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

10. Blood chemistry10.1 Estimation of glucose concentration in blood:o-toluidine method 1Estimates of the glucose (sugar) concentration in blood are required to help in thediagnosis of diabetes mellitus or any other condition in which there is abnormalcarbohydrate metabolism in the body. In patients with diabetes glucose is usuallyfound in the urine (see section 7.2.4).10.1.1 PrincipleThe proteins are first precipitated by trichloroacetic acid. The glucose in the filtratereacts with the o-toluidine reagent to give a green colour, which is measured usinga photoelectric colorimeter.10.1.2 Materials and reagents●●●●●ColorimeterConical centrifuge tubes and large test-tubes (to hold 20ml)Test-tube racksBlood (Sahli) pipettes, 0.2mlPipettes, 0.5ml, 5.0ml● Water-bath at 100°C● Glucose reagents (reagent no. 30)— trichloroacetic acid, 3% solution— o-toluidine reagent— benzoic acid, 0.1% solution— glucose stock reference solution (100mmol/l)— glucose working reference solutions (2.5, 5.0, 10, 20 and 25mmol/l)● Whole blood (capillary or venous), plasma or serum, taken from a fastingpatient 2●Control serum.A control serum should be used with each batch of tests. If the result of the testwith the control serum is correct, it can be assumed that the patient’s results willalso be correct.10.1.3 Method1. Pipette 1.8ml of trichloroacetic acid solution into each of three centrifuge tubes.Note: Trichloroacetic acid is corrosive. Use it with care.1This method is also used for estimating the glucose concentration in CSF (see section 8.3.4).2If venous blood is used, it is advisable to use fluoride oxalate (reagent no. 26) as the anticoagulant.This will prevent the glucose from being destroyed in the blood.322