Occurence of autoantibodies in healthy Omani individuals 13 ABSTRACT. Objectives : To investigate the occurence of various autoantibodies in the Omani population. Method: Sera from 392 healthy Omani individuals comprising 183 pregnant women and 209 blood donors (183 men and 26 women) were investigated. Autoanti- bodies were detected using immunofl uorescence, haemagglutination and latex agglutination techniques. Result : Low levels of autoan- tibodies were detected in approximately 50% of the subjects; very few subjects showed high autoantibody titres. Anti smooth muscle autoantibodies (ASMA) were the most prevalent, and were detected in 31.6% of the individuals. Anti thyroid microsomal autoantibodies (ATMA) and anti thyro globulin autoantibodies (ATA) were present in 5.9% and 4.9% of indivi duals respect ively. The other autoantibodies were detected much less frequently, viz. anti nuclear auto antibodies (ANA) in 1.5% , anti parietal cells autoantibodies (APCA) in 1.8% , anti reticulin autoantibodies patterns (ARAP) in 3.0% and rheumatoid factor (RF) in 1.0% of the subjects. Conclusion: The data indicate that autoantibodies do exist in healthy Omani individuals and the results of clinical tests for these autoantibodies must be interpreted with caution. Key Words : autoantibodies, Oman, healthy individuals, autoimmune diseases A utoa ntibodies can be present in sera of patients with or without autoimmune diseases. The litera- ture clearly defi nes their role in specifi c autoimmune conditions.1–3 Their presence merely supports, not confi rms the diagnosis of an autoimmune disease; for a clinical diag- nosis, evidence for clinical disease and/or tissue damage should be present. The defi nition of abnormality related to autoantibody levels is usually based on abnormal quantity rather than affi nity or avidity. Thus, abnormal autoanti- body levels can result in clinical or sub-clinical autoim- mune conditions.2 The demonstration and interpretation of autoanti- bodies in sera from patients is a diagnostic tool in autoim- mune diseases. Thus, the availability of data on the frequency and strength of autoantibodies within a normal population is important for determination of diagnostic levels.1–3 Many such population studies have been per- formed in healthy individuals in various communities.5–9 However, such data have not been described for Oman. This study aimed to establish baseline data for Oman and to provide clinicians with the level of occurence of auto- antibodies within this population. Thus, this is the fi rst report on the distribution of different auto antibodies within the normal Omani population. squ journal for scientific research: medical sciences 2001, 1, 13–19 ©sultan qaboos university Occurence of autoantibodies in healthy Omani individuals *Ali A. Al-Jabri, Elizabeth R. Richens Department of Microbiology & Immunology, College of Medicine, Sultan Qaboos University, P.O. Box 35, Al-Khod 123, Muscat, Sultanate of Oman *To whom correspondence should be addressed. E-mail: aaljabri@squ.edu.om a l - j a b r i e t a l14 Table 1. The number and percentage of Omani individuals positive for various autoantibodies Titre range Pregnant women (F=183) * Blood donors (M=183, F=26) ♦ Combined (n=392) Auto-antibody From To number positive % number positive % number positive % ASMA 1:20 1:160 63 34.5 61 29.2 124 31.6 ATMA 1:100 1:6400 11 6.0 12 5.7 23 5.9 ATA 1:10 1:1280 11 6.0 5 2.4 16 4.1 ARAP 1:20 1:20 3 1.6 9 4.3 12 3.1 APCA 1:20 1:640 6 3.3 1 0.5 7 1.8 AMA 1:20 1:20 0 0.0 5 2.4 5 1.3 ANA 1:20 1:20 0 0.0 6 2.9 6 1.5 RF 1:20 1:80 4 2.2 0 0.0 4 1.0 Total 98 53.6 99 47.4 197 50.3 *Age range 16–46 (Mean 26 years). ♦ Age range 17–58 (Mean 27 years); F= Number of women; M= Number of men ASMA: anti smooth muscle autoantibodies, ATMA: anti thyroid microsomal autoantibodies, ATA: thyroglobulin autoantibodies, AMA: antimitochondrial antibodies, ANA: antinuclear autoantibodies ARAP: antireticulin autoantibodies patterns, APCA: anti parietal cells autoantibodies, RF: rheumatoid factor M E T H O D Two different groups were investigated, pregnant women and blood donors. Individuals in both the groups, totalling 392, were all healthy with no history and/or symptoms of autoimmune diseases. All were residents of the capital area (Muscat), which has the highest population density in Oman. The pregnant group comprised 183 normal women, while the blood donor group numbered 209 individuals, 183 men and 26 women (Figure 1). All sera were kept frozen at –20°C and thawed only at the time of testing. Tests were conducted to detect the following eight autoantibodies: anti nuclear antibodies (A NA) , anti smooth muscle antibodies (ASM A), anti mitochondrial antibodies (A M A), anti parietal cells antibodies (A PCA), anti thy- roglobulin antibodies (ATA), anti thyroid microsomal antibodies (ATMA), anti reticu- lin antibodies patterns (AR AP) and rheumatoid factor (RF). Indirect Quantafl uor fl uorescent autoantibody tests10 were used for the detection and semiquantitation of all autoantibodies except ATA, ATM A and R F where hae- magglutination and latex agglutination assays, respectively, were used.11 Indirect immunofl uorescence (IIF) Sera were tested at a 1:20 dilution by standard immuno- fl uorescence techniques (Quantafl uor, Kallested, USA). Positive sera were titrated to determine the relative strength of the autoantibody under test. Agglutination assays Haemagglutination assays using turkey erythrocytes with bound microsomal antigens and bound thyroglobulin anti- gens were used to test for ATM A and ATA respectively (Thymune-M and Thymune T, Murex, UK). Sera were fi rst inactivated at 56°C for 30 minutes. Positive and negative controls were included in each microtitre plate. Sera and erythrocytes were mixed then incubated at room temperature out of direct sunlight for one hour in the case Figure 1. Age distribution of the Omani individuals studied The study population, which consisted of 392 health volunteers (183 men and 209 women), was divided according to age into 5 groups. 0 30 60 90 120 <20 21-30 31-40 41-50 51-60 Age groups (years) Nu m be r o f s ub je ct s Men Women a u t o a n t i b o d i e s i n o m a n i s 15 of ATM A and for 30 minutes in the case of ATA tests. The end point titre was taken as the highest dilution of the sample giving approximately 50% agglutination of the test cells. ATA tests were also performed by indirect immuno- fl uorescence using monkey thyroid as a substrate (Quan- tafl uor, Kallested, USA). Rheumatoid factor was determined by the latex agglu- tination method (Humatex RF detection kit, Germany). Sera were fi rst inactivated at 56°C and mixed with human gamma immunoglobulin coated latex particles. Macro- scopic agglutination was seen in sera where RF was present. The sera were fi rst diluted 1:20 in fresh buffer (containing glycine 0.1M sodium hydroxide 2.5mM sodium chloride 0.15M, sodium azide 0.1g/dl, pH 8.2); an equal volume of serum (50µl) was mixed with latex particles and checked for agglutination. Positive and negative controls were always included. Positive samples were titrated to determine their titres. R E S U L T S The results, summarized in Table 1, show that ASMA , the most frequent autoantibody present in this population, was detected in 63/183 (34.5%) and 61/209 (29.2%) of pregnant women and blood donors respectively (p < 0.05). However, only three pregnant women (1.6%) and two blood donors (0.9%) had titres greater than 1:100 for this autoantibody [Figure 2]. This titre is considered to be of clinical signifi cance in chronic autoimmune hepatitis.12–13 The remaining 60 pregnant women had low ASM A titres [Figure 2]. This was also the case for the blood donors, where 37/61 individuals had titres less than 1:20. Thyroid antibodies occurred in 11/183 (6%) for both ATA and ATM A in the pregnant women and in 5/209 (2.4%) and in 12/209 (5.7%) of blood donors for ATA and ATM A respectively [Table 1]. Of the twelve blood donors who tested positive for ATMA , nine were men and three were women. ATM A were detected at titres ranging between 1:100 and 1:1600. All men positive for ATM A had lower titres compared to women among whom one case of 1:1600 and two cases of 1:400 were seen. For ATA , three men and two women were shown to be positive. Women showed high titre of antibodies compared to men. The other antibodies occurred generally at lower fre- quencies. Thus A R A P occurred in 3/183 (1.6%) of the pregnant women and in 9/209 (4.3%) of the blood group donors; A M A were not detected in the pregnant women, but were found in 5/209 (2.4%) of the blood donors; A PCA occurred in 6/183 (3.3%) and in 1/209 (0.5%) of pregnant women and blood donors; R F in 4/183 (2.2%) of pregnant women but was not detected amongst blood donors [Table 1]. A NA were absent in pregnant women but found in 6/209 (2.9%) blood donors. Ten women (5.5%) tested positive for more than one of the autoantibodies [Table 2]. Although some of the titres were high, there were no clinical symptoms of autoimmune diseases in these persons. Considering the two groups together, autoantibodies have been detected in more than half of the subjects stud- ied (50.3%). Low titre autoantibodies occurred at high frequency in both groups, having been found in 98/183 (53.6%) and in 99/209 (47.4%) of the pregnant women and the blood donors respectively (p < 0.05); [Table 1]. The most common autoantibodies detected were ASM A (31.6%), ATM A (5.9%), ATA (4.1%), A R A P (3.1%), A PCA (1.8%), A NA (1.5%), and R F (1%). 0 10 20 30 40 50 20 40 80 >100 Titre No . o f S ub je ct s Women Men Women (n=63), Men (n=61) Figure 2. The number of Omani individuals positive for ASMA at different titres. Table 2. Ten pregnant Omani women positive for more than one autoantibody, with their titres Age ATA ATMA ASMA AMA APCA RF 20 1:40 1:400 – – – – 21 1:1280 1:400 – – – – 23 1:20 – – – – 1:20 24 1:320 1:400 1:20 – – – 25 1:320 – 1:20 – – – 26 – – 1:20 – – 1:80 27 – 1:1600 1:20 – – – 27 1:20 1:400 – – – – 32 1:1280 1:400 – – 1:320 – 35 – 1:400 – – 1:640 – a l - j a b r i e t a l16 D I S C U S S I O N Autoimmune phenomena can occur when tolerance to self- components fails and the immune system starts to produce autoantibodies. There is considerable evidence to suggest that autoantibodies may be present in the serum of appar- ently healthy individuals as well as of patients with autoim- mune diseases.4–8,15–16 Many factors are known to infl uence the production of autoantibodies, including genetic factors, sex, age and viral infections.14 In the present study, two groups of healthy Omani individuals were investigated for different autoantibodies: pregnant women and blood donors. Autoantibodies have previously been described in sera from normal pregnant women17–19 and it has been reported that there is no signifi - cant difference in the frequency of autoantibodies between pregnant and non-pregnant women.20 ASM A were fi rst found in the sera of patients with chronic active hepatitis and subsequently in those with other autoimmune liver diseases, viral infection, certain cancers, heroin addiction and female infertility.12 Now it is known that ASM A can occur at low levels in sera of healthy individuals. Our study confi rms this, by showing a preva- lence of 31.6% in our sample of normal Omanis [Figures 2 & 3]. Such high level may be attributed to genetic and/or environmental factors.14 ASM A occurred at a similar fre- quency in both pregnant females and blood donors. Thus it appears that there is no gender difference in the frequency of ASM A in the present study. Since the vast majority of our subjects were less than 40 years old, with only fi ve above 40, [Figure 1], we are unable to comment on the frequency of autoantibodies in older subjects (> 40 years). This should be addressed in future studies on this population. Baig and Shere,8 in their study of a Saudi Arabian popu lation, found A NA to be more prevalent in females than in males, whereas in our study only two women and four men among the blood donors and none among the pregnant women were positive for A NA [Figure 4]. The titres of A NA were all very low, at no more than 1:20 [Tables 1 & 3]; the prevalence of 1.5% of A NA in this study group is much lower than the prevalence of 4.5% found by Baig and Shere in their Saudi Arabian study.8 However, the prevalence of other autoantibodies with high titre (ATA and ATM A) is found higher in females than in males of a matched age group. Within the blood donors group, we noted a higher frequency of autoantibodies in older subjects. These fi ndings support those of other studies.8, 21 Sex and age factors are important when evalu- ating the presence and the titre of autoantibodies within patients.7 ATM A and ATA are also common autoantibodies among this group (occur in approximately 5%) and they come second after ASMA . Females tend to have high titres of ATA and ATMA . A NA is not very common within this population and R F is rarely detected. Only four pregnant Omani women were positive for R F. A R A P is common within both groups. There are many reasons for performing immunological laboratory investigations for autoantibodies. Occasionally , Figure 3. Positive immunofl uoresence of gastric smooth muscle Table 3.Clinically significant titres of autoantibodies among Omanis Autoanti- body Pregnant Women Blood Donors Combined ASMA >80 >80 >80 ANA ≥ 20 ≥ 20 ≥ 20 ATA > 40 > 40 > 40 ATMA > 400 >100 >100 AMA > 20 > 20 > 20 APCA >20 >20 >20 ARA >20 >20 >20 RF >20 >20 >20 The table shows cut-off values (titres), of the different autoantibodies studied, that may be clinically significant among the Omani population. a u t o a n t i b o d i e s i n o m a n i s 17 a diagnosis can be made from a positive test. More com- monly the test may contribute signifi cantly to diag nosis, confi rm a clinical diagnosis or exclude a possible diagnosis and are valu able for monitoring treatment. Low levels of many autoantibodies are not of diagnostic importance and may occur in otherwise normal individuals.4–6 For example, prevalence of autoantibodies increases steadily with age,7 and low titres of autoantibodies in the elderly are frequently of no clinical signifi cance. In general, both autoantibodies and autoimmune diseases can be found more frequently in woman, particularly Caucasians, than in men.3 Due to such variations, data on the frequency and strength of autoan- tibodies in normal individuals in a community are essen- tial as benchmarks for interpreting the autoantibody results of patients from that community. In the absence of this knowledge, such tests may not help in the management of patients. Physician seeking help from a laboratory without this knowledge must be resorting to the last refuge of the diagnostically destitute. The results of this study show clearly the existence of autoantibodies within normal healthy individuals as has been already shown by other investigators.4–8,22 The exist- ence of autoreactive antibodies, both in healthy subjects and in patients with autoimmune diseases, is a consequence of intra-thymic selective processes during the development of the T cell repertoire. Exposure of self-antigens to the thymic environment during foetal life results in the elimina- tion of specifi c anti-self T cells; the dominant mechanism is physi cal , that is, clonal deletion. Autoreactive T cells against antigens which do not passage through the thymus during developmental stages may be eliminated via functional inacti vation i.e. clonal anergy.23 Failure of either of these processes will result in the maturation of autoreactive T cells.24 There are many mechanisms which result in the breakdown of self tolerance. They include alteration in the control of apoptosis, cross reactivity and molecular mimicry, anti-idiotype antibodies that function as autoantibodies and the poly clonal stimulation of natural autoantibody-produc- ing cells that then progress via mutation and isotype switch- ing.25 These phenomena emphasize the importance of dis- tinguishing between autoimmune parameters and autoim- mune disease. The former is benign whereas the latter is potentially fatal. Autoimmunity often refl ected only by the presence of autoantibodies, a normal consequence of aging and readily inducible by drugs or infectious agents and potentially reversible, i.e., it may disappear when the offend- ing drug or agent is eradicated. Autoimmunity may be a normal physiological state: we are all probably autoimmune, but relatively few of us develop autoimmune diseases. These result when auto-reactive T and B cells, activated by genetic environmental triggers, cause actual tissue damage.26 The development of autoimmune disease is depend- ent on at least four factors. The two major ones are genetic and viral. A third factor is endocrine; oestrogen promotes autoimmune disease, whereas androgen acts as a natural immunosuppressive agent. These physiological effects of sex hormones on the normal immune response explain the marked female predominance of autoimmune diseases on susceptible genetic backgrounds. The fourth factor a. Human epithelial (Hep-2 ) cells negative for antinuclear antibodies Figure 4 . Indirect immunofl uoresence for the demonstration of antinuclear antibodies b. Antinuclear antibodies detected by Hep-2 cells a l - j a b r i e t a l18 is psychoneurological i.e. the infl uence of stress and neurochemicals on the immune response. A common feature of these factors is their ability to affect gene expression.27 This study suggests that the diagnosis of an autoim- mune disorder has to be made cautiously taking into con- sideration that, for all the above reasons, autoantibodies are present in low titres in the healthy population. The normal ranges for common autoantibodies that we have established for the Omani population may be a useful guide for diagnostic purposes [Table 3]. The establishment of normal cut-off values in clinically apparently healthy populations is important. Further structured epidemiologi- cal studies of autoantibodies throughout Oman should be undertaken. C O N C L U S I O N This study clearly shows that autoantibodies do exist in normal healthy individuals of Oman. This fact should be taken into account when assessing the titres of antibodies considered to be of clinical signifi cance in this population. a c k n ow l e d g e m e n t We thank Drs. Said Al-Dahry, Yaseen Al-Lawatia, Mr. Ibrahim Kutty, Mr. Steve Bishop and Mrs. Myrna Welsh for assisting in various aspects of this study. R E F E R E N C E S 1. De Rooij DJ, Van de Putte LB, Habets WJ, Verbeek A.L, Van Venrooij WJ. The use of immunoblotting to detect antibodies to nuclear and cytoplasmic antigens. Scand J Rheu 1988, 17, 353–64. 2. Tan EM. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Advanced Immunol 1989, 44, 93–151. 3. De Vlam K, De Keyser G, Verbruggen G, Vandenboss- che M, Vanneuville B, D’Haese D, Veys EM. Detec- tion and identifi cation of antinuclear autoantibodies in the serum of normal blood donors. Clin Exp Rheu 1993, 11, 393–7. 4. Hooper BS, Whittingham S, Mathew JD, Mackay IR, Gurnaw DH. Autoimmunity in a rural community. Clin Exp Immunol 1972, 12, 79–87. 5. Serafi n U, Torrigiani G, Masala C. The incidence of autoantibodies in normal population. In: Proceedings of the Vth International Congress of Allergology, Madrid, Spain 1964. 6. Whittingham S, Irwin J, Mackay IA, Marsch S, Cawl- ing DC. Autoantibodies in healthy subjects. Aust Ann Med 1969, 18, 130–4. 7. Willkens RF, Whitaker RR, Anderson RV, Berven D. Signifi cance of antinuclear factors in older persons. Ann Rheu Dise 1967, 26, 306–10. 8. Baig MM, Shere SJ. Prevalence of autoantibodies in Saudi population. J Med 1989, 20, 286–90. 9. Azizah MR, Shahnaz M, Zulkifl i MN, Nasuruddin BA. Anti-nuclear, anti-mitochondrial, anti-smooth muscle and anti-parietal cell antibodies in the healthy Malaysian population. Malaysia J Pathol 1995, 17, 83–6 10. Cavallaro JJ, Palmer DF, Bigazzi PE. Immunofl uores- cence detection of autoimmune diseases. Immunology series No.7, Centers for Disease Control, Atlanta GA, 1976. 11. Singer JM, Plotz CM. The latex fi xation test. I. Applica- tion to the serologic diagnosis of rheumatoid arthritis. Am J Med 1956, 21, 888. 12. Toh BH. Smooth muscle autoantibodies and autoantigens. Clin Exp Immunol 1979, 72, 621–8. 13. Brostoff J, Scadding GK, Male D, Roitt I. Clinical Immunology, Gower Medical Publishing, 1991, p30.6. 14. Shoenefeld Y, Schwartz RS. Genetic & immunologic factors in autoimmune diseases. N Engl J Med 1984, 311, 1019. 15. Hawkins BRR, O’connor KJ, Dawkins RL. Autoanti- bodies in an Australian population. J Clin Lab Immunol 1979, 2, 211. 16. El-Roeiy A, Gleicher N. Defi nition of normal auto- antibody levels in an apparently healthy population. Obstet Gynecol 1988, 72, 596. 17. Polishuk WZ, Bayth Y, Izak G. Antinuclear factor and LE cells in pregnant women. Lancet 1971, 2, 270. 18. Meles KA, Halfon V, Dobersen MJ, Ginsberg-Fellner F, Cowdery JS, Steinberg AD. Autoantibodies in preg- nancy. Lancet 1983, 1, 354. 19. Farrom J, Lavastida MT, Grant JA, Reddi RC, Daniles JC. Antinuclear antibodies in the serum of normal women: a prospective study. J Allergy Clin Immunol 1984, 73, 596–9. 20. Patton PE, Coulam CB, Bergstralh E. The prevalence of autoantibodies in pregnant and nonpregnant women. Am J Obstet Gynecol 1987, 157, 1345–50. 21. Manoussakis MN, Tzioufas AG, Silis MP, Pange PJE, Goudevenos J, Moutsopoulos HM. High prevalence of anti-cardiolipin and other autoantibodies in healthy elderly population. Clin Exper Immunol 1987, 69, 557–65. 22. Sakata S, Matsuda M, Ogawa T, Takuno H, Matsui I, Sarui H, et al. Prevalence of thyroid hormone autoanti- bodies in healthy subjects. Clin Endocrinol 1994, 41, 365–70. 23. Petrie HT, Livak F, Schatz DG, Strassr A, Crispe IN, Shortman K. Multiple arrangements in TCR α-chain maximizes the production of useful thymocytes. J Exp Med 1993, 178, 615–22. a u t o a n t i b o d i e s i n o m a n i s 19 24. Sinha AA, Lopez MT, McDevitt HO. Autoimmune diseases: the failure of self-tolerance. Science 1990, 248, 1380–8. 25. Steinberg AD, Krieg AM, Gourky MF. Theoretical and experimental approaches to generalized autoimmu- nity. Immunol Rev 1990, 118, 129–43. 26. Stevenson FK, Natvig J. Autoantibodies revealed: the role of B cells in autoimmune disease. Immunol Today 1999, 296–8. 27. Bijlsma JWJ, Cutolo M, Masi AT, Chickauza IC. The neuroendocrine basis of rheumatic disease. Immunol Today 1999, 298–301.