Validation and reproducibility of computerised cell-viability analysis of tissue slices

Background

The identification of live cells using membrane integrity dyes has become a frequently used technique, especially with articular cartilage and chondrocytes in situ where tissue slices are used to assess cell recovery as a function of location. The development of a reproducible computerised method of cell evaluation would eliminate many variables associated with manual counting and significantly reduce the amount of time required to evaluate experimental results.

Methods

To validate a custom computerised counting program, intra-person and inter-person cell counts of nine human evaluators (three groups – unskilled, novice, and experienced) were compared with repeated pixel counts of the custom program on 15 digitised images (in triplicate) of chondrocytes in situ stained with fluorescent dyes.

Results

Results indicated increased reproducibility with increased experience within evaluators [Intraclass Correlation Coefficient (ICC) range = 0.67 (unskilled) to 0.99 (experienced)] and between evaluators [ICC = 0.47 (unskilled), 0.85 (novice), 0.93 (experienced)]. The computer program had perfect reproducibility (ICC = 1.0). There was a significant relationship between the average of the experienced evaluators results and the custom program results (ICC = 0.77).

Conclusions

This study demonstrated that increased experience in cell counting resulted in increased reproducibility both within and between human evaluators but confirmed that the computer program was the most reproducible. There was a good correlation between the intact cell recovery determined by the computer program and the experienced human evaluators. The results of this study showed that the computer counting program was a reproducible tool to evaluate intact cell recovery after use of membrane integrity dyes on chondrocytes in situ. This and the significant decrease in the time used to count the cells by the computer program advocate its use in future studies because it has significant advantages.

     

Requiring help injecting as a risk factor for HIV infection in the Vancouver epidemic: implications for HIV prevention

BACKGROUND: Requiring help injecting was recently associated with syringe sharing, and later HIV-1 and HCV seroconversion among injection drug users (IDU) in Vancouver. This risk factor remains poorly understood. The present study investigates this risk factor among Vancouver IDUs. METHODS: We evaluated factors associated with requiring help injecting among participants enrolled in the Vancouver Injection Drug User Study (VIDUS) using univariate and logistic regression analyses. VIDUS participants who were followed-up during the period December 2000 to December 2001 were eligible for the present analyses. We also evaluated self-reported reasons for requiring help injecting. RESULTS: Overall, 661 active injection drug users were interviewed during the study period. Among this population, 151 (22.8%) had required help injecting during the last six months, whereas 510 (77.2%) indicated that they had not. Variables that were independently associated with requiring help injecting included borrowing a used syringe (adjusted odds ratio [AOR] = 2.18), frequent cocaine injection (AOR = 1.57), and female gender (AOR = 2.29). Among males, the most common reasons for requiring help injecting were: having no viable veins (77.1%), and anxiousness or being drug sick (42.9%). Among females, the most common reasons reported were: having no viable veins (71.6%), jugular injection or 'jugging' (45.7%), and being anxious or drug sick (27.2%). Almost twice as many females (13.6% vs 7.1%) reported not knowing how to inject as their reason for requiring help injecting. CONCLUSION: Although current public health approaches, such as needle exchange, are unable to address the concerns associated with requiring help injecting, available evidence suggests that safer injecting facilities have the potential to substantially mitigate this risk behaviour.     

Levonorgestrel concentrations during 7 years of continuous use of Jadelle contraceptive implants

Serum levonorgestrel concentrations were assayed in a multicenter, 7-year study of 199 users of Jadelle rod implants. We examined drug levels, patterns of changes, factors affecting drug levels, and concentrations at which pregnancies occurred. Mean levonorgestrel concentrations declined from 435 pg/mL at 1 month of use to 64% of that value (280 pg/mL) at the end of 3 years. Between the end of the third and fifth years neither mean nor median serum levels varied markedly. At 5 years the mean concentration was again 64% of the first month's mean. Declining levels were observed thereafter through the end of 7 years when the mean, 224 pg/mL, was 52% of the 1-month value. Last measured drug concentrations of women who became pregnant during Jadelle use had mean and median values of 152 and 144 pg/mL, respectively, and a maximum value of 180 pg/mL. Analyses indicated ponderal index, body weight, duration of use, and a single clinical center were the most important variables affecting measured levonorgestrel levels. Approximately one-third of assays in the sixth and seventh years were found to be below 180 pg/mL, suggesting that Jadelle levonorgestrel implants would not maintain sufficiently high levels of effectiveness against pregnancy after 5 years and that heavier women would then be at greater risk of pregnancy.     

Neuronal synthesized insulin roles on neural differentiation within fetal rat neuron cell cultures

We previously, described the production and secretion of insulin by fetal neurons in culture and demonstrated that neuronal synthesized insulin [I(n)] promoted neurofilament distribution and axonal growth. In this study we investigated the role of I(n) in promoting neural differentiation. Stem cells from 16 day gestational age rat brains were cultured in an insulin-free defined medium (IFDM) and treated with: 5, 20 or 100 ng/ml of exogenous insulin, 100 ng/ml insulin-like growth factor I (IGF-I) or an anti-insulin antibody. The neurons were studied at 1 and 3 days of incubation. The total number of cells showed no significant difference (P>0.05) in any of the media used, except the IFDM at day 3 of incubation treated with the anti-insulin antibody (P<0.05) and IGF-I to 20 ng/ml of insulin (P<0.05). No significant difference (P>0.05) was found in the number of differentiated neurons incubated in the IFMD, in which the neurons produce and secrete I(n), between days 1 and 3 of incubation, but neural differentiation decreased significantly (P<0.05) when treated with the anti-insulin antibody. Exogenous insulin significantly increased (P<0.05) the number of differentiated neurons compared to the IFDM. A significant reduction (P<0.05) of differentiated neurons was observed at day 3 of incubation with IGF-I compared to all the different media. Thus, I(n) has a role in promoting neural differentiation and growth, but exogenous insulin promoted neural differentiation and growth beyond I(n).     

The association of socioeconomic status with outcomes in cystic fibrosis patients in the United States

There is considerable variability in the clinical course of disease in cystic fibrosis (CF). Although currently unidentified modifier genes might explain some of this heterogeneity, other factors are probably contributory. Socioeconomic status (SES) is an important predictor of health status in many chronic polygenic diseases, but its role in CF has not been systematically evaluated. We performed a historical cohort analysis of pediatric CF patients in the United States using National Cystic Fibrosis Foundation Patient Registry (NCFPR) data for 1986 to 1994, and used Medicaid status as a proxy for low SES. The adjusted risk of death was 3.65 times higher (95% confidence interval [CI]: 3.03 to 4.40) for Medicaid patients than for those not receiving Medicaid. The percent predicted FEV(1) of surviving Medicaid patients was less by 9.1% (95% CI: 6.9 to 11.2). Medicaid patients were 2.19 times more likely to be below the 5th percentile for weight (95% CI: 1.91 to 2.51) and 2.22 times more likely to be below the 5th percentile for height (95% CI: 1.95 to 2.52) than were non-Medicaid patients. Medicaid patients were 1.60 times more likely to require treatment for a pulmonary exacerbation (95% CI: 1.29 to 1.98). There was no difference in the number of outpatient clinic visits for Medicaid and non-Medicaid patients. We conclude that low SES is associated with significantly poorer outcomes in children with CF. Barriers in access to specialty health care do not seem to explain this difference. Further study is indicated to determine what adverse environmental factors might cluster in CF patients of low SES to cause worse outcomes.     

Genetic testing: a physician's perspective

Progress in DNA diagnostics has been extremely rapid. We sought to determine attitudes, awareness, and knowledge of genetic testing by physicians affiliated with the Mount Sinai Medical Center. We surveyed 363 physicians within whose fields genetic testing for various diseases and disorders exist. Physicians' awareness of and opinions regarding testing, attitudes toward counseling, knowledge of the field, and interest in further education were assessed. Three hundred forty-one (341) physicians were determined to be eligible for the study and, of these, 89 (26%) returned completed surveys. Of the respondents, 71% rated their knowledge of genetics and genetic testing as "fair" to "poor"; only 37% read articles concerning genetic testing on a regular basis. Physician awareness of currently available testing produced a bell-shaped distribution. Knowledge regarding Mendelian genetics yielded a bimodal distribution, and knowledge reflecting an understanding of the mechanics behind genetic testing produced a bell-like curve, skewed to the right. Those who identified themselves as practicing within an "academic" setting scored significantly higher on the Mendelian genetics and testing mechanics sections than those practicing in a "private" setting. Ninety-eight percent (98%) of the physicians said they would refer their patients to a genetic counselor. Although 91% of the respondents were aware of the existence of genetic counseling services, only 71% were aware of the services available at major New York medical centers. Of those aware of counseling services, 53% had referred a patient to them, and 83% of those who referred were "mostly" to "very" satisfied with the counseling. Ninety-five percent (95%) of the physicians believed that the doctor, among others, has the responsibility to counsel patients about genetic testing, yet only 51% felt that they had the time. No statistically significant preference was found concerning the methods for gaining further education or information about genetic testing. Further education for physicians is required in order for them to accurately convey the risks and benefits of genetic testing to their patients. Furthermore, awareness of the counseling services available within the New York area needs to be heightened in order to provide physicians and patients with the specific services they desire. The most efficient and effective methods for providing information and for heightening awareness need to be determined through additional research.