Dermatology: Practical and Conceptual Original Article | Dermatol Pract Concept. 2024;14(3):e2024169 1 Frequency and Correlation of Peripheral and Tissue Eosinophilia with Clinical Manifestations in Patients with Bullous Pemphigoid Bahareh Abtahi-Naeini1,2, Farhad Zare-Mehrjerdi3, Zabihollah Shahmoradi1, Fereshte Rastegarnasab3, Mojtaba Akbari4, Azadeh Zolfaghari1, Asiyeh Heidari1, Fateme Mohaghegh1 1 Skin Diseases and Leishmaniasis Research Center, Isfahan University of Medical Sciences, Isfahan, Iran 2 Pediatric Dermatology Division of Department of Pediatrics, Imam Hossein Children’s Hospital, Isfahan University of Medical Sciences, Isfahan, Iran 3 Student Research Committee, Isfahan University of Medical Sciences, Isfahan, Iran 4 Department of Epidemiology, School of Health, Isfahan University of Medical Sciences, Iran Key words: Bullous pemphigoid, Eosinophilia, Eosinophils, BPDAI Citation: Abtahi-Naeini B, Zare-Mehrjerdi F, Shahmoradi Z, et al. Frequency and Correlation of Peripheral and Tissue Eosinophilia with Clinical Manifestations in Patients with Bullous Pemphigoid. Dermatol Pract Concept. 2024;14(3):e2024169. DOI: https://doi. org/10.5826/dpc.1403a169 Accepted: April 6, 2024; Published: July 2024 Copyright: ©2024 Abtahi-Naeini 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: Fateme Mohaghegh, Skin Diseases and Leishmaniasis Research Center, Isfahan University of Medical Sciences, Isfahan, Iran. Email: f.mohaghegh199@gmail.com Introduction: Bullous pemphigoid (BP) is an autoimmune disease involving the sub-epidermal layer. Eosinophilia may play a role in the pathogenesis of BP. Objectives: We aimed to investigate the correlation between dermal or peripheral eosinophilia with clinical presentations in patients with BP. Methods: This cross-sectional study was conducted on 108 BP patients from January 2010 to September 2019. Clinical data were recovered. Skin biopsies were re-evaluated, and the Bullous Pem- phigoid Disease Area Index (BPDAI) severity score was calculated. Finally, the relationship between clinical features of BP and dermal or peripheral eosinophilia was analyzed. Results: A total number of 108 patients were included in this study. Thirty-five were excluded due to our exclusion criteria. Finally, data from 73 patients were analyzed. Fifty-seven point five percent of the population was female. There was a significant direct correlation (r = 0.33) between BPDAI severity score and tissue eosinophilia (P = 0.03). No significant relationship was found between BPDAI severity score and peripheral eosinophilia (P = 0.52). There were significant positive correlations between tissue eosino- philia with absolute serum eosinophil count (P = 0.002; r = 0.49) and percentage (P < 0.0001; r = 0.89). ABSTRACT 2 Original Article | Dermatol Pract Concept. 2024;14(3):e2024169 Introduction Bullous pemphigoid (BP) is an autoimmune bullous dis- ease [1] more prevalent in the elderly with higher mor- bidity and mortality [2]. BP occurs due to immunological responses against hemi-desmosomal proteins responsible for dermo-epidermal cohesion [3]. Lesions mostly present on the lower abdomen, inner surface and flexors of the thighs, and forearms, localized or generalized. Mucosal involvement is rarely seen [4]. The metacognitive characteristics and bio- logical properties of eosinophils suggest a potential role in the onset or progression of BP [5]. Eosinophilic infiltration of the dermis is one of the crucial early events in the de- velopment of bullous lesions in BP [6]. Previous studies on the histopathological features of the lesions revealed the presence of extracellular eosinophil granules, degranulated eosinophils, and free granule proteins throughout the upper dermis or at the dermo-epidermal junction [7,8]. Peripheral eosinophilia has been reported in 50-60% of patients with BP and is associated with disease severity [9]. However, the relationship between peripheral eosinophilia and clinical presentations, and also the presence of tissue eosinophilia has not been studied in BP patients. Objectives This study aimed to investigate the possible relationship be- tween peripheral and tissue eosinophilia and clinical mani- festations in patients with BP to provide more insight into the disease mechanism. Methods Study Design This cross-sectional study was conducted among BP patients hospitalized in Al-Zahra Hospital, a referral center for der- matological disease in Isfahan, Iran, from January 2010 to September 2019. The study was approved by the ethical committee of Isfahan University of Medical Sciences (Ethical code: IR.MUI.MED.REC.1399.250). Inclusion Criteria All patients with a definite diagnosis of BP by an expert der- matologist were included in the study. The diagnosis of BP was based on suggestive clinical features, subepidermal blis- ter formation with dermal eosinophilic infiltration on histo- pathological evaluations, and linear IgG and/or C3 deposits along the dermo-epidermal junction by direct immunofluo- rescence [10]. Exclusion Criteria Patients with (I) incomplete documents, (II) hospitalization for less than 24 hours, and (III) peripheral eosinophilia related to other medical conditions (i.e. atopic dermatitis, asthma, other allergic conditions, parasitic diseases, and hyper-eosinophilic syndrome) were excluded from the study. Studied Variables The files of hospitalized patients were reviewed. Age, sex, pregnancy, site of involvement, hospitalization duration, concomitant diseases, past drug history, treatment proto- col, and the serum eosinophil count were recruited from the medical records. The Bullous Pemphigoid Disease Area Index (BPDAI) severity score was calculated for each patient. BPDAI is an index assessing cutaneous and mucosal involvement with a total score [11]. This index quantifies the size and number of the lesions altogether. In addition, the affected areas of the skin are weighted based on BPDAI with more emphasis on limbs and less on the face or scalp. The higher scores are attributed to worse conditions. The slides of skin biopsies were also re-evaluated by a blinded skilled dermatopathologist for histopathological findings including subepidermal blister, C3, IgG, tis- sue inflammation, peripheral eosinophilia, and dermal eosinophilia. The presence or absence of a cleft was registered. Tissue inflammation was assessed at grade 0 = 0%, grade 1 < 25%, grade 2 = 25% to 50%, grade 3 = 50% to 75%, and grade 4 > 75%, based on the proportion of light microscopic fields containing inflammatory cells. Four fields of ×40 power of each slide were evaluated, each field was di- vided into four quadrants (each 25%), and the tissue inflam- mation was scored based on the presence of inflammatory cells in each quadrant [12]. Finally, the average percentage in the four fields was considered as the final tissue inflam- mation score. Dermal eosinophilia was also scored as grade 0 = 0%, grade 1 < 25%, grade 2 = 25% to 50%, grade 3 = 50% to 75%, Conclusions: This study revealed significant relationships between tissue eosinophilia and BP sever- ity. These findings could be useful in clinical practice. The possible role of eosinophils in BP clinical features should be considered as a promising help for better diagnosis and treatment. Original Article | Dermatol Pract Concept. 2024;14(3):e2024169 3 and grade 4 > 75% [13]. Eosinophils were counted among the inflammatory cells and then the percentage was calcu- lated in the same four fields of ×40 power. The average per- centage in the four fields was considered the final dermal eosinophilia score. Peripheral eosinophilia is defined as the serum eosinophil count higher than 500 eosinophils per microliter. Periph- eral eosinophilia (serum eosinophilia) was graded as mild (500-1500 eosinophils per microliter), moderate (1500-5000 eosinophils per microliter), and severe (more than 5000 eo- sinophils per microliter) [14]. Statistical Analysis The data were analyzed using the Statistical Package for Social Science (SPSS) version 24 for Windows. Descriptive statistics (frequencies, percentages, means, and standard de- viations) were used for each item to describe the sample char- acteristics. Independent t-test, one-way ANOVA, chi-square, and Spearman-Pearson correlation test were performed. A multivariate linear regression test was also used to investi- gate and eliminate the effect of confounding factors affecting the severity of the disease. P-value < 0.05 was considered as the significance threshold. Results A total number of 108 patients were included in this study. Thirty-five were excluded due to our exclusion criteria. Fi- nally, data from 73 patients were analyzed. Among them, 45 patients had complete available histopathological data; 57.5% of the population was female. The mean age was 72.1 years. None of the patients were pregnant. Hyperten- sion was the most frequent medical history and 46.6% of patients had histories of cardiovascular drug consumption. One patient had BP lesions without blisters and one had localized BP. Limbs and trunk were the most frequent sites of involvement. The mean hospitalization duration was 11  days. Topical corticosteroids, systemic corticosteroids, and topical burrows were the most common treatment pro- tocols among the patients. The most common histopatholog- ical findings were subepidermal blisters and positive C3 in a linear pattern in the slide samples. The most common grade of tissue eosinophilia was grade 1. Analysis of patient charac- teristics between males and females is summarized in Table 1. Our results showed that patients with mucosal in- volvements had significantly lower serum eosinophil count (P = 0.02). Our study also showed that patients with higher grades of tissue eosinophilia had significantly higher grades of serum eosinophil count (P < 0.01) and also higher serum eosinophil percentage (P < 0.0001) (Table 2). There was a significant correlation between BPDAI se- verity score and tissue eosinophilia grade (P = 0.03; r = 0.33). Although, there was no significant correlation between BPDAI severity score and peripheral eosinophilia grade (P = 0.52; r = 0.76). There was also a significant relationship between dermal inflammation and serum eosinophil count (r = 0.95; P < 0.0001) and serum eosinophil percentage (r = 0.46; P = 0.003) (Table 3). Investigating the relationship between disease severity and other clinical and histopathological factors, only a signif- icant relationship was observed between the duration of the disease and the severity of the disease, but the designed re- gression model was not statistically significant (Co- efficient of determination [R2] = 0.06, F =1.53, degrees of freedom [df] = 3, P-value >0.05). In the regression model of patients medications, after the entering of anti-neoplastic drugs and oral corticosteroids into the model, the model was statistically significant and only the dose of corticosteroids was still statistically inversely related to the severity of the underlying disease (R2 = 0.11, F = 4.5, df = 2, P-value = 0.01). In the regression model of mucosal involvement and disease severity, after the inclusion of facial and trunk bullous involvement factors in the model, the model was statistically significant and no statistically significant relationship was observed between the sever- ity of the disease and trunk and facial bullous involvement (R2 = 0.1, F = 3.99, df = 2, P-value = 0.02). In the regression model of peripheral and tissue eosinophilia involvement, the tissue eosinophilia grade (P = 0.001) and severity of tissue inflammation (P = 0.04) had significant relationships with the severity of the disease (Table 4). Conclusions In this study, analysis of clinical and histopathological find- ings revealed a direct and significant relationship between BP severity and tissue eosinophilia, despite peripheral eosinophilia. Furthermore, a significant direct correlation was found between tissue eosinophilia and serum absolute eosinophil count and percentage. Eosinophils may play a significant role in the pathogene- sis of BP. Some serial histological observations in the studies support this hypothesis, such as eosinophilic dermal infiltra- tion, the presence of degranulated eosinophils, extracellu- lar eosinophilic granules, and free granular proteins in the lesions. Therefore, eosinophils infiltration may initiate and later the degranulation process may progress the formation of bullous lesions in BP [6]. There are some studies evaluating the association be- tween BP severity and related clinical and histopathological factors [15,16]. A recent prospective study was performed on 27 patients with newly diagnosed BP. This study reported that there is a correlation between dermal eosinophilia and tissue inflammation severity. Also, a significant direct 4 Original Article | Dermatol Pract Concept. 2024;14(3):e2024169 Table 1. Characteristics of the Study Population. Variable Total (N = 73) Male (N = 31) Female (N = 42) P-Value Age (years) Mean (SD) 72.1 (15.17) 70.81 (13.31) 73.10 (16.52) 0.53 Median (min-max) 76 (26-97) 72 (43-91) 76 (26-97) Past medical history N (%) Hypertension 33 (45.2) 10 (30.3) 23 (69.7) 0.05 Endocrine disease 28 (38.4) 10 (35.7) 18 (64.3) 0.33 Neurologic disease 16 (21.9) 6 (37.5) 10 (62.5) 0.57 Hyperlipidemia 7 (9.6) 0 (0.0) 7 (100) 0.01 Malignancy 3 (4.1) 2 (66.7) 1 (33.3) 0.77 Others 24 (32.9) 11 (45.8) 13 (54.2) 1 Past drug history N (%) Cardiovascular 34 (46.6) 14 (41.2) 20 (58.8) 0.81 Diabetes 12 (16.4) 4 (33.3) 8 (66.7) 0.53 Neurologic 11 (15.1) 6 (54.5) 5 (45.5) 0.51 Others 10 (13.7) 1 (10.0) 9 (90.0) 0.03 Site of involvement N (%) Limbs 72 (98.6) 32 (44.4) 40 (55.6) 1 Trunk 66 (91.7) 29 (43.9) 37 (56.1) 0.69 Mucus membrane 36 (49.3) 16 (44.4) 20 (55.6) 0.91 Acral 23 (31.5) 11 (47.8) 12 (52.2) 0.8 Head and neck 19 (26) 7 (36.8) 12 (63.2) 0.59 Hospitalization duration (days) Mean (SD) 11 (7.48) 10.16 (6.50) 11.69 (8.20) 0.39 Median (min-max) 9 (1-44) 9 (1-30) 10 (4-44) Treatment protocol N (%) Topical CS 69 (94.5) 30 (43.5) 39 (56.5) 1 Systemic CS 67 (93.1) 29 (43.3) 38 (56.7) 1 Topical Burrow 66 (90.4) 29 (43.9) 37 (59.1) 1 Topical antibiotic 61 (83.6) 29 (47.5) 32 (52.5) 0.2 Antihistamine 58 (79.5) 27 (46.6) 31 (53.4) 0.39 Systemic antibiotics 43 (58.9) 19 (44.2) 24 (55.8) 0.94 Antineoplastic 35 (47.9) 16 (45.7) 19 (54.3) 0.81 Histopathologic Finding N (%) Subepidermal blisters 35 (79.5) 16 (45.7) 19 (54.3) 0.71 C3 27 (79.5) 10 (37.0) 17 (63.0) 0.70 IgG 33 (75.0) 12 (36.4) 21 (63.6) 0.56 C3+IgG 24 (55.8) 9 (37.5) 15 (62.5) 0.55 Tissue eosinophilia N (%) Grade 1 23 (45.8) 8 (34.8) 15 (65.2) 0.45 Grade 2 13 (31.0) 5 (38.5) 8 (61.5) Grade 3 5 (11.9) 3 (60.0) 2 (40.0) Grade 4 1 (2.4) 1 (100) 0 (0.0) CS = corticosteroid; SD = standard deviation. relationship between the disease severity and serum eosino- phil is reported [10]. Another case-control study on 225 patients with BP in- vestigated the association between peripheral eosinophilia and BP clinical manifestations. It reported that patients with BP with serum eosinophilia were significantly older and had higher palmoplantar involvement. Patients with BP with a normal eosinophil count were younger and presented more Original Article | Dermatol Pract Concept. 2024;14(3):e2024169 5 An animal model has suggested that eosinophils can me- diate tissue injury in patients with BP by providing a relation between immunoglobulin-E auto-antibodies and skin blister- ing. As a result, higher tissue inflammations could be associ- ated with higher BP severity [18]. An experimental study also demonstrated the overex- pression of Thelper-2 cytokines like interleukin-31 on the injured skin of BP patients [19]. frequently with atypical clinical manifestations [6]. Al- though, we observed no significant relationship between age and eosinophilia. In 1983, Bushkell et al, reported a peripheral serum eo- sinophilia in patients with BP. They declared that peripheral blood eosinophilia was a common finding in their patients and BP should be included in the differential diagnosis of cutaneous bullous lesions with eosinophilia [17]. Table 2. Serum Eosinophil Count, Percentage, and BPDAI Severity Score Based on Different Variables. Variable Serum eosinophil count P-Value Serum Eosinophil Percentage P-value BPDAI Severity Score P-value Sex Male 1113.5±974.86 0.44 30.6±25.1 0.15 42.3±29.68 0.62 Female 1412.1±2426.15 21.3±17.15 40.6±30.44 Site of involvement Mucous yes 947.8±1011.97 0.02 27.7±20.66 0.52 39.3±30.29 0.42 no 1560.7±2278.69 23.5±21.86 43.4±29.83 Past medical history Autoimmune disease yes 899.7±499.36 0.6 19.7±16.58 0.16 45.8±27.9 0.51 no 1487.1±2235.73 28.8±23.27 38.5±31.11 Hypertension Yes 1113.5±974.86 0.31 20.3±17.4 0.17 44.2±27.78 0.31 No 1412.1±2426.15 29.1±23.32 38.9±31.76 Hyperlipidemia Yes 1459.1±540.23 0.42 25.4±21.96 0.99 43.8±31.95 0.81 No 1220.7±1849.54 25.3±21.4 41.1±29.94 Malignancy Yes 1303.3±1268.13 0.76 22.5±22.9 0.63 40.7±32.38 0.58 No 1228.8±1885.05 26.1±20.97 41.5±29.48 Neurologic disease Yes 1303.3±1268.13 0.61 17.4±20.21 0.50 58.3±16.07 0.31 No 1228.8±1885.05 25.9±21.4 40.6±30.23 Histopathologic findings Subepidermal blister Yes 1364 ±2177.73 0.75*** 25.2±19.26 0.92* 49.6±30.36 0.24 No 770±309.39 25.9±29.00 36.7±30.86 C3 Yes 1428.3±2604.77 0.19*** 24.5±19.6 0.19* 46.0±33.23 0.56*** No 1006.6±515.21 35.5±28.51 58.7±26.44 IgG Yes 1331.9±2326.14 0.66*** 25.2±21.88 0.31* 47.6±32.25 0.96*** No 1014.8±474.4 33.8±23.28 49.8±30.25 C3+IgG Yes 1448.4±2795.72 0.32*** 23.1±20.20 0.47* 41.9±33.09 0.19*** No 980.3±494.69 31.0±24.12 59.4±25.10 Peripheral eosinophilia Yes 1445.7±1880.36 <0.0001*** 25.0±21.12 0.8* 42.9±30.53 0.48*** No 309.7±111.02 27.4±23.72 33.5±26.37 Tissue eosinophilia grade 1 714.3 ±364.30 <0.01**** 10.5±6.01 <0.0001** 37.0±26.98 0.08**** 2 1176.5±415.24 34.6±7.61 61.4±30.4 3 1092.6±533.4 63.7±3.79 62.8±34.19 4 13200 83.7 - Dermal inflammation Mild 911.9 ±551.13 0.95**** 13.0±17.02 0.14** 50.5±31.42 0.86**** Moderate 964.5±469.64 25.0±20.38 43.1±23.38 Severe 1849.1±3295.53 31.8±22.41 47.8±29.36 BPDAI = Bullous Pemphigoid Disease Area Index. 6 Original Article | Dermatol Pract Concept. 2024;14(3):e2024169 and clinical findings severity in patients with BP. The limitations of this study are a restricted study popula- tion, unknown potential confounders, and also, we had inferior  level of evidence compared to prospective stud- ies. Therefore, more studies with larger populations are suggested. In conclusion, our study showed a significant correla- tion between histopathological findings in BP (such as tis- sue eosinophilia) and the disease severity. In conclusion, the possible role of eosinophils in BP clinical features should be considered as a promising help for better diagnosis and treatment. Although, Wakugawa et al, suggested that eotaxin and inter- leukin‐5 contribute to the blister formation but no correlations were reported between tissue eosinophilia and BP severity [20]. There are also a number of reports about BP and hyper-eosinophilia. A recent report in 2023, was a 16-year-old girl with generalized aggressive bullous lesions diagnosed as BP which was unresponsive to low dose prednisolone. More investigations revealed BP associated with hyper-eosinophilic syndrome. Finally, the patient was treated with high dose corti- costeroid [21]. Our study supports the possible association between histopathological markers like tissue hyper-eosinophilia Table 3. Relationships Between Quantitative Variables Related to BPDAI Severity. Variable Hospitalization Duration Serum eosinophil count Serum Eosinophil Percentage BPDAI Severity Score Age r −0.11 −0.09 −0.289 0.008 P 0.34 0.45 0.06 0.945 Hospitalization duration r 1 −0.03 0.003 −0.21 P - 0.77 0.984 0.08 Peripheral eosinophilia grade r −0.09 0.15 0.15 0.76 P 0.42 0.30 0.30 0.52 Tissue eosinophilia grade r −0.04 0.49 0.89 0.33 P 0.80 0.002 <0.000 0.03 Dermal inflammation r −0.01 0.95 0.46 0.22 P 0.95 <0.0001 0.003 0.06 BPDAI = Bullous Pemphigoid Disease Area Index. Table 4. Regression Model Analyses. Model Standardized beta Coefficients t P-Value 95% Confidence Interval for Beta Lower Bound Upper Bound 1 Constant 3.04 0.003 19.87 95.88 Age -0.02 -0.22 >0.05 -0.52 0.42 Sex (female/male) -0.02 -0.16 >0.05 -15.73 13.28 Hospitalization duration -0.25 -2.11 0.03 -2.00 -0.05 2 Constant 6.86 0.0001 45.51 82.83 Oral prednisolone -0.26 -2.33 0.02 -29.38 -2.33 3 Constant 5.45 0.0001 40.71 87.62 Facial bullous lesion (yes/no) -0.22 -1.93 0.05 -31.39 0.457 Trunk bullous lesion (yes/no) -0.19 -1.68 >0.05 -45.88 3.934 4 Constant 1.45 >0.05 -9.72 57.37 Tissue eosinophilia grade 0.61 3.65 0.001 16.25 57.40 Peripheral eosinophilia grade 0.21 1.45 >0.05 -4.67 28.06 Tissue inflammation grade -0.35 -2.08 0.04 -22.52 -0.24 Presence of subepidermal blister (yes/no) 0.12 0.88 >0.05 -11.56 29.11 C3 (positive/negative) 0.05 0.35 >0.05 -13.07 18.54 IgG (positive/negative) 0.31 1.86 >0.05 -1.73 38.04 Original Article | Dermatol Pract Concept. 2024;14(3):e2024169 7 11. 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