Characteristics of the participating laboratories and clinicians
Four hundred thirty-eight laboratory professionals and 248 clinicians (183 or 74% rheumatologists, for the other clinicians, specialty is not known) from 67 different countries worldwide responded to the surveys, of whom 358 (82%) and 84 (34%), respectively, completed the whole survey. Since the survey was also distributed through an open, web-based format we were unable to accurately estimate the response rate. Table 1 shows the geographic distribution of the respondents. Most of the respondents were from Europe (259/438 or 59% of laboratory professionals and 206/248 or 83% of the clinicians). Fifty percent (220/438) of the laboratory professionals that responded considered their laboratory as expert-level (i.e. recognize patterns that require more expertise) and 54% (135/248) of the clinicians worked in a tertiary hospital.
Reporting of ANA IIF patterns
Table 2 represents an overview of (i) the frequency of reporting of the various AC patterns, (ii) the estimation whether a pattern should be classified as competent, and (iii) the clinical relevance of the various AC patterns. For the clinical relevance score, 1 is the lowest and 5 the highest score.
For each question, the results per pattern, are presented as percentages of the total laboratory (competent and expert reported separately) and clinician respondents. The competent patterns are highlighted in grey. The reported range represents the minimum and maximum number of responders to the specific questions posed.
The competent-level patterns centromere (AC-3), homogeneous (AC-1), speckled (AC-2,4,5) and nucleolar (AC-8,9,10) patterns were used in ANA IIF test result reporting by >90% of competent-level and expert-level laboratories (Table 2). The nuclear dot patterns (AC-6,7) were reported by 85% of the laboratories and the DFS pattern (AC-2) by 62% of the laboratories. Clinicians reported comparable (or somewhat lower) frequencies of being acquainted with those patterns (Table 2).
Cytoplasmic AC patterns were less frequently used in ANA IIF test result reporting than nuclear AC patterns. Among the competent cytoplasmic patterns (fibrillar (AC-15,16,17), speckled (AC-18,19,20), reticular/ mitochondria-like (AC-21), polar/Golgi-like (AC-22), rods and rings (AC-23), the rods and rings pattern (AC-23) was the pattern that was least used (63% of the laboratories) (Table 2).
The competent-level DFS pattern (AC-2) and rods and rings pattern (AC-23) were significantly (p<0.0001) more used by expert-level laboratories than by competent-level laboratories (76% versus 48% for DFS and 73% versus 53% for rods and rings). Moreover, the DFS pattern was less reported by laboratories in North-America (44%) and Europe (57%) than by laboratories in the other continents (Table 3).
Distinction between competent and expert patterns
Laboratory professionals were also interrogated whether they would classify a pattern as competent or expert-level. For most of the nuclear patterns there was a good agreement (84-89%) between the ICAP classification and the provided responses, except for the DFS pattern. Only 50% of the respondents would classify this pattern as competent (Table 2). Of interest, 72% of the respondents considered the nuclear envelope pattern a competent pattern rather than an expert pattern.
For the cytoplasmic patterns considered competent-level by ICAP, 71% - 74% of the respondents consider the fibrillary, speckled and reticular/mitochondria-like pattern a competent pattern, 65% considered the polar/Golgi-like pattern a competent pattern and 57% considered the rods and rings pattern a competent pattern (Table 2).
Are cytoplasmic patterns considered ANA positive?
Sixty-one percent of the clinicians (n=105) and 54% of the laboratory professionals (n=346) considered cytoplasmic HEp-2 cell IIF staining as ANA IIF positive. There were more expert-level laboratory professionals (61%) than competent-level laboratory professionals (46%) that considered cytoplasmic patterns as ANA positive (p=0.0062). The fraction of laboratory professionals that considered cytoplasmic ANA patterns as ANA positive was higher in non-European countries (63%) than in European countries (48%) (p=0.0075) (Table 4.a). However, within Europe, differences between countries were observed. In Austria (15%; n=2/13), the Netherlands (12%; n=3/25), Sweden (13%; n=1/8), Switzerland (29%; n=2/7), Turkey (20%. n=1/5) and the United Kingdom (22%; n=2/9), cytoplasmic ANA IIF patterns were considered ANA positive by <30% of laboratory professionals, while in Belgium (65%; n=22/34), France (57%; n=4/7), Italy (61%; n=11/18), Portugal (78%; n=18/23) and Spain (67%; n=14/21), cytoplasmic patterns are considered ANA positive by >60% of the laboratory professionals (Table 4.b).
Sub-specification of patterns
Less than half of the clinicians and laboratory professionals found it important to sub-specify (i) nucleolar patterns into homogeneous, clumpy and speckled (AC-8,9,10) (respectively 45% and 34%), (ii) cytoplasmic fibrillary patterns into linear, filamentous and segmental (AC-15,16,17) (respectively 28% and 42%) and (iii) cytoplasmic speckled patterns into discrete dots, dense fine speckled and fine speckled (AC-18,19,20) (respectively 42% and 53%). Seventy-one percent of the clinicians and 86% of the laboratory professionals found it important to report the reticular/mitochondria-like cytoplasmic pattern (AC-21). Sixty-two percent of the laboratory professionals distinguished multiple nuclear dots from few nuclear dots (AC-6,7) (data not shown).
The most used sub-specifications in ANA IIF result reports are nuclear fine speckled AC-4 (63%), nuclear coarse speckled AC-5 (65%), multiple nuclear dots AC-6 (74%) and few nuclear dots AC-7 (69%). These sub-specifications are significantly more reported by expert-level than by competent-level laboratories (p<0.0001 for all sub-patterns). However, clinicians are not aware of such ANA IIF sub-specification and do not consider them clinically relevant (Table 2).
Confirmation of IIF by specific tests
There was no difference between competent-level and expert-level laboratories regarding follow-up testing for antibodies to extractable nuclear antigens (ENA) and to dsDNA in case of a positive ANA IIF test. A geographical difference, however, was observed (Supplemental data Table 5). Forty-one percent of European laboratories, but only 13% of North American and 10% of South American laboratories, systematically performed follow-up testing for anti-ENA antibodies and to dsDNA in case of a positive ANA IIF test or on clinical indication when ANA IIF is negative. Forty-seven percent of the North-American laboratories and 44% of the South-American ones, but only 16% of the European laboratories only performed confirmation testing when the tests were specifically requested (Supplemental data Table 5).
The majority of competent (72%; n=165) and expert laboratories (79%; n=179) review their ANA IIF results after ENA/dsDNA confirmation testing, but only a minority would change the results (respectively 8.0% and 9.6%) or add a comment if clinically relevant (respectively 36% and 46%).
Interpretation and clinical significance of ANA IIF patterns
Both clinicians (82%; n=246) and laboratory professionals (81%; n=352) considered the combination of an ANA IIF test result with a corresponding specific anti-ENA or anti-dsDNA test result as the most clinically relevant information.
The quantification of the nuclear ANA IIF fluorescence intensity was found clinically relevant by 79% of laboratory professionals and by 74% of clinicians (Supplemental data Table 6). Thirty-three percent of laboratory professionals and 22% of clinicians reported that one should not titrate cytoplasmic patterns. Forty-three percent of laboratory professionals and 33% of clinicians reported that mitotic patterns should not be titrated (Supplemental Table 6).
Fifty-four percent of the clinicians (n=107) reported that they would take medical decisions based on ANA IIF titer, whereas 87% of the clinicians reported that they would take medical decision based on results of specific testing for anti-ENA or anti-dsDNA.
Clinicians and laboratory professionals were also interrogated on how they appraise the clinical relevance of the various ANA patterns. Overall, clinicians scored the clinical relevance of the various patterns lower than laboratory professionals (Table 2). The highest scores for clinical relevance were obtained for the centromere (AC-3) pattern and the homogeneous (AC-1) pattern. Ninety-three percent of the laboratory professionals and 84% of the clinicians scored the clinical relevance of the centromere pattern high, and 90.2% of the laboratory professionals and 70.6% of the clinicians scored the clinical relevance of the homogeneous pattern high. Seventy-seven to 80% of the laboratory professionals and 61-64% of the clinicians scored the clinical relevance of the nuclear speckled pattern (AC-2,4,5) and nucleolar pattern (AC-8,9,10) high. In contrast, only 41.1% of laboratory professionals and 35.1% of clinicians scored the clinical relevance of the DFS pattern (AC-2) high.
Of the cytoplasmic patterns, the reticular/mitochondria-like pattern (AC-21) obtained the highest score for clinical relevance by laboratory professionals. The other competent cytoplasmic patterns received lower scores, with the lowest scores for polar/Golgi-like (AC-22) and rods and rings (AC-23).
The mitotic patterns obtained low scores for clinical relevance overall with ≤30% of the clinicians and laboratory professionals scoring these patterns as clinically relevant.