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

10. Blood chemistry 323Fig. 10.1 Delivering bloodunder trichloroaceticacid solution using ablood pipetteA: trichloroacetic acidsolution; B: blood.Fig. 10.2 Rinsing the bloodpipette withtrichloroacetic acidsolutionFig. 10.3 Expellingtrichloroacetic acidsolution into acentrifuge tube2. With a 0.2-ml blood pipette, deliver 0.2ml of the blood 1 specimen to the bottomof the first centrifuge tube (B in Fig. 10.1) — i.e. under the trichloroacetic acidsolution (A in Fig. 10.1). The trichloroacetic acid solution will become cloudywhere it makes contact with the blood specimen.3. Raise the pipette and draw clear trichloroacetic acid solution into it in order towash out all traces of the blood specimen (Fig. 10.2).4. Expel the trichloroacetic acid solution from the pipette into the centrifuge tube(Fig. 10.3).5. Mix well (the entire solution will become cloudy) and allow to stand for 5minutes.6. Using a clean 0.2ml blood pipette, deliver 0.2ml of distilled water and 0.2mlof glucose working reference solution to the second and third centrifuge tubes,respectively, as described in step 2. These tubes will be used to prepare thereagent blank and the glucose working reference standard, respectively.7. Centrifuge the three tubes at 3000g for 5 minutes. The precipitated proteins inthe tube containing the blood specimen will sediment and a clear supernatantfluid will be obtained.8. 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 Ptubes with the name or number of the patient.9. Pipette into each tube as follows:● Blank:— 0.5ml of fluid from the second centrifuge tube— 3.5ml of o-toluidine reagent.● Reference:— 0.5ml of from the third centrifuge tube— 3.5ml of o-toluidine reagent.● Patient:— 0.5ml of supernatant fluid from the first centrifuge tube— 3.5ml of o-toluidine reagent.Note: The o-toluidine reagent is corrosive.Fig. 10.4 Labelling test-tubes forthe testB: blank tube;R: reference tube;P: patient tube.1When this test is performed using CSF, the volume required in this step is greater (0.8ml).