Test Id : CTDC

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.

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.

• Connective Tissue Disease Cascade

0.7 mL

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

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.

The following diseases are often referred to as connective tissue diseases (CTD): rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis including CREST syndrome (calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), Sjogren syndrome, mixed connective tissue disease, and idiopathic inflammatory myopathies. CTD (also known as systemic rheumatic diseases) are characterized by immune-mediated inflammation that involves the joints, skin, and visceral organs. These diseases are also accompanied by antibodies to a host of nuclear and cytoplasmic autoantigens.

The diagnosis of any CTD is based on clinical signs and symptoms and characteristic radiographic, histopathologic, and serologic findings. Certain CTD are characterized by autoantibodies that are highly specific for individual diseases as outlined in the Table. CTD often present clinically with signs and symptoms that are nonspecific, including constitutional signs such as fever, weight loss, fatigue, and arthralgias.

Table. Autoantibodies and Clinical Associations with Specific Connective Tissue Diseases

In this testing cascade, serum is tested initially for the presence of antinuclear antibodies (ANA) and for cyclic citrullinated peptide (CCP) antibodies. The presence of CCP antibodies indicates a strong likelihood of rheumatoid arthritis (RA).(1) However, additional testing for rheumatoid factor, which is not included in the cascade, is important for optimal diagnosis as per the 2010 American College of Rheumatology/European League Against Rheumatism RA classification criteria.(2) The presence of ANA supports the possibility of a CTD, and the level of ANA is used to identify sera for second-order testing for antibodies to dsDNA and the other autoantigens. 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 very unlikely.(3) However, a negative ANA enzyme immunoassay result does not rule out a diagnosis of CTD as has been reported in a number of studies. Therefore, in patients with a strong clinical suspicion of CTD, testing for ANA by indirect immunofluorescence assay may be warranted.(4,5)

ANTINUCLEAR ANTIBODIES (ANA)

< or =1.0 U (negative)

1.1-2.9 U (weakly positive)

3.0-5.9 U (positive)

> or =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)

> or =60.0 U (strong positive) 

Reference values apply to all ages.

Interpretive comments are provided.

See individual test IDs for additional information.

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.

The results of tests for autoantibodies must be interpreted in the appropriate clinical context.

The presence of detectable levels of antinuclear antibodies (ANA) in solid-phase immunoassays, such as enzyme-linked immunosorbent assay (ELISA), is not specific for connective tissue disease.

Positive results may occur prior to onset of clinical diseases as well as in the absence of connective tissue disease.

Negative ANA by ELISA results may not be reliable for the evaluation of patients at-risk for systemic sclerosis and inflammatory myopathies.

Weak-to-moderate positive results for cyclic citrullinated peptide antibodies may occur in patients with connective tissue diseases other than rheumatoid arthritis.

1. Tomar R, Homburger H. Assessment of immunoglobulins and antibodies. In: Rich R, Fleisher T, Shearer W, et al, eds. Clinical Immunology: Principles and Practice. 2nd ed. Mosby-Year Book Inc; 2001:120.1-120.14

2. 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(9):2569-2581

3. 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

4. Pisetky DS, Spencer DM, Lipsky PE, Rovin BH. Assay variation in the detection of antinuclear antibodies in the sera of patients with established SLE. Ann Rheum Dis. 2018;77(6):911-913

5. Claessens J, Belmondo T, De Langhe E, et al. Solid phase assays versus automated indirect immunofluorescence for detection of antinuclear antibodies. Autoimmun Rev. 2018;17(6):533-540

6. van Boekel MA, Vossenaar ER, van den Hoogen FHJ, van Venrooij WJ. Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value. Arthritis Res. 2002;4(2):87-93

7. Kavanaugh A, Tomar R, Reveille J, Solomon DH, Homburger HA. 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

Antinuclear Antibodies:

The method used to detect antinuclear antibodies (ANA) is enzyme-linked immunosorbent assay (ELISA). A HEp-2 lysate supplemented with specific purified antigens (double-stranded [ds] DNA, histone, SS-A [Ro], SS-B [La], Smith, sm/RNP, Scl-70, Jo-1, and centromere B antigen) are coated onto microtiter plate wells. A dilution of patient serum is added to the well and incubated. After washing to remove unbound serum protein, an enzyme conjugated antihuman IgG antibody is added to detect human IgG bound to the microtiter plate well. After incubation and washing to remove unbound conjugate, a substrate to the enzyme is added to the well. After incubation, the enzyme substrate reaction is stopped. The complete assay is measured on a spectrophotometer plate reader. The optical density measured is proportional to the antibody present in the patient serum. Testing is performed on the Agility instrument by Dynex.(Package insert: ELISA kits. Bio-Rad Laboratories; 07/2014)

Cyclic Citrullinated Peptide:

Cyclic citrullinated peptide (CCP) antibodies in serum are detected by binding to the wells of a commercial microtiter plate coated with synthetic CCP. During the first incubation, serum antibodies bind to adsorbed, solid phase CCP. The wells are then washed to remove unbound serum constituents, and horse radish peroxidase-labeled goat anti-human IgG antibody is added. After further incubation and washing to remove unbound conjugate, substrate (3,3',5,5'-tetramethylbenzidine) is added and allowed to incubate. The reaction between enzyme and substrate is stopped and color in the wells is measured in a microtiter plate reader. The concentration of CCP antibodies is determined by comparison to a 5 point standard curve (15.6-250 U). Testing is performed on the Agility instrument by Dynex.(Package insert: Quanta Lite CCP3 IgG ELISA. INOVA Diagnostics; 02/2020)

dsDNA:

The test kit contains 12 micotiter strips each with 8 break-off reagent wells coated with double-stranded DNA (dsDNA). In the first reaction step, diluted patient samples, calibrators and controls are incubated in the wells. Anti-dsDNA antibodies will bind to the antigens coated in the microtiter wells. The wells are washed to remove any unbound proteins and non-specific antibodies. In a second reaction step, rabbit anti-human IgG HRP enzyme conjugate is added to each well. The enzyme conjugate will bind to any wells that have human IgG binding to the dsDNA antigen. The wells are washed to remove any unbound HRP enzyme conjugate. 3,3,5,5, tetramethylbenzidine (TMB) enzyme substrate is added. If the HRP enzyme is present in the well (positive reaction), the HRP enzyme will react with the TMB substrate and produce a blue color. After an additional incubation time to allow the color development, a stop solution is added which turns the blue color yellow and inhibits further color development to allow for stable spectrophotometric reading. The test strips are placed in a microplate reader and the optical density of the color is measured. The amount of antigen specific bound antibody is proportional to the color intensity.(Package insert: Anti-dsDNA-NcX ELISA (IgG). EUROIMMUN; 7/8/2020)

SS-A/Ro, SS-B/La, RNP, Sm, Scl 70, Jo 1, ribosome P, and centromere B antibodies are measured by a commercial multiplex flow immunoassay system. Recombinant or purified antigens are coupled covalently to polystyrene microspheres that are impregnated with fluorescent dyes to create unique fluorescent signatures, one microsphere type for each antigen. Diluted sera, calibrators, and controls are added to a mixture containing the antigen-coupled microspheres. Antibodies to each antigen bind to their homologous antigen-coupled microspheres. The microspheres are washed to remove extraneous serum proteins. Phycoerythrin (PE)-conjugated antihuman IgG antibody is then added to detect IgG antibodies bound to the microspheres. The microspheres are washed to remove unbound conjugate, and bound conjugate is detected by laser photometry. A primary laser determines the fluorescent signature of each microsphere, and a secondary laser reveals the level of PE fluorescence associated with the microsphere surface. Results are calculated for each antigen-coated microsphere type by comparing the median fluorescence response to a series of multipoint calibration curves.(Package insert: BioPlex 2200 ANA Screen. Bio-Rad Laboratories; 2019)

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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.

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)