Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

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

The antinuclear antibody HEp-2 indirect immunofluorescence assay: a survey of laboratory... Background: To evaluate the interpretation and reporting of antinuclear antibodies (ANA) by indirect immunofluo - rescence 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 nomen- clature [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 associ- ated with cytoplasmic patterns. Conclusions: Our study demonstrated significant accuracy for all participants in identifying the categorical nuclear stain- ing as well as traditional pattern assignments for nuclear patterns. However, there was less consistency in reporting cyto- plasmic and mitotic patterns, with implications for assigning competencies and training for clinical laboratory personnel. Keywords: Anti-nuclear antibodies, Cytoplasmic patterns, Performance survey, Indirect immunofluorescence, Mitotic patterns, Nuclear patterns *Correspondence: anne.tebo@hsc.utah.edu Department of Pathology, University of Utah, Salt Lake City, UT, USA Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. Tebo et al. Autoimmun Highlights (2021) 12:4 Page 2 of 10 HEp-2 IFA nuclear, cytoplasmic and mitotic patterns Introduction have been elucidated by ICAP and presented in a clas- The presence of antinuclear antibodies (ANA) is a sification tree (www.anapa ttern s.org). The ICAP guide - hallmark and classification criterion for a number of lines indicate that ‘expert-level’ laboratories would systemic autoimmune rheumatic diseases (SARD). report all the HEp-2 IFA patterns, whereas those des- ANA testing is usually performed as part of the initial ignated as ‘competent-level’ laboratories would report 6 diagnostic workup when suspicion of an underlying nuclear and 5 cytoplasmic HEp-2 IFA patterns [12]. autoimmune disorder is high. The indirect immuno - In a previously reported survey administered in coop- fluorescence antibody (IFA) technique on HEp-2 sub - eration with the Association of Medical Laboratory strate has been considered the traditional and preferred Immunologists (AMLI), a significant number of respond - method for detecting ANA by some [1]. It allows detec- ents were unaware of the ICAP initiative, although a tion of antibody binding to specific intracellular targets, majority agreed on the need to standardize the nomen- resulting in diverse staining patterns that are usually clature and reporting of HEp-2 IFA results [19]. Based on categorized based on the cellular components recog- the responses from this survey, a consensus to improve nized and the degree of binding, as reflected by the flu - ICAP awareness and further enhance HEp-2 IFA assess- orescence intensity or titer [2, 3]. As a screening tool, ment through increased collaboration between ICAP and the recognition of a well-defined HEp-2 IFA staining the clinical laboratory community was suggested with pattern may be helpful in determining the most likely emphasis on education and availability of reference mate- specific autoantibodies present, as well as suggesting rials. As others have also reported [20, 21], many labora- possible clinical associations for known specificities [3 , tories around the world are inclined to adopt the ICAP 4]. In this regard, a positive HEp-2 IFA screening pat- nomenclature and embrace the recommendations pro- tern can guide confirmatory testing and may also be vided in these consensus guidelines. useful for elucidating a specific clinical diagnosis or The objective of this study was to evaluate the perfor - prognosis. Thus, the provision of HEp-2 IFA patterns mance of HEp-2 IFA interpretation based on nuclear, and titers is considered to be clinically valuable with cytoplasmic and mitotic staining in an endeavor to favorable utility in comparison with other methods for characterize competency as outlined in the ICAP ANA detection [1–11]. classification. The nuclear IFA staining patterns most commonly recognized and reported by clinical laboratories include Materials and methods homogeneous, speckled, centromere, and nucleolar [1– Participants and recruitment 4, 12–14]. Use of HEp-2 cell substrates, permits detec- Participants for the survey were recruited via an email tion of additional nuclear staining patterns, as well as sent to the Association of Medical Laboratory Immunol- reactivity with cell constituents in compartments out- ogists (AMLI) membership (a professional organization side the nucleus (cytoplasmic) and cell components focused on immunological laboratory testing with 105 associated with mitosis (mitotic) [2, 4, 12–14]. How- active members) and 5 in  vitro diagnostics manufactur- ever, the reactivity and type of autoantigens associated ers (IVD). The requirements to participate included test - with these patterns may vary among HEp-2 substrates ing pre-defined specimens by IFA using HEp-2 substrate from different manufacturers [15]. Furthermore, the and reporting the patterns observed based on specific expertise required to identify the different patterns and “traditional” (as defined below) as well as ICAP (www. sub-classify their variants may not be universally avail- anapa ttern s.org) nomenclature for reading and reporting able in clinical laboratories. Traditional legacy recom- ANA patterns. Sixteen (n = 16) clinical laboratories (CL) mendations for reporting ANA patterns on HEp-2 cells and all 5 in vitro diagnostics manufacturers (IVD) agreed continue to significantly influence clinical laboratory to participate in the study. Three additional IVD (2 in the reporting [16, 17]. US and 1 in Europe), contacted the organizers to partici- The first I nternational Consensus on Antinuclear pate in the survey. Overall, 16 CL and 8 IVD participated Antibody (ANA) Patterns (ICAP) was published in in this performance survey (Additional file 1: Table S1). 2015 to systematize and update reporting of autoan- tibody patterns detected by IFA using HEp-2 cell sub- Study specimens and survey strates [12]. The goal of this initial and subsequent Twelve (n = 12) specimens were used in the survey. The publications was to optimize usage of HEp-2 IFA pat- specimens were chosen based on an assessment of need terns in patient care, by promoting standardization, (described below) by some of the authors (AET, LKP, harmonization and understanding of autoantibody test EKLC, MJF, MHW). The following attributes were taken nomenclature and providing guidelines for test inter- into consideration: 1) the three main categorical group pretation and reporting [4, 12–15, 18]. To-date, 30 T ebo et al. Autoimmun Highlights (2021) 12:4 Page 3 of 10 of HEp-2 IFA patterns, 2) the ICAP guidance for both on the ICAP classification tree (www.anapa ttern s.org), competent and expert levels, 3) the clinical significance which includes more detailed sub-pattern classifica - of patterns and 4) whether or not proficiency testing was tion than commonly reported. In addition, the partici- available for specific patterns. The authors also wanted to pants were requested to provide information about how evaluate how participants would interpret nuclear stain- the images were read and interpreted (manual and/or ing associated with anti-topoisomerase I antibodies given automation-assisted reading); the years of experience recent ICAP guidance for evaluating this complex pat- of the reading technologist(s); the manufacturer of the tern [22]. The twelve pre-specified HEp-2 IFA-positive HEp-2 substrate; the laboratory’s typical practice about specimens labeled ANA-001 through ANA-012 included reporting only nuclear patterns vs also reporting cyto- those positive for nuclear [ANA-002, ANA-003, ANA- plasmic and/or mitotic patterns when the ANA test is 005, ANA-006, ANA-007, ANA-010, ANA-011], cyto- requested; the screening dilution(s) of serum used for plasmic [ANA-004, ANA-008, ANA-009] and mitotic detection of ANA in performing the HEp-2 IFA; and the [ANA-001, ANA-012] categorical groups of IFA patterns titer of the ANA, based on serial dilution of the tested (Table 1). All specimens for the survey and their intended specimen. There were two types of participants: clinical responses were obtained from Plasma Services Group laboratories (CL) and in  vitro diagnostic manufactur- Inc. (PSG: Huntington Valley, PA, USA, https ://www. ers (IVD). After the results were tabulated, participants plasm aserv icesg roup.com/). Specimens were qualified were not afforded the opportunity to adjust or revise at PSG using routinely available methods and also veri- responses based on the responses of other respondents. fied in the laboratories of one or more expert members of Some of the participating CL included those directed ICAP (PSG, personal communication). by the authors, but the authors did not participate in Survey specimens were shipped to all participants in assigning the patterns reported from their laboratories. January 2020 with detailed instructions for testing as well as a report form to record and return results to one Data analyses of the organizers (AET). Parameters to be recorded by We compared participants’ HEp-2 IFA pattern clas- checking the survey form included the three categori- sification of specimens against a consensus classifica - cal groups of HEp-2 IFA patterns reported (nuclear, tion. The primary outcome was the percent accuracy cytoplasmic or mitotic); commonly used nomencla- between the participant and consensus classification. ture (also referred to as traditional in this investiga- We studied the impact of three factors on accuracy: tion) for 5 HEp-2 IFA nuclear patterns (homogeneous, pattern classification hierarchy or nomenclature, par - speckled, centromere, nucleolar, discrete nuclear dots), ticipant organization type, and participant experi- mitotic, cytoplasmic or ‘other’ in accord with legacy ence. We examined three hierarchial  nomenclatures: classification approaches [16, 17]; and a result based 1) group category (nuclear, cytoplasmic, mitotic); 2) Table 1 Survey specimens and their characteristics Specimen Cellular staining Traditional nomenclature ICAP nomenclature ICAP level ANA-001 Mitotic Mitotic Spindle fiber, AC-25 Expert ANA-002 Nuclear Discrete nuclear dots Multiple nuclear dots, AC-6 Expert ANA-003 Nuclear Speckled Coarse speckled, AC-5 Expert ANA-004 Cytoplasmic Cytoplasmic Reticular/AMA, AC-21 Competent ANA-005 Nuclear Centromere Centromere, AC-3 Competent ANA-006 Nuclear Nucleolar Homogeneous nucleolar, AC-8 Expert ANA-007 Nuclear Speckled DFS, AC-2 Competent ANA-008 Cytoplasmic Cytoplasmic DFS, AC-19 Expert ANA-009 Cytoplasmic Cytoplasmic Fine speckled, AC-20 Expert ANA-010 Nuclear Speckled Fine speckled, AC-4 Expert ANA-011 Nuclear Speckled/Other* Anti-topoisomerase I, AC-29 Expert ANA-012 Mitotic Mitotic NuMA-like, AC-26 Expert number, ICAP International Consensus on Antinuclear Antibody Pattern, AC anti-cell, AMA anti-mitochondrial antibodies, DFS dense fine speckled, NuMA nuclear mitotic apparatus protein. The AC-29 Anti-topoisomerase pattern I is a compound pattern, classified within ICAP as a speckled pattern. The complex pattern involves speckled nuclear staining, and also includes staining of the condensed chromatin, cytoplasmic staining, staining of the nucleolar organizing region in mitotic cells, and variable nucleolar staining of interphase cells. For this specimen, AC-25 was also considered acceptable Tebo et al. Autoimmun Highlights (2021) 12:4 Page 4 of 10 specific traditional pattern descriptions (e.g. speckled, Table 2 Characteristics of survey participants nucleolar, etc.); and 3) sub-pattern classification using a a Characteristics CL, N (%) IVD, N (%) the ICAP nomenclature. We refer to these as the group, HEp-2 kit Bio-Rad 4 (25.00) See legend traditional and ICAP classification methods. Each par - Euroimmun 5 (31.25) ticipant was classified according to the organizational Inova 6 (37.50) type and the reporting experience at their institution. MBL Bion 1 (6.25) There were two types of organizations: 1) clinical labo - Type of reader Manual only 9 (56.25) 5 (62.50) ratories (CL) and 2) in  vitro diagnostic manufacturers Manual and Auto- 7 (43.75) 3 (37.25) (IVD). Organizations were classified as experienced if mated they routinely reported all group categories and inex- Cut-off (titer) < 1:10 1 (6.25) 0 (00.00) perienced if they did not routinely report all three < 1:40 12 (75.00) 6 (66.67) group categories. Using these three factors and their < 1:80 3 (18.75) 2 (22.22) associated variables, we sought to answer the following < 1:100 0 (00.00) 1 (11.11) questions: Technologist 1–5 years 7 (33.30) 0 (00.00) experience 6–10 years 4 (12.10) 1 (11.11) 1. Was accuracy associated with the classification > 10 years 10 (47.60) 8 (88.89) method? Median (range), years 10 (1-45) 20 (2-51) 2. Was accuracy associated with experience? Nuclear only 3 (18.75) 0 (00.00) Patterns reported 3. Was accuracy associated with organization type Cytoplasmic and 2 (12.50) 0 (00.00) among experienced participants? nuclear 4. Was accuracy associated with categories within Mitotic and nuclear 3 (18.75) 0 (00.00) nomenclature methods? All patterns 8 (50.00) 8 (100.00) Number (N) of clinical laboratories (CL) or in vitro diagnostic manufacturers We used logistic regression to determine the asso- (IVD) unless otherwise stated. Participating IVD manufacturers included: ciation between accuracy and the three factors. Out- AESKU Diagnostics, Bio-Rad, Euroimmun, Inova, ImmunoConcepts, Scimedx, ThermoFisher and Zeus. CL labs using Inova HEp-2 substrate kits use < 1:40 comes were reported as odds ratios (OR). P-values were or < 1:80 as cut-off for ANA determinations. More than one technologist was adjusted for multiple comparisons using the method of involved in the reading and interpretation of the results in some laboratories. One laboratory in each group reports cytoplasmic pattern only as a comment Holm. Statistical analyses were performed using Stata 16.2 (Stata Corp LLP). they report patterns in all 3 categorical groups, however, Results all provided responses to all categories in the survey. Characteristics of survey participants There were 24 participants: 16 were CL (13 in the Performance of participants based on the categorical United States and 3 in Canada) and 8 IVD. Most of the HEp‑2 IFA groups CL used kits from three main IVD that also partici- The accuracy for reporting the nuclear pattern was 99% pated in the survey (Table  2). The majority of the CL (95% CI 95–100%) for all participants, while the cyto- read, interpreted and determined HEp-2 IFA patterns plasmic and mitotic group categories had accuracy of and titers manually using 1:40 as cut-off for HEp-2 78% (CI 66–87%) and 93% (CI 81–88%), respectively IFA determinations. The median years of experience (Table  3). The overall accuracy of IVD was greater than for technologists who participated in the survey was accuracy of CL (97% vs 91%, Additional file  2: Table S2a) 10  years for CL compared to 20  years for the IVD in assigning the HEp-2 IFA group categories of patterns participants. of all specimens. This difference was statistically signifi - The number of categorical groups of patterns typically cant (p = 0.04). Combined, the two organization types reported by the CL was variable. Among the 16 CL, 3 (CL and IVD) had an overall accuracy of 93% (95% CI indicated they reported only nuclear patterns, 3 indicated 89–96%) for determining the three categorical groups of they reported nuclear and mitotic patterns, 2 reported HEp-2 IFA patterns. nuclear and cytoplasmic patterns (with one of the 2 reporting the cytoplasmic pattern as a comment, not as Performance of participants based on “traditional” a ‘positive ANA’) and 8 indicated the laboratory reported and ICAP nomenclatures nuclear, cytoplasmic and mitotic patterns (with one of Participants were asked to report results based on sur- the 8 reporting the cytoplasmic patterns as a comment, vey-suggested classifications (referred here as “tradi - not as a ‘positive ANA’). Among the 8 IVD, 6 indicated tional”) as well as the ICAP nomenclature. The overall T ebo et al. Autoimmun Highlights (2021) 12:4 Page 5 of 10 Table 3 Performance of  Participants in  the  Three HEp-2 Several specimens yielded unexpected results. For IFA Group Categories example, ANA-010 was intended to represent a nuclear fine speckled pattern (AC-4), but that specimen was Group category Specimens Observations Accuracy (95% CI) (Number, n) (Number, n) reported as ICAP pattern AC-4 by only a minority of CL and IVD. The survey showed that all participants Nuclear 7 168 99 (95–100) reported this specimen as having a speckled nuclear pat- Cytoplasmic 3 63 78 (66–87) tern using traditional descriptions, but a majority (75% of Mitotic 2 45 93 (81–98) IVDs and 55% of CL) reported it as having coarse speck- Overall 12 276 93 (89–96) led nuclear staining (AC-5) rather than the expected CI confidence interval AC-4. Review of images from several participating labo- ratories revealed that the specimen produced patterns ranging from typical fine speckled to coarse speckled accuracy for the traditional nomenclature system was nuclear staining using different HEp-2 cell sources. Simi - 87% (95% CI 82–90%), Table  4. Only the specimen larly, the specimen (ANA-009) intended to represent a (ANA-011) with antibodies to DNA topoisomerase I was cytoplasmic fine speckled pattern (AC-20) was reported reported with accuracy less than 80% and that complex with other patterns by a majority of participants and mixed/compound pattern had not been included in many review of images from different laboratories showed a ANA pattern classification teaching schemes prior to its variable pattern of staining depending on the source of recent inclusion as a distinct ICAP pattern [20]. Among the HEp-2 substrate (data not shown). the traditional pattern reports, two specimens (ANA- The specimen with antibodies to topoisomerase I 003, centromere and ANA-006, nucleolar) were reported (topo-1, AC-29 pattern) was reported as homogeneous with accuracy of 100%. by most (56%) CL participants and as a mixed (homoge- The overall accuracy for the ICAP nomenclature neous and nucleolar) pattern by an additional 19% of CL reporting system was 74% (95% CI 68–79%), Table  4. using the common pattern descriptions. The IVD partici - For the ICAP nomenclature, 5 out of the 12 (41.7%) pants reported it as having a variety of mixed nucleolar specimens were reported with overall accuracy over patterns. Using ICAP nomenclature, 88% of IVD partici- 80%. These included AC-3: centromere, AC-6: multiple pants and 54% of responding CL participants assigned nuclear dots, AC-2: dense fine speckled (with 100% of the specimen as having the AC-29 (anti-topoisomerase I) IVD and 67% of responding CL accurately), AC-21: AMA pattern. and AC-26: NuMA-like. For ANA-012, participants Overall, for all participants, the accuracy was 13% reporting AC-25 and AC-26 were graded as having con- greater for the traditional nomenclature (87%, 95% CI sensus for the intended report. Table 4 Performance of participants in the traditional and ICAP nomenclature systems Specimen ID Traditional Observations Accuracy (95% CI) ICAP Observations Accuracy (95% CI) (n) (n) ANA-005 Centromere 24 100 (86–100) Centromere, AC-3 20 100 (83–100) ANA-002 DND 24 96 (73–99) MND, AC-6 20 85 (60–95) ANA-003 Speckled 24 100 (86–100) Nuclear CS, AC-5 18 67 (41–85) ANA-007 Speckled 24 96 (73–99) Nuclear DFS, AC-2 20 80 (55–93) ANA-010 Speckled 24 83 (62–94) Nuclear FS, AC-4 19 37 (18–61) ANA-011 Speckled/Other 24 42 (23–63) Anti-topo I, AC-29 21 62 (39–81) ANA-006 Nucleolar 24 100 (86–100) Homo nucleolar, AC-8 20 70 (46–87) ANA-004 Cytoplasmic 19 100 (86–100) AMA, AC-21 19 89 (66–97) ANA-008 Cytoplasmic 20 90 (65–98) Cytoplasmic DFS, AC-19 19 79 (53–92) ANA-009 Cytoplasmic 19 53 (30–74) Cytoplasmic FS, AC-20 19 42 (22–66) ANA-001 Mitotic 22 86 (64–96) Spindle fiber, AC-25 20 75 (50–90) ANA-012 Mitotic 24 92 (70–98) NuMA-like, AC-26 22 95 (71–99) Overall 250 87 (82–90) Centromere, AC-3 237 74 (68–79) ICAP International Consensus on Antinuclear Antibody Patterns). ID identification number, AC anti-cell, DND discrete nuclear dots, MND multiple nuclear dots, CS coarse speckled, DFS dense fine speckled, FS fine speckled, AMA anti-mitochondrial antibodies, homo homogeneous, anti-topo I anti-DNA topoisomerase I, CI confidence interval, NuMA nuclear mitotic apparatus. Variation in observation numbers in table reflects the fact that some laboratories did not report all cytoplasmic or mitotic categorical group patterns or ICAP patterns for some specimens Tebo et al. Autoimmun Highlights (2021) 12:4 Page 6 of 10 82–90%) compared to the ICAP nomenclature (74%, 95% was significantly less accurate than the group cat - CI 68–79%, Additional file  2: Table  S2b and Tables  3). egory nomenclature (OR = 0.48, p = 0.014). The aver - However, the accuracy for reporting the ICAP nomencla- age accuracy of the ICAP nomenclature was 74% (95% tures were lower for CL than IVD with an overall accu- CI 68–79%) which was significantly less than the group racy of 81% (95% CI 77–84%, Additional file 2: Table S2). category nomenclature (OR = 0.20, p = 0.002) and the The accuracy of classification was associated with par - traditional nomenclature (OR = 0.42, p = 0.002), Table 5. ticipant type (χ p < 0.0005) and nomenclature system Experienced participants had higher accuracy than (p < 0.0005). The accuracy of the CL group was 15% less nonexperienced participants (OR = 2.2, p < 0.0005). than the IVD group. The accuracy of experienced participants was greater To assess the performance of each organization type than the accuracy of nonexperienced participants for based on the accuracies for the main categorical, tradi- all nomenclatures. The difference was 6% for the group tional and ICAP nomenclature determinations, the data method, 10% for the traditional nomenclature and 13% were stratified, and frequencies of the correct intended for the ICAP nomenclature. responses estimated (data not shown). Both groups were effective in determining the intended nuclear staining, Impact of participant type however, the CL group demonstrated lower frequen- All IVD participants were experienced and 8 of the 16 CL cies of the expected responses for the different nomen - participants were experienced (with experience defined clatures. This was most pronounced for the ICAP as routinely reporting all group categories). Among expe- nomenclature. rienced participants, IVD had greater accuracy than CL (OR = 2.8, p = 0.002, Table  5). On average, the accuracy Impact of nomenclature and participant experience of the IVD participants was 92% (95% CI 89–95%) and on accuracy the accuracy of the experienced CL participants was Participants had the highest accuracy using the group 83% (95% CI 78–88%). The accuracy was associated with category nomenclature (Table  3). The average accu - nomenclature. The accuracy of the ICAP nomenclature racy associated with the group category nomenclature was 78% (95% CI 71–84%) which was significantly lower was 93% (95% CI 90–96%). For the traditional nomen- (OR = 0.16, p = 0.002) than the accuracy of the group clature, the average was 87% (95% CI 83–91%) which nomenclature (95%, 95% CI 92–98%) and significantly Table 5 Accuracy of classification based on experience and participant type Classification nomenclature Experience Observations (n) Accuracy (95% CI) Average (95% CI) P value Group category (n = 276) No 87 89 (82–95) 93 (90–96) Base Yes 189 95 (92–98) Traditional (n = 272) No 84 80 (71–89) 87 (83–91) 0.014 Yes 188 90 (86–94) ICAP (n = 232) No 67 64 (53–76) 74 (68–79) 0.002 0.002 Yes 170 77 (73–84) All (n = 785) No 238 79 (73–84) 85 (83–88) 0.002** Yes 547 88 (85–91) Group category (n = 189) IVD 96 97 (93–100) 95 (92–98) Base CL* 93 94 (88–99) Traditional (n = 188) IVD 96 97 (93–100) 90 (86–94) 0.05 CL* 92 83 (75–91) ICAP (n = 170) IVD 95 83 (76–91) 78 (71–84) 0.002 0.002 CL* 75 71 (60–81) All (n = 547) IVD 287 92 (89–95) 88 (85–91) 0.002** CL* 260 83 (78–88) HEp-2 cell IFA patterns were evaluated based on experience for all participants (yes or no), and experienced participant types (in vitro diagnostics manufacturers, IVD) and experienced clinical laboratories (CL*). Experienced CL defined as reporting all 3 main nomenclature categories. All IVD participants reported the three a b nomenclature categories and are rated experienced. CI: confidence interval, n number. ICAP vs Group category and Traditional vs ICAP **Indicates significant difference between groups T ebo et al. Autoimmun Highlights (2021) 12:4 Page 7 of 10 demonstrates competence for participants in identifying lower (OR = 0.35, p = 0.002) than the traditional nomen- and reporting common nuclear ANA patterns, but incon- clature (90%, 95% CI 86–94%). Among the CLs, use of sistency in the decision to report and pattern reporting of automation-assisted reading trended toward improved cytoplasmic and mitotic patterns. accuracy of pattern reporting for both traditional (87% In recent years, efforts to standardize interpretation vs 82% accuracy) and ICAP (70% vs 55% accuracy), but and reporting of HEp-2 patterns have led to a consen- these differences were not statistically significant. sus nomenclature presented by ICAP, a group of experts [4, 12–14] with the purpose of systematic reporting and Frequency distribution of end‑point titers optimizing the usage of HEp-2 IFA patterns in patient For each of the 12 specimens analyzed, the distribution care [4]. In a previous study, we identified increas - of the reported antibody titer was recorded. The screen - ing awareness of this guidance; availability of reference ing titer of determinations ranged from 1:10 to 1:80. The materials for training and collaboration between profes- titers were generally quite variable (Fig. 1, shown for CL). sional organizations, IVD and CL amongst others as key The specimens with cytoplasmic patterns were often not elements necessary for improved harmonization of the titered, particularly by laboratories that did not routinely HEp-2 IFA reporting [19]. report cytoplasmic patterns. Of these specimens, the titer In addition to accurately reporting binding of autoan- variability was most pronounced for ANA-004 (AMA, tibodies to defined cellular components, the survey also AC-21) with positive results demonstrating a bimodal evaluated responses based on “traditional” categoriza- response which spanned eight twofold titers ranging tion for nuclear patterns as well as the emerging ICAP from 1:80 1:10,240 for CL reporting this pattern. Nuclear nomenclature. As expected, all participants performed pattern staining titers also varied substantially, spanning better with the more widely used or common traditional from four to seven twofold titers in different specimens. HEp-2 IFA nomenclature, which has more emphasis on limited nuclear staining features than required to cor- Discussion rectly assign ICAP patterns. While the reason for this Using pre-tested and selected patient serum specimens, could be due to limited familiarity with ICAP, based on we report here the performance of 24 CL and IVD partic- the data, other reasons for this can be inferred. First, the ipants recruited from a professional organization focused “traditional” categorization which can also be referred to on immunological laboratory testing and accustomed to as the ICAP “competent-level” is broad and minimizes the the interpretation and reporting of HEp-2 IFA patterns. use of fine details and/or integrated pattern recognition The specimens included examples from all 3 main cat - in its interpretation. For example, most responders were egorical patterns (nuclear, cytoplasmic, or mitotic), were capable of identifying ANA-003 and ANA-007 as nuclear reported using ‘traditional’ and ICAP nomenclatures, speckled patterns but failed to accurately demonstrate and included patterns designated by ICAP as associated the intended ICAP nomenclatures, coarse speckled/ with both ‘competent’ and ‘expert’ laboratories. Our data Titer 1:10240 1:5120 1:2560 1:1280 1:640 1:320 1:160 1:80 1:40 Not Titered 1000000 10 9852 ICAP AC# 23456 8291920212526 Nuclear Patterns Cytoplasmic Patterns Mitotic Patterns Fig. 1 Distribution of end-point titers for survey specimens reported by clinical laboratory (CL) participants. The frequency distributions of titer values for the 12 samples as reported by 16 CL participants is graphically illustrated. The number of CL reporting titer (1:40 to 1:10,240) for each AC-numbered specimen is shown, as well as the number of clinical labs that did not titer the specimen. The distance between vertical lines represents 10 participants Tebo et al. Autoimmun Highlights (2021) 12:4 Page 8 of 10 AC-5, and dense fine speckled/AC-2, respectively. In nuclear dense fine speckled, AMA and NuMA-like sub- fact, a number of respondents classified the AC-2 DFS patterns. The NuMA-like pattern is considered uncom - specimen as a mixed nuclear homogeneous and nuclear mon, and expected to be recognized by “Expert” level speckled pattern, as might be expected for traditional laboratories, but it has a characteristic appearance, and classification based on speckled staining of the nucleo - has clinically significant associations with a number of plasm and intense chromatin staining. The combina - SARD [30]. Second, a significant group of participants tion requires integration to assign the nuclear DFS AC-2 could identify challenging ICAP-designated sub-pat- ICAP pattern, rather than describing mixed homogene- terns. These include the homogeneous nucleolar, cyto - ous and speckled staining pattern with which it might be plasmic dense fine speckled, spindle fiber, nuclear coarse confused. speckled, and anti-topoisomerase I patterns. Except for The specimen with antibodies to DNA topoisomer - the AMA pattern, the overall performance of the CL par- ase I and the AC-29 staining pattern also demonstrated ticipants for specimens with the cytoplasmic patterns remarkable challenges of consistent ANA pattern report- was more variable, and lower than the IVD group. These ing. Under ICAP, AC-29 is considered a sub-pattern of observations have implications for defining competency nuclear speckled staining [22], but only a minority of par- for CL for cytoplasmic and mitotic patterns. ticipants reported it as a nuclear speckled pattern using A minority of participants interpreted the nuclear traditional nomenclature. Using “traditional” classifica - fine speckled and cytoplasmic fine speckled sub-pat - tions, the pattern is a compound, mixed staining pattern tern specimens as intended. The data suggested that the in which the speckled component may not be perceived HEp-2 patterns generated by those specimens had a suf- as dominant, even though it is consistently observed. ficiently variable appearance, based on the kit manufac - In addition to the speckled nuclear staining, there is turer, and/or kit lot, to lead the specimens to appear as also staining of condensed chromatin in the mitotic different ICAP categories in the hands of different par - cells, making it difficult to distinguish from homogene - ticipants. That hypothesis was confirmed by our direct ous nuclear staining, as reported by majority of the CL, review of the appearance from different laboratories and nucleolar staining also is often present. Dellavance (data not shown). The observations reinforce the need for and colleagues [23] first reported on a composite of five harmonization of reagents, as well an enhanced training unique HEp-2 staining attributes associated with posi- in pattern interpretation, in order to generate consistent tivity for anti-topoisomerase I which may not be con- results. sistently observed in all HEp-2 substrates and/or serum Analyses of the performance of the participants dilutions [22]. Among the value of the ICAP classification showed that the average accuracy with the expected pat- scheme is that interpretation of complex mixed staining terns varied based on the hierarchical nomenclature cat- may be better reported as a single unifying pattern. In egories and rater groups (CL vs IVD). Combined, both support of this, laboratories accustomed to ICAP classi- group of participants exceeded 80% average accuracy fication correctly reported the ICAP AC-29 topoisomer - for two (nuclear and mitotic) of three group categorical ase pattern when asked to use the ICAP nomenclature, patterns. The performance for both groups was more although they may not have reported it as a speckled variable based on traditional and ICAP nomenclatures. ANA using traditional descriptions. However, the CL group had more varied average accu- A recent multicenter analysis to evaluate the inter- racy for both the traditional and ICAP nomenclatures pretation of HEp-2 IFA reported significant differences with the ICAP nomenclature demonstrating significantly among laboratories in terms of qualitative results, pat- lower performnace. This may reflect how HEp-2 IFA pat - terns, and titers, particularly at low levels and in those terns are reported in the CL and/or the experience of with speckled patterns [24]. HEp-2 IFA titer determina- these participants. Notably, the participants in the IVD tions have been reported to have clinical significance in group had more years of experience than those on the predicting risk for disease (healthy vs. disease) as well CL group. Furthermore, only half of the CL participants as association with specific autoantibodies [25–29]. routinely reported results for all three group categories, Our data confirm previous reports that ANA titers as and the accuracy of the CL participants that reported reported by individual laboratories  vary considerably, all group category patterns routinely was comparable to and point out another opportunity for harmonization of the accuracy of the IVD group. Based on this observa- ANA reporting. With respect to the ICAP nomenclature, tion, it is likely that a significant majority of participants our data demonstrated clusters of participants based on that report all three group categories developed compe- the HEp-2 patterns reported by the participants. First, tencies for the more challenging (expert-level) patterns. the majority of participants in this survey reliably read However, although automation of ANA reading holds the and interpreted the centromere, multiple nuclear dots, promise of improved consistency and accuracy of ANA T ebo et al. Autoimmun Highlights (2021) 12:4 Page 9 of 10 pattern recognition, automation-assisted reading in CL are commutable using different sources of HEp-2 rea - participants was not associated with a statistically signifi - gents. The relatively higher competencies of the IVD par - cant improvement in accuracy. ticipants relative to the CL participants is of interest as The ICAP guidance is recognized as a potential road - some laboratories depend on IVD for training as gleaned map towards the harmonization and standardization of from AMLI practice survey [19]. HEp-2 IFA nomenclature [31, 32]. It is understood by its members and opinion leaders that this guidance will Conclusion evolve, taking into consideration practical aspects for its This study highlights significant competency for all par - adoption in clinical laboratories; diverse experience, age- ticipants in identifying the nuclear main categorical ing workforce, variability in reagents, microscopy and HEp-2 IFA patterns. This observation validates the ICAP recent introduction of digital image readers [14, 19, 31]. competent-level classification for this group except for Along these lines, this investigation is not without limi- the anti-topoisomerase I antibody pattern. Our data also tations. First, the intended responses (traditional nomen- demonstrate opportunities for defining competencies clature) for specimens with the cytoplasmic and mitotic and training for CL personnel in recognition of cytoplas- patterns were not defined for specific sub-patterns (for mic and mitotic patterns. example, cytoplasmic speckled or NuMa). This was intentional as it was largely unknown how CL report Supplementary information both patterns. The results obtained from this survey Supplementary information accompanies this paper at https ://doi. validates the approach, as the minority of laboratories org/10.1186/s1331 7-020-00146 -w. reporting less than 3 main categorical patterns do report mitotic patterns considered expert-level on the ICAP Additional File 1: Participating clinical laboratories and in vitro diagnostic classification tree [www.anapa ttern s.org, 12]. Second, the manufacturers. intended responses were monospecific and did not take Additional File 2: Accuracy of HEp-2 IFA pattern reporting based on type mixed patterns into consideration. A number of partici- of nomenclature. pants reported mixed patterns for some of the specimens (data not shown), often with the intended dominant pat- Acknowledgements tern reported together with minor additional pattern We offer our sincere thanks to Maggie Fogel, the Association of Medical Labo - ratory Immunologists (AMLI), Kathryn Kohl and the Staff of PSG, Huntington variants. Such reports were considered appropriate and Valley, PA, USA for assistance with the survey. We also aknowledge all partici- in accordance for reporting patient results with more pating clinical laboraotories and IVD manufacturers for their involvement. than one pattern [2]. Third, the survey included a limited Availability of data and materials number of participating CL including those with a signif- Materials use in the survey was obatined from the Plasma Services Group Inc. icant interest and experience in ANA testing, which may (PSG), Huntington Valley, PA, USA. All data from the survey are in the posses- not reflect the experience of a wider spectrum of interna - sion AET and MHW. tional CL. Finally, some of the participants, particularly Ethics approval and consent to participate those in CL group, may have limited familiarity with the Formal consent not required for this study. ICAP nomenclature, despite being associated with expe- Consent for publication rienced laboratories. All authors have reviewed and approved of this submission. The data presented confirm that standardization of reporting has not been achieved in performance of Competing interests The authors declare that the research was conducted in the absence of any non-traditional HEp-2 patterns even by experienced commercial or financial relationships that could be construed as a potential and interested laboratories. This suggests the need and conflict of interest. opportunities for further training and consensus-build- Author details ing. Using the ICAP nomenclature may have benefits for 1 2 Department of Pathology, University of Utah, Salt Lake City, UT, USA. ARUP some sub-patterns and assigning competencies, nota- Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA. bly for the mitotic and cytoplasmic main categorical Immunopathology Laboratory, Robert J. Tomsich Pathology & Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH, USA. Department of Oral groups and our data clearly demonstrate that recogni- Biology, University of Florida, Gainesville, FL, USA. Department of Medicine, tion of the pattern associated with antibodies to topoi- Cumming School of Medicine, University of Calgary, Calgary, AB, Canada. somerase is linked to familiarity with ICAP patterns. Department of Laboratory Medicine and Pathology & Department of Medi- cine, University of Washington, Seattle, WA, USA. Furthermore, our data confirm previous observations that differences in the HEp-2 cell substrate can contrib - Received: 18 September 2020 Accepted: 12 November 2020 ute to inconsistency in ANA sub-patterns interpretation and reporting [22]. Clearly, consistent ICAP sub-pattern reporting by laboratories is most meaningful if patterns Tebo et al. Autoimmun Highlights (2021) 12:4 Page 10 of 10 References 19. Peterson LK, Tebo AE, Wener MH, Copple SS, Fritzler MJ. Assessment of 1. Meroni PL, Schur PH. ANA screening: an old test with new recommenda- antinuclear antibodies by indirect immunofluorescence assay: report tions. Ann Rheum Dis. 2010;69:1420–2. from a survey by the American Association of Medical Laboratory Immu- 2. Agmon-Levin N, Damoiseaux J, Kallenberg C, Sack U, Witte T, Herold M, nologists. Clin Chem Lab Med. 2020 Apr 8. [Epub ahead of print]. et al. International recommendations for the assessment of autoantibod- 20. Meroni PL, Borghi MO. Diagnostic laboratory tests for systemic autoim- ies to cellular antigens referred to as anti-nuclear antibodies. Ann Rheum mune rheumatic diseases: unmet needs towards harmonization. Clin Dis. 2014;73:17–23. Chem Lab Med. 2018;56(10):1743–8. 3. Pisetsky DS. Antinuclear antibody testing - misunderstood or misbegot- 21. Bogaert L, Van den Bremt S, Schouwers S, Bossuyt X, Van Hoovels L. Har- ten? Nat Rev Rheumatol. 2017;13:495–502. monizing by reducing inter-run variability: performance evaluation of a 4. Damoiseaux J, Andrade LEC, Carballo OG, Conrad K, Francescantonio PL, quality assurance program for antinuclear antibody detection by indirect Fritzler MJ, et al. Clinical relevance of HEp-2 indirect immunofluorescent immunofluorescence. Clin Chem Lab Med. 2019;57(7):990–8. patterns: the International Consensus on ANA patterns (ICAP) perspec- 22. Andrade LEC, Klotz W, Herold M, Conrad K, Rönnelid J, Fritzler MJ, von tive. Ann Rheum Dis. 2019;78:879–89. Mühlen CA, Satoh M, Damoiseaux J, de Melo Cruvinel W, Chan EKL; Exec- 5. Emlen W, O’Neill L. Clinical significance of antinuclear antibodies: utive Committee of ICAP. International consensus on antinuclear anti- comparison of detection with immunofluorescence and enzyme-linked body patterns: definition of the AC-29 pattern associated with antibodies immunosorbent assays. Arthritis Rheum. 1997;40:1612–8. to DNA topoisomerase I. Clin Chem Lab Med. 2018;56(10):1783-1788. 6. Homburger HA, Cahen YD, Griffiths J, Jacob GL. Detection of antinuclear 23. Dellavance A, Gallindo C, Soares MG, Silva NP, Mortara RA, Andrade LE. antibodies: comparative evaluation of enzyme immunoassay and indirect Redefining the Scl-70 indirect immunofluorescence pattern: autoanti- immunofluorescence methods. Arch Pathol Lab Med. 1998;122:993–9. bodies to DNA topoisomerase I yield a specific immunofluorescence 7. Tan EM, Smolen JS, McDougal JS, Butcher BT, Conn D, Dawkins R, et al. A pattern. Rheumatology. 2009;48:632–8. critical evaluation of enzyme immunoassays for detection of antinuclear 24. Turan Faraşat V, Ecemiş T, Doğan Y, et al. A Multicenter Analysis of Sub- autoantibodies of defined specificities. I. Precision, sensitivity, and speci- jectivity of Indirect Immunofluorescence Test in Antinuclear Antibody ficity. Arthritis Rheum 1999;42:455–64. Screening. Arch Rheumatol. 2019;34(3):326–33. 8. Tonuttia E, Bassetti D, Piazza A, Visentini D, Poletto M, Bassetto F, et al. 25. Tan EM, Feltkamp TE, Smolen JS, et al. Range of antinuclear antibodies in Diagnostic accuracy of ELISA methods as an alternative screening test to “healthy” individuals. Arthritis Rheum. 1997;40(9):1601–11. indirect immunofluorescence for the detection of antinuclear antibodies. 26. Egner W. The use of laboratory tests in the diagnosis of SLE. J Clin Pathol. Evaluation of five commercial kits. Autoimmunity. 2004;37:171–6. 2000;53(6):424–32. 9. Choi MY, Cui J, Costenbader K, Rydzewski D, Bernhard L, Schur P. Different 27. Sack U, Conrad K, Csernok E, et al. Autoantibody detection using indirect immunofluorescence ANA substrate performance in a diagnostic indirect immunofluorescence on HEp-2 cells. Ann N Y Acad Sci. setting of patients with SLE and related disorders: retrospective review 2009;1173:166–73. and analysis. Lupus Sci Med. 2020;7:e000431. 28. Banhuk FW, Pahim BC, Jorge AS, Menolli RA. Relationships among Anti- 10. Copple SS, Sawitzke AD, Wilson AM, Tebo AE, Hill HR. Enzyme-linked bodies against Extractable Nuclear Antigens, Antinuclear Antibodies, and immunosorbent assay screening then indirect immunofluorescence con- Autoimmune Diseases in a Brazilian Public Hospital. Autoimmune Dis. firmation of antinuclear antibodies: a statistical analysis. Am J Clin Pathol. 2018;2018:9856910. 2011;135:678–84. 29. Tanaka N, Muro Y, Sugiura K, Tomita Y. Anti-SS-A/Ro antibody determina- 11. Olsen NJ, Choi MY, Fritzler MJ. Emerging technologies in autoantibody tion by indirect immunofluorescence and comparison of different meth- testing for rheumatic diseases. Arthritis Res Ther. 2017;19:172. ods of anti-nuclear antibody screening: evaluation of the utility of HEp-2 12. Chan EK, Damoiseaux J, Carballo OG, Conrad K, de Melo Cruvinel W, cells transfected with the 60 kDa SS-A/Ro as a substrate. Mod Rheumatol. Francescantonio PL, et al. Report of the First International Consensus on 2008;18(6):585–92. Standardized Nomenclature of Antinuclear Antibody HEp-2 Cell Patterns 30. Betancur JF, Londoño A, Estrada VE, et al. Uncommon patterns of anti- 2014-2015. Front Immunol. 2015;6:412. nuclear antibodies recognizing mitotic spindle apparatus antigens and 13. Chan EK, Damoiseaux J, de Melo Cruvinel W, Carballo OG, Conrad K, clinical associations. Medicine (Baltimore). 2018;97(34):e11727. Francescantonio PL, et al. Report on the second International Con- 31. Tebo AE. Recent approaches to optimize laboratory assessment of anti- sensus on ANA Pattern (ICAP) workshop in Dresden 2015. Lupus. nuclear antibodies. Clin Vaccine Immunol. 2017;24(12):e00270-17. 2016;25:797–804. 32. Damoiseaux J. The perspective on standardisation and harmonisation: 14. Damoiseaux J, von Mühlen CA, Garcia-De La Torre I, Carballo OG, de Melo the viewpoint of the EASI president. Auto Immun Highlights. 2020;11(1):4. Cruvinel W, Francescantonio PL, Fritzler MJ, et al. International consensus on ANA patterns (ICAP): the bumpy road towards a consensus on report- Publisher’s Note ing ANA results. Auto Immun Highlights 2016;7:1. Springer Nature remains neutral with regard to jurisdictional claims in pub- 15. Hoffman IE, Peene I, Veys EM, De Keyser F. Detection of specific antinu- lished maps and institutional affiliations. clear reactivities in patients with negative anti-nuclear antibody immuno- fluorescence screening tests. Clin Chem. 2002;48:2171–6. 16. von Mühlen CA, Tan EM. Autoantibodies in the diagnosis of systemic rheumatic diseases. Semin Arthritis Rheum. 1995;24:323–58. 17. Wiik AS. Guidelines for Antinuclear Antibody Testing. EJIFCC. 2006;17(3):134–40. 18. Herold M, Klotz W, Andrade LEC, Conrad K, Cruvinel WM, Damoiseaux J, et al. International Consensus on Antinuclear Antibody Patterns: defining negative results and reporting unidentified patterns. Clin Chem Lab Med. 2018;56:1799–802. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Autoimmunity Highlights Springer Journals

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

Loading next page...
 
/lp/springer-journals/the-antinuclear-antibody-hep-2-indirect-immunofluorescence-assay-a-le9jtUAKhv

References (33)

Publisher
Springer Journals
Copyright
Copyright © The Author(s) 2021
ISSN
2038-0305
eISSN
2038-3274
DOI
10.1186/s13317-020-00146-w
Publisher site
See Article on Publisher Site

Abstract

Background: To evaluate the interpretation and reporting of antinuclear antibodies (ANA) by indirect immunofluo - rescence 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 nomen- clature [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 associ- ated with cytoplasmic patterns. Conclusions: Our study demonstrated significant accuracy for all participants in identifying the categorical nuclear stain- ing as well as traditional pattern assignments for nuclear patterns. However, there was less consistency in reporting cyto- plasmic and mitotic patterns, with implications for assigning competencies and training for clinical laboratory personnel. Keywords: Anti-nuclear antibodies, Cytoplasmic patterns, Performance survey, Indirect immunofluorescence, Mitotic patterns, Nuclear patterns *Correspondence: anne.tebo@hsc.utah.edu Department of Pathology, University of Utah, Salt Lake City, UT, USA Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. Tebo et al. Autoimmun Highlights (2021) 12:4 Page 2 of 10 HEp-2 IFA nuclear, cytoplasmic and mitotic patterns Introduction have been elucidated by ICAP and presented in a clas- The presence of antinuclear antibodies (ANA) is a sification tree (www.anapa ttern s.org). The ICAP guide - hallmark and classification criterion for a number of lines indicate that ‘expert-level’ laboratories would systemic autoimmune rheumatic diseases (SARD). report all the HEp-2 IFA patterns, whereas those des- ANA testing is usually performed as part of the initial ignated as ‘competent-level’ laboratories would report 6 diagnostic workup when suspicion of an underlying nuclear and 5 cytoplasmic HEp-2 IFA patterns [12]. autoimmune disorder is high. The indirect immuno - In a previously reported survey administered in coop- fluorescence antibody (IFA) technique on HEp-2 sub - eration with the Association of Medical Laboratory strate has been considered the traditional and preferred Immunologists (AMLI), a significant number of respond - method for detecting ANA by some [1]. It allows detec- ents were unaware of the ICAP initiative, although a tion of antibody binding to specific intracellular targets, majority agreed on the need to standardize the nomen- resulting in diverse staining patterns that are usually clature and reporting of HEp-2 IFA results [19]. Based on categorized based on the cellular components recog- the responses from this survey, a consensus to improve nized and the degree of binding, as reflected by the flu - ICAP awareness and further enhance HEp-2 IFA assess- orescence intensity or titer [2, 3]. As a screening tool, ment through increased collaboration between ICAP and the recognition of a well-defined HEp-2 IFA staining the clinical laboratory community was suggested with pattern may be helpful in determining the most likely emphasis on education and availability of reference mate- specific autoantibodies present, as well as suggesting rials. As others have also reported [20, 21], many labora- possible clinical associations for known specificities [3 , tories around the world are inclined to adopt the ICAP 4]. In this regard, a positive HEp-2 IFA screening pat- nomenclature and embrace the recommendations pro- tern can guide confirmatory testing and may also be vided in these consensus guidelines. useful for elucidating a specific clinical diagnosis or The objective of this study was to evaluate the perfor - prognosis. Thus, the provision of HEp-2 IFA patterns mance of HEp-2 IFA interpretation based on nuclear, and titers is considered to be clinically valuable with cytoplasmic and mitotic staining in an endeavor to favorable utility in comparison with other methods for characterize competency as outlined in the ICAP ANA detection [1–11]. classification. The nuclear IFA staining patterns most commonly recognized and reported by clinical laboratories include Materials and methods homogeneous, speckled, centromere, and nucleolar [1– Participants and recruitment 4, 12–14]. Use of HEp-2 cell substrates, permits detec- Participants for the survey were recruited via an email tion of additional nuclear staining patterns, as well as sent to the Association of Medical Laboratory Immunol- reactivity with cell constituents in compartments out- ogists (AMLI) membership (a professional organization side the nucleus (cytoplasmic) and cell components focused on immunological laboratory testing with 105 associated with mitosis (mitotic) [2, 4, 12–14]. How- active members) and 5 in  vitro diagnostics manufactur- ever, the reactivity and type of autoantigens associated ers (IVD). The requirements to participate included test - with these patterns may vary among HEp-2 substrates ing pre-defined specimens by IFA using HEp-2 substrate from different manufacturers [15]. Furthermore, the and reporting the patterns observed based on specific expertise required to identify the different patterns and “traditional” (as defined below) as well as ICAP (www. sub-classify their variants may not be universally avail- anapa ttern s.org) nomenclature for reading and reporting able in clinical laboratories. Traditional legacy recom- ANA patterns. Sixteen (n = 16) clinical laboratories (CL) mendations for reporting ANA patterns on HEp-2 cells and all 5 in vitro diagnostics manufacturers (IVD) agreed continue to significantly influence clinical laboratory to participate in the study. Three additional IVD (2 in the reporting [16, 17]. US and 1 in Europe), contacted the organizers to partici- The first I nternational Consensus on Antinuclear pate in the survey. Overall, 16 CL and 8 IVD participated Antibody (ANA) Patterns (ICAP) was published in in this performance survey (Additional file 1: Table S1). 2015 to systematize and update reporting of autoan- tibody patterns detected by IFA using HEp-2 cell sub- Study specimens and survey strates [12]. The goal of this initial and subsequent Twelve (n = 12) specimens were used in the survey. The publications was to optimize usage of HEp-2 IFA pat- specimens were chosen based on an assessment of need terns in patient care, by promoting standardization, (described below) by some of the authors (AET, LKP, harmonization and understanding of autoantibody test EKLC, MJF, MHW). The following attributes were taken nomenclature and providing guidelines for test inter- into consideration: 1) the three main categorical group pretation and reporting [4, 12–15, 18]. To-date, 30 T ebo et al. Autoimmun Highlights (2021) 12:4 Page 3 of 10 of HEp-2 IFA patterns, 2) the ICAP guidance for both on the ICAP classification tree (www.anapa ttern s.org), competent and expert levels, 3) the clinical significance which includes more detailed sub-pattern classifica - of patterns and 4) whether or not proficiency testing was tion than commonly reported. In addition, the partici- available for specific patterns. The authors also wanted to pants were requested to provide information about how evaluate how participants would interpret nuclear stain- the images were read and interpreted (manual and/or ing associated with anti-topoisomerase I antibodies given automation-assisted reading); the years of experience recent ICAP guidance for evaluating this complex pat- of the reading technologist(s); the manufacturer of the tern [22]. The twelve pre-specified HEp-2 IFA-positive HEp-2 substrate; the laboratory’s typical practice about specimens labeled ANA-001 through ANA-012 included reporting only nuclear patterns vs also reporting cyto- those positive for nuclear [ANA-002, ANA-003, ANA- plasmic and/or mitotic patterns when the ANA test is 005, ANA-006, ANA-007, ANA-010, ANA-011], cyto- requested; the screening dilution(s) of serum used for plasmic [ANA-004, ANA-008, ANA-009] and mitotic detection of ANA in performing the HEp-2 IFA; and the [ANA-001, ANA-012] categorical groups of IFA patterns titer of the ANA, based on serial dilution of the tested (Table 1). All specimens for the survey and their intended specimen. There were two types of participants: clinical responses were obtained from Plasma Services Group laboratories (CL) and in  vitro diagnostic manufactur- Inc. (PSG: Huntington Valley, PA, USA, https ://www. ers (IVD). After the results were tabulated, participants plasm aserv icesg roup.com/). Specimens were qualified were not afforded the opportunity to adjust or revise at PSG using routinely available methods and also veri- responses based on the responses of other respondents. fied in the laboratories of one or more expert members of Some of the participating CL included those directed ICAP (PSG, personal communication). by the authors, but the authors did not participate in Survey specimens were shipped to all participants in assigning the patterns reported from their laboratories. January 2020 with detailed instructions for testing as well as a report form to record and return results to one Data analyses of the organizers (AET). Parameters to be recorded by We compared participants’ HEp-2 IFA pattern clas- checking the survey form included the three categori- sification of specimens against a consensus classifica - cal groups of HEp-2 IFA patterns reported (nuclear, tion. The primary outcome was the percent accuracy cytoplasmic or mitotic); commonly used nomencla- between the participant and consensus classification. ture (also referred to as traditional in this investiga- We studied the impact of three factors on accuracy: tion) for 5 HEp-2 IFA nuclear patterns (homogeneous, pattern classification hierarchy or nomenclature, par - speckled, centromere, nucleolar, discrete nuclear dots), ticipant organization type, and participant experi- mitotic, cytoplasmic or ‘other’ in accord with legacy ence. We examined three hierarchial  nomenclatures: classification approaches [16, 17]; and a result based 1) group category (nuclear, cytoplasmic, mitotic); 2) Table 1 Survey specimens and their characteristics Specimen Cellular staining Traditional nomenclature ICAP nomenclature ICAP level ANA-001 Mitotic Mitotic Spindle fiber, AC-25 Expert ANA-002 Nuclear Discrete nuclear dots Multiple nuclear dots, AC-6 Expert ANA-003 Nuclear Speckled Coarse speckled, AC-5 Expert ANA-004 Cytoplasmic Cytoplasmic Reticular/AMA, AC-21 Competent ANA-005 Nuclear Centromere Centromere, AC-3 Competent ANA-006 Nuclear Nucleolar Homogeneous nucleolar, AC-8 Expert ANA-007 Nuclear Speckled DFS, AC-2 Competent ANA-008 Cytoplasmic Cytoplasmic DFS, AC-19 Expert ANA-009 Cytoplasmic Cytoplasmic Fine speckled, AC-20 Expert ANA-010 Nuclear Speckled Fine speckled, AC-4 Expert ANA-011 Nuclear Speckled/Other* Anti-topoisomerase I, AC-29 Expert ANA-012 Mitotic Mitotic NuMA-like, AC-26 Expert number, ICAP International Consensus on Antinuclear Antibody Pattern, AC anti-cell, AMA anti-mitochondrial antibodies, DFS dense fine speckled, NuMA nuclear mitotic apparatus protein. The AC-29 Anti-topoisomerase pattern I is a compound pattern, classified within ICAP as a speckled pattern. The complex pattern involves speckled nuclear staining, and also includes staining of the condensed chromatin, cytoplasmic staining, staining of the nucleolar organizing region in mitotic cells, and variable nucleolar staining of interphase cells. For this specimen, AC-25 was also considered acceptable Tebo et al. Autoimmun Highlights (2021) 12:4 Page 4 of 10 specific traditional pattern descriptions (e.g. speckled, Table 2 Characteristics of survey participants nucleolar, etc.); and 3) sub-pattern classification using a a Characteristics CL, N (%) IVD, N (%) the ICAP nomenclature. We refer to these as the group, HEp-2 kit Bio-Rad 4 (25.00) See legend traditional and ICAP classification methods. Each par - Euroimmun 5 (31.25) ticipant was classified according to the organizational Inova 6 (37.50) type and the reporting experience at their institution. MBL Bion 1 (6.25) There were two types of organizations: 1) clinical labo - Type of reader Manual only 9 (56.25) 5 (62.50) ratories (CL) and 2) in  vitro diagnostic manufacturers Manual and Auto- 7 (43.75) 3 (37.25) (IVD). Organizations were classified as experienced if mated they routinely reported all group categories and inex- Cut-off (titer) < 1:10 1 (6.25) 0 (00.00) perienced if they did not routinely report all three < 1:40 12 (75.00) 6 (66.67) group categories. Using these three factors and their < 1:80 3 (18.75) 2 (22.22) associated variables, we sought to answer the following < 1:100 0 (00.00) 1 (11.11) questions: Technologist 1–5 years 7 (33.30) 0 (00.00) experience 6–10 years 4 (12.10) 1 (11.11) 1. Was accuracy associated with the classification > 10 years 10 (47.60) 8 (88.89) method? Median (range), years 10 (1-45) 20 (2-51) 2. Was accuracy associated with experience? Nuclear only 3 (18.75) 0 (00.00) Patterns reported 3. Was accuracy associated with organization type Cytoplasmic and 2 (12.50) 0 (00.00) among experienced participants? nuclear 4. Was accuracy associated with categories within Mitotic and nuclear 3 (18.75) 0 (00.00) nomenclature methods? All patterns 8 (50.00) 8 (100.00) Number (N) of clinical laboratories (CL) or in vitro diagnostic manufacturers We used logistic regression to determine the asso- (IVD) unless otherwise stated. Participating IVD manufacturers included: ciation between accuracy and the three factors. Out- AESKU Diagnostics, Bio-Rad, Euroimmun, Inova, ImmunoConcepts, Scimedx, ThermoFisher and Zeus. CL labs using Inova HEp-2 substrate kits use < 1:40 comes were reported as odds ratios (OR). P-values were or < 1:80 as cut-off for ANA determinations. More than one technologist was adjusted for multiple comparisons using the method of involved in the reading and interpretation of the results in some laboratories. One laboratory in each group reports cytoplasmic pattern only as a comment Holm. Statistical analyses were performed using Stata 16.2 (Stata Corp LLP). they report patterns in all 3 categorical groups, however, Results all provided responses to all categories in the survey. Characteristics of survey participants There were 24 participants: 16 were CL (13 in the Performance of participants based on the categorical United States and 3 in Canada) and 8 IVD. Most of the HEp‑2 IFA groups CL used kits from three main IVD that also partici- The accuracy for reporting the nuclear pattern was 99% pated in the survey (Table  2). The majority of the CL (95% CI 95–100%) for all participants, while the cyto- read, interpreted and determined HEp-2 IFA patterns plasmic and mitotic group categories had accuracy of and titers manually using 1:40 as cut-off for HEp-2 78% (CI 66–87%) and 93% (CI 81–88%), respectively IFA determinations. The median years of experience (Table  3). The overall accuracy of IVD was greater than for technologists who participated in the survey was accuracy of CL (97% vs 91%, Additional file  2: Table S2a) 10  years for CL compared to 20  years for the IVD in assigning the HEp-2 IFA group categories of patterns participants. of all specimens. This difference was statistically signifi - The number of categorical groups of patterns typically cant (p = 0.04). Combined, the two organization types reported by the CL was variable. Among the 16 CL, 3 (CL and IVD) had an overall accuracy of 93% (95% CI indicated they reported only nuclear patterns, 3 indicated 89–96%) for determining the three categorical groups of they reported nuclear and mitotic patterns, 2 reported HEp-2 IFA patterns. nuclear and cytoplasmic patterns (with one of the 2 reporting the cytoplasmic pattern as a comment, not as Performance of participants based on “traditional” a ‘positive ANA’) and 8 indicated the laboratory reported and ICAP nomenclatures nuclear, cytoplasmic and mitotic patterns (with one of Participants were asked to report results based on sur- the 8 reporting the cytoplasmic patterns as a comment, vey-suggested classifications (referred here as “tradi - not as a ‘positive ANA’). Among the 8 IVD, 6 indicated tional”) as well as the ICAP nomenclature. The overall T ebo et al. Autoimmun Highlights (2021) 12:4 Page 5 of 10 Table 3 Performance of  Participants in  the  Three HEp-2 Several specimens yielded unexpected results. For IFA Group Categories example, ANA-010 was intended to represent a nuclear fine speckled pattern (AC-4), but that specimen was Group category Specimens Observations Accuracy (95% CI) (Number, n) (Number, n) reported as ICAP pattern AC-4 by only a minority of CL and IVD. The survey showed that all participants Nuclear 7 168 99 (95–100) reported this specimen as having a speckled nuclear pat- Cytoplasmic 3 63 78 (66–87) tern using traditional descriptions, but a majority (75% of Mitotic 2 45 93 (81–98) IVDs and 55% of CL) reported it as having coarse speck- Overall 12 276 93 (89–96) led nuclear staining (AC-5) rather than the expected CI confidence interval AC-4. Review of images from several participating labo- ratories revealed that the specimen produced patterns ranging from typical fine speckled to coarse speckled accuracy for the traditional nomenclature system was nuclear staining using different HEp-2 cell sources. Simi - 87% (95% CI 82–90%), Table  4. Only the specimen larly, the specimen (ANA-009) intended to represent a (ANA-011) with antibodies to DNA topoisomerase I was cytoplasmic fine speckled pattern (AC-20) was reported reported with accuracy less than 80% and that complex with other patterns by a majority of participants and mixed/compound pattern had not been included in many review of images from different laboratories showed a ANA pattern classification teaching schemes prior to its variable pattern of staining depending on the source of recent inclusion as a distinct ICAP pattern [20]. Among the HEp-2 substrate (data not shown). the traditional pattern reports, two specimens (ANA- The specimen with antibodies to topoisomerase I 003, centromere and ANA-006, nucleolar) were reported (topo-1, AC-29 pattern) was reported as homogeneous with accuracy of 100%. by most (56%) CL participants and as a mixed (homoge- The overall accuracy for the ICAP nomenclature neous and nucleolar) pattern by an additional 19% of CL reporting system was 74% (95% CI 68–79%), Table  4. using the common pattern descriptions. The IVD partici - For the ICAP nomenclature, 5 out of the 12 (41.7%) pants reported it as having a variety of mixed nucleolar specimens were reported with overall accuracy over patterns. Using ICAP nomenclature, 88% of IVD partici- 80%. These included AC-3: centromere, AC-6: multiple pants and 54% of responding CL participants assigned nuclear dots, AC-2: dense fine speckled (with 100% of the specimen as having the AC-29 (anti-topoisomerase I) IVD and 67% of responding CL accurately), AC-21: AMA pattern. and AC-26: NuMA-like. For ANA-012, participants Overall, for all participants, the accuracy was 13% reporting AC-25 and AC-26 were graded as having con- greater for the traditional nomenclature (87%, 95% CI sensus for the intended report. Table 4 Performance of participants in the traditional and ICAP nomenclature systems Specimen ID Traditional Observations Accuracy (95% CI) ICAP Observations Accuracy (95% CI) (n) (n) ANA-005 Centromere 24 100 (86–100) Centromere, AC-3 20 100 (83–100) ANA-002 DND 24 96 (73–99) MND, AC-6 20 85 (60–95) ANA-003 Speckled 24 100 (86–100) Nuclear CS, AC-5 18 67 (41–85) ANA-007 Speckled 24 96 (73–99) Nuclear DFS, AC-2 20 80 (55–93) ANA-010 Speckled 24 83 (62–94) Nuclear FS, AC-4 19 37 (18–61) ANA-011 Speckled/Other 24 42 (23–63) Anti-topo I, AC-29 21 62 (39–81) ANA-006 Nucleolar 24 100 (86–100) Homo nucleolar, AC-8 20 70 (46–87) ANA-004 Cytoplasmic 19 100 (86–100) AMA, AC-21 19 89 (66–97) ANA-008 Cytoplasmic 20 90 (65–98) Cytoplasmic DFS, AC-19 19 79 (53–92) ANA-009 Cytoplasmic 19 53 (30–74) Cytoplasmic FS, AC-20 19 42 (22–66) ANA-001 Mitotic 22 86 (64–96) Spindle fiber, AC-25 20 75 (50–90) ANA-012 Mitotic 24 92 (70–98) NuMA-like, AC-26 22 95 (71–99) Overall 250 87 (82–90) Centromere, AC-3 237 74 (68–79) ICAP International Consensus on Antinuclear Antibody Patterns). ID identification number, AC anti-cell, DND discrete nuclear dots, MND multiple nuclear dots, CS coarse speckled, DFS dense fine speckled, FS fine speckled, AMA anti-mitochondrial antibodies, homo homogeneous, anti-topo I anti-DNA topoisomerase I, CI confidence interval, NuMA nuclear mitotic apparatus. Variation in observation numbers in table reflects the fact that some laboratories did not report all cytoplasmic or mitotic categorical group patterns or ICAP patterns for some specimens Tebo et al. Autoimmun Highlights (2021) 12:4 Page 6 of 10 82–90%) compared to the ICAP nomenclature (74%, 95% was significantly less accurate than the group cat - CI 68–79%, Additional file  2: Table  S2b and Tables  3). egory nomenclature (OR = 0.48, p = 0.014). The aver - However, the accuracy for reporting the ICAP nomencla- age accuracy of the ICAP nomenclature was 74% (95% tures were lower for CL than IVD with an overall accu- CI 68–79%) which was significantly less than the group racy of 81% (95% CI 77–84%, Additional file 2: Table S2). category nomenclature (OR = 0.20, p = 0.002) and the The accuracy of classification was associated with par - traditional nomenclature (OR = 0.42, p = 0.002), Table 5. ticipant type (χ p < 0.0005) and nomenclature system Experienced participants had higher accuracy than (p < 0.0005). The accuracy of the CL group was 15% less nonexperienced participants (OR = 2.2, p < 0.0005). than the IVD group. The accuracy of experienced participants was greater To assess the performance of each organization type than the accuracy of nonexperienced participants for based on the accuracies for the main categorical, tradi- all nomenclatures. The difference was 6% for the group tional and ICAP nomenclature determinations, the data method, 10% for the traditional nomenclature and 13% were stratified, and frequencies of the correct intended for the ICAP nomenclature. responses estimated (data not shown). Both groups were effective in determining the intended nuclear staining, Impact of participant type however, the CL group demonstrated lower frequen- All IVD participants were experienced and 8 of the 16 CL cies of the expected responses for the different nomen - participants were experienced (with experience defined clatures. This was most pronounced for the ICAP as routinely reporting all group categories). Among expe- nomenclature. rienced participants, IVD had greater accuracy than CL (OR = 2.8, p = 0.002, Table  5). On average, the accuracy Impact of nomenclature and participant experience of the IVD participants was 92% (95% CI 89–95%) and on accuracy the accuracy of the experienced CL participants was Participants had the highest accuracy using the group 83% (95% CI 78–88%). The accuracy was associated with category nomenclature (Table  3). The average accu - nomenclature. The accuracy of the ICAP nomenclature racy associated with the group category nomenclature was 78% (95% CI 71–84%) which was significantly lower was 93% (95% CI 90–96%). For the traditional nomen- (OR = 0.16, p = 0.002) than the accuracy of the group clature, the average was 87% (95% CI 83–91%) which nomenclature (95%, 95% CI 92–98%) and significantly Table 5 Accuracy of classification based on experience and participant type Classification nomenclature Experience Observations (n) Accuracy (95% CI) Average (95% CI) P value Group category (n = 276) No 87 89 (82–95) 93 (90–96) Base Yes 189 95 (92–98) Traditional (n = 272) No 84 80 (71–89) 87 (83–91) 0.014 Yes 188 90 (86–94) ICAP (n = 232) No 67 64 (53–76) 74 (68–79) 0.002 0.002 Yes 170 77 (73–84) All (n = 785) No 238 79 (73–84) 85 (83–88) 0.002** Yes 547 88 (85–91) Group category (n = 189) IVD 96 97 (93–100) 95 (92–98) Base CL* 93 94 (88–99) Traditional (n = 188) IVD 96 97 (93–100) 90 (86–94) 0.05 CL* 92 83 (75–91) ICAP (n = 170) IVD 95 83 (76–91) 78 (71–84) 0.002 0.002 CL* 75 71 (60–81) All (n = 547) IVD 287 92 (89–95) 88 (85–91) 0.002** CL* 260 83 (78–88) HEp-2 cell IFA patterns were evaluated based on experience for all participants (yes or no), and experienced participant types (in vitro diagnostics manufacturers, IVD) and experienced clinical laboratories (CL*). Experienced CL defined as reporting all 3 main nomenclature categories. All IVD participants reported the three a b nomenclature categories and are rated experienced. CI: confidence interval, n number. ICAP vs Group category and Traditional vs ICAP **Indicates significant difference between groups T ebo et al. Autoimmun Highlights (2021) 12:4 Page 7 of 10 demonstrates competence for participants in identifying lower (OR = 0.35, p = 0.002) than the traditional nomen- and reporting common nuclear ANA patterns, but incon- clature (90%, 95% CI 86–94%). Among the CLs, use of sistency in the decision to report and pattern reporting of automation-assisted reading trended toward improved cytoplasmic and mitotic patterns. accuracy of pattern reporting for both traditional (87% In recent years, efforts to standardize interpretation vs 82% accuracy) and ICAP (70% vs 55% accuracy), but and reporting of HEp-2 patterns have led to a consen- these differences were not statistically significant. sus nomenclature presented by ICAP, a group of experts [4, 12–14] with the purpose of systematic reporting and Frequency distribution of end‑point titers optimizing the usage of HEp-2 IFA patterns in patient For each of the 12 specimens analyzed, the distribution care [4]. In a previous study, we identified increas - of the reported antibody titer was recorded. The screen - ing awareness of this guidance; availability of reference ing titer of determinations ranged from 1:10 to 1:80. The materials for training and collaboration between profes- titers were generally quite variable (Fig. 1, shown for CL). sional organizations, IVD and CL amongst others as key The specimens with cytoplasmic patterns were often not elements necessary for improved harmonization of the titered, particularly by laboratories that did not routinely HEp-2 IFA reporting [19]. report cytoplasmic patterns. Of these specimens, the titer In addition to accurately reporting binding of autoan- variability was most pronounced for ANA-004 (AMA, tibodies to defined cellular components, the survey also AC-21) with positive results demonstrating a bimodal evaluated responses based on “traditional” categoriza- response which spanned eight twofold titers ranging tion for nuclear patterns as well as the emerging ICAP from 1:80 1:10,240 for CL reporting this pattern. Nuclear nomenclature. As expected, all participants performed pattern staining titers also varied substantially, spanning better with the more widely used or common traditional from four to seven twofold titers in different specimens. HEp-2 IFA nomenclature, which has more emphasis on limited nuclear staining features than required to cor- Discussion rectly assign ICAP patterns. While the reason for this Using pre-tested and selected patient serum specimens, could be due to limited familiarity with ICAP, based on we report here the performance of 24 CL and IVD partic- the data, other reasons for this can be inferred. First, the ipants recruited from a professional organization focused “traditional” categorization which can also be referred to on immunological laboratory testing and accustomed to as the ICAP “competent-level” is broad and minimizes the the interpretation and reporting of HEp-2 IFA patterns. use of fine details and/or integrated pattern recognition The specimens included examples from all 3 main cat - in its interpretation. For example, most responders were egorical patterns (nuclear, cytoplasmic, or mitotic), were capable of identifying ANA-003 and ANA-007 as nuclear reported using ‘traditional’ and ICAP nomenclatures, speckled patterns but failed to accurately demonstrate and included patterns designated by ICAP as associated the intended ICAP nomenclatures, coarse speckled/ with both ‘competent’ and ‘expert’ laboratories. Our data Titer 1:10240 1:5120 1:2560 1:1280 1:640 1:320 1:160 1:80 1:40 Not Titered 1000000 10 9852 ICAP AC# 23456 8291920212526 Nuclear Patterns Cytoplasmic Patterns Mitotic Patterns Fig. 1 Distribution of end-point titers for survey specimens reported by clinical laboratory (CL) participants. The frequency distributions of titer values for the 12 samples as reported by 16 CL participants is graphically illustrated. The number of CL reporting titer (1:40 to 1:10,240) for each AC-numbered specimen is shown, as well as the number of clinical labs that did not titer the specimen. The distance between vertical lines represents 10 participants Tebo et al. Autoimmun Highlights (2021) 12:4 Page 8 of 10 AC-5, and dense fine speckled/AC-2, respectively. In nuclear dense fine speckled, AMA and NuMA-like sub- fact, a number of respondents classified the AC-2 DFS patterns. The NuMA-like pattern is considered uncom - specimen as a mixed nuclear homogeneous and nuclear mon, and expected to be recognized by “Expert” level speckled pattern, as might be expected for traditional laboratories, but it has a characteristic appearance, and classification based on speckled staining of the nucleo - has clinically significant associations with a number of plasm and intense chromatin staining. The combina - SARD [30]. Second, a significant group of participants tion requires integration to assign the nuclear DFS AC-2 could identify challenging ICAP-designated sub-pat- ICAP pattern, rather than describing mixed homogene- terns. These include the homogeneous nucleolar, cyto - ous and speckled staining pattern with which it might be plasmic dense fine speckled, spindle fiber, nuclear coarse confused. speckled, and anti-topoisomerase I patterns. Except for The specimen with antibodies to DNA topoisomer - the AMA pattern, the overall performance of the CL par- ase I and the AC-29 staining pattern also demonstrated ticipants for specimens with the cytoplasmic patterns remarkable challenges of consistent ANA pattern report- was more variable, and lower than the IVD group. These ing. Under ICAP, AC-29 is considered a sub-pattern of observations have implications for defining competency nuclear speckled staining [22], but only a minority of par- for CL for cytoplasmic and mitotic patterns. ticipants reported it as a nuclear speckled pattern using A minority of participants interpreted the nuclear traditional nomenclature. Using “traditional” classifica - fine speckled and cytoplasmic fine speckled sub-pat - tions, the pattern is a compound, mixed staining pattern tern specimens as intended. The data suggested that the in which the speckled component may not be perceived HEp-2 patterns generated by those specimens had a suf- as dominant, even though it is consistently observed. ficiently variable appearance, based on the kit manufac - In addition to the speckled nuclear staining, there is turer, and/or kit lot, to lead the specimens to appear as also staining of condensed chromatin in the mitotic different ICAP categories in the hands of different par - cells, making it difficult to distinguish from homogene - ticipants. That hypothesis was confirmed by our direct ous nuclear staining, as reported by majority of the CL, review of the appearance from different laboratories and nucleolar staining also is often present. Dellavance (data not shown). The observations reinforce the need for and colleagues [23] first reported on a composite of five harmonization of reagents, as well an enhanced training unique HEp-2 staining attributes associated with posi- in pattern interpretation, in order to generate consistent tivity for anti-topoisomerase I which may not be con- results. sistently observed in all HEp-2 substrates and/or serum Analyses of the performance of the participants dilutions [22]. Among the value of the ICAP classification showed that the average accuracy with the expected pat- scheme is that interpretation of complex mixed staining terns varied based on the hierarchical nomenclature cat- may be better reported as a single unifying pattern. In egories and rater groups (CL vs IVD). Combined, both support of this, laboratories accustomed to ICAP classi- group of participants exceeded 80% average accuracy fication correctly reported the ICAP AC-29 topoisomer - for two (nuclear and mitotic) of three group categorical ase pattern when asked to use the ICAP nomenclature, patterns. The performance for both groups was more although they may not have reported it as a speckled variable based on traditional and ICAP nomenclatures. ANA using traditional descriptions. However, the CL group had more varied average accu- A recent multicenter analysis to evaluate the inter- racy for both the traditional and ICAP nomenclatures pretation of HEp-2 IFA reported significant differences with the ICAP nomenclature demonstrating significantly among laboratories in terms of qualitative results, pat- lower performnace. This may reflect how HEp-2 IFA pat - terns, and titers, particularly at low levels and in those terns are reported in the CL and/or the experience of with speckled patterns [24]. HEp-2 IFA titer determina- these participants. Notably, the participants in the IVD tions have been reported to have clinical significance in group had more years of experience than those on the predicting risk for disease (healthy vs. disease) as well CL group. Furthermore, only half of the CL participants as association with specific autoantibodies [25–29]. routinely reported results for all three group categories, Our data confirm previous reports that ANA titers as and the accuracy of the CL participants that reported reported by individual laboratories  vary considerably, all group category patterns routinely was comparable to and point out another opportunity for harmonization of the accuracy of the IVD group. Based on this observa- ANA reporting. With respect to the ICAP nomenclature, tion, it is likely that a significant majority of participants our data demonstrated clusters of participants based on that report all three group categories developed compe- the HEp-2 patterns reported by the participants. First, tencies for the more challenging (expert-level) patterns. the majority of participants in this survey reliably read However, although automation of ANA reading holds the and interpreted the centromere, multiple nuclear dots, promise of improved consistency and accuracy of ANA T ebo et al. Autoimmun Highlights (2021) 12:4 Page 9 of 10 pattern recognition, automation-assisted reading in CL are commutable using different sources of HEp-2 rea - participants was not associated with a statistically signifi - gents. The relatively higher competencies of the IVD par - cant improvement in accuracy. ticipants relative to the CL participants is of interest as The ICAP guidance is recognized as a potential road - some laboratories depend on IVD for training as gleaned map towards the harmonization and standardization of from AMLI practice survey [19]. HEp-2 IFA nomenclature [31, 32]. It is understood by its members and opinion leaders that this guidance will Conclusion evolve, taking into consideration practical aspects for its This study highlights significant competency for all par - adoption in clinical laboratories; diverse experience, age- ticipants in identifying the nuclear main categorical ing workforce, variability in reagents, microscopy and HEp-2 IFA patterns. This observation validates the ICAP recent introduction of digital image readers [14, 19, 31]. competent-level classification for this group except for Along these lines, this investigation is not without limi- the anti-topoisomerase I antibody pattern. Our data also tations. First, the intended responses (traditional nomen- demonstrate opportunities for defining competencies clature) for specimens with the cytoplasmic and mitotic and training for CL personnel in recognition of cytoplas- patterns were not defined for specific sub-patterns (for mic and mitotic patterns. example, cytoplasmic speckled or NuMa). This was intentional as it was largely unknown how CL report Supplementary information both patterns. The results obtained from this survey Supplementary information accompanies this paper at https ://doi. validates the approach, as the minority of laboratories org/10.1186/s1331 7-020-00146 -w. reporting less than 3 main categorical patterns do report mitotic patterns considered expert-level on the ICAP Additional File 1: Participating clinical laboratories and in vitro diagnostic classification tree [www.anapa ttern s.org, 12]. Second, the manufacturers. intended responses were monospecific and did not take Additional File 2: Accuracy of HEp-2 IFA pattern reporting based on type mixed patterns into consideration. A number of partici- of nomenclature. pants reported mixed patterns for some of the specimens (data not shown), often with the intended dominant pat- Acknowledgements tern reported together with minor additional pattern We offer our sincere thanks to Maggie Fogel, the Association of Medical Labo - ratory Immunologists (AMLI), Kathryn Kohl and the Staff of PSG, Huntington variants. Such reports were considered appropriate and Valley, PA, USA for assistance with the survey. We also aknowledge all partici- in accordance for reporting patient results with more pating clinical laboraotories and IVD manufacturers for their involvement. than one pattern [2]. Third, the survey included a limited Availability of data and materials number of participating CL including those with a signif- Materials use in the survey was obatined from the Plasma Services Group Inc. icant interest and experience in ANA testing, which may (PSG), Huntington Valley, PA, USA. All data from the survey are in the posses- not reflect the experience of a wider spectrum of interna - sion AET and MHW. tional CL. Finally, some of the participants, particularly Ethics approval and consent to participate those in CL group, may have limited familiarity with the Formal consent not required for this study. ICAP nomenclature, despite being associated with expe- Consent for publication rienced laboratories. All authors have reviewed and approved of this submission. The data presented confirm that standardization of reporting has not been achieved in performance of Competing interests The authors declare that the research was conducted in the absence of any non-traditional HEp-2 patterns even by experienced commercial or financial relationships that could be construed as a potential and interested laboratories. This suggests the need and conflict of interest. opportunities for further training and consensus-build- Author details ing. Using the ICAP nomenclature may have benefits for 1 2 Department of Pathology, University of Utah, Salt Lake City, UT, USA. ARUP some sub-patterns and assigning competencies, nota- Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA. bly for the mitotic and cytoplasmic main categorical Immunopathology Laboratory, Robert J. Tomsich Pathology & Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH, USA. Department of Oral groups and our data clearly demonstrate that recogni- Biology, University of Florida, Gainesville, FL, USA. Department of Medicine, tion of the pattern associated with antibodies to topoi- Cumming School of Medicine, University of Calgary, Calgary, AB, Canada. somerase is linked to familiarity with ICAP patterns. Department of Laboratory Medicine and Pathology & Department of Medi- cine, University of Washington, Seattle, WA, USA. Furthermore, our data confirm previous observations that differences in the HEp-2 cell substrate can contrib - Received: 18 September 2020 Accepted: 12 November 2020 ute to inconsistency in ANA sub-patterns interpretation and reporting [22]. Clearly, consistent ICAP sub-pattern reporting by laboratories is most meaningful if patterns Tebo et al. Autoimmun Highlights (2021) 12:4 Page 10 of 10 References 19. Peterson LK, Tebo AE, Wener MH, Copple SS, Fritzler MJ. Assessment of 1. Meroni PL, Schur PH. ANA screening: an old test with new recommenda- antinuclear antibodies by indirect immunofluorescence assay: report tions. Ann Rheum Dis. 2010;69:1420–2. from a survey by the American Association of Medical Laboratory Immu- 2. Agmon-Levin N, Damoiseaux J, Kallenberg C, Sack U, Witte T, Herold M, nologists. Clin Chem Lab Med. 2020 Apr 8. [Epub ahead of print]. et al. International recommendations for the assessment of autoantibod- 20. Meroni PL, Borghi MO. Diagnostic laboratory tests for systemic autoim- ies to cellular antigens referred to as anti-nuclear antibodies. Ann Rheum mune rheumatic diseases: unmet needs towards harmonization. Clin Dis. 2014;73:17–23. Chem Lab Med. 2018;56(10):1743–8. 3. Pisetsky DS. Antinuclear antibody testing - misunderstood or misbegot- 21. Bogaert L, Van den Bremt S, Schouwers S, Bossuyt X, Van Hoovels L. Har- ten? Nat Rev Rheumatol. 2017;13:495–502. monizing by reducing inter-run variability: performance evaluation of a 4. Damoiseaux J, Andrade LEC, Carballo OG, Conrad K, Francescantonio PL, quality assurance program for antinuclear antibody detection by indirect Fritzler MJ, et al. Clinical relevance of HEp-2 indirect immunofluorescent immunofluorescence. Clin Chem Lab Med. 2019;57(7):990–8. patterns: the International Consensus on ANA patterns (ICAP) perspec- 22. Andrade LEC, Klotz W, Herold M, Conrad K, Rönnelid J, Fritzler MJ, von tive. Ann Rheum Dis. 2019;78:879–89. Mühlen CA, Satoh M, Damoiseaux J, de Melo Cruvinel W, Chan EKL; Exec- 5. Emlen W, O’Neill L. Clinical significance of antinuclear antibodies: utive Committee of ICAP. International consensus on antinuclear anti- comparison of detection with immunofluorescence and enzyme-linked body patterns: definition of the AC-29 pattern associated with antibodies immunosorbent assays. Arthritis Rheum. 1997;40:1612–8. to DNA topoisomerase I. Clin Chem Lab Med. 2018;56(10):1783-1788. 6. Homburger HA, Cahen YD, Griffiths J, Jacob GL. Detection of antinuclear 23. Dellavance A, Gallindo C, Soares MG, Silva NP, Mortara RA, Andrade LE. antibodies: comparative evaluation of enzyme immunoassay and indirect Redefining the Scl-70 indirect immunofluorescence pattern: autoanti- immunofluorescence methods. Arch Pathol Lab Med. 1998;122:993–9. bodies to DNA topoisomerase I yield a specific immunofluorescence 7. Tan EM, Smolen JS, McDougal JS, Butcher BT, Conn D, Dawkins R, et al. A pattern. Rheumatology. 2009;48:632–8. critical evaluation of enzyme immunoassays for detection of antinuclear 24. Turan Faraşat V, Ecemiş T, Doğan Y, et al. A Multicenter Analysis of Sub- autoantibodies of defined specificities. I. Precision, sensitivity, and speci- jectivity of Indirect Immunofluorescence Test in Antinuclear Antibody ficity. Arthritis Rheum 1999;42:455–64. Screening. Arch Rheumatol. 2019;34(3):326–33. 8. Tonuttia E, Bassetti D, Piazza A, Visentini D, Poletto M, Bassetto F, et al. 25. Tan EM, Feltkamp TE, Smolen JS, et al. Range of antinuclear antibodies in Diagnostic accuracy of ELISA methods as an alternative screening test to “healthy” individuals. Arthritis Rheum. 1997;40(9):1601–11. indirect immunofluorescence for the detection of antinuclear antibodies. 26. Egner W. The use of laboratory tests in the diagnosis of SLE. J Clin Pathol. Evaluation of five commercial kits. Autoimmunity. 2004;37:171–6. 2000;53(6):424–32. 9. Choi MY, Cui J, Costenbader K, Rydzewski D, Bernhard L, Schur P. Different 27. Sack U, Conrad K, Csernok E, et al. Autoantibody detection using indirect immunofluorescence ANA substrate performance in a diagnostic indirect immunofluorescence on HEp-2 cells. Ann N Y Acad Sci. setting of patients with SLE and related disorders: retrospective review 2009;1173:166–73. and analysis. Lupus Sci Med. 2020;7:e000431. 28. Banhuk FW, Pahim BC, Jorge AS, Menolli RA. Relationships among Anti- 10. Copple SS, Sawitzke AD, Wilson AM, Tebo AE, Hill HR. Enzyme-linked bodies against Extractable Nuclear Antigens, Antinuclear Antibodies, and immunosorbent assay screening then indirect immunofluorescence con- Autoimmune Diseases in a Brazilian Public Hospital. Autoimmune Dis. firmation of antinuclear antibodies: a statistical analysis. Am J Clin Pathol. 2018;2018:9856910. 2011;135:678–84. 29. Tanaka N, Muro Y, Sugiura K, Tomita Y. Anti-SS-A/Ro antibody determina- 11. Olsen NJ, Choi MY, Fritzler MJ. Emerging technologies in autoantibody tion by indirect immunofluorescence and comparison of different meth- testing for rheumatic diseases. Arthritis Res Ther. 2017;19:172. ods of anti-nuclear antibody screening: evaluation of the utility of HEp-2 12. Chan EK, Damoiseaux J, Carballo OG, Conrad K, de Melo Cruvinel W, cells transfected with the 60 kDa SS-A/Ro as a substrate. Mod Rheumatol. Francescantonio PL, et al. Report of the First International Consensus on 2008;18(6):585–92. Standardized Nomenclature of Antinuclear Antibody HEp-2 Cell Patterns 30. Betancur JF, Londoño A, Estrada VE, et al. Uncommon patterns of anti- 2014-2015. Front Immunol. 2015;6:412. nuclear antibodies recognizing mitotic spindle apparatus antigens and 13. Chan EK, Damoiseaux J, de Melo Cruvinel W, Carballo OG, Conrad K, clinical associations. Medicine (Baltimore). 2018;97(34):e11727. Francescantonio PL, et al. Report on the second International Con- 31. Tebo AE. Recent approaches to optimize laboratory assessment of anti- sensus on ANA Pattern (ICAP) workshop in Dresden 2015. Lupus. nuclear antibodies. Clin Vaccine Immunol. 2017;24(12):e00270-17. 2016;25:797–804. 32. Damoiseaux J. The perspective on standardisation and harmonisation: 14. Damoiseaux J, von Mühlen CA, Garcia-De La Torre I, Carballo OG, de Melo the viewpoint of the EASI president. Auto Immun Highlights. 2020;11(1):4. Cruvinel W, Francescantonio PL, Fritzler MJ, et al. International consensus on ANA patterns (ICAP): the bumpy road towards a consensus on report- Publisher’s Note ing ANA results. Auto Immun Highlights 2016;7:1. Springer Nature remains neutral with regard to jurisdictional claims in pub- 15. Hoffman IE, Peene I, Veys EM, De Keyser F. Detection of specific antinu- lished maps and institutional affiliations. clear reactivities in patients with negative anti-nuclear antibody immuno- fluorescence screening tests. Clin Chem. 2002;48:2171–6. 16. von Mühlen CA, Tan EM. Autoantibodies in the diagnosis of systemic rheumatic diseases. Semin Arthritis Rheum. 1995;24:323–58. 17. Wiik AS. Guidelines for Antinuclear Antibody Testing. EJIFCC. 2006;17(3):134–40. 18. Herold M, Klotz W, Andrade LEC, Conrad K, Cruvinel WM, Damoiseaux J, et al. International Consensus on Antinuclear Antibody Patterns: defining negative results and reporting unidentified patterns. Clin Chem Lab Med. 2018;56:1799–802.

Journal

Autoimmunity HighlightsSpringer Journals

Published: Feb 27, 2021

There are no references for this article.