Transcutaneous electrical neurostimulation relieves primary dysmenorrhea: A randomized, double-blind clinical study versus placebo.

INTRODUCTION: This randomized, crossover, double-blind, controlled trial evaluates the efficacy and safety of a preprogrammed transcutaneous electrical nerve stimulation (TENS) device versus placebo (SHAM) in women with primary dysmenorrhea (PD). MATERIAL: Forty women suffering from significant dysmenorrhea requiring the use of analgesics and/or non-steroidal anti-inflammatory drugs self-apply to the abdominal or lumbar region depending on the location of the pain, alternately according to randomization, the TENS device then the SHAM (dummy device) or conversely SHAM then TENS. The primary endpoint compares the evolution of pain intensity before and after application of TENS and SHAM. The speed of action, the persistence of the analgesic effect and the therapeutic savings are also evaluated. Adverse events (AEs) are collected. RESULTS: A statistically and clinically significant decrease in the pain of 53% (P<0.0001) is observed during the first 2 applications of TENS versus no analgesic effect (-5%, P=0.318) with SHAM. Over all 197 applications of TENS, the reduction of menstrual pain intensity by more than half is confirmed. The rapid relief, less than 20 minutes in 74% of cases, lasts on average more than 7 hours. A difference in analgesic consumption of -93% is observed in favor of TENS (P<0.01). Seven participants reported 10 non-serious AEs, 2 of which were possibly related to TENS. CONCLUSION: The TENS device tested represents a well-tolerated, rapidly and lastingly effective non-pharmacological analgesic solution, capable of replacing or being combined with analgesics in the management of PD.

[Psychometric validation of Convergence criteria PP]. / Validation psychométrique des critères de Convergences PP.

CONTEXT: The PP Convergences criteria group together 10 of the most significant clinical criteria for sensitization in the context of chronic pelvic pain. They are the result of a consensus of experts and represent to date the only clinical evaluation guide to identify patients with pelvic perineal pain in whom a pelvic sensitization component can be evoked. OBJECTIVE: This work concerns the psychometric validation of these criteria. The aim is to answer 3 questions: 1) is the instrument reliable (i.e., sensitive, specific and accurate)?; 2) can we define a screening score for pelvic-perineal pain by sensitization from the CPP criteria?; 3) can combinations of criteria be defined to predict pelvic-perineal sensitization from the CPP criteria? METHODOLOGY AND SUBJECTS: In total, 308 patients with pelviperineal pain were recruited during their medical consultation. PROCEDURE: Fifteen expert physicians were asked to judge the presence or absence of the 10 CPP criteria and to make a diagnosis of the presence or absence of pelviperineal sensitization in their patient. RESULTS: ROC curve analysis indicated that a score of 5 was the closest to a perfect score with a sensitivity of 95% and a specificity of 87%. They also indicate that the CPP criteria have a very good sensitivity (97%) and specificity (91%) and present globally a good reproducibility on all the criteria (Kappa>0.6). Finally, the statistical analyses reveal that the most discriminating criterion for predicting sensitization is Q8 (pain persisting after sexual activity). CONCLUSION: The CPP criteria represent a very good screening tool for pelvic sensitization. The score of 5 corresponds to the score at which the patient has sufficient clinical criteria to be classified as sensitized. LEVEL OF EVIDENCE: 4.

[Incidence of postural disorders in patients with chronic pelvic-perineal pain]. / Incidence des troubles posturaux chez les patients souffrant d'algies pelvipérinéales chroniques.

OBJECTIVE: Pelvic-perineal pain often accompanied by pain of the perineum and pelvi-trochanteric muscles, we sought to observe the frequency of postural disturbances in relation to the pelvi-perineal muscles in patients who consult for pelvic perineal pain compared to a control population free of these pain. MATERIAL AND METHODS: The prospective monocentric study was conducted during consultations of pelvic perineal pain in the urology department of Nantes and was based on 5 clinical tests successively looking for the presence of thoraco-lumbar hinge syndrome, myofascial syndrome in the pelvic diaphragm, pelvic instability, pelvic-pedic quadrilateral dysfunction and paravertebral muscle hypertonia. RESULTS: A total of 51 subjects were included in the study and divided into two populations: 26 patients, 25 controls. Thoraco-lumbar hinge syndrome was found in 28 % of patients vs 4 % of controls (P=0.024); myofascial syndromes were present in 68 % of patients vs 25 % of controls (P=0.005); pelvic instability concerned 76 % of patients vs 33 % of controls (P=0.002); the dysfunctions of the pelvic-pedic quadrilateral concerned 96 % of the patients vs 58 % of the controls (P=0.001); paravertebral muscle hypertonia was found bilaterally in 32 % of patients vs 4 % of controls (P=0.077) and unilaterally in 36 % of patients vs 0 % of controls (P=0.001). CONCLUSION: Patients with chronic pelvic perineal pain had significantly more posture problems than non-pain patients. It seemed relevant to us that the postural assessment was integrated into their usual clinical examination. LEVEL OF EVIDENCE: 4.

[Bladder cancer in neurogenic patients: A retrospective study of management and follow-up]. / Cancers de vessie chez les patients neurologiques : étude rétrospective de prise en charge et de suivi.

INTRODUCTION: The prevalence of bladder cancer (BC) in neurological patients seems to be similar to that of the general population. However, they are more aggressive with a higher rate of muscle-invasive forms and squamous cells carcinomas. The aim of the current study was to report etiologies, management and outcomes of BC in neurological population. MATERIAL AND METHOD: Were enrolled all neurological patients with a BC diagnosed between 2004 and 2017. The following data were retrospectively reported: age, gender, duration of the disease, mode of discovery, histological type, treatment and outcomes. RESULTS: In total, 27 patients were included: 11 spinal cord injuries, 7 Parkinson's disease, 5 multiple sclerosis, 3 head trauma, 3 brain strokes, 2 cerebral palsies and 1 spina bifida. The histological subtypes were as follows: 22 transitional cells carcinomas, 4 squamous cell carcinomas (SCC), one mucinous adenocarcinoma, one sarcomatoid and one neuroendocrine with 19 high-grade tumors and 15 muscle-invasive bladder cancer. Seven patients (26%) were diagnosed before 15 years history of neurogenic bladder. The mean follow-up was 14 months (1-210 months). Eight deaths were observed, with 5 related to bladder cancer. In our study, smoking habits, voiding mode, lithiasis or infection histories were not related with a more aggressive pattern, such as SCC. CONCLUSION: The high rate of muscle-invasive bladder cancer and aggressive patterns justify neuro-urological follow-up, even before 15 years of neurogenic bladder. LEVEL OF EVIDENCE: 4.

[Pelvi-perineal chronic pain, urgent demands of care and decision-making]. / Douleurs pelvipérinéales chroniques, demandes urgentes de soins et prise de décision.

OBJECTIVE: Some patients suffering from pelvi-perineal chronic pain express urgent, even vital, demands of care. The objective was to compare the profile and the psychological functioning of the patients, who have shown an imperious demand of care, in that of the patients who do not have an imperious demand of care. MATERIAL AND METHOD: From the medical consultations for chronic pelviperineal pain, we realized a comparative study including 26 patients (experimental group) expressing an urgent demand of care (i.e., patients who, during the last 3months, called an emergency service concerning the pains for which they consult in the service) and 28 patients (control group) without an urgent demand of care (i.e., patient who, during the last 3months, did not call an emergency service concerning the pains for which they consult in the service). All the patients were tested through a cognitive task of decision-making (Iowa Gambling Task) and through explicit measures of pain and its main psychological associated factors (anxiety, depression, impulsivity an catastrophism). RESULTS: In the first place, the patients from the experimental group possess decision-making abilities equivalent to the patients of the control group; however, both groups of patients show, in the beginning of the test, a deficit in the decision-making (F(4.208)=3.4116; P=.009). Secondly, the measures to questionnaires reveal that the patients of the control group have less severe scores in the scales of depression (t(52)=-2.068; P<04), catastrophism (amplification : t(52)=-3.069; P<0035; powerlessness: t(52)=-2.866, P<.006) and impulsivity (positive urgency: t(52)=-2.246, P<029; lack of premeditation: t(52)=-2.175, P<035) than the patients of the experimental group. CONCLUSION: The use of explicit measures (questionnaire) and implicit measures (experimental task) allowed to objectify more precisely the differences between the chronic pain patients in urgent demand of care and the other chronic pain patients. This psychological specificity obliges us to approach differently the caring of these patients in particular by proposing adapted cognitivo-behavioral techniques. LEVEL OF EVIDENCE: 3.

The injured heart: early cardiac effects of hematopoietic stem cell transplantation in children and young adults.

We hypothesized that subclinical cardiac injury in the peri-transplant period is more frequent than currently appreciated in children and young adults. We performed echocardiographic screening on 227 consecutive patients prior to hematopoietic stem cell transplantation (HSCT), and 7, 30 and 100 days after transplant. We measured cardiac biomarkers cardiac troponin-I (cTn-I), and soluble suppressor of tumorigenicity 2 (sST2) prior to transplant, during conditioning, and days +7, +14, +28 and +49 in 26 patients. We subsequently analyzed levels of cTn-I every 48-72 h in 15 consecutive children during conditioning. Thirty-two percent (73/227) of patients had a new abnormality on echocardiogram. New left ventricular systolic dysfunction (LVSD) occurred in 6.2% of subjects and new pericardial effusion in 27.3%. Eight of 227 (3.5%) patients underwent pericardial drain placement, and 5 (2.2%) received medical therapy for clinically occult LVSD. cTn-I was elevated in 53.0% of all samples and sST2 in 38.2%. At least one sample had a detectable cTn-I in 84.6% of patients and an elevated sST2 in 76.9%. Thirteen of fifteen patients monitored frequently during condition had elevation of cTn-I. Echocardiographic and biochemical abnormalities are frequent in the peri-HSCT period. Echocardiogram does not detect all subclinical cardiac injuries that may become clinically relevant over longer periods.

[Interest of infiltration of Impar node in rebel vulvodynia: About a series of 8 cases]. / Intérêt de l'infiltration du ganglion Impar dans les vulvodynies rebelles : à propos d'une série de 8 cas.

INTRODUCTION: Vulvodynia is a common and debilitating disease, for which treatments are often of limits efficacy. As the Impar node receives nociceptive afferents from pelvis and perineum, it is a potential therapeutic target to treat pain in this region. The objective of the study was to evaluate the relevance of ropivacaine Impar node infiltration in patients suffering from rebel vulvodyny. METHODS: This was a retrospective, single-center study. The Impar node infiltrations were performed by a single operator in eight patients suffering from rebel vulvodynia. Ropivacaine and iopamidol were administered in prone position with a lateral approach under scanner. The anaesthetic diagnostic block of the Impar node was positive in all eight patients included in the study. Thereafter these patients benefited of 2 additional therapeutic infiltrations. Subsequently, an infiltration of the node with 100UI of botulinum toxin was performed in two patients with a bilateral approach under scanner. The analgesic efficacy was evaluated by a Visual Analogic Scale (VAS) before, immediately after, and at day 15 following the infiltration. A subjective evaluation of pain comprising the percentage of overall improvement and duration of analgesic efficacy was performed after the third infiltration. RESULTS: Comparison of the VAS before and immediately after the Impar block showed in the first anesthetic block a significant decrease in pain median VAS from 51/100 to 16/100 (P=0.01). Similarly, for the second block, VAS decreased from 52.5/100 to 15/100 (P=0.02). The maximal pain reported on Day 15, was significantly lower after the third infiltration than that after the first (P=0.03). Five patients reported an overall improvement in their quality of life of over 50%, which lasted an average of six weeks. A long lasting effectiveness was obtained in the two patients who benefited of the botulinum toxin. CONCLUSION: The infiltration of Impar node is an interesting technique for patients suffering of rebel vulvodynia. LEVEL OF EVIDENCE: 4.

Visits to primary care physicians among persons who inject drugs at high risk of hepatitis C virus infection: room for improvement.

The role of primary care physicians (PCP) in hepatitis C virus (HCV) prevention is increasingly emphasized. Yet, little is known about the patterns of contacts with PCP among persons who inject drugs (PWID). We sought to assess the 6-month prevalence of PCP visiting among PWID at risk of HCV infection and to explore the associated factors. Baseline data were collected from HCV-seronegative PWID recruited in HEPCO, an observational Hepatitis Cohort study (2004-2011) in Montreal, Canada. An interviewer-administered questionnaire elicited information on socio-demographic factors, drug use patterns and healthcare services utilization. Blood samples were tested for HCV antibodies. Using the Gelberg-Andersen Behavioral Model, hierarchical logistic regression analyses were conducted to identify predisposing, need and enabling factors associated with PCP visiting. Of the 349 participants (mean age = 34; 80.8% male), 32.1% reported visiting a PCP. In the multivariate model, among predisposing factors, male gender [adjusted odds ratio (AOR) = 0.45 (0.25-0.83)], chronic homelessness [AOR = 0.08 (0.01-0.67)], cocaine injection [AOR = 0.46 (0.28-0.76)] and reporting greater illegal or semi-legal income [AOR = 0.48 (0.27-0.85)] were negatively associated with PCP visits. Markers of need were not associated with the outcome. Among enabling factors, contact with street nurses [AOR = 3.86 (1.49-9.90)] and food banks [AOR = 2.01 (1.20-3.37)] was positively associated with PCP visiting. Only one third of participating PWID reported a recent visit to a PCP. While a host of predisposing factors seems to hamper timely contacts with PCP among high-risk PWID, community-based support services may play an important role in initiating dialogue with primary healthcare services in this population.

Microstructural characterization of Ti-6Al-4V alloy subjected to the duplex SMAT/plasma nitriding.

In this study, microstructural characterization of Ti-6Al-4V alloy, subjected to the duplex surface mechanical attrition treatment (SMAT)/nitriding treatment, leading to improve its mechanical properties, was carried out through novel and original samples preparation methods. Instead of acid etching which is limited for morphological characterization by scanning electron microscopy (SEM), an original ion polishing method was developed. Moreover, for structural characterization by transmission electron microscopy (TEM), an ion milling method based with the use of two ions guns was also carried out for cross-section preparation. To demonstrate the efficiency of the two developed methods, morphological investigations were done by traditional SEM and field emission gun SEM. This was followed by structural investigations through selected area electron diffraction (SAED) coupled with TEM and X-ray diffraction techniques. The results demonstrated that ionic polishing allowed to reveal a variation of the microstructure according to the surface treatment that could not be observed by acid etching preparation. TEM associated to SAED and X-ray diffraction provided information regarding the nanostructure compositional changes induced by the duplex SMAT/nitriding process.

Surveillance of avian influenza in the Caribbean through the Caribbean Animal Health Network: surveillance tools and epidemiologic studies.

The Caribbean region is considered to be at risk for avian influenza (AI) due to a large backyard poultry system, an important commercial poultry production system, the presence of migratory birds, and disparities in the surveillance systems. The Caribbean Animal Health Network (CaribVET) has developed tools to implement AI surveillance in the region with the goals to have 1) a regionally harmonized surveillance protocol and specific web pages for AI surveillance on www.caribvet.net, and 2) an active and passive surveillance for AI in domestic and wild birds. A diagnostic network for the Caribbean, including technology transfer and AI virus molecular diagnostic capability in Guadeloupe (real-time reverse transcription-polymerase chain reaction for the AI virus matrix gene), was developed. Between 2006 and 2009, 627 samples from four Caribbean countries were tested for three circumstances: importation purposes, following a clinical suspicion of AI, or through an active survey of wild birds (mainly waders) during the southward and northward migration periods in Guadeloupe. None of the samples tested were positive, suggesting a limited role of these species in the AI virus ecology in the Caribbean. Following low pathogenic H5N2 outbreaks in the Dominican Republic in 2007, a questionnaire was developed to collect data for a risk analysis of AI spread in the region through fighting cocks. The infection pathway of the Martinique commercial poultry sector by AI, through introduction of infected cocks, was designed, and recommendations were provided to the Caribbean Veterinary Services to improve cock movement control and biosecurity measures. The CaribVET and its organization allowed interaction between diagnostic and surveillance tools on the one hand and epidemiologic studies on the other, both of them developed in congruence with regional strategies. Together, these CaribVET activities contribute to strengthening surveillance of avian influenza virus (AIV) in the Caribbean region and may allow the development of research studies on both AI risk analysis and on AIV ecology.

Self-organization of InAs/InP quantum dot multilayers: pseudophase diagram describing the transition from aligned to antialigned structures.

Based on experimental observations for the InAs/InP(001) system and atomistic strain calculations using Keating's valence force field method, we propose a pseudophase diagram describing the regimes of 3D self-organization in quantum dot (QD) multilayers. The combined experimental and theoretical analyses--varying the spacer thickness (H), QD height (h), base (b), and lateral spacing (D)--indicate that the vertically aligned to antialigned transition occurs for a critical value of H/D which increases weakly with b/D, while varying h has virtually no effect on the transition point.

The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles.

Chromokinesins have been postulated to provide the polar ejection force needed for chromosome congression during mitosis. We have evaluated that possibility by monitoring chromosome movement in vertebrate-cultured cells using time-lapse differential interference contrast microscopy after microinjection with antibodies specific for the chromokinesin Kid. 17.5% of cells injected with Kid-specific antibodies have one or more chromosomes that remain closely opposed to a spindle pole and fail to enter anaphase. In contrast, 82.5% of injected cells align chromosomes in metaphase, progress to anaphase, and display chromosome velocities not significantly different from control cells. However, injected cells lack chromosome oscillations, and chromosome orientation is atypical because chromosome arms extend toward spindle poles during both congression and metaphase. Furthermore, chromosomes cluster into a mass and fail to oscillate when Kid is perturbed in cells containing monopolar spindles. These data indicate that Kid generates the polar ejection force that pushes chromosome arms away from spindle poles in vertebrate-cultured cells. This force increases the efficiency with which chromosomes make bipolar spindle attachments and regulates kinetochore activities necessary for chromosome oscillation, but is not essential for chromosome congression.

Analytical evaluation of the testosterone assay on the Bayer Immuno 1 system.

OBJECTIVES: To evaluate the analytical performance of the testosterone assay performed on the Bayer immuno 1 system from Bayer Corporation. DESIGN AND METHODS: The precision was measured using three Bayer TESTpoint Ligand controls, three Medical Decision Pools and the Bayer SETpoint Testosterone calibrators. The linearity was verified by diluting two serum samples containing high testosterone concentrations with the zero calibrator and the minimum detectable concentration determined by repetitive analysis of the zero calibrator. The assay was correlated with the Diagnostic Products Corporation (DPC) Total Testosterone assay using 342 serum samples. The reference values were determined using serum samples from 75 women and 60 men. RESULTS: The assay showed within-run coefficients of variation (CVs) varying from 1.1-8.4% and between-day CVs from 1.5-4.9% for testosterone concentrations varying from 1.77 to 66.96 nmol/L. The minimum detectable concentration was estimated at 0.11 nmol/L. The assay linearity proved excellent. A good correlation between the Bayer Immuno 1 and the DPC assays was observed with different categories of serum samples (Immuno 1 = 1.11 x DPC-0.32, r = 0.989, Sy[symbol: see text]x = 2.07 nmol/L). The reference values were estimated at 0.3-3.2 nmol/L for females and 9.3-35.6 nmol/L for males. CONCLUSIONS: The Bayer Immuno 1 Testosterone assay demonstrates the analytical characteristics required for its utilization in the clinical laboratory.

Analytical performance of Bayer Immuno 1 estradiol and progesterone assays.

We evaluated the analytical performance of new estradiol and progesterone assays performed on the Bayer Immuno 1 system. Within-run and between-day CVs for estradiol at concentrations of 116.8-6645.8 pmol/L were < or = 6.4% and for 5.54-103.95 nmol/L progesterone were < or = 7.7%, thus meeting published analytical goals. The detection limits (2 SDs from mean of zero calibrator) were 27.1 pmol/L for estradiol (n = 72 over 20 days) and 0.51 nmol/L for progesterone (n = 47 over 20 days). The assays were linear to 9766 pmol/L and 113.0 nmol/L, respectively. Estradiol results agreed well with the Diagnostic Products Corporation (DPC) assays, except for serum samples from patients receiving estrogen replacement therapy; results for these samples agreed closely with the DPC estradiol-6 assay. The progesterone assay agreed closely with the DPC assay, except for samples from uremic patients. Reference values were estimated by the study of 29 women throughout the menstrual cycle with 15 samples per subject. We concluded that both assays demonstrate suitable precision, linearity, and intermethod agreement to allow their use in the clinical laboratory.

Development of an assay for urinary free cortisol determination on the Technicon Immuno 1 system.

OBJECTIVES: To develop and evaluate a method using an ethyl acetate extraction procedure for the determination of urinary free cortisol on the Technicon Immuno 1 system from Bayer Corporation. DESIGN AND METHODS: We tested the assay precision, linearity, and correlation with the Urinary Kallestad Quanticoat Cortisol radioimmunoassay. We also studied the efficiency of the extraction procedure, performed a cross-reactivity study with different cortisol metabolites, and determined the reference values. RESULTS: The assay shows within-run CVs varying from 1.6 to 5.3% and between-day CVs from 2.7 to 6.1% for urinary free cortisol concentrations from 58 to 1097 nmol/L. The assay demonstrates an excellent linearity and a very good correlation with the Kallestad Quanticoat Cortisol assay (slope = 0.94, y-intercept = 29 nmol/L, Sy/x = 54 nmol/L, r = 0.996). The reference values were estimated at 42-281 nmol/d. The extraction procedure shows an average recovery of 99.0% and minimal interference with the cortisol metabolites tested with the exception of cortisone. CONCLUSIONS: The evaluation shows that the developed assay has the analytical characteristics required for its utilization in a clinical laboratory.

Analytical evaluation of the CK-MB mass assay on the Technicon Immuno 1 system.

OBJECTIVES: To evaluate the performance of the Technicon Immuno 1 CK-MB mass assay. DESIGN AND METHODS: The precision was measured using the BioRad Liquichek CK-MB controls and the Technicon Immuno 1 SETpoint CK-MB calibrators. The linearity was verified by diluting 9 patient serum samples containing high CK-MB concentrations, and the minimum detectable concentration determined by repetitive analysis of the zero calibrator. The assay was compared with the Johnson & Johnson Vitros CK-MB activity assay using 111 patient serum samples. The reference range was determined using serum samples from 100 healthy adult volunteers. RESULTS: The assay shows within-run CVs varying from 1.5 to 7.5% and between-day CVs from 1.5 to 4.8% for CK-MB concentrations ranging from 3.9 to 99.0 micrograms/L. The linearity study gave correlation coefficients greater than 0.9961. There was a very good correlation between mass and activity assays (r = 0.985). The reference values were estimated at 0.0-4.2 micrograms/L and the minimum detectable concentration at 0.5 micrograms/L. CONCLUSIONS: The evaluation shows that the Technicon Immuno 1 CK-MB mass assay has the analytical characteristics suitable for its inclusion in the modern clinical repertoire.

Performance evaluation of automated immunoassays on the Technicon Immuno 1 system.

We performed an immunoassay evaluation for various analytes on a fully automated random-access analyzer, the Technicon Immuno 1 system from Bayer Corp. This system involves latex agglutination, magnetic separation sandwich, and magnetic separation competitive immunoassay configurations. The evaluated analytes were thyrotropin (TSH), triiodothyronine, thyroxine, free thyroxine, follitropin, lutropin, prolactin, beta subunit of human chorionic gonadotropin, cortisol, ferritin, alpha-fetoprotein, carcinoembryonic antigen, and prostate-specific antigen. We tested the assay precision, linearity, and correlation with comparison methods for these analytes. The functional sensitivity of the TSH assay and the sample-to-sample carryover were also studied. Excellent results were obtained for within-run and between-day precision studies, with most assays showing within-run CVs <4% and between-day CVs <6%. The linearity for all assays was acceptable and the correlation between Immuno 1 assays and comparison methods showed satisfactory results. The functional sensitivity of the TSH assay was estimated at 0.04 mU/L. No sample-to-sample carryover was detected.

Measurement of tumor necrosis factor activity by flow cytometry.

Tumor necrosis factor-alpha (TNF-alpha) is a monokine of 17 kDa produced by activated macrophages and various cells involved in the immune system. We propose a new method for the measurement of TNF activity using flow cytometry. After an incubation with TNF, L929 cells were harvested and treated with a calcein-AM and ethidium homodimer-1 solution. Nonfluorescent calcein-AM is hydrolyzed by intracellular esterases to yield fluorescent calcein. The ethidium homodimer-1 is a high-affinity red fluorescent DNA dye that is internalized only through altered cell membranes. A very good correlation was observed between the calcein fluorescence intensity and the number of viable cells as well as the ethidium fluorescence and the number of cells with altered membranes. The assay is sensitive, inexpensive, and correlates with the already reported crystal violet assay while measuring membrane alteration by TNF. It allows the simultaneous measurement of total living and dead cells. There is no interference with culture medium components. This method is rapid and may be used for routine measurement of TNF activity.

Improved fluorescent bioassay for the detection of tumor necrosis factor activity.

Tumor necrosis factor alpha (TNF-alpha) is a monokine of 17 kDa produced by activated macrophages and various cells involved in the immune system. We propose a new method for the measurement of TNF activity on mouse L929 fibroblast cells. After an incubation with TNF, the cells were stained with a solution of ethidium homodimer-1, a high-affinity red fluorescent DNA dye that is internalized only through altered cell membranes. The assay is sensitive, inexpensive and correlates with the already reported TNF assays while measuring the membrane alteration by TNF and not the cell detachment. It requires no rinsing before dye addition which may cause cell loss; there is no interference with culture medium components since the assay is performed in PBS. This method is more rapid and precise for routine measurement of TNF activity.