Evaluation of the effects of time, storage, and anticoagulant on venous blood gas samples in dogsABSTRACT
To analyze the effects of variables on venous blood specimens from dogs intended for blood gas analysis, four samples were collected from the jugular veins of 30 animals. Two were obtained using a commercial 3mL BD syringe containing lithium heparin (LH) and a 25x7 needle, and two using a conventional 3mL syringe prepared with sodium heparin (HS) and the same needle. Air bubbles were removed, and the samples were sealed. After the initial analysis (within 20 minutes - T0), one LH sample was stored in ice water (0-4 °C) and the other refrigerated; the same procedure was applied to the HS samples. Subsequent analyses were performed at 30, 120, 240, and 360 minutes after the initial test. Time influenced the values of most variables, except chloride, causing changes in bicarbonate and base excess levels. Therefore, immediate analysis is recommended. When it is not possible, storage in ice water is preferable to refrigeration. The use of HS led to variations in ionized potassium and calcium concentrations, which should be considered by the clinician. The commercial syringe resulted in fewer variations and is therefore recommended for preferred use.
Keywords:
lithium heparin; sodium heparin; blood gas analysis; temperature
RESUMO
Para analisar os efeitos de variáveis em espécimes de sangue venoso de cães, destinadas à gasometria, quatro amostras foram coletadas das jugulares de 30 animais. Duas foram coletadas com seringa comercial BD de 3mL, contendo heparina de lítio (HL), e agulha 25x7, e duas com seringa convencional de 3mL, preparada com heparina sódica (HS) e agulha 25x7. Bolhas de ar foram removidas, e as amostras seladas. Após a primeira análise (até 20min - T0), uma amostra da seringa contendo HL foi armazenada em água gelada (0-4°C) e outra refrigerada; o mesmo procedimento foi seguido para as seringas com HS. As análises subsequentes foram realizadas 30, 120, 240 e 360min após a análise inicial. O tempo influenciou os valores das variáveis, exceto cloretos, causando alterações nos valores de bicarbonato e no excesso de base; dessa forma, recomenda-se a análise imediata. Quando não for possível, recomenda-se o armazenamento em imerso em água gelada em vez de refrigerada. O uso de HS levou a variações nas concentrações de potássio e cálcio ionizado, cujos efeitos devem ser considerados pelo clínico; a seringa comercial acarretou menores variações, sendo recomendado o seu uso preferencialmente.
Palavras-chave:
heparina lítica; heparina sódica; análise de gases sanguíneos; temperatura
INTRODUCTION
Blood gas analysis is used to test critically ill patients and animals with suspected acid-based disorders. This exam helps the clinician assess underlying disease processes and the severity of the patient's current condition, guiding emergency decisions and therapeutic plan choices (Irizarry, 2009).
Properly requested and performed tests can help the clinician, but in the same way, their inappropriate use and/or execution can harm the patient. Therefore, it is necessary to understand the different phases of laboratory analysis to minimize undesirable effects on the results and to consider them when interpreting the test (Vasconcellos, 2020).
Studies on the preservation of blood gas samples in Veterinary Medicine are scarce and old (Haskins, 1977; Assal et al., 1978, 1980; Harsten et al., 1988; Madiedo et al., 1980; Szenci et al., 1991; Lisbôa et al., 2001; Hopper et al., 2005, Knowles et al., 2006; Leal et al., 2006, 2010; Tamura et al., 2015 ); and there is no consensus regarding the duration and temperature at which samples can be kept viable in dogs, often being extrapolated what is accepted for other species.
Considering the importance of samples for analysis, this study aimed to evaluate the effects of anticoagulants, time until analysis, and form of storage on venous blood gas values in dogs, facilitating an understanding of the influence of these factors on the results and minimizing errors in the pre- and post-analytical phases regarding the interpretation of the results.
ETHICAL ASPECTS
The study was submitted for evaluation and approved by the Ethics Committee on the Use of Animals (CEUA) of the Universidade Norte do Paraná (UNOPAR), Arapongas Campus, under protocol number 07/23, in accordance with national and institutional guidelines governing the ethical use of animals in research.
MATERIALS AND METHODS
The animals belonged to volunteer owners. They were of any breed, sex, and age (1-17 years), and could be healthy or have a systemic disease, as pre- analytical factors were analyzed. Patients with moderate to severe anemia (Packed Cell Volume (PCV) <20%), low weight (<3kg), and in shock and/or hypotension were excluded because of the volume of blood required.
Four venous blood samples were collected from the jugular veins of the 30 animals. Each sample was collected up to a 1.6-mL mark; two samples were collected with a commercial BD 3-mL blood gas syringe with a 25×7 gauge needle, which contained 80 IU of lithium heparin, dry spray-blasted, and balanced with calcium; and two other samples were collected with a conventional 3-mL syringe, with approximately 200IU of sodium heparin prepared as follows: heparin (0.5mL) was introduced to the syringe, the plunger was pulled up to the 3-mL mark so that the anticoagulant was in contact with the entire surface of the syringe, and all the material in the syringe was discarded; thus, residual heparin remained only in the center of the 25×7 gauge needle.
Air bubbles were removed from the samples immediately after collection. The needles were removed from the BD-A-Line® blood gas syringes, and a syringe cap was placed over the tip. In conventional syringes, the needles were occluded by placing a rubber stopper on them.
The dog’s temperatures were immediately measured with a thermometer intrarectally and recorded along with the animal’s identification data.
After collection, the blood samples were placed in a dry and clean Styrofoam container, sent to the Clinical Pathology Laboratory of the Veterinary Hospital at the State University of Londrina, and analyzed within 20min (T0) of collection at blood gas analyzer Rapid Point 500 (Siemens Healthineers, Berlim, Germany), which meets the recommendations of quality control standards. The concentrations of bicarbonate - HCO3 - and excess base (BE) were calculated and corrected for the animal temperature.
After the first analysis (T0), a sample from a commercial syringe containing lithium heparin (HL) was stored in an ice-water bath (0-4ºC) and the other in a refrigerator (2-8ºC). The same procedure was performed using a conventional syringe containing sodium heparin (HS). The ice-water bath consisted of water and recyclable ice in a Styrofoam box. The ice was changed every 60min, ensuring that the temperature remained between 0 and 4ºC. Subsequent analyses were performed at 30 (T30), 120 (T120), 240 (T240), and 360 (T360) min after the first analysis.
The data were analyzed using descriptive statistics in the Jamovi program, and normality (Shapiro-Wilk) and homogeneity of variance (Levene) tests were performed. Comparisons between treatments were performed using repeated measures analysis of variance over time (0, 30, 120, 240, and 360min) considering the storage mode (cold water/refrigerator) and anticoagulant used (HS or HL), as well as the interaction between the factors. Means were compared using Tukey test with a probability of error of 0.05. Data are presented as means and standard deviations.
RESULTS
The results are presented in Tables 1, 2, and 3, showing the behavior of the variables at the time points studied. Table 1 lists the variables that were affected by storage, Table 2 lists those that were affected by the type of anticoagulant, and Table 3 lists the variables that were not affected by storage or anticoagulants.
Variation of bicarbonate (HCO3-mmol/L), ionized calcium (iCa mg/dL), potassium (K+mmol/L), anion gap (AG mmol/L) and glucose (mg/dL) in venous blood samples from dogs (n=30) collected with a BD-A-Line® blood gas syringe (HL) and with a conventional syringe with a small amount of sodium heparin (HS), expressed as means of each time analyzed
The pH significantly differed (p<0.001) between the time points studied, regardless of the sample storage mode and type of anticoagulant used. Although the interaction between storage time and duration was significant (p<0.001), there was no difference between the two storage modes at the same time point (Table 1).
With respect to time, the mean values of partial pressure of carbon dioxide (pCO2) increased (p<0.001). Table 1 shows that this increase began at T240. This variable also showed a significant interaction between time and storage interaction (p=0.020). However, these differences were not observed when individual time points were considered.
The pO2 variable (partial pressure of oxygen) increased considerably over time (p<0.001). The average ranged from 44±20.5 mmHg at T0 to 73.5±44.1 mmHg at T360, regardless of storage mode (p=0.942) or anticoagulant type (p=0.865).
The average HCO3- concentration showed a statistically significant decrease over time (p<0.001), as well as in the interaction of time with the anticoagulant (p=0.003) and storage (p<0.001).
The time effect affected the means of the variables BE (p<0.001), sodium (Na+) (p<0.001), and potassium (K+). The mean K+ values were affected by the anticoagulant used (p=0.038). The chloride (Cl-) concentration was the only variable that did not show a statistically significant difference in relation to time (p=0.092), anticoagulant (p=0.37), and storage (p=0.48), nor in the interaction between them.The mean ionized calcium concentration (iCa), varied over time (p<0.001), along with the interaction of the effects of time × storage (p<0.001) and time × anticoagulant (p=0.003). The mean anion gap (AG) values showed differences in relation to the time effect (p<0.001) and the interaction of the time × anticoagulant effects (p=0.035) (Table 2).
The osmolality (mOsm) variable varied over time (p<0.001)-it decreased at T120 and increased again at T240 and T360. There was no significant difference when considering the interaction effects of time × storage (p=0.373), time × anticoagulant type (p=0.565), or the interactions between the three (p=0.334).
The mean lactate levels increased significantly, considering the time effect (p<0.001) and the interaction of the time and storage effects (p<0.001). The mean glucose concentration decreased over time (p<0.001), with a significant interaction between the time × storage mode (p<0.001) and time × anticoagulant type (p=0.024) effects.
DISCUSSION
Variations in blood pH values greater than 0.015 were considered above the acceptable limits reported (Haskins, 1977). This change occurred at each time point studied for the samples stored in the refrigerator, whereas for those stored in ice water, the difference began from T120 to T360. As physiological pH values do not vary greatly, with the reference range for venous blood in dogs be from 7.351 and 7.443 (DiBartola, 2012a), any change in this variable over time resulting from storage may be reflected in clinical interpretation.
Variations in pCO2 and pO2 should not exceed 3 and 5 mmHg, respectively (Haskins, 1977). Regardless of the storage method, the pCO2 values increased above the acceptable limit from T240 onwards. The mean pO2 values increased continuously at all time points studied.
Unlike the study by Harsten et al. (1988), in which arterial blood samples were collected in SH and stored in ice water for 60min and only blood pH values changed, the variations in pO2 were more evident in the present study.
The increase in pO2 was related with the interaction of the effects of time and plastic material of the syringe to the reduction in pCO2 with the interaction of the effects of time and glass material (Knowles et al., 2006). In this study, all samples were collected using plastic syringes, and similar changes were observed in both variables over time.
If the increase in pO2 is attributed to the exposure of the sample to atmospheric air (considering the semi-permeable syringe), a reduction in pCO2 should occur (Pruden et al., 1996); however, this was not observed in this study.
As observed in this study, the increase in pCO2 accompanied by a reduction in pH, a decrease in glucose, and an increase in lactate, may be explained by glycolysis reaction made by leukocytes, red blood cells, and platelets (Madiedo et al., 1980).
From a clinical perspective, venous stasis and muscular activity can result in the accumulation of acidic metabolites, and arterial blood gas analysis is the most suitable method for measuring pCO2 and pO2 (DiBartola, 2012a).
The HCO - concentration decreased over time, regardless of the storage method or anticoagulant used, starting at T30 and intensifying until T360. Bicarbonate variations can become relevant from a clinical point of view because this parameter is used to assess the presence of metabolic alterations, such as acidosis or alkalosis (DiBartola, 2012b). Inadequate judgment owing to a delay in performing the analysis can result in unnecessary therapies; therefore, a sample processed immediately after collection is the most reliable.
According to the behavior of the pH, HCO -, and pCO variables, it was observed that the bicarbonate buffer system remained active over time in a closed system. This should be considered by clinicians in cases of delayed sample analysis to ensure that inappropriate therapeutic decisions for the patient are not made.
With the reduction in the average HCO - concentration over time, it was expected that the average BE values would follow this trend, which occurred from T30 to T360. The variations of the values of these variables were not relevant from a clinical point of view, as the BE values ranged from -6.13 (T0) to -8.4 (T360), which did not cause major changes in the bicarbonate replacement calculation (Carlson and Bruss, 2012).
Cell membranes are permeable to sodium ions; sodium moves from the extracellular space to the intracellular space via a concentration gradient. The difference in concentration is maintained in vivo by the NA+-K+-ATPase pump, which expends energy. In vitro, the energy in collection syringes is limited. Over time, the plasma Na+ moves to the blood cells and lower values are measured (Wellman et al., 2012). In this study, the sodium concentration ranged over time at the time points analyzed. One hypothesis is that cell destruction occurs in vitro via the release of ions into the extracellular medium. However, despite the statistical difference, it is not sufficient to change clinical conduct because it varies within the physiological range. No differences were observed among the anticoagulants for this variable in samples containing SH, even at high concentrations.
The K concentration increased over time. The hypotheses for this are as follows: 1) the NA+-K+-ATPase pump decreased its activity in vitro when there was no more ATP, leading to the displacement of intracellular K+ to the extracellular medium, and 2) there was a consequence of hemolysis (Wellman et al., 2012).
Dogs have similar intra-erythrocyte and plasma K+ concentrations. Therefore, hemolysis does not cause hyperkalemia in this species, except in breeds with high intracellular K concentrations (DiBartola and Moraes 2012), However, in this study, in vitro hemolysis could not be excluded as a possible explanation for the changes observed in the Na+ and K+ concentrations.
Increases in the K+ concentration from T0 and T360 occurred regardless of the anticoagulant used, although they were less than 0.5mEq/L. If the animal presents with normokalemia, this variation will not cause harm; however, considering a patient with previous hypokalemia, a delay in the analysis may lead to insufficient replacement. The anticoagulant used interfered with the concentration of K. This difference could be attributed to the dilution of the sample, as approximately 0.039 mL of HS remained in the needle barrel (Hopper et al., 2005), corresponding to 2.4% of the final volume of the syringe. At each point, by subtracting 2.4% from the mean HL, a value similar to the mean HS was obtained. The difference in the values of this variable, when comparing the same time point with the different anticoagulants, was <0.2mEq/L, which is not clinically relevant and does not warrant a change in conduct.
In the study by Hopper et al. (2005), the addition of liquid heparin led to the dilution of the following variables: pCO2, pO2, BE, HCO3-, K+, Na+, Cl-, iCa, and lactate, unlike in this study, in which the concentrations of K+, iCa, AG, and glucose decreased.
According to Moraes and Biondo (2012), serum chloride levels can remain stable for months. In this study, this variable was measured in the blood, and its average values did not change, indicating stability in the blood for six hours.
The iCa concentrations in the samples stored in the refrigerator remained stable over time. The samples stored in ice water showed lower values at T360, showing a statistical difference. However, the difference in means from T360 to T0 was 0.1 mg/dL, which does not affect clinical management.
Over time, samples containing LH were stable until T360, when there was a slight decrease in calcemia. Samples containing HS as an anticoagulant remained stable during the analysis period, although with significantly lower values than those with LH, which should be considered by clinicians when analyzing results.
Regardless of whether heparin is Na, lytic, liquid, or lyophilized, it can chelate Ca, resulting in lower measurements (Hopper et al., 2005). In the BD-A-Line® blood gas syringes, this effect is already considered during their manufacture, as they are balanced with Ca, so the measured value is more reliable.
The AG originates from the difference between the main cations and anions in the extracellular space (DiBartola, 2012a). Among the ions considered in the calculation of the variable, there were changes in the K+ and HCO3- concentrations; therefore, the variation observed in the AG concentration over time reflected the oscillations in its components.
The osmolality of a solution refers to the concentration of osmotically active particles. Plasma osmolality can be calculated from sodium, urea nitrogen, and glucose concentrations (DiBartola, 2012c). The reduction in osmolality over time reflected decreases in the concentrations of Na and glucose. Although significant, this reduction was 5mOsm/kg, which is not clinically relevant when considering the physiological range of the species.
The mean lactate concentration increased over time, a finding similar to that observed by Assal et al. (1978). The anaerobic metabolism of blood cells causes glucose consumption and produces lactate. In turn, the glucose content decreased over time, demonstrating this effect.
Under experimental conditions, hemogasometric examination of venous blood from dogs should be performed immediately after collection, which differs from the results found by Rincon et al. (2023), who found stability for up to 60 minutes.
CONCLUSION
Under the conditions of this study, time altered most variables, except for chlorides.
The recommendation based on this study is that examinations should be performed immediately after collection whenever possible. If storage is necessary, ice water proved to be the best way to store canine venous blood samples for gasometric examination, as this method delayed in vitro changes.
SH syringes showed changes in the K+ and iCa concentrations. This preparation of syringes can be used but must be considered when analyzing the results.
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The research data are available within the article itself.
