American Journal of
EPIDEMIOLOGY
Volume 144
Copyright © 1996 by The Johns Hopkins University
Number 5
School of Hygiene and Public Hearth
September 1, 1996
Sponsored by the Society for Epldemlologlc Research
REVIEWS AND COMMENTARY
Guidelines of the National Heart, Lung, and Blood Institute Working Group
on Blood Drawing, Processing, and Storage for Genetic Studies
Melissa A. Austin,1 Jose M. Ordovas,2 John H. Eckfeldt,3 Russell Tracy,4 Eric Boerwinkle,5
Jean-Marc Lalouel,6 and Morton Printz7
Many large-scale epidemiologic studies are currently
obtaining blood samples for characterization of a variety
of coronary heart disease risk factors. With the rapid
advancements in molecular biology and the increasing
interest in genetic epidemiology, many investigators
would like to use these blood samples to obtain DNA for
genetic studies, including linkage analysis. However,
most epidemiologists are inexperienced in the laboratorybased procedures needed to obtain DNA for genetic
studies. The purpose of this paper is to provide guidelines
for epidemiologic investigators who wish to store blood
samples for genetic studies. These guidelines were first
developed in draft form at the meeting of the National
Heart, Lung, and Blood Institute (NHLBI) Working
Group on Blood Drawing, Processing, and Storage in
Bethesda, Maryland, in October 1994. Each of the authors participated in that meeting and in the completion
of this document
Epidemiologists should be aware that there are numerous ethical, social, and legal concerns involved in
many genetic studies, especially those that use stored
specimens. However, discussion of these issues is not
the purpose of this article. Investigators are referred to
the recent recommendations of the Joint Working
Group on Ethical, Legal, and Social Implications of
Human Genome Research (1) and the "ACMG Statement on Storage and Use of Genetic Materials" from
the American College of Medical Genetics (2). In
particular, the procedures described here assume that
proper informed consent has been obtained from all
study subjects providing specimens.
These guidelines focus on the white cells (buffy
coats) from blood samples that contain DNA. White
blood cells are generally obtained with one of two
goals in mind: to isolate DNA from the cells or to
develop immortalized cell lines, as discussed in the
first two sections of this paper. The final section
briefly describes alternative sources of genetic material. It is important to note that these guidelines are
limited to the procedures for obtaining, processing,
and storing blood samples. The actual isolation of
DNA and the establishment of cells lines should always be carried out in collaboration with an experienced molecular biologist. Investigators are referred to
Yates et al. (3) and Madisen et al. (4) for overviews of
DNA banking. Additional references are also provided
for investigators who may require more detail than is
presented here.
Received for publication May 30, 1995, and in final form May 20,
1996.
Abbreviations: DNA, deoxyribonucleic acid; EDTA ethylenediaminetetraacetjc acid; NHLBI, National Heart, Lung, and Blood Institute; PCR, polymerase chain reaction.
1
Department of Epidemiology, School of Public Health and
Community Medicine, University of Washington, Seattle, WA
2
LJpid Metabolism Lab, United States Department of Agriculture,
Human Nutrition Research Center on Aging at Tufts University,
Boston, MA
3
Department of Laboratory Medicine and Pathology, University
of Minnesota Hospital and Clinic, Minneapolis, MN.
4
Departments of Pathology and Biochemistry, University of Vermont, Colchester, VT.
5
Human Genetics Center, University of Texas, Houston, TX.
6
Howard Hughes Medical Institute, Research Laboratories, University of Utah, Salt Lake City, UT.
7
Department of Pharmacology, School of Medicine, University of
California, La Jolla, CA
Reprint requests to Dr. Melissa A Austin, Department of Epidemiology, Box 357236, School of Public Health and Community
Medicine, University of Washington, 1959 NE Pacific Avenue, Seattle, WA 98195.
437
438
Austin et al.
EXTRACTION OF DNA FROM BLOOD SAMPLES
Blood drawing
Blood should be drawn using a needle that avoids
hemolysis with atraumatic venipuncture whenever
possible and with limited use of the tourniquet.
The Working Group recommends using sterile vacutainer tubes with ethylenediaminetetraacetic acid
(EDTA, purple top tubes). If other types of tubes are
needed for specific measurements, such as citrate
(blue top) or heparin (green top), these are probably
acceptable. At least one study has reported the isolation of high-quality DNA from frozen citrated blood
(5). However, DNA extraction from cells collected in
these other anticoagulants should be pilot tested. Finally, it has been reported that DNA recovery from
clotted human blood (i.e., blood drawn without anticoagulants) is similar to yield obtained from citrated
blood (6).
Processing blood samples
Although it is ideal to extract DNA from fresh
samples, storage for up to 3 days at room temperature
does not seem to affect DNA recovery adversely.
Thus, shipment of blood to a central laboratory with
capacity to extract DNA within the same time frame is
perfectly acceptable. If samples are being shipped,
every attempt should be made to keep whole blood or
packed cells as close to normal room temperature as
possible, preferably under sterile conditions. Sterile
blood collection tubes should be used and, if possible,
should not be opened prior to shipment. Mailer kits
should include an insulated box with thermal buffering
material to maintain an even temperature, even when
the shipping package is in very warm or cold environments.
If DNA cannot be extracted within a few days of
collection, the buffy coat (or packed cells) should be
stored at — 80°C. Some investigators recommend adding
sterile glycerol freezing solution (see Appendix table 1),
if possible, to prevent the rupture of the cell membranes.
The following procedure is recommended:
1) Whole blood is centrifuged at approximately
1,500 X g (typically 2,000-3,000 rpm in most
bench-top centrifuges) for 30 minutes.
2) The plasma is removed, and the white cell layer
is transferred with a clean plastic transfer pipette.
It is acceptable to include some red blood cells in
the sample; efforts to avoid red cells can lead to
considerable loss of white cells and diminished
DNA yield.
3) If the glycerol freezing solution is used (Appendix table 1), the white cell layer should be added
to an aliquot of the solution. Mix the cells and
the solution gently by rocking the tube back and
forth. Combining the buffy coat with an equal
volume of the glycerol freezing solution is optimal (e.g., 1.5 ml of glycerol freezing solution to
1.5 ml of buffy coat from 20 ml of whole blood).
4) The samples should then be frozen at — 80°C in
cryovials (e.g., high-density polypropylene tube,
such as 3.6 ml Nunc no. 366524 (NUNC, Naperville, Illinois)) with screw tops and an O-ring.
Glass tubes are not suitable for mis purpose since
they will shatter when thawed. If short-term storage is anticipated, one aliquot is sufficient. However, for long-term storage, several aliquots
should be stored, preferably in two or more locations in case of a freezer or power failure. For
sample identification, cryogenically approved labels should be used with ink that is stable under
freezer conditions. Note that many laser-printed
labels fall off at — 80°C. Some laboratories use
thermal transfer printer labels and silk labels for
this purpose. The use of computer-generated labels should be considered to avoid possible transcription errors. If bar codes are used, the identification number should also be written directly
on the label.
Finally, if the procedures described above are not
feasible, anticoagulated whole blood, the entire packed
cell portion or buffy coats of anticoagulated blood, or
even blood clots can be stored at — 80°C, again preferably in several aliquots in different freezers. Although there is little documentation of expected DNA
yield under these circumstances, Ross et al. (7) note
that repeated freezing and thawing of blood collected
in EDTA had no deleterious effects on the integrity of
the DNA as assessed by DNA fingerprinting.
Extraction of DNA
As noted in the introduction, these procedures
should be performed in an experienced molecular biology laboratory. Three major methods for purification of large amounts of high-molecular-weight DNA
from white cells are currently available: 1) phenol/
chloroform method (8); 2) salt precipitation method
(9, 10) and recent "improvements" (11-15); and 3)
solid-phase extraction, used in some commercial kits
based on minicolumns (16-18). These methods are
fast and yield DNA of very good quality.
Some of these methods can be automated by using
DNA extractors, but this equipment can be very expensive to maintain. Several other methods of DNA
extraction have been used with other types of samples,
such as dried blood spots, blood clots, buccal smears,
semen, hair follicles, etc. Most of these methods use
Am J Epidemiol
Vol. 144, No. 5, 1996
NHLBI Guidelines on Blood Samples for Genetic Studies
some sort of DNA extraction with boiling water or
buffer. One of the problems has been breakdown of
DNA by endogenous metal ion catalysis at boiling
temperatures. This breakdown has been minimized by
use of EDTA in the boiling mixture or addition of
Chelex 100 (Sigma Chemical Co., St. Louis, Missouri), a cation exchange resin that binds tightly to divalent metal ions (19).
In particular, several large NHLBI projects are currently collecting and storing only clotted blood. Although there is DNA in the clot, specialized methods
are needed to extract it. A protocol that provides good
DNA yield and quality can be found in the paper by
Zeillinger et al. (20).
The Working Group recommends that investigators
validate that DNA yield and quality is acceptable with
the selected method before large-scale processing of
samples begins. Unfortunately, little data are currently
available on the effect of long-term storage conditions
on the quality and stability of DNA after extraction by
different isolation methods. Note that because the
methods referenced above are for nuclear DNA, they
cannot be used for mitochondrial DNA.
For all procedures, a dedicated pipette with an antiaerosol tip should be used. The same pipette should
never be used for pipetting polymerase chain reaction
(PCR) samples, reagents, or products. Wide-bored tips
are recommended to prevent breakage of the DNA and
to facilitate accurate dispensing of the viscous DNA
during aliquoting. Furthermore, every effort should be
made to minimize the possibility of specimen contamination, including limiting the number of times the
primary stock of DNA from a participant is entered.
Therefore, the Working Group recommends creating
multiple aliquots of the stock DNA at a working
dilution convenient for PCR reactions, as follows:
1) The primary stock DNA solutions should have a
concentration of 100-200 ng/ml of DNA and
should be made in Tris/EDTA buffer (see Appendix table 1).
2) The working DNA solutions should have a DNA
concentration of about 10-40 ng/ml and should
also be made in Tris/EDTA buffer. They should
be stored at 4°C. These working DNA solutions
can be used to provide samples to collaborators
for PCR amplification; usually only 1-2 ml
(20-40 ng of DNA per marker) of this working
DNA is needed for a typical PCR amplification.
Some investigators find it convenient to deliver
to collaborators DNA frozen in small, conical,
polypropylene tubes or 96-well microtiter plates
that fit directly into PCR thermocyclers, reducing DNA waste and the possibility of contamination.
Am J Epidemiol
Vol. 144, No. 5, 1996
439
3) Freeze primary stock and working DNA solutions at — 80°C or lower. Use screw-top vials
with O-ring seals that are known to be stable at
—80°C and be certain that labels are also stable
at — 80°C (see Processing blood samples,
above).
IMMORTALIZING CELL LINES
Immortalizing cell lines is the alternative to storing
DNA and again should be carried out in an experienced molecular biology laboratory or a specialized
commercial service laboratory. Although this procedure is more expensive and labor intensive, the Working Group noted three circumstances when this would
be the method of choice:
1) For detailed biochemical and molecular studies
in living cells. Although there are limited applications at this time, these types of studies are
likely to become important in the future. However, investigators should be cautioned that some
enzymes and receptors are poorly expressed or
are not expressed at all in lymphoblastoid cell
lines;
2) To enhance the available supply of DNA; cultured lymphoblastoid cell lines make it possible
to harvest virtually unlimited amounts of DNA
from a study subject;
3) For high-risk groups of patients from whom additional blood samples would be difficult or impossible to obtain, including those at high risk of
dying. In fact, obtaining two separate samples
from such key individuals has been recommended (21).
Blood drawing
The blood-drawing procedures described above
should be used, except that sterile vacutainer tubes
with acid citrate dextrose (yellow top tubes) are recommended. Lymphocytes in acid citrate dextrose anticoagulated whole blood are thought to retain viability
for as long as 4-5 days, significantly longer than
blood anticoagulated with other substances. Vacutainer tubes with heparin (green-top tubes) are an
alternative, although at least one report has indicated
that heparin may prevent reliable amplification of
DNA by PCR (22).
Processing blood samples
Since the success rate for immortalizing cell lines
declines with time, blood must be shipped as soon as
possible to a molecular biology laboratory with experience and facilities for tissue culture. Ideally, the
440
Austin et al.
sample should arrive within 48 hours, with a maximum of 72 hours. '
Tube(s) should be maintained at 25 °C until the
samples are delivered to the central laboratory. Mailer
kits should include insulation and thermal buffering to
maintain this temperature even in hot or cold environments, as described in detail above.
It is ideal to purify lymphocytes immediately. However, the Ficoll-Hypaque isolated lymphocytes can be
slowly frozen in a rate freezer with the addition of cell
culture medium (RPMI-1640, GibcoBRL, Life Technologies, Grand Island, New York), dimethyl sulfoxide, and heat-activated fetal calf serum (23). Under
these circumstances, a good yield can be obtained
when the lymphocytes are later thawed and immortalized.
be conducted in collaboration with an experienced
laboratory, a summary of the available methods with
appropriate references is given, and a method for the
extraction of DNA from clotted blood is referenced. In
addition, criteria for selecting study subjects for whom
immortalized cell lines are preferable to merely extracting DNA are presented. Finally, the use of alternative sources of genetic material, including cheek
swabs and dried blood spots, is described briefly.
ACKNOWLEDGMENTS
The authors thank Dr. Millicent Higgins for her guidance
and encouragement in developing these guidelines.
This work was performed during the tenures of Drs.
Austin and Boerwinkle as Established Investigators of the
American Heart Association.
ALTERNATIVE SOURCES OF GENETIC
MATERIAL
Although most NHLBI-sponsored studies involve
the collection of anticoagulated blood samples, alternative sources of genetic material are available, including cheek swabs and dried blood spots. While
cheek swabs do not provide large amounts of DNA,
they can be suitable for forensic purposes and for
candidate gene analyses using PCR methods.
The most common use of dried blood spots is for the
detection of metabolic disorders at birth. These samples are referred to as Guthrie cards and have been
used for many years by newborn screening laboratories. Recently, it has been demonstrated that Guthrie
cards collected as early as 1986 can be successfully
used for PCR (24, 25). Thus, these samples can be
used to detect specific mutations related to inborn
metabolic disorders and infectious diseases using a
few genetic markers. However, they are not suitable
for large-scale DNA extraction. Special care should be
taken to avoid contamination, including always handling the cards with gloves. It is also important to note
that the use of Guthrie cards for these purposes raises
specific ethical concerns, as described in the paper by
McEwen and Reilly (26).
SUMMARY
The guidelines presented here are intended for epidemiologic investigators who wish to store blood samples for genetic studies, either by extracting DNA
directly from white blood cells or from immortalized
cell lines. Recommended procedures for blood drawing and for processing samples are described. Protocols for freezing and storage of both white blood cells
and extracted DNA are provided. Although the extraction of DNA and immortalization of cell lines should
REFERENCES
1. Clayton EW, Steinberg KK, Khoury MJ, et al. Informed
consent for genetic research on stored tissue samples. JAMA
1995;274:1786-92.
2. American College of Medical Genetics Storage of Genetics
Materials Committee. ACMG statement on storage and use of
genetic materials. Am J Hum Genet 1995;57:1499-1500.
3. Yates JR, Malcolm S, Read AP. Guidelines for DNA banking.
Report of the Clinical Genetics Society working party on
DNA banking. J Med Genet 1989;26:245-50.
4. Madisen L, Hoar DI, Holroyd CD, et al. DNA banking: the
effects of storage of blood and isolated DNA on the integrity
of DNA. Am J Med Genet 1987;27:379-90.
5. Parzer S, Mannhalter C. A rapid method for the isolation of
genomic DNA from citrated whole blood. Biochem J 1991;
273 (Part 1):229-31.
6. Everson RB, Mass MJ, Gallagher JE. Extraction of DNA from
cryopreserved clotted human blood. Biotechniques 1993; 15:
18-20.
7. Ross RS, Haites NE, Kelly KF. Repeated freezing and thawing of peripheral blood and DNA in suspension: effects on
DNA yield and integrity. J Med Genet 1990;27:569-70.
8. Gross-Bellard M, Oudet P, Chambon P. Isolation of high
molecular-weight DNA from mammalian cells. Eur J Biochem 1973;36:32-8.
9. Dykes DD. The use of biotinylated DNA probes in parentage
testing: non-isotopic labeling and non-toxic extraction. Electrophoresis 1988;9:359-68.
10. Miller SA, Dykes DD, Polesky HF. A simple salting out
procedure for extracting DNA from human nucleated cells.
Nucleic Acids Res 1988;16:1215.
11. Lahiri DK, Numberger JI Jr. A rapid non-enzymatic method
for the preparation of HMW DNA from blood for RFLP
studies. Nucleic Acids Res 1991; 19:5444.
12. Nelson JE, Krawetz SA. Purification of cloned and genomic
DNA by guanidine thiocyanate/isobutyl alcohol fractionation.
Anal Biochem 1992 ;207:197-201.
13. Douglas AM, Georgalis AM, Benton LR, et al. Purification of
human leucocyte DNA: proteinase K is not necessary. Anal
Biochem 1992;201:362-5.
14. Lahiri DK, Schnabel B. DNA isolation by a rapid method
from human blood samples: effects of MgCl2, storage time,
and temperature on DNA yield and quality. Biochem Genet
1993;31:321-8.
15. Wang L, Hirayasu K, Ishizawa M, et al. Purification of
Am J Epidemiol
Vol. 144, No. 5, 1996
NHLBI Guidelines on Blood Samples for Genetic Studies
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
genomic DNA from human whole blood by isopropanolfractionation with concentrated Nal and SDS. Nucleic Acids
Res 1994;22:1774-5.
Boom R, Sol CJA, Salimans MMM, et al. Rapid and simple
method for purification of nucleic acids. J Clin Microbiol
199O;28:495-503.
McCormick RM. A solid-phase extraction procedure for DNA
purification. Anal Biochem 1989;181:66-74.
Hawkins TL, O'Connor-Morin T, Roy A, et al. DNA purification and isolation using a solid-phase. Nucleic Acids Res
1994;22:4543-4.
Walsh PS, Metzger DA, Higuchi R. Chelex 100 as a medium
for simple extraction of DNA for PCR-based typing from
forensic material. Biotechniques 1991;10:506-13.
Zeillinger R, Schneeberger C, Speiser P, et al. A simple
method for isolation of DNA from blood clots suited for use
in PCR. Biotechniques 1993;14:202-3.
Morrison PJ, Graham CA, Nevin NC. DNA storage and duplicate sampling: lessons leamt from testing for Huntington's
disease. (Letter). J Med Genet 1993;30:1042.
Satsangi J, Jewell DP, Welsh K, et al. Effect of heparin on
polymerase chain reaction. (Letter). Lancet 1994;343:
1509-10.
Louie LG, King M-C. A novel approach to establishing permanent lymphoblastoid cell lines: Epstein-Barr virus transformation of cryopreserved lymphocytes. (Letter). Am J Hum
Genet 1991;48:637-8.
Comeau AM, Hsu HW, Schwerzler M, et al. Identifying
human immunodeficiency virus infection at birth: application
of polymerase chain reaction to Guthrie cards. J Pediatr 1993;
123:252-8.
Raskin S, Phillips JA 3rd, Kaplan G, et al. Cystic fibrosis
genotyping by direct PCR analysis of Guthrie blood spots.
PCR Methods Appl 1992;2:154-6.
McEwen JE, Reilly PR. Stored Guthrie cards as DNA
"banks." Am J Hum Genet 1994;55:196-200.
Am J Epidemiol
Vol. 144, No. 5, 1996
441
APPENDIX TABLE 1
Gtycerol freezing solution for white blood cells
Molecular
weight
Reagent
Citric acid, sodium salt
Sodium phosphate,
monobasic, monohydiate
NaH,PO 4 -H,0
Sodium phosphate, dibasic,
anhydrous NaH,PO 4
99%gtycsrol
(9)
294
Amount
per
Her
Final
concentration
(mmoHSar)
14.7 g
50
138
2.76 g
20
142
2.84 g
400 ml
20
Note that all solutions should be prepared and stored under
sterile conditions.
• Adjust the volume of the solution to slightly less than 1 liter
using sterile distilled water. Add concentrate aocfium hydroxide to
bring pH to 7.4. Add sterile distilled water to bring volume to 1.00
fitor.
Trte-EDTA buffer
Reagent
Tris
Disodum ethylene diamine
tetraacetate 2 H 2 O
Molecular
weight
(g)
Amount
per
•er(g)
HnaJ
concentration
(mmoMter)
121.1
1.211
10
372.2
0.372
1
• Note that all solutions should be prepared and stored under
sterile conditions.
• Adjust pH to 8.0.
• Adjust the volume of the solution to 1.00 liter using sterile
dstBled water.