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

274 Manual of basic techniques for a health laboratoryTable 9.3 Preparing serial dilutions of haemiglobincyanide solutionTube Volume of haemiglobincyanide Volume of Drabkin Concentration ofnumber solution (ml) diluting fluid (ml) haemoglobin a (g/l)1 4.0 1.0 13.42 3.0 2.0 10.13 2.0 3.0 6.74 1.0 4.0 3.4aIn this example, it is assumed that the haemoglobin concentration of the haemiglobincyanide solutionis 168 g/l.8. Plot a graph of absorbance against haemoglobin concentration, using ordinarygraph paper. Draw a straight line starting at the origin passing as close to eachpoint as possible. Extend the line so that you can read absorbances for haemoglobinvalues greater than 168g/l.A reference table of values is prepared using the graphs obtained from either of theabove methods:● Draw up a table of absorbance readings starting from 0.00, 0.01, 0.02 and endingat 1.50.● Determine the haemoglobin concentrations for each of the absorbances fromthe graph.Precautions● Potassium cyanide is very poisonous. It must be kept in a locked cupboard at alltimes when not in use. Wash your hands immediately after handling it.● Store Drabkin diluting fluid in a brown reagent bottle because it decomposes onexposure to light. If a brown reagent bottle is not available, use a clear glassbottle carefully wrapped in silver foil.● Drabkin diluting fluid should be clear and pale yellow. If it becomes turbid, orloses its colour, it should be discarded. The clarity of the diluting fluid can bechecked by measuring its absorbance in a spectrophotometer at 540nm againstwater as a blank. The absorbance must read zero.● Once the haemiglobincyanide solution has been prepared, the haemoglobinestimation must be carried out within 6 hours.● Drabkin diluting fluid remains stable for several months when stored at cooltemperatures. If the room temperature exceeds 30°C, store it in a refrigeratorat 4–6 °C. Do not freeze, as this may cause decomposition of the compound.Always allow the diluting fluid to warm to room temperature before use.Method1. Pipette 5ml of Drabkin diluting fluid into a tube. Draw venous or capillary bloodto the 0.02-ml mark of a blood (Sahli) pipette. Do not allow air bubbles to enter.With venous blood ensure that it is well mixed by inverting the bottle containingit and the anticoagulant repeatedly for about 1 minute immediately beforepipetting it.2. Wipe the outside of the pipette. Check that the blood is still on the 0.02-ml mark(Fig. 9.25). Squeeze the bulb of the pipette to expel the blood into the Drabkindiluting fluid and rinse the pipette by drawing up and expelling the fluid in thetube three times.3. Mix the contents of the tube and leave for 5 minutes (see Fig. 9.23).