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Test Code LAB4195/6640/PARAN Paraneoplastic, Autoantibody Evaluation, Serum

Important Note

Send Outs - Mayo

Additional Codes

Mayo Code: PAVAL


Necessary Information


Provide the following information:

-Relevant clinical information

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



Specimen Required


Patient Preparation:

1. For optimal antibody detection, specimen collection is recommended prior to initiation of immunosuppressant medication or intravenous immunoglobulin (IVIg) treatment.

2. This test should not be requested in 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.

3. Patient should have no general anesthetic or muscle-relaxant drugs in the previous 24 hours.

 

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 plastic vial.


Method Name

AGN1S, AMPHS, AMPIS, ANN1S, ANN2S, ANN3S, CRMS, DPPIS, DPPTS, GABIS, GL1IS, GL1TS, NMDIS, PCAB2, PCABP, PCATR: Indirect Immunofluorescence Assay (IFA)

 

ARBI, CCPQ, GD65S, VGKC: Radioimmunoassay (RIA)

 

CRMWS: Western Blot (WB)

 

AGNBS, AMIBS, AN1BS, AN2BS, PC1BS, PCTBS: Immunoblot (IB)

 

AMPCS, CS2CS, DPPCS, GABCS, GL1CS, LG1CS, NMDCS: Cell-Binding Assay (CBA)

 

ACMFS: Flow Cytometry

Reporting Name

Paraneoplastic Autoantibody Eval, S

Specimen Type

Serum

Reject Due To

Gross hemolysis Reject
Gross lipemia Reject
Gross icterus Reject

Day(s) Performed

Profile tests: Monday through Sunday; Reflex tests: Varies

Report Available

10 to 17 days

Performing Laboratory

Mayo Clinic Laboratories in Rochester

Test Classification

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

CPT Code Information

83519

86596

86255 x 9

83519-ARBI (if appropriate)

86255 ACMFS (if appropriate)

84182-AGNBS (if appropriate)

86255-AMPCS (if appropriate)

86256-AMPIS (if appropriate)

84182-AMIBS (if appropriate)

84182-AN1BS (if appropriate)

84182-AN2BS (if appropriate)

86255-CS2CS (if appropriate)

84182-CRMWS (if appropriate)

86255-DPPCS (if appropriate)

86256-DPPTS (if appropriate)

86255-DPPIS (if appropriate)

86255-GABCS (if appropriate)

86256-GABIS (if appropriate)

86341-GD65S (if appropriate)

86255-LG1CS (if appropriate)

86255-GL1CS (if appropriate)

86256-GL1TS (if appropriate)

86255-GL1IS (if appropriate)

86255-NMDCS (if appropriate)

86256-NMDIS (if appropriate)

84182-PC1BS (if appropriate)

84182-PCTBS (if appropriate)

LOINC Code Information

Test ID Test Order Name Order LOINC Value
PAVAL Paraneoplastic Autoantibody Eval, S 43104-9

 

Result ID Test Result Name Result LOINC Value
80776 ANNA-2, S 94343-1
83137 ANNA-3, S 94344-9
81185 P/Q-Type Calcium Channel Ab 94349-8
83077 CRMP-5-IgG, S 94815-8
29347 Interpretive Comments 57771-8
83138 PCA-2, S 94351-4
9477 PCA-1, S 94350-6
83076 PCA-Tr, S 94352-2
89165 Neuronal (V-G) K+ Channel Ab, S 94816-6
89080 AGNA-1, S 94341-5
81722 Amphiphysin Ab, S 94340-7
80150 ANNA-1, S 94342-3
36349 Reflex Added 77202-0

Secondary ID

83380

Useful For

Serological evaluation of patients who present with a subacute neurological disorder of undetermined etiology, especially those with known risk factors for cancer

 

Directing a focused search for cancer

 

Investigating neurological symptoms that appear in the course of, or after, cancer therapy, and are not explainable by metastasis

 

Differentiating autoimmune neuropathies from neurotoxic effects of chemotherapy

 

Monitoring the immune response of seropositive patients in the course of cancer therapy

 

Detecting early evidence of cancer recurrence in previously seropositive patients

Clinical Information

Paraneoplastic autoimmune neurological disorders reflect a patient's humoral and cellular immune responses to cancer. The cancer may be new or recurrent, is usually limited in metastatic volume, and is often occult by standard imaging procedures. Autoantibodies specific for onconeural proteins found in the plasma membrane, cytoplasm, and nucleus of neurons, glia, or muscle are generated in this immune response and serve as serological markers of paraneoplastic autoimmunity. Cancers recognized in this context most commonly are small-cell lung carcinoma, thymoma, ovarian (or related mullerian) carcinoma, breast carcinoma, and Hodgkin lymphoma. Pertinent childhood neoplasms recognized thus far include neuroblastoma, thymoma, Hodgkin lymphoma, and chondroblastoma. An individual patient's autoantibody profile can predict a specific neoplasm with 90% certainty, but not the neurological syndrome.

 

Four classes of autoantibodies are recognized in this evaluation:

-Neuronal nuclear (ANNA-1, ANNA-2, ANNA-3)

-Anti-glial/neuronal nuclear (AGNA-1; also known as Sox1)

-Neuronal and muscle cytoplasmic (Purkinje cytoplasmic antibody [PCA]-1, PCA-2, PCA-Tr, collapsin response-mediator protein [CRMP]-5, amphiphysin, and striational)

-Plasma membrane cation channel, P/Q-type calcium channel, dendrotoxin-sensitive potassium channels, and muscle nicotinic acetylcholine receptors (AChR). These autoantibodies are potential effectors of neurological dysfunction.

 

Patients who are seropositive usually present with subacute neurological symptoms and signs such as encephalopathy, cerebellar ataxia, myelopathy, radiculopathy, plexopathy, or sensory, sensorimotor, or autoimmune neuropathy, with or without a neuromuscular transmission disorder: Lambert-Eaton syndrome, myasthenia gravis, or neuromuscular hyperexcitability. Initial signs may be subtle, but a subacute multifocal and progressive syndrome usually evolves. Sensorimotor neuropathy and cerebellar ataxia are common presentations, but the clinical picture in some patients is dominated by striking gastrointestinal dysmotility, limbic encephalopathy, basal ganglionitis, or cranial neuropathy (especially loss of vision, hearing, smell, or taste).

 

Cancer risk factors include past or family history of cancer, history of smoking, or social or environmental exposure to carcinogens. Early diagnosis and treatment of the neoplasm favor less neurological morbidity and offer the best hope for survival.

Interpretation

Antibodies directed at onconeural proteins shared by neurons, glia, muscle, and certain cancers are valuable serological markers of a patient's immune response to cancer. They are not found in healthy subjects and are usually accompanied by subacute neurological symptoms and signs. Several autoantibodies have a syndromic association, but no autoantibody predicts a specific neurological syndrome. Conversely, a positive autoantibody profile has 80% to 90% predictive value for a specific cancer. It is not uncommon for more than one paraneoplastic autoantibody to be detected, each predictive of the same cancer.

Cautions

Negative results do not exclude cancer.

 

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

 

This evaluation does not include Ma2 autoantibody (alias MaTa). Ma2 autoantibody has been described in patients with brainstem and limbic encephalitis in the context of testicular germ cell neoplasms. Scrotal ultrasound is advisable in men who present with unexplained subacute encephalitis. N-methyl-D-aspartate receptor antibodies have been reported in women with paraneoplastic encephalitis related to ovarian teratoma.

Clinical Reference

1. McKeon A, Pittock SJ: Paraneoplastic encephalomyelopathies: pathology and mechanisms. Acta Neuropathol. 2011 Oct;122(4):381-400. doi: 10.1007/s00401-011-0876-1

2. Horta ES, Lennon VA, Lachance DH, et al: Neural autoantibody clusters aid diagnosis of cancer. Clin Cancer Res. 2014 Jul;20(14):3862-3869. doi: 10.1158/1078-0432.CCR-14-0652

Method Description

Indirect Immunofluorescence Assay:

The patient's specimen is tested by a standardized indirect immunofluorescence assay (IFA) that uses a composite frozen section of mouse cerebellum, kidney, and gut tissues. After incubation with specimen 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 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.(Vernino S, Kryzer TJ, Lennon AV: Autoimmune autonomic neuropathy and neuromuscular hyperexcitability disorders. In: Rose NR, Hamilton RG, Detrick B, eds. Manual of Clinical and Laboratory Immunology. 6th ed. ASM Press; 2002:1013-1017; 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)

 

Sorting Assay/Flow Cytometry:

This method uses flow cytometry to measure the loss of acetylcholine receptor modulating (AChR) molecules expressed on the surface of live cells expressing AChR on the cell surface. The cell line used is an immortalized human rhabdomyosarcoma cell line that expresses endogenous muscle-type nicotinic AChR on its surface. Cells are plated in a 96-well plate and cultured 72 hours prior to the addition of patient serum for an additional 18 to 22 hours to enable internalization of AChR receptors (modulation). Modulation is then stopped by placing cells on ice. The amount of remaining AChRs on the cell surface is measured by flow cytometry. On ice, cells are incubated with a recombinant rat monoclonal antibody against alpha-subunit of the AChR followed by a secondary goat anti-rat IgG antibody conjugated with allophycocyanin (APC). The amount of AChR on the cell surface is proportional to the median fluorescence intensity (MFI) of APC. To calculate the amount of modulation (ie, % loss of AChR) the APC MFI is compared between cells treated with patient serum and cells treated with serum lacking AChR modulating antibodies. Background signal is established in each experiment utilizing cells stained with secondary antibody alone (no patient sera). The percent loss of AChR is calculated as 1-[(Patient MFI-Background MFI)/(Negative calibrator MFI - Background MFI)]*100%.(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, et al: CRMP-5 neuronal autoantibody: marker of lung cancer and thymoma-related autoimmunity. Ann Neurol. 2001 Feb;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)

 

Immunoblot:

All steps are performed at room temperature (18-28° 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 serums will bind to the purified recombinant antigen and negative serums will not bind. Strips are washed to remove unbound serum antibodies and then incubated with anti-human IgG antibodies (alkaline phosphatase-labelled) and incubated 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 (NBT/BCIP) 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)

 

Cell-Binding Assay:

Patient serum 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)

Specimen Retention Time

28 days

Specimen Minimum Volume

2 mL

Reference Values

Test ID

Reporting name

Methodology

Reference value

AMPHS

Amphiphysin Ab, S

IFA

<1:240

AGN1S

Anti-Glial Nuclear Ab, Type 1

IFA

<1:240

ANN1S

Anti-Neuronal Nuclear Ab, Type 1

IFA

<1:240

ANN2S

Anti-Neuronal Nuclear Ab, Type 2

IFA

<1:240

ANN3S

Anti-Neuronal Nuclear Ab, Type 3

IFA

<1:240

CRMS

CRMP-5-IgG, S

IFA

<1:240

VGKC

Neuronal (V-G) K+ Channel Ab, S

RIA

≤0.02 nmol/L

CCPQ

P/Q-Type Calcium Channel Ab

RIA

≤0.02 nmol/L

PCABP

Purkinje Cell Cytoplasmic Ab Type 1

IFA

<1:240

PCAB2

Purkinje Cell Cytoplasmic Ab Type 2

IFA

<1:240

PCATR

Purkinje Cell Cytoplasmic Ab Type Tr

IFA

<1:240

 

Reflex Tests:

Test ID

Reporting name

Methodology

Reference value

ARBI

ACh Receptor (Muscle) Binding Ab

RIA

≤0.02 nmol/L

AGNBS

AGNA-1 Immunoblot, S

IB

Negative

AMPCS

AMPA-R Ab CBA, S

CBA

Negative

AMPIS

AMPA-R Ab IF Titer Assay, S

IFA

<1:120

AMIBS

Amphiphysin Immunoblot, S

IB

Negative

AN1BS

ANNA-1 Immunoblot, S

IB

Negative

AN2BS

ANNA-2 Immunoblot, S

IB

Negative

CS2CS

CASPR2-IgG CBA, S

CBA

Negative

CRMWS

CRMP-5-IgG Western Blot, S

WB

Negative

DPPCS

DPPX Ab CBA, S

CBA

Negative

DPPIS

DPPX Ab IFA, S

IFA

Negative

DPPTS

DPPX Ab IFA Titer, S

IFA

<1:240

GABCS

GABA-B-R Ab CBA, S

CBA

Negative

GABIS

GABA-B-R Ab IF Titer Assay, S

IFA

<1:120

GD65S

GAD65 Ab Assay, S

RIA

≤0.02 nmol/L

Reference values apply to all ages

LG1CS

LGI1-IgG CBA, S

CBA

Negative

GL1CS

mGluR1 Ab CBA, S

CBA

Negative

GL1IS

mGluR1 Ab IFA, S

IFA

Negative

GL1TS

mGluR1 Ab IFA Titer, S

IFA

<1:240

NMDCS

NMDA-R Ab CBA, S

CBA

Negative

NMDIS

NMDA-R Ab IF Titer Assay, S

IFA

<1:120

PC1BS

PCA-1 Immunoblot, S

IB

Negative

PCTBS

PCA-Tr Immunoblot, S

IB

Negative

ACMFS

ACh Receptor Modulating FACS, S

Flow Cytometry

Negative

 

*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 amphiphysin, ANNA-1, ANNA-2, ANNA-3, AGNA-1, PCA-1, PCA-2, PCA-Tr, or CRMP-5-IgG may be reported as "unclassified antineuronal IgG." Complex patterns that include non-neuronal 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.

Forms

If not ordering electronically, complete, print, and send 1 of the following forms with the specimen:

-General Request (T239)

-Neurology Specialty Testing Client Test Request (T732)

Specimen Stability Information

Specimen Type Temperature Time Special Container
Serum Refrigerated (preferred) 28 days
  Frozen  28 days
  Ambient  72 hours

Profile Information

Test ID Reporting Name Available Separately Always Performed
PAINT Interpretive Comments No Yes
AMPHS Amphiphysin Ab, S No Yes
AGN1S Anti-Glial Nuclear Ab, Type 1 No Yes
ANN1S Anti-Neuronal Nuclear Ab, Type 1 No Yes
ANN2S Anti-Neuronal Nuclear Ab, Type 2 No Yes
ANN3S Anti-Neuronal Nuclear Ab, Type 3 No Yes
CRMS CRMP-5-IgG, S No Yes
VGKC Neuronal (V-G) K+ Channel Ab, S No Yes
CCPQ P/Q-Type Calcium Channel Ab No Yes
PCABP Purkinje Cell Cytoplasmic Ab Type 1 No Yes
PCAB2 Purkinje Cell Cytoplasmic Ab Type 2 No Yes
PCATR Purkinje Cell Cytoplasmic Ab Type Tr No Yes

Reflex Tests

Test ID Reporting Name Available Separately Always Performed
ARBI ACh Receptor (Muscle) Binding Ab Yes No
AGNBS AGNA-1 Immunoblot, S No No
AMPCS AMPA-R Ab CBA, S No No
AMPIS AMPA-R Ab IF Titer Assay, S No No
AMIBS Amphiphysin Immunoblot, S No No
AN1BS ANNA-1 Immunoblot, S No No
AN2BS ANNA-2 Immunoblot, S No No
CS2CS CASPR2-IgG CBA, S No No
CRMWS CRMP-5-IgG Western Blot, S Yes No
DPPCS DPPX Ab CBA, S No No
DPPIS DPPX Ab IFA, S No No
DPPTS DPPX Ab IFA Titer, S No No
GABCS GABA-B-R Ab CBA, S No No
GABIS GABA-B-R Ab IF Titer Assay, S No No
GD65S GAD65 Ab Assay, S Yes No
LG1CS LGI1-IgG CBA, S No No
GL1CS mGluR1 Ab CBA, S No No
GL1IS mGluR1 Ab IFA, S No No
GL1TS mGluR1 Ab IFA Titer, S No No
NMDCS NMDA-R Ab CBA, S No No
NMDIS NMDA-R Ab IF Titer Assay, S No No
PC1BS PCA-1 Immunoblot, S No No
PCTBS PCA-Tr Immunoblot, S No No
ACMFS AChR Modulating Flow Cytometry, S No No

Testing Algorithm

If immunofluorescence assay (IFA) patterns suggest antiglial nuclear antibody-1 (AGNA-1) antibody, then AGNA-1 immunoblot is performed at an additional charge.

 

If IFA patterns suggest amphiphysin antibody, then amphiphysin immunoblot is performed at an additional charge.

 

If IFA patterns suggest antineuronal nuclear antibodies (ANNA)-1 antibody, then ANNA-1 immunoblot is performed at an additional charge.

 

If IFA patterns suggest ANNA-2 antibody, then ANNA-2 immunoblot is performed at an additional charge.

 

If IFA patterns suggest Purkinje cytoplasmic antibody (PCA)-1 antibody, then PCA-1 immunoblot is performed at an additional charge.

 

If IFA patterns suggest PCA-Tr antibody, then PCA-Tr immunoblot is performed at an additional charge.

 

If IFA patterns suggest glutamic acid decarboxylase 65 (GAD65) antibody, then GAD65 antibody radioimmunoassay (RIA) is performed at an additional charge.

 

If IFA pattern suggests N-methyl-D-aspartate (NMDA)-receptor, then NMDA-receptor antibody cell-binding assay (CBA), and/or NMDA- receptor antibody titer is performed at an additional charge.

 

If IFA pattern suggests alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-receptor, then AMPA- receptor antibody CBA and/or AMPA- receptor antibody titer is performed at an additional charge.

 

If IFA pattern suggests gamma-aminobutyric acid B (GABA-B)-receptor, then GABA-B- receptor antibody CBA and/or GABA-B- receptor antibody titer is performed at an additional charge.

 

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

 

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

 

If voltage-gated potassium channels (VGKC) is above 0.00 nmol/L, then leucine-rich, glioma inactivated 1 (LGI1)-IgG CBA and contactin-associated protein-like 2 (CASPR2)-IgG are performed at an additional charge.

 

If collapsin response-mediator protein (CRMP) IFA is positive, then acetylcholine (muscle) receptor (AChR) binding antibody, CRMP-5-IgG Western blot, and ACh receptor (muscle) modulating antibody by fluorescence-activated cell sorting (FACS) will be performed at an additional charge.

 

CRMP-5-IgG Western blot is also performed by specific request for more sensitive detection of CRMP-5-IgG. Testing should be requested in cases of subacute basal ganglionic disorders (chorea, Parkinsonism), cranial neuropathies (especially loss of vision, taste, or smell) and myelopathies.

 

The following algorithms are available:

-Paraneoplastic Evaluation Algorithm-Serum

-Hereditary Peripheral Neuropathy Diagnostic Algorithm