The influence of anticoagulants on the measurement of total protein concentration in equine peritoneal fluid

The aim of this study was to evaluate the influence of two commonly used anticoagulants (K3EDTA and lithium heparin) on refractometric and spectrophotometric measurement of total protein (TP) concentration in equine peritoneal fluid samples. The influence of a commercial solution of K3EDTA, a solution of K3EDTA in distilled water and lithium heparin on the refractometric and spectrophotometric (biuret) quantification of TP content in peritoneal fluid samples was assessed. Total protein concentration measured by refractometry was consistently overestimated in samples with commercial K3EDTA. The solution of K3EDTA in distilled water only caused TP overestimation at high K3EDTA concentrations (>5 micromol/ml). By contrast, lithium heparin did not influence the refractometric values of TP. Neither anticoagulant modified TP values when measured by the biuret method. In conclusion, the use of K3EDTA as anticoagulant may result in a significant overestimation of TP values of peritoneal fluid samples measured by refractometry.     

Assessment of the effects of different concentrations of ethylenediaminetetraacetic acid (EDTA) on erythrocytic indices of Nigerian White Fulani cattle and West African Dwarf goat

The aim of the study was to evaluate the effects of varying concentrations of ethylenediaminetetraacetic acid (EDTA) on blood samples from White Fulani breed of cattle and West African Dwarf goat from Nigeria. Sample sizes of 20 animals were used for both species. Different concentrations of EDTA (2, 4, 8 and 16 mg/ml) were used. The packed cell volume (PCV), red blood cell (RBC) and haemoglobin (Hb) concentration of blood samples collected from White Fulani breed of cattle and West African Dwarf goat into bottles containing 16 mg/ml of EDTA were significantly lower (P < 0.05) than those samples collected from the same animals into bottle containing 2 mg/ml (control). Similarly, the PCV, RBC and Hb values of the West African Dwarf goats in bottles containing 8 mg/ml of EDTA were significantly lower than those of the samples in the control (2 mg/ml). This study has shown that high concentration of EDTA as an anticoagulant can lead to a false erythrocytic index especially the PCV. In collecting blood samples for evaluation of haematological parameters, therefore, the blood volume/anticoagulant ratio must be strictly adhered to prevent error in the evaluated parameters in cattle and goats. Taken together, there is tendency for haemolytic anaemia to occur in blood sampled at higher concentration of anticoagulants in West African Dwarf goat than in White Fulani breed of cattle.     

Effect of Manually Preheparinized Syringes on Packed Cell Volume and Total Solids in Blood Samples Collected from American Alligators (Alligator mississippiensis)

The hemodilution effect of manually preheparinized syringes was determined by evaluating the packed cell volume (PCV) and total solids (TS) from blood samples collected from 50 American alligators (Alligator mississippiensis). A volume of 0.2 mL of blood was drawn into 3 syringes of 1 mL: control with no heparin sodium, heparin sodium drawn to 0.1 mL and then expelled, and heparin sodium drawn to 0.2 mL and then expelled. PCV and TS values were determined from each syringe immediately after collection. Mean PCV and TS values were compared between the control and heparin groups using linear mixed modeling. The syringes coated with heparin resulted in a significantly lower mean PCV and TS values when compared with controls, with no significant difference between the heparin groups. This dilution effect was also found to be inconsistent and not accurate from one syringe to another. An adjunct method of obtaining 0.5 mL from already-collected blood samples into 1-mL syringes that were coated with heparin drawn to 0.2 mL and then expelled also showed a significant decrease in PCV and TS values when compared with the control samples, although to a lesser extent. As a result, it cannot be recommended to manually preheparinize syringes when collecting small volume of blood samples from American alligators because significant and unpredictable hemodilution is likely to occur.     

Temporal effects of 3 commonly used anticoagulants on hematologic and biochemical variables in blood samples from macaws and Burmese pythons

BACKGROUND: Few studies have been done to evaluate anticoagulants for use with blood samples from birds and reptiles. Heparin currently is the most commonly used anticoagulant in practice, but may adversely affect blood cell staining and quantitation. OBJECTIVE: The purpose of this study was to evaluate the effects of lithium heparin, K3-EDTA, and sodium citrate, with and without the addition of albumin, on hematologic variables in macaw (Ara sp) and python (Python molurus bivittatus) blood samples. METHODS: Blood samples from 10 macaws and 10 Burmese pythons were collected in heparin-coated syringes and placed into tubes containing either lithium heparin, K3-EDTA, or sodium citrate with and without the addition of 0.25 mL of a 22% bovine serum albumin solution. Cell lysis was determined by counting the number of lysed cells/200 WBCs in Wright's-Giemsa-stained blood smears and by qualitative evaluation of pink plasma in microhematocrit tubes. A CBC was done after 3, 12, and 24 hours of storage at 4 degrees C in anticoagulant-containing tubes and results were compared with those obtained at 0 hour for the heparin-coated syringe sample. A biochemical panel also was done at each time point in similarly stored lithium-heparin samples. RESULTS: Hemolysis was significantly increased in citrated samples from both macaws and pythons beginning at 12 hours. At 24 hours, 19 of 30 (63%) macaw samples in all anticoagulants had >100 lysed cells/200 WBCs. There were no significant differences in hematologic values in samples from pythons collected in heparin or EDTA at any time point. No significant differences were found in the number of lysed cells or in other hematologic data in samples with albumin. Glucose concentration decreased and potassium concentration increased significantly over time in heparinized blood samples. CONCLUSIONS: Based on the results of this study, whole blood samples anticoagulated with lithium heparin or EDTA should be evaluated within 12 hours (macaws) or 24 hours (pythons) of collection and stored at 4 degrees C for best results. Citrate should be avoided as it may result in increased cell lysis. The addition of albumin does not prevent cell lysis.     

Comparison of glucose concentrations in blood samples obtained with a marginal ear vein nick technique versus from a peripheral vein in healthy cats and cats with diabetes mellitus

OBJECTIVE: To compare blood glucose (BG) concentrations measured with a portable blood glucose meter in blood samples obtained with a marginal ear vein (MEV) nick technique, from a peripheral venous catheter, and by direct venipuncture in healthy cats and cats with diabetes mellitus. DESIGN: Prospective study. ANIMALS: 1 0 healthy cats and 11 cats with diabetes mellitus. Procedure-On day 1, blood samples were collected every hour for 10 hours by the MEV nick technique and from a peripheral venous catheter. On day 2, blood samples were collected every hour for 10 hours by the MEV nick technique and by direct venipuncture of the medial saphenous vein. RESULTS: For all cats, mean BG concentration for samples collected by the MEV nick technique was not significantly different from mean concentration for samples obtained from the peripheral venous catheter. For healthy cats, mean BG concentration for samples collected by the MEV nick technique was not significantly different from mean concentration for samples obtained by direct venipuncture. For cats with diabetes mellitus, mean BG concentration for samples collected by the MEV nick technique was significantly different from mean concentration for samples obtained by direct venipuncture; however, for the range of concentrations examined, this difference was not clinically important. Conclusions and Clinical Relevance: Results suggest that for the range of concentrations examined, the MEV nick technique is a reasonable alternative to venous blood collection for serial measurement of BG concentrations in cats.     

Comparison between tail and jugular venipuncture techniques for blood sample collection in common chameleons (Chamaeleo chamaeleon)

The most common technique used for collecting blood samples from chameleons, ventral tail caudal venipuncture (Reptile care. An Atlas of Diseases and Treatments, Vol. II, T.F.H. Publication, New Jersey, 1991) sometimes presents undesired effects. Here we compared tail versus jugular vein venipuncture techniques in the common chameleon. In the first experiment, we collected 0.25% of the chameleon's body mass in blood from either tail or jugular sites in size-matched pairs of animals to check for secondary effects. In a second experiment, we measured white blood cells (WBC), red blood cells (RBC), total plasma protein and uric acid in blood samples collected from both sites in the same individual. We found few secondary effects following the use of either venipuncture site although skin darkening was observed in few specimens when using the ventral tail vein. Blood profiles were similar between samples. The repeatability of measurements was similar in RBC and WBC counts and remarkably low (<0.90). Despite the few differences recorded, we recommend jugular venipunction in chameleons as skin darkening is unlikely, blood volumes are easily obtained and collection time is shorter.     

The comparison of thiobarbituric acid reactive substances (TBARS) concentrations in plasma and serum from dairy cattle

The aim of this study was to compare thiobarbituric acid reactive substances (TBARS) concentrations in serum, plasma with heparin (heparin plasma), and plasma with ethylenediaminetetraacetic acid disodium salt (EDTA plasma) as anticoagulants from dairy cattle. Serum, heparin plasma, and EDTA plasma TBARS were not sufficiently strongly correlated to allow accurate prediction of one set of values from the other. Heparin plasma TBARS concentrations were found to be lower, and were affected by the duration of mixing during the assay process. The results suggest that it is necessary to differentiate TBARS concentrations between different sample types such as serum, heparin plasma, and EDTA plasma. For measurements of TBARS concentrations in cattle, EDTA plasma samples may be more suitable than the other samples.     

Differences in hematocrit of blood samples obtained from two venipuncture sites in sharks

OBJECTIVE: To evaluate differences in Hct between 2 venipuncture sites in captive and free-ranging sharks. ANIMALS: 32 healthy adult captive sharks (Carcharhinus melanopterus, Carcharhinus plumbeus, Stegastoma fasciatum, Orectolobus japonicus, and Triaenodon obesus) and 15 captured free-ranging adult sharks (Carcharhinus limbatus and Carcharhinus acronotus). PROCEDURES: Blood samples were collected from the caudal tail artery followed by collection from the sinus located immediately caudal to the cranial dorsal fin. The Hct was determined for each sample and results were compared. Additionally, results for sharks that were highly active and used aerobic metabolism were compared with results for sharks that were less active and tolerant of anaerobic conditions. RESULTS: Mean Hct for all sharks was significantly less (8% less) in blood samples obtained from the cranial dorsal fin sinus, compared with the Hct for samples obtained from the caudal tail artery. When compared on the basis of metabolic class, sharks that were more tolerant of anaerobic conditions had lower Hct values and smaller differences between the 2 venipuncture sites. CONCLUSIONS AND CLINICAL RELEVANCE: Hct values were significantly lower in blood samples collected from the cranial dorsal fin sinus compared with values for samples collected from the caudal tail artery. It is important to recognize this difference when evaluating hematologic variables in sharks and when establishing reference ranges for Hcts for shark populations. Sharks that were more active and relied on aerobic metabolism had higher Hct values than did anaerobic-tolerant sharks, and the difference in Hct values between venipuncture sites was more pronounced.     

Comparison of Hematologic and Biochemical Results on Blood Obtained by Jugular Venipuncture as Compared with Intravenous Catheter in Adult Horses

Background: During hospitalization, horses typically undergo frequent blood sampling for diagnostic testing and monitoring. The need for numerous samples in hospitalized horses makes acquisition from an intravenous catheter (IVC) both convenient and less stressful to the patient. Hypothesis: We hypothesized that there would be no significant difference in the plasma chemistry and CBC variables from blood samples obtained from a jugular catheter as compared with direct jugular venipuncture. Animals: Fifty adult hospitalized horses; 25 receiving constant rate crystalloid therapy, and 25 receiving low volume IV medication. Methods: This study was conducted using a prospective, blinded, cross‐over design. Samples were obtained sequentially by direct venipuncture of the jugular vein and aspiration from an IVC in the contralateral vein after an appropriate presample of blood was obtained and discarded. Samples were submitted for blinded analysis including CBC, plasma chemistry analysis, stall side plasma glucose concentration, PCV, and total protein concentration. Data obtained were analyzed using a Student's t‐test with compensation for unequal variances between the 2 groups. Analyses were Bonferroni corrected for a 5% 2‐tailed hypothesis test. Results: There were no statistically significant or clinically relevant differences associated with sampling method (venipuncture versus catheter) regardless of fluid administration status in any of the 24 analytes measured. Conclusions and Clinical Importance: Blood samples obtained by IVC have clinically equivalent values to those taken by direct venipuncture in commonly performed analyses. Additional investigation is warranted to establish if this technique is associated with increased complications such as phlebitis or bacteremia.     

Hematologic evaluation of normal and anemic lambs with the Technicon H*1 using EDTA or heparin as anticoagulants

Abstract: A study was performed to evaluate blood from young lambs using the Technicon H*1 hematology analyzer, with emphasis on RBC parameters, comparison of tripotassium EDTA and heparin, and the effects of storage on heparinized blood. Blood samples from lambs 2 days to 18 weeks of age were analyzed within 6 hours, revealing a high precision, except for WBC counts in heparinized blood. The HCT values estimated by the H*1 correlated well (r²= .90) with those obtained by the microhematocrit method. Mean hematologic values obtained for heparinized blood differed by up to 4% from values obtained for blood collected into EDTA. WBC counts decreased 8.4% in heparinized blood stored at 4°C for 1 day, but differences observed in RBC counts were ≤ 2%. Problems occurred when analyzing blood from young lambs with low hemoglobin values because the H*1 incorrectly counted microcytes with volumes of < 10 fL as platelets. When the necessary corrections were performed, the H*1 was useful for analyzing RBC parameters in lamb blood collected both into EDTA and into heparin.     

High serum concentrations of iron, transferrin saturation and gamma glutamyl transferase in captive black rhinoceroses (Diceros bicornis)

Iron storage disease (haemochromatosis) is thought to be the cause of many disorders unique to captive black rhinoceroses (Diceros bicornis). To establish reliable reference ranges for iron parameters, serum samples from 27 eastern black rhinoceroses (Diceros bicornis michaeli) from a translocation programme in Kenya were analysed and compared with the samples from 17 captive individuals. The transferrin saturation, serum iron concentration and gamma glutamyl transferase were significantly higher in the captive rhinoceroses, but these elevations were not evident when the results were compared with previously published data.     

Agreement among three different equine venipuncture sites with regard to measurement of packed cell volume and total solids

The primary objective of this study was to determine the agreement between the packed cell volume (PCV) and total solids (TS) values in blood collected from the jugular vein (JV) in comparison with the cephalic vein (CV) and the transverse facial venous sinus (TFVS) in healthy adult horses. A total of 72 samples were taken from 24 horses. We found high agreement that reflects no clinically relevant differences between the PCV and TS values obtained from the CV or the TFVS in comparison with the JV in standing healthy adult horses.     

Phagocytic response of bovine polymorphonuclear leukocytes to different incubation conditions and following exposure to some effectors of phagocytosis and different anticoagulants in vitro.

The ability of bovine polymorphonuclear leukocytes (PMN) to phagocytose fluorescent beads in vitro was studied using flow cytometry. The effects of varying laboratory conditions (bead:PMN ratio, length of incubation, and temperature) were first determined, then the effects of lipopolysaccharide (LPS), phorbol myristate acetate (PMA), cytochalasin B, and formyl-met-leu-phe (fMLP) on phagocytosis were evaluated. The recommended bead:PMN ratio, incubation period, and incubation temperature are 20:1, 30 min, and 38.5 degrees C, respectively. Lipopolysaccharide increased phagocytosis at a relatively high minimum dose; PMA increased phagocytosis even at low doses; cytochalasin B increased and decreased phagocytosis at low and high doses, respectively; and fMLP had no significant effect on phagocytosis. Also, the effects of ethylene diamine tetraacetic acid (EDTA) and acid citrate dextrose (ACD) as anticoagulants were compared with heparin-treated blood PMNs. Both EDTA and ACD decreased phagocytosis. Although there are reports that demonstrated that heparin reduced PMN phagocytosis, at least among the 3 anticoagulants used, heparin remains to be the standard anticoagulant for the study of PMN phagocytosis.     

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.     

Effects of standard anticoagulants and storage procedures on plasma glucose values in seals.

Standard methods for the preservation of vertebrate blood samples for glucose analysis include collecting and storing the blood in evacuated tubes containing sodium fluoride (glycolytic inhibitor) and potassium oxalate (anticoagulant). We found that blood collected from 5 seals by venipuncture and transferred into these tubes had a significantly (P less than 0.05) lower plasma glucose value than blood transferred into tubes containing heparin. In species in which RBC glucose concentration is significantly less than that in the plasma, fluoride and oxalate-induced hemolysis dilutes the plasma with cytoplasm and lowers the measured concentration of glucose in plasma. Therefore, although plasma glucose is used extensively in experimental and clinical analyses, standard techniques for handling the blood may create errors that could confuse comparisons between individuals or species.     

Evaluation of EDTA hematology tubes for collection of blood samples for tests of secondary hemostasis in dogs

OBJECTIVE: To evaluate the use of EDTA tubes for collection of blood samples for assays of secondary hemostasis in dogs. ANIMALS: 108 dogs of various ages, breeds, and sexes (19 healthy and 89 with abnormalities of secondary hemostasis). PROCEDURES: Blood samples were collected via cephalic venipuncture and transferred to sodium citrate tubes and EDTA tubes. Plasma was harvested from each type of tube for assays of concentrations of fibrinogen and D-dimer as well as prothrombin time, activated partial thromboplastin time, and antithrombin activity. Intra-assay and interassay precision and correlation coefficients for all hemostatic tests were calculated for each type of plasma sample. The effect of storage conditions on assay results for the 2 types of plasma samples was also evaluated. RESULTS: Results of hemostatic tests were highly correlated between citrated and EDTA-treated plasma samples. Intra-assay imprecision for all hemostatic tests with the exception of D-dimer concentration was < 10% for both citrated and EDTA-treated plasma samples; interassay imprecision was higher for EDTA-treated versus citrated plasma samples. Storage of plasma samples for 1 hour did not result in significantly different assay results for either type of plasma sample, but storage for 2 hours significantly affected values for EDTA-treated plasma samples. CONCLUSIONS AND CLINICAL RELEVANCE: Although evaluation of the sensitivity and specificity of hemostatic tests that use EDTA-treated plasma samples is required, EDTA may be a suitable alternative to sodium citrate as an anticoagulant for use in hemostatic testing in conditions in which tests could be performed within 1 hour after sample collection.     

Anticoagulant‐dependent in vitro hemagglutination in a cat

Abstract: A 17‐year‐old domestic shorthaired cat was presented to the Cornell University Hospital for Animals for recheck of hyperthyroidism previously treated with radioiodine. Marked agglutination was noted in a blood sample collected into EDTA for a CBC; no other clinical or hematologic evidence of hemolysis was observed and none developed despite persistent agglutination in additional EDTA samples collected over a 2‐month period. Blood drawn into heparin and sodium citrate tubes did not have grossly or microscopically detectable agglutination, unless EDTA was added. Plasma from the cat induced agglutination of washed RBCs from a control cat in the presence of EDTA but not in the presence of heparin. Flow cytometric analysis of samples created by mixing plasma from the patient with washed RBCs from a control cat showed immunoglobulin coating of the control RBCs, predominantly by IgM. These findings suggested an anticoagulant‐dependent antibody‐mediated mechanism for the agglutination. EDTA‐dependent hemagglutination has not been reported previously in cats, although rare cases have been described in humans. The phenomenon needs to be recognized as an in vitro occurrence in order to prevent erroneous diagnosis of immune‐mediated hemolytic anemia.     

Assessment of the effect of dilution of blood samples with sodium heparin on blood gas, electrolyte, and lactate measurements in dogs

OBJECTIVE: To evaluate the effect of dilution of blood samples with sodium heparin on blood gas, electrolyte, and lactate measurements in dogs. Sample Population-Venous blood samples collected from 6 adult dogs of various breeds. PROCEDURE: Syringes were prepared with anticoagulant via 1 of 4 techniques, and the residual volume of liquid heparin in each type of prepared syringe was determined. Blood gas values and other selected clinicopathologic variables were measured in whole blood samples after collection (baseline) and after aliquots of the samples were diluted with heparin via 1 of the 4 manual syringe techniques. By use of a tonometer, whole blood samples were adjusted to 1 of 3 oxygen concentrations (40, 100, or 600 mm Hg) and the PO2 values were measured at baseline and subsequent to the 4 heparin dilutions. RESULTS: The 4 syringe techniques resulted in 3.9%, 9.4%, 18.8%, and 34.1% dilutions of a 1-mL blood sample. Compared with baseline values, dilution of blood samples with liquid heparin significantly changed the measured values of PCO2, PO2, and base deficit and concentrations of electrolytes and lactate. Of the variables assessed, measurement of ionized calcium concentration in blood was most affected by heparin dilution. CONCLUSIONS AND CLINICAL RELEVANCE: These findings in dogs indicate that dilution of blood samples with heparin can be a source of preanalytical error in blood gas, electrolyte, and lactate measurements. Limiting dilution of blood samples with heparin to < 4% by volume via an evacuation technique of syringe heparinization is recommended.     

Effects of collection methods and storage on the in vitro stability of canine plasma catecholamines

Norepinephrine (NE) and epinephrine (EPI) collected from dogs were sequentially and temporally measured in blood and plasma at 24 C. Heparin and EDTA anticoagulants, in combination with reduced glutathione and EDTA as a preservative, were also compared. Norepinephrine and EPI concentrations were measured by high-pressure liquid chromatography with electrochemical detection. In heparinized plasma, NE and EPI concentrations were relatively stable in the absence or presence of preservative after 24 hours at 24 C. In EDTA plasma, NE and EPI values were less stable when compared with those in heparinized samples. Norepinephrine concentrations in EDTA plasma without preservative decreased by 163.2 +/- 8.88 pg over 24 hours, compared with an 86.6 +/- 7.92 pg loss of NE in heparinized plasma. The degradation of EPI in EDTA plasma without preservative was also twofold greater, compared with that in heparinized plasma. Addition of preservative had no stabilizing effect on NE or EPI in heparinized or EDTA plasma. During long-term storage at -70 C, plasma NE and EPI values decreased less than 0.6 and less than 0.1 pg/d, respectively. Norepinephrine and EPI values were stable in heparinized blood for 6 hours but decreased to less than 25% and less than 6% of initial base line values, respectively, when plasma separation was delayed 24 hours.     

Acute intravascular hemolytic anemia in the black rhinoceros: hematologic and immunohematologic observations

To investigate the syndrome of acute intravascular hemolytic anemia in the black rhinoceros (Diceros bicornis), laboratory techniques used in the differential diagnosis of hemolytic anemias were performed on blood samples from 6 black rhinoceroses: 3 nonrelated healthy rhinoceroses, 1 rhinoceros with iron deficiency anemia, and 2 rhinoceroses with intravascular hemolysis. Osmotic fragility, erythrocyte membrane protein composition, hemoglobin electrophoresis, and hemoglobin stability did not distinguish between healthy and affected (anemia or hemolysis) rhinoceroses. Polyclonal antiglobulin reagents were prepared in rabbits, using whole rhinoceros serum and purified rhinoceros immunoglobulin G. These reagents were nonreactive against erythrocytes of the healthy and iron-deficient rhinoceroses. Reactions with RBC from the rhinoceros with fatal hemolytic anemia indicated increased membrane coating by the third component of complement; this was not evident in a second rhinoceros that survived a hemolytic event.