1、Designation: F1984 99 (Reapproved 2018)Standard Practice forTesting for Whole Complement Activation in Serum by SolidMaterials1This standard is issued under the fixed designation F1984; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revisio
2、n, 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 practice provides a protocol for rapid, in vitroscreening for whole complement activating properti
3、es of solidmaterials used in the fabrication of medical devices that willcontact blood.1.2 This practice is intended to evaluate the acute in vitrowhole complement activating properties of solid materialsintended for use in contact with blood. For this practice, thewords “serum” and “complement” are
4、 used interchangeably(most biological supply houses use these words synonymouslyin reference to serum used as a source of complement).1.3 This practice consists of two procedural parts. Proce-dureAdescribes exposure of solid materials to a standard lot ofhuman serum, using a 0.1-mL serum/13 x 100-mm
5、 disposabletest tube. Cellulose acetate powders and fibers are used asexamples of test materials. Procedure B describes assaying theexposed serum for significant functional whole complementdepletion as compared to control samples.1.4 This practice does not address function, elaboration, ordepletion
6、of individual complement components, nor does itaddress the use of plasma as a source of complement.1.5 This practice is one of several developed for theassessment of the biocompatibility of materials. Practice F748may provide guidance for the selection of appropriate methodsfor testing materials fo
7、r other aspects of biocompatibility.1.6 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.7 This international standard was developed in accor-dance with internationally recognized principles on standard-ization established in t
8、he Decision on Principles for theDevelopment of International Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2F748 Practice for Selecting Generic Biological Test Methodsfor Materials a
9、nd Devices2.2 ISO Document:ISO 10993-4: Biological Evaluation of Medical Devices,Part 4: Selection of Tests for Interactions with Blood33. Terminology3.1 Abbreviations:3.1.1 Abantibody (hemolysin).3.1.2 BBSbarbital buffered saline.3.1.3 BBS-Gbarbital buffered salinegelatin.3.1.4 BBS-GMbarbital buffe
10、red salinegelatin metals.3.1.5 Ccomplement.3.1.6 EDTAethylenediaminetetraacetic acid, disodiumsalt: dihydrate.3.1.7 HShuman serum.3.1.8 PVDFpolyvinylidene fluoride.3.1.9 RBCred blood cell(s).4. Summary of Practice4.1 Solid material specimens are exposed to contact with astandard lot of complement un
11、der defined conditions (Proce-dureA). Exposed serum then is tested for significant functionalcomplement depletion compared to controls under identicalconditions (Procedure B).5. Significance and Use5.1 Inappropriate activation of complement by blood-contacting medical devices may have serious acute
12、or chroniceffects on the host. This practice is useful as a simple,inexpensive screening method for determining functionalwhole complement activation by solid materials in vitro.1This practice is under the jurisdiction ofASTM Committee F04 on Medical andSurgical Materials and Devices and is the dire
13、ct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition approved Feb. 1, 2018. Published April 2018. Originallyapproved in 1999. Last previous edition approved in 2013 as F1984 99 (2013).DOI: 10.1520/F1984-99R18.2For referenced ASTM standards, visit the ASTM website,
14、 www.astm.org, orcontact ASTM Customer Service 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:/ww
15、w.ansi.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopme
16、nt of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.15.2 This practice is composed of two parts. In Part A(Section 11), human serum is exposed to a solid material.Complement may be depleted by the classical or a
17、lternativepathways. In principle, nonspecific binding of certain comple-ment components also may occur. The alternative pathway candeplete later acting components common to both pathways,that is components other than C1, C4, and C3 (1).4In Part B(Section 12), complement activity remaining in the ser
18、um afterexposure to the test material is assayed by classical pathway-mediated lysis of sensitized RBC.5.3 Assessment of in vitro whole complement activation, asdescribed here, provides one method for predicting potentialcomplement activation by medical materials intended forclinical application in
19、humans when the material contacts theblood. Other test methods for complement activation areavailable, including assays for specific complement compo-nents and their split products (see X1.3 and X1.4).5.4 This in vitro test method is suitable for adoption inspecifications and standards for screening
20、 solid materials foruse in the construction of medical devices intended to beimplanted in the human body or placed in contact with humanblood.6. Preparation of Buffers6.1 Buffers, are prepared according to detailed protocol(2).“Water” refers throughout to distilled, endotoxin-free water.The use of b
21、arbital (veronal) buffer is recommended. Barbitalis a class IVregulated substance and requires a DEA (3) licensefor purchase. The use of other buffer systems, such as, TRIS, ispermissible if they have been demonstrated not to activatecomplement(4).6.2 5X Stock BBS (barbital-buffered saline), is prep
22、ared byadding 20.75 g NaCl plus 2.545 g sodium barbital (sodium-5,5-diethyl barbiturate) to about 400 mL water. The pH isadjusted to 7.35 with 1 N HCl, then brought to a final volumeof 500 mL in a volumetric flask.6.3 Metals Solution, is prepared by making a 2.0 M solutionof MgCl2(40.66 g MgCl26H2O
23、into 100 mL distilledendotoxin-free water), and a 0.3 M solution of CaCl2(4.41 gCaCl22H2O into 100 mL distilled endotoxin-free water), andcombining the two solutions 1:1 (v:v). These solutions arestable one month at 4C.6.4 BBS-GM Working Solution, is prepared daily, by dis-solving 0.25 g gelatin in
24、50 mL endotoxin-free distilled waterthat is gently heated and stirred. The gelatin solution is addedto 50 mL 5X stock BBS plus 0.25 mL metals solution, broughtup to about 200 mL, then adjusted to pH 7.35 (with1NHClor 1 N NaOH) before bringing the final volume to 250 mL ina volumetric flask.6.5 BBS-G
25、 Working Solution, is prepared the same way, butthe addition of the metals solution is omitted.6.6 10X Stock EDTA (0.1 M disodium dihydrate EDTA), isprepared by adding 7.44 g disodium EDTA2 H2O to about 160mL water, adjusting the pH to 7.65 (with 1 N NaOH or 1 NHCl), then bringing the volume to 200
26、mL in a volumetricflask.6.7 BBS-G-EDTA (to be used in preparing RBC beforebeing washed out), is prepared by adding 10 mL of stock 10XEDTA to 90 mL of BBS-G in a volumetric flask.7. Preparation of Sheep RBC7.1 Commercially-obtained sheep red blood cells (RBC)preserved in Alsevers solution are stored
27、at 4C. The cells arediscarded after eight weeks or when the supernatant from thesecond wash contains hemoglobin by visual inspection.NOTE 1All centrifugations are at 4C. Except where indicated, allreagents, tubes, and cell preparations are kept on ice.7.2 Five mL of sheep RBC are centrifuged at 1 00
28、0xgfor10 min.7.3 The cell pellet is resuspended in 10 mL of coldBBS-G-EDTA and incubated for 10 min at 37C. The cells arecentrifuged, and the pellet resuspended in 10 mL of BBS-G-EDTA.7.4 The cells are centrifuged, the supernatant discarded(first wash), and the pellet resuspended in 10 mL of coldBBS
29、-GM. Repeat twice (total of three washes).7.5 Adjust cell count spectrophotometrically (where anabsorbance of 0.56 corresponds to 1.5 x 108sheep RBC/mL, ata wavelength of 412 nm and a 1.0-cm light path for 1 volumeof cells in BBS-GM plus 24 volumes of water) or count witha hemocytometer, preparing 1
30、0 mL of 1.5 x 108cells/mL incold BBS-GM.7.6 The washed, diluted RBC can be held on ice and usedfor at least 12 h.8. Absorption of Serum (Complement)8.1 The use of human complement is required since thereare species differences in the efficiency of complement activa-tion and the test materials are to
31、 be used in humans. Humanserum suitable as a source of complement may be purchasedfrom biological supply houses, and generally, is labeled asreagent-grade complement.8.2 Human serum may be absorbed with sheep RBC inorder to remove naturally-occurring anti-sheep RBC hemolyticantibodies, though for mo
32、st purposes, the amount of hetero-phile antibody in human serum is not enough to influence thereaction assuming the cells are optimally sensitized withhemolysin. The procedure is detailed in 8.3 8.8.8.3 Fresh human serum or a commercial lot of human serumis obtained and stored at 70C. Fresh serum is
33、 preferred aslyophilized complement often is not as active as fresh serum.8.4 The serum is thawed on ice or reconstituted (if ly-ophilized) with ice-cold (4C) distilled endotoxin-free water.8.5 All manipulations are done on ice, with ice cold reagentsand cells; centrifugations are carried out at 100
34、0xgat4C. Itis critical that this entire procedure be done in the cold to avoidactivation of complement in this step.4The boldface numbers in parentheses refer to the list of references at the end ofthis specification.F1984 99 (2018)28.6 Cold serum and cold, packed, washed sheep RBC aremixed, 0.1 mL
35、RBC/2.5 mL serum, incubated for 10 min onice, then centrifuged at 1 000 x g for 10 min at 4C. Thesupernatant is transferred carefully to a new container on ice.8.7 The procedure in 8.6 is repeated twice.8.8 The absorbed human serum is stored in 0.51.0-mLaliquots (convenient for one-experiment use),
36、in cold snap-capmicrofuge tubes and kept at 70C until used. Aliquots shouldbe thawed on ice, used on the day of thawing, and not berefrozen.9. Determination of Optimal Hemolysin Concentration9.1 Determination of optimal hemolysin concentration isnecessary in order to conserve expensive reagents and
37、to avoidprozone effects. Commercial rabbit anti-sheep RBC serum(Hemolysin) is obtained, thawed, or, if lyophilized, reconsti-tuted with distilled endotoxin-free water), heat-inactivated at56C for 30 min to inactivate the rabbit complement, aliquotedin convenient volumes, and stored at 70C until used
38、.9.2 To cold 13 x 100 mm disposable glass tubes, placed ina rack in an ice-bath, 0.1 mLof washed sheep RBC at 1.5 x 108cells/mL is added. If statistical evaluation of the results isdesired, three replicate tubes for each condition should be used.Otherwise, duplicates or even single dilution tubes ar
39、e suffi-cient. One set of three replicate tubes receives only 0.1 mL ofcold BBS-GM/tube (no RBC control, for complement color).9.3 To the RBC-containing tubes, one set of three tubes gets1.1 mL cold distilled H2O/tube (total lysis control), anothergets 0.1 mL BBS-GM (no hemolysin control), and the o
40、thersets get 0.1 mLeach of 1:2 serial dilutions of hemolysin (tests).Dilutions between 1:400 to 1:25 600 antibody arerecommended, with two sets of 1:400. The no RBC controlreceives 0.1 mL of additional BBS-GM.9.4 Each tube is mixed quickly by gentle shaking toresuspend cells, the rack is placed in a
41、 37C water bath,incubated 10 min, then returned to the ice bath.9.5 One of the two sets of 1:400 antibody gets 1.0 mL ofcold BBS-GM (no-complement control). All other tubes be-sides the total lysis control set get 0.5 mL cold BBS-GM, then0.5 mL of absorbed human serum (complement) diluted 1:100or 1:
42、200.NOTE 2For a particular lot of human serum, a 1:100 or 1:200 dilutionshould provide sufficient complement activity. Also, percent lysis in theno-hemolysin (complement only) control should not exceed 10 %. If lysiswith the 1:100 dilution of complement exceeds 10 %, use 1:200. If theno-hemolysin co
43、ntrol still exceeds 10 %, a different lot of serum will needto be tested.9.6 Tubes are shaken manually to suspend cells, then therack is incubated in a 37C water bath for 1h, and intermit-tently shaken to keep cells in suspension.9.7 At the end of 1h, the rack is placed on ice. The coldtubes then ar
44、e centrifuged at 1 000 x g for 10 min at 4C, andthe supernatants decanted to correspondingly numbered 13 x100-mm glass tubes.9.8 Absorbance of the supernatants is measured at 412 nm.Percent lysis is calculated for each test and control tube bysubtracting from the 412 nm absorbance the no RBC control
45、(mean of the three replicate tubes), dividing by the total lysiscontrol value (mean of the three replicate tubes), and multi-plying by 100.% lysis 5test absorbance 2 no RBC control absorbancetotal lysis absorbance3100 (1)9.9 Final % lysis for each condition is expressed as mean 6standard deviation o
46、f the three % lysis values for each three-replicate set.9.10 A titration curve is obtained by plotting the inverse ofthe hemolysin concentration versus % specific lysis. Twice theconcentration of hemolysin that is just on the plateau of thetitration curve is used for sensitizing RBC for subsequentas
47、says (optimal hemolysin concentration). Hemolysin isfreshly diluted from stock each day of use.10. Whole Complement Titration to Determine OptimalSerum Dilution10.1 If statistical evaluation of results is desired, all condi-tions should be assayed in triplicate, using three 13 x 100disposable glass
48、test tubes per condition. Otherwise, duplicatesor single tubes are sufficient. Tubes are numbered in advance.Conditions include total lysis, no complement (no C), tests(dilutions of human serumHS) with and without hemolysin,and no RBC (complement color control, at highest concentra-tion of serum use
49、d). All reagents, tubes, and manipulations aredone ice-cold, with tubes held in a rack in an ice slurry.10.2 Washed RBC are added to all tubes except no RBCtubes (0.1 mL/tube of a 1.5 x 108cells/mL suspension). NoRBC tubes get 0.1 mL cold buffer.10.3 Total lysis tubes get 1.1 mL distilled H2O. The no Cand test with hemolysin tubes get 0.1 mL optimal hemolysin(see 9.10), and no RBC tubes get 0.1 mL cold BBS-GM. Alltubes are shaken to resuspend cells, incubated in a 37C waterbath for 10 min, and placed back on ice.NOTE 3Another acceptable pr
