LECTURE 19: CLI NI CA L CHEM I STRY Marc Mate Composition of Blood Collection and Preservation of Samples Clinical Analyses: Common Determinations Immunoassays Whole blood can generally be broken down into; Plasma: which contains the serum and fibrinogen, Cellular elements: which contain the erythrocytes, leukocytes, and platelets. Plasma is the liquid portion of circulating blood Serum: the portion of plasma remaining after coagulation of blood, during which process the plasma protein fibrinogen is converted to fibrin and remains behind in the clot The majority of clinical analyses are performed on whole blood, plasma, or serum, and most of these use serum. Adapted from J. S. Annino, Clinical Chemistry. 3rd ed. Boston: Little, Brown and Company, 1960. Blood and urine samples are often collected after the patient has fasted for a period of time (e.g., overnight), particularly for cholesterol or glucose analysis One study indicates that an average breakfast has no significant effect on the concentration of the blood content of carbon dioxide, chloride, sodium, potassium, calcium, urea nitrogen, uric acid, creatinine, total protein, and albumin. Serum phosphorus is slightly depressed at 45 min after the meal then stabilizes later When serum is required for the analysis, the blood is collected in a clean and dry tube to prevent contamination and hemolysis. Hemolysis is the destruction of red cells, with the liberation of hemoglobin and other cell constituents into the surrounding fluid (serum or plasma). If plasma or whole blood is required for analysis, then the blood is collected in a tube containing an anticoagulant. A widely used anticoagulant is potassium oxalate , about 1 mg per mL blood. Oxalate precipitates blood calcium; the calcium is required in the clotting process. In CO2 analysis, the sample is kept anaerobically by adding mineral oil to the collection tube A preservative is added to the sample, usually along with an anticoagulant. NaF is widely used as a preservative for samples to be analyzed for glucose. This is an enzyme inhibitor that prevents the enzymatic break down of glucose, or glycolysis. One milligram sodium fluoride per milliliter blood is adequate. Since it also inhibits other enzymes, including urease, sodium fluoride should not be added to samples to be analyzed for enzymes or for urea based on urease catalysis. The major serum electrolytes like sodium, potassium, calcium, magnesium, chloride, and bicarbonate (CO2) are easily determined Blood glucose and blood urea nitrogen (BUN) are the two most frequently performed clinical tests The enzymatic determination of glucose and BUN are the most common methods Blood samples for glucose analysis should not be left for a long time. Both red cells and leukocytes contain glycolytic enzymes. Glucose will be consumed and the concentration of glucose in a sample of whole blood will decline with time. The rate of loss is generally said to be approximately 5% per hour, but may be as rapid as 40% in 3 hours. Consumption of glucose in whole blood samples can be prevented by adding sodium fluoride to the specimen to inhibit the glycolytic enzymes Rapid separation of the sample or cooling will also prevent glycolysis and will allow the sample to be used for other determinations. Unhemolyzed samples that have been separated within 30 minutes of drawing are generally considered adequate. Rapid cooling of the sample followed by centrifugation is even more effective in preventing glycolysis. Glucose concentration may be determined in whole blood, plasma, or serum samples. If whole blood is used, the concentration will be lower than if plasma or serum is used. This is due to the greater water content of the cellular fraction. Under usual circumstances, the concentration of glucose in whole blood is about 15% lower than in plasma or serum Blood glucose levels are measured by a procedure based upon the enzyme glucose oxidase. The enzyme is very specific for only D-glucose, and will not be subject to interferences from other molecules in the blood. The enzyme glucose oxidase catalyzes the oxidation of beta-D-glucose to D-gluconic acid Diatomic oxygen from the air is the oxidizing agent At the same time the oxygen in the presence of water is converted to hydrogen peroxide The hydrogen peroxide reacts with a second color producing chemical (o-Toluidine or 2-methylaniline) which reacts with the hydrogen peroxide using an enzyme called peroxidase to produce a color forming chemical. The concentration of the glucose can be related to the intensity of color produced. The more the intensity, the higher the concentration of glucose. A simple color chart can be used to "read" the concentration of the glucose. A blood urea nitrogen (BUN) test measures the amount of nitrogen in your blood that comes from the waste product urea. Urea is made in the liver and passed out of your body in the urine. A BUN test is done to see how well your kidneys are working. Normal human adult blood should contain 7 to 20 mg/dL (2.5 to 7.1 mmol/L) of urea nitrogen. If your kidneys are not able to remove urea from the blood normally, your BUN level rises. Heart failure, dehydration, or a diet high in protein can also make your BUN level higher. The enzyme Urease quantitatively converts urea to ammonia and carbon dioxide. The amount of urea is calculated from a determination of either the ammonia or the carbon dioxide produced, usually the former. This can be done by reacting the ammonia with a color reagent. Immunoassays are bioanalytical methods in which the quantification of the analyte depends on the reaction of an antigen (analyte) and an antibody. Immunoassays have been widely used in many important areas of pharmaceutical analysis such as diagnosis of diseases, therapeutic drug monitoring, clinical pharmacokinetics and in drug discovery. An immunoassay capitalizes on the specificity of the antibody-antigen binding found naturally in the immune system. The assay can be used to identify the presence of pathogens in a clinical sample, or it can be used to measure the amount of a target biomolecule. An antigen (e.g., a hormone) is a (foreign) substance capable of producing antibody formation in the body It is able to react with (bind to) that antibody. An antigen is always a large molecule such as a protein. An antibody (immunoglobulin) is a protein endowed with the capacity to recognize, by stereospecific association, a substance foreign to the organism it has invaded, for example, bacteria and viruses. The antibody will specifically react with an antigen to form an antigen-antibody complex. It is produced in the organism (where it will remain present for some time) only after the organism has had at least one exposure to the intruder (through vaccination, either spontaneous or artificial). An antibody is produced for use in immunoassay by injecting the antigen into an animal species to which it is foreign and recovering the serum that contains the resultant antibody (anti-serum). Immunoassay techniques generally involve a competitive reaction between an analyte antigen and a standard antigen that has been tagged (labelled), for limited binding sites on the antibody to that antigen. The label may be a radioactive tracer, an enzyme, or a fluorophore. RIA Principle: “Radio labelled antigen (Ag*) competes with the unlabelled antigens for the binding site of the fixed amount of antibody (Ab).” 100% RADIOACTIVITY 0 ng – 100% 1 ng – 90% The proportion of Ab-Ag* and Ab-Ag at equilibrium will be equal to the original proportion of Ag* to Ag The initial reaction vessel contains antibody solution (antiserum), labeled antigen, and the serum sample that may contain unlabeled (natural) antigen (the substance to be determined). Upon incubation, the antibody (Ab) will form an antigen-antibody immunocomplex (Ag-Ab). In the absence of unlabeled antigen, a certain fraction of the labeled antigen (Ag*) is bound (as Ag*-Ab). Increasing amounts of unlabeled antigen (Ag) are added, the limited binding sites of the antibody are progressively saturated and the antibody can bind less of the radiolabeled antigen. Following incubation, the bound antigens are separated from the unbound (free) antigens, and the labeled portion (radioactivity, fluorescence, etc.) is measured to determine the percent bound of the labeled antigen. The antibody solution is initially diluted so that, in the absence of unlabeled standard or unknown antigen, about 50% of the tracer dose of Ag* is bound. A diminished binding of labeled antigen when sample is added indicates the presence of unlabeled antigen. A calibration curve is prepared using antigen standards of known concentration by plotting either the percent bound labeled antigen or the ratio of percent bound to free (B/F) as a function of the unlabeled antigen concentration. From this, the unknown antigen concentration can be ascertained. NEXT LECTURE