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ASTM F2148-07(2012)

(Practice)

Standard Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA)

Standard Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA)

SIGNIFICANCE AND USE
5.1 The propensity of a material to stimulate delayed contact hypersensitivity must be assessed before clinical application of devices containing this material. Delayed hypersensitivity may occur anywhere in the body. Systemic delayed hypersensitivity may have a complex set of reactions and consequences depending on the actual tissue/organ site of reaction. Although the reactions are seldom life-threatening, severe tissue and organ damage my result over time. Skin is the usual test site to determine the propensity of a material to cause delayed hypersensitivity.  
5.2 The standard historical test methods have involved the use of guinea pigs with a cutaneous application and observation of the reaction site. The use of the murine local lymph node assay results in a numerical quantitation of stimulation, rather than subjective evaluation and could be used to determine dose responses.  
5.3 This practice may not be predictive of events occurring during all types of implant applications. The user is cautioned to consider the appropriateness of the method in view of the materials being tested, their potential applications, and the recommendations contained in Practice F748.
SCOPE
1.1 This practice provides a methodology to use an in-situ procedure for the evaluation of delayed contact hypersensitivity reactions.  
1.2 This practice is intended to provide an alternative to the use of guinea pigs for evaluation of the ability of a device material to stimulate delayed contact hypersensitivity reactions. This alternative is particularly applicable for materials used in devices that contact only intact skin. However, the guinea pig maximization test is still the recommended method when assessing the delayed hypersensitivity response to metals or when testing substances that do not penetrate the skin but are used in devices that contact deep tissues or breached surfaces. The guinea pig maximization test should be used for these substances.  
1.3 This practice consists of a protocol for assessing an increase in lymphocyte proliferation within the nodes draining the site of administration on the ears of mice.  
1.4 The LLNA has been validated only for low-molecular-weight chemicals that can penetrate the skin. The absorbed chemical or metabolite must bind to macromolecules, such as proteins, to form immunogenic conjugates.  
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 a specific application.  
1.6 Identification of a supplier of materials or reagents is for the convenience of the user and does not imply a single source. Appropriate materials and reagents may be obtained from many commercial supply houses.  
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.8 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.

General Information

Status
Historical
Publication Date
30-Sep-2012
ICS
07.100.10 - Medical microbiology
Technical Committee
F04 - Medical and Surgical Materials and Devices
Drafting Committee
F04.16 - Biocompatibility Test Methods
Current Stage
Ref Project

Relations

Replaces
ASTM F2148-07e1 - Standard Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA)
Effective Date
01-Oct-2012
Replaced By
ASTM F2148-13 - Standard Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA)
Effective Date
01-Jun-2013
Referred By
ASTM F748-16 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
Effective Date
01-Oct-2012
Referred By
ASTM F2212-20 - Standard Guide for Characterization of Type I Collagen as Starting Material for Surgical Implants and Substrates for Tissue Engineered Medical Products (TEMPs)
Effective Date
01-Oct-2012

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ASTM F2148-07(2012) - Standard Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA)ASTM F2148-07(2012) - Standard Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA)
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ASTM F2148-07(2012) - Standard Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA)
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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: F2148 − 07(Reapproved 2012)
Standard Practice for
Evaluation of Delayed Contact Hypersensitivity Using the
1
Murine Local Lymph Node Assay (LLNA)
This standard is issued under the fixed designation F2148; 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 1.8 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
1.1 This practice provides a methodology to use an in-situ
responsibility of the user of this standard to establish appro-
procedure for the evaluation of delayed contact hypersensitiv-
priate safety and health practices and determine the applica-
ity reactions.
bility of regulatory limitations prior to use.
1.2 This practice is intended to provide an alternative to the
2. Referenced Documents
use of guinea pigs for evaluation of the ability of a device
material to stimulate delayed contact hypersensitivity reac- 2
2.1 ASTM Standards:
tions. This alternative is particularly applicable for materials
F619 Practice for Extraction of Medical Plastics
used in devices that contact only intact skin. However, the
F720 PracticeforTestingGuineaPigsforContactAllergens:
guinea pig maximization test is still the recommended method
Guinea Pig Maximization Test
when assessing the delayed hypersensitivity response to metals
F748 PracticeforSelectingGenericBiologicalTestMethods
orwhentestingsubstancesthatdonotpenetratetheskinbutare
for Materials and Devices
used in devices that contact deep tissues or breached surfaces.
F750 Practice for Evaluating Material Extracts by Systemic
The guinea pig maximization test should be used for these
Injection in the Mouse
substances.
2.2 Other Document:
ICCVAM NIH Publication No: 99-4494 The Murine Local
1.3 This practice consists of a protocol for assessing an
3
increase in lymphocyte proliferation within the nodes draining Lymph Node Assay, 1999
the site of administration on the ears of mice.
3. Terminology
1.4 The LLNA has been validated only for low-molecular-
3.1 Definitions:
weight chemicals that can penetrate the skin. The absorbed
3.1.1 AOO, n—acetone olive oil solution (4:1 v/v) is a
chemical or metabolite must bind to macromolecules, such as
suitable nonpolar solvent.
proteins, to form immunogenic conjugates.
3.1.2 aqueous solvent, n—in this assay refers to the polar
1.5 This practice is one of several developed for the
solvent, saline.
assessment of the biocompatibility of materials. Practice F748
3.1.3 DMSO, n—dimethylsulfoxide (nonaqueous, suitable
may provide guidance for the selection of appropriate methods
organic solvent).
for testing materials for a specific application.
3.1.4 DNCB, n—2,4-dinitrochlorobenzene.
1.6 Identification of a supplier of materials or reagents is for
1
3.1.5 formalin, n—a ⁄10 dilution of 37 to 39 % formalde-
the convenience of the user and does not imply a single source.
hyde solution (formaldehyde) in PBS.
Appropriate materials and reagents may be obtained from
many commercial supply houses.
3.1.6 ICCVAM, n—Interagency Coordinating Committee on
the Validation of Alternative Methods.
1.7 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this 3.1.7 nonaqueous solvent, n—in this assay refers to the
standard.
organic or nonpolar solvent, which shall be dimethylsulfoxide
(DMSO) or acetone olive oil (AOO).
1 2
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Surgical Materials and Devices and is the direct responsibility of Subcommittee contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
F04.16 on Biocompatibility Test Methods. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved Oct. 1, 2012. Published October 2012. Originally the ASTM website.
ε1 3
approved in 2001. Last previous edition approved in 2007 as F2148 – 07 . DOI: Available from NICEATM, NIEHS, 79 Alexander Dr., Mail Drop EC-17,
10.1520/F2148-07R12. Research Triangle Park, NC 27709.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

---------------------- Page: 1 ----------------------
F2148 − 07 (2012)
4
3.1.8 PBS, n—phosphate buffered saline, pH 7.2. hydroxyethyl cellulose to each 10 mL of the aqueous vehicle
control and test solutions to aid in holding the solution to the
3.1.9 positive control, n—a substance capable of consis-
ear.
tently stimulating lymphocyte proliferation.
6.4 The final specimen to be extrac
...

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5.1 The propensity of a material to stimulate delayed contact hypersensitivity must be assessed before clinical application of devices containing this material. Delayed hypersensitivity may occur anywhere in the body. Systemic delayed hypersensitivity may have a complex set of reactions and consequences depending on the actual tissue/organ site of reaction. Although the reactions are seldom life-threatening, severe tissue and organ damage my result over time. Skin is the usual test site to determine the propensity of a material to cause delayed hypersensitivity.  
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SIGNIFICANCE AND USE
5.1 In-vitro osteoblast differentiation assays are one approach to screen progenitor stem cells for their capability to become osteoblasts. The extent of calcium deposits or mineralized matrix that form in vitro may be an indicator of differentiation to a functional osteoblast; however, expression of osteogenic genes or proteins is another important measurement to use in conjunction with this assay to determine the presence of an osteoblast.  
5.2 This practice provides a technique for staining, imaging, and quantifying the fluorescence intensity and area related to the mineralization in living cell cultures using the non-toxic calcium-chelating dye, XO. The positively stained area of mineralized deposits in cell cultures is an indirect measure of calcium content. It is important to measure the intensity to ensure that the images have not been underexposed or overexposed. Intensity and area do not correlate directly to calcium content.  
5.3 XO enables the monitoring of calcium deposits repeatedly throughout the life of the culture without detriment to the culture. There is no interference on subsequent measurements of the mineralized area due to dye accumulation from repeated application (1).3 Calcium deposits that have been previously stained may appear brighter, but this does not impact the area measurement. Calcein dyes may also be used for this purpose (1) but require a different procedure for analysis than XO (that is, concentration and filter sets) and are thus not included here. Alizarin Red and Von Kossa are not suitable for use with this procedure on living cultures since there is no documentation supporting their repeated use in living cultures without deleterious effects.  
5.4 The practice may be applied to cultures of any cells capable of producing calcium deposits. It may also be used to document the absence of mineral in cultures where the goal is to avoid mineralization.  
5.5 During osteoblast differentiation assays, osteogenic supplements are ...
SCOPE
1.1 This practice defines a method for the estimation of calcium content at multiple time points in living cell cultures that have been cultured under conditions known to promote mineralization. The practice involves applying a fluorescent calcium-chelating dye that binds to the calcium phosphate mineral crystals present in the live cultures followed by image analysis of fluorescence microscopy images of the stained cell cultures. Quantification of the positively stained areas provides a relative measure of the calcium content in the cell culture plate. A precise correlation between the image analysis parameters and calcium content is beyond the scope of this practice.  
1.2 Calcium deposition in a secreted matrix is one of several features that characterize bone formation (in vitro and in vivo), and is therefore a parameter that may indicate bone formation and osteoblast function (that is, osteoblastic differentiation). Calcium deposition may, however, be unrelated to osteoblast differentiation status if extensive cell death occurs in the cell cultures or if high amounts of osteogenic medium components that lead to artifactual calcium-based precipitates are used. Distinguishing between calcium deposition associated with osteoblast-produced mineralized matrix and that from pathological or artifactual deposition requires additional structural and chemical characterization of the mineralized matrix and biological characterization of the cell that is beyond the scope of this practice.  
1.3 The parameters obtained by image analysis are expressed in relative fluorescence units or area percentage (area%), for example, fraction of coverage of the area analyzed.  
1.4 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibili...

  • Standard
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  • Standard
    17 pages
    English language
    sale 15% off
ASTM
ASTM E1881-12(2020)(Guide)
Standard Guide for Cell Culture Analysis with SIMS

SIGNIFICANCE AND USE
5.1 The presence of cell growth medium complicates a direct analysis of cells with SIMS. Attempts to wash out the nutrient medium results in the exposure of cells to unphysiological reagents that may also alter their chemical composition. This obstacle is overcome by using a sandwich freeze-fracture method (1). This cryogenic method has provided a unique way of sampling individual cells in their native state for SIMS analysis.  
5.2 The procedure described here has been successfully used for imaging Na+ and K+ ion transport  (3), calcium alterations in stimulated cells (4,5), and localization of therapeutic drugs and isotopically labeled molecules in single cells (6). The frozen freeze-dried cells prepared according to this method have been checked for SIMS matrix effects  (7). Ion image quantification has also been achieved in this sample type (8).  
5.3 The procedure described here is amenable to a wide variety of cell cultures and provides a way for studying the response of individual cells for chemical alterations in the state of health and disease and localization of isotopically-labeled molecules and theraputic drugs in cell culture models.
SCOPE
1.1 This guide provides the Secondary Ion Mass Spectrometry (SIMS) analyst with a cryogenic method for analyzing individual tissue culture cells growing in vitro. This guide is suitable for frozen-hydrated and frozen-freeze-dried sample types. Included are procedures for correlating optical, laser scanning confocal and secondary electron microscopies to complement SIMS analysis.  
1.2 This guide is not suitable for cell cultures that do not attach to the substrate.  
1.3 This guide is not suitable for any plastic embedded cell culture specimens.  
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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 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.

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