Cost- and time-efficient diagnostic strategy for ZSS. Patients suspected of ZSS are screened by measurement of the concentration of very long chain fatty acids (VLCFAs) in serum/plasma and biosynthesis of plasmalogens in erythrocytes. If VLCFAs are elevated (▴) and plasmalogens are decreased (▾), the patient's DNA from EDTA whole blood sample will be extracted and sequenced for the two most common mutations in the most frequently affected PEX gene, PEX1. If the patient is heterozygotic for one of these two mutations, the entire PEX1 gene will be sequenced. If none of these two mutations are detected, we recommend cultivation of primary human fibroblasts (PHFs). To confirm the suspected diagnosis of ZSS on the cellular level, PHFs are analyzed by immunofluorescence with an anti-catalase antibody. If catalase is localized inside the peroxisome (see Figure 2a), ZSS is ruled out in the patient. If catalase is diffused in the cytosol of the patient's PHFs, diagnosis of ZSS is verified. Then, the patient's PHFs are transfected with all known human PEX genes (except PEX7) cloned in individual expression vectors and together with eGFP-SKL to screen for complementation. The PEX gene that complements the patient's PHFs will be sequenced. If none of the known human PEX genes complements the patient's PHFs, the patient potentially belongs to an unknown complementation group with a putative mutation in a novel, and so far unidentified PEX gene. The time bar on the left displays the approximate estimation of time needed for the different laboratory procedures used in this strategy. If concentrations of VLCFAs are within the normal range (n) and biosynthesis of plasmalogens in erythrocytes is also within the normal range or is only slightly decreased, but clinical presentation strongly points to a PBD, we recommend a skin biopsy to culture PHFs ( → ). Measurement of the concentration of VLCFAs, as well as biosynthesis of plasmalogens, should be repeated in these PHFs.