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Table of Contents
Year : 2022  |  Volume : 2  |  Issue : 1  |  Page : 36-39

Detection of antinuclear antibodies in oncology from slide to multiplexing: An overview

1 Department of Laboratory Medicine, Kokilaben Dhirubhai Ambani Hospital, Mumbai, Maharashtra, India
2 Oncquest, Dr G Path Labs, New Delhi, India
3 Dr. Anita's Laboratories, Karnal, Haryana, India
4 Department of Clinical Pathology, Ahhalia Hospital Musaffah Industrial, Abu Dhabi, UAE
5 Centre for Academic Research, HCG Cancer Center, Bengaluru, Karnataka, India

Date of Submission11-Jun-2021
Date of Decision09-Nov-2021
Date of Acceptance13-Mar-2022
Date of Web Publication03-May-2022

Correspondence Address:
Dr. Amrit Kaur Kaler
Consultant Molecular Pathologist, Department of Laboratory Medicine, Kokilaben Dhirubhai Ambani Hospital, Mumbai, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jpo.jpo_11_22

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Antinuclear antibodies (ANA) are autoantibodies considered as the immune biomarkers of systemic autoimmune diseases. ANAs are directed against antigens of the cell nucleus and are named after their biochemical characteristics (deoxyribonucleic acid [DNA], histones, ribonucleoprotiens), the disease is associated with the corresponding autoantibody, e.g., Sjögren syndrome antigen A [SS-A] and Systemic Sclerosis (SS-B); progressive systemic sclerosis, polymyositis, or occasionally after the patient in whom the corresponding antibody was first detected (Sm, Ro, La). Positive ANA can also be used as an aid to early diagnosis of solid tumors and prognosis in hematological malignancies such as non-Hodgkin's lymphoma patients. Positive ANA has been found in the sera from patients with head and neck, lung, breast cancers by multiple studies. Most commonly patients with chronic lymphocytic leukemia (CLL) frequently present with autoimmune disorders (AIDs) which include autoimmune hemolytic anemia (AIHA), immune thrombocytopenia, pure red cell aplasia and autoimmune granulocytopenia, and nonhematological AIDs such as paraneoplastic pemphigus, neuropathies, SS, rheumatoid arthritis, and systemic lupus erythematosus. The presence of AIHA was previously demonstrated to be a poor prognostic indicator and also proved the negative survival impact of positive direct antiglobulin test on CLL patients.

Keywords: Antinuclear antibodies, oncology, technologies, multiplexing

How to cite this article:
Kaler AK, Gaur R, Jain A, Rajan J, Vishal Rao U S. Detection of antinuclear antibodies in oncology from slide to multiplexing: An overview. J Precis Oncol 2022;2:36-9

How to cite this URL:
Kaler AK, Gaur R, Jain A, Rajan J, Vishal Rao U S. Detection of antinuclear antibodies in oncology from slide to multiplexing: An overview. J Precis Oncol [serial online] 2022 [cited 2023 Jun 8];2:36-9. Available from: https://www.jprecisiononcology.com//text.asp?2022/2/1/36/344532

  Introduction Top

Anti-nuclear antibodies (ANA) are a specific class of autoantibodies that have the capability to bind and destroy a variety of nuclear and cytoplasmic antigens within the cell.[1] Although the antibodies can be seen in low quantities in the normal population, a spurt in titers can be observed in patients with autoimmune disorders (AIDs); and even found associated with certain malignancies such as non-Hodgkin's lymphoma, hematological cancer, breast and lung cancer.[2] Several authors also found the connection between the subsequent risk of cancer in autoimmune diseases such as lung cancer in rheumatoid disorders; lymphoma in Sjogren's syndrome; gastrointestinal tumors in association with dermatomyositis.[3],[4],[5] This constitutes the basis for diagnosis, determine the prognosis and its significance in therapeutic monitoring of autoantibodies in certain malignant patients.

In cancer patients, autoantibodies may be broadly classified into two categories (I) anti-nuclear antibodies directed against antigens that play a role in the regulation of cell cycle and mitosis and not specific to the tumor, such as anti-ds deoxyribonucleic acid (DNA), anti-histone antibodies, anti-Sm antibodies, anti-La antibodies, anti-Ro antibodies, and anti-centromeric antibodies. (II) antibodies against specific tumor antigens, for example, tumor-associated antigens as oncoproteins and tumor suppression genes like p53.[6],[7],[8],[9],[10] Both types of antibodies have shown to have a protective role against tumor spread. The possible explanation of the anti-tumor activity of ANA is based on be antibody-dependent cell-mediated cytotoxicity, the release of cytokines that enhance the immune function, and reduction of the inhibitory effect of extracellular chromatin on natural killer cell activity through the binding of ANAs.[7] The present article focuses on the evolution of various diagnostic techniques in the diagnosis of ANA in a clinical setup. The table represents various malignancies associated with a rise in different ANA titers [Table 1].
Table 1: Tumors related to positive anti-nuclear antibodies titres[8]

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Anti-nuclear antibodies profiles

Most laboratories provide a cost-effective “ANA profile,” which includes information on DNA, histone, extractable nuclear antigens (Sjögren syndrome [SS]-A, SS-B, Sm, Scl-70, Jo-1, and ribonucleoprotiens), anti-ribosomal P antibodies, anti-centromere antibodies, topoisomerase I, and other circulating autoantibodies.

  Methods of Detection of Anti-Nuclear Antibodies Top

  1. Slide method: The diagnostic potential of ANAs originated with the discovery of Lupus Erythematosus cells in 1948 by Hargraves, who described them as mature polymorphonuclear leukocytes containing phagocytosed degraded nuclear material in the cytoplasm. The slides prepared from buffy coat produced after centrifugation of blood sample gives a specificity of 50%–70%.[9] Further research demonstrated that immunoglobulins (antibodies) from patient plasma binds to the nuclei of an injured cell, which in turn are phagocytosed by neutrophils.
  2. Indirect Immunofluorescence (IIF) remains the gold standard for the detection of anti-nuclear antibodies. It is based on a simple immunological principle that involves the incubation of patient sera with tissue substrate (Hep-2 cells) fixed on a glass slide and the adding fluorescein-labeled anti-human IgG.[10],[11],[12],[13],[14],[15],[16] The slide is viewed under a fluorescence microscope for the bound ANAs to the specific antigens [Figure 1].[16] The results are reported based on the staining pattern and titer. The International consensus on antinuclear antibody pattern defined and described six major patterns of staining, which include homogenous, speckled, centromere, or nucleolar, cytoplasmic, and nuclear dots, all of which suggest associations with certain autoimmune diseases.[12],[13]

    IIF is highly sensitive and has broad screening potential due to a large number of antigens, and it gives an ability to interpret the extensive data in a wide variety of clinical scenarios.[14] Other than the analytic challenges, the ANA testing can be labor-intensiveness, require highly trained staff, gives high positive results, and also yields discrepant inter-laboratory results. Several companies like Euroimmune, etc., have introduced automated computer-assisted pattern recognition systems, which include acquiring, storing, analyzing digital images of stained slides and displaying on the monitors for review.[15] This automation significantly minimizes the manual and analytic challenges, enhances ANA testing standardization, and helps reduce inter-laboratory variability. However, the laboratory community needs further evidence to determine whether the automated systems can be put into routine practice.
  3. Enzyme-linked Immunosorbent Assay (ELISA): It is one of the most versatile techniques available in laboratories. ELISA assays contain a mixture of known specific anti-nuclear antigens, coated on a multi-well plate to detect antibodies in the patient's serum, and the detection occurs through an enzyme-labeled anti-human immunoglobulin. The enzyme-linked to the detection antibody converts a colorless substrate to a colored product, the absorbance of which is compared to a standard curve generated by known concentrations of ANA. The results are reported as a numeric value of measurement, and each manufacturer establishes the units and analytical measuring range for its tests.[17],[18]

    ELISA is increasingly adopted as they can be automated, speed and simplicity of the assay, do not require a high level of skill to operate and reduces the number of subjective measurements. However, the clinical utility is limited as they carry defined antigen targets on the ELISA plate, which gives a lower sensitivity but excellent specificities. Hence, they may not be useful in the evaluation of target complex antibodies in autoimmune hepatitis. Sometimes, a conformational change in antigenic structures may result in inaccurate results, leading to strict quality control procedures. Differences in affinity to antibodies or contamination may lead to multiple assays to different antibodies.[2],[15]
  4. Electro chemiluminescence Assay (eCLIA): It has a larger analytic measurement range than ELISA, is more sensitive, and is easier to automate. It has been a preferred method for high throughput analysis compared to ELISA. ECLIA uses an enzyme to transform a substrate into a reaction product that emits a photon of light rather than producing a visible color as in ELISA[19]
  5. Multiple Immunoassay (MIA): It has a capacity to detect multiple independent antibodies associated with ANA. MIA utilizes a series of polystyrene bead sets, each coated with pure or recombinant antigens and showing distinct Immunofluorescence intensities [Figure 2].[16] Following exposure to diluted patient specimens, the presence of autoantibodies can be determined either qualitatively or quantitatively by determining the collective assessment of all the individual antigen specificities included in an assay. MIA is more efficient than conventional ELISA and technically less challenging than IF-ANA screening, decreases the false positivity, and removes the subjectivity. It also has the advantage of providing the simultaneous detection of several autoantibodies of diagnostic relevance.[20],[21]
  6. Newer techniques like flow cytometry and nanotechnology (Microarray) are completely automated and show increased the sensitivity and specificity but its usefulness is limited by its cost-effectiveness.[2]
Figure 1: Detection of anti-nuclear antibodies with Indirect Immunofluorescence[16]

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Figure 2: Detection by multi immune assay[16]

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  Conclusion Top

The changes in ANA titer may reflect early carcinogenic events or may reflect an autoimmune response to nuclear antigens related to the expression of particular antigens at different stages of tumor progression that are perturbed in cellular transformation. The progress of diagnosing ANA from the LE cell to fully automated ELISA/multiplexing, has been meteoric. Advances in computer-aided pattern recognition may be useful in the future for harmonizing ANA diagnosis. Finally, clinical laboratory analysis will benefit from advancements in fluorescent nanotechnology.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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Baecklund E, Smedby KE, Sutton LA, Askling J, Rosenquist R. Lymphoma development in patients with autoimmune and inflammatory disorders – What are the driving forces? Semin Cancer Biol 2014;24:61-70.  Back to cited text no. 4
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Vlagea A, Falagan S, Gutiérrez-Gutiérrez G, Moreno-Rubio J, Merino M, Zambrana F, et al. Antinuclear antibodies and cancer: A literature review. Crit Rev Oncol Hematol 2018;127:42-9.  Back to cited text no. 8
Hargraves MM, Richmond H, Morton R. Presentation of two bone marrow elements; the tart cell and the L.E. cell. Proc Staff Meet Mayo Clin 1948;23:25-8.  Back to cited text no. 9
McCarty GA. Autoimmunity and malignancy. Med Clin North Am 1985;69:599-615.  Back to cited text no. 10
Fernandez-Madrid F, Mattioli M. Antinuclear antibodies (ANA): Immunologic and clinical significance. Semin Arthritis Rheum 1976;6:83-124.  Back to cited text no. 11
Chan EK, Damoiseaux J, Carballo OG, Conrad K, de Melo Cruvinel W, Francescantonio PL, et al. Report of the first international consensus on standardized nomenclature of antinuclear antibody HEp-2 cell patterns 2014-2015. Front Immunol 2015;6:412.  Back to cited text no. 12
Tešija Kuna A, Đerek L, Drvar V, Kozmar A, Gugo K. Assessment of antinuclear antibodies (ANA): National recommendations on behalf of the Croatian society of medical biochemistry and laboratory medicine. Biochem Med (Zagreb) 2021;31:020502.  Back to cited text no. 13
Indirect Immunofluorescence: An Easy and Modern Method. Available from: https://www.euroimmun.us/technology/immunofluorescence. [Last accessed on 2021 Dec 20].  Back to cited text no. 14
Voigt J, Krause C, Rohwäder E, Saschenbrecker S, Hahn M, Danckwardt M, et al. Automated indirect immunofluorescence evaluation of antinuclear autoantibodies on HEp-2 cells. Clin Dev Immunol 2012;2012:651058.  Back to cited text no. 15
Sohn KY. ANA Testing: From Microscopy to Multiplexing. Clinical Laboratory News. Available from: https://www.aacc.org/cln/articles/2014/june/ana-testing. [Last accessed on 2021 Dec 20].  Back to cited text no. 16
Alsaed OS, Alamlih LI, Al-Radideh O, Chandra P, Alemadi S, Al-Allaf AW. Clinical utility of ANA-ELISA vs. ANA-immunofluorescence in connective tissue diseases. Sci Rep 2021;11:8229.  Back to cited text no. 17
Baronaite R, Engelhart M, Mørk Hansen T, Thamsborg G, Slott Jensen H, Stender S, et al. A comparison of anti-nuclear antibody quantification using automated enzyme immunoassays and immunofluorescence assays. Autoimmune Dis 2014;2014:534759.  Back to cited text no. 18
Kricka LJ, Thorpe GH, Stott RA. Enhanced chemiluminescence enzyme immunoassay. Pure and Applied Chemistry 1987;59:651-4. Available from: https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/chemiluminescence-immunoassays. [Last accessed on 2021 Dec 20].  Back to cited text no. 19
Aleksandrova EN, Verizhnikova ZG, Novikov AA, Panafidina TA, Popkova TV, Lukina GV. Clinical value of multiplex immune assay of antinuclear antibodies in systemic lupus erythematosus. Klin Lab Diagn 2018;63:434-8.  Back to cited text no. 20
Wei Q, Jiang Y, Xiao M, Zhang X, Qi J, Xie J, et al. Comparison of chemiluminescence microparticle immunoassay, indirect immunofluorescence assay, linear immunoassay and multiple microbead immunoassay detecting autoantibodies in systemic lupus erythematosus. Scand J Immunol 2020;91:e12849.  Back to cited text no. 21


  [Figure 1], [Figure 2]

  [Table 1]


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