1、Designation: F2382 04 (Reapproved 2010)F2382 17Standard Test Method forAssessment of Intravascular Circulating Blood-ContactingMedical Device Materials on Partial Thromboplastin Time(PTT)1This standard is issued under the fixed designation F2382; the number immediately following the designation indi
2、cates the year oforiginal adoption or, 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 c
3、ardiovascular circulating blood-contacting device materials for their ability toinduce blood coagulation via the intrinsic coagulation pathway. This assay should be part of the hemocompatibility evaluation fordevices and materials contacting human blood, as per ANSI/AAMI/ISO 10993-4.1.2 All safety p
4、olicies and practices shall be observed during the performance of this test method.1.3 All plasma and any materials that had contact with plasma will be bagged in a biohazard bag, properly labeledlabelled withthe contents, and disposed of by appropriate means. The plasma should be handled at the Bio
5、safety Level 2 as recommended inthe Centers for Disease Control/National Institutes of Health Manual Biosafety in Microbiological Laboratories.1.4 The normal pooled human plasma must have tested negative for Hepatitis B (HBV) or Human Immunodeficiency (HIV)viruses. The plasmas should be treated like
6、 any patient plasma using universalstandard precautions. The plasma should be handledat the Biosafety Level 2 as recommended in the Centers for Disease Control/National Institutes of Health Manual Biosafety inMicrobiological Laboratories.1.5 The values stated in SI units are to be regarded as standa
7、rd. No other units of measurement are included in this standard.1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety safety, health, and healthenvironmental prac
8、tices and determine theapplicability of regulatory limitations prior to use.1.7 This international standard was developed in accordance with internationally recognized principles on standardizationestablished in the Decision on Principles for the Development of International Standards, Guides and Re
9、commendations issuedby the World Trade Organization Technical Barriers to Trade (TBT) Committee.2. Referenced Documents2.1 ANSIANSI/AAMI Standard:ANSI/AAMI/ISO 10993-4 Biological Evaluation of Medical DevicesPart 4: Selection of Tests for Interactions with Blood22.2 Other Document:Centers for Diseas
10、e Control/National Institutes of Health Manual U.S. Department of Health and Human Services Biosafety inMicrobiological Laboratories, and Biomedical Laboratories (BMBL), 5th ed., 199933. Terminology3.1 Definitions:Definitions of Terms Specific to This Standard:3.1.1 activatora medical material which
11、 demonstrates a shortened clotting time; an initiator of the intrinsic coagulationpathway.1 This test method is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current e
12、dition approved June 1, 2010Sept. 1, 2017. Published September 2010September 2017. Originally approved in 2004. Last previous edition approved in 2004 asF2382 04. DOI: 10.1520/F2382-04R10.10.1520/F2382-17.2 Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New Y
13、ork, NY 10036, http:/www.ansi.org.3 Available from National Institute of Health (NIH), 9000 Rockville Pike, Bethesda, MD 20892.The BMBL 5th Edition (December 2009) is available from the GovernmentPrinting Office or https:/www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdfThis document is not an ASTM
14、standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all
15、cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.1.2 partial thromboplastin time (PTT) assaya modification of the Activated
16、Partial Thromboplastin Time (APTT) assay;unlike the APTT test, the PTT assay uses reagent (rabbit brain cephalin) without activating substances (silica, such as silica, kaolin,elagic acid.)acid. The material being tested acts as the activator.3.1.3 read timethe time during which data is collected to
17、 detect a clot.3.1.4 blank timea period at the beginning of an assay when no data is taken. This is done to eliminate interference frompremixing reagents, bubbles, and so forth.3.1.5 equilibration timethe time allowed for the plasma samples to warm to 37C. The fibrometer coagulation analyzer canbe s
18、et to zero if samples are pre-warmed to this temperature.3.1.6 duplicate flagthe agreement between the results of duplicate samples in percent. For example, if set to “15,” thedifference between the two channels must be less than or equal to 15 %. 15 %. If the variance in clot times exceeds this per
19、centage,an asterisk “*” will be printed by the average results on the report.4. Significance and Use4.1 The purpose of this test method is to determine the time citrated plasma exposed to medical materials takes to form a clotwhen exposed to a suspension of phospholipid particles and calcium chlorid
20、e. In this test method, the test article is the activator.The PTT assay is a general screening test for a medical materials ability to activate the intrinsic coagulation pathway. Materialsamples that show a shortened PTT are activators of the intrinsic coagulation pathway.4.2 Test samples that show
21、a shortened PTT are activators of the intrinsic coagulation pathway. The results are reported as apercent of the negative control. The The test article, reference materials, and controls are exposed to human plasma. The plasmais tested on a coagulation device. Each sample tube is assayed in duplicat
22、e. The results are reported as a percentage of the negativecontrol.5. Apparatus5.1 Polypropylene Test Tubes with Caps, 12 by 75 mm.5.2 Automatic Pipets and Tips, 100 and 1000 L.5.3 Ice Bath.5.4 Coagulation Analyzer (Automated Fibrometer). (Siemens BFT II analyzer or other).5.5 Agitating Water Bath,
23、37 6 2C, capable of 60 rpm.5.6 Coagulation Analyzer Cuvettes, or equivalent for specific analyzer.6. Reagents and Materials6.1 Calcium Chloride, 25 mm.mM.6.2 Citrated Human Blood Plasma, fresh (less than 4 h from draw) or freshly-frozen, maintained at minus 80C, pooled.6.3 Lyophilized Rabbit Brain C
24、ephalin (RBC).6.4 Positive Reference Control Material,Material (Optional), see Appendix X1.6.5 Positive Control Material, Control, glass (Pasteur pipette tips or glass beads).6.6 Negative Reference Material (e.g. High Density Polyethylene, HDPE).6.7 Marketed Comparator Device (Optional). A legally m
25、arketed, clinically acceptable device that has similar blood contactnature and clinical use as the material/device being investigated.NOTE 1It may be helpful to use a positive reference control material (n=1) per assay to assure continuity between runs.7. Hazards7.1 The human blood plasma should be
26、treated like any patient plasma using universalstandard precautions. The plasma shouldbe handled at the Biosafety Level 2 as recommended in the Centers for Disease Control/National Institutes of Health Manual USDepartment of Health and Human Services Biosafety in Microbiological and Biomedical Labor
27、atories.8. Preparation of Apparatus8.1 Prepare each test article in triplicate. The reference material(s), and the controls are prepared as singles. article, the negativereference materials, marketed comparator device (if used) and controls in triplicate. If a positive reference control material is
28、used,a single replicate is acceptable. All samples are prepared based on a ratio of 4 either 4 or, preferably 6 cm2 of material to 1 mLplasma and placed into polypropylene tubes. For device testing, if test sample quantity allows, use three separate devices,devices;otherwise, take three representati
29、ve samples from one device.8.2 Label duplicate polypropylene tubes and place in the ice bath.F2382 1728.3 Initialize Turn on the coagulation analyzer and allow it to warm up to 37 6 2C and equilibrate for at least 10 min.8.4 Program the analyzer to test under the APTT function with an equilibration
30、time of 60 s, activation time of 120 s, a | blanktime of 14 s, and a read time of 286 s.8.5 Print out test parameters and verify changes. Photocopy printout and attach to original data.8.5 Pre-warm analysis cuvettes (or cups, dependentdepending on analyzer selected) at 37 6 2C.8.6 Pre-warm calcium c
31、hloride at 37 6 2C.8.7 Rabbit Brain Cephalin (RBC) Preparation:8.7.1 Allow the RBC to come to room temperature.8.7.2 Reconstitute RBC with 10 mL reagent grade water/distilled water.the RBC as indicated by the RBC reagent manufacturer.8.7.3 Place in an agitating water bath set at 37 6 2C, at2C and 60
32、 rpm for 15 min to ensure complete rehydration of contents.8.7.4 Vortex 15 s after rehydration is complete.8.7.5 Place at 37 6 2C.8.8 If using frozen blood plasma, quick thaw the plasma at 37 6 2C and place on ice immediately.9. Procedure9.1 The test material(s), reference material(s), marketed comp
33、arator device (if used) and controls are placed into polypropylenetubes and exposed to the appropriate quantity of plasma, based on a ratio of 4 cm2, or preferably 6 cm2 of material to 1 mL plasma.The negative control is a polypropylene tube with 1 mL of plasma, without additional material.9.2 The s
34、amples are exposed to the plasma for 15 6 1 min in a 37 6 2C agitating water bath at 60 rpm.9.3 After 15 min of incubation, the tubes are immediately placed into the ice bath and immediately transferred intopre-chilledpre chilled new polypropylene tubes.9.4 Vortex each sample 15 s before each use/ru
35、n.9.5 Avoiding bubbles, transfer 100 LpL of the plasma into pre-warmed cuvettes and allow the plasma to equilibrate for 60 sat 37 6 2C.9.6 To each cuvette/cup, add 100 LpL warmed RBC preparation, initiating the 2 min activation step. (Invert RBC to mix priorto each use.)9.7 After activation, add 100
36、 LpL warmed 25 mmmM calcium chloride to each cuvette.9.8 Allow the analyzer to read the sample for the formation of clots (up to 5 min).9.9 Record the clotting time (seconds) for each sample, as well as the average clotting time of the duplicate samples.NOTE 2The volume of plasma, RBC reagent, and c
37、alcium chloride needed for the clotting time measurement may vary with different anticoagulationanalyzers. It is acceptable to use a plasma volume that is specific to the coagulation analyzer used, as long as the plasma to reagent ratio remains 1:1.10. Calculation or Interpretation of Results10.1 Ca
38、lculate the test sample result (% negative control) for test material, reference, and positive control sample mean.% negative control5 (1)Average clotting time s! of sampleAverage clotting time s! of negative control 3100F2382 17310.2 Test Sample Acceptance Criteria:% Negative Control Thrombogenicit
39、y100 Non-activator of intrinsic coagulationpathway75 to 100 Minimal activator50 to 74 Mild activator25 to 49 Moderate activator50 Pass10.3 As a comparison, the reference material(s) results are reported using Eq 1.10.4 The positive control result % negative control must be 80%. If the assay does not
40、 meet this specification, the experiment is to be repeated until the controls meet thesespecifications.10.2.5 The Coefficient of Variation (CV) between the duplicates for each sample must be 15 %. The duplicates of each testarticle sample are averaged and one value is reported as the clotting time.
41、This results in three clotting time values for each testarticle. The three values are then averaged to report a final average clotting time of the test article. The values for each test articlesample must be within 625 % of this average. If the values are greater than 25 % of the average of the run,
42、 the experiment needsto be repeated.11. Precision and Bias11.1 The precision and bias of this test method has not yet been determined.12. Keywords12.1 blood coagulation; blood compatibility; partial thromboplastin time; PTTF2382 174ANNEX(Mandatory Information)A1. VENDOR INFORMATIONA1.1 Rabbit Brain
43、Cephalin (RBC)Bio/Data,4 or equivalent vendor.A1.2 Coagulation AnalyzerCascade M-4, Helena LaboratoriesAble to reliably detect or equivalent instrument.clot formation,with a maximum clot detection time greater than 300 seconds.A1.3 Reference Control MaterialNatural latex tubing (Small Parts,(Fisher
44、Scientific5 Inc.,and McMaster-Carr6 or equivalentvendor) or black rubber stopper (Fisher5 or equivalent vendor). Alternate reference materials may be selected, once they havedemonstrated a consistent, ideally a mildly or moderately thrombogenic response. More than one reference material may be used.
45、APPENDIX(Nonmandatory Information)X1. RATIONALEX1.1 This test method allows assessment of intravascular medical device materials ability to induce blood coagulation via theintrinsic coagulation pathway. It should be part of the hemocompatibility evaluation for devices and materials contacting humanb
46、lood.REFERENCES(1) Sawyer, A., “In Vitro Hemocompatibility Screening Method for Biomaterials,” World Congress for Biomaterials Meeting Transactions, 1992, p. 669.(2) U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention and National Institute
47、s of Health,“Biosafety Services Biosafety in Microbiological and Biomedical Laboratories”, Fourth Edition, AprilLaboratories (BMBL), 5th ed., 19992009, p.21-27.ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this stan
48、dard. Users 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 revie
49、wed every 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 technical 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 Internati
