1、Designation: F2382 04 (Reapproved 2010)Standard Test Method forAssessment of Intravascular Medical Device Materials onPartial Thromboplastin Time (PTT)1This standard is issued under the fixed designation F2382; the number immediately following the designation indicates the year oforiginal adoption o
2、r, in the case of revision, the year of last 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 the screening of cardiovasculardevice materials for th
3、eir ability to induce blood coagulationvia the intrinsic coagulation pathway. This assay should be partof the hemocompatibility evaluation for devices and materialscontacting human blood, as per ANSI/AAMI/ISO 10993-4.1.2 All safety policies and practices shall be observedduring the performance of th
4、is test method.1.3 All plasma and any materials that had contact withplasma will be bagged in a biohazard bag, properly labeledwith the contents, and disposed by appropriate means. Theplasma should be handled at the Biosafety Level 2 as recom-mended in the Centers for Disease Control/National Instit
5、utesof Health Manual Biosafety in Microbiological Laboratories.1.4 The normal pooled human plasma must have testednegative for Hepatitis B (HBV) or Human Immunodeficiency(HIV) viruses. The plasmas should be treated like any patientplasma using universal precautions. The plasma should behandled at th
6、e Biosafety Level 2 as recommended in theCenters for Disease Control/National Institutes of HealthManual Biosafety in Microbiological Laboratories.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 pu
7、rport 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 ANSI Standard:ANS
8、I/AAMI/ISO 10993-4 Biological Evaluation of Medi-cal DevicesPart 4: Selection of Tests for Interactionswith Blood22.2 Other Document:Centers for Disease Control/National Institutes of HealthManual Biosafety in Microbiological Laboratories,199933. Terminology3.1 Definitions:3.1.1 activatora medical m
9、aterial which demonstrates ashortened clotting time; an initiator of the intrinsic coagulationpathway.3.1.2 partial thromboplastin time (PTT) assaya modifica-tion of the Activated Partial Thromboplastin Time (APTT)assay; unlike the APTT test, the PTT assay uses reagent (rabbitbrain cephalin) without
10、 activating substances (silica, kaolin,elagic acid.) The material being tested acts as the activator.3.1.3 read timethe time during which data is collected todetect a clot.3.1.4 blank timea period at the beginning of an assaywhen no data is taken. This is done to eliminate interferencefrom premixing
11、 reagents, bubbles, and so forth.3.1.5 equilibration timethe time allowed for the plasmasamples to warm to 37C. The fibrometer can be set to zero ifsamples are pre-warmed to this temperature.3.1.6 duplicate flagthe agreement between the results ofduplicate samples in percent. For example, if set to
12、“15,” thedifference between the two channels must be less than or equalto 15 %. If the variance in clot times exceeds this percentage,an asterisk “*” will be printed by the average results on thereport.4. Significance and Use4.1 The purpose of this test method is to determine the timecitrated plasma
13、 exposed to medical materials takes to form aclot when exposed to a suspension of phospholipid particlesand calcium chloride. In this test method, the test article is theactivator. The PTT assay is a general screening test for medicalmaterials ability to activate the intrinsic coagulation pathway.Ma
14、terial samples that show a shortened PTT are activators ofthe intrinsic coagulation pathway.1This test method is under the jurisdiction of ASTM Committee F04 on Medicaland Surgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current e
15、dition approved June 1, 2010. Published September 2010. Originallyapproved in 2004. Last previous edition approved in 2004 as F2382 04. DOI:10.1520/F2382-04R10.2Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036, http:/www.ansi.org.3Available fr
16、om National Institute of Health (NIH), 9000 Rockville Pike,Bethesda, MD 20892.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.4.2 Test samples that show a shortened PTT are activators ofthe intrinsic coagulation pathway. The results
17、are reported as apercent of the negative control. The test article, referencematerials, and controls are exposed to human plasma. Theplasma is tested on a coagulation device. Each sample tube isassayed in duplicate.5. Apparatus5.1 Polypropylene Test Tubes with Caps,12by75mm.5.2 Automatic Pipets and
18、Tips, 100 and 1000 L.5.3 Ice Bath.5.4 Coagulation Analyzer (Automated Fibrometer).5.5 Agitating Water Bath,376 2C, capable of 60 rpm.5.6 Coagulation Analyzer Cuvettes, or equivalent for spe-cific analyzer.6. Reagents and Materials6.1 Calcium Chloride,25mm.6.2 Citrated Human Blood Plasma, fresh (less
19、 than 4 h fromdraw) or freshly-frozen, maintained at minus 80C, pooled.6.3 Lyophilized Rabbit Brain Cephalin (RBC).6.4 Reference Control Material, see Appendix X1.6.5 Positive Control Material, glass (Pasteur pipette tips orglass beads).7. Hazards7.1 The human blood plasma should be treated like any
20、patient plasma using universal precautions. The plasma shouldbe handled at the Biosafety Level 2 as recommended in theCenters for Disease Control/National Institutes of HealthManual Biosafety in Microbiological Laboratories.8. Preparation of Apparatus8.1 Prepare each test article in triplicate. The
21、referencematerial(s), and the controls are prepared as singles. Allsamples are prepared based on a ratio of 4 cm2of material to1 mL plasma and placed into polypropylene tubes. For devicetesting, if test sample quantity allows, use three separatedevices, otherwise, take three representative samples f
22、rom onedevice.8.2 Label duplicate polypropylene tubes and place in icebath.8.3 Initialize coagulation analyzer and allow it to warm upto 37 6 2C and equilibrate for at least 10 min.8.4 Program the analyzer to test under the APTT functionwith an equilibration time of 60 s, activation time of 120 s, a
23、blank time of 14 s, and a read time of 286 s.8.5 Print out test parameters and verify changes. Photocopyprintout and attach to original data.8.6 Pre-warm analysis cuvettes (or cups, dependent onanalyzer selected) at 37 6 2C.8.7 Pre-warm calcium chloride at 37 6 2C.8.8 Rabbit Brain Cephalin (RBC) Pre
24、paration:8.8.1 Allow the RBC to come to room temperature.8.8.2 Reconstitute RBC with 10 mL reagent grade water/distilled water.8.8.3 Place in agitating water bath set at 37 6 2C, at 60rpm for 15 min to ensure complete rehydration of contents.8.8.4 Vortex 15 s after rehydration is complete.8.8.5 Plac
25、e at 37 6 2C.8.9 If using frozen blood plasma, quick thaw the plasma at37 6 2C and place on ice immediately.9. Procedure9.1 The test material(s), reference material(s), and controlsare placed into polypropylene tubes and exposed to theappropriate quantity of plasma, based on a ratio of 4 cm2ofmateri
26、al to 1 mL plasma. The negative control is a polypropy-lene tube with 1 mL of plasma, without additional material.9.2 The samples are exposed to the plasma for 15 6 1 minina376 2C agitating water bath at 60 rpm.9.3 After 15 min of incubation, the tubes are immediatelyplaced into the ice bath and imm
27、ediately transferred intopre-chilled new polypropylene tubes.9.4 Vortex each sample 15 s before each use/run.9.5 Avoiding bubbles, transfer 100 L of the plasma intopre-warmed cuvettes and allow the plasma to equilibrate for 60sat376 2C.9.6 To each cuvette/cup, add 100 L warmed RBC prepa-ration initi
28、ating the 2 min activation step. (Invert RBC to mixprior to each use.)9.7 After activation, add 100 L warmed 25 mm calciumchloride to each cuvette.9.8 Allow the analyzer to read the sample for the formationof clots (up to 5 min).9.9 Record the clotting time (seconds) for each sample, aswell as the a
29、verage clotting time of the duplicate samples.10. Calculation or Interpretation of Results10.1 Calculate the test sample result (% negative control)for test material, reference, and positive control sample mean.% negative control 5 (1)Average clotting time s! of sampleAverage clotting time s! of neg
30、ative control3 10010.2 Test Sample Acceptance Criteria:% Negative Control Thrombogenicity100 Non-activator of intrinsic coagulation pathway75 to 100 Minimal activator50 to 74 Mild activator25 to 49 Moderate activator50 Pass10.3 As a comparison, the reference material(s) results arereported using Eq
31、1.10.4 The positive control result % negative control must be50 %. If the assay does not meet this specification, theexperiment is to be repeated until the controls are within range.Reference material and positive control results should beequivalent (within the same thrombogenicity category) run tor
32、un.10.5 The variance between the duplicates for each samplemust be #15 %. The duplicates of each test article sample areaveraged and one value is reported as the clotting time. Thisresults in three clotting time values for each test article. Thethree values are then averaged to report a final averag
33、e clottingF2382 04 (2010)2time of the test article. The values for each test article samplemust be within 625 % of this average. If the values are greaterthan 25 % of the average of the run, the experiment needs to berepeated.11. Precision and Bias11.1 The precision and bias of this test method has
34、not yetbeen determined.12. Keywords12.1 blood coagulation; blood compatibility; partial throm-boplastin time; PTTANNEX(Mandatory Information)A1. VENDOR INFORMATIONA1.1 Rabbit Brain Cephalin (RBC)Bio/Data,4or equiva-lent vendor.A1.2 Coagulation AnalyzerCascade M-4, Helena Labo-ratories5or equivalent
35、instrument.A1.3 Reference Control MaterialNatural latex tubing(Small Parts, Inc.,6or equivalent vendor) or black rubberstopper (Fisher7or equivalent vendor). Alternate referencematerials may be selected, once they have demonstrated aconsistent, ideally a mildly or moderately thrombogenic re-sponse.
36、More than one reference material may be used.APPENDIX(Nonmandatory Information)X1. RATIONALEX1.1 This test method allows assessment of intravascularmedical device materials ability to induce blood coagulationvia the intrinsic coagulation pathway. It should be part of thehemocompatibility evaluation
37、for devices and materials con-tacting human blood.REFERENCES(1) Sawyer, A., “In Vitro Hemocompatibility Screening Method forBiomaterials,” World Congress for Biomaterials Meeting Transactions,1992, p. 669.(2) U.S. Department of Health and Human Services, Public HealthService, Centers for Disease Con
38、trol and Prevention and NationalInstitutes of Health, “Biosafety in Microbiological and BiomedicalLaboratories”, Fourth Edition, April 1999, p. 21-27.ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. User
39、s of this standard are expressly advised that determination of the validity of any such patent rights, 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
40、five years andif not revised, either reapproved or withdrawn. Your comments are invited either for revision 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 technica
41、l committee, which you may attend. If you feel that your comments have not received a fair hearing you 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,
42、 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). Permission rights to photocopy
43、 the standard may also be secured from the ASTM website (www.astm.org/COPYRIGHT/).4Available from Bio/Data, 155 Gilbraltar Rd., Horsham, PA 19044.5Available from Helena Laboratories, P.O. Box 752, Beaumont, TX 77704.6Available from Small Parts, Inc., 13980 NW 58th Ct., P.O. Box 4650, MiamiLakes, FL 33014.7Available from Fisher Scientific, 600 Business Center Dr., Pittsburgh, PA15205.F2382 04 (2010)3
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