ASTM F2888-19

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Designation: F2888 − 13 F2888 − 19
Standard Test Method Practice for
Platelet Leukocyte Count—An In-Vitro Measure for
Hemocompatibility Assessment of Cardiovascular Materials
This standard is issued under the fixed designation F2888; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This test method practice assists in the evaluation of cardiovascular device materials for their ability to induce thrombus
formation. Thrombus formation is assessed by means of a reduction in human platelets and leukocytes when consumed by
thrombus after activation on the material surface. This assay may be part of the hemocompatibility evaluation for devices and
materials contacting human blood, as per in accordance with ANSI/AAMI/ISO 10993–4. See also Test Method F2382.
1.2 All safety policies and practices shall be observed during the performance of this test method. practice. All human blood
and any materials that had contact with human blood shall be bagged in a biohazard bag, properly labeled aswith the contents, and
disposed of by appropriate means.
1.3 The human blood should be handled at Biosafety Level 2 (BSL-2) as recommended in the Centers for Disease
Control/National Institutes of Health Manualpublication, Biosafety in Microbiological Laboratories. and Biomedical Laboratories
(BMBL). The human blood donor must have tested negative for Hepatitis B (HBV) and Human Immunodeficiency (HIV) viruses.
The blood should be treated like any patient blood in and handled/manipulated using universalstandard precautions.
NOTE 1—The results of this in-vitro test may not correspond to actual human response.
1.4 The values stated in SI (International System of Units) units are to be regarded as standard. No other units of measurement
are included in this standard.
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use. Some specific hazards statements are given in Section 78 on Hazards.
1.6 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
F2382 Test Method for Assessment of Circulating Blood-Contacting Medical Device Materials on Partial Thromboplastin Time
(PTT)
2.2 Other Standards:
ANSI/AAMI/ISO 10993–4 Biological Evaluation of Medical Devices—Part 4: Selection of Tests for Interactions with Blood
Centers for Disease ControlBMBL /National Institutes of Health Manual Biosafety in Microbiological Laboratories, 1993and
Biomedical Laboratories, 5th ed., 2009
3. Summary of Test MethodPractice
3.1 This test method practice identifies materials which are capable of activating blood platelets and leukocytes on their surface
when exposed to freshly drawn human blood and causing the formation of thrombi on the material surface. A significant decrease
This test method practice is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of
Subcommittee F04.16 on Biocompatibility Test Methods.
Current edition approved Feb. 15, 2013Feb. 1, 2019. Published March 2013February 2019. Originally approved in 2013. Last previous edition approved in 2013 as F2888
– 13. DOI: 10.1520/F2888–13.10.1520/F2888–19.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
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http://www.cdc.gov.
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F2888 − 19
in the number of platelets and leukocytes when counted by a blood analyzer is an indication of these cells being entrapped in
thrombi. The materials are exposed to blood immediately after the blood fresh whole blood that is drawn with anticoagulant.
Another anticoagulant is added at the appropriate time (one hour) to stop the reaction. further thrombus formation. Different blood
analyzers may be used.
4. Significance and Use
4.1 The purpose of this test method practice is to determine if medical materials exposed to human whole blood will adversely
affect the thrombus formation has occurred by comparing platelet and leukocyte counts in whole blood. the blood exposed to the
test material relative to the blood cell counts in the control blood that has not been exposed to the test material. A large number
of platelets and leukocytes as part ofbecoming entrapped/incorporated in thrombi adhering to the material will be reflected by a
decrease in their counts in blood. Thrombogenic materials should not be used for cardiovascular medical devices, unless the
purpose of the device is to promote thrombosis.
5. Interferences
5.1 There is potential for interference if the materials of the test tubes used are thrombogenic (for example, glass tubes).
Therefore, polyethylene or polypropylene tubes should be used.
6. Apparatus
6.1 Hematology analyzer capable of determination of a complete 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 2ºC.
6.5 Pipettes and tips (non-glass).
7. Reagents and Materials
7.1 Cell count controls suitable for hematology analyzer.
7.2 Fresh whole human whole blood.
7.3 EDTA (ethylenediaminetetraacetic acid), 500 mM.
7.4 Saline, optional.
7.5 Positive reference control material (for example, black rubber, natural rubber latex, black rubber), optional.and glass).
7.6 Positive control material (for example, black rubber, natural rubber latex).
7.6 Negative reference control material (for example, high density polyethylene (HDPE)).
7.7 Marketed comparator device (a legally marketed device that has similar blood contact and clinical use as the material/device
being investigated), optional.
8. Hazards
8.1 Human blood should be treated with the proper universal precautions, including eye wear and laboratory gloves.handled
according to standard microbiological practices and techniques (for example, use of personal protective equipment) and specific
required precautions (for example, OSHA Bloodborne Pathogen Standard ).
9. Sampling, Test Specimens, and Test Units
9.1 Prepare each Each test sample, negative reference material, negative control, and positive control control material, positive
control material, and marketed comparator device (if applicable) shall be prepared in triplicate. All material samples are test
samples and controls, with the exception of, the negative control which is blood only, shall be prepared based on a ratio of 12 cm
of material to 1 mL of blood and placed into polypropylene tubes. Also, three empty test tubes shall be prepared for the negative
control (blood in test tubes without test materials).
9.2 Thirty-six square centimeterscentimetres of the test sample, positive and reference sample and each of the controls are
divided into three 12 cm samples, cut to maximize blood exposure, for triplicate testing.
NOTE 2—If this surface area cannot be achieved with the device, or other volumes of blood are used, the ratio of total surface area to blood volume
should remain at 12:1.12 cm :1 mL.
U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, and National Institutes of Health, “Section III—Principles of Biosafety,”
Biosafety in Microbiological and Biomedical Laboratories (BMBL), L. C. Chosewood and D. E. Wilson, eds. 2009.
U.S. Department of Labor, “Occupational Exposure to Bloodborne Pathogens,” Final Rule, 29 CFR, 1910.1030, 1991.
F2888 − 19
9.3 For each test sample, the percentage of test value (platelet count or leukocyte count) per negative control (blood not 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 negative control, and
C = percentage (%) of negative control.
When applicable, a comparison article should be used in the formula in place of the negative control.
10. Preparation of Apparatus
10.1 Initialize the hematology analyzer and allow it to perform internal self-checks. If no errors are noted, the analyzer is ready
for use.
10.2 To verify the analyzer is functioning properly, prior to analyzing samples, cell count controls shall be run to
conformconfirm that the results fall within the allowable ranges.
10.3 Fresh human blood is drawn from at least 3 donors should be used for the test system. to account for blood variability
among donors. Testing with blood from each donor separately or testing with pooled blood is acceptable. Blood should be from
donors who have not taken aspirin, acetaminophen, naproxen, warfarin, heparin, or ibuprofen for ten days. nonsteroidal,
anti-inflammatory drugs (NSAIDs; for example, ibuprofen, aspirin, naproxen), acetaminophen, or antithrombotic drugs (for
example, heparin, warfarin) for ten days prior to blood collection. Blood should be collected in a tube containing 3.2 %, 0.105 M
sodium citrate (at a ratio of 9:1 v/v blood to sodium citrate as per commercial blood collection tubes), pooled, ), gently mixed by
inversion, and stored on ice until use.at room temperature until use. Alternatively, blood should be collected in a tube containing
a low concentration of heparin (final concentration approximately 1 unit/mL). Other blood collection tubes/anticoagulant
concentrations may be used if validated for use in this practice, using two or more moderately thrombogenic positive control
materials (for example, black rubber, natural rubber latex, and glass) for the validation.
NOTE 3—Blood from an individual donor could be used directly to perform the test, as long as the test is repeated separately with blood collected from
2 additional donors. Alternatively, blood from 3 donors may be pooled for analyses, but in this case, blood type mismatch between donors should be
avoided. The test report should indicate the type and concentration of anticoagulant used, and whether the blood samples were pooled or not.
10.4 For each analysis, a single donor’s blood (or pooled blood, if applicable) will be exposed to the test sample, reference
material, negative control, and positive control negative control reference material, positive control material, and marketed
comparator device (when applicable) to provide a consistent test system to evaluate all reference and test materials.evaluate their
impact on platelet and leukocyte counts. It is recommended to pre-screen the blood to ensure the blood parameters fall within the
3 3
normal expected range (normal leukocyte count 3.4 to 8.37 × 10 /uL,/μL, normal platelet count 116 to 329 × 10 /uL)./μL). If the
donor blood parameters fall outside of the normal expected ranges, blood from another donor should be used.
11. Calibration and Standardization
11.1 Perform daily calibration procedures as per in accordance with instrument instructions. (Typically the instrument
self-calib
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