Sample Collection Processing and Storage EPI 243 ZuoFeng

Sample Collection, Processing and Storage EPI 243 Zuo-Feng Zhang, MD, Ph. D

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 • • 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 bio- specimens • 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 • • • 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 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 • • 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 p 4501 A 2 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 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 • • 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 • • Corticosteroids Antibodies to HIV-1 CYP 1 A 1 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