ASTM F1984-99(2003)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation:F1984–99 (Reapproved 2003)
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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope Part 4: Selection of Tests for Interactions with Blood
1.1 This practice provides a protocol for rapid, in vitro
3. Terminology
screening for whole complement activating properties of solid
3.1 Abbreviations:
materials used in the fabrication of medical devices that will
3.1.1 Ab—antibody (hemolysin).
contact blood.
3.1.2 BBS—barbital buffered saline.
1.2 This practice is intended to evaluate the acute in vitro
3.1.3 BBS-G—barbital buffered saline—gelatin.
whole complement activating properties of solid materials
3.1.4 BBS-GM—barbital buffered saline—gelatin metals.
intended for use in contact with blood. For this practice, the
3.1.5 C8—complement.
words “serum” and “complement” are used interchangeably
3.1.6 EDTA—ethylenediaminetetraacetic acid, disodium
(most biological supply houses use these words synonymously
salt: dihydrate.
in reference to serum used as a source of complement).
3.1.7 HS—human serum.
1.3 This practice consists of two procedural parts. Proce-
3.1.8 PVDF—polyvinylidene fluoride.
dureAdescribesexposureofsolidmaterialstoastandardlotof
3.1.9 RBC—red blood cell(s).
human serum, using a 0.1-mL serum/13 x 100-mm disposable
test tube. Cellulose acetate powders and fibers are used as
4. Summary of Practice
examples of test materials. Procedure B describes assaying the
4.1 Solid material specimens are exposed to contact with a
exposed serum for significant functional whole complement
standard lot of complement under defined conditions (Proce-
depletion as compared to control samples.
dureA).Exposedserumthenistestedforsignificantfunctional
1.4 This practice does not address function, elaboration, or
complement depletion compared to controls under identical
depletion of individual complement components, nor does it
conditions (Procedure B).
address the use of plasma as a source of complement.
1.5 This practice is one of several developed for the
5. Significance and Use
assessmentofthebiocompatibilityofmaterials.PracticeF748
5.1 Inappropriate activation of complement by blood-
may provide guidance for the selection of appropriate methods
contacting medical devices may have serious acute or chronic
for testing materials for other aspects of biocompatibility.
effects on the host. This practice is useful as a simple,
inexpensive screening method for determining functional
2. Referenced Documents
whole complement activation by solid materials in vitro.
2.1 ASTM Standards:
5.2 This practice is composed of two parts. In Part A
F748 Practice for Selecting Generic Biological Test Meth-
(Section 11), human serum is exposed to a solid material.
ods for Materials and Devices
Complement may be depleted by the classical or alternative
2.2 ISO Document:
pathways. In principle, nonspecific binding of certain comple-
ISO 10993-4: Biological Evaluation of Medical Devices,
mentcomponentsalsomayoccur.Thealternativepathwaycan
deplete later acting components common to both pathways,
that is components other than C1, C4, and C3 (1). In Part B
(Section12),complementactivityremainingintheserumafter
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland
exposure to the test material is assayed by classical pathway-
Surgical Materials and Devices and is the direct responsibility of Subcommittee
mediated lysis of sensitized RBC.
F04.16 on Biocompatibility Test Methods.
Current edition approved Nov. 1, 2003. Published December 2003. Originally
approved in 1999. Last previous edition approved in 1999 as F1984–99.
2 3
For referenced ASTM standards, visit the ASTM website, www.astm.org, or Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM 4th Floor, New York, NY 10036.
Standards volume information, refer to the standard’s Document Summary page on Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
the ASTM website. this specification.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F1984–99 (2003)
5.3 Assessment of in vitro whole complement activation, as 7.2 Five mL of sheep RBC are centrifuged at 1 000xgfor
described here, provides one method for predicting potential 10 min.
complement activation by medical materials intended for 7.3 The cell pellet is resuspended in 10 mL of cold
clinical application in humans when the material contacts the BBS-G-EDTAand incubated for 10 min at 37°C.The cells are
blood. Other test methods for complement activation are centrifuged, and the pellet resuspended in 10 mL of BBS-G-
available, including assays for specific complement compo- EDTA.
nents and their split products (see X1.3 and X1.4). 7.4 The cells are centrifuged, the supernatant discarded
(first wash), and the pellet resuspended in 10 mL of cold
5.4 This in vitro test method is suitable for adoption in
BBS-GM. Repeat twice (total of three washes).
specifications and standards for screening solid materials for
7.5 Adjust cell count spectrophotometrically (where an
use in the construction of medical devices intended to be
absorbanceof0.56correspondsto1.5x10 sheepRBC/mL,at
implanted in the human body or placed in contact with human
a wavelength of 412 nm and a 1.0-cm light path for 1 volume
blood.
of cells in BBS-GM plus 24 volumes of water) or count with
a hemocytometer, preparing 10 mL of 1.5 x 10 cells/mL in
6. Preparation of Buffers
cold BBS-GM.
6.1 Buffers, are prepared according to detailed protocol (2).
7.6 The washed, diluted RBC can be held on ice and used
“Water” refers throughout to distilled, endotoxin-free water.
for at least 12 h.
The use of barbital (veronal) buffer is recommended. Barbital
isaclassIVregulatedsubstanceandrequiresaDEA(3)license
8. Absorption of Serum (Complement)
forpurchase.Theuseofotherbuffersystems,suchas,TRIS,is
8.1 The use of human complement is required since there
permissible if they have been demonstrated not to activate
are species differences in the efficiency of complement activa-
complement(4).
tion and the test materials are to be used in humans. Human
6.2 5X Stock BBS (barbital-buffered saline), is prepared by
serum suitable as a source of complement may be purchased
adding 20.75 g NaCl plus 2.545 g sodium barbital (sodium-
from biological supply houses, and generally, is labeled as
5,5-diethyl barbiturate) to about 400 mL water. The pH is
reagent-grade complement.
adjusted to 7.35 with 1 N HCl, then brought to a final volume
8.2 Human serum may be absorbed with sheep RBC in
of 500 mL in a volumetric flask.
ordertoremovenaturally-occurringanti-sheepRBChemolytic
6.3 MetalsSolution,ispreparedbymakinga2.0Msolution
antibodies, though for most purposes, the amount of hetero-
of MgCl (40.66 g MgCl•6H O into 100 mL distilled
2 2 2
phile antibody in human serum is not enough to influence the
endotoxin-free water), and a 0.3 M solution of CaCl (4.41 g
2 reaction assuming the cells are optimally sensitized with
CaCl•2H Ointo100mLdistilledendotoxin-freewater),and
2 2
hemolysin. The procedure is detailed in 8.3-8.8.
combining the two solutions 1:1 (v:v). These solutions are
8.3 Freshhumanserumoracommerciallotofhumanserum
stable one month at 4°C.
is obtained and stored at −70°C. Fresh serum is preferred as
6.4 BBS-GM Working Solution, is prepared daily, by dis-
lyophilized complement often is not as active as fresh serum.
solving 0.25 g gelatin in 50 mL endotoxin-free distilled water
8.4 The serum is thawed on ice or reconstituted (if lyo-
that is gently heated and stirred. The gelatin solution is added
philized) with ice-cold (4°C) distilled endotoxin-free water.
to 50 mL5X stock BBS plus 0.25 mLmetals solution, brought
8.5 Allmanipulationsaredoneonice,withicecoldreagents
up to about 200 mL, then adjusted to pH 7.35 (with1NHCl
and cells; centrifugations are carried out at 1000xgat 4°C. It
or 1 N NaOH) before bringing the final volume to 250 mL in
iscriticalthatthisentireprocedurebedoneinthecoldtoavoid
a volumetric flask.
activation of complement in this step.
6.5 BBS-G Working Solution, is prepared the same way, but
8.6 Cold serum and cold, packed, washed sheep RBC are
the addition of the metals solution is omitted.
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
6.6 10X Stock EDTA (0.1 M disodium dihydrate EDTA),is
preparedbyadding7.44gdisodiumEDTA•2H Otoabout160 supernatant is transferred carefully to a new container on ice.
8.7 The procedure in 8.6 is repeated twice.
mL water, adjusting the pH to 7.65 (with 1 N NaOH or 1 N
HCl), then bringing the volume to 200 mL in a volumetric 8.8 The absorbed human serum is stored in 0.5–1.0-mL
aliquots (convenient for one-experiment use), in cold snap-cap
flask.
microfuge tubes and kept at −70°C until used.Aliquots should
6.7 BBS-G-EDTA (to be used in preparing RBC before
be thawed on ice, used on the day of thawing, and not be
being washed out), is prepared by adding 10 mLof stock 10X
refrozen.
EDTA to 90 mL of BBS-G in a volumetric flask.
9. Determination of Optimal Hemolysin Concentration
7. Preparation of Sheep RBC
9.1 Determination of optimal hemolysin concentration is
7.1 Commercially-obtained sheep red blood cells (RBC)
necessaryinordertoconserveexpensivereagentsandtoavoid
preserved inAlsever’s solution are stored at 4°C.The cells are
prozone effects. Commercial rabbit anti-sheep RBC serum
discarded after eight weeks or when the supernatant from the
(Hemolysin) is obtained, thawed, or, if lyophilized, reconsti-
second wash contains hemoglobin by visual inspection.
tuted with distilled endotoxin-free water), heat-inactivated at
56°Cfor30mintoinactivatetherabbitcomplement,aliquoted
NOTE 1—All centrifugations are at 4°C. Except where indicated, all
reagents, tubes, and cell preparations are kept on ice. in convenient volumes, and stored at −70°C until used.
F1984–99 (2003)
9.2 To cold 13 x 100 mm disposable glass tubes, placed in disposableglasstesttubespercondition.Otherwise,duplicates
arackinanice-bath,0.1mLofwashedsheepRBCat1.5x10 or single tubes are sufficient. Tubes are numbered in advance.
cells/mL is added. If statistical evaluation of the results is Conditions include total lysis, no complement (no C’), tests
desired,threereplicatetubesforeachconditionshouldbeused. (dilutions of human serum—HS) with and without hemolysin,
Otherwise, duplicates or even single dilution tubes are suffi- and no RBC (complement color control, at highest concentra-
cient. One set of three replicate tubes receives only 0.1 mL of tion of serum used).All reagents, tubes, and manipulations are
cold BBS-GM/tube (no RBC control, for complement color). done ice-cold, with tubes held in a rack in an ice slurry.
9.3 TotheRBC-containingtubes,onesetofthreetubesgets 10.2 Washed RBC are added to all tubes except no RBC
1.1 mL cold distilled H O/tube (total lysis control), another tubes (0.1 mL/tube of a 1.5 x 10 cells/mL suspension). No
gets 0.1 mL BBS-GM (no hemolysin control), and the other RBC tubes get 0.1 mL cold buffer.
setsget0.1mLeachof1:2serialdilutionsofhemolysin(tests). 10.3 Total lysis tubes get 1.1 mL distilled H O. The no C’
Dilutions between 1:400 to 1:25 600 antibody are recom- and test with hemolysin tubes get 0.1 mL optimal hemolysin
mended, with two sets of 1:400. The no RBC control receives (see 9.10), and no RBC tubes get 0.1 mL cold BBS-GM. All
0.1 mL of additional BBS-GM. tubes are shaken to resuspend cells, incubated in a 37°C water
9.4 Each tube is mixed quickly by gentle shaking to bath for 10 min, and placed back on ice.
resuspend cells, the rack is placed in a 37°C water bath,
NOTE 3—Another acceptable procedure is to make up one large batch
incubated 10 min, then returned to the ice bath.
of hemolysin-sensitized erythrocytes to cover all the tests planned within
9.5 One of the two sets of 1:400 antibody gets 1.0 mL of
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,
new sensitized cells are prepared. These sensitized cells are ready to use,
sides the total lysis control set get 0.5 mLcold BBS-GM, then
making the addition of hemolysin to each tube unnecessary, which
0.5 mLof absorbed human serum (complement) diluted 1:100
simplifies the experiment. Unsensitized RBC can be used as controls for
or 1:200.
nonspecific lysis.
NOTE 2—Foraparticularlotofhumanserum,a1:100or1:200dilution
10.4 To all but the total lysis tubes, a maximum volume of
should provide sufficient complement activity. Also, percent lysis in the
1.0 mL of cold BBS-GM is added, reduced by the amount of
no-hemolysin (complement only) control should not exceed 10%. If lysis
diluted serum (see 10.5), which will be added at a maximum
with the 1:100 dilution of complement exceeds 10%, use 1:200. If the
0.5 mL volume. The no C’ tubes get 1.0 mL BBS-GM.
no-hemolysincontrolstillexceeds10%,adifferentlotofserumwillneed
10.5 The cold serum is diluted in cold BBS-GM to the
to be tested.
desired concentration (with minimal agitation). It is recom-
9.6 Tubes are shaken manually to suspend cells, then the
mended to test the HS initially at 1:50 to 1:300. The diluted
rack is incubated in a 37°C water bath for 1h, and intermit-
serum is added to each test tube in a 0.5 mL volume. Final
tently shaken to keep cells in suspension.
volume in each tube should be made up to 1.2 mL with
9.7 At the end of 1h, the rack is placed on ice. The cold
BBS-GM.
tubes then are centrifuged at 1 000 x g for 10 min at 4°C, and
10.6 The tubes then are treated as detailed in 9.6-9.9.
the supernatants decanted to correspondingly numbered 13 x
10.7 Theoptimalhumanserumdilutionofaparticularlotof
100-mm glass tubes.
human serum is defined as that in which the nonspecific lysis
9.8 Absorbance of the supernatants is measured at 412 nm.
(HS + RBC, in absence of hemolysin antibody) is #10%,
Percent lysis is calculated for each test and control tube by
while specific lysis (total lysis [RBC + Ab + HS] minus
subtracting from the 412 nm absorbance the no RBC control
nonspecific lysis) is at least 20% but not greater than 80%,
(mean of the three replicate tubes), dividing by the tota
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