Positive Direct Immunofluorescence Research Articles

Risk Factors for Relapse in Patients With Bullous Pemphigoid in Clinical Remission

To identify prognostic factors for relapse in the first year after cessation of therapy in bullous pemphigoid (BP). Prospective, multicenter, cohort study (January 1, 2000, through December 31, 2006). Fifteen French dermatology departments. Patients Patients with BP in remission under low doses of topical or systemic corticosteroids. Interventions Cessation of corticosteroid treatment (day 0) followed by a systematic clinical and immunologic follow-up. The end point was clinical relapse within the first year after cessation of therapy. Associations of clinical, biological, and immunologic (including direct immunofluorescence, serum anti-basement membrane zone autoantibodies, and serum BP180 autoantibodies by enzyme-linked immunosorbent assay [ELISA] on day 0) variables with clinical relapse were assessed by means of univariate and multivariate analyses. On day 0, 30 of 114 patients (26.3%) still had a positive result of direct immunofluorescence, 63 of 112 (56.3%) had circulating anti-basement membrane zone autoantibodies, and 34 of 57 (60%) had anti-BP180 antibodies by ELISA. At month 12, 22 patients were dead (n = 11) or lost to follow-up (n = 11), 51 were in remission, and 45 had had relapses (mean interval to relapse, 3.2 months). Factors predictive of relapse within 12 months after cessation of therapy were a positive result of direct immunofluorescence microscopy (P = .02), a greater age (P = .01), and high-titer ELISA scores (P = .02) on day 0. In multivariate analysis, the only factor independently predictive of relapse was a high-titer ELISA score on day 0 (odds ratio, 11.00; 95% confidence interval, 1.29-93.76). High-titer anti-BP180 ELISA score and, to a lesser degree, a positive direct immunofluorescence finding are good indicators of further relapse of BP. At least 1 of these tests should be performed before therapy is discontinued.

A study on plucked hair as a substrate for direct immunofluorescence in pemphigus vulgaris

It has recently been demonstrated in a study on 15 patients that plucked hair can be used as a substrate for direct immunofluorescence (DIF) in pemphigus. Our aim was to assess the sensitivity of DIF on plucked hairs in pemphigus vulgaris (PV) patients with positive DIF of oral mucosa. One hundred and ten new PV patients were enrolled in the study. They all showed the typical clinical and histological findings as well as positive DIF of the oral mucosa, diagnostic for PV. Approximately 30 hairs were obtained in the same way as for the trichogram. The hairs with their outer root sheaths (ORS) were processed for DIF in order to detect immunoglobulin G and C3. Immunodeposits favouring PV were demonstrated in the ORS of 100 cases showing a sensitivity of 91%. Regarding the relatively high sensitivity of DIF on plucked hair in PV patients with positive oral mucosal DIF in our study, it seems that hair plucking is a suitable alternative to the more invasive techniques of skin or mucosal biopsy for obtaining specimens for DIF in PV.

Positive direct immunofluorescence and autoantibody profiles in psoriasis patients

Psoriasis and systemic lupus erythematosus are common, but their coexistence is thought to be infrequent. Each of these diseases has a broad clinical spectrum, so that diagnosis may not be straightforward. This study aimed to investigate the following immunological parameters in psoriasis patients: (i) direct immunofluorescence (DIF); (ii) antinuclear antibody; (iii) anti-double-stranded DNA (dsDNA); (iv) anti-Ro; and (v) anti-nuclear ribonucleoprotein (nRNP). Of 300 cases, comprising 189 men (62.9%) and 111 women (37.1%), 17 (5.7%; 10 men, seven women) were positive for at least one immunological parameter. Nine of 300 (3%; seven male, two female) biopsy specimens from sun-exposed psoriatic lesions demonstrated bright continuous bands of granular IF along the dermoepidermal junction with immunoreactant immunoglobulin (Ig)G, IgM, C3 and fibrinogen. The intensity of IF at the dermoepidermal junction was graded 3+ and 2+. Three cases demonstrated IgM, two had IgG, two fibrinogen and six cases showed C3. Three cases demonstrated more than one immunoreactant. One case demonstrated C3 at the vessels. No specimen demonstrated IgA deposition. Three hundred sera were obtained from patients with psoriasis, of which five demonstrated elevated antinuclear antibody (ANA) titer; dilution titer varied from 1:80-640. Three had a homogeneous pattern and two had a speckled pattern. None had a peripheral pattern. Five (1.7%) of 300 demonstrated anti-Ro, two had negative ANA, and three were positive ANA, two of which were speckled and the other homogenous. Anti-dsDNA, anti-Sm and anti-nRNP were not detected. Ten patients had positive DIF but negative ANA, while five had positive ANA; all had negative DIF results. Thus, the incidence of psoriasis and lupus erythematosus coexistence is low and a baseline immunological screening test for psoriasis might not prove worthwhile.

Direct immunofluorescence study in patients with lichen planus

Direct immunofluorescence (DIF) studies in patients with lichen planus (LP) show the deposition of multiple immunoglobulins (Igs) at the cytoid bodies (CBs) and fibrin at the dermoepidermal junction (DEJ). The deposition of Ig at the DEJ, as in patients with lupus erythematosus (LE), is occasionally found. For cases with no specific clinical and histologic characteristics, or with ambiguous features, DIF studies may be helpful in disease differentiation. From 1996 to 2004, data from 72 patients with LP, diagnosed on the basis of clinical and histologic criteria at the Department of Dermatology, Siriraj Hospital, Bangkok, Thailand, were collected. The results of DIF studies were analyzed. Deposits at the DEJ and CBs were detected in 53% and 60% of cases, respectively. A combination of DEJ (mostly fibrin) and CB (mostly IgM) deposits was found in 38% of cases. A combination of IgM and other immunoreactant deposits, including fibrin at the CBs, was found in 56% of cases. This study showed that the positive yield of DIF in LP was 75%. Shaggy fibrin deposition at the DEJ, which is the single best indicator in the diagnosis of LP, was found in 56% of cases. The presence of CBs only, which is a poorer indicator than the shaggy deposition of fibrin along the DEJ, was found in 22% of cases. There were no statistically significant differences in positive DIF yield between specimens derived from glabrous skin and oral lesions (P = 0.67). Forty-four per cent of cases had immunoreactants other than fibrin deposited along the DEJ, which resembled those of LE.

A Determination of the Range of Oral Conditions Submitted for Microscopic and Direct Immunofluorescence Analysis

Direct immunofluorescence (DIF) testing is a useful adjunct for the diagnosis of immune-mediated oral vesiculobullous diseases, helping to identify separate, histologically similar, but prognostically different, conditions. It is unknown how often biopsy of these lesions yields positive DIF results. A total of 270 consecutive archival cases submitted to a reference laboratory in Buffalo, New York, over a 2-year span were examined. These specimens were submitted to establish or rule out a diagnosis of a DIF-positive oral vesiculobullous disease. Demographic, clinical, and diagnostic information, based on conventional microscopic and DIF analysis, was tabulated. To assess the contribution of DIF to successful diagnosis, three pathologists examined the hematoxylin and eosin-stained slides of the known DIF-positive specimens without knowledge of the DIF results. Approximately 48% of the specimens demonstrated positive DIF findings and consisted of pemphigus vulgaris, mucous membrane pemphigoid, lichen planus, linear immunoglobulin A disease, and chronic ulcerative stomatitis. The remaining specimens had negative DIF findings and consisted of numerous non-specific inflammatory conditions. Of particular interest were several cases of epithelial dysplasia and squamous cell carcinoma. Of the DIF-positive cases, only pemphigus vulgaris could be diagnosed reliably by conventional microscopy alone. Approximately half of biopsies of oral conditions that clinically resembled typically DIF-positive vesiculobullous diseases did not yield positive findings on DIF testing. Instead, a wide range of oral diseases can mimic these lesions clinically. With the exception of pemphigus vulgaris, DIF is essential for establishing a definitive diagnosis for known DIF-positive diseases.

Predictability of the clinical course of juvenile dermatomyositis based on initial muscle biopsy: A retrospective study of 72 patients

Except when the diagnosis of juvenile dermatomyositis (DM) is in doubt, a case has not been made for routine muscle biopsy (MB). We sought to determine whether MB findings prior to systemic therapy have prognostic value. We reviewed the hospital records and slides prepared from the initial open MB of 72 patients treated at one center between 1977 and 2002 and followed for a minimum of 2 years. None of the patients had received a course of systemic corticosteroid therapy at the time of MB. Our approach to MB evaluation was based on recent discussions with muscle pathology experts to develop criteria for assessing inflammation, vasculopathy, myofiber atrophy, regeneration, acute and chronic myopathic change, and stromal changes. Using simple and multivariate logistic regression, we tested each MB parameter for ability to predict outcome using 2 published classification systems. Extensive active myopathic changes (excluding regeneration) and central nuclei without basophilia predicted chronic juvenile DM. Severe arteropathic change, positive arterial direct immunofluorescence, obvious foci of severe capillary loss/endomysial fibrosis, and muscle infarcts predicted chronic juvenile DM, particularly with ulceration. Other MB parameters, regardless of severity, were not significant predictors of chronic juvenile DM versus limited disease. A scoring system for evaluating pretreatment MB in juvenile DM that focuses on extent of necrotizing myopathy, severity of vasculopathy, and features of established chronicity such as central nucleation of nonbasophilic myofibers may provide a basis for stratification of therapeutic regimens according to risk for chronic disease. The validity of our findings should be prospectively tested.

Role of direct immunofluorescence on Tzanck smears in pemphigus vulgaris

The Tzanck smear is a simple, sensitive, and rapid test to diagnose pemphigus vulgaris (PV), a life threatening autoimmune blistering disorder. The presence of acantholytic cells in cytology is indicative of but not specific for PV. Hence, a direct Immunofluorescence (DIF) test to demonstrate immunoglobulin deposits on the acantholytic cells would make the Tzanck test more specific, in addition to being a rapid test. Twenty untreated patients with PV confirmed histopathologically were enrolled to evaluate the efficacy of using DIF technique using IgG on Tzanck smear samples. The DIF smears were compared with DIF on skin biopsies in the same patient. This prospective pilot study approved by the institutional ethics committee was carried out in a tertiary health care hospital in a developing country. Of the 15 patients presenting within 3 mo of onset of the illness, 40% (n = 6) showed DIF positivity on Tzanck smear, when compared with 46.67% (n = 7) on skin biopsy. On the other hand, of the five patients presenting beyond 3 mo of their illness, only 20% (n = 2) showed positivity on Tzanck, when compared with all 100% (n = 5) on skin biopsy. The study, thus, suggests that DIF on skin biopsy is comparable to biopsy in diagnosing early PV. This preliminary study proposes that the use of DIF on Tzanck smear is a simple, rapid, painless, and user-friendly out-patient procedure for the diagnosis of early PV, even for relatively inaccessible lesions in the oral cavity and flexural regions. This methodology would be of great help in outlying and rural facilities lacking proper histological equipment, thus avoiding the need for a surgical or punch biopsy or heavy investment in laboratory equipment and expertise. Probable reasons for DIF negativity on Tzanck smears are also discussed.

The Role of Direct Immunofluorescence in Differential Diagnosis Between Atypical Recurrent Aphtous Stomatitis and Pemphigus Vulgaris

Introduction. Vesiculobullous, erosive, or ulcerative disorders affecting the oral mucosa or gingivae like pemphigus vulgaris (PV) or atypical recurrent aphtous stomatitis (RAS) sometimes can be difficult to differentiate clinically. In addition, therefore, to a full history and examination, biopsy and appropriate histopathological and immunological investigations are frequently indicated. Biopsy of perilesional tissue, with histological and immunostaining examination, with direct immunofluorescence (DIF), are essential to diagnosis of PV. Otherwise, some authors have also found positive DIF in RAS patients.

Papular acantholytic dyskeratosis of the anogenital area with positive direct immunofluorescence results

Acantholytic dyskeratosis is a distinct histological pattern characterized by hyperkeratotic and parakeratotic epidermis with intraepidermal clefts harbouring acantholytic and dyskeratotic keratinocytes. This histopathological pattern is uncommon in dermatoses of the anogenital region. We report a 30-year-old woman who had numerous smooth whitish papules on the labia majora, perineum and perianal region, which coalesced into plaques in some areas. Microscopically, the lesions showed prominent suprabasal and intraspinous acantholysis with dyskeratotic keratinocytes. Direct immunofluorescence examination revealed intercellular Ig G and C(3) within the epidermis. We were unable to find a similar case of papular acantholytic dyskeratosis of the anogenital area with positive direct immunofluorescence findings reported in the literature, thus in this report, the clinicopathological features of a unique case are presented.

A new BAL fluid instillation and aspiration technique: A multicenter randomized study

Bronchoalveolar lavage (BAL) is a widely used clinical and research tool even though certain steps in the procedure have not been standarized. One step that is subject to variation is the technique used to instill and recover BAL fluid. Our aim was to assess whether attaching a plastic tube between the syringe and the working channel of the flexible bronchoscope allowed recovery of more fluid in comparison with recovery using only a syringe, and whether tubing had a favorable impact on diagnostic yield and complications. A randomized multicenter study was performed in eleven public hospitals in Catalonia (Spain). Patients scheduled for BAL were randomly assigned to tubing (n=140) or no-tubing groups (n=155). Flexible bronchoscopy and a 150 mL BAL in three 50 mL aliquots with or without tubing attached to the syringe. In the group with tubing, 8% more fluid was recovered. The difference was both statistically significant and clinically relevant given that 17.4% more diagnoses, 6.9% fewer complications, and 8.8% fewer technical failures were recorded. Based on these results, we recommend performing BAL using plastic tubing between the 50 mL syringe and the working channel of the flexible bronchoscope when carrying out manual instillation and suction.

Analyse des signes cliniques des malades atteints de pemphigoïde en fonction des antigènes reconnus par leur sérum en immunotransfert

Clinical features and extent of bullous pemphigoid lesions differed widely among patients. The pathogenic role of anti-BPAG2 antibodies has been recently demonstrated. The aim of this study was to analyze the relationship between clinical features of bullous pemphigoid patients and the antigens recognized by their serum. One hundred and twelve bullous pemphigoid patients were included in this prospective multicenter study. Inclusion criteria were the following: 1) diagnosis of bullous pemphigoid established on the presence of 3 of the 4 clinical features of bullous pemphigoid, histological picture of bullous pemphigoid and positive direct immunofluorescence; 2) serum available for immunoblotting studies. The clinical and biological findings were prospectively recorded on standard forms. Sera were collected and analyzed using indirect immunofluorescence and immunoblotting on human epidermal extracts. Analysis of patient's clinical features depending on the antigens recognized by their serum showed that patients whose serum contained anti-BPAG1 antibodies had more frequently pruritus, blisters on the lower limbs and a positive indirect immunofluorescence. Patients whose serum contained anti-BPAG2 antibodies had blisters more frequently localized on the head, and a more frequently negative indirect immunofluorescence. Patients whose serum was negative by immunoblotting had less frequently urticarial and/or eczematous lesions, bullae less frequently localized on the lower part of the trunk, abdomen and lower limbs, lower eosinophilia and a more frequently negative indirect immunofluorescence. Patients with circulating anti-BPAG1 antibodies exhibited the most typical, clinical and biological features of bullous pemphigoid.

A Case of Legionnaires' Disease with Extensive Concurrent Deep Vein Thrombosis

A previously healthy 37-y-old male presented with community-acquired pneumonia and extensive upper limb deep vein thrombosis. The diagnosis of Legionella pneumonia was made based on a positive direct immunofluorescence of the bronchial wash. An extensive investigation for hypercoagulable states was negative. The possible association between Legionella infection and deep vein thrombosis is highlighted.

Plasmapheresis as rescue therapy in accelerated acute humoral rejection.

Accelerated acute humoral rejection (AHR) continues to occur in renal transplantation despite improved crossmatching, with potentially devastating consequences. Between 1 June 1998 and 31 December 2000, 440 renal transplants were performed in our center. AHR was diagnosed by the demonstration of typical pathological features on renal histology and positive direct immunofluorescence or detection of anti-HLA antibodies in serum. AHR developed in 20 (4.5%) of our renal transplant recipients, nine male and eleven female at an average of 16.3 days post transplantation. All of these patients had a negative current cytotoxic crossmatch prior to transplantation. The median serum creatinine at diagnosis was 5.96 mg/dL, and 83% of these individuals developed oliguric renal failure requiring dialysis after having initially attained good graft function (median of best serum creatinine before AHR was 2.64 mg/dL). The 18 recipients who had not infarcted their grafts at the time of diagnosis of AHR received plasmapheresis in conjunction with intensification of their immunosuppressive regimen. This regimen was successful in reversing AHR in 78% of those treated with apheresis. In the 14 responders, graft survival at 6 months was 100% and at 12 months was 91%. Median serum creatinine at 6 and 12 months was 1.26 and 1.33 mg/dL, respectively. Patients received an average of 8.1 plasma exchanges. However, responders received a significantly higher frequency of plasmapheresis (P =.0053), despite undergoing a similar number of exchanges overall. Plasmapheresis appears to be an effective modality for reversing AHR and maintaining graft function.

Characterization of a Subgroup of Patients With Dermatitis Herpetiformis With Nonclassical Histologic Features.

Dermatitis herpetiformis (DH) is an autoimmune disease mediated by IgA antibodies. The diagnosis of DH is based on clinical presentation, biopsy for hematoxylin and eosin, and direct immunofluorescence. The chief hematoxylin and eosin finding is a subepithelial blister with neutrophils in dermal papillae. The direct immunofluorescence findings are deposition of IgA and sometimes C3 at the basement membrane with accentuation in dermal papillae. Immunofluorescence is thought to be a sensitive and specific assay in DH, and there are no other known diseases with this pattern of immunofluorescence. The aim of this project was to determine the prevalence of nonclassical histologic findings in DH. We studied 24 cases of DH received at our institution. All cases had clinical findings of DH as well as positive direct immunofluorescence with IgA. We found that 9 of 24 cases (37.5%) had nonspecific H&E findings of a lymphocytic infiltrate only with fibrosis in the dermal papillae and ectatic capillaries. The remaining 15 cases had classic findings of multilocular neutrophilic microabscesses in the dermal papillae. These findings were reproduced on step sectioning. These findings suggest that routine histology may be quite nonspecific in DH and direct immunofluorescence or other more specific immunologic assay is an essential adjunct to diagnosis.

Interaction between turkey monocytes and avian Chlamydia psittaci in the presence of Mycoplasma sp.: the importance of nitric oxide

The interaction between Chlamydia psittaci and turkey monocytes was studied in vitro. Purified monocytes were inoculated with C. psittaci, in the presence or absence of Mycoplasma hyorhinis. Whereas turkey monocytes produced high amounts of nitric oxide (NO) following the inoculation with M. hyorhinis, inoculation with C. psittaci did not induce NO production in these phagocytes. The monocytes strongly supported chlamydial growth, as demonstrated by the presence of inclusion forming units, the positive direct immunofluorescence staining and transmission electron microscopy. In contrast, upon co-inoculation of the monocytes with C. psittaci and M. hyorhinis, a reduced replication rate of C. psittaci was observed. N G-monomethyl- L-Arginine, a competitive inhibitor for the enzyme NO-synthase, inhibited the NO production and reversed the antichlamydial activity of the M. hyorhinis co-inoculated turkey monocytes. These results imply two considerations. First, as chlamydiae are obligate intracellular bacteria, special care should be taken to guard chlamydial cultures from mycoplasmal contamination, in order to prevent false results when investigating the response of immunomodulating cells to chlamydial infection. Secondly, as a mycoplasmal co-infection in vitro has the capacity of inducing antichlamydial activity in turkey monocytes, through the action of NO, it could be suggested that a similar interaction might take place in vivo. Moreover, it was shown that avian M. gallisepticum strains were also able to induce NO in turkey monocytes. Considering the high prevalence of both C. psittaci and Mycoplasma sp. in turkeys, this interaction, through the pivotal role of NO, might influence the outcome of respiratory diseases in turkeys.

Characterization of the antibody response in oesophageal cicatricial pemphigoid.

Cicatricial pemphigoid (CP) is a subepidermal, autoimmune bullous dermatosis. It is classified as a clinical subset of bullous pemphigoid (BP). However, it differs from BP in some significant ways: (i) in CP mucosal involvement with clinical scarring is prominent; (ii) there is a prominent IgA class antibody response alone or in addition to the IgG class antibody response; and (iii) there is a heterogeneous antibody response in CP, whereas in BP the majority of the antibodies are directed against a 180-kDa hemidesmosomal protein, bullous pemphigoid antigen 2 (BPAg2). Oesophageal involvement in CP is a rare, but often devastating manifestation. In this study we examined the humoral autoimmune response in oesophageal CP, in an attempt to characterize the autoantibody reactivity profile. We used direct and indirect immunofluorescence and Western immunoblotting using normal human skin and oesophagus substrates. We studied patient sera over time in order to search for evidence of epitope spreading in these patients. All patients had positive direct immunofluorescence of perilesional oesophageal epithelium. All patients had positive circulating antibasement membrane zone autoantibody titres. There was a significant IgA class in addition to an IgG class autoantibody response. IgA and IgG antibodies demonstrated significant reactivity with BPAg2 and the 97 kDa linear IgA disease antigen on Western immunoblot suggesting intraprotein epitope spreading. There was no evidence of interprotein epitope spreading over time. Our findings suggest that there is a heterogeneous antibody response in oesophageal CP with the predominant antigen being BPAg2.

Diagnostic value of IgM deposition at the dermo-epidermal junction.

Granular immunoglobulin M (IgM) deposits at the dermo-epidermal junction (DEJ) are common in lupus erythematosus (LE); however, weak, interrupted, linear and granular patterns of IgM at the DEJ have been reported in sun-exposed skin in normal adults and in a heterogeneous group of diseases. We analyzed 200 patients with positive IgM direct tissue immunofluorescence deposition at the DEJ, alone or in combination with other immunoreactants, in order to determine the diagnostic significant of IgM deposition at the DEJ. IgM deposition at the DEJ, commonly in combination with other immunoreactants, was associated with LE in 57.5% of patients. In cases with IgM deposition alone at the DEJ, only 12% had LE (strong intensities). IgM deposition at the DEJ was associated with other dermatoses in 42.5% of patients. In non-lupus patients, IgM deposition was the sole finding in 59% of cases, and was weaker and more focal than in LE patients. IgM deposition at the DEJ in patients with LE tends to exhibit a strong intensity and is commonly detected in combination with other immunoreactants. IgM deposition alone at the DEJ, especially of weak intensity or focal in pattern, is noted in non-lupus patients, and has less diagnostic significance.

Coexistence of Pemphigus and Bullous Pemphigoid

Widespread tense blisters developed on a 60-year-old Japanese man who had been diagnosed with pemphigus 11 years earlier, because of a history of pruritic erythema and erosions on his face, chest, and back, mild supra-basal layer blister formation found in a biopsy specimen, and a positive direct immunofluorescence test showing IgG deposition in the intercellular space. The histological findings showed subepidermal blister, and the immunoblot study detected 180kD bullous pemphigoid antigen. Direct immunofluorescence test revealed intercellular staining for IgG, and indirect immunofluorescence tests repeatedly demonstrated the presence of circulating antibodies to the intercellular space. From these observations, this case suggests the coexistence of pemphigus and bullous pemphigoid.

A retrospective analysis of tissue-fixed immunoreactants from skin biopsies maintained in Michel's medium.

656 skin biopsies with positive direct immunofluorescence from the UK and overseas were studied over a 2-year period. The length of time biopsies had remained in Michel's medium at pH 7.0 in various diseases (pemphigoid, pemphigus, linear IgA disease, epidermolysis bullosa acquisita, lupus erythematosus, vasculitis, amyloid, lichen planus and dermatitis herpetiformis) was analysed. We concluded that direct immunofluorescence remained positive at 6 months and that Michel's medium is a reliable long-term maintenance medium for skin biopsies.

Pemphigus associated with nail dystrophy

Two patients suffering from pemphigus vulgaris were found to have nail dystrophies which antedated the onset of mucocutaneous lesions by many years. The nail changes improved substantially on treatment of the bullous disease. One patient had nail matrix histology consistent with pemphigus, and both had positive direct immunofluorescence with intercellular IgG in the matrix epithelium, as well as at other body sites. We propose that dystrophic nails, as a non-specific indicator of autoimmune disease, are a genuine and relevant finding in pemphigus.