Microscopic examination of Plasmodium spp.
6.1 Microscopic examination
Fig. 2-2 Morphology of Plasmodium under microscope
（10×100）
Add a drop of cedar oil or special mirror oil to the stained blood film and examine it with an optical microscopy with 100×objective lens and 10× eyepiece. Observating a properly stained blood slide under microscopy, the red blood cells are light red, granules of eosinophils are orangered, the nuclei of neutrophils are violet blue, lymphocytes and Plasmodium cytoplasm are blue or light blue, and nuclei of Plasmodium are red. Malaria pigment can be found in all phases except for ring stage (except for Plasmodium malariae) (Fig. 2-2). The detection of Plasmodium is mainly based on thick blood film, while the identification of species of Plasmodium on thin blood film. It starts from the tip of the tongue to check the thin blood film and for the thick blood film it starts from the upper or lower end to check the thick blood film, following the pattern of movement shown in Fig. 2-3.
Fig. 2-3 Schematic diagram of reading sequence
6.2 Determination of microscopic examination results
6.2.1 Negative for Plasmodium spp.
Under oil immersion objective, if no malaria parasite can be detected in at least 100 fields or the whole thick blood film, it is negative.
6.2.2 Positive for Plasmodium spp.
If malaria parasites can be detected in blood films, it is positive. Moreover, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae (Plasmodium knowlesi), or mixed infection can be determined according to the morphology of Plasmodium spp.
6.3 Malaria parasite count
6.3.1 Plasmodium count on thick blood film
While making a thick blood film, count the number of white blood cells per microliter of blood. Then check the thick blood film under microscopy and count the number of malaria parasites and white blood cells in each field, counting to 100 white blood cells; when the number of malaria parasites is less than 100 in 100 white blood cells, continue to count to 500 white blood cells. Use the following formula to calculate the density of malaria parasite.
Number of malaria parasites ÷ number of white blood cells×number of white blood cells per microliter blood = number of malaria parasite/μL blood.
If a white blood cell count cannot be performed, it is calculated as 8,000 white blood cells/μL. In the case of mixed infection (infection of two or more species of malaria parasites), the density of asexual bodies of all infected parasites is usually calculated. For example, the parasite density of Plasmodium falciparum and Plasmodium vivax mix-infection is 23,720/μL blood (calculated as follows: 593 parasites÷200 white blood cells×8,000).
6.3.2 Plasmodium count on thin blood film (the density of Plasmodium in red blood cells)
While making thin blood film, count the number of red blood cells per microliter of blood, which can also be calculated as 5 million per microliter for men and 4.5 million per microliter for women. The thin blood film shall be examined under microscope, and the infection rate of red blood cells shall be calculated. The density of Plasmodium in red blood cells shall be calculated according to the following formula:
Plasmodium density in red blood cells = the infection rate of red blood cells×red blood cell count /μL blood.(https://www.daowen.com)
If the parasite density is high, 1,000 red blood cells can be examined. However, when the parasite density is low, more red blood cells should be examined. The number of red blood cells to be examined can be determined according to the formula N=45.5× (I-P)/P. Therein N is the number of red blood cells that need to be examined, P is the number of malaria parasites in 10,000 red blood cells, I is the unit of blood sample (calculated in terms of 10,000 red blood cells), 45.5 is a constant. In addition, the infection rate of red blood cell in different regions of the blood film is quite variable. Usually, the density of Plasmodium in the posterior and marginal parts of the blood film is often higher than that in the front half or the central part. As a consequence, microscopic examination should not only be aimed at one corner, but along the horizontal axis of the slide to review.
6.4 Quality control of microscopic examination
Monitor the whole process involved in the microscopic examination of blood slides of Plasmodium, including the standard operating procedures o?f examination methods, required instruments and equipment, reagents, materials, the qualification and ability of microscopists, and systematically manage the microscopic examination activities and report compilation. The objective is to ensure the accuracy, reliability and stability of microscopic examination results.
The quality control of microscopic examination can be divided into internal and external quality controls.
6.4.1 Internal quality control
6.4.1.1 Establish the standard operating procedures for microscopic examination. At the same time ensure that the laboratory microscopists strictly abide by the standard operating procedures for microscopic examination.
6.4.1.2 Establish a supplier catalogue of major instruments, equipment, reagents and materials involved in microscopic examination. Carry out a regular maintenance of the microscopy. Make standard blood slides using malaria positive blood to do a performance qualification for reagents purchased to ensure their effectiveness. Establish an account for the use of instruments and the preparation of reagents.
6.4.1.3 Establish the account records of the microscopic examination, which should include the details of the blood slides making, the preparation of dye solution, the results of microscopic examination, the species and density of parasite, the microscopist, and the date, etc.
6.4.1.4 Confirm the qualification of the microscopists and offer regular trainings to them to ensure that the microscopists who carry out the microscopic examination will be trained and certified in the past two years and have the corresponding qualifications.
6.4.1.5 Regularly carry out the proficiency testing for the microscopists to qualitatively evaluate the ability to ensure the accuracy of the daily microscopic examination results.
6.4.1.6 Establish a mechanism for rechecking the results of microscopic examination, such as cross-microscopic examination of blood slides or double-check by two microscopists. If there are differences between two microscopists for the same blood smear, they should be checked by expert microscopists. Sampling and reviewing: The expert microscopists in the laboratory shall randomly check the blood slides that have been completed on a regular or on irregular basis, calculate the agreement of the microscopic examination results to evaluate the quality of the microscopic examination.
6.4.1.7 Report and feedback of test results.
6.4.1.8 Establish a disposal mechanism for out-of-control of microscopic examination. The possible influencing factors can be traced and corrected when errors occur in microscopic examination.
6.4.2 External quality control
6.4.2.1 Regularly carry out outreach training and supportive supervision (OTSS) activities to evaluate the overall operation of the laboratory and all links. In addition, overall testing ability of the laboratory shall improve through supportive supervision.
6.4.2.2 Participate in regional or national laboratory network, regularly participate in external quality assessment to evaluate laboratory testing ability.
6.4.2.3 Regularly participate in external blind sample testing organized by relevant departments to evaluate the testing ability of microscopists.
6.4.2.4 The relevant departments at national or provincial level or a third party shall regularly verify the microscopic examination results of the laboratory, randomly pick out the samples already tested in the laboratory in proportion to evaluate the agreement of the test results.