This study was conducted in the Internal Medicine Department, Kasr El-Aini Hospital. A written informed consent was obtained from each participant or a responsible family member after explaining the possible complications of the diagnostic procedures. This study was submitted to and approved by the ethics committee of Kasr Alainy Hospital, Faculty of Medicine, Cairo University, with approval reference number [I-010316].
This observational case-control study included 90 subjects, 18 subjects were normal healthy individuals and 72 patients with liver disease (fibrosis/cirrhosis) due to the different etiologies such as post-hepatitis C infection, post-hepatitis B infection, NASH, and autoimmune hepatitis.
Patients were subjected to full clinical history and examination. They were classified into 4 groups: control group—composed of 18 normal healthy persons; group І—24 patients with chronic liver disease, Child A; group II—24 patients with chronic liver disease, Child B; and group III—24 patients with chronic liver disease, Child C.
Venous blood samples were taken for the analysis of the serum level of Nogo-B (enzyme-linked immunosorbent assay, ELISA, Human Nogo-B ELISA Kit, supplied by Chongqing Biospes Co., Ltd. (7F, Bldg B, High-tech Venture Park, Jiulongpo District, Chongqing, China), according to the manufacturer’s instructions), anti-bilharzial antibodies in the serum (Schistosoma mansoni IgG Human ELISA Kit, GenWay Biotech, Inc. 6777 Nancy Ridge Drive, San Diego, CA 9212, intended for the qualitative determination of IgG class antibodies against Schistosoma mansoni in human serum), HCV antibodies (ELISA, Murex anti-HCV (version 4.0) is an enzyme immunoassay for the detection of antibodies to hepatitis C virus (HCV) in human serum, Murex Biotech S.A. (Pty) Ltd., Kyalami Boulevard, Kyalami Business Park, Kyalami, Republic of South Africa), and serum ferritin (Human Ferritin Enzyme Immunoassay Test Kit, supplied by BIOCHECK, INC.(323 Vintage Park Dr, Foster City, CA 94404, USA, according to the manufacturer’s instructions).
Liver function tests and liver enzymes were also done: serum albumin, prothrombin time, concentration, INR, serum bilirubin, ALT, and AST, and renal function tests: serum urea, creatinine, and complete blood count.
Abdominal ultrasound and upper gastrointestinal endoscopy were done to all patients. Patients with other etiologies of liver affection rather than hepatitis C (HBV, AIH, NASH) were included in the study according to their past documented investigations.
The exclusion criteria included the following: patients have diseases which might affect plasma Nogo-B levels as cardiovascular disease (including hypertension), central nervous system disorders, chronic obstructive pulmonary diseases (COPD), and pulmonary artery hypertension.
Samples were withdrawn, left to clot at room temperature for 10 min, and then centrifuged at the speed of 2000–3000 rpm for 5 min, and the supernatant was collected and stored at − 20 °C till the time of analysis.
MELD score was calculated as 3.78[Ln serum bilirubin (mg/dL)] + 11.2[Ln INR] + 9.57[Ln serum creatinine (mg/dL)] + 6.43. MELD-Na = MELD + 1.59 [135 − Na]; APRI score: [(AST/upper limit of normal)/platelet count (109/L)] × 100; and FIB -4 index: (age [years] × AST [IU/L])/(platelet count [109/L] × (ALT [IU/L]) 1/2).
Statistical methods
Data were coded and entered using the statistical package SPSS (Statistical Package for the Social Sciences) version 24. Data was summarized using mean, standard deviation, median, minimum and maximum in quantitative data, and frequency (count) and relative frequency (percentage) for categorical data. Comparisons between quantitative variables were done using the non-parametric Kruskal-Wallis and Mann-Whitney tests [13]. For comparing categorical data, the chi-square (χ2) test was performed. The exact test was used instead when the expected frequency is less than 5. Correlations between quantitative variables were done using the Spearman correlation coefficient. ROC curve was constructed with the area under curve analysis performed to detect the best cutoff value of Nogo-B, MELD, APRI, and FIB-4 for the detection of patients [14].