Diagnostic Performance of Anti-dsDNA Tests by Indirect Immunofluorescence and Enzyme-linked Immunosorbent Assay in Patients with Systemic Lupus Erythematosus.

BACKGROUND: Several laboratory techniques for anti double-stranded (ds) DNA detection in systemic lupus erythematosus (SLE) are available, with variable diagnostic performance. We aimed to evaluate anti-dsDNA's diagnostic performance by indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (EIA). METHODS: We conducted a single-center retrospective (2015 to 2020) study. Patients with anti-dsDNA tests by IIF and EIA were included. We evaluated the indications, applications, concordance, positive predictive value (PPV) of anti-dsDNA to confirm SLE diagnosis or flares, and associations of disease manifestations with positivity with each technique. RESULTS: A total of 1368 reports of anti-dsDNA tests by IIF and EIA and the corresponding medical records of the patients were analyzed. The main indication for anti-dsDNA testing was to help in the diagnosis of SLE in 890 (65%) of the samples, and the main application after obtaining the results was SLE exclusion in 782 (57.2%) cases. The combination with the highest frequency was the negativity result by both techniques in 801 (58.5%) cases (Cohen kappa 0.57). Both methods were positive in 300 patients with SLE (Cohen kappa 0.42). The PPVs of anti-dsDNA tests to confirm diagnosis/flare was 79.64% (95% CI, 75.35-83.35) by EIA, 78.75% (95% CI, 74.27-82.62) by IIF, and 82% (95% CI, 77.26-85.93) when both were positive. CONCLUSIONS: Anti-dsDNA detection by IIF and EIA are complementary and may indicate different clinical patterns in patients with SLE. The detection of anti-dsDNA antibodies by both techniques has a higher PPV than either separately for confirming SLE diagnosis or flares. These results highlight the need for evaluating both methods in clinical practice.

Standardization and Quality Assessment Under the Perspective of Automated Computer-Assisted HEp-2 Immunofluorescence Assay Systems.

The recent availability of automated computer-assisted diagnosis (CAD) systems for the reading and interpretation of the anti-nuclear antibody (ANA) test performed with the indirect immunofluorescence (IIF) method on HEp-2 cells, has improved the reproducibility of the results and initiated a process of harmonization of this test. Furthermore, CAD systems provide quantitative expression of fluorescence intensity, allowing the introduction of objective quality control procedures to the monitoring of the entire process. The calibration of the reading systems and the automated image interpretation are essential prerequisites for obtaining reproducible and harmonized IIF test results and form the basis for standardization, regardless of the computer algorithms used in the different systems. The use of automated CAD systems, facilitating control procedures, represents a step forward for the quality certification of the laboratory.

[Autoimmunity accreditation: training, accreditation and maintenance of skills in indirect immunofluorescence]. / Accréditation en auto-immunité : formation, habilitation et maintien des compétences en immunofluorescence indirecte.

The ISO 15189 accreditation of biological analysis needs the validation of the analytical methods allowing the evaluation of their performance including all the factors that could influence the quality of their results. The field of autoimmunity includes many analyses and methods such as the indirect immunofluorescence technique (IIF) and the performance of this technique largely depends on the competency of staff members. For each staff member, the required levels of competency have to be precisely defined and evaluated after a period of formation before the final habilitation for the IIF technique. The French group of the international group called EASI (European autoimmunity standardisation initiative) proposes two habilitation forms to be filled with criteria, evidence and maintenance of target skills for the IIF preparation of slides and reading. These forms could be used as a model for the IIF formation and habilitation and have to be adapted to the routine practice of the laboratories.

Validation and field evaluation of a competitive inhibition ELISA based on the recombinant protein tSAG1 to detect anti-Neospora caninum antibodies in sheep and goats.

Neospora caninum is a protozoan parasite that causes abortion and reproductive failure in small ruminants. We validated and evaluated under field conditions a competitive inhibition ELISA based on the truncated SAG1 protein (tSAG1) from N. caninum for the detection of anti-N. caninum antibodies in sheep and goat flocks. The assay was validated using 80 positive and 142 negative serum samples from sheep and goats analyzed by IFAT and immunoblot (IB). ciELISAtSAG1 was then used to evaluate the prevalence of anti-N. caninum antibodies in 1449 goats from 143 flocks and 385 sheep from 40 flocks and compared to IFAT. The prevalence of anti-Toxoplasma gondii antibodies was evaluated by IFAT. The ciELISAtSAG1 cut-off was ≥ 36 percent inhibition, with a diagnostic sensitivity of 100.0 % (95 % CI = 95.4-100.0 %) and a diagnostic specificity of 98.6 % (95 % CI = 95.0-99.8 %) relative to the agreement between IFAT and IB. The field evaluation revealed a concordance between ciELISAtSAG1 and IFAT of 97.4 %, with an agreement (κ) of 0.90 for sheep sera, and a concordance of 96.5 % with κ = 0.85 for goat sera. The overall prevalence of anti-N. caninum antibodies in sheep was 14.3 % by IFAT and 15.8 % by ciELISAtSAG1. In goats, prevalence was 12.9 % by IFAT and 14.6 % by ciELISAtSAG1. The overall prevalence of anti-T. gondii antibodies was 28.8 % in goats and 43.8 % in sheep. The ciELISAtSAG1 could be useful for large-scale detection of anti-N. caninum antibodies in sheep and goats, and for seroepidemiological investigations due to its appropriate sensitivity and specificity, and the simplicity of production.

Clinical performance of different SARS-CoV-2 IgG antibody tests.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays are urgently needed for rapid diagnosis, contact tracing, and for epidemiological studies. So far, there is limited data on how commercially available tests perform with real patient samples, and if positive tested samples show neutralizing abilities. Focusing on IgG antibodies, we demonstrate the performance of two enzyme-linked immunosorbent assay (ELISA) assays (Euroimmun SARS-CoV-2 IgG and Vircell COVID-19 ELISA IgG) in comparison to one lateral flow assay (FaStep COVID-19 IgG/IgM Rapid Test Device) and two in-house developed assays (immunofluorescence assay [IFA] and plaque reduction neutralization test [PRNT]). We tested follow up serum/plasma samples of individuals polymerase chain reaction-diagnosed with COVID-19. Most of the SARS-CoV-2 samples were from individuals with moderate to the severe clinical course, who required an in-patient hospital stay. For all examined assays, the sensitivity ranged from 58.8 to 76.5% for the early phase of infection (days 5-9) and from 93.8% to 100% for the later period (days 10-18).

Assessment of antinuclear antibodies by indirect immunofluorescence assay: report from a survey by the American Association of Medical Laboratory Immunologists.

Background: The indirect immunofluorescence assay (IFA) using HEp-2 cell substrates is the preferred method by some for detecting antinuclear antibodies (ANA) as it demonstrates a number of characteristic staining patterns that reflect the cellular components bound as well as semi-quantitative results. Lack of harmonized nomenclature for HEp-2 IFA patterns, subjectivity in interpretation and variability in the number of patterns reported by different laboratories pose significant harmonization challenges. The main objectives of this study were to assess current practice in laboratory assessment of HEp-2 IFA, identify gaps and define strategies to improve reading, interpretation and reporting. Methods: We developed and administered a 24-item survey based on four domains: educational and professional background of participants, current practice of HEp-2 IFA testing and training, gap assessment and the perceived value of International Consensus on Antinuclear Antibody Patterns (ICAP) and other factors in HEp-2 IFA assessment. The Association of Medical Laboratory Immunologists (AMLI) and American Society for Clinical Pathology administered the survey from April 1 to June 30, 2018, to members involved in ANA testing. This report summarizes the survey results and discussion from a dry workshop held during the 2019 AMLI annual meeting. Results: One hundred and seventy-nine (n = 179) responses were obtained where a significant number were clinical laboratory scientists (46%), laboratory directors (24%), supervisors (13%) or others (17%). A majority of respondents agreed on the need to standardize nomenclature and reporting of HEp-2 IFA results. About 55% were aware of the ICAP initiative; however, among those aware, a significant majority thought its guidance on HEp-2 IFA nomenclature and reporting is of value to clinical laboratories. To improve ICAP awareness and further enhance HEp-2 IFA assessment, increased collaboration between ICAP and the clinical laboratory community was suggested with emphasis on education and availability of reference materials. Conclusions: Based on these suggestions, future efforts to optimize HEp-2 IFA reading, interpretation and reporting would benefit from more hands-on training of laboratory personnel as well as continuous collaboration between professional organizations, in vitro diagnostic manufacturers and clinical laboratories.

Performance evaluation of an indirect immunofluorescence kit for the serological diagnosis of dengue.

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.

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.

Evaluation of the automated indirect immunofluorescence test for anti-dsDNA antibodies.

BACKGROUND: Anti-dsDNA antibody is a specific antibody in systemic lupus erythematosus (SLE). Indirect immunofluorescence test (IIFT) is a highly specific method in detecting anti-dsDNA antibody. The application of automated system has gained better consistency than manual operation. This study detected anti-dsDNA antibodies using EUROPattern Computer-aided immunofluorescence microscopy (EPA), and evaluated the performance of the automated system. METHODS: The sera of 96 patients with suspected SLE and 102 control patients were examined using IIFT. The consistency between the EPA and manual reading was analyzed. RESULTS: Analysis of 198 samples showed that the overall consistency of the negative/positive results between the EPA and manual reading was 94.95%. Based on the manual reading results, the sensitivity and specificity of EPA were 95.70% and 94.29%, respectively. The analysis of 57 samples with non-specific fluorescence showed that the overall consistency of the negative/positive results was 96.49%. The analysis of the antibody titer of 89 positive samples showed that the consistency between the EPA and manual reading was 97.75%. CONCLUSION: EPA was consistent with the manual reading with regard to qualitative reading and antibody titer. With low-exposure function, EPA could read samples with non-specific fluorescence. EPA was superior to manual reading in automation and standardization.

Validation of an indirect immunofluorescence assay (IFA) for the detection of IgG antibodies against Coxiella burnetii in bovine serum.

There is limited knowledge of the true prevalence and distribution of coxiellosis in dairy and beef cattle populations in Australia. For this to occur, apparent prevalence estimates need to be reliably adjusted, accounting for diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the test used. However, there are few tests available with known diagnostic specifications suitable to inform screening and surveillance activities in the Australian context. We initially modified and optimised a human indirect immunofluorescence assay (IFA) test for the detection of IgG antibodies against phase I and/or phase II Coxiella burnetii in bovine sera and determined an optimal screening dilution cut-off to be 1:160. Direct comparison of the modified IFA with the commercial IDEXX enzyme-linked immunosorbent assay (ELISA) kit (Q Fever Ab Test IDEXX Laboratories, United States of America) was performed by testing 458 serum samples from four distinct cattle populations across the east coast of Australia and New Zealand. Cross classified test results were then analysed using Bayesian latent class modelling, to validate the tests in the absence of a gold standard reference test. Results from this analysis indicate that the IFA, at a 1:160 serum dilution, has an estimated DSe of 73.6% (95% Credible Interval (CrI) 61.1, 85.9) and DSp of 98.2% (95% CrI 95.1, 99.7). The commercial IDEXX ELISA kit was found to have a higher DSe of 87.9% (95% CrI 73.9, 96.4) and similar DSp of 97.7% (95% CrI 93.2, 99.7). Evaluation of the diagnostic performance of the IFA and ELISA methods, specifically for use in cattle will enable more accurate interpretation of prevalence estimates of C. burnetii exposure to be reported for cattle in Australia and other countries.

Harmonizing by reducing inter-run variability: performance evaluation of a quality assurance program for antinuclear antibody detection by indirect immunofluorescence.

Background The introduction of automated anti-nuclear antibody (ANA) indirect immunofluorescence (IIF) analysis may allow for more harmonized ANA IIF reporting, provided that a thorough quality assurance program controls this process. The aim of this study was to evaluate various quality indicators used for ANA IIF analysis with the final goal of optimizing the iQC program. Methods In an experimental setup, we introduced artificial errors, mimicking plausible problems during routine practice on a QUANTA-Lyser-NOVA View® system (Inova Diagnostics, San Diego, CA, USA). Predetermined quality indicators were evaluated against predefined acceptance criteria. In addition, we retrospectively investigated the applicability of the selected quality indicators in the daily routine practice during three pre-defined periods. Results Both the experimental as the retrospective study revealed that pre-analytical, analytical and post-analytical errors were not highlighted by company internal quality control (iQC) materials. The use of patient derived iQC samples, median fluorescence intensity results per run and the percentage of positive ANA IIF results as additional quality indicators ensured a more adequate ANA IIF quality assurance. Furthermore, negative and moderate positive sample iQC materials merit clinical validation, as titer changes of >1 correspond to clinically important shifts. Traditional Westgard rules, including a clinically defined stop limit, revealed to be useful in monitoring of the supplemental quality indicators. Conclusions A thorough ANA IIF quality assurance for daily routine practice necessitates the addition of supplemental quality indicators in combination with well-defined acceptance criteria.

Clinical relevance of HEp-2 indirect immunofluorescent patterns: the International Consensus on ANA patterns (ICAP) perspective.

The indirect immunofluorescence assay (IIFA) on HEp-2 cells is widely used for detection of antinuclear antibodies (ANA). The dichotomous outcome, negative or positive, is integrated in diagnostic and classification criteria for several systemic autoimmune diseases. However, the HEp-2 IIFA test has much more to offer: besides the titre or fluorescence intensity, it also provides fluorescence pattern(s). The latter include the nucleus and the cytoplasm of interphase cells as well as patterns associated with mitotic cells. The International Consensus on ANA Patterns (ICAP) initiative has previously reached consensus on the nomenclature and definitions of HEp-2 IIFA patterns. In the current paper, the ICAP consensus is presented on the clinical relevance of the 29 distinct HEp-2 IIFA patterns. This clinical relevance is primarily defined within the context of the suspected disease and includes recommendations for follow-up testing. The discussion includes how this information may benefit the clinicians in daily practice and how the knowledge can be used to further improve diagnostic and classification criteria.

Automated Processing and Evaluation of Anti-Nuclear Antibody Indirect Immunofluorescence Testing.

Indirect immunofluorescence (IIF) is considered by the American College of Rheumatology (ACR) and the international consensus on ANA patterns (ICAP) the gold standard for the screening of anti-nuclear antibodies (ANA). As conventional IIF is labor intensive, time-consuming, subjective, and poorly standardized, there have been ongoing efforts to improve the standardization of reagents and to develop automated platforms for assay incubation, microscopy, and evaluation. In this study, the workflow and performance characteristics of a fully automated ANA IIF system (Sprinter XL, EUROPattern Suite, IFA 40: HEp-20-10 cells) were compared to a manual approach using visual microscopy with a filter device for single-well titration and to technologist reading. The Sprinter/EUROPattern system enabled the processing of large daily workload cohorts in less than 8 h and the reduction of labor hands-on time by more than 4 h. Regarding the discrimination of positive from negative samples, the overall agreement of the EUROPattern software with technologist reading was higher (95.6%) than when compared to the current method (89.4%). Moreover, the software was consistent with technologist reading in 80.6-97.5% of patterns and 71.0-93.8% of titers. In conclusion, the Sprinter/EUROPattern system provides substantial labor savings and good concordance with technologist ANA IIF microscopy, thus increasing standardization, laboratory efficiency, and removing subjectivity.

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.

Comparison of 2 Anti-PLA2R Immunoassays for the Diagnosis of Primary Membranous Nephropathy.

BACKGROUND: Anti-phospholipase A2 receptor (anti-PLA2R) is a promising biomarker for diagnosis, activity evaluation, therapy monitoring, and prognostic estimation of primary membranous neuropathy (pMN). Difference in methodology may be one cause of the discrepancy in anti-PLA2R-positive percentages in reported studies. In this study, we evaluated the determination consistency of anti-PLA2R using indirect immunofluorescence assay (IIF) and enzyme-linked immunosorbent assay (ELISA). METHODS: A total of 113 patients with pMN, 34 with secondary membranous neuropathy (sMN), and 53 healthy control individuals were enrolled. We tested their circulating anti-PLA2R, along with other biochemical parameters, using IIF and ELISA. RESULTS: The sensitivity of anti-PLA2R for pMN was 70.8% by IIF and 67.3% by ELISA, with no statistically significant difference. The overall qualitative agreement of anti-PLA2R was 91.15% (95% confidence interval [CI], 85.91%-96.39%), and the correlation coefficient was 0.79 for IIF versus ELISA. The overall correlation coefficient of anti-PLA2R titers by IIF and concentration by ELISA was 0.85. Through ROC curve analysis, anti-PLA2R as measured by IIF and ELISA showed larger areas under the curve (AUCs) than other biochemical parameters. CONCLUSION: ELISA shows similar performance to IIF, with the advantages of quantitative results and suitability for high throughput.