title: The effect of hemolysis on plasma antithrombin activity as determined by a chromogenic method.
abstract: BACKGROUND
  The concentration of free hemoglobin (Hgb) in plasma can markedly affect the outcome of laboratory tests by interfering with the spectrophotometric absorbance of biochemical tests read at wavelengths within the absorbance range of Hgb (400-440 nm). Little is known about the effects of hemoglobinemia on antithrombin (AT) activity in human plasma samples, and we are unaware of data for canine plasma samples.
  
  
  OBJECTIVE
  The objective of this study was to determine the degree of interference by Hgb on plasma AT activity and to determine if the interference is proportional to the concentration of plasma Hgb.
  
  
  METHODS
  Two pools of test plasma, designated AT100 and AT70, were prepared. Hemolysate was prepared by washing and freeze-thawing packed red cells in a small volume of saline, followed by collection of the filtrate. Solutions of decreasing Hgb concentration were prepared and added to the test plasma pools to create a series of samples with final calculated and measured Hgb concentrations of 0, 0.5, 1.5, 2.5, 3.5, 4.5, and 5.5 g/L. AT activity, expressed as a percentage of normal human plasma, was determined using a functional chromogenic assay.
  
  
  RESULTS
  Increasing concentrations of Hgb resulted in a linear decrease in AT activity. Using linear regression analysis on the 70% and 100% plasma pools, the slopes for samples containing <1.5 g Hgb/L were not significantly different from zero. Slopes for samples containing Hgb concentrations >or=1.5 g/L were significantly (P > .0001) different from zero for both plasma pools, indicating interference with the assay.
  
  
  CONCLUSIONS
  The results of this study suggest it may be possible, using a conversion equation, to accurately determine AT activity in hemolyzed samples, facilitating evaluation of coagulation status in patients with intravascular hemolysis.
authors.affiliation: Department of Companion Animal Studies, Faculty of Veterinary Science, University of Pretoria, South Africa. lvdmerwe@op.up.ac.za
authors.forename: Liesel
authors.initials: L
authors.lastname: van der Merwe
authors.forename: Fred
authors.initials: F
authors.lastname: Reyers
mesh_terms: D000818:Animals; D000991:Antithrombins / Q000737:chemistry / Q000378:metabolism*; D002863:Chromogenic Compounds / Q000737:chemistry*; D004285:Dogs / Q000097:blood*; D006454:Hemoglobins; D006461:Hemolysis; D012680:Sensitivity and Specificity
chemical_list: D000991:Antithrombins; D002863:Chromogenic Compounds; D006454:Hemoglobins