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NEUROMUSCULAR CLINICAL LABORATORY

Contact Information Box 8111 - Neurology 660 South Euclid Avenue St. Louis, MO 63110 Phone: 314-362-2406 Fax: 314-362-3413 e-mail: pestronka@wustl.edu Instructions on sending   Antibody testing, or   Biopsies: Muscle or Nerve Phone: 314-362-2406 Laboratory certifications     CAP Lab # 19233-16     Medicare Provider # 26-8235     CLIA Certificate # 26D0652044     New York State 3499     California COS 00800679     Florida 800028650

Testing at Washington University   Laboratory: Specific tests   Requisition forms & Information     Serum (Antibody & Antigen)       Sending samples: Instructions     Biopsies: Muscle, Nerve & Skin       Muscle instructions         Freezing; Other       Nerve instructions         General; Surgery;         Freezing; Other technical       Skin instructions Antibodies   General principles   Classification   Binding to oligosaccharides     Glycoproteins & Glycolipids   Specific types   Testing methods   Survey

Charcot's clinic

Testing at Washington University Neuromuscular Laboratory

• Requisition form: PDF
• Directions for sending samples.
• Specific serum antibodies

• Muscle biopsies can be sent fresh or frozen: Instructions
• Tests performed
  • Histochemical stains include:
        H&E; GT; NADH; ATPase, pH9.4, 4.6 & 4.3; Congo red; VvG;
        Alkaline phosphatase; Acid phosphatase; Esterase; Cytochrome oxidase; SDH
        AMPDA; Phosphorylase; Phosphofructokinase; Sudan black; PAS
    
  • Dystrophy-related immunostains (Immunohistochemistry and/or Western blot) include:
        Dystrophin (Dys 1, 2, 3); Sarcoglycans (α, β, γ, δ); Dysferlin; α-Dystroglycan; Lamin A/C
        Merosin; Caveolin-3; Calpain-3; Desmin; Emerin; α-actinin; LAMP-2; MYH2; Actin (Phalloidin)
        Laminin-α2; laminin-B; Collagen VI & IV; Phosphorylated epitopes (Neurofilament & other; SMI-31 antibody)
  • Immune myopathy panel
        MHC Class I; Capillary morphology (Ulex); C_5b-9 (MAC);
        CD4; CD20; CD8; HAM56; CD163; Decorin; SMI-31; LC3; CA9; Alcian blue
  • Aggregate stains: Desmin; VCP; Phalloidin; Caveolin-3; Ubiquitinated proteins (FK2); SMI-31; TDP-43
  • Mitochondria: Oxidative-phosphorylation enzyme complex activities + Citrate Synthase + Coenzyme Q₁₀ levels
  • Glycogen pathway: Glycogen; Acid Maltase; Glycogen degradation (PFK, Phosphorylase, PGK, PGM1, PGAM1, LDH, PBK); PFK stain
  • Plastic sections (Toluidine blue stained): When necessary
  • Ultrastructure: When necessary
• Requisition form: Printable PDF
• Information about muscle biopsies
• Certification: Alan Pestronk MD

• Nerve biopsy specimens: Preparation
  • Size
    • Full thickness
    • Length: > 3 cm
  • Fresh: Transported in moist gauze on wood stick
  • Processed: 3 pieces of equal size
    • Frozen
    • Formalin fixed: 10% Buffered Formalin
    • Glutaraldehyde fixed
      • Modified Karnovsky's Fixative: (3% Glutaraldehyde, 1% Paraformaldehyde in 0.1M sodium cacodylate buffer)
• Nerve biopsies: Evaluation
  • Frozen sections
    • Stains: H&E; GT; Alkaline phosphatase; Acid phosphatase; Congo red; VvG; Neurofilament (Axons); NCAM (Schwann cells)
    • Immune features
  • Plastic sections: Toluidine blue stained
  • Ultrastructure: When necessary
  • Paraffin sections: H&E
• Requisition form: Printable PDF
• Nerve biopsies: Information
• Certification: Alan Pestronk MD

• Certification

Neuromuscular Autoantibodies: Classification & Features

AUTOANTIBODIES AND IMMUNE POLYNEUROPATHIES

• Serum factors associated with immune-mediated polyneuropathies
  • Monoclonal antibodies (M-proteins) without identifiable antigenic targets
  • Cryoglobulins
  • Monoclonal and polyclonal autoantibodies that bind to specific neural components
• Methodology: Optimal techniques increase the sensitivity & specificity of antibody detection.
• Clinical correlations
  • Serum autoantibodies
    • Are markers for immune-mediated, treatable disorders
      • Provide diagnostic information
      • Suggest adequacy of treatment: When titers reduced more than 60%
    • Suggest specific avenues of therapy
  • Many IgM autoantibody-associated neuropathies
    • Are difficult to treat with the more commonly used T-cell related immunosuppressants, including
      • Corticosteroids and Azathioprine
    • May improve after administration of IVIg or cytotoxic agents, such as
      • Cyclophosphamide
      • Rituximab

AUTOANTIBODIES with BINDING TO OLIGOSACCHARIDES:
  Glycoproteins or Glycolipids

Anti-GM1 Ganglioside Antibodies

Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1'Ceramide
                  3
                  |
           Neu5Acα2

Galβ1-3GalNAcβ1-4Galβ1-X
                  3
                  |
           Neu5Acα2

• General
  • Clinical syndromes: Associated with acute and chronic motor neuropathies ⁴
  • GM1 antibody cross-reactivity: GA1 or GD1b gangliosides
  • Co-GM1: GM1 ganglioside attached to ELISA plate by covalent linkage
  • NP9: GM1 ganglioside in lipid membrane environment
  • GalNACGD1a antibody: Associated with motor neuropathies with features similar to MMN or DLMN
• IgM anti-GM1 antibodies ³⁷
  • Clinical syndromes: Motor neuropathies
    • Subtypes
      • Multifocal motor neuropathy
      • Lower motor neuron syndrome, Distal
    • Motor neuropathy: Clinical & Lab features associated with anti-GM1 antibodies
      • Asymmetry
      • Weakness & Disability: More severe in MMN
      • Axon loss: More
  • Antibody
    • Type
      • Monoclonal: 15%; Associated with M-protein detected by immunofixation
      • Polyclonal: 85%
    • MMN correlations: More severe weakness & axon loss ⁴⁰
      • High innate activity of classical pathway of complement
      • High complement-activating capacity of IgM anti-GM1 IgM antibodies
  • Other reactivity in serums with IgM binding to GM1
    • GD1b ganglioside
      • Some motor neuropathy patients
      • Clinical association: More vibration sense loss
    • Asialo-GM1 ganglioside: ? Clinical associations
    • GM2 ganglioside
    • GalNAc-GD1a ganglioside
    • NS6S: Similar clinical features to GM1 associations
    • IgG or IgA vs GM1: Rare
  • IgM binding in motor neuropathies with no IgM binding to GM1
    • GalNAc-GD1a ganglioside: Occasional patients
    • NS6S
      • 43% of motor neuropathy patients
      • Similar clinical features to GM1 associations
    • NP9 antigen (GM1 ganglioside in lipid environment): 3%
    • GalNAc-GD1a ganglioside: 5%
    • GM2 ganglioside: Rare
    • IgG or IgA vs GM1: Rare
    • MAG (Western blot negative)
      • Frequency: 10%
      • IgM also binds to NS6S
• IgG anti-GM1 antibodies ⁵
  • Clinical: Associated with
    • Acute motor axonal neuropathies (AMAN)
      • Associated with preceding Campylobacter jejuni infection
      • Most frequent in China, Japan and third world countries
      • Uncommon in United States
    • Distal lower motor neuron syndrome (Chronic): Rare
  • Antibody type: Polyclonal
• Anti-GM1 antibodies: Possible pathogenic effects
  • Immunization of rabbits with GM1 ganglioside: Causes subclinical neuropathy ⁶
    • Conduction block
    • Mild axonal degeneration
    • IgM deposits at nodes of Ranvier
  • Injection into nerve of anti-GM1 antibody containing serums: May produce conduction block ⁷
  • Human anti-GM1 antibodies in vitro ⁸
    • Damage spinal motor neurons
    • Interfere with electrophysiological aspects of nerve function

• IgM vs GM1 ganglioside: Associated with chronic motor neuropathy syndromes
  • Optimal test panel: IgM vs Co-GM1; IgM vs Histone H3; IgM vs NP-9 antigen
  • Testing for IgM binding to this group of antigens provides
    • Sensitivity for IgM anti-GM1 antibodies in motor neuropathies: 43% to 50%
    • Specificity for treatable motor neuropathy syndromes: 97%
      • Multifocal motor neuropathy (MMN) & Lower motor neuron syndromes, acquired
    • False positive results: < 0.3% of patients without motor neuropathies in our clinical laboratory
  • Measurement of anti-GM1 antibodies
    • Helps to distinguish MMN from other Immune demyelinating polyneuropathies
    • Identifies motor syndromes that frequently improve with immunosuppression

• IgG vs GM1 ganglioside: Associated with acute and chronic motor neuropathy syndromes
  • Optimal test panel: IgG vs GM1 and IgG vs Sulfatide
  • IgG testing screens for antibodies that occur with acute and chronic lower motor neuron syndromes.
  • IgG antibodies that bind to GM1 but not to sulfatide occur in
    • Acute, usually axonal, motor neuropathies: Often associated with preceding Campylobacter jejuni infection
    • Chronic axonal lower motor neuron syndromes: Rare
  
  
• Testing Indications: Anti-GM1 antibodies
• Structure: GM1 ganglioside

Anti-Sulfatide antibodies

SO4-3Galactose-X

• General
  • Clinical syndromes: Predominantly sensory neuropathies ¹⁰
  • Axonal neuropathy: Polyclonal anti-Sulfatide antibodies; IgM or IgG
  • Demyelinating neuropathy:IgM; Monoclonal
  • GALOP syndrome: IgM or IgG
  • ? Association with HIV infection ²⁸: IgG or IgM
  • FOSMN: IgG
    
  • Treatment: Some neuropathies improve after immunosuppression
• Anti-Sulfatide antibody: Tissue binding ²⁹
  • Demyelinating polyneuropathies: Myelin; Schwann cell cytoplasm
  • Axonal polyneuropathies: Axons; Resident endoneurial macropahges
• Anti-Sulfatide antibody testing
  • Indications: Polyneuropathies with prominent sensory component; Especially > 50 years old
  • Antisulfatide antibodies: Testing indications

Anti-MAG antibodies

SO4-3Glucuronate-X

• General
  • Clinical syndromes: Chronic demyelinating sensory-motor polyneuropathy ⁹
  • Signs: Sensory loss; Gait ataxia; Tremor; Mild distal weakness
  • Treatments
    • Plasma exchange and intravenous cyclophosphamide: Monthly x 6
    • Rituxan
  • MAG antibody (IgM)
    • Cross-reactivity
      • Glycolipids: SGPG (90%); SGLPG
      • Heparan sulfate (70%)
      • ? Other glycoproteins: PMP-22; P₀
    • Type: Monoclonal IgM
    • Association with serum M-protein: 85%
• Testing for anti-MAG antibodies
  • Optimal test panel
    • IgM vs specially purified Myelin-associated Glycoprotein (Z-MAG) by ELISA method
    • Result confirmed, if positive, by Western Blot.
  • Anti-MAG antibody testing: Indications
• Anti-MAG antibodies: Possible pathogenic effects
  • Anti-MAG antibodies probably cause the associated demyelinating neuropathy ²²
  • Anti-MAG antibodies bind to ²⁷
    • Compact peripheral nervous system myelin
    • Schwann cell cytoplasm
    • Axons: Occasional
  • Deposits of IgM and complement can be found on myelin sheaths in biopsies from patients.
  • Injection into cat sciatic nerve of serum from patients with anti-MAG antibodies produces
    • Widening of myelin lamellae
    • Demyelination in experimental animals ²³
  • Passive transfer of human anti-MAG IgM into chicks
    • Produces pathology that is similar to the human polyneuropathy ²⁴
  • Clinical correlations
    • Presence of anti-MAG antibodies strongly correlates with the associated neuropathy
    • Anti-MAG antibodies found in the serum of asymptomatic patients predictive
    • Typical demyelinating neuropathy will develop during the next few years ²⁵
    • Long-term reduction in anti-MAG antibody titers correlates with improvement in associated neuropathy ²⁶

Anti-SGPG antibodies

SO4-3GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1'Ceramide

SO4-3Glucuronate-X

• SGPG: Sulfated glucuronyl paragloboside
• Clinical syndromes
  • Chronic demyelinating sensory-motor polyneuropathy: IgM binding to both SGPG and MAG
  • Motor neuropathy ²¹: IgM binding to SGPG but not MAG
  • Axonal sensory-motor neuropathy: IgM binding to SGPG but not MAG
• Antibody type: IgM

Anti-GQ1b & GT1a antibodies

                Galβ1-3GalNAcβ1-4Galβ1-X
                 3                3
                 |                |
Neu5Acα2-8Neu5Acα2                |
                 Neu5Acα2-8Neu5Acα2

                Galβ1-3GalNAcβ1-4Galβ1-X
                 3                3
                 |                |
Neu5Acα2-8Neu5Acα2                |
                           Neu5Acα2

• General
  • Epidemiology
    • Antibodies & MFS syndromes more common in Japan & some 3rd world nations
  • Signs associated with IgG vs GQ1b ¹¹
    • Ataxia
    • Ophthalmoplegia
    • Cranial neuropathies
  • Specific clinical syndromes
    • IgG binding to GQ1b
      • Ophthalmoplegia and ataxia as part of Miller Fisher Syndrome.
      • Acute onset extraocular palsies: Vertical and horizontal, or bilateral abducens.
      • Ataxic Guillain-Barré: More common in Japan & some 3rd world nations
      • Bickerstaff's brainstem encephalitis
      • Prodromal associations ¹⁵
        • Infection with specific serotypes (O-2 or O-10) of Campylobacter jejuni
      • Antibody: Cross-reactivity with GT1a ganglioside common
    • IgG binding to GT1a or GM1b gangliosides without binding to GQ1b
      • Acute neuropathies with bulbar dysfunction
        • Pharyngo-cervical-brachial variants ¹²
        • Bulbar variant
      • Prodromal associations: Within 4 weeks of illness
        • Diarrhea
        • Campylobacter jejuni infection
      • Antibody associations; GQ1b reactivity
        • Common: Especially in patients with GQ1b associated clinical phenotype
    • IgM vs GQ1b: CANOMAD; CANDA
• Testing for serum IgG binding to GQ1b or GT1a gangliosides
  • Is most useful with recent onset brainstem, or cranial nerve, syndrome
  • Clinical features may suggest either intrinsic or extrinsic disease
  • IgG binding to GQ1b or GT1a gangliosides suggests that a syndrome with brainstem signs
    • Is immune-mediated.
    • May respond to treatment with plasma exchange or IVIg,
    • Usually has a monophasic course with spontaneous recovery
  • Should be performed early: Antibodies may disappear within 2 to 4 weeks after onset
• Structure: GQ1b; GT1a & GM1b gangliosides
• IgG vs GQ1b ganglioside: Association with disease pathogenesis
  • IgG anti-GQ1b antibodies can bind to: Peripheral nerve & brainstem structures ¹⁴
  • IgG binding to GQ1b may produce: Presynaptic blockade of neurotransmitter release from motor nerve terminals ¹³

Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1'Ceramide
                  3
                  |
 Neu5Acα2-8Neu5Acα2

             Y-4Galβ1-X
                 3
                 |
Neu5Acα2-8Neu5Acα2

• Antibody targets
  • Antigenic group: Disialosyl groups (Neu5Acα2-8 Neu5Acα2-) attached to oligosaccharide sugar
  • Gangliosides: GD1b; GD3
  • IgM antibodies cross react with Pr2 antigens on red blood cells: May cause cold agglutination
• Clinical
  • IgM vs GD1b ganglioside or GQ1b, but not GM1 ganglioside, are associated with
    • Sensory ataxic neuropathy ± Cranial nerve syndromes
      • CANOMAD ¹⁶
      • CANDA: Chronic Ataxic Neuropathy with Disialosyl Antibodies
    • Motor neuropathies: IgM anti-GM1 antibodies may cross-react with GD1b
  • IgG antibodies that bind to GD1b ganglioside, but not GM1, have been described in
    • Acute sensory ± motor neuropathies ¹⁷: Some patients
• Anti-GD1b antibody testing useful: Sensory, ataxic neuropathy syndromes
• Association with disease pathogenesis
  • Antibodies that bind selectively to GD1b ganglioside can cause a sensory neuropathy ¹⁸
  • GD1b ganglioside is present in peripheral nerve on
    • Surface of sensory neurons in the dorsal root ganglion ¹⁹
    • Paranodal myelin
  • Antibodies to GD1b may be cytotoxic to sensory ganglion cells
  • Rabbits immunized with GD1b ganglioside: Develop ataxia and areflexia + Structural damage to sensory neurons

Anti-GM1b antibodies

      Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1'Ceramide
       3                
       |                
Neu5Acα2

• IgG and IgM anti-GM1b antibodies are associated with: Guillain-Barré-like syndromes ³³
  • Predominantly Motor (AMAN)
  • Weakness: Severe; Especially distal; Slower recovery
  • Axonal loss
  • Prodromal Campylobacter jejuni infection: Serology positive
  • Treatment: IVIg; Not plasma exchange
• Often associated with antibodies to GM1

Anti-GD1a antibodies

      Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1'Ceramide
       3                3
       |                |
Neu5Acα2         Neu5Acα2

      Galβ1-X
       3
       |
Neu5Acα2

• IgM anti-GD1a ganglioside antibodies are unusual.
  • High titers are associated with
    • Demyelinating motor neuropathy (with IgM M-protein) ³¹
    • Motor neuropathy & Lower motor neuron syndromes ³²
    • Demyelinating sensory-motor neuropathy ³⁰
  • Most serums with IgM binding to GD1a also show binding to GM1 ganglioside.
  • Low titers of IgM anti-GD1a antibodies occur in a some patients with ALS
• IgG anti-GD1a antibodies are associated with
  • Guillain-Barré-like syndromes: Predominantly Motor & Axonal (AMAN) ²⁰
    • Some "anti-GD1a antibodies" in GBS-like syndromes actually bind to a GalNAc-GD1a contaminant in GD1a.
  • Motor neuropathies (AMAN)
  • Dysarthria, Dysphagia & Sensory ataxia ³⁸
• Antibody cross-reactivity
  • GM1b, GT1b and GM3 gangliosides
  • Motor neuropathy: Binding to GM2; GalNAc-GD1a
• Association with disease pathogenesis
  • GD1a, GT1b & GM3 may be adhesion ligands for MAG
  • IgG anti-GD1a antibodies associated with prodromal Campylobacter jejuni infection
• GD1a structure

Anti-GalNAc-GD1a & GM2 ganglioside antibodies

GalNAcβ1-4Galβ1-X
           3
           |
    Neu5Acα2

• General
  • Associated clinical syndromes
    • GBS variants
    • Motor neuropathies
    • Sensory demyelinating neuropathy
  • Antibody cross-reactivity
    • Binding to GalNAc-GD1a and GM2 or TS-HDS gangliosides: Sensory > Motor neuropathy
    • Binding to GalNAc-GD1a, but not other, gangliosides or disaccharides: Motor neuropathy, acute or chronic
  • GalNAc-GD1a ganglioside localization ³⁵
    • Ventral root: Inner compact myelin; Periaxonal axolemma; Nodal & Paranodal regions
    • Dorsal root: Small diameter axons
    • Intramuscular nerves
    • Sural nerves: Small axons
• IgG anti-GalNAc-GD1a antibodies are associated with
  • Acute neuropathy ¹
    • Motor neuropathy with: Distal predominant weakness & Sparing of the cranial nerves
  • Chronic neuropathy ²
    • Chronic axonal motor neuropathy
    • Binding pattern: IgG anti-GalNAc-GD1a antibodies without cross-reactivity to GM2
• IgM anti-GalNAc-GD1a antibodies are associated with
  • Acute neuropathy
    • GBS-variant syndromes
      • Mild weakness, Facial paresis, Sensory loss & Good prognosis
      • Prior cytomegalovirus or Campylobacter jejuni infection
  • Chronic neuropathies
    • Chronic demyelinating polyneuropathy ³: Monoclonal IgM binding to GalNAc-GD1a and GM2 gangliosides
    • Chronic motor neuropathy: Polyclonal IgM binding to GalNAc-GD1a ganglioside
• Indications for testing
  • Chronic motor neuropathy syndromes
    • Anti-GalNAc-GD1a antibodies may be evidence that neuropathy is immune mediated and treatable.
  • Predominantly sensory demyelinating neuropathy syndrome
    • IgM binding to GM2 & GalNAc-GD1a gangliosides distinguishes neuropathy from CIDP and MAG neuropathies.

Anti-Galactocerebroside (GalC) antibodies ³⁹

Gal-Ceramide

• Epidemiology: 5% of patients with Guillain-Barré syndrome in Japan
• Type: IgG > IgM
• Associations
  • Prodrome: Mycoplasma pneumonia respiratory infection (43%)
  • Clinical features more frequent than other GBS
    • Sensory (87%): Loss; Paresthesias
    • Autonomic (28%): Orthostatic hypotension; Bladder; Rectal; Dyshidrosis
  • Other antiglycolipid antibodies: 25%
  • NCV
    • Demyelinating (80%) > Axon loss (20%) syndromes
    • Motor distal latencies: Prolonged
    • SNAP amplitudes: Reduced
• Other disease association: Acute disseminated encephalomyelopathy (ADEM)

Anti-Heparan Sulfate antibodies

• Clinical: Antibodies to Heparan sulfate occur in
  • 35% of patients with Guillain-Barré syndrome: IgM and IgG
  • Some patients with IgM anti-MAG M-proteins: IgM

GALOP Syndrome

• General
  • GALOP: Gait disorder; Autoantibody; Late-age Onset; Polyneuropathy
• Clinical syndrome
  • Gait disorder
  • Sensory neuropathy
  • Improvement after immunosuppression
• Laboratory
  • GALOP syndrome appears to be immune-mediated
  • Associated with a high titer of IgM binding to sulfatide in a lipid environment

• Clinical: IgM antibodies to Decorin (BJ antigen): Occur in myopathies with
  • Proximal weakness
  • Serum CK: Moderately high
  • Associated disorders
    • Serum M-protein: 50%
    • Waldenström's macroglobulinemia

OTHER NEUROLOGICAL- or NEUROPATHY-ASSOCIATED AUTOANTIBODIES

Anti-Tubulin Antibodies

• Patients with IgM anti-tubulin antibodies often have
  • CIDP-like syndromes with some asymmetry by history or on examination.
  • Syndromes that are somewhat more difficult to treat than other patients with CIDP
• Patients with IgG anti-tubulin antibodies also commonly have CIDP
• Patients with Leber's Hereditary Optic Neuropathy has been reported to be associated with IgG anti-tubulin antibodies.

Anti-Hu antibodies

• Paraneoplastic syndromes: Patients with anti-Hu antibodies in serum and CSF often have
  • Clinical
    • Pure sensory neuronopathy (ganglionopathy)
    • Gastrointestinal (autonomic) dysfunction
    • CNS: Limbic encephalitis, Cerebellar disorders
  • Associated neoplasms
    • Small cell: Lung (41%) & Other
• Lab testing
• Antibody binding
  • Hu family of proteins: 35 to 40 kD
  • Specifically located in: Neuronal nuclei; Small cell lung cancers

Lysoganglioside-GM1 (Lyso-GM1) antibodies

• Clinical: IgG anti-Lysoganglioside-GM1 antibodies are associated with
  • Sydenham chorea ³⁴: ? Present in serum & CSF
  • Acute motor neuropathies with IgG anti-GM1 antibodies

M-phase phosphoprotein-1 (Mphosph1; MPP1 ) antibodies ³⁶

• Clinical: Varied associated syndromes
  • Ataxia: Sensory or Cerebellar
    • Dysmetria (80%)
    • Nystagmus (40%)
  • Polyneuropathy
    • Sensory loss
      • Vibration
      • Joint position sense
      • Gait disorder
    • Axonal or Demyelinating
  • No malignancy
  • Male > Female
• Antibodies
  • IgG
  • Present in serum & CSF
  • Tissue staining: Nuclei in HEp-2 cells; Cytoplasm in some CNS cells
  • Present in other disorders: Occasional; Motor neuropathy, Polyneuropathy, MS

Polyspecific antibodies

• IgM and IgG antibodies that are polyspecific bind to
  • A wide range of glycolipids
  • Histone H3
• They occur in many disorders
• Do not predict a specific neurologic disorder or clinical syndrome
• They probably reflect the presence of
  • An ongoing immune phenomenon: Analogy to
    • Elevated sedimentation rate
    • Moderately positive ANA

Polyneuropathies associated with serum M-proteins

• This is a diverse group of neuropathies.
• When IgM M-proteins react with GM1 ganglioside, MAG, β-tubulin, GALOP antigen,
  or sulfatide, there is a correlation with specific neuropathy syndromes.
• The most distinctive syndrome associated with IgG and IgA M-proteins is
  POEMS (Polyneuropathy; Organomegaly; Edema or endocrinopathy; M-protein; Skin changes).
  Demyelinating features are common in the POEMS neuropathy.
• Plasmapheresis may be more effective in neuropathies associated with IgG and IgA, than with IgM M-proteins
  but POEMS syndrome does not respond well to any immunomodulating treatments.
• The initial procedure of choice for detecting serum M-proteins in patients with polyneuropathies is immunofixation.
• Also see: M-proteins and associated disorders

DETECTION OF ANTIBODIES: METHODOLOGIES

Antibody Testing Methods: General Principles

• Testing for specific autoantibodies in neuropathies
  • Antibody location: Serum or CSF
  • May be useful in: Diagnosis & Following effects of treatment
• General issues affect the utility of antibody testing in the evaluation of polyneuropathies
  • Serum antibodies often associated with specific clinical syndromes
  • Cost-effectiveness: Best when utilization is appropriately focused
    • Choose specific antibodies to search for
    • Avoid large, expensive pre-selected testing panels
  • Prevalence of antiganglioside antibodies may vary greatly in different geographic regions
    • Especially for antibodies associated with acute immune neuropathies
    • Example: IgG anti-glycolipid antibodies
      • Associated with specific prodromal infections
      • Common in patients with acute immune neuropathies in Japan and China
      • Rare in most parts of United States
• Routine antibody testing in acute neuropathies: More useful in regions with high prevalence
• Areas of low prevalence
  • Clinical evidence of typical antibody-associated syndrome: Antibody testing more likely to be useful
• Clinical validation of methodology
  • General principles of antibody testing are straightforward
  • However, assays are often difficult to carry out reproducibly in practice
    • Results of antibody testing differ among laboratories: Prominent interlaboratory variation
    • Each testing service should provide data on clinical correlations of results determined in their laboratory
  • "Gold standards" for antibody testing: Specificity; Sensitivity & Reproducibility
  • Results should be correlated with
    • Positive and negative control samples within the laboratory
    • Clinical features of the specific patient syndromes associated with positive test results
  • Pathogenic significance
    • For many antibodies pathogenic significance remains to be determined
    • Especially difficult to show pathologic effects of antibodies directed against intracellular antigens, such as Hu
    • Good evidence that antiMAG antibodies may play a pathogenic role in associated neuropathy
    • Some antibodies may be the result of antigenic cross-reactivity with a prodromal infectious agent
    • IgG anti-GM1 antibodies in motor neuropathies: Often associated with prior Campylobacter jejuni infection

ELISA Methods

• General utility: Determine the presence and amount of antibodies in a tissue fluid
• Method: Testing for antibody binding to an antigen that is immobilized in a plastic well.
• General procedures: ELISA involves three operations
  • Binding of an antibody to a target antigen that has been attached to a solid phase
  • Generation of a signal that can identify the presence of any bound antibody
  • Detection of the signal
• Specific methodologic steps
  1. Attachment: A defined amount of a target antigen is bound to a small plastic well
     • Methods of attachment may be simple
       • Antigen-containing solution is incubated in wells for a period of time
       • Antigen-containing solution is evaporated in the well: Dried antigen is attached to the plastic
     • Special methods of attaching antigens to the ELISA wells
       • May be necessary when
         • Aspects of the conformation or charge of an antigen are important
         • Only small amounts of a purified antigen are available
       • Special binding methods may include linking of the antigen to the well
         • Via covalent bonds
         • By monoclonal antibodies that are attached to the well prior to adding the antigen
  2. Blockade: Additional non-specific binding sites in the plastic wells are blocked
     • Blocking solution: Contains proteins such as albumin or animal, e.g. goat, serum
  3. Add patient serum or other tissue fluids to the wells for a period of hours
     • Allows binding of any serum antibodies to the antigen in the well
  4. Wash wells: To remove unbound serum antibodies
  5. Detection of antibody binding: Use second antibody that has
     • Specificity for the species being tested (e.g. anti-human)
     • Colored or fluorescent marker attached to it which can be measured in a "reading" device
  6. Controls
     • Control wells typically contain, either
       • Antigen with some structural similarities to the test antigen
       • Another control antigen, such as histone H3
         • Detects confounding polyspecific antibodies
           • Bind to a variety of antigens
           • Have no strong clinical correlations
       • Less optimally: No antigen
  7. Calculation of titers
     • At appropriate test serum dilution: Amount of antibody binding correlates with level of color that develops
     • Specific antibody binding is calculated
       • Subtract serum antibody binding to parallel sets of control wells
• Utility of ELISA assays
  • Sensitive screening of large numbers of serums
  • When the target antigen is available in purified form
  • Some degree of quantitation is desired
  • Typical target antigens: Glycolipids; Proteins
• Problems with ELISA assays
  • Poor sensitivity & specificity: When tertiary structural properties on target antigen needed for antibody binding
  • Target antigen must be highly purified
  • Serums may contain polyspecific antibodies
    • Bind non-specifically to a variety of substrates
    • May even bind to plastic wells of the ELISA plate
  • Minor methodological details may greatly alter reproducibility, sensitivity & specificity of the assay
  • Each laboratory provide its own data regarding positive and negative test values
    • Clinical correlations
    • Sensitivity
    • Specificity
    • Reproducibility
• External link: K&P Detailed protocol

Western Blotting Methods

• General utility: Evaluates for selective antibody binding to proteins of known molecular weight
• Method: Tests for antibody binding to antigens immobilized on a paper strip
• General procedures: A mixture of proteins is
  • Separated in a gel by size or charge
  • Transferred to a paper sheet
  • Exposed to tissue fluids to examine whether antibodies bind to any components of the mixture
• Specific methodologic steps
  1. Dissolve mixture of target tissue proteins: Often in detergent
  2. Separation of target tissue proteins
     • Method: Electrophoretic (polyacrylamide) gel
     • Principles of separation: Molecular weight, or Charge
  3. Transfer of separated proteins to paper
     • Method: Application of electric current
  4. Blockade: Non-specific binding sites on the paper are blocked
     • Blocking solution: Contains proteins such as albumin or animal, e.g. goat, serum
  5. Expose strips of the paper to patient serum or other tissue fluids
     • Allows binding of any serum antibodies to any of the antigens on the paper
  6. Wash paper: To remove unbound serum antibodies
  7. Detection of antibody binding to any protein on paper: Use second antibody that has
     • Specificity for the species being tested (e.g. anti-human)
     • Colored or fluorescent marker attached to it which can be measured in a "reading" device
• Utility of Western blot assays
  • Detection of antibodies to protein antigens
    • Examples: MAG; Hu; Tubulin
  • Specificity
    • Proteins on paper that remain unstained provide useful inherent control
    • Confirms specificity of antibody binding to protein antigens detected by other methods
• Problems with Western blot assays
  • Less sensitive than ELISA assays: Especially if
    • Variability in amount of the target antigen in tissue applied to the gel
    • Antigen on paper deteriorates with storage
  • Specificity reduced by: Presence of several protein antigens at similar molecular weight levels
  • Quantitation of results difficult
• External link: K&P Detailed protocol

Immunocytochemical Methods

• General utility: Detects anatomical patterns of antibody binding to
  • Specific cells
  • Sub-cellular structures
  • Other tissue components
• Method: Testing for antibody binding to an antigen in tissue sections
• General procedures
  • Section tissues
  • Binding of an antibody to a target antigen in tissue slice
  • Generation of a signal that can visually identify the presence of any antibody bound to the tissue
• Specific methodologic steps
  1. Tissue sections: Cut thin (~10μM) sections from tissue
     • Tissue may be fixed or frozen
     • Sections are placed on clean glass slides or cover slips
       • Glass may be pre-treated to increase adhesion of sections
  2. Blockade: Non-specific binding sites on the tissue sections are blocked
     • Blocking solution: Contains proteins such as albumin or animal, e.g. goat, serum
  3. Expose tisue sections to patient serum or other tissue fluids
     • Serum is diluted to a concentration that provides: Good signal & Minimum background
  4. Detection of antibody binding to tissue slice: Use second antibody that has
     • Specificity for the species being tested (e.g. anti-human)
     • Colored or fluorescent marker attached to it which can be visualized
     • If antibodies to a tissue protein are present in the serum, they will bind in an anatomically appropriate pattern
• Utility of Immunocytochemistry
  • Inexpensive
  • Rapid procedure
  • Useful for initial antibody screening
  • Commonly used for
    • Test antigens difficult to purify in quantities needed for other testing methods
    • Nuclear antigens
  • Specific tests: Hu; Yo; Anti-nuclear antibodies
  • Semi-quantitation possible with serial dilutions of serum
  • Identification of antibody binding to conformation-specific epitopes
• Problems with Immunocytochemistry
  • Poor specificity
    • Several different antigens can have similar distributions in tissue
    • Antibodies may be mistaken for one another

Thin Layer Chromatography Methods

• General utility: Evaluates for selective antibody binding to lipid antigens
• Method: Tests for antibody binding to lipids separated, by solubility, on a silica gel
• General procedures: A mixture of lipids is
  • Separated in a thin layer of silica on a plate by solubility in a solvent
  • Exposed to tissue fluids to examine whether antibodies bind to any components of the mixture
• Specific methodologic steps
  1. Dissolve mixture of lipids: Often in mixture of organic solvents
  2. Placement: Lipid mixture is placed in a dot at the bottom of flat plates coated with silica gel
  3. Bottom of plates is placed in solvent
     • Solvent moves up through the gel
     • Lipids are carried different varying distances up the plate according to their solubility
       • Lipids with more solubilty move further up the plate
  4. Antibody binding: The plate is then dried and exposed to diluted serum
  5. The bound antibodies are stained and visualized using a second antibody
  6. Interpretation: If an antibody to one of the lipids is present in the serum
     • It will bind to a band at the appropriate region on the plate
     • Other lipids that remain unstained provide a control for non-specific antibody binding
• Utility of TLC assays
  • Tests for, or confirms, antibody binding to lipid antigens
  • Especially useful when lipids are difficult to purify because they are present in very small amounts in tissues
  • Examples of antigens: Sulfated glucuronyl paragloboside (SGPG)
• Problems with TLC assays
  • Cumbersome to perform
  • May produce variable results
  • Quantitation difficult

Immunofixation Electrophoresis Methods

• General utility
  • Most sensitive method for identifying & characterizing monoclonal immunoglobulins (M-proteins)
    • Neuropathies with monoclonal gammopathy of unknown significance (MGUS)
      • Quantity of serum M-proteins may be very small
    • Immunofixation is the preferred method in the laboratory evaluation of neuropathies
    • Other methods, such as serum protein electrophoresis, may not detect some M-proteins
  • Specimen to be tested can be serum, urine, or other body fluids
• Specific methodologic steps
  1. Separation
     • Six aliquots of the test specimen are applied to an agarose gel
     • Proteins in the specimen are separated by electrophoresis according to net charge
  2. Fixation
     • Five electrophoresis lanes individually stained: Antibodies to IgG, IgA, IgM, and κ and λ light chains
       • Formation of antigen-antibody complexes precipitates & fixes proteins in the gel
     • Protein fixative is applied to the sixth lane
     • Gel is then fixed & washed with deproteination solution (NaCl)
       • Removes non-precipitated proteins from the gel
     • Antigen-antibody complexes & fixed proteins in 6th lane stained: Amido black

Sydenham chorea

• Epidemiology
  • Previous infection: Streptococcus, Group A
  • Frequency: 10% to 30% of acute rheumatic fever
  • Onset: Up to 6 months after Streptococcal pharyngitis
• Clinical features
  • Involuntary movements: Chorea
  • Psychiatric syndromes: Obsessive compulsive; Hyperactivity; Emotional lability
• Laboratory
  • Antibody: IgG vs Lysoganglioside-GM1
    • Immune target: N-acetyl-β-D-glucosamine
    • Location: Serum & CSF
• Treatment: ? Immunomodulation, Plasma exchange
• External link: PANDAS

Glutamate & GABA Receptor Antibodies

Antibody	Age & Sex	Syndrome	Neoplasm
NMDAR	Age   37%<18 years   5%>44 years Sex: 80% female	Panencephalitis Psychiatric at onset	Ovary teratoma (35%)   Young fertile women     12–44 years(55–60%)
AMPAR	Age: 23–87 years Female predominant	Limbic encephalitis Psychosis, rare	Thymoma Small cell lung Breast
GABAAR	Age: 3–63 Male predominant	Limbic encephalitis Seizures, refractory	None
GABABR	Age: 16–85 years Male = Female	Limbic encephalitis Seizures, refractory	Small cell lung
mGluR1	Age: 19–65 years	Cerebellar degeneration	Hodgkin's lymphoma
mGluR5	Age: 15–46 years Male = Female	Limbic encephalitis	Hodgkin's lymphoma

Alan Pestronk MD: Neuromuscular Pathology Certification

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