Whilst the assay is not sufficiently sensitive to identify a biochemical defect associated with some very well-characterised mtDNA – or rare catalytic defects affecting either mtDNA-encoded or nuclear-encoded CI subunits – we believe that a combination of the quadruple IHC assay, in tandem with full mitochondrial genome sequencing and standard biochemical assays, can be used to investigate likely genetic causes of CI deficiency in patients with mitochondrial disease, especially when muscle biopsy sample sizes are small necessitating the analysis of cryosectioned material. The gene discussed is NDUFB6; the disease is mitochondrial disease.