Sensitivity Of Indirect Immunofluorescence Research Articles

P015 Improving the diagnostic performance of ANCA testing: evaluating the use of a gating strategy for ANCA test requests

Abstract Background/Aims Anti-neutrophil cytoplasmic antibody (ANCAs) associated vasculitis (AAV) encompasses: Granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA). The 1999 international consensus for ANCA testing recommended the use of indirect immunofluorescence (IIF) to screen for ANCAs, with immunoassays for proteinase-3 (PR3)-ANCAs and myeloperoxidase (MPO)-ANCAs in positive samples. In 2017 this consensus was revised, recommending the implementation of a strict gating strategy for ANCA requests, followed by high-quality immunoassays as the primary screening method, to reduce the volume of tests requested, reducing the false-positive rates, thereby improving the diagnostic performance of ANCA testing. Testing at the Royal Wolverhampton Trust is based on the 1999 consensus. Tests perform poorly when not carried out in the correct setting due to low pre-test probability; we aimed to implement a gating strategy to filter inappropriate ANCA test requests to improve the diagnostic performance of ANCA testing at our centre. We assessed the use of IIF and antigen specific assays in the diagnosis of AAV. Methods We reviewed the records of patients that had ANCA tests requested in June 2019. A gating strategy in the form of an indication selection ‘pop-up box’ directing clinicians to select an appropriate clinical indication for the request was implemented. We re-audited ANCA requests made post-intervention Feburary 2020. ANCA requests were assessed against clinical indications in the 1999 consensus for appropriateness; we assessed the diagnostic performance by the proportion of ANCA tests correctly identifying AAV and calculating the sensitivity and specificity of IIF and immunoassays for MPO/PR3-ANCAs in detecting AAV. Results Post-intervention, 105 fewer ANCA requests were made (298v403). Due to insufficient information, 345 patients pre-gating and 252 patients post-gating-strategy were used for analysis. The proportion of appropriate requests made was greater post-intervention, 215/252 (85.3%) vs 119/345(34.5%). Pre-intervention AAV was identified in 13 (3.8%) patients; AAV was identified in 21 (8.3%) patients following the gating-strategy introduction. IIF was found to have sensitivity of 91.7% pre and 76.2% post-intervention; specificity was 53.0% pre and 58.9% post-intervention. MPO/PR3 immunoassay testing was found to have a sensitivity of 91.7% pre and 57.1% post-intervention; specificity was 97.4% pre and 97.9% post-intervention. Conclusion The introduction of a gating-strategy for ANCA testing at our centre effectively reduced the overall number of ANCA requests by 26.1% over a 1-month period and significantly increased requests with an appropriate recorded indication. The proportion of tests correctly identifying AAV diagnosis increased following the introduction of the gating strategy indicating an improvement in diagnostic performance of ANCA testing. These results indicate antigen specific testing is more specific for a diagnosis of AAV and sensitivity of IIF was reduced following introduction of the gating strategy suggesting IIF is of no additional benefit when an effective gating strategy is employed. Disclosure N. Cleaton: None. N. Barkham: None. T. Adizie: None.

A hierarchical bivariate meta-analysis of diagnostic test accuracy to provide direct comparisons of immunoassays vs. indirect immunofluorescence for initial screening of connective tissue diseases.

To compare indirect immunofluorescence (IIF) for antinuclear antibodies (ANA) against immunoassays (IAs) as an initial screening test for connective tissue diseases (CTDs). A systematic literature review identified cross-sectional or case-control studies reporting test accuracy data for IIF and enzyme-linked immunosorbent assays (ELISA), fluorescence enzyme immunoassay (FEIA), chemiluminescent immunoassay (CLIA) or multiplex immunoassay (MIA). The meta-analysis used hierarchical, bivariate, mixed-effect models with random-effects by test. Direct comparisons of IIF with ELISA showed that both tests had good sensitivity (five studies, 2321 patients: ELISA: 90.3% [95% confidence interval (CI): 80.5%, 95.5%] vs. IIF at a cut-off of 1:80: 86.8% [95% CI: 81.8%, 90.6%]; p = 0.4) but low specificity, with considerable variance across assays (ELISA: 56.9% [95% CI: 40.9%, 71.5%] vs. IIF 1:80: 68.0% [95% CI: 39.5%, 87.4%]; p = 0.5). FEIA sensitivity was lower than IIF sensitivity (1:80: p = 0.005; 1:160: p = 0.051); however, FEIA specificity was higher (seven studies, n = 12,311, FEIA 93.6% [95% CI: 89.9%, 96.0%] vs. IIF 1:80 72.4% [95% CI: 62.2%, 80.7%]; p < 0.001; seven studies, n = 3251, FEIA 93.5% [95% CI: 91.1%, 95.3%] vs. IIF 1:160 81.1% [95% CI: 73.4%, 86.9%]; p < 0.0001). CLIA sensitivity was similar to IIF (1:80) with higher specificity (four studies, n = 1981: sensitivity 85.9% [95% CI: 64.7%, 95.3%]; p = 0.86; specificity 86.1% [95% CI: 78.3%, 91.4%]). More data are needed to make firm inferences for CLIA vs. IIF given the wide prediction region. There were too few studies for the meta-analysis of MIA vs. IIF (MIA sensitivity range 73.7%-86%; specificity 53%-91%). FEIA and CLIA have good specificity compared to IIF. A positive FEIA or CLIA test is useful to support the diagnosis of a CTD. A negative IIF test is useful to exclude a CTD.

Evaluation of the value of indirect immunofluorescence on different substrates in the diagnosis of autoimmune subepidermal bullous diseases

Objective To evaluate the value of indirect immunofluorescence (IIF) on three different substrates including normal human skin (NS) , monkey esophagus (ME) and salt-split human skin (SS) in the diagnosis of autoimmune subepidermal bullous diseases. Methods A total of 56 patients with autoimmune subepidermal bullous diseases, including 47 with bullous pemphigoid (BP) , 6 with epidermolysis bullosa acquisita (EBA) , 2 with linear IgA bullous dermatosis, and 1 with anti-P200 pemphigoid, were diagnosed in and enrolled from Department of Dermatology, Institute of Dermatology, Chinese Academy of Medical Sciences between January 2015 and December 2016. Seventy patients with pemphigus, 15 patients with chronic eczema and 15 healthy adults served as controls. Blood samples collected from these patients and controls were subjected to IIF on three different substrates including NS, ME and SS, and the fluorescence deposition was observed. The sensitivities and specificities of IIF in the diagnosis of different subepidermal bullous diseases were compared. Statistical analysis was carried out with SPSS 13.0 software by using chi-square test for the comparison of enumeration data. Results IIF on NS or ME in the serum of patients with BP showed linear deposition of fluorescent material along the basement membrane zone. IIF on SS showed linear deposition of fluorescent material in the epidermis in the patients with BP, but in the dermis in the patients with EBA and anti-P200 pemphigoid. The sensitivities of IIF on NS, ME or SS in the diagnosis of subepidermal bullous diseases were 73.2%, 60.7% and 94.6% respectively, and the specificities were 98.0%, 100% and 97.1% respectively. There were significant differences among the sensitivities (χ2 = 18.2, P 0.05) . The diagnostic sensitivity of IIF on SS was significantly higher than that of IIF on NS or ME (χ2 = 8.0, 16.7, both P < 0.05) . Conclusion In the diagnosis of autoimmune subepidermal bullous diseases, IIF on SS is superior to IIF on ME or NS. Key words: Fluorescent antibody technique, indirect; Pemphigoid, bullous; Autoimmune subepidermal bullous disease; Diagnosis; Sensitivity; Specificity

Hygienic monitoring of Mongolian gerbils: which mouse viruses should be included?

The Mongolian gerbil (Meriones unguiculatus) serves as an animal model for a wide range of diseases. A practical limitation in its use is the definition of the hygienic status, as not much is known about viruses that potentially infect gerbils and might be transmitted to other rodents. As successful re-derivation was recently described for gerbils, we now aimed at investigating which mouse viruses induce seroconversion in gerbils and might be transmitted to mice. Gerbils were inoculated with viral agents of mice and co-housed with mouse contact sentinels. Seroconversion in gerbils was observed after oronasal inoculation with Sendai virus (SeV), mammalian orthoreovirus serotype 3 (Reo-3) and rotavirus A (RV-A, EDIM), seroconversion to RV-A also in sentinel mice. Pneumonia virus of mice (PVM) was not detected by serology but by polymerase chain reaction in gerbils and respective sentinel mice. No seroconversion towards or transmission of murine hepatitis virus, murine norovirus, minute virus of mice or mouse cytomegalovirus was detected. Anti-gerbil IgG antibodies did not increase sensitivity of indirect immunofluorescence (IFA) compared with anti-mouse IgG. In conclusion, seroconversion to SeV, Reo-3 and RV-A as well as transmission of RV-A and PVM indicate that these agents should be included in health monitoring of gerbils. Furthermore, anti-mouse IgG is suitable as a secondary antibody for IFA with gerbil serum.

Update on anti-NMO IgG antibodies

Neuromyelitis optica (NMO) or Devic’s syndrome is a severe idiopathic and relapsing, demyelinating disease characterised by optic neuritis (ON) and longitudinally extensive transverse myelitis (LETM). 1 Neuropathologically, NMO is characterised by extensive demyelination with partial necrosis of the spinal cord white and grey matter, acute axonal injury, neutrophil and eosinophil infiltrates, IgG and IgM deposition and perivascular complement activation. NMO is distinguished from classical multiple sclerosis (MS) by clinical, neuroimaging, CSF and serological criteria. 1 The identification of NMO-IgG by indirect immunofluorescence (IIF) was initially described as a highly specific marker with the titre paralleling the disease activity. However, the sensitivity of IIF is variable (58–75%) for clinical NMO. Consequently, numerous methods using aquaporin 4 (AQP4) as the target antigen have been developed in an attempt to improve sensitivity. Apart from cell-based assays, these have failed to improve on the sensitivity of IIF. Recently, it has been demonstrated that NMO-IgG targets mainly the M-23 isoform of AQP4 that structurally organises itself into orthogonol arrays of particles in cell membranes. As AQP4-Ab recognises conformational epitopes, the lower sensitivity of all but the cell-based assays may be due to changes resulting during purification, fixation or other causes of NMO, i.e., parainfectious NMO. As indirect immunofluorescence is the most widely available method for detecting NMO-IgG, the Pathology Queensland experience has raised issues of the ‘true’ sensitivity of NMO-IgG testing for NMO as well as identifying other autoantibodies (including anti-reticulin and anti-nuclear antibodies) that cause interference and make IIF interpretation difficult. These issues may contribute to both false positive and false negative IIF results. Some authors have suggested 2 that the published sensitivities of NMO-IgG may be artificially high and this has prompted the proposed Australian and New Zealand NMO study designed to evaluate the sensitivity and specificity of all currently available methods for NMO-IgG.

Sensitivity of indirect immunofluorescence and ELISA in detecting intercellular antibodies in endemic pemphigus foliaceus (Fogo Selvagem)

Since 1967 dermatology has used the classic technique of indirect immunofluorescence (IFI) for the detection of autoantibodies against antigens of the skin in diseased people with endemic pemphigus foliaceus. Thirty years later enzyme-linked immunosorbent assays--ELISA (rDsg1 and rDsg3) appeared as a viable option. A group of highly recognized researchers have concluded that ELISA is a simple, sensitive and highly specific method, allowing for diagnostic differentiation between pemphigus vulgaris (PV) and endemic pemphigus foliaceus (EPF). Scientific literature certifies that both ELISA and IIF bear high sensitivity in spite of the fact that a direct comparison between the ELISA and IIF tests has never been performed. This study was conducted to compare the sensitivity of these tests in detecting antibodies in the EPF. Thirty-two serum samples were collected from patients with EPF. The control serum of 15 healthy individuals was tested to detect the presence of antibodies of EPF by indirect immunofluorescence and ELISA (rDsg1 and rDsg3). The IIF was performed, taking human skin as a substrate. Antibodies in patients with EPF were detected more commonly by the ELISA (rDsg1) (91%) compared with IIF (81%). The ELISA (rDsg1) is slightly more sensitive than IIF in detecting antibodies related to EPV. However, according to our results, we do not currently possess a test with 100% accuracy in differentiating EPF from PV. Although previous studies have associated Dsg3 with PV, the tests performed during this study showed that 12% (4/32) of patients with EPF (cutaneous diseases only) also had Dsg3 antibodies.

Detection of anti-Giardia lamblia serum antibody among children of day care centers.

To detect anti-Giardia lamblia serum antibodies in healthy children attending public day care centers and to assess serological tests as tools for estimating the prevalence of G. lamblia in endemic areas. Three separate stool specimens and filter paper blood samples were collected from 147 children ranging from 0 to 6 years old. Each stool sample was processed using spontaneous sedimentation and zinc sulfate flotation methods. Blood samples were tested by indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA) for Giardia IgG. Of 147 individuals tested, 93 (63.3%) showed Giardia cysts in their feces. Using IIF and ELISA, serum antibodies were detected in 93 (63.3%) and 100 (68%) samples, respectively. Sensitivity of IIF and ELISA was 82% and 72%, respectively. However, ELISA revealed to be less specific (39%) than IIF (70%). IIF also showed a higher concordance with microscopic examination than ELISA.

Sensitivity of indirect immunofluorescence, substrate specificity, and immunoblotting in the diagnosis of pemphigus

Background: Several assays are available to detect pemphigus antibodies. The most commonly used are indirect immunofluorescence (IIF) and immunoblotting (IB). Objective: Our purpose was to compare the sensitivity of these assays in detecting pemphigus antibodies. Methods: Fifty-two sera from 41 patients with pemphigus vulgaris (PV) and 22 sera from 18 patients with pemphigus foliaceus (PF) were tested concurrently for the presence of pemphigus antibodies. The IIF studies were conducted with two different substrates: monkey and guinea pig esophagus. Results: Pemphigus antibodies were detected with equal sensitivity by IIF in patients with PV and PF (i.e., positive in 87% and 86% of sera, respectively). By contrast, IB assay was much less sensitive in PF than in PV (i.e., positive in 45% vs 83% of sera, respectively). The antibodies in PV generally reacted more strongly against monkey esophagus, whereas those in PF reacted more strongly against guinea pig esophagus. All patients with intercellular antibodies that reacted more strongly against monkey than guinea pig esophagus had PV, whereas all those with intercellular antibodies that reacted more strongly against guinea pig than monkey esophagus had PF. Conclusion: IIF is a more sensitive assay than IB for detecting antibodies associated with PF. The substrate specificity of the antibodies provides a simple means to distinguish between PV and PF. (J Am Acad Dermatol 1997;37:211-6.)

Detection of Bacteroides fragilis, Bacteroides thetaiotaomicron, and Bacteroides ovatus in clinical specimens by immunofluorescence with a monoclonal antibody to B. fragilis lipopolysaccharide

A total of 1,897 clinical specimens (1,019 aspirates and 876 swabs) were studied by indirect immunofluorescence (IF) with a mouse monoclonal antibody (MAb) against a D-galactose oligomer of Bacteroides fragilis lipopolysaccharide. The MAb has been shown to react with 96% of clinical B. fragilis isolates and with about 50% of Bacteroides ovatus and Bacteroides thetaiotaomicron isolates but not with other aerobic or anaerobic organisms tested. The sensitivity of IF in comparison with culturing was 78.9% for all three species. Of the 32 strains originating from culture-positive, IF-negative specimens, 13 lacked the target determinant for the MAb. Sensitivity was highest with specimens taken from the perineal area (87.1%) and lowest with those taken from undefined sites (56.6%). Sensitivity was better with aspirates (86.8%) than with swabs (72.6%). The specificity of IF was 95.6% for all of the material. Positive and negative predictive values were 51.1 and 98.0%, respectively. Neither long transportation times of specimens nor antimicrobial therapy seemed to correlate with the occurrence of IF-positive, culture-negative specimens. This study shows that a single MAb can be used to establish an IF assay that can complement isolation in the detection of these three members of the B. fragilis group.

Respiratory syncytial virus detection by immunofluorescence in nasal secretions with monoclonal antibodies against selected surface and internal proteins.

Specimens containing respiratory tract epithelial cells from infants and children with acute respiratory disease were evaluated by using an indirect immunofluorescence technique with two specific respiratory syncytial virus monoclonal antibodies. One (RS/HN 13-1) was directed against a cell surface viral antigen, and the other (RS/HN 25-2) was directed against viral antigen present in large cytoplasmic inclusions. The same results on presence or absence of respiratory syncytial virus were obtained by cell culture and immunofluorescence in 93% of 252 patients tested adequately by both methods. The sensitivity of indirect immunofluorescence was approximately equal to that of cell culture. A total of 84 specimens were positive for RSV by immunofluorescence; 82 of them were positive with both monoclones, and the remaining 2 were positive only with the monoclone directed against the internal protein. The fluorescence pattern of the latter monoclone was unique and easily recognized. Indirect immunofluorescence testing with monoclonal antibodies to respiratory syncytial virus proved to be a very useful diagnostic technique, and results could be obtained within 4 h of specimen collection.