Free insulin (FI) measurements obtained by polyethylene glycol (PEG) precipitation within 3 min of drawing the blood sample (FI3) from four insulin-treated diabetic subjects with a wide range of insulin antibodies were compared with published methods of FI estimation. Comparison of FI values obtained by PEG precipitation in assays of replicate samples of the same specimens (N = 9) stored at 4°C for 24 h (EFI) and FI3 were 4.76 ± 1.5 μU/ml (mean ± SEM) and 17.13 ± 4.7 μU/ ml, respectively (P < 0.005). Comparison of FI values obtained in six groups of replicate samples (N = 12, 18 per group, a total of 91 specimens) from these four patients assayed immediately after thawing to 18°C, and incubated at 37°C for 30, 60, and 120 min, and FI3 showed a significant difference in at least oneof these four comparisons (mean ± SEM) in each of these six sample groups. In 18 of 24 comparisons there was a loss of FI when stored samples were used with or without incubation (12 of these were significant at the P < 0.05–0.001 level), but in 6 of the 24 comparisons there was an increase in the FI against FI3 (3 of these 6 significant at the P < 0.05–0.01 level). There was a trend toward a greater loss of FI in stored samples with higher FI3 content. Loss of FI during incubation occurred in all groups irrespective of the FI3 content. The three comparisons showing a significant increase in FI during incubation were all from the same patient with high levels of insulin antibodies. We conclude that, in the presence of insulin antibodies, only immediately processed fresh serum samples can give a best approximation of in vivo FI. This observation may account, in part, for the conflicting data and views in the literature on the usefulness of FI measurements in IDDM.

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