Dermatology: Practical and Conceptual Original Article | Dermatol Pract Concept. 2022;12(4):e2022160 1 Diagnostic Role of Direct Immunofluorescence Assay in Determining The Etiology of Erythroderma: Experience in a Tertiary Referral Hospital Ecem Bostan MD1, Ozay Gokoz2, Sibel Dogan1, Duygu Gulseren1, Neslihan Akdogan1, Basak Yalici-Armagan1, Sibel Ersoy-Evans1, Gonca Elcin1, Aysen Karaduman1 1 Department of Dermatology and Venereology, Hacettepe University, Faculty of Medicine, Ankara, Turkey 2 Department of Pathology, Hacettepe University, Faculty of Medicine, Ankara, Turkey Key words: erythroderma, fluorescent antibody technique, immunobullous diseases, pathology Citation: Bostan E, Gokoz O, Dogan S, et al. Diagnostic Role of Direct Immunofluorescence Assay in Determining The Etiology of Erythroderma: Experience in a Tertiary Referral Hospital. Dermatol Pract Concept. 2022;12(4):e2022160. DOI: https://doi.org/10.5826/ dpc.1204a160 Accepted: January 14, 2022; Published: October 2022 Copyright: ©2022 Bostan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution- NonCommercial License (BY-NC-4.0), https://creativecommons.org/licenses/by-nc/4.0/, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original authors and source are credited. Funding: None. Competing interests: None. Authorship: All authors have contributed significantly to this publication. Corresponding author: Ecem Bostan, MD, Hacettepe University, Faculty of Medicine Department of Dermatology and Venereology, Ankara, Turkey, Phone:03123051704, Fax: 0 3123054127, E-mail: bostanecem@gmail.com Introduction: Erythroderma is a life-threatening dermatologic emergency which is characterized by diffuse erythema and exfoliation affecting more than 90% of the body surface area. Most common cutaneous diseases associated with erythroderma are systemic contact dermatitis, psoriasis, drug eruption and atopic dermatitis. Clinical-pathological correlation is used to determine the underlying disease. In addition, direct immunofluorescence (DIF) may provide significant clues for etiology of erythroderma especially in the case of autoimmune bullous skin diseases (ABSDs). Objectives: In our study, we aimed to analyze the demographic data, clinical pre-diagnoses, final di- agnosis, histopathological and DIF examination findings, accompanying systemic signs and laboratory abnormalities of erythrodermic patients. Methods: We conducted a retrospective study of 31 erythroderma patients in a referral hospital be- tween 2014 and 2021. Cutaneous biopsies were taken from all patients for H&E and DIF examination. Results: Average age was 54.6 ± 23 years, 48.4% of the patients were female (N = 15) whereas 51.6 % of the patients were male (N = 16). Average time between the onset of rash and biopsy was 18.8 days. DIF analysis showed immune deposits in 19.4% (N = 6) of the patients; whereas no immune deposits were detected in 80.6% (N = 25) of the patients. The most frequent final diagnosis was adverse cuta- neous drug eruption followed by ABSDs. Conclusions: Our findings suggest that DIF may be used in conjunction with clinical-pathologic and clinical findings to reveal the associated skin diseases in erythrodermic patients. Erythrodermic pa- tients presenting with clinical findings of ABSD should be considered for DIF examination. ABSTRACT 2 Original Article | Dermatol Pract Concept. 2022;12(4):e2022160 Introduction Erythroderma (exfoliative dermatitis) is defined as wide- spread erythema and exfoliation involving more than 90% of body surface area [1]. Men seem to be more affected compared to women male-to-female ratio ranging from 1.5 to 2.8 [1-3]. Average age at the onset differs among vari- ous studies and reported to be 50.7 and 57 years in two recent reports respectively.3,4 Even though skin involve- ment is the predominant clinical picture of the condition, life- threatening systemic complications such as tachycardia, electrolyte imbalance, edema and unstable body temperature may accompany [3]. Erythroderma generally arises from the exacerbation and generalization of a pre-existing skin diseases, even though new-onset cutaneous eruptions such as adverse cutaneous drug eruptions (ACDE), psoriasis, systemic allergic con- tact dermatitis (SACD), autoimmune bullous skin diseases (ABSD) such as bullous pemphigoid, pemphigus foliaceus may also be the cause [3,4]. Identification of the associated cutaneous disease is not always that easy and requires lon- gitudinal evaluation of the patient to reveal the underlying cause and manage the complications. Histopathological examination of the lesional skin pro- vides significant clues related to the etiology and thus may be the most fundamental evaluation to enlighten the pathogen- esis of erythroderma [5]. Direct immunofluorescence (DIF), on the other hand, might prove to be quite useful in cases of ABSD and leukocytoclastic vasculitis as the suspected causes of exfoliative dermatitis by revealing the specific immune deposition pattern in the biopsies taken from the perilesional and lesional skin respectively [5,6]. In our study, we aimed to determine the diagnostic role of DIF in identifying the etiology of erythroderma and show the relationship between the presence of immune deposits and underlying diseases of exfoliative dermatitis. Methods The present study was a retrospective study conducted by re- view of electronic medical data records and histopathologic slide images belonging to 31 patients in a tertiary referral hos- pital between January 2014 and September 2020. Ethics com- mittee approval was obtained (project number: GO 20/1099, decision number: 2020/19-45, decision date: November 17, 2020) and informed consent was taken from the participants for the study. All the patients were diagnosed as erythroderma and cutaneous biopsies were taken for histopathological exam- ination and DIF analysis. Patients without histopathological and DIF examination were excluded from the study. For DIF assay, skin tissue samples were frozen in a cryostat and then sectioned with a thickness of 5 µm. Then, fluorescein-labeled antisera against human IgM (Dako, dilution ratio: 1/20-1/40), IgG (Bio SB, dilution ratio: 1/25-1/100], IgA (Dako, dilution ratio: 1/20-1/40) and C3 (Diagnostic BioSystems Inc., dilution ratio: 1/75-1/100) were applied to sections and incubated. Presence of any positive immunofluorescence staining with IgM, IgG, IgA and C3 antisera was evaluated under immu- nofluorescence microscopy. Sex, age, dermatologic exam- ination findings, accompanying systemic symptoms, clinical pre- diagnoses, laboratory findings, elapsed time between the onset of the rash and biopsy procedure, histopathologic find- ings, presence of immune deposits in DIF analysis, final diag- nosis, treatment given and were evaluated and recorded. The underlying etiologies of erythroderma were divided into 4 cat- egories as follows: ‘category 1 (ABSD)’, ‘category 2 (ACDE)’, ‘category 3 (vascular skin diseases)’ and ‘category 4 (miscel- laneous other skin diseases) (Table 1). For cases with a final diagnosis of ACDE including drug rash with eosinophilia and systemic symptoms (DRESS) syndrome, maculopapular drug eruption (MDE), Stevens-Johnson syndrome/toxic epidermal necrolysis spectrum (SJS-TEN) and vasculitic drug eruption, the most probable inciting drug/drugs were determined and time between the onset of the erythroderma and first drug intake was also evaluated. Statistics Statistical analyses were performed with the IBM SPSS for Windows Version 22.0 and MS Excel. Categorical variables were given as frequencies and percentages. Numerical vari- ables were summarized as mean ± standard deviation or me- dian (minimum-maximum). Results Demographical, clinical, pathological characteristics; pre-diagnoses and final diagnoses of the all study subjects along with DIF findings and laboratory abnormalities are shown in Supplementary File 1. The average age of the subjects was 54. 6 ± 23 years (range: 3- 86 years, median: 61 years). 48.4% of the patients were female (N = 15) whereas, 51.6 % of the patients were male (N = 16). All patients presented with erythema and scaling covering > 90% of body surface area, 48.4 % of the patients (N = 15) had also one or more mucosal area (oral, anogenital and ocular) involvement. All patients with SJS (N = 4), five patients with MDE, two patients with DRESS, one patient with bullous mycosis fungoides, one patient with pemphigus foliaceus and two patients with SACD had mucosal involvement. The most common clinical presenta- tions of oral mucosal involvement were hemorrhagic-crusted plaques covering the lips, erosions on the buccal and palatal mucosa which were predominantly observed in cases with SJS. Mucopurulent conjunctivitis, eyelid margin ulceration Original Article | Dermatol Pract Concept. 2022;12(4):e2022160 3 and vulvovaginal/penile erosions were also present in pa- tients with SJS. Patients with MDE, DRESS and SACD most commonly had superficial erosions on the lips as the mu- cosal manifestation. The most frequent systemic symptoms and signs associated with erythroderma were fever (67.7%, N = 21), followed by pruritus (38.7 %, N = 12). Hypoten- sion, pain, irritability, facial/peripheral edema, lymphade- nopathy, malaise, arthralgia and myalgia were also present. 29% of the patients (N = 9) had eosinophilia (> 500/mm3 cells); other accompanying laboratory anomalies are shown in Supplementary Table 1. The average elapsed time between the onset of rash and performing the biopsy was 18.8 ± 28.3 days (range: 1-150 days). DIF analysis showed immune de- posits in 19.4% (N = 6) of the patients; whereas no immune deposits were detected in 80.6% (N = 25) of the patients. The final diagnoses of the underlying cutaneous diseases were classified as ‘category 1 (ABSD)’, ‘category 2 (ACDE)’, ‘category 3 (vascular skin diseases)’ and ‘category 4 (miscel- laneous)’. Category 1 consists of bullous pemphigoid, pem- phigus foliaceus; category 2 consists of ACDEs including SJS/TEN spectrum, DRESS, MDE, AGEP (acute generalized exanthematous pustulosis), fixed drug eruption, vasculitic Table 1. Underlying etiologies of erythroderma and direct immunofluorescence findings. Number of Patients N (%) Presence of any accompanying bulla, vesicle pustule, erosion, crusting or necrosis Direct Immunofluorescence Findings Total (N, %) Category 1 (Autoimmune bullous skin disorders) 3 (9.7%) Bullous Pemphigoid 2 (2.9%) Bulla, crusting and erosion Linear IgG and C3 deposition at the dermoepidermal junction Pemphigus Foliaceus 1 (1.4%) Flaccid bulla, vesicle and crusting Intercellular IgG deposition Category 2 (Adverse Cutaneous Drug Eruptions) 20 (64.5%) Stevens-Johnson Syndrome/ Toxic Epidermal Necrolysis 4 (5.8%) Erosion, crusting and Nikolsky (+) bulla None Maculopapular drug eruption 10 (14.5%) - Only one patient had linear IgM, granular IgG and IgA deposition at the dermoepidermal junction Drug rash with eosinopilia and systemic symptoms 3 (4.3%) - None Vasculitic drug eruption 1 (1.4%) - None Fixed drug eruption 1 (1.4%) Bulla, erosion, crusting None Acute generalized exantematous pustulosis 1 (1.4%) Pustule None Category 3 (Vascular Skin Diseases) 3 (9.7%) Purpura Fulminans 1 (1.4%) Hemorrhagic bulla, necrosis None Antiphospholipid syndrome 1 (1.4%) Hemorrhagic bulla, necrosis None Leukocytoclastic vasculitis 1 (1.4%) - None Category 4 (Miscellaneous) 5 (16.1%) Mycosis fungoides 1 (1.4%) Bulla Interrupted C3 deposition along dermal vessels and dermoepidermal junction Psoriasis 1 (1.4%) - None Psoriasiform dermatisis (finally diagnosed as idiopathic erythroderma) 1 (1.4%) Pustule Linear C3 deposition at the basal membrane Systemic allergic contact dermatitis 2 (2.9%) - None 4 Original Article | Dermatol Pract Concept. 2022;12(4):e2022160 from the intact skin just at the periphery of a bulla that had a pre-diagnosis of bullous pemphigoid or pemphigus foliaceus, whereas skin samples were obtained from a vesicle, bulla or pustule for the subjects who had pre-diagnosis of vasculitis or drug eruption. All 3 cases of erythrodermic patients in the category 1 diagnosed with either bullous pemphigoid (N = 2) or pemphigus foliaceus (N = 1), 1 patient from category 2 diagnosed with MDE and 2 cases from category 4 diagnosed with bullous mycosis fungoides and psoriasiform dermatitis, respectively, showed positive immunofluorescence in DIF as- say. Two cases of bullous pemphigoid showed linear IgG and C3 deposition at the dermoepidermal junction (Figure 4), whereas intercellular IgG deposition was detected in pem- phigus foliaceus. Conclusions To our knowledge, this is the first study which evaluates di- agnostic significance of DIF examination in clarifying the etiopathogenesis of exfoliative dermatitis. Immunofluores- cence microscopy is a well-developed, advanced technique which is utilized for the detection of tissue-fixed antibodies. For cutaneous disorders, DIF is used for designation of the antibodies bound to a specific antigen in the skin [7]. DIF assay simply involves the application of fluorescein-labeled secondary antibodies to a frozen section of sample tissue fol- lowed by examination of the issue for the deposition of im- mune reactants under immunofluorescence microscopy [7]. DIF is generally considered to be an auxiliary tool which aids drug eruption; category 3 encompasses purpura fulminans, antiphospholipid syndrome and leukocytoclastic vasculi- tis, whereas category 4 consists of miscellaneous causes of erythroderma including, bullous mycosis fungoides, SACD, psoriasis and psoriasiform dermatitis (Table 1). Patients in category 3 (purpura fulminans, antiphospholipid syndrome and leukocytoclastic vasculitis) were accepted to present an erythrodermic-purpuric form of the disease described, since >90% of the body surface area were covered with erythema accompanied by hemorrhagic bullae and ecchymotic plaque. The most frequent final clinical-pathological diagnosis was ACDE category (category 2) (64.5 %, N = 20) followed by bullous pemphigoid (6.5%, N = 2) and SACD (6.5 %, N = 2) (Table 1). The only patient diagnosed histopathologi- cally with ‘psoriasiform dermatitis’ was accepted to have an idiopathic form of erythroderma. Clinical and histopatho- logical pictures of erythrodermic patients with different un- derlying etiologies are shown in Figures 1-5. Presence of any accompanying pustule, bulla, vesicle, necrosis, erosion and crusting were also determined as the part of dermatological examination as shown in table 1. For the category 2, the mean elapsed time between the onset of the rash and most probable inciting drug intake was 14.6 ± 13.9 days (range: 0-42 days). The most common probable causes of erythro- derma were antimicrobials (40%, N = 8), followed by allopurinol (20%, N = 4), phenytoin (15%, N = 3), hydroxy- chloroquine (10%, N = 2), intravenous contrast media (10%, N = 2), chemotherapy agents (10%, N = 2) and others (15%, N = 3). The sample tissue for DIF examination was taken Figure 1. (A) Presentation of erythroderma in a patient diagnosed with mycosis fungoides widespread erythema and scaling. (B) Yellow/brown color change and subungal hyperkeratosis of the fingernails. (C) Diffuse dermo-epidermal atypical lymphoid infiltrate which shows epidermotropism (H&E, x200). Original Article | Dermatol Pract Concept. 2022;12(4):e2022160 5 Figure 2. (A) Dusky red, violaceous targetoid plaque and bulla formation in a patient with SJS. (B) In the same patient skin biopsy, vacuolar degeneration, necrotic keratinocytes and pigment incontinence compatible with Stevens-Johnson syndrome are observed (H&E, x200). (C) Widespread annular, polycyclic-erythematous plaques with scale involving the trunk and the extremities, (inset) closer view of the plaques in a patient finally diagnosed with idiopathic erythroderma. (D) Histopathological examination of the patient revealed psoriasiform dermatitis with sub-corneal pustule formation (H&E, x200). (E) Widespread erythematous, confluent patches and plaques on the trunk in a patient with drug eruption. (F) The same patient’s skin biopsy showed vacuolar degeneration at the basal layer, capillary congestion and perivascular eosinophilic and neutrophilic inflammation compatible with drug eruption. Figure 3. (A) Erythroderma in a patient with psoriasis. Histopathological findings in an erythrodermic patient diagnosed with psoriasis. (B) Subcorneal neutrophilic pustule (Kogoj pustule) formation is seen (arrowhead, H&E, x100). (C) Hypo-granulosis and psoriasiform acanthosis are present (H&E, x100). to reach the accurate diagnosis of various dermatologic dis- orders especially when supportive histopathological changes are minimal. In dermatology practice, incorporating DIF findings with routine pathological findings are particularly useful in the patients pre-diagnosed with ABSD, connective tissue diseases and cutaneous vasculitis [7]. A study by Buch et al showed that the sensitivity of DIF was 94.44% and 84% in the pemphigus vs bullous pemphigoid group respec- tively [8]. DIF was shown to have diagnostic significance in the classification of cutaneous small vessel vasculitides espe- cially in IgA vasculitis and lupus vasculitis [9]. In erythrod- ermic patients, expeditious diagnosis of the underlying cause is the essential step which enables the accurate intervention. In our study, we aimed to determine the diagnostic utility of DIF in patients with exfoliative dermatitis. Erythroderma appears to affect men more than women even though in some studies no sex predilection is showed [3,10]. In line with the present data in the literature, our study also showed a slight male predominance with a male-to-female ratio of 1.07. The mean age of affect study subjects in our study was 54.6 ± 23 years (range: 3- 86 years, median: 61 years). In a retrospective study of 49 erythroder- mic patients, the average age was reported to be 50.7 ± 17.9 years which was in concordance with our findings [4]. As an acquired-adulthood disease, erythroderma may have various underlying etiologies or may be idiopathic in at least 25% of 6 Original Article | Dermatol Pract Concept. 2022;12(4):e2022160 dermatoses leading causes being psoriasis, eczema and atopic dermatitis, in contrast to our study [4,12,13]. In our retro- spective study, we only included erythrodermic patients with available DIF examination results: this factor might explain the discrepancy between the results of our study and other ones. Our results show that skin samples for DIF examina- tion were mostly taken from the patients with a suspected the cases [11]. In the present study, the most frequent under- lying etiology of erythroderma was ACDE followed by ABSD, SACD, psoriasis and mycosis fungoides. In two patients with diagnoses of mycosis fungoides and psoriasis, generalization/ accentuation of the pre-existing dermatoses had evoked the erythrodermic status. In different studies, the most common diseases associated with erythroderma were pre-existing Figure 5. (A) Subcorneal/intragranular blister formation with neutrophils and few acantholytic cells ( arrow). Dermal edema with mixed cellular infiltrate bearing eosinophil leukocytes (H&E, x200). (B) Basal cell vacuolization, spongiosis, necrotic keratinocytes, and lymphocyte exocytosis in the epidermis. Lymphohistiocytic inflammation and a few extravasated erythrocytes in the dermis (H&E, x200). Figure 4. (A) Histopathological findings in an erythrodermic patient with bullous pemphigoid: sub-epidermal cleavage (arrowhead), eosinophilic spongiosis, superficial dermal edema and interstitial eosinophilic infiltration (H&E, x200). DIF revealed linear (B) C3 and (C) IgG deposition at dermoepidermal junction (DIF, x200). Original Article | Dermatol Pract Concept. 2022;12(4):e2022160 7 formation with neutrophils and few acantholytic cells which was compatible with pemphigus foliaceus (Figure 5). With the aid of DIF examination which revealed intercellular IgG deposition, the diagnosis of pemphigus foliaceus was con- firmed. On the other hand Joly et al reported three black African men diagnosed with lichenoid erythrodermic bullous pemphigoid [17]. In these patients, histopathological exam- ination showed subepidermal bulla with lichenoid infiltrate along with linear deposits of C3 along the basal membrane.17 Two erythrodermic patients in our study presented with widespread intact/flaccid bullae, erosion and crust forma- tion. Histopathological examination revealed sub-epidermal bulla, basal vacuolar degeneration and eosinophil-rich infil- trate. With the help of DIF analysis which detected linear deposits of IgG and C3 along the dermo-epidermal junction; the final diagnosis was bullous pemphigoid; the etiology of exfoliative dermatitis was unraveled. In the category 4, the study subject with a final diagnosis of bullous MF showed interrupted C3 deposition along der- mal vessels and dermoepidermal junction in the perilesional skin, whereas the case with the histopathological diagnosis of psoriasiform dermatitis and final clinical diagnosis of idio- pathic erythroderma showed linear C3 deposition at the basal membrane in the perilesional skin. We believe that these C3 deposition are non-specific, thus does not seem to carry any diagnostic significance as reported in a study by Leibold et al [18]. In this study, 41 non-lesional, sun-exposed skin samples obtained from Mohs surgery sites, 21 specimens demonstrated interrupted, weak linear or granular staining with IgM, IgG, IgA, Clq and C3 antisera [18]. On the other hand, the two other patients with final diagnoses of leukocytoclastic vascu- litis and vasculitis drug eruption did not show any immune deposition which might be linked to the long elapsed time between the onset of rash and biopsy (14 days) for both cases. DIF analysis is suggested to be performed within the first 24 hours to yield the best result [19]. Immunoreactants can not be shown efficiently 24-48 hours after the lesion formation. Even though our study has limitations, in that it was a retrospective study and only small number of erythrodermic patients who had undergone both histopathological exam- ination and DIF analysis were included, we would like to underline that DIF assay may be used as an auxiliary tool in enlightening etiopathogenesis of exfoliative dermatitis. References 1. Pal S, Haroon TS. Erythroderma: a clinico-etiologic study of 90 cases. Int J Dermatol. 1998;37(2):104-107. DOI: 10.1046/j.1365-4362.1998.00228.x. PMID: 9542663. 2. Cesar A, Cruz M, Mota A, Azevedo F. Erythroderma. A clini- cal and etiological study of 103 patients. J Dermatol Case Rep. 2016(1);10:1-9. DOI: 10.3315/jdcr.2016.1222. PMID: 27119000. PMCID: PMC4844528. diagnosis of bullous/vesicular or vasculitic skin diseases such as bullous pemphigoid, SJS, AGEP, FDE and leukocytoclas- tic vasculitis etc. So we most likely missed other causes of erythroderma for which DIF analysis was not performed in our center, which could be considered as a selection bias which is the limitation of our study. In ACDE category, the most common causes of cutane- ous eruption were antimicrobials followed by allopurinol, phenytoin, hydroxychloroquine, intravenous contrast media, chemotherapy agents and others. In concordance with our results, anticonvulsants, beta-lactams, allopurinol, rifam- picin, trimethoprim-sulfamethoxazole and non-steroidal anti-inflammatory drugs are reported to be the leading causes of acquired erythroderma in multiple studies [4,13]. 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Clin Exp Dermatol. 1990;15(4):293-295. DOI: 10.1111/j.1365-2230.1990. tb02093.x. PMID: 2145100. 15. King T, Helm TN, Valenzuela R, Bergfeld WF. Diffuse in- traepidermal deposition of immunoreactants on direct im- munofluorescence: a clue to the early diagnosis of epidermal necrolysis. Int J Dermatol. 1994;33(9):634-636. DOI: 10.1111/j.1365 -4362.1994.tb02922.x. PMID: 8002159. 16. Grekin SJ, Fox MC, Gudjonsson JE, Fullen DR. Psoriasiform pemphigus foliaceus: a report of two cases. J Cutan Pathol. 2012;39(5):549-553. DOI: 10.1111/j.1600-0560.2012.01866.x. PMID: 22449394. 17. Joly P, Tanasescu S, Wolkenstein P, et al. Lichenoid erythrodermic bullous pemphigoid of the African patient. J Am Acad Dermatol 1998;39(5 Pt 1):691-697. DOI: 10.1016/s0190-9622(98)70040-4. PMID: 9810884. 18. Leibold AM, Bennion S, David-Bajar K, Schleve MJ. Occur- rence of positive immunofluorescence in the dermo-epidermal junction of sun-exposed skin of normal adults. J Cutan Pathol. 1994;21(3):200-206. DOI: 10.1111/j.1600-0560.1994. tb00261.x. PMID: 7962822. 19. Palit A, Inamadar AC. Vasculitis: approach to diagnosis and ther- apy. Indian J Dermatol Venereol Leprol. 2006;72(5):334-345. DOI: 10.4103/0378-6323.27748. PMID: 17050926.