|Basic Malaria Microscopy (part I and II) (WHO - OMS, 1991, 72 p.)|
By the end of this Unit you should be able to:
· list the components of normal blood
· examine thick and thin blood films with the oil immersion objective and the x 7 ocular
· recognize and classify the normal components of blood
· recognize artefacts (contaminants, etc.) that may be mistaken for malaria parasites and so cause confusion in the diagnosis of malaria.
Components of normal blood
When taken directly from a vein, and collected in a test-tube, blood is a red liquid.
If the test-tube is allowed to stand for 5-20 minutes, you will see that the blood separates into two different layers, as shown in the diagram below. The serum layer is a pale yellow fluid. The blood clot is a semisolid substance that is now dark red or almost black in colour. It contains red blood cells, white blood cells and platelets. These components are much too small to see without the aid of your microscope and can be seen clearly only after making blood films which are then stained and dried.
Appearance of normal blood components
You need to be able to recognize the various components found in blood films. Their appearance differs slightly in thin and thick films.
Blood in thin films
When you examine thin blood films with the x 100 objective and x 7 ocular, you will see the following:
· red blood cells (or erythrocytes)
· white blood cells (or leukocytes)
· platelets (or thrombocytes).
You will see examples of all these blood components in thin films. You may use Plate 2 to help you to identify the various types of white blood cell. Your tutor will help you throughout this exercise.
Red blood cells
The shape of the red blood cell, or erythrocyte, is described as a biconcave disc; this is illustrated in the diagram on the right. The erythrocyte is the commonest cell that you will see in the thin blood film. There are about 5 000 000 red blood cells in each microlitre (µl) of blood.1 With good Giemsa staining, the red cell should be a pale greyish-pink. It measures about 7.5 micrometres (µm) in diameter.1
1 A microlitre is one-millionth (1/1 000 000) of a litre; it used to be referred to as a cubic millimetre (mm3). A micrometre is one-millionth of a metre, and is sometimes also known as a micron (µ)
Red blood cell or erythrocyte
The red cell does not have a nucleus. However, some cells may contain material that has stained differently; such cells may appear larger than the normal red blood cells, or normocytes (see Plate 2).
You will often see the abbreviation RBC used for red blood cells.
White blood cells
The total number of white blood cells or leukocytes in a microlitre of blood is about 6000 - 8000, which is much lower than the number of red blood cells. There are several different types of leukocytes, which stain differently from each other. With practice, it is quite easy to distinguish between them.
Before you go any further in this exercise, you need to know the main parts of a white blood cell. These are illustrated in the diagram below.
You will see that every leukocyte has a nucleus surrounded by cytoplasm. Sometimes the cytoplasm is granular in appearance. Some leukocytes have a multilobed nucleus. The leukocytes can be divided into two groups, as follows.
Group 1. Multilobed (polymorphonuclear) leukocytes
Neutrophils make up 65% of the total white cell count in the blood of a healthy person. They have well-defined granules in the cytoplasm and nuclei that stain deep purple. In cases of malaria it is common to see neutrophils containing malaria pigment, which is all that remains of the malaria parasite eaten or phagocytosed by the neutrophils.
Eosinophils make up about 1-4% of the total white cell count in the blood of a healthy person. The granular nature of the cytoplasm is very distinctive, with the granules taking on the pinkish colour of eosin. (If you are not familiar with this colour, you will see examples in the practical exercises.)
Basophils are rare leukocytes, usually making up less than 1% of the total. Large blue or mauve granules can be seen in the cytoplasm after staining.
Group 2. Non-multilobed leukocytes
Monocytes are the largest of the white blood cells - about 12-18 µm in diameter. The large nucleus is kidney- or bean-shaped and the cytoplasm may contain a few granules that stain pinkish or red. Monocytes make up 2 - 10% of the total white cell count in the blood of a healthy person; like neutrophils, they can phagocytose malaria parasites.
The two types of lymphocytes - large and small - make up 20-45% of the total white cell count.
The nucleus of the large lymphocyte is round and appears a deep mauve colour in well-stained blood films. The large amount of cytoplasm stains a clear water-blue and may contain a few mauve-staining granules.
The small lymphocyte is slightly larger than a normal red blood cell. It has very little cytoplasm and its nucleus stains a dark blue-black colour.
Platelets are small, red-staining bodies of irregular shape and without nuclei; they number about 100000 per microlitre of blood. They often appear in groups of 5-10 but may clump together in larger numbers if a blood film has been poorly made. It is important to be able to identify them; they may be confused with malaria parasites by inexperienced microscopists.
Blood in thick films
When you examine thick blood films with the x 100 objective and the x 7 ocular, you will see the following:
· the remains of red blood cells
· white blood cells (or leukocytes)
· platelets (or thrombocytes).
You will remember that a thick blood film consists of many dehaemoglobinized red blood cells packed together in a thick mass. When the thick film is stained with Giemsa stain, the water in the stain acts on the unpreserved red blood cell: the contents of the cell dissolve in the water. The main component of the red blood cell is called haemoglobin and so this process is called dehaemoglobinization. You can see this take place if you put a thick blood film into a dish or staining tray containing water. Within 1-2 minutes the red colour of the haemoglobin starts to flow out of the thick film, which becomes pale and opalescent.
This process happens during staining, so that all that remains on completion of staining are the remnants of the red blood cells, plus white cells and platelets.
The white blood cells and platelets have an appearance very similar to that in thin films. Because they have not been spread in a single layer on the slide, the white blood cells appear to be smaller, with the cytoplasm more compact around the nuclei. This can be seen in Fig. 2.
In this exercise you will learn to recognize the different kinds of leukocytes, the platelets, and the red cell remnants as they appear in Plate 2. If at any time you see something that you think is abnormal, bring it to the attention of your tutor or facilitator. A number of features that may appear in blood films and confuse you are illustrated in Plate 3; you will learn more about these artefacts in a later Learning Unit.