(1)
Office of Research and Development, United States Environmental Protection Agency, Washington DC, USA
Abstract
Every clinical pathology measurement can be undermined by poor sample collection, and there are many ways that samples can be compromised. A long list of tests can be ruined by red blood cell hemolysis and the presence of bilirubin because of improper blood collection technique. Similarly, a failure to properly fast animals can cause serum to be lipemic, which can interfere with many tests. Blood should be drawn from the same site, and at the same time of day throughout serial samplings. This is essential in rodents since their leukocyte counts fluctuate widely due to circadian effects and sampling site. Cell counts in all species differ depending on the collection site. When an animal is stressed during blood collection, there is a release of corticosteroids, splenic contraction, and the release of additional cells into circulation; and blood chemistries are also affected. Four methods for collecting urine are described along with their strengths and weaknesses. There is no ideal way to preserve urine so the means used are dictated by the evaluations needed.
3.1 Blood Sampling
Whenever drawing blood from animals, care should be taken to minimize subcutaneous hematomas, vascular damage, and thrombophlebitis. The animal should be at rest since activity and stress can significantly alter cell counts. It is generally prudent to draw duplicate samples, though this may not be possible with small animals.
Blood can be drawn with syringes, capillary tubes, and evacuated blood collection tubes (e.g. Vacutainers®). Collecting blood with a syringe is a two-step process. First the blood is drawn through a needle of appropriate size, then the needle is removed (to minimize hemolysis) and the blood is transferred to a tube. Capillary tubes gently remove blood from an exposed capillary bed or a lanced site. They run the risk of contamination with body fluids, dirt, hair, dander, and microbes unless the site is carefully cleaned.
Evacuated blood collection tubes are the most popular way to collect blood. They offer a one-step process that allows one or more tubes to be filled with a single venipuncture. They can cause small veins to collapse, however. Once blood is collected, tubes that contain a precise amount of anticoagulant should be inverted several times to allow the anticoagulant to mix with the blood. Capillary tubes are also available with premeasured anticoagulant. Syringes may be prewetted with anticoagulant before sampling. Excessive dilution of blood with anticoagulants can be avoided by always drawing a full volume of blood, or by using dry anticoagulants.
Pink tinted serum is a sign of hemolysis and the release of the pigment hemoglobin. These samples should be rejected because the release of cellular material will interfere with measurements of potassium, magnesium, calcium, phosphorus, iron, chloride, bromide, blood urea nitrogen, bilirubin, albumin, pyruvic acid, lactate dehydrogenase, alkaline phosphatase, bromsulphalein, ALT, AST, arginase, acid phosphatase, corticotrophin, icterus index, lactic acid, lipase, NPN (nonprotein nitrogen), sex hormone-binding globulin (SHBG), and pH. The major causes of hemolysis during sampling are excessive turbulence or vacuum when drawing blood into or emptying a syringe, vigorous shaking, chilled glassware, vigorous ringing of clots prior to centrifuging, high temperatures (e.g. during shipping), and wet or dirty needles, syringes, and glassware. The use of evacuated blood collection tubes minimizes these problems.
Capillary blood, such as that drawn from digits or heel pricks, can differ from venous blood. Serum or plasma calcium, potassium, and protein are lower, glucose is higher, and there tends to be more hemolysis. Lipemic serum often results from bleeding unfasted animals. This serum should be rejected since it may interfere with measurements of ALT, AST, total protein, albumin, cholesterol, bilirubin, icterus index, amylase, thymol, turbidity, platelet count, and uric acid. Prolonged contact with clots can affect measurements of glucose, alkaline phosphatase, alanine and aspartate aminotransferases, lactate dehydrogenase, and iron.
Serum is the cell-free portion of blood from which fibrinogen has been removed by clotting. It is obtained by allowing blood to sit at room temperature for 30–45 min to remove the fibrinogen by clotting. Plasma (serum that contains fibrinogen) is collected with a syringe or tube containing an anticoagulant to prevent clotting. Blood samples are centrifuged (>3,000 G for >15 min) and the plasma or serum is pipetted from the top of the tube into a clean container. Improper centrifugation will not remove all platelets so lactate dehydrogenase (LDH) may be released from lysed platelets. Alkaline phosphatase (ALP) and urate measurements may also be affected. The following anticoagulants are most frequently used for tests requiring whole blood or plasma:
EDTA (disodium and tripotassium salts of ethylenediaminetetraacetic acid) is a chelating agent that inhibits clotting by binding with calcium to form insoluble calcium salts. It is the most commonly used anticoagulant. EDTA does not affect blood cell morphology or staining.
Heparin (sodium, lithium, and ammonium heparin) has the least effect on clinical chemistry measurements and erythrocyte morphology because it is a natural product. It inhibits thrombin and thromboplastin, so it is not suitable for agglutination or prothrombin time tests. It must always be used when collecting blood for blood gas analyses. It is not recommended for blood smears because it interferes with the staining of leukocytes, and may cause leukocyte clumping.
Oxalates (ammonium, potassium, lithium, and sodium oxalates) form insoluble calcium oxalate that interferes with blood coagulation. The oxalates are not used for coagulation studies. Because they inhibit lactate dehydrogenase (LDH), they cannot be used for electrolyte and nitrogen measurements. Leukocytic degeneration begins after approximately 1 h.
Potassium, sodium, and lithium oxalates cause cell shrinkage (6–8 %), and ammonium oxalate causes cell swelling. Heller’s and Paul’s double oxalate is a mixture of ammonium and potassium oxalates. This mixture can be used for most hematology studies including morphology because the shrinkage caused by one oxalate is offset by the other.
Citrates (trisodium and lithium citrates) combine with calcium to form the insoluble salt calcium citrate. They are used for coagulation studies and to preserve blood for transfusion. Sodium citrate causes cell shrinkage. Lithium citrate is generally not used except for measurements of minerals in blood.
Sodium Fluoride/Thymol (10:1) is generally not used except to inhibit glycolysis (glucose test).
ACD Solution B (Acid Citrate Dextrose) is used to preserve blood for transfusions.
It is important to use the proper anticoagulant to prevent chemical interference. Blood collection tubes are color-coded according to the additives they contain. Every laboratory has its preferred tube choices. A Vacutainer tube guide wall chart may be downloaded at this website: http://www.bd.com/vacutainer/pdfs/plus_plastic_tubes_wallchart_tubeguide_vs5229.pdf.
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