Effects of storage temperature and time on canine plasma ammonia concentrations

Stability of ammonia in canine plasma was determined, with regard to temperature and time of storage. Heparinized venous blood samples were collected from 8 healthy dogs, immediately placed in an ice water bath, and centrifuged at 5 C. Plasma was harvested from the blood samples, and the initial analysis of each sample for plasma ammonia was performed within 30 minutes after collection. Separate aliquots of the plasma from each dog were stored at 21 C, 4 C, -15 C, or -40 C. Ammonia concentrations of the aliquots stored at the various temperatures were determined at 24, 48, and 96 hours after collection. Statistical analysis of the data from each dog did not indicate a significant relationship between the initial concentration of plasma ammonia and subsequent determinations. Correlation or significance was not found among samples stored at similar temperatures and evaluated at similar times.     

Effect of storage conditions on prothrombin time, activated partial thromboplastin time and fibrinogen concentration on canine plasma samples

The present study was to assess the effect of storage conditions on prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen concentration in blood samples of healthy dogs. Thirty-five dogs of various breeds were included in the study. Citrated blood samples were obtained and plasma was divided into four aliquots to assess selected clotting parameters by means of a coagulometer. The first aliquot was analysed within 1 h after collection, while the remaining 3 were stored at 8℃ for 4, 8 and 24 h, respectively. One-way repeated measures analysis of variance documented a significant decreasing effect on PT at 24 h compared to 8 h and on fibrinogen concentration after 8 and 24 h compared to sampling time and at 4 and 24 h compared to 8 h post sampling. In conclusion, the results of this study indicate that only fibrinogen appears prone to significant decrease. In fact, aPTT is not substantially affected by refrigeration for at least 24 h post sampling and PT showed a statistical difference that does not necessary indicate biological significance as the results obtained were within reference intervals for the dog.     

Effect of storage conditions on hemostatic parameters of canine plasma obtained for transfusion

OBJECTIVE: To evaluate the effects of various storage conditions on one-stage prothrombin time (OSPT), activated partial thromboplastin time (APTT), and fibrinogen concentration of canine plasma collected for transfusion. SAMPLE POPULATION: Plasma from 9 dogs. PROCEDURE: Whole blood was collected from dogs by means of jugular venipuncture and centrifuged at 7,300 X g for 20 minutes at 0 C. A plasma extractor was then used to generate plasma. Aliquots of plasma were collected in segments of plastic tubing and in microcentrifuge tubes, and plasma collection bags, tubing segments, and microcentrifuge tubes were immediately frozen at -30 C. Additional tubing segments and microcentrifuge tubes were stored at 2 C. After 1 week of storage, all samples were thawed, and OSPT, APTT, and fibrinogen concentration were measured. Collection bags and microcentrifuge tubes were refrozen at -30 C, and values were measured again 30 days after blood collection. RESULTS: Values for OSPT, APTT, and fibrinogen concentration did not vary significantly with storage time, temperature, or container. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that storage for up to 30 days and at 2 C versus -30 C did not have any significant effect on hemostatic parameters of canine plasma obtained for transfusion.     

Effects of season and sample handling on measurement of plasma alpha-melanocyte-stimulating hormone concentrations in horses and ponies

OBJECTIVE: To investigate effects of sample handling, storage, and collection time and season on plasma alpha-melanocyte-stimulating hormone (alpha-MSH) concentration in healthy equids. ANIMALS: 11 healthy Standardbreds and 13 healthy semiferal ponies. PROCEDURE: Plasma alpha-MSH concentration was measured by use of radioimmunoassay. Effects of delayed processing were accessed by comparing alpha-MSH concentrations in plasma immediately separated with that of plasma obtained from blood samples that were stored at 4 degrees C for 8 or 48 hours before plasma was separated. Effects of suboptimal handling were accessed by comparing alpha-MSH concentrations in plasma immediately stored at -80 degrees C with plasma that was stored at 25 degrees C for 24 hours, 4 degrees C for 48 hours or 7 days, and -20 degrees C for 30 days prior to freezing at -80 degrees C. Plasma alpha-MSH concentrations were compared among blood samples collected at 8:00 AM, 12 noon, and 4:00 PM. Plasma alpha-MSH concentrations were compared among blood samples collected in January, March, April, June, September, and November from horses and in September and May from ponies. RESULTS: Storage of blood samples at 4 degrees C for 48 hours before plasma was separated and storage of plasma samples at 4 degrees C for 7 days prior to freezing at -80 degrees C resulted in significant decreases in plasma alpha-MSH concentrations. A significantly greater plasma alpha-MSH concentration was found in September in ponies (11-fold) and horses (2-fold), compared with plasma alpha-MSH concentrations in spring. CONCLUSIONS AND CLINICAL RELEVANCE: Handling and storage conditions minimally affected plasma alpha-MSH concentrations. Seasonal variation in plasma alpha-MSH concentrations must be considered when evaluating pituitary pars intermedia dysfunction in equids.     

Hemostatic response to surgical neutering via ovariectomy and ovariohysterectomy in dogs

Objective-To investigate the hemostatic response to surgery and compare the response for ovariohysterectomy with that for ovariectomy and to evaluate the usefulness of thromboelastography on plasma samples. Animals-42 female dogs. Procedures-Dogs were assigned to undergo ovariohysterectomy or ovariectomy. Blood samples were collected immediately before and 1, 6, and 24 hours after surgery and stored at -80°C for subsequent analysis. Plasma samples were subjected to thromboelastography after thawing. In addition, coagulation variables were measured, including concentrations of von Willebrand factor antigen, fibrinogen, antithrombin, and protein C; activity of factor VIII; activated partial thromboplastin time; prothrombin time; and thrombin time. The fibrinolytic response was assessed via concentrations of D-dimer, plasminogen, and α-2-antiplasmin (plasmin inhibitor). Results-Substantial hemostatic and fibrinolytic activation was evident after surgery in both groups, as characterized by significantly increased global clot strength and an overall hypercoagulable state at 4 hours after surgery in addition to decreases in von Willebrand factor antigen and factor VIII concentrations and shortened prothrombin and thrombin times. The dogs also typically had activation of the fibrinolytic system, as evidenced by increased postoperative concentrations of D-dimer, plasminogen, and plasmin inhibitor. Differences between the 2 groups could not be detected for any variables. Conclusions and Clinical Relevance-Elective surgery with limited tissue trauma induced hemostatic activation in dogs, which led to hypercoagulability after surgery. A difference between the ovariohysterectomy and ovariectomy groups was not detected. Thromboelastography can be used on plasma samples and may be useful for evaluating patterns over time.     

Endotoxin-induced changes in the hemostatic system in neonatal calves: the effect of antiserum to a mutant Escherichia coli (J-5)

Changes in the hemostatic system were studied in 22 neonatal calves given a small dosage of Escherichia coli endotoxin (0.5 microgram/kg) by slow (5-hour) IV infusion. The effect of pretreatment with an antiserum to mutant of E coli O111:B4 (J-5) was evaluated. The platelet count, plasma fibrinogen concentration, prothrombin time, and activated partial thromboplastin time changed significantly from base line during and after endotoxin infusions in all calves. The mean platelet count was significantly decreased from 1 through 24 hours after endotoxin infusion was started. Mean plasma fibrinogen was decreased 2 through 12 hours after endotoxin infusion was started. The mean prothrombin time and activated partial thromboplastin time were significantly greater than base line at 3 to 6 hours and 3 to 12 hours, respectively, after endotoxin infusion was started. Serum concentration of fibrinolytic degradation products remained less than 10 micrograms/ml. Bovine J-5 antiserum did not prevent the endotoxin-induced changes in the hemostatic system of these neonatal calves.     

Evaluation of the effect of storage at -70 degrees C for six months on hemostatic function testing in dogs.

Freezing is a routine method of storage for plasma that is to be used in evaluating certain aspects of hemostatic function in many species. The purpose of this study was to evaluate the effect of storage at -70 degrees C for 6 mo on canine plasma samples. On fresh and frozen plasma from 12 clinically healthy dogs, prothrombin time, activated partial thromboplastin time, thrombin clotting time, fibrinogen determination, antithrombin III activity, fragment D and E assay, and protamine sulfate test were performed. Clinical agreement analysis was utilized to determine the effect of such storage on all assays. Individual differences detected between fresh and frozen samples were all within 2 standard deviations of the mean difference. With the exception of the activated partial thromboplastin time, storing canine plasma at -70 degrees C for 6 mo has no significant effect on hemostatic function, as assessed by these tests.     

Stability of stored canine plasma for hemostasis testing

BACKGROUND: A review of the literature revealed limited information about the stability of samples for coagulation testing in dogs. OBJECTIVE: The aim of this study was to evaluate the stability of individual coagulation factors, clotting times, and other parameters of hemostasis in stored canine plasma. METHODS: Citrated plasma samples were obtained from 21 dogs. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen concentration, and factor I, II, V, VII, VIII, IX, X, XI, and XII activities were measured on an automated coagulation analyzer with commercially available reagents. Antithrombin (AT) activity and D-dimer concentration were measured on an automated chemistry analyzer using validated kits. Samples were analyzed within 1 hour after collection (initial analysis) and once daily for 2 or 4 consecutive days following storage at room temperature (RT) or 4 degrees C, respectively. RESULTS: Storage time at either temperature did not have any effect on PT, factor II, V, VII, X, or XII activities, D-dimer concentration, or AT activity. In contrast, aPTT was significantly prolonged after 72 and 96 hours at 4 degrees C; fibrinogen concentration was decreased after 48 hours at RT; the activities of factors VIII and IX were decreased after 48, 72, and 96 hours at 4 degrees C; and factor XI activity was decreased after 72 hours at 4 degrees C. CONCLUSIONS: Results suggest that storage of canine plasma for 2 days at RT does not have a significant effect on hemostasis test results with the exception of a slight decrease in fibrinogen concentration. In contrast, aPTT and factors VIII, IX, and XI were unstable in refrigerated plasma after 48 or 72 hours of storage.     

Preoperative and postoperative hemostatic profiles of dogs undergoing ovariohysterectomy.

Hemostatic profiles were evaluated in 15 healthy dogs immediately before and 24 hours after celiotomy for routine ovariohysterectomy. Prothrombin time, activated partial thromboplastin time, fibrinogen, fibrin degradation products, antithrombin III activity, platelet count, and hemogram were measured. There were no significant changes in prothrombin time, activated partial thromboplastin time, fibrin degradation products, antithrombin III activity, or platelet count. Fibrinogen concentration was significantly higher following surgery. Postoperative leukocyte differential counts were typical of stress leukograms, and were characterized by leukocytosis, neutrophilia, lymphopenia and eosinopenia. Mild decreases in packed cell volume, red blood cell count and hemoglobin concentration were consistent with minor blood loss during surgery or fluid retention and hemodilution postoperatively. It was concluded that celiotomy and routine ovariohysterectomy in healthy dogs did not alter hemostatic profiles 24 hours after surgery. Abnormal postoperative hemostatic profiles should not be attributed to surgery alone; other causes of abnormal hemostatic profiles should be investigated.     

Effects of haemolysis, lipaemia and bilirubinaemia on prothrombin time, activated partial thromboplastin time and thrombin time in plasma samples from healthy dogs

Interferences caused by haemolysis, lipaemia and bilirubinaemia on prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT in normal canine plasma samples were studied using commercially available reagents and a steel ball coagulometer. Haemolysis significantly interfered with APTT (P = 0.0076) and TT (P = 0.0292). Regression analysis showed that TT was significantly shortened as haemoglobin concentrations increased. Lipaemia increased as demonstrated by regression analysis. Bilirubin significantly interfered with PT (P=0.0003) and APTT (P=0.002). Although statistically significant, none of the differences found were of clinical relevance.     

Comparison of prothrombin time, activated partial thromboplastin time, and fibrinogen concentration in blood samples collected via an intravenous catheter versus direct venipuncture in dogs

OBJECTIVE: To compare prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen concentration in canine blood samples collected via an indwelling IV catheter and direct venipuncture. ANIMALS: 35 dogs admitted to an intensive care unit that required placement of an IV catheter for treatment. PROCEDURES: Blood samples were collected via IV catheter and direct venipuncture at the time of catheter placement and 24 hours after catheter placement. Prothrombin time, APTT, and fibrinogen concentration were measured. RESULTS: 5 dogs were excluded from the study; results were obtained for the remaining 30 dogs. Agreement (bias) for PT was -0.327 seconds (limits of agreement, -1.350 to 0.696 seconds) and 0.003 seconds (limits of agreement, -1.120 to 1.127 seconds) for the 0- and 24-hour time points, respectively. Agreement for APTT was -0.423 seconds (limits of agreement, -3.123 to 2.276 seconds) and 0.677 seconds (limits of agreement, -3.854 to 5.207 seconds) for the 0- and 24-hour time points, respectively. Agreement for fibrinogen concentration was -2.333 mg/dL (limits of agreement, -80.639 to 75.973 mg/dL) and -1.767 mg/dL (limits of agreement, -50.056 to 46.523 mg/dL) for the 0- and 24-hour time points, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Agreement between the 2 techniques for sample collection was clinically acceptable for PT, APTT, and fibrinogen concentration at time 0 and 24 hours. It is often difficult or undesirable to perform multiple direct venipunctures in critically ill patients. Use of samples collected via an IV catheter to monitor PT and APTT can eliminate additional venous trauma and patient discomfort and reduce the volume of blood collected from these compromised patients.     

An improved fluorimetric determination of alpha-mannosidase activity in bovine plasma.

This paper describes a manual fluorimetric method for the assay of acidic alpha-mannosidase activity in bovine plasma. The optimum conditions for the assay of this enzyme were studied. The assay method devised includes the addition of zinc to the substrate, which stimulates activity by approximately twofold, and reduces the optimum substrate concentration. This latter feature affords considerable cost saving in each test. We have also shown that the alpha-mannosidase activity in lithium heparin plasma, EDTA plasma and blood serum is the same whether the plasma/serum is separated from the cells/clot at 6, 20 or 25 hours after sample collection. This has eliminated the previous necessity of having to deliver whole blood samples to the laboratory within 6 hours of collection. Furthermore samples for the supplementary neutrophil assay can now be taken at the same time as those for the plasma test, and both samples forwarded together. The plasma alpha-mannosidase assay is a rapid and reliable screening test for the mannosidosis genotype and for detecting carrier animals. Carrying out this plasma assay in conjunction with the more definitive neutrophil assay provides a reliable method of distinguishing homozygotes and heterozygotes from normal animals.     

Effect of temperature,storage time,and sample type on sorbitol dehydrogenase activity in llama serum and plasma

Abstract: Serum and heparinized plasma samples were collected from 11 adult, clinically healthy llamas. Aliquots were assayed for sorbitol dehydrogenase (SDH) activity after storage at room temperature (20°C), 4°C, or −20°C for defined time intervals up to 1 week postcollection. Sorbitol dehydrogenase activity in all samples was within reference intervals for our laboratory. No difference was found between serum and plasma SDH activity when measured immediately (within 1 hour) after collection. Sorbitol dehydrogenase activity decreased to 79% of initial activity by 24 hours in serum stored at room temperature; plasma had 94% of initial SDH activity under the same conditions. Sorbitol dehydrogenase activity was stable in both plasma and serum stored for up to 1 week at 4°C or −20°C. With the exception of serum stored at 20°C for > 8 hours, in vitro stability of llama SDH was adequate for its use in diagnostic testing.     

Whole blood re-calcification time in equine colic

Whole blood re-calcification times were evaluated as a measure of endotoxin-associated coagulopathy in horses. First, the effects of endotoxin concentration and duration of in vitro incubation of citrated whole blood with endotoxin on the whole blood re-calcification time of blood collected from healthy horses were determined. Increasing concentrations or incubation times of endotoxin accelerated the whole blood re-calcification time. This effect was attributed mainly to increased monocyte thromboplastin activity. Second, whole blood re-calcification time, a clotting profile, plasma factor VII activity and plasma endotoxin concentration on blood samples obtained from 35 equine colic patients and 10 healthy horses were determined. Compared with healthy horses, colic patients had a longer mean whole blood re-calcification and prothrombin time, lower per cent factor VII activity and higher mean fibrin degradation products concentration. Within the colic patient group, horses that did not survive had detectable endotoxin in plasma, longer whole blood re-calcification and prothrombin times, and lower plasma factor VII activity, compared with colic patients that survived. These data indicate that colic patients with endotoxaemia experience hypercoagulable states, followed by consumptive coagulopathy. Although the cause of endotoxin-associated coagulopathy is likely multi-factorial, increased expression of monocyte thromboplastin activity may be involved in the pathogenesis of coagulopathy. The whole blood recalcification time is a simple, fast and inexpensive way to detect coagulopathy during endotoxaemia and determine the prognosis for survival.     

Changes in canine ionised calcium under three storage conditions

OBJECTIVES: To determine the ionised calcium concentration following aerobic collection of blood and to compare ionised calcium concentration and pH of heparinised whole blood and plasma at 48 hours following collection under three different storage conditions to assess if ionised calcium concentration can be measured retrospectively. METHODS: Blood was collected from 17 dogs for analysis of ionised calcium concentration and pH using a Rapidpoint 400 (Bayer) blood gas analyser. Blood was collected into a commercial preheparinised syringe and into a plain syringe, with subsequent transfer to a commercially available heparinised sample tube. Samples were analysed within 10 minutes, and the remainder was divided for storage. One aliquot was set-aside at room temperature for 48 hours, and the other was immediately centrifuged and the plasma divided for storage at room temperature and at 4 degrees C for 48 hours each. In all samples, ionised calcium concentration and pH were measured again at 48 hours after storage. RESULTS: There was no significant difference in ionised calcium concentration or pH between anaerobically and aerobically collected heparinised whole blood analysed within 10 minutes of collection. At 48 hours, ionised calcium concentrations had decreased under all storage conditions irrespective of the direction of pH change. CLINICAL SIGNIFICANCE: Ionised calcium concentration can be measured in aerobically collected samples within 10 minutes and at 48 hours after collection under the conditions described.     

The effect of handling methods on subsequent plasma progesterone levels in sheep

The mean progesterone concentration in the plasma of 10 adult Ethiopian Highland sheep obtained immediately after slaughter was 10.56±3.98 ng/ml. Samples were subsequently incubated at 4°C, room temperature (19–22°C) or 26°C as either plasma or intact but citrated blood. Failure to separate plasma affected the progesterone content at 2–72 h at room temperature or 26°C (p<0.01-p<0.0001). Incubation temperature affected the plasma concentration at 18 h (p<0.05) and 24 h (p<0.001). Although progesterone values were generally higher in separated plasma, disparity with the values from plasma separated from incubated citrated blood was small (r=0.76–0.98). Progesterone concentration declined haphazardly after collection but sometimes exceeded the initial readings. This kept the average concentration of progesterone in plasma separated immediately after collection fairly constant and within 15% of zero time samples during the first 48 h.     

Studies on ionized calcium in serum and plasma from normal cows. Its relation to total serum calcium and the effects of sample storing.

Ionized calcium has been determined with a new improved instrument on serum samples from 111 Swedish red-and-white cows. Simultaneous sampling of plasma and serum was performed in 32 cows for comparison of the ionized calcium level. Multiple sampling of plasma and serum from seven cows was performed to evaluate the effect of storage at 4°C and room temperature.The normal range for ionized calcium found in this study implies that the ionized calcium fraction comprises for 43.4 ± 3.0 % (mean ± 2 s) of the total serum calcium. Simultaneous analyses on plasma and serum revealed that the plasma level of ionized calcium was generally 0.ι05 mιmol/1 lower than the serum value. pH changes in stored blood samples have a direct effect on the ionized calcium levels and is therefore to be avoided. Storing samples in vacutainers for five days at 4°C or for two days at room temperature was accompanied by only small decreases of serum or plasma ionized calcium.The new instrument used in this study enables rapid analyses, and most of earlier drawbacks with calcium-ion-selective analyzers have been eliminated.     

Hemostatic abnormalities in equine colic

Hemostatic profiles were determined in 30 horses with clinical colic. Blood samples were obtained at the time of the animal's admission, and the following hemostatic tests were done: blood platelet count, plasma fibrinogen, plasma antithrombin, prothrombin time, partial thromboplastin time, thrombin time, protamine sulfate test for soluble fibrin monomer, and fibrin-fibrinogen degradation products. The patients were categorized in retrospect, according to the cause of the colic: group 1--colic associated with colitis and/or severe diarrhea, group 2--colic associated with torsion or obstruction of the intestine, and group 3--colic associated with impaction of the intestine or the presence of enteroliths. Of the 30 horses with colic, 28 had at least 1 abnormality in their coagulogram--the most frequent abnormalities being high plasma fibrinogen concentration, high circulating soluble fibrin monomer, or a long partial thromboplastin time or thrombin time. The horses in group 1 seemed to have the most severe coagulopathies, as indicated by the average number of demonstrable abnormalities. The horses in group 3 showed the fewest abnormalities--usually a high plasma concentrations of fibrinogen and/or soluble fibrin monomer. The results indicated that hemostatic abnormalities are not uncommon in horses with gastrointestinal disease and colic--the degree of severity depending to some extent on the cause of the colic.     

Effect of oral administration of unfractionated heparin (UFH) on coagulation parameters in plasma and levels of urine and fecal heparin in dogs

The effects of heparin administration, by the oral route, were evaluated in dogs. In single and multiple dose studies (single 7.5 mg/kg, multiple 3 × 7.5 mg/kg per 48 h), plasma, urine, and fecal samples were collected at various times up to 120 h after oral administration of unfractionated heparin. Changes in plasma and urine anti-Xa activity, plasma and urine anti-IIa activity, plasma activated partial thromboplastin time (APTT) and antithrombin (ATIII), and chemical heparin in urine and feces were examined with time. There was support for heparin absorption, with significant differences in APTT, heparin in plasma as determined by anti-Xa activity (Heptest) in the single dose study and plasma anti-Xa activity, anti-IIa activity and ATIII; and chemical heparin in urine in the multiple dose study. No clinical evidence of bleeding was detected in any dog during the studies. Oral heparin therapy may be applicable for thromboembolic disease in animals. Further studies are warranted to determine the effects of oral heparin at the endothelial level in the dog.     

Ontogeny of bovine hemostasis

Ontogeny of selected hemostatic system components was studied in 120 bovine fetuses which had been divided into eight monthly gestational age groups. Fetal blood was subjected to the following tests: platelet count, partial thromboplastin time, prothrombin time, thrombin time, fibrinogen quantitation, and assays for prothrombin and factors V and VIII. Platelet numbers corresponding to adult numbers were in fetal blood at least as early as gestation day 60, and their numbers varied only slightly thereafter. Bovine blood was incapable of in vitro coagulation at gestation day 90, with all samples coagulating by gestation day 150. Fetal coagulation screening test times (partial thromboplastin time, prothrombin time, and thrombin time) shortened during gestation and were near times of adults at birth. Of the four individual coagulation factors tested, only factor VIII reached adult values in the fetus in utero. Amounts of fibrinogen, prothrombin, and factors V and VIII in the neonate exceeded that of normal adult cattle.