Guidance for Industry

Source Animal, Product, Preclinical, 

and Clinical Issues Concerning the Use 

of Xenotransplantation Products in 

Humans

FINAL GUIDANCE

Additional copies of this guidance document are available from the Office of Communication, Training 

and Manufacturers Assistance (HFM-40), 1401 Rockville Pike, Rockville, MD 20852-1448, or by 

calling 1-800-835-4709 or 301-827-1800, or from the Internet at 

http://www.fda.gov/cber/guidelines.htm.

For questions on the content of this document contact Eda Bloom, Ph.D. at 1-800-835-4709.

U.S. Department of Health and Human Services

Food and Drug Administration

Center for Biologics Evaluation and Research (CBER)

April 2003
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TABLE OF CONTENTS

I. INTRODUCTION ............................................................................................................1

A. Purpose of the Document .........................................................................................1

B. Background .............................................................................................................2

C. Definitions and Abbreviations ...................................................................................3

II. REGULATORY RESPONSIBILITY..............................................................................7

III. SOURCE ANIMAL CHARACTERIZATION ...............................................................8

A. General Considerations ............................................................................................8

B. Animal Welfare Concerns.........................................................................................8

C. Animal Origin...........................................................................................................9

D. Animal Health and Husbandry................................................................................11

E. Harvest of Nonhuman Live Cells, Tissues or Organs for Use in Producing 

Xenotransplantation Products.................................................................................17

F. Source Animal History for Xenogeneic Cell Lines...................................................20

G. Disposal of Animals and Use of Byproducts............................................................20

IV. CHARACTERIZATION OF XENOTRANSPLANTATION PRODUCTS................21

A. General Considerations ..........................................................................................21

B. Considerations for Classes of Xenotransplantation Products....................................21

V. MICROBIOLOGICAL TESTING OF XENOTRANSPLANTATION PRODUCTS23

A. General Considerations ..........................................................................................23

B. Considerations for Classes of Xenotransplantation Products....................................25

C. Assay Design for the Detection of Infectious Agents................................................26

VI. MANUFACTURING AND PROCESS-RELATED GMP CONSIDERATIONS FOR 

HARVEST AND PROCESSING OF XENOTRANSPLANTATION PRODUCTS ..30

A. General Considerations ..........................................................................................30

B. Contamination/Cross-Contamination Precautions....................................................31

C. Validation and Qualification....................................................................................32

VII. PRECLINICAL CONSIDERATIONS FOR XENOTRANSPLANTATION.............34

A. General Considerations ..........................................................................................34

B. Issues Related to Infectious Agents.........................................................................35

C. Xenotransplantation Product-Host Interactions .......................................................36

D. Considerations for the Use of Heterogeneous Xenotransplantation Products............38

E. In Vitro and In Vivo Tumorigenicity Models for Xenotransplantation Products Intended 

for Transplantation.................................................................................................39

F. Combinations of Xenotransplantation Products with Devices...................................40

VIII. CLINICAL ISSUES IN XENOTRANSPLANTATION...............................................42

A. General Considerations ..........................................................................................42

B. Clinical Protocol Review ........................................................................................42
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C. Xenotransplantation Site.........................................................................................42

D. Criteria for Patient Selection...................................................................................42

E. Risk/Benefit Assessment.........................................................................................43

F. Screening for Infectious Agents...............................................................................43

G. Patient Follow-up...................................................................................................49

H. Archiving of Patient Plasma and Tissue Specimens..................................................49

I. Health Records and Data Management...................................................................50

J. Informed Consent ..................................................................................................52

K. Responsibility of the Sponsor in Informing the Patient of New Scientific Information.56

IX. REFERENCES ...............................................................................................................56
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GUIDANCE FOR INDUSTRY

Source Animal, Product, Preclinical and Clinical Issues Concerning 

the Use of Xenotransplantation Products in Humans

This guidance document represents FDA’s current thinking on this topic. It does not create or 

confer any rights for or on any person and does not operate to bind FDA or the public. An 

alternative approach may be used if such approach satisfies the requirements of the applicable 

statutes and regulations.

I. INTRODUCTION

A. Purpose of the Document

This document is to provide guidance on the production, testing and evaluation of products 

intended for use in xenotransplantation. The guidance includes scientific questions that should 

be addressed by sponsors during protocol development and during the preparation of 

submissions to the Food and Drug Administration (FDA), e.g., Investigational New Drug 

Application (IND) and Biologics License Application (BLA). This guidance document finalizes 

the draft guidance of the same title dated February 2001.

For the purpose of this document, xenotransplantation refers to any procedure that involves the 

transplantation, implantation, or infusion into a human recipient of either (a) live cells, tissues, or 

organs from a nonhuman animal source, or (b) human body fluids, cells, tissues or organs that 

have had ex vivo contact with live nonhuman animal cells, tissues or organs. For the purpose of 

this document, xenotransplantation products include live cells, tissues or organs used in 

xenotransplantation. (See Definitions in section I.C.)

This document presents issues that should be considered in addressing the safety of viable 

materials obtained from animal sources and intended for clinical use in humans. The potential 

threat to both human and animal welfare from zoonotic or other infectious agents warrants 

careful characterization of animal sources of cells, tissues, and organs. This document 

addresses issues such as the characterization of source animals, source animal husbandry 

practices, characterization of xenotransplantation products, considerations for the 

xenotransplantation product manufacturing facility, appropriate preclinical models for 

xenotransplantation protocols, and monitoring of recipients of xenotransplantation products. 

This document recommends specific practices intended to prevent the introduction and spread 

of infectious agents of animal origin into the human population. FDA expects that new methods 

proposed by sponsors to address specific issues will be scientifically rigorous and that sufficient 
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data will be presented to justify their use.

B. Background

Recent advances in technology and pharmacology, which have been important for achieving 

success in allotransplantation, have led to the proposal that xenotransplantation, initially 

attempted nearly a century ago (reference 35), may provide a solution to the shortage of human 

allografts (reference 36). Proposed xenotransplantation protocols include implantation in 

humans of live organs, tissues, or cells from a nonhuman animal source, and procedures in which 

human cells or fluids that are intended for administration to human recipients have had ex vivo 

contact with live nonhuman cells, tissues, or organs. Examples of procedures involving 

xenotransplantation products include: 

• transplantation of xenogeneic hearts, kidneys, or pancreatic tissue to treat organ failure, 

• implantation of neural cells to ameliorate neurological degenerative diseases, 

• administration of human cells previously cultured ex vivo with live nonhuman animal 

antigen-presenting or feeder cells, and 

• extracorporeal perfusion of a patient’s blood or blood component perfused through an 

intact animal organ or isolated cells contained in a device to treat liver failure. 

The use of the different xenotransplantation products has the potential for transmission of 

infectious disease from nonhuman animals to humans. 

Potential public health risks posed by the use of xenotransplantation products include: 

(1) transmission of infectious agents that are pathogenic for humans but may not be 

pathogenic or even detectable in the source animal host; 

(2) transmission of organisms that may not normally be pathogenic in humans but can 

become so in the immunosuppressed or immunocompromised individual; and, 

(3) recombination or reassortment of infectious agents, particularly viruses, with 

nonpathogenic or endogenous human infectious agents, to form new pathogenic 

entities. 

Furthermore, it is difficult to predict the infectious agents that may cause disease in a recipient of 

a xenotransplantation product solely on the basis of analysis of naturally occurring zoonoses 

because there are major differences between normal contact of humans with animals and 

contact of a recipient with a xenotransplantation product. For example, the physical barrier or 

distance is eliminated in the recipient due to transplantation and vascularization of 

xenotransplantation products, or even due to implantation of nonvascularized cells or tissues, or 

ex vivo manipulations that cause intimate proximity or contact of xenotransplantation product 

materials with recipient cells, tissues, or fluids. The potential for viral adaptation in 

immunocompromised or iatrogenically immunosuppressed hosts and the potential for undetected 

spread of previously latent viral infections are of particular concern.
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For these reasons, during product development it is important to consider the safety, not only of 

recipients and their contacts, but also of the public. Public discussion of these issues is 

important and will continue to take place through the FDA Biological Response Modifiers 

Advisory Committee-Subcommittee on Xenotransplantation, the Department of Health and 

Human Services (DHHS) Secretary’s Advisory Committee on Xenotransplantation, and other 

public fora.

Many issues relevant to the use of live materials obtained from nonhuman animal sources are 

addressed in previous FDA guidance documents (see Guidance Documents in References). In 

addition, the International Conference on Harmonisation (ICH) has published a number of 

documents that are relevant to the use of xenotransplantation products in humans (see ICH 

guidelines in References). FDA adopts ICH documents as guidance documents, and you 

should also consult them.

The United States Public Health Service (PHS) has previously made recommendations 

regarding the infectious disease risks posed by use of xenotransplantation in humans. In 1996, 

the PHS published a “Draft PHS Guideline on Infectious Disease Issues in Xenotransplantation” 

(61 FR 49920, September 23, 1996). Based on comments received and advances in fields 

relating to xenotransplantation, the PHS updated and revised the draft guideline (reference 1) 

(hereafter referred to as “PHS Guideline”). This FDA guidance document reiterates many of 

the concepts in the PHS Guideline, but in addition includes specific advice regarding all aspects 

of xenotransplantation product development and production, and xenotransplantation clinical 

trials.

FDA anticipates that its approach to regulation of xenotransplantation products will evolve as 

the scientific knowledge in the area of xenotransplantation continues to accumulate. Thus, this 

document, as with other guidance documents, will change as knowledge and experience 

pertinent to xenotransplantation accumulates. In addition, FDA realizes that it may not be 

appropriate to apply every aspect of the guidance to every xenotransplantation product. For 

example, some of the recommendations for animal husbandry may not be needed for 

xenotransplantation products consisting of well-characterized, long-established animal cell 

culture lines or human cells co-cultured with such lines.

C. Definitions and Abbreviations

Act: The Federal Food, Drug, and Cosmetic Act (21 U.S.C. 321 et seq.).

AAALAC: Association for Assessment and Accreditation of Laboratory Animal Care, 

International. This organization inspects and accredits biomedical animal facilities.

Agents of concern: For the purpose of this document, agents of concern are infectious agents 

that may pose a risk to the recipient and/or public, i.e., agents that can infect, potentially could 
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infect, or have an inadequately defined ability to infect humans.

ATCC: American Type Culture Collection.

BLA: Biologics License Application.

BSL: BioSafety Level.

CDC: Centers for Disease Control and Prevention.

CDRH: Center for Devices and Radiological Health.

cGMP: Current good manufacturing practice. For drugs, including biological drugs, cGMP 

regulations can be found at 21 CFR Parts 210 and 211. For biological products, see 21 CFR 

Part 600 Subpart B and Part 610. For blood and blood components, additional regulations can 

be found at 21 CFR Part 606. For devices, quality system regulations can be found at 21 CFR 

Part 820. 

Closed herd or colony: Herd or colony governed by Standard Operating Procedures (SOPs) 

that specify criteria restricting admission of new animals to assure that all introduced animals are 

at the same or higher health standard compared to the residents of the herd or colony.

CPE: Cytopathic effects. An effect on nucleated cells in vitro caused by some viruses that are 

observable microscopically.

DPF: Designated pathogen free. This term is used to describe animals, animal herds, or animal 

facilities that have been rigorously documented to be free of specified infectious agents, and that 

are maintained using well-defined routines of testing for designated pathogens, and utilizing 

rigorous SOPs and practices of herd husbandry and veterinary care to assure the absence of the 

designated pathogens.

EM: Electron microscopy. A method used to visualize very small objects, such as subcellular 

particles, or infectious agents such as viruses.

FDA: Food and Drug Administration.

FSIS: Food Safety Inspection Service, Department of Agriculture.

Gnotobiotic: The science of rearing laboratory animals, the microflora and microfauna of which 

are specifically known in their entirety.

GVHD: Graft versus host disease.
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HEPA: High Efficiency Particulate Air.

IACUC: Institutional Animal Care and Use Committee. A local institutional committee 

established to oversee the institution’s animal program, facilities, and procedures. An IACUC 

carries out semiannual program reviews and facility inspections and reviews all animal use 

protocols and any animal welfare concerns. (See PHS Policy on Humane Care and Use of 

Laboratory Animals, September 1986; reprinted March 1996.)

IBC: Institutional Biosafety Committee. A local institutional committee established to review 

and oversee basic and clinical research conducted at that institution. The IBC assesses the 

safety of the research and identifies any potential risk to public health or the environment. (See 

section IV-B-2 of the NIH Guideline for Research Involving Recombinant DNA Molecules, 

reference 17.)

ICH: International Conference on Harmonisation.

IDE: Investigational device exemption application. These are applications containing requests 

to use an unapproved device in clinical tests using human subjects. The statutory requirement is 

at section 520(g) of the Act (21 U.S.C. 360(g)), and the implementing regulations can be found 

at 21 CFR Part 812.

IND: Investigational new drug application. These applications are required for persons who 

intend to conduct clinical investigations involving unapproved drug products, including those 

subject to section 505(i) of the Act (21 U.S.C. 355(i)) or to the licensure provisions of section 

351 of the PHS Act (42 U.S.C. 262). The statutory requirement is at section 505(i) of the Act 

(21 U.S.C. 355(i)), and the IND regulations are found at 21 CFR Part 312.

IRB: Institutional Review Board. A board, committee or other group designated by an 

institution established to review and approve biomedical and behavioral research involving 

human subjects in order to protect the rights and welfare of human subjects (See 21 CFR Part 

56, Institutional Review Boards.

Lot: Defined in 21 CFR 210.3(b)(10) as a batch, or a specific identified portion of a batch, 

having uniform character and quality within specified limits, and in 21 CFR 600.3(x) as that 

quantity of uniform material identified by the manufacturer as having been thoroughly mixed in a 

single vessel. Each lot of final product is subjected to appropriate tests to ascertain adherence 

to specifications prior to release of the product for clinical use. Licensed biological products 

may be subject to lot release as described in 21 CFR 610.2(a). Often in the case of 

xenotransplantation products, an entire lot is used for treating a single recipient. 

Master File: Master Files are submitted to FDA and contain information regarding a product, 

such as product manufacture or general procedures. Procedures and information contained in 

the Master File can be cross-referenced in INDs and IDEs on written permission from the 
6

Master File sponsor, but confidentiality of the information within the Master File is maintained. 

(See 21 CFR 314.420.)

PBMC: Peripheral blood mononuclear cells.

PCR: Polymerase chain reaction. An enzymatic technique, using a thermophyllic enzyme to 

catalyze synthesis of short DNA sequences, that allows detection of nucleic acids by 

amplification of specific DNA sequences.

PERV: Porcine endogenous retrovirus(es).

PHS Act: The Public Health Service Act (42 U.S.C. 201 et seq.). 

PMA: Premarket approval application. This is a marketing application for certain devices 

under section 515 of the Act. The regulations for PMAs can be found at 21 CFR Part 814.

Recipient: An individual who receives or who undergoes ex vivo exposure to a 

xenotransplantation product (as defined in xenotransplantation).

RT: Reverse Transcriptase. An enzyme found particularly in retroviruses, that catalyzes the 

synthesis of DNA from RNA.

SAF: Source animal facility.

Sentinel animal: Those animals, usually of the same species as the resident herd or colony, 

housed in direct contact with the animals being monitored, and tested for the purpose of 

detecting adventitious agents in the herd or colony including viral, fungal and bacterial diseases 

as well as endo- and ectoparasites. 

SOP: Standard operating procedure.

Source animal: An animal from which cells, tissues, and/or organs for use in xenotransplantation 

are obtained.

TSE: Transmissible spongiform encephalopathy. TSEs are fatal, subacute degenerative 

diseases of humans and animals with characteristic neuropathology (spongiform change and 

deposition of an abnormal form of a prion protein present in all mammalian brains). TSEs are 

experimentally transmissible by inoculation or ingestion of diseased tissue. The abnormal prion 

protein is hypothesized to be the agent of transmission. Alternatively, other unidentified cofactors or an as-yet-unidentified viral agent may be necessary for transmission.

USDA: United States Department of Agriculture.
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WFI: Water for injection.

Xenotransplantation: For the purpose of this document, any procedure that involves the 

transplantation, implantation, or infusion into a human recipient of either (a) live cells, tissues, or 

organs from a nonhuman animal source, or (b) human body fluids, cells, tissues or organs that 

have had ex vivo contact with live nonhuman animal cells, tissues or organs.

Xenotransplantation product(s): For the purpose of this document, xenotransplantation 

products include live cells, tissues or organs used in xenotransplantation (defined above).

Zoonosis: A disease of animals (e.g., brucellosis, rabies) that may be transmitted to humans 

under natural conditions.

Zoonotic: Relating to diseases that arise from the transfer of infectious agents by normal 

contacts between animals and humans.

II. REGULATORY RESPONSIBILITY

FDA has regulatory oversight of xenotransplantation products, including live organs, tissues, or cells 

from a nonhuman source, or xenotransplantation product materials used in encapsulated form or in 

which nonhuman live organs, tissues or cells have ex vivo contact with human body fluids, cells, tissues 

or organs that are subsequently given to a human recipient. If xenotransplantation products are to be 

used in clinical investigation, they require an appropriate investigational application be in place (e.g., 21 

CFR Part 312). FDA will regulate most xenotransplantation products as biological products. CBER 

regulates biological products, including cellular therapies, under authority of section 351 of the PHS Act 

(42 U.S.C. 262), and the Act (21 U.S.C. 321 et seq.). You will find regulations for drugs, biological 

products, and devices in Title 21 of the Code of Federal Regulations (e.g., 21 CFR Part 312 for 

regulations governing INDs, 21 CFR Part 812 for regulations governing IDEs, and 21 CFR Part 601 

for regulations governing licensing of biological products). The primary responsibility for designing and 

monitoring the conduct of xenotransplantation clinical trial rests with the sponsor (e.g., 21 CFR 

312.23(a)(6)(iii)(d) and 312.50). In this document “you” refers to the sponsor, the clinical investigator, 

or to any party designated by the sponsor to fulfill the recommended function (e.g., Source Animal 

Facility staff, laboratory personnel, etc.).

Some products may be combination products consisting of a biologic and a device, such as xenogeneic 

cells contained in a device used for extracorporeal hemoperfusion. Refer to 21 CFR Part 3 for issues 

regarding regulation and assignment for premarket review of combination products.
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III. SOURCE ANIMAL CHARACTERIZATION 

A. General Considerations

The cross-species infectious potential of specific animal pathogens should be a major 

consideration in the selection of the source animal species. Anatomic and physiologic 

considerations are also of importance. For example, whether an organ is of appropriate size 

and will function adequately across species barriers are considerations, as are certain 

immunologic concerns including the suitability of current regimens in prevention of rejection of 

the nonhuman live cells, tissues or organs. Please note that the species you use may be subject 

to other federal laws and regulations (see, e.g., 16 U.S.C. 1538) such as those covering 

endangered or protected species. You should consult all relevant PHS and FDA guidance 

documents on this subject prior to submitting an application, and specifically should consult the 

document, “Guidance for Industry: Public Health Issues Posed by the Use of Nonhuman 

Primate Xenografts in Humans” (reference 2) before submitting an application to FDA that 

involves the use of nonhuman primates as sources of a xenotransplantation product. (The term 

“xenograft” in the above referenced document is synonymous with the term “xenotransplantation 

product” in current use by DHHS and in this guidance.)

Due to potential infectious disease risks associated with the use of xenotransplantation products, 

you should develop appropriate source animal qualifications. These qualifications should include 

herd management and programs for prevention and screening for infectious agents. Although 

testing of the final xenotransplantation product for infectious agents is crucial, appropriate 

control of animal sources and husbandry provides important additional assurance for the safety 

of such products by controlling infections by both known and potentially even unknown agents. 

Therefore, the specific information supplied by the sponsor regarding animal husbandry including 

housing, feeding, veterinary care, drug and biologic treatment of source animal herds and 

individual source animals, will be crucial for FDA evaluation of the potential for safe use of cells, 

tissues, or organs from such source animals.

The SAF, production process, and records are subject to FDA inspection under section 704 of 

the Act (21 U.S.C. 374) and section 351(c) of the PHS Act (42 U.S.C. 262(c)). 

B. Animal Welfare Concerns

Another area of consideration for SAFs and manufacturers of xenotransplantation products is 

the welfare of the source animals. Procedures for animal husbandry, tissue harvesting, and 

termination of animals should be approved by an appropriate Institutional Animal Care and Use 

Committee, in accordance with the Animal Welfare Act (7 U.S.C. 2131, et seq.). In cases 

where funds are received from the PHS, procedures must also comply with the PHS Policy on 

Humane Care and Use of Laboratory Animals according to section 495 of the PHS Act (42 

U.S.C. 289(d)). We recommend that the SAF be accredited by the AAALAC. Standards for 

accredited facilities for when funds are received from the National Institutes of Health are 
9

provided in the National Research Council’s Guide for the Care and Use of Laboratory 

Animals (reference 4). 

C. Animal Origin

1. Animal and Herd Qualification

You should derive source animals only from closed herds with documented health 

screening programs. Individuals with expertise in infectious diseases of the species 

involved should develop a list of viruses, bacteria (including the rickettsiae), 

mycoplasma, fungi, transmissible spongiform encephalopathies (TSEs), and parasites for 

which the herd is screened and supply this information to FDA as part of the application

to FDA (e.g., IND). You should consider all infectious agents known to infect the 

source species. You should justify the rationale for omitting agents that are found in the 

source animal species from the herd screening program in the application to FDA 

requesting investigational use (e.g., IND). For example, the geographic location of the 

herd may allow exclusion of certain infectious agents. You should obtain source animals 

from TSE-susceptible species only from closed herds that are documented to be free 

from TSE diseases or TSE-associated agents (see also section III.C.3.c.). You should 

not use source animals obtained from geographic areas in which TSEs are known to 

exist in the source species. In the application to FDA requesting investigational use of 

the xenotransplantation product (e.g., IND), you should describe and justify the 

frequency of the screening, the method of assay, and the method of identifying which 

and what proportion of animals are sampled. As data are accumulated that 

demonstrate product safety, you may modify the screening program in consultation with 

the FDA.

2. Animal History

The sponsor should document the geographic origin, species, strain, and genealogy of 

the source animal(s) and herd(s). The documentation of source animal history should 

describe factors that may pose risks to recipients, such as possible exposure of the 

predecessor animals to sources of TSEs or other adventitious or infectious agents of 

concern (see Definitions, section I.C.). Source animals should be bred and raised in 

captivity and be derived from closed herds. You may use artificial insemination, embryo 

transfer, cloning, or hysterotomy plus foster feeding to establish animal herds with fewer 

endemic pathogens. In particular, the PHS Guideline suggests that breeding programs 

use caesarean-derived animals whenever possible (reference 1). You should document 

the use of these procedures in the animal history. 

3. Source Animals from Outside the U.S. 

a. You should not use animals from outside the U.S. or their first 
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generation offspring as sources for the production of xenotransplantation 

products unless they are of a species or strain not available in the United States, 

or have specific qualities that provide a unique and scientifically justified clinical 

advantage, such as transgenic animals. 

b. If the use of source animals from outside the U.S. is necessary and 

justifiable, you should apply the same considerations for these animals as for 

source animals bred in the U.S. (e.g., see section III.D. for Animal Health and 

Husbandry). You should use a quarantine period of sufficient length to 

demonstrate the absence of infectious agents of concern, and you should 

perform extensive screening of the animals. In the application to FDA 

requesting investigational use of the xenotransplantation product (e.g., IND), 

you should submit thorough documentation to demonstrate that such source 

animals have been derived from closed herds, have been housed under 

appropriate conditions and subjected to recommended health maintenance 

procedures and screenings, and have not been fed rendered or recycled 

mammalian materials for at least two generations. You should include in the 

screening agents that are endemic in the country of origin. In the application to 

FDA requesting investigational use of the xenotransplantation product (e.g., 

IND) you should describe methods and conditions of transport of imported 

animals. Descriptions should include means of transport and husbandry during 

transport including isolation, caging, handling, animal treatment, and presence of 

other animals of the same or different species. If animals from countries outside 

the U.S. are needed, you should use them as founders for a domestic herd that 

will be well-characterized for an extended period of time before use, using 

procedures sufficient to validate the herd’s acceptability as source animals. 

c. You should not import source animals from any country or geographic 

region where TSEs are known to be present in the source species. The USDA 

has identified countries from which the importation of ruminants and some 

ruminant products are restricted or prohibited (9 CFR Parts 94 and 96).

d. You should consult the USDA and, when appropriate, FDA’s Center 

for Veterinary Medicine (CVM) and CDC for their requirements regarding 

importation of animals or animal tissues.

4. Range and Wild Animals

You should not use as source animals those that are raised under free-ranging 

conditions. Such animals have a higher likelihood of harboring infectious agents due to 

potential exposure of the source animal to other animals, birds, insects, or other 

uncontrolled environmental factors.
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You should not use wild-caught animals as source animals. 

5. Animal Sources Obtained from Slaughterhouses or Abattoirs

Animals from slaughterhouses or abattoirs are unsafe for use as source animals. 

Appropriate documentation and histories of animals from slaughterhouses may not be 

available because the animals are often obtained from geographically divergent farms or 

markets, and exposure to other animals or potential sources of infectious agents during 

transit or after arrival at the slaughterhouse are unknown. Therefore, you should not use 

such animals as source animals.

6. Semen Donors

You should apply the same source animal considerations to semen donors, whether or 

not they are members of the herd including, for example, screening for infectious agents 

that may be transmitted by semen.

D. Animal Health and Husbandry

Production of animals as sources of live cells, tissues, or organs for use in xenotransplantation 

products involves an adequately designed facility and a program for the operation of the facility 

to minimize the animals' exposure to infectious agents.

You should obtain source animals exclusively from SAFs. Your application or submission to 

FDA should include detailed plans for maintaining source animals. These plans should include 

standard operating procedures detailing the containment and housing of animals, feeding and 

obtaining feed, water and bedding, performance and monitoring of the health screenings, 

removal from production and disposal of the animals and their byproducts, and identifying 

individual animals and recording their movements to, through, and out of the facility. These 

procedures should take into consideration the source animal species and xenotransplantation 

product(s) as appropriate. 

1. Facilities

You should house the animals in facilities built and operated in accordance with 

recommendations described in the National Research Council’s Guide for the Care and 

Use of Laboratory Animals (reference 4) and accredited by the AAALAC. You 

should not locate SAFs in geographic proximity to manufacturing or agricultural 

activities that could compromise the facility’s biosecurity by providing or enabling a 

source of infections.

SAFs are subject to the regulations in 21 CFR Part 600, Subpart B, on establishment 

standards, including the requirements regarding animals and personnel in §§ 600.10 and 
12

600.11. SAFs also are subject to the regulations in 21 CFR Part 600, Subpart C, 

regarding inspections. These facilities are subject to inspection by designated 

representatives of the clinical protocol sponsor and public health agencies.

You should include a detailed description of the facilities and procedures for housing 

source animals with the FDA submission (e.g., IND or Master File). The information 

provided should include plans for the shelters, the feeding areas, the washing areas, the 

fencing, air handling systems (particularly in quarantine areas), lighting, temperature, and 

other physical attributes of the animal environment. Facility descriptions should also 

include information on physical barriers and operational measures intended to eliminate 

or minimize exposure to insects, birds, or other animals that may transmit disease to the 

source animals. You should keep records of any biological or physical compromise of 

the animal environment as well as measures taken in response to this problem. These 

descriptions should also cover the procedures and schedules followed for cleaning and 

other routine maintenance of the animal enclosure. You should include procedures for 

elimination of animal wastes. Include in the description how qualified source animals will 

be housed (for example as a batch or individuals) and the methods used to 

decontaminate the housing after the source animals are used.

The SAF staff should include veterinarians with expertise in the infectious diseases and 

agents prevalent in the particular animal species being raised in the facility. If a 

veterinarian with expertise in infectious diseases is not on staff, you should provide 

documentation in the investigational application (e.g., IND) that an individual with the 

appropriate expertise is available for consultation. Staff should also include adequate 

numbers of caretaker personnel with appropriate training in the care and health of the 

species being housed (e.g., §§ 600.10 and 600.11).

2. Maintenance of Source Animals 

a. General 

You should maintain source animals in accordance with standard operating 

procedures appropriate to the species, xenotransplantation product, and the 

intended clinical application. SOPs should provide for admission of new 

animals to the SAF and source animal pool, for quarantine, and for removal, 

isolation, or elimination of diseased animals. You should provide this 

information to FDA in the application for investigational use (e.g., IND). You 

should not reintroduce animals that have been removed from the source animal 

pool due to illness or infection. 

You should develop procedures to identify incidents that negatively affect the 

health of the herd or colony. This information is relevant to the safety review of 

every xenotransplantation product application. You should report such 
13

information to FDA, as well as the procedures to collect the information, as part 

of the application for investigational use (e.g., IND).

b. Health Screening

i. You should maintain source animals in barrier facilities that are 

considered free of designated pathogens. For the purpose of this 

document, such facilities are termed “Designated Pathogen Free” 

(DPF), and animals derived from them are termed “DPF animals.” 

Initial screening and routine monitoring are important to validate that 

such facilities maintain DPF status. Protocols for monitoring the herd 

for disease and infectious agents should exist, and you should include a 

copy or a summary of the SOPs in the FDA submission requesting 

investigational use (e.g., IND). You may modify the frequency of 

testing as the reliability of the production system is established using 

data from earlier screens. You should consult appropriate experts, such 

as infectious disease consultants, virologists, microbiologists, accredited 

microbiological laboratories, and veterinarians, to generate a list of 

agents for which all source animals should be screened and a list of 

appropriate diagnostic tests. In addition to screening for specific 

infectious agents, you should use more general assays for detection of 

classes of agents. For example, you should examine feces from source 

animal herds on a regular basis for evidence of parasitic infections. If 

infectious agents, including normal flora that could potentially be 

infectious in an immunosuppressed recipient, have been identified in 

source animals, the use of such animals should be avoided. You should 

consult with CBER if the use of such animals is contemplated (see, for 

example, section V.C.4.d.). Techniques for introducing new animals, 

such as artificial insemination, caesarean section, cloning, or novel 

gnotobiotic techniques, should be fully described.

ii. Subclinical infections of source animals may not be apparent at 

the time of harvest of the nonhuman live cells, tissues, or organs, and 

may be identified only retrospectively. Sampling of individual animals 

from the herd of origin for screening, and the use of sentinel animals, 

should help minimize this problem and may help identify infections in 

recipients post transplantation. You should consider establishing a 

sentinel animal program that includes periodic necropsy and extensive 

histology and pathology evaluations. The screening procedures should 

be appropriate for the animal species, the xenotransplantation product, 

and the clinical application. Specific screening procedures should 

include appropriate physical examination and laboratory tests, and 

should be able to detect zoonoses known to exist in the species or 
14

geographical regions in which the source animals originate and are 

maintained. 

iii. You should quarantine and screen individual source animals 

before harvest of cells, tissues, or organs, as discussed in section 

III.D.4.b. below and in the PHS Guideline (reference 1).

c. Health Care

The herd health surveillance system should include comprehensive 

documentation of all veterinary care received by source animals. This includes 

documentation of all illnesses, medical care, procedures, drugs administered, 

vaccinations, routine physical exams, and any treatments received by each 

animal. You should carefully document use of antimicrobial agents due to 

potential risk to allergic recipients receiving unprocessed nonhuman live cells, 

tissues or organs. You should validate residual drug levels to be insignificant in 

cells, tissues, or organs taken from source animals that previously have received 

medications. Exclusive use of killed vaccines generally is warranted both in the 

source animal and in the herd with which it is associated. You should use live 

vaccinations only when alternative immunogens for vaccinations are not 

available, and only if scientific evidence exists to support that the live cells, 

tissues, or organs from the vaccine-treated animal will not pose a risk of 

infection for the human recipient. You should describe the procedures to deal 

with illnesses or other incidents that affect the health of the herd in your 

application to FDA for investigational use (e.g., IND). You should not use as 

source animals those animals requiring treatment with blood, blood products, or 

tissues obtained from animals outside the closed herd and you should remove 

these animals from the herd. You should use aseptic techniques and sterile 

equipment for all parenteral interventions including vaccinations, treatment with 

drugs or biologics, phlebotomy, and biopsies. If animals within the closed herd 

have been treated with a biological product (e.g., vaccine, monoclonal 

antibody), you should document the treatment in the application to FDA 

requesting investigational use (e.g., IND), and you should provide copies of 

package inserts or labeling. You should document and maintain records 

showing the treatment of animals with drugs for any reason, including the 

withdrawal period following drug treatment. You should develop procedures 

for disposal of dead animals (see section III.G.).

d. Feed

You should describe the storage and delivery of feed, water, and other 

consumables in the application to FDA for investigational use (e.g., IND). 

Records should include manufacturer, batch numbers, and other pertinent 
15

information, and you should describe recordkeeping procedures in an SOP. 

You should record in the individual source animal’s records the vendor and 

contents of feed given to a source animal for at least two generations before use 

as a source for live cells, tissues, or organs used in xenotransplantation. You 

should not use feeds containing rendered or recycled mammalian material, or 

significant drug contamination or pesticide or herbicide residues for source 

animals. You should not use natural, non-sterile, foods such as hay to minimize 

potential risks of exposure to pests or infectious agents. Water should be of 

sufficient quality to prevent unnecessary exposure of animals to infectious or 

adventitious agents, drugs, pesticides, herbicides, and fertilizers. You may 

include pasteurized milk products in feeds. You should feed newborn animals 

colostrum or milk from dams only if the dams have been specifically qualified by 

the same procedures used for herd qualification.

e. Caretakers 

You should provide SOPs for animal caretakers in the FDA submission 

requesting investigational use (e.g., IND), and you should include entry and exit 

procedures, clothing requirements and all interactions with the animals, e.g., 

feeding, watering, exercising, delivery of immunizations and medications, etc., 

(e.g., § 600.11). There should be a documented training program for personnel 

as described in the cGMP regulations (§ 211.25).

You should monitor the health of humans in contact with animals on a routine 

basis (reference 5). You should predetermine and customize the program for 

screening and monitoring of caretaker and other staff to maximize screening 

information, and you should describe the program in an SOP. Health 

monitoring of humans who come into contact with the animals should include 

physical exams with baseline and, if indicated, periodic sampling and storage of 

serum or plasma for individuals having frequent and close contact with source 

animals, to less rigorous monitoring for those with occasional contact. You 

should obtain baseline samples from all caretakers. 

3. Animal and Personnel Traffic Through the Source Animal Facility

You should develop SOPs for entry and exit of animals and they should include 

transportation of animals to and from the facility. You should subject all animals 

entering the facility to a defined quarantine period allowing for completion of any 

screening procedures. The minimum quarantine period for animals used in manufacture 

is seven days (§ 600.11(f)(2)). However, you should use longer quarantine periods that 

extend beyond the incubation period for infectious agents in the source animal species 

for animals entering a SAF. You should devise a tracking system that allows unique 

identification of each individual animal in the facility. You should minimize entry and exit 
16

of animals and human staff to avoid exposures to transmissible infectious agents. We 

encourage the use of an ‘all in/all out’ or batch approach for moving qualified source 

animals as a method of minimizing the potential for infectious agent transmission.

You should describe personnel traffic patterns in the FDA submission requesting 

investigational use (e.g., IND), and should minimize transmission of infectious agents. 

Caretakers should not work in more than one animal facility or with more than one 

species of animal. Caretakers should not work with more than one isolated group of 

animals or more than one herd within any given day unless validated SOPs for caretaker 

decontamination and disinfection are used.

4. Individual Source Animal Qualification

a. Testing for infectious agents

You should screen all individual source animals for presence of the same 

infectious agents used for herd qualification. In addition, you should perform 

further laboratory tests for infectious agents as described in section V. for 

testing of the xenotransplantation product (e.g., viral cocultivation assays,) on 

appropriate samples of source animal blood or tissue. When fetal or neonatal 

animals will be used as source animals, you should conduct testing of the 

mothers which may supplant testing of the fetus or neonate if technical and 

temporal difficulties render such testing unfeasible. 

When feasible, you should examine a biopsy of the live animal cells, tissue or 

organ or other relevant tissue (e.g., contra-lateral kidney as a surrogate for a 

kidney graft or thoracic tissue as a surrogate for a heart) by histopathology and 

tested for infectious agents by appropriate assays. You should take care in 

performing biopsies to use fastidious sterile technique so as not to introduce 

new microbial contaminants. You should archive remaining biopsy tissue as 

described in section III.E.3. 

You should perform all tests at a time as close as possible to the date of harvest 

of the live cells, tissues, or organs, but which allows the results to be obtained 

before their use. If more than 3 months have elapsed since the initial testing or 

biopsy of the source animal, you should repeat tests before harvest.

You should consider the nature, timing, and results of surveillance of the herd 

from which the individual animal is procured in designing appropriate screening 

of individual animals.
17

b. Quarantine

You should generally quarantine individual source animals for a minimum of 

three weeks before harvest of their live cells, tissues, or organs. It may be 

appropriate to modify individual quarantine periods depending on the 

characterization and surveillance of the source animal herd, the design of the 

facility, and the clinical indication. If the quarantine is shorter, you should 

provide justification in the application to FDA for investigational use (e.g., 

IND). During the quarantine period, in addition to tests for infectious agents, 

source animals should undergo physical examination by a veterinarian including 

complete blood count, peripheral blood smear, and fecal exam for parasites. 

E. Harvest of Nonhuman Live Cells, Tissues or Organs for Use in Producing 

Xenotransplantation Products

1. Harvest and Documentation

You should describe the procedures and physical facilities used for harvesting of live 

cells, tissues or organs from source animals in detail in the application to FDA 

requesting investigational use (e.g., IND). Qualified and controlled procedures for 

avoiding the introduction of infectious agents during harvesting should be in place. 

Qualification of the procurement and screening procedures should include documented 

performance of the processes, with documented results supporting successful harvest of 

live cells, tissues or organs from source animals that meet lot release criteria including 

identity, potency (or activity) and safety (e.g., microbiological sterility). Source animal 

anesthetic agents should not be harmful to the human recipient. A summary of the health 

records (e.g., health status and microbiological screening reports, results of lot release 

tests, and anesthetic used, if relevant) regarding the source animal should accompany 

the xenotransplantation product, and you should incorporate them into the recipient’s 

records.

SOPs should permit rapid, accurate, and facile tracking of tissue from an individual 

source animal to the recipient.

2. Transportation

Transportation of source animals may expose them to risks not encountered in closed 

herds and should be avoided if possible. Therefore we recommend that, when feasible, 

and particularly in cases where source animal tissues or cells are going to be processed 

further prior to use, live cells, tissues, or organs should be procured at the animal facility 

prior to shipping. In some cases, particularly when the xenotransplantation product is a 

whole organ intended for immediate transplantation, it may be necessary to ship live 
18

animals. In those cases where transportation is necessary, you should maintain barriers 

equivalent to, or better than, those in place at the SAF during transit to ensure that 

source animal contamination does not occur en route. Transportation should occur in 

dedicated vehicles in which source animals are not exposed to any other animals, and 

you should document the method in the submission to FDA (e.g., in the IND). If there 

is any question regarding the effectiveness of the transportation and containment 

procedures, you should quarantine and rescreen animals in a fashion comparable to that 

used for entry of new animals into a closed herd.

You should develop and implement procedures for avoiding shipping errors, avoiding 

contamination, and documenting transfer of animal materials to the correct patient. You 

should describe in detail the method of transporting the live animal cells, tissues or organ 

from harvest site to the clinical xenotransplantation site in the application to FDA 

requesting investigational use (e.g., IND). 

3. Source Animal Sample Archive

a. Timing of Sample Acquisition

If the source animal is sacrificed at the time its live cells, tissues, or organs are 

harvested, you should conduct a full necropsy including gross, histopathological, 

and microbiological evaluation, and you should obtain archival samples including 

portions of the product for storage as described in section III.E.3.b.

If the source animal is not sacrificed at the time its cells, tissues, or organs are 

harvested, you should archive portions of the harvested material and plasma and 

leukocytes from the source animal, and you should monitor the health of the 

source animal for life. 

When source animals die or are euthanitized, you should perform a full 

necropsy, and you should obtain archival samples for storage as described in 

section III.E.3.b.

b. Samples to be Archived and Storage Conditions

You should store archived samples of source animal tissues and body fluids at -

70°C or lower temperatures, as appropriate for preserving the sample, or you 

should maintain fixed samples at room temperature. Section 3.7.1 of the PHS 

Guideline (reference 1) recommends that you cryopreserve at least ten 0.5 cc 

aliquots of citrated- or EDTA-anticoagulated plasma and at least five aliquots of 

viable leukocytes (1x107/aliquot, for subsequent isolation of nucleic acids and 

proteins or for use as a source of viable cells for co-culture or other tissue 

culture assays). You should select the conditions of cryopreservation and 
19

storage for viable samples to maintain cell viability for the period of storage (see 

section III.E.3.c.). You should collect appropriate tissue samples for formalin 

fixation and paraffin-embedding and for cryopreservation from source animals 

at the time the live cells, tissues, or organs are procured. You should collect 

and cryopreserve tissue samples representative of major organ systems of 

source animals (e.g., spleen, liver, bone marrow, central nervous system, lung,) 

at necropsy. As appropriate to the xenotransplantation product, you should 

archive other body fluids, such as cerebrospinal fluid, at the time of procurement 

of the product and/or necropsy. If sentinel animals are used, you should also 

archive tissue samples and body fluids obtained at necropsy.

c. Archive Rationale, Duration, and Responsibility 

The PHS Guideline (reference 1) recommends that a sufficient quantity of 

materials be harvested and cryopreserved for three different uses:

(i) dedicated sample(s) for use by the PHS (see reference 1),

(ii) for use if needed for recipient diagnosis and care, and 

(iii) for use by the sponsor, as appropriate. 

You should include detailed plans for obtaining and storing the archive samples 

in the application to FDA requesting investigational use (e.g., IND). The PHS 

Guideline (reference 1) recommends that samples should be stored for 50 years 

from the time of sample acquisition. You should clearly designate responsibility 

for the archives and access to the specimens.

4. Herd Records

You should keep records pertaining to the source animals and facilities. These 

records are subject to inspection, and you should maintain the records for 50 

years beyond the date of procurement of the nonhuman live cells, tissues, or 

organs for use in xenotransplantation. 

5. Disposition of Records on Closing of a Source Animal Facility

If a SAF ceases operation, all records and archived samples should be 

transferred to the respective sponsors or the sponsors should be notified of a 

new archive site. You should make provisions for all records to be maintained 

for the requested period in the event that the establishment ceases operation. If 

you are a sponsor who plans to cease doing business, you should consult FDA 

regarding the disposition of records and archive samples. 
 20

F. Source Animal History for Xenogeneic Cell Lines

Cell lines from animals may be established and used in the production of xenotransplantation 

products. You should include the history of the cell line in the application to FDA requesting 

investigational use (e.g., IND). Especially for long-term cultures, it need not always include all 

the detailed information about the source animal and source animal facility mentioned above. 

However, it should include at a minimum the species and tissue of derivation. You should 

include, whenever possible, information such as age and sex of the source animal, laboratory of 

derivation, date of derivation, and the immediate provider of the cell line. For short-term 

cultures (e.g., derived less than one year previously), it should also include a description of the 

husbandry and health status of the particular source animal and herd or colony. The history of 

the cell line should also include the above information on feeder cells or animals used for 

passage in vivo, if such techniques were used to develop the cell line. Information regarding the 

commercial source and country of origin of any animal products used in deriving or maintaining 

the cell line should also be included in the history. You should characterize and test the final 

product as described in section V. You may also consult the “Points to Consider in the 

Characterization of Cell Lines Used to Produce Biologicals” (reference 25) for pertinent 

recommendations regarding the production, identification and characterization of cell lines used 

in manufacture. Additional information may be found in relevant International Conference on 

Harmonisation (ICH) Guidelines (references 32 and 33).

FDA has considered how this guidance relates to human embryonic stem (hES) cell lines that 

were in existence prior to August 9, 2001. These hES cell lines had used murine feeder 

layer cells and thus fit the definition of xenotransplantation used in this guidance and the PHS 

guideline. FDA has had several meetings with some of the derivers of certain of these hES cell 

lines. FDA does not intend that the agency's regulation of xenotransplantation will preclude the 

use of these hES cell lines. It may be necessary for a sponsor who wishes to investigate a stem 

cell product derived from existing hES cell lines in a clinical trial to demonstrate to FDA that the 

hES cell line is free from infectious agents, including murine infectious agents. Given current 

technology, a sponsor should be able to do this without undue burden. For example, it will not 

be necessary for a sponsor to provide FDA with complete animal husbandry information for 

mice from which the mouse feeder layers were derived. If continued expansion on murine 

feeder cell layers is planned, FDA may request that a sponsor demonstrate that the mouse 

colonies, from which such murine feeder layers are to be obtained, are maintained appropriately 

to ensure safety. (See section III of this guidance and section 3 of the PHS Guideline on 

Infectious Disease Issues in Xenotransplantation (reference 1)). The same recommendations 

apply to other xenotransplantation products that contain human cells with a history of co-culture 

with nonhuman animal cells. 

G. Disposal of Animals and Use of Byproducts

There is a need for advance planning for the ultimate disposition of source animals, including 

those animals in which the insertion of genetic information failed (“no-takes”), and sentinel 
21

animals bred for use in producing xenotransplantation products, especially animals of species 

ordinarily used to produce food. Food or feed derived from such animals may be adulterated 

under the Act. Generally, you should not use such animals as sources of human food via milk or 

meat or as ingredients of feed for other animals. You should not use such animals as pets or 

breeding animals because of the potential for them to enter the food/feed chain, either directly or 

through the rendering process. You should dispose of source animals in a manner consistent 

with the disposal of medical waste in compliance with federal, state, and local requirements.

There may be infrequent situations where animals from xenotransplantation facilities can be 

considered safe for human food use or as feed ingredients when disposed of through rendering. 

Persons wishing to offer animals into the human food or animal feed supply or who have food 

safety questions should first consult with FDA's Center for Veterinary Medicine. CBER will 

refer food safety issues from sponsors to CVM, or you may contact CVM directly through the 

Division of Compliance, HFV-235, FDA, Center for Veterinary Medicine, 7500 Standish 

Place, Rockville, MD 20855, 301-827-0181.

IV. CHARACTERIZATION OF XENOTRANSPLANTATION PRODUCTS

A. General Considerations

In general, you should test the xenotransplantation product for safety, identity, purity, and 

potency. 21 CFR Part 610 describes types of assays that are required for licensed biologics. 

You should use similar tests during investigational stages of product development. We discuss 

in more detail assays for safety testing including infectious agents tests, and tests for endotoxin, 

in section V. of this document. Assays for testing identity and potency will depend on the 

product itself. Assays for purity should include tests for endotoxin or pyrogen, and for certain 

xenotransplantation products should include measurements of cell populations in the 

xenotransplantation product. For further guidance in this section, see references 6, 31, 32, and 

33. 

Additional recommendations and comments regarding microbiologic tests are found in section 

V. of this document. 

B. Considerations for Classes of Xenotransplantation Products

1. Xenotransplantation Products Used Immediately after Procurement from the 

Source Animal

When xenotransplantation products are transplanted directly after removal from a 

source animal, it may not be possible to perform all tests on the final product and have 

the results available before use. However, you should use a biopsy of the organ or a 

relevant surrogate sample (e.g., adjacent tissues or contra-lateral organs) for assay of 
22

the xenotransplantation product. Safety analyses should include fungal and bacterial 

sterility, mycoplasma and virus testing. You should also perform tests for endotoxin or 

pyrogen. Although we realize that results of these tests will not be available before 

transplantation, you should still complete assay or culture periods and record the results. 

As appropriate, you may use histology, performed on a retention sample or biopsy of 

the xenotransplantation product, to document identity of the product.

2. Stored or Processed Xenotransplantation Products

For live xenogeneic cells or tissues that are stored, processed, or expanded ex vivo, in 

addition to safety testing, you should perform additional product characterization to 

measure identity, purity, and potency. As much as possible, results of these assays 

should be available before xenotransplantation, and used for lot release. You should 

also apply these same product characterization steps to xenotransplantation products 

comprised of human cells that have had ex vivo contact, for example by co-culture, with 

cells or tissues of nonhuman origin.

a. Safety

We discuss tests for bacterial and fungal sterility, mycoplasma, and viral agents, 

generally considered safety tests, in detail in section V. of this document.

b. Identity

You should develop a means to assess identity of the active component of the 

xenotransplantation product. This may include identification of relevant cell or 

tissue types using immunological, immunohistological or biochemical cell 

markers. In some cases, it may be possible to use histological evaluation. 

Depending on the manufacturing process, verification of the species or strain 

identity of the final product may be necessary, such as when the SAF handles 

more than one strain or species of animal.

c. Purity

If the final product is a heterogeneous xenotransplantation product, i.e., a tissue 

possessing several types of cells, or a cellular implant containing extraneous 

tissues or cells which may be incompletely removed during tissue dissection, cell 

processing or ex vivo culture, the determination of the purity of the cell 

population is especially important. You should develop a quantitative method to 

assess the presence of the putatively active cell type as well as contaminating 

cell types in the final product. You may achieve this, for example, using 

morphologic, histologic, molecular genetic, biochemical and/or 

immunocytochemical techniques to identify contaminating cells and/or their 
23

products. For xenotransplantation products comprised of human cells that have 

had ex vivo contact with cells or tissues of nonhuman origin, you should perform 

quantitative assays to assess the presence of nonhuman cells in the final product. 

You should validate purity assays. Purity assays are important for production of 

a consistent product. You should use results of such tests as a lot release 

specification if possible. In those cases where the final product is a purified 

population of cells of a single or few types, such as an established cell line, you 

should still test the product for purity, and you should develop tests for identity 

of the cells. 

You should measure endotoxin levels on the final product and results should be 

available for use as a lot release specification. We also discuss tests for 

endotoxin in the context of tests for infectious agents (section V.C.3.).

d. Potency

You should perform potency assays that measure and reflect the intended 

biological activity of the final product. For example, potency assays may 

measure biologically active molecules secreted/produced by the 

xenotransplantation product, such as cytokines, hormones or neurotransmitters. 

If necessary, development of appropriate potency assays may proceed along 

with product development. In addition, you should assay cell viability and use 

the results for lot release.

V. MICROBIOLOGICAL TESTING OF XENOTRANSPLANTATION PRODUCTS

A. General Considerations

1. Framework

This section of the guidance document provides a general framework for the 

microbiological testing of xenotransplantation products. We suggest some specific 

examples of tests and infectious agents. However, we encourage sponsors to consider 

all available up-to-date information regarding potential pathogens and testing strategies, 

to evaluate their own systems, to perform experiments to identify potentially infectious 

agents, and to propose and validate appropriate tests in consultation with CBER. 

During the initial stages of investigations, complete validation for all assays may not be 

necessary, with the exception of standard sterility tests. However, you should establish 

the specificity, sensitivity and reproducibility for all procedures used to detect infectious 

agents to the extent possible.

2. General Biological Products Standards
24

For general standards on testing of biologics for infectious agents, refer to 21 CFR Part 

610 (see references 6, 7, 25, 32, and 33). 

You can also find additional guidance on these issues as they relate to 

xenotransplantation in section 3.3 of the PHS Guideline (reference 1).

3. Inactivation or Removal of Infectious Agents

Whenever possible, without compromising the integrity and effectiveness of the 

xenotransplantation product, we encourage you to develop and incorporate validated 

procedures for inactivation or removal of adventitious agents, infectious agents, or other 

microbiological contaminants into the manufacture of the xenotransplantation product.

4. Archiving

You should cryopreserve and archive for further testing, as needed, samples of all final 

xenotransplantation products (i.e., cells or tissues, or biopsies of organs), whether fresh 

or from culture ex vivo. In some cases, for example if the xenotransplantation product 

is a whole intact organ, it may be acceptable to archive a relevant surrogate sample 

(e.g., adjacent tissues or contra-lateral organ). If the final product consists of human 

cells, you should archive tissues or organs that have been in contact ex vivo with live 

nonhuman cells, tissues or organs, samples of both the final product and the nonhuman 

animal cells tissues or organs. As in the case of the animal source samples (see section 

III.E.3.c.), you should harvest and cryopreserve sufficient quantities and numbers of 

replicates of the xenotransplantation product for three different uses: 

(a) dedicated sample(s) for use only by PHS (see reference 1), 

(b) for use if needed for recipient diagnosis and care, and 

(c) for use by the sponsor, as appropriate. 

You should include detailed plans for obtaining and storing archive samples in the 

application to FDA requesting investigational use (e.g., IND). You should store

samples for 50 years from the time of manufacture of the xenotransplantation product. 

You should clearly describe responsibility for the archives and access to the specimens. 

5. If you are the sponsor, you should provide for all samples and attendant records 

to be maintained for the requested period of time in the event that an establishment 

ceases operation.
25

B. Considerations for Classes of Xenotransplantation Products

1. Xenotransplantation Products Used Immediately after Procurement from the 

Source Animal

In procedures in which the xenotransplantation product is transplanted immediately after 

removal from the source animal, such as xenotransplantation of whole organs, results of 

testing of the xenotransplantation product may not be available before its clinical use. In 

such cases, testing of the source animal itself may be all the testing that is possible 

before the procedure. Testing of samples taken from such xenotransplantation products 

or appropriate relevant biological surrogates, e.g., adjacent tissues or contra-lateral 

organs, is also warranted even though the results will not be available before use of the 

xenotransplantation product because results may contribute to patient management and 

to development of a scientific data base. (See also section IV.B.1.)

2. Stored or Processed Xenotransplantation Products

For xenogeneic cells or tissues that are stored, processed, or expanded ex vivo, you 

should accomplish or, at a minimum, initiate testing for infectious agents before 

xenotransplantation. If cells or tissues are maintained in culture, you should validate cell 

culture procedures and reagents for maintenance of microbial sterility, including both 

xenogeneic infectious agents and other cell culture adventitious agents. You should 

perform testing periodically during the culture period. Performance of all tests at every 

time point may not be necessary, but you should provide FDA, as part of the 

application for investigational use (e.g., IND), with a scientific rationale to support the 

selection of tests performed at each given time. As an example, you may test samples:

(a) at the initiation of culture ex vivo, 

(b) before cryopreservation if performed as a step in manufacture, 

(c) as late as possible during culture such that final results (or useful preliminary 

results) will be available before the release and use of the product, 

(d) two to three days before clinical use (e.g., for microbiological cultures used 

in lot release), and 

(e) at the time of final product harvest, though results may not be available 

before clinical use. 

3. Xenotransplantation Product/Device Combination Products

In certain biologic/device combination products, physical barriers may separate the 

xenogeneic component from human fluids or tissues and might prevent or reduce 

transmission of certain classes of infectious agents. If such claims are to be made, or 

are implicit, or if the existence of the physical barrier is to be used in lieu of certain other 

precautions to lower the risk of transmission, you should provide to FDA as part of the 
26

application for investigational use (e.g., IND), the results of validation studies that 

demonstrate the inhibition of transmission of specific infectious agents and the 

maintenance of device/barrier integrity. For specific guidance on the design of these 

types of studies, see reference 33. For example, if such claims are to be made in the 

patient informed consent document, you should provide the results of these studies in 

the application to FDA requesting investigational use; if such claims are to be made 

during marketing, you should provide the data in the marketing application. The design 

of these studies should take into consideration the following parameters:

(a) Conditions of normal physiologic use of the xenotransplantation 

product/device combination product, and conditions under which the 

combination is subjected to physical and biological stress;

(b) Use of infectious agents that are representative of those potentially present 

in the xenotransplantation product. Note that the size and plasticity of 

selected agents should be considered; and

(c) Use of agents that would demonstrate the physical properties of the barrier 

(i.e., permeability to viruses or other particles with differing properties such 

as size, charge, hydrophobicity, shape, etc.). 

Without supporting data obtained from such studies, you should not assume that 

xenotransplantation products contained within a barrier present lesser risk of infection to 

humans than xenotransplantation products not contained within a barrier.

C. Assay Design for the Detection of Infectious Agents

1. General

The choice of tests will vary depending on the animal source, including the species, 

strain and geographic origin, the type of tissue, the processing of tissue before use, and 

the proposed use or clinical indication. You should give special consideration to 

infectious agents known to infect the source animal and those known to cause zoonoses. 

You should base the list of infectious agents to be tested for on that used for individual 

source animal qualification. We encourage discussions with CBER. You should include 

data in the FDA application for investigational use to document the specificity, 

sensitivity, and reproducibility of novel assays used to detect infectious agent(s).

2. Tests for Bacteria, Fungi, and Mycoplasma

You can find standards concerning the types of methods used for detection of bacteria, 

fungi, and mycoplasma in licensed biologics in 21 CFR Part 610. You may use 

alternative methods during product development but you should support use of such 

methods by data on the sensitivity, specificity, and reproducibility of the method. For 

xenotransplantation products, you should obtain such data using infectious agents 
27

appropriate to the source animal species, geographic origin of the source animal, and 

the cells, tissues or organ(s) to be used. You should submit these data to FDA in the 

application for investigational use (e.g., IND).

In addition to testing the final product for viable organisms, you should perform Gram 

stains on appropriate samples of all final xenotransplantation products. The results of 

these stains should be available before use of the product in humans, and you should set 

a negative Gram stain as a lot release criterion. 

3. Endotoxin Test

During the product development phase, you may perform a bacterial endotoxin test in 

lieu of the rabbit pyrogen test as described for licensed products (§ 610.13(b)). You 

should describe the type of endotoxin assay, and its specificity and sensitivity in the 

application for investigational use submitted to FDA (e.g., IND). If you intend to use an 

endotoxin assay in lieu of the rabbit pyrogen test after licensure, you should demonstrate 

equivalency with the pyrogen test for the specific xenotransplantation product at or 

before the time of license application (§ 610.9). 

Since it is possible to perform an endotoxin assay within a few hours, you should select 

and perform an appropriate assay on the final product. Results should be available 

before use for any xenotransplantation product that has been cultured, stored, or 

processed for more than the few hours required to perform the assay. You should use 

these results as a lot release specification. Consult the “Guideline on Validation of the 

Limulus Amoebocyte Lysate Test as an End-Product Endotoxin Test for Human and 

Animal Parenteral Drugs, Biological Products, and Medical Devices,” December 1987 

(reference 7), for additional guidance on endotoxin assays.

4. Viruses

a. Culture Assays 

You should test xenogeneic cells used for xenotransplantation (fresh or cultured) 

by co-culture with a panel of appropriate indicator cells to amplify potential viral 

contaminants. The panel of cells used in this analysis should include a cell line 

representative of the source animal species, a cell line representative of the 

animal tissue(s) type used in the manufacture of the xenotransplantation product, 

and a human cell line. For additional guidance see the “Points To Consider in 

the Characterization of Cell Lines Used to Produce Biologicals” (reference 25). 

This reference provides especially useful information for testing rodent 

xenotransplantation products for viral adventitious agents. You may find 

additional information in “Q5A Viral Safety Evaluation of Biotechnology 

Products Derived From Cell Lines of Human or Animal Origin” 9/24/98
28

(reference 33). When possible, you should also co-cultivate manipulated 

and/or unmanipulated source animal cells with recipient cells, such as peripheral 

blood cells. You should routinely observe co-cultivation cultures for CPE, 

focus formation, RT activity, and changes in cell growth or other unexpected 

changes. We recommend visualization of co-cultures by EM to identify 

morphologic changes or to recognize certain viruses. You should try to identify 

any viruses detected using immunoassay, PCR, or other assays using virusspecific probes. At the end of the culture period, you should test cultures for 

hemagglutination and hemadsorption with erythrocytes of three different species 

(reference 25). Additional efforts may be necessary to characterize viruses that 

are detected that may be novel or for which specific probes may not yet be 

available.

You should base lot release specifications on available data. You should use 

these specifications for release of xenotransplantation products for which results 

can be available before administration of the product to humans, such as for 

products that can be cryopreserved. For cells that are manipulated ex vivo, if 

time allows, you should perform viral tests during the period of culture or 

manipulation, so that the results are available before delivery of the product to 

the recipient. If it is not possible to obtain the results prior to use, you should 

still test samples of each product lot. In these instances, you should qualify 

assay procedures and obtain data on a number of representative final product 

lots before beginning human trials.

b. Activation of Latent Viruses

You should give special consideration to testing for viruses known to occur in 

the latent state. Transmission of viruses with long clinical latencies is of concern 

due to the possibility of transmission of these viruses from the recipient to the 

recipient's contacts in the absence of symptoms or signs of disease. 

Immunosuppression and transplantation, either alone or in combination, may 

activate latent viruses (reference 42). Manipulation or culture of cells ex vivo 

may also activate latent viruses (see references 43 and 44).

Which experiments might be appropriate to detect latent viruses in animal cells, 

tissues, or organs would depend upon the tissue type and the virus in question. 

Examples of experiments that have been used to detect viral activation and may 

be useful in the xenotransplantation product setting, include the following: 

• the expression of endogenous retroviruses is induced by culturing in 

vitro, or by treatment with iododeoxyuridine or demethylating 

agents such as 5-aza-cytidine (reference 37); and 

• cultivation in vitro of ganglia latently infected with Herpes simplex 
29

virus results in the production of infectious virus (reference 38). 

In certain cases, positive results may not necessarily preclude use of such tissue 

(see section V.C.4.d. for information regarding xenotransplantation products 

containing porcine endogenous retrovirus (PERV)), but the identification and 

characterization of the resulting virus may provide useful information and 

materials for monitoring the recipient of the xenotransplantation product for the 

presence of the activated virus (see section VIII.F.3.).

You should attempt to evaluate the potential of the processing or clinical use of 

the xenotransplantation product to activate latent viruses (e.g., PERV) before 

use.

c. In Vivo Assays for the Detection of Viruses

You should test xenotransplantation products by assay in vivo for detection of 

certain viruses that may not be found by culture methods in vitro. For example, 

many serotypes of Coxsackie A virus are only detected upon inoculation of 

newborn mice (reference 38). Therefore, we recommend that if there are no 

reliable in vitro assays, appropriate in vivo assays should be applied (see 

“Points To Consider in the Characterization of Cell Lines Used to Produce 

Biologicals,” reference 25 and “Q5A Viral Safety Evaluation of Biotechnology 

Products Derived From Cell Lines of Human or Animal Origin” 9/24/98, 

reference 33). Certain in vivo assays, such as antibody production assays, may 

be particularly useful for testing for rodent viruses (references 25 and 33).

d. Assays Suitable for the Detection of Porcine Endogenous Retroviruses 

(PERV)

All live cells, tissues, or organs derived from pigs contain sequences for porcine 

endogenous retroviruses in their genome (reference 39). It has been 

demonstrated that in some instances, and in some primary porcine cells, that 

these sequences are expressed, resulting in production of infectious retrovirus 

(references 40, 41, 43, and 44). In light of data demonstrating that PERV can 

infect human cell lines in vitro (references 43, 44, and 45), we recommend that 

all porcine-derived xenotransplantation products be evaluated using appropriate 

assays for the production of infectious retrovirus. You should assess several lots 

of each xenotransplantation product. Each time major manufacturing changes 

occur, such as use of a new source or herd or method of product procurement, 

you should repeat testing for infectious PERV. You should test 

xenotransplantation products (e.g., a fresh sample of the xenotransplantation 

product or relevant surrogate tissue, e.g., adjacent tissues or contra-lateral 

organs or the cultured xenogeneic cells) by co-culture with appropriate indicator 
30

cells to amplify any infectious retrovirus(es). Indicator cells that have been 

demonstrated to be permissive for PERV replication, include the human 

embryonic kidney cell line 293 (American Type Culture Center (ATCC CRL1573)), mink lung fibroblasts (ATCC CCL-64), certain feline cell lines (such as 

PG-4, ATCC CRL-2032), and a swine testis cell line ST (ATCC CRL-1746). 

You should choose one or more of these cell lines for initial analysis of the 

porcine xenotransplantation product or appropriate relevant surrogate tissue. 

After co-culture for a period of at least 30 days or 10 cell passages, you should 

analyze the cells for the transfer of PERV from the porcine cells to the indicator 

cell by either an optimized RT assay (reference 46) or use of PERV-specific 

primers to amplify, by PCR, reverse-transcribed viral RNA or cellular RNA 

(references 43, 44, and 45). Evidence for virus production will not necessarily 

result in the xenotransplantation product being considered unsuitable for clinical 

use. Rather, you should pursue additional characterization of the virus in 

consultation with CBER in order to ensure appropriate reagents are available 

for recipient follow-up (section VIII.F.). Additional characterization may 

include analysis for the cell substrate most sensitive to infection by the particular 

strain of PERV present in the xenotransplantation product and sequence 

analysis of the infectious virus produced by the xenotransplantation product. 

These steps will provide important information and development of diagnostic 

tools to optimize the protocols for follow-up of recipients for evidence of 

infection (section VIII.F.).

VI. MANUFACTURING AND PROCESS-RELATED GMP CONSIDERATIONS FOR 

HARVEST AND PROCESSING OF XENOTRANSPLANTATION PRODUCTS

A. General Considerations

You should use facilities for the harvest and/or processing of xenotransplantation products that 

have been designed to minimize the potential for contamination of the harvested and/or 

processed xenogeneic cells, tissue, or organs and cross-contamination between lots of these 

cells, tissues, or organs. 

For sponsors of investigational trials, you should phase in the validation activities described in 

this section during the investigational phase, as the clinical studies progress toward submission of 

an application for approval to market (e.g., BLA). The exception to this is sterility assurance 

validation, which you should complete before initiating clinical trials. Manufacturing process 

controls should be in adherence to cGMP regulations (21 CFR Parts 210 and 211). The IND 

regulations (21 CFR 312.23(a)(7)) allow that some controls may be introduced as appropriate 

for the phase of development.
31

B. Contamination/Cross-Contamination Precautions

You should take precautions to prevent contamination/cross-contamination during harvest and 

manipulation of xenogeneic cells or tissues. You should give consideration to:

• personnel, animal, material and waste flows into and out of the facility; 

• proposed air cleanliness classifications; 

• cleaning/sanitizing agents used and demonstration of their efficacy in relation to 

facility isolates, viruses and other potential adventitious agents; and

• environmental monitoring and gowning procedures.

1. Flows

Your facilities should be designed such that personnel, animal, material, product, and 

waste flows into and out of the facility exclude mixing of “clean” and “dirty” activities. 

Ideally, flows should be one way so that personnel, animals, materials and product enter 

and exit separately. Using this design, waste would only exit through designated 

airlocks, pass-throughs, and/or autoclaves. Alternatively, you may accomplish 

segregation of activities procedurally and/or temporally. In this case, you should take 

special care to avoid contamination or cross-contamination. For example, more 

stringent cleaning and sanitization schedules should be in place. 

Of special concern is the transfer of animals to the harvesting area (i.e., operating 

room). You should ensure that animals are prepared in such a way as to exclude 

potential surface contaminants, which may be carried from the animal facility.

2. Cleaning and Sanitizing Agents

You should demonstrate that agents used for cleaning and sanitizing work surfaces and 

equipment, as well as other surfaces in the harvesting and processing areas (e.g., floors, 

walls), are effective against facility isolates, viruses, and other potential adventitious 

agents. You should establish cleaning schedules which maintain acceptable control in 

relation to the activities performed in the specified area. We expect that validation 

studies demonstrating the efficacy of the agents used will be performed as the trial 

progresses towards submission of an application for premarket approval, e.g., BLA.

3. Environmental Monitoring

You should establish a program for monitoring the environment in the harvesting and 

processing areas based on the criticality of the manufacturing process involved. 

You should perform nonviable particulate monitoring to verify air cleanliness 

classifications in the harvesting and processing areas (see section VI.C.1. for 
32

recommended air cleanliness classifications). This verification should include laminar 

flow areas in the harvesting area and biological safety cabinets in the processing area 

(reference 8). After initial verification, you should perform nonviable particulate 

monitoring at established intervals to demonstrate maintenance of the assigned air 

cleanliness classification.

You may monitor viable particulates, i.e., microbes, using a variety of techniques. The 

use of settling plates during harvesting and processing activities, while not quantitative, 

provides some assurance that the quality of the environment has not been compromised. 

You should establish quantitative methods as clinical trials progress towards submission 

of a marketing application, e.g., BLA. You should monitor surfaces, including those of 

personnel performing production activities (e.g., gloved hands), using contact plates or 

swabs to demonstrate the continued efficacy of the cleaning regimen, and maintenance 

of asepsis for personnel. We recommend that personnel engaged directly in harvesting 

and processing activities be monitored at the conclusion of each critical activity (e.g., 

surgery, aseptic surgery). Additionally, you may randomly sample operators performing 

cell expansion activities.

4. Changeover Procedures

You should establish, follow, and document changeover procedures designed to 

prevent contamination between harvests of xenotransplantation products. These 

procedures should include clearance of all materials and waste from the operating room 

or cell processing cabinet, and cleaning/sanitization of surfaces. In addition, you should 

address segregation procedures, if multiple lots of xenogeneic cells or tissues are 

processed at the same time. Adequate labeling of processing vessels (e.g., tissue 

culture flasks) and dedication of equipment or portions of equipment (e.g., shelves 

within incubators) are examples of such segregation procedures. Centrifuges used for 

processing are of particular concern in terms of cross-contamination. We recommend 

that only one lot of xenogeneic cells or tissues be centrifuged at a time. You should 

demonstrate integrity of centrifuge tubes or closed systems employed, when possible. 

You should adequately clean centrifuges between each lot operation.

C. Validation and Qualification

As noted previously, validation and qualification efforts should be ongoing as clinical trials 

progress. Minimally, we expect assurance that systems and equipment are functioning as 

needed. You should submit validation protocols and data summaries to FDA for review as part 

of the ongoing investigational file (e.g., IND).

1. Air Handling Systems

Heating, Ventilation and Air Conditioning (HVAC) systems should be designed to 
33

provide adequate air quality for harvesting and processing of xenotransplantation 

products. You may use laminar flow units above the operating table to provide high 

quality air during harvesting operations. You may use Biological Safety Cabinets to 

maintain aseptic conditions during processing. We expect that this equipment be 

capable of producing Class 100 conditions for the most critical of processes, although 

we understand that maintenance of these conditions may be difficult during harvesting. 

Minimally, the environment surrounding the Class 100 laminar flow units and/or 

biological safety cabinets should be Class 100,000. Proceeding towards licensure, 

areas surrounding critical Class 100 processes should meet Class 10,000 conditions.

Validation of these systems and units should include verification of air changes and 

pressure differentials, and that the desired cleanliness level is achieved (see section 

VI.B.3.). Testing of the High Efficiency Particulate Air (HEPA) filters contained in the 

system should address integrity and efficiency. 

Routine environmental monitoring (see section VI.B.3.), pressure differential checks and 

recertification of HEPA filters should demonstrate maintenance of the desired 

conditions.

2. Water 

We expect that water used to formulate necessary reagents, or for critical cleaning 

purposes (i.e., equipment and surfaces in the harvesting and processing areas), will meet 

the United States Pharmacopoeia (USP) XXIV monograph for Water for Injection 

(WFI) (reference 9). If WFI is purchased, you should perform lot specific testing and 

validate hold times for open containers. If WFI is generated at the facility, you should 

properly validate and routinely monitor the system to ensure continued quality. 

3. Equipment

You should adequately calibrate and qualify equipment used for harvesting and/or 

processing of xenogeneic cells and tissues. You should then routinely monitor 

temperature-controlled equipment, such as refrigerators/freezers and incubators, to 

assure proper conditions. Carbon dioxide supplied to incubators used for cell 

expansion should be 0.2 micron filtered to minimize the potential for contamination. If 

water baths are used, you should use maintenance procedures for water quality. This 

may include the addition of agents to control contamination.

4. Aseptic Processing

Generally, manipulation or expansion of xenogeneic cells or tissues is an entirely aseptic 

process, i.e., there is no final sterilizing filtration of the product. In order to validate this 

process, you should perform media fills (substitution of media for product) to 
34

demonstrate that sterility may be maintained consistently. Assurance of sterility of the 

final product is necessary from the very beginning of the clinical studies (reference 10). 

You should adequately train and monitor personnel performing these functions to assure 

consistent performance during normal production.

All product contact equipment should be sterile and free of pyrogens when aseptically 

processing cells or tissues. You may use disposable labware (e.g., flasks), where 

possible. The sterility and depyrogenicity of the containers and closures used for the 

final product are of particular importance. For equipment and components that are to 

be sterilized, there should be evidence that the autoclave cycle(s) is validated to provide 

an acceptable level of sterility assurance. Minimally, you should establish and follow 

basic load configurations, and you should place biological indicators within each load to 

verify lethality. As studies progress, we expect that formal validation of all 

sterilizing/depyrogenating processes will be performed.

5. Process Validation

Ultimately, before licensure or product approval, you should validate all critical 

processes used to manufacture the product. We have previously defined process 

validation (reference 11). We expect that process validation, when performed, will be 

prospective and at full scale, with the exception of studies performed to demonstrate 

viral clearance (removal/inactivation). Laboratory studies may also help to establish 

appropriate operating and process parameters and may be used in support of the formal 

study. We expect that information on the validation protocol(s) and summaries of data 

resulting from its execution will be included in the license application.

VII. PRECLINICAL CONSIDERATIONS FOR XENOTRANSPLANTATION 

A. General Considerations

This section provides a general framework for the preclinical testing of xenotransplantation 

products before use in clinical trials. You may also apply the general principles as set forth in 

the document generated by the ICH on the safety of biotechnology-derived pharmaceuticals to 

these products (reference 34). In general, studies to support the safety characterization of 

therapeutic agents should focus on the intended alteration to the human pathophysiologic state 

(i.e., activity), as well as unintended effects (i.e., toxicity) to the host system. Such studies serve 

to assess the potential for clinical risks and constitute an important component of a FDA 

application (e.g., IND). Preclinical studies are particularly valuable for gaining insight into safety 

issues which cannot be evaluated in human recipients for ethical or practical reasons. 

Consequently, you should design strong preclinical safety programs, and also consult the ICH 

guidance documents related to acute and chronic drug safety characterization 

(http://www.ich.org/ich5.html under “ICH Safety” [S1-S5] or “Joint Safety/Efficacy” [M] 
35

headings). 

Specific considerations in the design of preclinical studies that are intended to support the safety 

of xenotransplantation products should include: 

(1) the animal source for the xenotransplantation product; 

(2) the tissue’s anatomic and physiologic similarity to its human homologue; 

(3) the determination of function of the xenotransplantation product; 

(4) the animal model system; 

(5) the integrity of the device components (if a device is used); 

(6) the dose levels (based on tissue mass, as well as pharmacologic/metabolic activity 

or release kinetics of bioactive molecules); 

(7) the route of administration (site of implantation/injection, extracorporeal or ex vivo 

use); 

(8) the study duration (as related to potential human exposure); 

(9) reactions between source animal and host immune systems; 

(10) interspecies extrapolation (i.e., cross-species activity of secreted 

proteins/hormones at receptors); and 

(11) device biocompatibility.

Because a primary intent of the preclinical animal and in vitro studies is to identify potential 

clinical risk factors, these evaluations should focus on maximizing the similarity between animal 

and human testing strategies in test substance, route of administration, and dosing regimen. 

Animal models of human xenotransplantation should utilize a xenotransplantation system 

evaluating the cell, tissue or organ type being examined for use in humans, and should utilize 

clinically relevant immunosuppressive therapy. Rigorous preclinical program design is needed to 

ensure comparability of preclinical to clinical study design and is important for planning the 

clinical testing program including selecting an appropriate clinical indication, inclusion/exclusion 

criteria, recipient monitoring scheme, dose, concomitant therapies, as well as for advising 

potential recipients of risks (informed consent). 

B. Issues Related to Infectious Agents

Since the transfer of infectious agents that are pathogenic, latent, or even non-pathogenic in their 

natural animal host may cause serious disease in an immunosuppressed patient, you should 

consider the microbiologic burden carried by the xenotransplantation product as well as the 

immune status of the recipient in preclinical study designs. Additionally, designs of preclinical 

studies should incorporate: 

(1) careful veterinary monitoring of animals, taking note of any early signs of infection; 

and 

(2) procedures needed to assign a cause of mortality (using appropriate serologic or 

immunohistochemical identification of pathogens). 
36

To prevent the spread of demonstrable or potential infectious agents, you should care for 

animals with appropriate precautions, including isolation if necessary. Deaths from infections in 

animal models may occur due to immunosuppressive regimens that may be intentionally more 

extreme than expected for use in humans in order to avoid rejection of the xenogeneic live cells, 

tissues, or organs, and to obtain proof-of-concept data. Therefore, data identifying cause of 

death (e.g., xenogeneic infectious agents or activation of latent host infection) could assist in 

interpreting human risk, may be helpful in refining animal experimental models, and may identify 

pathogenic infectious agents in the source animal. Animal models of xenotransplantation, while 

exploring these issues, are limited by uncertainties in extrapolation of cross-species infectivity 

information; e.g., data indicating no infections in animal, even primate species, are not adequate 

to assure that humans will not be susceptible to infections transmitted by the xenotransplantation 

product. 

Additional insight into refinements of animal immunosuppressant regimens may come from 

evaluation of host resistance. You may evaluate host immunocompetence by measuring 

resistance to infection by various pathogens including those that may be contained within the 

xenotransplantation product.

C. Xenotransplantation Product-Host Interactions

1. Immunologic Rejection

You should assess survival of the xenogeneic cells, tissues, or organs in animal models, 

with attention given to: 

(a) identifying infiltration of immune or inflammatory cells into the 

xenotransplantation product or alteration of such cells in other relevant 

compartments such as the blood and cerebrospinal fluid; 

(b) fibrotic encapsulation of the xenotransplantation product, e.g., resulting in 

impaired function or xenotransplantation product loss;

(c) xenotransplantation product necrosis;

(d) any evidence of Graft Versus Host Disease (GVHD);

(e) in vivo function and durability of encapsulation or barriers intended to 

diminish rejection or inflammatory responses; 

(f) any special concerns regarding the site and nature of the xenotransplantation 

product; and

(g) if relevant to the particular xenotransplantation product, the possibility that 

rejection of that product might predispose the recipient to rejection of 

subsequent xenotransplantation products or allotransplants.
37

2. Immunosuppression

Preclinical animal studies in which xenotransplantation is used in an immunosuppressed 

host may raise questions regarding the relevance of the model to clinical pharmacology, 

toxicology, or immunology. Consideration of how both the host and source species 

handle the immunosuppressive drugs may be necessary (for instance, where a 

nephrotoxic drug is metabolized by hepatic enzymes), but intra-species differences in 

metabolism exist. Immunosuppressive drugs often have very restricted therapeutic 

indices, so that pharmacokinetics and metabolism may markedly affect the activity 

and/or toxicity of the agents in the host or xenotransplantation products. You should 

attempt to delineate toxicities due to immunosuppressive drugs from toxicities due to the 

xenotransplantation product.

You should consider and study, where appropriate, relative activity of 

immunosuppression on the source species of the live xenogeneic cells, tissues or organs, 

since immunosuppressive treatment that selectively suppresses immunity in the host 

species may be permissive to GVHD. This might occur in cases where immunologically 

active cells are contained, either intentionally or inadvertently, within the 

xenotransplantation product.

3. Tumorigenicity in the Immunosuppressed Host

In addition, the tumorigenic potential of the xenotransplantation product, perhaps due to 

altered cell growth regulation or to immunosuppression of the host, is an important 

concern (refer to section VII.E.).

4. Cross-Species Compatibility of Bioactive Molecules

For xenotransplantation products where it is intended that the product synthesize and 

provide bioactive molecules, such as cytokines or hormones, you should provide to 

FDA, as part of the investigational application (e.g., IND), data from preclinical 

experiments that support that the molecules produced will be active in humans. 

Experiments to address this issue should evaluate concentration-response issues. You 

should perform the experiments in vitro, and/or in appropriate preclinical models in vivo.

Even when the xenotransplantation product is composed of a single cell type, the 

product may secrete unintended molecules that could alter normal host physiology. 

Moreover, host substances might affect product function. Therefore, preclinical models 

should evaluate the overall health of the recipient (i.e., clinical signs, gross pathology, 

and histopathology) as well as markers of activity of the xenotransplantation product. 

You may use combination toxicity and activity studies to evaluate both potential 

therapeutic and constitutive functions of the xenotransplantation product. In some 

instances, the ability to biopsy xenotransplantation products periodically is potentially a 
38

valuable tool for evaluating the histopathologic status of the product and host immune 

response, especially when evaluated in conjunction with clinical chemistries. You might 

also perform control experiments to test the in vivo effects of live xenogeneic cells, 

tissues, or organs taken from anatomic sites other than those used for therapeutic 

procurement of the xenotransplantation product, but lacking the therapeutic cell or tissue

type and its anticipated pharmacologic activity. (Please note that this advice also 

pertains to heterogeneous xenotransplantation products as discussed in section 

VII.D.2.)

5. Migration of Xenogeneic Cells

Cells from xenotransplantation products may migrate within the host, thus presenting 

clinical concerns regarding adverse reactions deriving from displaced, bioactive cells or 

unexpected anatomical impediments. This may be especially true for incompletely 

differentiated cells (see section VII.D.3.) and may be evaluated in animals using 

histopathology, possibly coupled with enhancing techniques such as fluorescent dye 

loading and/or species-specific antibodies, or more sensitive techniques such as PCR.

D. Considerations for the Use of Heterogeneous Xenotransplantation Products

You should apply the following principles to the development of appropriate preclinical testing 

of heterogeneous xenotransplantation products in order to assess potential adverse effects. A 

xenotransplantation product may be considered heterogeneous if it is, for example: 

• a tissue or solid organ, possessing many varieties of cells; or 

• a cellular implant, containing extraneous tissues or cells which may be incompletely 

removed during tissue dissection or present in short term cultures ex vivo. 

1. Characterization of Constituent Cell Types in a Heterogeneous 

Xenotransplantation Product

The procedures used in preclinical studies for the collection, isolation, and, where 

relevant, activation or expansion of the xenotransplantation product, should mimic the 

procedures intended for use in clinical trials, and you should characterize cell types in 

the product being tested in an analogous fashion to the proposed clinical 

xenotransplantation product. See section IV.B.2.c. regarding recommendations for the 

evaluation of purity of heterogeneous xenotransplantation products.

2. Secretion of Biologically Active Molecules by Xenotransplantation Products

Uncharacterized cells or tissues present in the xenotransplantation product may produce 

biologically active molecules with unintended activities. You should perform 

experiments to identify released, bioactive substances of potential biologic significance 

and appropriate to the particular xenotransplantation product (e.g., neurotransmitters, 
39

hormones, cytokines), whether by intended or extraneous cell types in the 

xenotransplantation product. For example, you may maintain or culture in vitro samples 

of tissues being prepared for transplantation, and test the supernatants for activities or 

relevant bioactive substances. You should consult the ICH guidance document on safety 

preclinical evaluations in biotechnology (reference 34) for additional guidance on these 

studies.

In addition to assessment in vitro, you should evaluate heterogeneous 

xenotransplantation products in appropriate animal models. (See section VII.C.4. for 

additional discussion of this topic). 

3. Differentiation in Heterogeneous Xenotransplantation Products

Xenotransplantation products derived from fetal animal sources, dedifferentiated cells or 

tissues, or cells expanded ex vivo may comprise a heterogeneous population with 

regard to cell maturity. The degree of heterogeneity may depend on the cell or tissue 

type from which the xenotransplantation product is obtained, the period of fetal 

development during which the tissue is procured, and/or the time in culture. For such 

products, preclinical studies should compare the viable cell types initially transplanted 

with those that exist subsequently in the xenotransplantation product. This comparison 

may warrant preclinical studies with sequential sacrifice groups, or biopsies. Techniques 

such as immunohistochemical staining, trypan blue exclusion, bioassays, or PCR assays 

may be useful in identifying heterogeneous cell differentiation. You should develop 

models to evaluate the effects of differentiation on the function of the xenotransplantation 

product, using, for example, measurements of release or secretion of biologically active 

molecules including those that may not be intended for efficacy of the 

xenotransplantation product but that it may produce. Viable products may change over 

time as they respond to, adapt to, and functionally integrate with the host environment. 

Therefore, you may monitor cell viability, morphology, and functional endpoints (e.g., 

endocrine, behavioral, or immunological) over time to guide development of clinical 

monitoring regimens. 

E. In Vitro and In Vivo Tumorigenicity Models for Xenotransplantation Products 

Intended for Transplantation

Tumorigenicity is an important part of preclinical testing for certain xenotransplantation products, 

such as those manipulated ex vivo. For further guidance applicable to this topic, see references 

6, 25, and 34.

Xenotransplantation products may be tumorigenic in a new species because of various factors, 

such as transgenic manipulations, endogenous viruses, ex vivo culture, and immunosuppression 

of the host. Therefore, for xenotransplantation products intended for implantation, you should 

consider evaluation of tumorigenicity in vivo and in vitro.
40

1. Multiple models exist for testing tumorigenesis in vivo. The role of immune 

challenge, immunosuppressive drugs, and exposure to certain infectious agents comprise 

an important set of safety concerns that may be addressed preclinically. Preclinical 

experiments should include careful evaluation of controls, background tumor growth 

rates, tumor incidence and type, location, and time of appearance of tumors over an 

extended period. These should make use of histopathologic evaluation as a primary 

endpoint. 

2. Colony formation in soft agar (clonogenic assays) and growth in organ culture 

may be useful in vitro assays of the tumorigenic potential, particularly for cell lines. 

These tests may provide information on stability or abnormal characteristics of cell lines, 

and may substitute for testing in animals if you demonstrate that the tests have equivalent 

sensitivity in your hands.

For xenotransplantation products consisting of cells that have been expanded ex vivo, a 

change in cellular growth pattern, morphology, or growth factor dependence may 

suggest transformation and a need for more rigorous investigation.

F. Combinations of Xenotransplantation Products with Devices

A number of products for therapeutic use are combinations of xenotransplantation products and 

device components, either for use as implants or extracorporeally. All of the preceding 

recommendations in section VII. applies to such products. These products also warrant further 

preclinical characterization for bioreactivity and biocompatibility of the device components. 

Preclinical testing often will include characterization of the device intended for human use, rather 

than a homologous product that has been made in scale with a small laboratory species. This in 

turn may dictate that the device be studied in an animal species with blood volume and size, and 

possibly, anatomic structures, similar to those of humans.

Device elements may be reviewed jointly by staff in CBER and the Center for Devices and 

Radiological Health (CDRH). Failure of device components (e.g., membranes and filters) that 

serve to isolate animal tissue from the recipient is an important aspect of safety assessment 

addressed by review staff in CDRH. Additional device toxicity issues, also considered by staff 

at CDRH, are covered in the biocompatibility guidance published by the International Standards 

Organization (reference 21). 

Implanted devices may be intended for permanent or extended residence in the human body. 

Since these are considered chronic therapies, long-term risks (such as chronic inflammation, 

carcinogenicity, consequences of re-implantation, and local/systemic toxicities) of implanted 

xenotransplantation product/device combinations may necessitate evaluation before product 

premarket approval, and to some extent, before initiation of investigational studies in humans. 

Studies before initiation of clinical investigational studies would usually last a minimum of 3 
41

months. 

Membranes with pores may partially isolate xenogeneic tissue housed in devices from attack by 

host immune cells, but proteins and pathogens from the xenogeneic tissue may still be released 

into the host along with desired pharmacologically active molecules. Such devices may reduce, 

but may not eliminate, a risk of xenogeneic infections. They also may act as a stimulus leading 

to local inflammation and fibrin deposition. Adhesions and granulomas may form in host tissue, 

and deposits on implants may interfere with activity and implanted cell viability. You should 

evaluate encapsulated xenotransplantation products intended to be permeable to bioactive 

substances (such as encapsulated islets) for preimplantation activity, and you should retrieve and 

assess the products for activity, capsule integrity, and tissue viability after various periods of time 

in the animals. 

You should conduct extended animal studies (e.g., 12 to 24 months) using the clinical route of 

administration (e.g., implant site) and clinical grade materials. You should design studies to 

include groups that elucidate reactions to the biomaterials alone, as well as groups exposed to 

clinical and supraclinical doses of the complete product. Toxicology studies for implanted 

biomaterials which have previously been utilized in non-cellular devices may be relevant to 

safety determinations of the xenotransplant/device combination products, but cannot completely 

satisfy the need for toxicity evaluation of the new product in its complete clinical form. You 

should be aware that later changes in manufacture of the xenotransplantation product may 

necessitate new toxicology studies.

For devices used for extracorporeal hemoperfusion, you should conduct studies to evaluate the 

hemodynamic effects of establishing and discontinuing the extracorporeal circuit, the products 

released from the tissues housed in the device (e.g., proteins that could cause anaphylactic 

responses or stimulate unintended autoimmunity), the deposition of blood cells (such as 

platelets) on device tubing or other components, coagulation or complement activation, and 

removal of drugs from the recipient’s circulation through filtration or device-localized cellular 

metabolism. Assessment of the biologic activity of the combination product is often a 

component of preclinical safety evaluations. For instance, studies should evaluate the duration 

and predictability of cellular (e.g., cell cartridge) activity, so that you may replace the biologic 

component of the device at appropriate intervals to maintain life-supporting pharmacologic or 

metabolic activity. 

In summary, you should design animal studies of xenotransplantation product/device 

combinations, as with other preclinical experiments, taking into consideration all aspects of the 

clinical trial and proposed patient population, the need to study both desired and undesired 

activities of the xenotransplantation product, and toxicities at the local and systemic levels. 
42

VIII. CLINICAL ISSUES IN XENOTRANSPLANTATION

A. General Considerations 

This section provides general principles rather than specific guidance. Because the available 

basic knowledge and clinical experience with xenotransplantation are limited, current issues may 

be resolved as new knowledge is acquired and new concerns may emerge.

B. Clinical Protocol Review

Sponsors are responsible for ensuring reviews, as appropriate, by local review bodies, including 

Institutional Review Boards (IRBs), Institutional Animal Care and Use Committees (IACUCs), 

and Institutional Biosafety Committees (IBCs) (see reference 1).

In addition to the human subjects protection issues traditionally addressed by local IRBs, 

institutional review of xenotransplantation clinical trial protocols should also address: 

(1) the potential risks of infection for the contact populations (including health care 

providers, family members, friends, and the community at large); 

(2) source animal husbandry (e.g., screening program, animal quarantine); and 

(3) adequacy of the proposal to address issues related to human and veterinary 

infectious diseases (including virology, laboratory diagnostics, epidemiology, and 

risk assessment).

C. Xenotransplantation Site

The PHS Guideline recommends that all clinical xenotransplantation procedures be performed in 

transplantation centers with appropriate experience and expertise for comparable 

allotransplantation procedures and with the capability to culture and to identify viral agents using 

in vitro and in vivo methods either on-site or through active and documented collaborations 

(reference 1). 

D. Criteria for Patient Selection 

Because of the potentially serious public health risks of possible zoonotic infections, you should 

limit xenotransplantation to patients with serious or life-threatening diseases for whom 

adequately safe and effective alternative therapies are not available except when very high 

assurance of safety can be demonstrated. You should limit candidates to those patients who 

have potential for a clinically significant improvement with increased quality of life following the 

procedure. You should also consider the patient's ability to comply with public health measures 

as stated in the protocol, including long-term monitoring.
43

E. Risk/Benefit Assessment

We understand that the lack of other therapeutic options and the severity of disease may raise 

the benefit-to-risk ratio for some individuals. However, consideration and evaluation of risks 

and benefits of xenotransplantation should address both recipient and public health concerns. 

You should consider the following in providing a benefit-to-risk analysis. Infectious disease is 

among the potential risks both to the recipient and to the public posed by the use of 

xenotransplantation products. You should describe and examine the likelihood and uncertainties 

associated with any potential recipient and public health consequences of each of the following 

events. Transmission of microbial agents from xenotransplantation products could lead to 

systemic disease (for example, infection or neoplasia) or failure of the xenotransplantation 

product in the recipient. In addition, transmission of infectious agents could result in outbreaks of 

zoonotic disease, silent transmission of latent viruses, or emergence of new strains of pathogens. 

Experience has shown that widespread horizontal or vertical transmission of new pathogens is 

possible before the pathogens are recognized (e.g., Human Immunodeficiency Virus). You 

should describe and examine the possibilities and uncertainties associated with any other types 

of adverse events that could have potential, significant recipient or public health impact arising 

from the xenotransplantation product. Similarly, you should describe any immunological risks 

including rejection of the live xenogeneic cells, tissues, or organs, and, in some cases, GVHD.

F. Screening for Infectious Agents

Consult the PHS Guideline (reference 1) for additional guidance and information on testing 

recipients of xenotransplantation products.

1. Infectious Agents of Concern

Infectious agents of concern will differ among source animal species and among cell or 

tissue types within each species. Therefore, you should individualize clinical tests for the 

specific xenotransplantation product in question. The categories of infectious agents of 

concern include bacteria (including the rickettsiae), fungi, mycoplasma, viruses, and the 

agent(s) causative for TSEs. Tests should be available for agents known potentially to 

be present, including those that are pathogenic in the source animal species and agents 

that are known to infect human cells in vivo or in vitro. You should have the capability 

to test for latent viruses or pathogens, and you should be prepared to develop and 

validate clinical tests for new pathogens that may not be recognized at the time of 

xenotransplantation. You should identify specific infectious agents for which tests will 

be performed. (See section VIII.F.3. for additional information on testing.) 

2. Collection and Analysis of Clinical Samples

You should only perform xenotransplantation at clinical centers with available state-of-
44

the-art virology and microbiology laboratories that include a staff with knowledge and 

experience in the isolation and identification of unusual pathogens. In addition, there 

should be access to laboratory facilities where viral cultures can be done in vivo, such as 

in embryonated eggs and suckling mice. You should place specimens into viral 

transport medium at the bedside, store at 4°C, and inoculate into cell cultures as soon 

as possible and always within 24 hours of collection. The sample(s) selected for culture 

will depend upon clinical evaluation of the recipient. In your investigational application 

to FDA (e.g., IND), you should describe tissue cell culture systems. These may include 

primary monkey, primary human embryonic kidney, semicontinuous human diploid, and 

continuous human heterodiploid cells. If isolation remains difficult, then inoculation in 

vivo, e.g., into embryonated hen's eggs and/or suckling mice, may be necessary. In 

addition to culture, you may examine tissue by EM. Immunohistopathology, 

immunofluorescent antibody, radioimmunoassay, Enzyme-Linked Immunosorbent 

Assay, and PCR may be helpful when appropriate antibodies and probes are available.

3. Testing and Scheduling of Testing of Recipients for Infectious Agents 

In your investigational application to FDA (e.g., IND), you should describe tests of 

clinical specimens from recipients for specific agents of concern. The tests may include, 

for example, serological and culture assays. You should also describe tests for latent 

agents known to be in the source animal species (e.g., retroviruses, herpesviruses). 

Assays should be able to distinguish between an infectious agent derived from the 

source species and a related infectious agent present in humans (i.e., porcine vs. human 

Cytomegalovirus (CMV)). Data should be available to demonstrate specificity, 

sensitivity and reproducibility for all tests not in widespread use, or for newly developed 

tests. In some cases, completion of development of new tests, which have already 

demonstrated some level of utility, specificity, and reproducibility, may proceed 

concurrently with the clinical trial.

You should also describe the schedule for screening of recipients for infections in the 

application for investigational use (e.g., IND).

a. Acute Infections 

Recipients are at risk for the same infections that are common among individuals 

who have received allografts. In general, these infections will be related to the 

use of immunosuppression and will arise from the recipient's endogenous flora, 

reactivation of latent infections, and environmental sources. The detection 

methods useful for these diseases will not differ from the methods used to detect 

infections after allotransplantation.

In addition to being at risk for these infections, a recipient may be at risk for 

infection by agents contained in the xenotransplantation product. Little clinical 
45

experience exists with xenogeneic infectious agents infecting humans from 

xenotransplantation products. We anticipate that a recipient will be at greatest 

risk for infection during the first few months after the procedure. However, 

there may be significant delay in the clinical manifestations of infection in some 

cases. The timing of occurrence of infectious episodes may vary depending on 

immunosuppression. It is important that you collect relevant data during the 

clinical trial and for the lifetime of the recipient and that investigation of acute 

infectious episodes includes appropriate tests. It is difficult to predict the 

diagnostic symptoms and signs of such infections in the immunosuppressed 

patient. When the source of a recipient's post-transplant illness remains 

obscure, you should perform testing on appropriate fluid and tissue samples. 

Such testing should include the use of serology as well as various cell and 

microbial culture systems and in vivo systems. Culturing may detect infections 

that serologic testing has missed (for example, when immunosuppressed 

transplant recipients are unable to mount the usual immunological response to a 

pathogen). 

Patient care workers who work with acutely ill recipients should follow 

recommended procedures for handling and disinfection/sterilization of medical 

instruments and disposal of infectious waste (references 22, 23, and 24). 

When there is a suspicion of a possible xenogeneic infection, you should 

notify FDA promptly if a non-xenogeneic causative infectious agent is 

not readily identified, and notify FDA immediately if a potentially 

xenogeneic, causative infectious agent is identified.

b. Chronic Infections 

An immunosuppressed recipient will be at risk for infection by the pathogens 

most commonly associated with allotransplantation. In addition, you should 

consider pathogens potentially derived from the source animal. With adequate 

preclinical and xenotransplantation product testing before the procedure, the 

most likely chronic pathogens from the animal may be endogenous or 

exogenous viruses, although parasites such as Toxoplasma should be 

considered.

c. Routine Screening for Clinically Inapparent Infections and 

Seroconversions

In addition to diagnostic testing when a recipient appears ill, it is important to 

establish ongoing recipient screening programs. Sponsors should describe and 

validate their screening programs, taking into consideration the source animal 

species and type of cell, tissue, or organ used.
46

i. Passive Screening Program

In passive screening programs, appropriate clinical samples, such as 

blood, plasma, urine, etc., are obtained periodically and archived for 

possible future testing. In the event of a diagnosed infection, or the 

onset of symptoms that may represent infection in one recipient, these 

samples are then available for retrospective screening of persons 

regardless of the presence or absence of symptoms, who shared a 

common or similar exposure to a xenotransplantation product. We 

recommend that a passive screening program be accomplished through 

an established schedule for routine sample collection and storage of 

samples from asymptomatic recipients. Such a passive screening 

program would be in addition to the collection and archiving of biologic 

specimens designated for PHS use as described in the PHS Guideline 

(reference 1). However, the time points identified by the PHS 

Guideline as appropriate for archiving specimens designated for PHS 

use also provide guidance on the minimal frequency with which 

specimens should be obtained and stored as part of a passive screening 

program. These time points include:

(a) before xenotransplantation (two samples, one month apart), 

(b) at the time of transplantation, 

(c) in the immediate post-transplant period, 

(d) at one month and six months after transplant, 

(e) annually for the first two years, and 

(f) every five years subsequently. 

In certain cases, more frequent acquisition of samples may be 

appropriate. The sponsor should consider the animal source and type 

of product in proposing the schedule and tests to be used in the passive 

screening program.

See section VIII.H. for recommendations regarding the number, size, 

use, and duration of storage of collected samples.

ii. Active Screening

In addition to a passive screening program, you should consider an 

active screening program. Possible mechanisms of active screening 

range from testing of samples immediately after collection from 

recipients to periodic performance of specific additional laboratory tests 

on a subset of the samples collected in the passive screening program. 

In addition, you should consider implementation of centralized review of 
47

routinely collected clinical data to detect trends suggestive of emerging 

diseases. Section 4.1.1.2 of the PHS Guideline (reference 1) suggests 

an active screening program for agents known to be in the 

xenotransplantation product with testing of samples acquired at 2, 4, 

and 6 weeks after the patient receives the xenotransplantation product. 

A significant advantage of such a program is screening prospectively for 

evidence of infection in the absence of symptoms provides for a 

prospective understanding of the patterns of infection and disease that 

may be occurring in recipients. Active screening could allow potential 

detection of a novel infection in the asymptomatic recipient and enable 

implementation of infection control practices to contain it before 

secondary human to human transmission or widespread dissemination in 

the general public, even in the absence of manifestation of associated 

disease (which may be absent altogether or simply delayed in onset). If 

a xenotransplantation product known to harbor an infectious agent is 

used for xenotransplantation, you should implement active screening for 

that infectious agent. For example, you should assess all recipients of 

xenotransplantation products involving the use of porcine cells, tissues, 

or organs for evidence of infection by PERV. Recipient screening for 

PERV should include analysis by multiple methods. Ideally, you should 

use all of the following detection methods:

(a) PCR of recipient’s PBMC for PERV DNA sequence, 

(b) serologic analysis for PERV-specific antibodies, and 

(c) assays capable of detecting plasma virions, such as RTPCR for detection of viral RNA or highly sensitive methods 

for detection of RT activity (reference 47).

You should collect a sufficient quantity of each sample in the active 

screening program to permit archiving for future use should the need 

arise. See section VIII.H. for recommendations regarding the number, 

size, use, and duration of storage of collected samples.

d. Identification of Xenogeneic Retroviruses in Recipients 

One particular concern is the potential transmission of xenogeneic retroviruses, 

such as PERV in the case of recipients of porcine products. If you are a 

sponsor of porcine xenotransplantation product clinical trials, you should 

develop a plan to address the possibility that a recipient tests positive for the 

presence of PERV or other similar xenogeneic infectious agents. The plan 

should include the following:

(i) Strategies to identify the source of a positive signal in the screening 
48

test (e.g., infection vs. false positive). For example, in the case of 

porcine xenotransplantation products, we recommend PCR of 

DNA isolated from recipient PBMC for detection of PERV genetic 

sequences. However, if a positive result is obtained from this 

analysis, one possible explanation would be the presence of porcine 

cells. Therefore, you should perform additional DNA PCR for a 

repetitive porcine genetic element, to determine whether the source 

of the positive result may be from microchimerism for pig cells, 

rather than from human cells infected with a pig retrovirus. If this 

analysis suggests the latter possibility, additional analysis should 

include an attempt to isolate the virus from relevant recipient 

specimens in an appropriate co-culture assay; 

(ii) Determination of infectivity of the agent using appropriate assays 

(e.g., co-cultivation) and additional characterization of the agent as 

necessary;

(iii) A plan to notify FDA and relevant sponsors and investigators and 

appropriate IRBs;

(iv) A contingency plan to modify the clinical trial (including suspension 

or termination of enrollment);

(v) Provisions for acute and follow-up medical care and counseling of 

the patients in the study; and 

(vi) Additional actions if required for the safety of the recipient and 

intimate contacts and to address possible public health risks.

e. Postmortem Detection of Agents and Archiving of Autopsy Samples

You should request a complete postmortem examination including 

histopathology and cultures of all recipients. At postmortem, you should fix and 

embed samples of body tissue for examination by light and electron microscopy. 

You should obtain samples from the xenotransplantation product and, as

appropriate, all major organs related to the product or to clinical syndromes that 

either resulted in the recipient’s death, were deemed to have been serious, or 

were of unexplained etiology. You should archive tissue and fluid samples at -

70°C or lower, as appropriate for preserving the sample, for 50 years beyond 

the recipient’s death as discussed in section VIII.H.1.

4. Infections in Recipient Contacts

We recommend that you develop a program to educate and monitor health care 

providers and to monitor other intimate contacts of recipients (e.g., persons with whom 

recipients repeatedly engage in activities that could result in intimate exchange of body 

fluids). In these groups, passive screening (see section VIII.F.3.c.i.) may be 

appropriate. You should obtain baseline samples of plasma and archive them at -70°C, 
49

and you should obtain leukocytes and archive them in liquid nitrogen, for health care 

personnel, for example, when they join the clinical teams. You should also ensure that 

such contacts are advised and counseled regarding potential risks.

G. Patient Follow-up

The sponsor should propose and submit a plan for clinical follow-up of recipients in a 

xenotransplantation protocol in the FDA application requesting investigational use (e.g., IND). 

This plan should take into account the timetable for collection and storage of specimens for the 

passive screening program and should extend for the life of the recipient (see section 

VIII.F.3.c.i.). The frequency of follow-up will decrease with time post-procedure. It is 

reasonable to plan for a tapering frequency of clinical monitoring and follow-up, with the 

flexibility to increase the frequency for individual recipients or trial participants as a whole, if 

events occur to make this appropriate.

H. Archiving of Patient Plasma and Tissue Specimens 

1. Before patients are treated, you should create protocols or SOPs for archiving 

all samples of patient tissue and fluids, including samples archived as part of 

recipient screening, post-mortem samples, and samples for PHS use.

a. You should follow appropriate biosafety precautions in collection of 

clinical samples from recipients. You should follow standard precautions in 

obtaining blood from recipients (reference 22). The PHS Guideline (reference 

1) recommends the use of at least a BioSafety Level (BSL) -2 containment 

facility with BSL-3 practices for any manipulation of clinical samples. 

b. For the schedule for archiving biological specimens recommended by 

PHS, see reference 1 and section VIII.F.3.c.i. of this document. The specific 

protocol or the recipient’s medical course may indicate more frequent archiving.

c. Plans should exist to maintain all archived samples according to the 

procedures recommended in the PHS Guideline (reference 1), including those 

obtained from patients during acute infectious episodes, and from health care 

workers. 

d. You should store patient blood and plasma samples in volumes and 

quantities according to the recommendations for animal plasma and blood cell 

samples (see section III.E.3.b.).

e. In addition to recipient samples collected during screening programs or 

post mortem, when xenotransplantation recipient tissues are collected for any 

medical use, such as a biopsy for diagnostic purposes, you should also archive 
50

samples of such tissues. You should store samples at -70°C or lower as 

appropriate for preserving the sample. 

2. Archive Samples

a. You should follow the PHS Guideline (see reference 1 and also section 

VIII.F.3.c.i. of this document) recommendations regarding archiving of plasma, 

blood, and other specimens. You should collect, archive, and reserve samples 

for use by PHS should the need for a PHS-led investigation arise. The PHS 

Guideline (reference 1) recommends that biologic specimens for PHS use be 

maintained for 50 years beyond the date of xenotransplantation, based on the 

latency periods of known human pathogenic persistent viruses and the 

precedents established by the U.S. Occupational Safety and Health 

Administration with respect to recordkeeping requirements.

b. In addition to the designated PHS samples, the sponsor should archive 

separate samples of patient plasma, blood cells, xenotransplantation product, or 

other tissues for clinical follow-up and for storage as part of a passive screening 

program, as detailed above. (See section VIII.H.1).

c. You should not use samples archived for use by PHS (see section 

VIII.H.2.a.) or for monitoring of the recipient through a passive screening 

program (see section VIII.H.2.b.) for other purposes, such as research. 

I. Health Records and Data Management

1. You should ensure that the recipient’s medical record contains information on 

the recipient’s health, and all xenotransplantation related information including 

procedures, a description of the xenotransplantation product, and any 

xenotransplantation product-related adverse events. In addition, you should develop an 

appropriate tracking system for all recipients of their xenotransplantation products and 

use this tracking information to facilitate notification in the case of a serious adverse 

event related to a xenotransplantation product. You should collect information when 

events occur, such as a xenotransplantation procedure or an adverse event, and at the 

time of clinical follow-up examinations. 

Reporting forms should be uniform and include information relevant to the recipient. We 

recommend that the information to be collected and tracked include, at a minimum, the 

following:

a. Facility information - Sponsors should record information regarding 

their animal facilities, manufacturing facilities, and clinical centers 

associated with each source animal, xenotransplantation product, and 
51

recipient.

b. Recipient information - You should identify recipients by code number 

or other identifier to link the recipient to relevant information in the 

tracking system.

c. Procedure information – You should record information about each 

xenotransplantation procedure. This information should include, but is 

not limited to: 

(i) recipient identifiers, 

(ii) the date of the procedure, 

(iii) the clinical center where the procedure was performed, 

(iv) the physician or investigator who performed the procedure, 

(v) the clinical indication for the xenotransplantation procedure,

(vi) medications and therapies administered at the time of the 

procedure, 

(vii) a description of the xenotransplantation product(s), 

(viii) identification of the animal source(s), 

(ix) animal facilities for each animal source,

(x) xenotransplantation product manufacturing facilities, and 

(xi) other pertinent clinical information

d. Adverse Event Reports - A sponsor must record adverse events and 

report the events to FDA pursuant to existing regulation (21 CFR 

312.32). Sponsors should keep records of each event. 

e. Recipient clinical follow-up examinations – You should periodically 

collect clinical status information for recipients of xenotransplantation 

products (see section VIII.F.). This information should include, but is 

not limited to: 

(i) the date of the clinical follow-up examination, 

(ii) the location of the clinical follow-up examination, 

(iii) the status of the xenotransplantation product in the recipient, 

(iv) any new significant co-morbidities or inter-current conditions, 

and 

(v) any hospitalizations since the recipients last clinical follow-up 

examination.

f. Animal Health Events - Animal facilities should record animal health 

events. These events include, but are not limited to: 
52

(i) breaks in the environmental barriers of the secured animal 

facility, 

(ii) disease outbreaks, and 

(iii) sudden, unexplained, or unexpected animal deaths. 

The animal facility should report animal health events to the IND 

sponsor. The sponsor should include this information in its tracking 

system for recipients and in reports to the FDA.

g. Recipient Death Reports - Sponsors should maintain death reports on 

recipients. This information should include recipient identifying 

information, the date of death, and the cause of death. You should 

record death certificate and autopsy information, if available. You 

should also report deaths to FDA.

2. FDA, together with other PHS agencies, is developing a computerized National 

Xenotransplantation Database intended to assist in data monitoring and tracking of 

recipients for Public Health Service needs. We may request sponsors to submit 

information to this database when it is mature.

3. You should maintain health records for at least 50 years beyond the date of 

transplantation. 

4. The sponsor should make provisions for all records and samples (including 

post-mortem samples) to be maintained for the requested period in the event that the 

establishment ceases operation.

J. Informed Consent

1. General Comments

The informed consent document must include the standard contents (see 21 CFR Part 

50).

2. Specific Issues

Within the general outline of the informed consent document, you should address certain 

specific issues regarding recipients. 

a. Participation in the Study

i. Because the zoonotic, opportunistic, and xenogeneic infectious 

risks to the recipient may extend to the recipient's family or intimate 
53

contacts, the patient should consent to inform his or her current and 

future intimate contacts of their potential risks from the source animal 

species, and of their deferral from blood donation. Intimate contacts of 

xenotransplantation product recipients include persons who have 

engaged repeatedly in activities that could result in intimate exchange of 

body fluids, including blood or saliva, with a xenotransplantation 

product recipient. Examples of intimate contacts include, but are not 

limited to, sexual partners, household members who share razors or 

toothbrushes and health care workers or laboratory personnel with 

repeated percutaneous, mucosal, or other direct exposures. Mere 

sharing of housing or casual contact, such as hugging or kissing without 

the exchange of saliva, would not be interpreted as intimate contact. 

You should offer the recipient assistance with this education process, if 

desired. This discussion should include the recipient's potential to 

transmit zoonotic or opportunistic infections if such an infection were to 

occur, and the possibly increased risk of such transmission to individuals 

who may be at increased risk for zoonotic or opportunistic pathogens 

such as infants, pregnant women, the elderly, chronically ill or 

immunosuppressed individuals.

ii. Pending further information clarifying risks, and further public 

consultation and discussion, xenotransplantation product recipients and 

their intimate contacts (see section VIII.J.2.a.i.) should be deferred 

from donation of Whole Blood, blood components, including Source 

Plasma and Source Leukocytes, tissues, breast milk, ova, sperm, or any 

other body parts for use in humans. For more detailed information, 

please consult the FDA draft guidance document “Guidance for 

Industry: Precautionary Measures to Reduce the Possible Risk of 

Transmission of Zoonoses by Blood and Blood Products from 

Xenotransplantation Product Recipients and Their Intimate Contacts,” 

which has been published for public comment (reference 12). 

iii. You should counsel the recipient regarding other behavioral 

modifications. You should provide, as appropriate, advice on the use 

of barriers to transmission of infectious agents during sexual activity and 

the use of appropriate precautions for nonsexual contacts.

iv. The informed consent document should contain information 

about the proposed life-long surveillance for all recipients and the need 

for clinical and laboratory monitoring throughout. You should explain, 

to the extent possible, the schedule for such clinical and laboratory 

monitoring.
54

v. The document should address the need for archiving plasma 

and tissue specimens from the source animal and the recipient for 

analysis in the case of xenogeneic disease concerns. The document 

should explain that such specimens may be tested in the future by the 

sponsor or PHS agencies as needed to evaluate concerns regarding 

xenogeneic infections.

vi. The document should inform the recipient of the responsibility to 

inform the investigator or his/her designee of any change in address or 

telephone number for the purpose of enabling life-long health 

surveillance.

vii. The document should inform the recipient of the long term need 

for access by the appropriate public health agencies (e.g., FDA, CDC) 

to the recipient’s medical records. To the extent permitted by 

applicable laws and/or regulations, you should maintain the 

confidentiality of medical records.

viii. You should include a request for autopsy in the informed 

consent document signed by the intended recipient or his/her 

appropriate representative.

b. Risks to the Recipient and his/her Intimate Contacts

i. The informed consent document should address the specific and 

known risks of all protocol-related activities not directly associated with 

source animal issues as well as the known and unknown zoonoses that 

may be associated with the source species. It should mention the 

uncertainty of the risks of infection or its transmission, and of the risk of 

tumorigenesis. It should mention the possibility of a long latency period 

before detection of possible adverse effects. It should specify the need 

for, and risks from, prophylactic antimicrobial, antiviral, or other 

chemo- or immunotherapy. It should provide in an attachment for the 

recipient and the recipient's family the reasoning behind the use of any 

prophylactic treatments.

ii. In addition, it should describe the possible need for 

confinement, reverse isolation or other specialized medical housing, 

including the estimated duration of such confinement. It should describe 

any specialized dietary, travel or other precautions in as much detail as 

possible.

iii. It should include any known time course for the risks of disease 
55

development and transmission. It should discuss infectious diseases 

with protracted incubation periods including TSEs and other unusual 

pathogens.

iv. In the specific case of xenotransplantation products from 

porcine sources, the informed consent document should include the 

following information:

(a) PERV can be transmitted from pig cells to human cells in 

culture and this virus can be transmitted from a human cell 

line to other human cell lines in culture.

(b) The clinical significance, if any, of this observation is 

unknown and is an area of active research; however, it is 

known that infection by certain type C retroviruses, similar 

in structure to PERV, can cause neurological disorders and 

diseases, such as lymphomas and other malignancies, in 

certain animal models.

c. Potential Benefits

It should be clear in the informed consent whether xenotransplantation is being 

studied as a first-line, second-line, or salvage therapy of the condition for which 

it is being proposed for the individual recipient. It should clearly convey the 

specific desired benefits, e.g., limited prolongation of survival, improved specific 

organ function, xenotransplantation product support until allograft becomes 

available, or experimental use without known or anticipated benefit.

d. Alternative Treatments

The informed consent document should also explain in detail the anticipated 

therapeutic options available to participants in the event of failure of the 

xenotransplantation product. 

e. Possible Consequences and Subsequent Treatment Options

The informed consent document, to the extent possible, should explain the 

consequences to the patient should the product fail or undergo irreversible 

rejection, including clear and unambiguous statements about the options that 

may not be possible after rejection of the xenotransplantation product, e.g., 

allotransplantation.

f. Confidentiality Issues
56

The informed consent should inform the patient that all data, including data 

collected during the follow-up period, could be made available to PHS 

agencies. 

K. Responsibility of the Sponsor in Informing the Patient of New Scientific 

Information

You should commit to providing recipients with updated information as soon as possible in the 

event that new data on risks, benefits or the need for additional treatments relevant to the 

recipient's clinical course becomes available or necessary. You should be willing to make a 

long-term commitment to provide information to the recipient’s families in the event that a 

recipient has died and new safety information of relevance to their potential exposures becomes 

known. If you are the sponsor, you should ensure that the investigators are also willing to 

commit to providing new information to recipients and their families.

IX. REFERENCES

Guidance Documents 

1. PHS Guideline on Infectious Disease Issues in Xenotransplantation. 

http://www.fda.gov/cber/guidelines.htm. January 29, 2001 (66 FR 8120).

2. Guidance for Industry: Public Health Issues Posed by the Use of Nonhuman Primate Xenografts 

in Humans, April 1999. 

3. The Public Health Policy on Humane Care and Use of Laboratory Animals, Office for the 

Protection from Research Risks, 9000 Rockville Pike, Building 31, Room 4B09, Bethesda, 

MD 20892, 301-496-7005.

4. Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, 

National Research Council, 2101 Constitution Ave. NW, Washington, D.C., 20418.

5. Occupational Health and Safety in the Care and Use of Research Animals, National Research 

Council, National Academy Press, 1997.

6. Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy, March 

1998.

7. Guideline on Validation of the Limulus Amoebocyte Lysate Test as an End-Product Endotoxin 

Test for Human and Animal Parenteral Drugs, Biological Products, and Medical Devices, 

December 1987, FDA, CBER.
57

8. Federal Standard 209E Airborne Particulate Cleanliness Classes in Cleanrooms and Clean 

Zones, General Services Administration, September 11, 1992.

9. United States Pharmacopoeia XXIII, 1995; Fifth Supplement, 1996.

10. Guideline on Sterile Drug Products Produced by Aseptic Processing, FDA, 6/87.

11. Guideline on General Principles of Process Validation, FDA, 5/87.

12. FDA Draft Guidance for Industry: Precautionary Measures to Reduce the Possible Risk 

of Transmission of Zoonoses by Blood and Blood Products from Xenotransplantation 

Product Recipients and Their Intimate Contacts, February 2002.

13. Proposed Approach to Regulation of Cellular and Tissue-Based Products, 2/28/97.

14. Guidance for the Submission of Chemistry, Manufacturing and Controls Information and 

Establishment Description for Autologous Somatic Cell Therapy Products, January 1997.

15. Guidance for Industry - Screening and Testing of Donors of Human Tissue Intended for 

Transplantation, July 1997.

16. Guidance for Industry for the Submission of Chemistry, Manufacturing, and Controls 

Information for a Therapeutic Recombinant DNA-Derived Product or a Monoclonal Antibody 

Product for In Vivo Use, August 1996.

17. Guideline for Research Involving Recombinant DNA Molecules, 1994, Office of Recombinant 

DNA Activities, NIH, 6006 Executive Blvd., Suite 323, MSC-7052, Bethesda, MD, 20892-

7052, 301-496-7005.

18. Final Policy Statement for Research and Regulation of Biotechnology Processes and Products, 

USDA, 6/26/86.

19. Livestock and Poultry Connected with Biotechnology Research, USDA, 12/27/91.

20. Update on Livestock and Poultry Connected with Biotechnology Research, USDA, 3/17/94.

21. ISO 10993: Biological Evaluation of Medical Devices. International Organization for 

Standardization, Geneva, Switzerland.

22. CDC Guideline for Isolation Precautions in Hospitals. Infection Control and Hospital 

Epidemiology 1996;17:53-80.

23. CDC. Notice to Readers NIOSH Guidelines for Protecting the Safety and Health of Health-
58

Care Workers. MMWR 1990; 39: 417.

24. CDC. Update: Universal Precautions for Prevention of Transmission of HIV, Hepatitis B Virus, 

and Other Blood Borne Pathogens in Health-Care Settings. MMWR 1988; 37: 377-88.

Points to Consider

25. Points to Consider in the Characterization of Cell lines Used to Produce Biologicals, 1993, 

FDA, CBER.

26. Points to Consider in the Manufacture and Testing of Therapeutic Products for Human Use 

Derived from Transgenic Animals, 1995, FDA, CBER.

27. Points to Consider in the Production and Testing of New Drugs and Biologicals Produced by 

Recombinant DNA Technology, 1985, FDA, CBER.

28. Supplement to the Points to Consider in the Production and Testing of New Drugs and 

Biologicals Produced by Recombinant DNA Technology: Nucleic Acid Characterization and 

Genetic Stability, 1992, FDA, CBER.

29. Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for 

Human Use, 1997, FDA, CBER.

30. Points to Consider in the Food Safety Evaluation of Transgenic Animals from Transgenic 

Animal Research, 1997, USDA, FSIS.

International Conference on Harmonisation (ICH) Guidelines

31. Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological 

Products, 8/18/99.

32. Q5D Quality of Biotechnological/Biological Products: Derivation and Characterization of Cell 

Substrates Used for Production of Biotechnological/Biological Products, 9/21/98.

33. Q5A Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or 

Animal Origin, 9/24/98.

34. S6 Safety Preclinical Evaluation of Biotechnology – Derived Pharmaceuticals for 

Biotechnological Product, 11/18/97. 
59

Other Publications

35. Reemtsma K, McCracken BH, Schlegel JV, Pearl M: Heterotransplantation of the kidney: two 

clinical experiences. Science 1964; 143:700-702.

36. Auchincloss H, Jr., Sachs DH: Xenogeneic transplantation. Annu Rev Immunol 1998; 16:433-

470.

37. Coffin, J. 1984. Endogenous Viruses, p. 1109-1204. In R. Weiss, N. Teich, H. Varmus, and J. 

Coffin (ed.), RNA Tumor Viruses, 2nd ed. Cold Spring Harbor Laboratories, Cold Spring 

Harbor.

38. McIntosh, K. “Diagnostic Virology” p. 411-440 in Virology, 2nd Edition, vol. 1. B.N. Fields, D. 

M. Knipe, R.M. Chanock, M. S. Hirsch, J.L. Melnick, T.P. Monath and B. Roizman, eds., 

Raven Press, New York, New York, 1990.

39. Benveniste, R. E., and G. J. Todaro. 1975. Evolution of type C viral genes: preservation of 

ancestral murine type C viral sequences in pig cellular DNA. Proceedings of the National 

Academy of Sciences USA. 72(10):4090-4094.

40. Armstrong, J. A., J. S. Porterfield, and A. T. D. Madrid. 1971. C-type virus particles in pig 

kidney cell lines. Journal of General Virology. 10:195-198. 

41. Strandstrom, H., P. Veijalainen, V. Moennig, G. Hunsmann, H. Schwarz, and W. Schafer. 

1974. C-type particles produced by a permanent cell line from a leukemic pig. I. Origin and 

properties of the host cells and some evidence for the occurrence of type-C-like particles. 

Virology. 57:175-178. 

42. Michaels, M. G., and R. L. Simmons. 1994. Xenotransplant-associated zoonoses. 

Transplantation. 57:1-7.

43. Martin, U., V. Kiessig, J. H. Blusch, A. Haverich, K. V. D. Helm, T. Herden, and G. 

Steinhoff. 1998. Expression of pig endogenous retrovirus by primary porcine endothelial cells 

and infection of human cells. The Lancet. 352:692-694. 

44. Wilson, C., S. Wong, J. Muller, C. E. Davidson, T. Rose, and P. Burd. 1998. Type C 

retrovirus released from porcine primary peripheral blood mononuclear cells infects human cells. 

Journal of Virology. 72(4):3082-3087.

45. Patience, C., Y. Takeuchi, and R. A. Weiss. 1997. Infection of human cells by an endogenous 

retrovirus of pigs. Nature Medicine. 3(3):282-286. 
60

46. Phan-Thanh, L., B. Kaeffer, and E. Bottreau. 1989. Porcine retrovirus reverse transcriptase: 

optimal conditions for its determination. Develop. Biol. Standard. 72:111-116.

47. Maudru, T. and Peden, K.1997. Elimination of background signals in a modified polymerase 

chain reaction-based reverse transcriptase assay. J. Virol. Meth. 66:247-261.