Assessment of Ideal Serum Dilution for Screening of Antinuclear Antibodies by an Indirect Immunofluorescence Method in Diagnosis of Autoimmune Disorders

Overview

Journal Med J Armed Forces India

Specialty General Medicine

Date 2022 Jan 17

PMID 35035044

Authors

Vivek Vasdev

S K Patnaik

D S Bhakuni

K Shanmuganandan

A Bhayana

G Mullick

A Hegde

Ashwini Kumar

Abhishek Kumar

R Singh

Affiliations

Soon will be listed here.

Abstract

Background: Time and cost constraints lead to majority of clinical laboratories deviating away from an ideal practice of checking for antinuclear antibodies (ANAs) by indirect immunofluorescence (IIF) at multiple dilutions. Usage of screening dilution of 1:40 recommended by most manufacturers of commercial ANA kits results in numerous false positive-tests and misdiagnosis of connective tissue disorders (CTDs). We sought to study the ideal screening dilution for ANA by IIF for a diagnosis of ANA-related CTDs.

Methods: Serum samples of patients with ANA-related conditions (n = 233) and healthy controls (n = 154) were evaluated by IIF using Immuno Concepts Hep-2000 ® ANA kits at dilutions from 1:40 to 1:640. Accuracy for diagnosis of CTDs for each serum dilution was assessed by receiver operating curve (ROC) analysis.

Results: Antinuclear antibodies (ANA) positivity was observed in 19.5%, 10.4%, 4.55%, 0.65%, and 0% of healthy controls at dilutions of 1:40, 1:80, 1:160, 1:320, and 1:640, respectively. ANA positivity at 1:40 dilution was observed among 26.4% cases with mimics of CTDs. Prevalence of ANA positivity in ANA-related CTDs was 97.3%, 96.4%, 89.3%, 83.9%, and 71.4% at dilutions of 1:40, 1:80, 1:160, 1:320, and 1:640, respectively. ROC analysis revealed best test performance for distinction between healthy and ANA-related CTD populations at a serum dilution of 1 in 80.

Conclusions: Antinuclear antibodies (ANA) positivity at low titers (1:40) is highly prevalent in healthy population (19.5%) as well as amongst mimics of CTD (26.4%). Our study suggests a higher screening dilution of 1:80 for ANA by IIF for diagnosis of CTD maybe better. Combination of 1:80 and 1:160 dilutions provides optimum sensitivity and specificity for diagnosis of ANA-related disorders.

References

Divate S, Hardikar P, Bichile L, Rajadhyaksha A . Clinical utility of screening for antinuclear antibodies by enzyme immunoassay--a preliminary study. J Assoc Physicians India. 2005; 52:290-3. View

Koshy M, Mathew J, Alex R, Jude J, Ralph R, Sudarsanam T . Antinuclear antibodies in scrub typhus: Transient occurrence during acute illness. J Vector Borne Dis. 2018; 55(1):52-57. DOI: 10.4103/0972-9062.234627. View

Solomon D, Kavanaugh A, Schur P . Evidence-based guidelines for the use of immunologic tests: antinuclear antibody testing. Arthritis Rheum. 2002; 47(4):434-44. DOI: 10.1002/art.10561. View

Tan E, FELTKAMP T, Smolen J, Butcher B, Dawkins R, Fritzler M . Range of antinuclear antibodies in "healthy" individuals. Arthritis Rheum. 1997; 40(9):1601-11. DOI: 10.1002/art.1780400909. View

Minz R, Kumar Y, Saikia B, Anand S, Varma S, Singh S . Use of panel testing for detection of antinuclear antibody in a resource-limited setting: an appraisal. Postgrad Med. 2016; 128(8):869-874. DOI: 10.1080/00325481.2016.1220808. View

Aggarwal A . Clinical application of tests used in rheumatology. Indian J Pediatr. 2002; 69(10):889-92. DOI: 10.1007/BF02723714. View

Wallace D . New methods for antinuclear antibody testing: does it cut costs and corners without jeopardizing clinical reliability?. Nat Clin Pract Rheumatol. 2006; 2(8):410-1. DOI: 10.1038/ncprheum0253. View

Phan T, Wong R, Adelstein S . Autoantibodies to extractable nuclear antigens: making detection and interpretation more meaningful. Clin Diagn Lab Immunol. 2002; 9(1):1-7. PMC: 119916. DOI: 10.1128/cdli.9.1.1-7.2002. View

Narain S, Richards H, Satoh M, Sarmiento M, Davidson R, Shuster J . Diagnostic accuracy for lupus and other systemic autoimmune diseases in the community setting. Arch Intern Med. 2004; 164(22):2435-41. DOI: 10.1001/archinte.164.22.2435. View

10.

Bonaguri C, Melegari A, Ballabio A, Parmeggiani M, Russo A, Battistelli L . Italian multicentre study for application of a diagnostic algorithm in autoantibody testing for autoimmune rheumatic disease: conclusive results. Autoimmun Rev. 2011; 11(1):1-5. DOI: 10.1016/j.autrev.2011.06.006. View

11.

Kavanaugh A, Tomar R, Reveille J, Solomon D, Homburger H . 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(1):71-81. DOI: 10.5858/2000-124-0071-GFCUOT. View

12.

Campos-Gonzalez I, Viveros M, Cardiel M . [Clinical utility of specialized immunologic testing in rheumatology in a secondary level hospital in Mexico]. Reumatol Clin. 2011; 3(3):110-6. DOI: 10.1016/S1699-258X(07)73677-X. View

13.

Fernandez S, Lobo A, Oliveira Z, Fukumori L, P rigo A, Rivitti E . Prevalence of antinuclear autoantibodies in the serum of normal blood dornors. Rev Hosp Clin Fac Med Sao Paulo. 2004; 58(6):315-9. DOI: 10.1590/s0041-87812003000600005. View

14.

Marin G, Cardiel M, Cornejo H, Viveros M . 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(7):325-9. DOI: 10.1097/RHU.0b013e3181bb971b. View

15.

Ferrari R . Evaluation of the Canadian Rheumatology Association Choosing Wisely recommendation concerning anti-nuclear antibody (ANA) testing. Clin Rheumatol. 2015; 34(9):1551-6. DOI: 10.1007/s10067-015-2985-z. View

16.

Kumar Y, Bhatia A, Minz R . Antinuclear antibodies and their detection methods in diagnosis of connective tissue diseases: a journey revisited. Diagn Pathol. 2009; 4:1. PMC: 2628865. DOI: 10.1186/1746-1596-4-1. View

17.

Wanchu A . Antinuclear antibodies: clinical applications. J Postgrad Med. 2000; 46(2):144-8. View

18.

Ghosh P, Dwivedi S, Naik S, Agarwal V, Verma A, Aggarwal A . Antinuclear antibodies by indirect immunofluorescence : optimum screening dilution for diagnosis of systemic lupus erythematosus. Indian J Med Res. 2007; 126(1):34-8. View

19.

Minz R, Kumar Y, Anand S, Singh S, Bamberi P, Verma S . Antinuclear antibody positive autoimmune disorders in North India: an appraisal. Rheumatol Int. 2011; 32(9):2883-8. DOI: 10.1007/s00296-011-2134-1. View

20.

Gupta R, Agarwal S, Jain M, Malhotra V, Sarin S . Autoimmune hepatitis in the Indian subcontinent: 7 years experience. J Gastroenterol Hepatol. 2001; 16(10):1144-8. DOI: 10.1046/j.1440-1746.2001.02602.x. View