1、Designation: F756 13Standard 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
2、 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 conta
3、ct 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 wi
4、th 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 provid
5、e 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
6、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
7、 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
8、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 conce
9、ntration(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 unde
10、r 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 hemolysisd
11、estruction 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 mm 60 cm2:20.0mL 21cm2:7.0mL1.0 mmor intricate geom-etry4.0g:20.0mL 1.4g:7.0mL9.2.2 Samples are cu
12、t 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 ea
13、ch tube containing the nonex-tracted sample. Place 7.0 mLof 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.NOTE 4This procedure calls for preparing
14、 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 cont
15、rols aswell as the test specimens.9.4 Maintain tubes in a suitable test tube rack for at least 3hat376 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 necessar
16、y 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 whic
17、h 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 Supernatant Hemoglobin Determination:9.8.1 Add 1.0 mL of
18、supernatant to 1.0 mL of cyanmethe-moglobin reagent, or validated diluent.9.8.2 Allow the sample to stand for 15 min for Drabkins or3 to 5 min for cyanmethemoglobin reagent. Read the absor-bance of the solution with a spectrophotometer at a wavelengthof 540 nm.9.8.3 In the unlikely event that A540ex
19、ceeds 2, this maysignify a procedural or background problem; the problemshould be identified and addressed, and the testing repeated.9.8.3.1 Determine the hemoglobin concentration in eachsupernatant from 9.8 using the calibration curve.9.8.3.2 The hemoglobin concentration of supernatant fromthe test
20、 sample or control tubes is calculated as follows (usingthe absorbance value obtained in 9.8.2 and correcting for thedilution factor of 2): :S 5 AS3 F 32 (4)The hemoglobin concentration of the blank tube is calcu-lated as follows:B 5 AB3 F 32 (5)9.8.3.3 Calculate the % hemolysis or hemolytic index a
21、s:% hemolysis 5supernatant hemoglobin concentration 3100%total hemoglobin concentration in tube(6)In the above equation, the “total hemoglobin concentrationin tube” is calculated by dividing the total blood hemoglobinconcentration obtained in 8.4.4 by 8 to account for the blooddilution in PBS in the
22、 test tubes. Use of this equation assumesthat background interference from endogenous plasma and freehemoglobin, and from the extracts, is negligible. This assump-tion can be verified by measuring the supernatant absorbanceof the extract solutions and of blood diluted in a test tubecontaining 7 mLof
23、 PBS and 1 mLof diluted blood (10 mg/mL)which has been incubated along with the test sample tubes.9.8.3.4 The % hemolysis is calculated by correcting for thebackground from the blank sample:Blank corrected % hemolysis 5S 2 BT/8! 2 B3100% (7)By following the dilution factors set out in subsections 8.
24、4.4and 9.8.1, Eq 7 can be simplified as follows:Blank corrected % hemolysis 5AS2 ABAT2 AB3100% (8)It should be noted that Eq 8 is only applicable if the dilutionsas set out in subsections 8.4.4 and 9.8.1 are strictly followed;otherwise, corrective dilution factors need to be introduced intoEq 7.10.
25、Report10.1 Express results in the form of the corrected % hemoly-sis index as described in 9.8.3.4.10.2 The final report, as a minimum shall include thefollowing:10.2.1 Detailed sample and control preparations includinggeneric or chemical names, catalog number, lot or batchnumber, and other pertinen
26、t available designations or descrip-tions.F756 13410.2.2 Detailed sample and control preparations, includingsample size, thickness, configuration of test specimens, andmethod of sterilization.10.2.3 Age of blood and type and concentration of antico-agulant used.10.2.4 Method of hemoglobin determinat
27、ion.10.2.5 Tabulation of total supernatant hemoglobin levels.10.2.6 % Hemolysis for the test samples, the negativecontrols, the positive controls, and the blanks. Include meanand standard deviation for each of the replicate samples,blanks, and positive and negative controls.10.2.7 Other pertinent ob
28、servations of the experiment.10.3 Conversion of % Hemolysis for reporting purposesThis practice provides a method for determining the propensityof a material to cause hemolysis. Pass/fail criteria for thematerial are subject to consideration of the nature of the tissuecontact, duration of contact, a
29、nd surface area to body ratios,and the nature of the device. Historically a hemolytic grade hadbeen assigned. However, the hemolytic grade is an arbitrarilyderived scale, has not been validated, and is based on previousresults using a slightly different procedure. If the assignment ofa hemolytic gra
30、de is required, the mean hemolytic index of theblank should be subtracted from the mean hemolytic index ofthe controls and the test samples. The results of the test sampleshould be compared to the results of the negative control.Hemolytic Index abovethe negative controlHemolytic Grade02 nonhemolytic
31、25 slightly hemolytic5 hemolyticIn addition, if the mean from the replicate test samples is lessthan 5 but one or more samples gave a hemolytic index ofgreater than 5, then the test should be repeated with double thenumber of test articles.11. Precision and Bias11.1 PrecisionThe precision of this te
32、st method is beingestablished. Although this method has been shown to haveintralaboratory repeatability, especially with regards to classi-fication of hemolytic response, interlaboratory variation is stillsignificant.11.2 BiasThe bias of this test method includes the quan-titative estimates of the u
33、ncertainties of the calibration of thetest equipment and the skill of the operators. At this time,statements of bias should be limited to the documentedperformance of particular laboratories.12. Keywords12.1 biocompatibility; blood compatibility; direct contact;extract; hemoglobin; hemolysis testing
34、APPENDIX(Nonmandatory Information)X1. RATIONALEX1.1 The presence of hemolytic material in contact withblood may produce increased levels of blood cell lysis andincreased levels of plasma hemoglobin. This may induce toxiceffects or other effects which may stress the kidneys or otherorgans.X1.2 This p
35、ractice is presented as a screening procedure forcomparing the hemolytic potential of a material with that of anegative control material which is generally acknowledged tobe appropriate for blood contact applications. Materials with ahemolytic potential above that of the specified negative controlma
36、terial, which is known to have excellent performance inblood contacting situations, should be carefully considered foruse since they may or may not be a potential cause of in vivohemolysis.X1.3 The procedure as presented is intended as a routinereproducible screening procedure. It is not to be repre
37、sented asbeing the most sensitive nor the most specific procedure forassessing the hemolytic potential of all materials in all useapplications. The results obtained with this procedure areintended to be used in conjunction with the results of other testsin assessing the blood compatibility of the te
38、st material.ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the riskof infringement of such
39、 rights, are entirely their own responsibility.This standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or for additi
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