As the statistical analysis used by Meyer et al. had proved to be biased in the comparison between patients and controls, the question remained whether or not the APS1 patients showed autoantibody responses against higher numbers of self-proteins than healthy subjects. We first looked at the distribution of autoantibody signals among patients and controls by applying different fixed signal cutoffs. The number of elevated IgG signals was higher in the APS1 patients than in the healthy controls, although the difference was small compared to that found by Meyer et al. (A). For example, at an arbitrary cutoff of 10,000 arbitrary fluorescence intensity units the APS1 patients displayed autoantibody responses against an average of 22 unique proteins as compared to 15 proteins in the healthy controls. Furthermore, the difference was almost completely eliminated when we excluded already known autoantigens – decreasing the number of autoantibody signals to an average of 16 proteins in the APS1 patients compared to the unchanged average of 15 proteins in the healthy controls (B). The results were similar for other cutoff values. We also compared the number of autoantibody signals between patients and controls by applying Z-score cutoffs. To allow for an unbiased detection of autoantibody signals, equally prone to register signals among patients and controls, we did not base the Z-score on the healthy controls as in the study by Meyer et al. but instead on the whole cohort. We further excluded the 10% highest and 10% lowest signals before calculating the cutoff, so that the cutoff level would not be affected by outlier values. This type of analysis is expected to detect elevated signals that occur in one or a few subjects, however, autoantibody signals that are present in many subjects are overlooked as the cutoff is consequently increased. Again we observed a small difference in the number of autoantibody signals between the patients and controls (C), which was further reduced by excluding the known autoantigens (D). Thereby, in contrast with results by Meyer et al. specifically identifying high-level autoantibody signals among the APS1 patients, we found only a small difference in the number of autoantibody signals between patients and controls. We also calculated the statistical significance that the number of autoantibody signals in APS1 differed from permutated data by comparing the number of distinct proteins with at least one hit (Z ≥ 3) among the APS1 cases to what is expected by random (when 21 samples are randomly assigned to represent controls and the remaining 51 represent cases) (E). The p-value was computed as the fraction of the permutations that result in as many distinct proteins as observed or more. At higher z-scores the numbers were borderline significant, showing a trend towards that the APS1 patients display higher numbers of autoantibody signals than the permutated data ‘cases’. The results were in line with the investigations in A-D. In all panels, the complexity factor was calculated as the number of distinct proteins divided by the number of individuals divided by the average number of proteins per individual. In panel A and B: Pat = APS1 patients, BG = healthy controls. In panel C and D: p=APS1 patients, C = healthy controls.