ASTM F1906-98
(Practice)Standard Practice for Evaluation of Immune Responses In Biocompatibility Testing Using ELISA Tests, Lymphocyte, Proliferation, and Cell Migration
Standard Practice for Evaluation of Immune Responses In Biocompatibility Testing Using ELISA Tests, Lymphocyte, Proliferation, and Cell Migration
SCOPE
1.1 This practice covers the introduction of a foreign substance into mammalian body that may induce the formation of an immune response. The immune response may lead to inadvertent tissue damage and be an undesirable event. In the standard protocols for biocompatibility testing, various studies in animals are done. These animals or their blood and tissues could be used to determine if immune responses have occurred and what types have occurred. At the current time, the immunologic testing in biocompatibility protocols is very limited. Techniques can be developed in the future which are simple, reliable, and sensitive.
1.2 It is the purpose of this practice to delineate some possible test methods. It must be remembered that these are protocols for use in biocompatibility testing, they are not diagnostic tests for evaluation of human conditions. Diagnostic test for use on humans must go through evaluation at the regulatory agencies. The tests described here are clearly adaptable for use in humans and can be used for research purposes and provide data in clinical trials, but are not necessarily cleared for diagnostic purposes. This practice presents selected methods. Other validated methods may be equally applicable.
1.3 The values state in SI units are to be regarded as the standard.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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:F1906–98
Standard Practice for
Evaluation of Immune Responses In Biocompatibility
Testing Using ELISA Tests, Lymphocyte Proliferation, and
Cell Migration
This standard is issued under the fixed designation F 1906; 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 F 719 Practice for Testing Biomaterials in Rabbits for
Primary Skin Irritation
1.1 This practice covers the introduction of a foreign sub-
F 720 Practice for Testing Guinea Pigs for Contact Aller-
stance into mammalian body that may induce the formation of
gens: Guinea Pig Maximization Test
an immune response. The immune response may lead to
F 748 Practice for Selecting Generic Biological Test Meth-
inadvertent tissue damage and be an undesirable event. In the
ods for Materials and Devices
standard protocols for biocompatibility testing, various studies
F 763 Practice for Short-Term Screening of Implant Mate-
in animals are done. These animals or their blood and tissues
rials
could be used to determine if immune responses have occurred
F 981 Practice for Assessment of Compatibility of Bioma-
and what types have occurred. At the current time, the
terials for Surgical Implants with Respect to Effect of
immunologic testing in biocompatibility protocols is very
Materials on Muscle and Bone
limited. Techniques can be developed in the future which are
simple, reliable, and sensitive.
3. Summary of Practice
1.2 It is the purpose of this practice to delineate some
3.1 Immunologic testing is done using specimens from
possible test methods. It must be remembered that these are
animals being tested according to the Practice F 748 matrix for
protocols for use in biocompatibility testing, they are not
irritation and sensitivity, or for implantation. Blood, organs, or
diagnostic tests for evaluation of human conditions. Diagnostic
tissues from the animals may be used. Blood or biopsies from
tests for use on humans must go through evaluation at the
patients in a clinical trial may also be used.Animals (rabbits or
regulatory agencies. The tests described here are clearly
mice) are also immunized with various antigens in this
adaptable for use in humans and can be used for research
practice. Humans may be immunized with an approved vac-
purposes and provide data in clinical trials, but are not
cine.
necessarily cleared for diagnostic purposes. This practice
3.2 Immunologic testing is done using materials, known
present selected methods. Other validated methods may be
components of the materials, or extracts prepared according to
equally applicable.
Practice F 619. These materials, components, or extracts may
1.3 The values stated in SI units are to be regarded as the
be used for in vivo tests or for the in vitro tests.
standard.
1.4 This standard does not purport to address all of the
4. Significance and Use
safety concerns, if any, associated with its use. It is the
4.1 This practice is to be used to help evaluate the biocom-
responsibility of the user of this standard to establish appro-
patibility of materials used in medical devices in terms of the
priate safety and health practices and determine the applica-
immune response.
bility of regulatory limitations prior to use.
4.2 The appropriateness of the methods should be carefully
2. Referenced Documents considered since not all materials or applications need to be
tested by this practice.
2.1 ASTM Standards:
4.3 The testing suggestions in Practice F 748 and in the
F 619 Practice for Extraction of Medical Plastics
matrices of recommended tests issued by regulatory agencies
may be considered before proceeding with these tests.
This practice is under the jurisdiction ofASTM Committee F-4 on Medical and 4.4 These tests require the use of blood. Procedures for
Surgical Materials and Devices and is the direct responsibility of Subcommittee
obtaining whole blood or serum should follow the recommen-
F04.16 on Biocompatibility Test Methods.
dations of the animal research committee of the institution
Current edition approved June 10, 1998. Published August 1998.
Annual Book of ASTM Standards, Vol 13.01.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
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.
F1906
responsible for the animals. In general serum and plasma in this protocol. The pH should be adjusted to pH 8.5 to 9.5
behave the same in these tests, but it should be noted which where possible without precipitation of the extract.
was used. 5.1.4 Add 100 uL of this to each of the wells except the
4.5 The Testing Protocols—These will be divided into the control wells. Incubate the plates in a humid environment at
two specific areas of humoral immunity and cell mediated room temperature (18 to 22°C) or 37°C for 1 h, place in the
immunity,andsubdividedfromthere.Thetestsforthehumoral cold (4 to 8°C) overnight. Wash the plates with phosphate
immune responses will be based on solid phase immunoassays buffered saline with Tween 20 (PBS/T) at least three times.
for use with enzyme linked immunoassays (ELISA) tech- Wash at least twice with coating buffer that is the PBS/T with
niques. a protein source such as 1 % gelatin, egg albumin, or serum.
4.6 Abbreviations: This blocks other combining sites on the plates and reduces the
background. The plates may be used immediately or stored in
4.6.1 RPMI 1640—Specific growth medium (Roswell Park
Memorial Institute). the cold until used.
5.1.5 Add 100 uL of the appropriate serum samples or
4.6.2 FCS (FBS)—Fetal Calf Serum (Fetal Bovine Serum).
buffer control to the wells. The test serum samples should be
4.6.3 NCS—Newborn Calf Serum.
run in at least two dilutions. It is recommended that eight wells
4.6.4 MTT—(3-[4,5-Dimethylthiazol-2-yL]-2,5-
receive only buffer and serve as an antigen-second antibody
diphenyltetrazolium bromide: Thiazolyl blue.
control. Incubate at room temperature or 37°C for 1 to 2 h.
4.6.5 LIF—Leukocyte Migration Inhibition Factor.
Wash well with PBS/T.
4.6.6 Ig—Immunoglobulin.
5.1.6 Add 100 uL of the appropriate antiserum to all of the
4.6.7 MIF—Macrophage Migration Inhibition Factor.
wells. This second antibody should be labeled with an enzyme
4.6.8 PEC—Peritoneal Exudate Cells.
(horse radish peroxidase or alkaline phosphatase are recom-
4.6.9 PMNS (POLYS)—Polymorphonuclear Leukocytes.
mended). These antisera can be purchased from scientific
4.6.10 PHA—Phytohemagglutinin.
supply houses and should be used at the dilution recommended
4.6.11 ConA—Conconavalin A.
by the manufacturer. It is recommended that polyvalent antis-
4.6.12 PBS—Phosphate Buffered Saline.
eradirectedagainstIgG,IgM,IgAoftheappropriatespeciesof
4.6.13 PBS/T—Phosphate Buffered Saline with Tween 20.
the animal be used and that IgE specific antisera be used
4.6.14 MW—Molecular Weight.
separately.The plates should be incubated at room temperature
4.6.15 MED 199—Medium 199, a specfic growth medium.
or 37°C for 1 to 2 h. They should then be washed well with
PBS/T.
5. Tests for Production of Humoral Immune Responses
5.1.7 Add 100 uL of the appropriate substrate to all wells.
5.1 These tests are generally done with serum or plasma.
The substrate specific for the enzyme label and at the correct
The use of peritoneal or ascites fluid is also permissible.
concentration should be used. This information should be
5.1.1 Response to Immunogenic Substances—Proteins and
supplied by the supplier of the antisera. Incubate at room
mostcarbohydrateswilladheretopolystyrene,especiallywhen
temperature (usually in the dark) for the recommended time
dissolved in buffer at pH 8 to 9. Other materials also usually
(usually 20 to 30 min). Read the optical density at the
will adhere. Solid materials may be used as the solid support
appropriate wave length for the substrate.
substrate with appropriate controls for nonspecific binding.
5.1.8 Analysis of the Data—The results of the optical
5.1.2 The use of polystyrene 96-well microtiter plates is
density achieved with the test serum samples should be
recommended. There are several manufacturers (for example,
compared to the control samples. Significant elevations or
Costar, Falcon, Nunc) and they are available from standard
depressions would be signified by values outside two standard
supply houses (for example, Fisher, VWR, Thomas). No
deviations of the control. If known standards were included,
specific source is recommended, however the choice should be
theresultscanbeexpressedfromcomparisonwiththestandard
documented in the report.
curve.
5.1.2.1 The configuration of the testing protocol is up to the
6. Response to Haptenic Substances
evaluator but control wells must be included in the matrix. It is
recommended that 8 wells receive no antigen and serve as 6.1 The immune response to haptens (low molecular weight
reagent controls.Anegative control using serum from matched materials) is similar to the immune response already described.
animals not receiving the material should be used in at least However, this response will be dealt with separately here since
four wells. If known positive antisera is available, this should there are substantial differences in testing methodologies.
beusedinatleastfourwells.Ifquantitationisdesired,standard 6.1.1 The production of an immune response to low mo-
solutionsofatleastthreedifferentconcentrationsshouldberun lecular weight degradation, wear, or elution products from
in triplicate and a buffer control run in triplicate. Each
materials is an important issue in biocompatibility. These low
specimen to be tested should be run at two different dilutions molecular weight substances may bind to host tissue or protein
and each in triplicate.
andbecomeimmunogenic.Itisnotapparentthatdetermination
5.1.3 Dissolve the substance in an aqueous based solution. of responses to haptenic materials can be determined using a
Adjust the pH to between 8.5 and 9.5 or as high as possible simple modification of the procedure in Section 5. It is
before it precipitates.Aphosphate buffer is recommended. The necessary to first coat the plates with a carrier to bind the
final concentration of the substance should be 0.5 mg/mL. hapten. For most haptens, albumin serves as an appropriate
(Any extract prepared according to Practice F 619 may be used carrier.
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.
F1906
6.2 Step one is to add 100uL of carrier (0.5mg/mL) bovine fortheseteststhatstrictasepticprecautionsbeused.Theuseof
serum albumin is recommended) to the wells in the plate. antibiotics other than penicillin/streptomycin or gentamicin is
Incubate for 1 to2hat room temperature or 37°C and then notadvisedbecausetheirmodeofactionmayinterferewiththe
overnightinthecold(4-8°C).WashtheplateswellwithPBS/T. cell responses.
6.3 Add 100 uL of the solution containing the hapten. PBS
8.1.1 Lymphocyte Transformation Tests—This detects the
alone should be used as a negative control. Incubate for 1 to 2 production of a cytokine that stimulates other lymphocytes to
h at room temperature. Wash well with the blocking wash
divide. Thus one lymphocyte responding specifically to an
(PBS/T containing gelatin). antigen or nonspecifically to a mitrogen will be activated and
6.4 Proceed as described in 5.1.5-5.1.7.
stimulate other cells to divide, thus amplifying the response.
6.5 Analysis of the Data—The results of the optical density
8.1.1.1 Isolate lymphocytes by differential sedimentation
achieved with the test serum samples should be compared to
and suspend in growth medium (RPMI 1640 with 10 % fetal or
the control samples. Significant elevations or depressions
newborn calf serum (FCS or NCS)) at a concentration of 10
wouldbesignifiedbyvaluesoutsidetwostandarddeviationsof
cells/ mL.
the control. If known standards were included, the results can
8.1.2 Dispense 1 mL of lymphocyte suspension into test
be expressed from comparison with the standard curve.
tubes for each of four tests. It is recommended that each be run
in triplicate.
7. Stimulation of Immune Responses to Unrelated
8.1.2.1 Cell control,
Antigens
8.1.2.2 Cells plus mitogen (PHA or ConA),
7.1 It is now known that various substances, especially oils,
8.1.2.3 Cells plus antigen (material or extract) at first
gels, and colloidal suspensions, can stimulate the immune
concentration, and
response to unrelated antigens. This is not necessarily a
8.1.2.4 Cells plus antigen (material or extract) at second
harmful situation, and may be beneficial. The ability of the
concentration.
material or extract to do this needs to be documented.
8.1.3 The tests should be incubated at 37°C in 5 % CO for
7.1.1 For this procedure animals should be given an unre-
seven days. The response to mitogen peaks at four days, the
lated antigen (such as bovine serum albumin) with and without
response to antigen peaks at seven days. After seven days of
the material.The antibody levels should be measured at 7 to 10
incubation add tritiated thymidine or I-125 thymidine to the
days, 21 days, and 8 weeks. The antibody levels should be
cultures. Incubate for 4 to 6 h. Collect the cells (usually by
measured using the techniques in 5.1.
filter wash) and determine the uptake of radioisotope. MTT is
7.1.2 Protocol:
being used increasingly as a simple dye marker for prolifera-
7.1.2.1 Control animals receiving only the antigen,
tion assays. Alamar Blue may also be used.
7.1.2.2 Test animals receiving the antigen mixed with the
8.1.4 The use of antigen and mitogen together will detect
material or extract, and
immunosuppression.
7.1.2.3 Testanimalsreceivingtheantigeninonesiteandthe
8.1.5 Data Analysis—The uptake of radioisotope in the
material or extract in another site.
presence of antigen should be compared to that of the cell
7.1.3 The following controls are recommended but not
control. The use of the mitogen confirms the test system was
required:
working.Howevercomparisonoftheresponsetoantigentothe
7.1.3.1 Animals receiving only saline and no material,
response to the mitogen is permissible.
extract, or antigen,
8.2 Leukocyte Migration Inhibition (LIF)—This test is lim-
7.1.3.2 Animals receiving only the material or extract and
ited to human and rabbit peripheral blood. It does not work
not antigen, and
with peritoneal exudate cells and may
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