ASTM F756-2008 Standard Practice for Assessment of Hemolytic Properties of Materials《材料溶血性能评估的标准实施规程》.pdf

1、Designation: F 756 08Standard Practice forAssessment of Hemolytic Properties of Materials1This standard is issued under the fixed designation F 756; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A numbe

2、r in parentheses indicates the year of last reapproval. Asuperscript epsilon () 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

3、 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 mater

4、ial 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 F 748may

5、 provide guidance for the selection of appropriate methodsfor testing materials for a specific application.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 concer

6、ns, 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:2E 691 Practice for Conducting an Interlab

7、oratory Study toDetermine the Precision of a Test MethodF 619 Practice for Extraction of Medical PlasticsF 748 Practice for Selecting Generic Biological Test Meth-ods for Materials and Devices3. Terminology3.1 Definitions:3.1.1 plasma hemoglobinamount of hemoglobin in theplasma.3.1.2 % hemolysisfree

8、 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 s

9、uch 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 PracticeF 619, in contact with t

10、he blood.3.1.6 hemolysisdestruction of erythrocytes resulting inthe liberation of hemoglobin into the plasma or suspensionmedium.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 geometry4.0g:20.0mL 1.4g:7.0

11、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 3 125 mm. However the tube size may be anysuch that the specimen is covered by 7.0 mL of PBS liquid.Pla

12、ce 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.F7560839.3 TestAdd 1.0 mL of blood prepared according to 8.4.4to each tube containing extract, each tube containing a speci-men, and the blanks. Cap all tubes.NOTE 4

13、This 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 i

14、s 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 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 complicated

15、configurations, 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 disturb

16、ingany 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 Supernatant Hemoglobin

17、Determination:9.8.1 Add 1.0 mL of 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

18、 In the unlikely event that A540exceeds 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 concent

19、ration of supernatant fromthe test 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 3 2 (4)The hemoglobin concentration of the blank tube is calcu-lated as follows:B 5 AB3 F 3 2 (5)9.8.3.3 Calculate

20、the % hemolysis or hemolytic index as:% hemolysis 5supernatant hemoglobin concentration 3 100 %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 accou

21、nt for the blooddilution in PBS in the 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 d

22、iluted in a test tubecontaining 7 mLof 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 BT/8! B3 100 % (7)By following the diluti

23、on factors set out in subsections 8.4.4and 9.8.1, Eq 7 can be simplified as follows:Blank corrected % hemolysis 5AS ABAT AB3 100 % (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

24、 need to be introduced intoEq 7.10. 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, lo

25、t or batchnumber, and other pertinent available designations or descrip-tions.10.2.2 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 Met

26、hod of hemoglobin determination.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 contro

27、ls.10.2.7 Other pertinent observations 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 tissuecon

28、tact, duration of contact, and 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

29、assignment ofa hemolytic grade 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 controlHe

30、molytic Grade02 nonhemolytic25 slightly hemolytic5 hemolyticIn addition, if the mean from the replicate test samples is lessthan 5 but one or more samples gave a hemolytic index ofF756084greater than 5, then the test should be repeated with double thenumber of test articles.11. Precision and Bias11.

31、1 PrecisionThe precision of this test 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 t

32、he quan-titative estimates of the uncertainties 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;ext

33、ract; hemoglobin; hemolysis testingAPPENDIX(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

34、 kidneys or otherorgans.X1.2 This practice 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

35、of the specified negative controlmaterial, 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 screeni

36、ng procedure. It is not to be represented 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 assessin

37、g the blood compatibility of the test 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,

38、and the riskof infringement of such 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 revi

39、sion of this standard or for additional standardsand should be addressed to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you

40、shouldmake your views known to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org).F756085