Effect of blood acidification on hemolysis-induced insulin degradation

Hemolysis is a well-known preanalytical source of interference in insulin measurement, primarily due to the release of insulin-degrading enzyme (IDE) from erythrocytes. Given the pH-dependent activity of IDE, this study aimed to investigate whether blood acidification could reduce hemolysis-induced insulin degradation.

Two experimental approaches were employed. First, blood samples from 30 healthy volunteers were collected into FC-Mix tubes and serum tubes. The plasma and serum samples were spiked with hemolysate to achieve different hemolysis index (HI) levels. Second, residual serum pools were acidified with citric acid-citrate buffers of increasing molarity (0.1 to 0.5 M) before hemolysate addition. Insulin was measured using the Roche Cobas Pro e801 analyzer, and percentage bias relative to non-hemolyzed controls was compared against the desirable limits based on biological variation.

In serum samples, insulin concentrations declined progressively with increasing hemolysis, with the lowest hemolysis group (median HI = 51) already exhibiting a 14.1 % decrease exceeding the desirable bias (10.5 %). At the highest hemolysis level (median HI = 1022), the reduction further intensified, reaching 90.8 %. FC-Mix plasma maintained stable median insulin concentrations across all hemolysis levels, with biases ranging from-0.3 % to -1.8 %, within desirable bias limits. Direct citrate buffer acidification attenuated hemolysis-induced insulin decline, resulting in biases of -4.8 and - 6.3 % at 0.4 and 0.5 M, respectively, below the desirable limits.

Acidification of blood using FC-Mix tubes or direct citrate buffer addition markedly reduces hemolysis-induced negative interference in insulin measurements, offering a practical strategy to improve assay reliability.