Test Id : MAC1

If the indirect immunofluorescence assay (IFA) pattern suggests anti-glial nuclear antibody (AGNA-1), then AGNA-1 immunoblot and AGNA-1 IFA titer will be performed at an additional charge.

If the IFA pattern suggests amphiphysin antibody, then amphiphysin immunoblot and amphiphysin IFA titer will be performed at an additional charge.

If the IFA pattern suggests anti-neuronal 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 ANNA-2 antibody, then ANNA-2 immunoblot, ANNA-2 IFA titer, and ANNA-1 immunoblot will be performed at an additional charge.

If client requests or the IFA pattern suggests ANNA-3 antibodies, then ANNA-3 IFA titer will be performed at an additional charge.

If the IFA pattern suggests adaptor protein 3 beta 2 (AP3B2) antibodies, then AP3BC cell-binding assay (CBA) and AP3BC IFA titer will be performed at an additional charge.

If collapsin response-mediator protein 5 (CRMP-5)-IgG Western blot is positive, then CRMP-5-IgG IFA titer is performed at an additional charge. 

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

If the IFA pattern suggests PCA-2 antibody, then PCA-2 IFA titer will be performed at an additional charge.

If the IFA pattern suggests N-methyl-D-aspartate (NMDA) receptor antibody, then NMDA-receptor antibody CBA and NMDA-receptor antibody IFA titer will be performed at an additional charge.

If gamma-aminobutyric acid B (GABA-B) receptor antibody CBA is positive, then GABA-B-receptor antibody IFA titer will be performed at an additional charge.

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

If the IFA pattern suggests metabotropic glutamate receptor 1 (mGluR1) antibody, then mGluR1 antibody CBA and mGluR1 antibody IFA titer will be performed at an additional charge.

If the IFA pattern suggests glial fibrillary acidic protein (GFAP) antibody, then GFAP antibody CBA and GFAP antibody IFA titer will be performed at an additional charge.

If the neuromyelitis optica/aquaporin-4-IgG (NMO/AQP4-IgG) fluorescence-activated cell sorting (FACS) screen assay requires further investigation, then NMO/AQP4-IgG FACS titration assay will be performed at an additional charge.

If the IFA pattern suggests neuronal intermediate filament (NIF) antibody, then alpha internexin CBA, NIF heavy chain CBA, NIF light chain CBA, and NIF antibody IFA titer will be performed at an additional charge.

If the IFA pattern suggests neurochondrin antibody, then neurochondrin antibody CBA and neurochondrin IFA titer will be performed at an additional charge.

If the IFA pattern suggests septin 7 antibody, then septin 7 CBA and septin 7 IFA titer will be performed at an additional charge.

For more information see Autoimmune/Paraneoplastic Myelopathy Evaluation Algorithm-Spinal Fluid.

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

Container/Tube: Sterile vial

Preferred: Vial number 1

Acceptable: Any vial

Specimen Volume: 4 mL

• Autoimmune/Paraneoplastic Myelopathy Evaluation Algorithm-Spinal Fluid

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

2 mL

Evaluating patients with suspected autoimmune myelopathy, myelitis, and paraneoplastic myelopathy using spinal fluid specimens

If the indirect immunofluorescence assay (IFA) pattern suggests anti-glial nuclear antibody (AGNA-1), then AGNA-1 immunoblot and AGNA-1 IFA titer will be performed at an additional charge.

If the IFA pattern suggests amphiphysin antibody, then amphiphysin immunoblot and amphiphysin IFA titer will be performed at an additional charge.

If the IFA pattern suggests anti-neuronal 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 ANNA-2 antibody, then ANNA-2 immunoblot, ANNA-2 IFA titer, and ANNA-1 immunoblot will be performed at an additional charge.

If client requests or the IFA pattern suggests ANNA-3 antibodies, then ANNA-3 IFA titer will be performed at an additional charge.

If the IFA pattern suggests adaptor protein 3 beta 2 (AP3B2) antibodies, then AP3BC cell-binding assay (CBA) and AP3BC IFA titer will be performed at an additional charge.

If collapsin response-mediator protein 5 (CRMP-5)-IgG Western blot is positive, then CRMP-5-IgG IFA titer is performed at an additional charge. 

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

If the IFA pattern suggests PCA-2 antibody, then PCA-2 IFA titer will be performed at an additional charge.

If the IFA pattern suggests N-methyl-D-aspartate (NMDA) receptor antibody, then NMDA-receptor antibody CBA and NMDA-receptor antibody IFA titer will be performed at an additional charge.

If gamma-aminobutyric acid B (GABA-B) receptor antibody CBA is positive, then GABA-B-receptor antibody IFA titer will be performed at an additional charge.

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

If the IFA pattern suggests metabotropic glutamate receptor 1 (mGluR1) antibody, then mGluR1 antibody CBA and mGluR1 antibody IFA titer will be performed at an additional charge.

If the IFA pattern suggests glial fibrillary acidic protein (GFAP) antibody, then GFAP antibody CBA and GFAP antibody IFA titer will be performed at an additional charge.

If the neuromyelitis optica/aquaporin-4-IgG (NMO/AQP4-IgG) fluorescence-activated cell sorting (FACS) screen assay requires further investigation, then NMO/AQP4-IgG FACS titration assay will be performed at an additional charge.

If the IFA pattern suggests neuronal intermediate filament (NIF) antibody, then alpha internexin CBA, NIF heavy chain CBA, NIF light chain CBA, and NIF antibody IFA titer will be performed at an additional charge.

If the IFA pattern suggests neurochondrin antibody, then neurochondrin antibody CBA and neurochondrin IFA titer will be performed at an additional charge.

If the IFA pattern suggests septin 7 antibody, then septin 7 CBA and septin 7 IFA titer will be performed at an additional charge.

For more information see Autoimmune/Paraneoplastic Myelopathy Evaluation Algorithm-Spinal Fluid.

Patients with autoimmune myelopathy present with subacute onset and rapid progression of spinal cord symptoms with one or more of the following: weakness, gait difficulties, loss of sensation, neuropathic pain, and bowel and bladder dysfunction. Clinical history and examination, spinal cord magnetic resonance imaging, and cerebrospinal fluid (CSF) testing may provide clues to an autoimmune diagnosis. Autoimmune myelopathy evaluation of both serum and CSF can assist in the diagnosis (paraneoplastic or idiopathic autoimmune) and aid distinction from other causes of myelopathy (multiple sclerosis, sarcoidosis, vascular disease). Early testing may assist in early diagnosis of occult cancer, prompt initiation of immune therapies, or both.

Reflex Information:

*Methodology Abbreviations:

Immunofluorescence assay (IFA)

Cell-binding assay (CBA)

Flow Cytometry (FCM)

Radioimmunoassay (RIA)

Immunoblot (IB)

Western blot (WB)

Neuron-restricted patterns of IgG staining that do not fulfill criteria for ANNA-1, ANNA-2, ANNA-3, CRMP-5-IgG, PCA-1, or PCA-2  may be reported as "unclassified anti-neuronal IgG." Complex patterns that include nonneuronal elements may be reported as "uninterpretable."

A positive result is consistent with a diagnosis of autoimmune myelopathy in the appropriate clinical context.

Negative results do not exclude a diagnosis of autoimmune myelopathy.

1. Dubey D, Pittock SJ, Krecke KN, et al: Clinical, radiologic, and prognostic features of myelitis associated with myelin oligodendrocyte glycoprotein autoantibody. JAMA Neurol. 2019 Mar 1;76(3):301-309. doi: 10.1001/jamaneurol.2018.4053

2. Zalewski NL, Flanagan EP: Autoimmune and paraneoplastic myelopathies. Semin Neurol. 2018 Jun;38(3):278-289. doi: 10.1055/s-0038-1660856

3. Flanagan EP, Hinson SR, Lennon VA, et al: Glial fibrillary acidic protein immunoglobulin G as biomarker of autoimmune astrocytopathy: Analysis of 102 patients. Ann Neurol. 2017 Feb;81(2):298-309. doi: 10.1002/ana.24881

4. Keegan BM, Pittock SJ, Lennon VA: Autoimmune myelopathy associated with collapsin response-mediator protein-5 immunoglobulin G. Ann Neurol. 2008 Apr;63(4):531-534. doi: 10.1002/ana.21324

5. Weinshenker BG, Wingerchuk DM, Vukusic S, et al: Neuromyelitis optica IgG predicts relapse after longitudinally extensive transverse myelitis. Ann Neurol. 2006 Mar;59(3):566-569. doi: 10.1002/ana.20770

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, 02/25/2019)

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 the patient specimen are incubated with (125)-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 specimens 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, et al, 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 Dec;73[12]:1161-1166. doi: 10.4065/73.12.1161; 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)

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 Feb;49[2]:145-154; Dubey D, Jitprapaikulsan J, Bi H, et al: Amphiphysin-IgG autoimmune neuropathy: a recognizable clinicopathologic syndrome. Neurology. 019 Nov 12;93[20]:e1873-e1880. doi: 10.1212/WNL.0000000000008472)

Immunoblot:

All steps are performed at room temperature (18-28 degrees C) utilizing the EUROBlot One instrument. Diluted patient specimens (1:12.5) are added to test strips (strips containing recombinant antigen manufactured and purified using biochemical methods) in individual channels and incubated for 30 minutes. Positive specimens will bind to the purified recombinant antigen, and negative specimens will not bind. Strips are washed to remove unbound antibodies and then incubated with anti-human IgG antibodies (alkaline phosphatase-labeled) 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 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 Apr 4;6[3]:e552. doi: 10.1212/NXI.0000000000000552)

NMO-IgG Fluorescence-Activated Cell Sorting Assay/Flow Cytometry:

Human embryonic kidney cells (HEK 293) are transfected transiently with a plasmid (pIRES2-Aequorea coerulescens green fluorescent protein [AcGFP]) encoding both green fluorescent protein (AcGFP) and AQP4-M1. After 36 hours, a mixed population of cells (transfected expressing AQP4 on the surface and AcGFP in the cytoplasm and nontransfected lacking AQP4 and AcGFP) are lifted and resuspended in live cell binding buffer. Patient specimen is then added to cells at a 1 in 5 screening dilution. After incubation and washing, the cells are resuspended in secondary antibody (AlexaFluor 647-conjugated goat-antihuman IgG; 1:2000 in LCBB), held on ice, washed, fixed with 4% paraformaldehyde, and analyzed by flow cytometry (BD FACSCanto; Becton, Dickinson, and Co). Two populations are gated based on AcGFP expression: positive (high AQP4 expression) and negative (low or no AQP4 expression). The median Alexafluor 647 fluorescence intensity (MFI) for the AcGFP-positive population indicates the relative abundance of human IgG potentially bound to AQP4 surface epitopes; MFI for the GFP-negative population indicated nonspecifically-bound IgG. The IgG binding index is calculated as the ratio of the average MFI for duplicate aliquots of each cell population (MFI GFP positive/MFI GFP negative). We established conservative cutoff IgG binding index values of 2.00 for M1-FACS.(Fryer JP, Lennon VP, Pittock SJ, et al: AQP4 autoantibody assay performance in clinical laboratory service. Neurol Neuroimmunol Neuroinflamm. 2014 May 22; 1[1]:e11. doi: 10.1212/NXI.0000000000000011)

If the fluorescence-activated cell sorting (FACS) assay is positive at screening dilution, FACS titer assay is performed at an additional charge. The patient specimen is titrated to endpoint. The dilution where the IgG binding index is greater than or equal to 2, is considered the endpoint dilution. If a patient is positive at a 1:5 dilution, but negative at 1:10 dilution, the endpoint will be reported as 5.

Profile tests: Monday through Sunday; Reflex tests: Varies

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• 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 15

86053

86341

84182

84182 AGNBC (if appropriate)

86256 AGNTC (if appropriate)

86255 AINCC (if appropriate)

84182 AMIBC (if appropriate)

84182 AN1BC (if appropriate)

86256 AN1TC (if appropriate)

84182 AN2BC (if appropriate)

86256 AN2TC (if appropriate)

86256 AN3TC (if appropriate)

86255 APBCC (if appropriate)

86256 APBTC (if appropriate)

86256 APHTC (if appropriate)

86256 CRMTC (if appropriate)

86255 DPPCC (if appropriate)

86256 DPPTC (if appropriate)

86256 GABIC (if appropriate)

86255 GFACC (if appropriate)

86256 GFATC (if appropriate)

86255 GL1CC (if appropriate)

86256 GL1TC (if appropriate)

86255 NCDCC (if appropriate)

86256 NCDTC (if appropriate)

86255 NFHCC (if appropriate)

86255 NFLCC (if appropriate)

86256 NIFTC (if appropriate)

86255-NMDCC (if appropriate)

86256-NMDIC (if appropriate)

86053 NMOTC (if appropriate)

84182 PC1BC (if appropriate)

86256 PC1TC (if appropriate)

86256 PC2TC (if appropriate)

86255 SP7CC (if appropriate)

86256 SP7TC (if appropriate)