A multi-centre study for standardization of antinuclear antibody indirect immunofluorescence screening with automated system.

INTRODUCTION: ndirect immunofluorescence assay (IFA) using HEp-2 as substrate plays a consolidate role for the detection and measurement of ANA, which is currently considered as the reference method for detection. Manual operation is still very common in China, therefore, the need of standardization and automation for ANA-IFA detecting has been highlighted. OBJECTIVE: The current multi-center study is aimed to evaluate if HELIOS (AESKU Diagnostics, Wendelsheim, Germany) contributes to comparability of ANA screening results among different labs，and establish application specification of HELIOS for standardization of ANA detection. METHODS: ANA detection by manual IFA method and HELIOS on 230 clinical serum samples in eight laboratories. The performance to discriminate positive/negative screening results, endpoint titer estimation and pattern recognition were evaluated in HELIOS and manual visual. RESULTS: The positive coincident rate for ANA detection by manual IFA ranges from 87.7% to 97.8%, the negative coincidence rate ranges from 68.8% to 100%, the correctly estimated titer evaluation were 80 to 171 cases, the correct pattern in 146 to 161 cases, respectively. The positive coincident rate of HELIOS for ANA detection ranges from 91.2% to 97.7%, the negative coincidence rate ranges from 96.5% to 100%, the correctly estimated titer evaluation were 145 to 157 cases, the correct pattern in 123 to 140 cases, respectively. CONCLUSION: HELIOS could provide accurate diagnostic results, this include not only positive/negative results, but also endpoint titer, common patterns. The application of this system can help to promote standardization of ANA detection.

The direct and indirect effects of the pneumococcal conjugated vaccine on carriage rates in children aged younger than 5 years in Latin America and the Caribbean: a systematic review / Efeitos diretos e indiretos da vacina pneumocócica conjugada no estado de portador em crianças menores de 5 anos, na América Latina e no Caribe: uma revisão sistemática

ABSTRACT Objective To demonstrate the impact of pneumococcal conjugate vaccine in Streptococcus pneumoniae carriage status in children younger than 5 years in Latin America and the Caribbean. Methods A systematic literature review was carried out on the direct and indirect effects of pneumococcal vaccine in the carriage status, after implementation in childhood immunization programs. Studies carried out in children younger than 5 years were selected from the PubMed® and Virtual Health Library databases, and data collected after implementation of pneumococcal vaccine in Latin America and the Caribbean, between 2008 and 2018. Results From 1,396 articles identified, 738 were selected based on titles and abstracts. After duplicate removal, 31 studies were eligible for full-text reading, resulting in 6 publications for analysis. All selected publications were observational studies and indicated a decrease in the carriage and vaccine types, and an increase in the circulation of non-vaccine serotypes, such as 6A, 19A, 35B, 21 and 38. We did not identify changes in the antimicrobial resistance after vaccine implementation. Conclusion A decrease in the carriage status of vaccine types and non-vaccine types was detected. The continuous monitoring of pneumococcal vaccine effect is fundamental to demonstrate the impact of the carriage status and, consequently, of invasive pneumococcal disease, allowing better targeting approaches in countries that included pneumococcal vaccine in their immunization programs. Our study protocol was registered in PROSPERO (www.crd.york.ac.uk/prospero) under number CRD42018096719.

RESUMO Objetivo Demonstrar o impacto das vacinas pneumocócicas conjugadas no estado de portador de Streptococcus pneumoniae em crianças menores de 5 anos na América Latina e no Caribe. Métodos Foi realizada revisão sistemática da literatura sobre os efeitos diretos e indiretos da vacina pneumocócica no estado de portador em crianças menores de 5 anos, após a implantação da vacina nos calendários de imunização infantil. A partir de dados da PubMed®e da Biblioteca Virtual da Saúde, foram selecionados estudos de portador em crianças menores de 5 anos, com dados coletados após implementação da vacina de 2008 a 2018, na América Latina e no Caribe. Resultados Dos 1.396 artigos identificados, 738 foram selecionados mediante leitura de títulos e resumos. Após a extração dos duplicados, 31 foram elegíveis para leitura do texto completo, restando 6 artigos para análise. Todos os estudos selecionados eram observacionais e indicavam diminuição do portador e tipos vacinais, e aumento da circulação de sorotipos não vacinais, como 6A, 19A, 35B, 21 e 38. Não foi observada alteração na resistência antimicrobiana após a introdução da vacina. Conclusão Detectou-se redução no estado de portador, dos tipos vacinais e não vacinais. O monitoramento contínuo do efeito das vacinas pneumocócicas é fundamental, para demonstrar o impacto do estado de portador e, consequentemente, da doença pneumocócica invasiva, permitindo o melhor direcionamento nas ações em saúde para os países que incluíram a vacina no calendário de imunização. Nosso protocolo de estudo foi registrado no PROSPERO (www.crd.york.ac.uk/prospero) sob o número CRD42018096719.

Performance evaluation of an indirect immunofluorescence kit for the serological diagnosis of dengue / Avaliação do desempenho de kit de imunofluorescência indireta para o diagnóstico sorológico de dengue

ABSTRACT Objective: To evaluate the performance of indirect immunofluorescence for serological diagnosis of dengue virus in a population with high prevalence of arboviruses. Methods: Two-hundred serum samples from patients with clinical suspicion of dengue fever were tested by immunoenzymatic and indirect immunofluorescence assay BIOCHIP® mosaic. Specificity, sensitivity and Kappa coefficient were calculated. Discordant samples were tested by polymerase chain reaction for confirmation. Results: Of the 200 samples, 20% were positive and 80% negative for anti-dengue virus IgM antibodies in the immunoenzymatic test. Of the 40 positives, 25% were negative in indirect immunofluorescence. Of these ten discordant results, only 20% were also negative in the polymerase chain reaction (PCR). Of the 160 negatives in the immunoenzymatic test, 5% were positive in indirect immunofluorescence. Of these nine discordant results, 33% were positive in the PCR. The Kappa coefficient was 0.7 (0.572-0.829). Sensitivity and specificity of indirect immunofluorescence were respectively 75% and 94%. For anti-dengue virus IgG antibodies, of the 200 samples, 15.5% were positive and 84.5% were negative in the immunoenzymatic test. Of the 31 positives, 12.9% were negative in indirect immunofluorescence. Of these four discordant results, 25% were negative in the PCR. Of the 169 negatives, 8% were positive in indirect immunofluorescence. Of these 14 discordant results, 64% were also positive in the PCR. The Kappa coefficient was 0.695 (0.563-0.83). Sensitivity and specificity of indirect immunofluorescence were 87.1% and 91.7%, respectively. Conclusion: For diagnosis of acute infection, the immunoenzymatic test is enough, and the use of additional methods is not warranted. Replacing the immunoenzymatic test by indirect immunofluorescence would compromise the sensitivity for IgM. However, indirect immunofluorescence can distinguish three arboviruses simultaneously, an advantage during concomitant epidemics.

RESUMO Objetivo: Avaliar o desempenho da imunofluorescência indireta no diagnóstico sorológico de dengue em uma população com alta prevalência de arboviroses. Métodos: Duzentas amostras de soro de pacientes com suspeita clínica de dengue foram testadas por ensaio imunoenzimático e imunofluorescência indireta mosaico BIOCHIP®. Foram calculados especificidade, sensibilidade e coeficiente Kappa. Nas amostras discordantes, realizou-se reação em cadeia da polimerase como método confirmatório. Resultados: Das 200 amostras, 20% foram positivas e 80% negativas para IgM antivírus da dengue no ensaio imunoenzimático. Das 40 positivas, 25% foram negativas na imunofluorescência indireta. Destas dez negativas, apenas 20% eram também negativas na reação em cadeia da polimerase. Das 160 negativas no ensaio imunoenzimático, 5% foram positivas na imunofluorescência indireta. Por fim, dentre as nove discordantes, 33% tiveram vírus da dengue detectado na reação em cadeia da polimerase. O coeficiente Kappa foi 0,70 (0,57-0,82). Sensibilidade e especificidade por imunofluorescência indireta foram, respectivamente, 75% e 94%. Para IgG antivírus da dengue, de 200 amostras, 15,5% foram positivas e 84,5% negativas no ensaio imunoenzimático. Das 31 positivas, 12,9% foram negativas na imunofluorescência indireta. Destas quatro discordantes, 25% apresentaram vírus da dengue não detectado na reação em cadeia da polimerase. Das 169 negativas, 8% foram positivas na imunofluorescência indireta. Destas, 64% foram positivas também na reação em cadeia da polimerase. O coeficiente Kappa foi 0,695 (0,56-0,83). Sensibilidade e a especificidade por imunofluorescência indireta foram, respectivamente, 87,1% e 91,7%. Conclusão: Ensaio imunoenzimático seria suficiente para diagnóstico sorológico de infecção aguda, não justificando a incorporação da imunofluorescência indireta. Substituir ensaio imunoenzimático pela imunofluorescência indireta poderia comprometer a sensibilidade para IgM. Contudo, a imunofluorescência indireta auxilia diferenciar três arboviroses simultaneamente, sendo vantajoso em epidemias concomitantes.

Performance Evaluation of a Prototype Architect Antibody Assay for Babesia microti.

The tick-borne protozoan Babesia microti is responsible for more than 200 cases of transfusion-transmitted babesiosis (TTB) infection in the United States that have occurred over the last 30 years. Measures to mitigate the risk of TTB include nucleic acid testing (NAT) and B. microti antibody testing. A fully automated prototype B. microti antibody test was developed on the Architect instrument. The specificity was determined to be 99.98% in volunteer blood donors (n = 28,740) from areas considered to have low endemicity for B. microti The sensitivity of the prototype test was studied in experimentally infected macaques; a total of 128 samples were detected as positive whereas 125 were detected as positive with an indirect fluorescent antibody (IFA) test; additionally, 83 (89.2%) of the PCR-positive samples were detected in contrast to 81 (87.1%) using an IFA test. All PCR-positive samples that tested negative in the prototype antibody test were preseroconversion period samples. Following seroconversion, periods of intermittent parasitemia occurred; 17 PCR-negative samples drawn in between PCR-positive bleed dates tested positive both by the prototype test (robust reactivity) and IFA test (marginal reactivity) prior to the administration of therapeutic drugs, indicating that the PCR test failed to detect samples from persistently infected macaques. The prototype assay detected 56 of 58 (96.6%) human subjects diagnosed with clinical babesiosis by both PCR and IFA testing. Overall, the prototype anti-Babesia assay provides a highly sensitive and specific test for the diagnosis of B. microti infection. While PCR is preferred for detection of window-period parasitemia, antibody tests detect infected subjects during periods of low-level parasitemia.

Increasing the accuracy and scalability of the Immunofluorescence Assay for Epstein Barr Virus by inferring continuous titers from a single sample dilution.

High Epstein Barr Virus (EBV) titers detected by the indirect Immunofluorescence Assay (IFA) are a reliable predictor of Nasopharyngeal Carcinoma (NPC). Despite being the gold standard for serological detection of NPC, the IFA is limited by scaling bottlenecks. Specifically, 5 serial dilutions of each patient sample must be prepared and visually matched by an evaluator to one of 5 discrete titers. Here, we describe a simple method for inferring continuous EBV titers from IFA images acquired from NPC-positive patient sera using only a single sample dilution. In the first part of our study, 2 blinded evaluators used a set of reference titer standards to perform independent re-evaluations of historical samples with known titers. Besides exhibiting high inter-evaluator agreement, both evaluators were also in high concordance with historical titers, thus validating the accuracy of the reference titer standards. In the second part of the study, the reference titer standards were IFA-processed and assigned an 'EBV Score' using image analysis. A log-linear relationship between titers and EBV Score was observed. This relationship was preserved even when images were acquired and analyzed 3days post-IFA. We conclude that image analysis of IFA-processed samples can be used to infer a continuous EBV titer with just a single dilution of NPC-positive patient sera. This work opens new possibilities for improving the accuracy and scalability of IFA in the context of clinical screening.

Antineutrophil cytoplasmic antibody (ANCA) testing: Audit from a clinical immunology laboratory.

AIM: Anti-neutrophil cytoplasmic antibodies (ANCA) are associated with small vessel vasculitis now termed 'ANCA associated vasculitis' (AAV). ANCAs are reported in diverse diseases where they have no clinical utility. We carried out an audit in a clinical immunology laboratory and assessed if use of ordering practices could have improved utility of ANCA. METHODS: All samples received for ANCA testing during 2014 were tested by indirect immunofluorescence (IIF) and automated enzyme-linked immunosorbent assay (ELISA). Clinical records of all samples positive by one or more assays were retrieved. We assessed the effect of applying proposed test ordering guidelines on performance of the tests. RESULTS: Of 1590 samples, 108 (6.8%) had a positive result by at least one method. IIF showed perinuclear pattern in 72 (21 were antinuclear antibody positive), cytoplasmic in 22, six had atypical pattern and eight were negative. By ELISA anti-myeloperoxidase antibodies were present in 33 samples, anti-proteinase 3 in 24, while five sera had both antibodies. ELISA and IIF were concordant in 45 samples. Twenty-seven patients had AAV of which 23 were both ELISA and IIF positive. Among these 27 with AAV all had at least one ordering criteria, while in 81 patients without AAV but with positive test, 38 had no ordering criteria. CONCLUSION: Reduction in false positive can be achieved by considering only those samples as ANCA positive that test positive both on IIF and ELISA and by following ordering guidelines before requesting ANCA testing, and by use of ordering criteria by clinicians.

A historical essay on detection of anti-neutrophil cytoplasmic antibodies.

In this essay we describe a number of the known and not so known experiences of the early anti-neutrophil cytoplasmic antibodies (ANCAs) days, explaining why and how we reached consensus on the standard indirect immunofluorescence (IIF) techniques, the naming of the two principal C- and P-ANCA patterns, why we chose to use IIF as the standard technique, how the solid phase assays have developed and where we stand today, the use of ANCA for diagnosis and the importance of using several techniques for that purpose, how ANCA titres are related to disease activity and the clinical impact of this, and finally the implications of ANCA being a natural, polyclonal antibody response against various epitopes in relation to diagnostics and disease patterns.

Evaluation of indirect immunofluorescence antibody test and enzyme-linked immunosorbent assay for the diagnosis of infection by Leishmania infantum in clinically normal and sick cats.

Cats that live in areas where canine and human leishmaniosis due to Leishmania infantum is endemic may become infected and may develop anti-Leishmania antibodies. In this study 50 clinically normal and 50 cats with cutaneous and/or systemic signs that lived in an endemic area and had been previously examined for infection by L. infantum using PCR in four different tissues were serologically tested for the presence of anti-Leishmania IgG (IFAT and ELISA) and IgM (IFAT). The aim was to compare the results of IFAT, ELISA and PCR and to investigate the possible associations between seropositivity to Leishmania spp and signalment, living conditions, season of sampling, health status of the cats, and seropositivity to other infectious agents. Low concentrations of anti-Leishmania IgG were detected by IFAT in 10% of the cats and by ELISA in 1%, whereas anti-Leishmania IgM were detected by IFAT in 1%. There was disagreement between the results of IFAT and ELISA for anti-Leishmania IgG (P = 0.039) and between all serological tests and PCR (P < 0.001). The diagnostic sensitivity of all serological tests, using PCR as the gold standard, was very low, but ELISA and IFAT for anti-Leishmania IgM had 100% specificity. The diagnostic sensitivity of all serological tests could not be improved by changing the cut-off values. Seropositivity for Leishmania spp was not associated with signalment, living conditions, season of sampling and health status of the cats or with seropositivity to feline leukemia virus, feline immunodeficiency virus, feline coronavirus, Toxoplasma gondii and Bartonella henselae. In conclusion, because of their low sensitivity and very high specificity two of the evaluated serological tests (ELISA for anti-Leishmania IgG and IFAT for anti-Leishmania IgM) may be useless as population screening tests but valuable for diagnosing feline infection by L. infantum.

Benchmarking HEp-2 cells classification methods.

In this paper, we report on the first edition of the HEp-2 Cells Classification contest, held at the 2012 edition of the International Conference on Pattern Recognition, and focused on indirect immunofluorescence (IIF) image analysis. The IIF methodology is used to detect autoimmune diseases by searching for antibodies in the patient serum but, unfortunately, it is still a subjective method that depends too heavily on the experience and expertise of the physician. This has been the motivation behind the recent initial developments of computer aided diagnosis systems in this field. The contest aimed to bring together researchers interested in the performance evaluation of algorithms for IIF image analysis: 28 different recognition systems able to automatically recognize the staining pattern of cells within IIF images were tested on the same undisclosed dataset. In particular, the dataset takes into account the six staining patterns that occur most frequently in the daily diagnostic practice: centromere, nucleolar, homogeneous, fine speckled, coarse speckled, and cytoplasmic. In the paper, we briefly describe all the submitted methods, analyze the obtained results, and discuss the design choices conditioning the performance of each method.

Anti-NMDA-receptor antibody encephalitis: performance evaluation and laboratory experience with the anti-NMDA-receptor IgG assay.

BACKGROUND: Antibodies targeting the NR1 subunit of the N-methyl-d-aspartate-receptor (NMDAR) are considered diagnostic for a novel form of autoimmune encephalitis. We report the validation of a qualitative indirect immunofluorescence antibody (IFA) test for the detection of anti-NMDAR IgG and describe the attributes of antibody-positive patients. METHODS: The anti-NMDAR IgG assay (Euroimmun Diagnosika, Lübeck, Germany) was validated with serum and cerebrospinal fluid (CSF) specimens from 30 healthy and 50 disease controls as well as 5 anti-NMDAR IgG-positive individuals. Consecutive specimens (n=1671) for anti-NMDAR IgG antibodies were evaluated and positive specimens titrated to end-point [starting dilutions: CSF; 1:1 and serum; 1:10]. In a subset of antibody-positive patients, we sought clinical information for correlation with diagnostic and treatment outcomes. RESULTS: The assay demonstrated excellent performance characteristics in all groups evaluated. Of the 1671 specimens tested, 1389 were unique cases with a positivity rate of 9.0% (n=123). For the antibody-positive samples, the female to male ratio was 2:1 with a prevalence of 46% in the pediatric population (≤17 years). Antibody titers were titrated to end-point for 106/123 specimens [45 CSF, 41 sera, and 20 CSF and serum pairs] with more than 75% having titers greater than 1:10 (CSF) and 1:20 (serum). Overall, high levels of these antibodies showed correlation to disease severity with variable response to treatment in the subset of patients evaluated. CONCLUSION: Our data suggests a high prevalence for anti-NMDAR antibody encephalitis irrespective of age and gender in our unselected disease cohort with support for measuring antibody titers in the evaluation of these patients.

Automated indirect immunofluorescence evaluation of antinuclear autoantibodies on HEp-2 cells.

Indirect immunofluorescence (IIF) on human epithelial (HEp-2) cells is considered as the gold standard screening method for the detection of antinuclear autoantibodies (ANA). However, in terms of automation and standardization, it has not been able to keep pace with most other analytical techniques used in diagnostic laboratories. Although there are already some automation solutions for IIF incubation in the market, the automation of result evaluation is still in its infancy. Therefore, the EUROPattern Suite has been developed as a comprehensive automated processing and interpretation system for standardized and efficient ANA detection by HEp-2 cell-based IIF. In this study, the automated pattern recognition was compared to conventional visual interpretation in a total of 351 sera. In the discrimination of positive from negative samples, concordant results between visual and automated evaluation were obtained for 349 sera (99.4%, kappa = 0.984). The system missed out none of the 272 antibody-positive samples and identified 77 out of 79 visually negative samples (analytical sensitivity/specificity: 100%/97.5%). Moreover, 94.0% of all main antibody patterns were recognized correctly by the software. Owing to its performance characteristics, EUROPattern enables fast, objective, and economic IIF ANA analysis and has the potential to reduce intra- and interlaboratory variability.

Primer Consenso Argentino para la Estandarización de la Determinación de Anticuerpos Anti-Nucleares por Inmunof uorescencia Indirecta-HEp-2 / First Argentine Consensus for Standardization of Antinuclear Antibodies by Indirect Immunofluorescence-HEp-2 / Primeiro Consenso Argentino para a Padronizagáo da Determinagáo de Anticorpos Antinucleares por Imunofluorescéncia Indireta-HEp-2

La presencia de anticuerpos antinucleares (AAN) es el denominador común de muchas enfermedades autoinmunes sistémicas y su significancia clínica depende de la metodología utilizada en su determinación. En la actualidad, la inmunofluorescencia indirecta (IFI) utilizando células HEp-2 como sustrato es la técnica más usada. Siendo un procedimiento subjetivo se deben optimizar los métodos de estandarización de las distintas variables involucradas, para asegurar la calidad de los resultados obtenidos. El uso de este sustrato permite la descripción no sólo de patrones de fluorescencia nucleares sino también citoplasmáticos y de diferentes organelas. El 29 de agosto de 2008 se llevó a cabo en Buenos Aires el Primer Consenso Argentino para la Estandarización de la Determinación de AAN por IFI-HEp-2, con la participación de 28 expertos. Se discutieron los aspectos metodológicos más importantes y se decidió llamar a la determinación "anticuerpos anti núcleo-citoplasmáticos". Se consensuó la sigla representativa de la determinación, el nombre en español de los diferentes patrones y el uso de controles de calidad internos y externos. La unificación de criterios llevará a la optimización de los resultados y a su correcta interpretación.

Antinuclear antibodies (ANA) are a common feature of many systemic autoimmune diseases. Their clinical significance depends on working conditions. Nowadays, indirect immunofluorescence (IFI), using HEp-2 cells as substrate, is the most used assay. Since IFI is a subjective method, different variables should be strictly standardized to assure the quality of the results. By using HEp-2 cells, not only a nuclear fluorescent pattern will be described but also cytoplasmic and different organelle patterns. In order to standardize the operative procedures, the 1st Argentine Consensus for AAN IFI HEp-2 determination took place in Buenos Aires on August 29, 2008, with 28 experts as participants. The most important methodological aspects were discussed and the assay was decided to be named: anti nuclear- cytoplasmic antibodies. Consesus was also CAICYThed about the acronym for the determination, the Spanish names for the different patterns and the use of internal and external quality controls. Using common criteria will improve the quality of the results and optimize assay interpretation.

A presenga de anticorpos antinucleares (AAN) é o denominador comum de muitas doengas autoimunes sistémicas e sua significancia clínica depende da metodologia utilizada em sua determinagáo. Na atualidade, a imunofluorescéncia indireta (IFI) utilizando células HEp-2 como substrato é a técnica mais utilizada. Sendo um procedimento subjetivo devem ser otimizados os métodos de padronizagáo das diferentes variáveis envolvidas, para garantir a qualidade dos resultados obtidos. O uso deste substrato permite a descrigáo náo apenas de padróes de fluorescéncia nucleares mas também citoplasmáticos e de diferentes organelas. Em 29 de agosto de 2008, foi levado a cabo, em Buenos Aires, o Primeiro Consenso Argentino para a Padronizagáo da Determinagáo de AAN por IFI-HEp-2, com a participagáo de 28 especialistas. Foram discutidos os aspectos metodológicos mais importantes e se decidiu chamar a determinagáo de "anticorpos antinúcleo-citoplasmáticos". Foi estabelecida por consenso a sigla representativa da determinagáo, o nome em espanhol dos diferentes padróes e o uso de controles de qualidade internos e externos. A unificagáo de critérios levará a otimizago dos resultados e a sua correta interpretagáo.

Novel immunofluorescence protocol for multimarker assessment of putative disseminating breast cancer stem cells.

PURPOSE: The phenotypical and functional variety of breast cancer cells is well recognized. This variety is evident in primary tumors and in disseminated tumor cells (DTCs) and solid metastases as shown for recognized prognostic factors, such as estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2/neu and also for cancer stem cell markers such as CD44, CD24, or aldehyde dehydrogenase (ALDH). For the development of new therapeutic strategies, the identification and characterization of disseminated breast cancer cells are needed. This requires the use of multiple antibodies (ie, cytokeratin, Her2/neu, ALDH1, CD44, and CD24) labeled with fluorochromes of different colors and spectral image analysis to separate different color spectra. METHODS: We have focused here on putative breast cancer stem cell markers and evaluated the feasibility of triple and quadruple labeling of breast cancer cells. Using breast cancer cell lines we have developed a method optimized for multimarker analysis by employing novel DyLight Technology. Single marker immunofluorescence was performed in 6 replicates, and reproducible results had to be obtained before proceeding to multimarker immunofluorescence. RESULTS: Three of the markers, CD44, ALDH1, and cytokeratin have been directly conjugated with DyLight dyes. CD24 could not be conjugated directly to the fluorescent dye. A labeled secondary antibody was used for visualization. Single and multimarker immunofluorescence gave consistent results throughout the replicates. CONCLUSIONS: This novel protocol will facilitate detection and phenotypical characterization of disseminated tumor cells. In addition, by adding additional markers, distinct subpopulations could be evaluated for the expression of particular therapeutic targets.

A Bayesian evaluation of two dip-stick assays for the on-site diagnosis of infection in calves suspected of clinical giardiasis.

The objective of the present study was to evaluate two commercially available dip-stick assays for the diagnosis of Giardia infections in faecal samples from calves suspected of clinical giardiasis. The dip-stick assays provide an on-site and hence quick alternative to laboratory diagnosis. A three-test Bayesian model was used, including the test results of the Coris Giardia strip (Coris Bioconcept, Gembloux, Belgium), the Speed Giardia or BVT dip-stick (Bio Veto Test/Virbac, La Seyne sur Mer, France), and the Meridian immunofluorescence assay (IFA: Meridian Diagnostics Inc., Cincinnati, OH). In total, 421 faecal samples were examined with the three diagnostic assays between October 2008 and November 2009. Overall, the number of positive samples was markedly higher using the IFA compared to both dip-stick assays, resulting in a high sensitivity (se: 88%, with a 95% probability interval (PI) 60-99%) compared to the Coris dip-stick assay (se: 28%; PI: 16-41%) and the BVT dip-stick assay (se: 26%, PI: 16-35). The specificities of all the three assays were very high (IFA sp: 94%, PI: 90-99%; Coris sp: 92%, PI 86-98%; BVT sp: 93%, PI 88-98%). A positive diagnosis by the dip-stick assays was significantly correlated with a higher cyst excretion level, as measured by IFA. The majority (76%) of the positive samples in the present study contained less than 5000cyst per gram of faeces, even though all these animals displayed clinical symptoms of diarrhea potentially due to Giardia. The low level of cyst excretion in these samples might in part explain the poor sensitivity of both dip-stick assays. Although multiple samplings might be an option to increase the sensitivity of the dip-stick assays, the laboratory based IFA seems at current to be the best option for clinical diagnosis of Giardia in calves.

Standardization of HER2 immunohistochemistry in breast cancer by automated quantitative analysis.

CONTEXT: There is critical need for standardization of HER2 immunohistochemistry testing in the clinical laboratory setting. Recently, the American Society of Clinical Oncology and the College of American Pathologists have submitted guidelines recommending that laboratories achieve 95% concordance between assays and observers for HER2 testing. OBJECTIVE: As a potential aid to pathologists for achieving these new guidelines, we have conducted an examination using automated quantitative analysis (AQUA analysis) to provide a standardized HER2 immunohistochemistry expression score across instruments (sites), operators, and staining runs. DESIGN: We analyzed HER2 expression by immunohistochemistry in a cohort (n = 669) of invasive breast cancers in tissue microarray format across different instruments (n = 3), operators (n = 3), and staining runs (n = 3). Using light source, instrument calibration techniques, and a new generation of image analysis software, we produced normalized AQUA scores for each parameter and examined their reproducibility. RESULTS: The average percent coefficients of variation across instruments, operators, and staining runs were 1.8%, 2.0%, and 5.1%, respectively. For positive/negative classification between parameters, concordance rates ranged from 94.5% to 99.3% for all cases. Differentially classified cases only occurred around the determined cut point, not over the entire distribution. CONCLUSIONS: These data demonstrate that AQUA analysis can provide a standardized HER2 immunohistochemistry test that can meet current guidelines by the American Society of Clinical Oncology/College of American Pathologists. The use of AQUA analysis could allow for standardized and objective HER2 testing in clinical laboratories.

Autoantibody detection using indirect immunofluorescence on HEp-2 cells.

The detection of autoantibodies is an important element in the diagnosis and monitoring of disease progression in patients with autoimmune diseases. In laboratory diagnostic tests for connective tissue and autoimmune liver diseases, indirect immunofluorescence on HEp-2 cells plays a central role in a multistage diagnostic process. Despite the high quality of diagnostics, findings at different laboratories can differ considerably due to a lack of standardization, as well as subjective factors. The present paper formulates recommendations for the standardized processing and interpretation of the HEp-2 cell test for the detection of non-organ-specific (especially antinuclear) antibodies. It provides requirements regarding the diagnostic tests used, instructions for laboratory procedure and evaluation, and recommendations for interpretation. For an optimal laboratory diagnostic process, it is useful to have an informative, tentative clinical diagnosis and an experienced laboratory diagnostician. In addition, the following key elements are recommended: initial screening using indirect immunofluorescence on carefully chosen HEp-2 cells beginning with a serum dilution of 1:80 and evaluation under a microscope with powerful illumination; results from a titer of 1:160 upwards being considered positive; internal laboratory quality control; and standardized interpretation. The aim is to improve diagnostic tests and care of patients with autoimmune diseases as a central concern of the European Autoimmunity Standardization Initiative (EASI).

Role of antineutrophil cytoplasmic antibodies and glomerular basement membrane antibodies in the diagnosis and monitoring of systemic vasculitides.

Systemic vasculitis, although rare, is often diagnosed late and long after the onset of symptoms. The small vessel vasculitides are recognized clinically by their multisystem presentation, markers of inflammation and evidence for an acute glomerulonephritis (GN), with the most apparent organ involved directing referral to secondary care. Routine laboratory tests are usually non-specific in systemic vasculitis but the use of anti-neutrophil cytoplasmic antibodies (ANCAs) and glomerular basement membrane (GBM) antibodies can aid diagnosis, treatment and monitoring decisions. These antibodies are detected and quantified by indirect immunofluorescence (IIF) and antigen-specific enzyme-linked immunosorbent assay (ELISA), usually in combination for ANCA, and ELISA systems (or direct IIF on kidney biopsy) for GBM antibodies. The presence or absence of ANCA does not confirm or exclude the diagnosis of systemic vasculitis but negative and positive predictive values will be strongly influenced by clinical presentation. Various large studies have been unable to conclude that following serial ANCA titres has great clinical utility in each case but each patient must be considered on its own merits; for example the reappearance of ANCA in a patient who was rendered ANCA negative following treatment is more likely to indicate relapse. The adoption of consensus guidelines that direct testing towards patients with rapidly progressive GN, pulmonary haemorrhage, persistent and destructive ear, nose and upper airways problems, such as subglottic tracheal stenosis, a retro-orbital mass and cutaneous vasculitis with systemic features or peripheral neuropathy, will greatly increase the clinical utility and positive predictive value of these tests.

Rapid development of polyclonal antisera against infectious salmon anaemia virus and its optimization and application as a diagnostic tool.

Infectious salmon anaemia is an important disease of Atlantic salmon. One of the current methods of diagnosis is the indirect fluorescent antibody test (IFAT), using a monoclonal antibody specific to the haemagglutinin of the virus. The conformationally dependent nature of this antibody could be a drawback in its usefulness in other tests. This study describes the development and optimization of a polyclonal antiserum against infectious salmon anaemia virus, including a method of separating virus from cell culture components within culture supernatant. The antiserum was subsequently optimized for use in a variety of immunological diagnostic tests, including IFAT and an alkaline phosphatase-based immunoassay, and Western blot.