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

278 Manual of basic techniques for a health laboratoryFig. 9.26 Calibration carve for determining haemoglobin concentration2. Arrange the test-tubes in a test-tube rack: one for each sample to be tested, onefor the blank and two for the control samples.3. Using a grease pencil, label the test-tubes with the appropriate laboratory numbersof the samples to be measured, B for the blank, and C1 and C2 for thecontrol samples.4. Pipette 3ml of AHD reagent into each test-tube.5. Pipette 20ml of blood collected in EDTA from a patient into the AHD reagentof the appropriate tube. Flush the pipette carefully five times with the AHDreagent.6. Pipette 20ml of AHD standard into test-tubes C1 and C2.7. Plug all the test-tubes with a clean cork or rubber stopper and mix by inversion.Leave the tubes to stand for 2–3 minutes.8. Pour the AHD solution from test-tube B into a clean cuvette. Dry the outside ofthe cuvette with cotton wool or gauze. Make sure that there are no air bubbles inthe solution. Place the cuvette in the cuvette chamber and adjust the spectrophotometeror haemoglobinometer to read zero.9. Repeat the procedure with the solution in test-tubes C1 and C2, respectively. Ifthe readings of the two controls differ by less than 2.5%, measure the haemoglobinconcentration of all the test samples. Record all the results.Method using a colorimeterThe AHD method is also applicable using a colorimeter. The measurement procedureis the same as that described for a spectrophotometer or haemoglobinometer.However, the absorbance in a colorimeter does not increase linearly with haemoglobinat elevated concentrations. Therefore, a calibration curve must be used torelate the absorbance readings to the haemoglobin concentration, as described above.ResultsReport the results in g/l. Example: “haemoglobin = 89g/l”.