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Background Flow Cytometry Standard (FCS) was initially created to standardize the software researchers use to analyze, transmit, and store data produced by flow cytometers and sorters. Because of the clinical utility of flow cytometry, it is necessary to have a standard consistent with the requirements of medical regulatory agencies. Methods We extended the existing mapping of FCS to the Digital Imaging and Communications in Medicine (DICOM) standard to include list-mode data produced by flow cytometry, laser scanning cytometry, and microscopic image cytometry. FCS list-mode was mapped to the DICOM Waveform Information Object. We created a collection of Extensible Markup Language (XML) schemas to express the DICOM analytical cytologic text-based data types except for large binary objects. We also developed a cytometry markup language, CytometryML, in an open environment subject to continuous peer review. Results The feasibility of expressing the data contained in FCS, including list-mode in DICOM, was demonstrated; and a preliminary mapping for list-mode data in the form of XML schemas and documents was completed. DICOM permitted the creation of indices that can be used to rapidly locate in a list-mode file the cells that are members of a subset. DICOM and its coding schemes for other medical standards can be represented by XML schemas, which can be combined with other relevant XML applications, such as Mathematical Markup Language (MathML). Conclusions The use of XML format based on DICOM for analytical cytology met most of the previously specified requirements and appears capable of meeting the others; therefore, the present FCS should be retired and replaced by an open, XML-based, standard CytometryML. Cytometry Part A 54A:56–65, 2003. © 2003 Wiley-Liss, Inc.

Background The performance of a fully automated scanning cytometer incorporating previously reported high-precision autofocus and accurate image segmentation was evaluated for the detection of “ultra-rare” cells using a model of fetal nucleated red blood cells (fnRBCs) in the maternal circulation. These distinctive scanning cytometry techniques were expected to markedly improve sensitivity and specificity. Methods Normal adult blood and fetal red blood cells were stained with fluorescein isothiocyanate–conjugated anti-fetal hemoglobin and 4,6-diamidino-2-phenylindole, a nuclear dye. Adult cells were spiked with fetal cells to create ratios of about 1 fnRBC in 107 nucleated cells and deposited in monolayers on slides using centrifugal cytology. Rare-event performance parameters were reviewed, formalized, and applied to test the new instrument using this cell model. Results Fifteen slides were analyzed to establish performance by comparison with manual detection, and four sets of four slides each were then scanned to explore the limit of detection. Results were an average sensitivity of 91%, an average specificity error of 12.3 false-positives per million cells, and repeatability of 100% at a cell analysis rate of 862 Hz. With addition of a quick interactive step subsequent to scanning, the false-positive rate dropped to a total of only one artifact over the 15 experiments. The instrument succeeded at locating 1 fnRBC in 20 million adult cells, the lowest limit of detection tested. Conclusion This consistently high performance, coupled with the capability of scanning arbitrarily large numbers of cells, validates the considerable potential of precise high-speed autofocus and accurate real-time image segmentation for ultra-rare event detection. Cytometry 39:285–294, 2000 © 2000 Wiley-Liss, Inc.

Because this article is a bit of history rather than ascientific paper, we will depart from the formality of ajournalarticle.Thisarticlehastwopurposes.Thefirstistogive credit to two deceased individuals, Albert Castro,M.D., and Howard Gratzner, Ph.D. (1). Castro was the keyperson who was able to produce a polyclonal antibodythat was specific for bromodeoxyuridine (BrdU). For 10years, Gratzner took the lead in converting an idea into afunctional assay that affected DNA analysis and led to thedevelopment of many other assays with similar method-ologies. The second is to inform young scientists that (a)inventions or creations have utility in fields other than thepurpose for which they were created; (b) it is possible todo useful work in an institution that certainly would nothave been classified as one of the best; and (c) the pres-ence of talented people who are willing to collaborate isinvaluable.Scientifically, Gratzner is best known for the develop-ment of the first monoclonal antibody (mAb) to BrdU andiododeoxyuridine. This work was the culmination of pre-vious efforts with Robert Leif to develop polyclonal anti-bodies, which detected BrdU with variable specificity.Collaborations with scientists at the Lawrence LivermoreLaboratory encouraged the development of a widely usedflow cytometric test for the simultaneous measurementsof DNA and BrdU. The use of mAbs to halogenated pyri-midines was a significant scientific advance and currentlyis an integral tool for the analysis by flow and digitalmicroscopy of the cell cycle.On a personal level, Gratzner’s enthusiasm for scienceand his intelligence, curiosity, loyal interactions with fel-low collaborators, and friendship are sorely missed by allwhoknewhim.Hewasgenuinelyawarmandnicepersonwho was liked by almost everyone with whom he came incontact (1).Castro was a jovial man who interacted well with hiscollaborators. He was generous and very knowledgeable.He ran an immunodiagnostics laboratory at the universi-ties of Oregon and Miami and was an expert in the pro-duction of antibodies in animals. His warm smile, gener-ous giving of his knowledge, common sense, enthusiasmfor scientific research, and ability to get the job done wereessential to this project.Because this is a story of events that happened approx-imately 30 years ago and the existing records are limited,the accuracy of this article is limited by our memory andthose of our colleagues who have kindly helped with thisproject. Of course, this is our version of the events.Cell division and DNA replication are fundamental tobiology and specifically to the biology of cancer. Thisarticledescribeshowasimpleprocedureforthedetectionand quantitation of DNA synthesis was developed. Theprecise determination of when S phase occurs in the cellcycle is of use to maximize the selective killing of tumorcells with cycle-specific chemotherapeutic drugs. Ofgreater significance, studies of the control of the cellcycle, including its detour into apoptosis, can provideextremely useful insights into the creation of new thera-peutic regimens.The original process to detect S-phase cells included theuse of tritiated thymidine with autoradiography to mea-sure the labeling index (percentage of S-phase cells)and/or the fraction of labeled mitotic cells. Before and atthe infancy of flow cytometry, it was believed that thequantitative DNA analysis of normal and neoplastic cellsmight provide an objective marker for the diagnosis ofneoplasia. These measurements were performed with amicroscope on Feulgen-stained cells. In a normal popula-tion, it was established that there were two predominantpeaks in the DNA distribution, with a ratio of 1:2 in DNAcontent, and that cells that were synthesizing DNA (Sphase) were scattered in between, with a relative DNAcontent between 1 and 2. The detection of tritiated thy

Fluorescence in situ hybridization (FISH) uses labeled DNA probes to determine the copy number of the specific DNA sequence targeted. After denaturation of the probe and target DNA, the probe is allowed to hybridize to an intact nucleus. The hybridization can then be visualized by direct conjugation with fluorescent dyes, or indirect conjugation, such as biotinavidin systems.

The electrokinetic properties of doxorubicin (DOX)-resistant (P388/R) and -sensitive (P388/S) murine leukemic cells were studied in a free-flow electrophoresis (FFE) system. The electrophoretic mobilities (EM) of P388/S and P388/R cells were 1.07 and 1.35 x 10(-4) cm2 V-1 s-1, respectively, suggesting a higher net negative charge on the P388/R cells. Neuraminidase treatment decreased the EM of both the P388/S and P388/R cells by 15-20% but had no effect on cellular doxorubicin retention. Total and cell surface sialic acid contents were similar in both the cell lines. Our studies show that no direct correlations may exist among surface charge, cell surface sialic acid content, and doxorubicin retention in DOX-resistant and -sensitive P388 cells; however, differences in cell surface charge between these cell types were used to separate them by preparative FFE.

The hydrodynamic artifacts of turning over and droplet formation associated with isopycnic swinging-bucket rotor centrifugation have been virtually eliminated by increasing the viscosity of the layering solution. A 0.25% DNA solution was utilized for this purpose. Since the cells are diluted as they enter the gradient column, they enter and act as single particles. Two sizes (8 ml and 25 ml) of special centrifuge tubes are described. Polyethylene washers which contain the sample and float on top of the gradient column are utilized to restrict the radial trajectory of the cells so that they miss the walls of the centrifuge tube. The utilization of a special swinging-bucket rotor together with programmed acceleration eliminates swirling of the gradients. Thus it is now possible to achieve virtually artifact-free centrifugation with a swinging-bucket rotor. This is demonstrated by the results of a rebanding experiment.

A special peristaltic pump is described that has functioned as part of a system for density gradient formation and fractionation. Twenty-five pumping tubes are actuated on both the top and bottom of the pump. A Mylar diaphragm interposed between the rollers and the pumping tubes filters out the horizontal, stretching component of the forces imparted to the tubes. This greatly prolongs tube life, increases the allowable pressures that can be achieved with such a pump, and thus permits accurate delivery of viscous solutions. An explanation of the cause of the pulsations produced by peristaltic pumps is presented and the virtual elimination of these pulsations is demonstrated. Both velocity and direction of flow of the pump are controlled. By means of two independent bidirectional digital counters, preset volumes of fluid can be delivered and the total volume of liquid determined. Studies demonstrating the relative independence of fluid volume delivered at a preset count versus flow velocity and composition are presented. Other possibilities for use of the pump in automating density gradient analysis are discussed. Possibilities for employment of the pump for autoanalysis and in artificial organs are indicated.

The Committee on Nomenclature of the Society for Analytical Cytology presents guidelines for the analysis of DNA content by cytometry. These guidelines cover staining of DNA, cytogenetic and cytometric terminology, DNA index, resolution of measurements, and cytometric standards.

Bovine adenohypophysial tissue was dissociated by sequential enzymatic incubation in a continuous flow system. Dispersed cells separated into discrete fractions after centrifugation in isopycnic bovine serum albumin gradients. The dispersed and separated cells were prepared for microscopic identification and differential counts by centrifugal cytology. Radioimmunoassays for LH, FSH, TSH, and Prl were used to corroborate the differential counts and determine the homogeneity of the fractions. The thyrotrophs banded at an average density (\(\bar \rho \)) of 1.0417, the FSH-secretory cells at\(\bar \rho = 1.0597\), the LH-secretory cells at\(\bar \rho = 1.0458\), and the Prl-secretory cells at\(\bar \rho = 1.0126\). A 7–16 fold enrichment of different cell populations was possible. In bovine hypophyses each hormone appears to be formed by specific cells: the average TSH concentrations of the thyrotrophs were 5.1 pg/cell and the average LH and FSH concentrations were 4.7 and 4.9 pg/cell for LH-and FSH-secreting cells, respectively. The average Prl concentration was 4.9 pg/cell for Prl-secreting cells.

An immunofluorescence method for monitoring DNA synthesis in single cells has been developed for flow cytometry. With antiserum which is specific for 5-bromodeoxyuridine (BrdUrd) and a second fluorescent label, BrdUrd-incorporation pulses of 30 min are detectable. The fluorescence intensity of the incorporated BrdUrd, as determined by immunofluorescence, is related to the amount of BrdUrd incorporated, as shown by isotopic methods and cell sorting. Thus, the technique may be applicable to determining rates of replication per cell. Multiple samples of as few as 1 X 10(5) cells can be fixed, hydrolyzed and treated with the anti-BrdUrd antiserum. Nuclear-bound IgG is localized by fluorescein-labeled avidin-D. Since the technique uses whole cells, other parameters such as light scatter and DNA content can be simultaneously monitored so that cohorts of "labeled" cells can be followed through the cell cycle.

International audience; We report two novel applications of multiphoton microscopy for pharmacological studies of unstained cardiovascular tissue. First, we show that second harmonic generation (SHG) microscopy of unstained cardiac myocytes can be used to determine the sarcomere length with sub-resolution accuracy, owing to the remarkable contrast of the SHG signal originating from myosin filaments. A measurement precision of 20 nm is achieved, taking the sample variability into account. We used this technique to measure sarcomere contracture in the presence of saxitoxin, and results were in agreement with mechanical measurements of atrial tissue contracture. Second, we characterized multiphoton microscopy of intact unlabeled arteries. We performed simultaneous detection of two-photon-excited fluorescence (2PEF) from elastin laminae and SHG from collagen fibers upon 860 nm excitation. Combined 2PEF/SHG images provide a highly specific, micron scale description of the architecture of these two major components of the vessel wall. We used this methodology to study the effects of lindane (a pesticide) on the artery wall structure and evidenced structural alteration of the vessel morphology. © (2005) COPYRIGHT SPIE-The International Society for Optical Engineering