Antimicrobial Exposures in Critically Ill Patients Receiving Extracorporeal Membrane Oxygenation.

Rationale: Data suggest that altered antimicrobial concentrations are likely during extracorporeal membrane oxygenation (ECMO). Objectives: The primary aim of this analysis was to describe the pharmacokinetics (PKs) of antimicrobials in critically ill adult patients receiving ECMO. Our secondary aim was to determine whether current antimicrobial dosing regimens achieve effective and safe exposure. Methods: This study was a prospective, open-labeled, PK study in six ICUs in Australia, New Zealand, South Korea, and Switzerland. Serial blood samples were collected over a single dosing interval during ECMO for 11 antimicrobials. PK parameters were estimated using noncompartmental methods. Adequacy of antimicrobial dosing regimens were evaluated using predefined concentration exposures associated with maximal clinical outcomes and minimal toxicity risks. Measurements and Main Results: We included 993 blood samples from 85 patients. The mean age was 44.7 ± 14.4 years, and 61.2% were male. Thirty-eight patients (44.7%) were receiving renal replacement therapy during the first PK sampling. Large variations (coefficient of variation of ⩾30%) in antimicrobial concentrations were seen leading to more than fivefold variations in all PK parameters across all study antimicrobials. Overall, 70 (56.5%) concentration profiles achieved the predefined target concentration and exposure range. Target attainment rates were not significantly different between modes of ECMO and renal replacement therapy. Poor target attainment was observed across the most frequently used antimicrobials for ECMO recipients, including for oseltamivir (33.3%), piperacillin (44.4%), and vancomycin (27.3%). Conclusions: Antimicrobial PKs were highly variable in critically ill patients receiving ECMO, leading to poor target attainment rates. Clinical trial registered with the Australian New Zealand Clinical Trials Registry (ACTRN12612000559819).

Population Pharmacokinetics of Vancomycin in Critically Ill Adult Patients Receiving Extracorporeal Membrane Oxygenation (an ASAP ECMO Study).

Our study aimed to describe the population pharmacokinetics (PK) of vancomycin in critically ill patients receiving extracorporeal membrane oxygenation (ECMO), including those receiving concomitant renal replacement therapy (RRT). Dosing simulations were used to recommend maximally effective and safe dosing regimens. Serial vancomycin plasma concentrations were measured and analyzed using a population PK approach on Pmetrics. The final model was used to identify dosing regimens that achieved target exposures of area under the curve (AUC0-24) of 400-700 mg · h/liter at steady state. Twenty-two patients were enrolled, of which 11 patients received concomitant RRT. In the non-RRT patients, the median creatinine clearance (CrCL) was 75 ml/min and the mean daily dose of vancomycin was 25.5 mg/kg. Vancomycin was well described in a two-compartment model with CrCL, the presence of RRT, and total body weight found as significant predictors of clearance and central volume of distribution (Vc). The mean vancomycin renal clearance and Vc were 3.20 liters/h and 29.7 liters respectively, while the clearance for patients on RRT was 0.15 liters/h. ECMO variables did not improve the final covariate model. We found that recommended dosing regimens for critically ill adult patients not on ECMO can be safely and effectively used in those on ECMO. Loading doses of at least 25 mg/kg followed by maintenance doses of 12.5-20 mg/kg every 12 h are associated with a 97-98% probability of efficacy and 11-12% probability of toxicity, in patients with normal renal function. Therapeutic drug monitoring along with reductions in dosing are warranted for patients with renal impairment and those with concomitant RRT. (This study is registered with the Australian New Zealand Clinical Trials Registry [ANZCTR] under number ACTRN12612000559819.).

Population pharmacokinetics of cefepime in critically ill patients receiving extracorporeal membrane oxygenation (an ASAP ECMO study).

OBJECTIVES: This study aimed to describe the population pharmacokinetics (PK) of cefepime during extracorporeal membrane oxygenation (ECMO) and through dosing simulations, identify a maximally effective and safe dosing strategy. METHODS: Serial cefepime plasma concentrations were measured in patients on ECMO, and data were analysed using a population PK approach with Pmetrics®. Dosing simulations were used to identify the optimal dosing strategy that achieved target trough concentrations (Cmin) of 8-20 mg/L. Six patients were enrolled, of which one was receiving renal replacement therapy. Cefepime was best described in a two-compartment model, with total body weight and creatinine clearance (CrCL) as significant predictors of PK parameters. The mean clearance and central volume of distribution were 2.42 L/h and 15.09 L, respectively. RESULTS: Based on simulations, patients with CrCL of 120 mL/min receiving 1 g 8-hourly dosing achieved a 40-44% probability of efficacy (Cmin > 8 mg/L) and 1-6% toxicity (Cmin > 20 mg/L). Patients with CrCL 30 mL/min and 65 mL/min receiving 1 g 12-hourly dosing achieved an 84-92% and 46-53% probability of efficacy and 8-44% and 1-8% probability of toxicity, respectively. Simulations demonstrated a lower probability of efficacy and higher probability of toxicity with decreasing patient weight. CONCLUSION: This study reported reduced cefepime clearance in patients receiving ECMO, resulting in an increased risk of cefepime toxicity. To avoid drug accumulation, modified dosing regimens should be used in critically ill patients on ECMO. Clinicians should adopt therapeutic drug monitoring when treating less susceptible organisms and in patients with reduced renal clearance on ECMO.

Population Pharmacokinetics of Piperacillin and Tazobactam in Critically Ill Patients Receiving Extracorporeal Membrane Oxygenation: an ASAP ECMO Study.

Our study aimed to describe the population pharmacokinetics (PK) of piperacillin and tazobactam in patients on extracorporeal membrane oxygenation (ECMO), with and without renal replacement therapy (RRT). We also aimed to use dosing simulations to identify the optimal dosing strategy for these patient groups. Serial piperacillin and tazobactam plasma concentrations were measured with data analyzed using a population PK approach that included staged testing of patient and treatment covariates. Dosing simulations were conducted to identify the optimal dosing strategy that achieved piperacillin target exposures of 50% and 100% fraction of time free drug concentration is above MIC (%fT>MIC) and toxic exposures of greater than 360 mg/liter. The tazobactam target of percentage of time free concentrations of >2 mg/liter was also assessed. Twenty-seven patients were enrolled, of which 14 patients were receiving concurrent RRT. Piperacillin and tazobactam were both adequately described by two-compartment models, with body mass index, creatinine clearance, and RRT as significant predictors of PK. There were no substantial differences between observed PK parameters and published parameters from non-ECMO patients. Based on dosing simulations, a 4.5-g every 6 hours regimen administered over 4 hours achieves high probabilities of efficacy at a piperacillin MIC of 16 mg/liter while exposing patients to a <3% probability of toxic concentrations. In patients receiving ECMO and RRT, a frequency reduction to every 12 hours dosing lowers the probability of toxic concentrations, although this remains at 7 to 9%. In ECMO patients, piperacillin and tazobactam should be dosed in line with standard recommendations for the critically ill.

Co-testing for detection of high-grade cervical intraepithelial neoplasia and cancer compared with cytology alone: a meta-analysis of randomized controlled trials.

BACKGROUND: Human papillomavirus (HPV) DNA testing combined with cytology has been recommended as a primary cervical cancer screening strategy. METHODS: PubMed/MEDLINE, Embase, the Cochrane Library and the NIH trial registry were searched for randomized controlled trials comparing co-testing with cytology alone for the detection of high-grade CIN lesions and cancers. Of 1156 articles identified, four met inclusion criteria. The performance of co-testing and cytology alone was compared at baseline screening, second round screening and overall. Cumulative meta-analysis, Begg's test, Egger's test and sensitivity analysis were performed. RESULTS: At baseline, co-testing was associated with a significantly higher detection rate of CIN 2+ [risk ratio (RR) = 1.41, 95% confidence interval (CI): 1.12, 1.76] and a non-significantly higher CIN 3+ detection rate (RR = 1.15, 95% CI: 0.99, 1.33). At second round screening, co-testing was associated with significantly lower detection rates of both CIN 2+ and CIN 3+ (RR = 0.77, 95% CI: 0.63, 0.93; RR = 0·68, 95% CI: 0.55, 0.85). The overall detection rate did not differ between co-testing and cytology alone for CIN 2+ (RR: 1·19, 95% CI: 0.99, 1.46) or CIN3+ (RR: 0.99, 95% CI: 0.87, 1.14). CONCLUSION: Co-testing increases the detection of CIN2+ lesions at baseline and significantly decreases the detection rates of CIN2+ or CIN3+ lesions at subsequent screening compared with cytology alone.

Survey of the clinical assessment and utility of near-infrared cerebral oximetry in cardiac surgery.

OBJECTIVES: Near-infrared cerebral oximetry increasingly is used for monitoring during cardiac surgery. Nonetheless, the scientific basis for incorporating this technology into clinical practice, the indications for when to do so, and standard diagnostic and treatment algorithms for defining abnormal values are yet to be rigorously defined. The authors hypothesized that there would be (1) variation in clinical use and practices for near-infrared spectroscopy (NIRS), and (2) variation in management of patients when clinicians are provided with NIRS information. In order to test this hypothesis, they sought to assess the nature and strength of response heterogeneity among anesthesiologists and cardiac perfusionists when provided with cardiac surgery patient scenarios and cerebral oximetry data. DESIGN: A prospectively collected survey. SETTING: A hospital-based, multi-institutional, multinational study. PARTICIPANTS: By e-mail, the authors surveyed the membership of the Society of Cardiovascular Anesthesiologists and the online Cardiovascular Perfusion Forum. INTERVENTIONS: This survey was focused on ascertaining what actions clinicians would take in each scenario, given case information and cerebral oximetry tracings. Questions were based on 11 patient scenarios selected to represent small, large, symmetric, or asymmetric decreases in measured regional cerebral oxygen saturation (rScO2) encountered during cardiac surgery. Information on the respondents' (n = 796; 73% anesthesiologists) clinical practice, demography, and cerebral oximetry utilization was collected. An index of dispersion was used to assess response heterogeneity overall and within demographic subgroups. MEASUREMENTS AND MAIN RESULTS: The majority of respondents indicated that cerebral oximetry monitoring was either useful or an essential monitor, especially perfusionists and clinicians who used cerebral oximetry most frequently. There were marked differences in responses between perfusionists and anesthesiologists for 4 of the 6 scenarios (p<0.005 for each of these 4 scenarios) occurring during cardiopulmonary bypass. Scenarios having greatest rScO2 reduction or asymmetry in rScO2 were associated with the highest dispersion, indicating least agreement in management. Scenarios with mild or moderate rScO2 reduction were associated with the lowest dispersion, indicating greater agreement in management. CONCLUSIONS: Although experimental data gradually are accumulating to support the role for cerebral oximetry monitoring during cardiac surgery, the results of the present survey support the view that its role remains poorly defined, and consensus for its appropriate use is lacking. Importantly, the authors observed marked variation in the use, perceived utility, and management of patients for 4 of the 6 CPB scenarios between perfusionists and anesthesiologists who share the management of CPB. These findings support the need for well-designed, adequately-powered clinical trials examining the value of this technology.

The palmitoylation state of PMP22 modulates epithelial cell morphology and migration.

PMP22 (peripheral myelin protein 22), also known as GAS 3 (growth-arrest-specific protein 3), is a disease-linked tetraspan glycoprotein of peripheral nerve myelin and constituent of intercellular junctions in epithelia. To date, our knowledge of the post-translational modification of PMP22 is limited. Using the CSS-Palm 2.0 software we predicted that C85 (cysteine 85), a highly conserved amino acid located between the second and third transmembrane domains, is a potential site for palmitoylation. To test this, we mutated C85S (C85 to serine) and established stable cells lines expressing the WT (wild-type) or the C85S-PMP22. In Schwann and MDCK (Madin-Darby canine kidney) cells mutating C85 blocked the palmitoylation of PMP22, which we monitored using 17-ODYA (17-octadecynoic acid). While palmitoylation was not necessary for processing the newly synthesized PMP22 through the secretory pathway, overexpression of C85S-PMP22 led to pronounced cell spreading and uneven monolayer thinning. To further investigate the functional significance of palmitoylated PMP22, we evaluated MDCK cell migration in a wound-healing assay. While WT-PMP22 expressing cells were resistant to migration, C85S cells displayed lamellipodial protrusions and migrated at a similar rate to vector control. These findings indicate that palmitoylation of PMP22 at C85 is critical for the role of the protein in modulating epithelial cell shape and motility.

Impedance of novel therapeutic technologies: the case of stem cells.

Embryonic stem cell (ES) technology has advanced considerably within the past three decades and has gained prominent distinction within the emerging field of regenerative medicine. As it now enters the nascent stages of clinical application, many hopes and expectations arise along with questions as to where the technology will go. This paper evaluates the technical and practical obstacles that must be overcome before it can fully translate into the clinical context, the existence of strong opposition to the technology, political and legal barriers that have impeded its progression, and the role of healthcare reform in creating new social and economic priorities. In contrast to the technological imperative, a driving force seeking to implement the most recent scientific advances into medical practice, we refer to such translational obstacles as "technological impedance." Rather than expending inordinate effort to preserve existing systems that continue to possess major hurdles, we advocate fostering interdisciplinary approaches in the development of new generation platforms and embracing disruptive innovations that create solutions to technological impedance and move us forward in healthcare delivery. Clin Trans Sci 2012; Volume 5: 422-427.

Antiproliferative factor-induced changes in phosphorylation and palmitoylation of cytoskeleton-associated protein-4 regulate its nuclear translocation and DNA binding.

Cytoskeleton-associated protein 4 (CKAP4) is a reversibly palmitoylated and phosphorylated transmembrane protein that functions as a high-affinity receptor for antiproliferative factor (APF)-a sialoglycopeptide secreted from bladder epithelial cells of patients with interstitial cystitis (IC). Palmitoylation of CKAP4 by the palmitoyl acyltransferase, DHHC2, is required for its cell surface localization and subsequent APF signal transduction; however, the mechanism for APF signal transduction by CKAP4 is unknown. In this paper, we demonstrate that APF treatment induces serine phosphorylation of residues S3, S17, and S19 of CKAP4 and nuclear translocation of CKAP4. Additionally, we demonstrate that CKAP4 binds gDNA in a phosphorylation-dependent manner in response to APF treatment, and that a phosphomimicking, constitutively nonpalmitoylated form of CKAP4 localizes to the nucleus, binds DNA, and mimics the inhibitory effects of APF on cellular proliferation. These results reveal a novel role for CKAP4 as a downstream effecter for APF signal transduction.

Humanin, a cytoprotective peptide, is expressed in carotid atherosclerotic [corrected] plaques in humans.

OBJECTIVE: The mechanism of atherosclerotic plaque progression leading to instability, rupture, and ischemic manifestation involves oxidative stress and apoptosis. Humanin (HN) is a newly emerging endogenously expressed cytoprotective peptide. Our goal was to determine the presence and localization of HN in carotid atherosclerotic plaques. METHODS AND RESULTS: Plaque specimens from 34 patients undergoing carotid endarterectomy were classified according to symptomatic history. Immunostaining combined with digital microscopy revealed greater expression of HN in the unstable plaques of symptomatic compared to asymptomatic patients (29.42±2.05 vs. 14.14±2.13% of plaque area, p<0.0001). These data were further confirmed by immunoblot (density of HN/ß-actin standard symptomatic vs. asymptomatic 1.32±0.14 vs. 0.79±0.11, p<0.01). TUNEL staining revealed a higher proportion of apoptotic nuclei in the plaques of symptomatic patients compared to asymptomatic (68.25±3.61 vs. 33.46±4.46% of nuclei, p<0.01). Double immunofluorescence labeling revealed co-localization of HN with macrophages (both M1 and M2 polarization), smooth muscle cells, fibroblasts, and dendritic cells as well as with inflammatory markers MMP2 and MMP9. CONCLUSIONS: The study demonstrates a higher expression of HN in unstable carotid plaques that is localized to multiple cell types within the plaque. These data support the involvement of HN in atherosclerosis, possibly as an endogenous response to the inflammatory and apoptotic processes within the atheromatous plaque.

The science and ethics of induced pluripotency: what will become of embryonic stem cells?

For over a decade, the field of stem cell research has advanced tremendously and gained new attention in light of novel insights and emerging developments for regenerative medicine. Invariably, multiple considerations come into play, and clinicians and researchers must weigh the benefits of certain stem cell platforms against the costs they incur. Notably, human embryonic stem (hES) cell research has been a source of continued debate, leading to differing policies and regulations worldwide. This article briefly reviews current stem cell platforms, looking specifically at the two existing pluripotent lines available for potential therapeutic applications: hES cells and induced pluripotent stem (iPS) cells. We submit iPS technology as a viable and possibly superior alternative for future medical and research endeavors as it obviates many ethical and resource-related concerns posed by hES cells while prospectively matching their potential for scientific use. However, while the clinical realities of iPS cells appear promising, we must recognize the current limitations of this technology, avoid hype, and articulate ethically acceptable medical and scientific goals.

Humanin preserves endothelial function and prevents atherosclerotic plaque progression in hypercholesterolemic ApoE deficient mice.

OBJECTIVE: Humanin (HN) is a cytoprotective peptide derived from endogenous mitochondria, expressed in the endothelial layer of human vessels, but its role in atherogenesis in vivo is not known. In vitro study, however, HN reduced oxidized low-density lipoprotein induced formation of reactive oxygen species and apoptosis. The present study tested the hypothesis that long term treatment with HN will have a protective role against endothelial dysfunction and progression of atherosclerosis in vivo. METHODS AND RESULTS: Daily intraperitonial injection of the HN analogue HNGF6A for 16 weeks prevented endothelial dysfunction and decreased atherosclerotic plaque size in the proximal aorta of ApoE-deficient mice fed on a high cholesterol diet, without showing direct vasoactive effects or cholesterol-reducing effects. HN was expressed in the endothelial layer on the aortic plaques. HNGF6A treatment reduced apoptosis and nitrotyrosine immunoreactivity in the aortic plaques without affecting the systemic cytokine profile. HNGF6A also preserved expression of endothelial nitric oxide synthase in aorta. CONCLUSIONS: HN may have a protective effect on endothelial function and progression of atherosclerosis by modulating oxidative stress and apoptosis in the developing plaque.

Long-term survival with metastatic carcinoid tumors: a case report and review of the literature.

Post hoc studies investigating patients with metastatic carcinoid tumors consistently have demonstrated outcomes that are not all too promising in terms of long-term survival, particularly for those with multiple metastases to the small bowel, mesentery, and liver. Here we present a patient with these clinical findings who, contrary to current expectations, has survived much longer than anticipated. This case is a sobering reminder for physicians not only to evaluate carefully and care for their patients but also to choose their words carefully in all communication with them.

Identification of targets and inhibitors of protein palmitoylation.

IMPORTANCE OF THE FIELD: Palmitoylation is the post-translational addition of a 16-carbon fatty acid, palmitate, to specific cysteines of proteins via a labile thioester bond. The transfer of palmitate to a substrate is mediated by palmitoyl acyltransferases (PATs). Nearly a third of the 23 genes that encode PATs are linked to human diseases, in particular cancer, and as such represent important targets for drug development. AREA COVERED IN THIS REVIEW: In this review, we summarize recent technical advances in the field of palmitoylation, how they will affect our ability to understand palmitoylation-related signaling, and outline a general strategy for the discovery of selective and potent palmitoylation inhibitors. WHAT THE READER WILL GAIN: The goals of this review are to increase awareness of the importance of palmitoylation in disease as well as our general lack of understanding of the complexity of the fundamental mechanisms of PAT regulation and specificity, and finally to suggest general strategies for the development of PAT inhibitors. TAKE HOME MESSAGE: Any reasonable hope of developing therapeutically useful, pharmacological modulators of palmitoylation will require that they be developed within the context of PAT-related signaling systems that are more extensively characterized than any we currently know. The successful creation of potent, specific drugs in other similarly complex systems suggests that development of useful drugs targeting PATs is certain.

Palmitoyl acyltransferases, their substrates, and novel assays to connect them (Review).

Thio-palmitoylation is the post-translational addition of the 16-carbon fatty acid, palmitate, to the thiol side chain of cysteine residues by a labile thioester bond. Palmitoylation increases the lipophilicity of a protein resulting in dramatic changes in its subcellular distribution such as moving from the endoplasmic reticulum to the plasma membrane or in subtle changes like an increased affinity for cholesterol-rich lipid rafts in membranes. Palmitoylation is also dynamic, making it unique among post-translational protein lipid modifications. Discovering the molecular identity of palmitoyl acyltransferases (PATs) was a watershed event that dramatically accelerated the pace of discovery in the field. Likewise, there has been increased interest in palmitoylation partly because many of the genes encoding PATs have been linked to cancer and other diseases. Now, with a greater understanding of how palmitate is enzymatically attached to proteins, some of the most interesting questions include: What are the substrates of each PAT?; how does a PAT recognize and palmitoylate a substrate?; how are PATs regulated?; and, how is depalmitoylation regulated? The answers to these questions are beginning to unfold due to the recent development of novel assays as well as the expansion and refinement of existing assays. Our ability to understand palmitoylation and its importance to human health and disease is only as good as the methods we use to test our hypotheses. The continued development of methods with increased sensitivity and selectivity is critical to this venture.

Palmitoylation of cytoskeleton associated protein 4 by DHHC2 regulates antiproliferative factor-mediated signaling.

Previously, we identified cytoskeleton-associated protein 4 (CKAP4) as a major substrate of the palmitoyl acyltransferase, DHHC2, using a novel proteomic method called palmitoyl-cysteine identification, capture and analysis (PICA). CKAP4 is a reversibly palmitoylated and phosphorylated protein that links the ER to the cytoskeleton. It is also a high-affinity receptor for antiproliferative factor (APF), a small sialoglycopeptide secreted from bladder epithelial cells of patients with interstitial cystitis (IC). The role of DHHC2-mediated palmitoylation of CKAP4 in the antiproliferative response of HeLa and normal bladder epithelial cells to APF was investigated. Our data show that siRNA-mediated knockdown of DHHC2 and consequent suppression of CKAP4 palmitoylation inhibited the ability of APF to regulate cellular proliferation and blocked APF-induced changes in the expression of E-cadherin, vimentin, and ZO-1 (genes known to play a role in cellular proliferation and tumorigenesis). Immunocytochemistry revealed that CKAP4 palmitoylation by DHHC2 is required for its trafficking from the ER to the plasma membrane and for its nuclear localization. These data suggest an important role for DHHC2-mediated palmitoylation of CKAP4 in IC and in opposing cancer-related cellular behaviors and support the idea that DHHC2 is a tumor suppressor.

Identification of CKAP4/p63 as a major substrate of the palmitoyl acyltransferase DHHC2, a putative tumor suppressor, using a novel proteomics method.

Protein palmitoylation is the post-translational addition of the 16-carbon fatty acid palmitate to specific cysteine residues by a labile thioester linkage. Palmitoylation is mediated by a family of at least 23 palmitoyl acyltransferases (PATs) characterized by an Asp-His-His-Cys (DHHC) motif. Many palmitoylated proteins have been identified, but PAT-substrate relationships are mostly unknown. Here we present a method called palmitoyl-cysteine isolation capture and analysis (or PICA) to identify PAT-substrate relationships in a living vertebrate system and demonstrate its effectiveness by identifying CKAP4/p63 as a substrate of DHHC2, a putative tumor suppressor.

Plasma membrane assays and three-compartment image cytometry for high content screening.

High throughput image cytometers analyze individual cells in digital photomicrographs by first assigning pixels within each image to plasma membrane, cytoplasm, nucleus, or other regions. In this study, we report on a novel algorithm that: 1) identifies plasma membrane regions to measure changes in plasma membrane-associated proteins (protein kinase C [PKC] alpha, N-cadherin, E-cadherin, vascular endothelium [VE]-cadherin, and pan-cadherin) that regulate cell division, migration, and adhesion and 2) delineates the cell for generalized three-compartment image cytometry. Validation assays were performed for these proteins on cells cultured in 96-well plates and also for tissue sections obtained from transgenic and chemical carcinogenic models of skin cancer. The algorithm successfully quantified phorbol 12-myristate 13-acetate (PMA)-induced plasma membrane localization of PKCalpha in HeLa cells (Z' of 0.88). Additionally, PMA activated translocation to the plasma membrane at P < .01 of N-cadherin (in HeLa cells), E-cadherin (in A431 cells), and VE-cadherin (in human dermal microvascular endothelial cells), suggesting a relationship between PKCalpha activity and cadherin localization. For VE-cadherin, a Z' of 0.52 was obtained between serum-free medium, which increased VE-cadherin, and EGTA, which diminished VE-cadherin at the plasma membrane. For sections obtained from the transgenic skin cancer model, analysis of images with the plasma membrane algorithm revealed that tumor cells exhibited cadherin expression that was just 34% of that expressed by surrounding normal tissue; furthermore, tumor cells expressed elevated DNA content, consistent with development of aneuploidy. In contrast, increased DNA content did not occur for tumor cells produced by chemical carcinogenesis. The results demonstrate that this new algorithm for plasma membrane image cytometry enables statistically significant analyses in a variety of applications in both cultured cells and tissue sections.