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

Domain 1: Educational and professional background of respondents

Almost half of the 179 respondents were medical technologists (MT), medical laboratory scientists (MLS) or medical laboratory technologists (MLT); 24% were laboratory directors, and 13% were laboratory supervisors. The ‘other’ write-in responses included a phlebotomist, a technical consultant, trainees (fellows and residents), a laboratory manager and a laboratory assistant. Approximately two thirds of respondents worked in hospital laboratories, while 12% were employed by private reference laboratories, 6% by university or academic laboratories and 4% by IVD manufacturers or distributors. More than half of respondents (57%) had worked in their current roles for over 10 years; approximately one quarter (26%) for 1 to 5 years; 12% for 6 to 10 years and 5% for less than 1 year (Table 1).

Domain 2: Current practice of ANA testing

Eighty-three percent (83%) of the respondents indicated that they were personally involved in ANA testing. Of those, three quarters reported utilizing microscopic (IFA or indirect immunoperoxidase) detection assays. Over one half of the respondents were involved in reviewing HEp-2 IFA results for quality control and quality assurance purposes or for supervising or training of staff performing ANA by microscopy. Approximately one third of the respondents were involved in the selection of ANA methods/kits, consulting with providers about ANA results and/or writing ANA interpretive results or comments. Relatively few respondents were involved in ordering tests as a clinician or involved in IVD manufacturing (Table 1). Most of the respondents’ institutions screen for ANA by IFA using HEp-2 or HEp-2000 substrates. Approximately one third of these laboratories used an automated HEp-2 IFA platform for this testing [10]. Other methods used for ANA screening include multi-analyte arrays (18%), enzyme-linked immunosorbent assays (ELISA; 17%) and indirect immunoperoxidase assays using HEp-2 or HEp-2000 substrates (8%). With respect to formal training on HEp-2 IFA testing, approximately 65% of respondents indicated they received training on the job, while a minority received training in formal training programs (Medical Laboratory Science [MLS]: <30%, MD, MD/PhD or PhD fellowship: <20%). Approximately 10% of the respondents reported that they did not receive formal training. The ‘other’ write-in responses specified that other respondents received training in workshops and meetings and by colleagues. Some respondents questioned the terminology used, indicating that they were not sure that they would regard their training as ‘formal’. When asked about external resources used by their laboratories to train on HEp-2 IFA assessment, approximately 45% indicated they used ICAP, 28% used an IVD manufacturer, 17% did not know if external resources were used and 15% reported that their laboratories had not used external resources to train on HEp-2 IFA assessment. The ‘other’ write-in responses included the following: CAP surveys/survey samples; workshops; vendor websites, promotional material and training documents (charts, booklets, online ANA training modules, etc.); training books and CDs; medical training website, University of Washington; Birmingham Online ANA Quiz; IfQ-Lübeck; reference laboratory and MLS student instruction only. Regarding competency for HEp-2 IFA testing, the majority (~90%) of the respondents reported using external proficiency testing such as the College America Pathologists (CAP) program, while 30–40% of individuals indicated they used internal proficiency and training modules. Less than 10% of the responders reported using programs provided by IVD manufacturers or specimen exchange (Figure 1).

Figure 1:

Resources used to evaluate competency for ANA testing at respondents’ institutions.

CAP, College of American Pathologists; IVD, in vitro diagnostic.

Domain 3: Gap assessment

The vast majority of respondents indicated that their laboratories reported homogenous (anti-cell [AC]-1; 98%), speckled (AC-2, 4, 5, 29; 97%), centromere (AC-3; 96%) and nucleolar (AC-8, 9, 10; 95%) nuclear HEp-2 IFA patterns. Fewer labs reported other nuclear patterns such as discrete nuclear dots (AC-6, 7; 41%), dense fine speckled (AC-2; 32%), pleomorphic (PCNA-like) (AC-13, 14; 28%) and the nuclear envelope (AC-11, 12; 27%) patterns (Figure 2). Overall, fewer respondents indicated that their laboratories reported both cytoplasmic and mitotic patterns. Of the cytoplasmic patterns, respondents indicated that their laboratories report speckled (AC-18, 19, 20; 43%), reticular/anti-mitochondrial antibody (AMA, AC-21; 36%), polar/Golgi (AC-22; 30%), rods and rings (AC-23; 17%) and fibrillar (AC-15, 16, 17; 17%) cytoplasmic patterns (Figure 3). Regarding the mitotic patterns, respondents indicated their laboratories reported spindle fibers (NuMA-like, AC-25, 26; 39%), centrosome (AC-24; 32%), mitotic chromosomal (AC-28; 21%) and intercellular bridge (AC-27; 11%) patterns (Figure 4).

Figure 2:

HEp-2 indirect immunofluorescence assay (IFA) patterns.

Comparison between the nuclear HEp-2 IFA patterns reported by respondents’ laboratories, those that respondents indicated must be reported and those that respondents indicated that they are uncomfortable reporting.

Figure 3:

Cytoplasmic HEp-2 IFA patterns.

Comparison between the cytoplasmic HEp-2 IFA patterns reported by respondents’ laboratories, those that respondents indicated must be reported and those that respondents indicated that they are uncomfortable reporting.

Figure 4:

Mitotic HEp-2 IFA patterns.

Comparison between the mitotic HEp-2 IFA patterns reported by respondents’ laboratories, those that respondents indicated must be reported and those that respondents indicated that they are uncomfortable reporting.

When asked which HEp-2 IFA patterns should be reported by clinical laboratories, for nuclear HEp-2 IFA patterns the responses tended to mirror the previous responses of those patterns reported by their laboratories. The majority of respondents indicated the homogenous (AC-1; 95%), speckled (AC-2, 4, 5, 29; 95%), centromere (AC-3; 92%) and nucleolar (AC-8, 9, 10; 90%) nuclear ANA patterns must be reported. Fewer respondents answered pleomorphic (PCNA-like) (AC-13, 14; 37%), discrete nuclear dots (AC-6, 7; 36%), dense fine speckled (AC-2; 34%) and the nuclear envelope (AC-11, 12; 33%) patterns must be reported (Figure 2). Overall, fewer respondents indicated cytoplasmic and mitotic ANA patterns must be reported. Of the cytoplasmic ANA patterns, respondents thought the reticular/anti-mitochondrial antibody (AMA, AC-21; 42%), speckled (AC-18, 19, 20; 41%), polar/Golgi (AC-22; 28%), rods and rings (AC-23; 22%) and fibrillar (AC-15, 16, 17; 17%) cytoplasmic ANA patterns must be reported (Figure 3). For the mitotic ANA patterns, respondents indicated the spindle fibers (NuMA-like AC-25, 26; 31%), centrosome (AC-24; 29%), mitotic chromosomal (AC-28; 23%) and intercellular bridge (AC-27; 14%) patterns must be reported (Figure 4).

We asked respondents to indicate the settings in which additional training would be most useful. Eighty percent of respondents indicated that additional training requirements for HEp-2 IFA assessment would be useful in the clinical laboratories performing the testing. Three quarters of respondents thought that additional training requirements would be useful in medical laboratory scientist or technologist training programs. Just over one half of the respondents (51%) answered additional training would be useful in physician/healthcare provider continuing education programs, and 42% indicated that additional training requirements for HEp-2 IFA assessment would be useful during postgraduate training programs for laboratory professionals. Four percent of respondents did not know where additional training requirements for HEp-2 IFA assessment would be most useful. One respondent indicated that they would be useful in continuing education programs for laboratory professionals, and one respondent did not think that any further training initiatives were necessary.

Nearly all (95%) respondents indicated that their laboratories reported HEp-2 IFA results as negative or positive along with titers and reference ranges (qualitative and semi-quantitative). However, 2% indicated their laboratory reported HEp-2 IFA results qualitatively as positive or negative (and/or indeterminate). Three percent reported that they did not know whether their laboratory reported titers and reference ranges for HEp-2 IFA. Almost half (48%) of respondents indicated that HEp-2 IFA or solid phase assay reports from their laboratory provide a statement about the clinical significance of the results and 38% provided recommendations for additional testing. In contrast, 33% of the respondents reported that their laboratories do not provide either recommendations for additional testing or information about the clinical significance of HEp-2 IFA or solid phase assays. About 10% of the respondents did not know whether HEp-2 IFA or solid phase reports include additional information about clinical relevance or follow-up testing.

Domain 4: Role of ICAP and other organizations in providing guidance on ANA testing

Fifty-five percent (55%) of respondents answered that they were aware of the ICAP initiatives. Even more (67%) thought ICAP guidance on ANA nomenclature and interpretation provides value to the clinical laboratory, whereas 31% answered that they did not know and only 3% answered no. Respondents were asked to provide their own answer for the value provided by the ICAP guidance: 61% answered that it provides value in promoting standardization and/or consistency, 24% answered that it provides value as a reference and/or educational tool, 10% indicated it increases clinical correlation and fills a gap and 5% did not know.

Since the ICAP HEp-2 IFA classification tree (www.anapatterns.org) includes nuclear, cytoplasmic and mitotic patterns, with designations of competent level or expert level, survey respondents were asked to indicate which of the patterns listed in the survey they were not comfortable identifying. Respondents were most comfortable identifying the homogeneous (AC-1), speckled (AC-2, 4, 5, 29), nucleolar (AC-8, 9, 10) and centromere (AC-3) nuclear HEp-2 IFA patterns. These patterns are considered competent level by ICAP (Figure 2). Approximately 20% of respondents were not comfortable identifying the discrete nuclear dot pattern (AC-6, 7), and nearly half of respondents were not comfortable identifying the dense fine speckled pattern (AC-2), both considered competent level by ICAP. Of the expert-level ICAP nuclear ANA patterns, 40% and 55% of respondents were not comfortable identifying the nuclear envelope (AC-11, 12) and pleomorphic (PCNA-like, AC-13, 14) patterns, respectively. Overall, respondents were less comfortable identifying cytoplasmic and mitotic HEp-2 IFA patterns than they were identifying nuclear patterns. Of the cytoplasmic patterns queried, respondents were least comfortable identifying the fibrillar (AC-15, 16, 17; 61%) and rods and rings (AC-23; 57%) patterns, while for polar/Golgi (AC-22), speckled (AC-18, 19, 20) and reticular/anti-mitochondrial antibody (AMA, AC-21) patterns the number of respondents not comfortable identifying the patterns ranged from 34% to 39% (Figure 3). Of the expert-level mitotic patterns, nearly three quarters of respondents were not comfortable identifying the intracellular bridge pattern (AC-27), 60% were not comfortable identifying the mitotic chromosomal pattern (AC-28), and 37% and 39% of respondents were not comfortable identifying the spindle fibers (NuMA-like, AC-25, 26) and centrosome (AC-24) patterns, respectively (Figure 4).

Responses to specific competencies for optimal microscopic analysis of HEp-2 IFA by the respondents indicated areas where guidance would be beneficial (Figure 5). More than 80% of participants indicated a need for guidance in classification of HEp-2 IFA staining and patterns with other suggestions for guidance on cytoplasmic and mitotic patterns, minimum requirements for performing ANA by IFA testing, troubleshooting discrepant ANA results and suggesting confirmatory testing based on the HEp-2 IFA pattern.

Figure 5:

Responses on specific areas where additional guidance on HEp-2 IFA analysis would be beneficial to laboratories.

Only 8% of respondents were aware of other initiatives to improve HEp-2 IFA assessment. When respondents were asked to provide their own answers for other initiative(s) for improving HEp-2 IFA assessment they were aware of, responses included the CAP Diagnostic Immunology and Flow Cytometry Committee, the American Association of Clinical Chemistry, the University of Washington (Seattle) training module on ANA (https://www.medtraining.org) and the European Autoimmunity Standardization Initiative.