Indian J Med Res 126, July 2007, pp 34-38

Antinuclear antibodies by indirect immunofluorescence : Optimum
screening dilution for diagnosis of systemic lupus erythematosus

P. Ghosh, S. Dwivedi, Sita Naik, Vikas Agarwal, Anupam Verma*, Amita Aggarwal & Ramnath Misra

Departments of Immunology & *Transfusion Medicine, Sanjay Gandhi Postgraduate Institute of Medical
Sciences, Lucknow, India

Received October 9, 2006

Background & objectives: Antinuclear antibodies (ANA) are serological hallmark of systemic lupus
erythematosus (SLE). Conventionally, the test is carried out on human epithelial cells (HEp2) by
indirect immunofluorescence (IIF) technique. Since culturing and maintaining HEp2 cells in the
laboratory are labour intensive, in-house assays have given way to kits manufactured by commercial
companies. The reference screening dilutions provided by the manufacturers are based on different
ethnic population than ours. Therefore, it becomes mandatory for every laboratory to have its own
screening dilutions for the local population that distinguishes best between healthy and diseased
state. As, there is paucity of such data, we aimed to define the optimum screening dilution that
distinguishes the patient with SLE from healthy individuals.
Methods: Sera of patients fulfilling ACR criteria for diagnosis of SLE, idiopathic inflammatory
polymyositis/dermatomyositis (PM/DM) and rheumatoid arthritis (RA), and age and sex matched
healthy individuals were tested for ANA by IIF using a commercial kit (Euroimmun, Germany) at
5 dilutions, namely 1:40, 1:80, 1:160, 1:320 and 1:640. Receiver operator characteristics (ROC)
curve were constructed to define the optimum dilution that distinguished healthy sera from the
diseased ones.
Results: Test was performed on 213 sera from 94 healthy individuals, and 43 SLE, 37 RA and 39
DM/PM patients. In healthy individuals, ANA at dilutions 1:40, 1:80, 1:160, 1:320 and 1:640 was
positive in 13.8, 4.3, 2.1, 2.1 and 0 per cent respectively, whereas in SLE it was positive in 95.3, 95.3,
65.1, 53.5 and 23.3 per cent respectively.
Interpretation & conclusion: ROC curves analysis showed that at 1:40 dilution, sera of 95.3 per cent
of SLE and 13.8 per cent of normal individuals were (ANA) positive, whereas at 1:80 dilution it was
95.3 per cent for SLE and 4.3 per cent for healthy individuals. A fluorescent intensity of ³2 was
more specific for SLE. The best discrimination between healthy individuals and the SLE patients
was found at screening dilution of 1:80 and fluorescent intensity of ³2 in our laboratory.

Key words Antinuclear antibodies - indirect immunofluorescence - screening dilutions - systemic lupus erythematosus

Antinuclear antibodies (ANA) are the serological immunofluorescent assay with rodent liver tissue as the
hallmark of systemic lupus erythematosus (SLE), substrate 1. With the increasing awareness among
originally described in 1957 using an physicians of autoimmune connective tissue diseases,
34

1:160. Sanjay Gandhi Postgraduate Institute of Medical Sciences. which are being used for diagnostic in reading ANA slides. a written informed consent was undertaken been recent advances in the technology of coating HEp2 from all healthy volunteers and SLE patients who cells onto glass slides that is being adopted by some participated in the study. Immunology. This has the advantages of increased blood was collected. rheumatoid and wash buffer. Both positive and negative control sera were with other connective tissue diseases. antibody conjugates and reference sera. Germany) were purchased sensitivity of different kits. The kit contained HEp2 diseases besides SLE. antibody conjugate. 1 + (faint green). control sera disorder. There have also However. This is especially important in view of the fluorescent microscope (Olympus. Again the slide was washed to influence autoantibody production5. The standardization of these kits has been for 5 min. ANA was defined as positive at a particular patient with SLE from normal individuals and establish dilution if it was read positive by both the observers at the frequency of ANA positivity in well defined patients that dilution. the (1:40) were layered on HEp-2 cell spots and incubated frequency increasing with age4. manufacturers and this adds to the variability in the All the kits (Euroimmun. for 30 min. Stored sera (-80°C) from patients reactivity and recognition of antibody specificities that with dermatomyositis/polymyositis (DM/PM)7 and react with antigens associated with dividing cells2. Hence. study was not submitted to the institutional ethics Hence. Briefly. The slide was washed by flush of phosphate Currently all the kits being used in Indian laboratories buffer saline (PBS)–Tween and then immersed in PBS are imported. Lucknow.80. blinded to the diagnosis and the tests in our population. should be validated using serum interpretation of the other observer read the slides. provided by the manufacturer. it may be expected in PBS-Tween for 5 min as described earlier. the technique has gave no history of any illness and had no physical or undergone considerable methodological modifications mental disability were selected from among the staff of and the rodent tissue substrate has been replaced with human epithelial cell lines such as the human epithelial the hospital and members of their family. The is labour intensive and requires considerable expertise. GHOSH et al : ANA SCREENING DILUTION 35 this test is frequently used by clinicians since it is the American College of Rheumatology (ACR) criteria for mainstay of diagnosis of SLE. ‘in house’ assays have been replaced by committee for the approval because this was a validation commercial kits. Healthy volunteers from among the staff was as follows: 4+ (very bright green). between July 2004 to The criterion for assigning intensity of fluorescence December 2005. 1:320 and 1:640 dilutions. Healthy volunteers between the ages of 18 and 65 yr. exercise undertaken to improve the quality of the test. embedded that the prevalence in healthy individuals will be different with 10 ml of embedding medium and read under the in our country. . Japan). The screening dilution recommended by The study was conducted at the department of the manufacturer was 1:100. 25 ml of diluted sera ANA positivity is also seen in normal individuals. such as mixed connective tissue slides. human globulin was added to each spot and slide Since both genetic and environmental factors are known incubated for another 30 min. 3+ (bright green). The assay was performed according to arthritis and in malignancies and infectious diseases3. and who Since its original description. The rheumatoid arthritis (RA)8 who fulfilled the diagnostic culture and maintenance of cell lines in the laboratory criteria of each of these diseases were also tested. We therefore carried out this study in several sessions of reading ANA slides to agree on a to define the dilution to be used for distinguishing the consensus. These kits contain HEp2 coated slides. All sera that were positive Material & Methods at the 1:40 dilution were tested at 1. Any increase in sensitivity and tests were carried out without any knowledge or usually compromises specificity and this is particularly obligation to the manufacturer. The study was performed relevant in the case of ANA detection since the test is with the same batch of the kit from the manufacturer and also positive in a large variety of connective tissue as per the instructions provided. easier identification of different patterns of stored at -20°C till use. of the hospital and consecutive patients who fulfilled 2 + (green). Prior samples from local population with a view to define the to the actual test both the investigators had participated screening dilutions. it is relevant Two independent observers (SN and VA) experienced that the imported kits. diagnosis of SLE6 were included in the study. Later 20 ml of fluorescein-labeled (goat) anti- done using normal and diseased sera of non Indian origin. sample diluents. Five ml of (HEp2) cells. high infection load in our population. and serum separated within 4 h and sensitivity. manufacturer’s instruction. primary Sjogren’s syndrome.

ANA was positive in 13. subjects in the 56-65 yr group was less than in other age Determining the dilution that distinguishes healthy groups. range 23-47). of Dilutions This is perhaps the first study from our country to groups indivi.905 (95%CI.972) ANA positive. range 18-61). ANA in healthy subjects : There was no difference in ANA positivity among different age groups (Table I).5 version). This indicated that 1:80 dilution all the three dilutions. 95. 0.7% vs 90.8.95] at 1:40 dilution. The chi-square test of significance was used to find out any difference in ANA positivity in different age groups of healthy controls. At a dilution of 1:80. ROC analysis was also done to assess which definition of the fluorescent positivity (³1 or ³2) gives better cut-off to differentiate healthy individuals from patients with SLE. SLE was no different between fluorescence intensity of ³1 or ³2 but there was better specificity at a fluorescence intensity of ³2 (95.0. 1. 56-65 5 1 1 0 0 0 Our observation of 1:80 being the best screening Total 94 13 4 2 1 0 dilution for diagnosis of SLE is in contrast to the .3 per cent of the at 1:80 dilution and 0. While generating ROC curves larger test results were taken to indicate more positive test and standard error of area with distribution assumption as nonparametric and confidence levels of 95 per cent were used. median age of sera positive for ANA in systemic autoimmune 35 yr.1 and 0 of ³1 or ³2 is a better cut-off for ANA positivity : At a per cent of healthy controls at dilutions 1:40. ROC analysis to determine whether fluorescent intensity Overall. Interclass correlation coefficient was used to look for inter-observer variability.1:40 1:80 1:160 1:320 1:640 determine the optimum screening dilution for ANA using (yr) dual a commercial kit. This validation has important clinical 18-25 19 2 0 0 0 0 implications as ANA is being widely used due to ease of 26-35 33 4 1 1 1 0 availability as well as increasing awareness among 36-45 19 4 2 1 0 0 46-55 18 2 0 0 0 0 medical fraternity. 4. [95% confidence interval (95% CI) patients and 13.0. 1. 0. 2.953.938. 2. ANA positivity in sera from healthy controls in different age groups Discussion Age No. dilution 1:80 the sensitivity of ANA for diagnosis of 1:320 and 1:640 respectively. was the best screening dilution to distinguish SLE patients from healthy individuals. collected from 94 healthy controls (59 females. ANA positivity at different dilutions in patients with SLE. JULY 2007 Statistical analysis : All statistical analyses were carried out on SPSS software (11.95) at 1:160 SLE patients and 4. Very few patients of RA median age 44 yr. individuals from SLE patients : The ROC curve (Fig. Table I. 43 SLE (35 females.821.1. 37 RA (26 females.92-0.3. diseases is shown in Fig. 2) Variability : Inter-observer variability gave an interclass showed that at a dilution of 1:40. median age 35 yr. Test for ANA was performed on 213 serum samples DM/PM and RA.36 INDIAN J MED RES. median ANA in systemic autoimmune disorders :The percentage age 33 yr.8 per cent of healthy individuals were 0. 2. 95. 1:160. the inter-observer variability was low at ANA positive (Table II). Results Fig. range 25-65) and 39 DM/PM patients (27 females. The age distribution of had ANA positivity at 1:320 or 1:640 in contrast to SLE healthy controls (Table I) shows that the number of and DM/PM. range 35-64).964 (95%CI. 0. Table II). 1:80.4%) as compared to intensity of ³1 (Fig.3 per cent of SLE coefficient of 0. The receiver operator characteristic (ROC) analysis was used to determine the cut-off level that differentiates between healthy and diseased individuals. Thus.3 per cent of healthy individuals were dilution.

7 per cent of healthy persons were ANA laboratory use ³1:40 as a cut-off. The percentage of ANA positivity in our reflected in a survey9 by the college of American study at 1:40 was lower than that reported by Tan et al Pathologists which.2 89.7 97. Sensitivity and specificity taking intensity of fluorescence (IIF)³1 and ³2 as positive Sensitivity (%) Specificity (%) Disease Dilution IIF intensity³I IIF intensity³2 IIF intensity³I IIF intensity³2 SLE 1:40 95. ( ) 1:80 positive.4 95.9 1:640 23.6 97.8 95.9 1:320 69.Systemic lupus erythematosus DM/PM – Dermatomyositis/polymyositis RA – Rheumatoid arthritis prevalent practice of using sera at 1:40 dilution. found that 59.4 1:80 95.1 93.4 1:80 48.3 93 86.9 1:640 2.9 43.3 90.3 95.2 86. ROC curves of ANA for diagnosis of SLE (a) taking intensity of fluorescent (IF) > 1.9 1:320 8.7 1:160 46.2 41 93. 23. Table II.4 90.7 1:160 10.9 1:640 53.5 23.1 95. This is individuals. our percentage was more that those dilution which distinguished SLE patients from healthy reported from Middle East countries where it is reported . 14 per cent use ³1:160 and rest use other cut.7 97.2 89. Tan et al10 have shown that 1:160 was the best factors. However.9 1:320 38.4 95. ( ) 1:320 positive.3 100 100 DM/PM 1:40 51.7 2. The lower percentage in our study could be ³1:80. ( ) 1:640 positive).5 5.2 90.1 93.6 97. 2.4 95.8 53.8 8.4 1:80 13.1 per cent use positive.6 97.1 8.2 89.7 100 100 SLE . and (b) taking intensity of fluorescent > 2 as positive.5 95. ( ) 1:160 positive.5 30.3 86.7 46. explained by difference in ethnicity and environmental offs.4 65.( ) 1:40 positive.7 97.3 51.6 per cent of the where 31. GHOSH et al : ANA SCREENING DILUTION 37 (a) (b) Fig.7 1:160 88.1 17.9 100 100 RA 1:40 45.

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