ASTM F2065-00(2010)
(Practice)Standard Practice for Testing for Alternative Pathway Complement Activation in Serum by Solid Materials (Withdrawn 2016)
Standard Practice for Testing for Alternative Pathway Complement Activation in Serum by Solid Materials (Withdrawn 2016)
SIGNIFICANCE AND USE
Inappropriate activation of complement by blood-contacting medical devices may have serious acute or chronic effects on the host. Solid medical device materials may activate the complement directly by the alternative pathway. Unlike the classical complement activation pathway (see Practice F1984), antibodies are not required for alternative pathway activation. This practice is useful as a simple, inexpensive screening method for determining alternative whole complement activation by solid materials in vitro.
This practice is composed of two parts. In Part A (Section 10) C4(-)GPS is exposed to a solid material. Since C4 is required for classical pathway activation, activation of complement in C4(-)GPS can only occur by the alternative pathway (1). In principle, nonspecific binding of certain complement components to the materials may also occur. In Part B (Section 11), complement activity remaining in the serum after exposure to the test material is assayed by alternative pathway-mediated lysis of rabbit RBC.
Assessment of in vitro whole complement activation as described here provides one method for predicting potential complement activation by solid medical device 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 in human serum (X1.3 and X1.4).
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 outside the body.
SCOPE
1.1 This practice provides a protocol for rapid, in vitro screening for alternative 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 alternative 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 C4-deficient guinea pig serum [C4(-)GPS], using 0.1-mL serum per 13 × 100-mm disposable glass test tubes. Sepharose CL-4B is used as an example of test materials. Procedure B describes assaying the exposed serum for significant functional alternative pathway complement depletion as compared to control samples. The endpoint in procedure B is lysis of rabbit RBC in buffer containing EGTA and excess Mg++.
1.4 This practice does not address function, elaboration, or depletion of individual complement components except as optional additional confirmatory information that can be acquired using human serum as the complement source. 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 F748 may provide guidance for the selection of appropriate methods for testing materials for other aspects of biocompatibility. Practice F1984 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.
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
WITHDRAWN RATIONALE
This practice provided a protocol for rapid, in vitro screening for alternative pathway complement activating properties of solid materials used in the fabrication of medical devices that contact blood.
Formerly under the jurisdiction of Committee F04 on Medical and Surgical Materials and Devices, this practice was withdrawn in April 2016. This standard is being withdraw...
General Information
Relations
Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: F2065 − 00(Reapproved 2010)
Standard Practice for
Testing for Alternative Pathway Complement Activation in
Serum by Solid Materials
This standard is issued under the fixed designation F2065; 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 1.6 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
1.1 This practice provides a protocol for rapid, in vitro
standard.
screeningforalternativepathwaycomplementactivatingprop-
erties of solid materials used in the fabrication of medical
2. Referenced Documents
devices that will contact blood.
2.1 ASTM Standards:
1.2 This practice is intended to evaluate the acute in vitro
F748PracticeforSelectingGenericBiologicalTestMethods
alternative pathway complement activating properties of solid
for Materials and Devices
materials intended for use in contact with blood. For this
F1984Practice for Testing for Whole Complement Activa-
practice, “serum” is synonymous with “complement.”
tion in Serum by Solid Materials
1.3 This practice consists of two procedural parts. Proce-
2.2 Other Document:
dureAdescribesexposureofsolidmaterialstoastandardlotof
ISO 10993-4:Biological Evaluation of Medical Devices.
C4-deficient guinea pig serum [C4(-)GPS], using 0.1-mL
Part 4: Selection of Tests for Interactions with Blood
serumper13×100-mmdisposableglasstesttubes.Sepharose
CL-4B is used as an example of test materials. Procedure B
3. Terminology
describesassayingtheexposedserumforsignificantfunctional
3.1 Definitions of Terms Specific to This Standard:
alternative pathway complement depletion as compared to
3.1.1 water—distilled, endotoxin-free.
control samples.The endpoint in procedure B is lysis of rabbit
++
3.2 Abbreviations:
RBC in buffer containing EGTA and excess Mg .
3.2.1 Ab—antibody (hemolysin)
1.4 This practice does not address function, elaboration, or
3.2.2 BBS—barbital buffered saline
depletion of individual complement components except as
3.2.3 BBS-G—barbital buffered saline – gelatin
optional additional confirmatory information that can be ac-
quired using human serum as the complement source. This
3.2.4 BBS-G-EGTA/Mg (Mg Buffer)—barbital buffered sa-
++
practice does not address the use of plasma as a source of
line – gelatin EGTA Mg
complement.
3.2.5 BBS-GM (Ca Buffer)—barbital buffered saline – gela-
1.5 This practice is one of several developed for the tin metals
assessment of the biocompatibility of materials. Practice F748
3.2.6 C'—complement
may provide guidance for the selection of appropriate methods
3.2.7 C4(-)GPS—C4-deficient guinea pig serum [serum
for testing materials for other aspects of biocompatibility.
from guinea pigs genetically incapable of producing C4, the
PracticeF1984providesguidancefortestingsolidmaterialsfor
fourth component of complement]
whole complement activation in human serum, but does not
3.2.8 EDTA—ethylenediaminetetraacetic acid, disodium
discriminate between the classical or alternative pathway of
salt: dihydrate
activation.
3.2.9 EGTA—ethylene glyco-bis(β-aminoethyl ether)-N,N,
N',N'-tetraacetic acid, tetrasodium salt
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland
Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.16 on Biocompatibility Test Methods. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved Sept. 1, 2010. Published November 2010. Originally contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
approved in 2000. Last previous edition approved in 2006 as F2065–00 (2006). Standards volume information, refer to the standard’s Document Summary page on
DOI: 10.1520/F2065-00R10. the ASTM website.
2 4
Sepharose is a registered trademark of Pharmacia, Inc. (now GE Healthcare), Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, U.K. 4th Floor, New York, NY 10036.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2065 − 00 (2010)
3.2.10 HAGG—heat aggregated gamma globulin 6. Preparation of Buffers
3.2.11 HS—human serum
6.1 Buffers are prepared in accordance with established
protocols (1, 2). “Water” refers throughout to distilled,
3.2.12 I—control tube with serum but no material, kept on
ice. endotoxin-free H O. The use of barbital (veronal) buffer is
recommended. In the United States, barbital is a class IV
3.2.13 M—tube containing serum plus a test material
regulated substance and requires a DEA (3) license for pur-
3.2.14 NM—tube containing serum but no material
chase. The use of other buffer systems (such as TRIS) is
3.2.15 PVDF—polyvinylidene fluoride
permissible if they have been demonstrated not to activate
complement (4). These solutions are stable for one month at
3.2.16 RBC—red blood cell(s)
4°C unless otherwise indicated.
4. Summary of Practice
6.2 The 5X stock BBS (barbital-buffered saline) is prepared
4.1 Solid material specimens are exposed to a standard lot by adding 20.75 g of NaCl plus 2.545 g of sodium barbital
of C4(-)GPS complement under defined conditions, in parallel (sodium-5,5-diethyl barbiturate) to about 400 mL water. The
to appropriate controls (Procedure A). If the alternative pH is adjusted to 7.35 with 1 N HCl, then brought to a final
complement pathway is activated by the material, complement volume of 500 mL in a volumetric flask.
componentswillbedepletedfromtheserum.Exposedserumis
6.3 Metals solution is prepared by making a 2.0-M solution
then tested for remaining functional complement activity, by
of MgCl (40.66 g MgCl ·6 H O up to a final volume of 100
2 2 2
determining complement mediated lysis of rabbit RBC in
mLwater),anda0.3-MsolutionofCaCl (4.41gCaCl·2H O
++
2 2 2
buffer containing EGTA and excess Mg (Procedure B).
up to a final volume of 100 mL of water), and combining the
two solutions 1:1 (v:v).
5. Significance and Use
6.4 The Ca buffer (BBS-GM working solution) is prepared
5.1 Inappropriate activation of complement by blood-
daily, by dissolving 0.25 g of gelatin (type A: Porcine Skin,
contacting medical devices may have serious acute or chronic
Approx. 300 Bloom, such as available from Sigma [ G-1890])
effectsonthehost.Solidmedicaldevicematerialsmayactivate
in 50 mLof water that is gently heated and stirred.The gelatin
thecomplementdirectlybythealternativepathway.Unlikethe
solutionisaddedto50mL5XStockBBSplus0.25mLmetals
classical complement activation pathway (see Practice F1984),
solution, brought up to about 200 mLthen adjusted to pH 7.35
antibodies are not required for alternative pathway activation.
(with 1 N HCl or 1 N NaOH) before bringing the final volume
This practice is useful as a simple, inexpensive screening
to 250 mL in a volumetric flask. The Ca buffer contains both
method for determining alternative whole complement activa-
++ ++
Mg and Ca , which allows both classical and alternative
tion by solid materials in vitro.
pathway complement. activation to occur.
5.2 This practice is composed of two parts. In Part A
6.5 The BBS-G working solution is prepared the same way,
(Section10)C4(-)GPSisexposedtoasolidmaterial.SinceC4
but omitting addition of the metals solution.
is required for classical pathway activation, activation of
complement in C4(-)GPS can only occur by the alternative
6.6 10X Stock EDTA(0.1-M disodium dihydrate EDTA) is
pathway (1). In principle, nonspecific binding of certain
preparedbyadding7.44gdisodiumEDTA·2H Otoabout160
complement components to the materials may also occur. In
mLof water, adjusting the pH to 7.65 (with 1 N NaOH or 1 N
Part B (Section 11), complement activity remaining in the
HCl), then bringing the volume to 200 mL in a volumetric
serum after exposure to the test material is assayed by
flask.
alternative pathway-mediated lysis of rabbit RBC.
6.7 The 0.1 M EGTA (tetrasodium salt, EGTA·4.5 H O) is
5.3 Assessment of in vitro whole complement activation as
preparedbyadding4.683gtetrasodiumEGTAtoabout80mL
described here provides one method for predicting potential
ofwater,adjustingthepHto7.35(with1 NNaOHor1 NHCl),
complement activation by solid medical device materials
then bringing the volume to 100 mL in a volumetric flask.
intended for clinical application in humans when the material
contacts the blood. Other test methods for complement activa- 6.8 BBS-G-EDTA (to be used in preparing RBC before
tion are available, including assays for specific complement being washed out) is prepared by adding 10 mL of stock 10X
componentsandtheirsplitproductsinhumanserum(X1.3and EDTA to 90 mL of BBS-G in a volumetric flask.
X1.4).
6.9 The Mg Buffer (BBS-G-EGTA/Mg working solution) is
5.4 This in vitro test method is suitable for adoption in
prepareddaily,bydissolving0.25ggelatinin50mLwaterthat
specifications and standards for screening solid materials for
is gently heated and stirred.The gelatin solution is added to 50
use in the construction of medical devices intended to be
mL5X stock BBS, plus 0.625 mL2.0 M MgCl,plus4mLof
implanted in the human body or placed in contact with human
0.1 MEGTA,broughtuptoabout200mL,thenadjustedtopH
blood outside the body.
7.35 (with 1 N HCl or 1 N NaOH) before bringing the final
volume to 250 mL in a volumetric flask. The Mg buffer has
++ ++
EGTA to bind Ca . The presence of Mg allows the
alternativepathwayactivationtoproceed,whiletheabsenceof
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
++
this specification. Ca prevents activation of the classical pathway.
F2065 − 00 (2010)
A
TABLE 1 Percent Lysis of Rabbit RBC in Mg Buffer by Human
7. Preparation of Sheep and Rabbit RBC
Serum or C4(-)GPS Pre-exposed1hat37°C, in 100-µL volumes,
to Different Amounts of Sepharose CL-4B
7.1 Commercially obtained sheep RBC preserved inAlsev-
B
µL Sepharose, CL-4B
C D E
er’s solution and defibrinated rabbit RBC are stored at 4°C.
HS prep. 1 HS prep. 2 C4(-)GPS
[HS/C4(-)GPS]
The sheep cells are discarded after eight weeks or when the
50/12.5 3.3 ± 1.2 3.4 ± 1.7 6.5 ± 2.6
supernatant from the second wash contains hemoglobin (red
25/6.25 14.2 ± 0.6 11.8 ± 0.8 21.3 ± 3.3
color)byvisualinspection(aslotsofRBCsage,theyaremore 12.5/3.13 23.6 ± 5.2 19.6 ± 2.8 20.1 ± 0.9
6.25/1.57 33.2 ± 2.8 29.4 ± 3.8 27.3 ± 1.4
sensitive to complement lysis in parallel with increased spon-
0/0 [37°C control] 49.9 ± 0.2 45.3 ± 4.6 41.3 ± 0.7
taneous lysis). The rabbit cells are more fragile than the sheep
0/0 [Ice control] 51.4 ± 1.6 52.9 ± 0.7 58.4 ± 0.5
cells, and should be discarded after four weeks or when the
A
Mean plus or minus standard deviation of three replicate tubes.
B
supernatant from the second wash contains hemoglobin by
The indicated volume of Sepharose CL-4B was added to 100 µL of HS or
C4(-)GPS.
visual inspection.
C
Whole human serum [Quidel (NHSC)], diluted 1:8 in Mg buffer.
D
Reconstituted lyophilized human serum [Sigma (S1764)], diluted 1:4 in Mg
NOTE 1—All centrifugations are at 4°C. Except when indicated, all
buffer.
reagents,tubes,andcellpreparationsarekeptcoldinchippediceoranice
E
Whole guinea pig serum, C4-deficient [Sigma (C1038)], 1:3 in Mg buffer.
slurry.
7.2 FivemillilitresofsheeporrabbitRBCarecentrifugedat
1000 × g, at 4°C, for 10 min.
matoryimmunoassaysthatdetectcomplementcomponentsand
7.3 The cell pellet is resuspended in 10 mL of cold
split-products indicative of alternative pathway activation in
BBS-G-EDTAand incubated for 10 min at 37°C.The cells are
whole human serum. The C4(-)GPS suitable as a source of
centrifuged, and the pellet resuspended in 10 mL of 4°C
complement may be purchased from biological supply houses,
BBS-G-EDTA.
and is generally labeled as reagent-grade complement.
7.4 The cells are centrifuged, the supernatant discarded
8.2 Serum may be absorbed with sheep RBC and rabbit
(first wash), and the pellet resuspended in 10 mL of cold RBC in order to remove naturally occurring anti-sheep and
BBS-GM (Ca buffer) or BBS-G-EGTA/Mg (Mg buffer) (cells
anti-rabbit RBC hemolytic antibodies. The procedure is as
tobeusedinabsorbingserumarewashedinCabuffer;cellsto follows:
be used for detecting alternative pathway C' depletion, Proce-
8.3 Commercially available C4(-)GPS is stored at −70°C.
dure B, are washed and suspended in Mg buffer). Repeat twice
8.4 The serum is thawed on chipped ice or reconstituted (if
(total of three washes.)
lyophilized) with ice-cold water.
7.5 Adjust cell count spectrophotometrically (where an
8.5 All manipulations are done in chipped ice or in an ice
absorbance of 0.75 for sheep RBC and 1.30 for rabbit RBC
slurry, with ice cold (4°C) reagents and cells. Centrifugations
corresponds to 2.0 × 10 RBC/mL, at a wavelength of 412 nm
are carried out at 1000×gat 4°C. It is critical that this entire
and a 1.0-cm light path for 1 volume of cells in BBS-GM or
procedure be done in the cold to avoid activation of comple-
BBS-G-EGTA/Mg plus 24 volumes of water) or count with a
ment in this step.
hemocytometer. Prepare 10 mL of 2.0 × 10 cells/mL in 4°C
BBS-GM.
8.6 Cold serum and 4°C, Ca buffer-washed RBC (a 1:1
mixed volume of sheep and rabbit packed RBC) are gently
7.6 The washed, diluted RBC can be held on ice and used
mixed (by slow rocking), 0.1 mL of packed RBC/2.5 mL of
for at least 12 h.
serum, incubated for 10 min on ice, then centrifuged at 1000 ×
g for 10 min at 4°C. The supernatant liquid is carefully
8. Absorption of Serum (Complement)
transferred to a new container on ice.
8.1 Although human serum would be preferable to guinea
8.7 The procedure in 8.6 is repeated twice.
pig serum for alternative pathway complement activation by
8.8 TheabsorbedC4(-)GPSisstoredin0.5–1.0-mLaliquots
materials to be used in medical devices intended to contact
(convenient for one-experiment use), in cold snap-cap mi-
patient blood, genetically deficient human sera are not rou-
crofuge tubes and kept at −70°C until used.Aliquots should be
tinely available. Human sera depleted of components by
thawed in chipped ice or an ice slurry, used on the day of
antibodyabsorptiononcolumnsareunsuitableforthispurpose
thawing, and not refrozen.
forthefollowingtworeasons:(1)specificcomponentdepletion
is incomplete, so significant classical pathway activation re-
9. Whole Complement Titration to Determine Optimal
mains; and (2) column material may activate the alternative
Serum Dilution
pathway,depletingfunctionalactivity.Serumfromguineapigs
genetically deficient in C4 [C4(-)G
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.