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.

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.

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.

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.

Performance analysis of automated evaluation of Crithidia luciliae-based indirect immunofluorescence tests in a routine setting - strengths and weaknesses.

BACKGROUND: Antibodies directed against dsDNA are a highly specific diagnostic marker for the presence of systemic lupus erythematosus and of particular importance in its diagnosis. To assess anti-dsDNA antibodies, the Crithidia luciliae-based indirect immunofluorescence test (CLIFT) is one of the assays considered to be the best choice. To overcome the drawback of subjective result interpretation that inheres indirect immunofluorescence assays in general, automated systems have been introduced into the market during the last years. Among these systems is the EUROPattern Suite, an advanced automated fluorescence microscope equipped with different software packages, capable of automated pattern interpretation and result suggestion for ANA, ANCA and CLIFT analysis. METHODS: We analyzed the performance of the EUROPattern Suite with its automated fluorescence interpretation for CLIFT in a routine setting, reflecting the everyday life of a diagnostic laboratory. Three hundred and twelve consecutive samples were collected, sent to the Central Diagnostic Laboratory of the Maastricht University Medical Centre with a request for anti-dsDNA analysis over a period of 7 months. RESULTS: Agreement between EUROPattern assay analysis and the visual read was 93.3%. Sensitivity and specificity were 94.1% and 93.2%, respectively. The EUROPattern Suite performed reliably and greatly supported result interpretation. CONCLUSIONS: Automated image acquisition is readily performed and automated image classification gives a reliable recommendation for assay evaluation to the operator. The EUROPattern Suite optimizes workflow and contributes to standardization between different operators or laboratories.

Automated antinuclear immunofluorescence antibody analysis is a reliable approach in routine clinical laboratories.

BACKGROUND: Indirect immunofluorescence (IIF) assays are recommended as the gold standard method for the detection of antinuclear antibodies (ANAs). This study aimed to investigate the reliability of an automated system. METHODS: We compared 3745 serum samples using NOVA View archived images with manual analysis via microscopy. A custom cutoff value was established to distinguish ANA titers and was validated in two clinical laboratories. The automatic ANA pattern recognition system was evaluated, and all ANA-positive sera were subjected to two commercial ANA IIF kits to compare the consistency of the pattern interpretation results. For inconsistent patterns, a third ANA IIF testing kit was utilized. RESULTS: Agreement of the interpretation of the ANA IIF test using the platform of NOVA View and manual microscopy was 96.9%. The local cutoff value to discriminate ANA titers in four main ANA patterns was calculated based on 1390 serum samples. In our laboratory, the titer prediction accuracy was superior to the preset cutoff in NOVA View (p<0.01); the performance was similar in another laboratory (p=0.11). The automatic pattern recognition accuracies of speckled, homogeneous, centromere, nucleolar and nuclear dot patterns were 62.7%, 57.4%, 92.6%, 30.5% and 27.3%, respectively. The consistency of the pattern interpretation results between INOVA and MBL kits was 95.3%. CONCLUSIONS: It is necessary to establish a custom value-added ANA report. However, confirmation of the digital immunofluorescence images by expert technicians was essential, and suspect results of an ANA pattern should be reconfirmed by another commercial ANA IIF kit to achieve more reliable results.

Chromosome Banding in Amphibia. XXXIII. Demonstration of 5-Methylcytosine-Rich Heterochromatin in Anura.

An experimental approach using monoclonal anti-5-methylcytosine (5-MeC) antibodies and indirect immunofluorescence was elaborated for detecting 5-MeC-rich chromosome regions in anuran chromosomes. This technique was applied to mitotic metaphases of 6 neotropical frog species belonging to 6 genera and 4 families. The hypermethylation patterns were compared with a variety of banding patterns obtained by conventional banding techniques. The hypermethylated DNA sequences are species-specific and located exclusively in constitutive heterochromatin. They are found in centromeric, pericentromeric, telomeric, and interstitial positions of the chromosomes and adjacent to nucleolus organizer regions. 5-MeC-rich DNA sequences can be embedded both in AT- and GC-rich repetitive DNA. The experimental parameters that have major influence on the reproducibility and quality of the anti-5-MeC antibody labeling are discussed.

Evaluation of serological cross-reactivity and cross-neutralization between the United States porcine epidemic diarrhea virus prototype and S-INDEL-variant strains.

BACKGROUND: At least two genetically different porcine epidemic diarrhea virus (PEDV) strains have been identified in the United States (U.S. PEDV prototype and S-INDEL-variant strains). The current serological assays offered at veterinary diagnostic laboratories for detection of PEDV-specific antibody are based on the U.S. PEDV prototype strain. The objectives of this study were: 1) isolate the U.S. PEDV S-INDEL-variant strain in cell culture; 2) generate antisera against the U.S. PEDV prototype and S-INDEL-variant strains by experimentally infecting weaned pigs; 3) determine if the various PEDV serological assays could detect antibodies against the U.S. PEDV S-INDEL-variant strain and vice versa. RESULTS: A U.S. PEDV S-INDEL-variant strain was isolated in cell culture in this study. Three groups of PEDV-negative, 3-week-old pigs (five pigs per group) were inoculated orally with a U.S. PEDV prototype isolate (previously isolated in our lab), an S-INDEL-variant isolate or virus-negative culture medium. Serum samples collected at 0, 7, 14, 21 and 28 days post inoculation were evaluated by the following PEDV serological assays: 1) indirect fluorescent antibody (IFA) assays using the prototype and S-INDEL-variant strains as indicator viruses; 2) virus neutralization (VN) tests against the prototype and S-INDEL-variant viruses; 3) PEDV prototype strain whole virus based ELISA; 4) PEDV prototype strain S1-based ELISA; and 5) PEDV S-INDEL-variant strain S1-based ELISA. The positive antisera against the prototype strain reacted to and neutralized both prototype and S-INDEL-variant viruses, and the positive antisera against the S-INDEL-variant strain also reacted to and neutralized both prototype and S-INDEL-variant viruses, as examined by IFA antibody assays and VN tests. Antibodies against the two PEDV strains could be detected by all three ELISAs although detection rates varied to some degree. CONCLUSIONS: These data indicate that the antibodies against U.S. PEDV prototype and S-INDEL-variant strains cross-reacted and cross-neutralized both strains in vitro. The current serological assays based on U.S. PEDV prototype strain can detect antibodies against both U.S. PEDV strains.

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.

Automated tests of ANA immunofluorescence as throughput autoantibody detection technology: strengths and limitations.

Anti-nuclear antibody (ANA) assay is a screening test used for almost all autoimmune rheumatic diseases, and in a number of these cases, it is a diagnostic/classification parameter. In addition, ANA is also a useful test for additional autoimmune disorders. The indirect immunofluorescence technique on monolayers of cultured epithelial cells is the current recommended method because it has higher sensitivity than solid phase assays. However, the technique is time-consuming and requires skilled operators. Automated ANA reading systems have recently been developed, which offer the advantage of faster and much easier performance as well as better harmonization in the interpretation of the results. Preliminary validation studies of these systems have given promising results in terms of analytical specificity and reproducibility. However, these techniques require further validation in clinical studies and need improvement in their recognition of mixed or less common staining patterns.

Antinuclear antibodies: two-step detection strategy.

In order to evaluate the performance of the chemiluminescent immunoassay (CLIA) in antinuclear antibodies (ANA) testing, using indirect fluorescent antibody (IFA) on HEp-2 cells as a standard, 209 samples were studied from September to October/2010. The tests were conducted according to the procedures recommended by the manufacturers of the reagents. The interpretation of the IFA results was done according to the Brazilian standards. The charts of patients showing different results between the two techniques were analyzed. The CLIA efficiency was 89%, with a sensitivity of 65%, and a specificity of 94.7%. Nine (4.3%) false-positive and 14 (6.7%) false-negative results were detected. Of these, 13 (93%) represented no risk for the diagnosis and therapeutic management of the patients. The CLIA methodology reduced the need for the IFA manual technique by 77% (160/209). The ANA screening test proved to be a fast and acceptable methodology in the studied population. We established the following criteria for the introduction of an automated ANA screening: (1) Positive results must be confirmed by IFA to characterize the pattern and titer of the antibody. (2) Negative results are issued with a notice to request a new test by IFA when the clinical suspicion of autoimmune disease persists.

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.

Comparison of an indirect fluorescent antibody test with Western blot for the detection of serum antibodies against Encephalitozoon cuniculi in cats.

Current clinical research indicates that Encephalitozoon (E.) cuniculi infections in cats may be underdiagnosed, especially in animals with typical ocular signs (cataract/anterior uveitis). Although molecular detection of the pathogen in tissue appears promising, serology remains the major diagnostic tool in the living animal. While serological tests are established for the main host of E. cuniculi, the rabbit, the routine serological diagnosis for cats still needs validation. The aim of the study was to evaluate the consistency of indirect fluorescence antibody test (IFAT) and Western blot (WB) for the detection of IgG antibodies against E. cuniculi in the serum of 84 cats. In addition, PCR of liquefied lens material or intraocular fluid was performed in those of the cats with a suspected ocular E. cuniculi infection. Twenty-one cats with positive PCR results were considered as a positive reference group. Results obtained by IFAT and WB corresponded in 83/84 serum samples, indicating a very good correlation between both serological methods. Using WB as the standard reference, sensitivity and specificity for the detection of antibodies against E. cuniculi by the IFAT were 97.6 and 100%, respectively. The positive and negative predictive values for the IFAT were 100 and 97.7%, respectively. The accuracy (correct classified proportion) for the detection of IgG antibodies against E. cuniculi in cats was 98.8%. The comparison of both serological methods with the PCR results also revealed a good agreement as 20 out of 21 PCR-positive samples were seropositive both in IFAT and WB. Both tests can be considered as equally reliable assays to detect IgG antibodies against E. cuniculi in cats. As the IFAT is quicker and easier to perform, it is recommended for routine use in the diagnosis of feline encephalitozoonosis.

Original approach for automated quantification of antinuclear autoantibodies by indirect immunofluorescence.

INTRODUCTION: Indirect immunofluorescence (IIF) is the gold standard method for the detection of antinuclear antibodies (ANA) which are essential markers for the diagnosis of systemic autoimmune rheumatic diseases. For the discrimination of positive and negative samples, we propose here an original approach named Immunofluorescence for Computed Antinuclear antibody Rational Evaluation (ICARE) based on the calculation of a fluorescence index (FI). METHODS: We made comparison between FI and visual evaluations on 237 consecutive samples and on a cohort of 25 patients with SLE. RESULTS: We obtained very good technical performance of FI (95% sensitivity, 98% specificity, and a kappa of 0.92), even in a subgroup of weakly positive samples. A significant correlation between quantification of FI and IIF ANA titers was found (Spearman's ρ = 0.80, P < 0.0001). Clinical performance of ICARE was validated on a cohort of patients with SLE corroborating the fact that FI could represent an attractive alternative for the evaluation of antibody titer. CONCLUSION: Our results represent a major step for automated quantification of IIF ANA, opening attractive perspectives such as rapid sample screening and laboratory standardization.