Diphenylcyclopropenone and platelet‐rich plasma in the management of severe or recalcitrant alopecia areata

Alopecia areata (AA) is a common disease characterized by hair loss with an autoimmune background. There are many lines of therapy, but no standard line for all cases. Consequently, treating severe forms of AA is challenging.

AA is not easily treated; unfortunately, no universally accepted treatment exists for all cases.However, British and Japanese treatment guidelines for AA have suggested topical immunotherapy (dinitrochlorobenzene (DNCB), diphenylcyclopropenone (DPCP), and squaric acid dibutylester (SADBE)) as one of the most effective options. 7pical immunotherapy involves inducing allergic contact dermatitis by applying potent contact allergens to the affected skin.
Contact sensitizers are believed to act through the skin's and its appendages' immunomodulation at several different points. 8atelet-rich plasma (PRP) therapy is a relatively new approach to tissue regeneration that promotes healing during many procedures.
The platelets actively secrete growth factors (GFs) within 10 minutes after activation.PRP contains more than 20 types of GFs, including platelet-derived growth factors (PDGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF-B1). 9tiapoptotic effects of activated PRP have been suggested to significantly contribute to stimulating hair growth.PRP-induced activation of antiapoptotic regulators prolongs the survival of dermal papilla cells during the hair cycle.In addition, it appears to increase the perifollicular vascular plexus.It occurs through increased VEGF and PDGF levels that have angiogenic potential.It is postulated that platelet GFs may act on stem cells in the bulge area of follicles.They bind to their respective receptors located in stem cells, stimulating the development of new follicles and promoting new vascularization. 10

| PATIENTS AND ME THODS
A randomized clinical trial was conducted on patients with severe and recalcitrant AA who attend the dermatology outpatient clinics at Sohag University hospitals.Informed written consent was obtained from all patients.Moreover, the study was approved by the Ethical and Research committees at Sohag Faculty of Medicine, Sohag University, Egypt.Registration number: Soh-Med-22-07-30.

| Inclusion criteria
The study included both sexes, aged more than 10 years, and patients who had severe AA (more than 40% of the scalp affected by AA) and refractory AA(patients who had not responded to standard therapies for at least one year). 11

| Exclusion criteria
Patients with one or more of the following criteria were excluded: pregnancy and breastfeeding, active infection or inflammation at the site of AA, anemia (hemoglobin level < 10 mg/dL), or thrombocytopenia (platelet count <100 000/ml).Moreover, patients with coagulopathies, patients on anticoagulant therapy, patients with iron deficiency, or patients who received previous treatment for AA over the last three months were excluded.

| Study design
All patients were subjected to the following:

| History recording
It included personal history (age, sex, residence, occupation, marital status, and unique habits of medical importance), disease history (onset, course, and duration), medical history (atopy, psoriasis, vitiligo, thyroid disorders, anemia, diabetes mellitus (DM), and autoimmune connective tissue diseases), therapeutic history (previous treatment modalities and response to them), family history, and history of similar conditions.

| General examination
Pallor, pulse, blood pressure, and temperature were examined.

| Local examination
The examination included hairy areas (scalp and other hairy areas of the body), the number and size of alopecic patches, and the presence of an exclamation mark.Grading of severity was performed using the Severity of Alopecia Tool (SALT) score, proposed in the guidelines of the National Alopecia Areata Foundation. 12It measures the disease's severity and response to treatment.It is calculated as follows: The scalp is divided into the following four areas: 1. Vertex: 40% (0.4) of scalp surface area.
The SALT score is the sum of the percentages of hair loss in all the mentioned areas.

| Trichoscopy
To detect trichoscopy findings and in the follow-up of patients. 13on consent, simple randomization with a coin was used to assign the participants into two groups (A and B).Patients in the first group (A) were treated with DPCP alone, while patients in the second group (B) were treated with a combination of DPCP and PRP.
Our study procedure was as follows: DPCP powder (Fluka, Sigma-Aldrich Corp, St. Louis, MO, USA) was diluted in acetone at concentrations of 0.001%, 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, and 2.0%.The application of DPCP involved two phases: the sensitization phase and the elicitation phase for both groups.The scalp was the usual sensitization site.A cotton-tipped applicator saturated with 2% DPCP in acetone was applied to an area of at least 4 4 × cm 2 .
Patients were advised to avoid washing the area and protect it from sunlight for 48 h.After 72 h, patients were evaluated for an eczematous reaction to detect whether sensitization to DPCP had occurred.
Two weeks after sensitization, the scalp was mapped for applying DPCP.Then six consecutive concentrations of DPCP (0.0001%, 0.001%, 0.01%, 0.05%, 0.1%, and 0.5%) were applied to the scalp over 3 × 3 cm 2 areas separated by a distance of 4 cm.DPCP was left on the scalp for 24 h and then washed off with gentle shampoo. 14ter one week, the concentration that created a mild eczematous reaction was chosen to be the first applied.The starting concentration was applied to only half of the affected scalp area, left on for 24 h, and then washed off.The patients were exposed to DPCP every week for 24 weeks.
The concentration of DPCP was adjusted every week to maintain a tolerable eczematous reaction.An eczematous response (which may occur after five days or more) indicates sensitization.The patients who failed to sensitize by 2% DPCP were excluded.Each patient was exposed to a concentration at which mild contact eczema developed every week for 24 weeks and was observed for an additional three months.
The combination of PRP and DPCP was applied to patients in the second group (B).The DPCP was applied as described above; the starting concentration was applied to only half of the affected scalp area, left on for 24 h, and then washed off.The patients were exposed to DPCP every week for 24 weeks.PRP injection was prepared by ordinary Prothrombin Time (PT) tubes as follows: 10 mL of blood was drawn from each patient, placed in PT tubes, and then centrifuged at 3000 rpm (revolutions per minute) for 10 min.
Three layers are formed because of their density.The bottom layer consists of red blood cells (RBCs), the middle layer consists of platelets and white blood cells (WBCs) (buffy coat or rich platelet plasma), and the top layer is platelet-poor plasma (PPP).Both PRP and PPP were drawn with an insulin syringe and injected intradermally at a 1 cm distance between injections in the affected scalp area every month.

| Laboratory investigations
A complete blood count (CBC) analysis was performed.

| Therapeutic response evaluation
All patients were evaluated before, during, and after treatment as follows: 1. Clinical assessment: The response was evaluated clinically by using the following: 1a.SALT score.

Photographing:
Photographing was performed before treatment and monthly after that.

Safety evaluation:
Safety and tolerability were evaluated throughout the study by assessing adverse events reported by the patient or the physician.

| Statistical analysis
Data were analyzed using the Statistical Package for the Social Sciences (SPSS) software program (version 16).Qualitative variables were recorded as frequencies and percentages and compared by the chi-squared test.In addition, quantitative variables were presented as means ± standard deviation (SD) and compared using a student t-test.Regression analysis and correlations between different variables were performed when indicated.A p-value of <0.05 was considered significant.

| RE SULTS
The study included 30 patients with severe or refractory AA; however, six did not complete the study.The remaining 24 patients were randomly divided into two study groups.Group A consisted of 13 patients treated with DPCP alone (eight males and five females), and group B comprised 11 patients treated with DPCP and PRP (seven males and four females).They completed the study in six months with.Follow-up was continued for another three months.

| Personal data and socio-demographic characteristics
The primary personal data and socio-demographic characteristics among the studied populations are shown in Table 1.

| Clinical characteristics
Clinical characteristics in the studied population are shown in Table 2.

| The number and percentages of improved patients according to the SALT score
Group A: Seven patients (53.85%) improved, and six (46.15%) did not.Group B: Six patients (54.5%) improved, and five (45.5%) did not.
As regards the comparison between the two groups, there is no significant difference between the two groups.

| The improvement according to the regrowth scale between the two study groups
In group A, seven patients showed improvement with a total response rate of (53.85%); five (38.46%) were grade 4 (excellent TA B L E 1 Comparisons of personal data and socio-demographic characteristics between the two study groups.response); one (7.69%)was grade 3 (satisfactory response); and one (7.69%)was grade 2 (fair response).

| The improvement according to the McDonald Hull and Norris regrowth score between the two study groups
In group A, five patients (38.46%) were grade 4 (regrowth of terminal hair on the scalp); two (15.3%) were grade 3 (regrowth of terminal hair with patches of alopecia); one (7.69%)was grade 1 (regrowth of vellus hair); and five (38.46%) were grade 0 (no vellus hair).

| Satisfaction score
The results of patient satisfaction are shown in (Table 5).
Photos of the patients, both clinically and by trichoscopy, are presented in (Figures 1-8).

| DISCUSS ION
Hair loss is a worldwide disease caused by many reasons.After AGA as the most common cause of hair loss, AA is the second most common cause.Many treatments can be used for AA, including topical immunotherapy, topical and intralesional corticosteroids, PUVA therapy, and systemic immunosuppressants.Moreover, systemic corticosteroids like MTX, cyclosporine A, and azathioprine may be utilized.In addition, biological agents such as IL-12/IL-23p40 blockers and JAK inhibitors can be used. 17Regarding group A (13 patients), the response rate was 53.85%; an excellent response was found in 38.46% of patients.
9][20][21][22][23][24][25][26] A study was done by Sotiriadis et al. in 2007 in which significant hair regrowth was observed in 15 of the 38 patients (39.5%) at 6 months. 24Another retrospective study was done in the USA on 50 patients with AA.The duration of DPCP treatment ranged from 6 months to 15 years, with a median of 3 years; a total response rate of 78% was achieved. 26ese differences in response rates may be attributed to many factors.The main factors include variations in the methodology, TA B L E 3 Comparison of improvement according to the regrowth scale between the two study groups.

TA B L E 4 Comparison of improvement according to McDonald
Hull and Norris score between the two study groups.including inclusion/exclusion criteria, the definition of clinical response, treatment duration, and immunotherapy protocol.
Regarding group B (11 patients), the response rate was 54.5%; an excellent response was found in 27.36% of patients.There is no published data on the use of DPCP and PRP in the same patient.Activated platelets in PRP release numerous growth factors and cytokines from their alpha granules, including platelet-derived endothelial growth factor (PDGF), transforming growth factor β (TGFβ), fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and glial cell line-derived neurotrophic factor (GDNF), which play a significant role in stimulating hair growth through cell proliferation, differentiation, and angiogenesis. 9Therefore, adding PRP may increase the chance of hair growth in the recalcitrant cases in our study.
After reviewing the literature, many studies evaluated PRP alone in the treatment of AA. [27][28][29][30] A double-blinded, placebo-controlled study evaluated the efficacy of PRP in treatment of AA.In this study 60% of patients achieved complete remission.These results were in agreement with our results. 27Another study was done to compare the efficacy of PRP with minoxidil, A significant regrowing hair was seen in patchy alopecia in 70% of patients and in 30% of cases of alopecia universalis. 30ese results were higher than the current study in cases of patchy AA.
And this was explained by that the cases of patchy AA have better prognosis than our cases that were complaining of severe and recalcitrant AA.Also, the current study revealed higher response rate than the previous study in cases of alopecia totalis and universalis, and this may be due to the use of combination therapy of DPCP and PRP in our study.
Durdu et al., in a retrospective case series in 2015, reported that using DPCP in combination with anthralin is effective in treating AA.
Their results have shown that 88% (higher than our results) of the patients who received both DPCP and anthralin achieved 50% or more significant terminal hair growth. 15other prospective study in 2019 reported that using DPCP combined with anthralin effectively treats AA.Their results have shown that 63.7% (higher than our results) of the patients who received both DPCP and anthralin achieved 50% or more remarkable terminal hair growth. 31   who received DPCP alone and 15.79% in patients who received a combination of DPCP and anthralin. 33 regards the comparison between the two groups in our study, in group A, the regrowth scale results were 53.85%, while in group B, the regrowth scale results were 54.5%.Although the response rate of group B is higher than that of group A, there is no statistically significant difference between the two groups.In our study, according to the safety and tolerability of both treatment modalities, there were no serious complications apart from severe vesiculation and bullae in a few patients, as exposure to DPCP pushed them to leave the study.Erythema and itching were the most frequent, while bullae, vesiculation, and lymphadenopathy were less frequent.Postinflammatory hyperpigmentation was the least frequent.
Because of the small sample size, this study had some limitations.
Therefore, our recommendations in future studies are to increase the sample size and the duration of the treatment.

ACK N OWLED G M ENTS
The authors thank their patients for their participation in the study also we extend our thanks to our colleagues in the Department of Dermatology.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declared that there wasn't any conflict of interest to be declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S S TATEM ENT
Approval to perform this work was obtained from the Institutional
Our randomized clinical trial included 24 patients who completed the study.The patients were divided into two groups; group A included 13 patients (DPCP alone), and group B included 11 patients (DPCP and PRP).The patients in both groups completed the study in six months.Our study used the regrowth scale, SALT score, and McDonald-Hull and Norris scores to assess the response rate.
Nasimi et al., in a retrospective case series in 2019, reported that using DPCP in combination with anthralin in F I G U R E 1 Patient with alopecia areata treated with DPCP alone with excellent response (A-C) according to the Regrowth scale.(D-F) According to McDonald-Hull and Noris Regrowth Scale.TA B L E 5 Comparison of satisfaction scores between the studied groups.

Note:F I G U R E 4
Group A: Alopecia areata patients that received DPCP alone.Group B: Alopecia areata patients that received DPCP + PRP.Data are presented as frequencies and percentages.p-value <0.05 is statistically significant.The satisfaction of each patient was conducted by asking the patient to rate his overall satisfaction: 1 = very unsatisfied, 2 = unsatisfied, 3 = neutral, 4 = satisfied, and 5 = very satisfied.treatingDPCP nonresponder AA patients is effective.Their results have shown that 27.27% of the patients who received both DPCP and anthralin achieved 50% or more significant terminal hair growth.32Our results were better than those of a recent study in which they used a combination therapy of DPCP and anthralin to treat moderate alopecia areata.The response rate was 18.75% in patients F I G U R E 2 Patient with alopecia areata treated with DPCP alone with grade 3 response (A-C) according to the Regrowth scale.(D-F) According to McDonald-Hull and Noris Regrowth Scale.F I G U R E 3 Patient with alopecia areata treated with DPCP alone with grade 2 response (A-C) according to the Regrowth scale.(D-F) According to McDonald-Hull and Noris Regrowth Scale.Patient with alopecia areata treated with DPCP alone with grade 0 response (A-C) according to the Regrowth scale.(D-F) According to McDonald-Hull and Noris Regrowth Scale.F I G U R E 5 Patient with alopecia areata treated with DPCP and PRP with grade 4 response (A-C) according to the Regrowth scale.(D-F) according to McDonald-Hull and Noris Regrowth Scale.

F I G U R E 6
Patient with alopecia areata treated with DPCP and PRP with grade 3 response (A-C) according to the Regrowth scale.(D-F) According to McDonald-Hull and Noris Regrowth Scale.F I G U R E 7 Patient with alopecia areata treated with DPCP and PRP with grade 2 response (A-C) according to the Regrowth scale.(D-F) According to McDonald-Hull and Noris Regrowth Scale.Regarding the nonresponder patients to treatment in our study, type and extent of alopecia, duration of the disease, age at DPCP onset, family history of AA, and presence of other medical diseases such as atopic dermatitis, DM, and thyroid disorders are the prognostic factors that affect DPCP treatment outcome.Wasserman et al. reported that the prognosis and treatment responses are poor in patients with severe AA, a positive family history of AA, a history of atopic dermatitis or other autoimmune diseases, and a more extended period from symptom development to the start of treatment.34

Finally, we can
conclude from our clinical trial that DPCP alone or combined with PRP is an effective and safe method for treating severe or recalcitrant AA.AUTH O R CO NTR I B UTI O N SWafaa Mohamed Abd El-Magid conceived the study, carried out its designing, coordinated the implementation, helped to perform the statistical analysis and drafted the manuscript.Reham Ezz-Eldawla Elsharkawy participated in the analysis and interpretation of data and revised the statistics and final draft of the manuscript.Raghda Alaa-Eldeen Mohamed collected the data.All authors read and approved the final manuscript.

ResearchF I G U R E 8
Ethical Committee.Prior to initiation of the study, every participant was informed about the study and a written consent was obtained.O RCI D Wafaa Mohamed Abd El-Magid https://orcid.org/0000-0002-3328-1967 Patient with alopecia areata treated with DPCP and PRP with grade 0 response (A-C) according to the Regrowth scale.(D-F) According to McDonald-Hull and Noris Regrowth Scale.

related data Group A (n = 13) Group B (n = 11) p-value
Note: Group A: Alopecia areata patients that received DPCP alone.Group B: Alopecia areata patients that received DPCP + PRP.Data are presented as frequencies and percentages.p-value<0.05 is statistically significant.TA B L E 2 Comparison of clinical characteristics between the two study groups.Disease-Note: Group A: Alopecia areata patients that received DPCP alone.Group B: Alopecia areata patients that received DPCP + PRP.Data are presented as frequencies and percentages.p-value <0.05 is statistically significant.