Manual of basic techniques for a health laboratory - libdoc.who.int

238 Manual of basic techniques for a health laboratoryMethodCollection of specimensRandom, timed or 24-hour urine specimens should be used (see section 7.1.1). Nopreservatives should be added to the specimens. Specimens that are collected over24 hours should be stored at 4–8°C during the period of collection, to preventbacterial growth.Collected specimens should be kept at 4°C until analysis. If analysis is likely to bedelayed for more than 24 hours, however, the specimens should be stored at-20°C.Technique1. Add 1.6ml of the urine specimen to each of two test-tubes (test and test blank).Repeat the process with the working standard and the control.2. Add 0.4ml of trichloroacetic acid solution to all of the test-tubes and mix well.Leave to stand at room temperature for 10 minutes.3. Centrifuge the test blanks at 2000g for 10 minutes.4. Using the spectrophotometer, measure and record the optical density of thetests and blanks at 620nm. The spectrophotometer should be set to zero withdistilled water before any measurements are taken. It should also be calibrated,as described below. The analytical range of measurement using this method is100–1000mg/l.CalculationCalculate the concentration of protein in the urine specimen using the followingformula:OD - OD ¥ CT TBOD - ODRRBwhere:C = concentration of the calibration solutionOD R= optical density of the working standardOD RT= optical density of the working standard test blankOD T= optical density of the test specimenOD TB= optical density of the test specimen blank.Note:● If a serum-based control is used for calibration purposes, an independent materialmust be used for the purpose of quality control.● Because the amount of protein excreted in the urine may vary greatly, any positiveresults should always be confirmed by repeating the test on one or more separatesamples.● If this method is used to screen for microproteinuria (which may be correlatedwith microalbuminuria in the absence of tubular damage, urinary infections andtreatment with certain drugs) in high-risk populations such as patients withdiabetes, the following modifications should be applied to steps 2 and 4:2. Leave all the tubes to stand at room temperature for 35 minutes after mixing.4. Using the spectrophotometer, measure and record the optical density of thetests and blanks at 405nm.