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BackgroundVibrio parahaemolyticus,in response to environmental conditions, may be present in a viable but nonculturable state VBNC, which can still be responsible for cases of infectious diseases in humans.MethodsThe characterization of the cellular states of V. parahaemolyticusduring entry into, persistence in, and resuscitation from the VBNC state, was assessed through plate culture method and epifluorescence microscope evaluation of actively respiring cells. Flow cytometry FCM in combination with SYBR Green I SG and propidium iodide allowed us to distinguish between viable, dead, and damagedcells. Immunofluorescence labeling detected by FCM was used to study changes in antibody affinity.ResultsTwo groups of bacteria, one with High Nucleic Acid HNA and one having Low Nucleic Acid LNA content, were differentiated using SG and FCM and each was correlated with cell viability. With the aging of the microcosm, the LNA bacteria population increased while the HNA population gradually disappeared. Cytofluorimetric immunofluorescence analyses showed that the bacterial cell levels dropped from 95 at day 0 to 40 at day 26 and by day 29, antibody affinity was virtually lost. FCM analyses of light scatter signals expressed by cell population highlighted morphological changes indicating a reduction in cell size, as also shown by scanning electron microscopy images and variations in cell structure.ConclusionsThe methodology used has provided useful data in relation to the state transitions of V. parahaemolyticusregarding cell viability, antigenic surface components, and the quantification of morphological variations during its entry into the VBNC state. © 2008 Clinical Cytometry Society

The enumeration of absolute levels of cells and their subsets in clinical samples is of primary importance in human immunodeficiency virus (HIV)+ individuals (CD4+ T- lymphocyte enumeration), in patients who are candidates for autotransplantation (CD34+ hematopoietic progenitor cells), and in evaluating leukoreduced blood products (residual white blood cells). These measurements share a number of technical options, namely, single- or multiple-color cell staining and logical gating strategies. These can be accomplished using single- or dual-platform counting technologies employing cytometric methods. Dual-platform counting technologies couple the percentage of positive cell subsets obtained by cytometry and the absolute cell count obtained by automated hematology analyzers to derive the absolute value of such subsets. Despite having many conceptual and technical limitations, this approach is traditionally considered as the reference method for absolute cell count enumeration. As a result, the development of single-platform technologies has recently attracted attention with several different technical approaches now being readily available. These single-platform approaches have less sources of variability. A number of reports clearly demonstrate that they provide better coefficients of variation (CVs) in multicenter studies and a lower chance to generate aberrant results. These methods are therefore candidates for the new gold standard for absolute cell assessments. The currently available technical options are discussed in this review together with the results of some cross-comparative studies. Each analytical system has its own specific requirements as far as the dispensing precision steps are concerned. The importance of precision reverse pipetting is emphasized. Issues still under development include the establishment of the critical error ranges, which are different in each test setting, and the applicability of simplified low-cost techniques to be used in countries with limited resources. Cytometry (Comm. Clin. Cytometry) 42:327–346, 2000. © 2000 Wiley-Liss, Inc.

The need for a rapid and reliable marker for the engraftment potential of hematopoietic stem and progenitor cell (HPC) transplants has led to the development of flow cytometric assays to quantitate such cells on the basis of their expression of CD34. The variability associated with enumeration of low-frequency cells (i.e., as low as 0.1% or 5 cells/μl) is exceedingly large, but recent developments have improved the accuracy and precision of the assay. Here, we review and compare the major techniques. Based on the current state of the art, we recommend 1) bright fluorochrome conjugates of class II or III monoclonal antibodies (mAbs) that detect all glycoforms of CD34, 2) use of a vital nucleic acid dye to exclude platelets, unlysed red cells, and debris or use of 7-amino actinomycin D to exclude dead cells during data acquisition, 3) counterstaining with CD45 mAb to be included in the definition of HPC, 4) during list mode data analysis, Boolean gating to resolve the CD34⁺ HPCs from irrelevant cell populations on the basis of the low levels of CD45 expression and low sideward light-scatter signals of HPCs, 5) inclusion of CD34^dim and CD34^bright populations in the CD34⁺ cell count, 6) omission of the negative control staining, and 7) for apheresis products, enumeration of at least 100 CD34⁺ cells to ensure a 10% precision. Unresolved technical questions are 1) the replacement of conventional dual-platform by single-platform assay formats, i.e., derivation of absolute CD34⁺ cell counts from a single flow cytometric assessment instead of from combined flow cytometer (percent CD34⁺) and hematology analyzer (absolute leukocyte count) data, 2) the cross-calibration of the available single-platform assays, and 3) the optimal method for sample preparation. An important clinical question to be addressed is the definition of the precise phenotypes and required numbers of HPCs responsible for short- and long-term recovery to optimize HPC transplant strategies. Cytometry (Comm. Clin. Cytometry) 34: 128–142, 1998. © 1998 Wiley-Liss, Inc.

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