Biopsy For Direct Immunofluorescence Research Articles

Direct Immunofluorescence Findings and Factors Affecting Conjunctival Biopsy Positivity in Ocular Mucous Membrane Pemphigoid.

The aim of this study was to describe the direct immunofluorescence (DIF) findings and factors affecting conjunctival biopsy positivity in patients clinically diagnosed with ocular mucous membrane pemphigoid (OMMP). This retrospective observational case series included patients with clinical OMMP who underwent conjunctival biopsy for DIF in at least 1 eye between 2018 and 2021 in an institutional setting. The primary outcome measures were association of age and chronic ocular complications with biopsy positivity. Of 61 patients, DIF positivity was seen in 33 (54.1%) clinically suspected cases of OMMP. Of 39 patients who underwent bilateral biopsy, 23 (59%) were positive, of which 12 (52%) were positive in both eyes while 11 (48%) were positive in 1 eye. Of 22 patients who underwent unilateral biopsy, 10 (45%) were positive. Of the 100 biopsied eyes, 45 (45%) were DIF positive. Among the immunoreactants studied, linear deposition of C3 was seen in all 45 positive eyes (100%). Increasing age was significantly associated with higher likelihood of biopsy negativity ( P = 0.032), whereas a greater Sotozono chronic ocular complication score, indicative of disease severity, was associated with low likelihood of biopsy positivity ( P = 0.0042) and lower overall expression of immunoreactants on DIF ( P = 0.0007). Older patients and patients with more severe ocular surface disease sequelae are likely to have negative DIF results. To optimize the chances of confirming the diagnosis of OMMP by DIF, both eyes should be biopsied early in the disease course. If 1 eye is being biopsied, the less affected eye must be chosen.

Utility of C3d and C4d immunohistochemical staining in formalin-fixed skin or mucosal biopsy specimens in diagnosis of bullous pemphigoid and mucous membrane pemphigoid

Bullous pemphigoid (BP) and mucous membrane pemphigoid (MMP) sometimes have overlapping clinical, histopathological, and direct immunofluorescence (DIF) features in the early stages. Complement deposition is an intrinsic component of the patho-mechanism of BP in contrast to MMP. Hence immunohistochemistry (IHC) for C3d and C4d may be helpful in differentiating the two disorders. Seventy-four patients of BP and 18 patients of MMP along with 10 negative controls were enrolled in this study. C3d and C4d IHC was performed in formalin-fixed skin biopsy specimens. C3d IHC staining in BP/MMP had a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 59.2%/41.2%, 100%/100%, 100%/100%, 25.6%/50.0%, respectively. C4d IHC staining in BP/MMP had a sensitivity, specificity, PPV and NPV of 26.8%/17.6%, 100%/100%, 100%/100% and 16.1%/41.7%, respectively. Receiver operator analysis showed utility of C3d in diagnosing both BP [Area under curve (AUC) = 0.8, p = 0.0001] and MMP (AUC = 0.71; p = 0.001). C4d was useful in diagnosis of BP (AUC = 0.5; p = 0.0001), but not MMP (AUC = 0.6; p = 0.064). Hence, C3d is a better diagnostic modality for BP as compared to C4d, whereas C3d and C4d have lower diagnostic importance in MMP. C3d IHC can be employed in diagnosing BP when a second biopsy for direct immunofluorescence (DIF) is not possible or where a facility for IF microscopy does not exist.

Assessment of Clinical and Laboratory Use of the Cutaneous Direct Immunofluorescence Assay

Dermatologists submit direct immunofluorescence (DIF) biopsies on a daily basis, using an assay detecting immunoreactant deposition with a panel that has traditionally comprised immunoglobulin (Ig) G, IgA, IgM, C3, and fibrin, with or without albumin antibodies. To evaluate and compare the frequency of immunoreactants in DIF biopsies submitted over an 8-year period and assess use by dermatologists based on clinical impression. A quality improvement study was conducted in a community outreach reference laboratory associated with a large academic medical center. Results of 2050 consecutive DIF skin biopsies submitted to the laboratory between April 1, 2012, and June 12, 2020, were analyzed by final pathologic diagnosis and antibody subtype positivity, in comparison with clinical impression. Biopsies in which the submitting physician had not performed the biopsy were excluded. Histopathologic findings and the results of DIF biopsies using the standard 6-antibody panel were evaluated in correlation with the submitted clinical diagnosis to assess immunoreactivity of the assay. Of 2050 DIF biopsies submitted, 367 (17.9%) were positive; IgG, IgA, and C3 alone identified all primary immunobullous disease cases (pemphigoid, pemphigus, linear IgA, and dermatitis herpetiformis), and IgA, C3, and fibrin antibodies alone identified all vasculitis cases. A panel of IgG, IgA, IgM, and fibrin identified all cases of lupus erythematosus. DIF results were positive in less than half of cases of hematoxylin and eosin biopsy-confirmed lupus erythematosus (23 of 47 [49%]). A total of 247 biopsies were submitted for clinical diagnoses not optimally supported on DIF: lichen planus, porphyria, and connective tissue disease. The findings of this study suggest that there is a knowledge gap among dermatologists relating to the opportunity for high-value, cost-conscious use of DIF. The practice of reflexive antibody testing using a 6-antibody panel for all DIF biopsies is likely unnecessary. DIF protocols tailored to the clinical diagnosis may enhance cost-effectiveness without loss of test sensitivity or specificity.

False-Negative Rate of Direct Immunofluorescence on Lower Extremities in Bullous Pemphigoid.

Bullous pemphigoid (BP) is the most common autoimmune blistering disorder of the skin. It is typified by tense blisters with a subepidermal split and mixed dermal inflammatory infiltrate on histology. Biopsy of the perilesional skin for direct immunofluorescence (DIF) has become the gold standard in the diagnosis of BP. Currently there is a pervasive clinical opinion that the lower extremity is a site with a high false-negative rate (FNR) for DIF in the diagnosis of BP. This notion is primarily based on 2 early studies from the 1980s without more recent confirmatory studies. To readdress this question regarding the lower extremities, a retrospective study from 2012 to 2018 was performed in our institution that evaluated the FNR of DIF by an anatomical site in the diagnosis of BP. Cases of BP were identified using standard criteria (clinical and histological data reviewed in cases with negative DIF), and overall, 79 patients were included in the study. A total of 4 false-negative DIF biopsies were verified. Two negative DIF were from the lower extremity yielding a FNR of 10% compared with 4% on the trunk and 3% from the upper extremity, with no statistically significant difference by anatomical sites. Our study fails to demonstrate a high FNR of DIF from the lower extremity in the diagnosis of BP.

Keratinocyte Binding Assay Identifies Anti-Desmosomal Pemphigus Antibodies Where Other Tests Are Negative.

The serological diagnosis of pemphigus relies on the detection of IgG autoantibodies directed against the epithelial cell surface by indirect immunofluorescence (IIF) on monkey esophagus and against desmoglein 1 (Dsg1) and Dsg3 by ELISA. Although being highly sensitive and specific tools, discrepancies can occur. It is not uncommon that sera testing positive by ELISA give a negative result by IIF and vice versa. This brings diagnostic challenges wherein pemphigus has to be ascertained or ruled out, especially when no biopsy is available. We utilized the ability of anti-Dsg3 and anti-Dsg1 IgG to bind in specific desmosomal patterns to living cells to investigate these discrepancies between IIF and ELISA. Living cultured primary normal human keratinocytes were grown under differentiating conditions to induce adequate expression of Dsg1 and Dsg3, incubated with patient serum for 1 h, and then stained to visualize bound IgG. We investigated two different groups; sera from patients with a positive direct immunofluorescence (DIF) and inconsistent serological findings (n = 43) and sera with positive ELISA or IIF but with negative DIF (n = 60). As positive controls we used 50 sera from patients who fulfilled all diagnostics criteria, and 10 sera from normal human subjects served as negative controls. In the DIF positive group, IgG from 39 of the 43 sera bound to the cells in a desmosomal pattern while in the DIF negative group none of the 60 sera bound to the cells. This shows that for pemphigus patients, ELISA and IIF can be negative while anti-desmosomal antibodies are present and vice versa that ELISA and IIF can be positive in non-pemphigus cases. In absence of a biopsy for DIF, such findings may lead to misdiagnosis.

Bullous Pemphigoid: Use of C4d Immunofluorescent Staining in a Case With Repeated Negative Conventional Direct Immunofluorescence Studies

Direct immunofluorescence (DIF) using frozen section material from a fresh/preserved perilesional biopsy is the gold standard for the immunopathologic diagnosis of bullous pemphigoid (BP). DIF in BP shows linear dermoepidermal junction (DEJ) staining for C3, with or without staining for IgG. In some situations, only a formalin-fixed lesional biopsy is obtained (with no fresh/preserved perilesional biopsy for DIF). In this setting, paraffin section C4d immunohistochemistry has proven to be diagnostically useful, demonstrating linear DEJ positivity for C4d. We present a novel use of C4d staining for the diagnosis of BP, specifically analyzing C4d perilesional frozen section DIF in a case where standard perilesional frozen section DIF for IgG/C3 was available, but was negative. An 80-year-old woman presented with a pruritic bullous lesion on her left upper extremity, clinically thought to represent BP. Lesional histologic findings were typical for BP, but perilesional frozen section DIF staining was negative for IgG and C3. A second set of biopsies processed at a different laboratory yielded the same result. A diagnosis of bullous scabies was considered. Subsequently, perilesional frozen section DIF for C4d was obtained, which showed strong linear DEJ positivity, confirming the diagnosis of BP. DIF for C4d is widely used in transplant pathology, since C4d is persistent in tissue, versus C3. Our case demonstrates that perilesional frozen section DIF staining for C4d may be positive and diagnostic in BP, even when conventional DIF staining for IgG and C3 is negative.

Indications and procedures for direct immunofluorescence biopsies of the oral mucosa.

A definitive diagnosis is crucial for management of any oral mucosal disease. Direct immunofluorescence (DIF) is a valuable diagnostic aid for immune-mediated blistering diseases and systemic connective tissue diseases of the skin and the mucosa. This paper gives an overview of the DIF biopsy technique for oral lesions and provides a background for the clinician to optimize the utilization of DIF biopsy. The key characteristic diagnostic findings of DIF of specific mucosal diseases are also discussed.

A refractory rash in a seborrheic distribution

A 70-year-old man presented to our clinic in April, 2014, with a 4 month history of painful, pruritic rash involving the scalp, face, chest, and back. He had a history of colon cancer and deep venous thrombosis. A biopsy sample of the rash had been interpreted as eczema, but he had had only minimum improvement with short courses of both topical and systemic corticosteroids and antibiotics. Examination showed a healthy-looking man with erythematous papules and plaques displaying cornfl akelike scale in a seborrheic distribution (fi gure). Oral and ocular mucosae were normal. Previous serological studies, including antinuclear antibody (ANA), complement, and serum protein electrophoresis, were normal. We did lesional punch biopsy for routine histology (appendix) and perilesional punch biopsy for direct immunofl uorescence (fi gure). Clinicopathological fi ndings were diagnostic of pemphigus foliaceus. Pemphigus foliaceus is a rare autoimmune bullous disease characterised by pathogenic autoantibodies against desmoglein-1 (a desmosomal protein) that cause disruption of intercellular adhesion between keratinocytes in the epidermal granular layer (acan tholysis). This results in fragile bullae that rupture easily to form erosions with characteristic scale crust. Pemphigus foliaceus often begins on the upper body in a seborrheic distribution, but progresses to widespread involvement or erythroderma if not appropriately treated. Pruritus and burning are common and can be exacerbated by UV exposure. Unlike pemphigus vulgaris, pemphigus foliaceus is rarely associated with mucosal involvement. Diagnosis is made with biopsies for routine histology and direct immunofl uorescence. Pemphigus foliaceus typically arises in middle age and can be drug-induced, such as by penicillamine and angiotensin-converting enzyme inhibitors. An endemic form, Fogo Sevelagem (“wild fi re”), occurs in South America and has been linked with exposure to black fl ies. Before eff ective treatment was available pemphigus foliaceus fatality reached 60% due to dehydration and sepsis. Rituximab, an anti-CD20 monoclonal antibody, has recently gained favour as treatment of choice for pemphigus foliaceus and other autoimmune bullous diseases because of relatively high rates of induced remission compared with classic treatments. We prescribed 30 mg prednisolone daily and rituximab (375 mg/m2 intravenously once weekly for 4 weeks) for our patient, and triamcinolone 0·1% cream twice daily. At follow-up 2 months after treatment with rituximab most skin lesions were healed and we could reduce prednisolone to 20 mg daily (appendix). He will be followed up every 3 months until prednisolone is tapered off completely.

Biopsy location for direct immunofluorescence in patients with suspected bullous pemphigoid impacts probability of a positive test result.

Bullous pemphigoid (BP) is an autoimmune polymorphic skin disease characterized by erythematous papules and plaques and tense bullae. A skin biopsy for direct immunofluorescence (DIF) is used to detect autoantibodies and complement proteins. We sought to determine which location would provide the highest probability of obtaining a positive DIF result. We undertook a retrospective chart review of 1,423 DIF biopsies. Biopsies with a clinical suspicion of BP were designated as either lesional, perilesional, or indeterminate. Fifty percent of lesional DIF biopsies were positive, whereas 22% of perilesional and 12% of indeterminate biopsies had a positive DIF result. The odds ratio of a positive DIF from a lesional versus perilesional biopsy site was found to be 3.45 (95% CI 1.44-8.29). Clinicians are more likely to obtain a positive DIF result from a lesional nonbullous skin biopsy than from a perilesional or normal skin biopsy.

Recognizing presentations of pemphigoid gestationis: a case study.

Introduction. Pemphigoid gestationis (PG) is an autoimmune blistering disease that occurs in approximately 1 in 50,000 pregnancies. Failing to recognize PG may lead to inadequate maternal treatment and possible neonatal complications. Case Report. At 18 weeks of gestation, a 36-year-old otherwise healthy Caucasian G4P1 presented with pruritic papules on her anterior thighs, initially treated with topical steroids. At 31 weeks of gestation, she was switched to oral steroids after her rash and pruritus worsened. The patient had an uncomplicated SVD of a healthy female infant at 37 weeks of gestation and was immediately tapered off steroid treatment, resulting in a severe postpartum flare of her disease. Discussion. This case was similar to reported cases of pruritic urticarial papules followed by blisters; however, this patient had palm, sole, and mucous membrane involvement, which is rare. Biopsy for direct immunofluorescence or ELISA is the preferred test for diagnosis. Previous case reports describe severe postdelivery flares that require higher steroid doses. Obstetrical providers need to be familiar with this disease although it is rare, as this condition can be easily confused with other dermatoses of pregnancy. Adequate treatment is imperative for the physical and psychological well-being of the mother and infant.

The Utility of C4d Immunohistochemistry on Formalin-Fixed Paraffin-Embedded Tissue in the Distinction of Polymorphic Eruption of Pregnancy From Pemphigoid Gestationis

Polymorphic eruption of pregnancy (PEP), formerly known as pruritic urticarial papules and plaques of pregnancy, is a dermatosis of pregnancy that must be distinguished from pemphigoid gestationis (PG). Although this differential diagnosis may be possible on routine histology, an additional biopsy for direct immunofluorescence (DIF) is often needed. Recent studies have demonstrated the utility of anti-C4d or anti-C3d antibodies in the diagnosis of bullous pemphigoid (BP) in formalin-fixed paraffin-embedded tissue (FFPE). We investigated the utility of routine immunohistochemistry (IHC) for anti-C4d in FFPE tissue in the specific differential diagnosis of PEP versus PG in known, DIF-proven cases. We performed C4d IHC on PEP (n = 11), PG (n = 8), DIF-proven BP (n = 12), and other common dermatoses (n = 12) that are typically DIF negative. None of the PEP cases (0/11) or the other common dermatoses (0/12) demonstrated C4d positivity at the basement membrane zone. In comparison, 100% of PG cases (8/8) and 83.3% of BP cases (10/12) showed linear C4d immunoreactant deposition along the basement membrane zone. The results demonstrate the potential utility of C4d IHC in FFPE tissue for distinguishing PEP from PG, thus potentially obviating the need of a repeat biopsy for DIF, particularly in C4d-negative cases where there is a low suspicion of PG on both clinical and histological grounds. Also, patients with positive C4d-positive immunoreactivity may also potentially proceed directly to less invasive serological confirmatory testing, such as BP180 NC16a enzyme-linked immunoabsorbent assay.

Bullous Tinea Pedis With Direct Immunofluorescence Positivity

Skin biopsy for direct immunofluorescence (DIF) testing is an essential tool in the diagnosis of blistering diseases. In the majority of cases, positive epidermal immunofluorescent staining is indicative of an autoimmune bullous disease (AIBD). We identified 2 patients with bullous dermatophyte infection diagnosed on hematoxylin- and eosin-stained sections who had positive DIF findings on biopsy of perilesional skin. We subsequently reviewed the literature regarding positive DIF findings in conditions other than AIBD. Other infections, including herpesviridae, scabies, and orf, have rarely been reported to yield positive DIF findings, with positive staining at the dermoepidermal junction. Some genodermatoses and many inflammatory skin diseases, including lichen planus, psoriasis, graft-versus-host disease, among others, may also have DIF findings mimicking those of both intra- and subepidermal AIBD. Although rare, positive DIF results occur in conditions other than AIBD. In many instances, the pathophysiological mechanisms behind immunoreactant deposition in these conditions are poorly understood. Misleading DIF results may lead to delay in correct diagnosis and treatment. Clinicians should be aware of potential alternate sources of positivity when there is lack of clinical correlation with immunofluorescence findings.

C4d immunohistochemical stain is a sensitive method to confirm immunoreactant deposition in formalin‐fixed paraffin‐embedded tissue in bullous pemphigoid

Bullous pemphigoid (BP) is characterized clinically by the onset of pruritic urticarial plaques, vesicles and bullae in a predominantly elderly population. While the diagnosis may be suspected on routine hematoxylin and eosin histology of formalin-fixed paraffin-embedded tissue, fresh-frozen tissue must be used to show the immunologic nature of the bullous process by direct immunofluorescence (DIF). The diagnosis is further confirmed and separated from epidermolysis bullosa acquisita (EBA) by subsequent serologic studies to detect antibodies directed against BP180 and BP230 antigens and characteristic antibody deposition on salt-split skin. Using a polyclonal complement fragment 4d (C4d) antibody, we stained formalin-fixed paraffin-embedded skin biopsy specimens from cases of BP and controls. We showed characteristic linear basement membrane deposition of C4d in formalin-fixed paraffin-embedded tissue in seven of nine cases diagnosed as BP vs. EBA by DIF on fresh-frozen tissue. None of the four controls for which we had adequate tissue were positive. These results indicate that formalin-fixed paraffin-embedded tissue can be stained for the immunoreactant C4d to show characteristic immunoreactant deposition, potentially obviating the need for repeat biopsy for DIF and allowing clinicians to proceed to serologic confirmation of BP.

Clinical experience in pemphigoid gestationis: Report of 10 cases

Pemphigoid gestationis is a rare autoimmune blistering disease that occurs during pregnancy. This study reviewed our clinical experience with pemphigoid gestationis. We reviewed medical records of 10 patients with pemphigoid gestationis seen at Mayo Clinic, Rochester, Minnesota, between 1976 and 2004. Urticarial papules were the most frequent clinical presentation followed by blisters and rash. Pruritus was the cardinal symptom. Lesions presented initially on the legs, thighs, back, and chest. Direct immunofluorescence had the highest diagnostic test sensitivity. Systemic corticosteroids were the mainstay of treatment. Fetal and maternal outcome was good in all cases. This was a retrospective, single-institution study. This condition can be easily confused with other dermatoses of pregnancy, for example, pruritic urticarial papules of pregnancy. Biopsy for direct immunofluorescence is the preferred test for confirmation of diagnosis. On the basis of good patient outcomes, conservative treatment seems warranted.

Conjunctival involvement in the autoimmune blistering dermatoses: a clinical and histopathological study

The conjunctiva was examined by slit lamp microscopy and biopsy for direct immunofluorescence (IF) in patients with cicatricial pemphigoid (CP), bullous pemphigoid (BP), pemphigoid gestationes (PG), linear IgA dermatosis (LAD), pemphigus and dermatitis herpetiformis (DH). In CP, five of 13 patients had definite scarring, seven equivocal, and one no signs. IF showed linear deposition of IgG and/or C3 along the BMZ in 45%. In BP, six of 18 patients had fine conjunctival scarring. IF showed linear IgG IgA and/or C3 in 73 %. Scarring was not observed in one PG patient. In LAD, three of seven patients had conjunctival scarring, one with marked symblepharon. IF in five patients showed linear IgG without IgA in three. In pemphigus, neither of two patients had scarring. IF in both showed IgG and/or C3 between epithelial cells. In DH, one of three patients had fine scarring. These findings demonstrate that conjunctival involvement may occur in autoimmune bullous dermatoses other than CP and LAD.

The histopathology of localized lipoatrophy.

We studied 11 patients with the clinical diagnosis of lipoatrophy and found two histopathological subsets. Six patients presented with a distinctive picture, which we termed 'involutional' fat, consisting of lobules of small lipocytes embedded in hyaline connective tissue with numerous capillaries. Five of these six patients had a single lesion, usually of the upper arm. Serological studies were normal, and direct immunofluorescence, performed in three cases, showed immunoreactants in the blood vessels in only one. The four patients with inflammation of the fat had multiple areas of localized lipoatrophy. Three had biopsies for direct immunofluorescence and all three showed immunoreactants involving the basement membrane zone (two cases) or blood vessels (one case); and three had serological abnormalities. We suggest that the involutional histopathological pattern is a distinctive subset of localized lipoatrophy.

The distribution of immunoglobulins and the C3 component of complement in multiple biopsies from the uninvolved and perilesional skin in pemphigus.

Summary The distribution of immunoglobulins and the C3 component of complement (C3) in the skin of 29 patients with various forms of pemphigus was studied. Biopsies, from clinically uninvolved skin, were taken from three different sites and perilesional skin was taken when lesions were present. Thirteen patients (45%) who were studied had intercellular deposits of IgG and C. There was no significant variation between sites. Ten patients (34%), in cutaneous remission, were negative at all sites. We conclude that in pemphigus, a biopsy for direct immunofluorescence can be taken from any suitable site on the skin surface.