MINI REVIEW

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

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

Amrit Kaur Kaler¹, Ravi Gaur², Anita Jain³, Junu Rajan⁴, US Vishal Rao^5
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

Correspondence Address:
Dr. Amrit Kaur Kaler
Consultant Molecular Pathologist, Department of Laboratory Medicine, Kokilaben Dhirubhai Ambani Hospital, Mumbai, Maharashtra
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jpo.jpo_11_22

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

Introduction

Table 1: Tumors related to positive anti-nuclear antibodies titres[8] Click here to view

Methods of Detection of Anti-Nuclear Antibodies

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] Click here to view

Figure 2: Detection by multi immune assay[16] Click here to view

Conclusion

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