ASTM F2567-06(2010)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: F2567 − 06(Reapproved 2010)
Standard Practice for
Testing for Classical Pathway Complement Activation in
Serum by Solid Materials
This standard is issued under the fixed designation F2567; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 1.7 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
1.1 This practice provides a protocol for rapid, in vitro
responsibility of the user of this standard to establish appro-
functionalscreeningforclassicalpathwaycomplementactivat-
priate safety and health practices and determine the applica-
ing properties of solid materials used in the fabrication of
bility of regulatory limitations prior to use.
medical devices that will contact blood.
2. Referenced Documents
1.2 This practice is intended to evaluate the acute in vitro
classical pathway complement activating properties of solid
2.1 ASTM Standards:
materials intended for use in contact with blood. For this
F748PracticeforSelectingGenericBiologicalTestMethods
practice, “serum” is synonymous with “complement.”
for Materials and Devices
F1984Practice for Testing for Whole Complement Activa-
1.3 This practice consists of two procedural parts. Proce-
tion in Serum by Solid Materials
dureAdescribesexposureofsolidmaterialstoastandardlotof
F2065Practice forTesting forAlternative Pathway Comple-
human serum [HS], using 0.1 mL serum per 13×100 mm
ment Activation in Serum by Solid Materials
disposable glass test tube. Procedure B describes assaying the
2.2 Other Document:
exposed serum for significant functional classical pathway
ISO10993-4BiologicalEvaluationofMedicalDevices,Part
complementdepletion(decreaseinamountofC4)ascompared
4: Selection of Tests for Interactions with Blood
to control serum samples not exposed to the material. The
endpointinProcedureBislysisofsheepredbloodcells(RBC)
3. Terminology
coated with antibody (hemolysin).
3.1 Definitions of Terms Specific to This Standard:
1.4 This practice does not address the use of plasma as a
3.1.1 water—distilled, endotoxin-free.
source of complement.
3.2 Abbreviations:
1.5 This practice is one of several developed for the
3.2.1 Ab—antibody (hemolysin)
assessment of the biocompatibility of materials. Practice F748
3.2.2 BBS—barbital buffered saline
may provide guidance for the selection of appropriate methods
3.2.3 BBS-G—barbital buffered saline–gelatin
for testing materials for other aspects of biocompatibility.
PracticeF1984providesguidancefortestingsolidmaterialsfor
3.2.4 BBS-GM (Ca Buffer)—barbital buffered saline–gelatin
whole complement activation in human serum, but does not
metals
discriminate between the classical or alternative pathway of
3.2.5 C'—complement
activation. Practice F2065 provides guidance for testing solid
3.2.6 C4—the fourth component of complement
materials for alternative pathway complement activation in
3.2.7 C4(-)GPS—C4-deficient guinea pig serum [serum
serum.
from guinea pigs genetically incapable of producing C4]
1.6 The values stated in SI units are to be regarded as
3.2.8 EDTA—ethylenediaminetetraacetic acid, disodium
standard. No other units of measurement are included in this
salt, dihydrate
standard.
3.2.9 HAGG—heat aggregated gamma globulin
1 2
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Surgical Materials and Devices and is the direct responsibility of Subcommittee contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
F04.16 on Biocompatibility Test Methods. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved Sept. 1, 2010. Published November 2010. Originally the ASTM website.
approved in 2006. Last previous edition approved in 2006 as F2567–06. DOI: Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
10.1520/F2567-06R10. 4th Floor, New York, NY 10036, http://www.ansi.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2567 − 06 (2010)
3.2.10 HS—human serum specifications and standards for screening solid materials for
use in the construction of medical devices intended to be
3.2.11 I—“ice” control tube with serum but no material,
implanted in the human body or placed in contact with human
kept on ice
bloodoutsidethebody.Itisdesignedtobeusedinconjunction
3.2.12 M—tube containing serum plus a test material
with Practice F1984 for function-based whole complement
3.2.13 NM—tube containing serum but no material
activation screening, and Practice F2065 for function-based
alternative pathway activation screening.
3.2.14 RBC—red blood cell(s)
5.4 Assessment of in vitro classical complement activation
4. Summary of Practice
as described here provides one method for predicting potential
4.1 ThispracticeisbasedonamethodpublishedbyGaither
complement activation by solid medical device materials
et al, 1974 (1).
intended for clinical application in humans when the material
4.2 Solid material specimens are exposed to a standard lot
contacts the blood. Other test methods for complement activa-
of human C' (specially-prepared, commercial human serum
tion are available, such as immunoassays for specific comple-
[HS]) under defined conditions, in parallel with appropriate
ment components (including C4) and their split products in
controls (ProcedureA).Iftheclassicalcomplement pathwayis
human serum (see X1.3 and X1.4).
activated by the material, C4 will be depleted from the serum.
5.5 If nonspecific binding of certain complement
Exposed serum is then tested for remaining C4 functional
components, including C4, to the materials occurs in partAof
activity. An appropriate dilution of the HS, which by itself is
this practice, a false positive for classical pathway activation
toodilutetolysesensitizedsheepRBC,isaddedtohemolysin-
will be observed in step B. Classical pathway complement
coated sheep RBC in the presence of C4(-)GPS in which all
activation by the test material may be confirmed by demon-
complement components save the missing C4 are present in
strating an absence of C4 bound to the material following
excess (Procedure B). Hemolysis in Procedure B provides a
removal of the serum, and/or production of complement
quantitativemeasureoftheC4remaininginHSexposedtotest
split-products such as C4d in the serum (as determined by
material in Procedure A. Depletion of hemolysis indicates
immunoassay).Although immunoassay could be done in place
specific classical pathway activation in the human serum
of this screening procedure, determination of C4d production
caused by exposure to the test material.
alone may not be functionally significant. This practice does
5. Significance and Use not detect trivial amounts of classical activation unable to
affect functional lysis of sensitized RBC.
5.1 Inappropriate activation of complement by blood-
contacting medical devices may have serious acute or chronic
6. Preparation of Buffers
effectsonthehost.Solidmedicaldevicematerialsmayactivate
complement directly by the alternative pathway, or indirectly
6.1 Buffers are prepared according to established protocols
because of antigen-bound antibodies (as with immuno-
(2, 3). “Water” refers throughout to distilled, endotoxin-free
adsorption columns) by the classical pathway. This practice is
H O. The use of barbital (veronal) buffer is recommended. In
useful as a simple, inexpensive, function-based screening
theUnitedStates,barbitalisaclassIVregulatedsubstanceand
method for determining complement activation by solid mate-
requiresaDEA (4)licenseforpurchase.Theuseofotherbuffer
rials in vitro by the classical pathway.
systems (such as TRIS) is permissible if they have been
demonstrated not to activate complement (5).
5.2 This practice is composed of two parts. In part A
(Section 11), HS is exposed to a solid material. If complement
6.2 5X Stock BBS (barbital-buffered saline) is prepared by
activationoccursbytheclassicalpathway,C4willbedepleted.
adding 20.75 g NaCl plus 2.545 g sodium barbital (sodium-5,
Activation by the alternative pathway will not deplete C4. In
5-diethyl barbiturate) to about 400 mL water. The pH is
part B (Section 12), C4 activity remaining in the serum after
adjusted to 7.35 with 1 N HCl, then brought to a final volume
exposure to the test material is assayed by diluting the serum
of 500 mL in a volumetric flask.
below the concentration needed to lyse antibody-coated sheep
6.3 Metals Solution is prepared by making a 2.0 M solution
RBC on its own, then adding the diluted HS to C4(-)GPS
ofMgCl (40.66gMgCl·6H Ointo100mLwater),anda0.3
2 2
(whichisitselfatadilutionwhereallcomplementcomponents
MsolutionofCaCl (4.41gCaCl ·2H Ointo100mLwater),
are in excess save the missing C4). Lacking C4, the C4(-)GPS 2 2 2
and combining the two solutions 1:1 (v:v).These solutions are
does not lyse the antibody-coated sheep RBC unless C4 is
stable for one month at 4°C.
present in the added HS. The proportion of lysis remaining in
the material-exposed HS sample versus the 37°C control HS
6.4 CaBuffer(BBS-GMWorkingSolution)isprepareddaily,
sample (which was not exposed to the test material) indicates
by dissolving 0.25 g gelatin in 50 mL water that is gently
the amount of C4 present in the HS, loss of which correlates
heated and stirred. The gelatin solution is added to 50 mL 5X
with classical pathway activation.
StockBBSplus0.25mLMetalsSolution,broughttoabout200
mL, then adjusted to pH 7.35 (with 1 N HCl or 1 N NaOH)
5.3 This function-based in vitro test method for classical
before bringing the final volume to 250 mL in a volumetric
pathway complement activation is suitable for adoption in
++ ++
flask. Ca buffer contains both Mg and Ca , which allows
both classical and alternative pathway complement activation
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this standard. to occur.
F2567 − 06 (2010)
6.5 BBS-G Working Solution is prepared the same way, but cappedtubeseveraltimes.Thecell/serummixtureisincubated
omitting addition of the metals solution. for 10 min on ice, then centrifuged at 1000× g for 10 min at
4°C. The supernatant liquid is carefully transferred to a fresh
6.6 10× Stock EDTA (0.1 M disodium dihydrate EDTA) is
glass tube on ice.
prepared by adding 7.44 g disodium EDTA.·2H O to about
160 mL water, adjusting the pH to 7.65 (with 1 N NaOH or 1 8.6 The procedure in 8.5 is repeated twice, exposing the
N HCl), then bringing the volume to 200 mL in a volumetric cold serum to three fresh preparations of cold cells.
flask.
8.7 The absorbed HS is stored in 0.5 to 1.0 mL aliquots
6.7 BBS-G-EDTA (to be used in preparing RBC before (convenient for one experiment), in pre-chilled, cold snap-cap
beingwashedoutwithCabuffer)ispreparedbyadding10mL microfuge tubes immediately placed at –70°C until used.
of stock 10X EDTAto 90 mLof BBS-G in a volumetric flask. Aliquots should be thawed cold, on ice (not allowed to warm
higher than 4°C), used on the day of thawing, and not
7. Preparation of Sheep RBC re-frozen.
7.1 Commercially-obtained sheep RBC preserved inAlsev-
9. Determination of Optimal Hemolysin Concentration
er’s solution are stored at 4°C. The sheep cells are discarded
after eight weeks or when the supernatant liquid from the
9.1 Determination of optimal hemolysin concentration is
second wash contains hemoglobin by visual inspection (as lots necessaryinordertoconserveexpensivereagentsandtoavoid
of RBCs age, they increase in sensitivity to complement lysis prozone effects. Commercial rabbit anti-sheep RBC serum
in parallel with increased spontaneous lysis).
(hemolysin) is thawed (or, if lyophilized, reconstituted with
NOTE 1—All centrifugations are at 4°C. Except when indicated, all
distilled endotoxin-free water), heat-inactivated at 56°C for 30
reagents, tubes, and cell preparations are kept on ice or in an ice slurry. In
min to inactivate the rabbit complement, aliquoted in conve-
subsequent sections where the word “cold” is used, that denotes tubes in
nient volumes, and stored at –70°C until used.
ice or sitting in an ice slurry.
9.2 To cold 13×100 mm disposable glass tubes, placed in a
7.2 Five mL of sheep RBC are centrifuged at 1000× g, at
rack in an ice-slurry, 50 µL of washed sheep RBC at 3×10
4°C, for 10 min.
cells/mL is added directly to the bottom of each tube. If
7.3 The cell pellet is resuspended in 10 mL of cold
statistical evaluation of the results is desired, three replicate
BBS-G-EDTAand incubated for 10 min at 37°C.The cells are
tubes for each condition should be used. Otherwise, duplicates
centrifuged, and the pellet resuspended in 10 mL of BBS-G-
or even single dilution tubes are sufficient. One set of three
EDTA.
replicate tubes receives only 50 µLof cold Ca buffer/tube (“no
7.4 The cells are centrifuged, the supernatant discarded RBC” control, for complement color).
(first wash), and the pellet resuspended in 10 mL of cold
9.3 TotheRBC-containingtubes,onesetofthreetubesgets
BBS-GM (Ca Buffer). This step is repeated twice more for a
0.35 mLcold distilled H O/tube (“total lysis” control), another
total of three washes.
gets 50 µL mL Ca buffer (“no hemolysin” control), and the
7.5 Adjust cell concentration by counting with a other sets get 50 µL mL each of 1:2 serial dilutions of
hemocytometer,andprepare10mLof3.0×10 cells/mLincold
hemolysin (“tests”). Dilutions between 1:200 to 1:25600
BBS-GM. antibody are recommended, with two sets of 3 tubes each for
1:200. The “no RBC” control receives 50 µL of additional
7.6 The washed, diluted RBC can be held on ice and used
BBS-GM instead of hemolysin. All tubes except “total lysis”
for at least 12 h.
controls should each contain at this point a total 0.1 mL.
8. Absorption of Serum (Complement)
9.4 Each tube is quickly mixed by gentle shaking to
resuspend cells, the rack is placed in a 37°C water bath,
8.1 Serum should be absorbed with sheep RBC in order to
incubated 10 min, then returned to the ice-slurry.
remove any naturally-occurring anti-sheep hemolytic antibod-
ies. The procedure is as follows.
9.5 One of the two 3-tube sets of 1:200 hemolysin gets 0.1
mL of cold Ca buffer (“no-complement” control). All other
8.2 Commercially-available HS and C4(-)GPS are stored at
tubes besides the “total lysis” control set get 0.1 mL cold
–70°C. Both sera should be absorbed separately.
absorbed HS (C') diluted 1:100 or 1:200.
8.3 Serum is thawed on ice or
...