Urinary cortisol metabolites are reduced in MDR1 mutant dogs in a pilot targeted GC-MS urinary steroid hormone metabolome analysis.

P-glycoprotein (P-gp) is the gene product of the multidrug resistance gene (MDR1, syn. ABCB1) that normally restricts the transfer of cortisol across the blood-brain barrier. In the absence of P-gp, cortisol access to the hypothalamus is increased and, by feedback inhibition, this finally leads to lower endogenous plasma cortisol levels in dogs with homozygous nt230(del4) MDR1 mutation (MDR1-/- mutant dogs). While a previous study only focused on plasma cortisol levels, the present study used urinary steroid hormone metabolites to analyze cortisol metabolism in MDR1-/- mutant dogs. Morning void urine was collected from 23 MDR1-/- mutant and 16 MDR1+/+ normal dogs and was subjected to targeted GC-MS steroid hormone metabolome analysis. Seven cortisol metabolites, cortisol itself, and 13 other steroid metabolites were detected. In general, all cortisol metabolites were lower in the urine of the MDR1-/- mutant dogs, with allo-tetrahydro-cortisol and ß-cortol reaching the level of significance. In addition, 11-keto-pregnanetriol levels were significantly lower in the urine of the MDR1-/- mutant dogs, indicating that also the 17alpha-OH-progesterone-derived metabolism was altered. In conclusion, the present study provides the first steroid hormone metabolome analysis in the urine of MDR1-/- mutant dogs. Significant differences in the steroid metabolome of MDR1-/- mutant dogs point to a significant role of P-gp for cortisol metabolism and excretion and so indirectly also for hypothalamic-pituitary-adrenal axis regulation in dogs.

Extremely high canine C-reactive protein concentrations > 100 mg/l - prevalence, etiology and prognostic significance.

BACKGROUND: In human medicine, extremely high CRP (C-reactive protein) concentrations > 100 mg/l are indicators of bacterial infection and the need of antibiotic treatment. Similar decision limits for septic pneumonia are recommended for dogs but have not yet been evaluated for other organ systems. The aim of the retrospective study was to investigate the prevalence and evaluate dogs with CRP concentrations > 100 mg/l regarding the underlying etiology, the affected organ system and the prognostic significance. RESULTS: Prevalence of CRP > 100 mg/l was investigated in dogs presented between 2014 and 2015 and was 12%. For evaluation of etiology and organ systems, dogs with CRP > 100 mg/l presented between 2014 and 2016 were enrolled. Dogs were classified into 4 main disease categories, i.e. inflammatory, neoplastic, tissue damage or "diverse". Diseases were assigned to the affected organ system. If an organ classification was not possible, dogs were classified as "multiple". 147 dogs with CRP 101-368 mg/l were included and classified into disease categories: 86/147 (59%) with inflammatory etiology (among these, 23/86 non-infectious, 44/86 infectious (33/44 bacterial), 19/86 inflammation non-classifiable), 31/147 (21%) tissue damage, 17/147 (12%) neoplastic (all malignant) and 13/147 (9%) diverse diseases. The affected organ systems included 57/147 (39%) multiple, 30/147 (20%) trauma, 21/147 (14%) gastrointestinal tract, 10/147 (7%) musculoskeletal system, 8/147 (5%) respiratory tract, 7/147 (5%) urinary/reproductive tract, 6/147 (4%) skin/subcutis/ear, 6/147 (4%) central/peripheral nervous system and 2/147 (1%) heart. The disease group (p = 0.081) or organ system (p = 0.17) did not have an impact on CRP. Based on CRP, a detection of bacterial infection was not possible. The prognostic significance was investigated by determining the 3-months survival and hospitalization rate in a subgroup with known outcome. The 3-months survival rate was 46/73 (63%) while the majority 66/73 (90%) of patients was hospitalized. CONCLUSIONS: CRP concentrations > 100 mg/l are occasionally seen in a clinic population. They indicate a severe systemic disease of various etiologies with guarded prognosis. Extremely high CRP concentrations do not allow a conclusion of the underlying etiology or an identification of bacterial inflammation.

Haemostatic, fibrinolytic and inflammatory profiles in West Highland white terriers with canine idiopathic pulmonary fibrosis and controls.

BACKGROUND: Canine idiopathic pulmonary fibrosis (CIPF) is a progressive interstitial lung disease mainly affecting old West Highland white terriers (WHWTs). The aetiology of CIPF is currently unknown and pathogenesis poorly understood. A genetic basis is strongly suspected based on the breed predisposition. CIPF shares clinical and pathological features with human IPF. In human IPF, coagulation disorders favouring a local and systemic pro-thrombotic state have been demonstrated in association with disease severity and outcome. The aim of this study was to compare the systemic haemostatic, fibrinolytic and inflammatory profiles of WHWTs affected with CIPF with breed-matched controls (CTRLs). Additionally, data collected in both groups were interpreted with regard to the reference intervals (when available) to assess possible pro-thrombotic features of the WHWT breed that may be related to CIPF predisposition. A total of 14 WHWTs affected with CIPF and 20 CTRLs were included. RESULTS: WHWTs affected with CIPF had prolonged activated partial thromboplastine time in comparison with CTRLs (12.2 ± 0.9 s vs. 11.5 ± 0.7 s, P = 0.028), whereas results obtained in both groups were all within reference ranges. There was no significant difference between groups for the other factors assessed including plasmatic concentrations of fibrinogen, D-dimers concentration, antithrombin III activity, protein S and protein C activities, anti-factor Xa activity, activated protein C ratio, serum C-reactive protein concentration, and rotational thromboelastometry indices. Platelet count and plasmatic fibrinogen concentration were found to be above the upper limit of the reference range in almost half of the WHWTs included, independently of the disease status. CONCLUSIONS: Results of this study provide no clear evidence of an altered systemic haemostatic, fibrinolytic or inflammatory state in WHWTs affected with CIPF compared with CTRLs. The higher platelet counts and fibrinogen concentrations found in the WHWT breed may serve as predisposing factors for CIPF or simply reflect biological variation in this breed.

Osteocyte Regulation of Receptor Activator of NF-κB Ligand/Osteoprotegerin in a Sheep Model of Osteoporosis.

Osteoporosis induction in a sheep model by steroid administration combined with ovariectomy recapitulates decreased bone formation and substandard matrix mineralization in patients. Recently, the role of osteocytes has been frequently addressed, with focus on their role in osteoclastogenesis. However, the quantification of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) signaling in osteocytes was not studied in sheep. The current study reproduced the sheep model of osteoporosis to study the RANKL/OPG ratio correlation to the method of osteoporosis induction. We investigated the induction of osteoporosis after 8 months using 31 female merino land sheep divided into four groups: control, ovariectomy, ovariectomy with dietary limitation, and ovariectomy with dietary limitation and steroid injection. In accordance to previous reports, the present study showed trabecular thinning, higher numbers of apoptotic osteocytes, and imbalanced metabolism, leading to defective mineralization. The global RANKL/OPG ratio in the spine after 8 months of steroid and dietary treatment was not different from that of the control. Interestingly, assessment of the osteocyte-specific RANKL/OPG ratio showed that the steroid-induced osteoporosis in its late progressive phase stimulates RANKL expression in osteocytes. Sclerostin is suggested to induce RANKL expression in osteocytes. The findings of this study can contribute to further explain the success of sclerostin antibodies in treating osteoporotic patients despite increased osteocyte-expressed RANKL.

Evaluation of a novel quantitative canine species-specific point-of-care assay for C-reactive protein.

BACKGROUND: Species-specific point-of-care tests (POCT) permit a rapid analysis of canine C-reactive protein (CRP), enabling veterinarians to include CRP in clinical decisions. Aim of the study was to evaluate a novel POCT for canine CRP (Point Strip™ Canine CRP Assay) run on a small in-house-analyzer (Point Reader™ V) using lithium heparin plasma and to compare assay performance to an already established canine CRP assay (Gentian Canine CRP Immunoassay) run on two different bench top analyzers serving as reference methods (ABX Pentra 400, AU 5800). Linearity was assessed by stepwise dilution of plasma samples with high CRP concentrations. Limit of quantification (LoQ) was determined by repeated measurements of samples with low CRP concentrations. Coefficient of variation (CV) at low (10-50 mg/l), moderate (50-100 mg/l), and high (100-200 mg/l) CRP concentrations was investigated as well as possible interferences. Method comparison study was performed using 45 samples of healthy and diseased dogs. Quality criteria were fulfilled if the total observed error (TEobs = 2CV% + bias%) was below the minimal total allowable error of 44.4% (TE min). Additionally, a reference range (n = 60 healthy dogs) was established. RESULTS: Linearity was present at CRP concentrations of 10-132 mg/l (≙ 361 mg/l CRP with reference method) with a LoQ set at 10 mg/l. At moderate to high CRP concentrations, intra- and inter-assay CVs were ≤ 8% and ≤ 11% respectively, while CVs ≤ 22% and ≤ 28% were present at low concentrations. No interferences were observed at concentrations of 4 g/l hemoglobin, 800 mg/l bilirubin and 8 g/l triglycerides. Method comparison study demonstrated an excellent correlation with both reference methods (r = 0.98 for ABX Pentra 400; 0.99 for AU 5800), though revealing a proportional bias of 19.7% (ABX Pentra 400) and 10.7% (AU 5800) respectively. TEobs was 26.7-31.9% and 16.7-21.9% and thus < TEmin. Healthy dogs presented with CRP values ≤11.9 mg/l. CONCLUSIONS: The POCT precisely detects canine CRP at clinically relevant moderate and high CRP concentrations. The assay correlates well with both reference methods. Due to the bias, however, follow-up examinations should be performed with the same assay and analyzer.

Preliminary reference intervals and the impact of citrate storage time for thrombelastography in cats including delta and the velocity curve.

BACKGROUND: Thrombelastography is a useful tool in assessment of hemostasis. Beside the traditional variables, the velocity curve and the variable delta have lately earned attention. The velocity curve provides knowledge about the speed of clot formation including information about thrombin generation. Delta, which only reflects enzymatic coagulation, allows the determination of the origin of hypercoagulability when compared to clot rigidity, a variable that reflects both platelet and enzymatic activity. The aim was to establish preliminary reference intervals for feline thrombelastography including the velocity curve variables and delta obtained after 60 min of storage including the assessment of coefficients of variation. Furthermore, the effect of citrate storage time (30 versus 60 min) on feline thrombelastography will be determined. RESULTS: Prolonged storage times significantly reduced reaction (R) (P = 0.019) and clotting (K) (P = 0.008) times, split point (SP) (P = 0.019) and time to maximum rate of thrombus generation (TMRTG) (P = 0.023) values whereas maximum rate of thrombus generation (MRTG) significantly increased (P = 0.040). Preliminary reference intervals: R (min): 2.7-18.1; K (min): 0.8-3.9; alpha (°): 27.6-75.2; maximum amplitude (mm): 18.5-62.5; clot rigidity (dyn/cm2): 1.2-8.2; coagulation index: -4.6 - 2.6; SP (min): 2.4-15.4; delta (min): 0.3-3.1; thrombus generation (mm/min): 255.3-751.2; MRTG (mm/min): 4.0-19.3; TMRTG (min): 3.5-22.0; maximum rate of lysis (mm/min): 0.0-4.7 and time to maximum rate of lysis (min): 0.4-55.8. CONCLUSION: Storage for 60 versus 30 min induces hypercoagulable tracings including the velocity curve, some of which variables (MRTG, TMRTG) might function as sensitive markers for changes in the coagulation activity. Because of the impact of citrate storage time on thrombelastography, reference intervals have to be established using a specific and constant storage time in each laboratory.

Evaluation of a species-specific C-reactive protein assay for the dog on the ABX Pentra 400 clinical chemistry analyzer.

BACKGROUND: A canine-specific immunoturbidimetric CRP assay, Gentian Canine CRP Immunoassay) with species-specific controls and calibrators was introduced and recently evaluated on the clinical chemistry analyzer Abbott Architect c4000 as well as on the Olympus AU600. Aims of our study were 1) to independently evaluate the canine-specific CRP assay on the ABX Pentra 400 clinical chemistry analyzer in comparison to the previously validated human-based immunoturbidimetric assay (Randox Canine CRP assay) and 2) to assess the impact of different sample types (serum versus heparinized plasma) on the results. Imprecision, accuracy, interference and the prozone effect were determined using samples from healthy and diseased dogs (n = 278). The Randox Canine CRP assay calibrated with canine specific control calibration material served as a reference method. Additionally, the impact of the sample type (serum and lithium heparin) was evaluated based on samples of healthy and diseased dogs (n = 49) in a second part of the study. RESULTS: Linearity was present for CRP concentrations ranging from 4 to 281 mg/l. For clinically relevant CRP concentrations of 7-281 mg/l, recovery ranged between 90 and 105% and intra- and inter-assay CVs ranged between 0.68% - 12.12% and 0.88% - 7.84%, respectively. CV was thus lower than 12.16%, i.e. the desired CV% based on biological variation. Interference was not present up to a concentration of 5 g/l hemoglobin, 800 mg/l bilirubin and 10 g/l triglycerides. No prozone effect occurred up to 676 mg/l CRP. Method comparison study revealed a Spearman's rank correlation coefficient of rs = 0.98 and a mean constant bias of 5.2%. The sample type had a significant (P = 0.008) but clinically not relevant impact on the results (median CRP of 30.9 mg/l in lithium heparin plasma versus 31.4 mg/l in serum). CONCLUSIONS: The species-specific Gentian Canine CRP Immunoassay reliably detects canine CRP on the ABX Pentra 400 clinical chemistry analyzer whereby both serum and heparin plasma can be used. The quality criteria reached on the Abbott Architect c4000 and Olympus AU600 could be met.

Optimized gating and reference ranges of reticulated platelets in dogs for the Sysmex XT-2000iV.

BACKGROUND: Canine reticulated platelets (r-PLTs) i.e., juvenile PLTs reflecting thrombopoiesis can be measured automatically with the hematology analyzer Sysmex XT-2000iV using manual gating options. However, the impact of interferences on r-PLT measurements performed with the gates published previously (Pankraz et al., Vet Clin Path 38:30-38, 2009; Gelain et al., High fluorescent platelets fraction in macrothrombocytopenic Norfolk terrier, 2010) is largely unknown. The aim was to compare different published gates for measurement of r-PLTs with the Sysmex XT-2000iV with an own, optimized gate ("Oellers-gate") and to establish reference intervals (RIs) in > 120 dogs. Data of 362 measurements of diseased and healthy dogs were analyzed retrospectively. Several gates were applied and RIs for r-PLTs and platelet indices were established for pet dogs and a group of 153 healthy Beagles kept under defined housing conditions. Intra-assay precision (CV) was also assessed. RESULTS: In 30/362 samples, interferences consistent with small erythrocytes/reticulocytes were seen in the previously published gates but not in the "Oellers-gate". Good correlation was found between the different gates (rs: 0.88-1.00). RIs for the "Pankraz-gate", the "Gelain-gate", and the "Oellers-gate" were 0.0-1.2, 0.2-3.7 and 0.2-3.9 % respectively. CVs were ranging between 22 and 41 %. CONCLUSIONS: Optimization of previously published gates minimized interferences of small erythrocytes with r-PLT measurements.

Impaired extracellular matrix structure resulting from malnutrition in ovariectomized mature rats.

Bone loss is a symptom related to disease and age, which reflects on bone cells and ECM. Discrepant regulation affects cell proliferation and ECM localization. Rat model of osteoporosis (OVX) was investigated against control rats (Sham) at young and old ages. Biophysical, histological and molecular techniques were implemented to examine the underlying cellular and extracellular matrix changes and to assess the mechanisms contributing to bone loss in the context of aging and the widely used osteoporotic models in rats. Bone loss exhibited a compromised function of bone cells and infiltration of adipocytes into bone marrow. However, the expression of genes regulating collagen catabolic process and adipogenesis was chronologically shifted in diseased bone in comparison with aged bone. The data showed the involvement of Wnt signaling inhibition in adipogenesis and bone loss due to over-expression of SOST in both diseased and aged bone. Further, in the OVX animals, an integrin-mediated ERK activation indicated the role of MAPK in osteoblastogenesis and adipogenesis. The increased PTH levels due to calcium and estrogen deficiency activated osteoblastogenesis. Thusly, RANKL-mediated osteoclastogenesis was initiated. Interestingly, the data show the role of MEPE regulating osteoclast-mediated resorption at late stages in osteoporotic bone. The interplay between ECM and bone cells change tissue microstructure and properties. The involvement of Wnt and MAPK pathways in activating cell proliferation has intriguing similarities to oncogenesis and myeloma. The study indicates the importance of targeting both pathways simultaneously to remedy metabolic bone diseases and age-related bone loss.

Bone matrix, cellularity, and structural changes in a rat model with high-turnover osteoporosis induced by combined ovariectomy and a multiple-deficient diet.

In estrogen-deficient, postmenopausal women, vitamin D and calcium deficiency increase osteoporotic fracture risk. Therefore, a new rat model of combined ovariectomy and multiple-deficient diet was established to mimic human postmenopausal osteoporotic conditions under nutrient deficiency. Sprague-Dawley rats were untreated (control), laparatomized (sham), or ovariectomized and received a deficient diet (OVX-Diet). Multiple analyses involving structure (micro-computed tomography and biomechanics), cellularity (osteoblasts and osteoclasts), bone matrix (mRNA expression and IHC), and mineralization were investigated for a detailed characterization of osteoporosis. The study involved long-term observation up to 14 months (M14) after laparotomy or after OVX-Diet, with intermediate time points at M3 and M12. OVX-Diet rats showed enhanced osteoblastogenesis and osteoclastogenesis. Bone matrix markers (biglycan, COL1A1, tenascin C, and fibronectin) and low-density lipoprotein-5 (bone mass marker) were down-regulated at M12 in OVX-Diet rats. However, up-regulation of matrix markers and existence of unmineralized osteoid were seen at M3 and M14. Osteoclast markers (matrix metallopeptidase 9 and cathepsin K) were up-regulated at M14. Micro-computed tomography and biomechanics confirmed bone fragility of OVX-Diet rats, and quantitative RT-PCR revealed a higher turnover rate in the humerus than in lumbar vertebrae, suggesting enhanced bone formation and resorption in OVX-Diet rats. Such bone remodeling caused disturbed bone mineralization and severe bone loss, as reported in patients with high-turnover, postmenopausal osteoporosis. Therefore, this rat model may serve as a suitable tool to evaluate osteoporotic drugs and new biomaterials or fracture implants.

Rate of manual leukocyte differentials in dog, cat and horse blood samples using ADVIA 120 cytograms.

BACKGROUND: Modern automated haematology instruments are capable of performing leukocyte differentials faster, cheaper and with a higher precision than the traditional 100-cell manual differential count. Thus, in human laboratories, criteria are defined for performing a manual review of the blood smear resulting in a marked reduction of manual differential counts. While common in human laboratories, this approach to reducing the number of manual differentials in veterinary laboratories is still not commonly performed. Thus, our aim was to determine the rate and causes of manual leukocyte differentials in a university clinical pathology laboratory using the automated laser-based haematology analyser ADVIA 120. Overall, 14,953 complete blood cell counts from dogs, cats and horses were reviewed. Manual leukocyte differentials were requested if abnormal ADVIA peroxidase and baso cytograms were detected (i.e. suspicion of left shift or atypical lymphocytes/blasts, inappropriate separation of cell populations). RESULTS: In 21% of canine, 32% of feline and 20% of equine samples, a manual differential was requested. Indistinct separation of the cell population was present in 10% to 15% of the cases. Depending on the species, atypical lymphocytes were suspected in 2% to 12%, left shift in 13% to 25% and suspicion of blasts was present in less than 0.4% of the cases. CONCLUSIONS: The obtained results are comparable to those published for human medicine and the rate of manual differentiation could be markedly reduced in veterinary laboratories if microscopic examination was used as a validation procedure rather than as a reflexive substitute for automated differentiation.

Development and validation of a questionnaire to assess health-related quality-of-life in cats with hyperthyroidism.

BACKGROUND: Health-related quality-of-life (HRQoL) assessment tools are becoming increasingly important for the assessment of diseases in veterinary medicine. OBJECTIVES: To develop a tool to assess the HRQoL of hyperthyroid cats and their owners. ANIMALS: Cats with hyperthyroidism (n = 229) and without hyperthyroidism (n = 322). METHODS: Cross-sectional study design. A preliminary list of 28 questions relating to the HRQoL of hyperthyroid cats and the influence their cat's disease might have on owners was created. Each question consisted of 2 subquestions: (1) "how often does the item apply"; (2) "how strongly does the item affect HRQoL." The questionnaire was refined based on statistical analysis, including Mann-Whitney-U tests on each item, comparing the results from cats with and without hyperthyroidism. Internal consistency and reliability of the questions were measured by Cronbach's alpha (α). P < .05 was considered significant. RESULTS: Overall, 25/28 questions were retained within the final HRQoL tool, which had an excellent internal consistency (α = .92). The tool produced a score between 0 and 382 (lower scores meaning better HRQoL). The median HRQoL score was 87.5 (range, 2-348) for cats with hyperthyroidism, and 27 (range, 0-249) for cats without (P < .001), suggesting the HRQoL was poorer in hyperthyroid cats. CONCLUSIONS AND CLINICAL IMPORTANCE: This validated HRQoL tool is useful to reliably quantify the influence of hyperthyroidism on the quality-of-life of affected cats and their owners. In the future, it could be considered of assistance in the clinical assessment of cats with hyperthyroidism.

Evaluation of canine and feline leukocyte differential counts obtained with the scil vCell 5 compared to the Advia 2120 hematology analyzer and a manual method.

The vCell 5 (scil Animal Care), a point-of-care hematology analyzer (POCA), was recently introduced to veterinary laboratories. This laser- and impedance-based analyzer is capable of providing a CBC with 5-part WBC differential count (Diff) along with WBC cytograms and flags serving as interpretation aids for numerical results. We compared the scil POCA-Diff to reference methods (i.e., manual differential count, Advia 2120 hematology analyzer [Siemens]) for canine and feline blood samples and considered WBC cytograms and flags. Total observed error (TEo), calculated from CV and bias%, was compared to total allowable error (TEa). Data were analyzed before and after a review process (exclusion of flagged and samples with invalid cytograms). For both species, correlation was good-to-excellent (rs = 0.81-0.97) between both analyzers for all variables, except for feline monocytes (rs = 0.21-0.63) and canine monocyte% (rs = 0.50). Smallest biases were seen for neutrophils (dog: -5.7 to 0.8%; cat: 1.5-9.4%) with both reference methods. Quality requirements (TEo < TEa) were fulfilled for canine and feline neutrophils (TEo = 5.3-10.6%, TEa = 15%) and eosinophils (TEo = 67.1-83%, TEa = (90)-50%) considering at least one reference method. Our review process led to mildly higher rs-values for most variables. Although not completely satisfactory, the scil POCA provides reliable results in compliance with ASVCP quality goals for canine and feline neutrophils and eosinophils. Analyzer flag and cytogram analysis served as useful tools for QA, indicating the necessity for manual review of blood smears, and contributed to improvement of scil POCA performance.

Simplified Light's Criteria and Acute Phase Proteins Reflect Aetiology of Feline Body Cavity Effusions Better than the Traditional Classification Scheme.

The traditional veterinary classification (TVC) of effusions based on cell count and total protein (TP) does not adequately reflect the aetiology. Light's criteria (LC) (activity of lactate dehydrogenase [LDH] in the effusion [LDHef], effusion/serum LDH ratio [LDHr], effusion/serum TP ratio [TPr]), serum-effusion albumin gradient (ALBg), acute phase proteins (APPs) [serum amyloid A (SAA), α1-acid glycoprotein (AGP), haptoglobin] might aid classification. The aim was to evaluate the utility of these parameters except LDHr in differentiating exudates from transudates. Sixty-five cats with effusions (33 peritoneal, 31 pleural, 1 pericardial), with 18 transudates and 47 exudates based on aetiological classification (AC), were included. The sensitivity, specificity and accuracy of several parameters to identify exudates (based on AC) was assessed. APPs were compared between exudates and transudates based on AC and TVC, with receiver operating characteristics analysis identifying the best APP to recognise exudates. Simplified LC (LDHef, TPr) had an accuracy of 79% and TVC of 48%. ALBg had the highest sensitivity (98%) and LDHef the highest specificity (83%) in identifying exudates in cats. All APPs but effusion SAA could differentiate exudates from transudates based on AC (effusion AGP had the largest area under the curve 0.79) but not TVC. All parameters were better than TVC in identifying exudates. The conformity of APPs with AC but not TVC favours the use of AC to classify effusions.

Evaluation of a novel moving threshold gating strategy for assessment of reticulated platelets in dogs using the ADVIA 2120 analyzer.

BACKGROUND: A novel method using a moving threshold (r-PLTmt) to determine canine r-PLTs (reticulated platelets) has been introduced for ADVIA 2120 software v6.11.7. OBJECTIVES: We aimed to evaluate absolute (ar-PLTmt) and percent (%r-PLTmt) prior to and after visual inspection of scattergrams (ar-PLTmtv, %rPLTmtv) compared with flow cytometry (flow) and to determine reference intervals (RIs) in 120 dogs. METHODS: For method comparison, 42 blood samples of healthy and thrombocytopenic dogs were included. Calculation of Spearman's rho, Bland-Altman, and Passing-Bablok analysis was performed. Coefficients of variation (CVs) were determined for three concentration levels. RESULTS: Moderate correlations between %r-PLTmt and %r-PLTmtv (rs 0.75-0.76) were seen compared with flow cytometry. The CV for medium %r-PLTs counts assessed with flow cytometry was 12.9%. Comparable CVs were obtained for ar-PLTmt (14.4%) and %r-PLTmt (15.7%), and ar-PLTmtv and %r-PLTmtv (10.9% and 12.9%, respectively). At low and high concentration levels, CVs for % and absolute r-PLTmt/rPLTmtv ranged between 23%-30% and 15%-20%. In patients with microcytic hypochromic erythrocytes, CVs for ar-PLTmt and %r-PLTmt were 36%-66%. Visual inspection of scattergrams resulted in a marked decrease in CV ranging between 15% and 20%. A proportional bias of 10.8% between %r-PLTmt and flow cytometry became lower (9.7%) after visual validation of scattergrams. Passing-Bablok analysis showed proportional and constant error. RIs for r-PLTmt and r-PLTmtv were 0.2%-3.8% and 0.6-10.2 × 109 /L and 0.3%-4.5% and 1.1-10.3 × 109 /L, respectively. Median values for %r-PLTmtv were higher in young adults (≤2 years) than in older dogs (P = 0.03). CONCLUSIONS: r-PLTmt and r-PLTmtv were moderately correlated with flow cytometry. Visual inspection of scattergrams is recommended.

[Myxosarcoma in the cervical region in a teddy bear hamster (Mesocricetus auratus)]. / Myxosarkom in der Halsregion eines Teddy-Hamsters (Mesocricetus auratus).

This case report describes a rare case of a myxosarcoma in a 1-year-old teddy bear hamster presenting with a mass in the cervical region. The fine-needle aspiration cytology revealed high numbers of pleomorphic spindle-shaped cells found in a viscous mucinous background. The presumptive cytological diagnosis was malignant spindle cell neoplasia based on marked criteria of malignancy of the mesenchymal cell population. The abundant matrix in the background was suggestive of a myxosarcoma. The hamster died during surgery and a necropsy was performed. Histopathology was in complete agreement with the cytological report. Immunohistochemistry revealed the tumour to be vimentin positive with alcian-blue positive matrix and confirmed the presumptive diagnosis of a myxosarcoma. This case shows that fine-needle aspiration cytology can be utilized as a minimally invasive diagnostic tool in small mammals to classify mass lesions. However, so far little is known about the biological behaviour of myxosarcoma in the hamster as case descriptions are rare.

Cat alpha-1-acid glycoprotein enzyme-linked immunosorbent assay: performance characteristics and reference intervals.

OBJECTIVES: The aim of this study was to evaluate performance of a feline-specific ELISA for the measurement of alpha-1-acid glycoprotein (AGP) and to establish a reference interval (RI). METHODS: Surplus serum samples with low (~200 µg/ml), medium (~450 µg/ml) and high (~745 and 930 µg/ml) AGP concentrations were used to assess the intra- and inter-assay coefficients of variation (CVs). The quality goal for bioanalytical method validation was a CV of <20%. Linearity was assessed by serial dilution of a sample with a high AGP concentration. Spike recovery was evaluated by mixing samples with low, medium and high AGP concentrations at different ratios. To establish the RI, residual serum samples from 51 healthy adult cats that were presented for health examinations or blood donation between August 2020 and June 2021 were included. RESULTS: The intra-assay CV was 8.5%, 4.3% and 4.0%, and the inter-assay CV was 18.8%, 15.5% and 11.5% for serum samples with low, medium and high AGP concentrations, respectively. Excellent linearity (R2 = 0.98) was demonstrated for AGP concentrations ranging between 251.6 and 954.4 µg/ml. Average recovery was 95.0-99.7%. The right-sided RI for AGP was 328 µg/ml (90% confidence interval 300-354). Age had a statistically significant impact (increasing values with older age, P = 0.0026), but sex did not (P = 0.44), on AGP concentrations. CONCLUSIONS AND RELEVANCE: The ELISA was accurate and showed acceptable precision with the modification of dilution used in this study. AGP concentrations in this population appeared to increase with increasing age.

[Cytologic evidence of hepatocytotropic T-cell-lymphoma in a 15-year-old male cat]. / Zytologischer Nachweis eines hepatozytotropen T-Zell-Lymphoms bei einem 15 Jahre alten Kater.

This case report describes the rare phenomenon of emperipolesis-like invasion of lymphatic blasts into the hepatocytes of a 15-year-old European Shorthair cat. The cat presented with nonspecific clinical signs (inappetence and weight loss). Cytologic examination of an ultrasound-guided fine-needle aspirate of the liver showed a subset of hepatocytes with emperipolesis-like invasion by lymphatic blasts. Few extracellularly located lymphatic blasts exhibited erythrophagia. Following the cytological diagnosis of large cell lymphoma and 2 weeks of monotherapy with prednisolone, the patient was euthanized due to his poor general condition. A post-mortem sample was obtained from the liver to confirm the suspected cytological diagnosis of hepatocytotropic lymphoma. Histopathology subsequently confirmed the cytologic findings. Immunohistochemically, the lymphatic blasts were positive for CD3 leading to a diagnosis of hepatocytotropic T-cell-lymphoma, which has rarely been described so far.

Method Validation and Establishment of Reference Intervals for an Insulin-like Growth Factor-1 Chemiluminescent Immunoassay in Cats.

Previously, radioimmunoassay (RIA) has been the only assay to measure insulin-like growth factor-1 (IGF-1) to diagnose hypersomatotropism (HS). Due to radiation concerns, availability, and the cost of IGF-1 RIA, validation of assays for automated analysers such as a chemiluminescent immunoassay (CLIA) is needed. The aim of this study was to validate a CLIA for measurement of feline IGF-1 (IMMULITE 2000® XPi, Siemens Medical Solutions Diagnostics, Malvern, PA, USA) compared to IGF1 RIA, establish reference interval (RI), and determine a cut-off value for diagnosis of HS in diabetic cats. Validation of assay performance included precision, linearity, and recovery studies. Right-sided RI was determined using surplus serum of 50 healthy adult cats. Surplus serum samples of diabetic cats with known IGF-1 concentration with (n = 32/68) and without HS (n = 36/68) were used for method comparison with RIA. The cut-off for diagnosis of HS was established using receiver operating characteristic (ROC) analysis. The intra-assay coefficient of variation (CV) was ≤4.7%, and the inter-assay CV was ≤5.6% for samples with low, medium, and high IGF-1 concentration. Linearity was excellent (R2 > 0.99). The correlation between CLIA and RIA was very high (rs = 0.97), with a mean negative bias for CLIA of 24.5%. The upper limit of RI was 670 ng/mL. ROC analysis showed an area under the curve of 0.94, with best cut-off for diagnosis of HS at 746 ng/mL (sensitivity, 84.4%; specificity, 97.2%). The performance of CLIA was good, and the RI and cut-off for HS diagnosis established in this study allow for CLIA to be used in routine work-up of diabetic cats.

Evaluation of the scil vCell 5, a novel laser- and impedance-based point-of-care hematology analyzer, for use in dogs and cats.

A novel laser- and impedance-based point-of-care hematology analyzer (POCA), the vCell 5 (scil Animal Care), providing a complete blood count with 5-part leukocyte differential count has recently been introduced to veterinary laboratories. We evaluated the analyzer for use in dogs and cats including method comparison and assessment of linearity, carryover, and precision. Fresh blood samples from 192 healthy and diseased dogs and 159 cats were analyzed, and results were compared to reference methods (i.e., microhematocrit [PCV], Advia 2120 hematology analyzer). Total observed error (TEo) was calculated from CV, obtained at 3 concentrations, and bias%, and compared to total allowable error (TEa). For both species, excellent correlation (rs = 0.93-0.99) was seen between methods for WBC and RBC, hematocrit, hemoglobin, and platelet counts (PLT), except for feline PLT (rs = 0.79). Quality requirements (TEo < TEa) were fulfilled for WBC (TEo = 8.6-11.1%; TEa = 20%) and RBC (TEo = 3.5-7%; TEa = 10%), hematocrit (TEo = 5.7-9.4%; TEa = 10%), PCV (cat TEo = 7.8%; TEa = 10%), mean corpuscular volume (cat TEo = 5.1%; TEa = 7%), and PLT (TEo = 13.1-24.1%; TEa = 25%). Excellent linearity was demonstrated for WBC, RBC, and PLT, and hemoglobin. CVs of <2% for WBC, RBC, hematocrit, hemoglobin, and of <5% (dog) and 8% (cat) for PLT were demonstrated for values within the RI. Except for calculated variables and well-known species-specific deviations in feline PLT, scil POCA results were correlated favorably with reference method results and complied with quality requirements for cats and dogs.