FIGURE 2 - uploaded by Aida Valencia
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A 74-year-old man with a verrucous plaque on the groin. A, Pseudoepitheliomatous hyperplasia with an intense inflammatory cell infiltrate containing numerous eosinophils and intraepidermal eosinophilic microabscesses suspicious for PEM vegetans (·40, H&E stain). B, DIF-P showing IC IgG staining (·200).
Source publication
Direct immunofluorescence (DIF) on frozen tissue (DIF-F) is the method of choice for the identification of immune deposits present in skin and other tissues. DIF can also be performed on formalin-fixed paraffin-embedded tissue (DIF-P) after antigen retrieval with proteases and has proven to be of value in renal pathology. However, its utility in sk...
Context in source publication
Context 1
... (IC) epidermal staining for IgG and/or C3 could be demonstrated in 3 of the 7 (43%) PEM cases on formalin-fixed tissue ( Fig. 2A-B). Table 5 shows that 1 case showed a similar pattern of IgG and C3 IC staining but with the addition of IgA on DIF-P only (case 1). The remaining 2 positive cases revealed the addition of C3 (case 2) and lack of C3 (case 5) IC staining on DIF-P in comparison to ...
Citations
... DIF on frozen tissue (DIF-F) can be the method of choice for immune deposits present in the skin and other tissues. DIF can also be performed on formalin-fixed paraffin-embedded tissue (DIF-P) after antigen retrieval with protease [9,10]. Nasr et al. have proven the value of DIF-P in renal pathology [11]. ...
... Valencia-Guerro et al. in 2017 compared the findings of DIF on paraffin-embedded specimens and fresh frozen specimens of 60 skin biopsies and found that DIF on paraffin-embedded blocks was overall less sensitive than on fresh frozen tissue; however, it was found to be a tool with a valuable technique that could aid the diagnosis of vasculitis and immune-bullous and connective tissue disorders when fresh tissue is unavailable [9]. ...
Background
Autoimmune vesiculobullous diseases (AIBDs) are a group of diseases characterized by blisters of the skin/mucosa due to the presence of circulating autoantibodies against antigens in the epidermis or the dermo-epidermal junction. Direct immunofluorescence (DIF) for immunoglobulin (Ig)G, IgC3, and IgA on fresh-frozen tissue is the gold standard diagnostic test for AIBDs. However, DIF in the absence of frozen tissue is challenging for the diagnosis of AIBDs. This study aimed to analyze the practical utility of DIF using paraffin-embedded skin biopsy rather than fresh frozen tissue for the diagnosis of AIBDs.
Methodology
This cross-sectional comparative study included 30 cases of AIBDs. DIF for IgG and IgA was performed on paraffin-embedded tissue (PE-DIF) after proteinase digestion on histopathologically confirmed 15 pemphigus vulgaris (PV), three pemphigus foliaceous (PF), four bullous pemphigoid (BP), three dermatitis herpetiformis (DH), three subcorneal pustular dermatosis (SCPD), and one case each of linear IgA disease and pemphigoid gestationis (PG). PE-DIF staining pattern was compared with the DIF on fresh frozen tissue (FF-DIF).
Results
All cases of PV and PF showed an intercellular IgG chicken wire staining pattern similar to FF-DIF. However, background staining was more intense in PV cases while less intense in PF cases. Three BP cases showed linear IgG staining in PE-DIF. DH, SCPD, linear IgA disease, and PG cases did not show IgG positivity. Out of three DH cases, two cases showed granular IgA positivity while linear IgA positivity along the basement membrane was seen in a single case of linear IgA disease. Negative IgG staining was observed in SCPD. Immunofluorescence in PE-DIF was rapidly deteriorating than in FF-DIF.
Conclusions
DIF done on paraffin-embedded tissue can be used as a supplement and salvage technique with histopathology for the diagnosis of AIBDs, particularly when a cryostat facility for frozen tissue is not available and the patient is unable to undergo a second biopsy procedure.
... In recent years, it has been reported that paraffin immunofluorescence (PIF) examination can be performed with various special protocols in cases where immunofluorescence examination cannot be performed or when there is insufficient glomerulus in nephropathology practice; this method has been recognized as a "salvage method" in nephropathology (3,4). In skin pathology, Valencia-Guer-rero et al.'s study in 2018 is the only study published on PIF in autoimmune bullous diseases (5). This study has demonstrated that this technique is valuable, though less sensitive than routine frozen examination. ...
... The paraffin immunofluorescence (PIF) method has been used as a salvage technique in nephropathology for quite some time (3,4). However, its use in skin pathology has only been reported once (5). In this study, PIF for the diagnosis of autoimmune bullous disease was performed on samples representing non-lesional skin. ...
Objective:
The gold-standard method for assessment of autoimmune bullous disease is direct/indirect immunofluorescence (IF) examination applied to fresh frozen tissue. Since the sensitivity of IF is greatly reduced in formalin-fixed paraffin-embedded (FFPE) tissues, IF cannot be relied upon in these samples. However, immunohistochemistry with the C4d antibody is a promising marker used as a surrogate for immune complex deposition, in nephropathology practice, and the paraffin IF method is also used as a "salvage" technique when fresh frozen tissue is not available or lacks glomeruli. We aimed to investigate whether it is possible to obtain immunofluorescence data from FFPE tissues diagnosed with bullous pemphigoid (BP) and pemphigus vulgaris (PV) and its relationship with inflammatory parameters in the skin.
Material and methods:
Eighty-nine in-house cases with both IgG and C3 positivity by routine immunofluorescence examination were included in the study. Inflammation parameters were evaluated in hematoxylin-eosin sections. Immunofluorescence study with IgG protease digestion and C4d immunohistochemistry were performed.
Results:
Results of 83 biopsies were obtained by paraffin immunofluorescence with IgG. There were positive reactions in 28 (34%) of these 83 biopsies. Five of the 28 positive results belonged to BP (18%), and 23 were PV (82%). Ten positive results were on lesional skin (36%), and 18 (64%) were on non-lesional skin. In the immunohistochemical study with C4d, 84 biopsy results were obtained. There were positive reactions in 34 (40.4%) of 84 biopsies. Of the 34 positive results, 12 belonged to BP (35.3%) and 22 to PV (64.7%). Again, 22 (64.7%) of 34 positive results belonged to lesional skin, and 12 (35.3%) belonged to non-lesional skin. When both techniques were used together, 44 (54%) of 81 biopsies yielded positive results for at least one of the two studies, while in 37 (46%), both tests showed negative results.
Conclusion:
The sensitivity of both IgG and C4d was less than in the literature, especially in BP-diagnosed biopsies. Positive samples were mostly PV. In conclusion, obtaining immunofluorescence data in FFPE samples is possible and is independent of the related skin being lesional or not, however, negative results should not be relied upon.
... In this study, the authors detected nucleoplasmin/B23 after a short proteinase treatment to allow immunostaining in the nucleolar structure. Proteinase treatment has been used successfully before to improve immunofluorescence methods [4][5][6]. Despite that, we decided to include Svistunova's manuscript due to the innovative use of proteinase to locate proteins deep into the nucleolar structure. ...
We have carefully read the interesting explanatory comment by Eugene V. Sheval [...]
... Antigen retrieval with proteinase on formalin-fixed and paraffin-embedded tissue has been used in renal pathology as a salvage technique when no glomeruli or frozen sections are available [1,2]. Recently, the value of direct immunofluorescence on proteinase-digested formalin fixed paraffinembedded skin biopsies was investigated, and it was found out to be a less sensitive but still valuable technique for the identification of immune deposits on skin [3]. In addition, H&E stained sections have been evaluated under a florescent microscope in the case of alopecia, melanoma and fibrous proliferations [4][5][6][7][8]. ...
... The proteinase digestion protocol applicable for formalin-fixed paraffin embedded tissues was carried out as it has been previously [3]. 5µ-thick sections were cut from representative blocks and placed on charged slides. ...
... It is known to be used in nephropathology practice when a frozen section is not available [1,2]. Recently, this technique has been applied to formalin-fixed paraffin-embedded skin tissues [3]. Although in this research the aim was not to detect immune accumulation, the protease digestion protocol was preferred. ...
Autofluorescence (AF) or naïve-florescence is the natural emission of light by biomolecules. During florescence microscope examination, we realized that elastic tissue is brighter or more autoflourescent than collagen and other biomolecules/cells in the skin. Consequently, we decided to review elastic tissue-related pathologies under a florescence microscope and to report the possible benefits of this technique from selected cases from the paraffin-block archive, by using the protease digestion immunofluorescence method. Selected and clinic-pathologically confirmed 3 elastofibroma dorsi, 3 pseudoxanthoma elasticum, 3 anetoderma, 3 arteriovenous malformations, 3 temporal arteritis, 3 scar tissue and 3 highly solar-damaged samples of skin from 2014-2019 were retrieved. Under the fluorescent microscope, coarse, thick and globularly-fragmented elastic fibers of elastofibroma dorsi, shortened, irregular and convoluted elastic fibers of pseudoxanthoma elasticum, internal elastic membranes of arteries and their integrity was visualized. None of the anetoderma cases had any signal representing elastic tissue. It was shown that elastic tissue can be observed easily under fluorescence microscope in the case of FFPE tissues. The resulting autofluorescence can be useful in recognizing elastic tissue-related pathologies, and it may be used as an ancillary or an alternative method to routine histochemical techniques.
... This technique was proposed first by Mera et al. in 1980 [83]. Later in 2018, another study was performed by Valencia-Guerrero et al. to evaluate the sensitivity of the method on formalin-fixed paraffin-embedded tissue after antigen retrieval with proteases [84]. Although in both studies, the authors have claimed the acceptable sensitivity for the diagnosis of pemphigus or other immunobullous diseases, given lack of enough information, few samples enrolled in both studies, and variability in interpretation, the results may not be reliable, and false-negative results are still significant. ...
... Nevertheless, this method may have some values when the only tissue available for study is paraffin blocks, or the patients are not accessible for the second biopsy. Overall, the sensitivity of this method for diagnosing pemphigus is about 40% [84]. ...
Introduction
Pemphigus vulgaris (PV) is an intraepidermal autoimmune bullous disease (AIBD) characterized by autoantibodies against desmosomal adhesion proteins, most commonly desmoglein (Dsg)3, leading to the suprabasal cleft formation and acantholysis.
Areas covered
Direct immunofluorescence (DIF) and indirect immunofluorescence (IIF) studies display the intercellular deposition of IgG/C3 throughout the epidermis and presence of circulating autoantibodies respectively, as a net-like pattern. These methods help to differentiate intraepithelial from subepidermal blistering diseases. However, the target antigen remains unknown using immunofluorescence techniques. Thanks to the development of Dsg ELISA, using recombinant technology, circulating antibodies against Dsg1 and 3 could be detected sensitively. It is possible to differentiate PV from pemphigus foliaceus (PF) using this assay. BIOCHIP mosaic and multivariant ELISA are two novel serologic methods with the added value of the ability to screen several AIBDs simultaneously.
Non-Dsg1/3 antigens are also involved in the pathogenesis of PV and investigated more deeply thanks to the protein microarrays technique. Additionally, patients with even high values of anti-Dsg1/3 may be lesion-free, suggesting the presence of non-pathogenic autoantibodies.
Expert opinion
Newer diagnostic methods to replace traditional techniques should possess high sensitivity and specificity and be widely available, noninvasive, and relatively cheap. The newly developed methods need to be further evaluated before they are recommended for routine use.
... Nötrofillerden salınan serbest oksijen radikalleri ve lizozomal proteolitik enzimler vasküler duvarda hasar ve fibrinoid nekroz oluşturur (6,9,15). İD'lerin DİF ile tespiti bazı kutanöz hastalıklarda olduğu kadar klinik öneme sahip olmasa da altta yatan etyolojik nedenleri göstermesi açısından (6,8,(9)(10)(11)13,17). Çalışmalarda en çok depolanan immunglobülin IgA (%30.6, %34.5, %64.7), en çok depolanan İD C3 (%54.5, %80.4, ...
... Daha önce bazı merkezlerde yapılan çalışmalarda, İD'lerden en az bir tanesinin bulunması pozitif kabul edildiğinde, kutanöz vaskülitlerde ve LKV'lerde değişken oranlarda (%20-%70,5) DİF pozitifliği bildirildi (6,8,(9)(10)(11)17). Tüm KV'ler için en düşük oran %20 ile Feasel ve arkadaşlarının geniş vaka serisi içeren (1318 vaka) çalışmasında tespit edilirken (6), en yüksek oran Guerraro ve arkadaşlarının 20 vaka analizinde %70 olarak tespit edilmiştir (17). ...
... Daha önce bazı merkezlerde yapılan çalışmalarda, İD'lerden en az bir tanesinin bulunması pozitif kabul edildiğinde, kutanöz vaskülitlerde ve LKV'lerde değişken oranlarda (%20-%70,5) DİF pozitifliği bildirildi (6,8,(9)(10)(11)17). Tüm KV'ler için en düşük oran %20 ile Feasel ve arkadaşlarının geniş vaka serisi içeren (1318 vaka) çalışmasında tespit edilirken (6), en yüksek oran Guerraro ve arkadaşlarının 20 vaka analizinde %70 olarak tespit edilmiştir (17). Vaka sayısı 100'ün üzerindeki çalışmalarda tüm KV'ler değerlendirildiğinde DİF pozitifliklerinin %40'ı aşmadığı görüldü. ...
Giriş: Tamamında depolanma gözlenmese de, dermatologlar pekçok kutanöz vaskülit (KV) ön tanılı hastaları için direkt immunflorosan mikroskopi (DİF) altında immun depolanmaların bulunup bulunmadığını öğrenmek ister. Dünya literatüründe çeşitli pozitiflik oranları bildiren çalışmalar mevcuttur. Bu çalışma ile kliniğimize ait küçük damar KV’lerin DİF mikroskopi sonuçlarını sunmayı amaçladık.Gereç ve yöntem: Vaskülit öntanısı ile biopsi ve DİF tetkiki yapılan, histopatolojik olarak küçük damar KV’i mevcut olan toplam 121 vaka retrospektif olarak çalışmaya dahil edildi. Olgular klinik verileri ve Chapel Hill Consensus Conference vaskülit sınıflaması gözönünde bulundurularak toplam 6 gruba ayrıldı. Bazal membran yada perivasküler (PV) alanda en az bir depolanma ‘DİF pozitif’ olarak kabul edildi. Tüm olgularda DİF IgG, IgM, IgA ve Compleman C3 depolanmalarının dağılımları, oranları ve gruplarda en az bir immun depozitin bulunma durumu belirlendi. Lökositoklazis bulunduran yada eozinofil bulunduranlar ayrı grup yapılarak diğerleri ile immun depolanmalar açısından istatistiksel olarak karşılaştırıldı.Bulgular: Tüm olgularda DİF pozitifliği %58.7 (n:71/121) idi. Lökositoklastik vaskülit olgularının %50.9’unda (n:28/55), nonspesifik KV olgularının %67.4’ünde (n:31/46), ürtikeryal vaskülit olgularının %44.4 (n:4/9)’ünde, livedoid vaskülit olgularının %75 (n:3/4)’inde, henoch schonlein purpurası (HSP) olgularının (n:5/5) %100’ünde DİF pozitifti. 2 vaskülopati olgusunda depolanma yoktu. Lökositoklazis ve eozinofil mevcudiyeti ile immun depolanmalar arasında herhangi bir ilişki yoktu. En fazla biriken depozit C3 iken, HSP olgularında IgA depolanma oranı %100’dü. Sonuç: Özellikle HSP olgularında DİF ile IgA depozit tespiti tanı için oldukça önemlidir. Diğer küçük damar KV’lerinde %100 olmasa da gözardı edilemeyecek yüksek DİF pozitiflik oranları (özellikle C3) tespit edildi. KV’lerde DİF tetkikinin, klinik ve histopatolojik incelemeye ek olarak uygulanması faydalıdır.
Background
When immunofluorescence on the frozen section is insufficient or unavailable, salvage immunofluorescence techniques can be used on formalin-fixed, paraffin-embedded tissue. The goal of the current investigation was to evaluate the diagnostic value of paraffin immunofluorescence following proteinase K digestion on skin biopsy samples in comparison to fresh frozen immunofluorescence.
Materials and Methods
It was standardized and compared to the immunofluorescence on fresh frozen tissue (IF-Frozen) for paraffin immunofluorescence by proteinase K digestion of paraffin-embedded skin biopsies (IF-FFPE). The study included 50 native skin biopsy cases, and fluorescein isothiocyanate-labeled IgA, IgG, IgM, and C3 intensity levels were evaluated in each case.
Results
A total of 50 cases of native skin biopsy were included in the study, and their intensities for IgA, IgG, IgM, and C3 antibodies were compared. The average staining intensities in each disease group for the antibodies had equal intensity or had a minor difference (1+)/significant difference (2+). Paraffin immunofluorescence, proteinase K digestion had the best correlation, that is, had either equal or minor difference (1+) with fresh frozen immunofluorescence. The difference of 2+ intensity of antibodies between IF-FFPE and IF-Frozen was noted mainly in C3 antibody on bullous pemphigoid (0.5%). IF-FFPE showed a sensitivity of 100%, 97.6%, 100%, and 81.6% for IgA, IgG, IgM, and C3, respectively, whereas the specificity was 100% for IgA, IgG, IgM, and C3.
Limitations
Small sample size and and the employment of one method of antigen retrieval in IF-FFPE.
Conclusion
Immunofluorescence techniques done on formalin-fixed paraffin-embedded tissue can serve as salvage techniques in cases where immunofluorescence on the frozen section may not be adequate or may not be available.
Objective
To determine the diagnostic utility of C4d immunohistochemical marker in cases of bullous pemphigoid by calculating the sensitivity, specificity, positive predictive value and negative predictive value.
Methods
We conducted an exploratory study (retrospectively and prospectively) from January 2017 to June 2022. All direct immunofluorescence proven cases of bullous pemphigoid were included in the study while cases with inadequate tissue for immunohistochemistry studies were excluded.
Results
Among the 57 cases of bullous pemphigoid, 49 showed positivity for C4d marker. All the ten control cases of inflammatory dermatoses were negative for C4d staining. A sensitivity of 86%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value of 55.56% were calculated with a confidence interval of 95%.
Conclusion
Direct immunofluorescence on fresh or frozen skin tissue remains the gold standard. But in circumstances where direct immunofluorescence facilities are not available, C4d immunohistochemistry marker staining on formalin-fixed paraffin-embedded material submitted for standard microscopic investigation can, in most cases, confirm the diagnosis of bullous pemphigoid, obviating the need for a second biopsy.
Limitation
It is a single centre study. Selection bias may come into play.
