[Downloaded free from http://www.indianjrheumatol.com on Saturday, November 2, 2019, IP: 114.125.233.

31]

Original Article

Standardizing Initial Dilution Titers of Antinuclear Antibodies for the

Screening of Systemic Lupus Erythematosus
Joseph Sushil Rao, Vineeta Shobha1, Tinku Thomas2, Usha Kini3
Final Year Medical Student, Departments of 1Rheumatology and 3Pathology, St. John’s Medical College Hospital, 2Department of Biostatistics,
St. John’s Research Institute, Bengaluru, Karnataka, India

Received: December, 2018

Accepted: May, 2019
Published: June, 2019
Address for correspondence:
Dr. Usha Kini,
Department of Pathology, St.
John’s Medical College Hospital,
Bengaluru ‑ 560 034, Karnataka,
India.
E‑mail: drushakini@gmail.com
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.
Key Words: Antinuclear antibody, dilution titers, indirect immunofluorescence, systemic lupus
erythematosus

Introduction SLE. Its timely diagnosis avoids disease progression to end

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
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

This is an open access journal, and articles are distributed under the terms of the Creative
Access this article online Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which allows others to
Quick Response remix, tweak, and build upon the work non‑commercially, as long as appropriate credit is
Code given and the new creations are licensed under the identical terms.
Website:
www.indianjrheumatol.com
For reprints contact: reprints@medknow.com

DOI: 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
10.4103/injr.injr_172_18
2019;14:211-7.

211 © 2019 Indian Journal of Rheumatology | Published by Wolters Kluwer - Medknow

[Downloaded free from http://www.indianjrheumatol.com on Saturday, November 2, 2019, IP: 114.125.233.31]
Rao, et al.: Initial ANA dilutional titers in diagnosis of SLE
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
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
Indian Journal of Rheumatology  ¦  Volume 14  ¦  Issue 3  ¦  September 2019
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.
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
212
[Downloaded free from http://www.indianjrheumatol.com on Saturday, November 2, 2019, IP: 114.125.233.31]

Rao, et al.: Initial ANA dilutional titers in diagnosis of SLE

patients from healthy individuals and analyze which of Tamil Nadu, 36 from Kerala, and 48 from Andhra Pradesh
the fluorescent positivity  (+1 or  +2) gives better cut‑off with a mean age group of 37.2 ± 11.8 years and
to differentiate healthy individuals from patients. The female:male ratio of 1:7.4. Most patients had fairly
standard area of distribution was estimated to compare active disease with high SLEDAI scores at the time of
the intensity under the three dilutions. ANA assessment  (mean 16.8  ±  7.1) and the details are
mentioned in Table 2.
Results
Clinical manifestations
Over an 8‑month study period, of the 1032 test serum
samples received for ANA testing to the Department of Dermatological lesions were predominantly alopecia
Pathology, 292  samples from 238  patients fulfilling the and photosensitivity noted in 71%  (n  =  169)
SLICC criteria were included in the study, of which 54 were of the test individuals followed by synovitis in
repeat samples. The ANA tests were also performed on 66.8% (n  =  159). Menstrual disturbances were noted
300 age‑matched healthy controls which were included in in 60.5% (n  =  144/210 women). Hematological
the study. manifestations  (thrombocytopenia  ±  leucopenia) in
37.4% (n = 89), lupus nephritis in 23.9% (n = 57), and pleural/
Control group pericardial effusion in 7.6% (n = 18) as shown in Table 2.
Three hundred healthy controls were from the four Serological results
districts of South India, namely Karnataka (n  =  106), Tamil
Nadu (n = 73), Kerala (n = 58), and Andhra Pradesh (n = 63). About 97.9% of SLE patients were positive at 1:100
Their age group and distribution with their ANA dilution in this study. The distribution of various ANA
positivity  (2%; n  =  6) is shown against dilution titer in patterns in the test group is shown in Table 3. The
Table  1. The fluorescence noted was predominantly
nuclear dense fine‑speckled pattern. Table 1: Antinuclear antibody positivity in three dilutions
Test group in healthy controls of different age groups
Age groups Number of individuals (n=300) Dilutions
Two hundred and thirty‑eight patients who fulfilled the 1:40 1:80 1:100
SLICC criteria and had assessment for SLEDAI score were 14-25 44 1 0 0
recruited into the study; 96 were from Karnataka, 58 from 26-35 76 1 1 0
36-45 89 1 1 0
46-55 70 1 0 0
Patients (18 – 60 years, newly diagnosed SLE) Controls (Healthy individuals)
56-65 21 0 0 0
SLICC and SLEDAI score 300 4 2 0
Total 6
3ml of whole blood in plain serum tube

Serum separated (1 to 4 hours) and stored at 2°C (Stored for a maximum of 48 hours from collection)
Table 2: Clinical manifestations in antinuclear antibody
positive patients of test group (n=238)
Coded samples run in batches; Diluted and processed for ANA-IF
Clinical manifestations n (%)
Mean SLEDAI score 16.8±7.1
ANA-HEp2-2010/liver biochip (Monkey) conjugated with specific Anti-Human IgG (EUROIMMUN AG)
Synovitis 159 (66.8)
Dermatological 169 (71)
1:40 1:80 1:100 Mucosal ulceration 90 (37.8)
Hematology 89 (37.4)
Slides read by two trained personnel/pathologists blinded to clinical diagnosis
Neurological 16 (6.7)
Immunofluorescence intensity* and pattern of staining reported Renal 57 (23.9)
*Intensity of IF assessed based on the following criteria: Pleural/pericardial 18 (7.6)
3+: Bright green with fluorescence
2+: Green fluorescent Menstrual disturbance 144 (60.5)
1+: Green (Stained, but not fluorescence)
0: No fluorescence ANA 238 (100)
Anti‑dsDNA 43 (18.1)
Anti‑smith 27 (11.3)
Data obtained and analysed statistically
Antiphospholipid antibodies 13 (5.5)
ROC analysis
Low C3/C4 levels 58 (24.4)
Sensitivity and Specificity Chi square test of significance
Direct coombs test 53 (22.3)
Kappa Co-efficient: Inter observer variability
SLEDAI: Systemic lupus erythematosus disease activity index,
Figure 1: Algorithmic approach of methodology ANA: Antinuclear Antibody

213 Indian Journal of Rheumatology  ¦  Volume 14  ¦  Issue 3  ¦  September 2019

[Downloaded free from http://www.indianjrheumatol.com on Saturday, November 2, 2019, IP: 114.125.233.31]
Rao, et al.: Initial ANA dilutional titers in diagnosis of SLE
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].
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].
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.
Table 3: Distribution of antinuclear antibody patterns in
three dilutions for the test group
ANA pattern Dilution
1:40 (%) 1: 80 (%) 1:100 (%)
Speckled 112 (47.1) 123 (51.6) 124 (52.1)
Homogeneous 50 (21) 68 (28.6) 89 (37.4)
Rim 2 (0.8) 3 (1.3) 1 (0.4)
Centromere 10 (4.2) 13 (5.4) 10 (4.2)
Nucleolar 7 (2.9) 9 (3.8) 12 (5)
Mixed 57 (23.9) 22 (9.2) 2 (0.8)
Total 238 238 238
ANA: Antinuclear antibody
Indian Journal of Rheumatology  ¦  Volume 14  ¦  Issue 3  ¦  September 2019
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
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[15‑19] 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.
Figure 2: Receiver operator characteristic curve for diagnosis of systemic
lupus erythematosus using antinuclear antibody
214
[Downloaded free from http://www.indianjrheumatol.com on Saturday, November 2, 2019, IP: 114.125.233.31]

Rao, et al.: Initial ANA dilutional titers in diagnosis of SLE

Table 4: Interobserver variability and significance in test multicentric study. Leuchten et  al.[22] in their systematic
group for the three dilutions for antinuclear antibody literature review of ANA in SLE of 25 years conclude that in
testing healthy individuals, the ANA positivity increases with age,
ANA dilutions κ P
reaching up to 23%; thus, proving that positive ANA will
1:40
yield far higher specificity for SLE in a population of young
healthy persons than in the typical patient population of
Intensity 0.78 <0.001
a rheumatology clinic. The nuclear dense fine‑speckled
Pattern 0.88 <0.001
ANA pattern noted exclusively in healthy individuals in this
1:80
study was also noted by Au.[26]
Intensity 0.82 <0.001
Pattern 0.89 <0.001 Test group
1:100
ANA positivity was seen in 97.9% of SLE patients at 1:100
Intensity 0.82 <0.001 dilution in this study. This is to be noted against 95%
Pattern 0.94 <0.001 positivity at 1:160 dilution by Tan et  al.[10] and 97.8% at
ANA: Antinuclear antibody 1:80 dilution noted by systematic review by Leuchten
et  al.[22] The finding of 100% specificity for ANA at a tire
Table 5: Sensitivity and specificity taking intensity of of 1:100  (2+) contrasts with 74.7% specificity at a titer of
fluorescence and dilution titers in test patients 1:80 by Leuchten et  al.[22] The latter with their systematic
Dilution IIF intensity evaluation of different ANA titer for classification of SLE
Sensitivity (%) Specificity (%) has shown a robust negative likelihood ratio of ANA below
1+ 2+ 1+ 2+ 1:80 in IIF on HEp‑2 cells.
1:40 77 68 97 99 With 1:100 dilution, the sensitivity with 1 + fluorescence of
1:80 77 70 97 99 81% is higher than in 1:80 or 1:40 dilution (77%). However,
1:100 81 98 98 100 sensitivity and specificity are at its best  (98% and 100%),
IIF: Indirect immunofluorescence respectively, with 2+  IF intensity at 1:100 dilution. 1:100
dilution was the highest screening dilution available in the
Control group commercial kits used in this study, and hence, when found
negative, none of the samples were tested again at higher
ANA positivity in healthy controls constituting dilutions. The speckled ANA patterns were noted in  >50%
2.1% (n  =  6/300)  (1.3% at 1:40, 0.6% at 1:80 and zero of test patients in this study as well as by Tan et al.[10] and
at 1:100) in this study is at a lower level in comparison Ghosh et al.[11]
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 The presence of mixed ANA pattern, predominantly of
of 1:40, 1:80, 1;160, 1:320, and 1:640, respectively, in homogeneous and speckled in a sample, is well known,
their study on North Indian population. The difference especially at lower dilutions  (23.9% at 1:40), and specific
in the percentages of ANA positivity between the patterns become more discernible at higher dilutions (5.5%
two populations of North and South India in healthy at 1:100). This result highlights that higher dilution of the
controls may probably be due to difference in ethnicity, sample facilitates the identification of multiple patterns
environmental factors, and infection load. The percentage and allows each pattern for identification. Rare situations
of ANA positivity in this study is also less than those can arise when ANA can be negative at lower dilutions due
reported from Oman and Saudi population where reports to the so‑called prozone  (hook) effect.[27] This is the range
vary between 4.2% and 7.6% or in par  (1%–3%) with the of relatively high antibody concentration within which no
normal population from Malaysia and New  Zealand.[23,24] reaction occurs. This can occur because of antigen excess
Interestingly, Marin et  al.[25] detected ANA positivity in when both capture and detection antibodies become
54.3% blood donors, hospital personnel, and relatives saturated.
of patients with autoimmune diseases and is attributed Nevertheless, a negative ANA test in the context of high
to occupation  (exposure to pesticides, herbicides, and clinical suspicion for SLE is both a clinical challenge. Since
solvents), genetic influence, and handling of blood from the establishment of HEp-2 cells as a substratre for ANA-
patients with SLE hypothesizing that a transmissible agent testing all over the world, the prevalance of ANA-negative
causing autoantibodies may exist in patients with SLE. SLE has decreased considerably.[28]Nevertheless, a small
A certain degree of variability noted in various centers was subgroup of truly ANA‑negative SLE population may
considered to be due to variability in the ANA test kits, pose difficulties; it may also be due a false‑negative ANA
the substrate  (using rodent liver instead of primate liver) test due to laboratory technical error and interobserver
staining protocol, and interpretation as attributed by Tan variability. In the absence of strong clinical suspicion, a
et  al.[10] who had a higher value of 31.7% at 1:40 dilution, negative ANA test would render SLE unlikely, but never
13.3% at 1:80, 5% at 1:160, and 3.3% at 1:320 in their rule it out, given that approximately 2% may have true
215 Indian Journal of Rheumatology  ¦  Volume 14  ¦  Issue 3  ¦  September 2019
[Downloaded free from http://www.indianjrheumatol.com on Saturday, November 2, 2019, IP: 114.125.233.31]
Rao, et al.: Initial ANA dilutional titers in diagnosis of SLE
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
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.
References
1. Rees F, Doherty M, Grainge MJ, Lanyon P, Zhang W. The
worldwide incidence and prevalence of systemic lupus
erythematosus: A  systematic review of epidemiological studies.
Rheumatology (Oxford) 2017;56:1945‑61.
2. D’Cruz  DP, Khamashta  MA, Hughes  GR. Systemic lupus
erythematosus. Lancet 2007;369:587‑96.
3. Petri  M, Orbai  AM, Alarcón GS, Gordon  C, Merrill  JT, Fortin  PR,
et al. Derivation and validation of the systemic lupus international
Indian Journal of Rheumatology  ¦  Volume 14  ¦  Issue 3  ¦  September 2019
collaborating clinics classification criteria for systemic lupus
erythematosus. Arthritis Rheum 2012;64:2677‑86.
4. Wichainun R, Kasitanon N, Wangkaew S, Hongsongkiat S,
Sukitawut  W, Louthrenoo  W. Sensitivity and specificity of
ANA and anti‑dsDNA in the diagnosis of systemic lupus
erythematosus: A  comparison using control sera obtained from
healthy individuals and patients with multiple medical problems.
Asian Pac J Allergy Immunol 2013;31:292‑8.
5. Hochberg  MC. Updating the American college of rheumatology
revised criteria for the classification of systemic lupus
erythematosus. Arthritis Rheum 1997;40:1725.
6. Copple SS, Giles SR, Jaskowski TD, Gardiner AE, Wilson AM,
Hill HR, et  al. Screening for IgG antinuclear autoantibodies by
HEp‑2 indirect fluorescent antibody assays and the need for
standardization. Am J Clin Pathol 2012;137:825‑30.
7. Mengeloglu Z, Tas T, Kocoglu E, Aktas G, Karabörk S.
Determination of anti‑nuclear antibody pattern distribution and
clinical relationship. Pak J Med Sci 2014;30:380‑3.
8. Chan EK, Damoiseaux J, de Melo Cruvinel W, Carballo OG,
Conrad K, Francescantonio PL, et  al. Report on the second
international consensus on ANA pattern  (ICAP) workshop in
dresden 2015. Lupus 2016;25:797‑804.
9. College of American Pathologists. Summary Report CAP
Diagnostic Immunology S‑A. Chicago: College of American
Pathologists; 2001. p. 14.
10. Tan EM, Feltkamp TE, Smolen JS, Butcher B, Dawkins R,
Fritzler MJ, et  al. Range of antinuclear antibodies in “healthy”
individuals. Arthritis Rheum 1997;40:601‑11.
11. Ghosh P, Dwivedi S, Naik S, Agarwal V, Verma A, Aggarwal A,
et  al. Antinuclear antibodies by indirect immunofluorescence:
Optimum screening dilution for diagnosis of systemic lupus
erythematosus. Indian J Med Res 2007;126:34‑8.
12. Ramsey‑Goldman R, Isenberg DA. Systemic lupus erythematosus
measures: British Isles Lupus Assessment Group  (BILAG),
European Consensus Lupus Activity Measurement  (ECLAM),
Systemic Lupus Activity Measure  (SLAM), Systemic Lupus
Erythematosus Disease Activity Measure  (SLEDAI), and Systemic
Lupus International Collaborating Clinics/American College of
Rheumatology‑Damage Index  (SLICC/ACR‑DI; SDI). Arthritis
Rheum 2003;49:S225‑3.
13. nMaster 2.0 Sample Size Software designed and developed by
Department of Biostatistics, Christian Medical College, Vellore;
Serial no. 1C80-00M0-19S5-MA2X-4L04-3K46-EK90.
14. Sebastian  W, Roy  A, Kini  U, Mullick  S. Correlation of antinuclear
antibody immunofluorescence patterns with immune profile
using line immunoassay in the Indian scenario. Indian J Pathol
Microbiol 2010;53:427‑32.
15. Malaviya AN, Singh RR, Kumar A, De A, Kumar A, Aradhye S.
Systemic lupus erythematosus in Northern India: A  review of
329 cases. J Assoc Physicians India 1988;36:476‑80, 484.
16. Madhavan R, Porkodi R, Ramakrishnan S,
Krishnamurthy V, Parthiban M, Chandrasekaran AN. Systemic
Lupus Erythematosus - The Madras Experience. J Assoc Phys
India 1988;36:481-4.
17. Amin SN, Angadi SA, Mangat GK, Joshi AD, Halankar SA,
Salgaonkar DS, et  al. Clinical profile of systemic lupus
erythematosus in Western India. J Assoc Physicians India
1988;36:473‑5.
18. Vaidya S, Samant RS, Nadkar MY, Borges NE. Systemic lupus
erythematosus  –  A review of 220  patients. J  Indian Rheumatol
Assoc 1997;5:14‑8.
19. Kumar A. Indian guidelines on the mangaement of SLE. J Indian
Rheumatol Assoc 2002;10:80-96.
216
[Downloaded free from http://www.indianjrheumatol.com on Saturday, November 2, 2019, IP: 114.125.233.31]

Rao, et al.: Initial ANA dilutional titers in diagnosis of SLE

20. Kumar  Y, Bhatia  A, Minz  RW. Antinuclear antibodies and their
detection methods in diagnosis of connective tissue diseases:
A journey revisited. Diagn Pathol 2009;4:1.
21. Kosaraju  K, Shenoy  S, Suchithra  U. A  cross‑sectional
hospital‑based study of autoantibody profile and clinical
manifestations of systemic lupus erythematosus in south Indian
patients. Indian J Med Microbiol 2010;28:245‑7.
22. Leuchten N, Hoyer A, Brinks R, Schoels M, Schneider M,
Smolen J, et  al. Performance of antinuclear antibodies for
classifying systemic lupus erythematosus: A systematic literature
review and meta‑regression of diagnostic data. Arthritis Care
Res (Hoboken) 2018;70:428‑38.
23. Al‑Jabri  AA, Al Belushi  MS, Nsanze  H. Frequency and levels
of autoantibodies in healthy adult Omanis. Ann Saudi Med
2003;23:372‑5.
24. Baig  MM, Shere  SJ. Prevalence of autoantibodies in Saudi
population. J Med 1989;20:286‑90.
25. Marin GG, Cardiel MH, Cornejo H, Viveros ME. Prevalence
of antinuclear antibodies in 3 groups of healthy individuals:
Blood donors, hospital personnel, and relatives of patients with
autoimmune diseases. J Clin Rheumatol 2009;15:325‑9.
26. Au  EY. ANA testing: What should we know about the methods,
indication and interpretation? Hong Kong Bull Rheumatic Dis
2017;2:53-7.
27. Butch  AW. Dilution protocols for detection of hook effects/
prozone phenomenon. Clin Chem 2000;46:1719‑21.
28. Cross  LS, Aslam  A, Misbah  SA. Antinuclear antibody‑negative
lupus as a distinct diagnostic entity  –  Does it no longer exist?
QJM 2004;97:303‑8.
29. Schmajuk G, Hoyer BF, Aringer M, Johnson SR, Daikh DI,
Dörner T. Multi-center Delphi Exercise Reveals Important Key
Items for Classifying Systemic Lupus Erythematosus. Arthritis
Care & Research 2017. doi:10.1002/acr.23503.
30. Kavanaugh A, Tomar R, Reveille J, Solomon DH,
Homburger HA. Guidelines for clinical use of the antinuclear
antibody test and tests for specific autoantibodies to nuclear
antigens. American college of pathologists. Arch Pathol Lab
Med 2000;124:71‑81.
31. Fabrizio C, Fulvia C, Carlo P, Lora M, Elisa M, Francesca M, et al.
Systemic lupus erythematosus with and without anti ds‑DNA
antibodies: Analysis from a large monocentric cohort. Mediators
Inflamm 2015;2015:328078.

217 Indian Journal of Rheumatology  ¦  Volume 14  ¦  Issue 3  ¦  September 2019