Test Id : DYS2

If the ganglionic acetylcholine receptor (alpha 3) flow screen assay result requires further investigation, then the Alpha3 flow titration assay will be performed at an additional charge.

If the indirect immunofluorescence assay (IFA) patterns suggest collapsin response-mediator protein (CRMP)-5-IgG, then the CRMP-5-IgG IFA titer and CRMP-5-IgG Western blot will be performed at an additional charge.

If the IFA pattern suggests antineuronal nuclear antibody type 1 (ANNA-1), then the ANNA-1 immunoblot, ANNA-1 IFA titer, and ANNA-2 immunoblot will be performed at an additional charge.

If the IFA pattern suggests adaptor protein 3 beta 2 (AP3B2) antibody, then the AP3B2 cell-binding assay (CBA) and AP3B2 IFA titer will be performed at an additional charge.

If the dipeptidyl-peptidase-like protein-6 antibody (DPPX) antibody CBA result is positive, then DPPX IFA titer will be performed at an additional charge.

If the IFA pattern suggests Purkinje cell cytoplasmic antibody type 2 (PCA-2), then the PCA-2 titer and microtubule-associated protein 1B (MAP1B) immunoblot will be performed at an additional charge.

For more information see Autoimmune/Paraneoplastic Dysautonomia Evaluation Algorithm.

Multiple neurological phenotype-specific autoimmune/paraneoplastic evaluations are available. For more information as well as phenotype-specific testing options, refer to Autoimmune Neurology Test Ordering Guide.

When more than one evaluation is ordered on the same order number, the duplicate test will be canceled.

For a list of antibodies performed with each evaluation, see Autoimmune Neurology Antibody Matrix.

This test should not be requested for patients who have recently received radioisotopes, therapeutically or diagnostically, because of potential assay interference. The specific waiting period before specimen collection will depend on the isotope administered, the dose given, and the clearance rate in the individual patient. Specimens will be screened for radioactivity prior to analysis. Radioactive specimens received in the laboratory will be held for 1 week and assayed if sufficiently decayed or canceled if radioactivity remains.

Provide the following information:

-Relevant clinical information

-Ordering healthcare professional's name, phone number, mailing address, and email address

Patient Preparation: For optimal antibody detection, collect specimen before starting immunosuppressant medication or intravenous immunoglobulin treatment.

Supplies: Sarstedt Aliquot Tube, 5 mL (T914)

Collection Container/Tube:

Preferred: Red top

Acceptable: Serum gel

Submission Container/Tube: Plastic vial

Specimen Volume: 4 mL Serum

Collection Instructions: Centrifuge and aliquot serum into a plastic vial.

• Autoimmune/Paraneoplastic Dysautonomia Evaluation Algorithm

If not ordering electronically, complete, print, and send a Neurology Specialty Testing Client Test Request (T732) with the specimen.

Serum: 2.5 mL

Investigating idiopathic dysautonomic symptoms

Directing a focused search for cancer in patients with idiopathic dysautonomia

Investigating autonomic symptoms that appear in the course or wake of cancer therapy and are not explainable by recurrent cancer or metastasis (detection of autoantibodies in this profile helps differentiate autoimmune dysautonomia from the effects of chemotherapy)

If the ganglionic acetylcholine receptor (alpha 3) flow screen assay result requires further investigation, then the Alpha3 flow titration assay will be performed at an additional charge.

If the indirect immunofluorescence assay (IFA) patterns suggest collapsin response-mediator protein (CRMP)-5-IgG, then the CRMP-5-IgG IFA titer and CRMP-5-IgG Western blot will be performed at an additional charge.

If the IFA pattern suggests antineuronal nuclear antibody type 1 (ANNA-1), then the ANNA-1 immunoblot, ANNA-1 IFA titer, and ANNA-2 immunoblot will be performed at an additional charge.

If the IFA pattern suggests adaptor protein 3 beta 2 (AP3B2) antibody, then the AP3B2 cell-binding assay (CBA) and AP3B2 IFA titer will be performed at an additional charge.

If the dipeptidyl-peptidase-like protein-6 antibody (DPPX) antibody CBA result is positive, then DPPX IFA titer will be performed at an additional charge.

If the IFA pattern suggests Purkinje cell cytoplasmic antibody type 2 (PCA-2), then the PCA-2 titer and microtubule-associated protein 1B (MAP1B) immunoblot will be performed at an additional charge.

For more information see Autoimmune/Paraneoplastic Dysautonomia Evaluation Algorithm.

Autoimmune dysautonomia encompasses disorders of peripheral autonomic synapses, ganglionic neurons, autonomic nerve fibers, and central autonomic pathways mediated by neural-specific IgG or effector T cells. These disorders may be idiopathic or paraneoplastic, subacute or insidious in onset, and may present as a limited disorder or generalized pandysautonomia. Pandysautonomia is usually subacute in onset and severity and includes impaired pupillary light reflex, anhidrosis, orthostatic hypotension, cardiac arrhythmias, gastrointestinal dysmotility, sicca manifestations, and bladder dysfunction. Limited dysautonomia is confined to one or just a few domains, is often mild, and may include sicca manifestations, postural orthostatism and cardiac arrhythmias, bladder dysfunction, or gastrointestinal dysmotilities. Diagnosis of limited dysautonomia requires documentation of objective abnormalities by autonomic reflex testing, thermoregulatory sweat test, or gastrointestinal motility studies.

The most frequently encountered autoantibody marker of autoimmune dysautonomia is the neuronal ganglionic alpha-3-acetylcholine receptor (AChR) autoantibody. This autoantibody to date is the only proven effector of autoimmune dysautonomia.

Importantly, cancer is detected in 30% of patients with alpha-3-AChR autoantibody. Cancers recognized include small-cell lung carcinomas, thymoma, lymphoma, and adenocarcinomas of breast, lung, prostate, and gastrointestinal tract. Cancer risk factors include a previous or family history of cancer, history of smoking, or social or environmental exposure to carcinogens. Early diagnosis and treatment of the neoplasm favor neurologic improvement and lessen morbidity.

Autoantibodies to other onconeural proteins shared by neurons, glia, or muscle (eg, antineuronal nuclear antibody-type 1 [ANNA-1], collapsin response-mediator protein-5 neuronal [CRMP-5-IgG]) serve as additional markers of paraneoplastic or idiopathic dysautonomia. A specific neoplasm is often predictable by the individual patient's autoantibody profile.

 Reflex Information:

*Methodology abbreviations:

Cell-binding assay (CBA)

Flow cytometry (FC)

Immunoblot (IB)

Immunofluorescence assay (IFA)

Western Blot (WB)

Antibodies directed at onconeural proteins shared by neurons, muscle, and glia are valuable serological markers of a patient's immune response to cancer. These autoantibodies are not found in healthy subjects and are usually accompanied by subacute neurological signs and symptoms. It is not uncommon for more than one autoantibody to be detected in patients with autoimmune dysautonomia. These include:

-Plasma membrane cation channel antibodies (neuronal ganglionic [alpha-3]); These autoantibodies are potential effectors of autonomic dysfunction.

-Antineuronal nuclear antibody-type 1 (ANNA-1)

-Neuronal and muscle cytoplasmic antibodies (collapsin response-mediator protein-5 neuronal: CRMP-5 IgG)

A rising autoantibody titer in previously seropositive patients suggests cancer recurrence.

Negative results do not exclude autoimmune dysautonomia or cancer.

Intravenous immunoglobulin (IVIg) treatment prior to the serum collection may cause a false-positive result.

1. Cutsforth-Gregory JK, McKeon A, Coon EA, et al. Ganglionic antibody level as a predictor of severity of autonomic failure. Mayo Clin Proc. 2018;93(10):1440-1447. doi:10.1016/j.mayocp.2018.05.033

2. Tobin WO, Lennon VA, Komorowski L, et al. DPPX potassium channel antibody: frequency, clinical accompaniments, and outcomes in 20 patients. Neurology. 2014;83(20):1797-1803.doi:10.1212/WNL.0000000000000991

Cell Binding Assay:

Patient sample is applied to a composite slide containing transfected and nontransfected cells. After incubation and washing, fluorescein-conjugated goat-antihuman IgG is applied to detect the presence of patient IgG binding.(Package insert: IIFT: Neurology Mosaics, Instructions for the indirect immunofluorescence test. EUROIMMUN; FA_112d-1_A_UK_C13, 02/2019)

Indirect Immunofluorescence Assay:

The patient's sample is tested by a standardized immunofluorescence assay that uses a composite frozen section of mouse cerebellum, kidney, and gut tissues. After incubation with sample and washing, fluorescein-conjugated goat-antihuman IgG is applied. Neuron-specific autoantibodies are identified by their characteristic fluorescence staining patterns. Samples that score positive for any neuronal nuclear or cytoplasmic autoantibody are titrated to an endpoint. Interference by coexisting non-neuron-specific autoantibodies can usually be eliminated by serologic absorption.(Honorat JA, Komorowski L, Josephs KA, et al. IgLON5 antibody: neurological accompaniments and outcomes in 20 patients. Neurol Neuroimmunol Neuroinflamm. 2017;4[5]:e385. doi:10.1212/NXI.0000000000000385)

Flow Cytometry:

This assay is a live cell-based binding assay using flow cytometry to detect ganglionic acetylcholine receptor (AChR) binding antibodies in patient serum. Human embryonic kidney cells (HEK293) transiently expressing human alpha-3 beta-4 ganglionic AChR subunits, along with accessory proteins required for receptor expression, are used as the antigen source. Receptor expression is enhanced prior to analysis under controlled culture conditions. Cells are incubated with diluted patient serum to allow binding of AChR-specific antibodies to surface-expressed receptors. Bound human IgG is detected using a fluorophore-conjugated anti-human IgG secondary antibody, with a viability dye included to exclude non-viable cells. Fluorescence intensity is measured by flow cytometry, and antibody binding is assessed by comparing signal from AChR-expressing cells to control cells lacking receptor expression. A humanized AChR-specific monoclonal antibody is used as an assay calibrator. The IgG Binding Index (IBI) of patient samples is compared to the assay calibrator to calculate a Cut-off Index (COI). Patient samples with a COI = 1.0 are reported as positive. Positive samples are further titrated to determine the end-point dilution, defined as the highest serum dilution that remains above the calibrator.(Unpublished Mayo method)

Western Blot:

Neuronal antigens extracted aqueously from adult rat cerebellum, full-length recombinant human collapsin response-mediator protein-5 (CRMP-5) or full-length recombinant human amphiphysin protein is denatured, reduced, and separated by electrophoresis on 10% polyacrylamide gel. IgG is detected autoradiographically by enhanced chemiluminescence.(Yu Z, Kryzer TJ, Griesmann GE, Kim K, Benarroch EE, Lennon VA. CRMP-5 neuronal autoantibody: marker of lung cancer and thymoma-related autoimmunity. Ann Neurol. 2001;49[2]:146-154; Dubey D, Jitprapaikulsan J, Bi H, et al. Amphiphysin-IgG autoimmune neuropathy: A recognizable clinicopathologic syndrome. Neurology. 2019;93[20]:e1873-e1880. doi:10.1212/WNL.0000000000008472)

Immunoblot for ANNA-1 and ANNA-2:

All steps are performed at room temperature (18 to 28 degrees C) utilizing the EUROBlot One instrument. Diluted patient sample is added to test strips (strips containing recombinant antigen manufactured and purified using biochemical methods) in individual channels and incubated for 30 minutes. Positive samples will bind to the purified recombinant antigen and negative samples will not bind. Strips are washed to remove unbound antibodies and then incubated with anti-human IgG antibodies (alkaline phosphatase-labelled) for 30 minutes. The strips are again washed to remove unbound anti-human IgG antibodies and nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate substrate (NBT/BCIP) is added. Alkaline phosphatase enzyme converts the soluble substrate into a colored insoluble product on the membrane to produce a black band. Strips are digitized via picture capture on the EUROBlot One instrument and evaluated with the EUROLineScan software.(O'Connor K, Waters P, Komorowski L, et al. GABAA receptor autoimmunity: A multicenter experience. Neurol Neuroimmunol Neuroinflamm. 2019;6[3]:e552. doi:10.1212/NXI.0000000000000552)

Immunoblot for MAP1B:

Test strips are incubated with appropriately diluted sample, prepared to meet the assay's required end-dilution for each specimen type. Samples and controls are added directly to the strips, ensuring they are fully covered with dilution buffer, then mixed gently and incubated for 60 minutes at room temperature on a rocking platform. After incubation, the strips undergo a series of wash steps using diluted wash buffer, followed by a 30-minute incubation with a ready-to-use alkaline phosphatase-labeled IgG conjugate. The strips are washed again using the same procedure, then incubated with a ready-to-use substrate solution for 20 minutes at room temperature. The reaction is stopped by rinsing with distilled water through several wash cycles. Strips are then placed on absorbent material and allowed to dry fully, generally requiring at least two hours. Results are interpreted only after the strips have dried completely, and dried strips are stored protected from light.(Package insert: PCA-2 line assay. ravo Diagnostika GmbH; 05/2023)

Profile tests: Monday through Sunday; Reflex tests: Varies

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• Clients without access to Test Prices can contact Customer Service 24 hours a day, seven days a week.
• Prospective clients should contact their account representative. For assistance, contact Customer Service.

This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.

86255 x 8

86256 AAGTS (if appropriate)

84182 AN1BS (if appropriate)

86256 AN1TS (if appropriate)

84182 AN2BS (if appropriate)

86255 APBCS (if appropriate)

86256 APBTS (if appropriate)

86256 CRMTS (if appropriate)

84182 CRMWS (if appropriate)

86256 DPPTS (if appropriate)

84182 MP1BS (if appropriate)

86256 PC2TS (if appropriate)