Antinuclear Antibody and Rheumatoid Factor Estimations in the Diagnosis of Rheumatic Diseases in Northern India * Anand N. Malaviya, M.D. Subhashis Banerjee, M.D. Ramnath N. Misra, M.D. Rashmi Kaul, M.Sc. Udaya N. Bhuyan, M.D. Auto-immune serum markers, namely antinuclear antibody (ANA) SUMMARY One hundred serum samples from North Indian patients with sys­ and rheumatoid factor (RF), are temic lupus erythematosus (SLE) and rheumatoid arthritis (RA) and 51 samples useful laboratory parameters in the from patients with other systemic connective tissue diseases (OCTO) such as sys­ diagnosis, differential diagnosis and temic sclerosis, overlap syndrome and dermatomyositis were studied for the pre­ follow-up of patients with systemic valence and titre of antinuclear antibody and rheumatoid factor expressed in In­ lupus erythematosus (SLE), rheu­ ternational units. The results were compared with those obtained in 50 patients rna toid arthritis (RA) and 0 ther with non-rheumatic diseases (disease controls) and 120 healthy controls. None of systemic connective tissue diseases the controls, diseased or healthy, showed an ANA titre of 15 SEAPAL units (1: (OCTD).I-s However, it has been 20) or greater. With increasing ANA titre beyond this level. there was a greater realised that these markers may also possibility of the disease being SLE or OCTO but not RA. However, the titre of be detected in varying amounts in ANA per se could not discriminate between SLE and OCTO. Similarly for RF, a normal healthy controls as well as titre of 30 IU/ml or more could be used to discriminate between connective in people with other unrelated dis­ tissue disorders and other diseases; the diagnosis of rheumatoid arthritis was eases. 6 Moreover, there is a possibi­ most Iikely at a titre of R F above 60 IU/ml. lity of ethnic and geographical ASIAN PACIFIC J ALLERG IMMUN 1984; 2 : 232-236. variations in these parameters. 7 Therefore, it is essential to investi­ gate the distribution of ANA and In the present study, an attempt years for males and from 6 to 70 RF in normal healthy individuals of has been made to standardise these years for females; the median age a certain ethnic group in a given tests, using availa ble in temational was 30 years. Of the subjects, 9 per geographic area and also to find out reference standards, in Northern In­ cen t were in their flfSt decade, 6 the extent of the parameters in dian population which is more or per cent in their second decade, 58 rheumatic-immunological and non­ less a homogeneous ethnic group of per cent in their third decade, 14 rheuma tic-nonimmunological d is­ brown Caucasians. per cent in their fourth decade, 2 eases in the same ethnic group in a per cent in their fifth decade, 5 per given geographic area. Such studies PATIENTS cent in their sixth and seventh de­ are essential for finding out the cades and 1 per cent in the eighth normal levels, relevant c ut-off The subjects studied included the points and the sensitivity and speci­ following groups: ficity of these tests in relation to (a) 120 normal healthy controls: *From the ainical Immunology Division, De­ partment of Medicine, and Department of the rheuma tic-immu nological d is­ There were 68 males and 52 fema­ Pathology, All-India Institute of Medical eases. les ; their ages ranged from 3 to 75 Sciences, New Delhi-1l0029, India. 232 ANA AND RF IN DIAGNOSIS OF RHEUMATIC DISEASES 233 decade. These subjects included 15 Sjogren's syndrome (2), diagnosed Prof. R.L. Dawkins, Department of staff members working in various as per standard text-book criteria. II Clinical Immunology, Royal Perth areas of the All-India Institute of There were 47 females and four Hospital, Perth, Australia), referred Medical Sciences, 50 healthy blood males, ranging in age from 15 to 55 to as SEAPAL units in this paper, donors, 30 subjects undergoing a years. The median age was 35 years were initially prepared in different rou tine serological test for syphilis and the majority were in their third dilutions to plot the standard curve. (VORL testing), 13 healthy elderly and fourth decades. Serum samples The reciprocal of the highest dilu­ individuals undergoing cataract sur­ were collected from patients after tion of the test sera as converted gery and 12 healthy children having they were diagnosed to have SLE, into units and, when necessary, the a rou tine medical check-u p and re­ RA or OCTO. None of the patients standard curve was extrapolated. ceiving vaccinations. was on or had been on steroid The highest dilutions in which the (b) 50 disease controls: There therapy when the samples were standards of 7.5 units/ml, 15 units/ were 19 males ranging in age from drawn. ml and 30 units/ml were positive, 4 to 79 years and 31 females were 1:10, 1:20 and 1:40 respec­ ranging in age from 19 to 65 years; METHODS tively; the standard of 2 .5 units/ml the median age was 40 years. The was negative even when applied un­ distribu tion of these su bjects in Indirect Immunofluorescence Test diluted. All control and test sera successive decades from the first to for ANA were screened at an initial dilution the eighth decade was 8%, 8%, 20%, Standard indirect immunofluo­ of I: 10. If a serum was negative 10%, 18%, 24%, 10% and 2% re­ rescent test was used throughout at I: 10, the test was repeated spectively. The subjects were pa­ the study. Unfixed cryostat sec­ (neat). If ANA was positive in neat tients with a variety of common tions of rat liver and kidney were serum only, the result was express­ non-rheumatic and non-immunolo­ used as substrate antigens. Serum ed as < 7.5 SEAPAL units/m!. gical diseases commonly seen in a dilutions were layered on these big general hospital ; the diseases tissue sections on microscopic Latex fIxation test for RF included tu berculosis, essen tial slides, inclubated for 30 minutes in Latex-RF kits (some purchased hypertension, coronary artery a humidified chamber and washed from M/s Wellcome Reagents, U.K., disease, pyelonephritis, non-Hodg­ three times in phosphate buffered and others generously provided by kin's lymphoma etc. saline (pH 7.40, 0 . 1 M) ; kept in M/s Hoechst Pharmaceuticals Ltd., (c) 100 patients with systemic motion by a mechanical stirring Behring Diagnostics, India) were lupus erythematosus (SL£): The device. Polyspecific antihuman im­ used throughout this study. A diagnosis of 92 females and eight mu noglobulins (lgG, IgA, IgM) an­ known reference standard provided males ranging in age from 8 to 60 tiserum raised in goats and conju­ by the manufacturers, as well as a years was made according to the gated with fluorescein isothyocya­ WHO reference laboratory standard 1971 American Rheumatism Asso­ nate (FITC) was obtained commer­ for RF (generously provided by ciation classification criteria from cially (M/s Immunodiagnostics, Del­ WHO International Laboratory for SLE.8 The median age was 24 years hi, India). Optimal dilution was de­ Biological Standards, Statens Serum and the majority of patien ts were in termined by "chequerboard" titra­ Institute, Copenhagen, Denmark) their second and third decades. tion with a known antinuclear posi­ were simultaneously run with each (d) 100 patients with rheuma­ tive serum. 12 The highest dilution batch of tests and the results ex­ toid arthritis (RA): The diagnosis of FITC-conjugate giving a clear, pressed in international units (lU)/ of 16 males and 84 females ranging positive test was considered "opti­ m!. in age from 19 to 70 years was as mal" and used in the test. .The per the 1958 revised ARA criteria serum-treated and -washed tissue RESULTS with the majority in the category of sections were then I ayered with " definite" RA after careful exclu­ optimally diluted FITC conjugate ANA was not detected in any sion of non-RA in flamma tory po­ and incubated for 30 minutes in a normal control at the minimum de­ lyarthritides.9 The median age was humidified chamber and washed as tection level of 7.5 SEAPAL units/ 40 years and the majority of these above. After the final wash, the ml (I : 10 dilution) or less. In the patients were in their third, fourth slides were mounted in glycerol disease control group, five (10%) of and fifth decades. saline buffer and read under the subjects showed a positive ANA (e) 51 patients with other syste­ epifluorescent light using a Nikon test; two of them were positive at mic connective tissue diseases 'Optophot' microscope. a titre of less than 7.5 SEAPAL (OCTD): including progressive sys­ A set of four reference standard units/m!. One of the latter subjects temic sclerosis (30), diagnosed as sera containing 2.5 units/ml, 7.5 was a 24-year-old male with per ARA criteria,lO undifferentiated units/ml, 15 units/ml and 30 units/ untreated malignant hypertension connective tissue disease (19) and ml respectively (kindly provided by and the second was a 51-year-old 234 MALAVIYA, ET AL. '­ male who had undergone a coronary Table 1 Antinuclear antibody in healthy and diseased Indians. bypass procedure. Three patients, a 58-year-old male with non-Hodg­ Titres Unrelated Normal subjects RA SLE OCTD kin's lymphoma , a 53-year-old male (SEAPAL diseases (n=120) (n=100) (n=lOO) (n=51) with renal calculus and a 56-year­ unit/ml) (n=50) old female with generalized tuber­ culosis, were positive at a titre of < 7.5 100* 65 2 94 7.5 SEAPAL units/m!. The highest 7.5 0 20 5 25 6 incidences of ANA were seen in 15 0 0 2 0 0 SLE and OCTO (Table I).