Potential impurities in drug substances: Compound-specific toxicology limits for 20 synthetic reagents and by-products, and a class-specific toxicology limit for alkyl bromides.

This paper provides compound-specific toxicology limits for 20 widely used synthetic reagents and common by-products that are potential impurities in drug substances. In addition, a 15 µg/day class-specific limit was developed for monofunctional alkyl bromides, aligning this with the class-specific limit previously defined for monofunctional alkyl chlorides. Both the compound- and class-specific toxicology limits assume a lifetime chronic exposure for the general population (including sensitive subpopulations) by all routes of exposure for pharmaceuticals. Inhalation-specific toxicology limits were also derived for acrolein, formaldehyde, and methyl bromide because of their localized toxicity via that route. Mode of action was an important consideration for a compound-specific toxicology limit. Acceptable intake (AI) calculations for certain mutagenic carcinogens assumed a linear dose-response for tumor induction, and permissible daily exposure (PDE) determination assumed a non-linear dose-response. Several compounds evaluated have been previously incorrectly assumed to be mutagenic, or to be mutagenic carcinogens, but the evidence reported here for such compounds indicates a lack of mutagenicity, and a non-mutagenic mode of action for tumor induction. For non-mutagens with insufficient data to develop a toxicology limit, the ICH Q3A qualification thresholds are recommended. The compound- and class-specific toxicology limits described here may be adjusted for an individual drug substance based on treatment duration, dosing schedule, severity of the disease and therapeutic indication.

A ten-color tube with dried antibody reagents for the screening of hematological malignancies.

INTRODUCTION: The workflow in clinical flow cytometry laboratories must constantly be reviewed to develop technical procedures that improve quality and productivity and reduce costs. Using the Beckman Coulter dry coating technology, we customized a ten-color tube with dried antibody reagents, designated the Duraclone screening tube (DST), for screening hematological malignancies. Here, we compared the applicability, clinical and numerical equivalence, and cost and time required for the technical procedures between the liquid reagents and the DST. METHODS: The DST contains CD4 + Kappa-FITC, CD8 + Lambda-PE, CD3 + CD14-ECD, CD33-PE-Cy5.5, CD20 + CD56-PE-Cy7, CD34-APC, CD19-APC-AlexaFluor700, CD10-APC-AlexaFluor750, CD5-Pacific Blue, and CD45-Krome Orange. We evaluated 20 bone marrow samples, 13 peripheral blood samples, 6 lymph node biopsy samples, 5 fine-needle aspirate samples, 5 cerebrospinal fluid samples, and 1 pleural fluid sample. RESULTS: The DST was useful for more than 60% of our samples. It was able to enumerate the majority of the populations in all types of samples with a statistically acceptable correlation with the liquid reagents. The use of the DST translated into significant time and cost savings of 15.8% and 12.3%, respectively, compared with the use of the liquid reagent. The cost was reduced by $14.36 per sample. CONCLUSIONS: The DST is an efficient solution for screening hematological malignancies with improved quality, productivity, standardization, and sustainability. These improvements could benefit patients by providing faster diagnoses using a higher quality and lower cost reagent.

Evaluation of Bacillus oleronius as a Biological Indicator for Terminal Sterilization of Large-Volume Parenterals.

In the production of large-volume parenterals in Japan, equipment and devices such as tanks, pipework, and filters used in production processes are exhaustively cleaned and sterilized, and the cleanliness of water for injection, drug materials, packaging materials, and manufacturing areas is well controlled. In this environment, the bioburden is relatively low, and less heat resistant compared with microorganisms frequently used as biological indicators such as Geobacillus stearothermophilus (ATCC 7953) and Bacillus subtilis 5230 (ATCC 35021). Consequently, the majority of large-volume parenteral solutions in Japan are manufactured under low-heat sterilization conditions of F0 <2 min, so that loss of clarity of solutions and formation of degradation products of constituents are minimized. Bacillus oleronius (ATCC 700005) is listed as a biological indicator in "Guidance on the Manufacture of Sterile Pharmaceutical Products Produced by Terminal Sterilization" (guidance in Japan, issued in 2012). In this study, we investigated whether B. oleronius is an appropriate biological indicator of the efficacy of low-heat, moist-heat sterilization of large-volume parenterals. Specifically, we investigated the spore-forming ability of this microorganism in various cultivation media and measured the D-values and z-values as parameters of heat resistance. The D-values and z-values changed depending on the constituents of large-volume parenteral products. Also, the spores from B. oleronius showed a moist-heat resistance that was similar to or greater than many of the spore-forming organisms isolated from Japanese parenteral manufacturing processes. Taken together, these results indicate that B. oleronius is suitable as a biological indicator for sterility assurance of large-volume parenteral solutions subjected to low-heat, moist-heat terminal sterilization.

Development of a novel, hemolysis-resistant reagent for assessment of α-amylase in biological fluids.

BACKGROUND: Although the assessment of α-amylase is an essential part of the diagnostic workout of several pancreatic and extra-pancreatic disorders, its enzymatic activity is significantly reduced in the presence of cell-free hemoglobin such as in samples with spurious hemolysis, due to chemical and spectrophotometric interference. We developed a new reagent that provides reliable results on hemolyzed biological specimens. METHODS: All tests were performed on Beckman Coulter AU5822. Intra-assay imprecision was assessed on three serum samples with low, intermediate and high α-amylase concentration. Linearity was assessed by serially diluting two samples with low and high values of α-amylase. The comparison with commercial reagent was performed on 40 serum samples. Hemolysis studies were carried out by mechanically hemolysis of 20 lithium-heparin samples. RESULTS: The intra-assay imprecision was comprised between 1.3% and 2.2%. The linearity was excellent (r=0.998), and highly significant agreement was observed with the commercial assay (r=1.00; mean bias -3.8%). Although a significant correlation between non-hemolyzed and hemolyzed specimens was found with both assays (p<0.001), a much greater agreement was observed with the experimental method (r=0.997 vs. 0.818). No measurement exceeded the total allowable error with the experimental assay, whereas the threshold was exceeded in 85% of samples using the commercial method. CONCLUSIONS: The clinical applications of the experimental reagent include α-amylase assessment in hemolyzed samples, in urine and other biological fluids contaminated with lysed erythrocytes, or in patients under frequent transfusions and hemoglobin-based blood substitutes therapy. The formulation of this reagent could be adapted for other clinical chemistry or immunochemistry assays.

[Study on the supervision of in-vitro diagnostic reagents].

The regulatory history and status of in vitro diagnostic reagents (IVD) at home and abroad are introduced. Suggestions are also provided on the administration of IVD.

Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR.

AIMS: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. METHODS AND RESULTS: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. CONCLUSIONS: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms.

1st NCI annual meeting on Clinical Proteomic Technologies for Cancer.

The National Cancer Institute of the US National Institutes of Health established a Clinical Proteomic Technologies Initiative for Cancer (CPTI) in 2006. The first annual meeting organized by the CPTI program provided up-to-date information on the research activities and achievements at its first anniversary of this program. Presentations were made by leaders from the five centers nationwide of the Clinical Proteomic Technology Assessment for Cancer (CPTAC), and other principal investigators funded by the CPTI.

Affordable diagnostics--changing the paradigm in India.

A successful strategy for developing affordable diagnostics begins with a shift in viewpoint. Diagnostics is a tool for generating clinical information. Amassed experience in different parts of the globe has shown that this process of generating and utilizing clinical information is not only different among various countries but also different in microenvironments within the same country. The development of affordable diagnostics requires consideration not only of the component costs such as hardware and consumables but also other related costs that contribute to the generation and delivery of that information. It is important to recognize that these costs associated with public health in resource-poor settings cannot remain at the mercy of charitable contributions from western nations. Therefore, the challenge of technological innovation is to create solutions that are locally affordable and sustainable in the long run within the local macroeconomic constraints. The solutions should permit generation of local economic activity that will reinforce long-term economic sustainability. For this reason it is essential not only to analyze the diagnostic process but also to define a pathway by which local healthcare systems in recipient nations can be endowed with elements that empower them to acquire and practice up-to-date modern diagnostic skills. The objective of this paper is to provide a wider view of diagnostic cost components and to show how solutions developed and delivered locally have resulted in economically affordable as well as sustainable products.

Evaluation of a single-platform microcapillary flow cytometer for enumeration of absolute CD4+ T-lymphocyte counts in HIV-1 infected Thai patients.

BACKGROUND: Various assays are used to enumerate peripheral blood absolute CD4+ T-lymphocytes. Flow cytometry is considered the gold standard for this purpose. However, the high cost of available flow cytometers and monoclonal antibody reagents make it difficult to implement such methods in the resource-poor settings. In this study, we evaluated a cheaper, recently developed single-platform microcapillary cytometer for CD4+ T-lymphocyte enumeration, the personal cell analyzer (PCA), from Guava Technologies. METHODS: CD4+ and CD8+ T-lymphocyte counts in whole blood samples from 250 HIV-1 infected Thais were determined, using a two-color reagent kit and the Guava PCA, and compared with the results obtained with two reference microbead-based methods from Becton Dickinson Biosciences: the three-color TruCOUNT tube method and the two-color FACSCount method. Statistical correlations and agreements were determined using linear correlation and Bland-Altman analysis. RESULTS: Absolute CD4+ T-lymphocyte counts obtained using the Guava PCA method highly correlated with those obtained using TruCOUNT method (R(2) = 0.95, mean bias +13.1 cells/microl, limit of agreement [LOA]-117.9 to +144.1 cells/microl) and the FACSCount method (R2 = 0.94, mean bias = +33.2 cells/microl, LOA-101.8 to +168.3 cells/microl). Absolute CD8+ T-lymphocyte counts obtained using the Guava PCA method also highly correlated with those obtained with the two reference methods (R(2) = 0.92 and 0.88, respectively). CONCLUSION: This study shows that the enumeration of CD4+ T-lymphocytes using the Guava microcapillary cytometer PCA method performed well when compared with the two reference bead-based methods. However, like the two reference methods, this new method needs substantial technical expertise.

A comparison of three stool tests for colorectal cancer screening.

The annual guaiac or immunochemical fecal occult blood test (FOBT) is one of the five colorectal cancer (CRC) screening regimens recommended by the American Cancer Society (Smith, Cokkinides, & Eyre, 2005). Stool-based deoxyribonucleic acid (DNA) testing for CRC is considered a promising technology (Smith, Cokkinides, & Eyre, 2003). Numerous features of three noninvasive stool tests for CRC are compared.

From guaiac to immune fecal occult blood tests: the emergence of technology in colorectal cancer screening.

Colorectal cancer is the second leading cause of cancer deaths in the United States for both men and women. Colorectal cancer screening is an important means for reducing morbidity and mortality. The American Cancer Society recommends five different screening regimens for adults of average risk, age 50 years and older. The optimal effectiveness of a screening program is dependent on the accuracy of the screening test used. An accurate screening test would have high sensitivity (positive) when an adenomatous polyp or cancer is present and high specificity (negative) in their absence. In April 2002, the American Cancer Society Colorectal Cancer Advisory Group concluded that the immunochemical fecal occult blood test has some advantages that merit revision of their guideline statement for fecal occult blood testing, to include the immunochemical fecal occult blood test. The advantages cited were the possibility of improved sensitivity and specificity and the lack of required dietary restrictions, which make it a more patient-friendly test. Several types of immunochemical fecal occult blood tests are discussed in this article, including their advantages and disadvantages compared with those of the traditional guaiac fecal occult blood testing.

A multicentre assessment of the endogenous thrombin potential using a continuous monitoring amidolytic technique.

This study assessed the inter-laboratory imprecision associated with the measurement of the endogenous thrombin potential (ETP). The initial studies used techniques that had evolved in each of the participating laboratories. Samples from normal healthy subjects (n = 10), two patients receiving coumarin therapy [International Normalized Ratio approximately 2.0 and approximately 4.0] and a further two subjects receiving treatment with unfractionated heparin (anti-Xa 0.07 i.u./ml and 0.31 i.u./ml) were assayed relative to a lyophilized normal plasma that had arbitrarily been assigned a potency of 100%. Considerable variation in potency estimates was observed between the centres, although individual laboratories using fully automated techniques achieved acceptable levels of imprecision as assessed by the coefficient of variation (CV) (intra-assay CV < 9.5%, inter-assay CV < 12.5%). A second study to assess a similar range of samples, using a standardized assay protocol and incorporating appraisal of two chromogenic substrates, CBS.0068 or Pefachrom TG, demonstrated markedly improved agreement in potency estimates between centres and good correlation (r > 0.96) between the chromogenic substrates. Our data demonstrates that an automated ETP method can be standardized between laboratories and suitable levels of imprecision achieved, using different analysers (COBAS Mira at two centres and an ACL-300R) and two thrombin substrates. This indicates that more widespread use of ETP measurements in clinical laboratories is feasible.

[Primary antisera before and after the expiration date. Comparative immunohistochemical observations and analysis of data sheets and labels]. / Antisieri primari prima e dopo la data di scadenza: osservazioni immunoistochimiche comparative e analisi dei fogli illustrativi e delle etichette.

By means of positive and negative controls, the immunostaining properties of a series (B) of 78 primary antibodies (PAB) that had expired 7-77 months previously (mean, 26.3 months) were evaluated in comparison those of the same non expired (functioning) PAB. Qualitatively, no significative difference was observed in the specificity and sensitivity. Among all of the PAB (with the exception of one), no immunonegativity was observed. With special reference to immunohistochemical methods, dilution and retrieval procedures, as suggested on data sheets, were additionally considered. Moreover the residual availability of the reagents was checked. In fact 58 PAB were still available for further examination with probable prolongation of the duration of validity. Other observations are analytically reported as far as polyclonal, monoclonal, concentrated and predilluted expired PAB are concerned. In the same way, duration of available validity before the expiration date was examined for the expired PAB and for an additional series of 90 nonexpired PAB. Finally textual information (including intended use) reported on data sheets and labels has been scrutinized in detail. In conclusion, for the diagnostically applied immunohistochemistry on the basis of these findings and the recent American and European rules, the following propositions should be considered: (1) surveillance on methodological technical approach and diagnostic evaluation, with emphasis on accurate standardization and primary responsibility of the pathologist; (2) opportunity of a continuous feed-back between laboratories-customers and producers-traders, in order to render more uniform the information and establish more realistic parameters of utilization; and (3) possibility of cost reduction according to limited financial support from the health care administration.