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Table of Content Volume 9 Issue 2 - February 2019


Detection of prevalence of antinuclear antibodies in clinically suspected cases of systemic autoimmune diseases by indirect immunofluorescence test in a tertiary care hospital


Jabeen Begum1*, V V Shailaja1, Rajeshwar Rao2


1*Consultant Microbiologist, Department of Microbiology, AIG Hospital, Mindspace Rd, Gachibowli, Hyderabad-500032, Telangana, INDIA.

1Associate Professor, 2Professor, Department of Microbiology, Gandhi Medical College, Secunderabad Telangana India 500003

Email: dr.jabeenbegum@gmail.com


Abstract                  Background: Systemic autoimmune diseases (SAD) are the diseases where multiple organs are involved in the presence of auto antibodies directed against sub-cellular structures or molecules and are characterized by presence of Antinuclear antibodies (ANA). Indirect immunofluorescence test (IIFA) on Hep-2 (human epithelial cell tumour line) is a “gold standard” technique for detection of ANA Purpose: 1) To detect the Prevalence of Antinuclear antibodies (ANA) in clinically suspected cases of Systemic autoimmune diseases (SAD) by Indirect Immunofluorescence test(IIFA)on Hep2 cells Methodology: A total of 150 clinically suspected cases of SAD of both sexes and above 18yrs of age from various departments were included in the study and blood samples collected were subjected to Indirect Immunofluorescence test on Hep-2 cells coated slides and slides were visualized in fluorescent microscope using blue green filter (450nm). Results: 150 samples were analysed for ANA by IIFA. Out of 150 samples, 54 samples were positive by IIFA. Prevalence of ANA among clinically suspected case of SAD by IIFA was found to be 36%. Female predominance was seen and most common age group of presentation was 20-30yrs. The homogenous pattern was the most common (n=26; 48.14%) pattern followed by speckled pattern (n = 19; 35%). Conclusions: Systemic autoimmune disorders are chronic conditions with no cure and are growing day by day. With the increase in prevalence of systemic autoimmune diseases there is a need for early and accurate diagnostic techniques. Therefore, early detection of ANA by Gold Standard Indirect Immunofluorescence test in a Clinically suspected cases helps to reduce disease morbidity and mortality

Key Words: Systemic Autoimmune diseases, Antinuclear antibody test, Indirect immunofluorescence test.





Systemic autoimmune diseases are characterized by tissue injury due to breakdown of one or more of the basic mechanisms regulating immune tolerance leading to immunological reaction of the organism against its own tissues and are characterised by involvement of multiple organs with presence of a large variety of auto antibodies directed against sub-cellular structures or molecules. Diseases in this group includes Systemic lupus erythematosis (SLE), Systemic sclerosis/Scleroderma (SSc), undifferentiated connective tissue diseases or Mixed Connective tissue diseases (MCTD), Dermatomyositis / Polymyositis, Sjogren’s syndrome (SS/SjS)1. Systemic autoimmune disorders are characterized by presence of Antinuclear antibodies (ANA) in the blood of patients. ANA are a specific class of autoantibodies that have the capability of binding and destroying certain structures within the nucleus of the cells and considered to be a serological hallmark of connective tissue diseases2. The prevalence of Systemic autoimmune diseases in India is increasing over years as it was 12.3% in 1996-2006, 18.9% in 2006-2007 as per retrospective North Indian study3, 38.2% in 2010 from Vellore4 and 35% in 2017 from Bhubaneswar5. This increase in prevalence is due to development and utilization of advanced diagnostic facilities for diagnosis of Systemic autoimmune diseases. ANA prevalence increases with age, with peak at 20–30 years of age and are more prevalent among females than males. No significant associations were seen with education, family income, alcohol use, smoking history6. The American College of Rheumatology (ACR) stated that ANA detection by IIFA on Hep-2 cells is considered as the gold standard7. In a Clinically suspected cases of connective tissue disorders, ANA test is done, if positive further tests are performed for the diagnosis of specific systemic autoimmune diseases. If negative no further autoantibody testing is performed8,9.



A cross-sectional study was conducted over a period of one and half year from March 2016 – Sept 2017 and 150 blood samples of Clinically suspected cases of Systemic autoimmune diseases of both sexes and more than 18yrs of age, were included in the study after informed consent.

Ethical Consideration: Ethical clearance was taken from institute’s Ethical Committee. Detailed clinical and epidemiological and laboratory data were recorded using structured proforma. Under aseptic precautions 3ml of Blood samples was collected, Serum separated out and alliquoted. ANA detection by IIFA was performed on HEp-2000 cells according to the instructions provided by the manufacturer (Immuno Concepts, Sacramento, CA).

Procedure: Positive and Negative controls were run with each test daily. Serum was diluted in 1:80 ratio (serum: diluent) (10 μl serum +790 μl diluent). 30 μl of the diluted serum was then put on each wells. This was then incubated at room temperature for 30 min. This step allowed the antibodies in the serum to react with the antigens coated on the wells. The slide (wells) was then washed carefully and then dipped into the PBS for 10 min to remove the unbound antibodies. In the next step, FITC conjugate (Anti-human IgG conjugated to fluorescein isothiocyanate) was added to wells, to get bound to the antibodies and emit fluorescence. The FITC was again washed off carefully and dipped in PBS (in dark) for 10 min, to remove the unbound conjugate. The wells were then mounted using mounting medium. The visualization of the slide was then done under the fluorescence microscope at 40X. Based on the fluorescent intensity, samples were graded positive (+, ++, +++) and negative.

Negative: A serum was considered negative for antinuclear antibodies if nuclear staining was less than or equal to the negative control well with no clearly discernible pattern. The cytoplasm may demonstrate weak staining, with brighter staining of the non-chromosomal region of mitotic cells, but with no clearly discernible nuclear pattern.

Positive: A serum was considered positive if the nucleus shows a clearly discernible pattern of staining in a majority of the interphase cells. The positive sample showed bright apple green fluorescence in the nuclei of the cells, with a clearly discernible pattern characteristic of the control serum that was used.

Hep-2000 cells are transfected with the gene for SSA-60, which makes these cells more sensitive for SSA antibody detection. Serum samples were screened in a 1: 80 dilutions. FITC-conjugated goat anti-human IgG antibody was used for detection of ANA. Five staining patterns are commonly reported: homogenous, speckled, centromere, nucleolar and cytoplasmic. In caseof mixed-patterns, the pattern with the highest titre was included in the present study. Slides were evaluated with a fluorescent microscope (Axioskop, Carl Zeiss MicroscopyGmbH, Jena, Germany) with LED light source at 450nm using Blue- green

Statistical analysis: EPI INFO 7 version statistical soft ware package was used forstatistical analysis. Chi-Square test was applied to test whether differences between values were significant. p values <0.05 was considered as statistically significant andp value >0.05 as insignificant.



In the present study out of 150 samples from various departments majority of samples were from Rheumatology Department (43%), followed by Medicine Department (30.4%) and Dermatology Department (30%) as shown in table 1. The prevalence of ANA by IIFA was 36% as shown table 2. In the present study, majority of patients were found to be in the age group of 20yrs to 30yrs followed by 41yrs-50yrs. There was a significant association between age group 20-30years with ANA positivity (p value<0.05) as shown in table 3 and female preponderance was seen with female to male ratio of ANA positive cases were 6.5:1. as shown table- 4. Out of 54 ANA positive samples Homogenous is most common pattern on IIFA followed by Speckled. Various pattern of ANA on IIFA with their percentage prevalence is shown in table 5. Most common clinical manifestation was Joint pains seen in 122 cases (81.3%) followed by chronic low grade fever seen in 86 cases as shown in table 6. Among other laboratory investigation anaemia was most common finding, followed by elevated ESR as shown in table 7


Table 1: Department wise distribution of samples from clinically suspected cases of Systemic autoimmune diseases (SAD).


Total number


ANA positive


Percentage (%)






























Table 2: Detection of ANA by IIFA in Clinically suspected cases of SAD

Total no

ANA positive by


ANA negative by


Percentage Prevalence (%)






Table 3: Age wise distribution of samples from clinically suspected cases of Systemic autoimmune diseases (SAD)

Age Limit

Total Number (n=150)

ANA Positive (n=53)

Percentage (%)

20-30 Year




31-40 Year




41-50 Year




51-60 Year




Table 4: Sex wise distribution of samples from clinically suspected cases of SAD


Number of clinically suspected Cases (n=150)

ANA Positive(n=53)








Table 5: Various Patterns of ANA on IIFA with their Prevalence







Speckled + Cytoplasmic








Nucleolar + Homogenous


                                                Figure 1                                                               Figure 2                                          Figure 3

Figure 4:                                                              Figure 5:                                              Figure 6:



Figure1: Homogenous pattern; Figure 2: Speckled pattern; Figure 3: Cytoplasmic+ Speckled pattern; Figure 4: Centromere pattern;

Figure 5: Nucleolar pattern; Figure 6: Cytoplasmic pattern



Table 6: Clinical manifestations in clinically suspected cases of SARD

Symptoms in clinically suspected cases of SARD

Number of cases (n=150)

Percentage (%)

Joint pains



Chronic low grade fever



Muscle Fatigue



Weight Loss






Hair Loss






Mouth ulcers



Raynaud’s phenomenon




Table 7: Other Laboratory investigations in clinically suspected cases of SARD


Number of Clinically suspected cases of SARD

Percentage (%)




TLC < 4000/mcl



PLATELETS <1.5lakhs/mcl



ESR Raised



RA Factor




In the present study, Prevalence of ANA by IIFA was found to be 36% which was correlating with following other studies of Sarojini Raman et al, Shaily Garg et al, Wendy Sebastine et al, Akmatov et al, J. Angel Thomas et al. Most common Age group of presentation was 20-30yrs followed by 41-50yrs which is correlating with other studies of Sarojini Raman et al, Asaithambi et al, J. Angel Thomas et al,Mengeloglu. Z et al, Thomas Y. Avery et al and the probable reason can be due to physiologic stage of Reproductive age group and perimenopause implying that oestrogen may play an important role and genetic and hormonal factors may have an influence on disease manifestation. In the present study, clinically suspected cases of SAD is more predominant in females than in males (2.3:1) correlating with studies of (table-7) Sarojiniraman et al, M.E. Soto et al. The likely explanation for this female preponderance is probably related to exogenous and endogenous hormonal change, Homogenous pattern (48.14%) is most common pattern detected by IIFA correlating with studies of Wendy Sebastine et al (45.5%) from Vellore, J.Angel Thomas et al (44%) from Tamil Nadu, Asaithambi et al (44.4%) from Tamil Nadu, Thomas Y. Avery et al (40%) from Netherland, Sarojiniraman et al (52.6%)from Bhubaneswar. Homogenous pattern mostly associated with autoantibodies to dsDNA which is diagnostic criteria for SLE. Hence indirectly indicating that SLE is the most common systemic autoimmune disease reported



Systemic autoimmune disorders are chronic conditions with no cure and with the increase in prevalence of systemic autoimmune diseases there is a need for early and accurate diagnostic techniques. Ther+efore, early detection of ANA by Gold Standard Indirect Immunofluorescence test in a Clinically suspected cases helps to reduce disease morbidity and mortality



The authors are thankful to Dr. K. Nagamani Professor and Head of Department of Microbiology, to faculty, technicians, non-teaching staff of Department of Microbiology, Gandhi Medical College, Secunderabad, Telangana, India, for providing facilities to carry out the research work. We are also thankful to Medicine, Rheumatology and SPM departments for their support to carry out this work.



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