Re-Evaluating the Current ANCA Screening Method: Multiplex Indirect Immunofluorescence Assay in ANCA Testing
Bibliographic record
Abstract
Objective:The Indirect Immunofluorescent Test (IIFT) method is a crucial component of Anti-Neutrophil Cytoplasmic Antibodies (ANCA) diagnostics as it is the only method that can be used to detect not only small vessel vasculitis related ANCA, but also ANCAs in Chronic Inflammatory Bowel Disease (CIBD) and other disease conditions.The conventional IIFT method only uses ethanol fixed granulocytes for initial testing, which results in a lack of specificity.The purpose of this study is to outline an improvement in the screening and interpretation of ANCA by IIFT, using a 3-chip combination per incubation well.With the inclusion of an ethanol fixed granulocyte chip, formalin fixed granulocyte chip, and mixed cell chip containing both ethanol fixed granulocytes and HEp-2 cells, we aim to demonstrate a reduction in the number of false positives and false negatives, increase ANCA testing specificity without the loss of sensitivity, as well as simplify the reporting.Design/Method: 261 serum samples were obtained from the serum bank of Euroimmun Canada.Each sample was tested for both Anti-Nuclear Antibodies (ANA) and ANCA (3-chip combination) using IIFT, and was also tested using Anti-MPO/PR3 ELISA.ANCA profile (including six antigens) ELISA was used as well.All reagents were from Euroimmun.Results: Distinct ANCA patterns were identified according to our simplified scheme, including: ANCA (c-or p-ANCA), atypical ANCA (atypical c-or p-ANCA) and atypical inconclusive ANCA.The use of a 3-chip combination eliminated ANA interference and resulted in a significant reduction of 75.6% in the unsure "positive" ANCA results that were initially determined from using ethanol fixed granulocytes only.The ethanol fixed granulocyte chip and formalin fixed granulocyte chip are important for pattern classification, while the mixed cell chip is crucial for differentiating true positive ANCA from ANA interference.Noticeably, this IIFT 3-chip combination testing revealed 5% more true ANCA positive samples, which were ANCA negative when using ethanol fixed granulocytes only as the initial screening. Conclusion:The use of a 3-chip combination multiplex IIFT approach is necessary for an accurate interpretation and analysis of patient serum for ANCA screening.This proposed inclusion of additional substrates in the IIFT ANCA diagnostic procedure has shown to result in increasing specificity of the IIFT, while maintaining a high sensitivity.The simplified reporting also makes the ANCA result standardization a reality.ANCA IIFT method continues to be very useful in routine testing for different groups of diseases.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.010 | 0.011 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.002 | 0.001 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.003 | 0.002 |
| Open science | 0.002 | 0.001 |
| Research integrity | 0.002 | 0.002 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".