ASTM F1984-99(2018)
(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.
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
General Information
Relations
Standards Content (Sample)
This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: F1984 − 99 (Reapproved 2018)
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 2. Referenced Documents
1.1 This practice provides a protocol for rapid, in vitro 2.1 ASTM Standards:
screening for whole complement activating properties of solid F748PracticeforSelectingGenericBiologicalTestMethods
materials used in the fabrication of medical devices that will for Materials and Devices
contact blood. 2.2 ISO Document:
ISO 10993-4:Biological Evaluation of Medical Devices,
1.2 This practice is intended to evaluate the acute in vitro
Part 4: Selection of Tests for Interactions with Blood
whole complement activating properties of solid materials
intended for use in contact with blood. For this practice, the
3. Terminology
words “serum” and “complement” are used interchangeably
3.1 Abbreviations:
(most biological supply houses use these words synonymously
3.1.1 Ab—antibody (hemolysin).
in reference to serum used as a source of complement).
3.1.2 BBS—barbital buffered saline.
1.3 This practice consists of two procedural parts. Proce-
3.1.3 BBS-G—barbital buffered saline—gelatin.
dureAdescribesexposureofsolidmaterialstoastandardlotof
human serum, using a 0.1-mL serum/13 x 100-mm disposable
3.1.4 BBS-GM—barbital buffered saline—gelatin metals.
test tube. Cellulose acetate powders and fibers are used as
3.1.5 C'—complement.
examples of test materials. Procedure B describes assaying the
3.1.6 EDTA—ethylenediaminetetraacetic acid, disodium
exposed serum for significant functional whole complement
salt: dihydrate.
depletion as compared to control samples.
3.1.7 HS—human serum.
1.4 This practice does not address function, elaboration, or
3.1.8 PVDF—polyvinylidene fluoride.
depletion of individual complement components, nor does it
address the use of plasma as a source of complement.
3.1.9 RBC—red blood cell(s).
1.5 This practice is one of several developed for the
4. Summary of Practice
assessment of the biocompatibility of materials. Practice F748
4.1 Solid material specimens are exposed to contact with a
may provide guidance for the selection of appropriate methods
standard lot of complement under defined conditions (Proce-
for testing materials for other aspects of biocompatibility.
dureA).Exposedserumthenistestedforsignificantfunctional
1.6 The values stated in SI units are to be regarded as
complement depletion compared to controls under identical
standard. No other units of measurement are included in this
conditions (Procedure B).
standard.
5. Significance and Use
1.7 This international standard was developed in accor-
dance with internationally recognized principles on standard-
5.1 Inappropriate activation of complement by blood-
ization established in the Decision on Principles for the
contacting medical devices may have serious acute or chronic
Development of International Standards, Guides and Recom-
effects on the host. This practice is useful as a simple,
mendations issued by the World Trade Organization Technical
inexpensive screening method for determining functional
Barriers to Trade (TBT) Committee.
whole complement activation by solid materials in vitro.
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 Feb. 1, 2018. Published April 2018. Originally the ASTM website.
approved in 1999. Last previous edition approved in 2013 as F1984–99 (2013). Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
DOI: 10.1520/F1984-99R18. 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 (2018)
5.2 This practice is composed of two parts. In Part A mL water, adjusting the pH to 7.65 (with 1 N NaOH or 1 N
(Section 11), human serum is exposed to a solid material. HCl), then bringing the volume to 200 mL in a volumetric
Complement may be depleted by the classical or alternative flask.
pathways. In principle, nonspecific binding of certain comple-
6.7 BBS-G-EDTA (to be used in preparing RBC before
mentcomponentsalsomayoccur.Thealternativepathwaycan
being washed out), is prepared by adding 10 mLof stock 10X
deplete later acting components common to both pathways,
EDTA to 90 mL of BBS-G in a volumetric flask.
that is components other than C1, C4, and C3 (1). In Part B
(Section12),complementactivityremainingintheserumafter
7. Preparation of Sheep RBC
exposure to the test material is assayed by classical pathway-
7.1 Commercially-obtained sheep red blood cells (RBC)
mediated lysis of sensitized RBC.
preserved inAlsever’s solution are stored at 4°C.The cells are
5.3 Assessment of in vitro whole complement activation, as
discarded after eight weeks or when the supernatant from the
described here, provides one method for predicting potential
second wash contains hemoglobin by visual inspection.
complement activation by medical materials intended for
NOTE 1—All centrifugations are at 4°C. Except where indicated, all
clinical application in humans when the material contacts the
reagents, tubes, and cell preparations are kept on ice.
blood. Other test methods for complement activation are
available, including assays for specific complement compo-
7.2 Five mL of sheep RBC are centrifuged at 1 000xgfor
nents and their split products (see X1.3 and X1.4). 10 min.
5.4 This in vitro test method is suitable for adoption in
7.3 The cell pellet is resuspended in 10 mL of cold
specifications and standards for screening solid materials for BBS-G-EDTAand incubated for 10 min at 37°C.The cells are
use in the construction of medical devices intended to be
centrifuged, and the pellet resuspended in 10 mL of BBS-G-
implanted in the human body or placed in contact with human EDTA.
blood.
7.4 The cells are centrifuged, the supernatant discarded
(first wash), and the pellet resuspended in 10 mL of cold
6. Preparation of Buffers
BBS-GM. Repeat twice (total of three washes).
6.1 Buffers, are prepared according to detailed protocol(2).
7.5 Adjust cell count spectrophotometrically (where an
“Water” refers throughout to distilled, endotoxin-free water.
absorbanceof0.56correspondsto1.5x10 sheepRBC/mL,at
The use of barbital (veronal) buffer is recommended. Barbital
a wavelength of 412 nm and a 1.0-cm light path for 1 volume
isaclassIVregulatedsubstanceandrequiresaDEA (3)license
of cells in BBS-GM plus 24 volumes of water) or count with
forpurchase.Theuseofotherbuffersystems,suchas,TRIS,is
a hemocytometer, preparing 10 mL of 1.5 x 10 cells/mL in
permissible if they have been demonstrated not to activate
cold BBS-GM.
complement(4).
7.6 The washed, diluted RBC can be held on ice and used
6.2 5X Stock BBS (barbital-buffered saline), is prepared by
for at least 12 h.
adding 20.75 g NaCl plus 2.545 g sodium barbital (sodium-5,
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
preparedbyadding7.44gdisodiumEDTA•2H Otoabout160
8.5 Allmanipulationsaredoneonice,withicecoldreagents
and cells; centrifugations are carried out at 1000xgat 4°C. It
iscriticalthatthisentireprocedurebedoneinthecoldtoavoid
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this specification. activation of complement in this step.
F1984 − 99 (2018)
8.6 Cold serum and cold, packed, washed sheep RBC are subtracting from the 412 nm absorbance the no RBC control
mixed, 0.1 mL RBC/2.5 mL serum, incubated for 10 min on (mean of the three replicate tubes), dividing by the total lysis
ice, then centrifuged at 1 000 x g for 10 min at 4°C. The control value (mean of the three replicate tubes), and multi-
supernatant is transferred carefully to a new container on ice. plying by 100.
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
standard deviation of the three %lysis values for each three-
microfuge tubes and kept at −70°C until used.Aliquots should
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
2 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-hem
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