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Evaluation of the Blood Smear

Blood film review by a trained professional is critical to produce complete and accurate results of the patient’s hemogram. With the advancement of technology with our automated analyzers, many of the manual tasks of a complete blood count can be performed quickly by this equipment. However, there are limitations to an analyzer’s ability, and their generated values should be confirmed by a trained individual. In addition to reviewing a blood smear, the RVT should be familiar with the function and limitations of their analyzer and compensate for those appropriately.


A properly made blood smear creates the foundation for an accurate examination. This ensures consistent cellular distribution and minimizes cell distortion. It is important to note that this review is not intended to be a tedious task, but a manual confirmation of the automated data generated by the analyzer. An RVT proficient in this task can perform the review efficiently in a minimal amount of time. The complete review should take no more than two to three minutes.


Begin by examining the smear under 100X magnification, using the 10X objective lens. Three areas of the film should be scanned: the feathered edge, the monolayer and the body of the film. Larger microscopic structures will be pushed out to the feathered edge. The RVT scans here for evidence of platelet clumping, or the clumping of leukocytes. Microfilaria, if present, may be identified in this area of the smear. Within the monolayer of the film, the density of the erythrocytes and the leukocytes are observed and compared with the totals counts provided by the analyzer. Moving into the body of the film, the cellular concentration is higher. The arrangement of the cells is observed in the body of the smear. Agglutination appears as clumps of red cells, while rouleaux is noted when the red cells are stacked, and create a chain-like pattern, or “stack of coins” (Table 2).


Figure 3: Eosinophilia in a feline patient (eosinophils shown with arrows) viewed under 400X magnification

Following the low power review, the 40X objective is moved into place, and the smear is scanned under 400X magnification. With a closer view of the cells, the leukocyte differential generated by the analyzer is validated. A full differential count is not performed, rather an estimation of the count is made, and trends can be confirmed. For example, if the analyzer had reported a neutrophilia or eosinophilia, this trend would be evident on the film by the presence of high numbers of neutrophils or eosinophils, respectively (Figure 3). These cells should be common, and quickly identified by the RVT. Under this level of magnification, the morphologies of the erythrocytes, leukocytes, and thrombocytes can also be scanned.

The cellular morphology can give insight into a potential disorder, the severity of the disease, or provide clues to the causes of the symptoms present in the patient. Automated analyzers cannot provide this information in depth, and cannot replace a trained eye in this instance. Some examples of erythrocyte morphologies that are reviewed include the evaluation of chromasia (cell color), anisocytosis (cell size) and poikilocytosis (cell shape). The presence of cellular inclusions and parasites should also be noted, if they are found. (Table 2) Examples of leukocyte morphologies that are noted include the degree of segmentation of the neutrophils (ex. band neutrophils vs. hypersegmentation), or the staining intensity of the lymphocytes (ex. reactive lymphocytes). (Table 3)

Figure 4: Megathrombocytes (red arrows) and mild platelet aggregate (blue arrow) (feline sample, 400X magnification)

The thrombogram is evaluated for the presence of megathrombocytes; any platelet clumping can also be further investigated. (Figure 4) Megathrombocytes can pose a problem for impedance-based analyzers. The technology used with these analyzers requires cells to be classified according to their size. If the size of a platelet overlaps with the size of an erythrocyte, as seen with megathrombocytes, the accuracy of the count is affected. In these instances, a manual platelet estimate can be performed on the blood smear.


The final step in the review requires the use of oil immersion under 1000X magnification. A full platelet estimate could be performed, or at a minimum, the platelet count should be validated. A finding of 8-10 platelets per field at this magnification is normal. Any closer examination of erythrocyte morphology that may be required can also be done at this time.


TABLE 2: Example of erythrocyte morphologies and their significance (non-exhaustive)

Figure 5: Schistocytes from a canine patient with hemangiosarcoma (1000X magnification)

TABLE 3: Examples of leukocyte morphologies and their significance (non-exhaustive)


As mentioned previously, the intent of the blood smear review is to validate the reported results of the analyzer, and to note findings that analyzers may be unable to determine. The review should be quick and efficient. It is recommended to review all blood samples for the following reasons: performing the activity regularly will allow the RVT to become more proficient at the task, and improve their speed and efficiency. The saying “practice makes perfect” certainly holds true in this case. By reviewing many samples, what appears as “normal” in healthy patients becomes ingrained in one’s mind, causing abnormal findings to be more obvious when encountered. The RVT will also become more skillful in preparing a blood smear of diagnostic quality, since they are preparing them regularly for each review.


The preparation of a blood film and subsequent microscopic examination is a task that should be completed as part of a standard protocol in practice. While automated analyzers can certainly alleviate much of the labour required to process many blood samples, they cannot fully replace a trained RVT. Results reported as part of a complete blood count should be just that – complete, and this includes a full description of cell populations and morphologies, regardless of the type of analyzer used. It is part of a best-practice protocol that should be implemented, to assist in the delivery of high-quality care to the patient.

 

References:

  1. Adewoyin, A S, and B Nwogoh. “Peripheral blood film - a review.” Annals of Ibadan postgraduate medicine vol. 12,2 (2014): 71-9.

  2. Metzger Jr, Fred, DVM DABVP, and Alan H. Rebar DVM PhD DACVP. "Three-minute peripheral blood film evaluation: Preparing the film." (2004).

  3. Gulati, Gene et al. “Purpose and criteria for blood smear scan, blood smear examination, and blood smear review.” Annals of laboratory medicine vol. 33,1 (2012): 1-7.



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