Serological Auto Antibodies in Health and Liver Disease in a Nigerian Population- A Preliminary Study

Research Article

J Hepat Res. 2014;1(1): 1002.

Serological Auto Antibodies in Health and Liver Disease in a Nigerian Population- A Preliminary Study

Otegbayo JA1* and Arinola OG1

Department of Medicine and Chemical Pathology/Immunology, University of Ibadan, Nigeria

*Corresponding author: Otegbayo J.A, Department of Medicine and Chemical Pathology/Immunology, University of Ibadan, Nigeria

Received: July 03, 2014; Accepted: July 29, 2014; Published: July 30, 2014

Abstract

Introduction: Some autoantibodies are useful in the diagnosis of autoimmune liver diseases. There is dearth of information on the prevalence, pattern of autoantibodies in black population of Africans with liver diseases. To the knowledge of the authors, there is no such information among Nigerians. This study determined the prevalence, pattern and significance of serological autoantibodies among patients with liver diseases and apparently healthy individuals in Nigeria.

Materials and Methods: The seroprevalence of antinuclear antibodies (ANA), antimitochondrial antibodies (AMA), anti-liver kidney microsomal antibodies (Anti-LKM-1), anti-soluble liver antigen/liver pancreas (Anti-SLA-LP), perinuclear anti-neutrophil cytoplasmic antibodies (pANCA), were determined in age and sex-matchedpatients with liver diseases and apparently healthy controls using ELISA method over a two-year period. There were one hundred and twenty six patients with liver diseases consisting of 91 (72.2%) males and 35 (27.8%) females) and 82 apparently normal control subjects, made up of 59 (72%) males and 23 (28%) females.Appropriate statistical methods were used to determine Odds ratio, Pearson Chi square and Students't-test. Significant statistical difference was specified at p<0.05.

Results: The patients consisted of hepatocellular carcinoma (HCC) 77 (61.1%), liver cirrhosis 32 (25.4%), chronic hepatitis 10 (7.9%), acute viral hepatitis 4 (3.2%), alcoholic cirrhosis 1 (0.8%) and primary biliary cirrhosis 2 (1.6%).

.

Of all the autoantibodies analysed (126 cases and 82 controls), only AMA was significantly higher among cases compared with controls. Antimitochondrial antibodies were present in 76 (60.3%) of the cases compared with 36 (43.9%) controls (p<0.05), while ANA were present in 42 (39.3%) of cases compared with 27 (39.7%) controls (p=0.68). Anti-soluble liver antigen (anti-SLA/LP) and pANCA were absent among cases and controls.

Though few in number, chronic hepatitis had the highest frequency of AMA, being positive in 9 (90%) of the 10 cases, compared to HCC, in which AMA was present in 48 (62.3%).

Conclusion: The prevalence of serological autoantibodies was similarly high in both liver diseases and in health, except for AMA. Serum autoantibodies, therefore, appear to be insignificant and insufficient for the diagnosis of autoimmune liver disease in Nigerians. Other parameters should be considered whenever there is a clinical suspicion of autoimmune liver disease among Nigerians.

Keywords: Human stem cells; Progenitor; β-cells; Insulin; Differentiation; De-differentiation; Culture; Re-differentiation

Introduction

Autoantibodies are immunoglobulins that react with normal host proteins and may be physiologic or pathologic [1]. The physiologic autoantibodies, also known as polyreactive antibodies, do not fix complements and are produced by normal humans and animals. They are found in low concentrations in the serum of normal humans of all ages, though commoner in women than men [2]. It is suggested that, physiologic or natural autoantibodies may constitute the antibodies secreted by B cells prior to encountering foreign antigens [3]. They are usually low affinity IgM isotype, though IgA and IgG isotypes are also found. CD5+ B cells, which represent 10-25% of circulating B lymphocytes, have been found to produce natural autoantibodies [4]. Pathologic autoantibodies on the other hand are produced by CD5- B cells which are usually monoreactive, have high affinity and are typically detectable only in autoimmune individuals [5,6]. The use of ELISA technique to measure the liverrelated autoantibodies is unique in that hitherto, the cumbersome indirect immunofluorescence technique with use of rat kidneys was employed in most studies [7,8]. The use of ELISA, which has been found to be sensitive, specific, objective and rapid would facilitate standardized approach to measurement of autoantibodies and afford comparability of studies globally.

There are several studies on pathological autoantibodies in the Caucasian populations but there is dearth of scientific literature on the prevalence and pattern of autoantibodies in black populations of Africa with liver diseases, and none from Nigeria. This study determined the prevalence, pattern and significance of autoantibodies among patients with liver diseases in Nigeria.

.

Materials and Methods

A cross sectional study to determine the seroprevalence of liverrelated autoantibodies among age and sex-matched consenting adults with liver diseases who were admitted or seen at the clinic of the hospital and compared to apparently normal controls was carried out at the University College Hospital, Ibadan, Nigeria over a two-year period. The control group was selected randomly and they consisted of volunteering relations of patients, medical, nursing and support staff of the hospital. Ethical approval was sought and obtained from the University of Ibadan/University College Hospital, Institutional Review Board (IRC protocol number: UI/IRC/07/0027).

The patients consisted of hepatocellular carcinoma (HCC) 77 (61.1%), liver cirrhosis 32 (25.4%), chronic hepatitis 10 (7.9%), acute viral hepatitis 4 (3.2%), alcoholic cirrhosis 1 (0.8%) and primary biliary cirrhosis 2 (1.6%), while the mean ages of the cases and the controls were 47.5±14.4yrs and 39.6±16.5yrs respectively.

Sera of the consecutive 126 patients consisting of 91(72.2%) males and 35(27.8%) females with liver diseases and 82 apparently normal controls consisting of 59 (72%) males and 23 (28%) females, (p>0.05), were analysed for antimitochondrial antibodies (AMA), anti-liver kidney microsomal antibodies (Anti-LKM-1), anti-soluble liver antigen/liver pancreas (Anti-SLA-LP), perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) and antinuclear antibodies (ANA were analysed in only 107 liver cases and 67 controls), using qualitative Enzyme-linked Immunosorbent Assay (ELISA) method with kits from AESKU Diagnostics, GmBH, Germany. The AESKU Diagnostics kit consisted of an ELISA plate with 96 microwells, Negative control, Positive control, Cut-off control, Test and Control sera, and Conjugate among others.

Briefly, after incubation of diluted sera (1:101) in microplates coated with specific antigen, patient's antibodies, if present in the specimen, bind to the antigen. Unbound fraction was washed off. Incubated anti-human immunoglobulins conjugated to Horseradish peroxidase (conjugate) reacted with the antigen-antibody complex of the samples in the microplates. Unbound conjugate was washed off. Addition of TMB-substrate generated an enzymatic colorimetric (blue) reaction, which was stopped by diluted acid (colour changes to yellow). The rate of colour formation from the chromogen is a function of the amount of conjugate bound to the antigen-antibody complex and this is proportional to the initial concentration of the respective antibody in the patient's sample. The positive samples were those with reading values greater than the value of the Cut-off control, while the negative samples were samples with reading values lower than the Cut-off control.

Other laboratory tests such as liver function tests, prothrombin time, and alphafetoprotein among others were carried out using standard laboratory methods. Specifically, liver function tests were determined with the clinical chemistry autoanalyser (Hitachi 912), using enzymatic method, while alphafetoprotein was carried out by using radioimmunoassay technique. Prothrombin time (INR) was done manually using commercially prepared thromboplastin.

Diagnosis of liver diseases was made by relevant clinical features and laboratory tests for liver function, prothrombin time, alphafetoprotein, ultrasonography and liver biopsy.

Statistical analysis was carried out with SPSS statistical software version 11.0 for windows. Prevalence rates of autoantibodies and other analytes were calculated to reflect the relative frequency of each liver disease. Odds ratio (OR) and ninety five percent confidence interval (95% CI) were calculated. Pearson Chi square test was used to compare proportions while Students't-test was used to compare means. Where numerical values were low, Fischer's exact test and medians were used. Significant statistical difference was specified at p≤ 0.05.

Results

The mean levels of bilirubin, gamma-glutamyltransferase, alkaline phosphatase, globulin, albumin, PTR and alpha-fetoprotein were high among some of the liver cases. Higher ALT 123 iu/l vs. 12.2 and AST 196.6 iu/l vs. 24.3 were recorded among cases compared with controls (p<0.01) as shown in Table 1. Among the test subjects, hepatomegaly occurred in 99 (78.6%), ascites in 72(57.1%) and jaundice in 62 (49.2%).