Pre-analytical and analytical variability of reticulocyte counts in dogs.

BACKGROUND: Automated fluorescence-based haematology analysers are now available for reticulocyte enumeration in veterinary medicine, but manual counting is still largely used. This study aimed to evaluate potential sources of analytical and pre-analytical errors when performing automated and manual counts. METHODS: Automated and two-operator double-blind manual reticulocyte counts were performed on 15 blood samples. The intra-assay variation of the automated and manual counts and the interoperator variation in the manual counts were then calculated. In addition, the effects of storage were evaluated using samples refrigerated at 4°C or stored at room temperature for 2, 4, 12, 24, 48 or 72 hours after sampling. RESULTS: Intra-assay coefficients of variation were lower for automated counts than for manual counts. Comparison between automated and mean total manual reticulocyte count showed no significant differences. In both refrigerated samples and those stored at room temperature, an increase in reticulocyte count was recorded only after 72 hours. Staining artefacts occurred only in one stored sample counted manually. LIMITATIONS: The presence of cytoplasmic particles other than RNA can cause misinterpretation of cells, leading to an erroneous reticulocyte count. CONCLUSION: The use of an automated analyser is preferable for reticulocyte enumeration in dogs. Common storage conditions seem to minimally affect reticulocyte evaluation; however, it is recommended to perform the analysis as soon as possible after sampling.

Comparison of six microscopic methods and two operators for estimation of white and red blood cells in canine urine sediment.

BACKGROUND: Little information is currently available about the analytical variability of urinalysis. OBJECTIVE: We aimed to compare results obtained by two operators using six microscopic methods in the quantification of urinary leukocytes (WBC) and erythrocytes (RBC). METHODS: Forty urine samples (10 mL) were centrifuged (450g, 5 minutes) and resuspended in 0.5 mL of supernatant. Two operators with different expertise in urinalysis interpreted sediment results using the six methods, obtained by combining the use of microscope slides (Slide) or counting chambers (Chamber) with three different techniques: bright-field (BF) microscopy, phase-contrast (PC) microscopy, and stained sediment (SS) evaluations. The mean WBC and RBC counts from 10 fields (Slide) or squares (Chamber) observed at 400× were used to calculate the difference and agreement between operators and methods. We also estimated the concordance between methods in classifying microhematuric or pyuric samples. RESULTS: Operator 2 counted significantly lower WBC counts using Slide+BF (P = 0.009) and Slide+PC (P = 0.001) than Operator 1, whereas no inter-operator differences were recorded for RBC counts. The concordance between the operators ranged from "good" to "very good." No differences or biases were found for WBC counts among the methods, and concordances were "good" to "very good"; proportional biases were found for RBC counts between Slide+BF vs Slide+SS and Slide+PC vs Slide+SS. Concordance measurements for RBC counts ranged from "good" to "very good." CONCLUSIONS: All methods yielded good reproducibility among operators, although stained SS evaluations allowed better identification of WBC by the inexperienced operator. However, we suspected that the SS preparations affected RBC counts. All other methods yielded reproducible WBC and RBC counts.

Ovarian Neuroglial Choristoma in a Bitch.

Neuroglial choristomas are rare malformations consisting of heterotopic mature neural tissue at a site isolated from the brain or spinal cord. In human medicine, neuroglial choristomas are predominantly reported in the head and in the neck, except for one recent case reported in a foot of a child. In domestic animals, neuroglial choristomas are exceedingly rare, reported only in the retina of a dog, in the pharynx and in the skin of two kittens, and within the oropharynx of a harbor seal. A three-year-old intact female Jack Russell Terrier presented for elective ovariectomy exhibited a cystic lesion 2 cm in diameter expanding in the right ovary. Histological examination of the lesion revealed a mass composed of well-organized neuroglial tissue. Immunohistochemistry with primary antibodies against GFAP, NSE, and IBA-1 confirmed the neuroglial origin of the mass. At the time of this writing, 7 years after ovariectomy, the dog was clinically normal. Together with a recent case described in the foot of a child, this case confirms that neuroglial choristoma may also be found far from the skull or spine, supporting the hypothesis that they may arise from an early embryological migration defect.

Clinical evaluation and microbiota analysis in 9 dogs with antibiotic-responsive enteropathy: A prospective comparison study.

BACKGROUND: Antibiotic-responsive enteropathy (ARE) is diagnosed by excluding other causes of diarrhea and when there is a short-term response to administration of antibiotics. OBJECTIVES: To characterize the gut microbiota and clinical trend of dogs with suspected ARE and to evaluate the variation in microbiota before (T0), after 30 days (T30) of tylosin treatment, and 30 days after discontinuation of treatment (T60). A further objective was to evaluate whether changes in gut microbiota are related to relapses of diarrhea when the therapy is tapered. ANIMALS: Study sample (group A) was composed of 15 dogs with chronic diarrhea, group B was composed of 15 healthy dogs. Group A was given tylosin for 30 days. METHODS: A multicentric prospective study. Clinical Indexes, fecal score, and samples for microbiota analysis were collected at T0, T30, and T60 in group A and T0 and T30 in group B. The gut microbiota was analyzed via 16S ribosomal RNA gene. Qiime2 version 2020.2 was used to perform bioinformatic analyses, and Alpha- and Beta-diversity were computed. RESULTS: Diarrhea recurred after T30 in 9 of 14 dogs, which were classified as affected by ARE. At T0, a difference was noted in the beta-diversity between groups (Bray Curtis metric P = .006). A T0-T30 difference in alpha-diversity was noted in group A (Shannon index P = .001, Faith PD P = .007). CONCLUSIONS AND CLINICAL IMPORTANCE: Although tylosin influences the microbiota of dogs with ARE, we failed to find any specific characteristic in the microbiota of dogs with ARE.

Influence of preanalytical factors on feline proteinuria.

BACKGROUND: To date, little information is available about the effect of preanalytical factors on the urinary protein-to-creatinine (UPC) ratio in cats. OBJECTIVES: We aimed to evaluate the effect of a commercially available cat litter, creatinine measurements at three different dilutions of urine, and different storage conditions on the UPC ratio in cats. METHODS: Feline urine specimens were prospectively collected. Twenty-two whole-urine specimens were placed uncovered and in contact with cat litter for 1 hour; 25 urine supernatants were diluted 1:10, 1:20, and 1:100 for creatinine measurements. The correlation, difference, agreement, and concordance in classifying specimens according to International Renal Interest Society staging were determined. Storage effects on UPC ratios were assessed in specimens stored for 6 hours at +20℃ (n = 20), 1 week at +4℃ (n = 20), and 3 months at -20℃ (n = 25). Specimens were also subjected to four freeze-thaw cycles (n = 20). Results were compared, and clinical significance was assessed by comparing each UPC ratio to the inter-assay range of the baseline value. RESULTS: Exposure to cat litter did not affect UPC ratios. A positive proportional bias was found in the 1:100 dilution compared with the 1:20 dilution; however, concordance was high for all comparisons. At +20, +4℃, and after four repeated freeze-thaw cycles, UPC ratios were stable. Compared with baseline values, UPC ratios decreased (P < .01) after 8 and 12 weeks at -20℃. However, all UPC ratios were within the inter-assay variability of the baseline value. CONCLUSIONS: Exposure to cat litter did not affect UPC ratios, but further studies are necessary to evaluate other potential variables. The effects of the dilutions and storage conditions were clinically acceptable, although the 1:20 and 1:100 dilutions were not perfectly comparable.

Evaluation of Urinary Big Endothelin-1 in Feline Spontaneous CKD.

The endothelin-1 (ET-1) system has been implicated in the development and progression of chronic kidney disease (CKD). No information on big ET-1 in feline urine is available. The purpose of this study was to evaluate if urinary big endothelin-1 (bigET-1) is associated with feline CKD. Sixty urine samples were prospectively collected from 13 healthy cats at risk of developing CKD and 22 cats with CKD of different International Renal Interest Society (IRIS) stages (1-4). Urinary bigET-1 was measured using a commercially available ELISA. BigET-1 normalized to urine creatinine (bigET-1:UC) was compared amongst stages and substages, as proposed by IRIS, and correlated with serum creatinine concentration, proteinuria and blood pressure. BigET-1:UC at the time of inclusion was compared between cats that remained stable and cats that progressed after 12 months. BigET-1:UC was significantly higher (p = 0.002) in cats at IRIS stages 3-4 (median: 21.9; range: 1.88-55.6), compared to all other stages, and in proteinuric (n = 8, median: 11.0; range: 0.00-46.4) compared with nonproteinuric cats (n = 38 median: 0.33; range: 0.00-55.6) (p = 0.029). BigET-1:UC was not associated with CKD progression. Urinary bigET-1 increased in advanced stages of CKD and in proteinuric patients, suggesting that ET-1 may be indicative of the severity of feline CKD.

Electrophoretic patterns of proteinuria in feline spontaneous chronic kidney disease.

OBJECTIVES: The purpose of this study was to describe the electrophoretic patterns of proteinuria in cats at risk of and cats with chronic kidney disease (CKD), and to investigate whether the presence of high-molecular-weight (HMW) and low-molecular-weight (LMW) proteins were associated with CKD, proteinuria and/or disease progression. METHODS: Healthy cats at risk of developing renal disease (n = 17) and cats affected with CKD at different stages (n = 22) were prospectively enrolled and sampled over time. Seventy urine samples were included and assayed with a commercially available sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) method. Each sample (gel lane) was inspected to identify albumin, HMW and LMW proteins, and an electrophoretic pattern (albuminuria, glomerular, tubular, mixed or negative) was assigned accordingly. Fisher's exact test was used to assess the distribution of HMW and LMW proteins in cats grouped according to International Renal Interest Society stage and to the magnitude of proteinuria, and to assess if HMW and LMW proteins at the time of inclusion were associated with the development and progression of CKD. RESULTS: In samples of cats at risk, the most common pattern was glomerular (84.6%); glomerular pattern was also common in cats with CKD (54.2%), although mixed proteinuria and tubular proteinuria were also present (29.5% and 11.4%, respectively). The presence of LMW proteins was associated with CKD (P <0.0001) and to a urine protein:creatinine ratio >0.2 (P = 0.025). Both HMW and LMW proteins were not associated with progression of CKD within 6 months (n = 14). CONCLUSIONS AND RELEVANCE: Our results showed that HMW proteinuria is common in healthy cats at risk of developing CKD, although the pathological significance needs to be confirmed. The detection of LMW proteins in urine of cats suspected to be affected by CKD, especially in non-azotaemic, non-proteinuric or borderline proteinuric cats, suggests the presence of kidney damage.

Comparison of the performance of the IDEXX SediVue Dx® with manual microscopy for the detection of cells and 2 crystal types in canine and feline urine.

BACKGROUND: Microscopic evaluation of urine is inconsistently performed in veterinary clinics. The IDEXX SediVue Dx® Urine Sediment Analyzer (SediVue) recently was introduced for automated analysis of canine and feline urine and may facilitate performance of urinalyses in practice. OBJECTIVE: Compare the performance of the SediVue with manual microscopy for detecting clinically relevant numbers of cells and 2 crystal types. SAMPLES: Five-hundred thirty urine samples (82% canine, 18% feline). METHODS: For SediVue analysis (software versions [SW] 1.0.0.0 and 1.0.1.3), uncentrifuged urine was pipetted into a cartridge. Images were captured and processed using a convolutional neural network algorithm. For manual microscopy, urine was centrifuged to obtain sediment. To determine sensitivity and specificity of the SediVue compared with manual microscopy, thresholds were set at ≥5/high power field (hpf) for red blood cells (RBC) and white blood cells (WBC) and ≥1/hpf for squamous epithelial cells (sqEPI), non-squamous epithelial cells (nsEPI), struvite crystals (STR), and calcium oxalate dihydrate crystals (CaOx Di). RESULTS: The sensitivity of the SediVue (SW1.0.1.3) was 85%-90% for the detection of RBC, WBC, and STR; 75% for CaOx Di; 71% for nsEPI; and 33% for sqEPI. Specificity was 99% for sqEPI and CaOx Di; 87%-90% for RBC, WBC, and nsEPI; and 84% for STR. Compared to SW1.0.0.0, SW1.0.1.3 had increased sensitivity but decreased specificity. Performance was similar for canine versus feline and fresh versus stored urine samples. CONCLUSIONS AND CLINICAL IMPORTANCE: The SediVue exhibits good agreement with manual microscopy for the detection of most formed elements evaluated, but improvement is needed for epithelial cells.

Hematologic and biochemical reference intervals in Shetland Sheepdogs.

BACKGROUND: Several breeds have physiological peculiarities that induce variations in reference intervals (RIs) compared with the general canine population. Shetland sheepdogs (SSs) are reported to be more predisposed to different diseases (eg, hyperlipidemia, gallbladder mucocele, and hypothyroidism). Consequently, a breed-specific approach is more often required. OBJECTIVES: The aim of this study was to determine whether the RIs of the general canine population could be applied to that of SSs, and to generate breed-specific RIs, where appropriate. METHODS: Sixty clinically healthy and fasted SSs (36% of the population registered at the Italian Breed association) were examined. Routine hematology and biochemistry analyses were performed. The transference method was used to compare the results of SSs with the RIs of the general canine population. When these RIs were not validated, new RIs were generated according to the guidelines of the American Society of Veterinary Clinical Pathology. Differences associated with sex, age, coat color, and whether used as a pet, a herding dog, or an agility dog were also investigated. RESULTS: The transference method validated for 30/38 SS RIs. For 6 of the remaining 8 variables, the difference with the claimed RIs could depend on preanalytical or analytical artifacts, whereas for glucose and total cholesterol, these differences could depend on breed peculiarities. However, in all SSs, the concentration of cholesterol was <12.95 mmol/L. Relevant differences associated with sex, age, coat color, and use were not found. CONCLUSIONS: This study suggests that breed-specific RIs should be used for glucose and cholesterol in SSs.

Evaluation of the analytical variability of urine protein-to-creatinine ratio in cats.

BACKGROUND: Proteinuria quantification with the urinary protein-to-creatinine (UPC) ratio is part of the diagnostic process in feline patients suspected of chronic kidney disease (CKD). In affected cats, monitoring and substaging of the UPC according to the International Renal Interest Society (IRIS) guidelines is also necessary for appropriate patient management. No information is available about the possible effects of analytical variability on urinary proteins (UPs) and the UPC ratio in cats. OBJECTIVES: This study aimed to determine whether imprecision and method-dependent differences due to the two dye-binding methods, pyrogallol red-molybdate (PRM) and Coomassie brilliant blue (CBB), could affect IRIS substaging. METHODS: Urine samples were collected from proteinuric and nonproteinuric cats. Intra-assay and inter-assay repeatability were assessed with both the PRM and CBB methods. Urinary supernatants (n = 120) were tested using both methods. Agreement between the methods and concordance with sample classification according to IRIS guidelines were determined. RESULTS: On average, the PRM method yielded a higher CV (UP 8.4 ± 5.2%; UPC 9.5 ± 4.8%) than the CBB method (UP 5.6 ± 2.6%; UPC 7.2 ± 2.6%), but similar rates of misclassification were found in samples with UPC ratios close to the IRIS cut-off. Although the two methods were correlated, the CBB method tended to yield UPs and UPC ratios that were significantly higher (P < 0.0001) than that of the PRM method. The Passing-Bablok test also found constant and proportional errors between the PRM and CBB methods. Concordance in substaging samples according to IRIS was good (k coefficient = 0.62). CONCLUSIONS: The two methods were precise, but the higher UPC ratios obtained with the CBB methods might affect the interpretation using the IRIS guidelines and clinical decisions.

Evaluation of the analytical variability of dipstick protein pads in canine urine.

BACKGROUND: The dipstick is a first-line and inexpensive test that can exclude the presence of proteinuria in dogs. However, no information is available about the analytical variability of canine urine dipstick analysis. OBJECTIVES: The aim of this study was to assess the analytical variability in 2 dipsticks and the inter-operator variability in dipstick interpretation. METHODS: Canine urine supernatants (n = 174) were analyzed with 2 commercially available dipsticks. Two observers evaluated each result blinded to the other observer and to the results of the other dipstick. Intra- and inter-assay variability was assessed in 5 samples (corresponding to the 5 different semi-quantitative results) tested 10 consecutive times over 5 consecutive days. The agreement between observers and between dipsticks was evaluated with Cohen's k test. RESULTS: Intra-assay repeatability was good (≤3/10 errors), whereas inter-assay variability was higher (from 1/5 to 4/5 discordant results). The concordance between the operators (k = 0.68 and 0.79 for the 2 dipsticks) and that of the dipsticks (k = 0.66 and 0.74 for the 2 operators) was good. However, 1 observer and 1 dipstick overestimated the results compared with the second observer or dipstick. In any case, discordant results accounted for a single unit of the semi-quantitative scale. CONCLUSIONS: As for any other method, analytic variability may affect the semi-quantitation of urinary proteins when using the dipstick method. Subjective interpretation of the pad and, to a lesser extent, intrinsic staining properties of the pads could affect the results. Further studies are warranted to evaluate the effect of this variability on clinical decisions.

Serum symmetric dimethylarginine and creatinine in Birman cats compared with cats of other breeds.

Objectives The aim of this study was to assess whether, in contrast to serum creatinine, which is higher in Birman cats than in other breeds, the serum concentration of symmetric dimethylarginine (SDMA) is comparable in clinically healthy Birmans and in the general feline population. This could allow, in this breed, to better evaluate chronic kidney disease (CKD). Methods Serum creatinine and SDMA were measured in clinically healthy Birmans (n = 50) and in cats of other breeds (n = 46), and the results were statistically compared. A breed-specific reference interval (RI) was established for Birmans and compared with the RI for the general feline population (0.0-14.0 µg/dl). Results Creatinine (1.58 ± 0.36 mg/dl) and SDMA (12.2 ± 2.8 µg/dl) were higher ( P <0.001) in Birmans than in cats of other breeds (1.19 ± 0.17 mg/dl; 10.3 ± 2.5 µg/dl). In 20/50 Birman cats (40.0%) serum creatinine was higher than both the non-breed-specific RI of our laboratory and the threshold recommended to classify cats as IRIS stage 2 (1.6 mg/dl). The concentration of SDMA was higher than the pre-existing RI in 10/50 Birmans (20.0%) and in four cats of other breeds (8.7%). Among Birmans, the proportion of cats with SDMA >14 µg/dl was lower ( P <0.017) than the proportion of cats with creatinine >1.60 mg/dl. However, the deviation from the upper limit of the RI was lower than the analytical variability of the method in 7/10 Birmans and in 4/4 cats of other breeds. The breed-specific RI (3.5-18.7 µg/dl) overlapped with the pre-existing one. Conclusions and relevance SDMA may be a better marker of CKD in Birman cats than creatinine when non-breed-specific RIs are utilised. The coupled analysis of creatinine and SDMA could help prevent errors in diagnosing and staging CKD in Birman cats.