Canine reference intervals for the Sysmex XT-2000iV hematology analyzer

Background: The laser‐based Sysmex XT‐2000iV hematology analyzer is increasingly used in veterinary clinical pathology laboratories, and instrument‐specific reference intervals for dogs are not available. Objective: The purpose of this study was to establish canine hematologic reference intervals according to International Federation of Clinical Chemistry and Clinical and Laboratory Standards Institute guidelines using the Sysmex XT‐2000iV hematology analyzer. Methods: Blood samples from 132 healthy purebred dogs from France, selected to represent the most prevalent canine breeds in France, were analyzed. Blood smears were scored for platelet (PLT) aggregates. Reference intervals were established using the nonparametric method. PLT and RBC counts obtained by impedance and optical methods were compared. Effects of sex and age on reference intervals were determined. Results: The correlation between impedance (I) and optical (O) measurements of RBC and PLT counts was excellent (Pearson r=.99 and .98, respectively); however, there were significant differences between the 2 methods (Student's paired t‐test, P<.0001). Differences between sexes were not significant except for HCT, PLT‐I, and PLT‐O. WBC, lymphocyte, and neutrophil counts decreased significantly with age (ANOVA, P<.05). Median eosinophil counts were higher in Brittany Spaniels (1.87 × 10⁹/L), Rottweilers (1.41 × 10⁹/L), and German Shepherd dogs (1.38 × 10⁹/L) than in the overall population (0.9 × 10⁹/L). PLT aggregates were responsible for lower PLT counts by the impedance, but not the optical, method. Conclusion: Reference intervals for hematologic analytes and indices were determined under controlled preanalytical and analytical conditions for a well‐characterized population of dogs according to international recommendations.     

Comparative clinical study of canine and feline total blood cell count results with seven in‐clinic and two commercial laboratory hematology analyzers

Background: A CBC is an integral part of the assessment of health and disease in companion animals. While in the past newer technologies for CBC analysis were limited to large clinical pathology laboratories, several smaller and affordable automated hematology analyzers have been developed for in‐clinic use. Objectives: The purpose of this study was to compare CBC results generated by 7 in‐clinic laser‐ and impedance‐based hematology instruments and 2 commercial laboratory analyzers. Methods: Over a 3‐month period, fresh EDTA‐anticoagulated blood samples from healthy and diseased dogs (n=260) and cats (n=110) were analyzed on the LaserCyte, ForCyte, MS45, Heska CBC, Scil Vet ABC, VetScan HMT, QBC Vet Autoread, CELL‐DYN 3500, and ADVIA 120 analyzers. Results were compared by regression correlation (linear, Deming, Passing‐Bablok) and Bland–Altman bias plots using the ADVIA as the criterion standard for all analytes except HCT, which was compared with manual PCV. Precision, linearity, and carryover also were evaluated. Results: For most analytes, the in‐clinic analyzers and the CELL‐DYN performed similarly and correlated well with the ADVIA. The biases ranged from ?0.6 to 2.4 × 10⁹/L for WBC count, 0 to 0.9 × 10¹²/L for RBC count, ?1.5 to 0.7 g/dL for hemoglobin concentration, ?4.3 to 8.3 fL for MCV, and ?69.3 to 77.2 × 10⁹/L for platelet count. Compared with PCV, the HCT on most analyzers had a bias from 0.1% to 7.2%. Canine reticulocyte counts on the LaserCyte and ForCyte correlated but had a negative bias compared with those on the ADVIA. Precision, linearity, and carryover results were excellent for most analyzers. Conclusions: Total WBC and RBC counts were acceptable on all in‐clinic hematology instruments studied, with limitations for some RBC parameters and platelet counts. Together with evaluation of a blood film, these in‐clinic instruments can provide useful information on canine and feline patients in veterinary practices.     

Hematology and Serum Biochemistry of Feline Immunodeficiency Virus-Infected and Feline Leukemia Virus-Infected Cats

Background: Hematological and biochemical values in cats naturally infected by feline immunodeficiency virus (FIV) or feline leukemia virus (FeLV) are not completely documented. Objective: Report differences in laboratory values between FIV‐ or FeLV‐infected and noninfected and between FIV‐ and FeLV‐infected cats. Animals: Three thousand seven hundred and eighty client‐owned cats tested for FIV and FeLV. Methods: Retrospective study. Evaluation of clinicopathologic changes in cats with defined FIV and FeLV status and for which laboratory data were available. Results: FIV‐infected cats were more likely to be neutropenic (odds ratio [OR]=3.6, 95% confidence interval [95% CI] 2.1–6.2, P < .0001) and had lower serum activities of aspartate aminotransferase and glutamate dehydrogenase than control cats; serum total protein (8.1 ± 1.1 versus 7.6 ± 1.3 g/dL, P < .001) and γ‐globulin concentrations (2.2 ± 1.1 versus 1.7 ± 1.3 g/dL, P < .001) were higher than in uninfected cats. Compared with controls, FeLV‐infected cats had a higher risk of anemia (OR = 3.8, 95% CI 2.4–6.0, P < .0001), thrombocytopenia (OR = 5.0, 95% CI 3.0–8.4, P < .0001), neutropenia (OR = 3.6, 95% CI 2.1–6.1, P < .0001), lymphocytosis (OR = 2.8, 95% CI 1.6–4.8, P= .0002), and lower erythrocyte counts (6.13 ± 2.95 × 10³ versus 8.72 ± 2.18 × 10³/μL, P < .001), thrombocyte counts (253.591 ± 171.841 × 10³ versus 333.506 ± 156.033 × 10³/μL, P < .001), hematocrit (28.72 ± 12.86 versus 37.67 ± 8.90%, P < .001), hemoglobin and creatinine concentration. Conclusions and Clinical Importance: Hematologic abnormalities are common in FeLV‐infected but not in FIV‐infected cats. Clinicopathologic abnormalities are less frequent in FIV‐infected cats and might reflect an unspecific immunologic response.     

Characterization of blood cells in the Leopard Cat (Prionailurus bengalensis)

Background: The Leopard Cat (Prionailurus bengalensis) is the most frequently encountered wild cat in most of Southeast Asia. Limited hematologic investigation exists for this species. Objectives: The objectives of this study were to assess routine hematologic measurements and parameters and characterize the morphology, cytochemical staining, and ultrastructural features of blood cells in Leopard Cats. Methods: Blood samples were collected from 12 adult healthy captive Leopard Cats (7 males and 5 females). Complete blood counts were performed using an automated hematology analyzer and manual differential counts. Cytochemical staining (Sudan black B [SBB], peroxidase [PO], periodic acid‐Schiff [PAS], α‐naphthyl acetate esterase [ANAE], and β‐glucuronidase [BG]) and scanning and transmission electron microscopy were performed using standard methods. Results: Median (range) hematologic results were as follows: PCV 0.46 L/L (0.30–0.55 L/L), hemoglobin 136.5 g/L (100–183 g/L), WBC 9.0 × 10⁹/L (6.9–15.2 × 10⁹/L), band neutrophils 0.07 × 10⁹/L (0–0.30 × 10⁹/L), segmented neutrophils 2.9 × 10⁹/L (1.2–6.34 × 10⁹/L), lymphocytes 5.3 × 10⁹/L (2.7–8.1 × 10⁹/L), eosinophils 0.14 × 10⁹/L (0–0.73 × 10⁹/L), basophils 0/L (0–0.22 × 10⁹/L), and monocytes 0.08 × 10⁹/L (0–0.30 × 10⁹/L). Neutrophils stained strongly positive for SBB, PO, and PAS; lymphocytes had fine granular positivity for ANAE and BG; monocytes were weakly positive for ANAE and BG; and basophils were strongly positive for BG. Ultrastructurally, eosinophils contained many large rod‐shaped granules with prominent crystalloid core structures, ribosomes, and mitochondria. Basophils contained many round to oval specific granules with homogeneous contents. Low number of basophils contained a few small vacuoles that usually were not detected by light microscopy. Conclusion: These findings will facilitate interpretation of hematologic results for future investigative and diagnostic studies of this species.     

Comparison of flow cytometry with the Sysmex XT2000iV automated analyzer for the detection of reticulated platelets in dogs

Background: Immature (reticulated) platelets (r‐PLT) are not routinely assessed by hematology analyzers, but may be useful in the evaluation of the bone marrow response to thrombocytopenia. Objective: The aim of this study was to compare the Sysmex XT2000iV hematology analyzer with standard flow cytometry for the determination of r‐PLT percentage in dogs. Methods: Blood samples were obtained from 40 healthy dogs, 12 thrombocytopenic dogs, and 6 dogs with normal platelet counts but with disorders associated with increased thrombopoiesis. The percentage of r‐PLT was determined with a FACscan flow cytometer (r‐PLT[F]) using CD61‐phycoerythrin antibody and thiazole orange, and with the PLT‐O channel of the Sysmex analyzer (r‐PLT[S]). Mean platelet volume, platelet distribution width, and platelet large cell ratio were also determined on the Sysmex. Repeatability (intra‐assay precision) and effect of storage were tested for the automated analyzer. Results: The reference interval (mean±1.96 X SD) for r‐PLT(F) was 1.91±1.29% (range 0.78–3.68%) and for r‐PLT(S) was 0.56±0.82% (range 0.11–2.16%). For both flow cytometry and the Sysmex, the patient group had a significantly higher mean percentage of r‐PLT compared with the control group (P<.0001, unpaired Student's t‐tests). Fair correlation (r=0.71; Spearman's regression analysis) was found for r‐PLT results between the 2 methods, and a negative proportional systematic bias of ?6.26 was found for the Sysmex (Bland–Altman analysis). Based on receiver operating characteristic curves and a cut‐off of ≥0.975%, a sensitivity of 94.7% and a specificity of 85.7% were obtained for detecting r‐PLT on the Sysmex, using flow cytometry as the reference method. Blood samples stored at 4 °C and 25 °C had a significant increase in the percentage of r‐PLT after 24 and 48 hours, respectively. Conclusions: The PLT‐O channel of the Sysmex XT2000iV is capable of detecting immature platelets in healthy, thrombocytopenic, and nonthrombocytopenic ill dogs.     

Vortex mixing of feline blood to disaggregate platelet clumps

Abstract: Platelet clumping is a common cause of erroneous platelet counts in cats. Mixing of blood with a vortex mixer was evaluated as a method to disaggregate platelet clumps in feline blood and thus obtain accurate platelet counts. Whole blood samples from 42 cats with platelet clumping and 10 control cats without platelet clumping were mixed for 1 minute at the maximal setting using a standard vortex mixer. Blood smears (for subjective assessment of the type and amount of platelet clumping), platelet counts, and total leukocyte counts were evaluated before and after mixing. Vortex treatment of blood samples with platelet clumps caused an increased platelet count in all but 1 sample. Although most samples had strong increases in platelet counts after mixing, only a minority of samples (5 of 42) appeared to have all platelet clumps dispersed. Of 39 feline blood samples with platelet counts initially <200×10⁹ cells/L, 23 counts increased to >200×10⁹ cells/L and 34 counts increased to >100×10⁹ cells/L. Overall, mixing gave inconsistent and partial improvement in platelet counts. Total leukocyte counts were not significantly affected by vortex mixing. Vortex mixing of 10 feline blood samples without platelet clumping had no consistent effect on platelet or WBC counts. In conclusion, vortex mixing of feline blood does not appear to be a consistent means of correcting the problem of feline platelet clumping.     

Thrombocytopenia in the cavalier King Charles spaniel

Blood samples were collected for the determination of platelet counts from 102 cavalier King Charles spaniels which had no history of bleeding disorders. Thrombocytopenia was found in 31 per cent of the samples when the lower reference limit was set at 100 X 10⁹/litre. The platelet count of the males (mean ± SD, 147 X 10⁹/litre ± 15 X 10⁹/litre) was found to be significantly (P<0.05) lower than that of the females (mean ± SD, 202 X 10⁹/litre ± 13 X 10⁹/litre). The platelet counts from a reference group of 16 normal beagles, analysed identically, were not lower than 189 X 10⁹/litre and no significant difference was found between males (mean ± SD, 249 X 10⁹/litre ± 14 X 10⁹/litre) and females (mean ± SD, 282 X 10⁹/litre ± 20 X 10⁹/litre).     

Platelet aggregation responses in clinically healthy adult llamas

Background: Limited information exists regarding hemostasis in camelids despite the importance of platelet function testing in the accurate identification of platelet disorders. As further importation of llamas to North America is restricted, variability in breeding stock will continue to decrease, potentially leading to an increase in heritable bleeding disorders. Objective: The objective of this study was to measure platelet aggregation responses in clinically healthy llamas and provide baseline data to which abnormal platelet function may be compared in the future. Methods: Blood samples were collected from 39 healthy adult llamas, citrated, and centrifuged to produce platelet‐rich plasma (PRP). Within 4 hours of the blood draw, 20 μL of each agonist reagent were added to 180 μL of PRP. Final concentrations of agonists were 2 × 10^?5 M ADP, 0.19 mg collagen/mL PRP, 1 × 10^?4 M epinephrine, and 500 μg arachidonic acid/mL PRP. Results: Llama platelets were most responsive to ADP and collagen, with a maximum percent aggregation (mean±SD) of 71.3±18.6% and 55.8±19% and aggregation rates of 9.5±3.9 and 6.7±3.7 cm/min, respectively. Llama platelet aggregation in response to epinephrine and arachidonic acid was minimal to absent. Conclusions: This study is the first of its kind to establish baseline values for platelet aggregation in healthy adult llamas.     

Spurious reticulocyte profiles in a dog with babesiosis

A 9‐year‐old, female Maltese dog was referred to the Veterinary School of Toulouse with a 2‐day history of anorexia and weakness. On clinical examination, the dog had hyperthermia (39.7°C), abdominal discomfort, and polypnea. Significant laboratory findings included pigmenturia, hyperbilirubinemia, hypercreatininemia, hyperfibrinogenemia, abnormal Snap canine pancreas‐specific lipase, and pancytopenia with a nonregenerative anemia. A peripheral blood smear revealed numerous intraerythrocytic large Babesia but no polychromasia. There was a discrepancy between the absolute automated reticulocyte count (Sysmex reticulocyte count: 60 × 10⁹/L; RI 19.4–150.1 × 10⁹/L) and the manual reticulocyte count (3.6 × 10⁹/L) as well as the absence of polychromasia. The optical red blood cell scattergram showed an abnormal isolated reticulocyte cluster at the location of low‐fluorescence ratio cells. These findings were interpreted as erythrocytes parasitized by large Babesia. The discrepancy between the Sysmex reticulocyte count and the manual reticulocyte count has been reported previously in people with falciparum malaria and numerous intra‐erythrocytic Plasmodium falciparum organisms. This spurious reticulocyte profile and reticulocyte count were observed with the Sysmex XT‐2000iV and the ProCyte using the same fluorescent dye polymethine but not with the LaserCyte using new methylene blue which does not stain Babesia organisms on a blood smear performed for manual reticulocyte counting.     

Estimating leukocyte,platelet, and erythrocyte counts in rats by blood smear examination

Background: The CBC is an essential test for assessing the health of rats used in drug development studies. Because of limited blood volume, estimates of cell counts from a blood smear would be valuable when other analytical methods of enumerating cells are not possible or available. Objective: The purpose of this study was to develop a statistical model to accurately estimate WBC, platelet (PLT), and RBC counts in blood smears from rats. Method: Blood smears and quantitative cell counts were obtained from vehicle‐treated male and female Fischer 344 rats (n=65) involved in a variety of studies. The numbers of WBCs, PLTs, and RBCs were estimated in 10 fields in the monolayer of smears using × 20 (WBC) or × 100 (PLT, RBC) objectives. Using a statistical model and the quantitative cell counts obtained on an ADVIA 120 hematology analyzer, formulas were developed to predict the quantitative counts from the estimates. Results: Data were log‐transformed before analysis. A formula was derived using the slope and intercept of the regression line between cell estimates and ADVIA counts to predict WBC, PLT, and RBC counts based only on estimates. A second formula was developed for situations in which limited quantitative analyses may be available, and resulted in even more accurately predicted counts from smear estimates. Conclusion: The formulas developed in this study can be a valuable tool in estimating cell counts from a blood smear when cell counting instruments are not available or when an instrument cell count needs to be verified. These formulas may be useful in the assessment of rat blood in discovery and lead optimization studies.     

Influence of Inseminate Components on Porcine Leucocyte Migration In Vitro and In Vivo after Pre- and Post-Ovulatory Insemination

A post‐breeding migration of leucocytes (PMN) into the uterus is considered to be an important reason for sperm losses. Minimizing such effects may be necessary for successful insemination with low sperm numbers, as required with sex‐sorted spermatozoa. We examined the magnitude of PMN influx 3 h after pre‐ or post‐ovulatory insemination with various combinations of seminal plasma (SP), semen extender Androhep? (AH; Minitüb, Tiefenbach, Germany) and sperm preparations (S). Pre‐ovulatory inseminations with preparations containing 98% AH caused a massive influx of PMN, independent of whether spermatozoa were present (628 ± 189 × 10⁶ leucocytes/uterine horn) or not (580 ± 153 × 10⁶). Post‐ovulatory, 98% AH caused a comparable immigration only in the absence of sperm cells (AH: 569 ± 198 × 10⁶, AH+S: 162 ± 102 × 10⁶). The presence of SP significantly dampened the numbers of recruited uterine leucocytes. The reaction to all inseminates containing 98% SP both with and without spermatozoa, used before ovulation (SP: 14 ± 6 × 10⁶, SP+S: 73 ± 27 × 10⁶) and after ovulation (SP: 60 ± 32 × 10⁶, SP+S: 51 ± 33 × 10⁶) did not differ significantly from controls using phosphate buffered saline (PBS) (pre‐ovulatory: 1 ± 1 × 10⁶, post‐ovulatory: 11 ± 9 × 10⁶). Quantitative in vitro transmigration assays with blood‐derived PMN proved that AH‐induced leucocyte migration into the uterus to be not as a result of direct chemotaxis, because, on account of the chelator citrate, AH significantly inhibited the transmigration towards recombinant human Interleukin‐8 (rhCXCL8) (AH: 14 ± 5% migration rate vs controls: 37 ± 6%, p < 0.05). Supernatants of spermatozoa incubated in PBS for 1, 12 or 24 h showed neither chemoattractive nor chemotaxis‐inhibiting properties. SP at ≥0.1% [v/v] significantly inhibited the in vitro transmigration of PMN. With respect to in vivo migration of neutrophils, the striking difference in the results between semen extender and seminal plasma suggests that adaptation of extender composition is needed to reflect more closely the in vivo regulatory potential of natural seminal plasma.     

Validation of the Sysmex XT‐2000iV hematology system for dogs,cats, and horses. I. Erythrocytes,platelets, and total leukocyte counts

Background: The Sysmex XT‐2000iV is a laser‐based, flow cytometric hematology system that has been introduced for use in large and referral veterinary laboratories. Objective: The purpose of this study was to validate the Sysmex XT‐2000iV for counting erythrocytes, reticulocytes, platelets, and total leukocytes in blood from ill dogs, cats, and horses. Methods: Blood samples from diseased animals (133 dogs, 65 cats, and 73 horses) were analyzed with the Sysmex XT‐2000iV and the CELL‐DYN 3500. Manual reticulocyte counts were done on an additional 98 canine and 14 feline samples and manual platelet counts were done on an additional 73 feline and 55 canine samples, and compared with automated Sysmex results. Results: Hemoglobin concentration, RBC counts, and total WBC counts on the Sysmex were highly correlated with those from the CELL‐DYN (r≥0.98). Systematic differences occurred for MCV and HCT. MCHC was poorly correlated in all species (r=0.33–0.67). The Sysmex impedance platelet count in dogs was highly correlated with both the impedance count from the CELL‐DYN (r=0.99) and the optical platelet count from the Sysmex (r=0.98). The Sysmex optical platelet count included large platelets, such that in samples from cats, the results agreed better with manual platelet counts than with impedance platelet counts on the Sysmex. Canine reticulocyte counts on the Sysmex correlated well (r=0.90) with manual reticulocyte counts. Feline reticulocyte counts on the Sysmex correlated well with aggregate (r=0.86) but not punctate (r=0.50) reticulocyte counts. Conclusion: The Sysmex XT‐2000iV performed as well as the CELL‐DYN on blood samples from dogs, cats, and horses with a variety of hematologic abnormalities. In addition, the Sysmex detected large platelets and provided accurate reticulocyte counts.     

Pelger-Huët anomaly in an Arabian horse

Abstract Abstract: A 9‐year‐old Arabian mare was evaluated for a 7‐day history of malaise. Results of a CBC included a leukocyte concentration within the reference interval (8.4 × 10³/μL, reference interval 6.0–14.0 × 10³/μL) with an apparent degenerative left shift (segmented neutrophils 1.2 × 10³/μL, reference interval 2.5–7.5 × 10³/μL; hyposegmented neutrophils 1.8 × 10³/μL, reference interval 0.0–0.2 × 10³/μL). Serum clinical chemistry results included increased aspartate transaminase, alkaline phosphatase, and gamma‐glutamyltransferase activities. A presumptive diagnosis of hepatitis or cholangiohepatitis was made. The horse was treated with antimicrobials and the malaise quickly resolved. However, in a recheck CBC on day 13, the apparent degenerative left shift remained. Further evaluation of the blood smear revealed many hyposegmented granulocytes with coarse mature chromatin and normal cytoplasmic features. On the basis of the microscopic examination, the horse was diagnosed with Pelger‐Huët anomaly. The patient's offspring was subsequently also diagnosed with Pelger‐Huët anomaly on the basis of blood film examination. Neutrophil, eosinophil, and basophil mean nuclear scores in both affected horses (mare, range 1.5–2.6; offspring, range 1.6–3.2) were lower than those in 2 unrelated Arabian horses (range, 2.8–5.0) and 5 non‐Arabian control horses (range, 2.8–5.0). Results of immunophenotyping and phagocytosis/oxidative burst assays via flow cytometry showed no difference in the expression of myeloid–specific or adhesion molecules or in neutrophil function between affected and control horses. This is the second known report of equine Pelger‐Huët anomaly, both of which affected Arabian horses.     

Hematology and clinical chemistry of adult yellow-headed temple turtles (Hieremys annandalii) in Thailand

Background: Yellow‐headed temple turtles (YHT), Hieremys annandalii, native to Thailand, are protected from exploitation under the Wild Animal Reservation and Protection Act, also listed under Appendix II of the Convention on International Trade of Endangered Species and the International Union for the Conservation of Nature red list. Objectives: The objectives of this study were to describe quantitative, morphologic, and cytochemical features of blood cells and plasma biochemical analytes of clinically healthy YHT. Methods: Blood samples were collected from 40 adult YHT from October 2007 to February 2008. Hematologic and biochemical analyses, cytochemical staining, and ultrastructural evaluation were performed using standard methods. Results: Hematologic results (mean ± SD) included: RBC count, 0.275 ± .094 × 10⁶ cells/μL; WBC count, 11.7 ± 6.6 × 10³ cells/μL; heterophils, 29.4 ± 6.9%; eosinophils, 23.7 ± 5.3%; basophils, 21.2 ± 1.9%; lymphocytes, 14.8 ± 5.9%; and azurophils, 10.7 ± 5.3%. Erythrocytes stained dark red with peroxidase‐staining. Periodic acid‐Schiff stain could not differentiate between thrombocytes and lymphocytes. Thrombocytes contained cytoplasmic vacuoles, similar to mammalian platelets and those of birds and snakes. Heterophils and eosinophils were similar in structure and cytochemical staining characteristics to those of other turtles and reptiles. Structure of basophils was similar to avian basophils. Lymphocytes and azurophils had similar cytochemical staining compared with mammalian lymphocytes and monocytes. Mean MCHC, WBC counts, absolute azurophil counts, and plasma alanine aminotransferase activity were higher in male turtles than in females. Conclusion: Blood characteristics of YHT are species‐specific, and this study can be served as a reference for future clinical studies and medical care of YHT.     

Prevalence of low automated platelet counts in cats: comparison with prevalence of thrombocytopenia based on blood smear estimation

Abstract: True thrombocytopenia is uncommon in cats; however, low platelet counts frequently are found using automated cell counters. Although this discrepancy is a well known problem, the prevalence of low automated platelet counts in feline blood samples has not been documented. We retrospectively compared the prevalence of low automated platelet counts with low blood smear-estimated platelet counts in feline blood samples. Results of blood sample analysis from 359 cats during a 1-year period at the University of Glasgow Veterinary Haematology Laboratory were examined. Smear estimates of platelet number were done in those cases in which records did not indicate adequate platelet numbers. Platelet counts obtained with an impedance counter (Minos Vet, Abx Hematologie) were <200×10⁹ cells/L in 256 samples (71%) and <50×10⁹ cells/L in 43 samples (12%). However, based on estimation of platelet numbers from blood smears, only 11 samples (3.1%) had platelet counts of <200×10⁹ cells/L and 9 samples (2.5%) had counts of <50×10⁹ cells/L. Four cats with thrombocytopenia estimated by blood smear evaluation had clinical signs of a bleeding disorder. Disorders associated with thrombocytopenia included neoplasia, cytotoxic chemotherapy, and infectious diseases. There was no evidence that delay due to mailing of samples was associated with lower automated platelet counts than would have been obtained on the day of sampling. The high prevalence of apparent thrombocytopenia in automated platelet counts was attributed to a combination of platelet aggregation and the impedance method of cell differentiation by size. Vigilance and careful examination of blood smears is required to identify the few cats with true thrombocytopenia.     

Evaluation of a citrate-based anticoagulant with platelet inhibitory activity for feline blood cell Counts

Abstract: Aggregation of feline platelets in vitro results in difficulty assessing platelet number. A citrate-based anticoagulant containing the platelet inhibitors theophylline, adenosine, and dipyridamole (CTAD; Diatube-H, Becton Dickinson, Oxford, UK) has been developed for use in human platelet studies and heparin assays. To evaluate the efficacy of CTAD in reducing platelet aggregation in feline blood samples, aliquots of blood from 51 cats were anticoagulated with EDTA, CTAD, and for 12 samples, citrate solution. Samples preserved in CTAD had significantly higher (P ≤ .001) platelet counts, as determined by an impedance counter, hemacy-tometer, and smear estimation, than samples preserved in EDTA. In addition, subjective assessment of blood smears showed significantly fewer platelet aggregates (P<.001) in CTAD-treated samples compared with EDTA samples. Although values were similar, automated platelet counts and smear estimates of platelet number were significantly higher (P < .05) and platelet aggregation was significantly less (P < .05) in CTAD samples than in citrate samples. These results suggest that the platelet inhibitory activity of CTAD reduced feline platelet aggregation. Automated total WBC counts in CTAD samples were significantly lower (P<.001) than automated counts in EDTA samples but were similar to manual WBC counts in EDTA samples. Differences in both platelet and WBC counts between CTAD and EDTA or citrate samples were clinically relevant. Mean platelet volume and MCV were significantly lower (P< .05) in CTAD samples than in EDTA samples. No effect was seen on cell morphology or staining characteristics. The anticoagulant CTAD offers an advantage over both EDTA and citrate for feline hematologic analysis, by decreasing pseudothrombocytopenia and pseudoleukocytosis.     

Effect of hemolysis on canine kaolin‐activated thromboelastography values and ADVIA 2120 platelet activation indices

Background: The impact of hemolysis on thromboelastography (TEG) and platelet activation indices has not been evaluated. Objective: The aim of this study was to investigate the influence of hemolysis induced mechanically (HM) and hemolysis induced by freezing (HF) on TEG, platelet counts (PLT), and platelet activation indicators. Methods: Blood from 17 dogs was divided into the following samples: controls, HM, and HF. HM was induced by 20 repetitions of expulsion of blood through a 23 g needle. Freezing was at −80°C, followed by warming to 37° and dilution with equal parts room temperature blood at 22°C. TEG variables that were examined included reaction time (R), coagulation time (K), angle (α), maximum amplitude (MA), and clot rigidity (G). Platelet indices were measured with the ADVIA 2120 hematology analyzer. Results: Hematocrit (HCT) (mean±SD) for controls, HM, and HF were 0.41±0.02, 0.39±0.03, and 0.25±0.02 L/L, respectively, consistent with decreases in HCT of 4.8% (HM) and 39.0% (HF). HM resulted in decreased R (2.5±0.9 minutes compared with 5.2±1.9 minutes for controls; P<0.001), and HF resulted in increased K (15.2±8.6 minutes compared with 5.3±4.0 minutes in controls; P<0.01) and decreased α (20±11° compared with 46±17° in controls; P<0.001). MA was decreased more in HF samples (26±2 mm) than in HM (38±8 mm) or control samples (49±9 mm; P<0.0001). The same applied to G values. PLT decreased after HM but not after HF. Hemolysis of both types resulted in decreased mean platelet component (MPC) concentration: control, 19.3±2.0, HM 15.5±3.4, and HF 14.3±0.7 g/dL (P<0.0001). Conclusion: In hemolyzed samples decreased MPC and R suggested activated primary and secondary hemostasis, respectively, but decreased MA and G indicated reduced clot firmness, possibly due to hyporeactive platelets. TEG and platelet activation indices should be interpreted cautiously after hemolysis.     

Bacteriological evaluation of composted manure solids prepared from anaerobic digested slurry for hygienic recycled bedding materials for dairy cows

Changes in mastitis‐causing pathogens, pH and water content in composted manure solids (CMS) prepared from digested slurry were evaluated during turning at 2‐day intervals for 8 days (C1–C4). The numbers of streptococci, coagulase‐negative staphylococci and coliforms were 2.6 × 10¹, 1.7 × 10² and 1.0 × 10¹ colony‐forming units (cfu)/g in CMS (C4) (summer), and these counts were markedly lower (P < 0.05) than those in CMS (C0 and C1). The bacterial counts ranged from 10¹ to 1.7 × 10² cfu/g in CMS (C4) (summer) and were within approved levels, <1 × 10⁶ cfu/g, indicating a minimal mastitis risk. The temperatures in CMS (C1–C4) increased to 63°C–74°C in summer and 67°C–70°C in winter. The mean pH values in CMS (C0–C4) were 9.2 in summer and 8.7 in winter, and water contents ranged from 61.7% to 69.6% in summer and 73.2% to 66.2% in winter. The significant decrease of pathogenic bacteria in CMS appears to be closely related to temperature >63°C for 8 days, pH 8.7–9.2, and water content 62% to 73%. This study demonstrates that prepared CMS has value as a recycled material with the potential to alleviate udder health issues in dairy cows.     

Clinical evaluation of the CA530-VET hematology analyzer for use in veterinary practice

BACKGROUND: The CA530-VET is a completely automated impedance cell hematology analyzer, which yields a 16-parameter blood count including a 3-part leukocyte differential. OBJECTIVES: The aim of this study was to examine the operational potential of the CA530-VET and its value for use in veterinary practice. METHODS: The analyzer was tested for blood carry-over, precision, and accuracy. Comparison methods included the CELL-DYN 3500, microhematocrit centrifugation, manual platelet (PLT) counting for feline and equine species, and a 100-cell manual WBC differential. Blood samples for comparison of the methods were obtained from 242 dogs, 166 cats, and 144 horses. RESULTS: The carry-over ratio (K) was 0.28% for RBC, 0.59% for PLT, 0.32% for WBC, and 0.18% for hemoglobin (HGB) concentration. Coefficients of variation (CVs) for within-batch precision and duplicate measurement of blood samples were clearly within the required limits, except for duplicate platelet counts in cats (8.7%) and horses (9.5%). The WBC count was in excellent agreement for dogs and horses and RBC count was in excellent agreement for horses. The accuracy of feline WBC counts was not acceptable, with the exception of values at the high end of the range. RBC counts in dogs and cats, and HGB concentration and MCV in all 3 species were sufficiently accurate. The CA530-VET HCT results were in excellent agreement with microhematocrit results in horses but exceeded the maximum allowed inaccuracy for cats and dogs. In all species, PLT counts established mechanically and manually were not in adequate agreement. Large differences were found between the CA530-VET and the manual differential percentage for lymphocytes and "mid-sized cells" (monocytes and basophilic granulocytes). CONCLUSIONS: The CA530-VET can be considered useful for routine canine, feline, and equine blood cell analyses. It should not be considered accurate, however, for PLT counts, feline total WBC counts in the subnormal and normal range, and leukocyte differentials, except for granulocytes.     

Comparative study of the effects of acepromazine and structurally related compounds on the TNF-α release by monocytes stimulated by Chlamydia pneumoniae

Acepromazine (ACP), a member of the phenothiazine family, has antioxidant properties and interacts with reactive oxygen species produced by stimulated neutrophils ( Serteyn et al. 1999 ). We found that ACP reduced the differentiation of monocytes induced by an overnight incubation with a crude Chlamydia pneumoniae extract ( Serteyn et al. 2001 ). The same model was used to test the effects of phenothiazines on the TNF‐α release by activated monocytes. A crude Chlamydia pneumoniae extract was obtained by mechanical disruption and centrifugation (1 minute, 1500 r.p.m.) of 78 hours infected McCoy cells. Monocytes (THP1 cell line; 2 × 10⁶ cells by assay) were incubated overnight with 30 µL of Chlamydia pneumoniae crude extract (equivalent to an endotoxin charge of 3.5 pg) in the presence or absence of phenothiazines (from 10^?6 to 10^?4 M) ( Mouithys‐Mickalad et al. 2001 ). For estimation of TNF‐α release, the supernatants were collected, centrifuged (to eliminate the undifferentiated monocytes) and used for TNF‐α measurements (n = 6) (Quantikine HS human TNF‐α, R&D Systems, UK). Acepromazine was compared to other phenothiazines (chlorpromazine, trifluoperazine) or to structural analogues of phenothiazines (phenoxazine, thioxanthen‐9‐one and methylene blue). For each assay, cytotoxicity was evaluated by microscopic examination and blue trypan exclusion method. Mean values of TNF‐α were compared by a Student t‐test (p < 0.05). TNF‐α release by Chlamydia‐treated THP1 was significantly decreased by ACP in a dose‐dependent manner, 378 ± 30, 209 ± 38 and 189 ± 35 ng mL^?1 for 10^?6, 10^?5 and 10^?4 M compared to the control values 385 ± 9 ng mL^?1. Similar inhibitions of TNF‐α release were obtained with trifluoperazine (313 ± 25 and 265 ± 14 ng mL^?1 at 10^?6 and 10^?5 M) and chlorpromazine (323 ± 29 and 227 ± 13 ng mL^?1 at 10^?6 and 10^?5 M), but at 10^?4 M, these two drugs were cytotoxic. The other structurally parent compounds increased significantly the TNF‐α production: 630 ± 46 and 468 ± 60 ng mL^?1 for thioxanthen‐9‐one and 547 ± 17 and 331 ± 111 ng mL^?1 for methylene blue at 10^?5 and 10^?6 (M). At 10^?4 M, the two compounds were cytotoxic. Phenoxazine increased the TNF‐α production, slightly at 10^?6 and 10^?5 M (444 ± 39 and 424 ± 16 ng mL^?1, respectively) and significantly at 10^?4 M (959 ± 30 ng mL^?1). Further studies are needed to verify if the inhibition of TNF‐α release by some phenothiazines could be linked to a reduction of the signal transduction, especially the NF‐κB pathway. These results could be interesting for the anaesthesia or treatment of animals suffering from a systemic inflammatory reaction.