Blood Smear Tests

• Prepare the blood smear and air dry
• Fix by immersing in Solution A for 30 seconds
• Transfer, without rinsing or drying to Solution B and stain for 15-30 seconds. Gently agitate during this period
• Without rinsing transfer the slide to Solution C and stain for 15-30 seconds. Gently agitate during this period
• Rinse briefly in buffered water pH 6.8 and allow drying
• Examine under a microscope

Role in diagnosis

Blood smears can be used to diagnose many differing conditions and diseases. One of these is malaria. Diagnosing this is done by microscopically examining staines as examining four major parasitic species that form during malaria, these a the distinguishing characteristics of malaria.Thick films enable the pathologist to examine and sreen a larger volume of blood. These thick slide are also about eleven times more sensitive than the thin film, meaning picking up low levels of infection is easier on the thick film. However his can cause 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. Microscopic diagnosis can be difficult because the early trophozoites ("ring form") of the four variations 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

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 anticoagulants such as heparin or citrate are used. The anticoagulant that causes the least problems is EDTA. Romanowsky stain or a variant stain is usually used. Some laboratories mistakenly use the same staining pH as they do for routine haematology blood films (pH 6.8): malaria blood films must be stained at pH 7.2, or Schüffner's dots and James's dots will not be seen

Other disorders that can be diagnosed are;

• Neutrophil granulocytes 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 by phagocytosis and intracellular killing.
• Eosinophil granulocytes have granules that stain with eosin and play a role in allergy and parasitic disease. Eos have a multilobate nucleus.
• Basophil granulocytes are only seen occasionally. They are polymorphonuceated and their granules stain dark with alkaline stains, such as 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.
• Lymphocytes have very little cytoplasm and a large nucleus (high NC ratio) and are responsible for antigen-specific immune functions, either by antibodies (B cell) or by direct cytotoxicity (T cell). The distinction between B and T cells cannot be made by light microscopy.
• Plasma cells are mature B lymphocytes that engage in the production of one specific antibody. They are characterised by light basophilic staining and a very eccentric nucleus.
• Other cells are white cell precursors. When these are very abundant it can be a feature of infection or 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 (additional tests can aid the diagnosis).