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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

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Sample Collection, Processing and Storage

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  1. Sample Collection, Processing and Storage EPI243 Zuo-Feng Zhang, MD, PhD

  2. 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

  3. 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

  4. Quality Assurance Systematic Application of optimum procedures to ensure valid, reproducible, and accurate results

  5. 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

  6. Liquid Nitrogen Tank

  7. Ultima freezers: -140 degree C to –220 degree C

  8. -70 freezers

  9. 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.

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

  11. 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.

  12. bar code scanner

  13. bar code scanner

  14. Bar Code Printer

  15. Computer System

  16. Bar Code System

  17. 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.

  18. 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

  19. 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.

  20. 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.

  21. Types of Biospecimens: Blood • Plasma • Serum • Lymphocytes • Erythrocytes • Platelets

  22. 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.

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

  24. Blood Collection

  25. 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.

  26. 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.

  27. 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.

  28. 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

  29. 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.

  30. 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

  31. 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.

  32. 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

  33. 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

  34. 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

  35. 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

  36. 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.

  37. Lymphocytes

  38. 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

  39. single macrophage (monocyte) surrounded by several lymphocytes

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

  41. Erythrocytes (RBC) • Stored after washing with physical saline • Can be useful to study adducts of haemoglobin

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