1、Designation:E252408(Reapproved 2013)Standard Test Method forAnalysis of Hemolytic Properties of Nanoparticles1This standard is issued under the fixed designation E2524;the number immediately following the designation indicates the year oforiginal adoption or,in the case of revision,the year of last
2、revision.A number in parentheses indicates the year of last reapproval.Asuperscript epsilon()indicates an editorial change since the last revision or reapproval.1.Scope1.1 This test method covers assessing the effect of nanopar-ticulate materials on the integrity of red blood cells.1.2 This test met
3、hod uses diluted whole blood incubatedwith nanoparticulate material and the hemoglobin releasedfrom damaged red blood cells is determined.1.3 This test method is similar to Practice F756 with thevolumes reduced to accommodate nanoparticulate material.1.4 This test method is part of the in-vitro prec
4、linicalcharacterization and is important for nanoparticulate materialthat will contact the blood in medical applications.1.5 The values stated in SI units are to be regarded asstandard.No other units of measurement are included in thisstandard.1.6 This standard does not purport to address all of the
5、safety concerns,if any,associated with its use.It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2.Referenced Documents2.1 ASTM Standards:2F748 Practice for Selecting Gene
6、ric Biological Test Methodsfor Materials and DevicesF756 Practice for Assessment of Hemolytic Properties ofMaterialsF1877 Practice for Characterization of ParticlesF1903 Practice for Testing For Biological Responses toParticles In Vitro2.2 ISO Standard:3ISO 10993-4 Biological Evaluation of Medical D
7、evices Part4:Selection of Tests for Interactions with Blood3.Terminology3.1 Acronyms:3.1.1 Calcalibration standard3.1.2 CMHcyanmethemoglobin3.1.3 DPBSDulbeccos phosphate-buffered saline3.1.4 PEGpolyethylene glycol3.1.5 PFHplasma-free hemoglobin3.1.6 QCquality controls3.1.7 TBHtotal blood hemoglobin3
8、.1.8 TBHdblood sample diluted to 10 mg 6 1 mg/mL4.Summary of Test Method4.1 This test method describes a protocol for assessing acutein-vitro damage to red blood cells(that is,hemolysis)causedby exposure to nanoparticles.4.2 This test method is based on the quantitative determi-nation of hemoglobin
9、released into PFH as a percentage of theTBH concentration when blood is exposed to nanoparticulatematerials.4.3 Using an established colorimetric assay,4hemoglobinand its derivatives,such as sulfhemoglobin,are oxidized tomethemoglobin by ferricyanide in the presence of alkali.Astable CMH concentrati
10、on is measured using a plate readerspectrophotometer set at 540 nm.4.4 Hemoglobin standards are used to create a standardcurve covering the range from 0.025 to 0.8 mg/mL and preparequality control samples at low(0.0625-mg/mL),mid(0.125-mg/mL),and high(0.625-mg/mL)concentrations to monitorassay perfo
11、rmance.The required sample volume is 100 L pertest replicate.4.5 The results are expressed as percent hemolysis toevaluate the acute in-vitro hemolytic properties of nanopar-ticles.5.Significance and Use5.1 This test method is one of a series of tests listed inPractice F748 and ISO 10993-4 to assess
12、 the biocompatibilityof materials contacting blood in medical applications.1This test method is under the jurisdiction of ASTM Committee E56 onNanotechnology and is the direct responsibility of Subcommittee E56.03 onEnvironment,Health,and Safety.Current edition approved Sept.1,2013.Published Septemb
13、er 2013.Originallyapproved in 2008.Last previous edition approved in 2008 as E2524 08.DOI:10.1520/E2524-08R13.2For referenced ASTM standards,visit the ASTM website,www.astm.org,orcontact ASTM Customer Service at serviceastm.org.For Annual Book of ASTMStandards volume information,refer to the standar
14、ds Document Summary page onthe ASTM website.3Available from American National Standards Institute(ANSI),25 W.43rd St.,4th Floor,New York,NY 10036,http:/www.ansi.org.4International Committee for Standardization in Haemotology,Journal ofClinical Pathology,Vol 31,1978,pp.139143.Copyright ASTM Internati
15、onal,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 5.2 This test method is similar to Practice F756 but modifiedto accommodate nanoparticulate materials.6.Apparatus6.1 Pipettes covering range from 0.05 to 10 mL.6.2 Ninety-six well plates.6.3 Water bath set at 37 6
16、1C.6.4 Plate reader capable of measuring at 540 nm.6.5 Plate shaker.6.6 Plastic beakers.6.7 Microcentrifuge tubes,1.5 mL,translucent,not colored.6.8 Centrifuge set at 700 to 800 g.7.Reagents7.1 Purity of ReagentsReagent-grade chemicals shall beused in all tests.Unless otherwise indicated,it is intended thatall reagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are available.5Other grades may be used,provided it
