ASTM E2524-08

Standard Test Method for Analysis of Hemolytic Properties of Nanoparticles

SIGNIFICANCE AND USE
p>This test method is one of a series of tests listed in Practice F 748 and ISO 10993-4 to assess the biocompatibility of materials contacting blood in medical applications.
This test method is similar to Practice F 756 but modified to accommodate nanoparticulate materials.
SCOPE
1.1 This test method covers assessing the effect of nanoparticulate materials on the integrity of red blood cells.
1.2 This test method uses diluted whole blood incubated with nanoparticulate material and the hemoglobin released from damaged red blood cells is determined.
1.3 This test method is similar to Practice F 756 with the volumes reduced to accommodate nanoparticulate material.
1.4 This test method is part of the in-vitro preclinical characterization and is important for nanoparticulate material that will contact the blood in medical applications.
1.5 The values given in SI units are to be considered as the standard.
1.6 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 and health practices and determine the applicability of regulatory limitations prior to use.

General Information

• Status: Historical
• Publication Date: 31-Jan-2008
• ICS: 11.040.20 - Transfusion, infusion and injection equipment
• ICS: 71.100.01 - Products of the chemical industry in general
• Technical Committee: E56 - Nanotechnology
• Drafting Committee: E56.03 - Environment, Health, and Safety

Relations

• Referred By: ASTM F756-17 - Standard Practice for Assessment of Hemolytic Properties of Materials
• Effective Date: 01-Feb-2008

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E2524 − 08
StandardTest Method for
Analysis of Hemolytic Properties of Nanoparticles
This standard is issued under the fixed designation E2524; 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 3. Terminology
1.1 This test method covers assessing the effect of nanopar- 3.1 Acronyms:
ticulate materials on the integrity of red blood cells. 3.1.1 Cal—calibration standard
3.1.2 CMH—cyanmethemoglobin
1.2 This test method uses diluted whole blood incubated
with nanoparticulate material and the hemoglobin released
3.1.3 DPBS—Dulbecco’s phosphate-buffered saline
from damaged red blood cells is determined.
3.1.4 PEG—polyethylene glycol
1.3 This test method is similar to Practice F756 with the
3.1.5 PFH—plasma-free hemoglobin
volumes reduced to accommodate nanoparticulate material.
3.1.6 QC—quality controls
1.4 This test method is part of the in-vitro preclinical
3.1.7 TBH—total blood hemoglobin
characterization and is important for nanoparticulate material
3.1.8 TBHd—blood sample diluted to 10 mg 6 1 mg/mL
that will contact the blood in medical applications.
1.5 The values given in SI units are to be considered as the 4. Summary of Test Method
standard.
4.1 Thistestmethoddescribesaprotocolforassessingacute
1.6 This standard does not purport to address all of the
in-vitro damage to red blood cells (that is, hemolysis) caused
safety concerns, if any, associated with its use. It is the
by exposure to nanoparticles.
responsibility of the user of this standard to establish appro-
4.2 This test method is based on the quantitative determi-
priate safety and health practices and determine the applica-
nation of hemoglobin released into PFH as a percentage of the
bility of regulatory limitations prior to use.
TBH concentration when blood is exposed to nanoparticulate
materials.
2. Referenced Documents
4.3 Using an established colorimetric assay, hemoglobin
2.1 ASTM Standards:
and its derivatives, such as sulfhemoglobin, are oxidized to
F748 PracticeforSelectingGenericBiologicalTestMethods
methemoglobin by ferricyanide in the presence of alkali. A
for Materials and Devices
stable CMH concentration is measured using a plate reader
F756 Practice for Assessment of Hemolytic Properties of
spectrophotometer set at 540 nm.
Materials
4.4 Hemoglobin standards are used to create a standard
F1877 Practice for Characterization of Particles
curve covering the range from 0.025 to 0.8 mg/mLand prepare
F1903 Practice for Testing For Biological Responses to
quality control samples at low (0.0625-mg/mL), mid (0.125-
Particles In Vitro
mg/mL), and high (0.625-mg/mL) concentrations to monitor
2.2 ISO Standard:
assay performance. The required sample volume is 100 µLper
ISO 10993-4 Biological Evaluation of Medical Devices Part
test replicate.
4: Selection of Tests for Interactions with Blood
4.5 The results are expressed as percent hemolysis to
evaluate the acute in-vitro hemolytic properties of nanopar-
This test method is under the jurisdiction of ASTM Committee E56 on ticles.
Nanotechnology and is the direct responsibility of Subcommittee E56.03 on
Environment, Health, and Safety.
5. Significance and Use
Current edition approved Feb. 1, 2008. Published April 2008. DOI: 10.1520/
5.1 This test method is one of a series of tests listed in
E2524-08.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or Practice F748 and ISO 10993-4 to assess the biocompatibility
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
of materials contacting blood in medical applications.
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St., International Committee for Standardization in Haemotology, J. Clin. Pathol.
4th Floor, New York, NY 10036, http://www.ansi.org. Vol 31, 1978, pp. 139-143.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2524 − 08
TABLE 2 Quality Controls
5.2 ThistestmethodissimilartoPracticeF756butmodified
to accommodate nanoparticulate materials. Nominal
Level Conc., Preparation Procedure
mg/mL
6. Apparatus
QC 1 0.625 1.5 mL of stock solution + 0.42 mL of CMH reagent
6.1 Pipettes covering range from 0.05 to 10 mL.
QC 2 0.125 200 µL of QC1 + 800 µL of CMH reagent
QC 3 0.0625 100 µL of QC1 + 900 µL of CMH reagent
6.2 Ninety-six well plates.
6.3 Water bath set at 37 6 1°C.
8.1 Preparation of Calibration Standards—Volumes can be
6.4 Plate reader capable of measuring at 540 nm.
adjusted based on the need (see Table 1).
6.5 Plate shaker.
8.2 Preparation of Quality Controls—Volumes can be ad-
6.6 Plastic beakers.
justed based on the need (see Table 2).
6.7 Microcentrifuge tubes, 1.5 mL, translucent, not colored.
8.3 Preparation of Positive Controls—Dissolve poly-L-
6.8 Centrifuge set at 700 to 800 g.
lysine hydrobromide powder to a final concentration of 1 %
(10 mg/mL) in sterile distilled water. Prepare aliquots for daily
7. Reagents
use and store at –20 6 3°C for up to 2 months. Alternatively,
7.1 Purity of Reagents—Reagent-grade chemicals shall be a 10 % solution of Triton-X 100 in water can be used as a
used in all tests. Unless otherwise indicated, it is intended that
positive control.
all reagents conform to the specifications of the Committee on
8.4 Preparation of Negative Control—PEG is supplied as
Analytical Reagents of the American Chemical Society where
40 % stock solution in water. Use this solution as the negative
such specifications are available. Other grades may be used,
control. Store the stock solution at 4 6 3°C.
provided it is first ascertained that the reagent is of sufficiently
8.5 Preparation of Nanoparticulate Test Samples:
high purity to permit its use without lessening the accuracy of
8.5.1 For the initial dose, use the highest concentration of
the determination.
nanoparticlesthatiswelldispersedinaphysiologicsolution.If
7.2 CMH reagent.
the concentration in the end use application is known, that may
7.3 Hemoglobin standard. beusedasthestartingconcentration.Thematerialshallbewell
2+ 2+
characterized under physiological conditions according to
7.4 Ca /Mg -free DPBS.
standard methods including those recommended in Practices
7.5 Pooled normal human whole blood anticoagulated with
F1877 and F1903. The nanoparticulate material for testing
Li-heparin.
shall be in physiologic solution which is isotonic with a pH of
7.6 Poly-L-lysine hydrobromide, MW 150 000 to 300 000. 7.2 62.Thissolutionshallbedefinedandtheparticlesshallbe
characterized in this solution. The number of particles/mL and
7.7 PEG, average MW 8000.
mg/mL shall be indicated. Prior characterization shall be
7.8 Distilled water.
performed as appropriate to allow adequate data interpretation.
NOTE 1—The source of the reagents is shown for information purposes
For example, lot-to-lot variations in particle size and surface
only to aid laboratories initiating this procedure. Equivalent reagents from
characteristics of the particles could result in different assay
other suppliers may be used.
results. If the particles suspension is sterile, the method of
8. Preparation of Standards and Controls sterilization shall be indicated. The nanoparticulate material
NOTE 2—Aseptic precautions are not needed, but contamination of
and the buffer used for its storage/reconstitution shall be tested
reagents to be stored shall be avoided.
in the same assay. The assay requires at least 300 mL of test
material and enough for diluting. The starting suspension shall
be diluted in DPBS with serial one to five (1:5) dilutions at
least three times to give four test samples in the assay.
Reagent Chemicals, American Chemical Society Specifications, American
8.5.2 Since some nanoparticulate materials may absorb at
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
listed by the American Chemical Society, see Analar Standards for Laboratory
the designated 540-nm wavelength, it is suggested that a trial
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
of the material in DPBS be tested at 540 nm. If absorption is
and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,
evident, it is advisable to determine if high-speed centrifuga-
MD.
tion will pellet the particles and remove the interference.When
centrifugation is not applicable, an assay result obtained for a
TABLE 1 Calibration Standards
particle incubated with blood is adjusted by subtracting result
Nominal
obtained for the same particle in “minus blood” control (see
Level Conc., Preparation Procedure
mg/mL
10.4).
Cal 1 0.8 2 mL of stock solution
8.6 Preparation of Blood Sample:
Cal2 0.4 1mLofCal1+1mLofCMHreagent
Cal3 0.2 1mLofCal2+1mLofCMHreagent 8.6.1 Collect whole blood in tubes containing Li-heparin as
Cal4 0.1 1mLofCal3+1mLofCMHreagent
the anticoagulant from at least three donors. The blood can be
Cal5 0.05 1mLofCal4+1mLofCMHreagent
stored at 2 to 8°C for up to 48 h. On the day of assay, prepare
Cal6 0.025 1mLofCal5+1mLofCMHreagent
pooled blood by mixing equal proportions of blood from each
E2524 − 08
donor. If microaggregates of blood
...