Search Results
You are looking at 1 - 10 of 11 items for
- Author or Editor: K. Jane Wardrop x
- Refine by Access: All Content x
Abstract
Objective
To determine posttransfusion viability (PTV) of canine RBC stored for 35 days in an additive solution, using in vitro biotinylation and technetium-99m and chromium-51 (99mTc/51Cr) labeling techniques.
Sample Population
6 random source, adult dogs.
Procedure
RBC from dogs were labeled with N-hydroxysuccinimide biotin (NHS-biotin) or 99mTc/51Cr in a crossover design. One unit (450 ml) of whole blood was collected from each dog, processed into packed RBC, and stored for 35 days in an additive solution. The process was repeated at a later date, so that each dog had 2 units stored under similar conditions. Stored autologous RBC were then labeled with either NHS-biotin or 51Cr and reinfused. When 51Cr was used, labeled cells were infused simultaneously with freshly drawn cells labeled with 99mTc. Posttransfusion viability of labeled cells was determined by dividing counts per minute (99mTc/51Cr) or percentage of cells (NHS-biotin) labeled at 24 hours by counts per minute or percentage of cells labeled after infusion.
Results
Mean PTV of packed RBC stored for 35 days in an additive system was 80% when determined by biotinylation, 83% as determined by 99mTc/51Cr, and 81% as determined by 51Cr alone.
Conclusions
In vitro biotinylation provides an acceptable, nonradioisotopic means of determining PTV of stored canine packed RBC.
Clinical Relevance
NHS-biotin can be used to determine maximal storage time of canine RBC prepared for transfusion purposes. (Am J Vet Res 1998;59:397–400)
Abstract
Objective—To evaluate whether markers of platelet activation, including P-selectin expression, phosphatidylserine exposure, platelet-leukocyte aggregates, and microparticle formation, could be measured in nonstimulated and stimulated canine blood samples and develop a standardized protocol for detection of activated platelet markers in canine blood.
Sample population—Blood samples from 10 dogs.
Procedure—Platelet activation was determined by flow cytometric measurement of platelets with P-selectin expression, platelet-leukocyte aggregates, platelet microparticles, and platelets with phosphatidylserine exposure. Changes in specific markers of platelet activation in nonstimulated versus stimulated samples were assessed by use of varying concentrations of 2 platelet agonists, platelet-activating factor (PAF) and adenosine diphosphate. Flow cytometry was used to detect platelet CD61 (glycoprotein IIIa), CD62P (P-selectin), and the leukocyte marker CD45. Annexin V was used to identify exposed phosphatidylserine.
Results—A significant difference was detected in the percentages of platelets with P-selectin, plateletleukocyte aggregates, microparticles, and platelets with annexin V exposure (phosphatidylserine) in samples stimulated with 10nM PAF versus the nonstimulated samples, with platelet-leukocyte aggregates having the greatest difference.
Conclusions and Clinical Relevance—Platelet activation is essential for thrombus formation and hemostasis and may be potentially useful for evaluation of dogs with suspected thromboembolic disease. Prior to development of a thrombotic state, a prothrombotic state may exist in which only a small number of platelets is activated. Identification of a prothrombotic state by use of activated platelets may help direct medical intervention to prevent a thromboembolic episode.
Abstract
Objective—To determine the characteristics of an automated canine C-reactive protein (CRP) assay and evaluate 2 human CRP assays for use in dogs.
Animals—56 client-owned dogs with pyometra and 11 healthy control dogs.
Procedures—Samples from 11 dogs with high (> 100 mg/L) or low (< 10 mg/L) CRP concentrations (determined by use of a canine ELISA) were evaluated by use of the automated canine CRP assay. Intra- and interassay imprecision was determined (by use of those 2 plasma pools), and assay inaccuracy was assessed by use of logistic regression analysis of results obtained via ELISA and the automated canine CRP assay. Two automated human CRP assays were used to measure plasma CRP concentration in 10 dogs.
Results—By use of the ELISA, mean ± SD plasma CRP concentration was 96.1 ± 38.5 mg/L and 10.1 ± 23.2 mg/L in dogs with pyometra and control dogs, respectively. The automated canine assay had intra-assay coefficients of variation (CVs) of 7.8% and 7.9%, respectively, and interassay CVs of 11.1% and 13.1%, respectively. Results from the automated assay were highly correlated with results obtained via ELISA. The human assay results did not exceed 0.4 mg/L in any dog.
Conclusions and Clinical Relevance—The automated canine CRP assay had less interassay imprecision, compared with the ELISA. The 2 human CRP assays were not suitable for analysis of canine plasma samples. The automated canine CRP assay was more precise than the ELISA for serial evaluations of plasma CRP concentration in dogs.
Abstract
Objectives
To develop methods to isolate, culture, and characterize equine hoof endothelial cells (EC) and keratinocytes.
Sample Population
Cells harvested from the forelimbs of 8 horses.
Procedure
EC were obtained via catheters placed in the palmar digital arteries of the disarticulated lower portion of the forelimbs from 4 horses that had been heparinized prior to euthanasia. Phosphate-buffered saline solution was used to remove and discard RBC from blood vessels, and collagenase was used to loosen and flush EC from the vasculature. Hoof keratinocytes were obtained from 4 recently euthanatized horses by use of dispase/trypsin dissociation of the coronary band epidermis. Use of an extracellular matrix gel as a culture flask attachment factor was important to the success of hoof keratinocyte cultures.
Results
EC from the palmar digital arteries were successfully cultured and characterized by in vitro morphology, uptake of a fluorescence-labeled acetylated-low density lipoprotein, and lack of expression of von Willebrand factor and smooth muscle actin. Hoof keratinocytes were characterized by morphology in culture and expression of keratin proteins, as determined by immunochemical reaction. Keratinocyte cultures were also positive for vimentin expression.
Conclusions
Culture techniques to isolate and characterize hoof cells should aid investigators in their study of equine hoof pathobiologic features, especially as it relates to laminitis. (Am J Vet Res 1999;60:128–132)
Summary
Survival time of 51Cr-labeled pigeon rbc transfused into 5 raptors was determined. Mean ± sd estimated rbc survival time was 0.51 ± 0.19 days. This was considerably shorter than estimated survival time of autologous rbc in a Red-tailed Hawk (estimated survival, 35.1 days) and in a pigeon (estimated survival, 26.8 days). Estimated survival time after homologous transfusion of rbc from one pigeon to another was 7.1 days. Although single heterologous blood transfusions have been recommended as a safe and efficacious means of whole blood replacement in birds, results of this study suggest that heterologous rbc transfused from pigeons to selected raptor species are rapidly destroyed.
Abstract
Objective—To investigate the potential use of fluorescent- labeled annexin V, anti-human fibrinogen antibody, and anti-human thrombospondin antibody for detection of the activation of equine platelets by use of flow cytometry.
Sample Population—Platelets obtained from 6 Thoroughbreds.
Procedure—Flow cytometry was used to assess platelet activation as indicated by detection of binding of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-thrombospondin antibody to unactivated and ADP-, collagen-, platelet activating factor (PAF)-, and A23187-activated equine platelets. Human platelets were used as control samples. Determination of 14C-serotonin uptake and release was used to assess the extent of platelet secretion.
Results—Anti-human thrombospondin antibody failed to bind to equine platelets. Annexin V bound to platelets activated with PAF or A23187 when platelets had undergone secretion. Anti-human fibrinogen antibody bound to ADP-, PAF-, and A23817- activated platelets, but binding was not dependent on platelet secretion. The extent of binding of anti-fibrinogen antibody was less in equine platelets, compared with that for human platelets, despite maximal stimulation.
Conclusions and Clinical Relevance—Activation of equine platelets can be detected by use of fluorescent- labeled annexin V and anti-human fibrinogen antibody but not by use of anti-human thrombospondin antibody. These flow cytometric techniques have the potential for detection of in vivo platelet activation in horses at risk of developing thrombotic disorders. (Am J Vet Res 2002;63:513–519)
Abstract
Objective—To investigate the effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation.
Sample Population—Blood samples from 6 Thoroughbreds.
Procedure—The degree of fluorescence associated with binding of fluorescein isothiocyanate (FITC)-conjugated anti-human fibrinogen antibody and FITCannexin V in unactivated and adenosine diphosphate (ADP)-, platelet activating factor (PAF)-, and A23187- activated platelet samples in unfixed and 0.5, 1.0, and 2.0% formaldehyde-fixed samples was assessed by use of flow cytometry.
Results—In samples incubated with FITC-anti-human fibrinogen antibody prior to fixation, addition of 2.0% formaldehyde resulted in a 30% increase in total fluorescence in ADP- and PAF-activated samples and a 60% increase in A23187-activated samples. Fixation for 24 hours prior to addition of antibody resulted in reduced fluorescence of samples containing antihuman fibrinogen antibody for all 3 concentrations of formaldehyde in PAF-activated samples. The addition of all 3 concentrations of formaldehyde after incubation with FITC-annexin V resulted in significant increases in fluorescence in unactivated and activated platelet samples. As length of fixation time increased, there was a gradual increase in fluorescence that was significant at 24 hours.
Conclusion and Clinical Relevance—Because fixation with 2.0% formaldehyde results in significant changes in fluorescence in activated platelet samples containing anti-fibrinogen antibody, lower concentrations of formaldehyde should be used to fix equine platelet samples. Formaldehyde-fixed platelet samples should be analyzed within 12 hours of fixation to avoid artifactual increases in fluorescence. Fixation of samples containing FITC-annexin V should be avoided because of significant increases in fluorescence that may interfere with interpretation of results. (Am J Vet Res 2002;63:840–844)
Abstract
Objective—To investigate the effects of sodium citrate, low molecular weight heparin (LMWH), and prostaglandin E1 (PGE1) on aggregation, fibrinogen binding, and enumeration of equine platelets. Sample Population—Blood samples obtained from 4 Thoroughbreds.
Sample Population—Blood samples obtained from 4 Thoroughbreds.
Procedure—Blood was collected into syringes in the ratio of 9 parts blood:1 part anticoagulant. Anticoagulants used were sodium citrate, LMWH, sodium citrate and LMWH, or 300 nM PGE1/ml of anticoagulant. Platelet aggregation in response to ADP, collagen, and PGE1 was assessed, using optical aggregometry. Platelet activation was evaluated, using flow cytometry, to detect binding of fluorescein- conjugated anti-human fibrinogen antibody. Plasma concentration of ionized calcium was measured, using an ion-selective electrode.
Results—Number of platelets (mean ± SEM) in samples containing LMWH (109.5 ± 11.3 × 103 cells/µl) was significantly less than the number in samples containing sodium citrate (187.3 ± 30.3 × 103 cells/µl). Increasing concentrations of sodium citrate resulted in reductions in platelet aggregation and plasma concentration of ionized calcium. Addition of PGE1 prior to addition of an agonist inhibited platelet aggregation in a concentration-dependent manner, whereas addition of PGE1 4 minutes after addition of ADP resulted in partial reversal of aggregation and fibrinogen binding.
Conclusion and Clinical Relevance—A high concentration of sodium citrate in blood samples decreases plasma concentration of ionized calcium, resulting in reduced platelet aggregation and fibrinogen binding. Platelets tend to clump in samples collected into LMWH, precluding its use as an anticoagulant. Platelet aggregation and fibrinogen binding can be reversed by PGE1, which may result in underestimation of platelet activation. (Am J Vet Res 2001; 62:547–554)
Abstract
Objectives—To assess safety and determine effects of IV administration of formaldehyde on hemostatic variables in healthy horses.
Animals—7 healthy adult horses.
Procedure—Clinical signs and results of CBC, serum biochemical analyses, and coagulation testing including template bleeding time (TBT) and activated clotting time (ACT) were compared in horses given a dose of 0.37% formaldehyde or lactated Ringer’s solution (LRS), IV, in a 2-way crossover design. In a subsequent experiment, horses received an infusion of 0.74% formaldehyde or LRS. In another experiment, horses were treated with aspirin to impair platelet responses prior to infusion of formaldehyde or LRS.
Results—Significant differences were not detected in any variable measured between horses when given formaldehyde or any other treatment. Infusion of higher doses of formaldehyde resulted in adverse effects including muscle fasciculations, tachycardia, tachypnea, serous ocular and nasal discharge, agitation, and restlessness.
Conclusions and Clinical Relevance—Intravenous infusion of formaldehyde at doses that do not induce adverse reactions did not have a detectable effect on measured hemostatic variables in healthy horses. (Am J Vet Res 2000;61:1191–1196)
