ASTM F2567-06
(Practice)Standard Practice for Testing for Classical Pathway Complement Activation in Serum by Solid Materials
Standard Practice for Testing for Classical Pathway Complement Activation in Serum by Solid Materials
SCOPE
1.1 This practice provides a protocol for rapid, in vitro functional screening for classical pathway complement activating properties of solid materials used in the fabrication of medical devices that will contact blood.
1.2 This practice is intended to evaluate the acute in vitro classical pathway complement activating properties of solid materials intended for use in contact with blood. For this practice, "serum" is synonymous with "complement."
1.3 This practice consists of two procedural parts. Procedure A describes exposure of solid materials to a standard lot of human serum [HS], using 0.1 mL serum per 13100 mm disposable glass test tube. Procedure B describes assaying the exposed serum for significant functional classical pathway complement depletion (decrease in amount of C4) as compared to control serum samples not exposed to the material. The endpoint 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 a source of complement.
1.5 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F 748 may provide guidance for the selection of appropriate methods for testing materials for other aspects of biocompatibility. Practice F 1984 provides guidance for testing solid materials for whole complement activation in human serum, but does not discriminate between the classical or alternative pathway of activation. Practice F 2065 provides guidance for testing solid materials for alternative pathway complement activation in serum.
This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation:F2567–06
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. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
1.1 This practice provides a protocol for rapid, in vitro 2.1 ASTM Standards:
functionalscreeningforclassicalpathwaycomplementactivat- F748 Practice for Selecting Generic Biological Test Meth-
ing properties of solid materials used in the fabrication of ods for Materials and Devices
medical devices that will contact blood. F1984 Practice for Testing for Whole Complement Activa-
1.2 This practice is intended to evaluate the acute in vitro tion in Serum by Solid Materials
classical pathway complement activating properties of solid F2065 Practice for Testing for Alternative Pathway
materials intended for use in contact with blood. For this Complement Activation in Serum by Solid Materials
practice, “serum” is synonymous with “complement.” 2.2 Other Document:
1.3 This practice consists of two procedural parts. Proce- ISO 10993-4 Biological Evaluation of Medical Devices,
dureAdescribesexposureofsolidmaterialstoastandardlotof Part 4: Selection of Tests for Interactions with Blood
human serum [HS], using 0.1 mL serum per 133100 mm
3. Terminology
disposable glass test tube. Procedure B describes assaying the
exposed serum for significant functional classical pathway 3.1 Definition of Term Specific to This Standard:
3.1.1 water—distilled, endotoxin-free.
complementdepletion(decreaseinamountofC4)ascompared
to control serum samples not exposed to the material. The 3.2 Abbreviations:
3.2.1 Ab—antibody (hemolysin)
endpointinProcedureBislysisofsheepredbloodcells(RBC)
coated with antibody (hemolysin). 3.2.2 BBS—barbital buffered saline
3.2.3 BBS-G—barbital buffered saline–gelatin
1.4 This practice does not address the use of plasma as a
3.2.4 BBS-GM (Ca Buffer)—barbitalbufferedsaline–gelatin
source of complement.
1.5 This practice is one of several developed for the metals
3.2.5 C8—complement
assessment of the biocompatibility of materials. Practice F748
may provide guidance for the selection of appropriate methods 3.2.6 C4—the fourth component of complement
3.2.7 C4(-)GPS—C4-deficient guinea pig serum [serum
for testing materials for other aspects of biocompatibility.
PracticeF1984providesguidancefortestingsolidmaterialsfor from guinea pigs genetically incapable of producing C4]
3.2.8 EDTA—ethylenediaminetetraacetic acid, disodium
whole complement activation in human serum, but does not
discriminate between the classical or alternative pathway of salt, dihydrate
3.2.9 HAGG—heat aggregated gamma globulin
activation. Practice F2065 provides guidance for testing solid
materials for alternative pathway complement activation in 3.2.10 HS—human serum
3.2.11 I—“ice” control tube with serum but no material,
serum.
1.6 This standard does not purport to address all of the kept on ice
3.2.12 M—tube containing serum plus a test material
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro- 3.2.13 NM—tube containing serum but no material
3.2.14 RBC—red blood cell(s)
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Surgical Materials and Devices and is the direct responsibility of Subcommittee Standards volume information, refer to the standard’s Document Summary page on
F04.16 on Biocompatibility Test Methods. the ASTM website.
Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
Current edition approved Oct. 1, 2006. Published October 2006. DOI: 10.1520/
F2567-06. 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
4. Summary of Practice 5.4 Assessment of in vitro classical complement activation
as described here provides one method for predicting potential
4.1 This practice is based on a method published by Gaither
4 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 C8 (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). If the classical complement pathway is
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 compo-
Exposed serum is then tested for remaining C4 functional
nents, including C4, to the materials occurs in part A of this
activity. An appropriate dilution of the HS, which by itself is
practice, a false positive for classical pathway activation will
too dilute to lyse sensitized sheep RBC, is added to hemolysin-
be observed in step B. Classical pathway complement activa-
coated sheep RBC in the presence of C4(-)GPS in which all
tionbythetestmaterialmaybeconfirmedbydemonstratingan
complement components save the missing C4 are present in
absence of C4 bound to the material following removal of the
excess (Procedure B). Hemolysis in Procedure B provides a
serum,and/orproductionofcomplementsplit-productssuchas
quantitativemeasureoftheC4remaininginHSexposedtotest
C4d in the serum (as determined by immunoassay). Although
material in Procedure A. Depletion of hemolysis indicates
immunoassay could be done in place of this screening proce-
specific classical pathway activation in the human serum
dure, determination of C4d production alone may not be
caused by exposure to the test material.
functionally significant. This practice does not detect trivial
amounts of classical activation unable to affect functional lysis
5. Significance and Use
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
6.1 Buffers are prepared according to established protocols
complement directly by the alternative pathway, or indirectly
(2, 3). “Water” refers throughout to distilled, endotoxin-free
because of antigen-bound antibodies (as with immuno-
H O. The use of barbital (veronal) buffer is recommended. In
adsorption columns) by the classical pathway. This practice is
the United States, barbital is a class IVregulated substance and
useful as a simple, inexpensive, function-based screening
requiresaDEA(4)licenseforpurchase.Theuseofotherbuffer
method for determining complement activation by solid mate-
systems (such as TRIS) is permissible if they have been
rials in vitro by the classical pathway.
demonstrated not to activate complement (5).
5.2 This practice is composed of two parts. In part A
6.2 5X Stock BBS (barbital-buffered saline) is prepared by
(Section 11), HS is exposed to a solid material. If complement
adding 20.75 g NaCl plus 2.545 g sodium barbital (sodium-
activation occurs bytheclassicalpathway,C4willbedepleted.
5,5-diethyl barbiturate) to about 400 mL water. The pH is
Activation by the alternative pathway will not deplete C4. In
adjusted to 7.35 with 1 N HCl, then brought to a final volume
part B (Section 12), C4 activity remaining in the serum after
of 500 mL in a volumetric flask.
exposure to the test material is assayed by diluting the serum
6.3 Metals Solution is prepared by making a 2.0 M solution
below the concentration needed to lyse antibody-coated sheep
ofMgCl (40.66gMgCl·6H Ointo100mLwater),anda0.3
2 2
RBC on its own, then adding the diluted HS to C4(-)GPS
M solution of CaCl (4.41 g CaCl ·2H O into 100 mLwater),
2 2 2
(which is itself at a dilution where all complement components
and combining the two solutions 1:1 (v:v). These solutions are
are in excess save the missing C4). Lacking C4, the C4(-)GPS
stable for one month at 4°C.
does not lyse the antibody-coated sheep RBC unless C4 is
6.4 Ca Buffer (BBS-GM Working Solution)isprepareddaily,
present in the added HS. The proportion of lysis remaining in
by dissolving 0.25 g gelatin in 50 mL water that is gently
the material-exposed HS sample versus the 37°C control HS
heated and stirred. The gelatin solution is added to 50 mL 5X
sample (which was not exposed to the test material) indicates
StockBBSplus0.25mLMetalsSolution,broughttoabout200
the amount of C4 present in the HS, loss of which correlates
mL, then adjusted to pH 7.35 (with 1 N HCl or 1 N NaOH)
with classical pathway activation.
before bringing the final volume to 250 mL in a volumetric
++ ++
5.3 This function-based in vitro test method for classical
flask. Ca buffer contains both Mg and Ca , which allows
pathway complement activation is suitable for adoption in
both classical and alternative pathway complement activation
specifications and standards for screening solid materials for
to occur.
use in the construction of medical devices intended to be
6.5 BBS-G Working Solution is prepared the same way, but
implanted in the human body or placed in contact with human
omitting addition of the metals solution.
blood outside the body. It is designed to be used in conjunction
6.6 103 Stock EDTA (0.1 M disodium dihydrate EDTA) is
with Practice F1984 for function-based whole complement
prepared by adding 7.44 g disodium EDTA.·2H O to about
activation screening, and Practice F2065 for function-based
160 mL water, adjusting the pH to 7.65 (with 1 N NaOH or 1
alternative pathway activation screening.
N HCl), then bringing the volume to 200 mL in a volumetric
flask.
6.7 BBS-G-EDTA (to be used in preparing RBC before
being washed out with Ca buffer) is prepared by adding 10 mL
The boldface numbers in parentheses refer to the list of references at the end of
this standard. of stock 10X EDTAto 90 mL of BBS-G in a volumetric flask.
F2567–06
7. Preparation of Sheep RBC 9. Determination of Optimal Hemolysin Concentration
7.1 Commercially-obtained sheep RBC preserved inAlsev-
9.1 Determination of optimal hemolysin concentration is
er’s solution are stored at 4°C. The sheep cells are discarded necessary in order to conserve expensive reagents and to avoid
after eight weeks or when the supernatant liquid from the
prozone effects. Commercial rabbit anti-sheep RBC serum
second wash contains hemoglobin by visual inspection (as lots (hemolysin) is thawed (or, if lyophilized, reconstituted with
of RBCs age, they increase in sensitivity to complement lysis
distilled endotoxin-free water), heat-inactivated at 56°C for 30
in parallel with increased spontaneous lysis). min to inactivate the rabbit complement, aliquoted in conve-
nient volumes, and stored at –70°C until used.
NOTE 1—All centrifugations are at 4°C. Except when indicated, all
9.2 To cold 133100 mm disposable glass tubes, placed in a
reagents, tubes, and cell preparations are kept on ice or in an ice slurry. In
rack in an ice-slurry, 50 µL of washed sheep RBC at 3310
subsequent sections where the word “cold” is used, that denotes tubes in
ice or sitting in an ice slurry. cells/mL is added directly to the bottom of each tube. If
statistical evaluation of the results is desired, three replicate
7.2 Five mL of sheep RBC are centrifuged at 10003 g, at
tubes for each condition should be used. Otherwise, duplicates
4°C, for 10 min.
or even single dilution tubes are sufficient. One set of three
7.3 The cell pellet is resuspended in 10 mL of cold
replicate tubes receives only 50 µLof cold Ca buffer/tube (“no
BBS-G-EDTAand incubated for 10 min at 37°C. The cells are
RBC” control, for complement color).
centrifuged, and the pellet resuspended in 10 mL of BBS-G-
9.3 To the RBC-containing tubes, one set of three tubes gets
EDTA.
0.35 mLcold distilled H O/tube (“total lysis” control), another
7.4 The cells are centrifuged, the supernatant discarded
gets 50 µL mL Ca buffer (“no hemolysin” control), and the
(first wash), and the pellet resuspended in 10 mL of cold
other sets get 50 µL mL each of 1:2 serial dilutions of
BBS-GM (Ca Buffer). This step is repeated twice more for a
hemolysin (“tests”). Dilutions between 1:200 to 1:25 600
total of three washes.
antibody are recommended, with two sets of 3 tubes each for
7.5 Adjust cell concentration by counting with a hemocy-
1:200. The “no RBC” control receives 50 µL of additional
tometer, and prepare 10 mL of 3.0310 cells/mL in cold
BBS-GM instead of hemolysin. All tubes except “total lysis”
BBS-GM.
controls should each contain at this point a total 0.1 mL.
7.6 The washed, diluted RBC can be held on ice and used
9.4 Each tube is quickly mixed by gentle shaking to
for at least 12 h.
resuspend cells, the rack is placed in a 37°C water bath,
incubated 10 min, then returned to the ice-slurry.
8. Absorption of Serum (Complement)
9.5 One of the two 3-tube sets of 1:200 hemolysin gets 0.1
8.1 Serum should be absorbed with sheep RBC in order to
mL of cold Ca buffer (“no-complement” control). All other
remove any naturally-occurring anti-sheep hemolytic antibod-
tubes besides the “total lysis” control set get 0.1 mL cold
ies. The procedure is as follows. absorbed HS (C8) diluted 1:100 or 1:200.
8.2 Commercially-available HS and C4(-)GPS are stored at
NOTE 2—For a particular lot of human serum, a 1:100 or 1:200 dilution
–70°C. Both sera should be absorbed separately.
should provide sufficient complement activity. Also, percent lysis in the
8.3 Serum is thawed on ice or reconstituted (if lyophilized)
“no-hemolysin” (complement only) control should not exceed 10 %. If
with ice-cold (4°C) water. lysis with the 1:100 dilution of complement exceeds 10 %, use 1:200. If
the“no-hemolysin”controlstillexceeds10 %,adifferentlotofserumwill
8.4 All manipulations are done on ice, with ice-cold re-
need to be tested.
agents and cells. Centrifugations are carried out at 10003gat
4°C. It is critical that this entire procedure be done in the cold
9.6 All tubes except the “total lysis” controls receive an
to avoid activation of complement in this step.
addition
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