Immunoassays for the detection of IgA antibodies to tissue transglutaminase: significance of multiples of the upper limit of normal and inter-assay correlations

2016 ◽  
Vol 54 (2) ◽  
Author(s):  
Brenda B. Suh-Lailam ◽  
K. Wayne Davis ◽  
Anne E. Tebo
Keyword(s):  
Celiac Disease ◽  
Indirect Immunofluorescence ◽  
Tissue Transglutaminase ◽  
Immunofluorescence Assay ◽  
Indirect Immunofluorescence Assay ◽  
Healthy Individuals ◽  
Reference Test ◽  
Upper Limit ◽  
Iga Antibodies ◽  
Diagnostic Pathways

AbstractThe presence of IgA antibodies to tissue transglutaminase (anti-tTg) is associated with variable risk for celiac disease. The use of common multiples of the upper limit of normal (ULN) has been suggested to optimize diagnostic pathways as well as improve harmonization between assays.The characteristics of four anti-tTG IgA assays relative to endomysial IgA (EMA) by indirect immunofluorescence assay (IFA) as reference test were assessed. Commutability between anti-tTG immunoassays and/or EMA based on manufacturer’s recommended cut-off values and three common multiples of ULN (3×, 5× and 10×) was also investigated. Sera from 200 patients and 100 healthy individuals were analyzed.At manufacturer’s cut-off; the sensitivities for the tTG assays ranged from 72.5% to 98.6% and specificities from 60.3% to 99.2%. The percent positive agreements between any anti-tTG and EMA or any two anti-tTG immunoassays varied from 56.7% to 98.0% and 46.7% to 100.0%, respectively. At 3×, 5× or 10× ULNs, the inter-rater reliability as measured by Cohen κ between any two anti-tTG assays were quite variable and ranged from 0.28 to 0.96, 0.26 to 0.89 or 0.13 to 0.78, respectively. Furthermore, the percent positive agreements between any two anti-tTg IgA immunoassays ranged from 83.1% to 98.2%, 92.0% to 100%, or 100%, at 3×, 5× or 10×, respectively.Commutability between tTG IgA immunoassays or tTG IgA and EMA is kit-dependent and common multiples of the ULN are not sufficient to correct for inter-assay variations. Many factors influence the performance of anti-tTG IgA assays which limit their commutability.

scholarly journals Longitudinal Profile of Immunoglobulin G (IgG), IgM, and IgA Antibodies against the Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein in Patients with Pneumonia Due to the SARS Coronavirus

2004 ◽  
Vol 11 (4) ◽  
pp. 665-668 ◽  
Author(s):  
Patrick C. Y. Woo ◽  
Susanna K. P. Lau ◽  
Beatrice H. L. Wong ◽  
Kwok-hung Chan ◽  
Chung-ming Chu ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Indirect Immunofluorescence ◽  
Immunoglobulin G ◽  
Nucleocapsid Protein ◽  
Baseline Level ◽  
Immunofluorescence Assay ◽  
Indirect Immunofluorescence Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sars Coronavirus ◽  
Iga Antibodies

ABSTRACT By using a recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein-based enzyme-linked immunosorbent assay (ELISA) and serum specimens serially collected (from day 0 to day 240 after symptom onset) from patients with pneumonia due to SARS-CoV, we analyzed the longitudinal profiles of immunoglobulin G (IgG), IgM, and IgA antibodies against the SARS-CoV nucleocapsid protein in patients with pneumonia due to SARS-CoV. For IgG, the median optical density at 450 nm (OD450) turned positive at day 17 and a biphasic response was observed. At day 240, all patients were still positive for anti-nucleocapsid protein IgG antibody. For IgM, the median OD450 turned positive at day 20.5, peaked at about day 80, and fell to below the baseline level at about day 180. At day 240, 36% of the patients were still positive for anti-nucleocapsid protein IgM antibody. For IgA, the median OD450 turned positive at day 17, peaked at about day 50, and fell to below the baseline level at about day 180. At day 240, 36% of the patients were still positive for anti-nucleocapsid protein IgA antibody. The time of seroconversion detected by the recombinant SARS-CoV nucleocapsid protein-based ELISA and that detected by indirect immunofluorescence assay were similar. The median times of seroconversion for IgG, IgM, and IgA detected by the indirect immunofluorescence assay were 17 days (17 days by ELISA), 16.5 days (20.5 days by ELISA), and 17.5 days (17 days by ELISA), respectively, after disease onset. One, four, and one of the six patients who died did not produce any IgG, IgM, and IgA antibodies against the nucleocapsid protein of SARS-CoV, respectively, although these antibodies were detected in all six patients by the indirect immunofluorescence assay. Further studies should be performed to see whether SARS-CoV nucleocapsid protein antibody positivity has any prognostic significance.

scholarly journals Celiac disease screening in patients with scleroderma

2011 ◽  
Vol 48 (2) ◽  
pp. 163-164 ◽  
Author(s):  
Renato Nisihara ◽  
Shirley Rosa Utiyama ◽  
Pedro Ming Azevedo ◽  
Thelma Larocca Skare
Keyword(s):  
Celiac Disease ◽  
Autoimmune Disease ◽  
Indirect Immunofluorescence ◽  
Disease Screening ◽  
Immunofluorescence Assay ◽  
Indirect Immunofluorescence Assay ◽  
Clinical Indication ◽  
Single Individual ◽  
Autoimmune Etiology

Both celiac disease and scleroderma have autoimmune etiology and affect the bowel causing diarrhea. As an association of autoimmune disease in a single individual is not rare, it is important to know if a patient with scleroderma may also have celiac disease. To analyze this we studied 105 scleroderma patients and 97 volunteers for IgA-EmA by indirect immunofluorescence assay. We could not find a higher prevalence of this autoantibody in scleroderma patients. The authors conclude that there is no need to screen scleroderma patients with diarrhea for celiac disease unless there is a clear clinical indication for this.

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

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.

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

2020 ◽  
Vol 58 (9) ◽  
pp. 1489-1497 ◽  
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.

scholarly journals A case of human monocytic ehrlichiosis in Serbia

2014 ◽  
Vol 142 (1-2) ◽  
pp. 79-82 ◽  
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.

scholarly journals A Novel Engineered Single-Chain Antibody Fragment for Targeting Pediatric Philadelphia Chromosome-like Acute Lymphoblastic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 36-36
Author(s):  
Sara M.A. Mohamed ◽  
Keith Sia ◽  
Karl-Heinz Friedrich ◽  
Andreas Wohlmann ◽  
Savvas Savvides ◽  
...  
Keyword(s):  
Indirect Immunofluorescence ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Statistical Significance ◽  
Immunofluorescence Assay ◽  
Laser Scanning Microscopy ◽  
Indirect Immunofluorescence Assay ◽  
Single Chain ◽  
Genetic Characteristics ◽  
Significant Difference

Introduction: Philadelphia-like acute lymphoblastic leukaemia (Ph-like ALL) is a high-risk ALL subtype characterized by an inferior survival rate and chemotherapeutic drug resistance (Tasian et al, Blood 130: 2064-2072, 2017). Around 50% of Ph-like ALL cases harbour gene rearrangements leading to the overexpression of cytokine receptor-like factor 2 (CRLF2) (Loh et al, Blood 121: 485-488, 2013). CRLF2 (also known as thymic stromal lymphopoietin receptor, TSLPR) heterodimerizes with the interleukin-7 receptor alpha (IL-7Rα) subunit to form the functional TSLPR. Upon TSLP binding, CRLF2 activates the JAK/STAT signalling pathway, leading to enhanced proliferation and survival of leukemia cells resulting in poor outcomes in patients (Harvey et al, Blood 115: 5312-5321, 2010). The extracellular overexpression of CRLF2 and association with poor prognosis suggest the translational value of designing precision-based therapeutics targeting CRLF2 in Ph-like ALL. Although conventional immunotherapy using full-sized antibodies is a promising treatment strategy that can improve treatment efficiency and minimize off-target toxicity, their clinical translation is challenging due to the high production cost and large size affecting targeting efficiency (Holliger et al, Nat Biotech 23: 1126-1136, 2005). Herein, we validated a novel single-chain variable fragment against CRLF2 (CRLF2-ScFv) for targeting Ph-like ALL cells. Methods: A CRLF2-rearranged Ph-like ALL cell line (MHH-CALL-4) was lentivirally transduced with a CRLF2-targeting shRNA driven by an inducible promoter, enabling the inducible knockdown of CRLF2. CRLF2 expression in MHH-CALL-4 cells after shRNA induction (KD-CALL-4) was evaluated using fluorescence-activated cell sorting (FACS). The cellular association of the CRLF2-ScFv was determined in MHH-CALL-4 and KD-CALL-4 at 4° and 37°C using an indirect immunofluorescence assay. Confocal laser scanning microscopy was used to visualize and compare the cellular association of CRLF2-ScFv in MHH-CALL-4 and KD-CALL-4. The cellular association of CRLF2-ScFv was also investigated ex vivo using a small panel of Ph-like and non-Ph-like ALL patient-derived xenografts (PDXs), representing similar immunophenotype and genetic characteristics to their original disease subtypes (Liem et al, Blood 103: 3905-3914, 2004), and peripheral blood mononuclear cells (PBMCs) to investigate the non-selective association. CRLF2 expression in MHH-CALL-4 and Ph-like ALL PDX cells was quantified using indirect immunofluorescence assay. The downstream impact of CRLF2-ScFv on STAT5 phosphorylation (pSTAT5) was determined by FACS either with or without TSLP stimulation. The statistical significance was tested using Unpaired unequal variances t-test or one-way ANOVA followed by multiple comparisons test. Statistical significance was considered when P ≤ 0.05. All experiments were performed in triplicate. Results: KD-CALL-4 showed a 75% reduction in CRLF2 expression compared with MHH-CALL-4 cells (P = 0.0009). CRLF2-ScFv exhibited a 94% higher association with MHH-CALL-4 compared with KD-CALL-4 cells at 37°C (P = 0.0013). The association of CRLF2-ScFv with MHH-CALL-4 cells was reduced by 75% at the non-proliferating state of cells at 4°C compared to 37°C (P = 0.006). Orthogonally viewed confocal microscopy images showed 82% higher intracellular uptake of CRLF2-ScFv in MHH-CALL-4 compared to KD-CALL-4 cells (P = 0.0003). CRLF2-ScFv showed >80% higher association with a Ph-like PDX sample compared with a control CRLF2low PDX and PBMCs (P < 0.001). Of note, a Ph-like ALL PDX exhibited only one-third of the association with CRLF2-ScFv compared with MHH-CALL-4 cells (P = 0.04), corresponding to the significant difference in CRLF2 surface expression (P = 0.01). CRLF2-ScFv significantly reduced pSTAT5 expression in MHH-CALL-4 cells (P = 0.05) and prevented TSLP-induced STAT5 phosphorylation (P = 0.01), suggesting competition with the TSLP binding site. Conclusion: CRLF2-ScFv is a selective targeting moiety for CRLF2 with a significant internalization potential and receptor antagonistic effect, highlighting the therapeutic implications for targeting Ph-like ALL. Keywords: CRLF2, ScFv, STAT5 phosphorylation, Patient-Derived Xenografts. Disclosures No relevant conflicts of interest to declare.

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