Ring Trial Research Articles

Inter-laboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction methods used for detection of infectious pancreatic necrosis virus in Chile

Background: Infectious Pancreatic Necrosis Virus (IPNV) is the etiological agent of a highly contagious disease that affects salmonids. In Chile, the second worldwide salmon producer, IPNV causes great economic loss and is one of the most frequently detected pathogens. Due to its high level of persistence and the lack of information about the efficiency of its diagnostic techniques, the National Reference Laboratory (NRL) for IPNV in Chile performed the first i nter laboratory ring trial, to evaluate the sensitivity, specificity and repeatability of the qRT-PCR detection methods used in the country. Results: Results showed 100% in sensitivity and specificity in most of the laboratories. Only three of the twelve participant laboratories presented problems in sensitivity and one in specificity. Problems in specificity (false positives) were most likely caused by cross contamination of the samples, while errors in sensitivity (false negatives) were due to detection problems of the least concentrated viral sample. Regarding r epeatability, many of the laboratories presented great dispersion of the results (Ct values) for replicate samples over the three days of the trial. Moreover, large differences in the Ct values for each sample were detected among all the laboratories. Conclusions: Overall, the ring trial showed high values of sensitivity and specificity, with some problems of repeatability and inter laboratory variability. This last issue needs to be addressed in order to allow harmonized diagnostic of IPNV within the country. We recommend the use of the NRL methods as validated and reliable qRT-PCR protocols for the detection of IPNV. Normal 0 false false false ES JA X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0cm; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi; mso-ansi-language:ES;}

Detection of Salmonella spp. in spices and herbs

The detection of microbial contaminations in spices and herbs is a challenging task due to their strong antimicrobial effects, which potentially increase the risk for false-negative results. Therefore, the present study mainly focuses on the detection of Salmonella spiked to cinnamon and oregano. Both condiments completely inhibited the proliferation of Salmonella at a 1:10 (w/w) dilution. Consequently, the supplementation of the buffered peptone water with K2SO3 as well as the application of higher initial dilutions was investigated. While no detrimental effect of K2SO3 was observed during the growth of 14 different Salmonella isolates, it even improved their detection in condiments. An effect, which was also determined with increased dilution ratios. For detection, a quantitative approach via enumeration of the colony-forming units (CFUs), and qualitative approaches via the culture-based detection according to ISO 6579 and via the nucleic acid-based detection with the 3M Molecular Detection System (MDS) were performed. Subsequently, the limit of detection (LOD) was determined, which was <5 CFU 25 g−1 for both qualitative approaches. Furthermore, the persistence of Salmonella DNA spiked to parsley was determined with the MDS. Despite of the modifications, the LOD for Salmonella spiked to oregano was significantly lower prior than after enrichment, pointing to the requirement for further improvements. Last but not least, a ring trial was performed, which emphasized the importance for a reliable detection.

NIAID, NIEHS, NHLBI, and MCAN Workshop Report: The indoor environment and childhood asthma—implications for home environmental intervention in asthma prevention and management

Environmental exposures have been recognized as critical in the initiation and exacerbation of asthma, one of the most common chronic childhood diseases. The National Institute of Allergy and Infectious Diseases; National Institute of Environmental Health Sciences; National Heart, Lung, and Blood Institute; and Merck Childhood Asthma Network sponsored a joint workshop to discuss the current state of science with respect to the indoor environment and its effects on the development and morbidity of childhood asthma. The workshop included US and international experts with backgrounds in allergy/allergens, immunology, asthma, environmental health, environmental exposures and pollutants, epidemiology, public health, and bioinformatics. Workshop participants provided new insights into the biologic properties of indoor exposures, indoor exposure assessment, and exposure reduction techniques. This informed a primary focus of the workshop: to critically review trials and research relevant to the prevention or control of asthma through environmental intervention. The participants identified important limitations and gaps in scientific methodologies and knowledge and proposed and prioritized areas for future research. The group reviewed socioeconomic and structural challenges to changing environmental exposure and offered recommendations for creative study design to overcome these challenges in trials to improve asthma management. The recommendations of this workshop can serve as guidance for future research in the study of the indoor environment and on environmental interventions as they pertain to the prevention and management of asthma and airway allergies.

Consistency and reproducibility of next-generation sequencing and other multigene mutational assays: A worldwide ring trial study on quantitative cytological molecular reference specimens.

Molecular testing of cytological lung cancer specimens includes, beyond epidermal growth factor receptor (EGFR), emerging predictive/prognostic genomic biomarkers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral [v-ras] oncogene homolog (NRAS), B-Raf proto-oncogene, serine/threonine kinase (BRAF), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA). Next-generation sequencing (NGS) and other multigene mutational assays are suitable for cytological specimens, including smears. However, the current literature reflects single-institution studies rather than multicenter experiences. Quantitative cytological molecular reference slides were produced with cell lines designed to harbor concurrent mutations in the EGFR, KRAS, NRAS, BRAF, and PIK3CA genes at various allelic ratios, including low allele frequencies (AFs; 1%). This interlaboratory ring trial study included 14 institutions across the world that performed multigene mutational assays, from tissue extraction to data analysis, on these reference slides, with each laboratory using its own mutation analysis platform and methodology. All laboratories using NGS (n = 11) successfully detected the study's set of mutations with minimal variations in the means and standard errors of variant fractions at dilution points of 10% (P = .171) and 5% (P = .063) despite the use of different sequencing platforms (Illumina, Ion Torrent/Proton, and Roche). However, when mutations at a low AF of 1% were analyzed, the concordance of the NGS results was low, and this reflected the use of different thresholds for variant calling among the institutions. In contrast, laboratories using matrix-assisted laser desorption/ionization-time of flight (n = 2) showed lower concordance in terms of mutation detection and mutant AF quantification. Quantitative molecular reference slides are a useful tool for monitoring the performance of different multigene mutational assays, and this could lead to better standardization of molecular cytopathology procedures. Cancer Cytopathol 2017;125:615-26. © 2017 American Cancer Society.

Ring trial 2016 for Bluetongue virus detection by real-time RT-PCR in France.

Since the unexpected emergence of BTV‐8 in Northern Europe and the incursion of BTV‐8 and 1 in France in 2006–2007, molecular diagnosis has considerably evolved. Several real‐time RT‐PCR (rtRT‐PCR) methods have been developed and published, and are currently being used in many countries across Europe for BTV detection and typing. In France, the national reference laboratory (NRL) for orbiviruses develops and validates ‘ready‐to‐use’ kits with private companies for viral RNA detection. The regional laboratories network that was set up to deal with a heavy demand for analyses has used these available kits. From 2007, ring tests were organized to monitor the performance of the French laboratories. This study presents the results of 63 regional laboratories in the ring trial organized in 2016. Blood samples were sent to the laboratories. Participants were asked to use the rtRT‐PCR methods in place in their laboratory, for detection of all BTV serotypes and specifically BTV‐8. The French regional laboratories are able to detect and genotype BTV in affected animals. Despite the use of several methods (i.e. RNA extraction and different commercial rtRT‐PCRs), the network is homogeneous. The ring trial demonstrated that the French regional veterinary laboratories have reliable and robust BTV diagnostic tools for BTV genome detection.

Abstract ES4-3: Tumor-infiltrating lymphocytes (TILs) and genomic signatures of immune cells

Abstract Increased levels of tumor-infiltrating lymphocytes have been described in subsets of breast cancer, in particular triple-negative (TNBC) and HER2-positive (HER2+) tumors. Neoadjuvant therapy approaches in breast cancer have been used as a strategy to characterize subgroups of tumors with increased tumor-infiltrating lymphocytes (TILs). We have described tumor-infiltrating lymphocytes (TILs) as predictors of pathological complete response to neoadjuvant chemotherapy in breast cancer trials conducted by the German Breast Group. It has been shown that increased TILs are linked to an increased response rate to neoadjuvant therapy and improved prognosis after adjuvant therapy, in particular in triple-negative and HER2 positive breast cancer. In addition to tumor-infiltrating lymphocytes, immune mRNA markers have been studied in breast cancer and are associated with increased chemotherapy response and improved prognosis. These results suggest that some subtypes of breast cancer are immunogenic and that there is an ongoing low-level immune response present even in clinically progressing tumors. Therapeutic approaches to modulate tumor-associated inflammation by immune checkpoint inhibitors have shown clinical responses in a subset of triple-negative breast cancers, and some of these responses were durable. These targeted therapies have stimulated interest in the interaction between immune cells and tumor cells. Based on the concept that conventional chemotherapy might be immunogenic, some studies are evaluating the combination of an immune checkpoint inhibitor and a chemotherapy. Considering the fact that responses to immune checkpoint inhibitors are observed in only a subset of tumors, biomarkers that will be able to identify those tumors are needed. Possible biomarkers include the therapeutic targets, in particular PD-L1 and PD1, tumor-infiltrating lymphocytes, predictive mRNA signatures, immunogenic neoepitope signatures that are generated by mutations as well as other factors. Due to the low costs of a simple H&amp;E based test, tumor infiltrating lymphocytes might become a promising biomarker as part of the pathology report. In preparation for this, the international immune-oncology working group (TIL working group) has published standardized guidelines for TIL evaluation in breast cancer, and has conducted a first international TIL ring trial. This ring trial suggests that decentral TIL evaluation is feasible. In some situations, in particular in tumors with intermediate and heterogeneous TILs, additional tests, including gene expression signatures might be necessary. It should be emphasized that the clinical utility of TILs for current medical decisions is very limited at the present time, and the ultimate usefulness of these biomarker concepts will be obtained in the ongoing and future clinical immunotherapy trials. The presentation will provide an overview on recent results on immune markers for prediction of response to neoadjuvant chemotherapy and prognosis with a focus in standardized histomorphological evaluation of tumor-infiltrating lymphocytes as well as clinical trials concepts and biomarker strategies for future immune checkpoint inhibitor therapies in breast cancer. Citation Format: Denkert C. Tumor-infiltrating lymphocytes (TILs) and genomic signatures of immune cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr ES4-3.

UV-press method versus artificial digestion method to detect Anisakidae L3 in fish fillets: Comparative study and suitability for the industry

To screen the presence of Anisakidae third stage larvae (L3) in fish, fast methods such as the visual inspection and candling have been widely used by the industry over the last 50 years, and they are regulated by the European Parliament and the Council. These methods are ineffective to detect L3 embedded in fish muscles, consequently alternative methods, such as the artificial digestion (AD) and the UV-Press (UVP) are increasingly applied, but their performance needs to be evaluated. The aims of the present work were: 1) to compare the performance of AD and UVP. methods by a Ring Trial (RT) involving highly experienced laboratories; and 2) to evaluate the potential transferability of the best performing method to the industry by a collaborative study involving industrial partners (β-testing). For RT, each participating laboratory (n=5) received 6 samples of 100g of fish fillets spiked with 1 L3 (1 sample), 3 L3 (2 samples), 7 L3 (2 samples), and a negative control sample (without L3). In each positive sample, there were live Anisakis pegreffii L3 collected from a naturally infected fish. The result evaluation was based on the agreement between the number of reported and the number of spiked L3. No false positive sample was detected. The L3 number detected by the UVP method showed higher (90%) level of agreement with the number of spiked L3 than the number of L3 detected by the AD method (83.3%); however, no significant difference in terms of accuracy (p=0.32) was detected when the two methods were compared. Moreover, considering only the presence/absence of L3 in the samples, the UVP reached 100% of accuracy and 100% of sensitivity; whereas, AD showed 98% of accuracy and 96% of sensitivity. The variability of the UVP method was lower than that of the AD method, indicating a better reproducibility. On the basis of the RT results, the UVP method was selected for the β-testing. Each industrial partner (n=3) received 15 samples of 100g of fish fillet spiked with 1 L3 (2 samples), 2 L3 (2 samples), 3 L3 (2 samples), 4 L3 (2 samples), 5 L3 (2 samples) and 6 L3 (2 samples) and three negative control samples (without L3). The number of L3 counted in 34 out of 45 samples (75.6%) by the UVP method was in agreement with the number of spiked L3. One company reached 93.3% of agreement; whereas the other two companies reached an agreement of 66.7%. Two false negative results were found; whereas, no false positive results were obtained. Moreover, at the industrial level, considering only the presence/absence of larvae in the samples, the UVP reached 97% of accuracy, 94.4% of sensitivity, and 100% of specificity. However, the UVP method, in spite of its accuracy, needs further investigations to provide new time −temperature combinations that could allow a reduction of the testing time and its integration in the fishing deck.

In vitro eye irritation testing using the open source reconstructed hemicornea - a ring trial.

The aim of the present ring trial was to test whether two new methodological approaches for the in vitro classification of eye irritating chemicals can be reliably transferred from the developers' laboratories to other sites. Both test methods are based on the well-established open source reconstructed 3D hemicornea models. In the first approach, the initial depth of injury after chemical treatment in the hemicornea model is derived from the quantitative analysis of histological sections. In the second approach, tissue viability, as a measure for corneal damage after chemical treatment, is analyzed separately for epithelium and stroma of the hemicornea model. The three independent laboratories that participated in the ring trial produced their own hemicornea models according to the test producer's instructions, thus supporting the open source concept. A total of 9 chemicals with different physicochemical and eye-irritating properties were tested to assess the between-laboratory reproducibility (BLR), the predictive performance, as well as possible limitations of the test systems. The BLR was 62.5% for the first and 100% for the second method. Both methods enabled to discriminate Cat. 1 chemicals from all non-Cat. 1 substances, which qualifies them to be used in a top-down approach. However, the selectivity between No Cat. and Cat. 2 chemicals still needs optimization.

In vitro eye irritation testing using the open source reconstructed hemicornea – a ring trial_suppl

The aim of the present ring trial was to test whether two new methodological approaches for the in vitro classification of eye irritating chemicals can be reliably transferred from the developers’ laboratories to other sites. Both test methods are based on the well-established open source reconstructed 3D hemicornea models. In the first approach, the initial depth of injury after chemical treatment in the hemicornea model is derived from the quantitative analysis of histological sections. In the second approach, tissue viability, as a measure for corneal damage after chemical treatment, is analyzed separately for epithelium and stroma of the hemicornea model. The three independent laboratories that participated in the ring trial produced their own hemicornea models according to the test producer’s instructions, thus supporting the open source concept. A total of 9 chemicals with different physicochemical and eye-irritating properties were tested to assess the between-laboratory reproducibility (BLR), the predictive performance, as well as possible limitations of the test systems. The BLR was 62.5% for the first and 100% for the second method. Both methods enabled to discriminate Cat. 1 chemicals from all non-Cat. 1 substances, which qualifies them to be used in a top-down approach. However, the selectivity between No Cat. and Cat. 2 chemicals still needs optimization.

P2.06-039 Searching for Standards: Multicenter Ring Trials to Evaluate Technologies for the Enrichment of Circulating Tumor Cells: Topic: LAB, Other

Circulating tumor cells (CTCs), which can be found in the peripheral blood of cancer patients, represent a simple and minimal-invasive source for monitoring neoplastic evolution or response to anti-cancer therapy. In recent years, numerous technology platforms for the enrichment and molecular characterization of CTCs have emerged, but comparative results and data demonstrating clinical utility are lacking for most of these platforms. To overcome this, the Innovative Medicines Initiative (IMI) consortium CANCER-ID (www.cancer-id.eu), which represents a joint undertaking of experts from academia and pharmaceutical industry, joined forces to define standards in blood-based biomarkers including the evaluation of different CTC enrichment technologies. CTC enrichment technologies including the CellSearch system, Parsortix PR1 and the Siemens filtration device were evaluated in a multicenter ring trial by using standardized spike-in samples of non-small cell lung cancer (NSCLC) cell lines, which were selected based on their different molecular/genetic features. NSCLC cells were spiked into blood of healthy volunteers with informed consent. To increase the comparability of results, spike-in samples were generated in a centralized way following well-defined protocols for pre-analytic sample handling including sample fixation, storage and shipment. Spike-in samples were subsequently analyzed using different CTC enrichment technologies by at least three CANCER-ID partners in a blinded way according to standard operating procedures (SOPs). To reflect clinically relevant disease subtypes, NSCLC cell lines were extensively profiled for copy number aberrations, mutational status (e.g. KRAS, EGFR), expression of cell surface antigens (e.g. EPCAM) as well as cell size. Based on this, cells lines with different molecular/genetic profiles were used to generate complex spike-in samples modeling the heterogeneity of real-life patient material. Spike-in samples were subsequently analyzed by at least three different CANCER-ID partners to determine sensitivity and specificity of the different platforms. In addition, comparative data was generated using the FDA approved CellSearch system, which represents the gold-standard for CTC detection and enumeration. IMI CANCER-ID is a public-private partnership in the field of liquid biopsies with 37 partners from 13 countries providing access to a variety of CTC enrichment technologies and patient samples. Making use of this major advantage, we describe the first efforts to establish standards in CTC enrichment and molecular characterization by generating comparative data in a multicenter ring experiment. The results will be used to improve SOPs for the analysis of patient blood samples, which represents a promising tool to monitor disease progression and/or therapeutic response. Support: IMI JU & EFPIA (grand no. 115749).

Ring trial validation of single and multiplex real-time PCR methods for the detection and quantification of the allergenic food ingredients mustard, celery, soy, wheat and rye

We describe an interlaboratory ring trial validation for quantification methods of 3 food allergens using real-time PCR: a real-time PCR method for the joint simultaneous detection and quantification of brown and black mustard, a real-time PCR method for the detection and quantification of wheat and rye, and a multiplex real-time PCR method combining the detection and quantification of brown/black mustard, white mustard, celery and soybean. Mixtures with defined copy numbers of the target sequences from brown mustard, white mustard, celery, soybean and wheat were applied as calibrants for the real-time PCR-based quantification. Boiled sausages incurred with these allergenic ingredients in the range of 0–100 mg/kg were used as ring trial samples. Within the ring trial, we demonstrated that 40 mg/kg of the respective allergens (wheat/rye 80 mg/kg) can reliably be detected and quantified by all PCR systems even in boiled sausage meat. Brown/black mustard was detectable at the lowest concentration level of 10 mg/kg even after autoclaving. Results for brown/black mustard in terms of sensitivity, trueness and precision were comparable in single- and multiplex PCR. The results of the study revealed the importance of matrix-matched control samples for allergen analysis in terms of both conversion to mg/kg and of normalization variations caused by different extraction procedures.

Inter-laboratory study to characterize the detection of serum antibodies against porcine epidemic diarrhoea virus

Porcine epidemic diarrhea virus (PEDV) has caused extensive economic losses to pig producers in many countries. It was recently introduced, for the first time, into North America and outbreaks have occurred again in multiple countries within Europe as well. To assess the properties of various diagnostic assays for the detection of PEDV infection, multiple panels of porcine sera have been shared and tested for the presence of antibodies against PEDV in an inter-laboratory ring trial. Different laboratories have used a variety of “in house” ELISAs and also one commercial assay. The sensitivity and specificity of each assay has been estimated using a Bayesian analysis applied to the ring trial results obtained with the different assays in the absence of a gold standard. Although different characteristics were found, it can be concluded that each of the assays used can detect infection of pigs at a herd level by either the early European strains of PEDV or the recently circulating strains (INDEL and non-INDEL). However, not all the assays seem suitable for demonstrating freedom from disease in a country. The results from individual animals, especially when the infection has occurred within an experimental situation, show more variation.

Inter-Laboratory Robustness of Next-Generation Bile Acid Study in Mice and Humans: International Ring Trial Involving 12 Laboratories.

The increasing relevance of individual bile acids quantification in biological samples requires analytical standardization to guarantee robustness and reliability of laboratory results. We have organized the first international ring trial, carried out in 12 laboratories, to evaluate the newly developed LC-MS/MS-based test kit for bile acid analysis. Each laboratory received a Biocrates® Bile Acids Kit including system suitability test (SST) protocol. The kit is designed to analyze 16 individual human and 19 mouse bile acids. A set of 9 human and mouse plasma samples was measured in replicates. Laboratories were first required to pass the acceptance criteria for the SST. Within the subset of laboratories passing SST criteria, we evaluated how many laboratories met the target criteria of 80% of reported values with a relative accuracy within the 70%-130% range and analytical precisions (%CV) below 30%. A total of 12 of 16 participating laboratories passed the SST as the prerequisite to enter the ring trial. All 12 laboratories were then able to successfully run the kit and ring trial samples. Of the overall reported values, 94% were within 70%-130% relative accuracy range. Mean precision was 8.3% CV. The condition of CV <30% was fulfilled by 99% of the reported values. The first publically available interlaboratory ring trial for standardized bile acids quantification in human and mouse plasma samples showed very good analytical performance, within acceptance criteria typically applied in the preclinical environment. The kit is therefore suitable for standardized quantitative bile acid analysis and the establishment of reference values.

Standardization of allergen products: 3. Validation of candidate European Pharmacopoeia standard methods for quantification of major birch allergen Bet v 1.

The BSP090 project aims at establishing European Pharmacopoeia Reference Substances in combination with the corresponding ELISA methods for the quantification of major allergens in allergen products. Two sandwich ELISAs proved suitable for quantification of Bet v 1, the major birch pollen allergen, in preceding phases of BSP090. Two Bet v 1-specific ELISA systems were compared with respect to accuracy and precision in a ring trial including 13 laboratories. Model samples containing recombinant rBet v 1.0101 as well as native birch pollen extracts were measured independently at least three times in each facility. The assessment was completed with a comparative quantification of Bet v 1 in 30 marketed birch allergen products in one laboratory, simulating the future use as reference method. In the collaborative study, both candidate ELISAs confirmed their suitability to quantify recombinant and native Bet v 1. ELISA-A showed higher precision and lower interlaboratory variability, yet ELISA-B exhibited slightly higher accuracy. Subsequent parallel measurement of Bet v 1 in a panel of 'real-life' birch allergen products indicated better repeatability of ELISA-B. Both systems detected substantial differences in Bet v 1 content between allergen products, but the effect was more pronounced using ELISA-B due to persistently higher values compared to ELISA-A. In the collaborative study, no deciding differences were observed between the two candidate ELISAs. Further comparison under conditions simulating the intended use combined with the criterion of long-term availability enabled the selection of one Bet v 1-specific ELISA for proposal as European Pharmacopoeia standard method.

Interlaboratory ring trial to evaluate CFT proficiency of European laboratories for diagnosis of glanders in equids

To evaluate the routine complement fixation test (CFT) used to detect Burkholderia mallei antibodies in equine sera, an interlaboratory proficiency test was held with 24 European laboratories, including 22 National...

Intra- and inter-laboratory validation of an innovative huFcεRIα-RBL-2H3 degranulation assay for in vitro allergenicity assessment of whey hydrolysates

Cow's milk-derived whey hydrolysates are milk substitutes for cow's milk allergic infants. Safety assessment of these hydrolysates is crucial. Currently, huFcεRIα-RBL-2H3 cells, sensitized with serum IgE from cow's milk allergic patients, are used to assess in vitro residual allergenicity. However, limited availability and high inter-lot variation of sera impede the standardization of safety testing. Recently, we generated an oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (BLG) as an alternative for human serum. These antibodies demonstrated increased sensitivity, specificity and reproducibility. An inter-laboratory ring trial using our new degranulation assay with different whey-based hydrolysates was performed at four independent laboratories to investigate the robustness and reproducibility. RBL-2H3 cells expressing huFcεRIα were sensitized with our oligoclonal pool of anti-BLG chuIgE antibodies. The cells were subsequently incubated with an amino-acid based formula (AAF), two extensively hydrolyzed formulas (eHF) and three partially hydrolyzed formulas (pHF) to assess the degranulation upon challenge. Results demonstrated a very strong inter-laboratory correlation and the intra- and inter-laboratory variations were acceptable. The AAF and both eHFs showed no degranulation, whereas all pHFs demonstrated degranulation. The study showed that this degranulation assay is robust and reproducible within and between laboratories. This new in vitro degranulation assay seems predictive for allergenicity outcome and might therefore be considered as a relevant substitute for animal models.

Abstract P5-07-02: Systematic analysis and modulation of Ki67 interobserver variance in 9069 patients from three clinical trials – How much pathologist concordance is needed for meaningful biomarker results?

Abstract Background: Ki67 has been suggested as a marker for diagnosis of luminal A and B breast carcinomas. Interestingly, on one hand a multitude of studies have described significant results for Ki67 as a prognostic marker, while on the other hand the analytical validation and standardization of this marker has been a challenge. The best parameter for Ki67 interobserver performance is the interclass correlation coefficient (ICC). ICC values between 0.59 and 0.92 have been reported. Recently a minimum ICC of 0.8 has been suggested as a goal for the international ring trial and as a prerequisite for introduction of Ki67 into clinical practice. However, this suggested ICC is not derived from analysis of data, and the amount of pathologist variance that is allowed for meaningful biomarker results is still not defined. Methods: This study is based on a total of 9069 tumor samples from three large clinical cohorts (IBCSG VIII+IX, BIG1-98, and GeparTrio). In a systematic modeling approach, we introduced different amounts of variance to previously generated central pathology Ki67 datasets by simulation of a total of 1800 different pathologist evaluations for each study cohort. These evaluations were grouped into groups with defined ICCs, ranging from very good concordance (ICC=0.9) to extremely poor concordance (ICC=0.1). For each of the simulated pathologist evaluations, all possible Ki67 cutoffs were systematically evaluated using the web-based software Cutoff Finder (http://molpath.charite.de/cutoff/). As endpoints, we used DFS for all three study cohorts as well as pCR for the neoadjuvant cohort. Results: For the neoadjuvant GeparTrio study, the different groups with ICCs of 0.8, 0.6 and 0.4 showed a very similar performance resulting in significant analyses for prediction of pCR across a wide range of cutoffs. The odd ratios for pCR were slightly lower with lower ICC. Even with an extremely low ICC of 0.2, 99% of the analyses had one or more significant cutpoints. The survival endpoint DFS was shown to be very stable despite increased interpathologist variance in all three clinical cohorts. Even with a poor ICC of 0.4, the majority of cutpoints were significant for DFS. For IBCSG VIII+IX 85% of the analyses with an ICC of 0.4 had one or more significant cutpoints for Ki67. In the large BIG 1-98 dataset (n=6090) even an ICC of 0.2 resulted in one or more significant DFS cutpoints in 100% of the analyses. Comparable results were obtained if the analysis was restricted to luminal tumors. Conclusion: Our results suggest that Ki67 is extremely robust to pathologist variation. Even if less than 40% of the variance is attributable to true Ki67-based proliferation (ICC&amp;lt;0.4), this percentage of information is sufficient to obtain statistically significant differences. This stable performance of Ki67 might provide an explanation for the observation that many Ki67 studies achieve significant results despite the interobserver variance and heterogeneity issues. It might also suggest a relevant clinical utility for Ki67 despite considerable variation introduced in the evaluation. Ongoing efforts to further reduce interobserver variability, however, should be continued. Citation Format: Denkert C, Budczies J, Regan M, Loibl S, Dell'Orto P, von Minckwitz G, Mastropasqua M, Mehta K, Müller V, Kammler R, Pfitzner BM, Fasching PA, Viale G. Systematic analysis and modulation of Ki67 interobserver variance in 9069 patients from three clinical trials – How much pathologist concordance is needed for meaningful biomarker results?. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-07-02.

STANDARDIZATION OF FLOW CYTOMETRIC MINIMAL RESIDUAL DISEASE MONITORING IN CHILDREN WITH B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA. RUSSIA–BELARUS MULTICENTER GROUP EXPERIENCE

We developed and implemented in multicenter setting the standardized approach for flow cytometric minimal residual disease (MRD) monitoring in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Participation of multicenter group reference laboratories in several ring trial studies demonstrated high concordance rate between participants. Successful integration of one additional laboratory in the multicenter group has shown good level of our approach reproducibility. These results will allow implementing MRD detection in stratification system of pediatric ALL treatment protocols of Russia-Belarus multicenter group.

First External Quality Assessment of Molecular and Serological Detection of Rift Valley Fever in the Western Mediterranean Region.

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis which affects humans and a wide range of domestic and wild ruminants. The large spread of RVF in Africa and its potential to emerge beyond its geographic range requires the development of surveillance strategies to promptly detect the disease outbreaks in order to implement efficient control measures, which could prevent the widespread of the virus to humans. The Animal Health Mediterranean Network (REMESA) linking some Northern African countries as Algeria, Egypt, Libya, Mauritania, Morocco, Tunisia with Southern European ones as France, Italy, Portugal and Spain aims at improving the animal health in the Western Mediterranean Region since 2009. In this context, a first assessment of the diagnostic capacities of the laboratories involved in the RVF surveillance was performed. The first proficiency testing (external quality assessment—EQA) for the detection of the viral genome and antibodies of RVF virus (RVFV) was carried out from October 2013 to February 2014. Ten laboratories participated from 6 different countries (4 from North Africa and 2 from Europe). Six laboratories participated in the ring trial for both viral RNA and antibodies detection methods, while four laboratories participated exclusively in the antibodies detection ring trial. For the EQA targeting the viral RNA detection methods 5 out of 6 laboratories reported 100% of correct results. One laboratory misidentified 2 positive samples as negative and 3 positive samples as doubtful indicating a need for corrective actions. For the EQA targeting IgG and IgM antibodies methods 9 out of the 10 laboratories reported 100% of correct results, whilst one laboratory reported all correct results except one false-positive. These two ring trials provide evidence that most of the participating laboratories are capable to detect RVF antibodies and viral RNA thus recognizing RVF infection in affected ruminants with the diagnostic methods currently available.

KINFix--A formalin-free non-commercial fixative optimized for histological, immunohistochemical and molecular analyses of neurosurgical tissue specimens.

An optimal fixative should ideally combine the advantages of formalin fixation and freezing, allowing for good preservation of histology and molecular components, easy handling and storage, lack of toxicity, and low costs. Most of these criteria are fulfilled by ethanol-based solutions, and due to our good experience with the commercial RCL2 fixative, reflected by our published single-center trial, we initiated a multicenter ring trial. However, during its course, RCL2 was discontinued on the market. Therefore, we created our own agent, KINFix, composed of the same main constituents as RCL2, and employed it in our laboratory with similar results. Here we present our evaluation of the three fixatives formalin, RCL2, and KINFix from the perspective of histopathology as well as nucleic acid and protein analyses in comparison to fresh frozen tissues together with the multicenter ring trial data for RCL2. We observe that RCL2 and KINFix offer comparable histomorphology and superior template for molecular analyses than formalin. Moreover, KINFix as freely available fixative might overcome some of the difficulties related to the commercial agents. Therefore, we conclude that KINFix might be an attractive complement to formalin in tissue processing and advocate its use in neuropathological practice.