Evaluation of hemolysis interference and possible protective effect of N-phenyl maleimide on the measurement of small peptides

Objectives This study aims to demonstrate that proteolytic enzymes released as a result of hemolysis decrease the small peptide hormones, whereas N-phenyl maleimide (NPM) can be used to prevent hemolysis interference. Methods The serum/plasma pools were prepared at two different concentrations and divided into two equal volumes. NPM solution was added to one sample. The erythrocyte package was washed five times and then centrifuged after each wash. Hemolysis was induced by the osmotic shock method upon adding distilled water. 100 mu L of hemolysate stock solutions were added over 400 mu L of serum/plasma pool. Repeated five times. Results Negative interference was demonstrated on ACTH at 200 hemolysis index (HI) for low concentration, at 100 HI and 200 HI for high concentration, and insulin at 100 HI and 200 HI for high concentration. Addition of NPM which inactivates protease enzymes prevented the reduction of ACTH and insulin. No interference was observed at C-peptide and IGF-1 concentration until 200 HI. Conclusions Hemolysis was detected to degrade ACTH and insulin, which demonstrated how important the effect of hemolysis can be utilized in deciding whether to accept or reject samples. The addition of NPM can increase accuracy in clinical decisions as well as treatment.