Evaluation of a Fully Automated Antinuclear Antibody Indirect Immunofluorescence Assay in Routine Use

Indirect immunofluorescence assay (IFA) using HEp-2 cells as a substrate is the gold standard for detecting antinuclear antibodies (ANA) in patient serum. However, the ANA IFA has labor-intensive nature of the procedure and lacks adequate standardization. To overcome these drawbacks, the automation has been developed and implemented to the clinical laboratory. The purposes of this study were to evaluate the analytical performance of a fully automated Helios ANA IFA analyzer in a real-life laboratory setting, and to compare the time and the cost of ANA IFA testing before and after adopting the Helios system. A total of 3,276 consecutive serum samples were analyzed for ANA using the Helios system from May to August 2019. The positive/negative results, staining patterns, and endpoint titers were compared between Helios and visual readings. Furthermore, the turnaround time and the number of wells used were compared before and after the introduction of Helios system. Of the 3,276 samples tested, 748 were positive and 2,528 were negative based on visual readings. Using visual reading as the reference standard, the overall relative sensitivity, relative specificity, and concordance of Helios reading were 73.3, 99.4, and 93.4% (κ = 0.80), respectively. For pattern recognition, the overall agreement was 70.1% (298/425) for single patterns, and 72.4% (89/123) for mixed patterns. For titration, there was an agreement of 75.9% (211/278) between automated and classical endpoint titers by regarding within ± one titer difference as acceptable. Helios significantly shortened the median turnaround time from 100.6 to 55.7 h (P < 0.0001). Furthermore, routine use of the system reduced the average number of wells used per test from 4 to 1.5. Helios shows good agreement in distinguishing between positive and negative results. However, it still has limitations in positive/negative discrimination, pattern recognition, and endpoint titer prediction, requiring additional validation of results by human observers. Helios provides significant advantages in routine laboratory ANA IFA work in terms of labor, time, and cost savings. We hope that upgrading and developing softwares with more reliable capabilities will allow automated ANA IFA analyzers to be fully integrated into the routine operations of the clinical laboratory.

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The antinuclear antibody HEp-2 indirect immunofluorescence assay: a survey of laboratory performance, pattern recognition and interpretation

Autoimmunity Highlights ◽

10.1186/s13317-020-00146-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Anne E. Tebo ◽

Robert L. Schmidt ◽

Kamran Kadkhoda ◽

Lisa K. Peterson ◽

Edward K. L. Chan ◽

...

Keyword(s):

Indirect Immunofluorescence ◽

Clinical Laboratory ◽

Immunofluorescence Assay ◽

Indirect Immunofluorescence Assay ◽

Medical Laboratory ◽

Nuclear Staining ◽

International Consensus ◽

In Vitro Diagnostic ◽

Traditional Pattern

Abstract Background To evaluate the interpretation and reporting of antinuclear antibodies (ANA) by indirect immunofluorescence assay (IFA) using HEp-2 substrates based on common practice and guidance by the International Consensus on ANA patterns (ICAP). Method Participants included two groups [16 clinical laboratories (CL) and 8 in vitro diagnostic manufacturers (IVD)] recruited via an email sent to the Association of Medical Laboratory Immunologists (AMLI) membership. Twelve (n = 12) pre-qualified specimens were distributed to participants for testing, interpretation and reporting HEp-2 IFA. Results obtained were analyzed for accuracy with the intended and consensus response for three main categorical patterns (nuclear, cytoplasmic and mitotic), common patterns and ICAP report nomenclatures. The distributions of antibody titers of specimens were also compared. Results Laboratories differed in the categorical patterns reported; 8 reporting all patterns, 3 reporting only nuclear patterns and 5 reporting nuclear patterns with various combinations of other patterns. For all participants, accuracy with the intended response for the categorical nuclear pattern was excellent at 99% [95% confidence interval (CI): 97–100%] compared to 78% [95% CI 67–88%] for the cytoplasmic, and 93% [95% CI 86%–100%] for mitotic patterns. The accuracy was 13% greater for the common nomenclature [87%, 95% CI 82–90%] compared to the ICAP nomenclature [74%, 95% CI 68–79%] for all participants. Participants reporting all three main categories demonstrated better performances compared to those reporting 2 or less categorical patterns. The average accuracies varied between participant groups, however, with the lowest and most variable performances for cytoplasmic pattern specimens. The reported titers for all specimens varied, with the least variability for nuclear patterns and most titer variability associated with cytoplasmic patterns. Conclusions Our study demonstrated significant accuracy for all participants in identifying the categorical nuclear staining as well as traditional pattern assignments for nuclear patterns. However, there was less consistency in reporting cytoplasmic and mitotic patterns, with implications for assigning competencies and training for clinical laboratory personnel.

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

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2019-1262 ◽

2020 ◽

Vol 58 (9) ◽

pp. 1489-1497 ◽

Cited By ~ 1

Author(s):

Lisa K. Peterson ◽

Anne E. Tebo ◽

Mark H. Wener ◽

Susan S. Copple ◽

Marvin J. Fritzler

Keyword(s):

Indirect Immunofluorescence ◽

Antinuclear Antibodies ◽

Current Practice ◽

Clinical Laboratory ◽

Immunofluorescence Assay ◽

Indirect Immunofluorescence Assay ◽

Medical Laboratory ◽

International Consensus ◽

Clinical Laboratories ◽

Reading Interpretation

AbstractBackgroundThe 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.MethodsWe 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.ResultsOne 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.ConclusionsBased 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.

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A case of human monocytic ehrlichiosis in Serbia

Srpski arhiv za celokupno lekarstvo ◽

10.2298/sarh1402079a ◽

2014 ◽

Vol 142 (1-2) ◽

pp. 79-82 ◽

Cited By ~ 3

Author(s):

Bogdan Arsic ◽

Ana Gligic ◽

Elizabeta Ristanovic ◽

Branislav Lako ◽

Aleksandar Potkonjak ◽

...

Keyword(s):

Indirect Immunofluorescence ◽

Clinical Picture ◽

Peripheral Blood ◽

Immunofluorescence Assay ◽

High Fever ◽

Tick Bite ◽

Indirect Immunofluorescence Assay ◽

Ehrlichia Chaffeensis ◽

Serum Samples ◽

History Of

Introduction. Ehrlichiosis is a bacterial zoonosis transmitted by hematophagous arthropods - ticks. In humans, it occurs as monocytic, granulocytic, and ewingii ehrlichiosis. Pathological process is based on parasitic presence of Ehrlichia organisms within peripheral blood cells - monocytes and granulocytes. Case Outline. Fifty-two year old patient was admitted to hospital due to high fever of over 40?C that lasted two days, accompanied with chills, muscle aches, malaise, loss of appetite, headache, confusion, breathing difficulties, and mild dry cough. The history suggested tick bite that occurred seven days before the onset of disease. Doxycycline was introduced and administered for 14 days, causing the disease to subside. Indirect immunofluorescence assay was used to analyze three serum samples obtained from this patient for Ehrlichia chaffeensis antibodies, and peripheral blood smear was evaluated for the presence of Ehrlichia and Ehrlichia aggregation into morulae. Conclusion. Ehrlichiosis should be considered in each case where there is a history of tick bite together with the clinical picture (high fever, chills, muscle aches, headache, generalized weakness and malaise, and possible maculopapular rash). The presence of Ehrlichia chaffeensis antibodies was confirmed in a patient with the history of tick bite, appropriate clinical picture and indirect immunofluorescence assay. This confirmed the presence of human monocytotropic ehrlichiosis, a disease that is uncommonly identified in our country.

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Evaluation of Four Commercially Available Epstein-Barr Virus Enzyme Immunoassays with an Immunofluorescence Assay as the Reference Method

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.10.1.78-82.2003 ◽

2003 ◽

Vol 10 (1) ◽

pp. 78-82 ◽

Cited By ~ 40

Author(s):

Barbara C. Gärtner ◽

Ralf D. Hess ◽

Dirk Bandt ◽

Alexander Kruse ◽

Axel Rethwilm ◽

...

Keyword(s):

Indirect Immunofluorescence ◽

Epstein Barr Virus ◽

Reference Method ◽

Immunofluorescence Assay ◽

Parameter Analysis ◽

Indirect Immunofluorescence Assay ◽

Serum Samples ◽

Enzyme Immunoassays ◽

Barr Virus ◽

Epstein Barr

ABSTRACT Four commercially available enzyme immunoassays (EIAs) (Novitec, Biotest, Virotech, and DiaSorin) were evaluated, with an indirect immunofluorescence assay as the reference method, for Epstein-Barr virus (EBV) VCA (viral capsid antigen) immunoglobulin G (IgG), VCA IgM, or EBNA (EBV nuclear antigen) IgG at three different locations (Homburg, Stuttgart, and Dresden). Serum samples from 66 immunocompetent patients with infectious mononucleosis, 73 patients without prior EBV infection, and 96 patients with past EBV infections and 29 serum samples with possible cross-reactions to other herpesviruses were included. In addition, 25 samples from an extensively pretested panel that is commercially available (Boston Biomedica) were tested. Each sample was tested at only one location. The four EIAs varied considerably in performance. When analyzing for EBV diagnosis, the Novitec assay performed the best, with 4.9% discrepant diagnoses, followed by the Biotest, Virotech, and DiaSorin assays, with 6.8, 11.7, and 14.0% discrepant diagnoses, respectively. On the basis of single-parameter analysis, the Novitec assay also showed the lowest number of discrepant results, with 3.5%, compared with the Virotech, Biotest, and DiaSorin assays, which produced 5.4, 6.4, and 8.6% discrepant results, respectively. VCA assays using affinity-purified native antigens performed better than assays with recombinant or synthetic antigens. The synthetic EBNA-1s showed the lowest concordance with the reference compared to recombinant p72. Commercially available EBV EIAs differed considerably in performance; however, some proved to be reliable and convenient alternatives to the indirect immunofluorescence assay for routine diagnostics. Native antigens, rather than synthetic peptides, are favored for EBV serology testing.

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Establishment of a peptide-based enzyme-linked immunosorbent assay for detecting antibodies against PRRSV M protein

BMC Veterinary Research ◽

10.1186/s12917-021-03060-z ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Jun Zhao ◽

Rubo Zhang ◽

Ling Zhu ◽

Huidan Deng ◽

Fengqing Li ◽

...

Keyword(s):

Indirect Immunofluorescence ◽

Area Under The Curve ◽

Immunofluorescence Assay ◽

M Protein ◽

Indirect Immunofluorescence Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Inactivated Vaccine ◽

Serum Samples ◽

Swine Industry ◽

Peptide Elisa

Abstract Background Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating diseases affecting the swine industry globally. Evaluation of antibody responses and neutralizing antibody titers is the most effective method for vaccine evaluation. In this study, the B cell line epitopes of PRRSV M protein were predicted, and two peptide ELISA assays were established (M-A110-129 ELISA, M-A148-174 ELISA) to detect antibodies against PRRSV M protein. Field serum samples collected from pig farms were used to validate the peptide ELISA and compare it with an indirect immunofluorescence assay. Results The sensitivity and specificity of M-A110-129 ELISA and M-A148-174 ELISA were (111/125) 88.80%, (69/70) 98.57% and (122/125) 97.60%, (70/70) 100%, relative to indirect immunofluorescence assay. This peptide ELISA could detect antibodies against different genotypes of PRRSV including type 1 PRRSV, classical PRRSV, HP-PRRSV, and NADC30 like PRRSV, but not antibodies against other common swine viruses. The results of ROC analysis showed that the area under the curve (AUC) of the M-A110-129 ELISA and M-A148-174 ELISA were 0.967 and 0.996, respectively. Compared the concordance of results using two peptide ELISA assays, the IDEXX PRRSV X3 Ab ELISA and a virus neutralization test, were assessed using a series of 147 sera from pigs vaccinated with the NADC30-like PRRSV inactivated vaccine. The M-A148-174 ELISA had the best consistency, with a Cohen’s kappa coefficient of 0.8772. The concordance rates of the Hipra PRRSV ELISA kit, M-A110-129 ELISA and M-A148-174 ELISA in the field seropositive detection results were 91.08, 86.32 and 95.35%, relative to indirect immunofluorescence assay. Conclusions In summary, compared with M-A110-129 ELISA, the PRRSV M-A148-174 ELISA is of value for detecting antibodies against PRRSV and the evaluation of the NADC30-like PRRSV inactivated vaccine, but the advantage is insufficient in serological early diagnosis.

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Comparison of Enzyme-Linked Immunosorbent Assay, Western Blotting, Microagglutination, Indirect Immunofluorescence Assay, and Flow Cytometry for Serological Diagnosis of Tularemia

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.11.6.1008-1015.2004 ◽

2004 ◽

Vol 11 (6) ◽

pp. 1008-1015 ◽

Cited By ~ 65

Author(s):

Mustafa Porsch-Özcürümez ◽

Nele Kischel ◽

Heidi Priebe ◽

Wolf Splettstösser ◽

Ernst-Jürgen Finke ◽

...

Keyword(s):

Flow Cytometry ◽

Western Blotting ◽

Francisella Tularensis ◽

Immunosorbent Assay ◽

Indirect Immunofluorescence ◽

Immunofluorescence Assay ◽

Indirect Immunofluorescence Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Diagnostic Sensitivity And Specificity

ABSTRACT The serodiagnostic efficiencies of five different approaches to detecting antibodies (immunoglobulins G, A, and M) developed in clinically proven infections with Francisella tularensis have been assessed. Fifty serum samples from patients suffering from tularemia during an outbreak in Sweden were compared with samples from 50 healthy blood donors (controls) by using an enzyme-linked immunosorbent assay (ELISA), microagglutination (MA), Western blotting (WB), an indirect immunofluorescence assay (IIFA), and flow cytometry (FC). ELISA, WB, and FC were based on the use of preparations of lipopolysaccharides (LPS) of the live vaccine strain of Francisella tularensis subsp. holarctica (ATCC 29684) as a capture antigen. Whole methanol-fixed bacteria were used for IIFA and MA. Optimized protocols yielded a diagnostic sensitivity and specificity of 100% for WB, MA, and FC, 98% for ELISA, and 93% for IIFA. A total of 6,632 serum samples from individuals between the ages of 18 and 79 years, representatively recruited from all regions of Germany, were screened to estimate and confirm the positive predictive value (PVpos) of the ELISA. Serum samples from 15 (0.226%) individuals tested positive for F. tularensis-specific antibodies by ELISA and confirmatory WB. The resulting prevalence-dependent PVpos of 10.2% and specificity of 98.1% were consistent with our findings for tularemia patients and controls. We conclude that the combined usage of a screening ELISA and a confirmatory WB based on LPS as a common antigen, as well as the MA, is a suitable serodiagnostic tool, while the quality of the IIFA is hampered by subjective variations of the results. FC is a promising new approach that might be improved further in terms of multiplex analyses or high-throughput applications.

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Serological prevalence of Toxoplasma gondii infection in cats (Belém, Pará, Brazil)

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-29612020038 ◽

2020 ◽

Vol 29 (2) ◽

Author(s):

Katarine de Souza Rocha ◽

Michele de Souza Lima ◽

Thamillys Rayssa Marques Monteiro ◽

Betsy Emely Tavares Honorio ◽

Ana Paula Vilhena Beckman Pinho ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Indirect Immunofluorescence ◽

Environmental Variables ◽

Immunofluorescence Assay ◽

Indirect Immunofluorescence Assay ◽

Domestic Cats ◽

Serum Samples ◽

Toxoplasma Gondii Infection

Abstract We evaluated the prevalence of anti-Toxoplasma gondii antibodies in the serum samples collected from domestic cats in Belém, Pará, Brazil. We also correlated the presence of T. gondii antibodies with environmental variables and cat-owner habits. Four-hundred and forty-seven serum samples from domestic cats were analyzed. The sera were tested using an indirect immunofluorescence assay. Among the animals analyzed, 21.92% (98/447) were seropositive. A statistically significant association was found in relation to age and serology among the animals over 1 year old (p<0.01): in the group up to 1 year old, 12.82% (20/156) of the animals were positive, and in the group over 1 year old, 26.80% (78/291) were positive. Our results show that the cats in Belém, Pará region have anti-T. gondii antibodies, and their owners are not aware of toxoplasmosis or how to prevent its transmission.

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Serodiagnosis of cilia-associated respiratory bacillus infection by the indirect immunofluorescence assay technique

Laboratory Animals ◽

10.1258/002367787781363327 ◽

1987 ◽

Vol 21 (4) ◽

pp. 356-359 ◽

Cited By ~ 20

Author(s):

S. Matsushita ◽

M. Kashima ◽

H. Joshima

Keyword(s):

Indirect Immunofluorescence ◽

Sendai Virus ◽

Early Stage ◽

Natural Infection ◽

Immunofluorescence Assay ◽

Indirect Immunofluorescence Assay ◽

Post Inoculation ◽

Mycoplasma Pulmonis ◽

Serum Samples ◽

Antibody Titres

Antibody to cilia-associated respiratory (CAR) bacillus was detected by the indirect immunofluorescence assay (IFA) technique using tracheal sections of infected mice as antigen in serum samples collected from rats infected naturally and experimentally. Nine of 23 cases of natural infection were positive in IFA antibody, with titres ranging from 1:10 to 1:80, and all these antibody-positive cases were also histologically positive. The remaining 14 cases were negative in both IFA antibody and histological diagnosis, even though some of them were infected with Sendai virus and Mycoplasma pulmonis. In the experimental infection, serum samples collected from 18 rats on days 4, 7, 14, 21, 28 and 56 post-inoculation (PI) (three rats for each point) and examined for IFA antibody revealed that seroconversion occurred in one rat on day 14 PI and in three rats on day 21 PI. Antibody titres of 1:80 to 1:160 remained to the termination of the experiment. The IFA technique was useful for the diagnosis of CAR bacillus infection except in the early stage of the infection.

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