Indirect Immunofluorescence Results Research Articles

Discordant indirect immunofluorescence compared to multiplex bead immunoassay in ANCA associated vasculitides, a retrospective analysis

Abstract Based on the revised 2017 international consensus on testing for anti-neutrophil cytoplasmic antibodies (ANCA) in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA), we conducted a retrospective analysis to assess whether testing algorithm modification would be appropriate in our laboratory. Currently, at our institution, the primary screening method for ANCA associated vasculitides (AAV) remains indirect immunofluorescence (IIF), which is reflexed to immunoassay (IA) for confirmation. IA is performed by multiplex bead technology to detect anti-myeloperoxidase (MPO) and anti-proteinase 3 (PR3) antibodies. We reviewed cases from January 1, 2021, to November 30, 2022 using Softlab (laboratory information system) with the aim to find discordant results between IIF and IA (IIF-positive and IA-negative, and IA-positive and IIF-negative). We limited our analysis to in-house patients with clinical information available (EPIC electronic medical record). 204 cases met the criterion for positive IIF and negative IA (positive IA defined as index value >0.9), of which 5% were close to the cut off index value (0.7-0.9). 64% of these 204 patients were never concordant in results available for review (IIF-positive, IA-negative). Potential confounding factors for these never concordant cases include 44% historically or concurrently ANA-positive (10% with clinical diagnosis of lupus), 11% with inflammatory bowel disease, and 10% with an underlying ophthalmologic diagnosis, both overlapping with positive ANA. Notably 36% of the patients who were IIF negative over this period had historically positive MPO or PR3 testing, and the current testing window may reflect higher sensitivity of IIF methods, which in this population includes index values near cutoff but definitionally negative. In contrast, of 247 cases with IA-positive results, 51 cases were discordant with IIF results (IA-positive, IIF-negative). 57% of the discordant cases had a historical diagnosis of AAV who were receiving treatment or in remission. 34% of these AAV discordant cases were near the cut off index value (1.0-1.2). Furthermore, we found that all patients with a clinical diagnosis of AAV were monitored by serial laboratory testing at varying frequency, which included IIF with titers and IA (at least once). As there are no explicit guidelines for laboratory testing to monitor AAV patients, it is unclear whether ordering both assays is of clinical utility in management. With advantages of efficiency and faster turnaround time, IAs may be considered as the primary screening method. In our patient population, we did not identify patients who were IIF-positive only with clinically meaningful disease. Although there may be clinical scenarios that warrant to perform both assays simultaneously, for those with GPA and MPA we conclude that it may be appropriate to transition to use IAs as the primary screening method and use IIF as a confirmatory assay, which may not need to be repeated if positive once.

Internal audit of two antineuronal immunoblots and the role of indirect immunofluorescence in sample screening

Anti-neuronal antibodies are associated with a range of clinical phenotypes that are often preceded by malignancy. Most of these antibodies can be identified by indirect immunofluorescence (IIF) using commercial primate neural tissue slides. The EUROImmun 12 and upgraded RAVO 14 antigen line immunoblots were directly compared to replace a 9 antigen RAVO immunoblot. Initial assessment was with samples with known positive specificities by IIF. The second phase involved parallel testing of routine pooled negative IIF samples as per institutional standard antineuronal testing algorithm. IIF results and clinical correlation were used to arbitrate on discordant immunoblot results. Correlation in known positives was 80%. The EUROImmun immunoblot did not identify disease causing SOX1 and Recoverin antibodies. Routine pooled analysis did not identify discrepancy between the platforms. Assessment of 318 patient samples did not reveal any cases where a diagnosis was correctly identified by line immunoblot and not IIF. A single sample with positive Yo antibodies was identified by both platforms without clinical or IIF correlation, making this a likely false positive. Subsequent to this audit, Westmead ICPMR has migrated to the 14 antigen RAVO line blot for confirmation of positive IIF or for patients with equivocal results only.

Integrating quality assurance in autoimmunity: the changing face of the automated ANA IIF test.

Currently available computer-aided diagnosis (CAD) systems for the detection of anti-nuclear antibodies (ANA) by indirect immunofluorescence (IIF) assay enable a standardized measurement of system-specific fluorescent intensity (FI) measures. We aimed to evaluate an internal quality control (iQC) program that controls the total ANA IIF process in routine practice. In addition to the kit iQC materials, supplemental quality indicators were integrated in a total quality assurance (QA) program: patient-derived iQC's samples (negative, 1/160 fine speckled and 1/160 homogeneous), median sample FI per run and percentage of ANA IIF positive samples per run. Analytical rejection criteria were based on the imprecision of the positivity index (PI) measure of the Zenit PRO system (Menarini). Clinical rejection criteria were based on changes in FI that correspond to a change in ANA IIF titer of≥2. To evaluate the QA program, different artificial errors were introduced during the ANA IIF process. After every run, quality indicators were evaluated and compared to the pre-set target values. Rescanning the ANA IIF slides five times, using an old conjugate and a needle obstruction resulted in analytically and even clinically relevant errors in ANA IIF results. All errors were correctly detected by the different defined quality indicators. Traditional Westgard rules, including analytically (and clinically) defined rejection limits were useful in monitoring quality indicators. The integration of a total process iQC program in CAD systems, based on the specific FI measurands and performance criteria of the system, adds value to QA.

Comparison of C3d immunohistochemical staining to enzyme-linked immunosorbent assay and immunofluorescence for diagnosis of bullous pemphigoid

Bullous pemphigoid (BP), the most common autoimmune blistering disease, may be diagnostically challenging. Direct immunofluorescence (DIF), indirect immunofluorescence (IIF), enzyme-linked immunosorbent assay (ELISA), and recently, C3d immunohistochemistry (IHC), are used as adjuncts to diagnosis. To compare C3d IHC to DIF, IIF, and ELISA testing in BP diagnosis. C3d IHC was performed on skin biopsyspecimens from 51 patients (27 with BP and 24 with other blistering diseases) and compared to DIF and IIF, with anti-BP180 or anti-BP230 ELISA results used as the gold standard. We found C3d IHC, DIF, and IIF had similar sensitivity (74.1%, 63.1%, and 70.4%), specificity (95.8%, 100%, and 100%), positive predictive value (95.2%, 100%, and 100%), and negative predictive value (76.7%, 70.6%, and 75%) for BP. Cases with positive C3d IHC, DIF, and IIF had significantly higher anti-BP180 and anti-BP230 by ELISA than cases with negative testing (P<.0001). False-negative IIF results were associated with lower BP230 compared with true-positive results (P=.03). This was a single-center, retrospective study. Our study compared C3d IHC to DIF and IIF in BP diagnosis, demonstrating C3d IHC on fixed tissue provides similar diagnostic utility to immunofluorescence and ELISA.

Plucked Hair as a Substrate for Indirect Immunofluorescence in Cases of Pemphigus Vulgaris.

Introduction:Indirect immunofluorescence (IIF) is used to determine the circulating autoantibodies in cases of Pemphigus. For IIF in Pemphigus several substrates had been used. This study was done to determine whether plucked hair can be utilized as a substrate for IIF in cases of Pemphigus. Aim & Objectives: To determine the efficacy of utilizing the plucked hair as a substrate for indirect immunofluorescence among patients with Pemphigus vulgaris.Methodology:Thirty two consecutive patients with active and fresh diagnosed cases of pemphigus vulgaris (PV) who did not undergo any treatment and those patients who had positive DIF findings of characteristic fish net or chicken wire pattern of intercellular IgG deposits in the epidermis of perilesional skin were included in the study. A total of 32 control subjects without any auto immune disorders were selected and the blood samples were taken from these patients for IIF analysis. Anagen hair were collected from the healthy volunteers without pemphigus in the same way as for trichogram. Telogen hair were obtained by combing the hair and collecting the loose strands of hair on the comb and further confirmed under microscope. Three hairs each of anagen and telogen stage were collected from each subject to be utilized as a substrate for IIF.Results:Out of 32 samples 20 samples showed positive results for Ig G alone and 5 samples showed positivity for Ig G and C3. One sample showed positivity for C3alone. All these 26 samples were considered to have positive IIF test based on the intercellular pattern of deposit. Positive IIF results were observed in anagen hair samples and were negative in all the telogen hair samples. Six anagen hair samples did not show any positive findings in IIF for the study group. All the 32 control samples showed negative reports in IIF.Conclusion:In conclusion, IIF microscopy on plucked hair as a substrate is a more sensitive immunoassay for the detection of circulating intercellular autoantibodies in PV and the lower negative predictive value of this substrate is a limitation. Further large scale studies might provide better information regarding the practical utility.

Vaccination with Recombinant Baculovirus Expressing Ranavirus Major Capsid Protein Induces Protective Immunity in Chinese Giant Salamander, Andrias davidianus.

The Chinese giant salamander iridovirus (CGSIV), belonging to the genus Ranavirus in the family Iridoviridae, is the causative agent of an emerging infectious disease causing high mortality of more than 90% and economic losses in Chinese giant salamanders in China. In this study, a recombinant baculovirus-based vaccine expressing the CGSIV major capsid protein (MCP) was developed and its protective immunity in Chinese giant salamanders was evaluated. The recombinant Autographa californica nucleopolyhedrosis virus (AcNPV), expressing CGSIV MCP, designated as AcNPV-MCP, was generated with the highest titers of 1 × 108 plaque forming units/mL (PFU/mL) and confirmed by Western blot and indirect immunofluorescence (IIF) assays. Western blot analysis revealed that the expressed MCP reacted with mouse anti-MCP monoclonal antibodies at the band of about 53 kDa. The results of IIF indicated that the MCP was expressed in the infected Spodoptera frugiperda 9 (Sf9) cells with the recombinant baculovirus, and the Chinese giant salamander muscle cells also transduced with the AcNPV-MCP. Immunization with the recombinant baculovirus of AcNPV-MCP elicited robust specific humoral immune responses detected by ELISA and neutralization assays and potent cellular immune responses in Chinese giant salamanders. Importantly, the effective immunization conferred highly protective immunity for Chinese giant salamanders against CGSIV challenge and produced a relative percent of survival rate of 84%. Thus, the recombinant baculovirus expressing CGSIV MCP can induce significant immune responses involving both humoral and cell-mediated immunity in Chinese giant salamanders and might represent a potential baculovirus based vaccine candidate for Chinese giant salamanders against CGSIV.

The use of anti-intercellular substance antibody in diagnosing patients with clinical suspicion of pemphigus

Abstract Background Pemphigus is frequently diagnosed via indirect immunofluorescence (IIF) or skin biopsy with direct immunofluorescence staining. However, the accuracy of IIF to detect anti-intercellular substance autoantibodies (anti-ICS Ab) has not been evaluated in Asian populations. Method IIF was performed on 177 patient samples. Demographic data and underlying diseases were analyzed. Histopathology and direct immunofluorescence results were also reviewed. The false-positive group included 45 patients without pemphigus but with positive ICS Ab results. The other groups included true-negative (116 patients without pemphigus and negative ICS Ab results), true-positive (14 pemphigus patients with positive ICS Ab results), and false-negative (two pemphigus patients with negative ICS Ab results) results. Univariate and multivariate analysis were performed to exam the factors associated with false positivity. Results Anti-ICS Ab detected using IIF has been shown in 87.5% of patients with confirmed pemphigus. The specificity of IIF on pemphigus was 72.1%. The positive and negative predictive rates were 23.7% and 98.3%, respectively. The false positive rate was 25.4% ( n = 45/177). The titers of the false positive results were 1:20, 1:40, and 1:80, in 28, 10, and 7 patients, respectively. The false-positive results also diminished during sequential follow-ups in some patients after treatments. There was higher rates of patients with blood type O (66.7% vs. 36.3%; p = 0.02) and bullous pemphigoid (31% vs. 15%; p = 0.05) in the false-positive group, compared with the rest of the population. In the adjusted analysis, blood type O was found to be significantly associated with a false-positive IIF results when compared with blood types A and B. Conclusion This study provided practical statistics for the accuracy of anti-ICS IIF tests. Due to the high false-positive rate and low positive-predictive rate, the results of anti-ICS IIF for pemphigus diagnosis should be carefully interpreted.

Comparative Study of Human and Automated Screening for Antinuclear Antibodies by Immunofluorescence on HEp-2 Cells

Background: Several automated systems had been developed in order to reduce inter-observer variability in indirect immunofluorescence (IIF) interpretation. We aimed to evaluate the performance of a processing system in antinuclear antibodies (ANA) screening on HEp-2 cells. Patients and Methods: This study included 64 ANA-positive sera and 107 ANA-negative sera that underwent IIF on two commercial kits of HEp-2 cells (BioSystems® and Euroimmun®). IIF results were compared with a novel automated interpretation system, the “CyclopusCADImmuno®” (CAD). Results: All ANA-positive sera images were recognized as positive by CAD (sensitivity = 100%), while 17 (15.9%) of the ANA-negative sera images were interpreted as positive (specificity = 84.1%), κ=0.799 (SD=0.045). Comparison of IIF pattern determination between human and CAD system revealed on HEp-2 (BioSystems®), a complete concordance in 6 (9.37%) sera, a partial concordance (sharing of at least 1 pattern) in 42 (65.6%) cases and in 16 (25%) sera the pattern interpretation was discordant. Similarly, on HEp-2 (Euroimmun®) the concordance in pattern interpretation was total in 5 (7.8%) sera, partial in 39 (60.9%) and absent in 20 (31.25%). For both tested HEp-2 cells kits agreement was enhanced for the most common patterns, homogenous, fine speckled and coarse speckled. While there was an issue in identification of nucleolar, dots and nuclear membranous patterns by CAD. Conclusion: Assessment of ANA by IIF on HEp-2 cells using the automated interpretation system, the “CyclopusCADImmuno®” is a reliable method for positive/negative differentiation. Continuous integration of IIF images would improve the pattern identification by the CAD.

EUROPattern Suite technology for computer-aided immunofluorescence microscopy in autoantibody diagnostics.

Antinuclear autoantibodies (ANA) are highly informative biomarkers in autoimmune diagnostics. The increasing demand for effective test systems, however, has led to the development of a confusingly large variety of different platforms. One of them, the indirect immunofluorescence (IIF), is regarded as the common gold standard for ANA screening, as described in a position statement by the American College of Rheumatology in 2009. Technological solutions have been developed aimed at standardization and automation of IIF to overcome methodological limitations and subjective bias in IIF interpretation. In this review, we present the EUROPattern Suite, a system for computer-aided immunofluorescence microscopy (CAIFM) including automated acquisition of digital images and evaluation of IIF results. The system was originally designed for ANA diagnostics on human epithelial cells, but its applications have been extended with the latest system update version 1.5 to the analysis of antineutrophil cytoplasmic antibodies (ANCA) and anti-dsDNA antibodies.

Usefulness of specific anti-desmoglein 1 and 3 enzyme-linked immunoassay and indirect immunofluorescence in the evaluation of pemphigus activity.

The objective was to assess the relationship between enzyme-linked immunoassay (ELISA) values of desmoglein (Dsg) 1 and Dsg3 antibodies and indirect immunofluorescence (IIF) values of anti-epithelial antibodies with disease activity in patients with pemphigus. In a retrospective study, we analyzed 353 serum samples taken from 35 patients with pemphigus vulgaris (PV) and nine with pemphigus foliaceus (PF) during the course of the disease. In each sample, we measured anti-Dsg1 and anti-Dsg3 antibodies by ELISA. A receiver operating characteristics (ROC) curve was calculated to determine a cutoff value for anti-Dsg1 and anti-Dsg3 antibodies with optimal sensitivity and specificity. In 263 samples, we compared the ROC curves of anti-Dsg1 and anti-Dsg3 antibodies with the ROC curves of the IIF results. Activity of pemphigus was associated with a wide range of anti-Dsg1 and anti-Dsg3 antibody values. Levels of anti-Dsg1 antibodies showed a better relationship with cutaneous activity of pemphigus than levels of IIF anti-epithelial antibodies. The levels of IIF anti-epithelial antibodies showed a relationship with activity of mucosas similar to the levels of anti-Dsg3 antibodies. Abnormal values of anti-Dsg antibodies are not always associated with disease activity. ELISA detects both pathogenic and nonpathogenic anti-Dsg antibodies. Therapeutic strategies should not be based exclusively on anti-Dsg antibody values. Anti-Dsg1 antibodies showed a closer relationship with skin activity than IIF, while anti-Dsg3 antibodies showed a relationship with mucosal activity similar to the IIF test.

Relevance of Indirect Immunofluorescence Patterns and Autoantibodies Identified Via Line Immunoassay in Patients With Rheumatoid Arthritis

To analyze indirect immunofluorescence (IIF) patterns and autoantibodies identified by line immunoassay (LIA) in rheumatoid arthritis (RA) patients, and to compare the autoantibody profiles with serological markers to determine their relevance. A total of 153 specimens were obtained from RA patients for diagnostic or monitoring purposes. Erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), and anti-cyclic citrullinated peptide (anti-CCP) were measured, and autoantibody testing using IIF and LIA was performed. Frequencies of IIF patterns and autoantibodies identified by LIA were analyzed and compared with each other and with serologic markers of RF, including anti-CCP, ESR, and CRP. The overall positivity of IIF and LIA results was 49.7% and 34.0%, respectively. The most frequent IIF pattern in RA was homogenous (20.9%), followed by dense fine speckled (DFS) pattern (17%). Anti-SS-A and/or anti-Ro-52 were most frequently detected in RA irrespective of the observed IIF pattern. Positive rates and intensities of autoantibodies detected did not differ by therapeutic regimen or disease activity, as reflected by CRP levels or ESR. Only anti-CCP showed a tendency to increase depending on IIF intensity. RA patients did not display a specific or dominant IIF pattern or autoantibody presence, as identified by LIA. RA patients had lower frequencies of anti-SS-A and anti-Ro-52 compared with other systemic autoimmune diseases (serologically characteristic of RA). IIF and LIA appear insufficient for diagnosing RA and monitoring disease activity yet can be useful for screening and differentiating major systemic autoimmune diseases.

Anti-nuclear antibodies in daily clinical practice: prevalence in primary, secondary, and tertiary care.

For the diagnosis of systemic autoimmune rheumatic diseases (SARD), patients are screened for anti-nuclear antibodies (ANA). ANA, as assessed by indirect immunofluorescence (IIF), have a poor specificity. This hampers interpretation of positive results in clinical settings with low pretest probability of SARD. We hypothesized that the utility of positive ANA IIF results increases from primary to tertiary care. We retrospectively determined ANA, anti-ENA, and anti-dsDNA antibody prevalence in patient cohorts from primary (n = 1453), secondary (n = 1621), and tertiary (n = 1168) care settings. Results reveal that from primary care to tertiary care, ANA prevalence increases (6.2, 10.8, and 16.0%, resp.). Moreover, in primary care low titres (70% versus 51% and 52% in secondary and tertiary care, resp.) are more frequent and anti-ENA/dsDNA reactivities are less prevalent (21% versus 39% in secondary care). Typically, in tertiary care the prevalence of anti-ENA/dsDNA reactivities (21%) is lower than expected. From this descriptive study we conclude that positive ANA IIF results are more prone to false interpretation in clinical settings with low pretest probabilities for SARD, as in primary care. Whether alternative approaches, that is, immunoadsorption of anti-DFS70 antibodies or implementation of anti-ENA screen assays, perform better, needs to be determined.

Aquaporin 4 IgG Serostatus and Outcome in Recurrent Longitudinally Extensive Transverse Myelitis

Studies focused on recurrent longitudinally extensive transverse myelitis (rLETM) are lacking. To determine the aquaporin 4 (AQP4) IgG detection rate using recombinant human APQ4-based assays in sequential serum specimens collected from patients with rLETM categorized as negative by first-generation tissue-based indirect immunofluorescence (IIF) assay and to define the clinical characteristics and motor disability outcomes in AQP4-IgG-positive rLETM. A search of the Mayo Clinic computerized central diagnostic index (October 1, 2005, through November 30, 2011), cross-linked with the Neuroimmunology Laboratory database, identified 48 patients with rLETM, of whom 36 (75%) were positive and 12 (25%) negative for neuromyelitis optica (NMO) IgG (per IIF of serial serum specimens). Stored serum specimens from "seronegative" patients were retested with recombinant human AQP4-based assays, including enzyme-linked immunosorbent, transfected cell-based, and fluorescence-activated cell-sorting assays. Control patients included 140 AQP4-IgG-positive patients with NMO, of whom a subgroup of 20 initially presented with 2 attacks of transverse myelitis (rLETM-onset NMO). AQP4-IgG serostatus, clinical characteristics, and Expanded Disability Status Scale score. Six patients with negative IIF results were reclassified as AQP4-IgG positive, yielding an overall AQP4-IgG seropositivity rate of 89%. Fluorescence-activated cell-sorting, cell-based, and enzyme-linked immunosorbent assays improved the detection rate to 89%, 85%, and 81%, respectively. The female to male ratio was 2:3 for AQP4-IgG-negative rLETM and 5:1 for AQP4-IgG-positive patients. The AQP4-IgG-positive patients with rLETM or rLETM-onset NMO were similar in age at onset, sex ratio, attack severity, relapse rate, and motor disability. From Kaplan-Meier analyses, 36% of AQP4-IgG-positive patients with rLETM are anticipated to need a cane to walk within 5 years after onset. For patients with rLETM-onset NMO, the median time from onset to first optic neuritis attack (54 months) was similar to the median disease duration for AQP4-IgG-positive patients with rLETM (59 months). The median number of attacks was 3 for AQP4-IgG-positive patients with rLETM (range, 2-22), and the first optic neuritis attack for those with rLETM-onset NMO followed a median of 3 myelitis attacks (range, 2-19). Immunosuppressant therapy reduced the relapse rate in both AQP4-IgG-positive and AQP4-IgG-negative patients with rLETM. Recombinant antigen-based assays significantly increase AQP4-IgG detection in patients with rLETM, and AQP4-IgG-negative adults with rLETM are rare. Evolution to NMO can be anticipated in AQP4-IgG-positive patients. Early initiation of immunotherapy may result in a more favorable motor outcome.

AB1258 Characterization of the CDC anti-nuclear antibody (ANA) reference serum panel using multiplex and immunoprecipitation assays

BackgroundAnti-nuclear antibodies (ANA) represent serological hallmarks of systemic rheumatic diseases (SARD), and have been traditionally identified by indirect immunofluorescence (IIF) on HEp-2 cells, followed by antigen-specific confirmation assays. Several new...

Ocular Mucous Membrane Pemphigoid after Lyell Syndrome: Occasional Finding or Predisposing Event?

Ocular mucous membrane pemphigoid (OMMP) is an autoimmune disease involving the eye and characterized by subepithelial detachment resulting from an immunologic reaction against conjunctival basal membrane zone (BMZ) antigens. Lyell syndrome (LS) is a drug-induced, T cell-mediated, cytotoxic reaction involving the mucocutaneous areas. Two patients with LS are presented in whom OMMP developed. Report of 2 cases. Two male patients, 80 and 60 years old, with persistent corneal ulcerations, corneal melting, and inflammation some months after an LS episode. Conjunctival biopsy samples were obtained to perform direct immunofluorescence (DIF) and histologic analyses. Indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA) also were performed. Immunodeposit findings on the conjunctival BMZ obtained by DIF and IIF, inflammatory infiltration of the corneoconjunctival samples studied by histologic analysis, and autoantibodies of patient sera directed against BMZ antigens tested by ELISA. Direct immunofluorescence analyses showed immunoglobulin G and complement 3 component deposits along the BMZ in a linear pattern. Histologic analysis revealed the presence of eosinophils, neutrophils, and mast cells with fibrin deposition in the substantia propria of both patients; the data confirmed the clinical suspicion of OMMP. The IIF and ELISA results were negative. Chronic eye surface injury associated with LS may promote autoimmunization against ocular epithelial BMZ antigens, playing a strategic role in the subsequent onset of OMMP. The occurrence of OMMP after LS could be an occasional finding, or conversely, LS could be an underestimated predisposing factor in the development of OMMP.

Pemphigoïde anti-p200 : réponse spectaculaire à la dapsone

Types of subepidermal autoimmune bullous dermatosis (AIBD) are classified by anatomoclinical picture and target antigen. A new entity has recently been identified: anti-p200 pemphigoid. An 82-year-old man consulted for a profuse pruritic bullous eruption refractory to the standard treatments for bullous pemphigoid (BP). Direct immunofluorescence examination of a skin biopsy revealed linear deposits of IgG and of C3 at the dermal-epidermal junction, but Elisa screening for circulating anti-BP180 and anti-BP230 antibodies was negative. Indirect immunofluorescence (IIF) testing of cleaved skin revealed a deposit of IgG4 antibodies on the dermal side. Immunoblotting was negative for a dermal extract but showed an antibody directed against a 200-kD epidermal antigen. A diagnosis of anti-p200 pemphigoid was eventually made and the patient was successfully treated with dapsone. The diagnosis of anti-p200 pemphigoid was made in this case in spite of discrepancy between the IIF and immunoblotting results, and despite the fact that the target antigen in this disease is considered as being restricted to dermal sites. Anti-p200 pemphigoid usually begins in the second part of life and differs from standard bullous pemphigoid in terms of more frequent mucous membrane and cephalic involvement, as well as a greater degree of miliary scarring. This disease appears more prominent in males and is associated with psoriasis in around one third of cases. Autoantibodies recognize laminin gamma-1, an extra-desmosomal protein that contributes to dermal-epidermal adhesion. This recently described disease as probably under-diagnosed in France. It should be considered in atypical presentations of bullous disease. Diagnosis is confirmed by immunoblotting detection of autoantibodies directed against a 200-kD antigen normally present in the extract. Dapsone appears to be the most effective treatment.

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.

Role of BP180NC16a-enzyme-linked immunosorbent assay (ELISA) in the diagnosis of bullous pemphigoid in China

Autoantibodies of bullous pemphigoid (BP) patients react with two components of the hemidesmosome of stratified epithelia: the BP antigen 230 (BP230) and the BP antigen 180 (BP180). Recently, strong evidence has been provided that autoantibodies to BP180 play a key role in subepidermal blister formation in BP patients, and NC16A contains an important antigen determinant of BP. To study the role of BP180NC16a enzyme-linked immunosorbent assay (BP180NC16a-ELISA) in the diagnosis of BP in China. Sera from BP patients (n = 42) and control subjects (normal controls, n = 24; pemphigus patients, n = 18) were measured by BP180NC16a-ELISA. All BP sera were obtained at presentation from patients who had not received previous systemic treatment. The values of immunoglobulin G (IgG) antibody levels measured by ELISA were compared with those measured by indirect immunofluorescence (IIF) (gold standard for the diagnosis of BP) on salt-split skin. Using BP180NC16a-ELISA, 41 of the 42 BP sera were positive, whereas only one of the serum samples from 24 normal controls was positive and all the pemphigus sera showed a negative result. Thus, the sensitivity and specificity of BP180NC16a-ELISA were both 97.62%. There was no correlation between the mean ELISA values and IIF titers. The ELISA and IIF results were further compared and analyzed using a 2 x 2 contingency table, which showed that they were not significantly different. It is suggested that BP180NC16a-ELISA is a useful tool for the diagnosis of BP.

A comparison of anti‐desmoglein antibodies and indirect immunofluorescence in the serodiagnosis of pemphigus vulgaris

Indirect immunofluorescence (IIF) is the standard method for the detection of pemphigus autoantibodies. Commercially available enzyme-linked immunosorbent assays (ELISAs) have recently become available to measure serum antibodies (Abs) against desmoglein1 (Dsg1) and desmoglein3 (Dsg3). It has been suggested that patients with mucosal-dominant pemphigus vulgaris (PV) have serum Abs against Dsg3 only, patients with mucocutaneous PV have Abs to both Dsg1 and Dsg3, and patients with pemphigus foliaceus (PF) have Abs against Dsg1 only. To compare the sensitivity and specificity of the IIF and ELISA tests in the diagnosis of pemphigus and its subsets. Thirty-three patients with PV and five patients with PF were studied, and compared with 50 healthy individuals or patients with unrelated skin diseases. Monkey esophagus was used as a substrate for the IIF test. The IIF and ELISA tests were each positive in 26 of the 32 (81%) PV patients, and in none (0%) and 3 (6%) of the 50 controls, respectively. Both the IIF and ELISA results were concordant in 69% of the PV patients, and only one of these two tests was positive in the remaining 31% of patients. Forty-six per cent of the PV patients with a positive ELISA test did not have the PV phenotype (mucosal or mucocutaneous) predicted by their autoantibody profile. The IIF and ELISA tests may be used as complementary tests for the serologic diagnosis of pemphigus.

Diagnostic Features of Pemphigus vulgaris in Patients with Bullous Pemphigoid

Background: The simultaneous presence of features of pemphigus vulgaris (PV) in patients with bullous pemphigoid (BP) has previously been reported in the literature. Objective: The purpose of this retrospective study is to present 13 patients with an initial diagnosis of BP, who subsequently demonstrated coexistent serological features of both BP and PV. Methods: The following information on each patient was documented, at the time of initial diagnosis: clinical profile on presentation, histology, direct immunofluorescence, indirect immunofluorescence (IIF) using monkey esophagus as substrate, salt-split skin (SSS) and an immunoblot assay. Since all 13 patients failed to respond to conventional systemic therapy, intravenous immunoglobulin (IVIg) was used as an alternative treatment modality. Prior to initiating IVIg therapy, in all 13 patients, serological studies were performed. In addition to IIF using monkey esophagus, an immunoblot assay and SSS, an enzyme-linked immunosorbent assay (ELISA) was performed to detect antibodies to desmogleins. These different assays were done to identify pathological autoantibodies typical of BP and PV. A control group of 25 healthy normal individuals, 37 patients with BP, 17 patients with PV and 12 patients with pemphigus foliaceus were used for comparison of serological studies. Results: At the time of initial presentation, histological and immunopathological studies confirmed the diagnosis of BP in all 13 patients. Prior to the initiation of IVIg therapy, results of IIF using monkey esophagus as substrate demonstrated high levels of anti-intercellular cement substance (anti-ICS) or antikeratinocyte cell surface antibody. Sera of all 13 patients on SSS bound to the epidermal side of the split. In an immunoblot, using bovine gingival lysate as substrate, sera of 6 patients bound to both a 230-kD (BP Ag1) and 180-kD protein (BP Ag2), while 7 sera bound to only a 230-kD protein. All 13 patients had high levels of antibodies to desmoglein 3 on ELISA. In a pilot experiment, the anti-ICS antibody in sera from 6 random patients was found to be predominantly of the IgG4 subclass. Use of IVIg resulted in an effective clinical response and the maintenance of a prolonged clinical remission. Conclusion: In patients with BP, who are nonresponsive to conventional therapy, the presence of two autoimmune diseases or a dual diagnosis should be considered.