Current laboratory and clinical practices in reporting and interpreting anti?nuclear antibody indirect immunofluorescence (ANA IIF) patterns: results of an international survey

Background: The International Consensus on Antinuclear Antibody (ANA) Patterns (ICAP) has recently proposed nomenclature in order to harmonize ANA indirect immunofluorescence (IIF) pattern reporting. ICAP distinguishes competent-level from expert-level patterns. A survey was organized to evaluate reporting, familiarity, and considered clinical value of ANA IIF patterns. Methods: Two surveys were distributed by European Autoimmunity Standardization Initiative (EASI) working groups, the International Consensus on ANA Patterns (ICAP) and UK NEQAS to laboratory professionals and clinicians. Results: 438 laboratory professionals and 248 clinicians from 67 countries responded. Except for dense fine speckled (DFS), the nuclear competent patterns were reported by>85% of the laboratories. Except for rods and rings, the cytoplasmic competent patterns were reported by>72% of laboratories. Cytoplasmic IIF staining was considered ANA positive by 55% of clinicians and 62% of laboratory professionals, with geographical and expertise-related differences. Quantification of fluorescence intensity was considered clinically relevant for nuclear patterns, but less so for cytoplasmic and mitotic patterns. Combining IIF with specific extractable nuclear antigens (ENA)/dsDNA antibody testing was considered most informative. Of the nuclear competent patterns, the centromere and homogeneous pattern obtained the highest scores for clinical relevance and the DFS pattern the lowest. Of the cytoplasmic patterns, the reticular/mitochondria-like pattern obtained the highest scores for clinical relevance and the polar/Golgi-like and rods and rings patterns the lowest. Conclusion: This survey confirms that the major nuclear and cytoplasmic ANA IIF patterns are considered clinically important. There is no unanimity on classifying DFS, rods and rings and polar/Golgi-like as a competent pattern and on reporting cytoplasmic patterns as ANA IIF positive

Clinical and laboratory studies of the fate of intranasal allergen.

The precise way in which allergen is handled by the nose is unknown. The objective of this study was to determine recovery of Der p 1 allergen following nasal administration and to determine whether Der p 1 can be detected in nasal biopsies after natural exposure and nasal challenge to allergen.(1) 20 nonatopic non-rhinitics were challenged with Der p 1 and recovery was measured by ELISA in the nasal wash, nasal mucus and induced sputum up to 30 minutes. Particulate charcoal (<40 μm) served as control. (2) In 8 subjects (5 atopics), 30 to 60 minutes after challenge histological localisation of Der p 1 in the nasal mucosal epithelium, subepithelial mucous glands and lamina propria was performed. Co-localisation of Der p 1 with macrophages and IgE-positive cells was undertaken.(1) Less than 25% of total allergen was retrievable after aqueous or particulate challenge, most from the nasal mucus during 1-5 min after the challenge. The median of carbon particles recovered was 9%. (2) Prechallenge Der p 1 staining was associated with the epithelium and subepithelial mucous glands. After challenge there was a trend for greater Der p 1 deposition in atopics, but both atopics and nonatopics showed increases in the number of Der p 1 stained cells and stained tissue compartments. In atopics, increased eosinophils, macrophages and IgE positive cells co-localized with Der p 1 staining.Der p 1 allergen is detected in nasal tissue independent of atopic status after natural exposure. After challenge the nose effectively retains allergen, which remains mucosally associated; in atopics there is greater Der p 1 deposition and inflammatory response than in nonatopics. These results support the hypothesis that nasal mucus and tissue act as a reservoir for the inhaled Der p 1 allergen leading to a persistent allergic inflammatory response in susceptible individuals

Total recovery of Der p 1 after allergen challenge in 20 nonatopic subjects.

<p>*Each subject had two random sampling times</p><p>**Samples taken on one occasion only.</p><p>***Total allergen recovery—comparison between sampling times (p = 0.2; analysis of variance, Friedman test for multiple samples)</p><p>**** Total allergen recovery—comparison between aqueous or particulate at any time point (p≥0.2; Wilcoxon rank sum test)</p><p>N = Number of subjects; IQR = Interquartile Range</p><p>Total recovery of Der p 1 after allergen challenge in 20 nonatopic subjects.</p

Immunohistochemical detection of Der p 1, IgE and CD68, and the presence of eosinophil leukocytes in nasal biopsies before and after <i>in vivo</i> challenge with Der p 1 allergen.

<p>H-E staining for Eosinophils; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127477#sec006" target="_blank">Methods</a>.</p><p>*Double staining with Der p 1.</p><p>** Staining of cells and the mucous in the epithelium, lamina propria and the submucosal glands. An ordinal scale was used for intensity of Der p 1 staining. This allows for rank order (negative, 1+, 2+, 3+, 4+), but does not allow for measuring relative degree of difference between them.</p><p>***Comparison between non-challenged (pre) and challenged (post) tissue.</p><p>****Comparison between atopic and nonatopic (non-challenged tissue).</p><p>EP: nasal mucosal epithelium; LP: lamina propria; GL: subepithelial mucous glands.</p><p>Number of cells: Mean (range); NA: not available; Statistics: t-test was used for comparison between means.</p><p>Immunohistochemical detection of Der p 1, IgE and CD68, and the presence of eosinophil leukocytes in nasal biopsies before and after <i>in vivo</i> challenge with Der p 1 allergen.</p

Immunohistochemical staining of Der p 1 using mouse anti-Der p 1.

<p>Immunohistochemical staining of Der p 1 using mouse anti-Der p 1.</p