Multiparameter Flow Cytometer Research Articles

Highlighting the Prognostic Importance of Measurable Residual Disease Among Acute Myeloid Leukemia Risk Factors

Objective:The optimal timing of measurable residual disease (MRD) evaluation in acute myeloid leukemia (AML) patients has not been well defined yet. We aimed to investigate the impact of MRD in pre- and post-allogeneic hematopoietic stem cell transplantation (AHSCT) periods on prognostic parameters.Materials and Methods:Seventy-seven AML patients who underwent AHSCT in complete morphological remission were included. MRD analyses were performed by 10-color MFC and 10-4 was defined as positive. Relapse risk and survival outcomes were assessed based on pre- and post-AHSCT MRD positivity.Results:The median age of the patients was 46 (range: 18-71) years, and 41 (53.2%) were male while 36 (46.8%) were female. The median follow-up after AHSCT was 12.2 months (range: 0.2-73.0). The 2-year overall survival (OS) in the entire cohort was 37.0%, with a significant difference between patients who were MRD-negative and MRD-positive before AHSCT, estimated as 63.0% versus 16.0%, respectively (p=0.005). MRD positivity at +28 days after AHSCT was also associated with significantly inferior 2-year OS when compared to MRD negativity (p=0.03). The risk of relapse at 1 year was 2.4 times higher (95% confidence interval: 1.1-5.6; p=0.04) in the pre-AHSCT MRD-positive group when compared to the MRD-negative group regardless of other transplant-related factors, including pre-AHSCT disease status (i.e., complete remission 1 and 2). Event-free survival (EFS) was significantly shorter in patients who were pre-AHSCT MRD-positive (p=0.016). Post-AHSCT MRD positivity was also related to an increased relapse risk. OS and EFS were significantly inferior among MRD-positive patients at +28 days after AHSCT (p=0.03 and p=0.019).Conclusion:Our results indicate the importance of MRD before and after AHSCT independently of other factors.

Urinary Cellular Profile as a Biomarker for Lupus Nephritis.

A search for the ideal biomarker for lupus nephritis (LN) is still underway, one that can be used for early detection and correlate with the class and activity of LN. Urine is normally devoid of leukocytes; however, it has been observed that macrophages and T lymphocytes are routinely present in the urine of LN patients and those with other proliferative renal diseases. This provides the idea for their potential use as biomarkers for proliferative LN. Here, we measured the urinary CD4+, CD8+ T lymphocytes, and CD14+ monocytes in patients with systemic lupus erythematosus (SLE) as potential biomarkers for LN. A longitudinal case-control study included 30 SLE patients with LN, 30 SLE patients without past or current LN, and 20 healthy subjects as a control group. The flow cytometric analysis was done using BD FACS Calibur multiparameter flow cytometer equipped with BD CellQuest Pro software for data analysis. CD14+ cells were the most abundant cells in the urine of LN patients. The mean numbers of urinary CD8+, CD4+, and CD14+ cells/mL were significantly higher in patients with LN than in those without. The cell counts correlated significantly with proteinuria. Urinary CD14+ cells seem to occur in much higher counts in class IV than class III LN. Urinary CD8+, CD4+, and CD14+ cells are highly sensitive and specific markers for detecting proliferative LN. A low CD4:CD8 ratio provides a further clue. Urinary CD14 cell counts may be a potential biomarker to differentiate between the different classes of proliferative LN.

Detection of mouse natural killer T follicular helper (NKT(FH)) cells by flow cytometry.

Natural killer T follicular helper (NKT(FH)) cell, a recently identified B-cell helper innate cell population, can be easily missed due to its low frequency and the fact that it only forms upon immunization or infection with glycolipid-containing antigens or microbes. Here, we describe our in-house optimized protocol to detect these mouse NKT(FH) cells by multiparameter flow cytometer.

Evaluation of zebu nellore cattle blood samples using the Cell-Dyn 3500 hematology analyzer

The Cell-dyn 3500 is a multiparameter flow cytometer, which may analyze samples from several species performing several simultaneous analyses. It is able to perform white blood cells, red blood cells and platelet counts, besides differential leukocyte counts, packed cell volume and hemoglobin determination. Cell-Dyn 3500 performs total leukocyte count both optically and by impedance. The equipment may choose one or other method, based on the reliability of the results. Erythrocyte and platelet counts are determined by impedance. Leukocyte differentiation is based on an optical principle, using separation in multiangular polarized light. The objective of this study was to compare the results of complete blood count of Zebu Nellore heifers from Cell-dyn 3500, with those obtained from a semi-automated cell counter (Celm CC 510) and the manual technique. Blood samples were collected from the jugular vein in 5 mL EDTA vacuum tubes from 58 Nellore heifers, at 24 months of age. Samples were processed in parallel in the three different techniques. Results were analyzed using paired t test, Pearson's correlation and the Bland-Altmann method. There was a strong correlation for all parameters analyzed by Cell-Dyn 3500, manual method and semi-automated cell counter, except for basophils and monocytes counts. These results confirm that this analyzer is reliable for blood samples analysis of zebu cattle.

Prognostic Value Of Quantifying Circulating Plasma Cells By Multiparametric Flow Cytometry In Patients With Relapsed Multiple Myeloma

BackgroundThe presence of circulating plasma cells (PCs) in multiple myeloma (MM) is a known poor prognostic marker. Prior studies have utilized a technically challenging slide-based immunofluorescence technique to detect and quantify the presence of circulating PCs which was not widely adapted in clinical practice. More recently, the routine use of flow cytometry has provided the opportunity to quantitatively assess circulating PCs in MM patients with relative ease. We report the prognostic value of quantifying circulating clonal PCs using multiparametric flow cytometry in MM patients with relapsed disease. MethodsWe evaluated all MM patients seen at the Mayo Clinic, Rochester from 2009 to 2011 with previous or ongoing relapsed disease and who had their peripheral blood samples evaluated by flow cytometry. Each blood sample had its peripheral blood mononuclear cells isolated by ficoll gradient and stained with antibodies to CD45, CD19, CD38, CD138 and cytoplasmic Kappa and Lambda Ig light chains. A six-color multi-parameter flow cytometer (Becton Dickinson FacsCantos II) was used to examine each sample with a target of detecting 150,000 events (cells) that was then analyzed using the Facs Diva Software. Plasma cells were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Normal PC's were then separated from clonal plasma cells based on the differential expression of CD45, CD19 and polytypic Ig light chains. The clonal plasma cells detected were reported out as the number of clonal events/150,000 collected total events. For those samples where less than 150,000 events were gated or examined, the number of final clonal events was adjusted to 150,000 events. Survival analysis was performed by the Kaplan-Meier method and differences in survival assessed using the log rank test. ResultsThere were 647 consecutive patients with a history of treated MM who had a peripheral blood flow cytometry as part of their routine clinical evaluation. The median patient age was 62 years (29-88) and 55% were male. The median time since diagnosis was 12 months (range: 1-363) and the median number of lines of treatment received was 2 (1-11). There were 81 (13%) patients with clonal circulating PCs with a median of 368 cells (4 – 133,464). The 2-year overall survival (OS) for the 81 (13%) patients with any circulating PCs was 17% compared with 65% for those with none (P<0.001). The presence of circulating clonal PCs was associated with high-risk disease by FISH (P <0.001) as well as higher PCLI (P <0.001). We then correlated the presence of circulating clonal PCs with the disease status at flow cytometry assessment. Among the study patients, only 145 (22%) had actively relapsing disease with the remaining 502 (78%) in a plateau including patients in CR. Circulating clonal PCs were more likely to be detected in patients with actively relapsing disease compared with plateau phase (43% vs. 4%, P < 0.001); none of the CR patients and <5% of the remaining plateau phase patients had any detectable circulating clonal PCs.We then restricted the analysis to the actively relapsing patients; the best cutoff predicting 1-year mortality by ROC analysis was 100 events. Based on this, we defined >=100 events as a cutoff for defining the prognostic role of circulating clonal PCs in actively relapsing patients. The median OS for those with >=100 clonal PC events was 12 months compared with not reached for those with <100 events (p<0.001; Figure 1 [Display omitted] ). Among patients with actively relapsing disease, >= 100 circulating PCs was associated with a higher ISS stage, plasma cell labeling index and bone marrow PC% compared with those with <100 circulating PCs. In a multivariable model, only LDH > 222 (HR: 2.08, P = 0.045) and >= 100 circulating PCs (HR: 2.48, P = 0.048) were found to adversely affect OS in actively relapsing patients. ConclusionThe utilization of flow cytometry to quantify circulating clonal PCs in patients with relapsed MM appears to have significant prognostic relevance. In patients with actively relapsing disease, >= 100 circulating PCs predicted for worse OS with a median of 12 months. Future studies are needed to determine if this would allow an opportunity to develop a more risk adapted approach for patients with relapsed disease.▪ Disclosures:Kumar:Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding. [Display omitted]

Increased Circulating Plasma Cells On Multiparametric Flow Cytometry As An Independent Prognostic Biomarker In Newly Diagnosed Multiple Myeloma: Implications For Redefining High-Risk Myeloma

BackgroundPrior studies have shown that presence of increased circulating plasma cell (PCs) identified using a slide-based immunofluorescence method is an adverse prognostic marker for overall survival in multiple myeloma (MM), and increases the risk of progression in patients with MGUS and smoldering MM. However, utility in clinical settings has been limited by the cumbersome nature of the test and lack of widespread availability. We studied the prognostic value of circulating PCs using sensitive multiparametric flow cytometry that enable us to quantitatively assess circulating PCs in patients with MM. MethodsWe evaluated all newly diagnosed MM patients seen at the Mayo Clinic, Rochester from 2009 to 2011 who had their peripheral blood samples evaluated by flow cytometry prior to therapy. Patients with plasma cell leukemia were excluded. Each blood sample had its peripheral blood mononuclear cells isolated by ficoll gradient and stained with antibodies to CD45, CD19, CD38, CD138 and cytoplasmic Kappa and Lambda Ig light chains. A six-color multi-parameter flow cytometer (Becton Dickinson FacsCantos II) was used to examine each sample with a target of detecting 150,000 events (cells) that was then analyzed using the Facs Diva Software. PCs were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Normal PC's were then separated from clonal PCs based on the differential expression of CD45, CD19 and polytypic Ig light chains. The clonal PCs detected were reported out as the number of clonal events/150,000 collected total events. For those samples where less than 150,000 events were gated or examined, the number of final clonal events was adjusted to 150,000 events. Survival analysis was performed by the Kaplan-Meier method and differences assessed using the log rank test. ResultsThere were 158 consecutive patients with newly diagnosed MM who had their peripheral blood evaluated via flow cytometry as part of their routine clinical evaluation. The median age of this group was 66 years (39-95) and 59% were male. At the time of this analysis, 25 patients had died and the 2-year OS rate for the cohort was 83%. The 2-year OS for the 89 (55%) patients with any circulating PCs was 76% compared with 91% for those with none (P=0.02). The median number of circulating clonal PCs was 33 (range, 0-46,413)/ 150,000 gated events. Using a ROC analysis the best cutoff predicting 1 and 2-year mortality were 435 and 376 events, respectively. Based on this, we defined >=400 events as a cutoff for defining patients with high-risk disease. The median time-to-next-treatment (TTNT) for patients with circulating clonal PCs >=400 (n=37, 23%) was 14 months compared with 26 months for those with <400 events (n=121, 77%) (p<0.001; Fig 1a). The median OS for those with >=400 clonal PC events was 32 months compared with not reached for those with <400 events (p<0.001; Fig 1b). Patients with >= 400 circulating PCs had higher ISS stage, creatinine, LDH, PC labeling index and bone marrow PC% compared with the rest. In a univariate analysis examining the presence of circulating PC >=400, LDH, labeling index, marrow PC% and FISH risk status, only circulating PCs were prognostic for overall survival (P = 0.0011). Among the 89 patients with any circulating PCs, 26 (30%) had CD45+ clonal PCs. The TTNT in patients with a clonal CD45+ population was inferior to those with no CD45+ clonal PCs in circulation (median 11 vs. 19 months, P = 0.034) as was the OS (P = 0.039). [Display omitted] [Display omitted] [Display omitted] ConclusionQuantitative estimation of circulating clonal PCs in patients with newly diagnosed MM is a powerful predictor of early relapse from therapy and mortality. A cutoff of >=400 clonal events/150,000 gated mononuclear events predicts for a median TTNT of 14 months and OS of 32 months. This parameter is more powerful than the current high-risk parameters such as FISH risk status as well as traditional high-risk markers such as ISS stage and LDH levels, and is able to identify a group of patients with particularly poor outcome. Disclosures:Kumar:Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding.

Minimal residual disease (MRD) status pre- and post- high-dose therapy/autologous stem (HDC/ASCT) cell transplantation for multiple myeloma (MM) in the era of novel agents.

8605 Background: MRD assayed by multi-parameter flow cytometer (MFC), has prognostic significance after HDT/ASCT for MM (Paiva et. al. 2008). The frequency of MRD negativity (-) after induction therapy using novel agents such as immunomodulatory drugs like lenalidomide (IMiDs), and proteasome inhibitors like bortezomib, is unknown. The impact of HDT/ASCT on MRD status in this patient group has not been studied. Methods: We performed a retrospective study of all MM patients undergoing HDT/ASCT (January 2010 - December 2012) in our institution. No restrictions on inclusion were made based on the International Myeloma Working Group response criteria. All patients had novel agents as part of their initial induction regimen. Statistical analysis was by SPSS software (V 12.0). MRD status was determined by MFC on bone marrow samples pre- HDC/ASCT [M1] and post- HDC/ASCT (D100 [M2] and I year [M3]). MFC was done with antibodies against CD45, CD19, CD138, CD38, CD20, CD56, and anti-k and l cytoplasmic antibodies. Results: MRD status was available on 91 patients pre-transplant. Of these patients, 80 had MFC recorded at M2 and 17 patients had MFC recorded at M3. Fifty-eight percent were male and 76% were Caucasian. Forty percent received IMiDs, while 60% got proteasome based therapies. Of the 91 patients with MRD pre-HDC/ASCT, 58% (53/91) were MRD (-), and of these patients 89% (41/46) remained MRD (-) at M2. 48 patients were MRD positive (+) pre-HDC/ASCT, 58% (20/34) became MRD (-) at M2. Age, cytogenetic risk, disease stage, number of chemotherapy cycles or immunofixation status had no impact on MRD status. There were only 6 relapses in the cohort, thus the impact of MRD status on progression-free survival could not be studied. Conclusions: Novel agents improve depth of response pre-transplant. HDC/ASCT increases MRD negativity post-transplant. MRD status could aid better timing of HDC/ASCT or adoption of a risk-adapted strategy for high-risk patients. MRD status validation in a prospective cohort is underway at our center (NCT01215344). With future follow-up, the impact of MRD on progression-free survival in the era of novel agents will be determined.

Time encoded multicolor fluorescence detection in a microfluidic flow cytometer.

We describe an optical detection technique that delivers high signal-to-noise discrimination to enable a multi-parameter flow cytometer that combines high performance, robustness, compactness and low cost. The enabling technique is termed "spatially modulated detection" and generates a time-dependent signal as a continuously fluorescing (bio-) particle traverses an optical transmission pattern along the fluidic channel. Correlating the detected signal with the expected transmission pattern achieves high discrimination of the particle signal from background noise. Additionally, the particle speed and its fluorescence emission characteristics are deduced from the correlation analysis. Our method uses a large excitation/emission volume along the fluidic channel in order to increase the total flux of fluorescence light that originates from a particle while requiring minimal optical alignment. Despite the large excitation/detection volume, the mask pattern enables a high spatial resolution in the micron range. This allows for detection and characterization of particles with a separation (in flow direction) comparable to the dimension of individual particles. In addition, the concept is intrinsically tolerant of non-encoded background fluorescence originating from fluorescent components in solution, fluorescing components of the chamber and contaminants on its surface. The optical detection technique is illustrated with experimental results of multicolor detection with a single large area detector by filtering fluorescence emission of different particles through a patterned color mask. Thereby the particles' fluorescence emission spectrum is encoded in a time dependent intensity signal and color information can be extracted from the correlation analysis. The multicolor detection technique is demonstrated by differentiation of micro-beads loaded with PE (Phycoerythrin) and PE-Cy5 that are excited at 532 nm.

Rapid determination of hazardous compounds in food based on a competitive fluorescence microsphere immunoassay

Development of a microsphere-based competitive fluorescence immunoassay for the determination of hazardous low-molecular-weight compounds in food is described. In this method, antigens are covalently bound to carboxy-modified microspheres to compete monoclonal antibody with low-molecular-weight compounds in food samples; mouse IgG/fluorescein isothiocyanate conjugate is used as the fluorescent molecular probe. Thus, the hazardous low-molecular-weight compounds are quantified using a multiparameter flow cytometer. This method has been evaluated using clenbuterol as a model compound. It has a sensitivity of 0.01 ng/mL with dynamic range of 0.01–100 ng/mL, and the concentration of clenbuterol providing 50% inhibition (IC 50) is 1.1 ng/mL. The main advantages of this method are its high efficiency, biocompatibility, and selectivity, as well as ultralow trace sample consumption and low cost.

Microsphere-based duplexed immunoassay for influenza virus typing by flow cytometry

We have developed a rapid, duplexed microsphere-based immunoassay for the characterization of influenza virus types that has the potential to overcome many of the limitations of current detection methods. The assay uses microspheres of two sizes, each coupled to an influenza type A- or type B-specific monoclonal antibody (MAb), to capture influenza viruses in the sample. A cocktail of fluorescently labeled, influenza-specific polyclonal antibodies then binds the captured viruses. The sandwich complexes are measured using a multiparameter flow cytometer. The assay can distinguish between influenza types A and B in a single reaction with good reproducibility and high sensitivity. Detection sensitivity is much higher than that of commercially available influenza diagnosis quick kits: the FLU OIA (Thermo Biostar) kit and the Directigen Flu A+B kit (Becton Dickinson). The multiplexing capabilities of the current assay, which are not possible with enzyme-linked immunosorbent assay (ELISA) and the commercially available kits, reduce sample handling and consume fewer costly reagents. This assay represents a more efficient and sensitive method of characterizing influenza types. With inclusion of influenza subtype-specific antibodies as capture antibodies, this microsphere-based immunoassay can be expanded to differentiate among influenza types and subtypes in a single reaction to improve world-wide influenza surveillance.

Multiparametric flow cytometric analysis in a breast cancer cell line (MCF-7).

To investigate whether multiparameter flow cytometric analysis of solid tumor specimens, including gynecologic tumors, which were stained triply with phycoerythrin (PE), fluorescein isothiocyanate (FITC) and propidium iodide (PI), can be performed simultaneously without interference from normal diploid cell populations and spectral overlap on a standard flow cytometer. MCF-7 breast cancer cell lines and heterogeneous cell populations mixed with MCF-7 cells and human peripheral blood lymphocytes (PBL) were fixed with 1% paraformaldehyde and permeabilized with 100% methanol. Cytokeratin and several proliferation-associated cellular antigens (proliferating cell nuclear antigen (PCNA), p53, c-erbB/2 and c-myc) were labeled with PE and FITC, which was followed by DNA staining using PI. These labeled cells were measured on a standard FACScan flow cytometer equipped with a 488 nm single laser. The coefficient of variation (CV) of the G0G1 peak of MCF-7 cells was 4.3 and the cell cycle phase fractions of G0G1, S and G2M were 44.9, 45.9 and 9.2%, respectively. Fluorescein isothiocyanate, PE and PI fluorescences were detected without interference. The MCF-7 cells expressed cytokeratin, PCNA, p53, c-erbB/2 and c-myc antigen. In the heterogeneous population of MCF-7 cells mixed with PBL, two cellular populations were clearly separated into diploid PBL and aneuploid MCF-7 cells without interference. The CV of G0G1 peak of PBL was 2.3 and the G0G1, S and G2M phase fractions were 85.5, 2.7 and 11.8%, respectively. The DNA index of MCF-7 cells was 1.7, which indicated that the MCF-7 cell line was composed of tumor cells with aneuploid DNA. The CV of the G0G1 peak of the MCF-7 cells was 4.2, and the cell cycle phase fractions were 47.5% for G0G1, 42.3% for S, and 10.2% for G2M. The MCF-7 cells expressed cytokeratin, but the PBL did not. Multiparameter flow cytometer analysis was useful to determine DNA ploidy status, phase fraction of the cell cycle and expression of cellular antigens and selective cytokeratin expression allowed epithelial originated tumor cells to be differentiated from normal stromal cells. This analysis could be performed without interference of spectral overlaps of fluorochromes using software-based algorithmic compensation of spectral overlaps. Thus, this method offers new possibilities for multiparameter flow cytometric analysis and its use should be extended to future studies of the diagnosis, treatment and prediction of prognosis of the neoplasm.

Evaluation of the CELL-DYN ® 3500 Haematology Instrument for the Analysis of the Mouse and Rat Blood

The objective of this study was to evaluate the performance of the CELL-DYN® 3500 for rat and mouse blood analysis in a routine environment. The WBC (white blood cells), RBC (red blood cells), PLT (platelets) counts and the WBC differential were determined. In addition, the following aspects were studied: within-run precision, day-to-day precision, biasfree paired difference precision; extended ranges of linearity for RBC, HCT (haematocrit), WBC, PLT; carry-over, the fffect of blood ageing, cell stability with different anticoagulants; and the normal ranges, the out of range flagging and some typical pathology cases.The CELL-DYN® 3500 is a multiparameter flow cytometer which counts and differentiates WBC, based on the principle of multi-angle polarised light scatter separation. RBC and PLT are determined by the impedance method. The WBC count is evaluated by both, optical and impedance methods. Reference methods used were according to the ICSH recommendations on blood cell analysis, including manual counts of WBC and platelets, a centrifugal microhaematocrit method and a haemoglobin measurement by spectrophotometry using the WHO haemoglobin standard. All cell counts were compared with the results obtained by our routine blood cell analyser (Contraves AL820), and the WBC differential was compared with the manual microscopic differentiation of the 400 WBC (200 cells differentiated by two technicians).The following coefficients of variation were obtained: within-run precision was 1.2% and 2.7% for WBC; 1.0% and 1.0% for RBC; 1.3% and 0.9% for haematocrit; 2.1% and 2.7% for platelets (rats and mice respectively). Day-to-day precision was performed using human trilevel control blood, and the CVs were found to be <1.7% for WBC, <1.4% for RBC, <1.2% for haemoglobin and <6.3% for platelets.The following ranges of measurement were found to be linear in the rat: WBC: 0.10–20.20×103/μl; RBC: 0.016–14.3×106/μl; haemoglobin: 0.08–26.8 g/dl; haematocrit: 5.0%–77%; platelets: 14.0–1670.0×103/μl. Equal ranges were observed for mouse blood. Carry-over in rat blood was found to be 0.12% for WBC, 0.05% for RBC, 0.15% for haemoglobin and 0.46% for platelets. In mice, similar carry-over results were obtained. The correlation coefficients (Pearson, correlation coefficient) between the CELL-DYN® 3500 and Contraves AL 820 using linear regression analysis were as follows: 0.988 and 0.997 for WBC; 0.986 and 0.920 for RBC; 0.995 and 0.984 for haemoglobin; 0.958 and 0.85 for haematocrit; 0.958 and 0.963 for platelets, for rats and mice, respectively. Correlation coefficients between the CELL-DYN® 3500 and the manual differential of NEU (neutrophils) and LYM (lymphocytes) were higher than 0.8 in rats and higher than 0.9 in mice. Due to the relatively low absolute counts of MONO (monocytes), EOS (eosinophils) and BASO (basophils), only moderate correlation of methods was found.The CELL-DYN® 3500 was judged to be reliable, accurate and easy-to-use for counting and identifying normal and most of the pathological blood specimens obtained from mice and rats. By using the CELL-DYN® 3500, the time for blood sample analysis can be shortened significantly and provides extensive opportunities to characterise pathological samples.

A new multiparameter flow cytometer: optical and electrical cell analysis in combination with video microscopy in flow.

Flow cytometers, which are commercially available, do not necessarily meet all demands of actual biomedical research. This is the case for the investigation of mechanisms involved in cell volume regulation, which requires electrical volume measurement and ratiometric multichannel fluorescence analysis for the simultaneous assessment of different physiologic parameters (intracellular pH and the intracellular concentration of calcium ions, etc). We describe the construction of a new nonsorting flow cytometer designed for the simultaneous acquisition of seven parameters including fluorescence signals, forward and perpendicular light scatter, cell volume according to the electrical Coulter principle, and flow cytometric imaging. The instrument is equipped with three different light sources. A tunable argon-ion laser generates efficient excitation of the most standard fluorescent probes in the visible spectral range, and an arc lamp provides the means for ultraviolet excitation at low cost. Because of the spatial filtering by the excitation and detection optics, two independent sets of dual fluorescence measurements can be performed, a prerequisite for flexible ratiometric fluorescence analysis. A flow video microscope integrated into the optical system optionally generates either brightfield or phase images of selected flowing particles. Only particles whose individual datasets meet predefined gating conditions are imaged in real time. To avoid smear effects, the motion of the object to be imaged (speed approximately 8 m/s) is frozen on the target of a CCD camera by flash illumination. For this purpose, a high radiance gas discharge lamp with 25-mJ electric pulse energy provides an illumination time of 18 ns (full width half maximum). Test results obtained from latex spheres and cells are shown. Test results indicate that our instrument can perform Coulter measurements in combination with flexible optical analysis. Moreover, integration of an adapted video microscope into a flow cytometer is an approach to overcome the gap between flow and image cytometry.

Cell cycle and proliferation dynamics of adult rat oligodendrocytes.

Adult oligodendrocytes (OLGs) have been shown to be mitotically responsive when co-cultured with dorsal root ganglion. We have investigated the population dynamics of the OLG proliferative response, including the maximum percent of cells which will proliferate and the cell cycle duration time of the adult rat OLG. Adult OLGs were stimulated with pituitary extract and either continuously labeled or pulse labeled with bromodeoxyuridine (BrdU). The labeled cells were stained with a fluorescent anti-BrdU monoclonal antibody, and fluorescence measurements were made with a multiparameter flow cytometer. A maximum number of 53% of the adult OLGs entered the cell division cycle after 9 days. The cell division cycle duration time (Tc) was determined to be approximately 35.4 hr, and the durations of G1, S, and G2M found to be 12.1, 8.5, and 5.8 hr, respectively. A scenario was generated for adult OLG proliferation in which 2.2% of the initial population is stimulated to enter the cell division cycle in the first 24 hr. This initial population divides and continues in the cell division cycle. Another 2.2% of the initial population enters the cell division cycle during the second 24 hr period, divides, and continues in the cell division cycle. This entry of 2.2% of the cells into the proliferative phase continues until 53% of the initial population has left a quiescent state and entered the cell cycle.

Heterogeneity index score (HIS): New computerized method for classification of human bladder carcinomas using flowcytometry

The rationale for studying nuclear DNA may be its direct relationship to the aggressiveness of cancer. Recent flow cytometric studies (FCM) of cancer cells show the limitation of the current methods for the accurate determination of the degree of aneuploidy or proliferative characteristics of a tumor cell. Here we report a new methodology for a computerized determination which is well correlated with relative mean DNA content in cell populations analyzed by FCM (heterogeneity index, HI). A total of seventy-six tissue samples were examined. Twenty-two specimens were benign tissue while fifty-four were histologically malignant bladder tumors. Forty tumors were grade (G)I–II, ten G-III, and four carcinoma in situ. The samples were mechanically minced into a single cell suspension and stained with propidium iodide. An Ortho system 50-H multiparameter flow cytometer equipped with an Ortho 2150 computer was used to determine DNA content and cell number. HI was calculated using the following formulas: ▪ The mean HIS of twenty-two normal and benign tissues was 9.805 ± 5.6. The forty G-II tumors had a mean HIS of 23.576 ± 26.519. Statistical differences were observed between benign tissue and G-I-II tumors (P = 0.0196). G-III tumors had a marked increase in HIS of 160.965 ± 63.404. The limited study of four carcinoma in situ tumors showed a mean HIS of 45.4± 9.5. Our computer extrapolation of flow cytometric DNA analysis quantifies an objective description of FCM characteristics and histochemical index which may distinguish the degree of tumor malignancy.

Dual laser beam focussing for flow cytometry through a single crossed cylindrical lens pair.

Two laser beans (argon 488 nm blue, and krypton UV lines) have been focussed either to the same point or to two different points in a vertical plane in a multiparameter flow cytometer. The technique exploits the focal length shortening of the longer wavelength beam that occurs with spherical and astigmatic lens aberrations. Ethidium bromide-stained DNA is excited by both the argon 488 nm and krypton UV lines and the excitation intensities of both beams were set to give the same red fluorescence pulse height from ethidium bromide-stained nuclei. DNA histograms were obtained for sequential focussing of the two beams and these were virtually identical irrespective of which beam was the first excitor and irrespective of which beam the histogram was recorded from. Coincident focussing of the two exciting beams resulted in a histogram of double the emission intensity.