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Test Code PAC1 Paraneoplastic, Autoantibody Evaluation, Spinal Fluid

Useful For

Aids in the diagnosis of paraneoplastic neurological autoimmune disorders related to carcinoma of lung, breast, ovary, thymoma, or Hodgkin lymphoma in spinal fluid specimens

Profile Information

Test ID Reporting Name Available Separately Always Performed
PNEOI Paraneoplastic Interpretation, CSF No Yes
AGN1C Anti-Glial Nuclear Ab, Type 1 No Yes
AMPHC Amphiphysin Ab, CSF No Yes
ANN1C Anti-Neuronal Nuclear Ab, Type 1 No Yes
ANN2C Anti-Neuronal Nuclear Ab, Type 2 No Yes
ANN3C Anti-Neuronal Nuclear Ab, Type 3 No Yes
CRMC CRMP-5-IgG, CSF No Yes
PCA1C Purkinje Cell Cytoplasmic Ab Type 1 No Yes
PCA2C Purkinje Cell Cytoplasmic Ab Type 2 No Yes
PCTRC Purkinje Cell Cytoplasmc Ab Type Tr No Yes

Testing Algorithm

If indirect immunofluorescence assay (IFA) (ANN1C, ANN2C, ANN3C, PCA1C, PCA2C, PCTRC, AMPHC, CRMC, AGN1C) is indeterminate, then paraneoplastic autoantibody Western blot is performed at an additional charge.

 

If IFA pattern suggest NMO/AQP4-IgG, then NMO/AQP4-IgG FACS is performed at an additional charge.

 

If NMO/AQP4-IgG FACS screen assay requires further evaluation, then NMO/AQP4-IgG FACS titration assay is performed at an additional charge.

 

If IFA patterns suggest CRMP-5-IgG, then CRMP-5-IgG Western blot is performed at an additional charge.

 

If IFA patterns suggest GAD65 antibody, then GAD65 antibody radioimmunoassay is performed at an additional charge.

 

If IFA patterns suggest neuronal voltage-gated potassium channel-complex autoantibody, then VGKC-complex antibody IPA is performed at an additional charge.

 

If VGKCC >0.00, LGI1-IgG CBA, CSF and CASPR2-IgG CBA, CSF are performed at an additional charge.

 

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

 

If IFA pattern suggest NMDA-R, then NMDA-R antibody CBA and/or NMDA-R titer is performed at an additional charge.

 

If IFA pattern suggest AMPA-R, then AMPA-R antibody CBA and/or AMPA-R titer is performed at an additional charge.

 

If IFA pattern suggest GABA-B-R, then GABA-B-R antibody CBA and/or GABA-B-R titer is performed at an additional charge.

 

In patients with a history of tobacco use or other lung cancer risk, or if thymoma is suspected, PAVAL / Paraneoplastic Autoantibody Evaluation, Serum is also recommended.

 

See Paraneoplastic Autoantibody CSF Evaluation Algorithm in Special Instructions.

Method Name

ANN1C, ANN2C, ANN3C, PCA1C, PCA2C, PCTRC, AMPHC, CRMC, AGN1C, NMDIC, AMPIC, GABIC: Indirect Immunofluorescence Assay (IFA)

WBNC, ABLTC: Western Blot

VGKCC: Radioimmunoassay

NMOTC: Flow Cytometry

NMDCC, AMPCC, GABCC, LG1CC, CS2CC: Cell-Binding Assay (CBA)

Reporting Name

Paraneoplas Autoantibody Eval,CSF

Specimen Type

CSF


Necessary Information


Include relevant clinical information, name, phone number, mailing address, and e-mail address (if applicable) of ordering physician.



Specimen Required


Container/Tube: Sterile vial

Specimen Volume: 4 mL


Specimen Minimum Volume

2 mL

Specimen Stability Information

Specimen Type Temperature Time
CSF Refrigerated (preferred) 28 days
  Frozen  28 days
  Ambient  72 hours

Reject Due To

Hemolysis

Mild OK; Gross reject

Lipemia

Mild OK; Gross reject

Icterus

Mild OK; Gross reject

Other

NA

Clinical Information

Several antineuronal and glial autoantibodies are recognized clinically as markers of a patient's immune response to specific cancers (paraneoplastic autoantibodies). Seropositive patients present with neurologic symptoms and signs in more than 90% of cases. The cancers are most commonly small-cell lung carcinoma, ovarian (or related mullerian) carcinoma, breast carcinoma, thymoma, or Hodgkin lymphoma. The cancers may be new or recurrent, are usually limited in metastatic volume, and are often occult by standard imaging procedures. Detection of the informative marker autoantibodies allows early diagnosis and treatment of the cancer, which may lessen neurological morbidity and improve survival.

 

Serum is the preferred specimen for paraneoplastic autoantibodies. However, cerebrospinal fluid (CSF) results are sometimes positive when serum results are negative (especially for CRMP-5 and other inflammatory central nervous system autoimmunity). Additionally, CSF is more readily interpretable because it generally lacks the interfering nonorgan-specific antibodies that are common in the serum of patients with cancer. Because neurologists typically perform spinal taps in these patients, we recommend that CSF be submitted with serum, either for simultaneous testing or to be held for testing only if serum is negative.

 

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.

Reference Values

NEURONAL NUCLEAR ANTIBODIES

Antineuronal Nuclear Antibody-Type 1 (ANNA-1)

<1:2

Antineuronal Nuclear Antibody-Type 2 (ANNA-2)

<1:2

Antineuronal Nuclear Antibody-Type 3 (ANNA-3)

<1:2

Anti-Glial/Neuronal Nuclear Antibody-Type 1 (AGNA-1)

<1:2

 

NEURONAL AND MUSCLE CYTOPLASMIC ANTIBODIES

Purkinje Cell Cytoplasmic Antibody, Type 1 (PCA-1)

<1:2

Purkinje Cell Cytoplasmic Antibody, Type 2 (PCA-2)

<1:2

Purkinje Cell Cytoplasmic Antibody, Type TR (PCA-TR)

<1:2

Amphiphysin Antibody

<1:2

Collapsin Response-Mediator Protein-5 Neuronal (CRMP-5-IGG)

<1:2

 

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."

 

ISLET CELL ANTIBODIES

Glutamic Acid Decarboxylase (GAD65) Antibody Assay

≤0.02 nmol/L

 

WESTERN BLOT

Paraneoplastic Western Blot

Negative

CRMP-5-IgG Western Blot

Negative

Amphiphysin Western Blot

Negative

 

N-Methyl-D-aspartate receptor (NMDA-R)

CBA: Negative

IFA: <1:2

2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl) propanoic acid receptor (AMPA-R)

CBA: Negative

IFA: <1:2

Gamma-Amino Butyric acid-type B receptor (GABA-B-R)

CBA: Negative

IFA: <1:2

Leucine-Rich Glioma Inactivated Protein-1 IgG (LGI1) CBA

Negative

Contactin-Associated Protein-Like-2 IgG (CASPR2) CBA

Negative

Neuromyelitis Optica (NMO)/Aquaporin-4-Igg FACS Assay

Negative

 

VGKC-Complex Antibody IPA

≤0.02 nmol/L

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.

 

In patients with a history of tobacco use or other lung cancer risk, or if thymoma is suspected, PAVAL / Paraneoplastic Autoantibody Evaluation, Serum is also recommended.

Cautions

In patients with a history of tobacco use or other lung cancer risk, or if thymoma is suspected, PAVAL / Paraneoplastic Autoantibody Evaluation, Serum is also recommended.

Clinical Reference

1. Lucchinetti CF, Kimmel DW, Lennon VA: Paraneoplastic and oncological profiles of patients seropositive for type 1 anti-neuronal nuclear antibody. Neurology 1998;50:652-657

2. Graus F, Vincent A, Pozo-Rosich P, et al: Anti-glial nuclear antibody: marker of lung cancer-related paraneoplastic neurological syndromes. J Neuroimmunol 2005;154(1-2):166-171

3. Pittock SJ, Lucchinetti CF, Lennon VA: Anti-neuronal nuclear autoantibody type 2: paraneoplastic accompaniments. Ann Neurol 2003;53(5):580-587

4. Chan KH, Vernino S, Lennon VA: ANNA-3 anti-neuronal nuclear antibody: marker of lung cancer-related autoimmunity. Ann Neurol 2001 September;50(3):301-311

5. Hetzel DJ, Stanhope CR, O'Neill BP, Lennon VA: Gynecologic cancer in patients with subacute cerebellar degeneration predicted by anti-purkinje cell antibodies and limited in metastatic volume. Mayo Clin Proc 1990;65:1558-1563

6. Pittock SJ, Lucchinetti CF, Parisi JE, et al: Amphiphysin autoimmunity: paraneoplastic accompaniments. Ann Neurol 2005;58(1):96-107

Method Description

Indirect Immunofluorescence Assay (IFA):

The patient's sample is tested by a standardized IFA 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. Autoantibody of antineuronal nuclear antibody-type 1 (ANNA-1) specificity is pan-neuronal in reactivity; it binds to the cytoplasm and nucleus (sparing nucleolus) of central and peripheral nervous system neurons. Autoantibody of ANNA-2 specificity has a similar binding pattern but reactivity is restricted to neurons of the central nervous system; neurons of the peripheral nervous system are nonreactive. Autoantibody of Purkinje cell cytoplasmic antibody (PCA)-1 specificity bind distinctively to endoplasmic reticulum in Purkinje cells, molecular neurons, and other large neurons in the central and peripheral nervous system. Samples that are scored positive for any neuronal nuclear or cytoplasmic autoantibody are titrated to an endpoint on mouse Purkinje cells or peripheral neurons. Interference by coexisting non-neuron-specific autoantibodies can usually be eliminated by serologic absorption. Occasionally Western blot analysis is required.(Yu Z, Kryzer TJ, Griesmann GE, Kim KK, et al: CRMP-5 neuronal autoantibody: marker of lung cancer and thymoma-related autoimmunity. Ann Neurol 2001;49:146-154)

 

Western Blot:

Western blot testing is indicated in the infrequent situation that interference by coexisting non-neuronal-specific autoantibodies precludes IFA interpretation. It is not cost-effective for routine serologic screening. However, results obtained by Western blot analyses in Mayo Clinic's Neuroimmunology Laboratory have shown 100% concordance with IFA results for PCA-1, and superior sensitivity of the IFA for detection of ANNA-1 and ANNA-2.

 

An aqueous extract of adult rat cerebellar proteins is used as the source of neuronal antigens on the paraneoplastic autoantibody Western blot. Western blot is performed on denatured and reduced proteins separated by electrophoresis on 10% polyacrylamide gel.(Vernino S, Lennon VA: New Purkinje cell antibody [PCA-2]: marker of lung cancer-related neurological autoimmunity. Ann Neurol 2000 March;47[3]:297-305). Full-length recombinant human CRMP-5 antigen is used to confirm CRMP-50IgG.(Yu Z, Kryzer TJ, Griesmann GE, et al: CRMP-5 neuronal autoantibody: marker of lung cancer and thymoma-related autoimmunity. Ann Neurol 2001 February;49[2]:145-154). Denatured full-length recombinant human amphiphysin protein is used to confirm amphiphysin antibody.

 

Radioimmunoassay (RIA):

(125)I-labeled recombinant human GAD65 and nonimmune human serum are incubated with the patient's diluted cerebrospinal fluid. Antihuman IgG and IgM are then added to form an immunoprecipitate. After washing the precipitated immune complexes, specific antibodies are detected by counting gamma-emission from the pellet's bound I-GAD65.(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)

 

Cell Binding Assay (CBA):

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: EUROIMMUN AG. Stocker W. et al: Differenzierte Autoantikorper-Diagnostik mit BIOCHIP-Mosaiken. U Conrad, K. [Hrsg] Autoantikorper. Pabst-Verlag [1998] 78-99)

 

NMO-IgG Fluorescence-Activated Cell Sorting Assay (FACS):

Human embryonic kidney cells (HEK 293) are transfected transiently with a plasmid (pIRES2- Aequorea coerulescens green fluorescent protein [AcGFP]) encoding both green fluorescent protein (GFP) and AQP4-M1. After 36 hours, the mixed population of cells (transfected expressing AQP4 on the surface and GFP in the cytoplasm and nontransfected lacking AQP4 and GFP) are harvested with short exposure to trypsin. Patient CSF is then added to cells at a 1 in 2 screening dilution. After incubation and washing, the cells are resuspended with AlexaFluor 647-conjugated goat-antihuman IgG-gamma specific secondary antibody (Southern Biotech catalog No. 2040-31, 1:2000 in LCBB), held on ice, washed, fixed with 4% paraformaldehyde, and examined with flow cytometry (BD FACSCanto; Becton, Dickinson and Co). Two populations are gated on the basis of GFP 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 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 was 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.

 

If FACS assay is positive at screening dilution, FACS Titer Assay is performed at an additional charge. The patient CSF 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 1:2 dilution, but negative at 1:4, the endpoint will be reported as 2.

Day(s) and Time(s) Performed

ANNA-1, ANNA-2, ANNA-3, AGNA-1, PCA-1, PCA-2, PCA-Tr, Amphiphysin, CRMP-5-IgG, NMDIC, AMPIC, GABIC: Monday through Friday; 11:30 a.m. and 8 p.m.; Saturday and Sunday 8 a.m.

Paraneoplastic autoantibody Western blot confirmation, CRMP-5-IgG Western blot, amphiphysin Western blot: Monday, Wednesday, Friday; 8 a.m.

NMDCC, AMPCC, GABCC, LG1CC, CS2CC: Monday through Friday; 6 a.m.

GAD65: Monday to Friday; 6 a.m. and 4 p.m.

VGKC-complex antibody: Monday through Friday 11 a.m. and 6 p.m.; Saturday 6 a.m.; Sunday 6 a.m.

NMO/AQP4 IgG FACS: Monday, Tuesday, Thursday; 6:00 p.m.

Analytic Time

3 days if negative/5 days if positive

Specimen Retention Time

28 days

Performing Laboratory

Mayo Medical Laboratories in Rochester

Test Classification

See Individual Test IDs

CPT Code Information

86255-AGNA-1

86255-Amphiphysin

86255-ANNA-1

86255-ANNA-2

86255-ANNA-3

86255-CRMP-5-IgG

86255-PCA-1

86255-PCA-2

86255-PCA-Tr

83519-VGKCC (if appropriate)

84182-Amphiphysin Western blot (if appropriate)

84182-CRMP-5 Western blot confirmation (if appropriate)

84182-Paraneoplastic autoantibody Western blot confirmation (if appropriate)

86255-NMO/AQP4-IgG FACS (if appropriate)

86255-AMPCC (if appropriate)

86255-GABCC (if appropriate)

86255-NMDCC (if appropriate)

86256-AMPIC (if appropriate)

86256-GABIC (if appropriate)

86256-NMDIC (if appropriate)

86341-GAD65 confirmation (if appropriate)

86256-NMO/AQP4-IgG FACS titer (if appropriate)

86255-LG1CC (if appropriate)

86255-CS2CC (if appropriate)

LOINC Code Information

Test ID Test Order Name Order LOINC Value
PAC1 Paraneoplas Autoantibody Eval,CSF In Process

 

Result ID Test Result Name Result LOINC Value
89079 AGNA-1, CSF 53714-2
5906 Amphiphysin Ab, CSF 56531-7
3852 ANNA-1, CSF 24400-4
7472 ANNA-2, CSF 24401-2
21633 ANNA-3, CSF 35144-5
21650 CRMP-5-IgG, CSF 35385-4
3988 PCA-1, CSF 14248-9
21632 PCA-2, CSF 35143-7
21631 PCA-Tr, CSF 51748-2
34271 Paraneoplastic Interpretation, CSF 69048-7
36429 Reflex Added No LOINC Needed

Reflex Tests

Test ID Reporting Name Available Separately Always Performed
WBNC Paraneoplas Autoantibody WBlot,CSF No No
CRMWC CRMP-5-IgG Western Blot, CSF Yes No
GD65C GAD65 Ab Assay, CSF Yes No
ABLTC Amphiphysin Western Blot, CSF No No
NMOFC NMO/AQP4 FACS, CSF Yes No
NMOTC NMO/AQP4 FACS Titer, CSF No No
NMDCC NMDA-R Ab CBA, CSF No No
AMPCC AMPA-R Ab CBA, CSF No No
GABCC GABA-B-R Ab CBA, CSF No No
NMDIC NMDA-R Ab IF Titer Assay, CSF No No
AMPIC AMPA-R Ab IF Titer Assay, CSF No No
GABIC GABA-B-R Ab IF Titer Assay, CSF No No
VGKCC VGKC-complex Ab IPA, CSF No No
LG1CC LGI1-IgG CBA, CSF No No
CS2CC CASPR2-IgG CBA, CSF No No

Forms

If not ordering electronically, complete, print, and send a Neurology Specialty Testing Client Test Request (T732) with the specimen (http://www.mayomedicallaboratories.com/it-mmfiles/neurology-request-form.pdf)