1、Designation: E2524 08 (Reapproved 2013)Standard Test Method forAnalysis of Hemolytic Properties of Nanoparticles1This standard is issued under the fixed designation E2524; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of
2、 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 assessing the effect of nanopar-ticulate materials on the integrity of red blood cells.1.2 Th
3、is test method 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 F756 with thevolumes reduced to accommodate nanoparticulate material.1.4 This test method is part of the in
4、-vitro preclinicalcharacterization and is important for nanoparticulate materialthat will contact the blood in medical applications.1.5 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.6 This standard does not purport to addres
5、s all of thesafety 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.2. Referenced Documents2.1 ASTM Standards:2F748 Practice fo
6、r Selecting Generic Biological Test Methodsfor Materials and DevicesF756 Practice for Assessment of Hemolytic Properties ofMaterialsF1877 Practice for Characterization of ParticlesF1903 Practice for Testing For Biological Responses toParticles In Vitro2.2 ISO Standard:3ISO 10993-4 Biological Evaluat
7、ion of Medical Devices Part4: Selection of Tests for Interactions with Blood3. 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
8、 blood hemoglobin3.1.8 TBHdblood sample diluted to 10 mg 6 1 mg/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
9、-nation of hemoglobin released into PFH as a percentage of theTBH 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
10、. Astable CMH concentration is measured using a plate readerspectrophotometer 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) conce
11、ntrations to monitorassay performance. The required sample volume 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 inPracti
12、ce F748 and ISO 10993-4 to assess the biocompatibilityof materials contacting blood in medical applications.1This test method is under the jurisdiction of ASTM Committee E56 onNanotechnology and is the direct responsibility of Subcommittee E56.03 onEnvironment, Health, and Safety.Current edition app
13、roved Sept. 1, 2013. Published September 2013. Originallyapproved in 2008. Last previous edition approved in 2008 as E2524 08. DOI:10.1520/E2524-08R13.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStan
14、dards 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 Standardization in Haemotology, Journal ofClinical Pat
15、hology, Vol 31, 1978, pp. 139143.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States15.2 This test method is similar to Practice F756 but modifiedto accommodate nanoparticulate materials.6. Apparatus6.1 Pipettes covering range from 0.05 t
16、o 10 mL.6.2 Ninety-six well plates.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 beakers.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 chemical
17、s shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are available.5Other grades may be used,provided it is first ascertained that the re
18、agent is of sufficientlyhigh purity to permit its use without lessening the accuracy ofthe determination.7.2 CMH reagent.7.3 Hemoglobin standard.7.4 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, av
19、erage MW 8000.7.8 Distilled water.NOTE 1The source of the reagents is shown for information purposesonly to aid laboratories initiating this procedure. Equivalent reagents fromother suppliers may be used.8. Preparation of Standards and ControlsNOTE 2Aseptic precautions are not needed, but contaminat
20、ion ofreagents to be stored shall be avoided.8.1 Preparation of Calibration StandardsVolumes can beadjusted based on the need (see Table 1).8.2 Preparation of Quality ControlsVolumes can be ad-justed based on the need (see Table 2).8.3 Preparation of Positive ControlsDissolve poly-L-lysine hydrobrom
21、ide 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 months. 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 % st
22、ock solution in water. Use this solution as the negativecontrol. Store the stock solution at 4 6 3C.8.5 Preparation of Nanoparticulate Test Samples:8.5.1 For the initial dose, use the highest concentration ofnanoparticles that is well dispersed in a physiologic solution. Ifthe concentration in the e
23、nd use application is known, that maybe used as the starting concentration. The material shall be wellcharacterized under physiological conditions according tostandard methods including those recommended in PracticesF1877 and F1903. The nanoparticulate material for testingshall be in physiologic sol
24、ution 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 of particles/mL andmg/mL shall be indicated. Prior characterization shall beperformed as appropriate to allow adequate data interpretation.For example, lot
25、-to-lot variations in particle size and surfacecharacteristics of the particles could result in different assayresults. If the particles suspension is sterile, the method ofsterilization shall be indicated. The nanoparticulate materialand the buffer used for its storage/reconstitution shall be teste
26、din the same assay. The assay requires at least 300 mL of testmaterial and enough for diluting. The starting suspension shallbe diluted in DPBS with 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 att
27、he 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 to determine if high-speed centrifuga-tion will pellet the particles and remove the interference. Whencentrifugation is not applicable, an assay result o
28、btained for aparticle incubated with blood is adjusted by subtracting resultobtained for the same particle in “minus blood” control (see10.4).8.6 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 bestore
29、d at 2 to 8C for up to 48 h. On the day of assay, preparepooled blood by mixing equal proportions of blood from each5Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For suggestions on the testing of reagents notlisted by the American Chemical So
30、ciety, see Analar Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.TABLE 1 Calibration StandardsLevelNominalConc.,mg/mLPreparation ProcedureCal 1 0.8 2 mL of stock solu
31、tionCal2 0.4 1mLofCal1+1mLofCMHreagentCal3 0.2 1mLofCal2+1mLofCMHreagentCal4 0.1 1mLofCal3+1mLofCMHreagentCal5 0.05 1mLofCal4+1mLofCMHreagentCal6 0.025 1mLofCal5+1mLofCMHreagentTABLE 2 Quality ControlsLevelNominalConc.,mg/mLPreparation ProcedureQC 1 0.625 1.5 mL of stock solution + 0.42 mL of CMH re
32、agentQC 2 0.125 200 L of QC1 + 800 L of CMH reagentQC 3 0.0625 100 L of QC1 + 900 L of CMH reagentE2524 08 (2013)2donor. If microaggregates of blood 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 Colle
33、ct the supernatant. Keep at room temperaturewhile preparing the standard curve, quality controls, and totalhemoglobin sample. The collected supernatant (plasma) is usedto determine PFH.9. Determination of PFH and TBH in Native BloodSample9.1 Add 200.0 L of each calibration standard, qualitycontrol,
34、and blank CMH reagent per well on 96-well plate. Usetwo wells for each calibrator and four wells for each QC andthe blank so that test samples are bracketed 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 comb
35、ining 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 wells 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 C
36、over 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 thehemoglobin concentration. Remember to use a dilution factorof 2 for the PFH sample and a dilution factor of 251 for TBH.If the calculated PFH concen
37、tration 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 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
38、L of sample, reagent blank (PBS or otherbuffer used to dissolve nanoparticles), positive control solution,and negative control solution to microcentrifuge tubes. Preparesix tubes for each unknown sample, three tubes for the blank,two tubes for the positive control, and two tubes for thenegative cont
39、rol.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 test sample. In thesetubes, add 100 L of Ca2+/Mg2+-free DPBS. These samplesrepresent a “minus blood” control and are used to evaluatepotential inter
40、ference of nanomaterial with the assay (forexample, absorbance at or close to 540 nm, reactivity withCMH reagent, and so forth).10.5 Cover tubes and gently rotate to mix. At each 30-mininterval, observe whether the sample of nanoparticlesflocculate, disperse, sink, or float during testing.NOTE 3Vort
41、exing 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 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
42、 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 complete, examine the tubesand record any unusual appearance of the supernatant or pelletthat can indicate additional damage to the red blood cells or the
43、hemoglobin or adsorption of hemoglobin to the particles. SeeFig. 1.10.10 In Fig. 1, commercially available preparations ofpolystyrene nanoparticles with size 20 nm (Tube 1) and 50 nm(Tube 2) demonstrated hemolytic activity that can be observedby the color of the supernatant. Polystyrene nanoparticle
44、s withsize 80 nm were also hemolytic; however, they adsorbedFIG. 1 Example Demonstrating the Importance of Recording Sample Appearance After Centrifugation to Avoid False Negative ResultsE2524 08 (2013)3hemoglobin as can be determined by the pellet size and color(Tube 3). If the supernatant of this
45、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 have an a
46、bsorbance 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 Prepar
47、e 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 eachbl
48、ank and each quality control, and six wells for the TBHdsample. As before, position all test samples bracketed betweenquality controls on the plate.11.3 Add 100.0 L per well of test samples, positive andnegative controls, prepared in 10.9. Fill six wells for each testsample (two wells from each of t
49、hree 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 and 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 dilut
