Renal Consequences of Diabetes After Kidney Donation.

Whether diabetes after kidney donation is associated with an accelerated GFR decay in the remaining kidney has not been studied. We determined the incidence of diabetes in kidney donors, and compared GFR change over time in diabetic to nondiabetic donors, in addition to the effect of diabetes mellitus (DM) on the development of proteinuria, hypertension, and end-stage renal disease (ESRD). Of the 4014 donors, 309 (7.7%) developed diabetes at a median age of 56.0 years and after a median of 18 years after donation. The difference in annual estimated GFR (eGFR) change between diabetic and nondiabetic donors in the 7 years before the development of DM was -0.08 mL/min/year; p = 0.51. After DM development, the difference was -1.10 mL/min/year for diabetic donors with hypertension and proteinuria, p < 0.001; -0.19 for diabetic donors with hypertension but no proteinuria, p = 0.29; -0.75 mL/min/year for diabetic donors with proteinuria but no hypertension, p = 0.19; and -0.09 mL/min/year for diabetic donors without proteinuria or hypertension, p = 0.63. When DM was considered as a time-dependent covariate, it was associated with the development of proteinuria (hazard ratio [HR] 2.65, 95% confidence interval [CI] 1.89-3.70; p < 0.001) and hypertension (HR 2.19, 95% CI 1.74-2.75; p < 0.001). It was not, however, associated with ESRD. eGFR decline after DM development exceeds that of nondiabetic donors only in diabetic donors with concomitant proteinuria and hypertension.

Defining the Tipping Point in Surgical Performance for Laparoscopic Donor Nephrectomy Among Transplant Surgery Fellows: A Risk-Adjusted Cumulative Summation Learning Curve Analysis.

The United Network for Organ Sharing recommends that fellowship-trained surgeons participate in 15 laparoscopic donor nephrectomy (LDN) procedures to be considered proficient. The American Society of Transplant Surgeons (ASTS) mandates 12 LDNs during an abdominal transplant surgery fellowship. We performed a retrospective intraoperative case analysis to create a risk-adjusted cumulative summation (RACUSUM) model to assess the learning curve of novice transplant surgery fellows (TSFs). Between January 2000 and December 2014, 30 novice TSFs participated in the organ procurement rotation of our ASTS-approved abdominal transplant surgery fellowship. Measures of surgical performance included intraoperative time, estimated blood loss, and incidence of intraoperative complications. The performance of senior TSFs was used to benchmark novice TSF performance. Scores were tabulated in a learning curve model, adjusting for case complexity and prior TSF case volume. Rates of adverse surgical events were significantly higher for novice TSFs than for senior TSFs. In univariable analysis, multiple renal arteries, high BMI, prior abdominal surgery, male donor, and nephrolithiasis were correlated with higher incidence of adverse surgical events. Based on the RACUSUM model, high intraoperative time is mitigated after 28 procedures, incidence of intraoperative complications tends to diminish after 24 procedures, and improvement in estimated blood loss did not remain consistent. TSFs exhibit a tipping point in LDN performance by 24-28 cases and proficiency by 35-38 cases.

Biomarkers of Tolerance in Kidney Transplantation: Are We Predicting Tolerance or Response to Immunosuppressive Treatment?

We and others have previously described signatures of tolerance in kidney transplantation showing the differential expression of B cell-related genes and the relative expansions of B cell subsets. However, in all of these studies, the index group-namely, the tolerant recipients-were not receiving immunosuppression (IS) treatment, unlike the rest of the comparator groups. We aimed to assess the confounding effect of these regimens and develop a novel IS-independent signature of tolerance. Analyzing gene expression in three independent kidney transplant patient cohorts (232 recipients and 14 tolerant patients), we have established that the expression of the previously reported signature was biased by IS regimens, which also influenced transitional B cells. We have defined and validated a new gene expression signature that is independent of drug effects and also differentiates tolerant patients from healthy controls (cross-validated area under the receiver operating characteristic curve [AUC] = 0.81). In a prospective cohort, we have demonstrated that the new signature remained stable before and after steroid withdrawal. In addition, we report on a validated and highly accurate gene expression signature that can be reliably used to identify patients suitable for IS reduction (approximately 12% of stable patients), irrespective of the IS drugs they are receiving. Only a similar approach will make the conduct of pilot clinical trials for IS minimization safe and hence allow critical improvements in kidney posttransplant management.

Immunological profiling of haemodialysis patients and young healthy individuals with implications for clinical regulatory T cell sorting.

With the increasing interest in clinical trials with regulatory T cells (Tregs), immunological profiling of prospective target groups and standardized procedures for Treg isolation are needed. In this study, flow cytometry was used to assess peripheral blood lymphocyte profiles of young healthy individuals and patients undergoing haemodialysis treatment. Tregs obtained from the former may be used in haematopoietic stem cell transplantation and Tregs from the latter in the prevention of kidney transplant rejection. FOXP3 mRNA expression with accompanying isoform distribution was also assessed by the quantitative reverse transcriptase polymerase chain reaction. Flow-cytometric gating strategies were systematically analysed to optimize the isolation of Tregs. Our findings showed an overall similar immunological profile of both cohorts in spite of great differences in both age and health. Analysis of flow-cytometric gating techniques highlighted the importance of gating for both CD25high and CD127low expression in the isolation of FOXP3-positive cells. This study provides additional insight into the immunological profile of young healthy individuals and uraemic patients as well as in-depth analysis of flow-cytometric gating strategies for Treg isolation, supporting the development of Treg therapy using cells from healthy donors and uraemic patients.

Daily fluid intake and outcomes in kidney recipients: post hoc analysis from the randomized ABCAN trial.

Generous and even excessive fluid intake is routinely recommended to kidney transplant recipients despite minimal evidence to support this practice. We hypothesized that increased fluid intake, ascertained by 24-h urine volume output, may adversely affect graft outcomes as it would impose an extra workload on a limited number of nephrons. Kidney transplant recipients who were randomized to losartan vs. placebo in the Angiotensin II Blockade for Chronic Allograft Nephropathy (ABCAN) trial (n = 153) underwent baseline, five-yr biopsies, and annual iothalamate glomerular filtration rate assessment. Recipients with higher urine volume at randomization had higher urinary sodium and also higher urinary protein. The proportion using diuretics or CNI based regimens were similar across urinary volume tertiles. The highest urinary volume tertile (>2.56 L/d) did not predict the development of interstitial volume doubling or end-stage renal disease (ESRD) from interstitial fibrosis/tubular atrophy (OR = 3.52, 95% CI 0.4, 31.24, p = 0.26), interstitial volume doubling or all-cause ESRD (OR = 7.04, 95% CI 0.66, 74.87, p = 0.11), and was not associated with the conventional endpoint of doubling serum creatinine, all-cause ESRD, or death (OR = 0.89, 95% CI 0.21, 3.71, p = 0.87). These results suggest that the current practice of liberal fluid intake may not be beneficial in low risk and mostly Caucasian transplant recipients.

Obtaining regulatory T cells from uraemic patients awaiting kidney transplantation for use in clinical trials.

Adoptive transfer of regulatory T cells (T(regs)) has been proposed for use as a cellular therapy to induce transplantation tolerance. Preclinical data are encouraging, and clinical trials with T(reg) therapy are anticipated. In this study, we investigate different strategies for the isolation and expansion of CD4(+) CD25(high) CD127(low) T(regs) from uraemic patients. We use allogeneic dendritic cells (DCs) as feeder cells for the expansion and compare T(reg) preparations isolated by either fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS) that have been expanded subsequently with either mature or tolerogenic DCs. Expanded T(reg) preparations have been characterized by their purity, cytokine production and in-vitro suppressive ability. The results show that T(reg) preparations can be isolated from uraemic patients by both FACS and MACS. Also, the type of feeder cells used in the expansion affects both the purity and the functional properties of the T(reg) preparations. In particular, FACS-sorted T(reg) preparations expanded with mature DCs secrete more interleukin (IL)-10 and granzyme B than FACS-sorted T(reg) preparations expanded with tolerogenic DCs. This is a direct comparison between different isolation techniques and expansion protocols with T(regs) from uraemic patients that may guide future efforts to produce clinical-grade T(regs) for use in kidney transplantation.

Ex Vivo Generation of Donor Antigen-Specific Immunomodulatory Cells: A Comparison Study of Anti-CD80/86 mAbs and CTLA4-lg Costimulatory Blockade.

Adoptive transfer of alloantigen-specific immunomodulatory cells generated ex vivo with anti-CD80/CD86 mAbs (2D10.4/IT2.2) holds promise for operational tolerance after transplantation. However, good manufacturing practice is required to allow widespread clinical application. Belatacept, a clinically approved cytotoxic T-lymphocyte antigen 4-immunoglobulin that also binds CD80/CD86, could be an alternative agent for 2D10.4/IT2.2. With the goal of generating an optimal cell treatment with clinically approved reagents, we evaluated the donor-specific immunomodulatory effects of belatacept- and 2D10.4/IT2.2-generated immunomodulatory cells. Immunomodulatory cells were generated by coculturing responder human peripheral blood mononuclear cells (PBMCs) (50 × 106 cells) with irradiated donor PBMCs (20 × 106 cells) from eight human leukocyte antigen-mismatched responder-donor pairs in the presence of either 2D10.4/IT2.2 (3 µg/106 cells) or belatacept (40 µg/106 cells). After 14 days of coculture, the frequencies of CD4+ T cells, CD8+ T cells, and natural killer cells as well as interferon gamma (IFN-γ) production in the 2D10.4/IT2.2- and belatacept-treated groups were lower than those in the control group. The percentage of CD19+ B cells was higher in the 2D10.4/IT2.2- and belatacept-treated groups than in the control group. The frequency of CD4+CD25+CD127lowFOXP3+ T cells increased from 4.1±1.0% (preculture) to 7.1±2.6% and 7.3±2.6% (day 14) in the 2D10.4/IT2.2- and belatacept-treated groups, respectively (p<0.05). Concurrently, delta-2 FOXP3 mRNA expression increased significantly. Compared with cells derived from the no-antibody treated control group, cells generated from both the 2D10.4/IT2.2- and belatacept-treated groups produced lower IFN-γ and higher interleukin-10 levels in response to donor-antigens, as detected by enzyme-linked immunospot. Most importantly, 2D10.4/IT2.2- and belatacept-generated cells effectively impeded the proliferative responses of freshly isolated responder PBMCs against donor-antigens. Our results indicate that belatacept-generated donor-specific immunomodulatory cells possess comparable phenotypes and immunomodulatory efficacies to those generated with 2D10.4/IT2.2. We suggest that belatacept could be used for ex vivo generation of clinical grade alloantigen-specific immunomodulatory cells for tolerance induction after transplantation.

Noninvasive measurement of phenylalanine by iontophoretic extraction in patients with phenylketonuria.

Phenylketonuria is an autosomal recessive disorder characterized by elevated concentrations of phenylalanine. Elevated phenylalanine concentrations can impair intellectual functions and the disease is treated with a lifelong diet and frequent monitoring of plasma phenylalanine concentrations. Previous in vitro studies have demonstrated the feasibility of iontophoretically enhanced transdermal transport of phenylalanine. Here we evaluate the feasibility of transdermal iontophoretic extraction of phenylalanine in vivo. Phenylalanine was iontophoretically extracted from the skin of healthy volunteers and of patients with phenylketonuria for up to 6 h and concentrations were compared with those measured in plasma. The amount of phenylalanine iontophoretically extracted from the skin declined over time, suggesting contribution of phenylalanine from the skin in the initial extraction. Phenylalanine iontophoretically extracted from skin correlated with plasma phenylalanine levels at plasma levels above 300 micromol/L. This correlation supports the feasibility of iontophoretic phenylalanine extraction for monitoring phenylketonuria.

Sutures impregnated with carbazate-activated polyvinyl alcohol reduce intraperitoneal adhesions.

BACKGROUND: Intraperitoneal adhesions cause significant morbidity. They occur after peritoneal trauma, which induces oxidative stress with production of inflammatory cytokines, peroxidized proteins (carbonyls) and lipids (aldehydes). This study aimed to investigate if carbazate-activated polyvinyl alcohol (PVAC), an aldehyde-carbonyl inhibitor, can reduce intraperitoneal adhesions in an experimental model. MATERIAL AND METHODS: Male Sprague-Dawley rats (n=110) underwent laparotomy, cecal abrasion and construction of a small bowel anastomosis. They either were treated with intraperitoneal instillation of PVAC or were sutured with PVAC-impregnated sutures. Thromboelastography analysis was performed using human blood and PVAC. The lipid peroxidation product malondialdehyde (MDA) and inflammatory cytokines IL-1ß and IL-6 were quantified in peritoneal fluid. At day 7, bursting pressure of the anastomosis was measured and adhesions were blindly scored. RESULTS: PVAC in human blood decreased the production of the fibrin-thrombocyte mesh without affecting the coagulation cascade. MDA, IL-1ß and IL-6 were increased after 6h without significant difference between the groups. PVAC-impregnated sutures reduced intraperitoneal adhesions compared to controls (p=0.0406) while intraperitoneal instillation of PVAC had no effect. Anastomotic bursting pressure was unchanged. CONCLUSIONS: Intervention with an aldehyde-carbonyl inhibitor locally in the wound by PVAC-impregnated sutures might be a new strategy to reduce intraperitoneal adhesions.

Isolation of viable tumor cells following introduction of labelled antibody to an intracellular oncogene product using electroporation.

A method for labelling the intracellular ras oncogene product, p21, with a monoclonal antibody, in B16BL6 mouse melanoma cells for subsequent flow cytometric analysis and viable cell sorting is described. Permeabilization of the cells for introduction of labelled antibody was attempted using (1) lysolecithin treatment, and (2) electroporation, a much more highly controllable technique. Permeabilization was assessed using propidium iodide or calcofluor white M2R staining, while short-term cellular viability was determined using fluorescein diacetate staining and long-term viability by reculturing the sorted cells. We successfully introduced labelled antibody into the cells with both permeabilization techniques. Insufficient numbers of viable permeabilized cells were obtained lysolecithin treatment to warrant an attempt at viable cell sorting. On the other hand, good numbers of viable, permeabilized cells were obtained using electroporation and we successfully sorted viable tumor cell populations based on the intensity of their anti-p21ras staining. These sorted tumor cells retained their characteristic anti-p21ras staining intensity for at least 2 weeks of propagation in culture.

Respiratory symptoms and pulmonary function in an elderly nonsmoking population.

OBJECTIVE: To examine risk factors for chronic airway disease (CAD) in elderly nonsmokers, as determined by pulmonary function tests (PFTs), and to correlate reported respiratory symptoms with PFT measures. DESIGN: An observational survey. SETTING: Several communities in California. MEASUREMENTS: Exposures and respiratory history were assessed by standardized questionnaire. PFTs were performed and prediction equations derived. RESULTS: Significant risk factors for obstruction on PFTs in multiple logistic regression included reported environmental tobacco smoke (ETS) exposure (relative risk [RR]=1.44), parental CAD or hay fever (RR=1.47), history of childhood respiratory illness (RR=2.15), increasing age, and male sex. The number of years of past smoking was of borderline significance (RR=1.29 for 10 years of smoking; p=0.06). The prevalence of obstruction on PFTs was 24.9% in those with definite symptomatic CAD, compared with 7.5% in those with no symptoms of CAD. The prevalence of obstruction was 36.0% among those with asthma and 70.6% among those with emphysema. Also, symptomatic CAD correlated with reduction in lung function by analysis of covariance. The mean percent predicted FEV1 adjusted for covariates was 90.6% in persons with definite symptoms of CAD, compared with 97.8% in those without it (p < 0.001). CONCLUSIONS: Age, sex, parental history, childhood respiratory illness, and reported ETS exposures were significant risk factors for obstruction on PFTs. Self-reported respiratory symptoms also correlated significantly with PFTs.

A flow cytometric method for intracellular labeling and purification of rare neuronal populations: isolation of fixed neurophysin neurons.

A method is described for flow cytometric analysis and fluorescence-activated cell sorting of small populations of neurons following dissociation of fixed brain tissue and immunofluorescent labeling of intracellular antigens. This method has been successfully applied to neurophysin-containing magnocellular neurons of the rat supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. These neurons constitute a rare population in the context of flow cytometry, comprising less than 2% of all cells present in dissociated tissue punches of SON and PVN. Following labeling with anti-neurophysins sera and fluorescein-conjugated second antibody, a highly enriched population containing 80-85% neurophysin-positive neurons was isolated by fluorescence-activated cell sorting. Recovery of 29% of all neurophysin-containing neurons in the SON/PVN was achieved. Perikarya were recovered largely intact, frequently with attached proximal dendritic processes. Applications of this method include purification of specific neuronal types for use as immunogens in production of monoclonal antibodies to cell-type-specific antigens, and rapid surveys of fluorescent lectin or other ligand binding to cell populations identified by the presence of particular intracellular antigens.

Cell-cell and cell-surface interactions in an illuminated biofilm: implications for marine sediment stabilization.

Most wetted surfaces that are illuminated support a population of phototrophs. The marine sediment is no exception and there the major component of the microphytobenthic population is diatoms. These organisms are credited with stabilizing the sediment against physical disturbance by virtue of the extracellular carbohydrate polymers that they elaborate. However, diatoms synthesize and secrete several carbohydrate polymers and it is not certain which of them is involved in the stabilization process. In order to investigate this, we have constructed small glass bead-filled flow through bioreactors to mimic marine sediments. The flow rate through the bioreactors was found to reflect the physical stability of the bead bed. Thus flow rate was measured as a function of diatom growth and the production of three operationally-defined polymers, i.e., those soluble in the medium, those soluble in 0.5 M NaHCO3 at 90 degrees C and those not soluble in either solvent (matrix polymer). Growth of the diatoms did not change the hydraulic conductivity of the bioreactors. For Amphora coffeaeformis, neither did the production of medium-soluble nor NaHCO3-soluble polymers. However, matrix polymer accumulation was directly correlated with a reduction in flow (regression coefficient R2 = 0.96) and stabilization against physical disturbance. Results with species of Navicula were not as clear. Both NaHCO3-soluble and matrix polymers were involved in producing the flow reduction. In the same manner we also measured the effect of Pseudoalteromonas haloplanktis growth on bead bed hydraulic conductivity and bead bed stability. Growing alone, no effect was found, but in co-culture with a single diatom species, the bacteria reduced the diatom effect on flow through the bioreactors seen earlier, however did not reduce the extent of their growth. Confocal scanning laser microscopy of beads colonized with diatoms alone, or diatoms in co-culture with bacteria, revealed that P. haloplanktis was able to inhibit diatom adhesion to the beads. When the bacteria were present there was less matrix polymer evident. We speculate that this interference with diatom metabolic activity was either the result of less matrix polymer synthesis, or its hydrolysis by the bacteria. The results are applicable to mixed species biofilms of this type on surfaces other than sediments.

A modular six-directional force sensor for prosthetic assessment: a technical note.

A device designed to measure the forces and moments transmitted through prostheses of persons with lower limb amputation is presented. The sensing unit design is an advancement over previous prosthesis force measurement devices in that it is very thin (19 mm) and lightweight (527.5 g, including signal-conditioning instrumentation). The disk-shaped transducer fits between the socket and socket adapter of a standard modular prosthesis, measuring all six force and moment components at this location. Twelve strain gages were used, configured into six two-arm active Wheatstone bridge circuits. A 6X6 matrix was constructed from calibration data to relate the 6-component bridge-output vector to a 6-component force and moment vector. In a bench-test setting, the sensor was evaluated under typical load combinations encountered during the walking of a person with transtibial amputation (TTA) and shown to have errors less than 7.2% of the full-scale output for each direction. Data collected on a subject with TTA walking at different speeds are presented.

New challenges, new answers: pediatric nurse practitioners and the care of adolescents.

INTRODUCTION: Since the 1950s, patterns of morbidity and mortality among adolescents have shifted to social and environmental causes. This study examines pediatric nurse practitioners' (PNPs') self-assessed competencies in addressing the common health concerns of adolescents. METHOD: The analysis used a sample of 257 PNPs drawn from a larger national data set of 637 nurses randomly sampled from 3 nursing organizations. Factors associated with self-perceived knowledge or skill and interest in training for 28 common health concerns of adolescents were analyzed using Chi square, t test, and Pearson's correlation. Barriers and attractions to working with adolescents were also investigated. RESULTS: The greatest deficits in self-perceived knowledge or skill, as well as low interest in training and low perceived relevance to practice, were around issues of gangs, gay/lesbian/bisexual/transgender youth, HIV/AIDS, and counseling about a positive pregnancy test. Also, PNPs identified the lack of resources appropriate for adolescent referrals as the greatest barrier to working with this population. DISCUSSION: PNPs assessed their lowest competencies in some of the areas that present the greatest threats to adolescents' health and well-being. These deficits suggest needed curricular shifts in entry-level and advanced-level preparation of PNPs, as well as new priorities for continuing education.

Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa colonizes the pulmonary tissue of patients with cystic fibrosis (CF), leading to biofilm-associated infections. The pulmonary fluid of CF patients usually contains elevated concentrations of cations and may contain the P. aeruginosa redox-active pigment pyocyanin, which is known to disrupt calcium homeostasis of host cells. Since divalent cations are important bridging ions for bacterial polysaccharides and since they may play regulatory roles in bacterial gene expression, we investigated the effect of calcium ions on the extracellular matrix constituents of P. aeruginosa biofilms. For mucoid strain P. aeruginosa FRD1, calcium addition (1.0 and 10 mM as CaCl(2)) resulted in biofilms that were at least 10-fold thicker than biofilms without added calcium. Scanning confocal laser microscopy showed increased spacing between cells for the thick biofilms, and Fourier transform infrared spectroscopy revealed that the material between cells is primarily alginate. An algD transcriptional reporter demonstrated that calcium addition caused an eightfold increase in alg gene expression in FRD1 biofilms. Calcium addition also resulted in increased amounts of three extracellular proteases (AprA, LasB, and PrpL). Immunoblots of the biofilm extracellular material established that AprA was harbored within the biofilm extracellular matrix. An aprA deletion mutation and a mutation in gene for a putative P. aeruginosa calmodulin-like protein did not significantly affect calcium-induced biofilm structure. Two-dimensional gel electrophoresis showed increased amounts of phenazine biosynthetic proteins in FRD1 biofilms and in calcium-amended planktonic cultures. Spectrochemical analyses showed that the calcium addition causes a three- to fivefold increase in pyocyanin production. These results demonstrate that calcium addition affects the structure and extracellular matrix composition of mucoid P. aeruginosa biofilms, through increased expression and stability of bacterial extracellular products. The calcium-induced extracellular matrix of mucoid P. aeruginosa consists primarily of the virulence factor alginate and also harbors extracellular proteases and perhaps pyocyanin, a biomolecule that may further disrupt cellular calcium levels.

Introduction of antibody into viable cells using electroporation.

Conditions for labelling an intracellular antigen, p21ras, using electroporation to introduce a fluorescent antibody, are described. Following labelling, cells were evaluated for p21ras associated fluorescence by flow cytometry. Electroporation, sorting, and cell handling parameters were varied to determine optimal conditions for cell viability. Cells were best held in serum containing growth medium both before and after electroporation, while antibody introduction during the electroporation phase was most efficient when carried out in a balanced saline solution. For maximum efficiency of antibody internalization, the antibody needed to be present during electroporation, and medium needed to be replaced several times in the first few hours after electroporation to ensure good cell survival.

Flow cytometric measurement of pollutant stresses on algal cells.

The lichen Usnea fulvoreagens (Räs). Räs. was treated with four pH levels (5.5, 4.5, 3.5, and 2.5) of simulated acid rain (sulfuric acid, nitric acid, and a 1:1 combination of both) and automobile exhaust. The samples were dissociated and analyzed by a Becton-Dickinson FACS 440 flow cytometer. Analyses included measurement of chlorophyll autofluorescence and fluorescence due to uptake of fluorescein diacetate (FDA) and calcofluor white M2R (CFW). Cell parameters measured were esterase activity (FDA), membrane permeability (FDA, CFW), and intracellular pH (FDA). Mean fluorescence intensity from FDA staining and numbers of events were incorporated with autofluorescence information to produce a "stress index" of relative cell stress. Results indicated that highly stressed samples (lower pH treatments and greater exposure to exhaust) exhibited a low "stress index" of FDA fluorescence.

A rapid analytical technique for flow cytometric analysis of cell viability using calcofluor white M2R.

Analysis of dead versus live cells is shown to be possible using Calcoflour White M2R (CFW), a fluorescent brightener. Comparison of CFW with both propidium iodide (PI) and fluorescein diacetate (FDA) was performed on a FACS 440 dual laser flow cytometer on several populations of cultured rat and mouse cell lines, peripheral leukocytes, splenocytes, diatoms, and plant protoplasts. As a measure of cell viability, staining results with CFW were strongly associated with PI (correlation coefficient of 0.9886) and FDA (inverse correlation coefficient of 0.9647). With plant and algal cells, controls are necessary as CFW does stain live cells to some extent. CFW (excitation: UV, emission max: 435 nm) can be used in conjunction with two-color immunofluorescence analysis using fluorochromes excited at 488 nm with no interference.