INTRODUCTION:

Immunity is a natural phenomenon of human body to fightagainst infectious diseases.But when this function gets disturbed immune system attacks healthy tissue resulting in autoimmunity andauto antibodies are thus produced against self-antigens. These autoimmune disorders and auto antibodies are catching the attention of medical personnel all over the world and arerapidly becoming a major cause of concern in the field¹. Formerly, the generation of auto antibodies were thought to be linked only with the pathogenesis of autoimmune diseases.

But it has been seen through medical research thatauto antibodies have now been found to occur in a number of other scenarios like stroke² cancer³or complicated pregnancy². Furthermore, natural auto antibodiesof both IgG, and IgM classes with various antigenic specificities have been found to occur permanently in the blood of healthy individuals⁴. This finding has led us tounderstand the concept that autoreactive lymphocytes continuously synthesizea certain amount of auto antibodiesagainst different self-antigens in healthy individuals lacking any tissue or organ damage. However amount of the different auto-Abs present in serum of healthy adults vary intensely⁵. Whereas the concentration of auto-Abs with the same specificity is roughly equal in healthy adult individuals of both genders⁴. One understandable purpose of the natural autoimmunity is immune auto clearance. Pierre Grabar was the first one to propose this mechanism of clearing out the cell the debris by a-Abs of immune system⁶.

Antinuclear antibody (ANA) has a significant role in autoimmunity and itis directed against different nuclear antigens. Its elevated levels are found in autoimmune disorders but is also synthesized in healthy individuals^7,
8. It is used as an important diagnostic markerof various autoimmune diseases^9,10. In autoimmune diseases, there areabnormal variations in the rate of apoptosis and necrosisand as a result multiple a-Abs are abnormally expressed and secreted². Presence of these a-Abs aid in the diagnosis but still do not confirm the underlying pathology, therefore the causal tissue damage and clinical symptomsshould be carefully evaluated for accurate diagnosis¹¹. Reliable blood biomarkers are required for initial detection of autoimmune disorders and for identification of those who haven’t developed symptoms but are at risk to have these diseases. More research andstatistics on the incidence and frequency of a-Abs in healthy population is needed tocreate border lines to enhance the reliability of a-Abs assays to be used as diagnostic biomarker of autoimmune disorders ¹¹,¹². A number ofstudies have been conducted on healthy individuals of various populations but limited data is available about the frequency of a-Abs in Pakistan¹³.

MATERIAL AND METHOD:

This cross sectional study was carried out at AL-RAZI Health Care Centre, Lahore Pakistan. Blood samples of 587 (503 females and 84 males) individualswere taken through convenience sampling technique and these study participants were divide into four different age group (0-25, 26-50, 51-75 and 76-100 years. The inclusion criteria included all individuals visited to the Laboratory during the study period. Participants with autoimmune diseases were excluded from this study Three ml blood was collected and serum was separated via centrifugation at 5,000 rpm for 7 minutes. Determination of ANA antibodies was performed by Enzyme Linked Immunosorbent Assay (Cal Biotech, USA). The principle of assay was that individual serum was added to wells coated with nuclear antigens. ANA IgG specific antibody, if present, bound to the antigen. Wells were then washed to sweep away all other materials and the enzyme conjugate was then added to bind the antibody-antigen complex, if present. Extra enzyme conjugate was washed off and substrate was added. Then ELISA plate was incubated to allow the hydrolysis of the substrate by the enzyme. The concentration of the color produced was proportionate to the quantity of IgG specific antibody in the sample which was measured by spectrophotometer at a wavelength of 490nm.SPSS version 20.0 (IBM-SPSS, Inc, Armonk, New York) was used for statistical calculations. An association of autoantibodies in males and femaleswas calculated by χ2 test. whereas p≤0.05 was considered as a limit of statistically significance.

RESULT:

In this study total 581 individuals were included out of which 84 were male whose mean age was 36.5±15.6yrs and 503 were female whose mean age was 36.4±15.4 years, for the screening of ANA which are shown in figure 1.

Fig. 1: An overview of all the participants included in the study with horizontal axis showing age and vertical axis showing number of individuals.

Table 1. Describes the ANA screening results in male individuals included in this study. Only 01 out 24 individuals of age group 0-25 was positive for ANA screening and remaining 23 were negative. Similarly in 26-50 age group, all 52 individuals were negative whereas in 51-75 age group all 08 individuals were negative and in the last age group 76-100 only 01 individual was present which was also negative

Table 1: Comparison of ANA in different age groups of male

No. of cases	Age groups (years)
No. of cases	0-25	26-50	51-75	76-100
Positive	1	0	0	0
Negative	23	52	8	1
Total	24	52	8	1

Similarly Table 2 gives the description of all the female participants of this study. In the agegroup 0 to 25 years total females were 132 and out of which only 1 was positive and remaining 131 were negative. Similarly in 26 to 50 years age group total individuals were 277 and 2 were positive while 275 were negative. In the 51 to 75 years age group, 2 were positive among 89 individuals and 5 individuals were in the last group and all of them were negative.

Table 2: Comparison of ANA in different age groups of females

No. of cases	Age groups (years)
No. of cases	0-25	26-50	51-75	76-100
Pos.	1	2	2	0
Neg.	131	275	87	5
Total	132	277	89	5

Fig. 2: Both genders with positive and negative results

SPSS version 20.0 (IBM-SPSS, Inc, Armonk, New York) was used for statistical calculations. An association of autoantibodies in males and females was calculated by χ2 test and p-value at CL 95% was calculated. p≤0.05 was considered as statistically significant and result of this study shows the χ2 statistic 0.024 and the p-value 0.876984 which was non-significant as shown in Table 3.

Table 3: χ2 statistical analysis of both genders

	Negative	Positive	Marginal Row Total	Chi Square(χ2)	P-Value
Male	84	01	85	0.024	0.876
Female	498	05	503
Marginal Column Total	582	06	588 Grand Total

DISCUSSION:

ANA is the most significantly assayed biomarkers for autoimmunity and is the easiest to assessat the population level¹⁴. In this study ANA levels were evaluated in general population and were found positive in only 1% ofindividuals which is quite insignificant. No ANA was detected in 99% of subjectsthus its nonexistence among healthy people showsits exclusive association with disease process. In the present study, ANA was detected in 1% of healthy individuals which is higher than already documented i.e 0% and lower than reported incidenceof ANA in people with oral habits i.e2.2%¹⁵. Current studyis supported by the previously documented study in which ANA prevalence was 0.8% in healthy male adults of Pakistani population¹³. Present study shows the ANA is detected in 0.99% females while another study shows the 0% ANA prevalence in adult healthy females of Pakistan¹³. The result of current study are different from the results ofa number of other studies which document the prevalence of ANA in US population to be 13.8%, in Indians as 12.3%. whereas for Japanese it is reported to be 9.5%^14,16. this study showed 1% frequency of ANA which is contrary to thepreviously reportedANA frequency of 31.3% in healthy individuals¹⁷. Some other researchers report ANA positivity in 7.6% of general population in Oman¹⁸ and 4.2% in Arab population¹⁹. This difference in the frequency of ANA in healthy individuals could be due to different ethnic groups,geographic places, gender and age of the participants ¹⁹. Method of assay could be also responsible for the differences in results. In current study ANA assay was performed by ELISA whereas in Malaysia Azizah and coworkers performed ANA assay by immunofluorescence (IF) using mouse liver and HEp-2 cell substrates and reported 6.9% positivity for ANA at a titre of 1:40 with HEp-2 andonly 1.9% positive cases were detected with mouse liver²⁰. In current study ANA was not found to be associated with any specific gender which might be due to a certain reason that sample size of male individuals of this study was very less as compare to female individuals, while inaresearch study conducted on Arab population ANA was reported to befour times more prevalent in females as compared to males¹⁹.Some other studies have been conducted to find out relevance of ANA with parameters like obesity and smoking etc. A study reported inverse relationship betweenANA and obesity in females²¹.In addition, a study of chronic obstructive pulmonary disease reported that ANAs were not linked to smoking, but their incidence was common in under weighed people (BMI <22 kg/m2) compared to those withaddictive habits²².

CONCLUSION:

Research study to evaluate the incidence of ANA in general population is important to ensure its reliability as a biomarker of disease. Current study found very low occurrence of ANA in general population which indicates its exclusive association with the diseases especially autoimmune diseases. The one percent positive cases can be further followed up for being at risk of developing disease.

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Received on 20.06.2017 Modified on 11.08.2017

Research J. Pharm. and Tech 2018; 11(10): 4339-4342.

DOI: 10.5958/0974-360X.2018.00794.1