Table 1. Questions, offered and collected answers in CSMBLM WG survey.
| Question | Answers | Received answers, N (%) |
|---|---|---|
| First survey (N = 80) | ||
| 1. Please enter the data of your institution. | Descriptive answer | 80 (100) |
| 2. Please specify humoral immunodiagnostic analyses that are performed in your laboratory? | Systemic autoimmune rheumatic diseases (ANA, ENA, anti-dsDNA, anti-histones, anti-nucleosomes, CENP, anti-CCP, RF) | 30 (38) |
| Systemic vasculitis and glomerulonephritis (ANCA, anti-GBM, anti-PR3, anti-MPO) | 14 (18) | |
| Autoimmune liver diseases (AMA, AGLM, LKM, LC-1, SLA, ASGPR) | 6 (8) | |
| Antiphospholipid syndrome (aCL, β2GPI, LA, aPS, anti-PT/PS) | 12 (15) | |
| Autoimmune bowel diseases (ASCA, APS, EMA, anti-tTG, AGA, anti-DGP) | 11 (14) | |
| Autoimmune neurological disorder / paraneoplastic syndrome (anti-Hu, anti-Yo, anti-Ri) | 2 (3) | |
| None of the analysis stated above. | 47 (59) | |
| Question | Answers |
Received answers, N (proportion) |
| First survey for participating laboratories performing humoral immunodiagnostic analyses (N = 33)* | ||
| 3. What is the type of institution were your laboratory operates? | General/county hospital | 11 (0.33) |
| Specialized health institution | 6 (0.19) | |
| University hospital/University hospital centre | 9 (0.27) | |
| Private institution | 7 (0.21) | |
| Other (specify) | 0 (0) | |
| 4. What is the dynamics of providing laboratory reports? | 24 hours | 14 (0.42) |
| Up to 7 days | 4 (0.13) | |
| 7 - 30 days | 14 (0.42) | |
| > 30 days | 1 (0.03) | |
| 5. What is the total number of humoral immunodiagnostic tests per month in your laboratory? | < 500 | 24 (0.73) |
| 500-1000 | 4 (0.12) | |
| > 1000 | 5 (0.15) | |
| 6. Please state the methods and units next to the tests (from the list) that you perform. | Descriptive answer for ANA, ENA, anti-dsDNA, anti-histones, anti-nucleosomes, CENP, anti-CCP, RF, ANCA, anti-GBM, anti-PR3, anti-MPO, AMA, SMA, LKM, LC-1, SLA, ASGPR, aCL, anti-B2GPI, LA, APS, anti-PT/PS, ASCA, APC, EMA, anti-tTG, AGA, anti-DGP, anti-Hu, anti-Yo, anti-Ri. | 33 (1.0) (see text and Tables 3 - 7) |
| 7. If you use IIF for ANA determination, what is your initial dilution? | Descriptive answer | 7 (0.21) (details in Table 3) |
| 8. If you determine ANA with IIF, do you determine titre of autoantibodies? | Yes | 6 (0.18) |
| No | 1 (0.03) | |
| Depending on the fluorescence type | 0 (0) | |
| 9. If you determine ANA using IIF, do you describe the type of fluorescence? | Yes | 7 (0.21) |
| No | 0 (0) | |
| 10. If ANA screening is positive, do you automatically determine specific autoantibodies? | Yes, depending on the fluorescence pattern | 2 (0.06) |
| Yes, depending on the ANA titre | 4 (0.12) | |
| If requested, independently of ANA-screen test result | 2 (0.06) | |
| No | 6 (0.18) | |
| 11. If you use some other methods instead of IIF for ANA screen, do you specify the antigens included in the test on the laboratory report? | Yes | 5 (0.15) |
| No | 5 (0.15) | |
| 12. Do you determine the titre of autoantibodies for analysis (besides ANA) that are tested with IIF? If yes, please state which tests? | Yes | 6 (0.18) (details in text) |
| No | 2 (0.06) | |
| 13. If you perform ANCA screening test by IIF method, do you describe the type of fluorescence? If yes, please state which type of fluorescence you describe. | Yes | 3 (0.09) (details in text) |
| No | 0 (0) | |
| 14. If you determine anti-PR3 and anti-MPO with ANCA-screening, please choose the option that corresponds with your protocol. | They are determined in all samples regardless of the results of ANCA-screening test | 0 (0) |
| They are determined within ANCA-screening test | 0 (0) | |
| They are only determined in samples that are positive in ANCA-screening test. | 4 (0.12) | |
| 15. Do you determine the concentration of total IgA in serum/plasma prior to determination of EMA IgA / tTg IgA? | Yes | 5 (0.15) |
| No | 6 (0.18) | |
| 16. Do you participate in external quality assessment (EQA) with analysis stated in Question No. 6. If the answer is “partially”, please specify the analysis that are included in EQA. | Yes | 13 (0.39) |
| No | 12 (0.36) | |
| Partially (specify) | 5 (0.15) (details in text) | |
| 17. Please state the dynamics of participation in EQA schemes. | Monthly | 2 (0.06) |
| Every 2 - 4 months | 5 (0.15) | |
| Every 6 months | 9 (0.27) | |
| Once a year | 2 (0.06) | |
| Second survey (N = 19) | ||
| Questions | Answers | Received answers, N (proportion) |
| 1. When determining ANA-screen with IIF method, which types of fluorescence do you recognize and report? (Possibility of multiple answers). | Homogenous | 7 (0.37) |
| Speckled / granular | 7 (0.37) | |
| Centromeres | 7 (0.37) | |
| Nucleolar | 7 (0.37) | |
| Nuclear envelope | 5 (0.26) | |
| Mitotic apparatus | 4 (0.21) | |
| Fluorescence of cytoplasm | 6 (0.32) | |
| Other (specify) | 4 (0.21) (details in Table 3) |
|
| We do not perform ANA test with IIF | 12 (0.63) | |
| 2. When determining ANA-screen with IIF do you report different types of fluorescence in the same sample? | We report different types of fluorescence and the corresponding titres | 2 (0.11) |
| We report different types of fluorescence but only one titre | 4 (0.21) | |
| We do not report different types of fluorescence | 0 (0) | |
| Other (please describe) | 1 (0.05) (details in Table 3) |
|
| 3. When determining ANA-screen with IIF, slides are examined by: | One examiner | 1 (0.05) |
| Two examiners (results are reported in agreement) | 3 (0.16) | |
| One examiner in consultation with another if needed. | 3 (0.16) | |
| Other (please describe) | 0 (0) | |
| 4. What is the highest dilution when you determine ANA titre in IIF ANA screening? | Maximally up to: (please state a dilution) | 5 (0.26) (details in Table 3) |
| We dilute till no detectable fluorescence | 2 (0.11) | |
| 5. In which cases after ANA-screen test do you determine anti-dsDNA? | We do not determine anti-dsDNA | 1 (0.05) |
| Only when ordered, regardless of the ANA result | 6 (0.32) | |
| Only when ordered and ANA-screen result is positive | 1 (0.05) | |
| Only when ordered and ANA IIF screen result is positive with a specific type of fluorescence | 0 (0) | |
| Always with positive ANA result, even if anti-dsDNA was not ordered. | 4 (0.21) | |
| Only when ordered and ANA IIF screen result is positive with a titre higher then (please insert titre) | 1 (0.05) (details in Table 5) |
|
| 6. Does your laboratory perform ENA-screening test? | Yes | 4 (0.21) |
| No | 15 (0.79) | |
| 7. Which autoantibodies are included in your ENA-screening test? (Possibility of multiple answers.) | anti-SS-A (Ro60) | 4 (0.21) |
| anti-Ro52 (TRIM21) | 1 (0.05) | |
| anti-SS-B | 4 (0.21) | |
| anti-ribosome P protein | 1 (0.05) | |
| anti-U1RNP | 4 (0.21) | |
| anti-Sm | 4 (0.21) | |
| anti-Scl-70 (Topoisomerase-1) | 4 (0.21) | |
| anti-Jo-1 | 4 (0.21) | |
| anti-CENP | 1 (0.05) | |
| other (please specify) | 1 (0.05) (details in Table 4) |
|
| 8. What method do you use for ENA-screening test? | ELISA | 3 (0.16) |
| Immunoblot (Line blot) | 0 (0) | |
| Multiplex (Luminex) | 0 (0) | |
| FEIA | 1 (0.05) | |
| Other (please specify) | 0 (0) | |
| 9. Results of ENA-screening are expressed as: | Semiquantitative (ratio) | 1 (0.05) |
| Qualitative (positive/negative) | 3 (0.16) | |
| 10. Which specific ENA antibodies do you determine? (Possibility of multiple answers.) | We do not determine ENA specific antibodies | 7 (0.37) |
| anti-SS-A (Ro60) | 12 (0.63) | |
| anti-Ro52 (TRIM21) | 5 (0.26) | |
| anti-SS-B (La) | 12 (0.63) | |
| anti-ribosome P protein | 4 (0.21) | |
| anti-U1RNP | 11 (0.58) | |
| anti-Sm | 11 (0.58) | |
| anti-Scl-70 (Topoisomerase-1) | 12 (0.63) | |
| anti-Jo-1 | 11 (0.58) | |
| anti-CENP | 10 (0.53) | |
| other (please specify) | 6 (0.32) (details in Table 4) |
|
| 11. Which method do you use for ENA specific antibodies determination? | ELISA | 3 (0.16) |
| Immunoblot (Line blot) | 1 (0.05) | |
| Multiplex (Luminex) | 3 (0.16) | |
| FEIA | 4 (0.21) | |
| Other (please specify) | 1 (0.05) (details in Table 4) |
|
| 12. Do you use algorithm for anti-ENA determination? | No, we determine everything that has been ordered | 6 (0.32) |
| Yes, anti-ENA is analysed depending on the ANA-screening result | 4 (0.21) | |
| 13. Do you have a rule regarding the frequency of ENA-specificity repeat testing? If yes, please state the rule. | No | 18 (0.95) |
| Yes (state the rule) | 1 (0.05) (details in text) |
|
| 14. When only anti-dsDNA and/or anti-ENA have been ordered, you perform: | ANA-screen test first, and anti-dsDNA/anti-ENA only in the case of positive ANA | 3 (0.16) |
| Anti-dsDNA and/or anti-ENA are determined without ANA-screening | 9 (0.47) | |
| 15. Do you include interpretative comments on the laboratory report? | No | 14 (0.74) |
| Yes, in the case that fluorescence pattern on ANA-screen test indicates unusual antibodies (e.g. antibodies to Golgi complex, lysosomes, peroxisomes) | 2 (0.11) | |
| Yes, in the case that fluorescence pattern on ANA-screen test indicates other antibodies (e.g. AMA, SMA) | 3 (0.16) | |
| Other (please specify) | 1 (0.05) (details in Table 3) |
|
| 16. As a part of celiac disease screening you determine IgG class antibodies to: | tissue transglutaminase | 2 (0.11) |
| deamidated gliadin | 3 (0.16) | |
| native gliadin | 0 (0) | |
| endomysium (EMA) | 0 (0) | |
| we do not determine IgG class antibodies in celiac disease screening | 6 (0.32) | |
| we do not perform celiac disease screening | 8 (0.42) | |
| 17. When do you determine IgG class antibodies in celiac disease screening? | Only when there is a complete IgA deficiency | 0 (0) |
| When there is a complete IgA deficiency and in children ≤ 2 years | 3 (0.16) | |
| In case of decreased IgA or in a complete IgA deficiency | 0 (0) | |
| In case of decreased IgA or in a complete IgA deficiency and in children ≤ 2 years | 0 (0) | |
| Always, together with IgA class antibodies | 2 (0.11) | |
| 18. What is your starting dilution in ANCA IIF- screening test? | 1: 5 | 0 (0) |
| 1: 10 | 2 (0.11) | |
| 1: 20 | 1 (0.05) | |
| 1: 40 | 0 (0) | |
| We do not perform ANCA-screening test | 15 (0.79) | |
| 19. In ANCA IIF-screening test, do you use formalin-fixed granulocytes? | No | 1 (0.05) |
| Yes, always with ethanol-fixed granulocytes | 2 (0.11) | |
| Yes, only in case of ANCA positive test on ethanol-fixed granulocytes | 0 (0) | |
| Yes, only in case of pANCA positive test on ethanol-fixed granulocytes | 0 (0) | |
| Yes, only in case of atypical pANCA positive test on ethanol-fixed granulocytes | 0 (0) | |
| Yes, only if ANCA is ordered from gastroenterologist | 0 (0) | |
| Yes, only in case of positive ANA | 0 (0) | |
| 20. Do you determine other ANCA specificities except PR3 and MPO (elastase, lactoferrin, etc.)? If yes, please specify. | Yes (specify) | 0 (0) |
| No | 19 (1.0) | |
| 21. In the case of positive liver kidney microsome (LKM) antibodies with IIF method, do you automatically determine LKM-1 specificity? | Yes | 3 (0.16) |
| No | 0 (0) | |
| We do not determine anti-LKM with IIF method | 16 (0.84) | |
| 22. In the case of positive AMA with IIF method, do you automatically determine AMA-M2 specificity? | Yes | 4 (0.21) |
| No | 0 (0) | |
| We do not determine AMA with IIF method | 15 (0.79) | |
| 23. Do you use algorithm when antiphospholipid antibodies are ordered? | No, only aCL antibodies are determined | 2 (0.11) |
| No, only LA is determined | 3 (0.16) | |
| No, LA and aCL are determined simultaneously | 0 (0) | |
| No, aCL and anti-B2GPI are determined simultaneously | 3 (0.16)** | |
| No, aCL, anti-B2GPI and LA are determined simultaneously | 3 (0.16) | |
| Yes, anti-B2GPI antibodies are determined depending on the aCL results | 0 (0) | |
| We do not determine antiphospholipid antibodies | 7 (0.37) | |
| 24. Which isotypes of aCL do you determine? | IgG and IgM | 8 (0.42) |
| IgG, IgM and IgA | 0 (0) | |
| We do not determine aCL | 11 (0.58) | |
| 25. Which isotypes of anti-B2GPI do you determine? | IgG and IgM | 7 (0.37) |
| IgG, IgM and IgA | 0 (0) | |
| We do not determine anti-B2GPI | 12 (0.63) | |
| Question and answers obtained after personal communication with managers of 7 laboratories that perform IIF method for antibodies detection | ||
| 1. Who is authorised to perform examination of IIF slides in your laboratory? | specialist in medical biochemistry | 4 (0.57) |
| master of medical biochemistry and laboratory medicine | 2 (0.29) | |
| other (specify) | 1 (0.14) (details in Table 3) |
|
|
*Out of 80 participating laboratories, 47 declared to not perform humoral immunodiagnostic analyses (see question 2). **Including laboratory that simultaneously determines aCL, anti-B2GPI and aPS. ANA = antinuclear antibodies. ENA = extractible nuclear antibodies. anti dsDNA = anti-double stranded DNA. SS-A/Ro60 = Anti-Sjögren’s-syndrome-related antigen A. Ro52 (TRIM21) = Tripartite motif-containing protein 21. SS-B/La = Sjögren syndrome type B antigen. U1-RNP = U1 ribonucleoprotein. PM/Scl = Polymyositis/Scleroderma. PCNA = Pleomorphic cell nuclear antigen. Sm = Smith antigen. Scl-70 = topoisomerase I. Jo-1 = histidyl tRNA synthetase. CENP = antibodies targeting centromere proteins. anti-CCP = antibodies targeting synthetic cyclic citrullinated peptides. RF = rheumatoid factor. ANCA = anti-neutrophil cytoplasmic antibodies. anti-PR3 = anti-proteinase 3 antibodies. anti-MPO = anti-myeloperoxidase antibodies. anti-GBM = anti-glomerular basement membrane antibodies. AMA = antimitochondrial antibodies. AGLM = Anti-smooth muscle antibody. LKM = liver-kidney microsomal antibodies. LC-1 = liver cytosol. SLA = soluble liver antigen. ASGPR= asialoglycoprotein receptor. aCL = anticardiolipin antibodies. anti-B2GPI = anti-beta2 glycoprotein I antibodies. LA = lupus anticoagulant. aPS = antiphosphatidilserine antibodies. anti-PT/PS= anti-phosphatidylserine/prothrombin antibodies. ASCA = anti-saccharomyces cerevisiae antibodies. APCA = antibodies against parietal cells. EMA = anti-endomysium antibodies. anti-tTG = antibodies against tissue transglutaminase. AGA = antibodies against native gliadin. anti-DGP = antibodies targeting deamidated gliadin peptides. | ||