Measurement of platelet aggregation in ovine blood using a new impedance aggregometer

Background: Whole blood platelet aggregometry (impedance) is an important method to investigate platelet function disorders. Examination of hemostatic function in sheep is important with respect to their role as an animal model of human disease. Objective: The aim of this study was to evaluate and optimize selected methodological aspects (anticoagulant, agonist concentration) of impedance aggregometry in ovine blood using the new Multiplate 5.0 analyzer. Methods: Blood samples were collected in hirudin anticoagulant from 40 clinically healthy sheep. Samples from selected sheep were collected in citrate, with or without the addition of calcium chloride. The agonists adenosine diphosphate (ADP), collagen, ristocetin, arachidonic acid, and thrombin receptor‐activating peptide (TRAP) were added in several concentrations to induce aggregation. Results: Based on maximum aggregation values and internal precision, no significant difference was found between ADP concentrations of 3–10 μmol/L and collagen concentrations of 3–5 μg/mL (P>.05). The lowest interindividual variation of approximately 3–4‐fold was seen with 4 and 5 μmol/L ADP and 4 and 5 μg/mL collagen. Ristocetin, arachidonic acid, and TRAP did not induce significant aggregation at any concentration. Aggregation results were significantly lower when measured in citrate‐ vs hirudin‐anticoagulated blood, regardless of the presence of calcium chloride. Conclusions: Our results indicate that the multiplate impedance aggregometer is suitable for the measurement of platelet aggregation in sheep using optimal agonist concentrations of 4–5 μmol/L ADP and 4–5 μg/mL collagen. Hirudin‐anticoagulated blood is the preferred sample material.     

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.     

Heterozygosity for P2Y12 receptor gene mutation associated with postoperative hemorrhage in a Greater Swiss Mountain dog

A 3‐year‐old, female Greater Swiss Mountain dog developed a hemoperitoneum following an exploratory laparotomy and ovariohysterectomy. Platelet count, PT, APTT, and plasma von Willebrand factor antigen concentration were within RIs. A buccal mucosal bleeding time (BMBT) was prolonged. Given the probability of a hereditary thrombopathia, the dog was administered desmopressin, fresh platelet transfusions, and aminocaproic acid to control hemorrhage. Subsequently, DNA testing for the P2Y12 receptor gene mutation identified the dog as being a heterozygote (carrier). Further platelet function testing was performed following complete recovery. Results of a repeat BMBT and a point‐of‐care screening test using the Platelet Function Analyzer‐100 (collagen/adenosine‐diphosphate [ADP] test cartridge) were within RIs. Flow cytometric studies demonstrated a marked reduction in fibrinogen binding to the dog's platelets in response to ADP ‐ adenosine diphosphate activation. Likewise, turbidimetric aggregometry revealed a complete absence of platelet aggregation in response to ADP. However, there were a normal aggregation response to the platelet agonist convulxin and a mild reduction in amplitude in response to γ‐thrombin. This is the first report of a dog heterozygous for the P2Y12 receptor gene mutation exhibiting a bleeding tendency and having evidence of impaired platelet function in vitro in response to ADP activation. Given that the mutant allele for the P2Y12 thrombopathia appears to be widespread in the Greater Swiss Mountain dog breed, veterinarians need to be aware that both homozygotes and heterozygotes for this platelet receptor mutation are at risk of developing life‐threatening bleeding following trauma or surgery.     

Investigation into the causes of aspirin resistance in healthy dogs

Antiplatelet effects of acetylsalicylic acid (ASA, aspirin) may be poor in some individuals. Additionally, no method exists for predicting poor ASA response (resistance) in individual dogs. This study's main objective was to determine whether poor ASA response results from pharmacodynamic or pharmacokinetic causes. ASA concentrations causing 50% inhibition of platelet aggregation (in vitro IC50) were determined using whole blood collected from 21 drug‐free healthy dogs to evaluate intrinsic sensitivity of platelets to ASA. Dogs were then administered ASA at 4 mg/kg once orally. Percent decrease in platelet aggregation from baseline, and plasma ASA and salicylic acid (SA) concentrations (expressed as AUC values) were measured for up to 3 hr. By 3 hr, 13/21 (62%) dogs showed >50% aggregation inhibition, while 8/21 (38%) dogs showed <50% inhibition. Aggregation inhibition values were negatively correlated with in vitro IC50 values (Rs = ?0.49; p = 0.028) and positively correlated with ASA concentrations (Rs = 0.48; p = 0.03). Furthermore, ASA concentrations were strongly negatively correlated (Rs = ?0.88; p < 0.001) with SA/ASA concentration ratios, an index of ASA metabolism to SA by esterase enzymes. Multiple linear regression analysis indicated that 59% (p < 0.001) of interindividual variability in aggregation inhibition was explained by in vitro IC50 values (29% of variability) and ASA concentrations (29% of variability). Consequently, poor in vivo ASA response in these dogs resulted from both pharmacodynamic (decreased platelet sensitivity) and pharmacokinetic (lower ASA concentrations) causes. Lower ASA concentrations may be explained by reduced bioavailability associated with higher esterase activities.     

Validation of the Coulter AcT Diff hematology analyzer for analysis of blood of common domestic animals

Abstract: The objective of this study was to compare and assess the agreement between the Coulter AcT Diff hematology analyzer (CAD) and the Bayer Technicon H1 (H1) using blood samples from 391 animals of 4 species. The H1 has been used in veterinary laboratories for many years. Recently, Coulter modified the CAD and added veterinary software for hematologic analysis of feline, canine, and equine samples. A comparison of hemograms from dogs, cats, horses, and cattle was made using EDTA-anticoagulated blood samples. Both instruments were calibrated using human blood products. Performance characteristics were excellent for most values. The exceptions were MCV in canine samples (concordance correlation of .710), platelet counts for feline and equine samples (.258 and .740, respectively), feline and bovine WBC counts (.863 and .857, respectively), and bovine hemoglobin (.876).     

Evaluation of platelet function in dogs with cardiac disease using the PFA‐100 platelet function analyzer

Background: Cardiac disease has the potential to alter platelet function in dogs. Evaluation of platelet function using the PFA‐100 analyzer in dogs of multiple breeds and with a broad range of cardiac conditions would help clarify the effect of cardiac disease on platelets. Objectives: The objective of this study was to assess differences in closure time (CT) in dogs with cardiac disease associated with murmurs, when compared with that of healthy dogs. Methods: Thirty‐nine dogs with cardiac murmurs and turbulent blood flow as determined echocardiographically were included in the study. The dogs represented 23 different breeds. Dogs with murmurs were further divided into those with atrioventricular valvular insufficiency (n=23) and subaortic stenosis (n=9). Fifty‐eight clinically healthy dogs were used as controls. CTs were determined in duplicate on a PFA‐100 analyzer using collagen/ADP cartridges. Results: Compared with CTs in the control group (mean±SD, 57.6±5.9 seconds; median, 56.5 seconds; reference interval, 48.0–77.0 seconds), dogs with valvular insufficiency (mean±SD, 81.9±26.3 seconds; median, 78.0 seconds; range, 52.5–187 seconds), subaortic stenosis (71.4±16.5 seconds; median, 66.0 seconds; range, 51.5–95.0 seconds), and all dogs with murmurs combined (79.6±24.1 seconds; median, 74.0 seconds; range, 48.0–187 seconds) had significantly prolonged CTs (P<.01). Conclusions: The PFA‐100 analyzer is useful in detecting platelet function defects in dogs with cardiac murmurs, most notably those caused by mitral and/or tricuspid valvular insufficiency or subaortic stenosis. The form of turbulent blood flow does not appear to be an important factor in platelet hypofunction in these forms of cardiac disease.     

Development of gates to measure the immature platelet fraction in C57BL/6J mice using the Sysmex XN-V series multispecies hematology analyzer

The immature platelet fraction (IPF) is a measure of newly released platelets, which has been used as a marker of platelet production in multiple human studies but is not widely available in multispecies analyzers. We developed gates to measure the IPF in diluted and undiluted murine blood samples on the Sysmex XN-1000V multispecies hematology analyzer. IPF gates were created using undiluted and diluted (1/10) blood samples obtained from adult and newborn (postnatal day 10, P10) C57BL/6J wild-type (WT) mice, and from 3 murine models of thrombocytopenia: c-MPL^−/− mice, which lack the thrombopoietin receptor (hyporegenerative); antibody-mediated thrombocytopenia; and acute inflammation-induced thrombocytopenia. P10 mice were chosen because, at their size, we could consistently obtain (by terminal phlebotomy) the blood volume needed to run an undiluted sample. The undiluted blood IPF gate successfully differentiated between mechanisms of thrombocytopenia in both adult and P10 mice. For diluted samples, 2 IPF gates were generated: a thrombocytopenic (T) gate, which performed well in samples with platelet counts (PCs) <800 × 10⁹/L in adult mice and <500 × 10⁹/L in newborn mice, and a non-thrombocytopenic (NT) gate, which performed well in samples with PCs above these thresholds. PCs and IPFs measured in diluted blood using these gates agreed well with those measured in undiluted blood and had good reproducibility. These diluted gates allow for the accurate measurement of PCs and IPFs in small (10 µL) blood volumes, which can be obtained easily from adult and newborn mice as small as P1 to assess platelet production serially.     

A comparison of platelet parameters in EDTA- and citrate-anticoagulated blood in dogs

BACKGROUND: Platelet aggregates are a common artifact in canine blood. Aggregates may affect the accuracy of platelet counts, with important consequences for patient care. OBJECTIVES: The purpose of this study was to determine if platelet counts in dogs were more accurate if blood was collected into citrate instead of EDTA as an anticoagulant. METHODS: Blood was collected from 50 dogs with neoplasia admitted to the oncology service at Cornell University. EDTA and citrate Vacutainer tubes were filled with blood in random order. Platelet counts and parameters (mean platelet volume [MPV], platelet distribution width [PDW], mean platelet component concentration [MPC], platelet component distribution width [PCDW], and automated platelet clump count [APCC]) were determined using an optical-based hematology analyzer (ADVIA 120). Blood smears from each anticoagulated sample were scored visually for platelet aggregates. RESULTS: The median platelet count was significantly lower (median decrease, 27 x 10(9)/L) in citrate-anticoagulated blood compared with EDTA-anticoagulated blood. This was attributed to platelet activation and aggregation: significantly more aggregates were seen in smears of citrate- than of EDTA-anticoagulated blood. Aggregates were typically small and not detected by the analyzer. Also, the MPV and MPC (or density) were significantly higher (median increase, 3 fL) and lower (median decrease, 33 g/L) in citrate-anticoagulated samples, respectively. CONCLUSIONS: Platelets aggregate, likely from activation, when blood from dogs with neoplasia is anticoagulated with citrate for hematology testing, resulting in lower platelet counts. Citrate also yields inaccurate results for MPV and MPC, likely because of inadequate sphering of platelets. Thus, we recommend that citrate not be used as an anticoagulant when accurate platelet counts are desired in dogs.     

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.     

Validation of the sperm quality analyzer and the hypo-osmotic swelling test for frozen-thawed ram and minke whale (Balaenoptera bonarensis) spermatozoa

The object of the present study was to investigate the validation of the sperm quality analyzer (SQA) and the hypo-osmotic swelling (HOS) test with standard sperm analysis methods in frozen-thawed ram and minke whale spermatozoa. In rams, highly significant correlations were observed in the percentage of motile spermatozoa (P<0.01) and sperm concentration (P<0.01) between the standard and SQA methods. But, the percentage of morphologically normal spermatozoa did not significantly correlate between the standard and SQA methods. The percentages of swollen spermatozoa at 15 minutes by the HOS test were significantly correlated with the motility by the standard (P<0.05) and by the SQA (P<0.05) methods. For minke whale spermatozoa, the SVI (sperm viability index) values by the standard method were significantly (P<0.001) correlated with the sperm motility index (SMI) values by SQA. The percentage of motile spermatozoa was also significantly correlated (P<0.01) with the motility measured by SQA. Using different hypo-osmotic solutions and incubation times, the HOS test with 25, 100 and 150 mOsM did not show significant variations. Motility observed by the standard method and the percentage of swollen spermatozoa were significantly correlated (P<0.05). These results indicate that the SQA and HOS test can be utilized to assess the post-thawing motility of ram and minke whale spermatozoa, and that the SQA and HOS test values are significantly correlated in ram spermatozoa. However, sperm concentration and morphologically normal spermatozoa are not assessed accurately by SQA in minke whales.