Morphologic, cytochemical, and ultrastructural features of gray eosinophils in nine cats

Gray or “vacuolated” eosinophils (GE) are commonly reported in the Greyhound and other sighthound dog breeds, and occasionally in nonsighthound dog breeds, but they have not been reported in the cat. 1,2 In the dog, the term “gray eosinophil” reflects the presence of variably sized, unstained granules resembling clear vacuoles. 2 Ultrastructural characterization of canine gray eosinophils shows a decrease, not only in the overall secondary granule size but also in the dense matrix and outer rim width, resembling changes ob-served in people with eosinophil peroxidase deficiency. 3 Functional abnormalities have not yet been found, and their distinctive cy-tologic appearance is thought to be due to a difference in granule staining properties, which appears more prominent with aqueous Romanowsky stains such as Diff- Quik. 1,2 Accurate recognition of these cells is important in peripheral blood film examinations Abstract Background: Gray eosinophils, resembling those in sighthound dog breeds, have not previously been reported in cats. Objectives: We aimed to provide a morphologic, cytochemical, and ultrastructural description of gray eosinophils in cats. Methods: Blood films examined as part of routine hematology profiles in cats from May 2015 to July 2018 were evaluated for the presence of gray eosinophils. When identified with modified Wright stain, cells were morphologically assessed and ad-ditionally stained with Diff- Quik, ALP, Luna, and Luxol fast blue stains and compared with feline controls. Two cases were prepared for transmission electron microscopy (TEM) and compared with a feline control. Results: Gray eosinophils were identified in 9 of 2641 cats during the study period. Compared with typical feline eosinophils, these cells contained abundant round granules instead of the characteristic rod- shaped specific granules. These granules lacked the characteristic intense pink/red staining with Romanowsky stains and did not stain with ALP, Luna, or Luxol fast blue stains. On TEM, the classical electron- dense core of these granules was replaced by a core with fragmented or amorphous internal material. Typical eosinophils were not identified in any cat in which gray eosinophils were identified. Conclusions: The distinct morphologic, cytochemical, and ultrastructural changes in gray feline eosinophils might be associated with a reduction or lack of major basic protein (MBP) in specific granule cores. Similar to canine gray eosinophils, accurate recognition of these cells is essential to prevent their misclassification as toxic neutrophils.

where these cells can be mistaken for toxic neutrophils, especially in sighthounds, which might also have a concurrently lower WBC or neutrophil count compared with other canine breeds. 4 Inaccurate classification (underestimation) by hematology analyzers has also been reported, although for dogs with eosinophil concentrations within reference intervals, this is unlikely to be clinically significant. 1 Following identification of suspected gray eosinophils in a routine feline hematology sample from the initial case, the aim of this project was to morphologically, cytochemically, and ultrastructurally describe these cells, not previously reported in cats, and determine their clinical significance.
Ethical approval for the project was obtained (URN 2019(URN 1928. A total of 4257 blood films from 2641 cases, stained with modified Wright stain (automated stainer; Hema-tek 2000, Bayer) from routine feline hematology profiles were evaluated between May 2015 and July 2018 for the presence of gray eosinophils. When gray eosinophils were identified, additional blood smears were made from EDTA blood for further cytochemical staining and analysis. Two cases and a feline control were selected for transmission electron microscopy (TEM) evaluation.
Blood films from cats identified with gray eosinophils and feline controls were stained with an aqueous Romanowsky stain and cytochemical stains for alkaline phosphatase (ALP), Luna, and Luxol fast blue as previously described. 5 For TEM analyses, buffy coat preparations from two cats with gray eosinophils and one control cat were fixed with 3% glutaraldehyde (Sigma-Aldrich). In brief, samples were postfixed in 1% osmium tetroxide and, following dehydration in ascending ethanol concentrations, were infiltrated with and embedded in Agar 100 resin (Agar Scientific Ltd). The samples were then cured for at least 20 hrs at 60°C. Subsequently, ultrathin sections (90 nm) were cut and then stained with 0.5% uranyl acetate and 3% lead citrate. The sections were evaluated in a transmission electron microscope (H7500, Hitachi) at an accelerating voltage of 80kV. Gray eosinophils were identified based on size, shape, and granule structure, after exclusion of neutrophils and basophils and visually compared with feline control eosinophils.
Over the 3-year study period, gray eosinophils were identified in a total of nine (of 2641) cats presenting to the Queen Mother Hospital for small animals (QMHA) at the Royal Veterinary College (RVC). Cats were comprised of five males (four neutered; one intact) and four females (one neutered; three intact) with ages that ranged from 2 months to 12.5 years. Domestic shorthair (4/9) cats were most commonly represented, with British shorthair (BSH) cats accounting for one-third of cases (3/9). There was also one Ragdoll (1/9) and one domestic longhair (1/9) cat. Presenting clinical signs were related to the underlying clinical diseases, which included congenital extrahepatic portosystemic shunts (3/9), neoplasia (2/9; lymphoma and hemangiosarcoma), gastrointestinal disease (IBD; 1/9), peripheral vestibular disease (1/9), and two cases lacking a definitive diagnosis. Hematologic changes were mild in the majority of cases and included mild lymphopenia (3/9) attributed to a stress/steroid response, microcytosis (2/9) associated with portosystemic shunts (PSS), mild thrombocytopenia (1/9), or were unremarkable (2/9). The remaining cat had a mild to moderate Heinz body hemolytic anemia. This study describes the morphologic, cytochemical, and ultrastructural features of specific granules in a population of cats with gray eosinophils, not previously described in this species.
Eosinophils in most species are identified on light microscopy by the "eosin-loving" properties of their granules, the characteristic staining pattern a result of the high cationic protein content. The most numerous and cationic of these proteins is the major basic F I G U R E 1 Photomicrographs showing a population of granulocytes with small, round, nonstaining granules, and pale blue cytoplasm; peripheral blood smear; modified Wright stain; Bar = 10 µm protein (MBP), which forms the crystalline lattice structure of the core of eosinophil (specific/secondary) granules. It is this core that gives the eosinophil its unique ultrastructural feature. [6][7][8][9] Eosinophil-specific granule abnormalities are occasionally Luna staining is commonly used for histologic identification of eosinophils in many species and is described as highly specific. 11 Biebrich scarlet, the chromatophore used within the Luna staining protocol, has a high affinity for basic proteins and readily highlights eosinophil cytoplasmic granules as a result of their basic nature. 6,12 The suspected lack or marked reduction in MBP content in the appearance. An improper fixation method also appears unlikely, given the repeatability of the findings and the structural integrity of other leukocytes. Eosinophil nuclei were intact without evidence of fragmentation or pyknosis, and specific granule limiting membranes remained well preserved.
The significance of gray eosinophils in cats is not fully understood. Functional abnormalities were not suspected, given that affected cats did not appear to have clinical signs associated with eosinophil dysfunction or deficiency. However, if the lack of MBP is confirmed, functional abnormalities, such as reduced parasite immunity similar to CF null mice, could be considered. 15 In summary, in this study, we described the morphologic, cytochemical, and ultrastructural features of gray eosinophils in a series of cats. While not currently expected to be of clinical concern, in contrast to gray eosinophils in sighthounds, cells exhibit distinct cytochemical and ultrastructural changes. The absence of the characteristic electron-dense core in specific granules is proposed as the cause of the features identified, with a decrease in or complete lack of MBP content suspected. Similarly, to sighthounds, accurate recognition of these cells is important to prevent their classification as toxic neutrophils and an association with inflammation.