ASTM F561-19

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: F561 − 19
Standard Practice for
Retrieval and Analysis of Medical Devices, and Associated
Tissues and Fluids
ThisstandardisissuedunderthefixeddesignationF561;thenumberimmediatelyfollowingthedesignationindicatestheyearoforiginal
adoptionor,inthecaseofrevision,theyearoflastrevision.Anumberinparenthesesindicatestheyearoflastreapproval.Asuperscript
epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 1.4 Asignificant portion of the information associated with
aretrievedimplantdeviceisoftenatthedevice-tissueinterface
1.1 This practice covers recommendations for the retrieval,
or in the tissues associated with the implant and related organ
handling, and analysis of implanted medical devices and
systems.Attention should be given to the handling of adjacent
associatedspecimensthatareremovedfromhumanandanimal
tissues, so as not to interfere with study of the particles in the
subjects during revision surgery and at postmortem. This
adjacent tissue, a chemical analysis for the byproducts of
practice may be used for the analysis of any implant including
degradation of the implant, or a study of the cellular response
inert, bioactive, resorbable, and tissue engineered products.
to the implant.
This practice can also be used for analysis of specimens and
fluids from in vitro tests, including those from wear tests and 1.5 The values stated in SI units are to be regarded as
joint simulators. The aim is to provide guidance to minimize standard. No other units of measurement are included in this
iatrogenic damage during the recovery and handling of the standard.
associated specimens which could obscure the investigational
1.6 This standard may involve hazardous materials,
results.This practice is also intended to provide guidance as to
operations, and equipment. As a precautionary measure, ex-
gathering data at the proper time and circumstance.
planteddevicesshouldbesterilizedorminimallydisinfectedby
an appropriate means that does not adversely affect the
1.2 This practice offers guidelines for the analysis of re-
implant or the associated tissue that may be subject to
trieved implants to limit damage to them, and to allow
comparisons between investigational results from different subsequent analysis. A detailed discussion of precautions to be
used in handling of human tissues can be found in ISO
studies. The protocols are divided into three stages, where
Stage I is the minimum non-destructive analysis, Stage II is 12891-1. This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
more complete non-destructive analysis, and Stage III is
responsibility of the user of this standard to establish appro-
destructive analysis. Standard protocols for the examination
priate safety, health, and environmental practices and deter-
and collection of data are provided for specific types of
mine the applicability of regulatory limitations prior to use.
materialsinrelationtotheirtypicalapplications.Forparticular
1.7 This international standard was developed in accor-
investigational programs, additional, more specific, protocols
dance with internationally recognized principles on standard-
mayberequired.Ifspecialanalyticaltechniquesareemployed,
ization established in the Decision on Principles for the
the appropriate handling procedures must be specified. Note
Development of International Standards, Guides and Recom-
that regulations for handling of patient information, tissues,
mendations issued by the World Trade Organization Technical
and retrieved devices will vary by geography.
Barriers to Trade (TBT) Committee.
1.3 This practice should be applied in accordance with
pertinent regulations or legal requirements regarding the han-
2. Referenced Documents
dling of patient data as well as the handling and analysis of
2.1 ASTM Standards:
retrieved implants and excised tissues, especially with regard
A262Practices for Detecting Susceptibility to Intergranular
to handling devices which may become involved in litigation,
Attack in Austenitic Stainless Steels
as in accordance with Practice E860. Note that regulations for
A751Test Methods, Practices, and Terminology for Chemi-
handling of patient information, tissues, and retrieved devices
cal Analysis of Steel Products
will vary by geography
C20Test Methods forApparent Porosity, WaterAbsorption,
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland
Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.15 on Material Test Methods. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved Jan. 1, 2019. Published February 2019. Originally contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
approved 1978. Last previous edition approved in 2013 as F561–13. DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/F0561-19. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F561 − 19
Apparent Specific Gravity, and Bulk Density of Burned D1621Test Method for Compressive Properties of Rigid
Refractory Brick and Shapes by Boiling Water Cellular Plastics
C158Test Methods for Strength of Glass by Flexure (De-
D1622Test Method for Apparent Density of Rigid Cellular
termination of Modulus of Rupture) Plastics
C169Test Methods for Chemical Analysis of Soda-Lime
D1623Test Method for Tensile and TensileAdhesion Prop-
and Borosilicate Glass
erties of Rigid Cellular Plastics
C623Test Method for Young’s Modulus, Shear Modulus,
D1708TestMethodforTensilePropertiesofPlasticsbyUse
and Poisson’s Ratio for Glass and Glass-Ceramics by
of Microtensile Specimens
Resonance
D2240TestMethodforRubberProperty—DurometerHard-
C633Test Method for Adhesion or Cohesion Strength of
ness
Thermal Spray Coatings
D2842Test Method for Water Absorption of Rigid Cellular
C674Test Methods for Flexural Properties of Ceramic
Plastics
Whiteware Materials
D2857Practice for Dilute Solution Viscosity of Polymers
C730Test Method for Knoop Indentation Hardness of Glass
D2990Test Methods forTensile, Compressive, and Flexural
C1069Test Method for Specific SurfaceArea ofAlumina or
Creep and Creep-Rupture of Plastics
Quartz by Nitrogen Adsorption
D3016Practice for Use of Liquid Exclusion Chromatogra-
C1161Test Method for Flexural Strength of Advanced
phy Terms and Relationships
Ceramics at Ambient Temperature
D3418Test Method for Transition Temperatures and En-
C1198Test Method for Dynamic Young’s Modulus, Shear
thalpies of Fusion and Crystallization of Polymers by
Modulus, and Poisson’s Ratio forAdvanced Ceramics by
Differential Scanning Calorimetry
Sonic Resonance
D3835Test Method for Determination of Properties of
C1322Practice for Fractography and Characterization of
Polymeric Materials by Means of a Capillary Rheometer
Fracture Origins in Advanced Ceramics
D3919Practice for Measuring Trace Elements in Water by
C1326Test Method for Knoop Indentation Hardness of
Graphite Furnace Atomic Absorption Spectrophotometry
Advanced Ceramics
D4000Classification System for Specifying Plastic Materi-
C1327Test Method for Vickers Indentation Hardness of
als
Advanced Ceramics
D4001Test Method for Determination of Weight-Average
D256Test Methods for Determining the Izod Pendulum
Molecular Weight of Polymers By Light Scattering
Impact Resistance of Plastics
D4065Practice for Plastics: Dynamic Mechanical Proper-
D412TestMethodsforVulcanizedRubberandThermoplas-
ties: Determination and Report of Procedures
tic Elastomers—Tension
D4754Test Method for Two-Sided Liquid Extraction of
D624Test Method for Tear Strength of Conventional Vul-
Plastic Materials Using FDA Migration Cell
canized Rubber and Thermoplastic Elastomers
D5227TestMethodforMeasurementofHexaneExtractable
D638Test Method for Tensile Properties of Plastics
Content of Polyolefins
D695Test Method for Compressive Properties of Rigid
D5296Test Method for Molecular Weight Averages and
Plastics
Molecular Weight Distribution of Polystyrene by High
D732Test Method for Shear Strength of Plastics by Punch
Performance Size-Exclusion Chromatography
Tool
E3Guide for Preparation of Metallographic Specimens
D747Test Method for Apparent Bending Modulus of Plas-
3 E7Terminology Relating to Metallography
tics by Means of a Cantilever Beam (Withdrawn 2019)
E8Test Methods for Tension Testing of Metallic Materials
D785Test Method for Rockwell Hardness of Plastics and
[Metric] E0008_E0008M
Electrical Insulating Materials
E10Test Method for Brinell Hardness of Metallic Materials
D790Test Methods for Flexural Properties of Unreinforced
E18Test Methods for Rockwell Hardness of Metallic Ma-
and Reinforced Plastics and Electrical Insulating Materi-
terials
als
E45Test Methods for Determining the Inclusion Content of
D792Test Methods for Density and Specific Gravity (Rela-
Steel
tive Density) of Plastics by Displacement
E92Test Methods for Vickers Hardness and Knoop Hard-
D1004Test Method for Tear Resistance (Graves Tear) of
ness of Metallic Materials
Plastic Film and Sheeting
E112Test Methods for Determining Average Grain Size
D1238Test Method for Melt Flow Rates of Thermoplastics
E135Terminology Relating to Analytical Chemistry for
by Extrusion Plastometer
Metals, Ores, and Related Materials
D1239Test Method for Resistance of Plastic Films to
E353Test Methods for Chemical Analysis of Stainless,
Extraction by Chemicals
Heat-Resisting, Maraging, and Other Similar Chromium-
D1505Test Method for Density of Plastics by the Density-
Nickel-Iron Alloys
Gradient Technique
E354 Test Methods for Chemical Analysis of High-
Temperature,Electrical,Magnetic,andOtherSimilarIron,
Nickel, and Cobalt Alloys
The last approved version of this historical standard is referenced on
www.astm.org. E407Practice for Microetching Metals and Alloys
F561 − 19
E539TestMethodforAnalysisofTitaniumAlloysbyX-Ray 2.2 Other Document:
Fluorescence Spectrometry ISO12891-1,RetrievalandAnalysisofImplantableMedical
Devices, Part 1: Standard Practice for Retrieval and
E562Test Method for Determining Volume Fraction by
Handling
Systematic Manual Point Count
E860Practice for ExaminingAnd Preparing Items ThatAre
3. Terminology
Or May Become Involved In Criminal or Civil Litigation
E883Guide for Reflected–Light Photomicrography 3.1 Definition of Terms Specific to Issues of Microbial
Contamination:
E986Practice for Scanning Electron Microscope Beam Size
3.1.1 antiseptic—a germicide that is used on skin or living
Characterization
tissue for the purposes of inhibiting or destroying microorgan-
E1188Practice for Collection and Preservation of Informa-
isms.
tion and Physical Items by a Technical Investigator
E1479Practice for Describing and Specifying Inductively 3.1.2 decontamination—a process or treatment that renders
a medical device, instrument, or environmental surface safe to
Coupled Plasma Atomic Emission Spectrometers
handle. Ranges from sterilization to cleaning with soap and
F316Test Methods for Pore Size Characteristics of Mem-
water.
brane Filters by Bubble Point and Mean Flow Pore Test
F619Practice for Extraction of Medical Plastics
3.1.3 disinfectant—a germicide that is used solely for de-
F981Practice for Assessment of Compatibility of Biomate- stroying microorganisms on inanimate objects.
rials for Surgical Implants with Respect to Effect of
3.1.4 disinfection—a process or treatment using a disinfec-
Materials on Muscle and Insertion into Bone
tant. Disinfection is generally less lethal than sterilization. It
F1044Test Method for Shear Testing of Calcium Phosphate
eliminates virtually all recognized pathogenic microorganisms
Coatings and Metallic Coatings
but not necessarily all microbial forms (for example, bacterial
F1147Test Method for Tension Testing of Calcium Phos-
endospores) on inanimate objects. It does not ensure overkill.
phate and Metallic Coatings
3.1.5 sterilization—use of a physical or chemical procedure
F1635Test Method for in vitro Degradation Testing of
to destroy all microbial life; including large numbers of highly
HydrolyticallyDegradablePolymerResinsandFabricated
resistant bacterial spores.
Forms for Surgical Implants
4. Summary of Practice
F1854Test Method for Stereological Evaluation of Porous
Coatings on Medical Implants
4.1 This practice provides recommendations for collection
F1877Practice for Characterization of Particles
of clinical data, analysis of adjacent tissues, and the material
F2102Guide for Evaluating the Extent of Oxidation in
characterizations to be performed when an implant is retrieved
Polyethylene Fabricated Forms Intended for Surgical
as part of a clinical or an animal study. It also provides for
Implants
analysis of specimens and lubrication fluids from in vitro wear
F2182Test Method for Measurement of Radio Frequency
tests.
Induced Heating On or Near Passive Implants During
4.2 The clinical data to be recorded include a case history
Magnetic Resonance Imaging
review, roentgenogram reviews, tissue culture, and observa-
F2214Test Method forIn Situ Determination of Network
tions of the implant site.
Parameters of Crosslinked Ultra High Molecular Weight
4.3 Protocols are provided for the handling of the implant
Polyethylene (UHMWPE)
tissue interface, and adjacent tissues and fluids for subsequent
E2451Practice for Preserving Ignitable Liquids and Ignit-
analysis. These protocols are intended to facilitate (a) histo-
able Liquid Residue Extracts from Fire Debris Samples
logicandimmunohistochemicalexaminationofthetissues, (b)
F2502SpecificationandTestMethodsforAbsorbablePlates
chemical analysis of the tissues for identification and quanti-
and Screws for Internal Fixation Implants
fication of implant corrosion or degradation products, and (c)
F2739Guide for Quantifying Cell Viability within Bioma-
digestion of tissues and fluids for subsequent harvesting and
terial Scaffolds
analysis of particulate debris.
F2977Test Method for Small Punch Testing of Polymeric
4.4 The material characterizations include observation and
Biomaterials Used in Surgical Implants
description of the retrieved device and adjacent tissues, deter-
F2995Guide for Shipping Possibly Infectious Materials,
mination of chemical composition, macroscopic and micro-
Tissues, and Fluids
scopic examinations and mechanical property determinations.
F2979Guide for Characterization of Wear from the Articu-
The guidelines are separated in three stages. Stage I is
lating Surfaces in Retrieved Metal-on-Metal and other
considered to comprise an essential minimum analysis for
Hard-on-Hard Hip Prostheses
F3036Guide for Testing Absorbable Stents
F3129Guide for Characterization of Material Loss from 4
Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
Conical Taper Junctions in Total Joint Prostheses 4th Floor, New York, NY 10036, http://www.ansi.org.
F561 − 19
routine examination of all types of materials. Stage II is Subsequently,theplanmayneedtoberevisedbasedonresults
nondestructive but provides more detail and is intended for obtained throughout the analyses. Due to the potential inter-
special studies of devices with or without impaired function, ferences described in Section 6, protocols and methods should
made of various types of materials. Stage III includes destruc- be executed in a sequence such as to minimize the impact of
tive methods for and material-specific protocols for detailed interferences
failure, microstructural, and chemical analysis as well as
5.6 Anydestructiveanalysisofimplantsmustbedonesoas
determination of physical and mechanical properties. The
to not destroy any features that may become the subject of
flowchart below can be used to guide the type of analysis that
litigation, in accordance with Practice E860. This standard
can be completed (Fig. 1).
recommendation should be applied in accordance with state or
national regulations or legal requirements regarding the han-
5. Significance and Use
dling and analysis of retrieved implants and tissues.
5.1 The investigation of retrieved implantable medical de-
6. Interferences
vices and adjacent tissues can be of value in the assessment of
clinical complications associated with the use of a specific
6.1 Some critical features of the retrieved implant, tissue
prostheticdevicedesign;canexpandtheknowledgeofclinical
and the interface can only be accurately described by observa-
implantperformanceandinteractionsbetweenimplantsandthe
tion at the time of removal, and prior to fixation, sterilization,
body; provide information on implant performance and safety;
or disinfection. Such observation must be made using appro-
and thus further the development of biocompatible implant
priate aseptic precautions. Photomicrographs are recommend
materials and devices with improved performance. Compari-
at this stage of device retrieval.
son of wear patterns and wear particle morphology observed
6.2 Due to the destructive nature of some of the analysis
with retrievals and those observed with in vitro joint simulator
protocols provided in this practice, their use precludes any
tests can provide valuable insight into the validity of the in
other type of analysis. It is therefore essential that handling of
vitro simulation.
thedeviceandtissuesbedoneinconcertwiththerequirements
5.2 Asignificant portion of the information associated with
of all of the analyses to be performed, including analyses that
a retrieved implant is obtained with detailed studies of the
may be done in the future.
device-tissue interface healing response. Appropriate methods
6.2.1 For example, when harvesting tissues for subsequent
are provided to facilitate a study of the particles in the tissues,
chemical analysis, it is important to use tools that do not
and chemical analysis for the byproducts of degradation of the
contain the materials or elements of interest in the tissues.
implant, and histologic evaluation of the cellular response to
6.2.2 If possible, retrieved implants should be placed in
the implant.
bags or containers, unless the bone-implant interface will be
evaluated. There is possibility of material degradation follow-
5.3 For the analysis to be accurate, it is essential that the
ing exposure to formalin (or other fixatives such as glutaral-
deviceandassociatedtissuesberemovedminimizingasbestas
dehyde) such as in a Morse taper of a ceramic femoral head.
possible alteration of their form and structure. It is also
6.2.3 Soft tissue implants especially those with a lumen or
essential that the tissues be handled in such a way as to avoid
cavity that may be compressed during transport should be
microbial or viral contamination of the work place or the
placed in a hard wall container with a fluid tight lid.
investigator. The tissue-device interface may need to be stabi-
6.2.4 Ensure that air-fluid interfaces are minimized to avoid
lized with chemical fixation prior to separation of the device
focal desiccation of explanted tissue surfaces.
from it’s in-situ position. It is also highly recommended to
document detailed information about the tissue specimens,
7. Hazards
including location of extraction. Standard protocols for the
examination and collection of data are provided for retrieval 7.1 The handling of retrieved implants and tissues may
and handling of implantable medical devices, as well as for involvehandlingofinfectiousmaterial(bacterial,viral,fungal,
specific types of materials in relation to their typical applica- or protozoal).
tions.Forparticularinvestigationalprograms,additional,more
7.2 It is suggested that individuals handling blood, tissues,
specific, protocols may be required. If special analytical
or the devices, or combinations thereof be vaccinated against
techniques are employed, the appropriate procedures must be
Hepatitis B. As a precautionary measure, removed devices
specified.
could should be sterilized by an appropriate means that does
5.4 Inordertointerprettheanalysisofmaterialsandtissues, not adversely affect the implant.
it is also essential to capture a minimum data set regarding the
7.3 There are situations where tissues or implants cannot be
reason for device removal, method of removal, method and
sterilized or disinfected prior to analysis, for example, require-
timingpreservationandclinicalfindingsandlaboratorystudies
ments of specialized protocols in which sterilization will
documenting device performance.
adversely affect cell/tissue morphology and staining quality or
5.5 Planningoftheoverallretrievalanalysespriortoexecu- materialproperties.Insuchcases,extremecareshouldbetaken
tion of any of the protocols or methods within this practice is to use aseptic technique and disinfection. Where institutional
essential to maximize the overall effectiveness of the analyses. guidelines for the handling of septic material do not exist,
The plan shall be based on initial observations from the details for handling and sterilizing retrievals, and laboratory
available clinical information, tissues, and implants. practice recommendations can be found in ISO 12891-1. For
F561 − 19
FIG. 1 Retrieval Analysis Flowchart
F561 − 19
decontamination of metallic implants, the use of bleach should 8.5 Tofacilitatesubsequentanalysis,itisrecommendedthat
be considered and possibly avoided to prevent corrosion the device be removed with the tissue interface intact.
damage from the decontamination process. However, interface preservation should not jeopardize the
practice of medicine and patient safety. The investigator shall
7.4 Handling of explanted devices with an electrical energy
follow all rules and guidelines for implant and tissue handling
source such as implantable cardioverter defibrillators may be
established by the clinical center where the retrieval is per-
hazardous. It is recommended that manufacturer’s inactivation
formed.
procedures be followed.When all required equipment, such as
8.5.1 In cases of animal studies of tissue responses to
a device-specific wireless programmer, are not available it is
implants, the implant should be removed with at least a 4 mm
recommended that the device be disabled using a magnet or
thick layer of adjacent tissue, in accordance with Practice
other alternative method that is appropriate for the device.
F981.Careshouldbetakentoavoidunnecessarymanipulation
Handling with double gloves will reduce the risk of an
of the device-tissue interface before chemical stabilization.
unintended shock.
Trimming of tissues, orientation of blocks and the creation of
8. Clinical Information Gathered at the Time of Implant
histology slides should be sufficiently detailed and tracked to
Explantation
aid in the subsequent interpretation of any histology reactions.
8.5.2 When handling of devices with electrical energy
8.1 The extent of clinical information to be obtained will
source that have internal data storage such as implantable
depend in part on the type of implant and reasons for removal.
cardioverterdefibrillators,pacemakersorneurostimulators,the
Similarly, the amount of information provided about the
investigator is responsible for ensuring patient privacy and
implant site will depend on the circumstances regarding the
protecting patient data
removal. The investigator is responsible for ensuring patient
privacy and protecting patient data. The investigator shall
9. Packaging and Shipping of Explanted Devices, Tissues
follow all relevant regulations in the geography where the
and Fluids
retrieval and analysis is performed. Steps to enhance patient
protectionmayincludeobtainingpatientconsentandengaging 9.1 In the event that an implant is explanted at a facility
an Institutional Review Board (IRB) to review and approve where the device needs to be transported to a laboratory for
study details prior to initiation. Clinical data should be de- analysis, the components, tissues, and fluids must be packaged
identified at the earliest practical point. A detailed listing and in a manner that provides adequate protection to the items
formatfordocumentationoftheclinicalinformationassociated during the shipping process.
with removal are provided in Appendix X1. Standard patient
9.2 Care should be taken during any handling of the
evaluationscoringschemessuchasthosedevelopedbyclinical
retrieved components to avoid damage to the components (i.e.
societies may also be utilized.
rubbing together the articulating surfaces, dropping or knock-
8.2 As a minimum, the clinical information for device
ing the parts, or allowing tissues to dry out).
tracking should include the following information:
9.3 Disassembly of components beyond what is necessary
8.2.1 Date of implantation, and date of explantation.
for the explanation of the implanted device should be avoided.
8.2.2 Identification of hospitals, or physicians’ offices,
9.4 Each component should be packaged individually and
where device implantation and removal was performed.
devicepackagingshouldbedictatedbythetypeofanalysisthat
8.2.3 Confidential, unique, patient ID Code to link to
willbeperformed(forexample,formalinsubmersionfortissue
healthcare institution’s implantation and removal records.
ongrowth analysis of coated metallic components). All pack-
8.2.4 Device identification (manufacturer’s name and de-
aging should be labeled with a unique identification code.
vice catalogue number).
Double bagging is preferred and each bag should be labeled
8.2.5 Device lot and serial number.
with the unique identification code. The addition of an absor-
8.2.6 Indicationforuseandreasonforexplantation(clinical
bent material may also be required.
diagnosis).
9.5 Transportationofthedevicesandtissueforanalysismay
8.3 For purposes of implant retrieval studies, the following
be time sensitive. Anticipate worst-case scenario timelines to
information is considered essential:
makesurethesampleevaluationisnotcompromisedbydelays
8.3.1 Patient or animal age and sex.
in the transportation process. The potential for extremes of
8.3.2 A generic statement as to level of patient activity
temperature exposure and possible effects of specimen should
relative to the device.
also be considered.
8.3.3 Astatement as to any gross evidence of inflammation,
implant site infection, or tissue damage such as osteolysis.
9.6 Specific details with regard to the packaging and ship-
8.3.4 Orientation of the implant relative to the patient and
ping of medical devices, tissues, and fluids is covered in
original placement. It is suggested that the proximal end of the
Guidance F2995.
device or other critical landmark identified with a nondestruc-
10. Analysis of the Tissues and the Tissue-Implant
tive marking scheme.
Interface
8.4 More detailed clinical information should be gathered,
where feasible, as indicated in Appendix X1. Obtaining an in 10.1 Macroscopic Examination of Tissue:
situ, intraoperative photograph of the implant is highly desir- 10.1.1 Record a gross pathologic description of the tissue
able. immediately adjacent to the implant, as to consistency and
F561 − 19
color, as seen by the naked eye, or with a hand lens or 10.3 Immunohistochemistry Protocols:
dissecting microscope. Record any differences between the
10.3.1 IHC can be used to identify specific cell types and
implant-tissue interface and the tissues not in direct contact
ECM protein deposition in response to implantable materials
with the implant such as tissue downstream from an intravas-
and prosthetic devices. Typically, monoclonal or polyclonal
cularorintracardiacimplant.Describethespecimensizeeither
antibodies raised against a particular epitope are used to
by dimensions or weight.
identify cells, proteins or enzymes of interest that may aid in
10.1.2 Since the color and texture of the tissue is altered by
determining the cause of failure. For example, positive identi-
sterilizationandfixationmethods,itisrecommendedthatgross
ficationofB-cellsinatissuethatcontainsmaylymphocytesin
observations be made prior to fixation or sterilization. Such
additiontoonlysmallnumbersofplasmacellsandneutrophils
observations should be made utilizing appropriate blood borne
could aid in determining if a patient is sensitive to a metal
pathogen protective techniques and equipment. The type of
present in the retrieved implant. Other markers may be useful
fixativeandtimeoffixationshouldbeconsidereddependingon
to identify activated cells or chemokines. This field is con-
the type of pathologic analysis to be conducted. For example,
stantly changing as new markers are developed and new or
if simply routineH&E staining then fixation in appropriate
improved methods are developed that increase specificity of
volume of 10% buffered formalin for 24 to 72 hours may be
current markers. Therefore, it is not possible to provide
fine but if immunohistochemistry (IHC) is considered then
anything but a basic introduction, and a recommendation to
short duration (24 hr or<)in4% paraformaldehyde may be
follow published methods and methods provided by the manu-
required to maintain epitope antigenicity.
facturer of the antibody to be used whenever possible.
10.1.3 Where appropriate and feasible, obtain photographic
10.3.1.1 Methods for typical markers chosen for studies of
documentation of the explant and adjacent tissue, as well as a
humantissuessuchasanti-humanBcell,Tcell,ormonocytes/
photographic record of subsequent dissections. A scale and
macrophages markers are readily available from a number of
uniqueID#shouldbeincludedineachphotographifpossible.
reputable manufacturers. However, specific markers for other
10.2 Histopathological Analysis of Tissue: species (for example, rats, mice, rabbits, goats), may not be
readily available and may require extensive methods develop-
10.2.1 Process the excised tissue using standard laboratory
mentofdevelopmentofnewantibodiesspecificforthespecies
procedures for the histological processing to create stained
under investigation. It is essential that appropriate positive and
microscope slides for pathologic interpretation. Processing
negative controls be used to enable appropriate interpretation
may require that the device remains in the in-situ position
of the data. It is recommended that positive controls be
within the tissue. Alternatively, the device may have been
processed in the same manner as test tissue.
completely removed from the tissue. Depending on the study
objectives or diagnostic goals, these procedures may be for
10.3.1.2 IHC methods consist of a series of steps or reac-
frozen sections, paraffin embedding, methacrylate embedding
tions that have been developed to amplify the signal on the
orotherspecialprocedures.Routinestainingwithhematoxylin
markers. First, a primary antibody specific for the CD marker
and eosin (H & E), a trichrome or pentachrome stain, or
(forexample,mouseanti-human)isapplied.Then,asecondary
toluidine blue are recommended for light microscopy of most
antibody that binds specifically to the first antibody is applied
soft tissues and bone. Special stains (for example, von Kossa,
(for example, goat anti-mouse). This antibody is typically
Lendrum fibrin, Pearls iron, Picrosirius red, Massons
tagged with a reagent such as biotin, which serves as a marker
trichrome, Movat pentachrome, or other) may be utilized as
in this amplification phase of the reaction. In this example,
indicated and should be fully described.
strept-avidine peroxidase would then be added to bind to the
10.2.2 Provide a detailed histopathologic description of the biotin and immobilize the peroxidase. Finally, a substrate is
tissue-implantinterfaceaswellasalladjacenttissuespecimens
added that will react with the peroxidase, change color and
(for example, acute or chronic nature of the response, edema, precipitate at the location of the antigen. Diaminobenzidine
thrombosis, calcification the type and extent of extracellular
(DAB) is often used, although several substrates are available
matrix deposition, necrotic changes, thickness of fibrous for different kits or automatic systems. The end result in this
capsule, inflammatory cell types, giant cells, particulates, example is the peroxidase oxidation of DAB to give a
hyperplasia, dysplasia, type of inflammatory reaction). yellow-brown precipitate at the site of the reaction. The
sections can be stained with hematoxylin or other counterstain
10.2.3 If the implant material is porous, then tissue analysis
mustincludeevaluationofthereactionwithintheporesaswell to enhance the visibility of cells and contrast with the colored
reaction identifying the epitope.Additional amplification steps
as in the adjacent tissues. This should include the degree and
nature of tissue ingrowth, interstitial inflammation, and overall may be needed to ensure adequate visualization, or addition of
blocking steps such as addition of hydrogen peroxide or horse
biological fixation.
serum may be needed to reduce background labeling. Back-
10.2.4 For detailed studies of tissue reactions, the use of a
groundlabelingcanbeintroducedbybindingofthereagentsto
quantitative scoring scheme, such as that in Practice F981 is
endogenous proteins or enzymes (for example, peroxidase) or
recommended.
to block non-specific binding of minor or substantive known
10.2.5 Since some polymeric materials, for example,
contaminants in the reagents may be needed.
Polymethyl methacrylate (PMMA) bone cement, are altered or
dissolved by the solutions used for routine histology, special 10.3.1.3 It is best if an antibody is selected that has been
techniques may be indicated, or special note made of voids previously used successfully on tissues embedded in the same
formerly occupied by the material. manner as those being tested as not all methods that work on
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onetypeofsection(forexample,frozensections)willworkon 10.4.1.4 Tissues should be transferred to plastic or glass
another type of section (for example, paraffin-embedded or containers of high quality which have been thoroughly acid
MMA-embedded sections). This is because the embedding cleanedorunusedfromalottestedtobefreeofcontamination.
processes may mask, alter or destroy antigens. While antigen Acid cleaning which may etch the glass surfaces is not
retrieval processes (for example, citrate, heat and enzyme recommended for subsequent silicone analysis. Tissue transfer
digestion) can be used to unmask the antigen for which the should be done in a dust free environment.
antibody is specific, these methods are not always successful,
10.4.2 Test solutions should be analyzed in triplicate, either
can result in tissue falling off the slides and can even increase
as is or after dilution with 1% nitric acid to a concentration
background labeling.
which falls within the standards, and the results averaged.
10.3.1.4 Irrespective of the embedding technique or the
Concentrations are determined in µ/l (ppb), or µ/g of tissue
methodused,thedesiredoutcomeisonewheretheisloworno
(ppm). Results from solutions of known volumes from in vitro
nonspecific coloration of the tissues with the substrate used to
studies can be converted to total micrograms in solution.
localize the signal identifying the antigen of choice (for
10.4.2.1 Theconcentrationofmetallicspeciesintissuemay
example, CD-2), one where positive control tissues embedded
vary according to the location of the specimen relative to the
and labeled in the same manner show an expected pattern of
implant. It is therefore important to carefully record the
labeling and one where test sections labeled with all the same
location of the specimen.
methods except the primary antibody (no-antibody controls)
10.4.3 These methods of analysis require chemical diges-
are negative (for example, show no label). Some additional
tion of the tissue samples prior to analysis, and therefore the
generalconsiderationsareprovidedbelowthatwillapplytoall
samples cannot be used for any other analysis. The ability to
methods used.
digesttissueisinfluencedbythemethodoftissuefixation.Itis
10.3.2 Reagents and Other Considerations:
recommended that tissues be fixed in analytical grade 70%
10.3.2.1 Tissueshouldbefixedinreagentsthatareknownto
ethanol in analytical grade water. The methods of digestion
support the IHC methods you intend to use.
depend on the type of tissue to be analyzed.
10.3.2.2 HC reagents can be purchased from a variety of
10.4.3.1 Blood samples drawn from patients or animals
companies. It is advisable to use those recommended by the
should be done using polypropylene syringes. The blood can
manufacturer or shown in the literature to produce a strong
beallowedtoclotatroomtemperatureandcentrifugedat1850
label with low or no background.
g for 30 min to separate serum and clot fractions. Blood may
10.3.2.3 t is important to use a humidified chamber when
also be drawn in heparinized vacutainer tubes. The blood may
labeling to ensure that slides do not dry out during the
be allowed to settle so as to isolate red and white cells, or be
procedure.
centrifuged at 400 g and the plasma supernatant drawn off.
10.3.2.4 It is important to completely wash away the prior
Plasma is diluted at least 2× in 1% nitric acid.
reagent from the section with buffered saline or other recom-
10.4.3.2 Cells, either red blood cells or cells from cell
mended wash before adding the next reagent.
culture experiments may receive special treatment, such as
10.4 Chemical Analysis of Tissues By Flame Atomic Ab-
separation of cell contents and cell membranes. The cells are
sorption Spectroscopy (AAS), Graphite Furnace Atomic Ab-
washed and centrifuged 3 times with physiologic saline to
sorption Spectroscopy (GFAAS), by Inductively Coupled
remove trapped serum or growth media.The cell pellet is then
Plasma Optical Emission Spectroscopy (ICP-OES) or Mass lysed with 1% Triton X100 to release intracellular contents,
Spectroscopy (ICPMS):
centrifuged and the supernatant harvested. This solution is
10.4.1 Reagents and Materials: pipetted off, diluted 2× in 1% nitric acid, and referred to as
10.4.1.1 Standard Atomic Absorption Spectroscopy (AAS) “cell contents.” The pellet of cell membranes is then washed
and centrifuged 3 times in saline to remove the Triton and
grade solutions (MCB reagents, Fisher, and VWR) are used to
make calibration curves. Calibration solutions should be pre- remaining contents. The membranes are then digested in 50%
nitric acid, diluted in 0.5% nitric acid for analysis as “cell
pared according to Practice D3919, using the same matrix
solution as the test specimen. Solutions of low concentration membranes.”
should be made fresh daily. The sensitivity and possible
10.4.3.3 Tissue from implant sites or joint capsules should
interferences depend on the particular element.
be weighed and placed in plastic bags.
10.4.1.2 Any fixing agents, chemicals and solvents must be
(1)Forthemechanicaltissuedigestionappropriatebagsfor
of analytical purity. The use of 70% ethanol is recommended the machine should be used. These may then be frozen until
as a transport and storage solution.The use of double distilled,
use. For preparation, 5 mL of 50% nitric acid shall be added
deionized water is necessary. to each bag. The tissue is then homogenized in a mechanical
10.4.1.3 Handlingoftissuesforsubsequentchemicalanaly- blender.
sis requires special precautions to be taken to ensure that the (2)Alternatively, tissue should be placed in analytical
specimens are not contaminated with the elements to be grade 70% ethanol in analytical grade water in a proper
analyzed. Surgical knives or instruments used for tissue exci- container that will not allow cross-contamination.
sion shall be free of any contamination or loose particles. The (3)The tissue sample may be dried (15 min at 90°C) prior
use of ceramic or glass knives is recommended for preparation todigestion,todeterminethedryweight.Aciddigestionofthe
of specimens associated with metallic implants. Glass knives dry sample can then be accomplished with nitric acid. The
are not recommended for subsequent silicone analysis. dried tissue sample (~100 mg) should be mixed with 5 mL of
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low trace nitric acid (minimum 70% HNO ) and heated for 11. Analysis of Tissues and Fluids for Wear Particles
approximately 2 h (or more if needed) at 90°C. Alternatively,
11.1 Analysis for implant particles can be done histopatho-
the same solution can be placed in a microwave digestion
logically as in 10.2, or by tissue or fluid digestion and particle
bomb (that is, Parr Microwave Digestion Bombs Model No.
separation.Tissues subjected to digestion become dedicated to
4781 23 mL or 4782 45 mL), which is a chemically inert
this type of analysis.
vessel designed for high heat where venting may be required.
11.1.1 Preparation of tissue for drying and digestion will
These vessels can be placed in a household microwave for
depend on the state of the tissue. If the tissue was previously
more rapid sample dissolution (that is, 2 min at medium
embeddedinparaffin,thetissueblockshouldbedeparaffinized
power). There are also commercially available microwave
with xylene overnight at room temperature, and then washed
digestionsystemsavailablethatoperateathigherpressuresand
with 100% ethanol. If tissue is fixed in formalin, it should be
temperatures which also have vessels and carrousels as part of
dehydrated through a series of alcohols using standard histo-
the system. (See Note 1.)
logical protocol, and infiltrated with 100% ethanol. If tissue is
(4)The samples are then pipetted into the GFAAS or fixed in formalin, it should be dehydrated through a series of
ICPMS for analysis. Dilution as necessary is done with nitric alcohols using a standard histological protocol, and infiltrated
acid.Followingthedigestionprocedure,thesolutionmayneed with 100% ethanol. If tissue is fresh, it should be frozen and
to be diluted to a pre-determined amount (that is, 5:1) using free of embedding media.
de-ionized water. Dilutions may be necessary if the analytical 11.1.2 The tissue shall be weighed, or the fluid volume
determined before digestion. If a wet weight is desired (only
equipment cannot operate with samples containing high acid
applicableforthefreshtissue),0.3–0.5garetypicallyweighed
concentrations.
out. For a dry tissue weight, the tissue should be freeze dried,
NOTE 1—Microwave digestion is a good alternative to traditional acid
sliced with a ceramic knife, and 0.02–0.03 g weighed out with
digestion, since microwave dissolution is faster, safer, and simpler, and
a microbalance. Weighed tissue should then be placed in acid
provides more controlled reproducible conditions than conventional
washed polystyrene vials. Once weighed, the tissue is suitable
methods. Depending on the power ratings of the microwave, an appro-
for digestion.
priate level will need to be chosen in conjunction with the adequate time
to achieve full digestion of the tissue.
11.1.3 Several digestion protocols are described in the
literature.Thechoiceofprotocoldependsinpartonthetypeof
10.4.3.4 Bone specimens can be subjected to a two-stage
particle of interest, for example, metal versus polymer, and on
digestion procedure to separate them into two phases, miner-
the type of tissue. Four methods of digestion are described in
alized and demineralized (or organic).The bones are placed in
this recommended practice.
0.5NHClfor48hrsat4°Ctodemineralizethem.Therubbery
11.2 Reagents for Digestion:
demineralized samples are then placed in 50% nitric acid to
11.2.1 Ultrapure water—Distilled H O filtered with 0.2 µm
digesttheorganicportion.BoththeHClandHNO samplesare 2
filter.
diluted as necessary and analyzed by GFAAS.
11.2.2 Phosphate Buffer—3.55 g Na HPO , 3.45 g
2 4
10.4.4 Analysis with graphite furnace (GFAAS) should be
NaH PO , 0.744 g EDTA, in 100 ml ultrapure water.
2 4
done according to Practice D3919 using the manufacturer’s
11.2.3 Papain solution—1mlphosphatebuffer,100µLpure
specifications for analyte wavelength and slit width. Calibra-
papain, 3.26 mg N-acetylcysteine, 9 ml ultrapure water.
tionstandardsshouldbemadeupinthesamematrixasthetest
11.2.4 Strong bases—Tissues have been digested in solu-
specimens.
tions of sodium hydroxide ranging from 1–10 N NaOH (5–50
10.4.4.1 A multi-cycle protocol is recommended to ensure
ml/g of tissue), or in potassium hydroxide 2 N KOH (10 ml/g
complete d
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