Evaluation of the Role of High-Mobility Group Box 1 Protein in Patients With Keloids

A Case Control Study

Omar Ahmad Azzam, MD; Marwa Salah El-Mesidy, MD; Moataz Maher Kamel, MD; Amira Basyouny Nouh, MBBch

Disclosures

Wounds. 2019;31(7):179-183. 

In This Article

Materials and Methods

Case Group

Forty patients of both sexes with keloids of any size were recruited from the outpatient clinic of dermatology at Kasr Al Ainy Hospital (Cairo, Egypt). Patient skin type, according to the Fitzpatrick scale,[10] ranged from 2 to 4; 1 patient had albinism. A written informed consent form was obtained from each patient prior to study inclusion.

Patients with the following criteria were excluded: pregnancy, immunosuppression, long-term systemic corticosteroid therapy, diabetes, renal fibrosis, and history of the use of any treatment for keloids in the 4 weeks prior to the initiation of the study.

A full patient evaluation, including medical history, clinical scar history (duration and etiology), and previous therapy, was completed. Scar grade was determined as per the modified Vancouver scar scale.[11]

Control Group

Forty controls of both sexes with a skin type[10] ranging from 2 to 4 were recruited. Exclusion criteria consisted of no keloid scarring and no concurrent therapy trial. Before study inclusion, written informed consent was obtained from each patient. A full patient evaluation, including medical history, was recorded.

Methodology

A 3-mL sample of whole blood was obtained from 40 patients with keloids and 40 controls. The samples were immediately centrifuged; the resultant supernatant serum was kept frozen at -20°C for detection and quantification of HMGB1 using enzyme-linked immunosorbent assay (ELISA).

The Sandwich ELISA method was employed using ELISA kit (SinoGeneClon Biotech Co, Ltd, Hang Zhou, Zhejiang, China). The micro-ELISA plate provided in this kit was precoated with an antibody specific to HMGB1. Samples were added to the appropriate micro-ELISA plate wells and combined with the specific antibody. Then, a biotinylated detection antibody specific for HMGB1 and avidin-horseradish peroxidase (A-HRP) conjugate was successively added to each microplate well and incubated. Free components were washed away. The substrate solution was added to each well. Only those wells that contain HMGB1, biotinylated detection antibody, and A-HRP conjugate appeared blue in color. The enzyme-substrate reaction was terminated by the addition of a sulfuric acid solution and the color turned yellow. The optical density (OD) was spectrophotometrically measured at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of HMGB1. The concentration of HMGB1 was calculated in the samples by comparing the OD of the samples with the standard curve.

The stability of HMGB1 during ELISA practical work was ensured with the following procedures: incubation in dark at 37°C, wrapping the ELISA plate in aluminum foil, following all ELISA manual instructions, and duplication of each sample to avoid data disparity.

Ethics

Approval for the study was obtained from the Dermatology Research Ethical Committee Post conduction (Kasr Al Ainy Hospital).

Statistical Analysis

Results are expressed as mean ± standard deviation or as number (percentage). Comparison between categorical data was performed using chi-square test. According to normality test, comparison between values of different parameters in the 2 patient groups was performed using either unpaired t test or Mann-Whitney U test whenever appropriate. Receiver operating curve (ROC) was used to determine the diagnostic indices of HMGB1. Correlation between HMGB1 and both scar duration and scar severity in the case group was performed using Spearman's rank correlation coefficient test. Binary logistic regression test was used to study the predictive effect of HMGB1. For data analysis, SPSS for Windows, Version 16 (SPSS Inc, Chicago, IL), was used. A P value ≤ .05 was considered statistically significant.

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