A Starter Guide to Immunofluorescence Testing in Dermatology (original) (raw)
Related papers
Immunohistochemistry versus immunofluoresence in the diagnosis of autoimmune blistering diseases
Our Dermatology Online, 2013
Introduction: In patients with autoimmune skin blistering diseases (ABDs), the diagnostic gold standard has classically been direct and indirect immunofluorescence (DIF and IIF), despite inherent technical problems of autofluorescence. Aim: We sought to overcome autofluorescence issues and compare the reliability of immunofluorescence versus immunohistochemistry (IHC) staining in the diagnoses of these diseases. Methods: We tested via IHC for anti-human IgG, IgM, IgA, IgD, IgE, Kappa light chains, Lambda light chains, Complement/C3c, Complement/C1q, Complement/C3d, albumin and fibrinogen in 30 patients affected by a new variant of endemic pemphigus foliaceus in El Bagre, Colombia (El Bagre-EPF), and 30 control biopsies from the endemic area. We also tested archival biopsies from patients with ABDs whose diagnoses were made clinically, histopathologically and by DIF/IIF studies from 2 independent dermatopathology laboratories in the USA. Specifically, we tested 34 patients with bullous pemphigoid (BP), 18 with pemphigus vulgaris (PV), 8 with pemphigus foliaceus (PF), 14 with dermatitis herpetiformis (DH) and 30 control skin samples from plastic esthetic surgery reduction surgeries. Results: The diagnostic correlation between IHC and DIF-IIF was almost 98% in most cases. IHC revealed evidence of autofluorescence around dermal blood vessels, dermal eccrine glands and neurovascular packages feeding skin appendices in ABDs; this autofluorescence may represent a non-specific immune response. Strong patterns of positivity were seen also in endothelial-mesenchymal cell junction-like structures, as well as between dermal fibrohistiocytic cells. In PV, we noted strong reactivity to neurovascular packages supplying sebaceous glands, as well as apocrine glands with edematous changes. Conclusions: We suggest that IHC is as reliable as DIF or IIF for the diagnosis of ABDs; our findings further suggest that what has previously been considered DIF/IIF autofluorescence background may be of relevance in ABDs. Our discovery of reactivity against edematous dermal apocrine glands may be related to the fact that PV has a vegetant form, with lesions present in anatomic areas where these glands exist..
Background: Autoimmune vesiculobullous disorders represent a heterogeneous group of dermatoses whose diagnosis is made based on clinical history, histologic features, and immunopathologic features. The most commonly used techniques for the diagnosis of these diseases are direct and indirect immunofluorescence (DIF and IIF), including salt-split processing. NaCl split skin is used to determine the level of blister formation, and the localization of autoantibodies relative to the split. Classically, immunofluorescence has been performed with one fluorochrome in the diagnosis of autoimmune bullous skin diseases. Aims: To compare DIF and IIF of the skin, using a single fluorochrome versus multiple fluorochromes. Materials and Methods: We studied 20 autoimmune skin disease cases using fluorescein isothiocyanate (FITC) alone, in comparison to multiple fluorochromes (with or without DNA counterstaining). Results: The use of multiple fluorochromes helped to simultaneously visualize reactivity in multiple skin areas, in contrast to using FITC alone. Conclusions: Using multiple fluorochromes allows simultaneous labeling of two or more antigens within the same cell/or tissue section, assists in colocalization of unknown antigens with known molecules, and helps in ruling out “background” staining.
Direct immunofluorescence for immunobullous and other skin diseases
Expert Review of Clinical Immunology, 2015
A swift glance at ample evidence currently available about the assay clearly illustrates that the development of direct immunofluorescence (DIF), in which direct fluorescent antibodies are utilized to identify the target antigen, has been of immense importance. The immunoreactant deposits have been delineated by the DIF assay in three main locations, including throughout the epidermis, at the dermoepidermal junction (also known as the basement membrane zone) and in and/or around blood vessel walls. DIF testing can be conducted on several specimen sources, which are categorized according to feasibility of collection into invasive (e.g., skin) and non-invasive (e.g., hair). This review was intended to indicate that inspection of immunoreactant deposits via DIF is highly instrumental in diagnosing and monitoring the immunobullous and other diseases of the skin.
Update on immunohistochemical methods relevant to dermatopathology
Archives of pathology & laboratory medicine, 2009
Dermatopathology covers a large variety of entities, some having very similar histologic appearances. Immunohistochemistry is an incredibly helpful tool that is useful in diagnosis as well as prognosis of selected skin tumors. To provide a comprehensive review of recent trends and immunohistochemical stains used by dermatopathologists. Emphasis is placed on new stains as well as novel uses of existing stains. All data were gathered from published journal articles available through the National Center for Biotechnology Information PubMed database. New immunohistochemical targets are continually being found, contributing to more accurate diagnosis and classification of skin tumors. The presence of specific markers can be used to determine the aggressiveness of malignancies and design treatment strategies. In addition, application of existing stains can help determine intravascular spread of malignancy in primary cutaneous lesions. And use of rapid immunohistochemical staining techniqu...
Immunoflorescence: a Refined Investigative Method
Immunofluorescence (IF) is a reliable biochemical staining technique for the detection of antibodies, which are bound to antigen in the tissue or circulating in body fluids. Immunofluorescence techniques take advantage of the selectivity of antibodies for a specific antigen in order to detect target structures in cells and tissues. Fluorochromes, enzymes, radioactive and electro-opaque compounds are among the labelers most commonly used. These techniques are essential to supplement clinical findings and histopathology in the diagnosis of immunobullous disorders. The diagnosis of the subepidermal immunobullous diseases which include bullous pemphigoid, mucosal pemphigoid [cicatricial pemphigoid], epidermolysis bullosa acquisita, linear IgA bullous disease requires clinicopathological correlation; immunofluorescence methods provide a useful adjunct to light microscopy. They permit early diagnosis, treatment and subsequent monitoring of disease activity in patients with potentially life-threatening disorders. Immunofluorescence (direct and indirect) is a sensitive and specific method to detect in situ and circulating autoantibodies in autoimune vesicle-bullous and inflammatory dermatosis. It is considered to be an auxiliary laboratory tool in the conclusive diagnosis of the disease under investigation.
Context: Immunofluorescence (IF) is an immunological technique which is used for identifying antibodies against antigens that are bound to tissues or those circulating in body fluids. This study is an attempt to evaluate the dermatological lesions such as noninfectious vesiculobullous lesions, connective tissue disorders, vasculitis, and lichen planus using histopathology techniques and direct IF (DIF) studies. Furthermore, an attempt was made to evaluate the diagnostic utility of the IF technique in our hospital. Aims: (1) To determine the utility of IF in the diagnosis of skin lesions. (2) To correlate IF with histopathological fndings. Settings and Design: A cross‑sectional, observational study. Subjects and Methods: The present study was conducted in the Pathology Department, Coimbatore Medical College, Coimbatore, from August 2012 to August 2013. Fifty cases received during the period were included in our study. Two skin biopsies for each case ‑ one in formalin for routine histopathological examination and other in phosphate buffer solution for IF studies ‑ were received. Statistical Analysis Used: Fisher’s exact value, mean, and coeffcient of mean were calculated. Results: The lesions were diagnosed by both light microscopy and IF study. Out of ffty cases, 42 cases were positive with the IF technique and the overall sensitivity of IF was 84% in our study. When histopathological and IF fndings were correlated, statistically signifcant two‑tailed P = 0.0130 was obtained. Both light microscopic fndings and IF fndings complemented each other in our study. IF proved to be a necessary supplementary technique to both clinical and histopathological examinations, especially in cases of diagnostic dilemmas. Conclusions: IF is a rapid diagnostic procedure. IF was useful in early diagnosis, treatment, and monitoring of various disorders in our study. Positive and negative DIF had prognostic signifcance in certain cases. Clinicopathological correlation along with DIF studies proved to be a powerful tool in defnite diagnosis of the skin lesions
International Journal of Dermatology, 2004
Subepidermal autoimmune bullous diseases (SABD) comprise several disorders, such as bullous pemphigoid (BP), cicatricial pemphigoid (CP), epidermolysis bullosa acquisita (EBA), herpes gestationis (HG), and linear immunoglobulin A (IgA) dermatosis (LAD), and are characterized by antibody production against the basement membrane structures of the skin and mucosa. Although indirect immunofluorescence (IIF) on serum is a routine test for the detection of basement membrane zone antibodies, there have only been a few studies related to IIF on blister fluid. Aim To perform IIF on blister fluid and to compare the results with those of serum. IIF on salt-split skin was performed on the serum and blister fluid of 35 patients with SABD (25 bp, three EBA, three HG, three LAD, and one bullous systemic lupus erythematosus) with conjugated IgG, IgA, and C3. Twenty-eight of the 35 patients showed IIF-positive blister fluid with a titer similar or less than that of serum. In 25 patients with BP, the most common disease in this study, 23 cases (92%) had positive IIF on serum, 23 cases (92%) on blister fluid, and 24 cases (96%) on either serum or blister fluid. Immunoreactant titers in BP blister fluid and serum did not show significant differences (P > 0.05). Epidermal binding of immunoreactants was the most prevalent staining pattern of IIF on salt-split skin (92%) in BP. From the findings of this study, the blister fluid of patients with SABD can be used for IIF. Although IIF sensitivity on blister fluid is no more than that on serum, the performance of this test on blister fluid in addition to serum may reduce the number of false negative results of IIF found using either of these two substrates alone.
Central European Journal of Immunology, 2021
Introduction: Autoimmune bullous diseases (ABDs) are potentially life-threatening mucocutaneous illnesses that require diagnosis with direct immunofluorescence (DIF). In this study we compared the diagnostic accuracy of traditional DIF (DIFt; separate immunoglobulin (Ig) G, IgG1, IgG4, IgA, IgM and C3 deposits detection) and modified DIF (DIFm; simultaneous IgG + IgG4 deposits detection instead of separate IgG and IgG4 deposits detection) in routine diagnostics of ABDs. Material and methods: Eighteen patients with ABDs (7 with pemphigus dermatoses and 11 with subepithelial ABDs) were evaluated with DIFt and DIFm. Results: The agreement of detectability of IgG immunoreactants was obtained in 16 ABD cases (88.89%), as positive results in both DIFt and DIFm were obtained in 13 cases and negative results in both DIFt and DIFm were obtained in 3 cases. One ABD case (Brunsting-Perry pemphigoid) (5.56%) was negative in DIFm with a positive DIFt result (IgG1 deposits). One ABD case (bullous pemphigoid) (5.56%) had only C3 deposits in DIFt with a positive DIFm reading (IgG + IgG4 deposits). A statistically significant relationship (p = 0.0186) between DIFm and DIFt results was revealed using Fisher's exact test. Conclusions: Both DIFt and DIFm are useful methods to detect deposition of IgG immunoreactants, but it seems that the innovative DIFm method slightly increases the detectability of IgG/IgG4 immunoreactants in relation to DIFt. The introduction of DIFm into routine laboratory diagnostics of ABDs seems to be justified, as it enables the abandonment of separate FITC conjugates for IgG and IgG4, which is important for cost-effectiveness.