Accreditation and the use of validated/recognised methods to analyse human semen

Accreditation of laboratories who perform diagnostic semen analysis in Australia and New Zealand is a requirement of the healthcare system. Within the accreditation process laboratories are required to set ISO standards within their policies and procedures. In order to achieve their aims, laboratories need to be able to measure a number of defined semen parameters both accurately and repetitively, especially around the lower limit of the reference intervals. The methods documented in the WHO-manual are used almost universal as the laboratory standard. Some laboratories incorporate minor method variations into their procedures. As part of the ISO requirements all variations require validation using internally approved processes that are documented and that incorporate appropriate statistical analysis and comparison of results. Validation is an ongoing process and regular review is essential. Evidence of the validation must be available for review by external auditors during accreditation. Where any validated variant method returns results that are significantly different to any method within the WHO-manual, the laboratory needs to develop its own, in-house reference interval for that method.     

Collaborative trial of the direct epifluorescent filter technique (DEFT), a rapid method for counting bacteria in milk

The direct epifluorescent filter technique (DEFT) is a new rapid method which uses membrane filtration and epifluorescent microscopy for counting bacteria in milk. A collaborative trial of the DEFT was conducted between six laboratories. Each laboratory obtained a highly significant relationship between the DEFT count and plate count with a correlation coefficient generally > 0.9 but there were significant differences between these relationships. The repeatability of the DEFT, although ca 1·5 times worse than that of the plate count, was of a level acceptable in practice. Reproducibility of the DEFT was ca 3 times that of the plate count. This poor reproducibility was probably mainly due to counting errors. Possible reasons for this and ways of reducing counting errors are discussed.     

First colombian interlaboratory study for the determination of aflatoxin B1 in yellow corn

An interlaboratory study for the determination of aflatoxin B1 in yellow corn was conducted with 16 laboratories that analyze for aflatoxins in Colombia. Naturally contaminated ground yellow corn (Monsanto DK 4004) with an assigned reference value of 26.3 μg/kg aflatoxin B1 was distributed as double blind duplicates in sachets of 55 g. z-Scores were computed for each of the results; repeatability of the two replicate analysis was also calculated. Four of the participating laboratories used HPLC, seven used TLC, one used fluorometry, one ELISA and three a semi quantitative analytical technique (Aflacard®). Only 10 of the 26 quantitative results (39%) had satisfactory z-scores, two scores were questionable (8%) and 14 of the 26 results had unsatisfactory z-scores (54%). A total of 8 laboratories had satisfactory repeatability (62%), and 5 had unsatisfactory repeatability (39%). The present study indicates that only about one third of the results for aflatoxin reported by Colombian laboratories have good accuracy (as measured by the z-score of the result), although satisfactory precision (measured as repeatability) is achieved by about two thirds of the laboratories. These results indicate that an improvement in quality assurance is needed in Colombian laboratories. The routine use of reference standards and reference materials is highly recommended.     

Comparability of poliovirus neutralizing antibody tests.

A serology panel was used to assess the comparability of results of poliovirus neutralizing antibody assays. Five laboratories from three countries provided results with seven in-house methods. A high degree of within-laboratory reproducibility was seen for all laboratories and methods, with 94% of results for repeat titrations within plus or minus one twofold dilution. Considerable differences in sensitivity between methods were, however, observed. The period of serum/virus incubation prior to inoculation of cells was shown by one laboratory to have a 10-fold effect on titres, and another laboratory showed that use of Sabin or wild-type challenge virus significantly influenced results for types 1 and 3. Expression of results as relative potencies abolished these difference due to method. The difference in sensitivity between laboratories for end-point titres (if one very sensitive method was excluded) was around fourfold for types 1 and 2, and ninefold for type 3. This range increased to 15-fold for type 2; and 20-fold for types 1 and 3 if the most sensitive method was included. Expression of results relative to a standard considerably improved agreement with a residual variation of around fourfold. Therefore, expression of results from serological studies, including vaccine trials, in International Units (i.e. relative to the new International Standard) will assure that comparisons between studies can be made with confidence.     

Measurement of genotoxicity in wastewater samples with the in vitro micronucleus test: results of a round-robin study in the context of standardisation according to ISO

In the course of standardisation of the in vitro micronucleus test for analysis of effluents according to ISO, a national round-robin study was organised by the German Federal Institute of Hydrology (BfG), involving 10 laboratories of private companies, universities and public authorities. The micronucleus assay was performed with the permanently growing Chinese hamster lung fibroblast cell line V79. All participants tested four encoded samples from one municipal and one industrial wastewater treatment plant with and without metabolic activation by S9-mix. Two of these samples were spiked in advance with defined concentrations of the clastogenic substances cyclophosphamide and mitomycin C, respectively. Cyclophosphamide and ethyl methanesulfonate were used as positive controls. The defined assessment criterion for genotoxicity was the lowest dilution of a sample that does not show any significant induction of micronuclei. Cytotoxicity was judged by determining the cell-survival index, i.e. the percentage growth rate of the cells compared with the corresponding negative controls. As supplementary qualitative criteria, the mitotic index and the proliferation index were assessed. All participants successfully established the method within a few weeks and generated viable test results in time. The two non-genotoxic samples were detected as negative by 90% (with S9-mix) and 95% (without S9-mix) of the participants. The mitomycin C-spiked wastewater sample (expected to be positive without S9-mix supplementation) was correctly judged as positive by all laboratories. The cyclophosphamide-spiked sample (expected to be positive with S9-mix addition) was evaluated correctly as genotoxic by 80% of the laboratories. A post-test analysis found evidence that the false negative results were due to technical failure, but not of a methodological nature. In 94% of all tests the sample LID values (lowest ineffective dilution=dilution stage of the sample in the test at which a statistically significant increase in the micronucleus rate was not detectable any more) varied by no more than one dilution step around the median LID value. The survival index was proven to be a robust measure for estimation of toxicity. This round-robin study is the first inter-laboratory comparison of the in vitro micronucleus test using wastewater samples. The test system is intended to complement the already DIN- and ISO-standardised bacterial tests, i.e. the umu-test and the Ames plate-incorporation assay. The data provide evidence that the robust and practicable in vitro micronucleus test is suitable as a routine method for wastewater testing.     

A collaborative study of a method for enumeration of probiotic enterococci in animal feed

AIMS: Validation of an enumeration method to be used as an official control method in the framework of Council Directive 70/524/EEC for probiotic enterococci used as feed additives. METHODS AND RESULTS: Twenty laboratories in 12 European countries carried out a collaborative study. A plate count method using bile esculin azide (BEA) agar was used. Precision data in terms of repeatability (r) and reproducibility (R) of the method using different feeding stuffs and three inoculation levels were determined. Enterococci were present in the samples as a single component or in mixtures with other probiotic feed additives. The enumeration of enterococci on BEA agar showed a relative standard deviation (RSD)r of 1.5-3.6% and an RSD(R) between 2.9 and 7.4%. BEA agar was selective for enterococci in the presence of other probiotic micro-organisms such as pediococci, lactobacilli and yeast. CONCLUSIONS: For routine analysis of viable enterococci concentrations in feeding stuffs, the use of BEA is recommended. This methodology is not applicable for mineral feeds. SIGNIFICANCE AND IMPACT OF THE STUDY: An official control method for enumeration of authorized probiotic enterococci in feeding stuffs was validated. The results are intended for consideration for adoption as CEN and ISO standards.     

Multi-Site N-glycan mapping study 1: Capillary electrophoresis – laser induced fluorescence

An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra high-performance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Our results demonstrated low variability and high reproducibility, both, within any given site as well across all sites, which indicates that a standard N-glycan analysis platform appropriate for general use (clone selection, process development, lot release, etc.) within the industry can be established.     

Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI)

The Cancer Vaccine Consortium of the Sabin Vaccine Institute (CVC/SVI) is conducting an ongoing large-scale immune monitoring harmonization program through its members and affiliated associations. This effort was brought to life as an external validation program by conducting an international Elispot proficiency panel with 36 laboratories in 2005, and was followed by a second panel with 29 participating laboratories in 2006 allowing for application of learnings from the first panel. Critical protocol choices, as well as standardization and validation practices among laboratories were assessed through detailed surveys. Although panel participants had to follow general guidelines in order to allow comparison of results, each laboratory was able to use its own protocols, materials and reagents. The second panel recorded an overall significantly improved performance, as measured by the ability to detect all predefined responses correctly. Protocol choices and laboratory practices, which can have a dramatic effect on the overall assay outcome, were identified and lead to the following recommendations: (A) Establish a laboratory SOP for Elispot testing procedures including (A1) a counting method for apoptotic cells for determining adequate cell dilution for plating, and (A2) overnight rest of cells prior to plating and incubation, (B) Use only pre-tested serum optimized for low background: high signal ratio, (C) Establish a laboratory SOP for plate reading including (C1) human auditing during the reading process and (C2) adequate adjustments for technical artifacts, and (D) Only allow trained personnel, which is certified per laboratory SOPs to conduct assays. Recommendations described under (A) were found to make a statistically significant difference in assay performance, while the remaining recommendations are based on practical experiences confirmed by the panel results, which could not be statistically tested. These results provide initial harmonization guidelines to optimize Elispot assay performance to the immunotherapy community. Further optimization is in process with ongoing panels.     

Prevalidation study of the Syrian hamster embryo (SHE) cell transformation assay at pH 7.0 for assessment of carcinogenic potential of chemicals

The European Centre for the Validation of Alternative Methods (ECVAM) has organised an interlaboratory prevalidation study on the Syrian hamster embryo (SHE) cell transformation assay (CTA) at pH 7.0 for the detection of rodent carcinogens. The SHE CTA at pH 7.0 has been evaluated for its within-laboratory reproducibility, transferability and between-laboratory reproducibility. Four laboratories using the same basic protocol with minor modifications participated in this study and tested a series of six coded-chemicals: four rodent carcinogens (benzo(a)pyrene, 3-methylcholanthrene, 2,4-diaminotoluene and o-toluidine HCl) and two non-carcinogens (anthracene and phthalic anhydride). All the laboratories found the expected results with coded chemicals except for phthalic anhydride which resulted in a different call in only one laboratory. Based on the outcome of this study, it can be concluded that a standardised protocol is available that should be the basis for future use. This protocol and the assay system itself are transferable between laboratories and the SHE CTA at pH 7.0 is reproducible within- and between-laboratories.     

Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis

Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and trained health professionals. Furthermore, adequate care and transmission control depend on early and reliable pathogen detection. Here, we describe a qPCR test for routine diagnosis of leprosy-suspected patients. The reaction simultaneously amplifies two specific Mycobacterium leprae targets (16S rRNA and RLEP), and the human 18S rRNA gene as internal control. The limit of detection was estimated to be 2.29 copies of the M. leprae genome. Analytical specificity was evaluated using a panel of 20 other skin pathogenic microorganisms and Mycobacteria, showing no cross-reactivity. Intra- and inter-operator C_p variation was evaluated using dilution curves of M. leprae DNA or a synthetic gene, and no significant difference was observed between three operators in two different laboratories. The multiplex assay was evaluated using 97 patient samples with clinical and histopathological leprosy confirmation, displaying high diagnostic sensitivity (91%) and specificity (100%). Validation tests in an independent panel of 50 samples confirmed sensitivity and specificity of 97% and 98%, respectively. Importantly, assay performance remained stable for at least five months. Our results show that the newly developed multiplex qPCR effectively and specifically detects M. leprae DNA in skin samples, contributing to an efficient diagnosis that expedites the appropriate treatment.     

Validation and utility of novel volume reduction technique for determination of parallel conductance

The parallel conductance volume, created by the conductivity of structures surrounding the ventricular blood pool, can be estimated by using a saline dilution technique. This paper examines the use of a novel volume reduction method, during a standard vena caval preload reduction maneuver, as an alternative to the routinely used saline dilution method to calibrate conductance catheter measurements in the left (LV) and right ventricle (RV) of animals and humans. The serial reproducibility of both methods was examined by measurement of percent difference, and by assessing the coefficient of repeatability 1) between two measurements within the same subject, 2) between the two techniques, and 3) interobserver variability. The effect of ventricular size and contractile state on the volume reduction technique was also observed. It was essential to ensure the technique was not affected by inotropic state. The volume reduction technique and saline dilution method were repeated at three different loading states (baseline, 5, and 10 microg x kg(-1) x min(-1) of dobutamine). The coefficient of repeatability between serial measurements was similar for both the volume reduction and saline dilution methods, and good interobserver variability was demonstrated. The volume reduction technique was compared with the saline dilution technique over a large range of ventricular sizes. No significant difference was observed in the RV or LV of adult humans or in the LV of neonatal pigs and children. There was no significant effect on either the saline dilution or the volume reduction technique as the inotropic state increased. In conclusion, the volume reduction technique is neither affected by ventricular size nor contractile state, is repeatable between different observers, and can be used to substitute the saline dilution method when preload reduction of the ventricle is being employed.     

Precision and Agreement of Corneal Power Measurements Obtained Using a New Corneal Topographer OphthaTOP

Purpose

To evaluate repeatability and reproducibility of anterior corneal power measurements obtained with a new corneal topographer OphthaTOP (Hummel AG, Germany) and agreement with measurements by a rotating Scheimpflug camera (Pentacam HR, Oculus, Germany) and an automated keratometer (IOLMaster, Carl Zeiss Meditec, Germany).

Methods

The right eyes of 79 healthy subjects were prospectively measured three times with all three devices. Another examiner performed three additional scans with the OphthaTOP in the same session. Within one week, the first examiner repeated the measurements using the OphthaTOP. The flat simulated keratometry (Kf), steep K (Ks), mean K (Km), J₀, and J₄₅ were noted. Repeatability and reproducibility of measurements were assessed by within-subject standard deviation (Sw), repeatability (2.77 Sw), coefficient of variation (CoV), and intraclass correlation coefficient (ICC). Agreement between devices was assessed using 95% limits of agreement (LoA).

Results

Intraobserver repeatability and interobserver and intersession reproducibility of all measured parameters showed a 2.77 Sw of 0.29 diopter or less, a CoV of less than 0.24%, and an ICC of more than 0.906. Statistically significant differences (P<0.001) were found between the parameters analyzed by the three devices, except J₀ and J₄₅. The mean differences between OphthaTOP and the other two devices were small, and the 95% LoA was narrow for all results.

Conclusions

The OphthaTOP showed excellent intraobserver repeatability and interobserver and intersession reproducibility of corneal power measurements. Good agreements with the other two devices in these parameters were found in healthy eyes.     

Multiple Laboratory Comparison of the Doubly Labeled Water Technique*

A double-blind study was conducted to determine between-laboratory variability in the doubly labeled water method for measurement of total energy expenditure in humans, and to compare the accuracy and precision of three widely-used procedures for calculating rates of carbon dioxide production from the original isotope data. Eighteen laboratories from five countries participated in the study. All laboratories were provided with five water standards containing varying amounts of ²H and ¹⁸O, and in addition 11 laboratories were provided with urine and dose specimens from one (six laboratories) or two (five laboratories) healthy elderly subjects of normal height and weight undergoing a calorimetric validation of the doubly labeled water method. The data from the five water standards were analyzed to predict between-laboratory variability in the doubly labeled water technique in all laboratories. In addition, data from the subjects were analyzed using the “slope-intercept”, “2-point” and “modified” methods of calculation. The results confirm that the doubly labeled water method can be an accurate technique for the measurement of energy expenditure in adult human subjects in some laboratories. However, there was substantial between-laboratory variability in the results and some laboratories returned physiologically impossible results. There was no significant effect of calculation procedure on the accuracy of the technique in this limited comparison, although the slope-intercept procedure appeared to be more susceptible to analytical error than the other procedures. The isotope standards analyzed by participants in this study will be made available to other investigators on request.     

Validation and standardization of IS900 and F57 real-time quantitative PCR assays for the specific detection and quantification of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne's disease and may contribute to the onset and development of Crohn's disease in humans. Rapid detection of Map is fundamental because of its reported isolation from pasteurized milk and its potential for transmission through environmental sources. In this study, we developed two independent real-time quantitative PCR assays targeting the IS900 genetic insertion sequence and the F57 sequence, which proved capable of detecting and quantifying Map DNA. Validation and standardization of the developed methods were performed by evaluating diagnostic trueness, precision, and accuracy of the techniques. Specificity of the IS900 and F57 methods was verified in both in silico and experimental studies. The assays were found to be very accurate and precise with high repeatability and reproducibility. Moreover, the two real-time assays were very specific for Map, discriminating most of mycobacterial and nonmycobacterial species.     

Frequent major errors in antimicrobial susceptibility testing of bacterial strains distributed under the Deutsches Krebsforschungszentrum Quality Assurance Program

The Quality Assurance Program (QAP) of the Deutsches Krebsforschungszentrum (DKFZ) was a proficiency testing system developed to service the laboratory animal discipline. The QAP comprised the distribution of bacterial strains from various species of animals for identification to species level and antibiotic susceptibility testing (AST). Identification capabilities were below acceptable standards. This study evaluated AST results using the DKFZ compilations of test results for all bacterial strains showing the number of participants reporting the strain as resistant (R), sensitive (S) or intermediate susceptible (I) to each antibiotic substance used. Due to lack of information about methods used, it was assumed that what the majority of the participants reported (R or S) was the correct test result and that an opposite result was a major error (ME). MEs occurred in 1375 of 14,258 (9.7%) of test results and ME% ranged from 0% to 23.2% per bacterial group-agent group combination. Considerable variation in MEs was found within groups of bacteria and within groups of agents. In addition to poor performance in proper species classification, the quality of AST in laboratory animal diagnostic laboratories seems far below standards considered acceptable in human diagnostic microbiology.     

Intra- and inter-laboratory variability in Real Dynamic Respiration Index (RDRI) method used to evaluate the potential rate of microbial self heating of solid recovered fuel

Microbial activity acts as primer in the self combustion process of solid recovered fuels (SRF) during their storage or transport. Thus, EU gave mandate to the European Committee for Standardization (CEN) to develop biological methods, (i.e. respirometric method) able to assess the risk of potential self combustion of SRF. Real Dynamic Respiration Index (RDRI) was chosen as official method, and a validation procedure was requested, to assure the quality of the results, when the method is applied for official purpose, i.e. repeatability and reproducibility detection. Two SRF coming from full-scale plants were analyzed for RDRI by three laboratories in six replicates. Results indicated a good precision of the method proposed in agreement with other biological methods, i.e. relative standard deviations of repeatability ranged from 16.7% to 17.8%, and a relative standard deviations of reproducibility ranged from 17.5% to 23.9%.