The presence of myasthenia gravis (MG) is suspected in a patient with physical examination findings and a clinical history that are compatible with the diagnosis. Bedside techniques like the ice pack test are useful for a rapid assessment of the potential for an MG diagnosis; however, serologic or electrophysiologic testing may be required to confirm the diagnosis. 

Bedside Tests

The edrophonium (Tensilon) test was once historically useful to diagnose MG; however, it is no longer employed and no longer available in the United States and many other countries. Edrophonium is a short-acting acetylcholinesterase inhibitor that increases the amount of acetylcholine in the neuromuscular junction (NMJ) and briefly relieves the symptoms of MG. Because of high rates of false-positive results and the development of more sensitive diagnostic techniques, the FDA discontinued edrophonium in 2018.1  

The ice pack test is a cheap, safe, and useful bedside technique for evaluating a patient for a possible MG diagnosis, with good sensitivity and specificity. An ice pack is applied to the eyelid of a patient with ptosis for 2 to 5 minutes. If the patient has MG, the ptosis will improve or resolve. It is theorized that cooling the involved muscles reduces the activity of acetylcholinesterase, resulting in a temporary resolution of the ptosis. The sensitivity of the test is 76.9% at 2 minutes, and the specificity is 98.3% with the 5-minute application.2  

Serologic Tests

The detection of pathogenic antibodies against NMJ proteins in a patient with features suggestive of MG is diagnostic. Antibodies to 3 NMJ synaptic proteins have been identified as pathogenic: acetylcholine receptor antibody (AChR Ab), muscle-specific kinase antibody (MuSK Ab), and lipoprotein-related protein 4 antibody (LPR-4 Ab).3 

The detection of AChR Ab is the first recommended diagnostic step in the most recent MG guidelines. In the radioimmunoprecipitation assay (RIPA), the most widely used test, a radioactive isotope of iodine is attached to a highly selective molecule with an affinity for AChR Ab. The complex binds to and precipitates AChR Ab in the patient’s serum, which can then be quantified. An enzyme-linked immunosorbent assay (ELISA) is also available for AChR Ab detection, and the handling of radioactive compounds is not required. ELISA may not be as sensitive or specific as RIPA. A cell-based assay (CBA), which uses developed cell lines expressing highly concentrated clusters of AChR on their surface, is an option for detecting pathogenic antibodies in seronegative patients with clinical features suggestive of MG. An immunofluorescent technique is used to detect autoantibodies to the clustered AChR, and the result may be positive in 15% of previously seronegative patients.3 

Autoantibodies to the post-synaptic receptor for agrin (MuSK) are found in 6% to 8% of all patients with MG. Commercially available RIPA tests are available to detect MuSK Ab, and they are extremely specific. ELISA kits are also available but not widely used.3 

Autoantibodies to LPR-4 may be present in some patients with MG; however, because of the high rates of positivity for LPR-4 Ab in other diseases, such as amyotrophic lateral sclerosis and other neuroimmune diseases, its presence should be interpreted strictly within the clinical context. LPR-4 Ab is identifiable with CBA, RIPA, and ELISA.3 

Electrophysiologic Testing 

In the repetitive nerve stimulation (RNS) test, the compound muscle action potential (CMAP) of a muscle in response to supramaximal RNS is measured. A decremental response is observed in individuals with MG—that is to say, the area and amplitude of the CMAP decrease with each successive stimulation. In healthy muscle, successive CMAPs are identical in area and amplitude. RNS is useful for diagnosing seronegative MG and may distinguish between a cholinergic crisis and a myasthenic crisis.3 

Single-fiber electromyography (or a neuromuscular “jitter” study) records the action potentials of single muscle fibers in situ. The time between ACh release from the pre-synaptic neuron and the generation of an end plate potential (EPP) varies slightly (called a jitter) and is predictable in healthy muscle fibers. In patients with post-synaptic pathology, such as MG, the EPP is abnormally high because of deranged synaptic morphology. The jitter study result is abnormal in 98% to 100% of patients with generalized MG and in more than 90% of patients with ocular MG. It may also be abnormal in other synaptic disorders.3


1. Naji A, Owens ML. Edrophonium. StatPearls (Internet). Updated July 17, 2021. Accessed February 9, 2022.

2. Kearsey C, Fernando P, D’Costa D, Ferdinand P. The use of the ice pack test in myasthenia gravis. JRSM Short Rep. 2010;1(1):1-3. doi:10.1258/shorts.2009.090037

3. Rousseff RT. Diagnosis of myasthenia gravis. J Clin Med. 2021;10(8):1736. doi:10.3390/jcm10081736

Reviewed by Hasan Avcu, MD, on 2/20/2022.