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

316 Manual of basic techniques for a health laboratoryNote:False-negative results may occur if:— outdated reagents are used;— concentrations of haemoglobin S are low;— patients have moderate or severe anaemia.If the test is positive a thin blood film should be examined. Patients with sickle-cellanaemia have sickle cells, nucleated erythrocytes, target cells, marked poikilocytosisand often macrocytosis. Patients with sickle-cell trait are not usually anaemicand have a normal erythrocyte morphology. Whenever possible, electrophoresis ofthe haemoglobin should be carried out to confirm a diagnosis of sickle-cell disease.This can be done in a reference laboratory.Other methods● The test can be carried out on venous blood provided it is freshly collected(within 1–2 hours of the test) or collected into an anticoagulant (EDTA dipotassiumsalt, 10% solution (reagent no. 22)).● The test can also be carried out using a test-tube rather than a Petri dish. Commercialreagents are available for this method.9.12 Determination of the reticulocyte numberconcentration/fractionReticulocytes are immature erythrocytes that pass into the bloodstream from thebone marrow. The number of reticulocytes in the blood indicates the degree ofactivity of the bone marrow in the production of erythrocytes, and when the marrowis very active (as in anaemia) their number increases. Reticulocytes containfine, deep-violet granules arranged in a network (reticulum). They do not contain anucleus.9.12.1 PrincipleThe fine granules in reticulocytes can be stained with brilliant cresyl blue. A bloodfilm is stained with this dye and a certain number of erythrocytes observed underthe microscope. From this observation, either:— the number of reticulocytes per litre of blood, or— the proportion of erythrocytes that are reticulocytes is calculated.9.12.2 Materials and reagents●●●●●●●●●MicroscopeMicroscope slides (grease-free)Glass spreaderTest-tubesTest-tube rackFunnelFilter-paperTwo Pasteur pipettes with teatsHand tally counter, if available● Saturated solution of brilliant cresyl blue (reagent no. 13).