Clinical and clinicopathological features and outcomes of cats with suspected dietary induced pancytopenia

Background: After a strong epidemiological link to diet was established in an outbreak of pancytopenia in cats in spring 2021 in the United Kingdom, 3 dry diets were recalled. Concentrations of the hemato- and myelotoxic mycotoxins T-2, HT-2 and diacetoxyscirpenol (DAS) greater than the European Commission guidance for dry cat foods were detected in the recalled diets. Objectives: To describe clinical and clinicopathological findings in cats diagnosed with suspected diet induced pancytopenia. Animals: Fifty cats presenting with pancytopenia after exposure to a recalled diet. Methods: Multicenter retrospective case series study. Cats with known exposure to 1 of the recalled diets were included if presented with bi- or pancytopenia and underwent bone marrow examination. Results: Case fatality rate was 78%. Bone marrow aspirates and biopsy examination results were available in 23 cats; 19 cats had a bone marrow aspirate, and 8 cats had a biopsy core, available for examination. Bone marrow hypo to aplasia—often affecting all cell lines—was the main feature in all 31 available core specimens. A disproportionately pronounced effect on myeloid and megakaryocytic cells was observed in 19 cats. Myelofibrosis or bone marrow necrosis was not a feature. Conclusion and Clinical Importance: Mycotoxin induced pancytopenia should be considered as differential diagnosis in otherwise healthy cats presenting with bi- or pancytopenia and bone marrow hypo- to aplasia

Clinical, biochemical and immunological aspects of the canine C-reactive protein

Das cCRP spielt als major Akute-Phase-Protein beim Hund eine wichtige Rolle bei der Entstehung und Regulation von Entzündungsprozessen. Die Bildung und Freisetzung durch Hepatozyten aufgrund eines Interleukin-Stimulus folgt dem Entzündungsreiz innerhalb von 4 Stunden. Dieser schnelle Anstieg bei akuten Entzündungsreaktionen zeichnet CRP als nützlichen Biomarker für die Überwachung von Patienten und Therapieerfolgen aus – sowohl beim Hund wie auch beim Menschen. Ein Ziel der Arbeit war daher die Evaluierung verschiedener Schnelltestsysteme für die Messung des cCRP. Grundvoraussetzung, um gute diagnostische Testverfahren zu entwickeln, ist eine möglichst genaue Kenntnis über den zu untersuchenden Parameter. Daher war ein weiteres Ziel der vorliegenden Arbeit, den aktuellen Kenntnisstand über die strukturellen Eigenschaften des cCRP-Proteins sowie seine diagnostischen Einsatzmöglichkeiten zu erweitern. Da es sich bei dem cCRP-Protein um einen so genannten „Real-time“-Biomarker handelt, ist die Messung besonders bei Patienten in akuten Notfallsituationen diagnostisch wertvoll. Um solch eine Diagnostik durchführen zu können, sind entsprechende Schnelltestgeräte notwendig, die für den klinischen Einsatz geprüft sind. Im ersten Teil der Arbeit wurden daher drei verschiedene quantitative Schnelltestgeräte (TECO ©dogCRP-quant, EUROLyser solo cCRP, LifeAssays® canine CRP) für die Messung der cCRP-Konzentration in Serumproben von caninen Patienten der Klinik und Poliklinik für kleine Haustiere der Freien Universität Berlin evaluiert. Die Geräte wurden hinsichtlich ihrer Genauigkeit durch statistische Auswertung mit der Referenzmethode (ELISA) verglichen und darüber hinaus die Praktikabilität im klinischen Alltag bewertet. Dabei zeigten sich deutliche Unterschiede in Bezug auf die Genauigkeit und Reproduzierbarkeit der Ergebnisse (TETECOmedical 69%, TEEUROLyser 28,2%, TELifeAssays 30,3%), jedoch ist die Verwendung aller drei Schnelltestgeräte im klinischen Alltag möglich. Auch die Anwendbarkeit für Verlaufskontrollen ist möglich, allerdings ist hier zu beachten, dass immer dasselbe Testsystem verwendet werden sollte, da sich alle Geräte bezüglich Abweichung der absoluten cCRP-Konzentrationswerte unterscheiden. Der in einem dieser Schnelltestgeräte (TECOmedical) eingesetzte, neu entwickelte anticCRP- Antikörper wurde in biochemischen Analysen hinsichtlich seiner Qualität untersucht und auch für die meisten immunologischen Nachweisverfahren in dieser Arbeit verwendet. Dabei zeigte sich zum einen seine speziesspezifische Detektion von CRP bei Hundeartigen und zum anderen eine unspezifische Bindung an ein bisher nicht näher charakterisiertes bakterielles Protein. Im zweiten Teil der Studie wurden weitere Details über die Struktur des cCRP-Proteins generiert. Die bisher bekannten Daten aus in silico Studien zur RNA- und Aminosäuresequenz konnten durch die durchgeführten biochemischen Analysen von gereinigtem, nativem Serum-cCRP größtenteils bestätigt und darüber hinaus neue Struktureigenschaften charakterisiert werden. Es wurde einerseits eine Verkürzung der primären Aminosäuresequenz von bisher angenommenen 223 auf 204 Aminosäuren im nativen cCRP festgestellt, was möglicherweise eine Bedeutung bei der Freisetzung des Proteins aus Hepatozyten spielt. Außerdem wurden die spezifischen Proteinmodifikationen bezüglich Glykosylierungen analysiert und eine Ähnlichkeit mit dem humanen CRPGlykosylierungsmuster festgestellt. In einem dritten Teilprojekt wurde aufgrund der immunologischen Ähnlichkeit des cCRPs die Verwandtschaft mit dem anderer Tierarten untersucht. Hier gelang der Nachweis von immunreaktivem Protein mittels des anti-cCRP-Antikörpers in Einzeltieren nur aus der Gruppe der Caniformia. Bei den übrigen untersuchten Tierarten der Unterfamilie der Eutheria war durch den Antikörper kein CRP detektierbar. Es wäre aufgrund immunologischer Gemeinsamkeiten denkbar, die diagnostischen Möglichkeiten der cCRP-Analyse der veterinärmedizinischen Praxis zukünftig auch auf Wildtiere aus der Gruppe der Caniformia auszuweiten.Canine C-reactive protein is a major acute phase protein in dogs that plays an important role in initiation and regulation of inflammatory processes. It is produced in hepatocytes after interleukin stimulus and is released in the circulation where it can be detected in serum within 4 hours after initiation of the inflammatory process. Human medicine routinely uses CRP as a diagnostic measure and it has potential for the use in veterinary clinical practice as a biomarker for monitoring health status and response to therapy. Therefore, one objective of the study was to evaluate three different point-of-care (POC) systems for the measurement of cCRP. However, to establish its diagnostic possibilities, many details about the parameter cCRP still need to be elucidated. Another objective of this study was to increase knowledge about cCRP, with a focus on structural details and use within the clinical setting. Measuring of cCRP may be particularly useful for application in emergency medicine, as it can help determine if the underlying problem is caused by an inflammatory process. In addition, it may be used in conjunction with other diagnostic measures to help determine how severe the health condition is. Point-of-care testing devices can be used to provide rapid measurement of cCRP, however testing and evaluation of their performance is necessary due to the current lack of data regarding their applicability in veterinary clinical practice. For the first part of the study, three different quantitative POC (TECO©dogCRP-quant, EUROLyser solo cCRP, LifeAssays® canine CRP) systems for measuring the cCRPconcentration in serum samples of dogs presented to the Small Animal Clinic, Freie Universität Berlin were evaluated. All assays were tested for their clinical applicability and precision was compared to the gold standard (ELISA). Results revealed distinct variations in their accuracy as well as reproducibility of values (TETECOmedical 69%, TEEUROLyser 28,2%, TELifeAssays 30,3%). However, all POC systems provide potential for their clinical usage not only in one-point measuring but also to monitor patients. For disease-monitoring, it is important to measure all parameters with the same POC system, because the measured cCRP-concentration varies between the assays as well as between assay and reference method. The anti-cCRP-antibody used in the TECOmedical device was also used in the majority of immunological analyses performed, to examine its’ quality and specificity. It was found that CRP could be detected by the antibody in different species from the Caniformia family while it was not detected in unrelated species but also reacts with some bacterial proteins, which are not specified yet. In the second part of the study, further information about the cCRP was determined. Specifically, in silico data about mRNA and amino acid (aa) sequence of cCRP were confirmed by biochemical analysis and new structural properties were characterized. The primary amino acid sequence was found to be shorter in the serum cCRP (204 aa) than the mRNA codes for cCRP (223 aa). This might have an influence on cCRP release from hepatocytes as well as protein activation. Furthermore, the specific glycosylated protein modifications were analyzed and revealed similar structure to human CRP. The importance of and immunological similarity with CRP in other species was examined in the third part of the study. CRP-protein could be detected in serum samples of different Caniformia species by the anti-cCRP-antibody, but failed to be detected in several species of the Eutheria family. However, the detection of CRP in the serum of dog-related species might increase the diagnostic possibilities for wild animals from the Caniformia family

Deep Learning-Based Quantification of Pulmonary Hemosiderophages in Cytology Slides

Exercise-induced pulmonary hemorrhage (EIPH) is a common condition in sport horses with negative impact on performance. Cytology of bronchoalveolar lavage fluid by use of a scoring system is considered the most sensitive diagnostic method. Macrophages are classified depending on the degree of cytoplasmic hemosiderin content. The current gold standard is manual grading, which is however monotonous and time-consuming. We evaluated state-of-the-art deep learning-based methods for single cell macrophage classification and compared them against the performance of nine cytology experts and evaluated inter- and intra-observer variability. Additionally, we evaluated object detection methods on a novel data set of 17 completely annotated cytology whole slide images (WSI) containing 78,047 hemosiderophages. Our deep learning-based approach reached a concordance of 0.85, partially exceeding human expert concordance (0.68 to 0.86, mean of 0.73, SD of 0.04). Intra-observer variability was high (0.68 to 0.88) and inter-observer concordance was moderate (Fleiss’ kappa = 0.67). Our object detection approach has a mean average precision of 0.66 over the five classes from the whole slide gigapixel image and a computation time of below two minutes. To mitigate the high inter- and intra-rater variability, we propose our automated object detection pipeline, enabling accurate, reproducible and quick EIPH scoring in WSI

Clonality testing in the lymph nodes from dogs with lymphadenomegaly due to Leishmania infantum infection.

INTRODUCTION:In southern European countries, multicentric lymphoma and leishmaniosis are the main differential diagnoses in dogs presented with generalized lymphadenomegaly. The cytological examination is in some cases inconclusive and polymerase chain reaction (PCR) for antigen receptor rearrangement (PARR) has become a common method to confirm or rule out a lymphoproliferative neoplasia. According to the literature, leishmaniosis may lead to clonal arrangements and therefore to a false diagnosis of lymphoma, but this assumption is made from a single leishmania infected dog. Therefore, the objective of this study was to prospectively evaluate results from PARR in dogs with lymphadenomegaly due to clinical leishmaniosis at the moment of diagnosis. MATERIALS AND METHODS:31 dogs with a diagnosis of leishmaniosis based on the LeishVet guidelines were included in the study. Samples from enlarged lymph nodes were taken for cytological examination, clonality testing and Leishmania infantum PCR. RESULTS:All 31 dogs had medium to high positive antibody titers against Leishmania spp. and 30/31 had a positive Leishmania PCR from the lymph node. A polyclonal arrangement for B cells (immunoglobulin heavy chain gene) and T cells (T-cell receptor gamma chain gene) antigen receptors was found in 28/31 dogs. Two out of 31 dogs showed a monoclonal arrangement for Ig with high (1:2) and low (1:7) polyclonal background respectively; and one of the 31 dogs showed a monoclonal arrangement for T cell receptor with low (1:3) polyclonal background. CONCLUSION:Infections with Leishmania infantum resulted in clonal rearrangement, and therefore in a possible false diagnosis of lymphoma, in 3 out of 31 dogs (9.7%). Although, PARR is a useful method to differentiate lymphoma from reactive lymphoid hyperplasia in dogs with leishmaniosis, mono-/biclonal results should be interpreted carefully, especially in the presence of any degree of polyclonal background, and together with other clinicopathological findings