ASTM F1984-25 用固体材料检测血清中完整补体激活的标准试验方法

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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:F1984-25

INTERNATIONAL

Standard Practice for

Testing for Whole Complement Activation in Serum by Solid

Materials¹

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.A number in parentheses indicates the year of last reapproval.A

superscript epsilon(e)indicates an editorial change since the last revision or reapproval.

1.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.Proce-

dure A describes exposure of solid materials to a standard lot of

human serum,using a 0.1mL serum/13x100 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 accor-

dance with internationally recognized principles on standard-

ization established in the Decision on Principles for the

Development of International Standards,Guides and Recom-

mendations issued by the World Trade Organization Technical

Barriers to Trade(TBT)Committee.

IThis practice is under the jurisdiction of ASTM Committee F04 on Medical and

Surgical Materials and Devices and is the direct responsibility of Subcommittee

F04.16 on Biocompatibility Test Methods.

Current edition approved Feb.15,2025.Published March 2025.Originally

approved in 1999.Last previous edition approved in 2018 as F1984-99(2018).

DOI:10.1520/F1984-25.

2.Referenced Documents

2.1 ASTM Standards:²

F748 Practice for Selecting Biological Test Methods for

Materials and Devices

2.2 ISO Document:³

ISO 10993-4 Biological Evaluation of Medical Devices,Part

4:Selection of Tests for Interactions with Blood

3.Terminology

3.1 Abbreviations:

3.1.1 Ab—antibody (hemolysin).

3.1.2 BBS—barbital buffered saline.

3.1.3 BBS-G—barbital buffered saline—gelatin.

3.1.4 BBS-GM—barbital buffered saline—gelatin metals.

3.1.5 C'—complement.

3.1.6 EDTA—ethylenediaminetetraacetic acid,disodium

salt:dihydrate.

3.1.7 HS—human serum.

3.1.8 PVDF—polyvinylidene fluoride.

3.1.9 RBC—red blood cell(s).

4.Summary of Practice

4.1 Solid material specimens are exposed to contact with a

standard lot of complement under defined conditions(Proce-

dure A).Exposed serum then is tested for significant functional

complement depletion compared to controls under identical

conditions(Procedure B)

5.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.

²For referenced ASTM standards,visit the ASTM website,www.astm.org,or

contact ASTM Customer Service at www.astm.org/contact.For Annual Book of

ASTM Standards volume information,refer to the standard's Document Summary

page on the ASTM website.

³Available from American National Standards Institute(ANSI),25W.43rd St.

4th Floor,New York,NY 10036,http://www.ansi.org.

F1984-25

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 comple-

ment 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).⁴In 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 compo-

nents 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.

6.Preparation of Buffers

6.1 Buffers,are prepared according to detailed protocol(2).

“Water”refers throughout to distilled,endotoxin-free water.

The use of barbital (veronal)buffer is recommended.Barbital

is a class IV regulated substance and requires a DEA(3)license

for purchase.The use of other buffer systems,such as TRIS,is

permissible if they have been demonstrated not to activate

complement (4).

6.25X Stock BBS(barbital-buffered saline),is prepared by

adding 20.75g NaCl plus 2.545g sodium barbital(sodium-5,

5-diethyl barbiturate)to about 400 mL water.The pH is

adjusted to 7.35 with 1N HCl,then brought to a final volume

of 500 mL in a volumetric flask.

6.3 Metals Solution,is prepared by making a 2.0M solution

of MgCl₂(40.66 g MgCl₂·6H₂O into 100 mL distilled

endotoxin-free water),and a 0.3 M solution of CaCl₂(4.41g

CaCl₂·2H₂O into 100 mL distilled endotoxin-free water),and

combining the two solutions 1:1(v:v).These solutions are

stable one month at 4℃.

160 mL water,adjusting the pH to 7.65(with 1N NaOHor 1N

HCl),then bringing the volume to 200 mL in a volumetric

flask.

6.7 BBS-G-EDTA(to be used in preparing RBC before

being washed out),is prepared by adding 10 mL of stock 10X

EDTA to 90mL of BBS-G in a volumetric flask.

7.Preparation of Sheep RBC

7.1 Commercially obtained sheep red blood cells(RBC)

preserved in Alsever's solution are stored at 4℃.The cells are

discarded after eight weeks or when the supernatant from the

second wash contains hemoglobin by visual inspection.

NoTE 1—All centrifugations are at 4℃.Except where indicated,all

reagents,tubes,and cellpreparations are kept on ice.

7.2 Five mL of sheep RBC are centrifuged at 1000xg for

10 min.

7.3 The cell pellet is resuspended in 10 mL of cold

BBS-G-EDTA and incubated for 10 min at 37℃.The cells are

centrifuged,and the pellet resuspended in 10 mL of BBS-G-

EDTA.

7.4 The cells are centrifuged,the supernatant discarded

(first wash),and the pellet resuspended in 10 mL of cold

BBS-GM.Repeat twice (total of three washes)

7.5 Adjust cell count spectrophotometrically(where an

absorbance of 0.56 corresponds to 1.5x10⁸sheep RBC/mL,at

a wavelength of 412 nm and a 1.0 cm light path for 1 volume

of cells in BBS-GM plus 24 volumes of water)or count with

a hemocytometer,preparing 10 mL of 1.5x 10⁸cells/mL in

cold BBS-GM.

7.6 The washed,diluted RBC can be held on ice and used

for at least 12 h.

8.Absorption of Serum(Complement)

8.1 The use of human complement is required since there

are species differences in the efficiency of complement activa-

tion and the test materials are to be used in humans.Human

serum suitable as a source of complement may be purchased

from biological supply houses,and generally is labeled as

reagent-grade complement.

8.2 Human serum may be absorbed with sheep RBC in

6.4 BBS-GM Working Solution,is prepared daily by dissolv-

ing 0.25g gelatin in 50 mL endotoxin-free distilled water that

is gently heated and stirred.The gelatin solution is added to

50 mL5X stock BBS plus 0.25 mL metals solution,brought up

to about 200 mL,then adjusted to pH 7.35(with 1N HCl or

1N NaOH)before bringing the final volume to 250 mL in a

volumetric flask.

6.5 BBS-G Working Solution,is prepared the same way,but

the addition of the metals solution is omitted.

6.610X Stock EDTA(0.1 M disodium dihydrate EDTA),is

prepared by adding 7.44 g disodium EDTA·2H₂O to about

4The boldface numbers in parentheses refer to the list of references at the end of

this specification.

order to remove naturally occurring anti-sheep RBC hemolytic

antibodies,though for most purposes,the amount of hetero-

phile antibody in human serum is not enough to influence the

reaction assuming the cells are optimally sensitized with

hemolysin.The procedure is detailed in 8.3-8.8.

8.3 Fresh human serum or a commercial lot of human serum

is obtained and stored at-70 ℃.Fresh serum is preferred as

lyophilized complement often is not as active as fresh serum.

8.4 The serum is thawed on ice or reconstituted (if lyo-

philized)with ice-cold (4℃)distilled endotoxin-free water.

8.5 All manipulations are done on ice,with ice-cold re-

agents and cells;centrifugations are carried out at 1000xg at

4℃.It is critical that this entire procedure be done in the cold

to avoid activation of complement in this step.

8.6 Cold serum and cold,packed,washed sheep RBC are

mixed,0.1 mL RBC/2.5 mL serum,incubated for 10 min on

ice,then centrifuged at 1000x g for 10 min at 4℃.The

supernatant is transferred carefully to a new container on ice.

8.7 The procedure in 8.6 is repeated twice.

8.8 The absorbed human serum is stored in 0.5 to 1.0mL

aliquots(convenient for one-experiment use)in cold snap-cap

microfuge tubes and kept at-70℃until used.Aliquots should

be thawed on ice,used on the day of thawing,and not be

refrozen.

9.Determination of Optimal Hemolysin Concentration

9.1 Determination of optimal hemolysin concentration is

necessary in order to conserve expensive reagents and to avoid

prozone effects.Commercial rabbit anti-sheep RBC serum

(Hemolysin)is obtained,thawed,or if lyophilized,reconsti-

tuted with distilled endotoxin-free water,heat-inactivated at

56℃ for 30 min to inactivate the rabbit complement,aliquoted

in convenient volumes,and stored at-70℃ until used.

9.2 To cold 13x100 mm disposable glass tubes placed in a

rack in an ice bath,0.1 mL of washed sheep RBC at 1.5x 10⁸

cells/mL is added.If statistical evaluation of the results is

desired,three replicate tubes for each condition should be used.

Otherwise,duplicates or even single dilution tubes are suffi-

cient.One set of three replicate tubes receives only 0.1 mL of

cold BBS-GM/tube (no-RBC control,for complement color).

9.3 To the RBC-containing tubes,one set of three tubes gets

1.1 mL cold distilled H₂O/tube (total lysis control),another

gets 0.1 mL BBS-GM (no hemolysin control),and the other

sets get 0.1mL each of 1:2 serial dilutions of hemolysin(tests).

Dilutions between 1:400 to 1:25600 antibody are

recommended,with two sets of 1:400.The no-RBC control

receives 0.1 mL of additional BBS-GM.

9.4 Each tube is mixed quickly by gentle shaking to

resuspend cells,the rack is placed in a 37℃ water bath,

incubated 10 min,then returned to the ice bath.

9.5 One of the two sets of 1:400 antibody gets 1.0 mL of

cold BBS-GM (no-complement control).All other tubes be-

sides the total lysis control set get 0.5 mL cold BBS-GM,then

0.5 mL of absorbed human serum(complement)diluted 1:100

or 1:200.

NoTE 2—For a particular lot ofhuman serum,a 1:100 or 1:200 dilution

should provide sufficient complement activity.Also,percent lysis in the

no-hemolysin(complement only)control should not exceed 10%.If lysis

with the 1:100 dilution of complement exceeds 10%,use 1:200.If the

no-hemolysin control still exceeds 10%,a different lot of serum will need

to be tested.

9.6 Tubes are shaken manually to suspend cells,then the

rack is incubated in a 37℃ water bath for 1h,and intermit-

tently shaken to keep cells in suspension.

9.7 At the end of 1h,the rack is placed on ice.The cold

tubes then are centrifuged at 1000xg for 10 min at 4℃,and

the supernatants decanted to correspondingly numbered 13x

100 mm glass tubes.

9.8 Absorbance of the supernatants is measured at 412 nm.

Percent lysis is calculated for each test and control tube by

subtracting from the 412 nm absorbance the no-RBC control

(mean of the three replicate tubes),dividing by the total lysis

control value (mean of the three replicate tubes),and multi-

plying by 100.

9.9 Final %lysis for each condition is expressed as mean±

standard deviation of the three %lysis values for each three-

replicate set.

9.10 A titration curve is obtained by plotting the inverse of

the hemolysin concentration versus %specific lysis.Twice the

concentration of hemolysin that is just on the plateau of the

titration curve is used for sensitizing RBC for subsequent

assays (optimal hemolysin concentration).Hemolysin is

freshly diluted from stock each day of use.

10.Whole Complement Titration to Determine Optimal

Serum Dilution

10.1 If statistical evaluation of results is desired,all condi-

tions should be assayed in triplicate,using three 13x 100

disposable glass test tubes per condition.Otherwise,duplicates

or single tubes are sufficient.Tubes are numbered in advance.

Conditions include total lysis,no complement (no C'),tests

(dilutions of human serum—HS)with and without hemolysin,

and no RBC(complement color control,at highest concentra-

tion of serum used).All reagents,tubes,and manipulations are

done ice-cold,with tubes held in a rack in an ice slurry.

10.2 Washed RBC are added to all tubes except no-RBC

tubes(0.1 mL/tube of a 1.5x 10⁸cells/mL suspension).

No-RBC tubes get 0.1 mL cold buffer

10.3 Total lysis tubes get 1.1 mL distilled H₂O.The no-C'

and test with hemolysin tubes get 0.1 mL optimal hemolysin

(see 9.10 ),and no-RBC tubes get 0.1mL cold BBS-GM.All

tubes are shaken to resuspend cells,incubated in a 37℃ water

bath for 10 min,and placed back on ice.

NoTE 3—Another acceptable procedure is to make up one large batch

of hemolysin-sensitized erythrocytes to cover all the tests planned within

one week's time.These cells are made up at 5x10⁸/mL and are stored at

4℃.They are washed each time they are used,and ifhemolysis occurs,

new sensitized cells are prepared.These sensitized cells are ready to use,

making the addition of hemolysin to each tube unnecessary,which

simplifies the experiment.Unsensitized RBC can be used as controls for

nonspecific lysis.

10.4 To all but the total lysis tubes,a maximum volume of

1.0 mL of cold BBS-GM is added,reduced by the amount of

diluted serum(see 10.5 ),which will be added at a maximum

0.5 mL volume.The no-C'tubes get 1.0 mL BBS-GM.

10.5 The cold serum is diluted in cold BBS-GM to the

desired concentration(with minimal agitation).It is recom-

mended to test the HS initially at 1:50 to 1:300.The diluted

serum is added to each test tube in a 0.5 mL volume.Final

volume in each tube should be made up to 1.2 mL with

BBS-GM.

10.6 The tubes then are treated as detailed in 9.6-9.9.

10.7 The optimal human serum dilution of a particular lot of

human serum is defined as that in which the nonspecific lysis

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1ThisinternationalstandardwasdevelopedinaccordancewithinternationallyrecognizedprinciplesonstandardizationestablishedintheDecisiononPrinciplesfortheDevelopmentofInternationalStandards,GuidesandRecommendationsissuedbytheWorldTradeOrganizationTechnicalBarrierstoTrade(TBT)Committee.Designation:F1984-25...

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ASTM F1984-25 用固体材料检测血清中完整补体激活的标准试验方法

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