Effect of time delay and storage temperature on blood gas and acid-base values of bovine venous blood

The aim of this study was to investigate possible changes in the gas composition and acid-base values of bovine venous blood samples stored at different temperatures (+4, 22 and 37 degrees C) for up to 48 h. Five healthy cattle were used in the study. A total of 15 blood samples collected from the animals were allocated into three groups, which were, respectively, then stored in a refrigerator adjusted to +4 degrees C (Group I, n=5), at a room temperature of about 22 degrees C (Group II, n=5) and in an incubator adjusted to 37 degrees C (Group III; n=5) for up to 48 h. Blood gas and acid-base values were analysed at 0 (baseline), 1, 2, 3, 4, 5, 6, 12, 24, 36 and 48 h of storage. A significant decrease (p<0.001) was found, in the pH of the refrigerated blood after 5 h and its maximum decrease was recorded at 48 h as 0.04 unit. There were also significant alterations (p<0.001) in the blood pH of the samples stored at room temperature and in the incubator after 2 and 3 h, respectively. The maximum mean alteration in pCO(2) value for Group I was -0.72 kPa during the assessment, while for groups II and III, maximum alterations in pCO(2) were detected as +2.68 and +4.16 kPa, respectively. Mean pO(2) values increased significantly (p<0.001) for Group I after 24 h and for Group II after 6 h, while a significant decrease was recorded for Group III after 24 h (p<0.001). Base excess (BE) and bicarbonate (HCO(3)) fractions decreased significantly for all the groups during the study, compared to their baseline values. In conclusion, acid-base values of the samples stored at 22 and +4 degrees C were found to be within normal range and could be used for clinical purposes for up to 12 and 48 h, respectively, although there were small statistically significant alterations.     

Changes in blood gas and acid-base values of bovine venous blood during storage

The stability of blood gas and acid-base values in bovine venous blood samples (n = 22) stored on ice for 3, 6, 9, or 24 hours was studied. Values studied include pH, PO2 and PCO2 tensions, base excess, standard base excess, bicarbonate concentration, standard bicarbonate concentration, total carbon dioxide content, oxygen saturation, and hemoglobin. The results indicate that, except for PCO2, changes in blood gas and acid-base values during 24 hours of storage and differences between cattle of differing ages, rectal temperatures, and acid-base status were too small to be of clinical significance. Therefore, bovine venous blood samples stored up to 24 hours on ice are of diagnostic utility.     

Effect of storage on oxygen dissociation of canine blood.

Oxygen dissociation curve and adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) contents in red blood cells (RBC) were determined in canine blood stored in acid citrate dextrose (ACD) and citrate phosphate dextrose (CPD) solutions. The oxygen-unloading ability decreased, as shown by the left shift of oxygen dissociation curve during storage, and the shift correlated with decreasing DPG but not decreasing ATP concentrations. After 2 weeks of storage in ACD solution, oxygen dissociation curves were shifted significantly to the left. For blood stored in CPD solution, 4 weeks was required before the shift was significant. It was concluded that canine blood collected and stored in CPD solution is more efficient than that stored in ACD solution in delivering oxygen to the tissues.     

Cross-reactivity of a monoclonal antibody with bovine, equine, ovine, and porcine peripheral blood B lymphocytes

Ficoll-thrombin purified suspensions of bovine, equine, ovine, and porcine peripheral blood lymphocytes were fractionated on nylon-wool columns. The percentages of surface immunoglobulin (SIg+)-bearing lymphocytes in the adherent (B-cell enriched) and nonadherent (T-cell enriched) fractions were determined for individual animals using fluorescein isothiocyanate conjugated species-specific anti-Ig sera. Subsequently, the human leukocyte antigen DR-specific monoclonal antibody, H4, was tested for its ability to recognize a cross-reactive antigen on the fractionated lymphocytes, using the microcytotoxicity technique. The H4 plus complement killed a percentage of lymphocytes equivalent to the percentage of SIg+ lymphocytes in the adherent and nonadherent fractions. In a parallel experiment, a 2 fluorochrome technique was used to visualize bovine lymphocytes that were SIg+ and H4+. Lymphocytes that were SIg+ also stained with ethidium bromide (orange fluorescence) after complement-mediated cytotoxicity. Seemingly, H4 recognizes an evolutionarily conserved major histocompatibility complex encoded class-II-like determinant on the B lymphocytes of cattle, horses, sheep, and swine.     

The distributions of phytohemagglutinin-P and concanavalin A binding sites on equine, bovine and canine peripheral blood lymphocytes

The distributions of phytohemagglutinin-P (PHA) and concanavalin A (ConA) binding sites were investigated for equine, bovine and canine peripheral blood lymphocytes (PBL). Non-B lymphocytes were collected from each PBL using a fluorescence-activated cell sorter (FACS), and the numbers of PHA and ConA binding sites on their surfaces were counted. Most PHA binding sites on PBL of the three species were shown on the surfaces of non-B lymphocytes. On the other hand, the ConA binding sites on equine and canine PBL existed mainly on the surfaces of non-B lymphocytes, but B lymphocytes of these two species had many ConA binding sites. These results were confirmed by the results of two-parameter fluorescence analysis using FACS. It is, therefore, concluded that the different optimum concentrations of PHA and ConA in PBL blastogenic responses of each animal depended on the different distributions of their binding sites.     

Effects of storage on serum ionized calcium and pH from horses with normal and abnormal ionized calcium concentrations

It has been previously shown that Ca(I) concentration is stable in serum collected from healthy horses for 10 days if stored at 40 degrees C. This may not be true for horses with abnormal Ca(I) concentrations. Thus the stability of ionized calcium (Ca(I)) concentration and pH measurement in serum from horses with both normal and abnormal Ca(I) concentrations stored for various times at 40 degrees C and -10 degrees C was evaluated. Our results indicated that serum Ca(I) concentration was stable throughout 7 days of cold or frozen storage, after being received by the Clinical Chemistry Laboratory. Serum Ca(I) concentration showed a significant decrease by 14 days of frozen storage (-10 degrees C). Serum pH showed a statistically significant increase by 7 days of cold storage, and within 3 days of frozen storage. If equine serum is collected, handled and stored anaerobically, and kept cold or frozen, Ca(I) concentration can be accurately measured for approximately 7 days after collection, regardless of the health status of the animal. An accurate measurement of pH may be made within 3 days of cold or 1 day of frozen storage.     

Human recombinant interleukin-2(125) induced in vitro proliferation of equine, caprine, ovine, canine and feline peripheral blood lymphocytes

Equine, caprine, ovine, canine and feline peripheral blood lymphocytes were evaluated in a short term dose-response study for their in vitro blastogenic responsiveness to human recombinant interleukin-2(125) (HrIL-2(125] alone or in combination with phytohemagglutinin-P, concanavalin-A, and pokeweed mitogen. HrIL-2(125) induced lymphocyte proliferation in all of the animals tested. The magnitude of the proliferative response varied among the species of animal tested. In all cases the proliferative response was dependent on the concentration of HrIL-2(125). HrIL-2(125) at a minimum concentration of 10(2) Cetus Units (CU)/ml produced a significant proliferative response in isolated horse, goat and sheep lymphocytes. In cat and dog lymphocytes, a concentration of 10(3) CU/ml was necessary to induce a significant proliferative response. Maximal lymphocyte proliferation was reached in horses and sheep at a concentration of 10(4) CU/ml of HrIL-2(125). In goats, cats, and dogs a maximum proliferative response was found to be at a concentration equal to or greater than 10(4) CU/ml of HrIL-2. Co-stimulation of lymphocytes with mitogens and submaximal concentrations of HrIL-2(125) (10 CU/ml) induced a synergistic proliferative response which in nearly all cases was significantly greater (P less than 0.05) than the arithmetic sum of the responses induced by the same concentration of the mitogens and HrIL-2(125) alone. The two exceptions were co-stimulation of feline lymphocytes with concanavalin-A and co-stimulation of canine lymphocytes with pokeweed mitogen.     

Evaluation of an electrolyte analyzer for measurement of ionized calcium and magnesium concentrations in blood, plasma, and serum of dogs

OBJECTIVE: To evaluate an electrolyte analyzer for measurement of ionized calcium (Ca(i)) and magnesium (Mg(i)) concentrations in blood, plasma, and serum; investigate the effect of various factors on measured values; and establish reference ranges for Ca(i) and Mg(i) in dogs. ANIMALS: 30 healthy adult dogs of various breeds. PROCEDURE: Precision in a measurement series, day-to-day precision, and linearity were used to evaluate the analyzer. The effects of exposure of serum samples to air, type of specimen (blood, plasma, or serum), and storage temperature on sample stability were assessed. Reference ranges were established with anaerobically handled serum. RESULTS: The coefficient of variation for precision in a measurement series was < or = 1.5% for both electrolytes at various concentrations. The Ca(i) and Mg(i) concentrations were significantly lower in aerobically handled serum samples, compared with anaerobically handled samples. The Ca(i) and Mg(i) concentrations differed significantly among blood, plasma, and serum samples. In anaerobically handled serum, Ca(i) was stable for 24 hours at 22 degrees C, 48 hours at 4 degrees C, and 11 weeks at -20 degrees C; Mg(i) was stable for 8 hours at 22 degrees C, < 24 hours at 4 degrees C, and < 1 week at -20 degrees C. In anaerobically handled serum, reference ranges were 1.20 to 1.35 mmol/L for Ca(i) and 0.42 to 0.58 mmol/L for Mg(i). CONCLUSIONS AND CLINICAL RELEVANCE: The electrolyte analyzer was suitable for determination of Ca(i) and Mg(i) concentrations in dogs. Accurate results were obtained in anaerobically handled serum samples analyzed within 8 hours and kept at 22 degrees C.     

Validation of the bovine blood calcium checker as a rapid and simple measuring tool for the ionized calcium concentration in cattle

Point-of-care (POC) devices that veterinary practitioners can use to easily and rapidly measure blood ionized calcium (iCa) levels in cows immediately after withdrawing a blood sample on the dairy farm are needed. Aims of present studies was to compare the commercially available ion-selective electrode handheld iCa meter (bovine blood iCa checker) with the benchtop blood gas analyzer GEM premier 3500 and handheld analyzer i-STAT 1. Sixty-two paired-point whole blood samples were obtained from three cows with hypocalcemia experimentally induced by Na₂-EDTA infusion. Whole blood samples were also obtained from the 36 cows kept on a farm in field conditions. The results using the bovine blood iCa checker correlated with those using the GEM premier 3500 and i-STAT 1. Bovine blood iCa checker was “compatible” with the GEM premier 3500 and i-STAT 1 because the frequency of differences between the measurements within ± 20% of the mean were 100% (65/65, >75%) and 90.8% (59/65, >75%), respectively. In the field trial, the blood iCa concentration measured by the bovine blood Ca checker was significantly positively correlated with that measured by the i-STAT 1 portable analyzer. Bovine blood iCa checker was “compatible” with the i-STAT 1 because the frequency of differences between the measurements within ± 20% of the mean was 100% (36/36, >75%). Results from these findings, the bovine blood iCa checker may be applied as a simplified system to measure the iCa concentration in bovine whole blood.     

Validation of a rapid solid-phase radioimmunoassay for canine, bovine, and equine insulin

A rapid and convenient commercial radioimmunoassay kit, developed for quantifying hormones in specimens from human beings, was validated for use in measuring insulin in serum of dogs, cattle, and horses. The procedure uses polypropylene assay tubes treated with rabbit anti-porcine insulin serum and porcine [125I]iodoinsulin. Specificity was proven by demonstrating that standard solutions of porcine insulin and serial dilutions of canine, bovine, and equine sera and pancreatic extracts inhibited binding of [125I]iodoinsulin to the antibody in a parallel manner. Gel-filtration chromatography of pancreatic extracts yielded a major peak of immunoreactive material that eluted identically with [125I]iodoinsulin. Immunoreactivity was not associated with fractions that contain larger and smaller molecular weight peptides (eg, proinsulin and C-peptide, respectively). Biological specificity of the assay was shown by demonstrating increased insulin in serum after injection of glucose into heifers and glucagon into dogs and horses. Purified insulin and insulin in pancreatic extracts could be quantitatively recovered from serum, thereby demonstrating accuracy of the assay. Interassay precision of 5 control specimens run in 20 consecutive assays ranged from 6.7% to 20.1% (coefficient of variation) and intra-assay precision of 6 control specimens each assayed 10 times ranged from 4.4% to 10.7% (coefficient of variation). Sensitivity of the assay was 3.2 microIU/ml. This radioimmunoassay for insulin is ideal for veterinary research and diagnosis, because a single set of reagents and procedures can be used for at least 3 species.     

Binding of 125I-labeled endotoxin to bovine, canine, and equine platelets and endotoxin-induced agglutination of canine platelets

Endotoxin from Escherichia coli O127:B8, Salmonella abortus-equi and S minnesota induced clumping of some canine platelets (PLT) at a final endotoxin concentration of 1 microgram/ml. Endotoxin-induced clumping of canine PLT was independent of PLT energy-requiring processes, because clumping was observed with canine PLT incubated with 2-deoxy-D-glucose and antimycin A. The PLT responded to adenosine diphosphate before, but not after, incubation with the metabolic inhibitors. Endotoxin induced a slight and inconsistant clumping of bovine and equine PLT at high (mg/ml) endotoxin concentration. High-affinity binding sites could not be demonstrated on canine, bovine, and equine PLT, using 125I-labeled E coli O127:B8 endotoxin. Nonspecific binding was observed and appeared to be due primarily to an extraneous coat on the PLT surface that was removed by gel filtration. The endotoxin that was bound to PLT did not appear to modify PLT function. An attempt to identify plasma proteins that bound physiologically relevant amounts of endotoxin was not successful. The significance of the endotoxin-induced clumping or lack of it on the pathophysiology of endotoxemia is discussed.     

Effects of syringe material and temperature and duration of storage on the stability of equine arterial blood gas variables

OBJECTIVES: To evaluate the consistency of partial pressures (P) of arterial oxygen (aO(2)), arterial carbon dioxide (aCO(2)) and pH measurements in equine carotid arterial blood samples taken into syringes made from three different materials and stored at room temperature or placed in iced water for measurement at three different times. STUDY DESIGN: Prospective observational study over 19 days. ANIMALS: Four clinically normal Thoroughbred or Thoroughbred-cross horses (three geldings, one mare, mean age 6.25 years, range 5-7 years). METHODS: Identical blood samples were taken on two separate occasions from the carotid arteries of the four horses into syringes made of glass, plastic and polypropylene. PaO(2), PaCO(2) and pH determinations were performed on blood from each syringe type at 10, 60 and 120 minutes post-sampling with samples stored at room temperature (approximately 20 degrees C) or in iced water (approximately 0 degrees C). Data were analysed by anova and a split plot model fitting syringe within horse X pair and time within temperature within syringe. RESULTS: Syringe material, storage temperature and time before analysis all had significant effects on PaO(2) (p < 0.001). PaCO(2) was unaffected by syringe material or storage temperature. However, over 120 minutes, storage duration significantly (p = 0.002) affected values. Temperature of storage and duration prior to analysis both significantly affected pH values (p = 0.005 and p < 0.001, respectively), but syringe material did not. Several significant interactions between these variables were noted. CONCLUSIONS: Equine arterial blood gas determination has a different sensitivity to storage conditions compared to other veterinary species. CLINICAL RELEVANCE: For accurate equine arterial blood analysis, PaO(2) samples need to be analysed within 10 minutes or taken into glass syringes, stored on ice and analysed at 2 hours post-sampling. PaCO(2) and pH measurements can be performed on samples stored in glass, plastic or polypropylene syringes at room temperature for up to 1 hour post-sampling.     

Circadian changes in blood ionized calcium, sodium, potassium, and chloride concentrations and pH in cattle.

OBJECTIVE: To explore diurnal variation in blood ionized calcium, sodium, potassium, and chloride concentrations and pH in pregnant dairy cows. ANIMALS: 14 dairy cows in their third or later pregnancy approximately 6 weeks before expected parturition. PROCEDURE: Throughout a 24-hour period, blood samples were taken at 2-hour intervals and analyzed for ionized calcium (iCa2+), potassium (K+), sodium (Na+), and chloride (Cl-) concentrations and pH. Paired t-tests were used to compare initial and final values. Circadian changes were tested by use of repeated-measures ANOVA. Additionally, a nonparametric analysis was performed for each animal to determine minimal and maximal values for the variables. RESULTS: Significant differences were not detected between initial and final values of any variable. Repeated-measures ANOVA indicated significant diurnal fluctuations in all variables. With the exception of Cl- concentration, nonparametric analysis of individual values also revealed significant changes over time. CONCLUSIONS AND CLINICAL RELEVANCE: The magnitude of daily fluctuations does not make it necessary to standardize time of sample collection for routine diagnostic examination of blood iCa2+, Na+, and Cl-concentrations and pH. However, it may be important to standardize time of sample collection in comparative studies in which more discrete differences may be identified.