realtime acquisition and display software are examined; methods to increase the realtime speed are discussed.     

Improved FACS-Gal: flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs

The previously reported FACS-Gal assay (Nolan et al., Proc Natl Acad Sci USA 85:2603-2607, 1988) measures E. coli lacZ-encoded beta-galactosidase activity in individual viable eukaryotic cells for a variety of molecular and cellular biological applications. Enzyme activity is measured by flow cytometry, using a fluorogenic substrate, which is hydrolyzed and retained intracellularly. In this system, lacZ serves both as a reporter gene to quantitate gene expression and as a selectable marker for the fluorescence-activated sorting of cells based on their lacZ expression level. This report details the following improvements of the original assay: 1) use of phenylethyl-beta-D-thiogalactoside, a competitive inhibitor, to inhibit beta-galactosidase activity; 2) reduction of false positives by two-color measurements; and 3) inhibition of interfering mammalian beta-galactosidases by the weak base chloroquine. We found an exponential relationship between fluorescence generated by beta-galactosidase in this assay and the intracellular concentration of beta-galactosidase molecules. Finally, we report conditions for optimal loading of the substrate (FDG) and retention of the product, fluorescein. Under these conditions, we found uniform loading of FDG in all cells of a clone in individual experiments. Together, these improvements make FACS-Gal an extremely powerful tool for investigation of gene expression in eukaryotic cells.     

Flow cytometric determination of carbohydrates in human erythrocytes

Carbohydrate components known from biochemical analysis to be present in peripheral normal human erythrocytes so far could not be detected cytochemically. By periodic acid oxidation followed by Schiff pararosaniline (SO2) staining, however, a specific fluorescent signal can be obtained, strong enough to allow measurement by flow cytometry. Dimethylsuberimidate fixation results in low autofluorescence and low staining of unoxidized cells. By treating erythrocyte ghosts similarly, it is found that about 20% of the signal is present in the membrane, most probably due to glycophorins. The main signal resides in the matrix of the fixed erythrocyte and may be due to traces of glycogen and to the glycosylation of proteins, especially hemoglobin.     

Flow cytometric determination of carbohydrates in human erythrocytes

Summary Carbohydrate components known from biochemical analysis to be present in peripheral normal human erythrocytes so far could not be detected cytochemically. By periodic acid oxidation followed by Schiff pararosaniline (SO₂ sstaining, however, a specific fluorescent signal can be obtained, strong enough to allow measurement by flow cytometry. Dimethylsuberimidate fixation results in low autofluorescence and low staining of unoxidized cells. By treating erythrocyte ghosts similarly, it is found that about 20% of the signal is present in the membrane, most probably due to glycophorins. The main signal resides in the matrix of the fixed erythrocyte and may be due to traces of glycogen and to the glycosylation of proteins, especially hemoglobin.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Long-term storage of nuclear suspensions prepared from paraffin-embedded material for flow cytometric DNA analysis

Nuclear suspensions were prepared from archival material of cancer biopsies. DNA content analysis by flow cytometry was performed after storage at -80 degrees C for 24 h and 6 months, compared to 24 h storage at +4 degrees C. No significant variations in CV or DNA indices were observed. Repeated freezing and thawing did not reduce the DNA histogram quality.     

Determination of glucose metabolites in stored erythrocytes and in erythrocytes from patients with thalassemia by analytical isotachophoresis

Glycolysis is for some cells, such as erythrocytes, neutrophil granulocytes and many cancer cells, the only or most important source of energy (ATP) production. Based on previous studies we developed an isotachophoretic (ITP) method which allows, in principle, the simultaneous determination of all metabolites of glycolysis. Since glucose metabolites are small anions, mobility of some of them may overlap in isotachophoresis and, therefore, partial mixed zones are generated. By variation of the leading/terminating system, however, it is possible to separate the compounds of interest. In this communication, we describe a method for analysis of glucose metabolites in erythrocytes from healthy donors during storage in blood bags, and from patients with thalassemia, with special respect to intracellular 2,3 bisphosphoglycerate, lactate and ATP/ADP. The well known characteristic changes of glycolysis in erythrocytes during blood storage and in erythrocytes from thalassemia patients, which are often analysed by separate enzymatic assays, could be confirmed with this isotachophoretic procedure. The method is currently adapted for analysis of glycolysis in neutrophil granulocytes and cancer cells which requires some modifications of sample preparation and performance of the isotachophoretic analysis.