ASTM F1906-98(2003)
(Practice)Standard Practice for Evaluation of Immune Responses In Biocompatibility Testing Using ELISA Tests, Lymphocyte, Proliferation, and Cell Migration (Withdrawn 2011)
Standard Practice for Evaluation of Immune Responses In Biocompatibility Testing Using ELISA Tests, Lymphocyte, Proliferation, and Cell Migration (Withdrawn 2011)
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.
WITHDRAWN RATIONALE
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.
Formerly under the jurisdiction of Committee F04 on Medical and Surgical Materials and Devices, this practice was withdrawn in March 2011 due to lack of support for its continuance.
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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 (Reapproved 2003)
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 F1906; 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 (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
1.1 This practice covers the introduction of a foreign sub- 2.1 ASTM Standards:
stance into mammalian body that may induce the formation of F619 Practice for Extraction of Medical Plastics
an immune response. The immune response may lead to F719 Practice for Testing Biomaterials in Rabbits for Pri-
inadvertent tissue damage and be an undesirable event. In the mary Skin Irritation
standard protocols for biocompatibility testing, various studies F720 Practice for Testing Guinea Pigs for Contact Aller-
in animals are done. These animals or their blood and tissues gens: Guinea Pig Maximization Test
could be used to determine if immune responses have occurred F748 Practice for Selecting Generic Biological Test Meth-
and what types have occurred. At the current time, the ods for Materials and Devices
immunologic testing in biocompatibility protocols is very F763 Practice for Short-Term Screening of Implant Materi-
limited. Techniques can be developed in the future which are als
simple, reliable, and sensitive. F981 Practice forAssessment of Compatibility of Biomate-
1.2 It is the purpose of this practice to delineate some rials for Surgical Implants with Respect to Effect of
possible test methods. It must be remembered that these are Materials on Muscle and Bone
protocols for use in biocompatibility testing, they are not
3. Summary of Practice
diagnostic tests for evaluation of human conditions. Diagnostic
3.1 Immunologic testing is done using specimens from
tests for use on humans must go through evaluation at the
regulatory agencies. The tests described here are clearly animals being tested according to the Practice F748 matrix for
adaptable for use in humans and can be used for research irritation and sensitivity, or for implantation. Blood, organs, or
tissues from the animals may be used. Blood or biopsies from
purposes and provide data in clinical trials, but are not
necessarily cleared for diagnostic purposes. This practice patients in a clinical trial may also be used.Animals (rabbits or
mice) are also immunized with various antigens in this
present selected methods. Other validated methods may be
equally applicable. practice. Humans may be immunized with an approved vac-
cine.
1.3 The values stated in SI units are to be regarded as the
standard. 3.2 Immunologic testing is done using materials, known
components of the materials, or extracts prepared according to
1.4 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the Practice F619. These materials, components, or extracts may
be used for in vivo tests or for the in vitro tests.
responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica-
4. Significance and Use
bility of regulatory limitations prior to use.
4.1 This practice is to be used to help evaluate the biocom-
patibility of materials used in medical devices in terms of the
immune response.
4.2 The appropriateness of the methods should be carefully
considered since not all materials or applications need to be
tested by this practice.
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 Nov. 1, 2003. Published December 2003. Originally contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
approved in 1998. Last previous edition approved in 1998 as F1906 – 98. DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/F1906-98R03. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F1906–98 (2003)
4.3 The testing suggestions in Practice F748 and in the 5.1.3 Dissolve the substance in an aqueous based solution.
matrices of recommended tests issued by regulatory agencies Adjust the pH to between 8.5 and 9.5 or as high as possible
may be considered before proceeding with these tests. before it precipitates.Aphosphate buffer is recommended. The
4.4 These tests require the use of blood. Procedures for final concentration of the substance should be 0.5 mg/mL.
obtaining whole blood or serum should follow the recommen- (Any extract prepared according to Practice F619 may be used
dations of the animal research committee of the institution in this protocol. The pH should be adjusted to pH 8.5 to 9.5
responsible for the animals. In general serum and plasma where possible without precipitation of the extract.
behave the same in these tests, but it should be noted which 5.1.4 Add 100 uL of this to each of the wells except the
was used. control wells. Incubate the plates in a humid environment at
4.5 The Testing Protocols—These will be divided into the room temperature (18 to 22°C) or 37°C for 1 h, place in the
two specific areas of humoral immunity and cell mediated cold (4 to 8°C) overnight. Wash the plates with phosphate
immunity,andsubdividedfromthere.Thetestsforthehumoral buffered saline with Tween 20 (PBS/T) at least three times.
immune responses will be based on solid phase immunoassays Wash at least twice with coating buffer that is the PBS/T with
for use with enzyme linked immunoassays (ELISA) tech- a protein source such as 1 % gelatin, egg albumin, or serum.
niques. This blocks other combining sites on the plates and reduces the
4.6 Abbreviations: background. The plates may be used immediately or stored in
4.6.1 RPMI 1640—Specific growth medium (Roswell Park the cold until used.
Memorial Institute). 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 eight 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
F1906–98 (2003)
of responses to haptenic materials can be determined using a alveolar washes, or minced lymphoid organs. These tests are
simple modification of the procedure in Section 5.Itis based on the stimulation of lymphocytes by antigen with the
necessary to first coat the plates with a carrier to bind the release of various substances (cytokines and interleukins) that
hapten. For most haptens, albumin serves as an appropriate affect other cells. The polyclonal T cell stimulants (mitogen)
carrier. such as PHAor ConAserve as positive controls. It is essential
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 mat
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