Blood film Topics Blood film Topic Home A blood film or peripheral blood smear is a microscope slide Microscope slide made from a drop of blood Blood , that allows the cells to be examined microscopically. Blood films are usually done to investigate hematological Hematology problems (disorders of the blood) and, occasionally, to look for parasites Apicomplexa within the blood such as malaria Malaria and filaria Filariasis . Preparation Blood films are made by placing a drop of blood on one end of a slide, and using a spreader slide to disperse the blood over the slide's length. The aim is to get a region where the cells are spaced far enough apart to be counted and differentiated. The slide is left to air dry, after which the blood is fixed Fixation (histology) to the slide by immersing it briefly in methanol Methanol . The fixative is essential for good staining and presentation of cellular detail. After fixation, the slide is stained Staining (biology) to distinguish the cells from each other. Common blood film staining methods o Romanowsky stain Romanowsky stain o Giemsa stain Giemsa stain o Wright's stain Wright's stain o Jenner's stain Jenner's stain o Leishman stain Leishman stain o Field's stain Field's stain Disorders Characteristic red blood cell abnormalities are anemia Anemia , sickle cell anemia and spherocytosis Spherocytosis . Sometimes the microscopic investigation of the red cells can be essential to the diagnosis of life-threatening disease (e.g. TTP Thrombotic thrombocytopenic purpura ). White blood cells are classified according to their propensity to stain with particular substances, the shape of the nuclei and the granular inclusions. * Neutrophil granulocyte Neutrophil granulocyte s usually make up close to 80% of the white count. They have multilobate nuclei and lightly staining granules. They assist in destruction of foreign particles by the immune system Immune system by phagocytosis Phagocytosis and intracellular killing. Eosinophil granulocyte Eosinophil granulocyte s have granules that stain with eosin Eosin and play a role in allergy Allergy and parasitic disease. Eos have a multilobate nucleus Cell nucleus . Basophil granulocyte Basophil granulocyte s are only seen occasionally. They are polymorphonuceated and their granules stain dark with alkaline stains, such as haematoxylin Haematoxylin . They are further characterised by the fact that the granules seem to overlie the nucleus. Basophils are similar if not identical in cell lineage to mast cells, although no conclusive evidence to this end has been shown. Mast cells are "tissue basophils" and mediate certain immune reactions to allergens. * Lymphocyte Lymphocyte s have very little cytoplasm Cytoplasm and a large nucleus Cell nucleus (high NC ratio) and are responsible for antigen Antigen -specific immune functions, either by antibodies (B cell B cell ) or by direct cytotoxicity Cytotoxicity (T cell T cell ). The distinction between B and T cells cannot be made by light microscopy. Plasma cell Plasma cell s are mature B lymphocytes that engage in the production of one specific antibody Antibody . They are characterised by light basophilic Basophilic staining and a very eccentric nucleus Cell nucleus . Other cells are white cell precursors. When these are very abundant it can be a feature of infection Infection or leukemia Leukemia , although the most common types of leukemia (CML and CLL) are characterised by mature cells, and have more of an abnormal appearance on light microscopy (it should be noted that additional tests can aid the diagnosis). Use in diagnosing Malaria The preferred and most reliable diagnosis of malaria is microscopic examination of blood films, because each of the four major parasite species has distinguishing characteristics. Two sorts of blood film are traditionally used. Thin films are similar to usual blood films and allow species identification, because the parasite's appearance is best preserved in this preparation. Thick films allow the microscopist to screen a larger volume of blood and are about eleven times more sensitive than the thin film, so picking up low levels of infection is easier on the thick film, but the appearance of the parasite is much more distorted and therefore distinguishing between the different species can be much more difficult. From the thick film, an experienced microscopist can detect parasite levels down to as low as 0.0000001%. Microscopic diagnosis can be difficult because the early trophozoites ("ring form") of all four species look identical and it is never possible to diagnose species on the basis of a single ring form; species identification is always based on several trophozoites. Please refer to the chapters on each parasite for their microscopic appearances: P. falciparum Plasmodium falciparum , P. vivax Plasmodium vivax , P. ovale Plasmodium ovale , P. malariae Plasmodium malariae . The biggest pitfall in most laboratories in developed countries is leaving too great a delay between taking the blood sample and making the blood films. As blood cools to room temperature, male gametocytes will divide and release microgametes: these are long sinuous filamentous structures that can be mistaken for organisms such as Borrelia. If the blood is kept at warmer temperatures, schizonts will rupture and merozoites invading erythrocytes will mistakenly give the appearance of the accolé form of P. falciparum. If P. vivax or P. ovale is left for several hours in EDTA, the build up of acid in the sample will cause the parasitised erythrocytes to shrink and the parasite will roll up, simulating the appearance of P. malariae. This problem is made worse if anticoagulant Anticoagulant s such as heparin Heparin or citrate Citrate are used. The anticoagulant that causes the least problems is EDTA EDTA . Romanovski's stain or a variant stain Staining (biology) is usually used. Some laboratories mistakenly use the same stain as they do for routine haematology blood films (pH PH 7.2): malaria blood films must be stained at pH 6.8, or Schüffner's dots and James's dots will not be seen.