Lessons learned from a comprehensive electronic patient record procurement process-implications for healthcare organisations.

BACKGROUND: This study describes learning from procurement of a comprehensive electronic patient record (EPR/electronic health record (EHR)), system for a specialist clinical academic institution. METHOD: Retrospective review of procurement process in addition to evaluation of peer-reviewed literature in the field. RESULTS: Main lessons learned include the importance of detailed preparation of organisational requirements/specifications and organisational 'readiness'. Early staff involvement, resulting in ownership of the selected system by the organisation was a key achievement. The scoring process used required significant resource commitment but, despite being extensive in scope, provided relatively poor distinction between suppliers, despite significant variation in supplier self-scoring. Other elements, such as demonstrations and site visits, provided superior evaluation of functional abilities, and specification requirements should be regarded as threshold evaluation. CONCLUSION: While principles should be followed, the procurement process must be modified to meet the needs of the specific organisation, in terms of its clinical activities, digital maturity, existing infrastructure and budget.

Data file standard for flow cytometry, FCS 3.0.

This unit describes the data file format FCS 3.0 developed to permit the sharing of data among laboratories and to provide some uniformity in data formats from various instruments.

Sheath fluid control to permit stable flow in rapid mix flow cytometry.

BACKGROUND: Flow cytometry is a potentially powerful tool to analyze the kinetics of ligand binding, cell response and molecular assembly. The difficulty in adding reactant to cells, achieving adequate mixing, delivering those cells to the laser focal point and establishing stable flow, has historically limited flow cytometry to systems with reactions times longer than 5 s. With the advent of automated syringes and flow injection methods, sample injection times shorter than 1 s have become routine. However, an inherent problem in acquiring time courses starting under 1 s is that rapid sample introduction through the flow tip to the detection point perturbs laminar flow. The purpose of this work was to determine if stable flow could be reestablished more quickly if the sheath flow was reduced during sample introduction, returning to normal sheath and sample rates afterward. METHODS: We used programmable syringes and valves to control sample mixing as well as sheath and sample delivery through the flow tip to the detection point for stream-in-air detection. Stable flow was monitored by mean particle fluorescence during sample introduction. RESULTS: With no sheath reduction, stable flow recovered after more than 1 s. By reducing sheath flow during the short period (300 msec) of sample mixing and delivery, stable laminar flow recovered within 200 msec. CONCLUSIONS: This use of automated syringes to control both sheath and sample flow provides a potential for robust sample handling applicable to kinetic as well as high throughput flow cytometric analysis.

Biosafety guidelines for sorting of unfixed cells.

The International Society of Analytical Cytology (ISAC) Biohazard Working Group presents guidelines for sorting of unfixed cells, including known biohazardous samples, using jet-in-air, deflected-droplet cell sorters. There is a risk that personnel operating these instruments could become exposed to droplets and aerosols containing biological agents present in the samples. The following guidelines can aid in the prevention of exposures of laboratory personnel to pathogens contained in the sort samples. The document provides biosafety recommendations for sample handling, operator training and protection, laboratory facility design, and instrument setup and maintenance. In addition, it describes in detail methods for assessment of instrument aerosol containment. Recommendations provided here may also help laboratories to obtain institutional and/or regulatory agency approval for sorting of unfixed and known biohazardous samples.

Proposed new data file standard for flow cytometry, version FCS 3.0.

In 1984, the first flow cytometry data file format was proposed as Flow Cytometry Standard 1.0 (FCS1.0). FCS 1.0 provided a uniform file format allowing data acquired on one computer to be correctly read and interpreted on other computers running a variety of operating systems. That standard was modified in 1990 and adopted by the Society of Analytical Cytology as FCS 2.0. Here, we report on an update of the FCS 2.0 standard which we propose to designate FCS 3.0. We have retained the basic four segment structure of earlier versions (HEADER, TEXT, DATA and ANALYSIS) in order to maintain analysis software compatibility, where possible. The changes described in this proposal include a method to collect files larger than 100 megabytes (not possible in earlier versions of the standard), the inclusion of international characters in the TEXT portions of the file, a method of verifying data integrity using a 16-bit cyclic redundancy check, and increased keyword support for cluster analysis and time acquisition. This report summarizes the work of the ISAC Data File Standards Committee. The complete and detailed FCS 3.0 standard is available through the ISAC office [Sherwood Group, 60 Revere Drive, Ste 500, Northbrook, IL 60062, phone: (847) 480-9080 ext. 231, fax: (847) 480-9282, E-mail: isac@sherwood-group.com] or through the internet at the ISAC WWW site, http://nucleus.immunol.washington.edu/ISAC.ht ml.

Calcium metabolism in blood and peritoneal lymphocytes from continuous ambulatory peritoneal dialysis patients.

OBJECTIVE: Cellular immune function in peritoneal dialysis patients has been shown to be depressed, but the mechanism of this immunosuppression has not been ascertained. Because calcium is an important mediator of lymphocyte activation, this study was designed to investigate if there was an alteration of calcium metabolism in the lymphocytes of continuous ambulatory peritoneal dialysis (CAPD) patients. DESIGN: Sixteen CAPD patients were studied at the initiation of CAPD and after two months of treatment. Twenty-three normal controls were also enrolled in the study. Cytoplasmic calcium changes were investigated in response to the mitogen phytohemagglutinin (PHA) in peripheral blood and peritoneal lymphocytes, using the intracellular calcium probe indo-1 and flow cytometry. Baseline cytoplasmic calcium levels and changes in cytoplasmic calcium in response to PHA were assessed at the initiation of CAPD and after two months of therapy. RESULTS: Peripheral lymphocytes of patients and controls had similar calcium baseline levels, but the peritoneal lymphocytes had baseline cytoplasmic calcium levels averaging 81% higher than the corresponding calcium levels of the patients' peripheral blood lymphocytes. As compared to peripheral lymphocytes, the response to PHA stimulation was significantly less in the peritoneal lymphocytes, increasing an average of only 46.8% above baseline. Peripheral blood lymphocytes of the patients responded by an average increase of 78.9% over baseline. Control cells increased an average of 66.3% over baseline. Follow-up studies done two months after the initiation of CAPD indicated there were no significant changes (as compared to month 0) that occurred in baseline or stimulated intracellular calcium concentrations. CONCLUSIONS: While the peripheral lymphocytes of CAPD patients respond adequately to PHA, the high baseline calcium levels of the peritoneal lymphocytes suggest that these cells may be in a state of chronic activation and may respond minimally to an antigenic challenge.

Fluorescence drift detection as a novel QC procedure for DNA cell-cycle analysis.

The goal of this work was to develop an objective, quantitative, and reproducible method of detecting fluorescence drift which may have occurred during DNA cell-cycle data acquisition. Quality control software, "TruPloid," is described that analyzes list-mode files to detect and quantify fluorescence drift using three separate statistical tests. We show that fluorescence drift may lead to a variety of measurement artifacts including high coefficients of variation, obscuring of small populations and creation of distinct artificial peaks. Forty percent of 50 archived list-mode files displayed fluorescence drift, which demonstrates the need for detection methods to deal with this source of DNA cell-cycle histogram artifacts.

Progesterone but not estrogen depolarizes natural killer cells.

Natural killer (NK) cell tumoricidal and antimicrobial activities can be rapidly modulated by molecules that interact with membrane receptors. The discovery of NK cell depolarization induced by steroid-like Na+ channel agonists prompted a study of purified human NK cell excitability to a variety of steroids. Progesterone, but not estrogen, depolarized NK cells with concentration and time dependency. Excitability was measured by using flow cytometry and the anionic voltage-sensitive dye oxonol. Preincubation with the Na+ channel antagonist tetrodotoxin or removal of the extracellular Na+ blocked the response. Progesterone may rapidly change membrane potential, and eventually function, by acting on putative NK plasma membrane receptors coupled to Na+ conductances.

Heparin enhances the interaction of infective Leishmania donovani promastigotes with mouse peritoneal macrophages. A fluorescence flow cytometric analysis.

Human visceral leishmaniasis results from the infection of macrophages by the protozoan parasite Leishmania donovani. Both forms of the parasite, the extracellular promastigote and the obligate intracellular amastigote, require cell surface molecules to ensure their recognition and uptake by the host cell, the macrophage. We have proposed previously that the heparin-binding protein on the surface of promastigotes is an adhesion molecule. The present report provides experimental evidence to support this hypothesis. Fluorescence flow cytometry using FITC-heparin was employed to study the heparin-binding protein of L. donovani promastigotes and amastigotes. We demonstrate the presence of the heparin-binding protein on the surface of amastigotes and document the heparin specificity of the binding protein for both forms of the parasite. Two-color fluorescence analysis was performed to compare R-PNA reactivity and FITC-heparin binding during the parasite's 7-day growth curve. Using this strategy we show that the expression of heparin binding activity coincides with the differentiation of the noninfective promastigote into the infective metacyclic from of the parasite. Macrophages that were challenged for 30 min with heparin-treated, FITC-labeled parasites became 2.82-fold more fluorescent than their counterparts which were exposed to non-heparin-treated FITC-labeled promastigotes. Finally, using Kolmogorov-Smirnov analysis we show that the adhesion of promastigotes to mouse peritoneal macrophages is significantly enhanced in the presence of 3.3 microM heparin. The experiments described in the present report provide evidence for the hypothesis that L. donovani's heparin-binding protein is a virulence factor that functions as an adhesion molecule in the parasite-macrophage interaction.

Method to improve the sensitivity of flow cytometric membrane potential measurements in mouse spinal cord cells.

We have developed a technique to improve the sensitivity of relative membrane potential measurements in mouse spinal cord cells using the fluorescent, anionic, voltage sensitive dye, DiBa-C4(3) (Oxonol) and flow cytometry. In order to attribute cellular fluorescence primarily to membrane potential, signal variability due to cell size and shape was reduced by dividing the log fluorescence signal from each cell by either its log forward angle light scatter or log side scatter signals. The use of these ratios in place of log oxonol fluorescence reduced the coefficient of variation of the distributions while leaving the changes in mean fluorescence largely unaffected. Kolmogorov-Smirnov analysis of pre- vs. postkainate stimulation (an excitatory amino acid) showed improved sensitivity of the assay with the use of this ratio technique.

Inhibition of lymphocyte activation in splenic and gut-associated lymphoid tissues following oral exposure of mice to 7,12-dimethylbenz[a]anthracene.

The hypothesis that 7,12-dimethyl-benz[a]anthracene (DMBA) suppresses immune function in mice via an inhibition of lymphocyte activation was examined in these studies. Daily exposure of B6C3F1 mice to DMBA (cumulative doses of 1.4 to 140 mg/kg) via the oral route for 14 days was found to inhibit phytohemagglutinin (PHA) and lipopolysaccharide mitogen responses in lymphoid cells obtained from the spleen. Peyer's Patches, and mesenteric lymph nodes. The 14 mg/kg cumulative dose of DMBA produced no significant decrease in the number of recovered viable cells, yet mitogen responses were suppressed by approximately 50% in the spleen and mesenteric lymph nodes, and by greater than 70% in the Peyer's Patches. DMBA inhibited PHA-induced Ca+2 mobilization measured by flow cytometry in each of these three lymphoid tissues. There was no change in the percentage of T cells recovered from the spleen, mesenteric lymph nodes, or Peyer's Patches. Peyer's Patch lymphocytes obtained from the GI tract appeared to be slightly more sensitive to inhibition of mitogen responsiveness and perhaps Ca+2 mobilization, potentially due to the oral route of exposure to DMBA. These studies provide evidence that DMBA inhibits early events associated with lymphocyte activation in mice.

Human natural killer cells express Na+ channels. A pharmacologic flow cytometric study.

Voltage-gated excitability of purified human NK cells was studied by using flow cytometry and the voltage-sensitive dye, oxonol. Highly purified human NK cells (CD16 = 95 +/- 1%) from normal volunteers were prepared by using a negative panning technique. The Na(+)-channel agonists batrachotoxin (BTX) (1 to 4 microM) and veratridine (Ver) (100 to 400 microM) depolarized a population of highly purified human NK cells as determined by flow cytometry. BTX and Ver responses were concentration-, time-, temperature-, and Na(+)-dependent. The Na+ channel antagonist tetrodotoxin (1 microM) blocked BTX and Ver responses. Ver (100 microM) produced significant inhibition of cytotoxicity when purified NK cells were incubated with K562 tumor target cells in a 4-h 51Cr release cytotoxicity assay. The effect was blocked by tetrodotoxin. These results strongly suggest presence of functional Na+ channels in NK cells. Activation of voltage-dependent Na+ channels depolarizes cells and reduces their in vitro cytotoxic function.

The pattern of dihydrofolate reductase expression through the cell cycle in rodent and human cultured cells.

We have examined the pattern of dihydrofolate reductase (DHFR) enzyme and mRNA levels in cell cycle stage-specific populations obtained by centrifugal elutriation in Chinese hamster ovary cells and in a derivative line in which the dihydrofolate reductase gene is amplified approximately 50-fold. On a per cell basis, we observed a 2-fold increase in DHFR activity as cells progressed from G1 to G2/M with a concomitant 2-fold increase in the rate of protein synthesis and steady state level of mRNA. Analysis of DHFR mRNA levels in cell cycle stage-specific mouse 3T6 and human 143 tk- cells gave a similar pattern. We also demonstrate that simple alterations in growth conditions prior to elutriations can dramatically increase the levels of DHFR mRNA in all cell cycle states, thereby indicating that growth response associated with the DHFR gene functions independent of the cell cycle. We conclude that during periods of exponential growth the increases in dihydrofolate reductase activity, rate of protein synthesis, and steady state levels of mRNA parallel the general increases in cell volume and protein content associated with normal progression through the cell cycle, and therefore DHFR cannot be considered a cell cycle-regulated enzyme.

Thermal denaturation of DNA for immunochemical staining of incorporated bromodeoxyuridine (BrdUrd): critical factors that affect the amount of fluorescence and the shape of BrdUrd/DNA histogram.

Significant inter- and intraexperimental variations of the relative antibromodeoxyuridine fluorescence were found during measurement of DNA synthesis rates using flow cytometric analysis of 5-bromodeoxyuridine (BrdUrd)-labeled cells with an anti-BrdUrd antibody. Fluctuations in other endpoints associated with levels of denaturation (integrity of DNA and cell size) were also observed to vary widely among samples that were otherwise thought to have been treated identically. Therefore, the denaturation step has been carefully re-examined, and several critical factors were identified that influence the denaturation and subsequent binding of the anti-BrdUrd to the labeled DNA. These factors include cell density, volume of water, and pH of the sample during heating. Appropriate adjustments are now included in the protocol, resulting in more consistent anti-Brd-Urd measurements in the face of routine (and sometimes necessary) experimental variations.

Characterization by flow cytometry of fluorescein-methotrexate transport in Chinese hamster ovary cells.

We have studied by flow cytometry the transport of fluorescein-methotrexate in Chinese hamster ovary cells. Fluorescein-methotrexate appears to enter cells via a mechanism different from the carrier-mediated system for methotrexate. This conclusion is supported by the following observations: 1) Fluorescein-methotrexate is transported equally well into normal and mutant cells defective in the inward methotrexate uptake. 2) Folic acid and its reduced states, which competitively inhibit methotrexate uptake, do not alter fluorescein-methotrexate transport. 3) Fluorescein-methotrexate accumulation exhibits a low temperature coefficient (Q10 = 1.6) compared with the influx of methotrexate (Q10 = 6-8). 4) Initial rates of fluorescein-methotrexate uptake are concentration dependent but are not saturable. 5) Fluorescein-methotrexate uptake is very slow and reaches steady state after 8 h, whereas at an equimolar concentration methotrexate reaches saturation after 20 min. 6) Initial influx rates of fluorescein-methotrexate are not affected by the presence of methotrexate. 7) Sulfhydryl-reactive mercurials, which block methotrexate transport, do not reduce fluorescein-methotrexate influx, but rather stimulate it. Thus, based on the nonsaturability of fluorescein-methotrexate inward transport, its low temperature coefficient, and lack of inhibition with structural analogs, we conclude that fluorescein-methotrexate is accumulated in hamster cells by a passive diffusion process.

DNA flow cytometry and prognostic factors in 1331 frozen breast cancer specimens.

Breast cancer proliferative capacity as determined by the DNA thymidine labeling index, along with estrogen and progesterone receptor status, is highly predictive for risk of relapse and overall survival. Recently, DNA ploidy and proliferative capacity (S-phase fraction [SPF]) as determined by flow cytometry have also shown significant prognostic value. The authors have developed a technique which allows a 50 to 100 mg aliquot of the same frozen breast tumor specimen routinely employed in steroid receptor assays, to be assayed for both DNA ploidy and SPF by flow cytometry. Of the 1331 tumors examined, DNA histograms were evaluable for ploidy in 89% (1184) of specimens examined; 57% of these were aneuploid. Adapting a trapezoidal model to estimate SPF in both diploid and aneuploid tumors, the authors found 81% (1084) to be evaluable for SPF, with a median SPF of 5.8% for the entire population. The median SPF was significantly lower in diploid tumors (2.6%) than in aneuploid tumors (10.3%, P less than 0.0001). Both aneuploidy and high SPF were strongly associated with absence of steroid receptors. Aneuploid tumors showed more striking differences in the frequency of high S-phase values with respect to receptor status and age or menopausal status, whereas diploid but not aneuploid tumors showed lower SPF in node-negative versus node-positive patients. Because it is particularly important to identify the high-risk minority of node-negative patients, the authors examined the node-negative group separately. High SPF subgroups appeared in each category of receptor status and age or menopausal status within the node-negative group, suggesting that SPF will be an independent prognostic factor. With the DNA flow cytometric methods used here, it is now practical to determine ploidy and SPF for nearly every breast cancer patient. These factors, which show associations with established prognostic factors, such as receptor status can now be fully evaluated for their prognostic significance in broad patient populations.

Evaluation of a modeling system for S-phase estimation in breast cancer by flow cytometry.

Using software programs provided by Coulter Electronics, we have developed an analysis system that would address problems encountered in DNA flow cytometric analysis of heterogeneous solid tumor populations, especially where the G2-M phase of the diploid population contaminates the S-phase of the aneuploid population, causing an overestimation of cells in S phase. We used the PARA 1 and PARA 2 programs in concert and developed three analysis models: (a) for euploid tumors; (b) for hyperdiploid tumors with overlapping populations; and (c) for near-diploid aneuploid tumors. Our purpose in this paper is to determine the limits and reproducibility of this analysis system with an emphasis on tumors with overlapping populations. Aliquots of frozen, pulverized breast tumor tissue (50 to 100 mg), routinely used in the steroid receptor assay, were used for routine flow cytometric measurement of the DNA index and S-phase fraction. To determine the accuracy of the analysis when overlapping populations were present, we mixed an aneuploid breast cancer cell line with human blood lymphocytes in varying ratios. A 10% mixture of aneuploid cells, the lowest mixture tested, still allowed analysis results within 95% confidence limits. Reproducibility of the system was assessed on frozen breast tumor tissue by intra- and interassay variation studies measuring cell cycle parameters and coefficient of variation of the G0-G1 peak width. Within any sample the amount of variation (+/- 2 SD) for the G0-G1 value was +/- 4.40 for intraassay and +/- 4.60 for interassay, and the amount of variation for S phase was +/- 3.0 and +/- 3.2 for intraassay and interassay, respectively. There was no difference in the variation of estimates for G2-M (+/- 2.6 for both intra- and interassay). In this study, the coefficient of variation of the G0-G1 peak greater than 5% was defined as unacceptable for accurate analysis, with the conclusion that S-phase fractions in aneuploid tumors can be routinely analyzed in human breast tumor biopsies despite tumor cell heterogeneity.