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

326 Manual of basic techniques for a health laboratory● Blank reagent (reagent no. 11)●●Patient’s blood (treated with EDTA dipotassium salt, 10% solution (reagent no.22)), serum or plasmaControl serum.A control serum (of known concentration) should be used with each batch oftests. If the result of the test with the control serum is correct, it can be assumedthat the patient’s results will also be correct.10.2.3 Method1. Prepare the colour reagent immediately before use, using a 1:1 mixture of thediacetyl monoxime stock solution and acid reagent. Prepare at least 15ml ofcolour reagent for each test.Mix the colour reagent in a large test-tube or small flask.2. Pipette into a conical centrifuge tube 50ml of whole blood (treated with EDTAdipotassium salt solution), serum or plasma.3. Add 1ml of trichloroacetic acid solution and mix.Centrifuge at high speed (3000g) for 5 minutes to sediment the precipitatedproteins and obtain a clear supernatant fluid.4. Take three (or more if needed) large test-tubes and label as shown in Fig. 10.4:— blank tube (B)— reference tube (R)— patient tube (P).Note: If more than one estimation is being carried out, label each of the P tubeswith the name or number of the patient.5. Pipette into each tube as follows:● Blank:— 0.1 ml of blank reagent— 3.0 ml of freshly prepared colour reagent.● Reference:— 0.1 ml of working reference solution— 3.0 ml of freshly prepared colour reagent.● Patient:— 0.1 ml of supernatant fluid— 3.0 ml of freshly prepared colour reagent.6. Mix the contents of each tube. Place all the tubes in the water-bath at 100°C for15 minutes (see Fig. 10.5) to allow the red colour to develop.7. Remove the tubes and place them in a beaker of cold water until they havecooled to room temperature.8. Measure the colour produced in a colorimeter at a wavelength of 520nm.(a) Place the green filter in the colorimeter.(b) Fill the colorimeter test-tube or cuvette with the solution contained in thetube marked B (blank) and place in the colorimeter.(c) Adjust the reading of the colorimeter to zero with the cuvette containingsolution B in place.(d) Pour solution B out of the cuvette, rinse the cuvette with a small amount ofworking reference solution R (reference), pour this out, and fill the cuvettewith solution R; place the cuvette in the colorimeter and read the absorbance,A R.(e) Pour solution R out of the cuvette, rinse the cuvette with a small amount ofsolution P (patient), pour this out, and fill the cuvette with solution P; placethe cuvette in the colorimeter and read the absorbance, A P.