1、Designation: F2888 13Standard Test Method forPlatelet Leukocyte CountAn In-Vitro Measure forHemocompatibility Assessment of Cardiovascular Materials1This standard is issued under the fixed designation F2888; the number immediately following the designation indicates the year oforiginal adoption or,
2、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 assists in the evaluation of cardiovas-cular device materials fo
3、r their ability to induce thrombusformation. Thrombus formation is assessed by means of areduction in human platelets and leukocytes when consumedby thrombus after activation on the material surface. This assaymay be part of the hemocompatibility evaluation for devicesand materials contacting human
4、blood, as per ANSI/AAMI/ISO 109934. See also Test Method F2382.1.2 All safety policies and practices shall be observedduring the performance of this test method. All human bloodand any materials that had contact with human blood shall bebagged in a biohazard bag, properly labeled as the contents,and
5、 disposed of by appropriate means.1.3 The human blood should be handled at Biosafety Level2 as recommended in the Centers for Disease Control/NationalInstitutes of Health Manual Biosafety in MicrobiologicalLaboratories. The human blood donor must have tested nega-tive for Hepatitis B (HBV) and Human
6、 Immunodeficiency(HIV) viruses. The blood should be treated like any patientblood in using universal precautions.1.4 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.5 This standard does not purport to address all of thesafety
7、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. Some specifichazards statements are given in Section 7 on Hazards.2. Referenc
8、ed Documents2.1 ASTM Standards:2F2382 Test Method forAssessment of Intravascular MedicalDevice Materials on Partial Thromboplastin Time (PTT)2.2 Other Standards:ANSI/AAMI/ISO 109934 Biological Evaluation of Medi-cal DevicesPart 4: Selection of Tests for Interactionswith Blood3Centers for Disease Con
9、trol /National Institutes of HealthManual Biosafety in Microbiological Laboratories, 19933. Summary of Test Method3.1 This test method identifies materials which are capableof activating blood platelets and leukocytes on their surfacewhen exposed to freshly drawn human blood and causing theformation
10、 of thrombi on the material surface. A significantdecrease in the number of platelets and leukocytes whencounted by a blood analyzer is an indication of these cellsbeing entrapped in thrombi. The materials are exposed to bloodimmediately after the blood is drawn with anticoagulant.Another anticoagul
11、ant is added at the appropriate time (onehour) to stop the reaction. Different blood analyzers may beused.4. Significance and Use4.1 The purpose of this test method is to determine ifmedical materials exposed to human whole blood will ad-versely affect the platelet and leukocyte counts in whole bloo
12、d.A large number of platelets and leukocytes as part of thrombiadhering to the material will be reflected by a decrease in theircounts in blood. Thrombogenic materials should not be used1This test method is under the jurisdiction ofASTM Committee F04 on Medicaland Surgical Materials and Devices and
13、is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition approved Feb. 15, 2013. Published March 2013. DOI: 10.1520/F288813.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual
14、Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036, http:/www.ansi.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, W
15、est Conshohocken, PA 19428-2959. United States1for cardiovascular medical devices, unless the purpose of thedevice is to promote thrombosis.5. Interferences5.1 There is potential for interference if the materials of thetest tubes used are thrombogenic (for example, glass tubes).Therefore, polyethyle
16、ne or polypropylene tubes should beused.6. Apparatus6.1 Hematology analyzer capable of determination of acomplete blood count.6.2 Polypropylene test tubes with caps.6.3 Commercial blood collection tubes containing 3.2 %,0.105 M sodium citrate.6.4 Agitating water bath/incubator, 37 6 2C.6.5 Pipettes
17、and tips (non-glass).7. Reagents and Materials7.1 Cell count controls suitable for hematology analyzer.7.2 Fresh whole human blood.7.3 EDTA (ethylenediaminetetraacetic acid), 500 mM.7.4 Saline, optional.7.5 Positive reference control material (for example, naturalrubber latex, black rubber), optiona
18、l.7.6 Positive control material (for example, black rubber,natural rubber latex).7.7 Negative reference control material (for example, highdensity polyethylene (HDPE).8. Hazards8.1 Human blood should be treated with the proper univer-sal precautions, including eye wear and laboratory gloves.9. Sampl
19、ing, Test Specimens, and Test Units9.1 Prepare each test sample, reference material, negativecontrol, and positive control in triplicate. All material samplesare prepared based on a ratio of 12 cm2of material to 1 mL ofblood and placed into polypropylene tubes.9.2 Thirty-six square centimeters of th
20、e test sample, positiveand reference controls are divided into three 12 cm2samples,cut to maximize blood exposure, for triplicate testing.NOTE 1If other volumes of blood are used, the ratio of total surfacearea to blood volume should remain at 12:1.9.3 For each test sample, the percentage of test va
21、lue(platelet count or leukocyte count) per negative control (bloodnot exposed to a material) is calculated as follows:A B 3100 5 C (1)where:A = average count (platelets or leukocytes),B = average count (platelets or leukocytes) of negativecontrol, andC = percentage (%) of negative control.When appli
22、cable, a comparison article should be used in theformula in place of the negative control.10. Preparation of Apparatus10.1 Initialize the hematology analyzer and allow it toperform internal self-checks. If no errors are noted, theanalyzer is ready for use.10.2 To verify the analyzer is functioning p
23、roperly, prior toanalyzing samples, cell count controls shall be run to conformthat the results fall within the allowable ranges.10.3 Fresh human blood is drawn for the test system. Bloodshould be from donors who have not taken aspirin,acetaminophen, naproxen, warfarin, heparin, or ibuprofen forten
24、days. Blood should be collected in a tube containing 3.2 %,0.105 M sodium citrate (at a ratio of 9:1 blood to sodium citrateas per commercial blood collection tubes), pooled, gentlymixed by inversion, and stored on ice until use.10.4 For each analysis, a single donors blood will beexposed to the tes
25、t sample, reference material, negativecontrol, and positive control (when applicable) to provide aconsistent test system to evaluate all reference and test mate-rials. It is recommended to pre-screen the blood to ensure theblood parameters fall within the normal expected range (nor-mal leukocyte cou
26、nt 3.4 to 8.37 103/uL, normal platelet count116to329103/uL). If the donor blood parameters fall outsideof the normal expected ranges, blood from another donorshould be used.11. Calibration and Standardization11.1 Perform daily calibration procedures as per instrumentinstructions. (Typically the inst
27、rument self-calibrates uponinitiation.)12. Procedure12.1 The test sample(s), reference material, and positivecontrol materials are placed into polypropylene tubes andexposed to the appropriate quantity of blood, based on a ratioof 12 cm2of material to 1 mL blood. The negative control isblood only. O
28、ptionally, the test sample(s), referencematerials(s), and positive control material may be pre-wettedwith saline prior to exposure to blood by adding the same ratioof saline to each tube, allowed to sit for 30 s at roomtemperature, then all saline is removed prior to addition ofblood.12.2 The sample
29、s are exposed to blood for 1 h 6 5 min ina376 2C agitating water bath or incubator at approximately60 r/min.12.3 After the 1 h incubation period, EDTA is added to alltubes for a final concentration of 5 mM to stop any furtherreaction from occurring. Typically, 0.01 mL of EDTA (500mM) is added per 1.
30、0 mL of human whole blood to achieve thecorrect final concentration.12.4 All tubes are gently mixed, blood removed as com-pletely as possible from each sample, and transferred to new,F2888 132appropriately labeled tubes. All tubes are placed on ice untilassayed on the hematology analyzer.12.5 Each s
31、ample is gently mixed by rolling and invertingthe tube at least eight times and then the sample loaded on thehematology analyzer. This is repeated until all samples havebeen assayed.13. Calculation or Interpretation of Results13.1 The three test sample values, reference control, nega-tive control, a
32、nd positive control values for platelets and whiteblood cells (leukocytes) are averaged.13.2 The average count of the test sample, positive control,and positive and/or negative reference control materials iscompared to the negative control, or comparison article whenapplicable. In each case the perc
33、entage of the negative control/comparison article will be calculated:AB 3100 5 C (2)where:A = average count (platelet or leukocyte) of the test sample,B = average count (platelet or leukocyte) of negativecontrol/comparison article, andC = percentage (%) of negative control/comparison article.Referen
34、ce material values are calculated using the sameformula as above and should be used to monitor assayconsistency run to run.14. Assay Validity14.1 The individual results of the three readings for thepositive control, negative control, reference control and testsamples shall be within 620 % of the mea
35、n value of the threereadings. If this criterion is not met, the assay shall be repeatedusing fresh blood.14.2 The acceptance criteria for both platelet and leukocytecount for the negative reference material is 80 to 120 % of thenegative control. The acceptance criteria for the plateletpositive refer
36、ence control material or positive control is aminimum of 50 % reduction from the negative control. (Avisible clot is also considered a positive response.) Due todonor variability it is not possible to set an acceptance forleukocyte counts for the positive reference material.15. Test Evaluation15.1 A
37、t this time, there are no ranges or levels established asacceptable for this assay. Therefore it is recommended thateach biomaterial is assayed in comparison to a legally mar-keted device, or other appropriate material.15.2 All biomaterials have some potential to affect themake-up of the platelet an
38、d leukocyte components of theblood. Substantial decreases in platelet and leukocyte counts inbiomaterials exposed blood may be indicative of increasedplatelet and leukocyte activation.15.3 Additional evaluation may be needed when results areequivocal and test values fall within the range between the
39、negative and positive reference materials (for example, 21 to49 % reduction in platelet count for the negative control.16. Precision and Bias16.1 The precision and bias of this test method have not yetbeen determined.17. Keywords17.1 hemocompatibility; leukocyte; platelet; thrombosisANNEX(Mandatory
40、Information)A1. REFERENCE AND POSITIVE CONTROL MATERIALA1.1 Reference and Positive Control MaterialNaturallatex tubing or black rubber stopper. Alternative referencematerials may be selected, once they have demonstrated aconsistent, and ideally a mildly or moderately thrombogenicresponse.F2888 133AP
41、PENDIX(Nonmandatory Information)X1. RATIONALEX1.1 This test method allows assessment of intravascularmedical device materialsability to induce thrombus. It shouldbe part of the hemocompatibility evaluation/ material screeningfor devices and material contacting human blood.BIBLIOGRAPHY(1) Sefton M. V
42、., Sawyer, A., Gorbet, M., Black, J. P., Cheng, E.,Gemmell, C., Cooper, E., “Does Surface Chemistry Affect Throm-bogenicity of Surface Modified Polymers?,” J Biomed Mater Res,Vol 55, No. 4, June 15, 2001, pp. 447459.ASTM International takes no position respecting the validity of any patent rights as
43、serted 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 rights, are entirely their own responsibility.This standard is subject to revision at any time
44、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 additional standardsand should be addressed to ASTM International Headquarters. Your comments will rec
45、eive 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 Int
46、ernational, 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). Permission rights to photocopy the standard may also be secured from the ASTM website (www.astm.org/COPYRIGHT/).F2888 134
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