ASTM_F_756_-_13.pdf

Designation:F75613Standard Practice forAssessment of Hemolytic Properties of Materials1This standard is issued under the fixed designation F756;the number immediately following the designation indicates the year of originaladoption or,in the case of revision,the year of last revision.Anumber in parentheses indicates the year of last reapproval.Asuperscriptepsilon()indicates an editorial change since the last revision or reapproval.1.Scope1.1 This practice provides a protocol for the assessment ofhemolytic properties of materials used in the fabrication ofmedical devices that will contact blood.1.2 This practice is intended to evaluate the acute in vitrohemolytic properties of materials intended for use in contactwith blood.1.3 This practice consists of a protocol for a hemolysis testunder static conditions with either an extract of the material ordirect contact of the material with blood.It is recommendedthat both tests(extract and direct contact)be performed unlessthe material application or contact time justifies the exclusionof one of the tests.1.4 This practice is one of several developed for theassessment of the biocompatibility of materials.Practice F748may provide guidance for the selection of appropriate methodsfor testing materials for a specific application.Test MethodE2524 provides a protocol using reduced test volumes to assessthe hemolytic properties of blood-contacting nanoparticulatematerials;this may include nanoparticles that become unboundfrom material surfaces.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 thesafety 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:2E691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test MethodE2524 Test Method for Analysis of Hemolytic Properties ofNanoparticlesF619 Practice for Extraction of Medical PlasticsF748 Practice for Selecting Generic Biological Test Methodsfor Materials and Devices3.Terminology3.1 Definitions:3.1.1 plasma hemoglobinamount of hemoglobin in theplasma.3.1.2%hemolysisfree plasma hemoglobin concentration(mg/mL)divided by the total hemoglobin concentration(mg/mL)present multiplied by 100.This is synonymous withhemolytic index.3.1.3 comparative hemolysiscomparison of the hemolyticindex produced by a test material with that produced by astandard reference material such as polyethylene under thesame test conditions.3.1.4 direct contact testtest for hemolysis performed withthe test material in direct contact with the blood.3.1.5 extract testtest for hemolysis performed with anisotonic extract of the test material,as described in PracticeF619,in contact with the blood.3.1.6 hemolysisdestruction of erythrocytes resulting in theliberation of hemoglobin into the plasma or suspension me-dium.3.1.7 negative controlmaterial,such as polyethylene,thatproduces little or no hemolysis(0.50 mm60 cm2:20.0 mL21 cm2:7.0 mL1.0 mmor intricate geom-etry4.0 g:20.0 mL1.4 g:7.0 mL9.2.2 Samples are cut into appropriate pieces.Transfer eachof three nonextracted samples of test and control specimensinto individual tubes as described in 9.1.3.The recommendedtube size is 16 125 mm.However the tube size may be anysuch that the specimen is covered by 7.0 mL of PBS liquid.Place 7.0 mL of PBS into each tube containing the nonex-tracted sample.Place 7.0 mL of PBS into each of three tubes toserve as the blank.9.3 TestAdd 1.0 mL of blood prepared according to 8.4.4to each tube containing extract,each tube containing aspecimen,and the blanks.Cap all tubes.NOTE4This procedure calls for preparing the sample,adding thediluent to the sample and then adding the blood,which minimizes the timedifference for contact of the sample with blood.Alternatively,the bloodmay be added to the diluent and then the sample added to the preparedsolution.Whichever method is chosen must be used for the controls aswell as the test specimens.9.4 Maintain tubes in a suitable test tube rack for at least 3h at 37 6 2C in a water bath.Gently invert each tube twiceapproximately every 30 min to maintain contact of the bloodand material.In some cases of samples with complicatedconfigurations,it may be necessary to do more inversions toadequately mix the sample.9.5 At the end of the specified incubation time,transfer thefluid to a suitable tube and centrifuge at 700 to 800 G for 15min in a standard clinical centrifuge.9.6 Remove the supernatant carefully to avoid disturbingany button of erythrocytes which may be present.Place thesupernatant into a second screw cap tube.Record the presenceof any color to the supernatant and any precipitate.9.7 Analyze the samples from 9.6 for supernatant hemoglo-bin concentration using the method in 9.8.9.8