Sample%20Collection,%20Processing%20and%20Storage

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Sample Collection, Processing and Storage

• EPI243
• Zuo-Feng Zhang, MD, PhD

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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

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Information linked to Sample

• Time and date of collection
• Recent diet and supplement use,
• Reproductive information (menstrual cycle)
• Recent smoking
• current medication use
• Recent medical illness
• Storage conditions

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Quality Assurance

• Systematic Application of optimum procedures to
  ensure valid, reproducible, and accurate results

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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

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Liquid Nitrogen Tank
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Ultima freezers -140 degree C to 220 degree C
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-70 freezers
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Quality Assurance

• Storing each persons 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.

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Quality Assurance

• Sample should be selected from specimens that
  received the same treatment throughout the
  storage process or the same variations in
  handling.

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Quality Assurance Careful Record of disbursement

• Barcode system to check in and out the bio-
  specimens
• which specimen, how much material remains,
  documentation on factors such as thawing which
  could influence future use of the material.

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bar code scanner
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bar code scanner
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Bar Code Printer
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Computer System
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Bar Code System
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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.

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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

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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.

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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.

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Types of Biospecimens Blood

• Plasma
• Serum
• Lymphocytes
• Erythrocytes
• Platelets

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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.

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Sterile Blood needles Sterile Syringes Plain
Vacutainer Blood Tubes Alcohol Prep
Pads Tourniquet
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Blood Collection
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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.

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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.

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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.

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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
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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-citrate-dextrose
  (ACD) solution.
• Grey-top tubes contain a glycolytic inhibitor.

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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

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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.

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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

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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-molecular-weight
  DNA
• A quantity of 50 ul of dried blood can provide
  0.5 ug DNA, sufficient for multiple PCR-based
  assays

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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

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• 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

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• White blood cells
• are much less numerous than red (the ratio
  between the two is around 1700),
• 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.

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Lymphocytes
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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

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single macrophage (monocyte) surrounded by
several lymphocytes
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Granulocytes

• Granulocytes can serve as a source of DNA without
  sacrificing the lymphocytes

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Erythrocytes (RBC)

• Stored after washing with physical saline
• Can be useful to study adducts of haemoglobin

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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.

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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 group-specific average
  value.

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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.

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DNA Extraction

• DNA can be extracted from
• Whole blood
• Leukocytes
• Serum
• Plasma
• Blood clot

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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

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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

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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.

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Storage

• Samples stored on the top of the freezer may be
  exposed to more extreme temperature fluctuation
  then those stored at the bottom.

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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

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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.

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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.

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Urine Collection

• First morning
• Random
• Fractional
• timed

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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

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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

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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.

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Urine Collection

• Timed collection usually done over 12-24 hour
  period, eliminate the need to determine when
  excretion is optimal and allow day-to-day
  comparison.

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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

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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

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Tissue Collections

• Confirming clinical diagnosis by histological
  analysis
• Examining tumor characteristics at chromosome and
  molecular level

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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.

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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.

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Laboratory Techniques with Tissue
tissue
RT-PCR
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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.

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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.

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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)

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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

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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.

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Hair

• Hair analysis provides long-term information
  from months to years, concerning both the
  severity and pattern of drug use.

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Hair

• Hair roots can be optimal source of DNA for PCR
  analysis and permit easy collection,
  transportation and low overall costs.

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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

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Nail Clippings

• Trace elements
• Selenium levels
• Arsenic levels
• Less likely to be contaminated by environmental
  factors
• Involves more complicated processing

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Buccal cells

• No invasive
• Good for PCR-analysis
• Can measure both germline and somatic mutations

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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

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Measurements

• Corticosteroids
• Antibodies to HIV-1
• CYP1A1 phenotype
• Cotinine level

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Breast Milk

• Measuring hormones, exposures to chemicals and
  biological contaminants (Aflatoxin), selenium
  levels
• Cells of interests

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Feaces

• Certain cells of interest
• Infectious markers
• Oncogenes

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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.

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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

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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.

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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.

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Safety

• Protect specimen from contamination
• Workers safety, HIV, HBV

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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