ASTM F2065-00(2006)
(Practice)Standard Practice for Testing for Alternative Pathway Complement Activation in Serum by Solid Materials
Standard Practice for Testing for Alternative Pathway Complement Activation in Serum by Solid Materials
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 F 1984), 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).5 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™ 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.
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Designation: F2065 – 00 (Reapproved 2006)
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 2. Referenced Documents
1.1 This practice provides a protocol for rapid, in vitro 2.1 ASTM Standards:
screeningforalternativepathwaycomplementactivatingprop- F748 Practice for Selecting Generic Biological Test Meth-
erties of solid materials used in the fabrication of medical ods for Materials and Devices
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
alternative pathway complement activating properties of solid 2.2 Other Document:
materials intended for use in contact with blood. For this ISO 10993-4: Biological Evaluation of Medical Devices.
practice, “serum” is synonymous with “complement.” Part 4: Selection of Tests for Interactions with Blood
1.3 This practice consists of two procedural parts. Proce-
3. Terminology
dureAdescribesexposureofsolidmaterialstoastandardlotof
C4-deficient guinea pig serum [C4(-)GPS], using 0.1-mL 3.1 Definitions of Terms Specific to This Standard:
3.1.1 water—distilled, endotoxin-free.
serum per 13 3 100-mm disposable glass test tubes.
Sepharose CL-4B is used as an example of test materials. 3.2 Abbreviations:Abbreviations:
3.2.1 Ab—antibody (hemolysin)
Procedure B describes assaying the exposed serum for signifi-
cant functional alternative pathway complement depletion as 3.2.2 BBS—barbital buffered saline
3.2.3 BBS-G—barbital buffered saline – gelatin
compared to control samples. The endpoint in procedure B is
lysis of rabbit RBC in buffer containing EGTA and excess 3.2.4 BBS-G-EGTA/Mg (Mg Buffer)—barbital buffered sa-
++ ++
line – gelatin EGTA Mg
Mg .
3.2.5 BBS-GM (Ca Buffer)—barbital buffered saline – gela-
1.4 This practice does not address function, elaboration, or
depletion of individual complement components except as tin metals
3.2.6 C8—complement
optional additional confirmatory information that can be ac-
quired using human serum as the complement source. This 3.2.7 C4(-)GPS—C4-deficient guinea pig serum [serum
from guinea pigs genetically incapable of producing C4, the
practice does not address the use of plasma as a source of
complement. fourth component of complement]
3.2.8 EDTA—ethylenediaminetetraacetic acid, disodium
1.5 This practice is one of several developed for the
assessment of the biocompatibility of materials. Practice F748 salt: dihydrate
3.2.9 EGTA—ethylene glyco-bis(b-aminoethyl ether)-
may provide guidance for the selection of appropriate methods
for testing materials for other aspects of biocompatibility. N,N,N8,N8-tetraacetic acid, tetrasodium salt
3.2.10 HAGG—heat aggregated gamma globulin
PracticeF1984providesguidancefortestingsolidmaterialsfor
whole complement activation in human serum, but does not 3.2.11 HS—human serum
3.2.12 I—control tube with serum but no material, kept on
discriminate between the classical or alternative pathway of
activation. ice.
3.2.13 M—tube containing serum plus a test material
3.2.14 NM—tube containing serum but no material
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 March 1, 2006. Published April 2006. Originally contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
´1
approved in 2000. Last previous edition approved in 2000 as F2065–00 . DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/F2065-00R06. 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 (2006)
3.2.15 PVDF—polyvinylidene fluoride complement (4). These solutions are stable for one month at
3.2.16 RBC—red blood cell(s) 4°C unless otherwise indicated.
6.2 The 5X stock BBS (barbital-buffered saline) is prepared
4. Summary of Practice
by adding 20.75 g of NaCl plus 2.545 g of sodium barbital
(sodium-5,5-diethyl barbiturate) to about 400 mL water. The
4.1 Solid material specimens are exposed to a standard lot
pH is adjusted to 7.35 with 1 N HCl, then brought to a final
of C4(-)GPS complement under defined conditions, in parallel
volume of 500 mL in a volumetric flask.
to appropriate controls (Procedure A). If the alternative
6.3 Metals solution is prepared by making a 2.0-M solution
complement pathway is activated by the material, complement
of MgCl (40.66 g MgCl ·6 H O up to a final volume of 100
componentswillbedepletedfromtheserum.Exposedserumis
2 2 2
mLwater), and a 0.3-M solution of CaCl (4.41 g CaCl·2H O
then tested for remaining functional complement activity, by
2 2 2
up to a final volume of 100 mL of water), and combining the
determining complement mediated lysis of rabbit RBC in
++
two solutions 1:1 (v:v).
buffer containing EGTA and excess Mg (Procedure B).
6.4 The Ca buffer (BBS-GM working solution) is prepared
5. Significance and Use daily, by dissolving 0.25 g of gelatin (type A: Porcine Skin,
Approx. 300 Bloom, such as available from Sigma [ G-1890])
5.1 Inappropriate activation of complement by blood-
in 50 mLof water that is gently heated and stirred.The gelatin
contacting medical devices may have serious acute or chronic
solutionisaddedto50mL5XStockBBSplus0.25mLmetals
effectsonthehost.Solidmedicaldevicematerialsmayactivate
solution, brought up to about 200 mLthen adjusted to pH 7.35
thecomplementdirectlybythealternativepathway.Unlikethe
(with 1 N HCl or 1 N NaOH) before bringing the final volume
classical complement activation pathway (see PracticeF1984),
to 250 mL in a volumetric flask. The Ca buffer contains both
antibodies are not required for alternative pathway activation.
++ ++
Mg and Ca , which allows both classical and alternative
This practice is useful as a simple, inexpensive screening
pathway complement. activation to occur.
method for determining alternative whole complement activa-
6.5 The BBS-G working solution is prepared the same way,
tion by solid materials in vitro.
but omitting addition of the metals solution.
5.2 This practice is composed of two parts. In Part A
6.6 10X Stock EDTA(0.1-M disodium dihydrate EDTA) is
(Section10)C4(-)GPSisexposedtoasolidmaterial.SinceC4
preparedbyadding7.44gdisodiumEDTA·2H Otoabout160
is required for classical pathway activation, activation of 2
mLof water, adjusting the pH to 7.65 (with 1 N NaOH or 1 N
complement in C4(-)GPS can only occur by the alternative
HCl), then bringing the volume to 200 mL in a volumetric
pathway (1). In principle, nonspecific binding of certain
flask.
complement components to the materials may also occur. In
6.7 The 0.1 M EGTA (tetrasodium salt, EGTA·4.5 H O) is
Part B (Section 11), complement activity remaining in the
preparedbyadding4.683gtetrasodiumEGTAtoabout80mL
serum after exposure to the test material is assayed by
ofwater,adjustingthepHto7.35(with1 NNaOHor1 NHCl),
alternative pathway-mediated lysis of rabbit RBC.
then bringing the volume to 100 mL in a volumetric flask.
5.3 Assessment of in vitro whole complement activation as
described here provides one method for predicting potential 6.8 BBS-G-EDTA (to be used in preparing RBC before
being washed out) is prepared by adding 10 mL of stock 10X
complement activation by solid medical device materials
intended for clinical application in humans when the material EDTA to 90 mL of BBS-G in a volumetric flask.
contacts the blood. Other test methods for complement activa- 6.9 The Mg Buffer (BBS-G-EGTA/Mg working solution) is
tion are available, including assays for specific complement prepareddaily,bydissolving0.25ggelatinin50mLwaterthat
componentsandtheirsplitproductsinhumanserum(X1.3and isgentlyheatedandstirred.Thegelatinsolutionisaddedto50
X1.4). mL5X stock BBS, plus 0.625 mL2.0 M MgCl,plus4mLof
5.4 This in vitro test method is suitable for adoption in 0.1 MEGTA,broughtuptoabout200mL,thenadjustedtopH
specifications and standards for screening solid materials for 7.35 (with 1 N HCl or 1 N NaOH) before bringing the final
use in the construction of medical devices intended to be volume to 250 mL in a volumetric flask. The Mg buffer has
++ ++
implanted in the human body or placed in contact with human EGTA to bind Ca . The presence of Mg allows the
blood outside the body. alternativepathwayactivationtoproceed,whiletheabsenceof
++
Ca prevents activation of the classical pathway.
6. Preparation of Buffers
7. Preparation of Sheep and Rabbit RBC
6.1 Buffers are prepared in accordance with established
protocols (1, 2). “Water” refers throughout to distilled,
7.1 Commercially obtained sheep RBC preserved inAlsev-
endotoxin-free H O. The use of barbital (veronal) buffer is
er’s solution and defibrinated rabbit RBC are stored at 4°C.
recommended. In the United States, barbital is a class IV
The sheep cells are discarded after eight weeks or when the
regulated substance and requires a DEA (3) license for pur-
supernatant from the second wash contains hemoglobin (red
chase. The use of other buffer systems (such as TRIS) is
color)byvisualinspection(aslotsofRBCsage,theyaremore
permissible if they have been demonstrated not to activate
sensitive to complement lysis in parallel with increased spon-
taneous lysis). The rabbit cells are more fragile than the sheep
cells, and should be discarded after four weeks or when the
supernatant from the second wash contains hemoglobin by
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this specification. visual inspection.
F2065 – 00 (2006)
A
NOTE 1—All centrifugations are at 4°C. Except when indicated, all TABLE 1 Percent Lysis of Rabbit RBC in Mg Buffer by Human
Serum or C4(-)GPS Pre-exposed1hat37°C,in 100-µL volumes,
reagents,tubes,andcellpreparationsarekeptcoldinchippediceoranice
to Different Amounts of Sepharose CL-4B
slurry.
2 B
µL Sepharose, CL-4B
C D E
HS prep. 1 HS prep. 2 C4(-)GPS
7.2 FivemillilitresofsheeporrabbitRBCarecentrifugedat
[HS/C4(-)GPS]
1000 3 g, at 4°C, for 10 min.
50/12.5 3.3 6 1.2 3.4 6 1.7 6.5 6 2.6
7.3 The cell pellet is resuspended in 10 mL of cold
25/6.25 14.2 6 0.6 11.8 6 0.8 21.3 6 3.3
12.5/3.13 23.6 6 5.2 19.6 6 2.8 20.1 6 0.9
BBS-G-EDTAand incubated for 10 min at 37°C.The cells are
6.25/1.57 33.2 6 2.8 29.4 6 3.8 27.3 6 1.4
centrifuged, and the pellet resuspended in 10 mL of 4°C
0/0 [37°C control] 49.9 6 0.2 45.3 6 4.6 41.3 6 0.7
BBS-G-EDTA.
0/0 [Ice control] 51.4 6 1.6 52.9 6 0.7 58.4 6 0.5
A
7.4 The cells are centrifuged, the supernatant discarded
Mean plus or minus standard deviation of three replicate tubes.
B 2
The indicated volume of Sepharose CL-4B was added to 100 µL of HS or
(first wash), and the pellet resuspended in 10 mL of cold
C4(-)GPS.
BBS-GM (Ca buffer) or BBS-G-EGTA/Mg (Mg buffer) (cells
C
Whole human serum [Quidel (NHSC)], diluted 1:8 in Mg buffer.
D
tobeusedinabsorbingserumarewashedinCabuffer;cellsto
Reconstituted lyophilized human serum [Sigma (S1764)], diluted 1:4 in Mg
buffer.
be used for detecting alternative pathway C8 depletion, Proce-
E
Whole guinea pig serum, C4-deficient [Sigma (C1038)], 1:3 in Mg buffer.
dureB,arewashedand suspended in Mg buffer). Repeattwice
(total of three washes.)
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
correspondsto2.0 310 RBC/mL,atawavelengthof412nm
are carried out at 1000 3 g at 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 3 10 cells/mL in 4°C
8.6 Cold serum and 4°C, Ca buffer-washed RBC (a 1:1
BBS-GM.
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,incubatedfor10minonice,thencentrifugedat1000 3
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.
8.8 TheabsorbedC4(-)GPSisstoredin0.5–1.0-mLaliquots
pig serum for alternative pathway complement activation by
materials to be used in medical devices intended to contact (convenient for one-experiment use), in cold snap-cap mi-
crofugetubesandkeptat−70°Cuntilused.Aliquotsshouldbe
patient blood, genetically deficient human sera are not rou-
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
9. Whole Complement Titration to Determine Optimal
is incomplete, so significant classical pathway activation re-
Serum Dilution
mains; and (2) column material may activate the alternative
pathway,depletingfunctionalactivity.Serumfromguineapigs
9.1 If statistical evaluation of results is desired, all condi-
genetically deficient in C4 [C4(-)GPS] has no classical path-
tions should be assayed in triplicate, using three 13 3 100 mm
way complement activity but is fully competent for alternative
round-bottomed, disposable glass test tubes per condition.
pathway complement activation.Although the titers are differ-
Otherwise, single or duplicate tubes are sufficient. Tubes are
ent(agreaterconcentrationofC4(-)GPSisrequiredtoproduce
numbered in advance. Conditions include “total lysis,” “no
the same lysis of rabbit RBC as human serum)(5)C4(-)GPS
complement (RBC only)” (no C8), “tests” (dilutions of C4(-
gives equivalent results to human serum in detecting biomate-
)GPS) with and without hemolysin,
...
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