www.elsevier.com/locate/transci

Transfusion and Apheresis Science 29 (2003) 269–274

Tissue archives to track blood borne pathogens inpeople receiving blood products

Bruce C. Ritchie *

University of Alberta, 4-71 MSB, Edmonton, AB, Canada

Abstract

Tissue banks for surveillance of pathogens, research and transplantation have been established in a variety of set-

tings, and following a variety of guidelines. There are significant and changing ethical, moral, and legal issues in the

development and use of such archives. The Association of Hemophilia Clinic Directors of Canada has established an

archive of blood samples to test for known and emerging blood borne pathogens, and known and emerging blood

clotting genes. Key to the development of the tissue bank was the consent process for sample collection and handling,

the establishment of standard operating procedures for the handling of samples and data, and the establishment of an

oversight board.

� 2003 Elsevier Ltd. All rights reserved.

1. Introduction: A brief history of tissue banks

Tissue banks are critical resources in medicine.

They are used for transplantation, surveillance ofdisease, research, teaching, quality control, and

sometimes in the manufacture of therapeutic and

diagnostic aids (see review by Indech [1]). Both

living and cadaver sources of tissue are used, often

without thoughtful epidemiologic selection [2],

sometimes without consent [3,4], sometimes long

after the donors� death, often across borders, and

sometimes for uses other than originally intended[5]. Lack of informed consent [6,7], questionable

donor selection [2], and use for un-consented re-

search [5] pose major hurdles to using these valu-

able resources. In response to these issues,

* Tel.: +1-780-492-3550; fax: +1-780-492-0886.

E-mail address: bruce.ritchie@ualberta.ca (B.C. Ritchie).

1473-0502/$ - see front matter � 2003 Elsevier Ltd. All rights reserve

doi:10.1016/j.transci.2003.08.010

regulation of tissue banks has begun [8,9], but re-

mains formative, and untested [10].

2. A brief history of pathogen screening and

inactivation strategies

Blood and blood products have been critical

advances in the treatment of human disease.

However, the use of these has also been fraught

with adverse events, including hemolytic transfu-sion reactions, allergic reactions, and infection

with blood borne pathogens. The use of pooled

blood products is a key, first step in the production

of many useful products from coagulation prod-

ucts to intravenous immunoglobulin, IVIg. Pool-

ing of the products makes purification of blood

components practical and efficient. However,

pooling also potentially exposes the recipient topathogens from all donors to the pool [11].

d.

270 B.C. Ritchie / Transfusion and Apheresis Science 29 (2003) 269–274

The manufacturers of pooled blood products

have responded by screening for known pathogens

to minimize loads of potential pathogens, and then

using powerful viral inactivation techniques toremove any residual infectious agents. At present,

much of the western world is using ‘‘source

plasma’’ from the United States because of con-

cerns about variant Creutzfeld Jacob Disease

(vCJD). This plasma is collected in plasmapheresis

centers from carefully screened donors. The plas-

ma is not used until the donor returns at three

months for the next donation, and is re-screened,to make sure that the first collection was not done

during a ‘‘window period’’ when the donor may

have been infected with an agent, but before he/she

developed an antibody response that could be

detected by the screening tests.

Manufacturers of pooled blood products also

use powerful viral inactivation techniques, often in

combination, and validated with model viruses, toremove any pathogens not caught in the screening

process. These techniques include the purification

processes themselves, as well as treatment with

heat, solvents and detergents, and filtration. This

combination of strategies has produced safe

products, free from blood borne pathogens.

These strategies have not been foolproof. The

producers of recombinant Factor VIII stabilizedthis product with non-pasteurized albumin. The

albumin has been shown to transfer the virus

known as TTV [12]. Manufacturers now use viral

inactivation processes on these products after the

addition of stabilizing agents, and have moved

away from the use of human or animal products to

stabilize (generation 2 products) or to grow the

cells used to produce these products (generation 3products). Failure of viral inactivation techniques

in the early days, before strong validation pro-

cesses were used, have led to infection of recipients

with HIV and hepatitis A. Current pathogen in-

activation protocols are stringently validated be-

fore use.

More recently, concern has been raised about

the potential for transmission of prions diseases,which are not inactivated by heat, solvents, de-

tergents, or filtration. Reassurance comes from the

lack of transmission of experimental prion diseases

by blood, and documentation of the clearance of

model prions from blood through routine purifi-

cation steps, particularly centrifugation and pre-

cipitation. However, investigators continue to find

novel pathogens that raise concern about thesafety of the blood supply.

3. The blood borne pathogens archives

3.1. Introduction

The goal of a tissue sample archive is to save

time-stamped samples for testing at a later date,when there is new testing based on new technol-

ogy, new testing based on new knowledge, or to

preserve samples from subjects who may not sur-

vive till testing can be done. The strength of any

such tissue archive is critically dependent on the

accuracy of clinical data collected at the same

time, so that test results can be correlated with

clinical outcomes. The Blood Borne PathogensProject of the Association of Hemophilia Clinic

Directors of Canada (AHCDC) fulfills current

standards and is described below.

3.2. Goals

The goals of this archive project are to:

1. Detect new and emerging pathogens and deter-

mine the consequence of exposure.

2. Detect genetic elements that may worsen or im-

prove outcomes from a disorder. For example

co-inheritance of severe hemophilia and theFactor V Leiden mutation leads to lessening

of the bleeding tendency [13–15].

3.3. Consent process

The consent process for tissue research has re-

cently been clearly summarized by Riddick et al.

[16]. Their appendix provides a very useful

checklist of information that should be given to all

subjects, including the nature and amount of

samples, procedures used to obtain samples, risk

and benefits of obtaining samples, the nature of

treatment of complications, how the samples willbe used, where the samples will be kept, who will

B.C. Ritchie / Transfusion and Apheresis Science 29 (2003) 269–274 271

have access to the samples, how long the samples

will be kept, and the arrangements for disposal.

Where relevant, subjects should be informed:

whether the results will be relayed back to thesubject, if there may be adverse consequences

of disclosure of results and arrangements for

counseling, whether the results could reveal in-

formation about family relationships such as non-

paternity, whether the results will identify past or

current infectious disease, whether the results will

predict genetic predisposition to disease, whether

the results could affect employability or insurabil-ity, whether the research will involve embryos,

whether the research may lead to profit, who is

funding the research, and whether the sponsor is

paying the researcher for each subject.

All archiving of blood and testing for this pro-

ject is done under consent. Consent for sample

testing is obtained at the same time as consent for

the sample to be archived. In the Blood BornePathogens Project, we have asked for consent to

perform assays to detect known and emerging

blood borne pathogens, and known and emerging

coagulation genes. This provides some flexibility

without allowing unreasonably wide testing that

would be outside the scope of the project. The

research committee of the AHCDC first approves

projects, and then the research ethics boards atparticipating institutions are informed of testing

before each project begins to assure that it is

covered by the consent. An oversight board su-

pervises the overall running of the project. Mem-

bership includes: external representatives of

Health Canada, the Canadian Blood Services,

Hema Quebec, the Canadian Hemophilia Society,

and an ethicist. Encoding of samples with a reg-istry number at the time of sample collection and

re-encoding of aliquots with an inventory number

provide good security. The central lab will provide

results linked to the original registry number,

which will be electronically relayed to clinics where

the subjects can be identified and notified of their

results. Only the participating clinic will be able to

link results to individual patients. Samples will bekept indefinitely or until the death of the patient,

depending on the subjects� preference as indicated

on the consent form. Because of the number of

institutions involved, and the funding by public

resources, the developers of the project have

agreed not to seek intellectual protection of the

results, but instead to make summary results

widely available through peer reviewed publica-tion.

All patients are informed that their participa-

tion in the project means that they will be told the

results of testing. The decision to do this comes

from difficulties encountered with a previous study

in which patients were not told the results of HIV

testing because the implications of a positive test

were not known (i.e. a positive test may signalexposure, infection, or immunity). However, pa-

tients who were positive in such assays were not

given the option of protecting their sexual part-

ners. We have taken the approach that it is best to

deal with such information openly and fully.

Health Canada has agreed to provide help with

counseling. This project is unlikely to identify non-

paternity, but may identify genetic risks of bleed-ing or thrombosis, and subjects will be promptly

notified.

Subjects are re-consented at each annual as-

sessment clinic. At that time, they are asked if they

wish to continue their participation in the project

by donating another sample, if they would like

their samples destroyed in the event they are not

continuing in the project, and if they want tocontinue to have their samples tested for things

they have previously consented to, after their

death. Standard Operating Procedures cover all

eventualities. Subjects are informed that no testing

can be done which is not consented to. For studies

not covered in the original consent, new con-

sent will be needed. Published evidence suggests

that this will not be a difficult nor onerous process[17].

3.4. Ownership

There has been much written about the own-

ership of tissue samples [3,4,9,18–21]. There are

working models in blood banks and cord stem cell

banks. In most blood banks, the donated blood is

donated with no expectation that the blood will be

transfused into the donor, while in autologous

collection programs, the expectation is that thedonor will be given his/her own blood. Some

272 B.C. Ritchie / Transfusion and Apheresis Science 29 (2003) 269–274

confusion is caused by the different routes by

which tissue is acquired. Samples may be collected

following consent, or as ‘‘waste’’ products from

surgery or post-mortem. We collect all samplesfollowing consent, which in general, gives ‘‘own-

ership’’ to the investigators. However, we give the

subjects the opportunity to withdraw their samples

at any time. The samples are then destroyed fol-

lowing a standard operating procedure.

3.5. Samples collected for antibody based techniques

Samples collected depend on the technologyused to screen the samples. For instance, the use of

antibody based techniques depend on the collec-

tion of antibody bearing samples such as plasma

and serum. Detection of pathogen elements de-

pends on where they are found: in plasma, in pe-

ripheral blood cells, or as other markers (i.e.

certain hepatic transaminases are markers of viral

hepatitis).

3.6. Samples collected for DNA based techniques

Pathogens that are detected by DNA based

techniques, such as the polymerase chain reaction,

are often found in plasma, while some, such as the

human immunodeficiency virus, may be better

found in peripheral blood leukocytes. Both plasmaand peripheral blood leukocytes are collected for

processing.

3.7. Samples collected for archiving genomic DNA

Genomic DNA for archive purposes is often

stored in liquid phase either at 4 �C (refrigerator

temperatures) or frozen, usually at )20 �C (stan-

dard domestic freezer temperature). However,DNA is susceptible to degradation by hydrolysis,

which requires the presence of water. Saving DNA

in dried form is preferable. Museum samples with

DNA hundreds of years old have been analyzed

successfully using the polymerase chain reactions.

DNA in dehydrated samples in amber, much older

than museum samples, has also been detected and

characterized. DNA for archive purposes istherefore better preserved when dried on blotting

paper (Guthrie spots) or on ion exchange paper

(FTA paper). Intact cells are also stored to be

available for testing using new technologies.

3.8. Privacy protection and freedom of informa-

tion––sample integrity

Any archive must be able to protect the privacy

and integrity of the samples, and the data obtainedfrom such samples [22]. The integrity of the sam-

ples requires the use of validated storage condi-

tions, validated disaster recovery procedures, and

constant vigilance. Frozen samples are most effi-

ciently stored in liquid nitrogen, which has a sim-

ilar price as powdered drinks. Automated methods

for topping up storage containers and monitoring

the level of liquid nitrogen are required, but loss ofpower is of less severe consequence than when the

samples are stored in electric freezers. Automated

handling of samples labeled with machine readable

labels, such as bar codes, has a much lower error

rate, and is used to log the handling of samples to

permit error tracing.

3.9. Privacy protection and freedom of informa-

tion––data integrity

Aggregate data generated by archives and their

associated databases is a very powerful and useful

tool for groups funding and providing health care.

It can be used to predict economic outcomes ofdisease, predict future blood product require-

ments, and predict health care costs. The institu-

tions interested in this information do not want

data on individuals, which could only lead to

questions of data security, so data should not be

used to identify individuals. The use of encoded

labels in the central lab and the provision of

summary or aggregate data only to participatinginstitutions will protect the confidentiality of the

subjects.

Individual data, on the other hand, can be used

to tailor treatment to individual patients, and

therefore should be available to the treating clinic

and must be accurately linked to individuals. In-

dividual labs testing samples must have an accu-

rate unique identifier for each sample, but eachidentifier must not be linked to other data that

B.C. Ritchie / Transfusion and Apheresis Science 29 (2003) 269–274 273

could be used to identify the donor of a sample. In

the project described here, a label with a unique

identifier, in human and machine-readable form, is

attached at the time the blood sample is collected,so linkage of the test results to an individual can

only occur at the clinic. Unique identifiers, or

registry numbers, for individual bleeding disorder

patients are generated on a central server, when

they are registered with bleeding disorder clinics in

Canada. Only the clinic has the ability to decode

the registry number. Labeled samples are pro-

cessed using a robotic or other automated systemthat can read the machine-readable label, and

track the sample through processing and storage.

Stored samples are divided in aliquots, each la-

beled with a unique inventory number that is

linked to the CHR in the archive laboratory dat-

abase only. All data are recorded in a secure, en-

crypted database with no connection to the

Internet. Computer backups are performed regu-larly and automatically, and are stored off-site.

3.10. Sample testing

Sample testing is done under contract with the

testing lab agreeing to the specific testing to be

done, as well as agreeing to not share samples, to

not share data outside of the agreement, and to

not do testing that is not specifically contractedfor. The funding agency, Health Canada, and the

participating institutions are informed that the

testing will be done, and that counseling of clinic

staff and subjects may be necessary. Samples are

removed from storage for testing and sent to the

testing lab along with a sample database for en-

tering data.

3.11. Notification of results

Test results are returned to the archive labora-

tory, validated, decoded to link to the registry

number, then the registry number linked data is

sent as an encrypted electronic file to the individ-

ual clinics. The clinic is notified that new data are

available in their clinic database to be assessed,

and appropriate sources of counseling are identi-fied.

3.12. Dealing with data which has unknown conse-

quences

In Canada, Health Canada has agreed to helpwith counseling of patients found to be positive for

a new or emerging blood borne pathogen. Health

Canada has a good track record of dealing with

information such as this, as demonstrated in their

handling of exposure to the ‘‘Utah Donor’’. This

plasma donor contracted Creutzfeld Jacob Dis-

ease, after donating plasma that was used in the

culture of tissue culture cells that were engineeredto produce recombinant Factor VIII.

4. Conclusion

The Association of Hemophilia Clinic Directors

of Canada has developed a blood sample archive

to test for known and emerging blood borne

pathogens and known and emerging blood clotting

genes. Informed consent, validated sample anddata handling, and strict adherence to standard

operating procedures will assure the integrity and

usefulness of this critical resource.

Acknowledgements

The author has received research funding from

the following pharmaceutical companies: Bayer,

Baxter, Astra Zeneca, Sanofi, Pharmacia, andNovoNordisk. In addition, he has received funding

for travel and honoraria from Bayer, Baxter, Wy-

eth, Novo Nordisk, and Pharmacia. This work has

been supported by funding from Health Canada.

References

[1] Indech B. The international harmonization of human tissue

regulation: regulatory control over human tissue use and

tissue banking in select countries and the current state of

international harmonization efforts. Food Drug Law J

2000;55(3):343–72.

[2] Winn DM, Reichman ME, Gunter E. Epidemiologic issues

in the design and use of biologic specimen banks.

Epidemiol Rev 1990;12:56–70.

[3] Skene L. Who owns your body? Legal issues on the own-

ership of bodily material. Trends Mol Med 2002;8(1):48–9.

274 B.C. Ritchie / Transfusion and Apheresis Science 29 (2003) 269–274

[4] Skene L. Ownership of human tissue and the law. Nat Rev

Genet 2002;3(2):145–8.

[5] Dalton R. Tribe blasts �exploitation� of blood samples.

Nature 2002;420(6912):111.

[6] Marshall E. Policy on DNA research troubles tissue

bankers. Science 1996;271(5248):440.

[7] Basu S, Martin SW, Phillips RM, Puri R. Obtaining

archived pathological material for biomedical research.

Lancet 2003;361(9366):1394.

[8] Salter B, Jones M. Human genetic technologies, European

governance and the politics of bioethics. Nat Rev Genet

2002;3(10):808–14.

[9] Fox M, McHale J. Regulating human body parts and

products. Health Care Anal 2000;8(2):83–6.

[10] Helminski F. Formalities, good faith, and tissue donation.

Mayo Clin Proc 1994;69(10):985–6.

[11] Poon MC, Landay A, Prasthofer EF, Stagno S. Acquired

immunodeficiency syndrome with Pneumocystis carinii

pneumonia and Mycobacterium avium–intracellulare infec-

tion in a previously healthy patient with classic hemophilia.

Clinical, immunologic, and virologic findings. Ann Intern

Med 1983;98(3):287–90.

[12] Chen BP, Rumi MG, ColomboM, et al. TT virus is present

in a high frequency of Italian hemophilic patients trans-

fused with plasma-derived clotting factor concentrates.

Blood 1999;94(12):4333–6.

[13] Nichols WC, Amano K, Cacheris PM, et al. Moderation of

hemophilia A phenotype by the factor V R506Q mutation.

Blood 1996;88(4):1183–7.

[14] van �t Veer C, Golden NJ, Kalafatis M, Simioni P, Bertina

RM, Mann KG. An in vitro analysis of the combination of

hemophilia A and factor V (LEIDEN). Blood 1997;90(8):

3067–72.

[15] Lee DH, Walker IR, Teitel J, et al. Effect of the factor V

Leiden mutation on the clinical expression of severe

hemophilia A. Thromb Haemost 2000;83(3):387–91.

[16] Riddick AC, Barker C, Sheriffs I, et al. Banking of fresh-

frozen prostate tissue: methods, validation and use. BJU

Int 2003;91(4):315–23, discussion 323–4.

[17] Stegmayr B, Asplund K. Informed consent for genetic

research on blood stored for more than a decade: a

population based study. BMJ 2002;325(7365):634–5.

[18] Brahams D. Brain banks. Lancet 1990;335(8684):282–3.

[19] McHale J. Waste, ownership and bodily products. Health

Care Anal 2000;8(2):123–35.

[20] van Diest PJ. No consent should be needed for using

leftover body material for scientific purposes (For). BMJ

2002;325(7365):648–51.

[21] O�Brien C. U.K. panel weighs tissue ownership. Science

1995;268(5210):492.

[22] Strauss E. The tissue issue: losing oneself to science? Sci

News 1997;152(12):190–1.