ASTM F1984-99(2013)
(Practice)Standard Practice for Testing for Whole Complement Activation in Serum by Solid Materials
Standard Practice for Testing for Whole Complement Activation in Serum by Solid Materials
SIGNIFICANCE AND USE
5.1 Inappropriate activation of complement by blood-contacting medical devices may have serious acute or chronic effects on the host. This practice is useful as a simple, inexpensive screening method for determining functional whole complement activation by solid materials in vitro.
5.2 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 alternative pathways. In principle, nonspecific binding of certain complement components also may occur. The alternative pathway can deplete later acting components common to both pathways, that is components other than C1, C4, and C3 (1) .4 In Part B (Section 12), complement activity remaining in the serum after exposure to the test material is assayed by classical pathway-mediated lysis of sensitized RBC.
5.3 Assessment of in vitro whole complement activation, as described here, provides one method for predicting potential complement activation by medical materials intended for clinical application in humans when the material contacts the blood. Other test methods for complement activation are available, including assays for specific complement components and their split products (see X1.3 and X1.4).
5.4 This in vitro test method is suitable for adoption in specifications and standards for screening solid materials for use in the construction of medical devices intended to be implanted in the human body or placed in contact with human blood.
SCOPE
1.1 This practice provides a protocol for rapid, in vitro screening for whole 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 whole complement activating properties of solid materials intended for use in contact with blood. For this practice, the words “serum” and “complement” are used interchangeably (most biological supply houses use these words synonymously in reference to serum used as a source of complement).
1.3 This practice consists of two procedural parts. Procedure A describes exposure of solid materials to a standard lot of human serum, using a 0.1-mL serum/13 x 100-mm disposable test tube. Cellulose acetate powders and fibers are used as examples of test materials. Procedure B describes assaying the exposed serum for significant functional whole complement depletion as compared to control samples.
1.4 This practice does not address function, elaboration, or depletion of individual complement components, nor does it 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 F748 may provide guidance for the selection of appropriate methods for testing materials for other aspects of biocompatibility.
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: F1984 − 99 (Reapproved 2013)
Standard Practice for
Testing for Whole Complement Activation in Serum by Solid
Materials
This standard is issued under the fixed designation F1984; 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 F748PracticeforSelectingGenericBiologicalTestMethods
for Materials and Devices
1.1 This practice provides a protocol for rapid, in vitro
2.2 ISO Document:
screening for whole complement activating properties of solid
ISO 10993-4:Biological Evaluation of Medical Devices,
materials used in the fabrication of medical devices that will
Part 4: Selection of Tests for Interactions with Blood
contact blood.
1.2 This practice is intended to evaluate the acute in vitro
3. Terminology
whole complement activating properties of solid materials
3.1 Abbreviations:
intended for use in contact with blood. For this practice, the
3.1.1 Ab—antibody (hemolysin).
words “serum” and “complement” are used interchangeably
3.1.2 BBS—barbital buffered saline.
(most biological supply houses use these words synonymously
3.1.3 BBS-G—barbital buffered saline—gelatin.
in reference to serum used as a source of complement).
3.1.4 BBS-GM—barbital buffered saline—gelatin metals.
1.3 This practice consists of two procedural parts. Proce-
dureAdescribesexposureofsolidmaterialstoastandardlotof
3.1.5 C'—complement.
human serum, using a 0.1-mL serum/13 x 100-mm disposable
3.1.6 EDTA—ethylenediaminetetraacetic acid, disodium
test tube. Cellulose acetate powders and fibers are used as
salt: dihydrate.
examples of test materials. Procedure B describes assaying the
3.1.7 HS—human serum.
exposed serum for significant functional whole complement
3.1.8 PVDF—polyvinylidene fluoride.
depletion as compared to control samples.
3.1.9 RBC—red blood cell(s).
1.4 This practice does not address function, elaboration, or
depletion of individual complement components, nor does it
4. Summary of Practice
address the use of plasma as a source of complement.
4.1 Solid material specimens are exposed to contact with a
1.5 This practice is one of several developed for the
standard lot of complement under defined conditions (Proce-
assessment of the biocompatibility of materials. Practice F748
dureA).Exposedserumthenistestedforsignificantfunctional
may provide guidance for the selection of appropriate methods
complement depletion compared to controls under identical
for testing materials for other aspects of biocompatibility.
conditions (Procedure B).
1.6 The values stated in SI units are to be regarded as
5. Significance and Use
standard. No other units of measurement are included in this
standard.
5.1 Inappropriate activation of complement by blood-
contacting medical devices may have serious acute or chronic
2. Referenced Documents
effects on the host. This practice is useful as a simple,
2.1 ASTM Standards:
inexpensive screening method for determining functional
whole complement activation by solid materials in vitro.
1 5.2 This practice is composed of two parts. In Part A
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland
Surgical Materials and Devices and is the direct responsibility of Subcommittee
(Section 11), human serum is exposed to a solid material.
F04.16 on Biocompatibility Test Methods.
Complement may be depleted by the classical or alternative
Current edition approved Dec. 1, 2013. Published February 2014. Originally
pathways. In principle, nonspecific binding of certain comple-
approved in 1999. Last previous edition approved in 2008 as F1984–99 (2008).
mentcomponentsalsomayoccur.Thealternativepathwaycan
DOI: 10.1520/F1984-99R13.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
the ASTM website. 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
F1984 − 99 (2013)
deplete later acting components common to both pathways, 6.7 BBS-G-EDTA (to be used in preparing RBC before
that is components other than C1, C4, and C3 (1). In Part B being washed out), is prepared by adding 10 mLof stock 10X
(Section12),complementactivityremainingintheserumafter EDTA to 90 mL of BBS-G in a volumetric flask.
exposure to the test material is assayed by classical pathway-
7. Preparation of Sheep RBC
mediated lysis of sensitized RBC.
7.1 Commercially-obtained sheep red blood cells (RBC)
5.3 Assessment of in vitro whole complement activation, as
preserved inAlsever’s solution are stored at 4°C.The cells are
described here, provides one method for predicting potential
discarded after eight weeks or when the supernatant from the
complement activation by medical materials intended for
second wash contains hemoglobin by visual inspection.
clinical application in humans when the material contacts the
blood. Other test methods for complement activation are
NOTE 1—All centrifugations are at 4°C. Except where indicated, all
reagents, tubes, and cell preparations are kept on ice.
available, including assays for specific complement compo-
nents and their split products (see X1.3 and X1.4).
7.2 Five mL of sheep RBC are centrifuged at 1 000xgfor
10 min.
5.4 This in vitro test method is suitable for adoption in
specifications and standards for screening solid materials for
7.3 The cell pellet is resuspended in 10 mL of cold
use in the construction of medical devices intended to be BBS-G-EDTAand incubated for 10 min at 37°C.The cells are
implanted in the human body or placed in contact with human
centrifuged, and the pellet resuspended in 10 mL of BBS-G-
blood. EDTA.
7.4 The cells are centrifuged, the supernatant discarded
6. Preparation of Buffers
(first wash), and the pellet resuspended in 10 mL of cold
BBS-GM. Repeat twice (total of three washes).
6.1 Buffers, are prepared according to detailed protocol(2).
“Water” refers throughout to distilled, endotoxin-free water.
7.5 Adjust cell count spectrophotometrically (where an
The use of barbital (veronal) buffer is recommended. Barbital
absorbanceof0.56correspondsto1.5x10 sheepRBC/mL,at
isaclassIVregulatedsubstanceandrequiresaDEA (3)license
a wavelength of 412 nm and a 1.0-cm light path for 1 volume
forpurchase.Theuseofotherbuffersystems,suchas,TRIS,is
of cells in BBS-GM plus 24 volumes of water) or count with
permissible if they have been demonstrated not to activate 8
a hemocytometer, preparing 10 mL of 1.5 x 10 cells/mL in
complement(4).
cold BBS-GM.
6.2 5X Stock BBS (barbital-buffered saline), is prepared by
7.6 The washed, diluted RBC can be held on ice and used
adding 20.75 g NaCl plus 2.545 g sodium barbital (sodium-5,
for at least 12 h.
5-diethyl barbiturate) to about 400 mL water. The pH is
8. Absorption of Serum (Complement)
adjusted to 7.35 with 1 N HCl, then brought to a final volume
of 500 mL in a volumetric flask.
8.1 The use of human complement is required since there
are species differences in the efficiency of complement activa-
6.3 MetalsSolution,ispreparedbymakinga2.0Msolution
tion and the test materials are to be used in humans. Human
of MgCl (40.66 g MgCl•6H O into 100 mL distilled
2 2 2
serum suitable as a source of complement may be purchased
endotoxin-free water), and a 0.3 M solution of CaCl (4.41 g
from biological supply houses, and generally, is labeled as
CaCl•2H Ointo100mLdistilledendotoxin-freewater),and
2 2
reagent-grade complement.
combining the two solutions 1:1 (v:v). These solutions are
stable one month at 4°C.
8.2 Human serum may be absorbed with sheep RBC in
ordertoremovenaturally-occurringanti-sheepRBChemolytic
6.4 BBS-GM Working Solution, is prepared daily, by dis-
antibodies, though for most purposes, the amount of hetero-
solving 0.25 g gelatin in 50 mL endotoxin-free distilled water
phile antibody in human serum is not enough to influence the
that is gently heated and stirred. The gelatin solution is added
reaction assuming the cells are optimally sensitized with
to 50 mL5X stock BBS plus 0.25 mLmetals solution, brought
hemolysin. The procedure is detailed in 8.3 – 8.8.
up to about 200 mL, then adjusted to pH 7.35 (with1NHCl
or 1 N NaOH) before bringing the final volume to 250 mL in 8.3 Freshhumanserumoracommerciallotofhumanserum
a volumetric flask.
is obtained and stored at −70°C. Fresh serum is preferred as
lyophilized complement often is not as active as fresh serum.
6.5 BBS-G Working Solution, is prepared the same way, but
the addition of the metals solution is omitted. 8.4 The serum is thawed on ice or reconstituted (if ly-
ophilized) with ice-cold (4°C) distilled endotoxin-free water.
6.6 10X Stock EDTA (0.1 M disodium dihydrate EDTA), is
8.5 Allmanipulationsaredoneonice,withicecoldreagents
preparedbyadding7.44gdisodiumEDTA•2H Otoabout160
and cells; centrifugations are carried out at 1000xgat 4°C. It
mL water, adjusting the pH to 7.65 (with 1 N NaOH or 1 N
iscriticalthatthisentireprocedurebedoneinthecoldtoavoid
HCl), then bringing the volume to 200 mL in a volumetric
activation of complement in this step.
flask.
8.6 Cold serum and cold, packed, washed sheep RBC are
mixed, 0.1 mL RBC/2.5 mL serum, incubated for 10 min on
ice, then centrifuged at 1 000 x g for 10 min at 4°C. The
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this specification. supernatant is transferred carefully to a new container on ice.
F1984 − 99 (2013)
8.7 The procedure in 8.6 is repeated twice. testabsorbance 2noRBCcontrolabsorbance
%lysis 5 3100 (1)
totallysisabsorbance
8.8 The absorbed human serum is stored in 0.5–1.0-mL
aliquots (convenient for one-experiment use), in cold snap-cap 9.9 Final%lysisforeachconditionisexpressedasmean 6
microfuge tubes and kept at −70°C until used.Aliquots should standard deviation of the three %lysis values for each three-
be thawed on ice, used on the day of thawing, and not be replicate set.
refrozen.
9.10 Atitration curve is obtained by plotting the inverse of
the hemolysin concentration versus %specific lysis. Twice the
9. Determination of Optimal Hemolysin Concentration
concentration of hemolysin that is just on the plateau of the
9.1 Determination of optimal hemolysin concentration is titration curve is used for sensitizing RBC for subsequent
necessaryinordertoconserveexpensivereagentsandtoavoid assays (optimal hemolysin concentration). Hemolysin is
prozone effects. Commercial rabbit anti-sheep RBC serum freshly diluted from stock each day of use.
(Hemolysin) is obtained, thawed, or, if lyophilized, reconsti-
tuted with distilled endotoxin-free water), heat-inactivated at 10. Whole Complement Titration to Determine Optimal
56°Cfor30mintoinactivatetherabbitcomplement,aliquoted
Serum Dilution
in convenient volumes, and stored at −70°C until used.
10.1 If statistical evaluation of results is desired, all condi-
9.2 To cold 13 x 100 mm disposable glass tubes, placed in tions should be assayed in triplicate, using three 13 x 100
arackinanice-bath,0.1mLofwashedsheepRBCat1.5x10
disposableglasstesttubespercondition.Otherwise,duplicates
cells/mL is added. If statistical evaluation of the results is
or single tubes are sufficient. Tubes are numbered in advance.
desired,threereplicatetubesforeachconditionshouldbeused.
Conditions include total lysis, no complement (no C’), tests
Otherwise, duplicates or even single dilution tubes are suffi-
(dilutions of human serum—HS) with and without hemolysin,
cient. One set of three replicate tubes receives only 0.1 mL of
and no RBC (complement color control, at highest concentra-
cold BBS-GM/tube (no RBC control, for complement color).
tion of serum used).All reagents, tubes, and manipulations are
done ice-cold, with tubes held in a rack in an ice slurry.
9.3 TotheRBC-containingtubes,onesetofthreetubesgets
1.1 mL cold distilled H O/tube (total lysis control), another
10.2 Washed RBC are added to all tubes except no RBC
gets 0.1 mL BBS-GM (no hemolysin control), and the other
tubes (0.1 mL/tube of a 1.5 x 10 cells/mL suspension). No
setsget0.1mLeachof1:2serialdilutionsofhemolysin(tests).
RBC tubes get 0.1 mL cold buffer.
Dilutions between 1:400 to 1:25 600 antibody are
10.3 Total lysis tubes get 1.1 mL distilled H O. The no C’
recommended, with two sets of 1:400. The no RBC control
and test with hemolysin tubes get 0.1 mL optimal hemolysin
receives 0.1 mL of additional BBS-GM.
(see 9.10), and no RBC tubes get 0.1 mL cold BBS-GM. All
9.4 Each tube is mixed quickly by gentle shaking to
tubes are shaken to resuspend cells, incubated in a 37°C water
resuspend cells, the rack is placed in a 37°C water bath,
bath for 10 min, and placed back on ice.
incubated 10 min, then returned to the ice bath.
NOTE 3—Another acceptable procedure is to make up one large batch
9.5 One of the two sets of 1:400 antibody gets 1.0 mL of
of hemolysin-sensitized erythrocytes to cover all the tests planned within
one week’s time. These cells are made up at5x10 /mLand are stored at
cold BBS-GM (no-complement control). All other tubes be-
4°C. They are washed each time they are used, and if hemolysis occurs,
sides the total lysis control set get 0.5 mLcold BBS-GM, then
new sensitized cells are prepared. These sensitized cells are ready to use,
0.5 mLof absorbed human serum (complement) diluted 1:100
making the addition of hemolysin to each tube unnecessary, which
or 1:200.
simplifies the experiment. Unsensitized RBC can be used as controls for
nonspecific lysis.
NOTE2—Foraparticularlotofhumanserum,a1:100or1:200dilution
should provide sufficient complement activity. Also, percent lysis in the
10.4 To all but the total lysis tubes, a maximum volume of
no-hemolysin (complement only) control should not exceed 10%. If lysis
1.0 mL of cold BBS-GM is added, reduced by the amount of
with the 1:100 dilution of complement exceeds 10%, use 1:200. If the
diluted serum (see 10.5), which will be added at a maximum
no-hemolysincontrolstillexceeds10%,adifferentlotofserumwillneed
0.5 mL volume. The no C’ tubes get 1.0 mL BBS-GM.
to be tested.
10.5 The cold serum is diluted in cold BBS-GM to the
9.6 Tubes are shaken manually to suspend cells, then the
rack is incubated in a 37°C water bath for 1h, and intermit- desired concentration (with minimal agitation). It is recom-
mended to test the HS initially at 1:50 to 1:300. The diluted
tently shak
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