*4.1.2. Laboratory tests*

Muscle biopsy in conjunction with molecular genetic testing is required for effective diagnosis of MD [151]. A major feature of the histological result of the biopsy using the Gomori Trichrome stain shows >2% ragged red fibers that come from the sub-sarcolemmal mitochondrial accumulation. However, these ragged red fibers are present only in the late stage of the disease and commonly absent in children [132]. The key diagnostic feature is the presence of fibers that are deficient for cytochrome c oxidase (COX) activity [with >2% of COX negative fibers], reflecting low activity of complex IV of the respiratory chain, in patients less than 50 years [148, 151]. COX activity may be decreased in healthy older patients, so its use in diagnosis is limited to younger patients. Laboratory tests for the levels of creatine phosphokinase, pyruvate, albumin, lactate, transaminases, and blood count are also recommended [152]. An elevated postprandial lactate:pyruvate ratio (>20) is commonly found in MD patients; however, some MD patients may show normal ratio and thus other tests are required to confirm the disease [146]. Nextgeneration sequencing is also proposed for screening of the multiple mutations associated with MDs [152]. Additionally, fibroblast growth factor-21 (FGF-21) has been recently identified as a serum biomarker of MDs associated with both mtDNA and nDNA mutations [148], potentially simplifying the clinical diagnosis of MD.
