1、Designation: E 2524 08Standard Test Method forAnalysis of Hemolytic Properties of Nanoparticles1This standard is issued under the fixed designation E 2524; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision.
2、A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers assessing the effect of nanopar-ticulate materials on the integrity of red blood cells.1.2 This test method
3、uses diluted whole blood incubatedwith nanoparticulate material and the hemoglobin releasedfrom damaged red blood cells is determined.1.3 This test method is similar to Practice F 756 with thevolumes reduced to accommodate nanoparticulate material.1.4 This test method is part of the in-vitro preclin
4、icalcharacterization and is important for nanoparticulate materialthat will contact the blood in medical applications.1.5 The values given in SI units are to be considered as thestandard.1.6 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is t
5、heresponsibility 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 ASTM Standards:2F 748 Practice for Selecting Generic Biological Test Meth-ods for Materials and Devic
6、esF 756 Practice for Assessment of Hemolytic Properties ofMaterialsF 1877 Practice for Characterization of ParticlesF 1903 Practice for Testing For Biological Responses toParticles in vitro2.2 ISO Standard:ISO 10993-4 Biological Evaluation of Medical Devices Part4: Selection of Tests for Interaction
7、s with Blood33. Terminology3.1 Acronyms:3.1.1 Calcalibration standard3.1.2 CMHcyanmethemoglobin3.1.3 DPBSDulbeccos phosphate-buffered saline3.1.4 PEGpolyethylene glycol3.1.5 PFHplasma-free hemoglobin3.1.6 QCquality controls3.1.7 TBHtotal blood hemoglobin3.1.8 TBHdblood sample diluted to 10 mg 6 1 mg
8、/mL4. Summary of Test Method4.1 This test method describes a protocol for assessing acutein-vitro damage to red blood cells (that is, hemolysis) causedby exposure to nanoparticles.4.2 This test method is based on the quantitative determi-nation of hemoglobin released into PFH as a percentage of theT
9、BH concentration when blood is exposed to nanoparticulatematerials.4.3 Using an established colorimetric assay,4hemoglobinand its derivatives, such as sulfhemoglobin, are oxidized tomethemoglobin by ferricyanide in the presence of alkali. Astable CMH concentration is measured using a plate readerspe
10、ctrophotometer set at 540 nm.4.4 Hemoglobin standards are used to create a standardcurve covering the range from 0.025 to 0.8 mg/mL and preparequality control samples at low (0.0625-mg/mL), mid (0.125-mg/mL), and high (0.625-mg/mL) concentrations to monitorassay performance. The required sample volu
11、me is 100 L pertest replicate.4.5 The results are expressed as percent hemolysis toevaluate the acute in-vitro hemolytic properties of nanopar-ticles.5. Significance and Use5.1 This test method is one of a series of tests listed inPractice F 748 and ISO 10993-4 to assess the biocompatibilityof mater
12、ials contacting blood in medical applications.5.2 This test method is similar to Practice F 756 butmodified to accommodate nanoparticulate materials.6. Apparatus6.1 Pipettes covering range from 0.05 to 10 mL.6.2 Ninety-six well plates.1This test method is under the jurisdiction of ASTM Committee E56
13、 onNanotechnology and is the direct responsibility of Subcommittee E56.02 onCharacterization: Physical, Chemical, and Toxicological Properties.Current edition approved Feb. 1, 2008. Published April 2008.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Ser
14、vice at serviceastm.org. For Annual 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.4International Committee for St
15、andardization in Haemotology, J. Clin. Pathol.Vol 31, 1978, pp. 139-143.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.6.3 Water bath set at 37 6 1C.6.4 Plate reader capable of measuring at 540 nm.6.5 Plate shaker.6.6 Plastic beaker
16、s.6.7 Microcentrifuge tubes, 1.5 mL, translucent, not colored.6.8 Centrifuge set at 700 to 800 g.7. Reagents7.1 Purity of ReagentsReagent-grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents conform to the specifications of the Committee onAnalytical
17、 Reagents of the American Chemical Society wheresuch specifications are available.5Other grades may be used,provided it is first ascertained that the reagent is of sufficientlyhigh purity to permit its use without lessening the accuracy ofthe determination.7.2 CMH reagent.7.3 Hemoglobin standard.7.4
18、 Ca2+/Mg2+-free DPBS.7.5 Pooled normal human whole blood anticoagulated withLi-heparin.7.6 Poly-L-lysine hydrobromide, MW 150 000 to 300 000.7.7 PEG, average MW 8000.7.8 Distilled water.NOTE 1The source of the reagents is shown for information purposesonly to aid laboratories initiating this procedu
19、re. Equivalent reagents fromother suppliers may be used.8. Preparation of Standards and ControlsNOTE 2Aseptic precautions are not needed, but contamination ofreagents to be stored shall be avoided.8.1 Preparation of Calibration StandardsVolumes can beadjusted based on the need (see Table 1).8.2 Prep
20、aration of Quality ControlsVolumes can be ad-justed based on the need (see Table 2).8.3 Preparation of Positive ControlsDissolve poly-L-lysine hydrobromide powder to a final concentration of 1 %(10 mg/mL) in sterile distilled water. Prepare aliquots for dailyuse and store at 20 6 3C for up to 2 mont
21、hs. Alternatively,a 10 % solution of Triton-X 100 in water can be used as apositive control.8.4 Preparation of Negative ControlPEG is supplied as40 % stock solution in water. Use this solution as the negativecontrol. Store the stock solution at 4 6 3C.8.5 Preparation of Nanoparticulate Test Samples:
22、8.5.1 For the initial dose, use the highest concentration ofnanoparticles that is well dispersed in a physiologic solution. Ifthe concentration in the end use application is known, that maybe used as the starting concentration. The material shall be wellcharacterized under physiological conditions a
23、ccording tostandard methods including those recommended in PracticesF 1877 and F 1903. The nanoparticulate material for testingshall be in physiologic solution which is isotonic with a pH of7.2 6 2. This solution shall be defined and the particles shall becharacterized in this solution. The number o
24、f particles/mL andmg/mL shall be indicated. Prior characterization shall beperformed as appropriate to allow adequate data interpretation.For example, lot-to-lot variations in particle size and surfacecharacteristics of the particles could result in different assayresults. If the particles suspensio
25、n is sterile, the method ofsterilization shall be indicated. The nanoparticulate materialand the buffer used for its storage/reconstitution shall be testedin the same assay. The assay requires at least 300 mL of testmaterial and enough for diluting. The starting suspension shallbe diluted in DPBS wi
26、th serial one to five (1:5) dilutions atleast three times to give four test samples in the assay.8.5.2 Since some nanoparticulate materials may absorb atthe designated 540-nm wavelength, it is suggested that a trialof the material in DPBS be tested at 540 nm. If absorption isevident, it is advisable
27、 to determine if high-speed centrifuga-tion will pellet the particles and remove the interference. Whencentrifugation is not applicable, an assay result obtained for aparticle incubated with blood is adjusted by subtracting resultobtained for the same particle in “minus blood” control (see10.4).8.6
28、Preparation of Blood Sample:8.6.1 Collect whole blood in tubes containing Li-heparin asthe anticoagulant from at least three donors. The blood can bestored at 2 to 8C for up to 48 h. On the day of assay, preparepooled blood by mixing equal proportions of blood from eachdonor. If microaggregates of b
29、lood are observed, filter theblood through a 40-m blood filter.8.6.2 Take a 2- to 3-mL aliquot of the pooled blood andcentrifuge 15 min at 800 g.8.6.3 Collect the supernatant. Keep at room temperaturewhile preparing the standard curve, quality controls, and totalhemoglobin sample. The collected supe
30、rnatant (plasma) is usedto determine PFH.9. Determination of PFH and TBH in Native BloodSample9.1 Add 200.0 L of each calibration standard, qualitycontrol, and blank CMH reagent per well on 96-well plate. Usetwo wells for each calibrator and four wells for each QC and5Reagent Chemicals, American Che
31、mical Society Specifications, AmericanChemical Society, Washington, DC. For suggestions on the testing of reagents notlisted by the American Chemical Society, see Analar Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. P
32、harmacopeial Convention, Inc. (USPC), Rockville,MD.TABLE 1 Calibration StandardsLevelNominal Conc.,mg/mLPreparation ProcedureCal 1 0.8 2 mL of stock solutionCal2 0.4 1mLofCal1+1mLofCMHreagentCal3 0.2 1mLofCal2+1mLofCMHreagentCal4 0.1 1mLofCal3+1mLofCMHreagentCal5 0.05 1mLofCal4+1mLofCMHreagentCal6 0
33、.025 1mLofCal5+1mLofCMHreagentTABLE 2 Quality ControlsLevelNominal Conc.,mg/mLPreparation ProcedureQC 1 0.625 1.5 mL of stock solution + 0.42 mL of CMH reagentQC 2 0.125 200 L of QC1 + 800 L of CMH reagentQC 3 0.0625 100 L of QC1 + 900 L of CMH reagentE2524082the blank so that test samples are brack
34、eted by QCs; forexample, a sequence such as (blank, Cal1, Cal6, QC1, QC2,QC3, test samples, blank, QC1, QC2, QC3).9.2 Prepare the TBH sample by combining 20.0 L of thepooled whole blood and 5.0 mL of CMH reagent.After 15 min,add 200.0 L to each of six wells.9.3 Add 100.0 L of plasma (8.6.3) to six w
35、ells and add100.0 L of CMH reagent to each of these wells. Do not addCMH reagent to wells containing calibration standards andquality controls.9.4 Cover the plate with the plate sealer and gently shake ona plate shaker (medium speed settings 2 to 3).9.5 Read the absorbance at 540 nm to determine the
36、hemoglobin concentration. Remember to use a dilution factorof 2 for the PFH sample and a dilution factor of 251 for TBH.If the calculated PFH concentration is below 1 mg/mL, proceedto the next step. If it is above 1 mg/mL, the blood sample is notsuitable for the procedure.10. Procedure with the Test
37、 Material10.1 Dilute pooled whole blood with Ca2+/Mg2+-free DPBSto adjust TBH concentration to 10 6 1 mg/mL based on resultsfrom 9.5.10.2 Add 100.0 L of sample, reagent blank (PBS or otherbuffer used to dissolve nanoparticles), positive control solution,and negative control solution to microcentrifu
38、ge tubes. Preparesix tubes for each unknown sample, three tubes for the blank,two tubes for the positive control, and two tubes for thenegative control.10.3 Add 700.0 L of Ca2+/Mg2+-free DPBS to each tube.10.4 Add 100 L of the whole blood prepared in 10.1 toeach tube, except for three tubes of each
39、test sample. In thesetubes, add 100 L of Ca2+/Mg2+-free DPBS. These samplesrepresent a “minus blood” control and are used to evaluatepotential interference of nanomaterial with the assay (forexample, absorbance at or close to 540 nm, reactivity withCMH reagent, and so forth).10.5 Cover tubes and gen
40、tly rotate to mix. At each 30-mininterval, observe whether the sample of nanoparticles floccu-late, disperse, sink, or float during testing.NOTE 3Vortexing may damage erythrocytes and shall be avoided.10.6 Place the tubes in a water bath set at 37 6 1C andincubate for 3 h 6 15 min mixing the sample
41、by rotation every30 min.Alternatively, tubes may be incubated on a tube rotatorin an incubator set at 37 6 1C.10.7 Remove the tubes from water bath or incubator. If awater bath was used, dry the tubes with absorbent paper.10.8 Centrifuge the tubes for 15 min at 800 g.10.9 When centrifugation is comp
42、lete, examine the tubesand record any unusual appearance of the supernatant or pelletthat can indicate additional damage to the red blood cells or thehemoglobin or adsorption of hemoglobin to the particles. SeeFig. 1.10.10 In Fig. 1, commercially available preparations ofpolystyrene nanoparticles wi
43、th size 20 nm (Tube 1) and 50 nm(Tube 2) demonstrated hemolytic activity that can be observedby the color of the supernatant. Polystyrene nanoparticles withsize 80 nm were also hemolytic; however, they adsorbedhemoglobin as can be determined by the pellet size and color(Tube 3). If the supernatant o
44、f this sample is used in the assay,the absorbance at 540 nm will demonstrate a negative result.Sample #4 is the negative control. No hemolytic activity wasobserved in the supernatant, and the intact red blood cellsformed a tight dark red pellet on the bottom of the tube.10.11 If the nanoparticles ha
45、ve an absorbance at or close to540 nm, removal of these particles from the supernatant will berequired before proceeding to the next step (see 8.5). Thesesupernatants shall be transferred to fresh tubes and centrifuged30 min at 40 000 rpm or speed determined in 8.5.11. Determination of Hemolysis11.1
46、 Prepare fresh set of calibrators and quality controls asin 8.1 and 8.2.11.2 To a fresh 96-well plate, add 200.0 L of blank CMH,calibrators, quality controls, or diluted TBHd prepared bycombining 400.0 L of blood from 10.1 with 5.0 mL of CMHreagent. Fill two wells for each calibrator, four wells for
47、 eachblank and each quality control, and six wells for the TBHdsample. As before, position all test samples bracketed betweenquality controls on the plate.FIG. 1 Example Demonstrating the Importance of Recording Sample Appearance After Centrifugation to Avoid False Negative ResultsE252408311.3 Add 1
48、00.0 L per well of test samples, positive andnegative controls, prepared in 10.9. Fill six wells for each testsample (two wells from each of three tubes prepared in 10.2)and four wells for each control (two wells from each of twotubes).11.4 Add 100.0 L of CMH reagent to each well containingsample an
49、d controls. Do not add CMH reagent to wellscontaining calibration standards, quality controls, and TBHdthat already contain CMH.11.5 Cover the plate with plate sealer and gently shake on aplate shaker (medium speed settings 2 to 3).11.6 Read the absorbance at 540 nm to determine theconcentration of hemoglobin. Remember to use the dilutionfactor 18 for samples and controls and dilution factor 13.5 forTBHd.12. Calculation12.1 A four-parameter regression algorithm is used to buildthe calibration curve. The following parameters shall becalculated
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