Quality of hematology and clinical chemistry results in laboratory and zoo nonhuman primates: Effects of the preanalytical phase. A review.

Most errors in clinical pathology originate in the preanalytical phase, which includes all steps from the preparation of animals and equipment to the collection of the specimen and its management until analyzed. Blood is the most common specimen collected in nonhuman primates. Other specimens collected include urine, saliva, feces, and hair. The primary concern is the variability of blood hematology and biochemistry results due to sampling conditions with the effects of capture, restraint, and/or anesthesia. Housing and diet have fewer effects, with the exception of food restriction to reduce obesity. There has been less investigation regarding the impact of sampling conditions of nonblood specimens.

Improving the Quality of EDTA-treated Blood Specimens from Mice.

Nonterminal blood sampling in laboratory mice is a very common procedure. With the goal of improving animal welfare, different sampling sites and methods have been compared but have not achieved a consensus. Moreover, most of these studies overlooked the quality of blood specimens collected. The main preanalytical concern with EDTA-treated blood specimens for hematology analyses is platelet aggregation, which is known to cause analytical errors. Our objective was to find a nonterminal blood sampling method with minimal adverse effects on mice and few or no platelet aggregates. We tested and compared 2 collection sites, 4 sampling methods, and 3 antithrombotic drugs in 80 C57BL6/j male and female mice by evaluating platelet aggregates on blood smears and platelet, WBC, and RBC counts. In addition, the blood collection process was carefully evaluated, and adverse effects were recorded. Platelet aggregation was lower in specimens collected from the jugular vein than from the facial vein, with no effect of the sampling device or the presence of an antithrombotic additive. Highly aggregated specimens were significantly associated with lower platelet counts, whereas aggregation had no effect on WBC or RBC counts. Adverse events during sampling were significantly associated with more numerous platelet aggregates. The jugular vein is thus a satisfactory sampling site in mice in terms of both animal welfare and low platelet aggregation. Using antithrombotic agents appears to be unnecessary, whereas improving sampling conditions remains a key requirement to ensure the quality of EDTA-treated blood specimens from mice.

Reference Values for Hematology, Plasma Biochemistry, Bone Marrow Cytology and Bone Histology of NOD.Cg-Prkdcscid Il2rgtm1Wjl/ SzJ Immunodeficient Mice.

Highly immunodeficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) are commonly used as a models in preclinical studies for patient-derived engraftment. However, despite the frequency of their use, reference values for their clinical pathology markers have not been determined. In accordance with the American Society of Veterinary Clinical Pathology (ASVCP) recommendations, we established de novo reference values for hematologic and biochemical variables and evaluated bone marrow cytology and histology in forty 9-wk-old male and female NSG mice. Hematologic analyses were performed using 2 separate analyzers (IDEXX ProCyte Dx, Sysmex XT-2000iV) and biochemical values were measured using a Scil VetScan2. The primary hematologic characteristic seen in NSG mice was a very low white blood cell (WBC) count (below 1.6 109/L). Lymphocyte and monocyte counts were respectively over- and under-estimated by the analyzers, as compared with manual counts, likely due to misidentification of the very low concentrations of these cell types by the analyzers. This analytical bias highlights the need for confirmatory microscopic observation of blood smears from these mice for WBC differential identification. Results for all other hematology and biochemistry variables were similar to those previously reported in inbred mice, except for MPV and an unexpectedly high glucose concentration (11.5 to 19.0 mmol/L), potentially due to the nonfasting status of the animals. The differential bone marrow cell count and Myeloid:Erythroid ratio (median 1.76) were also established. Megakaryocyte and adipocyte count differed significantly between the femoral diaphysis and metaphysis and between genders. These results provide a reliable resource of baseline data for hematologic variables for researchers monitoring graft rejection studies in NSG mice.

Effects of storage conditions on results for quantitative and qualitative evaluation of proteins in canine urine.

OBJECTIVE To investigate effects of storage conditions on the canine urine protein-to-creatinine ratio (UPC) and on SDS-agarose gel electrophoresis (AGE) of urinary proteins. SAMPLE Urine specimens from 20 proteinuric (UPC > 0.5) and 20 nonproteinuric (UPC ≤ 0.2) dogs. PROCEDURES UPC and SDS-AGE were performed on urine specimens stored at room temperature (20°C) and 4°C for up to 5 days and at -20° and -80°C for up to 360 days; some specimens were subjected to 3 freeze-thaw cycles. Results were compared with those obtained for fresh urine specimens. RESULTS UPC was not affected by storage at room temperature or by freezing. A decrease in UPC was observed for specimens from nonproteinuric dogs after 5 days at 4°C (10%) and from both groups after 90 days at -20° and -80°C (≤ 20% and ≤ 15%, respectively). The SDS-AGE profiles revealed no visual changes regardless of duration of storage for specimens stored at room temperature, 4°C, and -80°C, except for 1 profile after 360 days at -80°C. Repeated freeze-thaw cycles did not affect SDS-AGE profiles. Appearance or strengthening of high-molecular-weight bands that could alter interpretation was evident in SDS-AGE profiles after storage at -20°C for ≥ 15 days (31/40 dogs). CONCLUSIONS AND CLINICAL RELEVANCE Storage of urine at -20° or -80°C for up to 1 year influenced the UPC without affecting clinical interpretation. Storage of urine specimens at -20°C impaired visual analysis of SDS-AGE. When SDS-AGE cannot be performed on fresh or recently refrigerated urine specimens, storage at -80°C is recommended.

Evaluation of CTAD (citrate-theophylline-adenosine-dipyridamole) as a universal anticoagulant in dogs.

CTAD (citrate-theophylline-adenosine-dipyridamole) has been shown to be an almost universal anticoagulant in human and feline medicine, allowing most hematology, coagulation, and biochemical analyses. Forty canine blood specimens were collected in CTAD, EDTA, heparin, and citrate for hematology, biochemistry, and coagulation analyses. CTAD partially limited platelet aggregation observed in EDTA blood smears. CTAD specimens gave similar and well-correlated results for most variables of a complete blood cell count, except for mean corpuscular volume, which was moderately higher, and mean corpuscular hemoglobin concentration, which was moderately lower in CTAD than in EDTA; reticulocyte and platelet indexes were poorly correlated. CTAD plasma gave similar results to citrate for fibrinogen, antithrombin, and D-dimers, and relatively similar results for prothrombin time, but activated partial thromboplastin time was poorly correlated. Triglycerides, cholesterol, glucose, total proteins, phosphate, iron, alanine aminotransferase, γ-glutamyl transferase, and lipase were similar and well correlated in CTAD and heparin plasmas. Urea, creatinine, albumin, alkaline phosphatase, amylase, and aspartate aminotransferase showed moderate-to-marked bias, but these variables could be measured in CTAD plasma if new reference intervals were determined. Creatine kinase activity, potassium, chloride, and total carbon dioxide measurements are not recommended in CTAD plasma. CTAD is a prospective candidate as an almost universal anticoagulant for routine hematology, some plasma coagulation, and many biochemistry variables in dogs. Definitive recommendations will require study of abnormal canine blood specimens.

Hematologic and Biochemical Biologic Variation in Laboratory Cats.

The biologic variation associated with a clinical pathology result is important to consider before reference intervals (RI) are used. Most available RI are population-based RI, in which the analytical variability, interindividual variability, and intraindividual variability are confounded. In addition, when the intraindividual variability is considerably less than the interindividual variability, a population-based RI is insufficiently sensitive to detect changes in a subject over time. Here we determined the biologic variation and reference change value (RCV) of hematologic and biochemical variables in laboratory cats. Blood specimens from 14 (7 females and 7 males) overnight-fasted laboratory cats sampled 7 times (days 1, 2, 7, 14, 31, 42, and 100) were analyzed regarding hematology and biochemistry variables. For each variable, analytical, intraindividual, and interindividual coefficients of variation were estimated prior to calculation of the index of individuality and the RCV. RBC variables (count, Hgb, Hct, MCV, MCH, MCHC, and RBC distribution width) and 5 biochemical analytes (cholesterol, creatinine, triglycerides, ALP, and calcium) exhibited marked individuality, therefore indicating that subject-based reference intervals or RCV would be preferable when monitoring these variables in laboratory cats. Population-based RI were shown to be adequate for glucose and sodium, and both types of population and individual RI were similarly efficient for albumin, total protein, urea, ALT, AST, creatine kinase, chloride, carbon dioxide, iron, magnesium, inorganic phosphate, and potassium and reticulocyte, WBC, neutrophil, lymphocyte, monocyte, eosinophil, and platelet counts. The RCV determined in the present study provide a valuable tool for monitoring hematologic and biochemical variables in healthy laboratory cats.

Changes in hematology measurements in healthy and diseased dog blood stored at room temperature for 24 and 48 hours using the XT-2000iV analyzer.

BACKGROUND: Changes in canine hematology measurements may occur when analyses are delayed due to shipment of specimens to a laboratory. OBJECTIVE: The purpose of this study was to report changes in hematologic variables in healthy and diseased canine blood measured with a Sysmex XT-2000iV during storage at room temperature for 24 and 48 hours. METHODS: EDTA-K3 blood samples from 42 healthy and diseased dogs were measured on a Sysmex XT-2000iV analyzer within one hour of sampling, and after storage for 24 and 48 hours at room temperature in the dark. RESULTS: Storage caused little or no change in RBC count, HGB concentration and MCH, while there was a moderate increase in HCT, MCV and reticulocytes count, and a moderate decrease in MCHC. Decreased platelet counts by impedance (PLT-I) and optical (PLT-O) measurements were associated with increased mean platelet volume (MPV), platelet-large cell ratio (P-LCR) and platelet distribution width (PDW), including a right shift in the platelet histogram and a dispersion of the platelet dot plot on the scattergram. The total and differential WBC count remained stable except for decreased monocyte counts. In the scatterplots, monocytes shifted into the lymphocyte population after 24 hours, and neutrophil population shifted to the right appearing in the eosinophil gate at 48 hours of storage. The disease status had only a small effect on storage-induced changes, and observed changes had no consequences for clinical decisions. CONCLUSIONS: Blood storage at room temperature was accompanied by moderate variations in some hematologic variables, awareness of which helps in avoiding misinterpretations.

Effects of intravenous, low-dose ketamine-diazepam sedation on the results of hematologic, plasma biochemical, and coagulation analyses in cats.

OBJECTIVE: To evaluate the effects of an IV, low-dose ketamine-diazepam combination used for short-duration chemical restraint on the results of clinicopathologic testing in cats and to assess its practicality and tolerance. DESIGN: Prospective case series. ANIMALS: 42 client-owned cats of various breeds, ages, and health status. PROCEDURES: Blood samples were obtained just prior to and just after IV injection of ketamine chlorhydrate (10 mg) and diazepam (0.5 mg). A CBC, plasma biochemistry panel, and coagulation profile were performed on each sample (ie, before and after chemical restraint). Practicality of the procedure was assessed, and cats were monitored for immediate and delayed effects. RESULTS: Significant changes were observed for most of the analytes tested. However, the magnitude of the observed changes was notably low and likely not of clinical relevance. The chemical-restraint procedure appeared effective, safe, and well tolerated. CONCLUSIONS AND CLINICAL RELEVANCE: The IV, low-dose ketamine-diazepam combination used for short-duration chemical restraint in the present study may be suitable to assist physical restraint for blood sampling for assessment of hematologic, serum biochemical, and coagulation parameters in cats.

Determination of reference intervals for plasma biochemical values in clinically normal adult domestic shorthair cats by use of a dry-slide biochemical analyzer.

OBJECTIVE: To establish reference intervals of plasma biochemical values in healthy adult domestic shorthair (DSH) cats by use of controlled conditions. ANIMALS: 95 healthy client-owned cats. PROCEDURES: Food was withheld from the cats overnight. All blood samples were obtained on the same day, at the same location, and by the same investigator. Blood samples were collected from a cephalic vein into lithium heparin tubes. After centrifugation of blood samples, plasma supernatants were harvested and stored at -20 degrees C until assayed for total proteins, albumin, creatinine, urea, glucose, calcium, phosphates, sodium, chloride, potassium, and CO2 concentrations and alkaline phosphatase and alanine aminotransferase activities. RESULTS: Reference intervals in healthy adult DSH cats were 65 to 85 g/L for total proteins, 27 to 39 g/L for albumin, 89 to 207 micromol/L for creatinine, 6.6 to 11.3 mmol/L for urea, 4.1 to 8.2 mmol/L for glucose, 2.4 to 2.9 mmol/L for calcium, 1.1 to 2.1 mmol/L for phosphates, 153 to 161 mmol/L for sodium, 120 to 127 mmol/L for chloride, 3.3 to 4.2 mmol/L for potassium, 15 to 21 mmol/L for CO2, 32 to 147 U/L for alkaline phosphatase, and 34 to 123 U/L for alanine aminotransferase. CONCLUSIONS AND CLINICAL RELEVANCE: This study provided reference intervals for plasma analytes in adult DSH cats. The influence of potential confounding factors was minimized through use of controlled preanalytic and analytic conditions. However, these results cannot be extrapolated to other feline breeds or used to interpret results from other biochemical analyzers.