Transcript Sample Collection, Processing and Storage

Sample Collection, Processing
and Storage
EPI243
Zuo-Feng Zhang, MD, PhD
Introduction
• Sample Collection, such as handling,
labeling, processing, aliquoting, storage,
and transportation, may affect the results of
the study
• If case sample are handled differently from
controls samples, differential
misclassification may occur
Information linked to Sample
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Time and date of collection
Recent diet and supplement use,
Reproductive information (menstrual cycle)
Recent smoking
current medication use
Recent medical illness
Storage conditions
Quality Assurance
Systematic Application of optimum
procedures to ensure valid, reproducible,
and accurate results
Quality Assurance
• Adoption of standardized operation
procedures for each aspect of biospecimen
handling
• Stored specimens should be tested on a
regular basis to detect sample deterioration
• Aliquoting material into multiple small vials
Ultima freezers: -140 degree C to –220 degree C
-70 freezers
Quality Assurance
• Storing each person’s specimen in at least
two different physical locations to avoid the
likelihood of loss of a large volume of
specimen as a result of accidental thawing
due to freezer failure or electronic blackout.
Quality Assurance
Sample should be selected from specimens
that received the same treatment throughout
the storage process or the same variations in
handling.
Quality Assurance: Careful
Record of disbursement
• Barcode system to check in and out the biospecimens
• which specimen, how much material
remains, documentation on factors such as
thawing which could influence future use of
the material.
bar code scanner
bar code scanner
Bar Code Printer
Computer System
Bar Code System
Types of Biospecimens
It is critical to collect samples not only for
main biomarkers of interest in your study,
but also to process and store material in a
way that allows the new biomarkers to be
tested in future.
Types of Biospecimens
Prospective collection and storage of
biospecimens at a low temperature before
the onset of the disease, which may provide
essential information on exposure to factors
not biased by the metabolic effects of the
illness
Types of Biospecimens
Collection of biospecimens from
individuals who already diagnosed as
having illness to characterize the history of
the disease. Many collections of tissues
including tumor and normal tissues. It will
be much better if the related
epidemiological data are also collected.
Types of Biospecimens: Blood
The use of skilled technicians and precise
procedures when perform phlebotomy are
important because painful, prolonged or
repeated attempts at venepuncture can cause
patient discomfort or injury and result in
less than optimum quality or quantity of
sample.
Types of Biospecimens: Blood
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Plasma
Serum
Lymphocytes
Erythrocytes
Platelets
Blood Sample Collection
• When a large amount of blood sample
needed, an evacuated tube system with
interchangeable glass tubes can be used to
avoid multiple venepunctures.
• Evacuated tubes are commercially prepared
with or without additives and with sufficient
vacuum to draw a predetermined blood
volume per tube.
Sterile Blood needles; Sterile Syringes; Plain
Vacutainer; Blood Tubes; Alcohol Prep Pads;
Tourniquet
Blood Collection: Color-code
Tubes
• Red-top tubes contain no additives. These
tubes are used for tests performed on serum
samples and DNA.
• When you use the red-top tubes, the sample
an be placed for 1-2 hours so that the serum
and blood clots will be separated. Blood
clots can be used for DNA analysis.
Blood Collection: Color-code
Tubes
• Lavender-top tubes contain EDTA,
commonly used clinically for complete
blood cell counts.
• This is the way to obtain lymphocytes for
DNA extraction, plasma for nutritional
analysis, and red blood cells for other
assays.
EDTA
EDTA is a anticoagulant. It works by
calcium chelation and is used clinically in
heamatology studies. It is well suited to
DNA-based assays, but has problems for
cytogenetic analysis.
Whole blood in
the collection
tube
Blood after
centrifugation
WBCs and
RBCs
fter plasma
removal
Top view of the
WBCs (buffy coat)
Top view of sample
after WBC removal
Blood Collection: Color-code
Tubes
• Green-top tubes contain heparin
• Blue-top tubes contain sodium citrate and
citric acid
• Black-top tubes contain sodium oxalate
• Yellow-top tubes contain acid-citratedextrose (ACD) solution.
• Grey-top tubes contain a glycolytic
inhibitor.
Heparin
• Heparin is an anticoagulant. There are some
reports of occasional problems with heparin
in PCR assays, studies generally find that
there are no major difference in the use of
EDTA or heparin
Citrate
• Citrate also works by calcium chelation and
is used in coagulation studies and blood
banking. It is optimal for assays conducted
on lymphocytes and DNA.
Dried Blood Spot
Dried blood spot specimens:
• Small quantities of blood adequate for the
characterization of DNA.
• Not require venepuncture or low
temperature condition during collection,
processing and storage
• Can be from whole blood or antocoagulated
with EDTA
Dried Blood Spot
• Blood specimen is spotted onto clean slides
or paper or cotton cloth.
• Transported and stored at room temperature
• Serves as a good source of high-molecularweight DNA
• A quantity of 50 ul of dried blood can
provide 0.5 ug DNA, sufficient for multiple
PCR-based assays
Blood Components
From 10 ml of blood:
• Plasma or serum 6-7 ml
• Lymphocytes and mononuclear cells 10-20
x 106 Cells/ml
• Erythrocyte (red blood cells) and other cells
– 5 x 106 cells/ul; 10-15 mg HB
• Blood is a liquid tissue. Suspended in the watery plasma
are seven types of cells and cell fragments.
• Red blood cells (RBCs) or erythrocytes
• plateletsor thrombocytes
• five kinds of white blood cells (WBCs) or leukocytes
• Three kinds of granulocytes: Neutrophils; Eosinophils;
Basophils
• The number
• Two kinds of leukocytes without granules in their
cytoplasm: lymphocytes and monocytes
• White blood cells
• are much less numerous than red (the ratio
between the two is around 1:700),
• have nuclei,
• participate in protecting the body from infection,
• consist of lymphocytes and monocytes with
relatively clear cytoplasm, and three types of
granulocytes, whose cytoplasm is filled with
granules.
Lymphocytes
Mononuclear leukocytes
• Mononuclear leukocytes are the only cell
type in blood capable of growth
• They can be cryopreserved for the
establishment of cell lines.
• Cryopreservation permits cell viability and
can be the only source to measure RNA
single
macrophage
(monocyte)
surrounded by
several
lymphocytes
Granulocytes
• Granulocytes can serve as a source of DNA
without sacrificing the lymphocytes
Erythrocytes (RBC)
• Stored after washing with physical saline
• Can be useful to study adducts of
haemoglobin
Plasma and Serum
• Can be used to measure microanalytes, diet
components, vitamins, xenobiotic exposures
and so on.
• Plasma can be obtained from an
anticoagulated blood sample through
separation from cell components.
• Serum is better for antibody measurements,
nutrients, etc.
Polled aliquots
• Polled aliquots of serum specimens have
been used for nutritional or other
biochemical studies, e.g., HIV antibody
testing.
• This requires merging aliquots of specimens
from each individual within a subgroup and
testing combined sample to obtain groupspecific average value.
Polled aliquots
• The approach requires only a small number
of laboratory tests to be performed, but
yields only a mean value without a variance
or information about the distribution of
results.
DNA Extraction
DNA can be extracted from
• Whole blood
• Leukocytes
• Serum
• Plasma
• Blood clot
Processing
• The sample processing depends on the
marker needed. Investigator must design
studies to fit the requirement of the critical
biomarkers.
• The stability of assay in relation to time and
temperature of storage has not been well
documented, but should be considered in
the context of specific studies
Processing
• Serum fatty acids should be measured
within 2 weeks at 4 degree C, within a few
months at –20 degree C, and within a year
at –80 degree C
Storage
It is critical to maintain careful records of
the identity and location of all materials,
with particular attention to storage history,
occurrence of temperature fluctuation and
monitoring of stored control specimen in
order to check the effects of storage
duration.
Storage
Samples stored on the top of the freezer
may be exposed to more extreme
temperature fluctuation then those stored at
the bottom.
Timing
• For studies of hormones, which have hourly,
daily and monthly cycles, timing of sample
collection is critical.
• It is critical to obtain information at the time
of specimen collection, e.g., time and date
of draw, volumes and type of specimen,
medical illness, medication use, menstrual
period, cigarette and alcohol consumption
Urine Collection
Urine is an ultrafiltrate of the plasma. It can
be used to evaluate and monitor body
metabolic disease process, exposure to
xenobiotic agents, mutagenicity, exfoliated
cells, DNA adducts, etc.
Urine Collection
Urine collection is not invasive and readily
obtainable. However, it is more
inconvenient than blood collection.
The type of urine selected and the collection
procedure used to depend on the tests to be
performed.
Urine Collection
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First morning
Random
Fractional
timed
Urine Collection
• Morning Urine. To collect a first morning
specimen, the subject voids before going to
sleep and immediately upon rising, collects
a urine specimen.
• The specimen must be preserved if not
delivered within 2 hours of collection
Urine Collection
• Random Urine can be collected at any time.
These specimens are usually satisfactory for
routine screening and for cytology studies.
• If a large amount of urine is needed, subject
will be asked to drink a lot of water 2 hour
before collection
Urine Collection
• Fractional Collection are use to compare the
concentration of an analyte in urine with its
concentration in the blood.
• The first morning urine (containing solutes
and metabolites from evening meal) is
discarded, but the second urine excreted
(fasting urine specimen) is collected.
Urine Collection
Timed collection usually done over 12-24
hour period, eliminate the need to determine
when excretion is optimal and allow day-today comparison.
Urine Collection
• Clean and dry plastic or glass containers
(50-3000 ml capacity)
• A preservative may be needed depending on
the proposed assay
• Total volume must be recorded
• The specimen well mixed to ensure
homogeneity
• Aliquots for specific assays
Urine Collection
• Time frame for measurement of proposed
assay.
• Microbial contamination
• Cost of storing large volumes of material
• Paucity of studies on the effect of long-term
storage on quantitative or qualitative
detection
Tissue Collections
• Confirming clinical diagnosis by
histological analysis
• Examining tumor characteristics at
chromosome and molecular level
Tissue Collections
• It requires to collect more materials than it
is necessary for pathological evaluation
• When possible, the tissue sample should
contain both tumor and normal tissues to
permit to study different characteristics of
the two tissues.
Tissue Collections
• Formalin-fixed paraffin-embedded tissue
specimens
• Frozen tissues (-70 degree C). The tissue is
embedded in frozen section support media
(OTC) and stored at –70 degree C
• Snap frozen tissues.
Laboratory Techniques with Tissue
tissue
RT-PCR
Adipose Tissue
• Adipose tissue may be quite feasible for
subject and involve low risk. The tissue
offers a relatively stable deposit of
triglyceride and fat-soluble substances such
as fat-soluble vitamins (vitamins A and D).
It represents the greatest reservoir of
carotenoids and reflect long-term dietary
intake of essential fatty acids.
Bronchoalveolar Lavage (BAL)
• BAL is used to assess and quantify asbestos
exposures
• Induced sputum sample and BALF can also
provide sufficient DNA for PCR assays.
Exhaled Air
• To evaluate exposure to different
substances, particularly solvents such as
benzene, styrene
• To be used as a source of exposure and
susceptibility markers (caffeine breath test
for p4501A2 activity)
• Breath urea (presence of urease positive
organisms such as H. pylori)
Hair
• Easy available biological tissue whose
typical morphology may reflect disease
conditions within the body
• Provides permanent record of trace
elements associated with normal and
abnormal metabolism
• A source for occupational and
environmental exposure to toxic metals
Hair
• Good marker for environment tobacco
smoke (ETS) exposure in children.
• The hair nicotine levels were shown to be
well correlated with cotinine creatinine
ratios in urine from the same individual.
Hair
Hair analysis provides long-term
information from months to years,
concerning both the severity and pattern of
drug use.
Hair
• Hair roots can be optimal source of DNA
for PCR analysis and permit easy collection,
transportation and low overall costs.
Nail Clippings
• Toenail or fingernail clippings are obtained
in a very easy and comfortable way.
• They do not require processing, storage and
shipping condition and thus suitable for
large epidemiological studies
Nail Clippings
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Trace elements
Selenium levels
Arsenic levels
Less likely to be contaminated by
environmental factors
• Involves more complicated processing
Buccal cells
• No invasive
• Good for PCR-analysis
• Can measure both germline and somatic
mutations
Saliva
• It is an efficient, painless and relatively
inexpensive source of biological materials
for certain assays
• It provides a useful tool for measuring
endogenous and xenobiotic compounds
Measurements
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Corticosteroids
Antibodies to HIV-1
CYP1A1 phenotype
Cotinine level
Breast Milk
• Measuring hormones, exposures to
chemicals and biological contaminants
(Aflatoxin), selenium levels
• Cells of interests
Feaces
• Certain cells of interest
• Infectious markers
• Oncogenes
Semen
• Evaluate the effects of exposures on
endocrine and reproductive factors.
• Sexual abstinence for at least 2 days but not
exceeding 7 days.
• Should reach the lab within one hour.
Temperature
• Specimen collection requires storage system that
capable of maintaining the optimal temperature for
the diverse type of specimens:
• -20 degree C, certain items stable, I.e., urine
• -70 degree C, DNA, Serum, Hormone, vitamins
• -120 degree C, hormones, corotenoids, other
nutrients
Storage
• Freezers may fail, leading to the necessity
for 24 hour monitoring for the facility
through a computerized alarm system to
alter personnel and activate backup
equipment.
• Monitoring fire, power loss, leakage, etc.
Shipping
• Sample shipping requirements depends on the
time, distance, climate, season, method of
transport, applicable regulations, type of specimen
and markers to be assayed.
• Polyurethane boxes containing dye ice are used to
ship and transport samples that require low
temperature. For samples require very low
temperature, liquid nitrogen container can be used
• The quantity of dry ice should be carefully
calculated, based on estimated time of trip.
Safety
• Protect specimen from contamination
• Workers safety, HIV, HBV
Procedures
• Standardized approaches in order to ensure
quality control
• Biological specimen collection manual
• Manuals for field trip preparation, packing
and shipping samples
• Protocols for lab assays