The presence of unusual two RBCs patterns (so-called “quatrefoil RBCs,” qRBCs) on canine blood smears at Optical Microscope (OM) was seen during routine evaluation of CBCs. Two consecutive retrospective investigations were arranged including about 7,000 CBCs and clinical records and laboratory data from dogs showing qRBCs. Few samples with qRBCs were prepared for Scanning Electron Microscope (SEM). qRBCs were found in 6.89% (139 of 2016) and 8.47% (133 of 1569) of dogs and in 3.89% (154 of 3,958) and 4.47% (138 of 3,081) of CBCs (some dogs were tested more than once). Statistical analysis was significant for age groups (Chi squared, ), decreased total leukocyte and neutrophil counts (ANOVA, ), RBCs anisocytosis, polychromasia, and Howell-Jolly bodies (ANOVA, , <0.005, and <0.003, respectively). qRBCs were distributed in the area of feathered edge and at the smear side of body-feathered edge area in blood films. SEM ruled out the possibility of an optical illusion or an accidental overlap. qRBCs are associated with ageing of dogs, total leukocyte and neutrophil counts, and RBC anisocytosis, polychromasia, and Howell-Jolly bodies. Few hypotheses were discussed to explain the origin and meaning of this RBC arrangement. 1. Introduction Evaluating blood films whenever a CBC is requested validates data generated by hematology analyzers and provides information related to RBCs, WBCs, and platelets (PLTs). Evaluation of RBCs morphology is a critical step in blood smear evaluation adding important information [1, 2]. Morphologic evaluation of RBCs from a well-made blood smear can sensitively detect abnormalities in size and hemoglobin concentration as well as provide information about abnormal cell shape (poikilocytosis) and the presence of inclusions, not detectable even with the most advanced analysers. This procedure is generally underutilized by practitioners largely because of the lack of confidence in preparing a well-made blood film and in being unable to identify important abnormalities. However, no other hematology procedure provides more valuable information yet requires so little additional time (two-three minutes) and expense. Indeed, a good experience is needed to evaluate a blood smear and in particular “abnormal” cells [3, 4]. A variety of causes can modify RBC shape including regenerative response, altered metabolism (iron and lipid above all, but also electrolyte depletion), oxidative injury, immune-mediate damage, mechanical fragmentation, sepsis, toxins, and drugs, and also artifactual agents like temperature, humidity, and errors in smear
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