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ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 4  |  Page : 222-229

A comparative study of immunofluorescence pattern and line immune assay profile in the detection of the antinuclear antibodies


1 Department of Microbiology and Immunology, Lucid Medical Diagnostics, Kharkhana, Hyderabad, Telangana, India
2 Department of Microbiology and Immunology, Osmania Medical College, Koti, Hyderabad, Telangana, India

Date of Submission07-Sep-2019
Date of Acceptance19-Mar-2020
Date of Web Publication6-Jan-2021

Correspondence Address:
Dr. S Y Boorgula
Lucid Medical Diagnostics, Plot no 203, Vasavi Nagar, Kharkhana, Hyderabad - 500 015, Telangana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JDRNTRUHS.JDRNTRUHS_89_19

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  Abstract 


Background and Aims: To compare the efficacy of the Immunofluorescence and Line Immune Assay profile in the detection of ANA antibody. To ascertain an association between ANA patterns and the specific antibodies. To correlate the obtained ANA patterns with the Immunoassay.
Settings and Design: This cross sectional study was done at a tertiary care center at Hyderabad Telangana. The samples were tested for ANA by Indirect Immunofluorescence (IIF) method and by Line immunoassay (LIA) methods.
Materials and Methods: Serum samples of patients with a clinical suspicion of autoimmune disease were tested for ANA by indirect immunofluorescence (IIF) method and Line immunoassay (LIA) during January 2018-December 2018. Serum samples for indirect immunofluorescence were processed in 1:100 dilution using HEp–2010/liver/stomach/kidney biochip (EUROIMMUN AG). The serum samples were further processed for the line immunoassay in 1:100 dilution on nylon strips coated with purified and recombinant antigens as discrete lines with plastic backing coated with the antigens nRNP/Sm, Sm, SSA, Ro-52, SSB, PM-Scl, Scl-70, PCNA, Jo-1, CENP-B, dsDNA, nucleosomes, ribosomal protein-P, histones, anti-mitochondrial antibodies (AMA-M2) along with the control band. The analysis was done by the comparison of the intensity of reaction with the positive control line by image analysis.
Results: Out of 236 samples tested, 77 were tested positive for Immunofluorescence and Line immuno assay, 115 were tested negative for Immunofluorescence and Immunoblot assay, only 1 was tested positive for Immunofluorescence and negative for Line immunoassay and 43 were tested positive for Line immunoassay and negative for Immunofluorescence.
Conclusion: Detection of ANA by IIF method is a cost effective method. It may be used for the screening purposes for patients with or without clinical criterion for the autoimmune disease in daily clinical practice. The fluorescent pattern can also predict presence of certain specific antibodies in the sera. This may regulate one from requesting for line immunoassay, which is expensive. The Line Immuno assay may be reserved for those patients who need them for prognostic significance.

Keywords: Autoimmune disease, immunofluorescence, line immunoassay


How to cite this article:
Boorgula S Y, Jyothi Lakshmi G. A comparative study of immunofluorescence pattern and line immune assay profile in the detection of the antinuclear antibodies. J NTR Univ Health Sci 2020;9:222-9

How to cite this URL:
Boorgula S Y, Jyothi Lakshmi G. A comparative study of immunofluorescence pattern and line immune assay profile in the detection of the antinuclear antibodies. J NTR Univ Health Sci [serial online] 2020 [cited 2021 Feb 26];9:222-9. Available from: https://www.jdrntruhs.org/text.asp?2020/9/4/222/306130




  Introduction Top


Autoimmune disease is a condition which causes tissue destruction or organ malfunction due to autoimmune mechanisms.[1] Since the direct role of the autoimmune reaction in the disease pathogenesis is not always apparent, it is usually interpreted as a disease accompanied by an autoimmune phenomenon. Anti-nuclear antibody (ANA) testing is a cornerstone for the diagnosis of the autoimmune diseases.[1]

ANA occurs in various autoimmune diseases; systemic autoimmune rheumatic diseases (SARD), Sjögren's syndrome (SjS), mixed connective tissue disease (MCTD), systemic lupus erythematosus (SLE), systemic sclerosis (SSc), polymyositis (PM), dermatomyositis (DM) and primary biliary cirrhosis (PBC). Detection and classification of ANA forms a major criterion for diagnosing and differentiating these diseases.[1]

The Anti-Nuclear Antibody test exhibits reactivity against the nucleus and the sub cellular structures and cell organelles including cell surfaces, cytoplasm, nuclei, or nucleoli.[1] These antigens are mainly proteins, protein–nucleic acid complexes, macromolecular complexes, nucleic acids and protein.[1]

Most of the clinically important antibodies target RNA–protein or DNA–protein complexes.[1] The staining may be purely nucleolar, as seen in few Systemic Sclerosis patients, or purely cytoplasmic, as in anti-Jo-1 positive Polymyositis/Dermatomyositis patients. Hence ANA test is not just for “nuclear” staining.[1]

Even though innumerable number of laboratory tests are available for the ANA detection, Enzyme linked immunosorbent assay (ELISA)/Enzyme immunoassay (EIA), Indirect immunofluorescence antinuclear antibody test (IF-ANA) and Immunoblot assay are frequently used in regular practice.[2] Indirect immunofluorescence test by using Hep-2 (human epithelial cell tumor line) is a traditional technique for ANA diagnosis and is also considered as the “gold standard” for ANA testing in clinical practice with high sensitivity.[2] Positive fluorescence staining denotes the presence of ANA, but accurate identification of these antibodies is not possible. For detection of the specific antibody, some specialized techniques like Western blotting, Line Immuno Assay (LIA) or ELISA are employed.[3]


  Methods Top


The study was conducted from January 2018 – December 2018 at an accredited tertiary care center. Serum samples of patients who were suspected for Autoimmune Diseases requesting for Immuno fluorescence and Line Immunoassay were subjected for ANA testing.

Fresh whole blood samples were collected after patient consent was obtained at the phlebotomy section. Serum separated from the clotted blood samples by centrifugation was stored at 4°C if testing was planned within 72 hours or stored at -20°C for testing after three days (without freezing and thawing). A fasting blood sample was advised in order to avoid lipemic serum as it could result in increased background fluorescence or unclear staining pattern.

Ethical Clearance

Ethical approval for this study (Ethical Committee V.1.0., 10/01/2020) was provided by the Ethical Committee Sweccha Independent Ethics Committee, Hyderabad on 10 October 2020.


  Method for Immunofluorescence Top


Euroimmun AG kit was used for testing the immunofluorescence pattern. Serum sample is diluted 1:100 in PBS –Tween. 30 μl/field volume of diluted sample is transferred on to the biochip slide and incubated at room temperature for 30 min. Biochip slide is rinsed with PBS –Tween and immersed in Couplin jar containing PBS-Tween for 5 minutes.

Defined volume of fluorescein labeled antihuman globulin is applied to each reaction field and incubated for next 30 minutes. Couplin jar is filled with new PBS-Tween wash and then the slide is put back for 5 min. The mounting medium is placed onto a cover glass- volume per reaction. The visualization of the slide is done under fluorescence microscope at 40 × magnification. Based on the fluorescent intensity, the samples were graded (+, ++,+++,++++) and NEGATIVE.

  • Negative: A serum was considered as negative for antinuclear antibodies if[4]


The nuclear staining was less than or equal to negative control well with no clear discernible pattern.

The cytoplasm demonstrating weak staining, with brighter staining of non-chromosomal region of the mitotic cells, but with no clear discernible nuclear pattern.

  • Positive: A serum was considered positive if nucleus[4]


Shows a clear discernible pattern of staining in a majority of 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.

The test result was discarded if positive control sample failed to show precise results.

Grading of the immunofluorescence

  • +4 Brilliant apple green fluorescence
  • +3 Bright apple green fluorescence
  • +2 Clearly distinguishable, positive fluorescence
  • +1 Lowest specific fluorescence that enables the staining to be easily differentiated from the background fluorescence.



  Line Immune Assay Top


The serum samples were processed further for line immunoblotassay. Nylon strips coated with purified and recombinant antigens as discrete lines with plastic backing (EUROIMMUN AG) coated with the following antigens

  • nRNP/Sm, Sm, SSA,
  • Ro-52,
  • SSB,
  • Scl-70,
  • PM-Scl,
  • PCNA,
  • Jo-1,
  • CENP-B,
  • dsDNA, histones, nucleosomes, ribosomal protein-P, anti-mitochondrial antibodies (AMA-M2) were used, along with a control band.


Each channel is filled with 1.5 ml of diluted sample and incubated at room temperature for 30 minutes on a rocking shaker. Liquid is aspirated off from each channel and washed with 1.5 ml wash buffer on a rocking shaker. 1.5 ml substrate solution is pipetted into the channels and incubated for 10 minutes at room temperature. Later, substrate liquid is aspirated from each channel and each strip is washed with distilled water 3 times for1 minute. The strip is then placed on the evaluation protocol, air dried and evaluated [Image 1] and [Image 2].




  Results Top





  Discussion Top


The presence of ANA is the hallmark of autoimmune disease. Besides the dsDNA antibodies, antibodies against a variety of the other nuclear antigens can also cause positive ANA-IIF.[3]

Even though some IIF patterns strongly suggest the distinct specificities, additional tests are requested to demonstrate the antibody reactivities against the specific nuclear and cytoplasmic antigens. These tests are used to support either the diagnosis (disease specificity) or to identify the subsets of patterns that are prone to a particular disease manifestation (prognostic marker).

Detection of ANA by using traditional assays such as hemagglutination reactions and immunodiffussion are being replaced by the newer technologies such as ELISA, ANA-HEp-2. The advantage of these tests are that they focus on short hands-on time and economical costs. HEp-2 cell lines are derived from the cultured human laryngeal epithelial carcinoma and are more sensitive than the animal (mouse line or rat kidney) tissue sections to detect the presence of antinuclear antibodies both in patients and controls.

But it has high sensitivity rates, which results in a high false positive rate for antinuclear antibodies, making the interpretation of a positive test result very difficult.[3]

A biochip incorporating HEp-2 cells and primate liver, rat stomach and rat kidney is used to overcome this problem, thus it incorporates the good of both the old and the new technologies. Due to this, false positives are markedly reduced by IIF method in this study.

For patients with suspected autoimmune disease a variety of tests are requested. These tests should be reliable, cost effective. In this study, diagnostic value and cost effectiveness of ANA pattern (costing approximately Rs 1130 per test) with line immunoassay (Rs 4800 per test) was also compared.

In this study [Table 1] and [Table 2] and [Figure 1] and [Figure 2], 236 serum samples satisfied the definite selection criteria and processed for both ANA and line immunoassay tests. 77/236 (32.6%) of serum samples were positive for both ANA and line immunoassay wherein the florescent ANA pattern could be correlated with the presence of specific antibodies delineated by the immunostrip. The most common ANA pattern observed in this study was Speckled pattern [Table 3] and [Table 4], [Figure 3] and [Figure 4] and [Image 3] (n = 47, 61.03%). SSA/Ro-52, SSB in all of the cases and in varying combinations with RIB and RNP/Sm was observed. Hence SSA/Ro-52 and SSB presence was seen in 100% of cases with the speckled pattern.
Table 1: Number of Samples Tested

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Table 2: Gender Preponderance

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Figure 1: Total Number of Samples tested

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Figure 2: Gender preponderance

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Table 3: Spectrum of ANA-IIF Patterns Observed in the Study Population

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Table 4: Immunoassay Details of ANA-IIF Speckled Pattern (Number: 47 Samples)

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Figure 3: Spectrum of ANA.IIF patterns observed in the study population

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Figure 4: Immunoassay details of ANA-IIF speckled pattern (Number: 47 samples) Others- SSA, dsDNA, Histones, AMA, Sm, Nucleosomes, Ribosomal pattern, Ro52, SS-B, PM-SCL

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The next most common ANA pattern observed in this study was Homogenous [Image 4] and [Table 5] and [Figure 5] (n = 24, 31.16%). It showed an association with dsDNA (n = 9), PCNA and nucleosomes (n = 4) with variable intensities of Sm, dsDNA (n = 3). Most of the cases with Homogenous pattern had antibodies against dsDNA, nucleosomes and histones.

Table 5: Immunoassay Details of 24 Samples with ANA-IIF Homogenous Pattern

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Figure 5: Immunoassay details of 24 samples with ANA-IIF Homogenous pattern

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The centromeric [Table 6] and [Image 6] homogenous pattern with nucleoli accentuation [Image 7] and nucleolar dots patterns [Image 5] were seen only in two cases each (2.59%). Fifty per cent of cases (n = 3) were positive for centromeric protein-B. Scl-70 (n = 1, 2%) was seen with the nucleolar pattern. Due to a small number, no definite correlation could be drawn between the nuclear pattern and the antibodies present in case of nucleolar pattern.
Table 6: Immunoassay Details of 8 Samples with ANA-IIF Other Patterns

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Out of 236, 77 were tested positive on IF and Line immunoassay whereas only one was tested positive on IIF, with line immunoassay being negative [Table 1]. Review of both IIF and line immunoassay was done and based on the clinical history, the pattern was not found to be significant. On the contrary, 43 were tested positive for Line immunoassay and negative for IIF. 7 of these sera tested positive for SSA/Ro-52 while 2 tested positive for Scl-70 and 4 tested positive for Jo-1.

A similar observation was made by Vos et al. and Hoffman et al.[5] This is explained by the fact that the line immunoassay is more sensitive to detect SSA/Ro-52 than ANA-IIF even when Hep-2000 cells were used. Scl-70 antibodies, which is of importance in polymyositis was also missed with Immunofluorescence. Scl-70 reactivity goes unreported with ANA-IIF as Scl -70 antibodies exhibit a cytoplasmic positivity rather than nuclear staining pattern on IIF they might be not reported and ANA could have been reported as negative. Hence, cytoplasmic staining pattern identification is equally important.

The ANA patterns with their corresponding antibodies are available for one's reference from the Western literature. The present study results are compared with the Western literature and Dr. Usha kini study [Table 7], which is an extensive study conducted on the South Indian population. The findings of the present study are comparable with those in Western literature and Dr. Usha kini study. There is no difference in correlation between speckled, homogenous and centromere patterns and their corresponding antibodies in sera. But few cases of homogenous with nucleoli accentuation and nuclear dot patterns were insufficient to draw a comparison with line immunoassay in our study. Patterns such as nuclear dots and homogenous with nucleoli accentuation were not observed in Dr. Usha Kini study.
Table 7: Comparison of Immunoassay Details of the Present Study Population with Western Literature

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In conclusion, detection of ANA by indirect immunofluorescence is the most cost - effective test in investigating autoimmune disease. Indirect Immunofluorescence can be done for screening purposes for patients with or without any clinical symptoms for autoimmune disease in routine clinical practice. The fluorescent pattern predicts the presence of certain specific antibodies in sera. These correlations are important for the diagnosis of a specific autoimmune disease and probably line immunoassay can be used as a special investigation especially for those patients who require them for the prognostic significance. Cytoplasmic pattern in ANA-IIF are equally important as the nuclear pattern though they are read as ANA-negative and those sera must be subjected for line immunoassay as they help in the diagnosis of a specific rheumatic disease or autoimmune disease.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient (s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Satoh M, Chan EKL, Sobel ES, Kimpel DL, Yamasaki Y, Narain S, et al. Clinical implication of autoantibodies in patients with systemic rheumatic diseases. Exp Rev Clin Immunol 2007;3:721-38.  Back to cited text no. 1
    
2.
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.  Back to cited text no. 2
    
3.
Sebastian W, Roy A, Kini U, Mullick S. Correlation of antinuclear antibody immunofluorescence patterns with immune profile using line immunoassay in the Indian scenario. Ind J Pathol Microbiol 2010;53:427-32.  Back to cited text no. 3
    
4.
Garg S, Srivastava A, Prasad S. Correlation of line immuno assay with indirect immunofluoresence assay for the detection of anti-nuclear antibodies in various autoimmune disorders. J Autoimmune Disord 2017;3:37.  Back to cited text no. 4
    
5.
Vos PA, Bast EJ, Derksen RH. Cost-effective detection of non-antidouble- stranded DNA antinuclear antibody specificities in daily clinical practice. Rheumatology (Oxford) 2006;45:629-35.  Back to cited text no. 5
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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Abstract
Introduction
Methods
Method for Immun...
Line Immune Assay
Results
Discussion
References
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