ASTM F2065-00e1
(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
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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e1
Designation: F 2065 – 00
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 (e) indicates an editorial change since the last revision or reapproval.
e NOTE—Editorial corrections were made throughout this standard in June 2001.
1. Scope F748 Practice for Selecting Generic Biological Test Meth-
ods for Materials and Devices
1.1 This practice provides a protocol for rapid, in vitro
F1984 Practice for Testing for Whole ComplementActiva-
screeningforalternativepathwaycomplementactivatingprop-
tion in Serum by Solid Materials
erties of solid materials used in the fabrication of medical
2.2 Other Document:
devices that will contact blood.
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
alternative pathway complement activating properties of solid
materials intended for use in contact with blood. For this
3. Terminology
practice, “serum” is synonymous with “complement.”
3.1 Definitions of Terms Specific to This Standard:
1.3 This practice consists of two procedural parts. Proce-
3.1.1 water—distilled, endotoxin-free.
dureAdescribesexposureofsolidmaterialstoastandardlotof
3.2 Abbreviations:Abbreviations:
C4-deficient guinea pig serum [C4(-)GPS], using 0.1-mL
3.2.1 Ab—antibody (hemolysin)
serum per 13 3 100-mm disposable glass test tubes.
3.2.2 BBS—barbital buffered saline
Sepharosey CL-4B is used as an example of test materials.
3.2.3 BBS-G—barbital buffered saline – gelatin
Procedure B describes assaying the exposed serum for signifi-
3.2.4 BBS-G-EGTA/Mg (Mg Buffer)—barbital buffered sa-
cant functional alternative pathway complement depletion as
++
line – gelatin EGTA Mg
compared to control samples. The endpoint in procedure B is
3.2.5 BBS-GM (Ca Buffer)—barbital buffered saline – gela-
lysis of rabbit RBC in buffer containing EGTA and excess
++
tin metals
Mg .
3.2.6 C8—complement
1.4 This practice does not address function, elaboration, or
3.2.7 C4(-)GPS—C4-deficient guinea pig serum [serum
depletion of individual complement components except as
from guinea pigs genetically incapable of producing C4, the
optional additional confirmatory information that can be ac-
fourth component of complement]
quired using human serum as the complement source. This
3.2.8 EDTA—ethylenediaminetetraacetic acid, disodium
practice does not address the use of plasma as a source of
salt: dihydrate
complement.
3.2.9 EGTA—ethylene glyco-bis(b-aminoethyl ether)-
1.5 This practice is one of several developed for the
N,N,N8,N8-tetraacetic acid, tetrasodium salt
assessment of the biocompatibility of materials. Practice F748
3.2.10 HAGG—heat aggregated gamma globulin
may provide guidance for the selection of appropriate methods
3.2.11 HS—human serum
for testing materials for other aspects of biocompatibility.
3.2.12 I—control tube with serum but no material, kept on
Practice F1984 provides guidance for testing solid materials
ice.
forwholecomplementactivationinhumanserum,butdoesnot
3.2.13 M—tube containing serum plus a test material
discriminate between the classical or alternative pathway of
3.2.14 NM—tube containing serum but no material
activation.
3.2.15 PVDF—polyvinylidene fluoride
2. Referenced Documents 3.2.16 RBC—red blood cell(s)
2.1 ASTM Standards:
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland
Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.16 on Biocompatibility Test Methods. Annual Book of ASTM Standards, Vol 13.01.
Current edition approved Nov. 10, 2000. Published February 2001. Available from American National Standards Institute, 11 W. 42nd St., 13th
Sepharosey is a registered trademark of Pharmacia, Inc. Floor, New York, NY 10036.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F 2065
4. Summary of Practice (sodium-5,5-diethyl barbiturate) to about 400 mL water. The
pH is adjusted to 7.35 with 1 N HCl, then brought to a final
4.1 Solid material specimens are exposed to a standard lot
volume of 500 mL in a volumetric flask.
of C4(-)GPS complement under defined conditions, in parallel
6.3 Metals solution is prepared by making a 2.0-M solution
to appropriate controls (Procedure A). If the alternative
of MgCl (40.66 g MgCl ·6 H O up to a final volume of 100
complement pathway is activated by the material, complement
2 2 2
mLwater), and a 0.3-M solution of CaCl (4.41 g CaCl·2H O
componentswillbedepletedfromtheserum.Exposedserumis
2 2 2
up to a final volume of 100 mL of water), and combining the
then tested for remaining functional complement activity, by
two solutions 1:1 (v:v).
determining complement mediated lysis of rabbit RBC in
++
buffer containing EGTA and excess Mg (Procedure B).
6.4 The Ca buffer (BBS-GM working solution) is prepared
daily, by dissolving 0.25 g of gelatin (type A: Porcine Skin,
5. Significance and Use
Approx. 300 Bloom, such as available from Sigma [ G-1890]
5.1 Inappropriate activation of complement by blood-
)in50mLofwaterthatisgentlyheatedandstirred.Thegelatin
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
classicalcomplementactivationpathway(seePracticeF1984),
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
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
prepared by adding 4.683 g tetrasodium EGTAto about 80 mL
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
6.8 BBS-G-EDTA (to be used in preparing RBC before
described here provides one method for predicting potential
being washed out) is prepared by adding 10 mL of stock 10X
complement activation by solid medical device materials
EDTA to 90 mL of BBS-G in a volumetric flask.
intended for clinical application in humans when the material
6.9 The Mg Buffer (BBS-G-EGTA/Mg working solution) is
contacts the blood. Other test methods for complement activa-
prepareddaily,bydissolving0.25ggelatinin50mLwaterthat
tion are available, including assays for specific complement
is gently heated and stirred.The gelatin solution is added to 50
componentsandtheirsplitproductsinhumanserum(X1.3and
mL5X stock BBS, plus 0.625 mL2.0 M MgCl,plus4mLof
X1.4). 2
0.1 MEGTA,broughtuptoabout200mL,thenadjustedtopH
5.4 This in vitro test method is suitable for adoption in
7.35 (with 1 N HCl or 1 N NaOH) before bringing the final
specifications and standards for screening solid materials for
volume to 250 mL in a volumetric flask. The Mg buffer has
use in the construction of medical devices intended to be
++ ++
EGTA to bind Ca . The presence of Mg allows the
implanted in the human body or placed in contact with human
alternativepathwayactivationtoproceed,whiletheabsenceof
blood outside the body.
++
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-
complement (4). These solutions are stable for one month at
taneous lysis). The rabbit cells are more fragile than the sheep
4°C unless otherwise indicated.
cells, and should be discarded after four weeks or when the
6.2 The 5X stock BBS (barbital-buffered saline) is prepared
supernatant from the second wash contains hemoglobin by
by adding 20.75 g of NaCl plus 2.545 g of sodium barbital
visual inspection.
NOTE 1—All centrifugations are at 4°C. Except when indicated, all
reagents,tubes,andcellpreparationsarekeptcoldinchippediceoranice
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this specification. slurry.
F 2065
7.2 FivemillilitresofsheeporrabbitRBCarecentrifugedat confirmatory immunoassays that detect complement compo-
1000 3 g, at 4°C, for 10 min. nents and split-products indicative of alternative pathway
7.3 The cell pellet is resuspended in 10 mL of cold activation in whole human serum. The C4(-)GPS suitable as a
BBS-G-EDTAand incubated for 10 min at 37°C.The cells are source of complement may be purchased from biological
centrifuged, and the pellet resuspended in 10 mL of 4°C supply houses, and is generally labeled as reagent-grade
BBS-G-EDTA. 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
to be used in absorbing serum are washed in Ca buffer; cells to follows:
be used for detecting alternative pathway C8 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
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.
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
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, and “no RBC (serum
rial complement activation (see Sepharosey example in Table
color)” (complement color control, at highest concentration of
1). Thus, C4(-)GPS is suitable as a screen for subsequent
serumused).Allreagents,tubes,andmanipulationsaredoneat
4°C, with tubes held in a rack in an ice slurry.
A
9.2 Two sets of tubes are prepared in accordance with
TABLE 1 Percent Lysis of Rabbit RBC in Mg Buffer by Human
Serum or C4(-)GPS Pre-exposed1hat37°C,in 100-µL volumes, 9.3–9.4. One set contains diluted sera placed on Mg buffer-
to Different Amounts of SepharoseY
...
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