title: Development of a novel, hemolysis-resistant reagent for assessment of α-amylase in biological fluids.
abstract: BACKGROUND
  Although the assessment of α-amylase is an essential part of the diagnostic workout of several pancreatic and extra-pancreatic disorders, its enzymatic activity is significantly reduced in the presence of cell-free hemoglobin such as in samples with spurious hemolysis, due to chemical and spectrophotometric interference. We developed a new reagent that provides reliable results on hemolyzed biological specimens.
  
  
  METHODS
  All tests were performed on Beckman Coulter AU5822. Intra-assay imprecision was assessed on three serum samples with low, intermediate and high α-amylase concentration. Linearity was assessed by serially diluting two samples with low and high values of α-amylase. The comparison with commercial reagent was performed on 40 serum samples. Hemolysis studies were carried out by mechanically hemolysis of 20 lithium-heparin samples.
  
  
  RESULTS
  The intra-assay imprecision was comprised between 1.3% and 2.2%. The linearity was excellent (r=0.998), and highly significant agreement was observed with the commercial assay (r=1.00; mean bias -3.8%). Although a significant correlation between non-hemolyzed and hemolyzed specimens was found with both assays (p<0.001), a much greater agreement was observed with the experimental method (r=0.997 vs. 0.818). No measurement exceeded the total allowable error with the experimental assay, whereas the threshold was exceeded in 85% of samples using the commercial method.
  
  
  CONCLUSIONS
  The clinical applications of the experimental reagent include α-amylase assessment in hemolyzed samples, in urine and other biological fluids contaminated with lysed erythrocytes, or in patients under frequent transfusions and hemoglobin-based blood substitutes therapy. The formulation of this reagent could be adapted for other clinical chemistry or immunochemistry assays.
authors: Department of Pharmacy, University of Parma, Parco Area delle Scienze, Parma, Italy.
authors: Luca
authors: L
authors: Ronda
authors: Andrea
authors: A
authors: Mozzarelli
authors: Rosalia
authors: R
authors: Aloe
authors: Giuseppe
authors: G
authors: Lippi
mesh_terms: D000328:Adult; D002138:Calibration; D004912:Erythrocytes / Q000737:chemistry*; D005260:Female; D006461:Hemolysis; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry / Q000592:standards*; D008297:Male; D008875:Middle Aged; D015588:Observer Variation; D011933:Reagent Kits, Diagnostic / Q000592:standards*; D015203:Reproducibility of Results; D000516:alpha-Amylases / Q000097:blood* / Q000652:urine*
chemical_list: D007202:Indicators and Reagents; D011933:Reagent Kits, Diagnostic; D000516:alpha-Amylases