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Test ID: CTDC Connective Tissue Diseases Cascade, Serum

Reporting Name

Connective Tissue Disease Cascade,S

Useful For

Evaluation of patients with signs and symptoms compatible with connective tissue diseases

 

Initial evaluation of patients in clinical situations in which the prevalence of disease is low (6)

 

This test is not recommended for:

-Testing in clinical situations in which there is a high prevalence of connective tissue diseases (eg, rheumatology specialty practice)

-Follow-up evaluation of patients with known connective tissue diseases

Clinical Information

Connective tissue diseases (CTD) constitute diverse disorders affecting the joints, skin, eyes, heart, lungs, and gastrointestinal tract.(1) The diseases may be caused mainly by inheritable disorders of the connective tissue or autoimmune mechanisms.(1,2) The most common autoimmune-mediated CTD include rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), systemic sclerosis (SSc) including CREST syndrome (calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), Sjogren syndrome (Sjs), mixed connective tissue disease (MCTD), and idiopathic inflammatory myopathies (IIM).(1-3) Collectively, these diseases are also referred to as antinuclear antibody (ANA)-associated CTD or ANA-associated systemic autoimmune rheumatic diseases (ASARD). These diseases are generally accompanied by antibodies to nuclear and cytoplasmic autoantigens when tested by indirect immunofluorescence assay (IFA) using HEp-2 substrate (HEp-2 IFA).(2,3) In addition to the ANA-CTD, RA (another CTD with overlapping clinical features) is an important consideration in the early detection of patients at-risk for CTD.(2,4,5)

 

The diagnosis of any CTD is based on the patient’s personal and family medical histories, clinical presentation, radiographic and histopathologic features, presence of specific autoantibodies, and other laboratory findings.(6-8) Certain CTD subsets are characterized by autoantibodies that are highly specific for individual diseases as outlined in the Table below. The detection of ANA in certain clinical subsets of CTD is generally dependent on the type of immunoassay employed and the reference intervals.(5,9) Clinically, patients with CTD often present with signs and symptoms that are nonspecific such as fever, weight loss, fatigue, and arthralgias.

 

Table. Autoantibodies and Clinical Associations with Specific Connective Tissue Diseases

Autoantibody

Connective tissue disease specificity

Cyclic citrullinated peptide antibodies

Rheumatoid arthritis (RA)

Double-Stranded DNA (dsDNA)

Systemic lupus erythematosus (SLE)

Smith (Sm)

SLE

Ribosome P

SLE

SS-B/La

Sjogren syndrome (SjS)

SS-A/Ro (Ro52 or Ro60)

SjS, SLE, systemic sclerosis (SSc), antisynthetase syndrome

RNP 68 and A (RNP)

Mixed connective tissue disease

Topoisomerase 1 (topo-1, Scl-70)

SSc (commonly the diffuse cutaneous SSc, dsSSc)

Histidyl tRNA synthetase (JO1)

Idiopathic inflammatory myositis (IIM), commonly associated with anti-synthetase syndrome

Centromere B

SSc (commonly  limited cutaneous lcSSc)

 

In the Connective Tissue Cascade, serum is tested initially for the presence of ANA and for cyclic citrullinated peptide (CCP) antibodies using solid-phase immunoassays (SPAs). The presence of significantly elevated CCP antibodies is suggestive of RA or risk for developing disease in at-risk individuals.(5,6) However, additional testing for rheumatoid factor (RF) and inflammatory markers, which are not included in the cascade, are important for optimal diagnosis as per the 2010 American College of Rheumatology/European League Against Rheumatism RA classification criteria.(6) The presence of ANA detected with SPAs such as enzyme-linked immunosorbent assay maybe useful in identifying patients with specific CTD subsets (ANA-associated CTD), notably Sjogren’s syndrome, mixed connective tissue disease MCTD), systemic lupus erythematosus.(2,3) The CTD cascade employs a cut-off based on degree of ANA positivity to identify sera for second-order testing for first-line antibodies (dsDNA. SSA, SSB, Scl 70. Centromere B, U1RNP, Smith) and anti-CCP antibodies allowing for identification of the common autoantibodies in the classification criteria for RA, SLE and SSc.(6-8) The decision threshold for performing second-order tests is based on empirical data derived from testing patients with varying levels of ANA and is chosen to minimize testing in situations in which positive results for dsDNA and other antibodies is less likely.(9) However, a negative ANA enzyme immunoassay result does not rule out a diagnosis of CTD as has been reported in a number of studies.(reviewed in 3,10) Therefore, in patients with a strong clinical suspicion of CTD, testing for ANA using HEp-2 IFA may be warranted.(3,10)

Interpretation

Interpretive comments are provided.

 

Differential testing for Ro52 and Ro60 antibodies in SS-A/Ro positive patients may be useful in the diagnosis of specific CTD clinical subset, disease stratification, and prognosis. Consider testing for Ro52 and Ro60 antibodies (ROPAN / Ro52 and Ro60 Antibodies, IgG, Serum) if the patient is positive for SS-A/Ro.

Profile Information

Test ID Reporting Name Available Separately Always Performed
ANA2 Antinuclear Ab, S Yes Yes
CCP Cyclic Citrullinated Peptide Ab, S Yes Yes
IM_01 Interpretation No Yes

Reflex Tests

Test ID Reporting Name Available Separately Always Performed
CMA Centromere Ab, IgG, S Yes No
CASMT ANA2 Cascade No No
RIB Ribosome P Ab, IgG, S Yes No
ENAE Ab to Extractable Nuclear Ag Eval,S Yes No
ADNA1 dsDNA Ab, IgG, S Yes No

Testing Algorithm

If antinuclear antibodies are greater than or equal to 3.0 U, then antibodies to double-stranded DNA (dsDNA), extractable nuclear antigen evaluation, ribosome P, and centromere are performed at an additional charge.

 

For more information see Connective Tissue Disease Cascade.

Report Available

3 to 4 days

Day(s) Performed

Monday through Saturday

Clinical Reference

1. Jog NR, James JA. Biomarkers in connective tissue diseases. J Allergy Clin Immunol. 2017;140(6):1473-1483

2. Pisetsky DS. Annals of the rheumatic diseases collection on autoantibodies in the rheumatic diseases: new insights into pathogenesis and the development of novel biomarkers. Ann Rheum Dis. 2023; 82(10):1243-1247

3. Bossuyt, X., De Langhe, E., Borghi, M.O. et al. Understanding and interpreting antinuclear antibody tests in systemic rheumatic diseases. Nat Rev Rheumatol. 2020;16(12):715-726

4. Stinton LM, Fritzler MJ. A clinical approach to autoantibody testing in systemic autoimmune rheumatic disorders. Autoimmun Rev. 2007;7(1):77-84

5. Anaparti V, Smolik I, Meng X, et al. Expansion of Alternative Autoantibodies Does Not Follow the Evolution of Anti-Citrullinated Protein Antibodies in Preclinical Rheumatoid Arthritis: An Analysis in At-Risk First Degree Relatives. Arthritis Rheumatol. 2021;73(5):740-749. doi:10.1002/art.41675

6. Aletaha D, Neogi T, Silman AJ, et al: 2010 Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum. 2010 ;62:2569-2581.

7. 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. Arthritis Rheum. 2013;65(11):2737-2747. doi:10.1002/art.38098

8. 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. 3

9. Deng X, Peters B, Ettore MW, et al: Utility of antinuclear antibody screening by various methods in a clinical laboratory patient cohort. J Appl Lab Med. 2016;1(1):36-46

10. Orme ME, Andalucia C, Sjölander S, Bossuyt X. A comparison of a fluorescence enzyme immunoassay versus indirect immunofluorescence for initial screening of connective tissue diseases: Systematic literature review and meta-analysis of diagnostic test accuracy studies. Best Pract Res Clin Rheumatol. 2018; 32(4):521-534

Method Name

Enzyme-Linked Immunosorbent Assay (ELISA)

Specimen Type

Serum


Specimen Required


Supplies: Sarstedt Aliquot Tube, 5 mL (T914)

Collection Container/Tube:

Preferred: Serum gel

Acceptable: Red top

Submission Container/Tube: Plastic vial

Specimen Volume: 1 mL

Collection Information: Centrifuge and aliquot serum into plastic vial.


Specimen Minimum Volume

0.7 mL

Specimen Stability Information

Specimen Type Temperature Time
Serum Refrigerated (preferred) 21 days
  Frozen  21 days

Special Instructions

Reference Values

ANTINUCLEAR ANTIBODIES (ANA)

≤1.0 U (Negative)

1.1-2.9 U (Weakly positive)

3.0-5.9 U (Positive)

≥6.0 U (Strongly positive)

Reference values apply to all ages.

 

CYCLIC CITRULLINATED PEPTIDE ANTIBODIES, IgG

<20.0 U (Negative)

20.0-39.9 U (Weak positive)

40.0-59.9 U (Positive)

≥60.0 U (Strong positive) 

Reference values apply to all ages.

Test Classification

This test has been cleared, approved, or is exempt by the US Food and Drug Administration and is used per manufacturer's instructions. Performance characteristics were verified by Mayo Clinic in a manner consistent with CLIA requirements.

CPT Code Information

86038

86200

83516-Centromere (if appropriate)

83516-Ribosome (if appropriate)

86225-ds-DNA AB IgG, Serum (if appropriate)

86235 x 6-RNP, Sm, SS-B, SS-A, Jo 1, and Scl 70 (if appropriate)

LOINC Code Information

Test ID Test Order Name Order LOINC Value
CTDC Connective Tissue Disease Cascade,S 95267-1

 

Result ID Test Result Name Result LOINC Value
ANA2 Antinuclear Ab, S 94875-2
CCP Cyclic Citrullinated Peptide Ab, S 33935-8
IM_01 Interpretation 69048-7
Mayo Clinic Laboratories | Neurology Catalog Additional Information:

mml-Behavioral, mml-Cerebrovascular, mml-Epilepsy, mml-Headache, mml-Movement-Disorders, mml-Demyelinating-Diseases, mml-Neuroimmunology, mml-Neuromuscular, mml-Autonomic, mml-Pediatric, mml-Spinal-Cord, mml-Neuro-ophthalmology