ANA, IFA, Cascade and Rheumatoid Arthritis Panel 2, With Reflexes

This test is used to evaluate patients with suspected autoimmune rheumatic disease.

Test Summary

ANA, IFA, Cascade and Rheumatoid Arthritis Panel 2, With Reflexes

Test code: 94954

Clinical use

Evaluate suspected autoimmune rheumatic diseases

Clinical background

Autoimmune rheumatic diseases are conditions in which the immune system attacks the joints and certain systems. They are often difficult to diagnose, as their symptoms can be vague, vary from patient to patient, and often overlap. Laboratory testing can provide useful information, but no single test provides a definitive diagnosis for any one rheumatic disease.

Diagnosis is most often based on a compilation of symptoms and signs, which include clinical information and laboratory test results. Testing for antinuclear antibodies (ANAs) using an immunofluorescence assay (IFA) is a good first approach for laboratory evaluation of patients suspected of having certain autoimmune rheumatic diseases.

ANAs are a group of autoantibodies directed against diverse nuclear and cytoplasmic antigens and are associated with several autoimmune rheumatic diseases (Table 1).1-4 These include systemic lupus erythematosus (SLE), Sjögren syndrome, systemic sclerosis (SSc), and mixed connective tissue disease (MCTD). Although various platforms can be used to detect ANAs, an IFA with HEp-2 cells remains the gold standard because of its high sensitivity for several of the autoimmune rheumatic diseases.5,6 The high sensitivity stems from the large number of autoantigens (up to 150) in HEp-2 cells.

The diagnostic value of ANA testing varies with the specific clinical condition (Table 1).1-4 For example, positive ANA results are required for diagnosis of drug-induced lupus and MCTD.2 When SLE or SSc is suspected, ANA testing is recommended.1,2 While ANA testing is less useful (ie, not specific) for diagnosing polymyositis or dermatomyositis, rheumatoid arthritis, and Sjögren syndrome, it can be used to screen for, or in the workup of, such diseases. When Sjögren syndrome is suspected, for example, testing can help clarify whether an underlying connective tissue disease exists.1,2

Table 1. Utility of Antinuclear Antibody IFA Test by Condition

Condition	Comments/recommendations1,2,7
Drug-induced lupus	Positive ANA part of the diagnostic criteria ANA useful for symptomatic people who are taking a drug associated with drug-induced lupus
Mixed connective tissue disease (MCTD)	Positive ANA part of the diagnostic criteria ANA recommended when clinical suspicion of MCTD Follow-up with RNP antibody recommended to confirm diagnosis
Autoimmune hepatitis	Positive ANA part of diagnostic criteria Positive ANA often seen in patients with diverse liver disease; does not exclude other hepatic diseases
Systemic lupus erythematosus (SLE)3	Positive ANA part of classification criteria ANA sensitivity, 93%; specificity, 57% Best initial test when clinical suspicion of SLE is strong SLE unlikely if ANA negative Specific antibody tests may be considered as follow-up to positive ANA
Systemic sclerosis (SSc)	ANA sensitivity, 85%; specificity, 54% ANA recommended when clinical suspicion of SSc If negative, consider other fibrosing illnesses (eg, eosinophilic fasciitis, linear scleroderma)
Sjögren syndrome4	ANA sensitivity, 48%; specificity, 52% (if ANA titer ≥320, sensitivity, 72.8% [95% CI, 67.5-77.7%]; specificity, 80.4% [95% CI, 76.9-84.0%]) Can help clarify whether an underlying connective tissue disease exists when Sjögren syndrome suspected
Polymyositis or dermatomyositis	ANA sensitivity, 61%; specificity, 63% Positive ANA provides weak evidence of disease even when combined with clinical suspicion Must consider other connective tissue diseases (SLE or overlap syndrome) regardless of ANA status

ANA, antinuclear antibody test; RNP, ribonucleoprotein.
a	The American College of Rheumatology Ad Hoc Committee on Immunologic Testing Guidelines7

Knowing the ANA titer can help interpret positive ANA results. A titer of at least 1:40 is considered positive, but low-positive titers (eg, 1:40) are not uncommon in healthy individuals.8 For example, about 20% to 30% of healthy individuals will have a positive ANA when screened at a 1:40 dilution, but this prevalence drops to 15% when screened at 1:80 and to 5% when screened at 1:160.2 Nevertheless, the sensitivity of ANA tests for SLE, systemic sclerosis, and Sjögren syndrome can be increased by using a low threshold of 1:40.9

For patients with positive ANA screening results, nuclear, cytoplasmic, and mitotic antibody fluorescence patterns can be used to evaluate the likelihood of certain autoimmune diseases. These patterns may inform the differential diagnosis, although they may not be specific for individual antibodies or diseases. For example, a homogeneous nuclear pattern may be associated with SLE, drug-induced SLE/vasculitis, or juvenile idiopathic arthritis (JIA), while a diffuse cytoplasmic pattern could be consistent with SLE or an inflammatory myopathy.6

Given the range of conditions associated with positive ANA IFA results, positive results are not highly specific for any single condition. Tests for individual antibodies may offer greater specificity and are recommended to help establish the diagnosis.2,5,10,11 The combination of the highly sensitive ANA IFA screen, followed by specific autoantibody testing when the IFA is positive, can help maximize sensitivity as well as specificity.5 Table 2 summarizes the sensitivity of individual antibodies across disorders. Testing tiers of specific antibodies following a positive ANA IFA result, as depicted in the Figure, can help speed the evaluation.5

Table 2. Antibody Prevalence in Rheumatic and Related Diseases and Healthy Individuals,a %

Analyte	SLE 3,12	MCTD 13	SSc 14	SS 4	PM/DM 15	CREST syndromeb	Neurologic SLE 16	HI
ANA2,17,18	90-95	90-100	85-95b	50-60	50-60	70		20-30
CENP-B2	2-5	2-5	20-40b,c	5-10	1-3	66		<3
Chromatin2	40-70	5-18	<3	<3	<3			<3
dsDNA2	40-70	<3	<3	<3	<3			<3
Jo-12	1-3	<2d	1-3	<2	15-30			<1
MCV19-21	12-36		10	10				≤5
Rib P2	10-30	<2	<2	<2	<2		10-47	<1
RNP2	10-40	100	5-15	<3	5-15			<3
Scl-702	0-5	<3e	20-40c	<3	<3			<1
Sm2	5-20	<2	<2	<1	<1			<1
Sm/RNP11,22	30	100	4	9	5
SS-A/Ro2	40-70	<3	3-10	60-90	<3			<3
SS-B/La2	15-30	<3	1-5	60-80	5-15			<3

ANA, antinuclear antibody; CENP-B, centromere B; dsDNA, double-stranded DNA; HI, healthy individuals; Jo-1, histidyl-tRNA synthetase; MCTD, mixed connective tissue disease; Rib P, ribosomal P; MCV, mutated citrullinated vimentin; PM/DM, polymyositis or dermatomyositis; RNP, ribonucleoprotein; Scl-70, scleroderma-70 (topoisomerase 1); SLE, systemic lupus erythematosus; Sm, Smith; Sm/RNP, Smith/ribonucleoprotein; SS, Sjögren syndrome; SS-A, SS-B, Sjögren antibodies A and B; SSc, systemic sclerosis.
a	Highlighted antibodies represent classification or diagnostic criteria for the disease. Note that antibodies included in criteria are not always those with the highest prevalence in that disease.
b	CREST syndrome is a variant of systemic sclerosis defined by the presence of calcinosis cutis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia. Also known as limited cutaneous scleroderma. ANA is present in 70% of CREST patients, and CENP-B is present in 90% to 95%.23
c	The presence of scleroderma-related antibodies (centromere, Scl-70, or RNA polymerase III antibodies) is not necessary or sufficient for diagnosis, but is useful for classification in the absence of diagnostic clinical findings (ie, "skin thickening of the fingers extending proximal to the metacarpophalangeal joints"14).
d	Especially in patients with features of muscle inflammation.
e	Especially in patients with features of systemic sclerosis.

Figure. ANA, IFA, Cascade with Rheumatoid Arthritis Panel 2, With Reflexes (test code 94954).5,12,19,24-28

The ANA, IFA, Cascade with Rheumatoid Arthritis Panel 2, with Reflexes (test code 94954) consists of 2 main components: (1) an ANA IFA component that reflexes to titer and specific antibody tests for autoimmune rheumatic diseases; and (2) an RA component (Figure). The Rheumatoid Arthritis panel 2 includes widely used laboratory markers for RA such as rheumatoid factor (RF) and cyclic citrullinated peptide (CCP) antibodies. However, 20% or more of patients with RA are seronegative (negative for both RF and CCP).29 For these patients, another antibody that can help diagnose RA is mutated citrullinated vimentin (MCV).19 Although not yet included in the classification criteria, MCV has demonstrated similar utility to the established markers and improves sensitivity for early RA when tested in conjunction with CCP (Table 3).19

Table 3. Sensitivity and Specificity of Serological Markers for Rheumatoid Arthritis

Marker	Early RA19,a,b (n=170)		All RA20,b,c
Marker	Sensitivity	Specificity		Sensitivity	Specificity
RF	72	80		77	73
CCP antibody	62	96		71	95
MCV antibody	78	93		71	89
MCV or CCP antibody	81	nd		77d/60e	ndf
MCV or RF antibody	nd	nd		64d	ndf

CCP, cyclic citrullinated peptide; MCV, mutated citrullinated vimentin; nd, not determined; RA, rheumatoid arthritis; RF, rheumatoid factor.
a	Early RA defined as disease history of <1 year.
b	Compared to healthy control individuals and patients with other rheumatic or infectious diseases.
c	Meta-analysis.
d	Assays performed in parallel.
e	Assays performed in serial.
f	Control groups not well defined.

Individuals suitable for testing

Individuals with signs and symptoms associated with autoimmune disease(s)

Methods

CCP antibody and MCV testing performed using an ELISA-based assays.
RF testing performed using immunoturbidimetry.
ANA screening conducted using an IFA performed with HEp-2 cells.

Negative ANA IFA results at the 1:40 dilution terminate the reflex cascade.
Positive ANA IFA results at the 1:40 dilution prompt reflex to
Reporting of the corresponding antibody fluorescence pattern.
Titer determined by serial dilution until the pattern cannot be observed, or to a dilution of 1:1,280; specimens may be titrated to endpoint upon request.
The 3-tier autoantibody reflex cascade (Figure) performed using a multiplex immunoassay.

Aside from ANA titer and pattern, panel components may be ordered separately (Table 4).
Reflex tests are performed at additional charge and are associated with an additional CPT code(s).

Table 4. Test Components Included in the ANA, IFA, Cascade and Rheumatoid Arthritis Panel 2, With Reflexes

Test code	Test name
16814	ANA Cascade (ANA, IFA With Reflex to Titer and Pattern, and Reflex to 11 Ab Cascade)a Includes ANA screen (IFA) with reflex to titer and pattern; and chromatin (nucleosomal), dsDNA, RNP, Sm, and Sm/RNP antibodies. If all 5 antibodies are negative, reflex to Jo-1, Scl-70, SS-A, and SS-B antibodies; if all 4 of these antibodies are negative, reflex to centromere B and ribosomal P antibodies.
249	ANA Screen, IFA, With Reflex to Titer and Patterna
Tier 1 antibodies
34088	Chromatin (Nucleosomal) Antibody
255	DNA (ds) Antibody
19887	RNP Antibody
38567	Sm/RNP Antibody
37923	Sm Antibody
Tier 2 antibodies
5810	Jo-1 Antibody
38568	Sjögren’s Antibody (SS-A)
38569	Sjögren’s Antibody (SS-B)
4942	Scleroderma Antibody (Scl-70)
Tier 3 antibodies
16088	Centromere B Antibody
34283	Ribosomal P Antibody
Rheumatoid arthritis antibodies
91472	Rheumatoid Arthritis Diagnostic Panel 2 Includes cyclic citrullinated peptide (CCP) antibody (test code 11173), mutated citrullinated vimentin (MCV) antibody (test code 13238), and rheumatoid factor (test code 4418).

a	Reflex tests are performed at additional charge and are associated with an additional CPT code(s).

Interpretive information

A positive ANA result in conjunction with clinical suspicion suggests, but does not necessarily confirm, the presence of an autoimmune disease. Positive results are not uncommon in healthy individuals (particularly as they age) and those with certain infectious diseases or cancer.12

A positive ANA IFA result with a positive result for 1 or more of the specific antibodies may suggest the presence of a certain autoimmune disease (Table 2). If the ANA is positive but the 11 antibodies tested in the cascade antibody tests are negative, the patient may still have an autoimmune disease other than those typically associated with the antibodies tested. Tests for other autoimmune diseases may be considered if clinically indicated; these include autoimmune hepatitis, primary biliary cholangitis, autoimmune thyroiditis, Addison's disease, pernicious anemia, autoimmune neuropathies, vasculitis, celiac disease, and bullous disease.

A negative ANA IFA result suggests the absence of many autoimmune diseases but does not rule them out. Thus, specific autoantibody testing may be considered for patients with negative ANA IFA results if clinical suspicion is high (eg, Jo-1 for suspected polymyositis or dermatomyositis, SS-A for suspected Sjögren syndrome).

A positive result on the RF, CCP antibody, or MCV antibody tests generally supports a diagnosis of early or established RA, or potential subclinical RA (Table 3). In ANA-negative patients, positive results are consistent with early RA.30 A negative result on all 3 tests is consistent with absence of RA though early RA cannot be ruled out (Table 3). If clinically indicated, a negative test result may also be followed up with tests for other autoimmune diseases.

References

Solomon DH, Kavanaugh AJ, Schur PH. Evidence-based guidelines for the use of immunologic tests: antinuclear antibody testing. Arthritis Rheum. 2002;47(4):434-444. doi:10.1002/art.10561
Kavanaugh A, Tomar R, Reveille J, et al. Guidelines for clinical use of the antinuclear antibody test and tests for specific autoantibodies to nuclear antigens. Arch Pathol Lab Med. 2000;124(1):71-81. doi:10.5858/2000-124-0071-GFCUOT
Aringer M, Costenbader K, Daikh D, et al. 2019 European League Against Rheumatism/American College of Rheumatology classification criteria for systemic lupus erythematosus. Arthritis Rheumatol. 2019;71(9):1400-1412. doi:10.1002/art.40930
Shiboski CH, Shiboski SC, Seror R, et al. 2016 American College of Rheumatology/European League Against Rheumatism classification criteria for primary Sjögren's syndrome: A consensus and data-driven methodology involving three international patient cohorts. Ann Rheum Dis. 2017;76(1):9-16. doi:10.1136/annrheumdis-2016-210571
Bossuyt X, De Langhe E, Borghi MO, et al. Understanding and interpreting antinuclear antibody tests in systemic rheumatic diseases. Nat Rev Rheumatol. 2020;16(12):715-726. doi:10.1038/s41584-020-00522-w
Agmon-Levin N, Damoiseaux J, Kallenberg C, et al. International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies. Ann Rheum Dis. 2014;73(1):17-23. doi:10.1136/annrheumdis-2013-203863
Kavanaugh AF, Solomon DH. Guidelines for immunologic laboratory testing in the rheumatic diseases: anti-DNA antibody tests. Arthritis Rheum. 2002;47(5):546-555. doi:10.1002/art.10558
Antinuclear antibodies (ANA). American College of Rheumatology. Updated February 2023. Accessed January 30, 2024. www.rheumatology.org/patients/antinuclear-antibodies-ana
Tozzoli R, Bizzaro N, Tonutti E, et al. Guidelines for the laboratory use of autoantibody tests in the diagnosis and monitoring of autoimmune rheumatic diseases. Am J Clin Pathol. 2002;117(2):316-324. doi:10.1309/Y5VF-C3DM-l8XV-U053
Damoiseaux J, Andrade LEC, Carballo OG, et al. Clinical relevance of HEp-2 indirect immunofluorescent patterns: the International Consensus on ANA patterns (ICAP) perspective. Ann Rheum Dis. 2019;78(7):879-889. doi:10.1136/annrheumdis-2018-214436
Satoh M, Vázquez-Del Mercado M, Chan EK. Clinical interpretation of antinuclear antibody tests in systemic rheumatic diseases. Mod Rheumatol. 2009;19(3):219-228. doi:10.1007/s10165-009-0155-3
Petri M, Orbai AM, Alarcón GS, et al. Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum. 2012;64(8):2677-2686. doi:10.1002/art.34473
Ortega-Hernandez OD, Shoenfeld Y. Mixed connective tissue disease: an overview of clinical manifestations, diagnosis and treatment. Best Pract Res Clin Rheumatol. 2012;26(1):61-72. doi:10.1016/j.berh.2012.01.009
van den Hoogen F, Khanna D, Fransen J, et al. 2013 classification criteria for systemic sclerosis: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Ann Rheum Dis. 2013;72(11):1747-1755. doi:10.1136/annrheumdis-2013-204424
Lundberg IE, Tjärnlund A, Bottai M, et al. 2017 European League Against Rheumatism/American College of Rheumatology classification criteria for adult and juvenile idiopathic inflammatory myopathies and their major subgroups. Ann Rheum Dis. 2017;76(12):1955-1964. doi:10.1136/annrheumdis-2017-211468
Manca E. Autoantibodies in neuropsychiatric systemic lupus erythematosus (NPSLE): can they be used as biomarkers for the differential diagnosis of this disease? Clin Rev Allergy Immunol. 2022;63(2):194-209. doi:10.1007/s12016-021-08865-2
Mahler M, Meroni PL, Bossuyt X, et al. Current concepts and future directions for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies. J Immunol Res. 2014;2014:315179. doi:10.1155/2014/315179
Rönnelid J, Turesson C, Kastbom A. Autoantibodies in rheumatoid arthritis - laboratory and clinical perspectives. Front Immunol. 2021;12:685312. doi:10.3389/fimmu.2021.685312
Liu X, Jia R, Zhao J, et al. The role of anti-mutated citrullinated vimentin antibodies in the diagnosis of early rheumatoid arthritis. J Rheumatol. 2009;36(6):1136-1142. doi:10.3899/jrheum.080796
Zhu JN, Nie LY, Lu XY, et al. Meta-analysis: compared with anti-CCP and rheumatoid factor, could anti-MCV be the next biomarker in the rheumatoid arthritis classification criteria? Clin Chem Lab Med. 2019;57(11):1668-1679. doi:10.1515/cclm-2019-0167
Alessandri C, Agmon-Levin N, Conti F, et al. Anti-mutated citrullinated vimentin antibodies in antiphospholipid syndrome: diagnostic value and relationship with clinical features. Immunol Res. 2017;65(2):524-531. doi:10.1007/s12026-017-8899-x
Scholz J, Grossmann K, Knütter I, et al. Second generation analysis of antinuclear antibody (ANA) by combination of screening and confirmatory testing. Clin Chem Lab Med. 2015;53(12):1991-2002. doi:10.1515/cclm-2015-0083
van Paassen P, Damoiseaux J, Tervaert JW. Laboratory assessment in musculoskeletal disorders. Best Pract Res Clin Rheumatol. 2003;17(3):475-494. doi:10.1016/S1521-6942(03)00029-9
Satoh M, Chan EK, Sobel ES, et al. Clinical implication of autoantibodies in patients with systemic rheumatic diseases. Expert Rev Clin Immunol. 2007;3(5):721-738. doi:10.1586/1744666X.3.5.721
Cappelli S, Bellando Randone S, Martinović D, et al. "To be or not to be," ten years after: evidence for mixed connective tissue disease as a distinct entity. Semin Arthritis Rheum. 2012;41(4):589-598. doi:10.1016/j.semarthrit.2011.07.010
Hoffman IEA, Peene I, Meheus L, et al. Specific antinuclear antibodies are associated with clinical features in systemic lupus erythematosus. Ann Rheum Dis. 2004;63(9):1155-1158. doi:10.1136/ard.2003.013417
Cervera R, Viñas O, Ramos-Casals M, et al. Anti-chromatin antibodies in systemic lupus erythematosus: a useful marker for lupus nephropathy. Ann Rheum Dis. 2003;62(5):431-434. doi:10.1136/ard.62.5.431
Choi MY, FitzPatrick RD, Buhler K, et al. A review and meta-analysis of anti-ribosomal P autoantibodies in systemic lupus erythematosus. Autoimmun Rev. 2020;19(3):102463. doi:10.1016/j.autrev.2020.102463
Scott DL, Wolfe F, Huizinga TW. Rheumatoid arthritis. Lancet. 2010;376(9746):1094-1108. doi:10.1016/s0140-6736(10)60826-4
Fraenkel L, Bathon JM, England BR, et al. 2021 American College of Rheumatology guideline for the treatment of rheumatoid arthritis. Arthritis Care Res (Hoboken). 2021;73(7):924-939. doi:10.1002/acr.24596

Content reviewed 03/2024

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This test is used to evaluate patients with suspected autoimmune rheumatic disease.

Test Guide List

ANA, IFA, Cascade and Rheumatoid Arthritis Panel 2, With Reflexes

Test Summary

ANA, IFA, Cascade and Rheumatoid Arthritis Panel 2, With Reflexes

Test code: 94954

Clinical use

Evaluate suspected autoimmune rheumatic diseases

Clinical background

Table 1. Utility of Antinuclear Antibody IFA Test by Condition

Condition	Comments/recommendations1,2,7
Drug-induced lupus	Positive ANA part of the diagnostic criteria ANA useful for symptomatic people who are taking a drug associated with drug-induced lupus
Mixed connective tissue disease (MCTD)	Positive ANA part of the diagnostic criteria ANA recommended when clinical suspicion of MCTD Follow-up with RNP antibody recommended to confirm diagnosis
Autoimmune hepatitis	Positive ANA part of diagnostic criteria Positive ANA often seen in patients with diverse liver disease; does not exclude other hepatic diseases
Systemic lupus erythematosus (SLE)3	Positive ANA part of classification criteria ANA sensitivity, 93%; specificity, 57% Best initial test when clinical suspicion of SLE is strong SLE unlikely if ANA negative Specific antibody tests may be considered as follow-up to positive ANA
Systemic sclerosis (SSc)	ANA sensitivity, 85%; specificity, 54% ANA recommended when clinical suspicion of SSc If negative, consider other fibrosing illnesses (eg, eosinophilic fasciitis, linear scleroderma)
Sjögren syndrome4	ANA sensitivity, 48%; specificity, 52% (if ANA titer ≥320, sensitivity, 72.8% [95% CI, 67.5-77.7%]; specificity, 80.4% [95% CI, 76.9-84.0%]) Can help clarify whether an underlying connective tissue disease exists when Sjögren syndrome suspected
Polymyositis or dermatomyositis	ANA sensitivity, 61%; specificity, 63% Positive ANA provides weak evidence of disease even when combined with clinical suspicion Must consider other connective tissue diseases (SLE or overlap syndrome) regardless of ANA status

ANA, antinuclear antibody test; RNP, ribonucleoprotein.
a	The American College of Rheumatology Ad Hoc Committee on Immunologic Testing Guidelines7

Table 2. Antibody Prevalence in Rheumatic and Related Diseases and Healthy Individuals,a %

Analyte	SLE 3,12	MCTD 13	SSc 14	SS 4	PM/DM 15	CREST syndromeb	Neurologic SLE 16	HI
ANA2,17,18	90-95	90-100	85-95b	50-60	50-60	70		20-30
CENP-B2	2-5	2-5	20-40b,c	5-10	1-3	66		<3
Chromatin2	40-70	5-18	<3	<3	<3			<3
dsDNA2	40-70	<3	<3	<3	<3			<3
Jo-12	1-3	<2d	1-3	<2	15-30			<1
MCV19-21	12-36		10	10				≤5
Rib P2	10-30	<2	<2	<2	<2		10-47	<1
RNP2	10-40	100	5-15	<3	5-15			<3
Scl-702	0-5	<3e	20-40c	<3	<3			<1
Sm2	5-20	<2	<2	<1	<1			<1
Sm/RNP11,22	30	100	4	9	5
SS-A/Ro2	40-70	<3	3-10	60-90	<3			<3
SS-B/La2	15-30	<3	1-5	60-80	5-15			<3

ANA, antinuclear antibody; CENP-B, centromere B; dsDNA, double-stranded DNA; HI, healthy individuals; Jo-1, histidyl-tRNA synthetase; MCTD, mixed connective tissue disease; Rib P, ribosomal P; MCV, mutated citrullinated vimentin; PM/DM, polymyositis or dermatomyositis; RNP, ribonucleoprotein; Scl-70, scleroderma-70 (topoisomerase 1); SLE, systemic lupus erythematosus; Sm, Smith; Sm/RNP, Smith/ribonucleoprotein; SS, Sjögren syndrome; SS-A, SS-B, Sjögren antibodies A and B; SSc, systemic sclerosis.
a	Highlighted antibodies represent classification or diagnostic criteria for the disease. Note that antibodies included in criteria are not always those with the highest prevalence in that disease.
b	CREST syndrome is a variant of systemic sclerosis defined by the presence of calcinosis cutis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia. Also known as limited cutaneous scleroderma. ANA is present in 70% of CREST patients, and CENP-B is present in 90% to 95%.23
c	The presence of scleroderma-related antibodies (centromere, Scl-70, or RNA polymerase III antibodies) is not necessary or sufficient for diagnosis, but is useful for classification in the absence of diagnostic clinical findings (ie, "skin thickening of the fingers extending proximal to the metacarpophalangeal joints"14).
d	Especially in patients with features of muscle inflammation.
e	Especially in patients with features of systemic sclerosis.

Figure. ANA, IFA, Cascade with Rheumatoid Arthritis Panel 2, With Reflexes (test code 94954).5,12,19,24-28

Table 3. Sensitivity and Specificity of Serological Markers for Rheumatoid Arthritis

Marker	Early RA19,a,b (n=170)		All RA20,b,c
Marker	Sensitivity	Specificity		Sensitivity	Specificity
RF	72	80		77	73
CCP antibody	62	96		71	95
MCV antibody	78	93		71	89
MCV or CCP antibody	81	nd		77d/60e	ndf
MCV or RF antibody	nd	nd		64d	ndf

CCP, cyclic citrullinated peptide; MCV, mutated citrullinated vimentin; nd, not determined; RA, rheumatoid arthritis; RF, rheumatoid factor.
a	Early RA defined as disease history of <1 year.
b	Compared to healthy control individuals and patients with other rheumatic or infectious diseases.
c	Meta-analysis.
d	Assays performed in parallel.
e	Assays performed in serial.
f	Control groups not well defined.

Individuals suitable for testing

Individuals with signs and symptoms associated with autoimmune disease(s)

Methods

CCP antibody and MCV testing performed using an ELISA-based assays.
RF testing performed using immunoturbidimetry.
ANA screening conducted using an IFA performed with HEp-2 cells.

Negative ANA IFA results at the 1:40 dilution terminate the reflex cascade.
Positive ANA IFA results at the 1:40 dilution prompt reflex to
Reporting of the corresponding antibody fluorescence pattern.
Titer determined by serial dilution until the pattern cannot be observed, or to a dilution of 1:1,280; specimens may be titrated to endpoint upon request.
The 3-tier autoantibody reflex cascade (Figure) performed using a multiplex immunoassay.

Aside from ANA titer and pattern, panel components may be ordered separately (Table 4).
Reflex tests are performed at additional charge and are associated with an additional CPT code(s).

Table 4. Test Components Included in the ANA, IFA, Cascade and Rheumatoid Arthritis Panel 2, With Reflexes

Test code	Test name
16814	ANA Cascade (ANA, IFA With Reflex to Titer and Pattern, and Reflex to 11 Ab Cascade)a Includes ANA screen (IFA) with reflex to titer and pattern; and chromatin (nucleosomal), dsDNA, RNP, Sm, and Sm/RNP antibodies. If all 5 antibodies are negative, reflex to Jo-1, Scl-70, SS-A, and SS-B antibodies; if all 4 of these antibodies are negative, reflex to centromere B and ribosomal P antibodies.
249	ANA Screen, IFA, With Reflex to Titer and Patterna
Tier 1 antibodies
34088	Chromatin (Nucleosomal) Antibody
255	DNA (ds) Antibody
19887	RNP Antibody
38567	Sm/RNP Antibody
37923	Sm Antibody
Tier 2 antibodies
5810	Jo-1 Antibody
38568	Sjögren’s Antibody (SS-A)
38569	Sjögren’s Antibody (SS-B)
4942	Scleroderma Antibody (Scl-70)
Tier 3 antibodies
16088	Centromere B Antibody
34283	Ribosomal P Antibody
Rheumatoid arthritis antibodies
91472	Rheumatoid Arthritis Diagnostic Panel 2 Includes cyclic citrullinated peptide (CCP) antibody (test code 11173), mutated citrullinated vimentin (MCV) antibody (test code 13238), and rheumatoid factor (test code 4418).

a	Reflex tests are performed at additional charge and are associated with an additional CPT code(s).

Interpretive information

References

Solomon DH, Kavanaugh AJ, Schur PH. Evidence-based guidelines for the use of immunologic tests: antinuclear antibody testing. Arthritis Rheum. 2002;47(4):434-444. doi:10.1002/art.10561
Kavanaugh A, Tomar R, Reveille J, et al. Guidelines for clinical use of the antinuclear antibody test and tests for specific autoantibodies to nuclear antigens. Arch Pathol Lab Med. 2000;124(1):71-81. doi:10.5858/2000-124-0071-GFCUOT
Aringer M, Costenbader K, Daikh D, et al. 2019 European League Against Rheumatism/American College of Rheumatology classification criteria for systemic lupus erythematosus. Arthritis Rheumatol. 2019;71(9):1400-1412. doi:10.1002/art.40930
Shiboski CH, Shiboski SC, Seror R, et al. 2016 American College of Rheumatology/European League Against Rheumatism classification criteria for primary Sjögren's syndrome: A consensus and data-driven methodology involving three international patient cohorts. Ann Rheum Dis. 2017;76(1):9-16. doi:10.1136/annrheumdis-2016-210571
Bossuyt X, De Langhe E, Borghi MO, et al. Understanding and interpreting antinuclear antibody tests in systemic rheumatic diseases. Nat Rev Rheumatol. 2020;16(12):715-726. doi:10.1038/s41584-020-00522-w
Agmon-Levin N, Damoiseaux J, Kallenberg C, et al. International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies. Ann Rheum Dis. 2014;73(1):17-23. doi:10.1136/annrheumdis-2013-203863
Kavanaugh AF, Solomon DH. Guidelines for immunologic laboratory testing in the rheumatic diseases: anti-DNA antibody tests. Arthritis Rheum. 2002;47(5):546-555. doi:10.1002/art.10558
Antinuclear antibodies (ANA). American College of Rheumatology. Updated February 2023. Accessed January 30, 2024. www.rheumatology.org/patients/antinuclear-antibodies-ana
Tozzoli R, Bizzaro N, Tonutti E, et al. Guidelines for the laboratory use of autoantibody tests in the diagnosis and monitoring of autoimmune rheumatic diseases. Am J Clin Pathol. 2002;117(2):316-324. doi:10.1309/Y5VF-C3DM-l8XV-U053
Damoiseaux J, Andrade LEC, Carballo OG, et al. Clinical relevance of HEp-2 indirect immunofluorescent patterns: the International Consensus on ANA patterns (ICAP) perspective. Ann Rheum Dis. 2019;78(7):879-889. doi:10.1136/annrheumdis-2018-214436
Satoh M, Vázquez-Del Mercado M, Chan EK. Clinical interpretation of antinuclear antibody tests in systemic rheumatic diseases. Mod Rheumatol. 2009;19(3):219-228. doi:10.1007/s10165-009-0155-3
Petri M, Orbai AM, Alarcón GS, et al. Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum. 2012;64(8):2677-2686. doi:10.1002/art.34473
Ortega-Hernandez OD, Shoenfeld Y. Mixed connective tissue disease: an overview of clinical manifestations, diagnosis and treatment. Best Pract Res Clin Rheumatol. 2012;26(1):61-72. doi:10.1016/j.berh.2012.01.009
van den Hoogen F, Khanna D, Fransen J, et al. 2013 classification criteria for systemic sclerosis: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Ann Rheum Dis. 2013;72(11):1747-1755. doi:10.1136/annrheumdis-2013-204424
Lundberg IE, Tjärnlund A, Bottai M, et al. 2017 European League Against Rheumatism/American College of Rheumatology classification criteria for adult and juvenile idiopathic inflammatory myopathies and their major subgroups. Ann Rheum Dis. 2017;76(12):1955-1964. doi:10.1136/annrheumdis-2017-211468
Manca E. Autoantibodies in neuropsychiatric systemic lupus erythematosus (NPSLE): can they be used as biomarkers for the differential diagnosis of this disease? Clin Rev Allergy Immunol. 2022;63(2):194-209. doi:10.1007/s12016-021-08865-2
Mahler M, Meroni PL, Bossuyt X, et al. Current concepts and future directions for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies. J Immunol Res. 2014;2014:315179. doi:10.1155/2014/315179
Rönnelid J, Turesson C, Kastbom A. Autoantibodies in rheumatoid arthritis - laboratory and clinical perspectives. Front Immunol. 2021;12:685312. doi:10.3389/fimmu.2021.685312
Liu X, Jia R, Zhao J, et al. The role of anti-mutated citrullinated vimentin antibodies in the diagnosis of early rheumatoid arthritis. J Rheumatol. 2009;36(6):1136-1142. doi:10.3899/jrheum.080796
Zhu JN, Nie LY, Lu XY, et al. Meta-analysis: compared with anti-CCP and rheumatoid factor, could anti-MCV be the next biomarker in the rheumatoid arthritis classification criteria? Clin Chem Lab Med. 2019;57(11):1668-1679. doi:10.1515/cclm-2019-0167
Alessandri C, Agmon-Levin N, Conti F, et al. Anti-mutated citrullinated vimentin antibodies in antiphospholipid syndrome: diagnostic value and relationship with clinical features. Immunol Res. 2017;65(2):524-531. doi:10.1007/s12026-017-8899-x
Scholz J, Grossmann K, Knütter I, et al. Second generation analysis of antinuclear antibody (ANA) by combination of screening and confirmatory testing. Clin Chem Lab Med. 2015;53(12):1991-2002. doi:10.1515/cclm-2015-0083
van Paassen P, Damoiseaux J, Tervaert JW. Laboratory assessment in musculoskeletal disorders. Best Pract Res Clin Rheumatol. 2003;17(3):475-494. doi:10.1016/S1521-6942(03)00029-9
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Content reviewed 03/2024

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Test Code 16088

Centromere B Antibody

Centromere B Antibody is diagnostic for the form of scleroderma known as CREST (calcinosis, Raynaud's phenomenon, esophageal immotility, sclerodactyly, and telangiectasia).

Test Code 16814

ANA Cascade (ANA, IFA with Reflex to Titer and Pattern, and Reflex to 11 Ab Cascade)

This test can be helpful in the diagnosis of autoimmune rheumatic diseases. Specimens with positive results for antinuclear antibodies (ANAs) on the immunofluorescence assay (IFA), performed on human epithelial type 2 (HEp-2) cells, are subsequently tested for a series of disease-specific autoantibodies in cascade tiers. The first tier focuses on autoantibodies associated with systemic lupus erythematosus (SLE) and mixed connective tissue disease; the second tier detects autoantibodies seen in S

Test Code 34088

Chromatin (Nucleosomal) Antibody

Chromatin Antibody plays a central role in the autoimmune response in systemic lupus erythematosus (SLE). Approximately 90% of patients with SLE have sera that will exhibit reactivity to nucleosomes.

Test Code 38568

Sjogren's Antibody (SS-A)

Sjögren's Antibodies (SS-B) is detected in approximately 15% of patients with Sjögren's Syndrome. Sjögren's Antibody (SS-B) is present only if Sjögren's Antibody (SS-A) is also detected. The presence of both antibodies (SS-A and SS-B) strengthen the diagnosis of Sjögren's Syndrome and conveys prognostic information.

Test Code 37923

Sm Antibody

Smith Antibody (Sm) is highly specific for systemic lupus erythematosus (SLE). Smith Antibody is also detected in approximately 15% of patients with SLE. Smith Antibody is detected in more than half of young African-American women with SLE.

Test Code 249

ANA Screen,IFA, with Reflex to Titer and Pattern

This immunofluorescence assay (IFA) is often ordered as part of an initial diagnostic evaluation of individuals with clinical suspicion of autoimmune diseases associated with antinuclear antibodies (ANAs). The American College of Rheumatology (ACR) recommends IFA on human epithelial type 2 (HEp-2) cells as the gold standard method for ANA testing because of its overall high sensitivity [1].

ANAs are associated with several autoimmune diseases, such as systemic lupus erythematosus, syst

Test Code 38567

Sm/RNP Antibody

Smith (Sm)/U1-RNP Antibody is detected in patients with mixed connective tissue disease (having features of systemic lupus erythematosus (SLE), scleroderma, and polymyositis).

Test Code 91472

Rheumatoid Arthritis Diagnostic Panel 2

Autoantibody testing has a major role in the diagnosis of RA. This panel includes Rheumatoid factor (RF), anti-cyclic citrullinated peptide (CCP) and anti-mutated citrullinated vimentin (MCV). RF is found in a majority of patients with RA but is not specific for the diagnosis. Anti-CCP antibody is more sensitive and highly specific. RF and anti-CCP are included in the most recent classification criteria for RA. The past several years have seen a search for biomarkers which might increase sensiti

Test Code 255

DNA (ds) Antibody

dsDNA Antibody is detected in patients with active systemic lupus erythematosus (SLE) and approximately 20% of patients with Mixed Connective Tissue Disease.

Test Code 5810

Jo-1 Antibody

Jo-1 Antibody occurs most frequently (31%) in patients with polymyositis, but has also been found in patients with dermatomyositis, and the polymyositis/scleroderma "overlap syndrome" (PM/SCL) or polymyositis/systemic lupus erythematosis "overlap syndrome" (PM/SLE).

Test Code 4942

Scleroderma Antibody (Scl-70)

Scl-70 antibody is present in approximately 40% of patients with progressive systemic sclerosis (PSS).

Test Code 19887

RNP Antibody

RNP Antibodies have been associated with Mixed Connective Tissue disease.

Test Code 94954

ANA, IFA, Cascade and Rheumatoid Arthritis Panel 2, with Reflexes

This test can be helpful in the diagnosis of autoimmune rheumatic diseases. Specimens with positive results for antinuclear antibodies (ANA) by indirect immunofluorescence assay (IFA) are subsequently tested for a series of ANA-specific autoantibodies in cascade tiers. The first tier focuses on autoantibodies associated with systemic lupus erythematosus (SLE) and mixed connective tissue disease; the second tier detects autoantibodies seen in Sjogren syndrome, systemic sclerosis and polymyositis;

Test Code 38569

Sjogren's Antibody (SS-B)

Sjögren's Antibody (SS-B) is detected in approximately 15% of patients with Sjögren's Syndrome. Sjögren's Antibody (SS-B) is present only if Sjögren's Antibody (SS-A) is also detected. The presence of both antibodies (SS-A and SS-B) strengthen the diagnosis of Sjögren's Syndrome and conveys prognostic information.

Test Code 34283

Ribosomal P Antibody

Ribosomal P antibody is present in 5-10% of patients with systemic lupus erythematosus (SLE).

IG/Reference Manual

Test Guide List

This page is a complete list of Test Guides created by Quest Diagnostics.

Test Summary

ANAlyzeR ANA, IFA with Reflex Titer/Pattern, Systemic Autoimmune Panel 1

This test is used to evaluate patients with suspected autoimmune rheumatic disease.

Clinical Focus

Autoimmune Rheumatic and Related Diseases

This document provides information about laboratory tests used in the diagnosis and classification of autoimmune rheumatic and related diseases. The document also includes a summary of symptoms and diagnosis/classification criteria for the various conditions.

Clinical Focus

Myasthenia Gravis and Autoantibodies

This Clinical Focus provides information relating to the appropriate use and interpretation of antibody tests for myasthenia gravis diagnosis and management.

Clinical Focus

Inflammatory Bowel Disease: Laboratory Support for Diagnosis and Management

This Clinical Focus reviews the selection and interpretation of laboratory tests used in the workup of symptomatic patients and in the management of patients with IBD.

Test Summary

Interstitial Lung Disease (ILD) Panel

This test is used to identify connective tissue disease (CTD) as a possible cause of interstitial lung disease (ILD).

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Reference ranges are provided as general guidance only. To interpret test results use the reference range in the laboratory report.

The tests listed by specialty and category are a select group of tests offered. For a complete list of Quest Diagnostics tests, please adjust the filter options chosen, or refer to our Directory of Services.

ANA, IFA, Cascade and Rheumatoid Arthritis Panel 2, With Reflexes

ANA, IFA, Cascade and Rheumatoid Arthritis Panel 2, With Reflexes

Test Summary

Test Summary

(15) Related Tests

(6) Related Guides