Sharon M. Dial Evaluation of a buffy coat has in the past been mostly associated with evaluating or staging canine mast cell tumors or systemic mastocytosis in the cat. It is now known that inflammatory diseases of the gastrointestinal or reproductive tract and hypersensitivity reactions are the most common causes of circulating mast cells in the dog and reviews of buffy coat preparation are less frequently done. Because of the focus on mast cell disease and buffy coat examination, the use of buffy coat evaluation for detection of blood‐borne infectious agents and to concentrate atypical cells present in low numbers on the blood film for further evaluation has been neglected. This procedure allows the concentration of leukocytes and platelets for further evaluation. Atypical cells and blood‐borne infectious agents that are often present in low numbers on a routine blood film will be concentrated in the buffy coat layer of the hematocrit tube. Circulating microfilaria will be concentrated just above the buffy coat and can be easily visualized by microscopy prior to breaking the tube and preparing the buffy coat film. Gross evaluation of the buffy coat can quickly provide visual information on the number of leukocytes and/or platelets present. The buffy coat is the small layer of leukocytes and platelets that concentrates above the red blood cell (RBC) column when whole blood is centrifuged in a microhematocrit tube. Gross inspection of the buffy coat should be a part of the evaluation of any blood sample centrifuged for a PCV and plasma protein evaluation. The preparation of a buffy coat slide is done when other tests suggest that close evaluation of leukocyte populations for atypical cells or infectious agents may be helpful or for submission of a sample for infectious disease polymerase chain reaction (PCR) or immunofluorescent tests. Buffy coat samples can be useful in dogs suspected of having canine distemper and cats for confirmation of persistent infection with feline leukemia virus. When the buffy coat appears grossly abnormal (larger or smaller than expected), performing this procedure in conjunction with the PCV and plasma TP will save time and inform the interpretation of the complete blood count as a whole. Visual inspection of the height of the buffy coat, the plasma just above the buffy coat, and preparation of a buffy coat film are all useful tests to assist in the interpretation of the complete blood count in an ill patient. Visual inspection of the height of the buffy coat can provide a heads up that the patient may have significant changes in leukocyte and platelet numbers prompting submission of a blood sample for an automated complete blood count and close evaluation of the blood film. The height of the buffy coat is a rough estimate of the combined leukocyte and platelet mass and can indicate significant leukocytosis or leukopenia and thrombocytosis or thrombocytopenia. Examination of the buffy coat enhances the chance of identifying atypical hematogenous and non‐hematogenous cells, hemoparasites, and other blood‐borne infectious agents. They are also excellent preparations to submit for more specific tests for infectious agents (PCR, fluorescent antibody tests). Since air‐dried buffy coats retain their staining quality for days to weeks when stored properly, a buffy coat film prepared at the same time as recording the PCV and TP can be stored for future testing if needed, based on history, clinical exam findings, complete blood count, and other diagnostic tests. It is important to remember that any test that depends on the recognition of a specific antigen on the cells may have a limited storage time since some antigen proteins can degrade over time. Examples of the infectious agents that can be difficult to find because they are present in low numbers on a blood film but will be concentrated in the buffy coat will be discussed below. Buffy coat preparations in patients with relatively low numbers of atypical cells can be used for further evaluation using special diagnostic tests, such as immunocytochemistry, cytochemistry, and PCR testing, where a concentrated sample will provide sufficient cells for interpretation. Note: While it is tempting to perform a differential cell count on a buffy coat when the patient is leukopenic, the results will not be valid. The distribution of leukocytes in the buffy coat will be affected by the centrifugation process and how well the cells are distributed in making the slide. A more appropriate method of performing a differential cell count on a leukopenic sample is to place a temporary coverslip on the blood film and, using the 10× and 40× objectives, find and identify at least 50 or more cells. Preparing the buffy coat slide. Figure 5.1 The equipment for preparing a buffy coat preparation includes whole blood with EDTA anticoagulant, gloves, microhematocrit tube without anticoagulant, sealant, glass microscope slides. (not shown is the Romanowski stain set up and distilled water). Source: Courtesy of Jeremy Bessett. Figure 5.2 The buffy coat in this case is increased in size (greater than 1% of the RBC column). This sample is from a cat with severe anemia and a marked leukocytosis due to marked numbers of large undifferentiated leukocytes consistent with acute lymphoid leukemia. Source: Courtesy of Jeremy Bessett.
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Gross and Microscopic Evaluation of the Buffy Coat
5.1 Procedural Definition: What Is This Test About?
5.2 Procedural Purpose: Why Should I Perform this Test?
5.3 Equipment
5.4 Procedural Steps: How Do I Perform this Test?
