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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 14  |  Issue : 3  |  Page : 211-217

Standardizing initial dilution titers of antinuclear antibodies for the screening of systemic lupus erythematosus


1 Final Year Medical Student, St. John's Medical College Hospital, Bengaluru, Karnataka, India
2 Department of Rheumatology, St. John's Medical College Hospital, Bengaluru, Karnataka, India
3 Department of Biostatistics, St. John's Research Institute, Bengaluru, Karnataka, India
4 Department of Pathology, St. John's Medical College Hospital, Bengaluru, Karnataka, India

Date of Web Publication30-Oct-2019

Correspondence Address:
Dr. Usha Kini
Department of Pathology, St. John's Medical College Hospital, Bengaluru - 560 034, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/injr.injr_172_18

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  Abstract 


Background: Systemic Lupus Erythematosus (SLE), a systemic autoimmune disease, is diagnosed by correlating clinical features with a positive Antinuclear Antibody (ANA) test. Detection of ANA by Indirect Immunofluorescence (IIF) is used for screening SLE and is dependent on initial dilution titers which require population-specific standardization. With lack of exclusive commercial kits for the South Indian population, it is necessary to standardize initial screening dilution titers for ANA to distinguish SLE and other rheumatic diseases from the healthy.
Methods: Newly diagnosed SLE patients between 18 and 60 years along with healthy controls from South India over 8 months (September 2015–April 2016) were selected for this prospective study. Serum samples were subjected to ANA-IIF in dilution titers of 1:40, 1:80, and 1:100 as per kit recommendations. IIF intensity and staining patterns were correlated clinically and statistically analyzed.
Results: ANA positivity in dilutions of 1:40 and 1:80 was seen in 2.1% of healthy controls and negative at 1:100. About 97.9% of SLE patients were positive at 1:100 dilution; speckled pattern being the most common (52.1%), followed by homogeneous (37.4%). The patterns were best appreciated in 1:100 dilution with high significant measure of agreement of kappa between the two pathologists for both patterns and intensity at all three dilutions.
Conclusion: 1:100 is the best screening dilution to distinguish SLE patterns from normal healthy individuals, and its main advantage is the delineation of various ANA patterns when positive, especially when mixed at this lower dilution.

Keywords: Antinuclear antibody, dilution titers, indirect immunofluorescence, systemic lupus erythematosus


How to cite this article:
Rao JS, Shobha V, Thomas T, Kini U. Standardizing initial dilution titers of antinuclear antibodies for the screening of systemic lupus erythematosus. Indian J Rheumatol 2019;14:211-7

How to cite this URL:
Rao JS, Shobha V, Thomas T, Kini U. Standardizing initial dilution titers of antinuclear antibodies for the screening of systemic lupus erythematosus. Indian J Rheumatol [serial online] 2019 [cited 2019 Nov 15];14:211-7. Available from: http://www.indianjrheumatol.com/text.asp?2019/14/3/211/261655




  Introduction Top


Systemic Lupus Erythematosus (SLE) is a prototype of the systemic autoimmune diseases, with a prevalence of 1 in 2500.[1] It predominantly affects 1 in 700 women during their fertility period with a female-to-male ratio of 9:1.[2] This chronic disease with remissions and exacerbations is characterized by multisystem involvement such as skin, joints, kidney, and serosal membranes and is diagnosed as per the Systemic Lupus International Collaborating Classification (SLICC)[3] criteria by the presence of Antinuclear Antibody (ANA) in the serum, a component of IgG class common in 95% of patients with SLE. Its timely diagnosis avoids disease progression to end organ failure.

ANA detected by Indirect Immunofluorescence (IIF) test on the Human Epithelial (HEp-2) cell lines and the preferred method for ANA screening as declared by the American College of Rheumatology[4],[5] has a high sensitivity (90%–98%) and specificity of 90% and is used as a screening tool when SLE is suspected. The initial screening dilution for ANA positivity employed in various laboratories varies from 1:40 to 1:100. All samples with results lower than screening dilution are reported as negative, and hence, the assay sensitivity and specificity vary depending on the screening dilution performed,[6],[7] and three major categories of fluorescent patterns are described, namely nuclear (true ANA), cytoplasmic, and mitotic fluorescence pattern using the International Consensus on ANA Patterns (ICAP).[8]

The College of American Pathologists, in their survey,[9] found 59.6% of the laboratories using ≥1:40 as an initial dilution; 23.1% use ≥1:80; 14% use ≥1:160; and the rest using other cut-off dilutions. Moreover, the commercially available ANA-IIF test kits are validated to their respective population,[10] but not to our local population; Ghosh et al.[11] demonstrated 1:80 dilution as the best screening dilution for the diagnosis of SLE in the North Indian population while 1:160 was used in earlier studies[10] to differentiate systemic autoimmune rheumatic diseases from healthy individuals but test kits with ready to use 1:160 dilution are not available in the market.

Due to high variability in dilution titers and its impact on diagnosis as noted, determination of the optimal screening dilution is essential and to be validated to its own population. Hence, the present study is aimed to standardize an optimum screening ANA-IIF dilution titer among the three dilutions available in commercially available kits, namely 1:40, 1:80, and 1:100 for South Indian population in differentiating ANA positive cases from healthy individuals. The screening dilution titers once validated can be implemented by laboratories in the region to have a uniformity in reporting ANA positivity.


  Methods Top


The study was conducted over an 8-month period from September 2015 to April 2016 at the Division of Rheumatology and the Department of Pathology of a tertiary medical center in South India. Newly diagnosed patients of SLE in the age group of 18–60 years hailing from South India (Karnataka, Kerala, Tamil Nadu and Andhra Pradesh) who fulfilled the SLICC criteria[3] were included in the study. These patients were recruited consecutively after taking a written informed consent and were assessed for SLE Disease Activity Index (SLEDAI) score[12] and treatment response thereafter. Those patients who were pregnant, lactating, positive for any malignancy and seropositive for hepatitis B virus surface antigen (HBsAg), hepatitis C virus (HCV), or HIV infection were excluded from the study. The study was approved by the Institutional Ethical Board. Written informed consent was obtained from all patients and healthy controls before the start of the study.

Students and staff (age matched) from the medical center were included as healthy controls after excluding pregnant/lactating women and those found positive for malignancy, HBsAg, HCV, or HIV infection.

The sample size required to estimate 90% sensitivity for the dilution 1:80, 1:100 versus the standard dilution of 1:40, with 10% precision and 95% confidence interval was 70 to include 35 SLE patients and 35 healthy controls.[11] This was calculated using nMaster version 2.0.[13]

Serum separated within six hours of collection from 3ml of blood was aliquoted and stored at 2°C in the refrigerator for batch-wise testing for a maximum of 48 hours. These test and control samples were coded and subjected to batch testing for ANA antibodies by IIF with HEp-20-10 cells. All the kits used were commercial and purchased from EUROIMMUN (Germany). The tests for this study were carried out without the manufacturer/dealer's knowledge thereby confirming no conflict of interest. The commercial ANA kits contained slides, each containing two biochips coated with HEp-20-10 cells and primate liver, sample diluents, antibody conjugates, control sera, and wash buffer. The assay was performed in dilutions of 1:40, 1:80, and 1:100 - the dilutions recommended by the commercial kits. 1:100 is the screening dilution recommended by the Euroimmun manufacturer. Volume of 25μl of the diluted serum in 1:40/1:80/1:100 was layered on wells with HEp-20-10 cells and stained as per the standardized protocol. During the staining procedure, the biochip was kept moist all through, and after the last step of staining, they were mounted with IF embedding medium and studied using Carl Zeiss Fluorescent Microscope by two qualified pathologists blinded to the coding of the samples under X400 magnification for fluorescence pattern using the ICAP[8] and intensity of fluorescence. The data obtained from subsequent dilutions of positive samples were not considered in this study.

The positive samples showed fluorescence intensity equal to or greater than the positive control on the HEp-20-10 cells and demonstrated a well-defined pattern of staining with fluorescence. Cases with divergent results were re-read and ranked after obtaining consensus from the reporting pathologists. The algorithm of the workflow and the criteria for assigning the intensity of fluorescence[14] (0 [no fluorescence], 1+ [green but no fluorescence], 2+ [green with fluorescence] and 3+ [bright green with fluorescence]) positivity at the recommended dilution of 1:40, 1:80 and 1:100 are shown in [Figure 1]. For convenience and data analysis, the four intensity scales were collapsed to three categories, namely 0 (negative), 1 + and 2+ (2 + and 3+) fluorescence.
Figure 1: Algorithmic approach of methodology

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Demographic characteristics and results obtained were statistically analyzed using IBM SPSS Statistics for Windows, Version 24.0. (IBM Corp., Armonk, NY). Along with the sensitivity and specificity, the Chi-square test of significance was employed to compare the differences between the three dilution titers. The interobserver agreement using kappa coefficient was calculated. P = 0.05 was considered statistically significant. The Receiver Operator Characteristic (ROC) analysis was employed to determine the cut-off level that differentiates patients from healthy individuals and analyze which of the fluorescent positivity (+1 or +2) gives better cut-off to differentiate healthy individuals from patients. The standard area of distribution was estimated to compare the intensity under the three dilutions.


  Results Top


Over an 8-month study period, of the 1032 test serum samples received for ANA testing to the Department of Pathology, 292 samples from 238 patients fulfilling the SLICC criteria were included in the study, of which 54 were repeat samples. The ANA tests were also performed on 300 age-matched healthy controls which were included in the study.

Control group

Three hundred healthy controls were from the four districts of South India, namely Karnataka (n = 106), Tamil Nadu (n = 73), Kerala (n = 58), and Andhra Pradesh (n = 63). Their age group and distribution with their ANA positivity (2%; n = 6) is shown against dilution titer in [Table 1]. The fluorescence noted was predominantly nuclear dense fine-speckled pattern.
Table 1: Antinuclear antibody positivity in three dilutions in healthy controls of different age groups

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Test group

Two hundred and thirty-eight patients who fulfilled the SLICC criteria and had assessment for SLEDAI score were recruited into the study; 96 were from Karnataka, 58 from Tamil Nadu, 36 from Kerala, and 48 from Andhra Pradesh with a mean age group of 37.2 ± 11.8 years and female:male ratio of 1:7.4. Most patients had fairly active disease with high SLEDAI scores at the time of ANA assessment (mean 16.8 ± 7.1) and the details are mentioned in [Table 2].
Table 2: Clinical manifestations in antinuclear antibody positive patients of test group (n=238)

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Clinical manifestations

Dermatological lesions were predominantly alopecia and photosensitivity noted in 71% (n = 169) of the test individuals followed by synovitis in 66.8% (n = 159). Menstrual disturbances were noted in 60.5% (n = 144/210 women). Hematological manifestations (thrombocytopenia ± leucopenia) in 37.4% (n = 89), lupus nephritis in 23.9% (n = 57), and pleural/pericardial effusion in 7.6% (n = 18) as shown in [Table 2].

Serological results

About 97.9% of SLE patients were positive at 1:100 dilution in this study. The distribution of various ANA patterns in the test group is shown in [Table 3]. The speckled pattern was the commonest (52.1%), with the homogeneous (37.4%) and nucleolar (5%) patterns better appreciated at 1:100 dilution. The homogeneous pattern was best seen in 1:80 and 1:100 dilutions while nucleolar pattern was best appreciated in 1:100. Mixed patterns were appreciated in 23.9% (n = 57) in 1:40, 9.2% (n = 22) in 1:80, and 0.8% (n = 2) in 1:100 dilution, comprising chiefly of homogeneous and speckled patterns. The primate liver present in the biochip supported the findings of the HEp-2 cells. These findings on ANA IIF tested samples were found to be significant with a measure of agreement of Cohen's kappa coefficient (k) between the two pathologists for dilution and intensity at 1:40, 1:80, and 1:100 dilution being high (all κ >0.78 with P < 0.001) [Table 4].
Table 3: Distribution of antinuclear antibody patterns in three dilutions for the test group

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Table 4: Interobserver variability and significance in test group for the three dilutions for antinuclear antibody testing

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ANA with speckled pattern showed a better area under the ROC curve for 1:100 dilution (0.97 [0.96,0.99]) compared to 1:40 and 1:80 [Figure 2]. At 1+ fluorescence, the sensitivity and specificity were estimated to be 81% and 98% respectively. However, at 2+ fluorescence in 1:100 dilution, the specificity and sensitivity increased to 100% and 98% repectively [Table 5].
Figure 2: Receiver operator characteristic curve for diagnosis of systemic lupus erythematosus using antinuclear antibody

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Table 5: Sensitivity and specificity taking intensity of fluorescence and dilution titers in test patients

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Speckled patterns were better appreciated in 1:100 and 1:80 dilution in 52.1% and 51.6%, respectively, while homogeneous pattern was best seen in 1:100 and 1:80 dilutions [Table 3]. These findings on ANA–IIF-tested samples found to be significant with a measure of agreement of kappa between the two pathologists for dilution and intensity at 1:40, 1:80, and 1:100 dilution being highly significant (P < 0.005) [Table 5].

The group of clinically suspected SLE patients with a SLEDAI score of ≥6 showed positivity for dsDNA, anti-Sm, Anti-Phospholipid Antibody (APLA), low C3/C4 levels, and direct Coombs test. However, five cases with SLEDAI score ≥6 were ANA negative (2.1%) which prompted dsDNA test which was positive and were subsequently treated as cases of SLE.

ds-DNA was found positive in 18.1%; 11.3% (n = 27) positive for anti-Sm; 24.4% (n = 58) showed low C3/C4 levels while 22.3% (n = 53) had direct Coomb's test positive and 5.5% (n = 13) were associated APLA syndrome.

A total of 54 (22.6%) patients diagnosed positive for ANA had mucocutaneous manifestations of SLE and were positive for ds-DNA. They showed fairly good response to treatment. They are being followed up meticulously for lupus nephritis.

The patients were treated with various combinations of methylprednisolone (95%), hydroxychloroquine (95%), methotrexate (55%), mycophenolate mofetil (77.5%), and azathioprine (40%) after obtaining all the relevant serological investigations for the study.

Those 5 (2.1%) ANA-negative SLE patients with dermal and renal involvement were treated with the combined drug regimen of prednisolone, hydroxychloroquine, methotrexate, mycophenolate mofetil, azathioprine, and leflunomide.


  Discussion Top


ANA is the immunological hallmark of SLE and is widely used for its diagnosis based on its high sensitivity, despite low specificity.[12] Indian data on ANA testing[15],[16],[17],[18],[19] for SLE, though limited, consider ANA as a good screening test with 95% of cases picked up at a titer of 1:80 or more. Kumar et al.[20] provide an overview on advancement in ANA detection methods and state that a titer of 1:160 was significant for the diagnosis of Connective Tissue Disorders (CTD) based on study by Ghosh et al.[11] while Kosaraju et al.[21] have employed 1:100 as the screening dilution titer for their 48 patients. A recent meta analysis by Leuchten et al.[22] demonstrated a sensitivity of 97.8% with acceptable specificity at cut off dilution titer of 1:80 in the IIF-HEp-2 ANA test. They considered ANA of this titer to constitute a resonable entry criterion for SLE classification. These data highlight the variability in screening dilution titers employed at various centers and the need for an optimal screening dilution validated to one's population. The present study is perhaps, the first study to aim at standardizing and validating the initial ANA dilution titers for the South Indian population.

Control group

ANA positivity in healthy controls constituting 2.1% (n = 6/300) (1.3% at 1:40, 0.6% at 1:80 and zero at 1:100) in this study is at a lower level in comparison to the data obtained by Ghosh et al.[11] who found ANA positivity of 13.8%, 4.3%, 2.1%, 2.1%, and 0%, at dilutions of 1:40, 1:80, 1;160, 1:320, and 1:640, respectively, in their study on North Indian population. The difference in the percentages of ANA positivity between the two populations of North and South India in healthy controls may probably be due to difference in ethnicity, environmental factors, and infection load. The percentage of ANA positivity in this study is also less than those reported from Oman and Saudi population where reports vary between 4.2% and 7.6% or in par (1%–3%) with the normal population from Malaysia and New Zealand.[23],[24] Interestingly, Marin et al.[25] detected ANA positivity in 54.3% blood donors, hospital personnel, and relatives of patients with autoimmune diseases and is attributed to occupation (exposure to pesticides, herbicides, and solvents), genetic influence, and handling of blood from patients with SLE hypothesizing that a transmissible agent causing autoantibodies may exist in patients with SLE. A certain degree of variability noted in various centers was considered to be due to variability in the ANA test kits, the substrate (using rodent liver instead of primate liver) staining protocol, and interpretation as attributed by Tan et al.[10] who had a higher value of 31.7% at 1:40 dilution, 13.3% at 1:80, 5% at 1:160, and 3.3% at 1:320 in their multicentric study. Leuchten et al.[22] in their systematic literature review of ANA in SLE of 25 years conclude that in healthy individuals, the ANA positivity increases with age, reaching up to 23%; thus, proving that positive ANA will yield far higher specificity for SLE in a population of young healthy persons than in the typical patient population of a rheumatology clinic. The nuclear dense fine-speckled ANA pattern noted exclusively in healthy individuals in this study was also noted by Au.[26]

Test group

ANA positivity was seen in 97.9% of SLE patients at 1:100 dilution in this study. This is to be noted against 95% positivity at 1:160 dilution by Tan et al.[10] and 97.8% at 1:80 dilution noted by systematic review by Leuchten et al.[22] The finding of 100% specificity for ANA at a tire of 1:100 (2+) contrasts with 74.7% specificity at a titer of 1:80 by Leuchten et al.[22] The latter with their systematic evaluation of different ANA titer for classification of SLE has shown a robust negative likelihood ratio of ANA below 1:80 in IIF on HEp-2 cells.

With 1:100 dilution, the sensitivity with 1 + fluorescence of 81% is higher than in 1:80 or 1:40 dilution (77%). However, sensitivity and specificity are at its best (98% and 100%), respectively, with 2+ IF intensity at 1:100 dilution. 1:100 dilution was the highest screening dilution available in the commercial kits used in this study, and hence, when found negative, none of the samples were tested again at higher dilutions. The speckled ANA patterns were noted in >50% of test patients in this study as well as by Tan et al.[10] and Ghosh et al.[11]

The presence of mixed ANA pattern, predominantly of homogeneous and speckled in a sample, is well known, especially at lower dilutions (23.9% at 1:40), and specific patterns become more discernible at higher dilutions (5.5% at 1:100). This result highlights that higher dilution of the sample facilitates the identification of multiple patterns and allows each pattern for identification. Rare situations can arise when ANA can be negative at lower dilutions due to the so-called prozone (hook) effect.[27] This is the range of relatively high antibody concentration within which no reaction occurs. This can occur because of antigen excess when both capture and detection antibodies become saturated.

Nevertheless, a negative ANA test in the context of high clinical suspicion for SLE is both a clinical challenge. Since the establishment of HEp-2 cells as a substratre for ANA-testing all over the world, the prevalance of ANA-negative SLE has decreased considerably.[28] Nevertheless, a small subgroup of truly ANA-negative SLE population may pose difficulties; it may also be due a false-negative ANA test due to laboratory technical error and interobserver variability. In the absence of strong clinical suspicion, a negative ANA test would render SLE unlikely, but never rule it out, given that approximately 2% may have true ANA-negative SLE.[29] Leuchten et al.[22] also state that ANA once positive (before therapy) will have to count as positive ANA. It is imperative that all ANA-negative patients with strong clinical suspicion of SLE need to have dsDNA and line immunoassay done.[13],[30]

The low rate of ds-DNA positivity of 18% in the SLE patients included in the study is probably related to the disease pattern. The presence of anti-dsDNA antibodies has been associated with severe disease pattern characterized by renal involvement, and titer increase may predict a disease prolapse.[31] This may explain the possibility that the study included only newly diagnosed SLE patients and that the disease might not have progressed much though the renal involvement was seen in 24% of the patients in our study. The follow-up of these patients may help in concluding the same.


  Conclusion Top


With the changes in the field of autoimmune diagnostics and lack of standardization in practice by different laboratories, the interpretation of ANA results could be complicated. With standardization of the initial dilution titer of 1:100 for the South Indian population and better understanding of the assay interpretation, we could aim at facilitating patient management, while minimizing unnecessary investigations and anxiety. In line with the kits recommendation, 1:100 dilution was appropriate as the initial screening dilution for ANA to clearly define positivity in autoimmune/connective tissue disease from normal individuals in the South Indian population and help delineate various ANA patterns when positive, especially when mixed patterns were identified in lower dilutions.

Acknowledgment

The study was supported by the Indian Council of Medical Research – Short Term Student's Program of the year 2015 (ICMR STS ID: 2015-02172) and intramural St. John's Research Society grant (RS/1/1416/15).

Financial support and sponsorship

  1. ICMR STS 2015 (ICMR STS ID: 2015- 02172)
  2. Intramural St. John's Research Society grant (RS/1/1416/15).


Conflicts of interest

There are no conflicts of interest.



 
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