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Background: Benefit of adding a second‐line immunosuppressive drug to glucocorticoids for the treatment of non‐associative immune‐mediated hemolytic anemia (naIMHA) in dogs has not been defined prospectively. Hypothesis/Objectives: Evaluate the effectiveness of different immunosuppressive protocols in naIMHA dogs. Animals: Forty‐three client‐owned dogs. Methods: Open label, randomized, clinical trial. Dogs were treated with methylprednisolone (M‐group), methylprednisolone plus cyclosporine (MC‐group) or methylprednisolone plus mycophenolate mofetil (MM‐group). Dogs were defined as responders by disappearance of signs of immune‐mediated destruction and hematocrit stabilization. Frequency of responders was compared between M‐group and combined protocols (MC and MM‐group evaluated together), and among the 3 different therapeutic groups at 14 (T14), 30 (T30), 60 (T60) days after admission. Frequency of complications, length of hospitalization and relapse were also compared. Death rate was evaluated at discharge, T60 and 365 (T365) days. Results: Proportion of responders was not significantly different between M‐group and combined protocols (MC and MM‐groups), nor among the 3 therapeutic groups at T14, T30, and T60 (P >.17). Frequency of relapse, complications, and length of hospitalization were not significantly different between M‐group and dogs treated with combined protocols, nor among the 3 treatment groups (P >.22). Death was significantly more common only for MM‐group compared with MC‐group at T60 (+42.8%; 95% CI: 11.5–67.4; P =.009), and at T365 (+50%; 95% CI: 17.5–73.2; P =.003). Conclusions and Clinical Importance: Combined immunosuppressive therapy did not improve hematological response in naIMHA. [ABSTRACT FROM AUTHOR]

Simple Summary: Vitamin D deficiency and toxicity are common and well-recognized conditions in dogs. The metabolite 25-hydroxyvitamin D3 is the main endogenous biomarker for the evaluation of vitamin D3 status. In this study, we developed a simple analytical method for 25-hydroxyvitamin D3 quantification in canine serum. The method was validated following the current European guidelines and yielded satisfactory results, representing a new tool for supporting clinicians in the correct diagnosis and monitoring of vitamin D3 status in dogs. Several studies have shown the importance of vitamin D3 supplementation in small animals. In dogs, a low vitamin D3 status is associated not only with bone metabolism but also with different kinds of disorders, such as congestive heart failure, gastrointestinal diseases, chronic kidney diseases, and some types of cancer. However, it is crucial to maintain balance and monitor the introduction of this essential nutrient through the diet because over-supplementation can result in toxicity. Due to the clinical importance of assessing the vitamin D3 status in small animal patients, a quick, simple, and highly performing analytical method for its measurement is needed. In this study, we describe the development of a novel liquid chromatography–tandem mass spectrometry method for 25-hydroxyvitamin D3 quantification in canine serum. The approach was successfully validated following current European guidelines, proving excellent linearity (R2 always ≥0.996), accuracy (always within ±13%) and precision (always <10%). The application of the validated approach to samples collected from 40 healthy dogs made possible the definition of a reliable reference interval for 25-hydroxyvitamin D3, the main biomarker of vitamin D3. In addition, variations below 5% in the results obtained quantifying the same samples using a water-based calibration curve demonstrated that a surrogate matrix may be used without affecting data accuracy. Thanks to its simplicity, the proposed technique represents a useful tool for supporting clinical routine and investigating correlations between serum concentrations of this metabolite and multiple diseases. Additionally, it could enable the monitoring of supplementation in small animal patients in veterinary clinical practice. [ABSTRACT FROM AUTHOR]