After 8 freeze-thaw cycles 99.6 ± 14.3% and 90.4 ± 1.6% of endogenous MANF was detected in T1D (n = 4) and autoantibody-negative sera (n = 4), respectively, compared to the samples with only 1 freeze-thaw cycle (Fig. 1c). This evidence concerns the gene MANF and type 1 diabetes mellitus.