Vet Clin Pathol
· 2025 Jun · PMID 40454744
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BACKGROUND: Pancreatic lipase assays are commonly utilized in the diagnostic approach to suspected pancreatitis in cats. The Vcheck fPL 2.0 assay is commercially available; however, analytical validation has yet to be re...BACKGROUND: Pancreatic lipase assays are commonly utilized in the diagnostic approach to suspected pancreatitis in cats. The Vcheck fPL 2.0 assay is commercially available; however, analytical validation has yet to be reported in the peer-reviewed literature. OBJECTIVE: To evaluate the analytic validity of a fluorescent immunoassay for the point-of-care quantification of feline pancreatic lipase immunoreactivity (Vcheck fPL 2.0 assay). METHODS: Linearity was assessed via dilutional parallelism. Intra- and interassay variability was assessed by calculating coefficients of variation (%CV) across replicates on the same day and across multiple days, respectively. Bilirubin, hemoglobin, and intralipid (intralipid 30% IV emulsion, VWR, Radnor, PA, USA) were utilized to evaluate the potential effects of interfering substances on the assay. RESULTS: Linearity was sub-optimal, with 12/15 diluted runs having an O/E ratio outside of the 80-120% target range. The mean %CV was 10.2% for intra-assay variability and 16.3% for interassay variability. Interfering substances had no significant effect on the results of the Vcheck fPL 2.0 assay (P = 0.888). CONCLUSIONS: The Vcheck fPL 2.0 appears to have poor linearity and suboptimal precision, limiting the accurate interpretation of results. Additional assay optimization should be considered prior to routine clinical use.
Bilhalva LC, Zebovitz LN, Nutter FB
… +4 more, Higgs A, Fox D, Cray C, Conrado FO
Vet Clin Pathol
· 2025 Jun · PMID 40454732
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BACKGROUND: The Atlantic sturgeon has faced severe population declines due to overfishing and environmental changes, resulting in its critical conservation status. There is concern today for the survival of this species,...BACKGROUND: The Atlantic sturgeon has faced severe population declines due to overfishing and environmental changes, resulting in its critical conservation status. There is concern today for the survival of this species, leading to the initiation of studies focused on its conservation. OBJECTIVES: The aims of this study were to establish reference intervals (RIs) for plasma protein fractions in Atlantic sturgeon using agarose gel electrophoresis (AGE), to evaluate whether changes occur during the spawning season, and to compare results with those obtained using capillary zone electrophoresis (CZE). METHODS: Between late May and June 2022, blood samples were collected from 81 spawning Atlantic sturgeon. Plasma from all individuals was analyzed using AGE, with a subset of 16 paired samples further evaluated using CZE for comparative analysis. RESULTS: AGE revealed six distinct protein fractions, while CZE identified seven fractions. Significant differences in the proportions of fraction 3 (traditionally referred to as alpha-1 globulins) were observed between early and late spawning seasons. CONCLUSION: Comparison of AGE and CZE results showed that while both methods provided useful data, CZE offered greater resolution and detection sensitivity. Our results provide important data for sturgeon health assessment; however, discrepancies between the two methods underscore the need for method-specific RI.
Brandon J, Reider H, Pabilonia KL
… +1 more, Moore AR
Vet Clin Pathol
· 2025 Jun · PMID 40454730
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BACKGROUND: The bromocresol green albumin assay (ALB) has been used in birds and reportedly is noncomparable with electrophoretic albumin (ALB) in many species. It is accepted for use in some species and rejected in othe...BACKGROUND: The bromocresol green albumin assay (ALB) has been used in birds and reportedly is noncomparable with electrophoretic albumin (ALB) in many species. It is accepted for use in some species and rejected in others. OBJECTIVES: We aimed to compare the performance of ALB and ALB methods within backyard chickens and compare the performance of ALB in chickens with other veterinary species where the ALB method is accepted and used clinically. METHODS: Chicken plasma collected during reference interval development and samples submitted for diagnostic biochemistry profile were evaluated using the ALB and ALB assays. Method comparison was performed according to current recommendations, including the use of Passing-Bablok and Bland-Altman analysis. ALB and ALB were also measured in other avian species, dogs, cats, horses, and domestic ruminants. Method comparison was evaluated within and between species, including clinical utility based on the percentage of cases discordantly interpreted as hypo-, normo-, or hyperalbuminemic by ALB and ALB. RESULTS: In chickens, ALB and ALB were not comparable, having a constant bias (-0.4 g/dL) and proportional bias. Similarly, the methods were not comparable in other species; > 10% of samples had > TE (15%) difference in all species. The clinical utility of albumin interpretation in chickens did not differ significantly from that in dogs and horses, as determined by ANOVA. CONCLUSIONS: The data suggest that ALB is not comparable with ALB and performs similarly across all tested species. There is no evidence to support the continued rejection of the ALB in chicken and other avians and acceptance in some mammals.
Mato-Martín V, Ginders J, Riond B
… +2 more, Hofmann-Lehmann R, Novacco M
Vet Clin Pathol
· 2025 Jun · PMID 40454728
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Scattergram evaluation is an important part of nonstatistical quality control in Veterinary Hematology. In this case report, we present two feline hematological cases with abnormal Sysmex XN-V scattergrams. Case 1 involv...Scattergram evaluation is an important part of nonstatistical quality control in Veterinary Hematology. In this case report, we present two feline hematological cases with abnormal Sysmex XN-V scattergrams. Case 1 involved a 16-year-old neutered female domestic shorthair cat referred for the investigation of a suspected abdominal mass, which revealed marked mastocythemia on blood smear evaluation. Case 2 involved a 9-year-old castrated male domestic shorthair cat that was presented for follow-up after cystostomy and perineal urethrostomy, which revealed marked basophilia on blood smear evaluation. In both cases, an abnormal population was observed on the white cell differential channel by fluorescence (WDF) scattergram using a Sysmex XN-V hematology analyzer. The abnormal population appeared as an oval cloud of dots between monocytes and eosinophils. Manual re-gating of these unusual populations on the WDF channel correlates with manually determined counts. Mast cells appeared as a lysis-resistant population on the white count and nucleated red blood cell (WNR) scattergram, whereas basophils did not show this feature. Therefore, the appearance of an unexpected population between the eosinophil and monocyte area on the WDF scattergram may indicate mastocythemia or basophilia in cats. Further assessment of the WNR scattergram, focusing on identifying lysis-resistant populations, can help differentiate between basophilia and mastocythemia. However, blood smear evaluation remains mandatory for the accurate enumeration of basophils and/or determination of mastocythemia. Manufacturer software adjustments within the WDF and WNR channels may improve the accuracy of basophil counts in veterinary hematology.
Oikonomidis IL, Rees P, Hernando Sanz J
… +1 more, Woods G
Vet Clin Pathol
· 2025 Jun · PMID 40454701
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BACKGROUND: Hypophosphatemia is commonly observed in unselected human intensive care unit (ICU) patients, and it has been associated, although inconsistently, with worse outcomes and longer duration of hospitalization (D...BACKGROUND: Hypophosphatemia is commonly observed in unselected human intensive care unit (ICU) patients, and it has been associated, although inconsistently, with worse outcomes and longer duration of hospitalization (DOH). The incidence of hypophosphatemia and its association with mortality and DOH in ICU canine patients is unknown. OBJECTIVES: The aims of this study were to determine the occurrence of hypophosphatemia in unselected ICU canine patients and its association with mortality and DOH. METHODS: The medical records of all dogs admitted to the Teaching Hospital ICU between January 2019 and December 2023 were retrospectively reviewed. Dogs with hypophosphatemia (serum phosphate < 0.9 mmol/L) were identified. For every hypophosphataemic dog included in this study, two age-matched control, non-hypophosphataemic dogs, closely admitted to the ICU in time, were included. RESULTS: In total, 3233 medical records were reviewed. Hypophosphatemia was noted in ≥ 1 day of hospitalization in 32 dogs (0.99%). The age-matched case and control groups had a median (range) age of 8.0 (1.0-14.0) years. The survival to discharge rates of both hypophosphataemic and control groups were 78% (25/32 and 50/64, respectively), and their DOH (median, 3.5 days; range 1.0-9.0 and median 3.0 days; range 1.0-25.0, respectively) were not significantly different (p = 0.557). Serum phosphate concentration was not correlated with the DOH (p = 0.649). CONCLUSIONS: Hypophosphatemia was noted in only 1% in this canine ICU patient population and was not associated with the survival to discharge and DOH.
Holland SN, Tinkle AK, Prescott JN
… +4 more, Blattman BL, Dog Aging Project Consortium, Creevy KE, Fajt VR
Vet Clin Pathol
· 2025 Mar · PMID 40406054
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BACKGROUND: The Dog Aging Project (DAP) is a large-scale longitudinal study for studying aging in dogs. For some dogs in the DAP, blood samples for plasma isolation are collected by non-DAP personnel. However, plasma sam...BACKGROUND: The Dog Aging Project (DAP) is a large-scale longitudinal study for studying aging in dogs. For some dogs in the DAP, blood samples for plasma isolation are collected by non-DAP personnel. However, plasma samples are sometimes inadequate, eg, insufficient volume for assays. OBJECTIVE: We aimed to examine three factors that may affect plasma yield: resting time after sample collection, centrifugation time, and level of operator or technician training. METHODS: We designed three experiments using a convenience sample of 5 dogs. Each experiment varied one of the three factors and held the other two constant. Experiment 1 examined 5 different resting times: 10 minutes and 1, 4, 24, and 72 hours. Experiment 2 compared centrifugation times of 7 and 14 minutes. Experiment 3 compared trained and untrained personnel. Sample resting was always under refrigeration. Experimental outcomes were total plasma volume, number of successful aliquots, hemolysis, and lipemia. RESULTS: A resting time of 72 hours yielded statistically significantly lower plasma volume than resting times ≤ 4 hours. Resting times of 24 and 72 hours also had statistically significantly higher hemolysis scores compared with other resting time points. In addition, trained operators or technicians yielded an average of 0.5 more aliquots. Outcomes were similar by centrifugation time in Experiment 2. CONCLUSION: To mitigate sample loss, we recommend shorter post-collection resting times and ensuring technician proficiency. Additionally, increasing the requested whole blood volume may improve sample yield.
Evans SJM, Allen B, Mollenkopf D
… +5 more, Truelove MP, Tebbe NA, Pannone S, Radin MJ, Santangelo KS
Vet Clin Pathol
· 2025 Mar · PMID 40022478
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BACKGROUND: Biochemistry analyzers, which utilize indirect potentiometry, are used to determine serum electrolyte concentrations in dogs. Artifactual increases or decreases in these electrolyte concentrations can be caus...BACKGROUND: Biochemistry analyzers, which utilize indirect potentiometry, are used to determine serum electrolyte concentrations in dogs. Artifactual increases or decreases in these electrolyte concentrations can be caused by alterations in the serum water fraction (SWF). Severe hypo- and hyperproteinemia cause changes in SWF, which can then result in incorrectly reported serum sodium concentrations. OBJECTIVES: The goals of this study were to determine an average actual SWF (SWF) in dogs and establish formulae to correct serum sodium concentration measured by indirect potentiometry in hypo- and hyperproteinemic patients. METHODS: Serum samples from 115 canine patients were analyzed for electrolytes measured by both indirect and direct potentiometry. Total protein, albumin, triglycerides, and cholesterol were also determined. Each serum sample was then lyophilized to determine SWF. A canine-specific formula to estimate SWF (SWF) was developed using a multivariable linear model and compared with the human-estimated formula (SWF). RESULTS: The mean SWF in this population of dogs was 92.7%, which was significantly different (p < .0001) than the mean (95.1%) calculated using SWF. The formula derived from SWF recapitulated SWF more accurately than SWF. Based on a slope closer to 1.0, the corrected sodium concentrations calculated using the canine formula correlated marginally better with the serum sodium measured by direct potentiometry than those calculated using the human formula. CONCLUSIONS: Applications of correction formulae are expected to limit the misinterpretation of electrolyte data from indirect potentiometry when altered SWF occurs. A case example of the potential utility of these correction formulae is presented.
Vet Clin Pathol
· 2025 Mar · PMID 39928986
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BACKGROUND: Total thyroxine (TT4) measurement is used to assess thyroid status in cats. OBJECTIVES: The aim of the prospective study was to evaluate the analytical performance of the point-of-care analyzer (POCA) Immuno...BACKGROUND: Total thyroxine (TT4) measurement is used to assess thyroid status in cats. OBJECTIVES: The aim of the prospective study was to evaluate the analytical performance of the point-of-care analyzer (POCA) Immuno AU10V using the v-T4 test kit for feline TT4 measurement. Additionally, method comparison with a benchtop analyzer (IMMULITE 2000) was done. METHODS: Validation included linearity, inter- and intra-assay precision, precision near the lower limit of quantification (LloQ), and interference testing for hemoglobin, lipid, and bilirubin. Correlation and bias were assessed. RESULTS: Linearity was given within the dynamic range. Coefficients of variation (CV) were ≤4% near the LloQ as well as for intra-and inter-assay precision. No interference was observed for lipid and bilirubin, while hemoglobin caused a negative bias of 28%. Method comparison included 74 samples within three TT4 concentration ranges (0.5-3.7, >3.7-5.13, >5.13-8 μg/dL). Correlation between POCA and reference method was excellent (r = 0.95) with a slight proportional bias of 4.5%. TE was between 7.0% and 9.8%. Despite substantial agreement, discordant results on thyroid status occurred in 15% of samples. CONCLUSIONS: The analytical performance of the POCA was excellent, as was its correlation with the reference method. Except for the interferent effect of hemoglobin, the TE was <TE for all analyses. Analysis of severely hemolytic samples is not advised. However, the relatively small dynamic range of the POCA precludes quantitative analysis of samples with TT4 >8 μg/dL, and de novo reference intervals need to be established.
Basso E, Chulla DR, Tubelli E
… +4 more, Wicks S, Flatt E, Beirne C, Whitworth A
Vet Clin Pathol
· 2025 Mar · PMID 39894936
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BACKGROUND: As obligate scavengers, New World vultures (Cathartiformes: Cathartidae) play a key role in carcass removal and disease control. Associated with this ecosystem service, vultures are exposed to the consumption...BACKGROUND: As obligate scavengers, New World vultures (Cathartiformes: Cathartidae) play a key role in carcass removal and disease control. Associated with this ecosystem service, vultures are exposed to the consumption of harmful substances such as poisons, heavy metals, antibiotics, or non-steroidal anti-inflammatory drugs. Monitoring the health status of vulture populations is a priority and an important conservation strategy, and hematologic analysis is a practical and effective method that can be useful for this purpose. OBJECTIVES: Report hematologic reference intervals in free-living King vultures (Sarcoramphus papa), one of the least studied New World vulture species. METHODS: Red blood cell concentration, packed cell volume, hemoglobin (Hb) concentration, MCV, MCH, MCHC, WBC concentration, differential count and absolute WBC concentration, heterophil to lymphocyte ratio, and total protein concentration were determined. We used a non-parametric method to calculate the reference intervals. In addition, we compared our results with the hematologic measurands reported for the Black vulture (Coragyps atratus), the Andean condor (Vultur gryphus) and the California condor (Gymnogyps californianus). RESULTS: With the exception of Hb, monocyte, basophil, and eosinophil concentrations, the mean hematologic measurands observed in the King vulture were similar to those recorded for the other New World vulture species, and the heterophil to lymphocyte ratio was lower in the King vulture compared to the Black vulture. CONCLUSIONS: In this study, we have reported for the first time the hematologic reference intervals in a free-living population of King vultures in Costa Rica. Future research should consider comparison among free-living New World vulture species.
Vet Clin Pathol
· 2025 Dec · PMID 39865558
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MicroRNAs (miRNAs or miRs) are small, non-coding RNAs that play a crucial role in gene regulation, making them potential biomarkers for various diseases. In the field of veterinary medicine, there is a growing interest i...MicroRNAs (miRNAs or miRs) are small, non-coding RNAs that play a crucial role in gene regulation, making them potential biomarkers for various diseases. In the field of veterinary medicine, there is a growing interest in exploring the diagnostic and therapeutic potential of miRNAs in kidney diseases affecting dogs and cats. This review focuses on the use of urinary miRNAs as biomarkers for chronic kidney disease (CKD) in these companion animals. We introduce miRNAs, their biogenesis, and their presence in biofluids, particularly within exosomes, and discuss studies investigating miRNAs in kidney tissue and urine. We acknowledge the challenges associated with miRNA studies, including preanalytical factors such as biological variation, sample collection/processing, storage conditions, and experimental design. We highlight the importance of technical considerations, such as sample pooling, sequencing depth, multiplexing, and the various steps of the miRNA experimental workflow. Furthermore, we discuss RNA isolation methods, small RNA sequencing data analysis, and the use of quantitative reverse transcription PCR (qRT-PCR) and droplet digital PCR for verification. We emphasize the importance of internal controls, spike-ins, and normalization methods to minimize technical variation and ensure reliable results in qRT-PCR analysis. This review concludes that while urinary miRNAs hold promise as non-invasive biomarkers for CKD in dogs and cats, addressing the challenges and standardization of protocols is vital for the successful translation of this research into clinical practice.
Pacholec C, Flatland B, Xie H
… +1 more, Zimmerman K
Vet Clin Pathol
· 2025 Dec · PMID 39843399
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Artificial intelligence (AI) is emerging as a valuable diagnostic tool in veterinary medicine, offering affordable and accessible tests that can match or even exceed the performance of medical professionals in similar ta...Artificial intelligence (AI) is emerging as a valuable diagnostic tool in veterinary medicine, offering affordable and accessible tests that can match or even exceed the performance of medical professionals in similar tasks. Despite the promising outcomes of using AI systems (AIS) as highly accurate diagnostic tools, the field of quality assurance in AIS is still in its early stages. Our Part I manuscript focused on the development and technical validation of an AIS. In Part II, we explore the next step in development: external validation (i.e., in silico testing). This phase is a critical quality assurance component for any AIS intended for medical use, ensuring that high-quality diagnostics remain the standard in veterinary medicine. The quality assurance process for evaluating an AIS involves rigorous: (1) investigation of sources of bias, (2) application of calibration methods and prediction of uncertainty, (3) implementation of safety monitoring systems, and (4) assessment of repeatability and robustness. Testing with unseen data is an essential part of in silico testing, as it ensures the accuracy and precision of the AIS output.
Vet Clin Pathol
· 2025 Mar · PMID 39734254
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BACKGROUND: Lipoprotein fractions are reported to be unstable in stored human samples, and there is a paucity of information on the analytical precision of electrophoretic separation of lipoproteins in canine serum sampl...BACKGROUND: Lipoprotein fractions are reported to be unstable in stored human samples, and there is a paucity of information on the analytical precision of electrophoretic separation of lipoproteins in canine serum samples. OBJECTIVE: The aim of this study was to assess the effects of intra- and inter-assay imprecision and of storage conditions on the electrophoretic separation of canine lipoproteins. METHODS: Imprecision was assessed by calculating the coefficient of variation (CV) of five replicates of six serum samples run in two sequential runs of agarose gel lipoprotein electrophoresis. The effect of storage was assessed with a Friedmann test by comparing the results of samples analyzed after sampling (T0) and after 24 and 48 h at room temperature or stored at 4°C and after 7, 14, 21 days, 1, 2, and 3 months at -20°C or at -80°C. Moreover, electrophoretograms obtained after storage were visually analyzed by two observers in a blind manner to assess whether storage alters the electrophoretic profile. RESULTS: The imprecision of high-density lipoproteins (HDL), low-density lipoproteins (LDL), very low-density lipoprotein (VLDL), and chylomicrons were respectively 0.8%-11.5%, 2.4%-22.7%, 2.3%-11.5%, and 12.5%-105.2%. Compared with T0, HDL significantly decreased, and LDL significantly increased over time in all the storage conditions, whereas VLDL significantly increased only in frozen samples, and chylomicrons did not significantly differ. In frozen samples, deviations from baseline values were lower than the imprecision of the method, and visual misclassifications of electrophoretograms were rare. CONCLUSIONS: Despite minimal variation in the percentage of some fractions, freezing does not influence the interpretation of canine lipidograms.