Estudio comparativo de la morfología de sangre periférica analizada mediante el microscopio y el CellaVision DM96 en enfermedades hematológicas y no hematológicas / Comparative study of peripheral blood morphology in manual microscopy and the CellaVision DM96® in hematological and non-hematological diseases

Introducción. La revisión de la citología de sangre periférica es un punto de partida imprescindible para el diagnóstico de la mayoría de las enfermedades hematológicas, e incluso no hematológicas. Objetivo. Evaluar la concordancia entre los resultados obtenidos al realizar el recuento diferencial leucocitario mediante el sistema de análisis digital CellaVision DM96 (DM96) y el microscopio óptico convencional. Material y métodos. Se analizaron 234 extensiones de sangre periférica, de pacientes del Hospital Clínic de Barcelona con cifras de leucocitos entre 1,12 y 282×109/L. 177 preparaciones correspondieron a pacientes con enfermedades hematológicas. Se compararon los porcentajes de neutrófilos, bandas, eosinófilos, basófilos, linfocitos, monocitos, células linfoides reactivas, metamielocitos, mielocitos, promielocitos, blastos, células plasmáticas y eritroblastos PRE y POST obtenidos con el DM96 y al microscopio. Resultados. La correlación de los resultados del DM96 PRE con respecto al microscopio fue excelente para neutrófilos, linfocitos, monocitos, y blastos (r>0,87<0,94 y p<0,0001) y aceptable para bandas, eosinófilos, basófilos y células plasmáticas (r>0,74<0,81 y p<0,0001). Después de la reclasificación celular, los coeficientes de concordancia fueron excelentes (> 0,7) para promielocitos y mielocitos, intermedios para células linfoides reactivas y eritroblastos (> 0,5 y<0,7), y bajos (< 0,5) para los metamielocitos. No se observaron falsos negativos en la detección de blastos por el DM96 (97 casos). Con excepción de las células linfoides reactivas y blastos linfoides, el equipo no preclasificó otras células linfoides atípicas, que debieron ser identificadas por el citólogo. Conclusiones. El análisis morfológico de sangre periférica mediante el equipo CellaVision DM96 muestra una buena concordancia con respecto al microscopio, y representa un avance tecnológico para el laboratorio de Hematología con un número elevado de muestras. Tiene ventajas adicionales, tales como mejorar las condiciones ergonómicas, mayor rapidez, asegurar la trazabilidad y facilitar la docencia (AU)

Introduction. Differential leukocyte counts of peripheral blood cells are an important diagnostic tool. Objective. We evaluated the CellaVision DM96 (CellaVision AB, Lund, Sweden), an automated system for digital peripheral blood cell analysis. Material and methods. We analysed 234 blood films in which leukocyte values were from 1.12 to 282×109/L. A total of 177 blood films were from patients with hematological diseases. Results. Correlation coefficients between results obtained from the CellaVision DM96 pre-classification and by conventional direct microscopy were excellent for segmented neutrophils, lymphocytes, monocytes and blasts (r>0.87<0.94 and P<.0001) and good for band neutrophils, eosinophils, basophils and plasma cells (r>0.74<0.81 and P<.0001). After the reclassification of the cells, very good concordance coefficients were observed for promyelocytes and myelocytes (> 0.7), intermediate for reactive lymphocytes and erythroblasts (>0.5 and<0.7) and low (<0.5) for metamyelocytes. Whatever the pathology and the number of blasts on the films, all 97 patients were positive for blast detection on the DM96. Pathological cells such as prolymphocytes, large granular lymphocytes, hairy cells, Sézary cells and other atypical lymphocytes were reclassified by the user. Conclusions. Advantages of the CellaVision DM96 over direct microscopy include, requires less time than manual differentiation, is a good tool for educational purposes, improve the traceability of the results, and can have an important role in a modern Hematology Laboratory (AU)

Mean platelet component as an indicator of platelet activation in foals and adult horses.

BACKGROUND: Mean platelet component (MPC) is a new platelet variable, measured by modern commercial complete blood count analyzers, that is reduced during platelet activation in humans and small animals. HYPOTHESIS: MPC decreases in horses with clinical conditions that cause platelet activation and disseminated intravascular coagulation (DIC). ANIMALS: We obtained 418 CBCs from 100 sick and 20 healthy neonates and 178 sick and 45 sound adult horses. Sick neonates were classified into septic and nonseptic, and DIC and non-DIC groups. Adults were grouped by diagnoses (systemic inflammatory disorders, gastrointestinal problems, and thrombocytopenia). METHODS: MPC together with platelet count, mean platelet volume, platelet distribution width, and platelet component distribution width were measured with a commercial analyzer and compared between the different disease and control groups in neonates and in adults. RESULTS: MPC values were significantly lower in the septic and nonseptic neonates (24.0 +/- 3.5 g/dL and 26.6 +/- 2.6 g/dL, respectively) than in the control group (28.1 +/- 1.7 g/dL). Neonates with DIC had the lowest MPC values (23.8 +/- 6.3 g/dL). MPC values in adult horses were significantly lower in the inflammatory (23.5 +/- 4.7 g/dL), gastrointestinal obstruction (23.0 +/- 5.0 g/dL), enteritis (23.6 +/- 4.6 g/dL), ischemic (23.9 +/- 5.1 g/dL), and thrombocytopenia (20.2 +/- 5.7 g/dL) groups when compared with control horses (26.2 +/- 3.5 g/dL). Other platelet variables were not different between the control and the disease groups. CONCLUSION AND CLINICAL IMPORTANCE: MPC might be a useful variable for quickly and easily detecting platelet activation in sick neonates and adult horses.

Effects of etamsylate on equine platelets: in vitro and in vivo studies.

The aim of this study was to investigate whether etamsylate produces equine platelet activation. In vitro and in vivo studies were designed in which seven and eight adult healthy horses were included, respectively. In the in vitro study, citrated blood was incubated with different concentrations of etamsylate, and P-selectin expression and annexin V binding were determined by flow cytometry. In the in vivo study, blood was collected before and 1 and 2h after IV administration of etamsylate, and P-selectin expression was evaluated. In the in vitro study, a significant increase in P-selectin expression, leukocyte-platelet aggregate formation and annexin V binding were observed. In the in vivo study, a marked increase in P-selectin expression and heterotypic aggregate formation was seen in two and five horses, respectively, although no significant differences were detected when analyzing results from all the animals together. The results of the in vitro study indicate that etamsylate produces a pre-activation state in equine platelets, but this fact could be confirmed by the in vivo study.

Assessment of platelet function in horses: ultrastructure, flow cytometry, and perfusion techniques.

We studied equine platelet function and activation using ultrastructural examination, flow cytometry, and perfusion. The main aim of the study was to evaluate hemostatic mechanisms in horses using these techniques. Ultrastructural observations were done on resting and activated platelets. Flow cytometry was used to evaluate binding of antibodies to major platelet glycoproteins (GPIIb-IIIa, GPIV, and GPIb) and activation-dependent antigens (P-selectin and lysosomal integral membrane protein [LIMP]). Perfusion techniques were used to evaluate the interaction between platelets and damaged subendothelium. Aggregation experiments were done to identify the best agonists for flow cytometry. Ultrastructural observations confirmed that equine platelets lack a developed open canalicular system and that release of granule contents occurs by fusion of adjacent granule membranes that ultimately connect with external membranes. Flow cytometry identified a 2-fold increase in binding of antibodies against GPIIb-IIIa and GPIV after activation. Binding of antibodies against P-selectin and LIMP increased from 2.12 and 1.74% to 15.5 and 11.6%, respectively, in response to thrombin and to 21.86 and 10.50%, respectively, in response to collagen. Annexin V binding increased moderately after activation. Perfusion experiments with citrated blood indicated that equine platelets react more strongly to subendothelium than do human platelets. When blood was anticoagulated with low molecular weight heparin, a marked impairment of platelet interactions was observed. In conclusion, although some differences were observed between human and equine platelet function, some techniques currently used to assess human platelet function may be useful to assess equine platelets.