Updated Method for In Vitro Analysis of Nanoparticle Hemolytic Properties

Methods Mol Biol. 2018;1682:91-102. doi: 10.1007/978-1-4939-7352-1_9.


Hemolysis is damage to red blood cells (RBCs), which results in the release of the iron-containing protein hemoglobin into plasma. An in vitro assay was developed and described earlier for the analysis of nanoparticle hemolytic properties. Herein, we present a revised version of the original protocol. In this protocol, analyte nanoparticles and controls are incubated in blood. Undamaged RBCs are removed by centrifugation and hemoglobin, released by the damaged erythrocytes, is converted to cyanmethemoglobin by incubation with Drabkin's reagent. The amount of cyanmethemoglobin in the supernatant is measured by spectrophotometry. This measured absorbance is compared to a standard curve to determine the concentration of hemoglobin in the supernatant. The measured hemoglobin concentration is then compared to the total hemoglobin concentration to obtain the percentage of nanoparticle-induced hemolysis. The revision includes updated details about nanoparticle sample preparation, selection of nanoparticle concentration for the in vitro study, updated details about assay controls and case studies about nanoparticle interference with the in vitro hemolysis assay.

Keywords: Hemoglobin; Hemolysis; Nanoparticles; Red blood cells (RBC).

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Blood Specimen Collection / methods
  • Centrifugation / methods
  • Erythrocytes / pathology*
  • Hemoglobins / analysis
  • Hemolysis*
  • Humans
  • Indicators and Reagents
  • Methemoglobin / analogs & derivatives
  • Methemoglobin / analysis
  • Nanoparticles / adverse effects*
  • Spectrophotometry / methods


  • Hemoglobins
  • Indicators and Reagents
  • cyanomethemoglobin
  • Methemoglobin