160 Chapter 5 To study the association of plasma lysoGb3 with the natural disease course, only data obtained before the start of ERT or any other Fabry specific treatment were included in this study. Laboratory measurements Plasma lysoGb3 (nmol/l) was measured by tandem mass spectrometry, as previously described [19]. For reproduction purposes, a detailed description of our assay is added as supplemental material (SM3). All available untreated lysoGb3 values were used to assess stability in individual patients over time. Only the last available untreated value of lysoGb3 was used to assess its association with markers of disease severity. For the sake of visualization only (figures 1-6) patients were grouped in 4 different groups. Groups are not included in any of the statistical models. The cutoffs of 2.3 nmol/L and 40 nmol/L were chosen as they differentiate between classical and non-classical disease in female and male patients respectively. 7.3 nmol/L was chosen to split the remaining groups (predominately men with non-classical FD and women with classical FD) into equal halves to visualize the effect within patient groups with the same disease phenotype. A separate table of patient characteristics for each visual group is added in the supplemental material (SM1). Glomerular filtration rate was estimated (eGFR) in mL/min/1.73m2 using CKDEPI formula. Patients <18 years were excluded from this analysis as the formula can be used in adults only. (Micro)albuminuria was assessed using the urinary albumin to creatinine ratio (uACR, mg/mmol). For analyses, uACR was included as a continuous variable. If urinary albumin was below the level of detection, uACR was set to be 0. The following patients were excluded from the analysis of all renal outcome parameters: patients with a renal transplantation at time of presentation (n=5), as well as patients (n=3) with a confirmed second renal disease that was considered to be the main reason for the decline in renal function (i,e, renal artery stenosis; acute glomerulonephritis and severe bilateral kidney atrophy of uncertain origin). Echocardiograms Left ventricular mass index (LVMI) was calculated using the Devereux formula and was corrected for body surface area (BSA) using the Dubois formula. For the calculation of LVMI and relative wall thickness (RWT), longitudinal data, extracted from clinical echocardiography reports, were used. In a subset of patients (those with at least two echocardiograms with a minimum of 5 years between them) extensive re-evaluation of a large number of echocardiography markers was performed (as part of a different study on the development of cardiac manifestations in FD) by a single observer (MES). If Fabry specific treatment was started between the first and last echocardiogram in this study, only the
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