In-clinic veterinary hematology has seen tremendous advances in recent decades, bringing added expertise to our day-to-day practice. Long gone are the days when in-clinic labs were limited to spun PCVs and manual microscopy as their only diagnostic tools. Practices now have access to affordable point-of-care hematology analyzers, many of which share some of the same advanced technologies present in reference lab systems. Such advances have automated many aspects of blood cell analysis, leading to faster turnaround times and more reproducible results with less hands-on technician time. Having this advanced technology in veterinary clinics allows for more in-depth and accurate complete blood count (CBC) reports, contributing to more comprehensive and rapid diagnoses, timely and targeted treatment options, and, when repeated, sensitive monitoring of responses to interventions. In-clinic hematology analysis The key to maximizing in-clinic hematology testing is having accurate, detailed information and an advanced understanding of its interpretation. We are all familiar with the numerical CBC data generated by automated hematology analyzers. There is less awareness and understanding, however, of the cytograms, or graphical representations of the cell analyses, that accompany the CBC report. These various histograms (curvilinear tracings of bar graphs) and scattergrams (dots plotted on 2D graphs) offer deeper insights into the cellular morphology and distribution that go beyond the basic counts–providing visual confirmation of numerical results, identifying subtle but significant abnormalities, and supporting diagnoses even before further investigations, like peripheral blood smears, are performed. The key to maximizing the value of individual cytograms is understanding the underlying technology contributing to their development. Impedance technology and histograms Electrical impedance is a traditional cell counting technology and remains a common methodology used in current in-clinic hematology analyzers. The foundation of impedance-based counters is the Coulter principle (Figure 1), in which cells are passed individually through a small aperture across which there is a measured charge differential. The electrical signal is disrupted by each cell’s presence, generating a pulse. The enumeration of those pulses determines the number of cells per fluid volume, thus providing an accurate measurement of cell concentrations. Further, the amplitude of each pulse is proportional to the volume of each cell, and this is used to determine the cells’ identity. Figure 1. Illustration of the Coulter principle used for impedance-based cell counting. When a blood cell in suspension passes through a small aperture separated by two electrodes, this creates a change in impedance proportional to the cell volume, enabling the enumeration of different cell types. Source: DeNicola DB. 2011. “Advances in Hematology Analyzers.” Topics in Companion Animal Medicine 26 (2): 52–61. Hematology histograms are graphical representations of the blood cell populations counted and measured via impedance analyzers (Figure 2). They can be reviewed by trained doctors and technicians to verify the reported cell counts and classification and help recognize underlying pathologic processes. Differentiation of cells based solely on size, however, can result in inaccurate cell classification, especially in veterinary medicine. Even in health, cats’ platelets (generally the smallest cell type) are more variable in size and shape, and their red blood cells are generally smaller compared with those of other species. That potential overlap in size can hinder accurate platelet and erythrocyte counts. Figure 2. Platelet (PLT) histograms with cell size in femtoliter (fL) on the x axis and relative number of cells is along the y axis. A healthy dog PLT histogram (A) shows a single platelet peak on the left with clear nadir separating the platelet population from the start of a larger erythrocyte peak starting on the right of the graph. Clear separation of the peaks on the PLT histogram provides confidence in the impedance analyzer’s ability to accurately identify and enumerate platelets for this patient. A healthy cat PLT histogram (B) shows the platelet peak on the left, but owing to more variable, often larger platelets and smaller erythrocytes, there is size overlap between these two cell populations and thus impedance technology alone may struggle to confidently distinguish, and thus accurately enumerate, feline platelets and erythrocytes. Additionally, differentiation among the various white blood cells can be difficult via impedance technology, even with the addition of lysing agents that differentially affect various cell lines to more accurately determine the WBC differential. Another limitation of impedance technology is that accuracy suffers when cells are pathologically altered in their size and structure, for instance left-shifted or toxic neutrophils and reactive or immature large lymphocytes. Flow cytometry and scattergrams Flow cytometry is a newer technology utilized in more advanced in-clinic hematology analyzers (Figure 3). The basic principle is the use of laminar flow to direct blood cells individually past a focused laser, and the individual cells absorb and scatter light as they travel through. Information about the light scatter is recorded at multiple angles, providing data on cell size, nuclear size and shape, and cytoplasmic complexity. The end result is a unique signature of data points for each interrogated cell that assists with more accurate cellular identification. Figure 3. Principle of flow cytometry using laser light scattering to analyze blood cells. A blood cell suspension is focused into a single-cell stream that is interrogated by a laser beam. As each cell passes through the laser, light is scattered in different directions, capturing information about the cell size and internal complexity or granularity. Photodetectors capture these signals for each cell, allowing for classification and enumeration. Source: Noris, P., & Zaninetti, C. (2017). Platelet counting and measurement of platelet dimensions. In P. Gresele, N. Kleiman, J. Lopez, & C. Page (Eds.), Platelets in thrombotic and non-thrombotic disorders (pp. 571–587). Springer. The collected data from the laser analysis can be used to create graphical depictions of the interrogated cells, called scattergrams (Figure 4), based on their measured coordinates from scattered light capture. When cells are populated in distinct clusters, this lends confidence to the reported cell counts and differentials (Figure 4A). Changes in cell clusters deviating from those of healthy pets provide sensitive and objective information about underlying pathologic changes. Additionally, there are numerous common scattergram patterns that, with training, allow the user to obtain significantly greater diagnostic information from abnormal cell populations. Figure 4. Annotated WBC scattergrams generated from flow cytometric analysis with added fluorescence staining. Each dot is an analyzed cell that is plotted on the graph, with cellular complexity (determined by side scatter, SS) assessed on the x axis and fluorescence (FL) on the y axis. A healthy dog WBC scattergram (A) shows relatively distinct clouds of same-color dots indicating confident identification and enumeration of the various WBC populations portrayed (identified by colored circles). The WBC scattergram from a sick dog (B) with increased bands (an inflammatory left shift) is identified as an extension of the turquoise cloud higher on the y axis (circled in white) as a reflection of the increased number of larger (more immature) neutrophils (i.e. bands). The addition of cytochemical and fluorescent staining in some analyzers provides more discernible separation between red blood cells and platelet populations on the RBC scattergram and an enhanced white blood cell differential on the WBC scattergram. The additional fluorescence information provides sensitive and precise identification and enumeration of reticulocytes (immature, non-nucleated red blood cells), reflecting an objective measurement of the bone marrow’s response to a peripheral demand for more red blood cells needed for proper characterization of anemia. Further, the RBC scattergram reveals information about reticulocyte size and their chronological development, which may provide important clinical insights, even in non-anemic patients. The additional fluorescent staining affords an enhanced white blood cell differential on the WBC scattergram. The resulting, more discriminating, five-part white blood cell differential allows for more accurate leukogram interpretations. Importantly, the generated WBC scattergram affords a visual identification of band neutrophils (Figure 4B), which is key in the identification of inflammation in sick patients. Further, once identified, trending of band neutrophils on the WBC scattergram provides sensitive feedback about a patient’s response to treatment or early identification of unanticipated complications. Cytograms in action Smooch is a five-year-old M/N domestic shorthair cat that presented with vomiting and anorexia. A CBC revealed a normal total leukocyte count (WBC=5.97; RI 3.46-17.50 109/L) and a normal neutrophil count (NEU=5.33; RI 1.95-11.50 109/L). Review of Smooch’s WBC scattergram (Figure 5A), however, and comparison to a healthy cat WBC scattergram (Figure 5B), identified the presence of increased bands (i.e. a left shift) supporting significant inflammatory disease that was, through additional imaging, attributed to a gastrointestinal foreign body. Figure 5.Smooch’s WBC scattergram (A), in comparison to a healthy cat WBC scattergram (B), identifies the presence of bands (white circle) supporting significant inflammatory disease. Smooch’s PLT histogram (C) identifies suspected platelet clumping, as indicated by the lack of an expected valley between the PLT and erythrocyte peaks along the curvilinear tracing that is present (arrow) in a PLT histogram from a healthy cat sample that lacks platelet clumping (D). Additionally, Smooch had a reported thrombocytopenia (PLT=99; RI 140-595 109/L). However, a review of Smooch’s PLT histogram (Figure 5C) identified suspected platelet clumping that was confirmed on blood film review. Information from the cytograms accompanying Smooch’s CBC provided essential diagnostic information that was not evident in the numerical data alone and guided appropriate ancillary testing and case management. Maximizing diagnostic information In-clinic hematology has evolved from simple cell counts to sophisticated, data-rich reports with increased accuracy and added clinical insights. The displayed cytograms are an invaluable tool, depicting objective information about tens of thousands of analyzed cells, providing validation of the numerical results, and offering insights into morphology changes not evident in the CBC numbers alone. As veterinary teams grow more adept at integrating these visual analytics into their daily practice, the opportunity for more timely and accurate diagnoses and improved patient care will grow. Holly Brown, DVM, PhD, DACVP, began her veterinary career in small animal practice and then transitioned back to academia for specialty training and her next role as a young faculty member. Dr. Brown then returned to practice, where she joined a large hybrid hospital as an on-staff clinical pathologist, contributing her diagnostic expertise on the patient side. Brown has always been passionate about providing continuing education around maximizing laboratory diagnostics, and she currently serves as chief veterinary educator for Antech Diagnostics.
