1、Designation: F 2065 00 (Reapproved 2006)Standard Practice forTesting for Alternative Pathway Complement Activation inSerum by Solid Materials1This standard is issued under the fixed designation F 2065; the number immediately following the designation indicates the year oforiginal adoption or, in the
2、 case of revision, the 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 vitroscreening for alternative pathway
3、 complement activating prop-erties of solid materials used in the fabrication of medicaldevices that will contact blood.1.2 This practice is intended to evaluate the acute in vitroalternative pathway complement activating properties of solidmaterials intended for use in contact with blood. For thisp
4、ractice, “serum” is synonymous with “complement.”1.3 This practice consists of two procedural parts. Proce-dureAdescribes exposure of solid materials to a standard lot ofC4-deficient guinea pig serum C4(-)GPS, using 0.1-mLserum per 13 3 100-mm disposable glass test tubes.Sepharose2CL-4B is used as a
5、n example of test materials.Procedure B describes assaying the exposed serum for signifi-cant functional alternative pathway complement depletion ascompared to control samples. The endpoint in procedure B islysis of rabbit RBC in buffer containing EGTA and excessMg+.1.4 This practice does not addres
6、s function, elaboration, ordepletion of individual complement components except asoptional additional confirmatory information that can be ac-quired using human serum as the complement source. Thispractice does not address the use of plasma as a source ofcomplement.1.5 This practice is one of severa
7、l 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 solid materialsfor whole complement activation in h
8、uman serum, but does notdiscriminate between the classical or alternative pathway ofactivation.2. Referenced Documents2.1 ASTM Standards:3F 748 Practice for Selecting Generic Biological Test Meth-ods for Materials and DevicesF 1984 Practice for Testing for Whole Complement Activa-tion in Serum by So
9、lid Materials2.2 Other Document:ISO 10993-4: Biological Evaluation of Medical Devices.Part 4: Selection of Tests for Interactions with Blood43. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 waterdistilled, endotoxin-free.3.2 Abbreviations:Abbreviations:3.2.1 Abantibody (hemolys
10、in)3.2.2 BBSbarbital buffered saline3.2.3 BBS-Gbarbital buffered saline gelatin3.2.4 BBS-G-EGTA/Mg (Mg Buffer)barbital buffered sa-line gelatin EGTA Mg+3.2.5 BBS-GM (Ca Buffer)barbital buffered saline gela-tin metals3.2.6 C8complement3.2.7 C4(-)GPSC4-deficient guinea pig serum serumfrom guinea pigs
11、genetically incapable of producing C4, thefourth component of complement3.2.8 EDTAethylenediaminetetraacetic acid, disodiumsalt: dihydrate3.2.9 EGTAethylene glyco-bis(b-aminoethyl ether)-N,N,N8,N8-tetraacetic acid, tetrasodium salt3.2.10 HAGGheat aggregated gamma globulin3.2.11 HShuman serum3.2.12 I
12、control tube with serum but no material, kept onice.3.2.13 Mtube containing serum plus a test material1This practice is under the jurisdiction ofASTM Committee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Cur
13、rent edition approved March 1, 2006. Published April 2006. Originallyapproved in 2000. Last previous edition approved in 2000 as F 2065 00e1.2Sepharose is a registered trademark of Pharmacia, Inc. (now GE Healthcare),Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, U.K.3For referenced ASTM
14、standards, visit the ASTM website, 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.4Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th F
15、loor, New York, NY 10036.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.2.14 NMtube containing serum but no material3.2.15 PVDFpolyvinylidene fluoride3.2.16 RBCred blood cell(s)4. Summary of Practice4.1 Solid material specimens ar
16、e exposed to a standard lotof C4(-)GPS complement under defined conditions, in parallelto appropriate controls (Procedure A). If the alternativecomplement pathway is activated by the material, complementcomponents will be depleted from the serum. Exposed serum isthen tested for remaining functional
17、complement activity, bydetermining complement mediated lysis of rabbit RBC inbuffer containing EGTA and excess Mg+(Procedure B).5. Significance and Use5.1 Inappropriate activation of complement by blood-contacting medical devices may have serious acute or chroniceffects on the host. Solid medical de
18、vice materials may activatethe complement directly by the alternative pathway. Unlike theclassical complement activation pathway (see Practice F 1984),antibodies are not required for alternative pathway activation.This practice is useful as a simple, inexpensive screeningmethod for determining alter
19、native whole complement activa-tion by solid materials in vitro.5.2 This practice is composed of two parts. In Part A(Section 10) C4(-)GPS is exposed to a solid material. Since C4is required for classical pathway activation, activation ofcomplement in C4(-)GPS can only occur by the alternativepathwa
20、y (1).5In principle, nonspecific binding of certaincomplement components to the materials may also occur. InPart B (Section 11), complement activity remaining in theserum after exposure to the test material is assayed byalternative pathway-mediated lysis of rabbit RBC.5.3 Assessment of in vitro whol
21、e complement activation asdescribed 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 complement activa-tion are available, including assays f
22、or specific complementcomponents and their split products in human serum (X1.3 andX1.4).5.4 This in vitro test method is suitable for adoption inspecifications and standards for screening solid materials foruse in the construction of medical devices intended to beimplanted in the human body or place
23、d in contact with humanblood outside the body.6. Preparation of Buffers6.1 Buffers are prepared in accordance with establishedprotocols (1, 2). “Water” refers throughout to distilled,endotoxin-free H2O. The use of barbital (veronal) buffer isrecommended. In the United States, barbital is a class IVr
24、egulated substance and requires a DEA (3) license for pur-chase. The use of other buffer systems (such as TRIS) ispermissible if they have been demonstrated not to activatecomplement (4). These solutions are stable for one month at4C unless otherwise indicated.6.2 The 5X stock BBS (barbital-buffered
25、 saline) is preparedby adding 20.75 g of NaCl plus 2.545 g of sodium barbital(sodium-5,5-diethyl barbiturate) to about 400 mL water. ThepH is adjusted to 7.35 with 1 N HCl, then brought to a finalvolume of 500 mL in a volumetric flask.6.3 Metals solution is prepared by making a 2.0-M solutionof MgCl
26、2(40.66 g MgCl26 H2O up to a final volume of 100mL water), and a 0.3-M solution of CaCl2(4.41 g CaCl22H2Oup to a final volume of 100 mL of water), and combining thetwo solutions 1:1 (v:v).6.4 The Ca buffer (BBS-GM working solution) is prepareddaily, by dissolving 0.25 g of gelatin (type A: Porcine S
27、kin,Approx. 300 Bloom, such as available from Sigma G-1890)in 50 mL of water that is gently heated and stirred. The gelatinsolution is added to 50 mL 5X Stock BBS plus 0.25 mL metalssolution, brought up to about 200 mL then adjusted to pH 7.35(with 1 N HCl or 1 N NaOH) before bringing the final volu
28、meto 250 mL in a volumetric flask. The Ca buffer contains bothMg+and Ca+, which allows both classical and alternativepathway complement. activation to occur.6.5 The BBS-G working solution is prepared the same way,but omitting addition of the metals solution.6.6 10X Stock EDTA (0.1-M disodium dihydra
29、te EDTA) isprepared by adding 7.44 g disodium EDTA2 H2O to about 160mL of water, adjusting the pH to 7.65 (with 1 N NaOH or 1 NHCl), then bringing the volume to 200 mL in a volumetricflask.6.7 The 0.1 M EGTA (tetrasodium salt, EGTA4.5 H2O) isprepared by adding 4.683 g tetrasodium EGTAto about 80 mLo
30、f water, adjusting the pH to 7.35 (with 1 N NaOH or 1 N HCl),then bringing the volume to 100 mL in a volumetric flask.6.8 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.6.9 The Mg Buffer (BBS-G-EGTA
31、/Mg working solution) isprepared daily, by dissolving 0.25 g gelatin in 50 mLwater thatis gently heated and stirred. The gelatin solution is added to 50mL 5X stock BBS, plus 0.625 mL 2.0 M MgCl2,plus4mLof0.1 M EGTA, brought up to about 200 mL, then adjusted to pH7.35 (with 1 N HCl or 1 N NaOH) befor
32、e bringing the finalvolume to 250 mL in a volumetric flask. The Mg buffer hasEGTA to bind Ca+. The presence of Mg+allows thealternative pathway activation to proceed, while the absence ofCa+prevents activation of the classical pathway.7. Preparation of Sheep and Rabbit RBC7.1 Commercially obtained s
33、heep RBC preserved in Alsev-ers solution and defibrinated rabbit RBC are stored at 4C.The sheep cells are discarded after eight weeks or when thesupernatant from the second wash contains hemoglobin (redcolor) by visual inspection (as lots of RBCs age, they are moresensitive to complement lysis in pa
34、rallel with increased spon-taneous lysis). The rabbit cells are more fragile than the sheepcells, and should be discarded after four weeks or when thesupernatant from the second wash contains hemoglobin byvisual inspection.5The boldface numbers in parentheses refer to the list of references at the e
35、nd ofthis specification.F 2065 00 (2006)2NOTE 1All centrifugations are at 4C. Except when indicated, allreagents, tubes, and cell preparations are kept cold in chipped ice or an iceslurry.7.2 Five millilitres of sheep or rabbit RBC are centrifuged at1000 3 g, at 4C, for 10 min.7.3 The cell pellet is
36、 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 4CBBS-G-EDTA.7.4 The cells are centrifuged, the supernatant discarded(first wash), and the pellet resuspended in 10 mL of coldBBS-GM (Ca buffer) or BBS-G-EGTA/Mg
37、(Mg buffer) (cellsto be used in absorbing serum are washed in Ca buffer; cells tobe used for detecting alternative pathway C8 depletion, Proce-dure B, are washed and suspended in Mg buffer). Repeat twice(total of three washes.)7.5 Adjust cell count spectrophotometrically (where anabsorbance of 0.75
38、for sheep RBC and 1.30 for rabbit RBCcorresponds to 2.0 3 108RBC/mL, at a wavelength of 412 nmand a 1.0-cm light path for 1 volume of cells in BBS-GM orBBS-G-EGTA/Mg plus 24 volumes of water) or count with ahemocytometer. Prepare 10 mL of 2.0 3 108cells/mL in 4CBBS-GM.7.6 The washed, diluted RBC can
39、 be held on ice and usedfor at least 12 h.8. Absorption of Serum (Complement)8.1 Although human serum would be preferable to guineapig serum for alternative pathway complement activation bymaterials to be used in medical devices intended to contactpatient blood, genetically deficient human sera are
40、not rou-tinely available. Human sera depleted of components byantibody absorption on columns are unsuitable for this purposefor the following two reasons: (1) specific component depletionis incomplete, so significant classical pathway activation re-mains; and (2) column material may activate the alt
41、ernativepathway, depleting functional activity. Serum from guinea pigsgenetically deficient in C4 C4(-)GPS has no classical path-way complement activity but is fully competent for alternativepathway complement activation. Although the titers are differ-ent (a greater concentration of C4(-)GPS is req
42、uired to producethe same lysis of rabbit RBC as human serum)(5)C4(-)GPSgives equivalent results to human serum in detecting biomate-rial complement activation (see Sepharose2example in Table1). Thus, C4(-)GPS is suitable as a screen for subsequentconfirmatory immunoassays that detect complement comp
43、o-nents and split-products indicative of alternative pathwayactivation in whole human serum. The C4(-)GPS suitable as asource of complement may be purchased from biologicalsupply houses, and is generally labeled as reagent-gradecomplement.8.2 Serum may be absorbed with sheep RBC and rabbitRBC in ord
44、er to remove naturally occurring anti-sheep andanti-rabbit RBC hemolytic antibodies. The procedure is asfollows:8.3 Commercially available C4(-)GPS is stored at 70C.8.4 The serum is thawed on chipped ice or reconstituted (iflyophilized) with ice-cold water.8.5 All manipulations are done in chipped i
45、ce or in an iceslurry, with ice cold (4C) reagents and cells. Centrifugationsare carried out at 1000 3 g at 4C. It is critical that this entireprocedure be done in the cold to avoid activation of comple-ment in this step.8.6 Cold serum and 4C, Ca buffer-washed RBC (a 1:1mixed volume of sheep and rab
46、bit packed RBC) are gentlymixed (by slow rocking), 0.1 mL of packed RBC/2.5 mL ofserum, incubated for 10 min on ice, then centrifuged at 1000 3g for 10 min at 4C. The supernatant liquid is carefullytransferred to a new container on ice.8.7 The procedure in 8.6 is repeated twice.8.8 The absorbed C4(-
47、)GPS is stored in 0.51.0-mLaliquots(convenient for one-experiment use), in cold snap-cap mi-crofuge tubes and kept at 70C until used.Aliquots should bethawed in chipped ice or an ice slurry, used on the day ofthawing, and not refrozen.9. Whole Complement Titration to Determine OptimalSerum Dilution9
48、.1 If statistical evaluation of results is desired, all condi-tions should be assayed in triplicate, using three 13 3 100 mmround-bottomed, disposable glass test tubes per condition.Otherwise, single or duplicate tubes are sufficient. Tubes arenumbered in advance. Conditions include “total lysis,” “
49、nocomplement (RBC only)” (no C8), “tests” (dilutions of C4(-)GPS) with and without hemolysin, and “no RBC (serumcolor)” (complement color control, at highest concentration ofserum used).All reagents, tubes, and manipulations are done at4C, with tubes held in a rack in an ice slurry.9.2 Two sets of tubes are prepared in accordance with9.39.4. One set contains diluted sera placed on Mg buffer-washed sheep RBC, and one set contains diluted sera placed onMg buffer-washed rabbit RBC.9.3 The Mg buffer-washed rabbit RBC are add
