Osmotic phenomena in application for hyperbaric oxygen treatment.

Hyperbaric oxygen (HBO) treatment defines the medical procedure when the patient inhales pure oxygen at elevated pressure conditions. Many diseases and all injuries are associated with a lack of oxygen in tissues, known as hypoxia. HBO provides an effective method for fast oxygen delivery in medical practice. The exact mechanism of the oxygen transport under HBO conditions is not fully identified. The objective of this article is to extend the colloid and surface science basis for the oxygen transport in HBO conditions beyond the molecular diffusion transport mechanism. At a pressure in the hyperbaric chamber of two atmospheres, the partial pressure of oxygen in the blood plasma increases 10 times. The sharp increase of oxygen concentration in the blood plasma creates a considerable concentration gradient between the oxygen dissolved in the plasma and in the tissue. The concentration gradient of oxygen as a non-electrolyte solute causes an osmotic flow of blood plasma with dissolved oxygen. In other words, the molecular diffusion transport of oxygen is supplemented by the convective diffusion raised due to the osmotic flow, accelerating the oxygen delivery from blood to tissue. A non steady state equation for non-electrolyte osmosis is solved asymptotically. The solution clearly demonstrates two modes of osmotic flow: normal osmosis, directed from lower to higher solute concentrations, and anomalous osmosis, directed from higher to lower solute concentrations. The fast delivery of oxygen from blood to tissue is explained on the basis of the strong molecular interaction between the oxygen and the tissue, causing an influx of oxygen into the tissue by convective diffusion in the anomalous osmosis process. The transport of the second gas, nitrogen, dissolved in the blood plasma, is also taken into the consideration. As the patient does not inhale nitrogen during HBO treatment, but exhales it along with oxygen and carbon dioxide, the concentration of nitrogen in blood plasma drops and the nitrogen concentration gradient becomes directed from blood to tissue. On the assumption of weak interaction between the inert nitrogen and the human tissue, normal osmosis for the nitrogen transport takes place. Thus, the directions of anomalous osmotic flow caused by the oxygen concentration gradient coincide with the directions of normal osmotic flow, caused by the nitrogen concentration gradient. This leads to the conclusion that the presence of nitrogen in the human body promotes the oxygen delivery under HBO conditions, rendering the overall success of the hyperbaric oxygen treatment procedure.

Changes in serum calcium, phosphate, and PTH and the risk of death in incident dialysis patients: a longitudinal study.

Elevated bone mineral parameters have been associated with mortality in dialysis patients. There are conflicting data about calcium, parathyroid hormone (PTH), and mortality and few data about changes in bone mineral parameters over time. We conducted a prospective cohort study of 1007 incident hemodialysis and peritoneal dialysis patients. We examined longitudinal changes in bone mineral parameters and whether their associations with mortality were independent of time on dialysis, inflammation, and comorbidity. Serum calcium, phosphate, and calcium-phosphate product (CaP) increased in these patients between baseline and 6 months (P<0.001) and then remained stable. Serum PTH decreased over the first year (P<0.001). In Cox proportional hazards models adjusting for inflammation, comorbidity, and other confounders, the highest quartile of phosphate was associated with a hazard ratio (HR) of 1.57 (1.07-2.30) using both baseline and time-dependent values. The highest quartiles of calcium, CaP, and PTH were associated with mortality in time-dependent models but not in those using baseline values. The lowest quartile of PTH was associated with an HR of 0.65 (0.44-0.98) in the time-dependent model with 6-month lag analysis. We conclude that high levels of phosphate both at baseline and over follow-up are associated with mortality in incident dialysis patients. High levels of calcium, CaP, and PTH are associated with mortality immediately preceding an event. Promising new interventions need to be rigorously tested in clinical trials for their ability to achieve normalization of bone mineral parameters and reduce deaths of dialysis patients.

Measuring reactive nitrogen emissions from point sources using visible spectroscopy from aircraft.

Accurate measurements of nitrogen dioxide (NO2), a key trace gas in the formation and destruction of tropospheric ozone, are important in studies of urban pollution. Nitrogen dioxide column abundances were measured during the Texas Air Quality Study 2000 using visible absorption spectroscopy from an aircraft. The method allows for quantification of the integrated total number of nitrogen dioxide molecules in the polluted atmosphere and is hence a useful tool for measuring plumes of this key trace gas. Further, we show how such remote-sensing observations can be used to obtain information on the fluxes of nitrogen dioxide into the atmosphere with unique flexibility in terms of aircraft altitude, and the height and extent of mixing of the boundary layer. Observations of nitrogen dioxide plumes downwind of power plants were used to estimate the flux of nitrogen oxide emitted from several power plants in the Houston and Dallas metropolitan areas and in North Carolina. Measurements taken over the city of Houston were also employed to infer the total flux from the city as a whole.

Morphometry of nucleoli and expression of nucleolin analyzed by laser scanning cytometry in mitogenically stimulated lymphocytes.

BACKGROUND: Various attributes of nucleoli, including abundance of the nucleolar product (rRNA), correlate with cell-proliferative status and are useful markers for tumor diagnosis and prognosis. However, there is a paucity of methods that can quantitatively probe nucleolus. The aim of the present study was to utilize the morphometric capacity of the laser scanning cytometer (LSC) to analyze nucleoli and measure expression of the nucleolar protein nucleolin (NCL) in individual cells and correlate it with their state of proliferation. MATERIALS AND METHODS: Human lymphocytes were mitogenically stimulated, and at different time points their nucleoli were detected immunocytochemically using NCL Ab. The frequency of nucleoli per nucleus, their area, and the level of expression of NCL, separately in the nuclear and nucleolar compartments, were estimated in relation to the G(0) to G(1) transition and the cell cycle progression. RESULTS: During the first 24 h of stimulation, when the cells underwent G(0) to G(1) transition, their RNA content was increased nearly 8-fold, the level of NCL per nucleus also increased 8-fold, the NCL per nucleolus increased 12-fold, nucleolear area increased 3-fold, and NCL/nucleolar area increased nearly 4-fold. During the subsequent 24-48 h of stimulation, when cells were progressing through S, G(2), and M and reentering the next cycle, the number of nucleoli per nucleus was increased and a massive translocation of NCL from nucleoli to nucleoplasm was observed; its overall level per nucleus, however, still remained high, at 6-fold above of that of G(0) cells. CONCLUSIONS: While high expression of NCL in the nucleolar compartment correlates with the rate of rRNA accumulation in the cell and is a sensitive marker of the G(0) to G(1) transition, the cells progressing through the remainder of the cycle are better distinguished from G(0) cells by high overall level of NCL within the nucleus. Such an analysis, when applied to tumors, may be helpful in obtaining the quantitative parameters related to the kinetic status of the tumor-cell population and tumor prognosis. The capability of LSC to measure the protein translocation between nucleolus and nucleoplasm can be used to study the function and regulatory mechanisms of other proteins that reside in these compartments.

Proliferation and apoptosis in solid tumors. Analysis by laser scanning cytometry.

OBJECTIVE: To examine the relationship between apoptosis and proliferation in a series of human solid malignant tumors, making use of objective, reproducible techniques newly developed for laser scanning cytometry (LSC). STUDY DESIGN: Apoptosis was detected by in situ end labeling of DNA strand breaks with FITC-conjugated nucleotide. Proliferation was detected by Ki-67 antibody. Two parameters were detected independently and simultaneously with DNA measurement on aliquots of cell suspensions obtained by mechanical dissociation of fresh tumors and placed on microscope slides. RESULTS: The number of cells undergoing apoptosis varied from 0.5% to 28.1% (average, 5.4 +/- 6.0). Aneuploid tumors showed a higher percentage of apoptotic cells (7.9 +/- 7.2) as compared to diploid tumors (3.4 +/- 4.0). Tumors with the greatest number of apoptotic cells on LSC also had the largest number of apoptotic cells on light microscopic examination. The number of cells labeled by Ki-67 ranged from 1.7% to 56.7% (average, 20.0 +/- 15.5). Aneuploid tumors were characterized by a higher Ki-67 index (average, 28.3 +/- 14.3%) than the diploid tumors (13.2 +/- 13.3%). CONCLUSION: Overall, there was a very weak or no correlation between apoptosis and proliferation. However, a subset of aneuploid tumors had a high percentage of cells positive for Ki-67 and low percentage of apoptotic cells. Diploid tumors did not show any correlation between apoptosis and proliferation, although many of those tumors had both low apoptotic and proliferative indices. Whether those differences are of prognostic significance remains to be determined in follow-up studies that include more cases and clinical data. Here we have shown that LSC is a powerful new tool of potential clinical value for fast, objective analysis of apoptosis, proliferation and DNA ploidy in solid malignant tumors.

Differential diagnosis of malignant lymphomas and related disorders by specific pattern of expression of immunophenotypic markers revealed by multiparameter flow cytometry (Review).

The introduction of monoclonal antibodies (mAbs) for cell immunophenotyping and use of flow cytometry with the progressively improving software for multivariate analyses have revolutionized the diagnosis and influenced the classification of hematologic neoplasms. In this review we focus on the practical application of flow cytometry in the diagnosis and classification of malignant lymphomas and related lymphoproliferative disorders with special emphasis on differential diagnosis. A general approach to the utilization of flow cytometry (FC) in hematopathology with an algorithm to diagnose the most common neoplasms is presented. We discuss precursor B-cell neoplasms, mature B-cell neoplasms (SLL/CLL, mantle cell lymphoma, marginal zone lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, plasma cell dyscrasias and lymphomas with plasmacytic differentiation), precursor T-lymphoblastic leukemia and mature (peripheral) T-cell neoplasms, including T-SLL/PLL, anaplastic cell lymphomas and large granular cell leukemia/lymphoma. The text is accompanied by characteristic FC scatterplots of the discussed entities.

SpinThin, a simple, inexpensive technique for preparation of thin-layer cervical cytology from liquid-based specimens: data on 791 cases.

BACKGROUND: Acceptance of liquid-based fixatives for cervical cytology has been limited by the more complex slide-preparation procedures, increased cost, and reports that increased sensitivity has been based largely on comparison with conventional cytology without histologic correlation. Here the authors describe and evaluate a technically simple and relatively inexpensive method (which they call SpinThin) for preparing Cytospin (Shandon Inc., Pittsburgh, PA) cervical cytology slides from samples in liquid fixative using a modified electric toothbrush holder to put the cells in suspension. Results are compared with conventional cytology and histologic biopsy. METHODS: A total of 791 cervical cytology specimens from 2 patient groups at high risk of uterine cervical neoplasia were entered into this study, and a spatula and cytobrush (174 specimens) or cytobroom (617 specimens) were used to collect conventional smears. The collection device with remaining cellular sample was placed in an alcohol-based fixative solution; the cells were put into suspension by a brief burst of vibration using a modified electric toothbrush holder, then cytocentrifuged on a slide and stained with the Papanicolaou technique. RESULTS: Specimen adequacy in SpinThin slides was better than that of conventional cytology smears. However, the prevalence of dysplasia, including atypical squamous cells of undetermined significance (ASCUS-D), in conventional smears and SpinThin slides was the same--27% and 25%, respectively--and excluding ASCUS-D, it was 20% in both. The prevalence of neoplasia (low or high grade squamous intraepithelial lesion, or carcinoma) histologically was 31% in the 647 cases biopsied, and agreement with histology was similar for SpinThin and conventional smears. CONCLUSIONS: Using a simple and relatively inexpensive new technique (Spin-Thin), slides prepared from fluid-based cervical cytology specimens obtained with the cytobrush or cytobroom correlated very well with the corresponding conventional smears within major diagnostic categories, and both correlated well with histology.

Lung cancer screening results in the National Cancer Institute New York study.

BACKGROUND: Anecdotal reports of radiologically occult early stage lung carcinomas detected by sputum cytology suggested that screening by cytology might lead to earlier diagnosis, more effective surgical therapy, and lower death rates from lung carcinoma. Thus, a randomized study was undertaken to evaluate sputum cytology as a lung carcinoma screening technique supplementing the chest X-ray. METHODS: Three major medical centers participated in the study, recruiting approximately 10,000 cigarette smoking men older than 45 years of age at each center: Memorial Sloan-Kettering Cancer Center (MSKCC) in New York, NY, Johns Hopkins Hospital in Baltimore, MD, and The Mayo Clinic in Rochester, MN. At MSKCC, the men were divided randomly into two groups: a dual screen group received four monthly sputum cytology examinations in addition to annual posteroanterior and lateral chest X-rays and an X-ray only group received annual chest X-rays but no sputum examinations. The men suspected of lung carcinoma because of radiologic or sputum cytology findings were referred immediately for evaluation, and those with operable lung carcinoma were recommended for surgery and treated with intent to cure. RESULTS: The men who entered into the study remained in the screening program for 5- 8 years, depending on their date of enrollment, and were followed for 2 years after screening. Follow-up was completed on more than 99%. There were 53 of the 10,040 men in the study who were found to have lung carcinoma on initial examination (prevalence): 23 were in the X-ray only group; of 30 found in the dual screen group, 9 (all with squamous cell carcinoma) were detected by cytology alone. During the entire study and the 2-year follow-up period, 354 of the 10,040 men developed lung carcinoma, equally divided between the dual screen and X-ray only groups. Nearly two-thirds (190 men) had lesions that were detected by screening, and over 50% (100 men) were in Stage I. Excluding oat cell carcinoma, during the screening period 175 of 250 carcinomas (70%) were detected by screening. In contrast, during the 2-year post-screening period, 61 lung carcinomas were diagnosed of which only 12 (20%) were Stage I. Chest X-ray was most effective in detecting peripheral adenocarcinomas of the lung, which were the most common cell type. Cytology was most effective in detecting early epidermoid carcinomas of major bronchi. The epidermoid carcinomas grew slowly, metastasized late, and after becoming visible by X-ray could be treated equally effectively as in the earlier occult stage. Forty percent of all the lung carcinomas were detected in Stage I, and at least two-thirds of the patients with Stage I lung carcinoma treated by complete resection did not die of their disease. Overall 5-year survival of all patients with lung carcinoma who had enrolled in the detection program was 35%, compared with 13% for the United States as a whole during this same time period. CONCLUSIONS: Sputum cytology and the chest X-ray complemented each other as lung carcinoma detection techniques. The chest X-ray best detected peripheral adenocarcinomas of the lung, which are the most common type of lung carcinoma. Sputum cytology detected epidermoid carcinomas arising in major bronchi, but these are slow growing tumors that can be resected and cured after becoming visible by chest X-ray. Thus, for subjects at risk of lung carcinoma who could be followed by annual chest X-rays, sputum cytology did not improve survival, but for high risk subjects who had only a single screening examination, sputum cytology increased the number of early lung carcinomas detected. The design of the current study did not permit evaluation of chest X-ray screening versus nonscreening for prevention of death from lung carcinoma. However, the large proportion of Stage I lung carcinomas and the high survival rate of patients in this study compared with Surveillance, Epidemiology, and End Results program data strongly suggested that screening for lung carcinoma in high risk populations is a valuable public health measure.

A human monoclonal antibody encoded by the V4-34 gene segment recognises melanoma-associated ganglioside via CDR3 and FWR1.

A heterohybridoma cell line producing the human monoclonal antibody (MoAb) MDT.1 has been established. The heavy chain of MoAb MDT.1 is encoded by the VH gene segment V4-34 (previously designated VH4-21), and the light chain is encoded by the V kappa 1-L12a gene segment, both in germline configuration. MDT.1 has reactivity against lipid A, double- and single-stranded DNA, red blood cell associated i antigen, and ganglioside antigens. In a panel of tumour cell lines, MDT.1 reacted specifically with melanoma cells and other tumour cells of neuroectodermal origin. Cellular recognition appears to be via tumour-associated ganglioside antigens, and may involve the minimal essential epitope NeuNac alpha 2-->3Gal beta 1-->-4Glc-. Binding to ganglioside antigen is inhibited by the monoclonal anti-idiotypic antibody 9G4. Since the 9G4 idiotope is located in framework region 1 (FWR1) of V4-34-encoded antibodies, this region is likely to be involved, either directly or indirectly, in ganglioside binding. The complementarity-determining region 3 (CDR3) of MDT.1 is arginine rich, with five out of 12 residues being arginine and these residues are candidates for interaction with the negatively charged ganglioside. The ability of MoAb MDT.1 to recognise ganglioside antigens is associated with potentially useful anti-tumour activity.

High affinity binding of fluorescein isothiocyanate to eosinophils detected by laser scanning cytometry: a potential source of error in analysis of blood samples utilizing fluorescein-conjugated reagents in flow cytometry.

BACKGROUND: In samples of peripheral blood cells processed using the commercial kits for detection of apoptosis based on DNA strand break labeling, a subpopulation of cells characterized by high green fluorescence, similar in intensity to that of apoptotic cells but more uniform, was consistently observed by flow cytometry. The labeled cells had no other features of apoptosis. The labeling was observed regardless of the fixative used and was evident in control samples lacking terminal deoxynucleotidyltransferase. Common to all the kits that generated this labeling pattern was the presence of fluorescein (f) conjugated reagents, f-dUTP, f-avidin, or f-antibody. METHODS: Laser scanning cytometry was used to identify the labeled cells and study the mechanism of labeling. Because it was suspected that the traces of unconjugated f-isothiocyanate (FITC) that may contaminate the reagents were responsible for the labeling, FITC binding affinity to white blood cells was studied. Gel electrophoresis was used to detect the presence of unconjugated FITC in the reagents. RESULTS: After staining with Giemsa, the strongly fluorescent objects were identified as eosinophils with normal morphology and no evidence of apoptosis. The fluorescence was localized exclusively within the cytoplasmic granules. Labeling of eosinophils was observed at 2 nM concentration of FITC, which was over three orders of magnitude lower than that needed to label neutrophils, monocytes, or lymphocytes. Gel electrophoresis of the f-conjugated reagents revealed only minor contamination with FITC. CONCLUSIONS: (1) Trace amounts of unconjugated FITC contaminating the reagents are adequate to strongly label eosinophils thereby introducing experimental bias in analysis of apoptosis and in other studies on blood cells utilizing f-labeled antibodies, e.g., in detecting cytokines. (2) FITC at concentration 2-500 nM can be used as a marker of eosinophiles; (3) Because of high affinity to FITC, eosinophiles (or the protein from these cells) may serve as a means of removing traces of unconjugated FITC from the reagents during their manufacture or prior to use.

Laser-scanning cytometry: A new instrumentation with many applications.

The laser-scanning cytometer (LSC) is a microscope-based cytofluorometer which has attributes of both flow and image cytometry. Laser-excited fluorescence emitted from fluorochromed individual cells on a microscope slide is measured at multiple wavelengths rapidly with high sensitivity and accuracy. Though the instrument has been available commercially for only 3 years, it is already used in a variety of different applications in many laboratories. This review focuses on the following unique analytical capabilities of LSC which complement those of flow cytometry and fluorescence image analysis: (a) the cells are positioned on slides during measurement so they may be examined repeatedly over time, a feature useful for studies of enzyme kinetics and other time-resolved processes; (b) sequential analysis of the same cells can be carried out using different immuno- or cytochemical stains or genetic probes, merging information on cell immunophenotype, cell functions, expression of particular proteins, DNA ploidy and cell cycle position, and/or cytogenetic profile for each measured cell; (c) any of the cells measured can be relocated to correlate with visual examination by fluorescence or brightfield microscopy or with any other parameter; (d) topographic distribution of fluorescence measurements within the cell, in cytoplasm vs nucleus, permits analysis of the translocation of regulatory molecules such as NFkappaB, p53, etc., and is essential for FISH analysis; (e) hyperchromicity of nuclear DNA as measured by maximal pixel fluorescence intensity allows one to identify cell types differing in degree of chromatin condensation such as mitotic or apoptotic cells; (f) analysis of tissue section architecture and of the constituents in transected cells within tissue sections by ratiometric assays normalized to DNA content extends applications of LSC in clinical pathology; (g) because cell loss during sample preparation and staining is minimal, samples with a paucity of cells can be analyzed; and (h) analyzed cells can be stored indefinitely, e.g., for archival preservation or additional analysis. Potential future applications of LSC are discussed.

Analysis of apoptosis by laser scanning cytometry.

Flow cytometry techniques that are widely used in studies of cell death, and particularly in the identification of apoptotic cells, generally rely on the measurement of a single characteristic biochemical or molecular attribute. These methods fail to recognize cell death lacking that attribute, as in some examples of atypical apoptosis. Since apoptosis was originally defined by morphologic criteria, we suggest that for any new cell system the cytometry-defined apoptosis be confirmed by morphologic examination. This quality assurance measure is now provided by laser scanning cytometry (LSC). LSC measurements of cell fluorescence are precise and highly sensitive, comparable to flow cytometry (FCM), and can be carried out on cells on slides, permitting cell by cell correlation of fluorescence cytometry with visual microscopic morphology. In this report we describe adaptations of various flow cytometry techniques for detection of apoptosis by laser scanning cytometry. We also describe features unique to LSC that are useful in recognizing apoptosis. Hyperchromicity of DNA, reflecting chromatin condensation, is evidenced by high maximal pixel values for fluorescence of the DNA-bound fluorochrome. Mitochondrial probes that have been adapted to LSC to measure the drop in mitochondrial transmembrane potential that occurs early in apoptosis include rhodamine 123, 3,3'-dihexiloxadicarbocyanine [DiOC6(3)], and the aggregate dye 5,5',6,6'tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). The changes in plasma membrane phospholipids and transport function, also early in apoptosis, are probed by a combination of the fluoresceinated annexin V and DNA fluorochromes such as propidium or 7-aminoactinomycin D. We also review methods of detection of apoptosis based on analysis of DNA fragmentation and their application to clinical oncology. Visual examination of the presumed apoptotic cells detected by cytometry makes it possible to discriminate those that are genuine from monocytes/macrophages that have ingested nuclear fragments via apoptotic bodies. Applications of flow cytometry and laser scanning cytometry in analysis of cell death are discussed and their respective advantages and disadvantages compared.

Laser scanning cytometry quantification of estrogen receptors in breast cancer.

OBJECTIVE: To describe the laser scanning cytometry (LSC) processing and analysis developed for the quantitative analysis of estrogen receptor (ER) content in routine paraffin sections of breast carcinomas. STUDY DESIGN: Histologic sections of archival, paraffin-embedded tissues from 30 breast carcinomas were labeled for ER with fluoresceinated monoclonal antibody. ER expression was quantified by LSC and expressed as percent positive tumor cells and as histogram distributions of receptor expression per cell. Duplicate sections of the same tumors were stained for ER by a conventional immunoperoxidase reaction and percent positive tumor cells counted visually. RESULTS: Percent ER-positive tumor cells by LSC of immunofluorescence-stained sections correlated well with conventional (visual) counts of immunoperoxidase-stained duplicate sections when the latter were categorized as low, intermediate or high percent of positive cells. In addition, the marked variation in relative number of ER binding sites per cell could be quantified by LSC and displayed in histogram distribution. CONCLUSION: LSC measurements are fast and objective and can be carried out on sections of paraffin-embedded tissue after routine processing in the pathology laboratory. In addition, LSC data provide the relative number of ER binding sites per unit of DNA; that may reveal clinically significant skewed distributions or subpopulations of tumor cells.

Analysis of apoptosis in solid tumors by laser-scanning cytometry.

Apoptosis (programmed cell death) plays an important role in tissue physiology and pathology. First, tumor development and progression is often associated with defective regulation of apoptotic pathways, and measurement of apoptosis might be equally important for the analysis of cell proliferation. Second, tumor cells die by apoptosis during chemotherapy or radiotherapy, so monitoring the level of apoptosis might prove useful in modulating treatment or in predicting the biologic behavior of the tumor. Flow cytometry (FC), in conjunction with DNA strand-break labeling assays, is commonly used to assess apoptosis, but unless combined with cell sorting, FC results cannot correlate with morphology. Also, analysis by FC is associated with cell loss during preparation of the specimen. In this study, we identified apoptotic cells by in situ DNA strand-break labeling in solid tumors and then analyzed those cells by laser-scanning cytometry (LSC). LSC provides data comparable to those obtained by FC, but because the cells are prepared and measured on a slide, there is no cell loss. Also, with LSC, each measured cell can be relocated and examined visually by conventional or fluorescence microscopy to confirm its identity. With use of this approach, we analyzed apoptosis in 30 primary solid tumors of different types. The number of cells with DNA strand breaks varied from tumor to tumor and ranged from 0.5 to 28.1% (average, 7.0%). FC on the same tumors gave similar results. More than 95% of the relocated cells with DNA strand breaks either had the typical appearance of late apoptosis or showed strong green fluorescence within or at the periphery of the nucleus. Our results suggest that different morphologic variants of apoptosis might be common in different tumor types and that cytometric quantification might provide biologically useful information not otherwise easy to obtain.

Enzyme kinetic reactions and fluorochrome uptake rates measured in individual cells by laser scanning cytometry.

This study was designed to explore the utility of a microscope-based cytofluorometer, the laser scanning cytometer (LSC), for time-resolved kinetic measurements. This instrument measures fluorescence of individual cells rapidly, with high sensitivity and accuracy. Also recorded in a list mode fashion are the spatial X-Y coordinates of the cell on the slide as well as the time of individual cell measurement. Repeated measurement of each of a group of cells within a selected area of the slide, thus, yields information on their fluorescence parameters (integrated value, maximal pixel intensity, or fluorescence area) as a function of time. Using the fluorogenic substrate di-(leucyl)-rhodamine 110, we measured the kinetic activity of L-aminopeptidase in HL-60 cells and in monocytes, granulocytes, and lymphocytes from human blood. Likewise, the rate of fluorescein diacetate (FDA) hydrolysis by esterases was measured in HL-60 cells. Also studied was the rate of uptake of the lysosomo-trophic fluorochrome acridine orange (AO) by human leukocytes. Several hundred cells per sample were measured rapidly with a time resolution of 20-60 s. The resolution was inversely proportional to the number of cells within the measured population. The kinetic curves constructed for individual cells had clearly defined slope and plateau regions. During data analysis the kinetic plots were matched with the respective cells; the latter were identified by their position on the slide and classified by their fluorescence or image after staining with Giemsa. Great intercellular variability was noted in enzyme kinetics among cells of the same type, and differences were seen in rate of AO uptake between granulocytes, monocytes, and lymphocytes. Fluorescence fading and recovery was observed especially in the case of FDA hydrolysis and AO uptake. Our results indicate that LSC can be used to rapidly measure the rate of uptake or accumulation of a particular fluorochrome or the kinetics of enzymatic reactions in individual cells of large populations to reveal intercellular variability or the presence of a cell subpopulation with different kinetic properties.

Cell cycle effects and induction of apoptosis caused by infection of HL-60 cells with human granulocytic ehrlichiosis pathogen measured by flow and laser scanning cytometry.

Human granulocytic ehrlichiosis (HGE) is an occasionally severe and even fatal disease caused by an agent closely related to Ehrlichia equi and Ehrlichia phagocytophila, which is transmitted by ticks. Little is known about the pathogen itself, which only very recently has been isolated. The agent can be cultivated in vitro because it replicates in human promyelocytic leukemic HL-60 cells. Using multiparameter flow cytometry and laser scanning cytometry (LSC) we have investigated changes in HL-60 cells following their infection with the pathogen. Its presence within the infected HL-60 cells was detected and its intracellular level measured inmmunocytochemically using antibodies obtained from HGE-infected patients. The percentage of the infected cells measured by flow cytometry or LSC correlated well with the estimates by microscopy on the Giemsa-stained specimens. In the infected cultures, the cells had diminished levels of cyclins D3 and E as well as the cyclin dependent kinase inhibitor p21WAF1/CIP1 and were arrested predominantly in G0/1. The apoptosis-associated regulatory proteins were also affected by cell infection: expression of Bcl-2 was decreased in the infected cells whereas expression of Bax become more variable, with some cells showing higher levels of this protein. The infected cells developed numerous DNA strand breaks characteristic of apoptosis. The presence of the pathogen was also detected by LSC in cells from peripheral blood of the infected patients; after relocation and visual inspection ("CompuSort") the pathogen-positive cells were identified as leukocytes. This unique ability of LSC to detect, quantify, and visualize HGE in infected cells made this instrument particularly useful to measure the degree of infection in peripheral blood of the patients and study effects of the infectious agent on the cell cycle and apoptosis of the host cells.