Thirdly, BCM is not recommended to be used for diagnostics of Plasmodium and Leucocytozoon infections. BCM fails to detect Plasmodium infections mainly because malaria parasites do not exflagellate when the blood is simply exposed to air, as is the case in all tested Haemoproteus species [ 40 , 41 ]. Haemoproteus parasites develop readily visible moving stages exflagellating microgametocytes and microgametes in preparations prepared using BCM Fig.
Intracellular blood stages of malaria parasites are often small and difficult to visualize in non-stained preparations [ 2 ]. Although the leucocytozoids were seen in a few samples Table 2 , they are often at low intensity and might be difficult to distinguish from leucocytes. In BCM, Leucocytozoon parasites can be readily identified mainly when gametocytes develop in fusiform host cells.
Such host-parasite complexes can be readily distinguished from all blood cells Fig. It is worth noting that although exflagellation of some Leucocytozoon spp. Absence of pigment granules in Leucocytozoon spp. Fourthly, buffy coat wet capillary preparations can be stained for further microscopical analysis, but we do not recommend using this material to perform any morphological analysis, particularly parasite species description.
The deformation of parasites after centrifugation was reported by Bennett [ 58 ] and we corroborate these findings. In Haemoproteus parasites, morphological changes might occur not only due to deformation during centrifugation, but also due to changes of mature gametocytes during sexual process and exflagellation that rapidly occur when the blood is exposed to air after withdrawal from birds Fig.
Another possible artefact, which might preclude microscopical examination in stained BCM preparations, might be due to the presence of high amounts of plasma, increasing the amount of protein and resulting in a dark pink staining of the background of the blood film.
Despite these disadvantages, BCM is useful for the rapid detection of infected birds, which is essential for parasite detection and for further use in precise studies without harming the avian host. When choosing a diagnostic method for parasite detection, it is important to known how sensitive and specific it is in comparison to other applied methods [ 63 ]. The present study shows that BCM can be recommended for use in diagnostic detection of Haemoproteus spp. In regard to Lankesterella infections, it is of relatively low sensitivity, but of high specificity Table 2.
However, BCM had the lowest sensitivity among encountered parasites for Leucocytozoon species despite of its high specificity. In regard to Trypanosoma and Plasmodium infections, we could not measure the BCM sensitivity because these parasites were only detected by one method, i.
BCM or ME, respectively. However, it is always desirable to reduce the time of sample screening during fieldwork, replacing the time-consuming techniques with less time-dependent methods, which certainly is the case with the BCM. The agreement between BCM and ME in detection of other parasites was lower than for Haemoproteus species; however, this study strongly indicates that the former method is markedly more sensitive in the diagnostics of Trypanosoma and microfilariae infections.
Results of diagnostics of human Plasmodium parasites using commercial kits containing capillary tubes with orange acridine were compared with data obtained using microscopical examination of Giemsa-stained thick blood films, which is considered the gold standard diagnose method for human malaria. Reported results were controversial; some studies showed a high sensitivity [ 74 , 84 , 85 ], while others reported a low sensitivity in the diagnostics [ 54 , 86 ].
The present study corroborates this conclusion, particularly because BCM provides opportunities to analyse samples and select birds for experimental studies more quickly than during ME. The quick detection of animals infected with parasites is often an important requirement in wildlife parasitology because it provides opportunities to minimize sampling time, resulting in less harm for individual animals and wildlife populations.
That is particularly true in experimental research with avian blood parasites, which need to be selected from wildlife populations, in which infection prevalence is low and many host individuals need to be tested before the appropriate infected animals are selected. This study shows that BCM is a quick, reliable and powerful diagnostics tool, which is recommended to be used for detection of infections in birds, particularly of Haemoproteus , Trypanosoma and microfilariae parasites.
This method is cheap, fast and simple to use, and thus is recommended for application during field studies even in remote areas. Importantly, this tool is sensitive for detection of blood parasite co-infections.
It could also be applied in studies of blood parasites in other vertebrates, with the aim to increase the speed of diagnosis and to rapidly initiate animal treatment and application of prophylactic measures in disease control.
All data generated or analysed during this study are included in this published article and its additional file. Molecular evidence for host specificity of parasitic nematode microfilariae in some African rainforest birds. Mol Ecol. PubMed Article Google Scholar. Avian malaria parasites and other haemosporidian. Google Scholar. Molecular characterization of the 18S rDNA gene of an avian Hepatozoon reveals that it is closely related to Lankesterella. J Parasitol. Microfilariae in Galapagos penguins Spheniscus mendiculus and flightless cormorants Phalacrocorax harrisi : genetics, morphology, and prevalence.
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Genetic diversity of avian blood parasites in SE Europe: cytochrome b lineages of the genera Plasmodium and Haemoproteus Haemosporida from Bulgaria. Acta Parasitol. Ecology and dynamics of the blood parasite, Hepatozoon tuatarae Apicomplexa , in tuatara Sphenodon punctatus on Stephens Island, New Zealand.
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Nested cytochrome b polymerase chain reaction diagnostics underestimate mixed infections of avian blood haemosporidian parasites: microscopy is still essential. A restriction site to differentiate Plasmodium and Haemoproteus infections in birds: on the inefficiency of general primers for detection of mixed infections.
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J Clin Microbiol. Parasitol Int. Comparative study of modified quantitative buffy coat and two rapid tests in comparison with peripheral blood smear in malaria diagnosis in Mumbai, India. Different types work together to identify foreign substances, eliminate threats to the body, and build a sustainable defense system against future infections. Platelets are the first responders of the human body, rushing to injured areas and clotting blood flow through adhesion.
Both WBCs and platelets are present and prevalent in the buffy coat. The high concentration of WBCs and platelets make the buffy coat a critical bio-fluid in the medical research field, enabling academic professionals to conduct experiments and study these essential cells and how they work in the body — which in turn can shape the direction of medicine and effective patient treatment.
Beyond its implications in research, the buffy coat also has a practical application in testing for certain diseases and treating patients. Because key immune cells concentrate in this layer, the buffy coat can be used diagnostically when looking for the presence of harmful diseases such as malaria.
The platelets can also be further purified and used therapeutically to increase counts in patients who may have low platelet levels. Blood banks often separate samples of whole blood to isolate the buffy coat and store it for therapeutic use in individuals who could need this type of therapy to treat their conditions. The term buffy coat is often used interchangeably with PBMC, however, there are minor differences in cell composition that can make a large difference.
A buffy coat is a mix of lymphocytes, monocytes, granulocytes, and platelets, isolated from plasma and RBCs by centrifugation. PBMCs, on the other hand, are individual fragmented lymphocytes and monocytes that separate from the rest of the whole blood sample through a process called density-gradient centrifugation. This process involves an additional density gradient that separates sample particles further based on their size and density.
By separating out granulocytes and platelets and creating a barrier between the WBCs and the RBCs, the sample is comprised of a more concentrated sample of monocytes and lymphocytes that can then be used for research or further isolated for specific downstream applications.
Although the contents of a buffy coat already exist within a whole blood sample, they are not aggregated until centrifugation occurs. Before centrifugation, researchers should take a variety of precautions to ensure the extraction process is successful. STREK tubes are commonly used for research looking to isolate DNA from blood, while EDTA tubes contain an EDTA anticoagulant used for processing whole blood samples without clotting, allowing for centrifugation into the different layers — including the buffy coat layer.
The prepared whole blood sample is placed into a centrifuge to fractionate the buffy coat and separate it from the plasma and RBC. Using a small pipette, the experimenter collects the buffy coat and moves it to a separate container. The target is to concentrate the leukocytes approximately 5-fold while maintaining an equivalent ratio of leukocytes to RBCs e. Document PR Version 1. Related Resources. Choosing a Cell Separation Method that Meets Your Research Needs Learn more about the cell separation methods outlined above to choose the best method for your application.
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