COMMON TESTS AFFECTED BY ADDITIVE CONTAMINATION

CONTAMINATING ADDITIVE
Citrate EDTA Potassium Protime Serum iron

TESTS POTENTIALLY AFFECTED

Alkaline phosphatase Calcium Phosphorus Alkaline phosphatase Calcium Partial thromboplastin Sodium Creatine kinase

Heparin (all formulations)

Activated clotting time Acid phosphatase Calcium (some test methods) Partial thromboplastin Sodium (sodium formulations) Lithium (lithium formulations) Acid phosphatase Alkaline phosphatase Amylase Calcium Lactate dehydrogenase Partial thromboplastin Potassium Red cell morphology

Oxalates

Silica (clot activator) Sodium fluoride

Partial thromboplastin time Sodium Urea nitrogen

Ref. McCall R, Tankersley C. Phlebotomy essentials. 4th ed. Baltimore, Md.: Lippincott Williams & Wilkins, 2008.

Sample collection
Certain assays require samples to be collected into particular anticoagulants. The table lists some of the common assays and their requirements. EDTA FlOx Citrate Li Plain Hep H,L H,L J,H,L Albumin ALT ALP Ammonia If in doubt check requirements with a laboratory Amylase Bile acids before obtaining samples. Consider effects of Bilirubin transport on samples. For most assays the Blood gases patient should be starved overnight prior to the blood sample being taken. The letters indicated Calcium Chloride that some methodologies may be affected by alterations to serum or plasma, which would give Cholesterol CK rise to artefactual lowering or raising of values. Clotting Contact specific laboratories to determine factors whether their assays are affected or not. Creatinine Enzymes + Preferred Fibrinogen Acceptable Globulin Unacceptable Glucose GGT A - affected by azotemia Hemoglobin B - affected by hyperproteinemia Lipase C - affected by hyperglycemia PCV D - affected by Phosphate hypoalbuminemia/hyperalbuminemia Potassium H - affected by hemolysis Sodium J - affected by jaundice Total Protein L - affected by lipemia Triglycerides Source: BSAVA Small Animal Formulary, 2nd Urea edition.

H,L H,L A,H,L H,L D,H,L B,J,L J,L H H,L J,H,L H,L

H,L H,J,L L H,L H H H,J,L A,B,C B, L A,H,J, J L

Additional information about anticoagulants - why do we need so many? Potassium EDTA EDTA chelated divalent cations. Potassium EDTA is the standard anticoagulant used for hematology, because it preserves the cellular components of the blood (but is not a fixative). This anticoagulant can be used for some biochemistry but is not suitable for:
Alkaline phosphatase - due to chelation of metallic cofactors which inhibits the enzyme activity. Potassium and sodium - due to the addition of potassium to the sample. Calcium and magnesium - these are chelated by the EDTA.

EDTA is also an unsuitable additive for samples requiring bacterial culture,

since the chelation of the divalent cations inhibits the growth of bacteria. EDTA is sometimes used to prevent cells clumping in fluid samples requiring cell counts to accompany a cytology evaluation but it does not actually 'fix' the cells. A sample fixed in formalin or alcohol/ethanol is required for accurate cytology examination. EDTA is not suitable for samples requiring virus isolation in cell/tissue culture because it forms a gel when added to the cell culture medium and this disrupts the cell monolayer. Plain (no anticoagulant) Plain (or clotted) samples are used to provide serum for serology and most biochemistry or endocrine assays. Serum is plasma without fibrinogen since the fibrinogen has been used' in the formation of the clot. Lithium Heparin Lithium heparin accelerates the action of antithrombin III which neutralizes thrombin and thus prevents the formation of fibrin from fibrinogen (clot formation). This effect makes heparin samples unsuitable for determination of fibrinogen or clotting factors. Lithium heparin is a standard anticoagulant used to obtain plasma for biochemistry analysis. Lithium heparin is the most suitable anticoagulant for the isolation of viruses in cell/tissue culture. This anticoagulant is not suitable for hematology as the heparin alters the cell morphology. Whilst measurement of hemoglobin, and blood cell counts can be obtained using this anticoagulant an accurate white cell differential and morphology comments are not possible. Fluoride/Oxalate Fluoride/oxalate samples are used for glucose (and lactate) determination only. Sodium fluoride functions by stabilizing the blood cell membrane and inhibiting the enzyme systems involved in glycolysis, which prevents red blood cells metabolising any glucose present in the sample. For this reason it is the only suitable sample for accurate glucose analysis. Fluoride is a potent inhibitor of many enzymes and the inhibition of glycolysis tends to cause fluid shifts. Fluoride is a weak anticoagulant on its own, so potassium oxalate (another powerful enzyme inhibitor) is usually added to supplement its action. Other plasma or serum samples may be used for glucose analysis ONLY if the plasma/serum is separated from the cells within 1 hour of sample collection. Without an antiglycolytic agent, the blood glucose concentration decreases by approximately 0.56 mmol/l per hour at 25C.

Sodium citrate Sodium citrate is the standard anticoagulant for coagulation assays. Citrate functions by chelating calcium. The effect is easily reversed by the addition of calcium to the sample. Other anticoagulants are not suitable for coagulation assays as they interfere with coagulation factors. Citrate also inhibits ALP, ALT and interferes with the measurement of phosphate.