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MYASTHENIA GRAVIS: DIAGNOSTIC TESTS

Antibodies   Anti-AChR   MuSK   Striational   Other General principles Electrodiagnostic   Oculo-Vestibular EMP   Repetitive CMAP   Repetitive nerve stimulation   Single fiber EMG Tensilon test Other diagnostic tests

Decremental response to RNS in Myasthenia Gravis

General Principles of Diagnostic Testing for MG

• Rationale
  • Essential to establish a firm diagnosis
    • Thymectomy or long term immunotherapy may be needed to treat MG
    • Avoids inappropriate treatments, & side effects, in patients without MG
  
  
• The mainstays of diagnostic testing have traditionally been
  • Pharmacological
  • Serological
  • Electrodiagnostic
  
  
• Strategy of diagnostic testing
  • As a general rule, a firm diagnosis is based upon
    • A characteristic history and physical examination, and
    • Two positive diagnostic tests, preferably serological and electrodiagnostic.
  • Diagnostic investigations of MG should usually include both
    • Testing for serum anti-AChR antibodies
    • Repetitive nerve stimulation studies
  
  
• Tensilon tests
  • May be readily performed at the bedside
  • Are not as sensitive, or specific, as the serological and electrophysiological studies
  
  
• Single fiber EMG: Reserved for selected patients in whom other tests have been negative or equivocal.
  
  
• Other diagnostic tests: Generally not required or indicated in majority of MG cases
  • Motor point muscle biopsies
    • Count AChRs at neuromuscular junctions
    • Evaluate neuromuscular transmission by in vitro electrophysiologic methods
    • Immunocytochemical staining of muscle endplates for immunoglobulin and complement
  • Tests of ocular movement
  • Genetic evaluation for defects in AChR subunits

Edrophonium (Tensilon) Testing

General Drug properties Action Inhibits acetylcholinesterase Prolongs presence of neurotransmitter, acetylcholine, in the NMJ Results in enhanced muscle strength Duration: Lasts for a few minutes Response In patients with NMJ dysfunction Not specific for MG Time course: Minutes; Rapid-onset; Short-acting Method Initially Dosing: 2 mg of edrophonium is administered intravenously as a test dose Monitoring heart rate: Bradycardia or ventricular fibrillation may develop Follow-up After observing for about 2 minutes, if no clear response develops Up to 8 additional mg of edrophonium is injected A double-blind protocol with a saline injection as placebo has been advocated Testing performed with patient free of all cholinesterase-inhibitor medications Cholinergic side effects of edrophonium May include Salivation & Lacrimation: Increased Sweating & Flushing Bladder urgency Fasciculations, perioral Atropine should be readily available To reverse effects of edrophonium if hemodynamic instability Extra precautions: Especially important in elderly patients Positive test Most myasthenic muscles respond in 30 to 45 seconds after injection Improvement in strength that may persist for up to 5 minutes Requires objective improvement in muscle strength. Do not over interpret Subjective or minor responses: Reduction of sense of fatigue Utility of Tensilon test Only useful in patients with objective, preferably measurable, findings on physical examination Rarely helpful in the diagnostic evaluation of equivocal cases of MG Sensitivity for MG is relatively low (60%) compared to other diagnostic tests Tensilon testing should not be used to determine adjustments in the dose of pyridostigmine False positive results Can occur in patients with LES, ALS or even localized, intracranial mass lesions Positive testing does not necessarily predict respose to a longer-acting anti-AChE drug

Cogan Tensilon test: Before (left); After (right)

Serum antibodies vs Acetylcholine Receptors

AChR Antibodies: Properties & Types   Type: IgG1-dominant Properties Binding Modulating Blocking vs AChR aggregates Low affinity Antibody Targets

Muscle AChR (Adult)

• Antibody (IgG) targets in MG: Nicotinic AChRs ⁷
  • α1-subunit of AChR
  • Myasthenia gravis: Adults
    • Males
    • Older; Lower AChR Ab titers
  • Epitope location: Main immunogenic region on extracellular tip of α1-subunit
  • Main immunogenic region (MIR) features
    • Cluster of overlapping epitopes
    • Conformation dependent
    • Also commonly a target of anti-AChR antibodies in
      • Experimental autoimmune MG (EAMG)
    • Cross-linking
      • Single bivalent antibody cannot bind to both MIRs on same AChR
      • Antibodies can cross-link adjacent AChRs
    • Clinical correlation
  • e-subunit
    • Acquired slow channel syndrome
    • Binding to Adult AChRs (containing e-subunit) ¹
      • 13% of serums with no IgG binding to fetal AChRs
      • Increases finding (sensitivity) of anti-AChR antibodies in MG by ~ 3%
      • Occur in patients with either ocular or generalized MG syndromes
  • γ-subunit
    • Younger Adult Females: Higher AChR Ab titers; More severe disease; Thymic hyperplasia
    • Neonatal MG, Transient
    • Arthrogryposis, Recurrent
• AChR binding antibodies: Measurement of serum IgG (& IgM) antibodies that bind to AChRs
  • AChR binding antibody testing: General
    • Method: Radio-Immunoprecipitation
  • Antigenic targets
    • AChRs from human skeletal muscle: Mixed innervated & denervated, or
    • Myogenic cell line: Expresses both adult & fetal AChRs
    • Target modification for immunoprecipitation assay
      • Human nicotinic AChRs from skeletal muscle labeled with ¹²⁵I-α-Bungarotoxin
  • Clinical relevance
    • Relatively specific & sensitive test for MG
    • Other types of AChR antibody testing
      • Modulating & Blocking antibodies
      • Specificity: Low
  • Anti-AChR binding antibodies occur in
    • Adults with generalized MG: 85 to 90%
    • Childhood MG: 50%
    • Ocular MG: 50% to 70%
      • Lower titers
        
      • Bind best to adult AChRs with e subunit
    • MG and thymoma: Nearly 100%
    • Some patients taking penicillamine with or without MG
    • "False" positives ⁶
      • General
        • Frequency: Up to 14%
        • False postives more common with
          • Low titer positive results
          • Patient features: Younger; Female
        • May be negative with
          • Repeat (subsequent) testing
          • Cell-based assay testing methods
      • Disorders
        • Thymoma without MG
        • Systemic
          • Immune liver
            
          • Rheumatologic
          • Ophthalmologic & Pseudo-ptosis
          • Primary lung cancer: 3%
        • Neuromuscular disorders
          • Lambert-Eaton syndrome (13%)
          • Neuromyotonia
          • Guillain-Barré (Ganglioside Ab+): May be transiently positive
        • Older patients (> 70 years): 1% to 3%
• Antibody effects on AChRs
  • AChR Modulating Antibodies
    • Mechanism
      • Anti-AChR antibodies cross-link AChRs on the post-synaptic membrane
      • Endocytosis & degradation of AChRs are accelerated
    • Test results
      • May occur in a rare patient when anti-AChR antibody binding is negative.
      • MG: Usual loss = 20% to 90% of AChRs (Normal < 20%)
      • MG with thymoma: > 90% loss
      • False positives: Common
        • Low specificity for MG
        • Specific causes: Hemolysis; Muscle relaxant drugs; Serum heating
  • AChR blocking antibodies: Block binding site for ACh on AChR (positive = 26% to 100% blocking).
    • Prevalence: Repeated arthrogryposis; 52% of generalized MG; 30% of ocular
    • 1% of patients with no binding or blocking antibodies
  • Complement binding
    • Damage to postsynaptic membrane: Simplification of postsynaptic folds
    • Widening of synaptic cleft
• Clinical correlations of MG & Anti-AChR antibodies
  • Absolute titer of AChR binding antibodies
    • Among patients: No relation with severity of MG
    • In individual patient: Improvement often seen with reduction in titer of > 50%
  • Titer of antibody binding to main immunogenic region (MIR) of AChRs ²
    • Correlates with disease severity: Ocular vs Generalized
    • MIR location: Extracellular end of 2 α subunits of pentameric AChR
    • > 50% of MG AChR Abs bind to MIR
  • Antibody blocking & modulation of AChRs
    • Some correlation with disease severity
    • Not diagnostically specific
  • Neonatal MG: Transient
    • High anti-fetal/anti-adult muscle anti-AChR antibody ratio
  • Recurrent arthrogryposis
    • IgG vs γ subunit of fetal AChR: Blocks AChR function
  • Slow channel syndrome: Acquired
    • IgG vs adult AChR
  • IgG vs Clustered AChRs ³
    • Present in
      • "Sero-Negative" Ocular or Generalized MG patients: 50%
      • AChR Ab+ patients by RIA: 98% positive
      • May be more specific for MG than RIA binding Ab
    • Complement fixing
    • Antibody subclass: IgG₁
    • Associated with thymic pathology: Lymphocytic infiltrates; Germinal centers
  • Neuronal AChR: α3 subunit
    • Autonomic PN (42%)
    • Isaacs (25%)
    • LEMS (12%)
  • Also see: AChR disorders
• Patients with MG but no anti-AChR antibodies
  • Rule out hereditary MG
  • Low frequency of thymic pathology & thymoma
  • May have antibodies to other neuromuscular junction antigens

Repetitive Nerve Stimulation (RNS): 2 to 3 Hz

Most frequently used electrodiagnostic test for MG Procedure Nerve to be studied with RNS Electrically stimulated six to ten times at 2 or 3 Hertz Compound muscle action potential (CMAP) Recorded with surface electrodes over muscle Nerves tested At least one proximal & one distal motor nerve Nerves innervating weak muscles If Decrement present Ensure decrement is reproducible Repeat RNS Test for: Post-exercise facilitation (Repair of decrement) Perform EMG Rule out Denervating, or other, disorder producing decrement Changes supporting NMJ disorder Motor unit action potentials: Small-short or Unstable If NO Decrement at baseline Look for: Post exercise exhaustion Appearance of decrement after exercise Perform: Single fiber EMG Original description Friedrich Jolly: Berliner Klinische Wochenschrift 1895;32;1

Decrement with RNS in MG: Orignial description

Normal muscles CMAP amplitudes: No change with repetitive nerve stimulation Myasthenia gravis CMAP amplitudes: Progressive decline during first 4 to 5 stimuli Caused by failure (Block) of increasing number of NMJs Positive RNS test features Decrement in CMAP amplitude Size: More than 10% in reduction in CMAP amplitude Measure from 1st to 4th or 5th potential in train Smallest CMAP is often 2nd or 3rd potential in train Post-tetanic potentiation (Post-exercise facilitation) Definition: Reduction in (Repair of) decrement after exercise Stimulus: Isometric exercise, brief (10 to 15 sec) Time course Onset: Immediate Duration: ~2 minutes Degree of repair: Partial or Complete Post-exercise exhaustion Definition Post exercise: Appearance, or Exacerbation, of decremental response Protocol Stimulus: Muscle exercise for 1 minute Repeat RNS after 1, 2, 3 and 4 minutes Time course Onset: Maximal 3 to 5 minutes after exercise Disappears by: 10 minutes after exercise RNS is positive in about 75% of patients with generalized MG, if: Proximal & Clinically involved muscles are tested Muscle is warm: Cooling reduces size of decrement More than one muscle is tested: Strong muscles often have less decrement Diagnostic issues Sensitivity of RNS for MG Sensitivity in generalized MG Overall: 75% to 80% Increased Proximal & Clinically involved muscles are tested Muscle is warm More than one muscle is tested Reduced Only distal muscles are tested Cooling: Reduces size of decrement Childhood MG Acute severe generalized or bulbar MG (≤ 4 weeks of disease) Decrement frequency: 11% to 40% positive 4 Often have abnormal jitter Strong muscles: Often have less decrement Ocular MG: 50% Diagnostic specificity A decremental response to RNS is not specific for MG Decrements may also be seen in Presynaptic disorders: Such as LEMS Motor neuron diseases: Including ALS Myopathies McArdle's Myotonia Becker MD

Myasthenia Gravis Repetitive Nerve Stimulation From: M Al-Lozi

• Rapid RNS
• Repetitive CMAP

Single fiber electromyography (SFEMG)

Principle: Muscle fibers innervated by a single axon Normal: Activated with consistent latencies NMJ disorders NMJ abnormal: Increased variability of latencies among muscle fibers in single motor unit NMJ fails completely: Muscle fiber potential may be blocked if transmission at NMJ fails completely   SFEMG Simultaneously records potentials of two muscle fibers innervated by an individual axon Measures Variability = "Jitter" Block: NMJ failure SFEMG is the most sensitive test for MG Sensitivity: > 95% positive in generalized & ocular MG When the test site includes facial muscles Abnormal jitter is not specific for MG May occur in other neuromuscular disorders, including ALS, polymyositis or LEMS More specific for MG if large degree of jitter occurs with mild or no other changes on EMG MuSK MG: Jitter may be present in absence of RNS decrement

Normal SFEMG Increased jitter: MG patient From: M Al-Lozi

• Stimulus: Repetitive (20 Hz) vibration stimuli to forehead
• Recording: Inferior oblique muscle; 2 surface electrodes placed beneath eyes
• Abnormal result: Decrement of 15% to 20%
• MG (Ocular or General) sensitivity: 89% unilateral; 63% bilateral