Biochemical Analysis of Blood - Essay Example

Introduction Body fluids are a good source of getting information about health status and blood is an ideal sample from which relevant diagnostic information can be collected. Numerous tests have been developed for blood analysis that can give information on health status of a patient. Sample selection depends upon the disease status for which the patient is being investigated. For that reason, either serum or plasma is chosen. Plasma is being free from the proteins that involved in the coagulation of blood when an anticoagulant agent has been added to the blood sample. If total proteins need to be evaluated, serum is therefore the ideal choice. Liver function can be appropriately evaluated by measuring the total protein and albumin in the plasma sample and if a low concentration is detected, that means liver functioning is compromised (Gerardo 1998). Therefore, the total protein and albumin levels have been tested in this particular patient to evaluate his liver function. The level of albumin present is also an important marker of the state of osmotic regulation in the bloodstream as its deficiency can lead to oedema (known as dropsy, the medical term for fluid retention in the body). Oedema occurs as a result of certain health conditions, such as heart failure or kidney failure (NHS 2012). Total protein can be measured by the Biuret method. The method is based on the reaction between peptide bonds, imines and the carbonyl moiety in blood proteins, and a colour change results after reaction with Copper ions in an alkaline solution. The colour intensity can be measured by a spectrophotometer at 550nm wavelength and total protein can be estimated by comparing the value of obtained readings with that of standards. Another method of measuring proteins in a serum sample is by ‘Serum Protein Electrophoresis’ which elucidates levels of individual protein fractions in blood, such as albumin, ?, ?, and ? globulin. This provides a broad hint of the relative proportion of each protein fraction within the patient’s total serum. Cellulose acetate is used as a support medium in the electrophoresis tub. The negatively charged protein molecules on the cellulose acetate move towards the positively charged end, when electrical current is passed through the solution. The movement of the molecules depends on the size and charge taken up by each type of protein (Kaneko et al, 2008). Bilirubin a breakdown product of RBC (Red Blood Cells/Erythrocytes) disintegration, is another metabolic product whose level in the serum can assist in diagnosing liver malfunction. Liver is the site where bilirubin breakdown takes place and in case of liver malfunction, excessive bilirubin enters the bloodstream. Normal level of bilirubin in the blood as detected by the direct bilirubin (conjugated) estimation method is between 0 to 0.3 mg/dL, and as estimated by the total bilirubin method is 0.3 to 1.9 mg/dL (NIH, 2012). Any excess than these values is indicative of liver disease. This practical aims to recognize the importance of biochemical analysis of blood proteins in detecting diseases and carry out standardized clinical measurements of serum proteins in a blood sample obtained from a patient. Material and Methods: Patient's details Surname: OKEREKE Forename: JERMAINE DOB: 23/01/1950 URN: X855210 Information of: quality control used in SPE; Control: Kentro lot no: 109177702 expiry: 2012-06. Randox: lot no: 4674 expiry: 2014-03 Jermaine Okereke, a 62 year old male, was the patient from whom blood samples were taken. Dr. Siva, the attending physician had recommended several tests for this patient. The tests done in this practical were: 1. Serum Protein Electrophoresis 2. Total Protein by Biuret Method 3. Albumin by Bromcresol Green Binding, and 4. Bilirubin by Jendrassik and Grof Method. Serum Protein Electrophoresis: The tank was prepared by adding barbital buffer in two reservoirs. Filter paper wicks were used at both edges of the bridge. Cellulose acetate strips were removed from barbital buffer with a pair of forceps and blotted between two paper towels to remove excess buffer. Te quality controls as well as the patient samples were applied to the non-shiny side of the cellulose acetate strips by means of a special applicator. The application was about 3 cm away from the cathode end. A pencil mark was made to check the orientation while placing the cellulose acetate strips into the tank. The strip was placed in the electrophoresis tank bridge with its non-shiny surface downwards. To promote sufficient flow, it was ensured that the strips always had a good contact with the wicks. Electrical current was switched on and its value adjusted to 2mA and the reaction was allowed to continue for 90 minutes. Power supply was then switched off and the strips removed from the tank. The strips were then stained and incubated with Ponceau S dye solution for 5-10 minutes. Excess dye was decolorized in 5% Acetic acid and the strips dried by gently pressing them between paper towels. Total Protein: Total protein was determined by the Biuret method. 0.1 ml each of the patient's sample was added by a micropipette into 2 identical test tubes and mixed with 2.4 ml of normal saline solution. Two blank tubes were prepared by putting 2.5 ml of normal saline solution in each. 3 ml of Biuret reagent was added to all tubes. The solutions were mixed thoroughly by shaking and left in a rack for 30 minutes for colour development. The saline blank tube was used to calibrate the spectrophotometer to show a '0' value and the actual sample reading taken then. A calibration curve was prepared to determine the value of total protein in the sample. Albumin by Bromcresol Green Binding: 20 µL each of the patient's serum samples were added to 2 test tubes and 4 ml of Bromocresol Green (BCG) solution was added in each. 2 blank tubes were prepared by adding just 4 ml of BCG solution in each. The sample tubes were shaken to mix the sample thoroughly and incubated for 10 minutes. The readings from the spectrophotometer were obtained for the samples as well as controls at 628 nm wavelength. A calibration curve was prepared and the values of albumin determined. Bilirubin – Jendrassik & Grof Method: Bilirubin levels in the patient’s serum sample were determined by the Jendrassik & Grof method. As above, the patient and quality control samples were prepared in duplicate. In the blank, control tubes, only 0.2 ml of distilled water (DW) and 0.05 ml of serum were added. In the direct tested samples 0.2 ml of DW, 0.05 ml of serum, 0.125 ml of the Diazo reagent were added. In the total Bilirubin test, 0.5 ml of Caffeine benzoate was added in addition to the Diazo reagent, serum and DW. All tubes were shaken to mix the contents thoroughly and incubated at room temperature for 10 minutes. 0.025 ml of Ascorbic acid was added to all the tubes. 0.125 ml of the Diazo reagent, 0.5 ml of Caffeine benzoate and 0.375 ml of Tartrate was added to the blank tubes. In the tubes containing the sample to be measured by the direct method, 0.5 ml of Caffeine benzoate and 0.375 ml of Tartrate were added. In the tubes that meant for measuring Total Bilirubin, only 0.375 ml of Tartrate was added. Contents of all tubes were carefully mixed and the absorbance reading for each was taken at 607 nm wavelength. The calibration curve was used to determine the total and direct Bilirubin levels in the patient's sample. Results Table 1: Quality Control (Randox): Expected values for Total Protein, Albumin and Bilirubin Total protein 45.4 g/L Albumin 27.0 g/L Bilirubin - direct 32.2 µM Bilirubin - total 91.1 µM Table2: Reference range data for Total Protein, Albumin and Bilirubin Analyte Reference Range Total protein 60-80 g/L Albumin 38-50 g/L Bilirubin – direct Read More