United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
vvEPA
Research and Development
EPA-600/S1 -81 -025 May 1 981
Project Summary
Pilot Program for the
National Environmental
Specimen Bank—Phase I
Sally H. Harrison, Rolf Zeisler, and Stephen A. Wise
This work was performed under a
joint NBS/EPA research program to
develop state-of-the-art protocols for
the sampling, storage, and analysis of
biological and environmental-type
matrices. This report summarizes the
procedures used in the initial phase of
a pilot program for determining the
feasibility of the National Environmental
Specimen Bank. A special clean-labo-
ratory/storage facility has been
designed and completed for use in this
program. Detailed protocols for sam-
pling, storage, and analysis of human
liver samples have been designed for
this study. The implementation of
these protocols for human liver sam-
ples is described in this report.
This Project Summary was developed
by EPA's Health Effects Research Lab-
oratory, Research Triangle Park, NC,
to announce key findings of the re-
search project that is fully documented
in a separate report of the same title
(see Project Report ordering informa-
tion at back).
Introduction
In response to the growing concern
for the potential dangers to human
health and the environment by the
increasing influx of man-made sub-
stances in our ecosystem, the U.S.
Environmental Protection Agency (EPA)
and the National Bureau of Standards
(NBS) are currently studying the feasi-
bility of establishing a National Environ-
mental Specimen Bank (NESB). The
program for such a bank would incorpo-
rate a well-defined system of collection,
analysis, and long-term storage of
selected environmental samples to
provide (1) real-time monitoring data for
pollutant trend analysis and (2) properly
collected and stored samples for retro-
spective analysis.
The National Bureau of Standards is
currently involved in a Pilot Environ-
mental Specimen Bank Program which
will provide actual working experience
in all stages of the banking effort:
specimen collection, processing, storage,
analysis, and data management. The
purposes of this pilot study are: (1) to
develop analytical protocols for sampling
and storage of four types of environ-
mental samples, (2) to improve analytical
methodologies for both trace elements
and trace organics, (3) to evaluate the
feasibility of long-term specimen storage
at various conditions (-25°C, -80°C,
-120°C, and freeze-dried at room tem-
perature), and (4) to provide a "bank" of
samples for retrospective analysis in
future years as analytical methodologies
improve. The experience gained in this
pilot study will be used to evaluate the
feasibility of the NESB Program. The
rationale for this specimen banking
program, a review of the NBS activities
relating to this program, and an outline
of the proposed pilot specimen bank
study at NBS have been described
previously. In these previous reports, the
preliminary plans for sample collection
and storage were reviewed. During this
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Donors 100/Y
Baltimore,
Maryland
Donors
Donors
Donors
1OO/Y
Minnesota
Donors 100/Y
Seattle,
Washington
Teflon Bag
LN2 Dry Shipper
Teflon Bag
LN2 Dry Shipper
Teflon Bag
LN2 Dry Shipper
N B S
Receiving Lab
Section A
Long Term Storage
-120°Cto-196°C
Section B
Storage Evaluation
Figure 1. Sample collection scheme for human livers.
initial year of the proposed pilot study,
the collection, processing, storage
evaluation, and trace element analysis
of the first environmental specimens,
human livers, were implemented. These
activities are described in detail in this
report. In addition, the design of the
clean laboratory/storage facility is
discussed.
Collection of Liver Samples
Contracts for procurement of liver
samples were negotiated and awarded
to three medical schools located in
Baltimore, Maryland; Minneapolis,
Minnesota; and Seattle, Washington.
Each contractor is providing 100 liver
samples this year for a total of 300
samples (see Figure 1).
Due to the extremely low levels of
trace elements and organic pollutants in
human liver tissue, the analytical chemist
must exercise extreme caution to avoid
possible contamination of the sample.
(The lack of contamination control has
made existing banks of samples useless
for general trace constituent analyses.)
A detailed sampling protocol, designed
from the viewpoint of analytical chemists,
is utilized to minimize contamination.
The written sampling protocol outlines
in detail the case selection criteria and
the steps to be followed by the contractor
in obtaining a "valid" sample. The
implementation of this protocol is a
result of extensive interaction of NBS
personnel with the contractors to insure
the quality of the samples.
The individual collecting the liver
samples uses (1) non-talced gloves, (2) a
special titanium-bladed knife for bisect-
ing the liver (to avoid contamination
from other elements, i.e., Cr, Ni, etc.), (3)
Teflon sheets upon which the sample is
placed, and (4) high-purity water to
rinse the sample. The samples are then
sealed in Teflon bags, frozen at liquid
nitrogen temperature, and shipped to
NBS. A data form, sent with each liver
sample, contains information about the
donor and specimen, e.g., date of birth,
sex, residence, ethnic group, height,
weight, smoker, occupation (if known),
diagnosis of autopsy, date and time of
death and autopsy, and weight of sample.
Approximately 100 samples have been
received and stored in the pilot bank
facility at NBS.
The schemes for the collection and
allocation of subsamples for storage
evaluation are shown in Figures 1 and
2. The liver specimens are received as
duplicate sections of the left lobe identi-
fied as sections "A" and "B." All "A"
sections are placed in liquid nitrogen
freezers for long-term storage, and the
"B" sections are used for storage
evaluation, i.e., real-time analysis and
long-term storage evaluation at different
conditions. Approximately 10 percent of
the samples will be homogenized using
a cryogenic brittle fracture technique.
This homogenization provides about 20
aliquots of 5-8 g which are stored in
Teflon jars. To solve the question of
Sample Storage
Evaluation
Cryogenic
Homogenization
Transfer to
20 Teflon PFA jars,
6-8g/iar
Figure 2. Allocation of subsamples for storage evaluation.
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•8
•§
0.2
20
100
*- N
=N umber of Livers Analyzed
Figure 3.
Probability of detecting change in the concentration due to storage
condition.
appropriate temperature for sample
storage, these sample aliquots will be
stored under four different conditions:
freeze-dried at room temperature, frozen
at -25°C, at -80°C, and in liquid nitrogen
vapor at -120°C to -190°C. The concen-
trations of trace constituents found
after storage under those conditions
will be compared to the data found in the
"real-time" analysis of each specimen
(Figure 2). The "A" sections are reference
samples which may be used to re-
evaluate results on a particular "B"
section. At the end of the pilot program,
the "A" sections stored at liquid nitrogen
temperatures will represent a valuable
bank of validated samples available to
the scientific community. In addition, a
large quantity of data, from the analyses
of the "B" sections, will be available on
many of these samples.
The success of the NESB will be
determined in a large part by the ability
to preserve the integrity of the trace
substances (i.e., organic, inorganic, and
organometallic species) in samples
during long-term storage. Changes in
the forms and concentrations of the
numerous environmentally important
substances in specimens stored for
extended periods may occur in several
ways. Processes such as surface ad-
sorption and sample degradation may
reduce the concentrations of various
components. In addition, continued bio-
chemical and enzymatic activity may
produce species which may not have
been present in the original sample. On
the other hand, contamination of the
specimen fluids could lead to apparent
increases in trace substances' concen-
trations. Superimposed on these
processes are the factors which will
affect the process rates, such as con-
tainer material, contact time and area,
storage temperature, pH, and initial
species concentration. All of these
factors are important considerations in
evaluating the suitability of long-term
storage.
The probability for the discovery of
changes in the concentrations of trace
constituents during storage has been
assessed by statistical treatment of the
expected experimental parameters,
e.g., number of specimens analyzed,
homogeneity of subsamples, and ana-
lytical error (K. R. Eberhardt and L.
DeRobertis, Center for Applied Mathe-
matics, Statistical Engineering Division,
NBS). Figure 3 consists of a series of
curves generated from the experimental
parameters of 5 percent inhomogeneity
and 5 percent analytical error. These
curves determine the probability of
detecting a particular percent change in
concentration (e.g., 6 and 4 percent for
curves 1.0 and 0.7, respectively) for a
given number of liver samples analyzed.
For example, we would have a 98 per-
cent probability of detecting a 6 percent
change (Point A) and 78 percent proba-
bility of detecting a 4 percent change in
concentration (Point B) if 30 livers are
analyzed. If the sample inhomogeneity
or analytical error increased to 10
instead of 5 percent, the percent change
detectable at Points A and B would be
12 and 8 percent, respectively. Based on
these curves, the number of samples to
be analyzed was set at 30. Analyses of a
smaller number of samples would sig-
nificantly reduce the probabilities of
detecting small changes, whereas in-
creasing the number of samples ana-
Table 1. Number of Livers to be Analyzed for Storage Evaluation Study
Year 80 81 82 83 84
Real Time Analysis 36
1 Year Storage LNZ
-85°C Samples
-25°C from '80
RT
2 Years Storage LNz
-85°C
-25°C
RT
3 Years Storage LN'.
-85°C
-25°C
RT
4 Years Storage LNZ
-85°C
-25°C
RT
30 30 30
12
12 Samples
12 from '83
12
Samples
from '82
Samples
from '81
Samples
from '80
30
30
30
30
30
30
30
30
30
30
30
30
24
24
24
24
Livers Analyzed/Year
36
78
30
30
456
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2 Staff Weeks
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( HG and BE J
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Figure 4. Analytical scheme for the determination of trace elements in human livers.
lyzed would not increase greatly the
probability of detecting changes.
The 30 specimens used for the storage
evaluation represent approximately 10
percent of the specimens collected per
year. A major limitation to this number
is the present analytical capacity. In
order not to exceed the currently avail-
able analytical capability, most of the
analytical work is deferred to the year
1984, when the program should have a
larger analytical capacity. For the evalu-
ation of the storage of human livers,
Table 1 gives the analytical needs in
terms of numbers of livers to be analyzed
each year.
Trace Element and Trace
Organic Analysis
The trace element analysis activities
of the specimen bank project have
concentrated on three main tasks this
year: (1) development of a comprehen-
sive analytical scheme, (2) evaluation of
the sample collection protocols, and (3)
initiation of the analysis of the first
year's collected samples.
The current analytical protocol is
directed toward the analysis of most of
the first-priority elements proposed by
the International Workshop on Monitor-
ing Environmental Materials and Speci-
men Banking. Additional elements
occurring as biological major elements
and trace elements in the samples are
included in the protocol because their
levels might supply information about
the "normal" state of the specimen. The
elements under consideration, their
priority, and the analytical techniques to
be used appear in Table 2. The analytical
scheme (Figure 4) incorporates the NBS
Center for Analytical Chemistry approach
of multi-technique analysis for quality
control. The first three techniques
listed, i.e., atomic absorption spectros-
copy (AAS), anodic stripping voltammetry
(ASV) or linear sweep voltammetry
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(LSV), and instrumental neutron activa-
tion analysis (INAA), comprise the
current routine analytical scheme.
Radiochemical neutron activation anal-
ysis (RNAA) will be performed on a
limited number of samples to provide
multi-technique quality control. Accord-
ing to the proposed storage evaluation
scheme, 36 livers are being analyzed
during the first year of sample collection.
Analytical methods for the analysis of
two classes of organic compounds, i.e.,
organochlorine pesticides and polycyclic
aromatic hydrocarbons (PAH) are cur-
rently being developed. Preliminary
results have been obtained using the
following analytical procedures: (1) ex-
traction with acetonitrile, (2) liquid-
liquid partition into hexane, (3) concen-
tration by evaporation, (4) high-perform-
ance liquid chromatography (HPLC) to
isolate the compounds of interest, and
(5) gas chromatography (GC) analysis on
capillary columns with selective electron
capture detection. An HPLC method,
using a chemically bonded amine column
has been developed to isolate the
organochlorine pesticides and PAH
from interfering organic constituents.
GC methods have been developed for
the determination of the 16 pesticides
on the priority pollutant list. Research is
continuing on the methodology for
quantitation of these compounds in liver
samples in order to initiate real-time
analyses for organics in 1981. Future
plans include (1) the evaluation of
homogeneity for organics, (2) the devel-
opment of procedures for comparison of
organochlorine content determined by
GC and determined by neutron activa-
tion analysis, and (3) expansion of the
pilot program to include development of
analytical protocols for the mussel/oyster
samples.
Safety Requirements
The handling of human liver samples
carries the potential risk of exposure to
infectious diseases, particularly hepatitis
B virus or "serum hepatitis." Asa result,
certain precautions are necessary to
insure protection of the analysts involved
in sample processing and analysis.
To eliminate potentially infectious
liver specimens from the specimen bank
samples, a blood sample is removed
from the donor at the time of autopsy to
be used for hepatitis B screening. In
addition, liver specimens from the right
lobe are removed for preparation of
listological slides. These slides are
examined by the pathologists for evi-
Table 2. Analysis of Trace Elements in Human Liver
Element Concentration (vg/gj* Analytical Methodb
Category^
Be
F
Al
V
Cr
Mn
Fe
Co
Ni
Cu
In
As
Se
Rb
Sr
Mo
Pd
Ag
Cd
Sn
Sb
Ba
Pt
Hg
71
Pb
ND
0.06 - 1.4
1.6 - 2.6
< 0.007- 0.09
0.005- 0.27
0.5 - 1.9
70 -210
0.017- 0.16
0.009- 0.32
3.2 - 14.7
31 - 80
0.006- 0.46
0.097- 0.68
7 - 12
0.01
0.4 - 1.6
ND
0.006- 0.07
0.5 - 4.9
0.08 - 0.65
0.01
0.01
ND
0.005- 0.25
0.001- 0.009
0.8 - 2.3
AAS
ASV/LSV
R
INAA
INAA
INAA
INAA
ASV/LSV
ASV/LSV, RNAA
ASV/LSV, INAA
RNAA
INAA, RNAA
INAA
RNAA, INAA
INAA
ASV/LSV. RNAA
R
INAA. RNAA
R
AAS
R
ASV/LSV
1
1
2
1
1
1
2
1
1
3
1
1
1
2
3
1
1
2
1
1
2
3
3
1
1
1
"Fresh weight, range of concentrations from reference 4.
''Techniques used at NBS for Pilot Specimen Bank Program:
AAS - atomic absorption spectroscopy,
ASV/LSV - anodic skipping voltammetry/linear sweep voltammetry,
INAA = instrumental neutron activation analysis,
RNAA = radiochemical neutron activation analysis.
C1) First priority element (3);
2) Trace elements available with applied methods to monitor "normal" body
concentrations for non-pollutants.
3) Trace elements of environmental importance not included in first priority list.
R - Research initiated ND = No data available
dence of infectious diseases. Liver
samples are placed in temporary storage
at liquid nitrogen temperatures at NBS
until the results of the screening are
received from the hospitals supplying
the livers.
A Safety Advisory Committee has
been established to advise the Center
for Analytical Chemistry concerning
suitable safety precautions for the
handling and processing of these liver
samples at NBS. Recommendations
were made by the committee regarding
procedures to safeguard personnel. The
Safety Advisory Committee will be
requested to review annually the pro-
cedures of the pilot specimen bank
program.
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Sally H. Harrison, Rolf Zeisler, and Stephen A. Wise are editors with the Center
for Analytical Chemistry, National Bureau of Standards, Washington, DC
20234.
George M. Goldstein is the EPA Project Officer (see below).
The complete report, entitled "Pilot Program for the National Environmental
Specimen Bank—Phase I." (Order No. PB81 -173 320; Cost: $8.00. subject to
change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, V'A 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
t, US GOVERNMENT PfllNTINO OFFICE-1»1 -757-012/7088
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