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DNA Banking in Epidemiologic Studies by Karen
K. Steinberg, Karen C. Sanderlin, Chin-Yih Ou, W. Harry Hannon,
Advances in genetic technology, such as the polymerase chain reaction (PCR) (1) and the work of the Human Genome Project to map and sequence the human genome (2-4), are expediting our understanding of the genetic determinants of common disease. For example, genetic factors are proving to be important in determining who is susceptible to infectious agents such as HIV (5), as well as in identifying populations who are at risk for cancer because of exposure to environmental hazards such as cigarette smoke and aniline dyes (6-9). DNA is now being used in epidemiologic investigations to study genetic risk factors (10). Genetic risk factors include rare gene mutations such as the BRCA1 mutation, which confers high risk for breast or ovarian cancer (11,12), as well as genetic polymorphisms, such as the polymorphic genes that code for carcinogen-metabolizing enzymes that affect the way people process carcinogens, thereby increasing or decreasing their risk for sporadic forms of cancer (13). Because of new technologies that have become a part of routine molecular methods in the last decade, especially PCR, specimens other than fresh, whole blood can be used, in many cases making specimen collection in the field possible for the first time (14). All nucleated cells, including cells from hair follicles, buccal swabs, and urine specimens, are suitable specimens for DNA analysis (15-20). The type of specimen that will be collected in the field is dictated by many factors including 1) the amount of DNA needed (which may depend on the number of mutations or polymorphisms to be studied in the future), the degree to which the mutations and polymorphisms have been characterized, and the molecular methods to be used for DNA analysis; 2) the resources that are available to obtain, process, and store specimens; and 3) the conditions for collecting specimens in the field. We first present a review
of methods for collecting, processing, and storing specimens for DNA banking in
epidemiologic studies. We then briefly discuss the specific experience of the Centers for
Disease Control and Prevention (CDC) with the Third National Health and Nutrition
Examination Survey (NHANES III) DNA Bank to illustrate the ethical and social questions
that can arise when creating DNA banks, and how CDC, with others, addressed these issues. Because obtaining buccal
swabs and hair follicles as sources of DNA cost less than obtaining blood, can increase
participation in studies, and is relatively easy, buccal swabs and hair follicles have
great potential as sources of DNA for epidemiologic studies. Hairs with visible roots are
collected and stored at room temperature (RT) in contamination-free containers such as
"sealable" plastic bags or clean envelopes. DNA can be extracted from the
specimen and PCR performed (18). Buccal cell specimens can be collected with sterile
cotton swabs and smeared onto alcohol-cleaned slides and allowed to air dry. Slides are
transported in contamination free holders (suitable to prevent breakage). Cells can be
removed from the slide using a sterile cotton swab wet with sterile saline. Cells can then
be concentrated by centrifugation and resuspended in buffer and stored at -20EC until
cells are lysed and DNA is extracted for PCR (18, 19). Buccal swabs have also been
collected using cytology brushes. Although longterm stability of DNA in buccal cells
collect in this way was not studied, PCR product yields from specimens stored at 4EC for
at least 1 month were similar to yields from fresh samples. DNA extracted from these
specimens was stable at 4EC for at least 10 months (19). Saline washes, sometimes called a
"swish and spit" technique, are also used to obtain buccal cells and are
reported to yield DNA suitable for PCR for at least 1 week stored at -20EC, 4EC, 25EC, or
37EC (20). The major disadvantage of buccal swabs and hair follicles is that numbers of
nucleated cells are small using these specimens compared to the numbers obtained from
whole blood and DNA can be exhausted fairly quickly. Blood spots The use of filter paper is the least costly method of collecting, shipping, and storing of blood specimens, and it is the method of choice for many epidemiologic studies, especially when blood specimens have to be collected from remote areas where refrigeration is not possible. Dried blood spots are used to quantify analytes that include metabolic products, enzymes, and hormones (21). In the last 10 years, DNA from blood spots has been used in newborn screening to identify gene mutations for diseases such as cystic fibrosis (22) and sickle cell disease (23, 24). Dried blood spots are especially suitable for PCR when the mutation or polymorphism of interest is well-defined (25). Residual dried blood spots, collected originally for newborn screening programs, now fill at least two large population-based DNA banks (26,27). Blood spots from military personnel are now being stored to serve as biologic "dog tags" (28). Dried blood spots are also a source from which to isolate and amplify messenger-RNA (mRNA). Analysis of mRNA offers two advantages: 1) it provides a means of confirming a genotype obtained by DNA analysis (29); and 2) it provides a means of studying only the coding regions of DNA, because only the regions of the genes that actually code for protein are transcribed into mRNA (30). Messenger-RNA analysis is thus especially helpful for large genes with long non-coding segments. However, if mutations are present in the noncoding regions of the genomic DNA, they will escape detection. Aside from the low cost of collection and storage, another advantage of using blood spots is that dried specimens are easier to transport than whole blood because they can be shipped through the U.S. mail with few precautions. HIV and certain other infectious agents lose viability on drying. Two disadvantages of dried blood spots have been the labor associated with obtaining "punches" from the filter paper for analysis and the difficulty in eluting DNA from filter paper to remove substances that inhibit the PCR reaction (21). Methods for automated punching of cards for large collections exist and are being improved (31). The National Committee for Clinical Laboratory Standards (NCCLS) has published guidelines on how blood is to be collected on filter paper for newborn screening programs (32). Two filter paper sources are approved by the Food and Drug Administration for blood collection: S&S grade 903 filter paper (Schleicher and Schuell, Keene, NH) and Whatman BFC180 filter paper, lot #6411 (Whatman International, LTD, Maidstone, Kent, UK). Experts in the field of newborn screening recommend that the NCCLS-approved standard be adopted as a standard of practice for all screening programs (33). The Council of Regional Networks for Genetic Services has recently issued guidelines for banking dried blood spots (34). The guidelines recommend that blood spots for DNA testing be stored at 4EC with a desiccant, in sealed bags of low gas permeability, and that spots be protected from cross-contamination by contact with each other. Whole blood Anticoagulated whole blood can be shipped to central laboratories where large quantities of DNA can be purified from the whole blood or from white cells obtained by low-speed centrifugation and then stored. Purification from whole blood requires that blood be processed quickly to prevent degradation of DNA (35). Whole blood also can be frozen at -20EC. If blood will be frozen in collection containers, glass containers should not be used. Although DNA can be extracted from blood clots, DNA yields are greater when blood is anticoagulated. Anticoagulants including heparin, acid citrate dextrose (ACD), and ethylenediaminetetraacetic acid (EDTA) have been used. We have used a stabilizing solution containing detergents and anticoagulant (0.05% NP40, 10mmol/L EDTA and 10 mmol/L Tris HCl, CH 8.3) in a 1:1 ratio with whole blood to inactivate infectious agents and to preserve the DNA at room temperature for at least 1 month. Only a few systematic studies to determine the optimal storage conditions for whole blood before DNA isolation or analysis have been done, and they have not all used the same conditions or reached the same conclusions. In one study comparing the effects of storage times and temperatures on DNA yield and quality, the authors reported that blood collected in EDTA and held at 4EC gave the highest yield of DNA, but all temperatures (-20EC, 4EC, 23EC, and 37EC) and all storage durations (3, 7, 14, and 28 days) studied resulted in high quality DNA suitable for PCR and restriction enzyme digestion (36). These investigators recommend shipping whole blood at 4EC unless the shipping time is less than 3 days and the ambient temperature is 23EC or less, in which case ambient temperatures were considered adequate. Other investigators have reported successfully performing PCR directly on EDTA-anticoagulated blood that had been stored frozen at -20EC for several months (37). Lahiri and Schnabel reported no significant change in DNA yield or quality whether blood specimens were stored for 24 hours at 45EC, 37EC, 25EC, 4EC, -20EC or -70EC, although subjecting whole blood to freeze-thaw cycles did degrade the DNA (38). Decreases in DNA yield of 30% to 40% have been reported for blood stored at 4EC for longer than 4 days or frozen at -20EC with no prior treatment (39). Madisen et al. reported that 1 in 6 specimens gave poor DNA yields after storage at -70EC for 8 weeks (40). In summary, most investigators have not reported problems in DNA purification from blood collected and held at RT (if RT is not more than 23EC) for 48 hours before processing. Although PCR can be performed on whole blood, preparation of whole blood for long-term storage usually includes isolation of lymphocytes or treatment to remove inhibitors to molecular methods of analysis (41). Lymphocytes can be isolated for immortalization by Epstein Bar virus (EBV) as discussed below, DNA can be extracted from lymphocytes, or isolated lymphocytes can be frozen for later immortalization or DNA extraction. DNA can be partially purified and stored frozen for years at -20EC. DNA purification: Many DNA purification methods call for a crude leukocyte isolation (42, 43), although others have used whole blood (40). Traditional purification methods have usually fallen into one of four categories: 1) those that use enzymes (including proteinase K and RNAse A), 2) those that use enzymes and organic solvents (phenol and chloroform), 3) those that use solvents only, and 4) those that use resins or affinity gels (41). Steps include either separation of leukocytes or red cell lysis with detergent (NP-40 or Triton X-100), nuclear lysis, deproteinization, RNAse A treatment, and DNA precipitation. Although a multitude of combinations of methods have been used, only a few systematic studies of the relative merits of different systems have been published (40, 41). Several commercial products are now available for rapid purification of high- quality, genomic DNA from blood spots, whole blood, and other sources. These products include: The WizardTM Genomic Purification Kit (Promega, Madison, WI) and the PureGeneTM kit (Gentra Systems Inc., Minneapolis, MN) both of which are methods that rely on separation of white cells from red cells, lysis of white cells, elimination of protein, and precipitation of DNA. Examples of kits that are used for purification of DNA from blood spots are the following: the GenerationTM method of Gentra Systems Inc. (Minneapolis, MN), removes proteins and other extraneous substances from the blood spot filter paper by washing punches a few times with a proprietary extraction solution. The DNA-containing punches can then be used directly or eluted for PCR. No protease digestion is required and a punch 1/8 inch in diameter can be used in five PCR reactions. A method developed by Qiagen Inc. (Chartsworth, CA) includes the elution of a proteinase-digested mixture from the punches and then the binding of DNA onto an affinity column. The column is washed to remove impurities, and DNA is eluted for PCR. The affinity matrix, IsoCodeTM, developed by Scheicher & Schuel (Keene, NH), uses a reverse principle. The solid matrix binds with stronger affinity to proteins than to DNA, thus allowing DNA to be eluted in 95EC water in one step and the eluate used directly for PCR. This is not a comprehensive list, and new products are constantly being introduced. Lymphocyte immortalization The specimen of choice for an unlimited source of stable, genomic DNA and for viable cells with which to perform biochemical and molecular studies is peripheral B-lymphocytes. Lymphocytes can be used either to immediately create lymphoblastoid cell lines or frozen for later immortalization. Establishing lymphoblastoid cell lines requires that mononuclear cells be separated from whole blood with a density gradient and then exposed to EBV to induce cell immortalization (44). After the cell lines are established, they are frozen in liquid nitrogen. Although methods have varied with respect to virus preparation, media content, cell-separation media, and the elimination of T-lymphocytes (45), most B-lymphocyte immortalization techniques are based on cell-separation methods as described by Boyum (44), and most obtain EBV from a Marmoset cell line (47). In large epidemiologic surveys, such as NHANES III, mononuclear cells cannot always be separated from whole blood at the collection location, and blood can not always be sent to a central laboratory within 12 to 24 hours. Lymphocytes can be immortalized when collected in ACD and held at RT for up to 3 days (46, 48). From our experience with the NHANES III collection, we found the Vacutainer-CPTTM (Bectin-Dickenson, NJ) to be ideal for collecting specimens for immortaliztion in the field. The Vacutainer-CPTTM is a cell-separation tube containing sodium citrate as an anticoagulant and a polyester gel and a density-gradient liquid for separating mononuclear cells from red blood cells and other white cells and has two major advantages over other separation products: 1) cell separation requires only one centrifugation step, and 2) the specimen can be shipped in the original collection tube, thus reducing the risk for sample contamination and worker exposure to hazardous biologic agents. Using this system, we were able to immortalize cells up to 72 hours after collection in the field for NHANES III. The methods that we adapted to immortalize approximately 8,500 cell lines in NHANES III using EBV and cyclosporin A are typical of this process and are instructive for investigators estimating labor and resources required for a large collection (46, 49). Lymphocytes are immortalized using marmoset B95-8 EBV. For immortalization, mononuclear cells must be depleted of T-lymphocytes which can interfere with the growth of the B-lymphocytes. One of two methods is commonly used to prevent T-lymphocyte overgrowth: exposing lymphocytes to phytohemagglutinin (PHA) to stimulate growth of B-lymphocytes or treatment of the cultures with cyclosporin A to inhibit the growth of T-lymphocytes (44, 50). After visible clumps appear in the medium, cultures are transferred to tissue culture flasks, and a culture medium without cyclosporin A is added. Cells are separated by centrifugation, resuspended at a concentration of about 5 X 106 cells/ mL in cold culture medium supplemented with 25% FCS, and 10% DMSO then dispensed in 1 mL amounts into cryotubes, frozen, and transferred to liquid nitrogen. Our transformation success rate using unfrozen cells agrees with that reported by others (49). When specimens are received and transformation attempted within 4 days after the specimen was collected, we have a 94% or greater success rate, but this success rate drops significantly for specimens held more than 4 days (Figure 1). Quality control and quality assurance The Clinical Laboratory Improvement Act (CLIA-1967) mandates that the federal government regulate laboratories that provide more than 100 commercial laboratory tests per year in interstate commerce. Neither CLIA-1967 nor the Clinical Laboratory Improvement Amendments (51) have established guidelines for quality control and quality assurance specifically for DNA banks or DNA-based laboratory methods. In the absence of other guidelines and in conjunction with future recommendations, we find that the recommendations of the Council of Regional Networks for Genetic Services provide useful guidelines for collecting and storing dried blood spots (34). For frozen specimens the
following general recommendations should be a part of the quality assurance protocol.
Specimens should be stored in duplicate in separate freezers, preferably in different
buildings. Freezers should have emergency power and liquid nitrogen sources and be
equipped with smoke and fire alarms. For all stored specimens, records containing the
sample identification number and sample location should be kept. For frozen specimens, any
history of unplanned defrosting should be recorded with the date and maximum temperature
reached. For immortalized cell lines, any manipulation of cell lines, passages, emergency
events, and contamination should also be recorded. Freezer checks should be recorded
daily, and alarm checks should be recorded weekly. Cell cultures should be monitored for
bacterial, fungal, and mycoplasma growth. Although ethical, legal and social issues are ably addressed in this issue (52), we would like to make some comments on these issues from the perspective of the NHANES III experience. NHANES is a family of surveys that the National Center for Health Statistics began conducting in 1966. The NHANES program has several objectives: 1) it estimates the prevalence of common diseases and risk factors for those diseases in the US population; 2) it detects secular trends for diseases and risk factors; and 3) it helps explain the causes and natural history of diseases; and 4) it provides normal values for the US population (53-57). NHANES III serves as an example of a large survey in which specimens were stored for later use to test new hypotheses about pathophysiology and risk factors for disease. We used the methods described above to establish a DNA Bank which now includes uncultured mononuclear cells stored in liquid nitrogen from 19,553 study participants, and immortalized lymphoblastoid cell lines from 8,532 participants. As part of the extensive informed consent for the survey, participants were told that a portion of their blood would be frozen and used for future research. Consent specific for genetic testing was not included. Even though the NHANES III informed consent conformed to Federal Regulations (51) and was standard for the time that it was written, CDC decided to review the informed consent in light of the potential for genetic testing and the current literature on informed consent. To address issues relating to informed consent and genetic testing, CDC drew on the resources of the National Human Genome Research Institute (NHGRI) through its Ethical, Legal, and Social Implications (ELSI) Branch. In July of 1994, CDC and NHGRI sponsored a workshop addressing issues of informed consent for genetic research on stored tissue samples and the situation of the NHANES III DNA specimens was specifically discussed. An article addressing issues of informed consent for genetic research on stored tissues samples was published based on the dialogue that occurred during and after the workshop (58). The recommendations from the document were: 1) informed consent is required for all genetic research using linkable samples unless conditions of Federal Regulations for limitation or waiver of this requirement are met (59); 2) informed consent is not required for genetic research using anonymous samples but may be considered appropriate if identifiers are to be removed from currently identifiable samples; and 3) Institutional Review Boards (IRBs) could usefully review all protocols that propose to use samples for genetic research. With regard to NHANES III, the informed consent did not discuss genetic testing, and conditions for waiver of informed consent were not met. According to recommendation 1, linked testing could only be done if subjects were recontacted and informed consent obtained. Given the number of subjects involved and the geographic distribution, recontacting would be a complex task requiring an extended period of time. In the interim, according to recommendation 2, "anonymized" testing could be considered in the current NHANES III situation. The American Society for Human Genetics distinguishes anonymous from "anonymized" specimens (60). Anonymous specimens are biological materials originally collected without identifiers and are impossible to link to their sources. Anonymized biologic materials are specimens that were initially identified but have subsequently been irreversibly stripped of all identifiers and are impossible to link to their sources. Participants of the workshop noted that testing of "anonymized" specimens is permissible under Federal Regulations, but recommended that IRBs consider the following during their assessment of proposals to anonymize samples: 1) whether the information can be obtained in a manner that allows individuals to consent; 2) whether the proposed research is scientifically sound; 3) how difficult it would be to recontact subjects; 4) whether the samples are finite; and 5) how the availability of effective medical interventions affects the appropriateness of pursuing anonymous research. The NHANES IRB conducted extensive deliberations of the recommendations of the workshop and the concept of anonymizing NHANES III tissue samples and concluded that it was appropriate to anonymize the samples. Much of the difficulty involved in the general issues of informed consent for genetic testing relate to the basic philosophical issue of whether DNA testing is different from other tests. Annas (61) cited three differences: DNA testing 1) provides a future diary of the patient's health, 2) gives information on other family members, and 3) has a history of misuse. These three differences are usually valid for situations in which highly-penetrant mutations that are associated with serious diseases are identified. However, examples can be given in each case to show that information derived from methods other than DNA-based testing has the same characteristics. For DNA testing that does not identify high-risk, high-penetrance mutations , the relevance of the above differences is less clear. For example, many genes are present in two or more alternative forms in populations. Variation may be benign, such as the variation that produces differences in eye color, or variation may influence risk for disease, such as the differences in carcinogen-metabolizing-enzymes have on cancer susceptibility. Genetic variants (called polymorphisms) often only slightly increase the risk for disease. In these situations, the altered risk for disease is similar to well-characterized risk factors, such as cholesterol. Clearly, generalizations can not be made which accurately characterize all DNA tests, and each situation must be carefully considered. Testing of identifiable specimens for relatively benign polymorphisms that have a small impact on risk for disease (with risk depending on other genes as well as environmental factors) should entail less risk for loss of insurance, psychological distress, or social stigmatization than testing for the genetic mutations that will almost ensure that a person will develop a serious disease. The requirement for pre-test and post-test counseling would be greater for the latter because of these substantial risks. Since the recommendations of the CDC-NHGRI were published, at least two professional groups have written guidelines for genetic testing of stored tissue samples (60, 62), and a first step toward insurance reform was taken (63). Because many issues surrounding informed consent for genetic testing remain unresolved, the NHANES III DNA specimens will only be provided for anonymized research at this time. By using this approach, genetic material from this representative sample of the U.S. population will be made available to the research community to contribute to our knowledge of the role of genes in health and disease.
DNA is now being used in epidemiologic investigations to study genetic risk factors for common disease. We first present a review of methods for collection, processing, and storage of blood specimens for DNA banking in epidemiologic studies. We include methods used by the Centers for Disease Control and Prevention (CDC) for the third National Health and Nutrition Examination Survey (NHANES III) DNA Bank. To illustrate the ethical and social questions that can arise when creating DNA banks, we will discuss the issues that arose as a result of the establishment of the NHANES III DNA Bank and how CDC, with others, addressed these issues.
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