Antinuclear and Rheumatoid Factor Estimations in the Diagnosis of Rheumatic 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 erythematosus (SLE) and rheumatoid (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 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 ( 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 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 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 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). 37.5 0 5 8 8 0 The distribution of ANA titres in 75 0 3 17 14 0 subjects in different categories, is 150 0 4 13 15 0 expressed in a composite way gra­ phically in Figure I using cumula­ 225 0 0 10 6 0 tive frequencies. A titre of 7.5 300 0 2 15 12 0 SEAPAL units/ml or more exclud­ 375 0 1 15 6 0 ed all normal subjects and 94 per 600 0 0 9 4 0 cent of the disease controls but yet 750 0 0 3 8 0 included 99 per cent of the SLE 900 0 0 1 0 0 and 98 per cent of the OCTO pa­ tients. Therefore, this was con­ 1,125 0 0 0 0 sidered as the optimal starting *Percentage screening titre of the serum samples which would exclude the 'back­ ground noise" and yet be highly sensitive for the diagnosis of SLE. - Normals (n=120) <>---<> RA (n=100) Similarly, a titre of 75 SEAPAL 6----i>SLE (n=100) units/ml (l: 100 dilution) or more ~ 100 If---¥ Other syst (n=51) 'ij conn. tis. dis. :::J - -- - Disease (n=50) was found to exclude up to 90 per 1;; 80 w controls cent of the RA patients and yet de­ ~ :e­ 60 tected 84 per .cent of the patients i;l with SLE and 65 per cent of those '0 40 with OCTD. *­ .~'" Table 2 gives the results of RF. ..!!! 20 :::J E The highest incidence and titres :::J '-' 0 were seen in RA. The distribution > 15 >37.5 > 75 >150 >225 >300 >600 > 1125 of RF titre in subjects in different ANA in Seapal units per ml categories is expressed in a compo­ site way graphically in Figure 2 as in Figure I. A titre of 30 or more Fig. 1 Antinuclear antibody IV/ml excluded 100 per cent of the normal subjects, 97 per cent of the SLE patients, 78 per cent of those Table 2 Rheumatoid factor in healthy and diseased Indians. with OCTO and 98 per cent of those with non-rheumatic nonim­ Titres Nonnal subjects RA SLE OCTD Unrelated munological diseases and yet it (International (n=120) (n= 100) (n=100) (n=37) diseases detected 69 per cent of the patients units/ml) (n=50) with RA. At a titre of 60 or more IV/ml, 53 per cent of the RA pa­ < 15 98* 10 88 70 92 tients were positive but almost all 15 2 21 9 8 6 other groups were excluded. Thus, a 30 0 16 2 5 0 titre of 30 IV/ml RF was considered 60 0 18 1 5 0 sufficiently sensitive for the detec­ 120 0 20 0 11 2 tion of RA and other related connec­ 240 0 9 0 0 0 tive tissue diseases, and a titre of 60 V I or more IV/ml was considered high­ 480 0 6 0 0 0 ly specific for seropositive RA. *Percentage , ANA AND RF IN DIAGNOSIS OF RHEUMATIC DISEASES 235

off-point would be very sensitive and therefore include some of the - Normals (n=120) SLE and OCTO patients as well as 0-0 RA (n=100) ." 100 "-" SLE (n=100) patients with unrelated diseases_ By :.;:;...... Other syst. (n=37) conn. tis. dis increasing the cut-off point to 60 ~ 80 ---­ Disease (n=50) IU/ml, the RF test becomes much u controls ., more specific and, to a large extent, f 60 excludes u nrela ted conditions. '0 15 >30 >60 > 120 >240 >480 milar positivity in North Indians as RF titre in ioU. per ml (logarithmic scale) they do in Western Caucasian po­ pulation studies, and more or less Fig. 2 Rheumatoid factor similar cut-off points for applying these tests to clinical situa tions ..1 ,2 These findings could be very diffe­ rent from that seen among Mongo­ DISCUSSION Such data collection for various im­ loid races where ANA has been re­ mu nological tests becomes imp era­ ported to be negative at the time of It has been well brought out in tive because of the known varia­ diagnosis of SLE becoming positive the past that medical laboratory im­ tions in these diseases as rela ted to much later in the course of the dis­ munology, as is the case wi th any ethnic origin,13 genetic variation ease.7 Therefore, similar studies in type of laboratory medicine, would and the variable prevalence of the other races would be of interest. become useful only if the specifici­ infections in different geographic ty and sensitivity of the test being areas. 14 The wo rkshops 0 n AN A ACKNOWLEDGEMENTS employed are well worked ou t and and RF held during the Fifth understood by the users of the SEAPAL Congress a t Bangkok Technical help provided by M/s test.3 Thus, it has been shown that brought out these facts very welL 7 Gopal Singh, Sube Singh, Titus Ver­ a test can be set up in such a way From this standpoint, the stan­ ghese and R. L. Taneja is gra tefully that it is positive in a large propor­ dardisation of ANA and RF tests acknowledged. tion of patients with a given disease for the North Indian population of This work was supported by the when the disease is in its active Delhi was carried out at this labora­ Western Australia Arthritis and form. However, if set up in such a tory. A cut-off point of 15 SEA­ Rheumatism Foundation (Inc.) and manner, the test could give a posi­ PAL units/ml (1:20 dilution) ex­ helped by Prof. R.L. Dawkins, De­ tive result for other unrelated dis­ cluded all unrelated and irrelevant partment of Clinical Immunology, eases as well as in some normal, clinical situations and detected the Royal Perth Hospital, Perth, Austra­ healthy individuals. While such a majority of patients with SLE and lia, for which we are grateful. test would be useful for diagnosing OCTO. High titres of ANA were /J a particulr disease, it would give a observed both in SLE and OCTO; high number of "false positives". the ANA titre alone could not dis­ On the other hand, if the same test tinguish between them. The evalua­ is set up with a higher cut-off point tion of the clinical picture and the it may exclude all the false positives complete auto-antibody profile, REFERENCES and yet include a significant pro­ such as the estimation of anti-DNA, 1. Carson DA. Rheumatoid factor- In: KeUy portion of the patients with the anti-Sm, anti-RNP, anti-SSA etc., WN, Harris ED, Ruddy S, Sledge CB, eds, disease in question. It is obvious, would be helpful in distinguishing Text-blook of . Toronto: therefore, that each laboratory in­ SLE from other related connective W_B_ Saunders, 1981; 677-90. 2_ Davis JS- Antinuclear (ANA)_ volved in patient-care-related labora­ tissue diseases, and further categori­ Idem: 691-700_ tory medicine would have to find sing the latteL 3_ Peter JB, Dawkins RL The value of im­ out the prevalence of the positivity Applied to the diagnosis of RA, munology tests_ Diagnost Med 1979; Feb of the relevant t est first among the lower cut-off point of RF 1-8. their own normal healthy popula­ which would be sensitive enough to 4. Bhuyan UN, MaJaviya AN- Antinuclear antibodies and patterns of nuclear im­ {f tions, second with regard to unre­ detect up to 69 per cent of the RA munofluorescence in systemic lupus lated diseases and third in those patients appears to be 30 IU/ml in erythematosus and other collagen vascular diseases for which the test is useful. this laboratory. However, this cut diseases_ Ind J Med Res 1976; 64: 895-902_ I 236 MALAVIYA, ET AL.

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