Test Id : DYS2

If the indirect immunofluorescence assay (IFA) patterns suggest collapsin response-mediator protein (CRMP)-5-IgG, then 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 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 AP3B2 cell-binding assay (CBA) and AP3B2 IFA titer will be performed at an additional charge.

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

If the IFA pattern suggests Purkinje cytoplasmic antibody type 2 (PCA-2), then PCA-2 titer is 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.

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

Provide the following information:

-Relevant clinical information

-Ordering provider name, phone number, mailing address, and e-mail address

Patient Preparation:

1. For optimal antibody detection, specimen collection is recommended before initiation of immunosuppressant medication or intravenous immunoglobulin treatment.

2. 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 1 week and assayed if sufficiently decayed or canceled if radioactivity remains.

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

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.

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 indirect immunofluorescence assay (IFA) patterns suggest collapsin response-mediator protein (CRMP)-5-IgG, then 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 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 AP3B2 cell-binding assay (CBA) and AP3B2 IFA titer will be performed at an additional charge.

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

If the IFA pattern suggests Purkinje cytoplasmic antibody type 2 (PCA-2), then PCA-2 titer is 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. A direct relationship has been demonstrated between antibody titer and severity of dysautonomia in both alpha-3-AChR-immunized animals and patients with autoimmune dysautonomia. Patients with high alpha-3-AChR autoantibody values (>1.0 nmol/L) generally have profound pandysautonomia. Dysautonomic patients with lower alpha-3-AChR autoantibody values (0.03-0.99 nmol/L) have limited 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 favors neurologic improvement and lessens 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:

Immunofluorescence assay (IFA)

Cell-binding assay (CBA)

Western blot (WB)

Radioimmunoassay (RIA)

Immunoblot (IB)

Neuron-restricted patterns of IgG staining that do not fulfill criteria for ANNA-1, CRMP-5-IgG, or PCA-2 may be reported as "unclassified anti-neuronal IgG." Complex patterns that include nonneuronal elements may be reported as "uninterpretable."

Note: CRMP-5 titers lower than 1:240 are detectable by recombinant CRMP-5 Western blot analysis. CRMP-5 Western blot analysis will be done on request on stored serum (held 4 weeks). This supplemental testing is recommended in cases of chorea, vision loss, cranial neuropathy, and myelopathy. Call 800-533-1710 to request CRMP-5 Western blot.

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. Vernino S, Low PA, Fealey RD, Stewart JD, Farrugia G, Lennon VA: Autoantibodies to ganglionic acetylcholine receptors in autoimmune autonomic neuropathies. N Engl J Med. 2000 Sep 21;343(12):847-855

2. O'Suilleabhain PO, Low PA, Lennon VA: Autonomic dysfunction in the Lambert-Eaton myasthenic syndrome: serologic and clinical correlates. Neurology. 1998 Jan;50(1):88-93

3. Dhamija R, Tan KM, Pittock SJ, Foxx-Orenstein A, Benarroch E, Lennon VA: Serological profiles aiding the diagnosis of autoimmune gastrointestinal dysmotility. Clin Gastroenterol Hepatol. 2008 Sep;6(9):988-992

4. McKeon A, Lennon VA, Lachance DH, Fealey RD, Pittock SJ: Ganglionic acetylcholine receptor autoantibody: oncological, neurological and serological accompaniments. Arch Neurol. 2009 Jun;66(6):735-741

5. McKeon A, Lennon VA, LaChance DH, Klein CJ, Pittock SJ: Striational antibodies in a paraneoplastic context. Muscle Nerve. 2013 Apr;47(4):585-587

Cell Binding Assay:

Patient specimen is applied to a composite slide containing transfected and nontransfected HEK-293 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 are scored 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 Jul 18;4(5):e385. doi: 10.1212/NXI.0000000000000385)

Radioimmunoassay:

Duplicate aliquots of patient specimen are incubated with (125)I-labeled antigen. Immune complexes, formed by adding secondary (goat) antihuman immunoglobulin, are pelleted by centrifugation and washed. Gamma emission from the washed pellet is counted, and mean counts per minute (cpm) are compared with results yielded by high positive and negative control sera. Specimen yielding cpm higher than the background cpm yielded by normal human specimen are retested to confirm positivity and titrated as necessary to obtain a value in the linear range of the assay. The antigen binding capacity (nmol per liter) is calculated from the cpm precipitated at a dilution yielding a linear range value.(Griesmann GE, Kryzer TJ, Lennon VA: Autoantibody profiles of myasthenia gravis and Lambert-Eaton myasthenic syndrome. In: Rose NR, Hamilton RG, eds. Manual of Clinical and Laboratory Immunology. 6th ed. ASM Press; 2002:1005-1012; Walikonis JE, Lennon VA: Radioimmunoassay for glutamic acid decarboxylase [GAD65] autoantibodies as a diagnostic aid for stiff-man syndrome and a correlate of susceptibility to type 1 diabetes mellitus. Mayo Clin Proc. 1998 December;73[12]:1161-1166; Jones AL, Flanagan EP, Pittock SJ, et al: Responses to and outcomes of treatment of autoimmune cerebellar ataxia in adults. JAMA Neurol. 2015 Nov;72[11]:1304-1312. doi: 10.1001/jamaneurol.2015.2378)

Immunoblot:

All steps are performed at room temperature (18 to 28 degrees C) utilizing the EUROBlot One instrument. Diluted patient serum (1:101) 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 serum 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 is added. Alkaline phosphatase enzyme converts the soluble substrate into a colored insoluble product on the membrane to produces 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 Apr 4;6[3]:e552. doi: 10.1212/NXI.0000000000000552)

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 February;49[2]:146-154; Dubey D, Jitprapaikulsan J, Bi H, et al: Amphiphysin-IgG autoimmune neuropathy: A recognizable clinicopathologic syndrome. Neurology. 2019 Nov 12;93(20):e1873-e1880. doi: 10.1212/WNL.0000000000008472)

Profile tests: Monday through Sunday; Reflex tests: Varies

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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.

83519

86255 x 7

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)

86255 DPPCS (if appropriate)

86256 DPPTS (if appropriate)

86256 PC2TS (if appropriate)