1、Designation: F 2567 06Standard Practice forTesting for Classical Pathway Complement Activation inSerum by Solid Materials1This standard is issued under the fixed designation F 2567; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, t
2、he year of last revision. 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 practice provides a protocol for rapid, in vitrofunctional screening for classical pathway complement
3、 activat-ing properties of solid materials used in the fabrication ofmedical devices that will contact blood.1.2 This practice is intended to evaluate the acute in vitroclassical pathway complement activating properties of solidmaterials intended for use in contact with blood. For thispractice, “ser
4、um” is synonymous with “complement.”1.3 This practice consists of two procedural parts. Proce-dureAdescribes exposure of solid materials to a standard lot ofhuman serum HS, using 0.1 mL serum per 133100 mmdisposable glass test tube. Procedure B describes assaying theexposed serum for significant fun
5、ctional classical pathwaycomplement depletion (decrease in amount of C4) as comparedto control serum samples not exposed to the material. Theendpoint in Procedure B is lysis of sheep red blood cells (RBC)coated with antibody (hemolysin).1.4 This practice does not address the use of plasma as asource
6、 of complement.1.5 This practice is one of several developed for theassessment of the biocompatibility of materials. Practice F 748may provide guidance for the selection of appropriate methodsfor testing materials for other aspects of biocompatibility.Practice F 1984 provides guidance for testing so
7、lid materialsfor whole complement activation in human serum, but does notdiscriminate between the classical or alternative pathway ofactivation. Practice F 2065 provides guidance for testing solidmaterials for alternative pathway complement activation inserum.1.6 This standard does not purport to ad
8、dress 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:2F 748 Practi
9、ce for Selecting Generic Biological Test Meth-ods for Materials and DevicesF 1984 Practice for Testing for Whole Complement Activa-tion in Serum by Solid MaterialsF 2065 Practice for Testing for Alternative PathwayComplement Activation in Serum by Solid Materials2.2 Other Document:ISO 10993-4 Biolog
10、ical Evaluation of Medical Devices,Part 4: Selection of Tests for Interactions with Blood33. Terminology3.1 Definition of Term Specific to This Standard:3.1.1 waterdistilled, endotoxin-free.3.2 Abbreviations:3.2.1 Abantibody (hemolysin)3.2.2 BBSbarbital buffered saline3.2.3 BBS-Gbarbital buffered sa
11、linegelatin3.2.4 BBS-GM (Ca Buffer)barbital buffered salinegelatinmetals3.2.5 C8complement3.2.6 C4the fourth component of complement3.2.7 C4(-)GPSC4-deficient guinea pig serum serumfrom guinea pigs genetically incapable of producing C43.2.8 EDTAethylenediaminetetraacetic acid, disodiumsalt, dihydrat
12、e3.2.9 HAGGheat aggregated gamma globulin3.2.10 HShuman serum3.2.11 I“ice” control tube with serum but no material,kept on ice3.2.12 Mtube containing serum plus a test material3.2.13 NMtube containing serum but no material1This practice is under the jurisdiction ofASTM Committee F04 on Medical andSu
13、rgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition approved Oct. 1, 2006. Published October 2006.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org.
14、 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.1Copyright ASTM International, 100 Barr Harbor Drive, P
15、O Box C700, West Conshohocken, PA 19428-2959, United States.3.2.14 RBCred blood cell(s)4. Summary of Practice4.1 This practice is based on a method published by Gaitheret al, 1974 (1).44.2 Solid material specimens are exposed to a standard lotof human C8 (specially-prepared, commercial human serumHS
16、) under defined conditions, in parallel with appropriatecontrols (Procedure A). If the classical complement pathway isactivated by the material, C4 will be depleted from the serum.Exposed serum is then tested for remaining C4 functionalactivity. An appropriate dilution of the HS, which by itself ist
17、oo dilute to lyse sensitized sheep RBC, is added to hemolysin-coated sheep RBC in the presence of C4(-)GPS in which allcomplement components save the missing C4 are present inexcess (Procedure B). Hemolysis in Procedure B provides aquantitative measure of the C4 remaining in HS exposed to testmateri
18、al in Procedure A. Depletion of hemolysis indicatesspecific classical pathway activation in the human serumcaused by exposure to the test material.5. Significance and Use5.1 Inappropriate activation of complement by blood-contacting medical devices may have serious acute or chroniceffects on the hos
19、t. Solid medical device materials may activatecomplement directly by the alternative pathway, or indirectlybecause of antigen-bound antibodies (as with immuno-adsorption columns) by the classical pathway. This practice isuseful as a simple, inexpensive, function-based screeningmethod for determining
20、 complement activation by solid mate-rials in vitro by the classical pathway.5.2 This practice is composed of two parts. In part A(Section 11), HS is exposed to a solid material. If complementactivation occurs by the classical pathway, C4 will be depleted.Activation by the alternative pathway will n
21、ot deplete C4. Inpart B (Section 12), C4 activity remaining in the serum afterexposure to the test material is assayed by diluting the serumbelow the concentration needed to lyse antibody-coated sheepRBC on its own, then adding the diluted HS to C4(-)GPS(which is itself at a dilution where all compl
22、ement componentsare in excess save the missing C4). Lacking C4, the C4(-)GPSdoes not lyse the antibody-coated sheep RBC unless C4 ispresent in the added HS. The proportion of lysis remaining inthe material-exposed HS sample versus the 37C control HSsample (which was not exposed to the test material)
23、 indicatesthe amount of C4 present in the HS, loss of which correlateswith classical pathway activation.5.3 This function-based in vitro test method for classicalpathway complement activation is suitable for adoption inspecifications and standards for screening solid materials foruse in the construc
24、tion of medical devices intended to beimplanted in the human body or placed in contact with humanblood outside the body. It is designed to be used in conjunctionwith Practice F 1984 for function-based whole complementactivation screening, and Practice F 2065 for function-basedalternative pathway act
25、ivation screening.5.4 Assessment of in vitro classical complement activationas described here provides one method for predicting potentialcomplement activation by solid medical device materialsintended for clinical application in humans when the materialcontacts the blood. Other test methods for com
26、plement activa-tion are available, such as immunoassays for specific comple-ment components (including C4) and their split products inhuman serum (see X1.3 and X1.4).5.5 If nonspecific binding of certain complement compo-nents, including C4, to the materials occurs in part A of thispractice, a false
27、 positive for classical pathway activation willbe observed in step B. Classical pathway complement activa-tion by the test material may be confirmed by demonstrating anabsence of C4 bound to the material following removal of theserum, and/or production of complement split-products such asC4d in the
28、serum (as determined by immunoassay). Althoughimmunoassay could be done in place of this screening proce-dure, determination of C4d production alone may not befunctionally significant. This practice does not detect trivialamounts of classical activation unable to affect functional lysisof sensitized
29、 RBC.6. Preparation of Buffers6.1 Buffers are prepared according to established protocols(2, 3). “Water” refers throughout to distilled, endotoxin-freeH2O. The use of barbital (veronal) buffer is recommended. Inthe United States, barbital is a class IV regulated substance andrequires a DEA(4) licens
30、e for purchase. The use of other buffersystems (such as TRIS) is permissible if they have beendemonstrated not to activate complement (5).6.2 5X Stock BBS (barbital-buffered saline) is prepared byadding 20.75 g NaCl plus 2.545 g sodium barbital (sodium-5,5-diethyl barbiturate) to about 400 mL water.
31、 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 MgCl 6 H2O into 100 mL water), and a 0.3M solution of CaCl2(4.41 g CaCl22H2O into 100 mL water),and combining the two solu
32、tions 1:1 (v:v). These solutions arestable for one month at 4C.6.4 Ca Buffer (BBS-GM Working Solution) is prepared daily,by dissolving 0.25 g gelatin in 50 mL water that is gentlyheated and stirred. The gelatin solution is added to 50 mL 5XStock BBS plus 0.25 mLMetals Solution, brought to about 200m
33、L, then adjusted to pH 7.35 (with 1 N HCl or 1 N NaOH)before bringing the final volume to 250 mL in a volumetricflask. Ca buffer contains both Mg+and Ca+, which allowsboth classical and alternative pathway complement activationto occur.6.5 BBS-G Working Solution is prepared the same way, butomitting
34、 addition of the metals solution.6.6 103 Stock EDTA (0.1 M disodium dihydrate EDTA) isprepared by adding 7.44 g disodium EDTA.2H2O to about160 mL water, adjusting the pH to 7.65 (with 1 N NaOH or 1N HCl), then bringing the volume to 200 mL in a volumetricflask.4The boldface numbers in parentheses re
35、fer to the list of references at the end ofthis standard.F25670626.7 BBS-G-EDTA (to be used in preparing RBC beforebeing washed out with Ca buffer) is prepared by adding 10 mLof stock 10X EDTA to 90 mL of BBS-G in a volumetric flask.7. Preparation of Sheep RBC7.1 Commercially-obtained sheep RBC pres
36、erved in Alsev-ers solution are stored at 4C. The sheep cells are discardedafter eight weeks or when the supernatant liquid from thesecond wash contains hemoglobin by visual inspection (as lotsof RBCs age, they increase in sensitivity to complement lysisin parallel with increased spontaneous lysis).
37、NOTE 1All centrifugations are at 4C. Except when indicated, allreagents, tubes, and cell preparations are kept on ice or in an ice slurry. Insubsequent sections where the word “cold” is used, that denotes tubes inice or sitting in an ice slurry.7.2 Five mL of sheep RBC are centrifuged at 10003 g, at
38、4C, for 10 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 coldB
39、BS-GM (Ca Buffer). This step is repeated twice more for atotal of three washes.7.5 Adjust cell concentration by counting with a hemocy-tometer, and prepare 10 mL of 3.03108cells/mL in coldBBS-GM.7.6 The washed, diluted RBC can be held on ice and usedfor at least 12 h.8. Absorption of Serum (Compleme
40、nt)8.1 Serum should be absorbed with sheep RBC in order toremove any naturally-occurring anti-sheep hemolytic antibod-ies. The procedure is as follows.8.2 Commercially-available HS and C4(-)GPS are stored at70C. Both sera should be absorbed separately.8.3 Serum is thawed on ice or reconstituted (if
41、lyophilized)with ice-cold (4C) water.8.4 All manipulations are done on ice, with ice-cold re-agents and cells. Centrifugations are carried out at 10003 gat4C. It is critical that this entire procedure be done in the coldto avoid activation of complement in this step.8.5 Sheep RBC are washed as in Se
42、ction 7, centrifuged10003 g for 10 min at 4C, and the cold supernatant removeddown to the pellet. The cold, packed RBC are then added to theserum in a glass tube on ice, 0.1 mL/2.5 mL serum. The cellsare mixed thoroughly into the serum slowly inverting thecapped tube several times. The cell/serum mi
43、xture is incubatedfor 10 min on ice, then centrifuged at 10003 g for 10 min at4C. The supernatant liquid is carefully transferred to a freshglass tube on ice.8.6 The procedure in 8.5 is repeated twice, exposing thecold serum to three fresh preparations of cold cells.8.7 The absorbed HS is stored in
44、0.5 to 1.0 mL aliquots(convenient for one experiment), in pre-chilled, cold snap-capmicrofuge tubes immediately placed at 70C until used.Aliquots should be thawed cold, on ice (not allowed to warmhigher than 4C), used on the day of thawing, and notre-frozen.9. Determination of Optimal Hemolysin Conc
45、entration9.1 Determination of optimal hemolysin concentration isnecessary in order to conserve expensive reagents and to avoidprozone effects. Commercial rabbit anti-sheep RBC serum(hemolysin) is thawed (or, if lyophilized, reconstituted withdistilled endotoxin-free water), heat-inactivated at 56C f
46、or 30min to inactivate the rabbit complement, aliquoted in conve-nient volumes, and stored at 70C until used.9.2 To cold 133100 mm disposable glass tubes, placed in arack in an ice-slurry, 50 L of washed sheep RBC at 33108cells/mL is added directly to the bottom of each tube. Ifstatistical evaluatio
47、n of the results is desired, three replicatetubes for each condition should be used. Otherwise, duplicatesor even single dilution tubes are sufficient. One set of threereplicate tubes receives only 50 L of cold Ca buffer/tube (“noRBC” control, for complement color).9.3 To the RBC-containing tubes, o
48、ne set of three tubes gets0.35 mL cold distilled H2O/tube (“total lysis” control), anothergets 50 L mL Ca buffer (“no hemolysin” control), and theother sets get 50 L mL each of 1:2 serial dilutions ofhemolysin (“tests”). Dilutions between 1:200 to 1:25 600antibody are recommended, with two sets of 3
49、 tubes each for1:200. The “no RBC” control receives 50 L of additionalBBS-GM instead of hemolysin. All tubes except “total lysis”controls should each contain at this point a total 0.1 mL.9.4 Each tube is quickly mixed by gentle shaking toresuspend cells, the rack is placed in a 37C water bath,incubated 10 min, then returned to the ice-slurry.9.5 One of the two 3-tube sets of 1:200 hemolysin gets 0.1mL of cold Ca buffer (“no-complement” control). All othertubes besides the “total lysis” control set get 0.1 mL coldabsorbed HS (C8) di
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