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Generally, a clinical diagnosis of ON is based on the history and examination findings.  Though demyelination is its most common identifiable cause, many other causes of optic neuropathy may resemble ON, and misdiagnosis is not uncommon. Diagnostic testing is typically directed toward excluding other causes of visual loss in atypical cases, and assessing the risk of subsequent MS. An early evaluation is essential to ensuring visual recovery has begun and to reconsider the diagnosis if it has not.

As mentioned, in typical cases of ON, visual improvement occurs within 2 to 3 weeks regardless of treatment.  Thus, in typical cases, which show no additional clinical signs and symptoms of a systemic disease, the value of diagnostic testing is fairly low.  However, if there are atypical signs and symptoms (i.e., bilateral presentation, younger than 15 years old, or possible infection) suggesting an alternative diagnosis, a complete assessment should be undertaken.

MRI is used to take images of the brain and orbits to confirm the diagnosis of ON. However, the real value of MRI in typical ON is not to image the optic nerves, but rather to image the brain as a prognostic indicator for the future development of MS. Often the brain MRI shows white matter abnormalities, or lesions, which are characteristic of MS – ovoid, periventricular, and larger than 3 mm lesions which indicate a higher risk of developing MS.

Lumbar puncture is usually not considered an essential diagnostic test in ON, but should be considered in atypical cases. Approximately 60 to 80 percent of ON diagnoses show nonspecific abnormalities in cerebrospinal fluid (CSF). Additionally, 56 to 69 percent of individuals also show oligoclonal bands (OCB) in their CSF, which implies a higher risk of developing MS. However, since OCB is closely associated with white matter lesions seen in MRI, the presence of OCB is not of high prognostic importance.

Finally, optical coherence tomography (OCT) is also commonly used to detect ON.  OCT measures the thickness in the retinal nerve fiber layer and detects thinning in 85 percent of patients with ON. While lower values correlate with impaired visual outcome, its usefulness as a prognostic tool is limited due to the fact that abnormal values do not show up until early swelling disappears. OCT is also important as a number of studies have found that a greater severity of optic nerve injury, seen on OCT, suggests NMO rather than ON associated with multiple sclerosis.

Additional diagnostic tests used to detect ON or assess the risk of other conditions include: fluorescein angiography, visual evoked response, and Aquaporin-4-specific serum autoantibody to rule out NMO.