• View in gallery
    Figure 1—

    Least squares mean and SE values for the effects of type of syringe, storage temperature, and duration of storage on measured blood Po2. Values reported are for tonometer-equilibrated blood samples collected in a 3-mL GS and stored at 0°C (reference method; white circles), a 3-mL GS and stored at 22°C (black circles), a 3-mL GPPS and stored at 0°C (white triangles), a 3-mL GPPS and stored at 22°C (black triangles), a 3-mL PSBGA and stored at 0°C (white squares), and a 3-mL PSBGA and stored at 22°C (black squares). *Within a time point, value differs significantly (P < 0.05) from the value for blood stored in a GS at 0°C.

  • View in gallery
    Figure 2—

    Least squares mean and SE values for the effects of type of syringe, storage temperature, and duration of storage on measured blood Pco2. See Figure 1 for key.

  • View in gallery
    Figure 3—

    Least squares mean and SE values for the effects of type of syringe, storage temperature, and duration of storage on measured blood pH. See Figure 1 for key.

  • View in gallery
    Figure 4—

    Least squares mean and SE values for the effects of type of syringe, storage temperature, and duration of storage on calculated plasma Tco2. See Figure 1 for key.

  • View in gallery
    Figure 5—

    Least squares mean and SE values for the effects of type of syringe, storage temperature, and duration of storage on calculated BEecf. See Figure 1 for key.

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Effects of syringe type and storage conditions on results of equine blood gas and acid-base analysis

Sarah A. KennedyDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.

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Peter D. ConstableDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.

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Ismail SenFaculty of Veterinary Medicine, University of Selcuk, Konya, Turkey.

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Laurent CouëtilDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.

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Abstract

Objective—To determine effects of syringe type and storage conditions on blood gas and acid-base values for equine blood samples.

Sample—Blood samples obtained from 8 healthy horses.

Procedures—Heparinized jugular venous blood was equilibrated via a tonometer at 37°C with 12% O2 and 5% CO2. Aliquots (3 mL) of tonometer-equilibrated blood were collected in random order by use of a glass syringe (GS), general-purpose polypropylene syringe (GPPS), or polypropylene syringe designed for blood gas analysis (PSBGA) and stored in ice water (0°C) or at room temperature (22°C) for 0, 5, 15, 30, 60, or 120 minutes. Blood pH was measured, and blood gas analysis was performed; data were analyzed by use of multivariable regression analysis.

Results—Blood Po2 remained constant for the reference method (GS stored at 0°C) but decreased linearly at a rate of 7.3 mm Hg/h when stored in a GS at 22°C. In contrast, Po2 increased when blood was stored at 0°C in a GPPS and PSBGA or at 22°C in a GPPS; however, Po2 did not change when blood was stored at 22°C in a PSBGA. Calculated values for plasma concentration of HCO3 and total CO2 concentration remained constant in the 3 syringe types when blood was stored at 22°C for 2 hours but increased when blood was stored in a GS or GPPS at 0°C.

Conclusions and Clinical Relevance—Blood samples for blood gas and acid-base analysis should be collected into a GS and stored at 0°C or collected into a PSBGA and stored at room temperature.

Abstract

Objective—To determine effects of syringe type and storage conditions on blood gas and acid-base values for equine blood samples.

Sample—Blood samples obtained from 8 healthy horses.

Procedures—Heparinized jugular venous blood was equilibrated via a tonometer at 37°C with 12% O2 and 5% CO2. Aliquots (3 mL) of tonometer-equilibrated blood were collected in random order by use of a glass syringe (GS), general-purpose polypropylene syringe (GPPS), or polypropylene syringe designed for blood gas analysis (PSBGA) and stored in ice water (0°C) or at room temperature (22°C) for 0, 5, 15, 30, 60, or 120 minutes. Blood pH was measured, and blood gas analysis was performed; data were analyzed by use of multivariable regression analysis.

Results—Blood Po2 remained constant for the reference method (GS stored at 0°C) but decreased linearly at a rate of 7.3 mm Hg/h when stored in a GS at 22°C. In contrast, Po2 increased when blood was stored at 0°C in a GPPS and PSBGA or at 22°C in a GPPS; however, Po2 did not change when blood was stored at 22°C in a PSBGA. Calculated values for plasma concentration of HCO3 and total CO2 concentration remained constant in the 3 syringe types when blood was stored at 22°C for 2 hours but increased when blood was stored in a GS or GPPS at 0°C.

Conclusions and Clinical Relevance—Blood samples for blood gas and acid-base analysis should be collected into a GS and stored at 0°C or collected into a PSBGA and stored at room temperature.

Anaerobic collection of blood into a GS and storage in ice water is the reference method for the collection and storage of blood samples for blood gas and acid-base analysis.1,2 Glass syringes are impermeable to O2 and CO2, and storage at 0°C in ice water slows aerobic metabolism by leukocytes and platelets (which consume O2 and produce CO2, thereby decreasing blood pH)3,4 and slows anaerobic metabolism by erythrocytes and leukocytes3,4 (which consume glucose and produce lactic acid, thereby decreasing the blood pH, HCO3 concentration, Tco2, and BEecf). Despite their advantages, GSs are rarely used for collection of samples for blood gas and acid-base analysis because the syringes are expensive, fragile, difficult to use, and not widely available. Moreover, GSs must be sterilized before use. Furthermore, coagulation of the sample is usually inhibited by wetting the syringe barrel with liquid sodium heparin, which can dilute the sample and thereby decrease the measured values for Pco2 and Po2 and the calculated values for the HCO3 concentration, Tco2, and BEecf.5–7

Disposable plastic syringes offer an attractive alternative method to the use of GSs for the collection and storage of blood because they are inexpensive, resistant to breakage, easy to use, and widely available in sterile packaging. Plastic syringes were first marketed in the 1960s and initially manufactured from polystyrene, but after 1965, polystyrene syringes were replaced by polypropylene syringes because the latter were lighter, stronger, less permeable to oxygen, and more impact resistant.8,9 A number of 1- to 3-mL polypropylene syringes are currently marketed for use in blood gas and acid-base analysis. These syringes contain lyophilized lithium heparin as the anticoagulant, thereby mitigating the potential effect of liquid heparin on decreasing the measured values for Pco2 and Po2 and calculated values for the HCO3 concentration, Tco2, and BEecf.10

Despite their many advantages, polypropylene syringes that are marketed for use in blood gas analysis have a major disadvantage in that they are semipermeable to gases, thereby permitting the movement of O2 and CO2 along partial pressure gradients between a blood sample and the syringe wall or ambient air4,8,11–14; the magnitude and direction of change depend on the initial Po2 of the blood sample, temperature of storage, duration of storage, the ratio of surface area to volume of the syringe barrel, and wall thickness and material of the syringe barrel.1,3,8,14–17 An underappreciated phenomenon is that blood samples collected in small-volume polypropylene syringes and stored in ice water have greater increases in Po2 during storage because of their higher surface area-to-volume ratio and thinner walls of the syringe barrel than do larger-volume polypropylene syringes.15,16,18

The effect of storage conditions on blood gas and acid-base values has been extensively investigated in blood samples obtained from humans3,5,12,14,15,19–22; fewer studies have been completed in cattle,23–26 sheep,27,28 goats,29 pigs,30–33 dogs,16,34 and horses.35–38 Results of studies35–39 in horses indicate that accurate measurement of Po2 in arterial blood samples requires the analysis to be completed within 10 minutes after blood has been collected in a 1- to 2-mL GPPS and stored at room temperature or within 2 hours after blood has been collected in a GS or 10-mL GPPS and stored in ice water. Although these studies were valuable in identifying optimal storage conditions for equine blood collected in 1- to 2-mL or 10-mL polypropylene syringes, anecdotal evidence suggests that most samples for blood gas and acid-base analysis in adult horses are collected in 3-mL polypropylene syringes. Because the blood volume in a syringe has an effect on the stability of Po2 and Pco2 during storage,16,18,20 we were interested in accurately characterizing the effect of storage conditions and syringe type on blood gas and acid-base values in equine blood samples collected by use of 3-mL syringes.

In contrast to procedures in 4 other studies in horses,35–38 a tonometer was used to standardize equilibration of the Pco2 and Po2 values at a time of 0 minutes and therefore increased our ability to detect a change in Pco2 and Po2.39 For the study reported here, we hypothesized that blood gas and acid-base values would remain stable for 2 hours when tonometer-equilibrated equine blood was collected in a 3-mL GS, GPPS, or PSBGA and stored in ice water or at room temperature. Because polypropylene syringes made by different manufacturers differ in their permeability to oxygen,3,4,12,15,40 we also hypothesized that a polypropylene syringe developed specifically for use in blood gas and pH analysis would be less permeable to oxygen than would a GPSS.

Materials and Methods

Sample—Blood samples were obtained from 8 horses (5 mares and 3 geldings; 3 Quarter Horses, 3 Standardbreds, 1 Arabian, and 1 Thoroughbred) that ranged from 10 to 23 years of age (mean, 16.3 years). Horses were selected from a university teaching herd for use in the present study. Horses were maintained on pasture and provided supplemental grass hay and grain when necessary. Horses were judged to be healthy on the basis of results of routine physical examination, hematologic analysis, and serum biochemical analysis. None of the horses had clinical signs of disease during the 3 months before the start of the study. The study was approved by an institutional animal care and use committee.

Experimental method—A 70% alcohol gauze swab was used to clean a venipuncture site over a jugular vein of each horse. A 20-gauge, 1-inch needlea and evacuated tube holder were used to collect 120 mL of venous blood into 10-mL plastic partial vacuum tubes that contained 144 U of lyophilized lithium heparin.b The designated filling line on each tube was approximately 9.1 mL of blood. In accordance with the manufacturer's instructions, tubes were inverted 8 to 10 times immediately after blood collection to ensure anticoagulation. Atmospheric air was prevented from entering the tubes by removing each tube from the needle before the needle was removed from the vein. Blood samples were stored in a vertical position in an insulated container at approximately 22°C during the 10-minute transport to the laboratory.

At the laboratory, tubes of blood were warmed to 37°C in a heated water bath and gently inverted 5 times to ensure a homogeneous sample. Then, 7 mL was gently transferred into a temperature-controlled rotating thin-film tonometer.c Each 7-mL sample was equilibrated at 37°C with a calibration gasd that comprised 12% O2, 5% CO2, and 83% N2 at a flow rate of 400 mL/min. Results of preliminary experiments indicate that 7 mL of blood would be equilibrated within 15 minutes at this flow rate.

Sample collection method and storage conditions for each tonometer-equilibrated blood sample were assigned with a random number generator.e Samples (3 mL) of equilibrated blood were aspirated directly into a 3-mL GSf or 2 commercially available 3-mL polypropylene syringes (GPPSg and PSBGAh). The GPPS was selected for investigation because it is widely used for collection of samples for blood gas and acid-base analysis in veterinary medicine. Furthermore, we intended to compare results of the present study with results of another study,17 in which investigators examined the effect of storage conditions on blood gas and acid-base analysis in canine blood samples collected by use of this syringe. The PSBGA was selected for investigation because it had the least change in Po2 when blood was stored in ice water for 60 minutes, compared with results for 4 other commercially available polypropylene syringes.i

Each syringe was used to aspirate gas from the tonometer and expel it (aspiration and expulsion repeated 3 times) immediately before anaerobic collection of the equilibrated sample to ensure atmospheric air was removed from the syringe barrel and to assist in equilibrating the polypropylene syringe barrel with the gas in the tonometer.8 An aliquot (3 mL) of equilibrated blood was collected from the tonometer bowl; a 20-gauge, 1-inch needlej was attached firmly to the syringe; and all visible air bubbles were expelled within 20 seconds after sample aspiration by holding the syringe vertically and tapping the syringe barrel while simultaneously expelling a small volume of blood.22,41 The end of the needle was then inserted into a cork to maintain the anaerobic status of the sample. Each 3-mL aliquot of blood was subsequently stored in accordance with its randomization assignment in ice water at 0°C or at room temperature (approx 22°C) for 0, 5, 10, 15, 30, 60, or 120 minutes. Storage in ice water was used because this storage method provides a faster and more predictable rate of cooling than does storage by placing samples on ice cubes.22

Blood gas analysis—Each day, the blood gas and pH analyzerk was calibrated in accordance with the manufacturer's recommendations. This included 2-point calibrations of pH, Pco2, and Po2 every 4 hours and a 1-point calibration of pH every 30 minutes. Barometric pressure was measured by use of a barometer in the blood gas analyzer; the accuracy of this barometer was assessed monthly against that of a mercury barometer. Three aqueous quality-control standardsl were analyzed each day; these standards represented low, medium, and high values within the physiologic range for pH, Pco2, and Po2 and were used to confirm the linearity of the blood gas analyzer (R2 > 0.999 for pH and Pco2 and > 0.990 for Po2 for all analytic assays). The combined interassay and intra-assay coefficient of variation for median pH values for aqueous quality-control standards of 7.11, 7.37, and 7.61 was 0.2%, 0.4%, and 0.5%, respectively, for all analytic assays. The combined interassay and intra-assay coefficient of variation for Pco2 and Po2 in 180 equilibrated blood samples was 4.4% and 3.0%, respectively.

At the end of the allotted storage time, samples were homogenized by inversion and rolling of the syringes 10 times. Samples stored in plastic syringes were aspirated directly into the blood gas analyzer via the hub of the storage syringe. Because the hub of the GS was too small to permit direct aspiration by the blood gas analyzer, 1 mL of blood was carefully aspirated from the GS via a 20-gauge, 1-inch needle attached to a 1-mL plastic Luer-tip tuberculin syringe.m Air bubbles were expelled from each 1-mL tuberculin syringe within 20 seconds after sample aspiration as described previously, and the sample was immediately analyzed.

Duplicate measurements of pH, Pco2, and Po2 in all samples were obtained by use of the blood gas and pH analyzer. Care was taken to maintain an anaerobic state between analyses by removing air bubbles from the syringe after the first aspiration and immediately replacing the needle on the syringe and capping it with a cork. The plasma concentration of HCO3 was calculated with the Henderson-Hasselbalch equation, measured values for pH and Pco2, and established values2 for the negative logarithm of the apparent dissociation constant (pK1′ = 6.095) for plasma H2CO3 and solubility of carbon dioxide (ie, S = 0.0307 mmol•L−1•mm Hg−1) in plasma at 37°C, whereby the concentration of HCO3 = S × Pco2 × 10(pH − pK1′). The Tco2 was calculated with the Henderson-Hasselbalch equation,2 whereby Tco2 = S × Pco2 × (10(pH − pK1′) + 1). The BEecf was calculated from the measured pH and Pco2 and established valuesecf2 for pK1′ and S, whereby BE f = (S × Pco2 × 10[pH − pK1′]) − 24.8 + (16.2 × [pH −7.40]).

Statistical analysis—Multivariable regression analysisn was used to determine the linear association (y = a•x + b) or curvilinear (quadratic) association (y = a•x2 + bx + c) between measured variables (pH, Pco2, and Po2) or calculated variables (concentration of HCO3, Tco2, and BEecf) and time by use of a dummy variable coding for each horse. Values of P < 0.05 were considered significant.

A curvilinear relationship was investigated because Po2 in human blood appears to decrease in a curvilinear manner during storage for 1 to 2 hours8,13,19 and because it takes 18 to 30 minutes for a 2- to 3-mL blood sample to cool from 37°C to room temperature22,42 and presumably longer to cool to 0°C in ice water. An ANCOVA approach accounts for between-subject variability, thereby increasing the precision with which slope and intercept coefficients for the linear regression line can be estimated.43 Dummy variables (H1 through HN) were defined as described elsewhere.39

Results

Blood Po2—Blood Po2 remained stable for 2 hours when samples were stored at 0°C in a GS (Figure 1; Table 1). In contrast, a significant (P < 0.001) clinically relevant increase in Po2 was detected during 2 hours for samples stored at 0°C in polypropylene syringes. A curvilinear increase in Po2 was detected when blood was stored 0°C in a GPPS, whereas a linear increase in Po2 over time (ΔPo2/Δt = 5.5 mm Hg/h) was detected when blood was stored at 0°C in a PSBGA.

Figure 1—
Figure 1—

Least squares mean and SE values for the effects of type of syringe, storage temperature, and duration of storage on measured blood Po2. Values reported are for tonometer-equilibrated blood samples collected in a 3-mL GS and stored at 0°C (reference method; white circles), a 3-mL GS and stored at 22°C (black circles), a 3-mL GPPS and stored at 0°C (white triangles), a 3-mL GPPS and stored at 22°C (black triangles), a 3-mL PSBGA and stored at 0°C (white squares), and a 3-mL PSBGA and stored at 22°C (black squares). *Within a time point, value differs significantly (P < 0.05) from the value for blood stored in a GS at 0°C.

Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.979

Table 1—

Results for linear and quadratic regression analysis of the relationship between duration of storage and measured blood gas values (pH, Pco2, and Po2) or calculated acid-base values (concentration of HCO3, Tco2, and BEecf) for blood samples obtained from 8 horses, tonometer-equilibrated with gas (12% O2, 5% CO2, and 83% N), collected in 3 types of syringes, and stored at 2 temperatures for up to 2 hours.

Variable0°c22°c
R2InterceptLinear coefficient P valueLinear coefficient valueQuadratic coefficient P valueQuadratic coefficient valueR2InterceptLinear coefficient P valueLinear coefficient valueQuardratic coefficient P valueQuardratic coefficient value
GS
   pH0.917.462 ± 0.0020.150.004 ± 0.002NSNA0.657.462 ± 0.0040.10−0.007 ± 0.004NSNA
   Pco2 (mm Hg)0.6037.2 ± 0.20.0120.41 ± 0.15NSNA0.1737.8 ± 0.40.220.54 ± 0.43NSNA
   Po2 (mm Hg)0.2691.7 ± 0.50.69−0.21 ± 0.51NSNA0.7491.3 ± 0.8< 0.001−7.3 ± 0.9NSNA
   HCO3 (mmol/L)0.8126.6 ± 0.20.0060.53 ± 0.18NSNA0.7127.1 ± 0.30.86−0.05 ± 0.27NSNA
   Tco2 (mmol/L)0.8027.8 ± 0.20.0060.54 ± 0.18NSNA0.7028.2 ± 0.30.91−0.03 ± 0.28NSNA
   BEecf (mmol/L)0.842.8 ± 0.20.0100.59 ± 0.22NSNA0.743.3 ± 0.30.60−0.16 ± 0.29NSNA
GPPS
   pH0.697.468 ± 0.0030.670.001 ± 0.003NSNA0.767.463 ± 0.0030.0013−0.012 ± 0.003NSNA
   Pco2 (mm Hg)0.2637.1 ± 0.30.0190.70 ± 0.28NSNA0.2137.3 ± 0.40.0880.65 ± 0.37NSNA
   Po2 (mm Hg)0.6089.4 ± 1.1< 0.00115.6 ± 3.40.0029−5.3 ± 1.70.3991.3 ± 0.90.0187.28 ± 2.930.0083−3.92 ± 1.42
   HCO3 (mmol/L)0.7026.9 ± 0.20.0340.43 ± 0.20NSNA0.6926.7 ± 0.30.34−0.27 ± 0.28NSNA
   Tco2 (mmol/L)0.6928.1 ± 0.20.0300.46 ± 0.20NSNA0.6827.9 ± 0.30.39−0.25 ± 0.29NSNA
   BEecf (mmol/L)0.743.3 ± 0.20.0690.41 ± 0.22NSNA0.733.0 ± 0.30.14−0.47 ± 0.31NSNA
PSBGA
   pH0.827.472 ± 0.0030.081−0.005 ± 0.003NANA0.777.461 ± 0.0030.031−0.008 ± 0.004NSNA
   Pco2 (mm Hg)0.5837.3 ± 0.20.0180.60 ± 0.24NSNA0.4137.2 ± 0.20.00040.98 ± 0.26NSNA
   Po2 (mm Hg)0.5491.2 ± 1.0< 0.0015.5 ± 1.1NSNA0.5292.9 ± 0.60.35−0.64 ± 0.68NSNA
   HCO3 (mmol/L)0.6327.3 ± 0.20.500.14 ± 0.20NSNA0.7026.6 ± 0.20.450.19 ± 0.25NSNA
   Tco2 (mmol/L)0.6228.4 ± 0.20.460.15 ± 0.21NSNA0.7027.7 ± 0.20.390.22 ± 0.26NSNA
   BEecf (mmol/L)0.703.7 ± 0.20.810.06 ± 0.23NSNA0.732.8 ± 0.30.840.06 ± 0.30NSNA

Values reported are the estimate ± SE.

The effect of syringe type or storage temperature on the coefficient for the linear and curvilinear relationship was determined by use of a dummy variable coding for each horse. The GS at 0°C is considered the reference method.

NA = Not applicable. NS = Not significant (P ≥ 0.05)

Blood Po2 decreased linearly over time (ΔPo2/Δt = −7.3 mm Hg/h) when blood was stored at 22°C for 2 hours in a GS (Figure 1; Table 1). In contrast, a curvilinear increase in Po2 was detected when blood was stored at 22°C in a GPPS, whereas blood Po2 remained constant when blood was stored at 22°C in a PSBGA.

Blood Pco2—Samples stored at 0°C had a linear increase in blood Pco2 over time (ΔPco2/Δt = 0.41, 0.70, and 0.60 mm Hg/h) when stored for 2 hours in a GS, GPPS, and PSBGA, respectively (Figure 2; Table 1). Similar numeric increases in blood Pco2 over time (ΔPco2/Δt = 0.54, 0.65, and 0.98 mm Hg/h) were detected when samples were stored for 2 hours at 22°C in a GS, GPPS, and PSBGA, respectively; however, the increase was significant (P < 0.001) only for samples stored in a PSBGA.

Figure 2—
Figure 2—

Least squares mean and SE values for the effects of type of syringe, storage temperature, and duration of storage on measured blood Pco2. See Figure 1 for key.

Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.979

Blood pH—Samples stored at 0° or 22°C in a GS maintained a stable pH for 2 hours (Figure 3; Table 1). Blood pH also remained stable in samples stored at 0°C in polypropylene syringes. However, blood pH decreased linearly over time when stored at 22°C in a GPPS (ΔpH/Δt = −0.012 pH units/h) or a PSBGA (ΔpH/Δt = −0.008 pH U/h).

Figure 3—
Figure 3—

Least squares mean and SE values for the effects of type of syringe, storage temperature, and duration of storage on measured blood pH. See Figure 1 for key.

Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.979

Calculated values for the concentration of HCO3, Tco2, and BEecf—Calculated values for the concentration of HCO3, Tco2, and BEecf remained constant when blood was stored for 2 hours at 22°C in all 3 syringe types (Figures 4 and 5; Table 1). The calculated value for the concentration of HCO3 increased linearly over time when blood was stored at 0°C in a GS (ΔHCO3/Δt = 0.53 [mmol/L]/h) or a GPPS (ΔHCO3/Δt = 0.43 [mmol/L]/h) but not when stored in a PSBGA. Similarly, the calculated value for Tco2 increased linearly over time when blood was stored at 0°C in a GS (ΔTco2/Δt = 0.54 [mmol/L]/h) or GPPS (ΔTco2/Δt = 0.46 [mmol/L]/h) but not when stored in a PSBGA. The calculated value for BEecf increased linearly over time when blood was stored at 0°C in a GS (ΔBEecf/Δt = 0.59 [mmol/L]/h) but not when stored in polypropylene syringes.

Figure 4—
Figure 4—

Least squares mean and SE values for the effects of type of syringe, storage temperature, and duration of storage on calculated plasma Tco2. See Figure 1 for key.

Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.979

Figure 5—
Figure 5—

Least squares mean and SE values for the effects of type of syringe, storage temperature, and duration of storage on calculated BEecf. See Figure 1 for key.

Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.979

Comparison with other reported values—Values determined for the horses of the present study were compared with values determined for other domestic species (Table 2). Values for the tonometer-equilibrated equine blood of the present study were compared with values determined for dogs, cattle, sheep, goats, pigs, and horses.

Table 2—

Summary of change in pH, Pco2, and Po2 over time for blood samples collected from various species and stored in glass or polypropylene syringes at various temperatures for up to 3 hours.

SpeciesNo. of samplesSample typeSyringe (blood volume)Storage temperature (°C)ΔpH/Δt (U/h)ΔPco2/Δt (mm Hg/h)ΔPo2/Δt (mm Hg/h)Reference
Canine5Tonometer-equilibratedGlass (5 mL)0−0.0050.6NS16
6ArterialPolypropylene (1 mL)4−0.005NS5.117
5Tonometer-equilibratedPolypropylene (3 mL)0−0.0040.41.516
6ArterialPolypropylene (3 mL)4−0.005NS4.117
5Tonometer-equilibratedPolypropylene (5 mL)0−0.0050.51.216
12ArterialPolypropylene (not indicated)0−0.0040.31.034
6VenousPolypropylene (1 mL)4NSNSNS17
6VenousPolypropylene (3 mL)4NSNSNS17
15VenousPolypropylene (10 mL)4−0.001NSNS27
Bovine4ArterialPolypropylene (20 mL)21–24−0.005NS−6.023
22VenousPolypropylene (5 mL)0−0.001NS0.224
15VenousPolypropylene (10 mL)4−0.003NSNS27
14VenousPolypropylene (10 mL)0–4NSNSNS25
5VenousPolypropylene (10 mL)4NS−1.8NS26
4VenousPolypropylene (20 mL)0–4NSNSNS23
14VenousPolypropylene (10 mL)23–30−0.0070.8NS25
5VenousPolypropylene (10 mL)22−0.001NS1.026
5VenousPolypropylene (10 mL)37−0.002NSNS26
Ovine15VenousPolypropylene (10 mL)4−0.002NSNS27
12VenousPolypropylene (10 mL)0–3NSNSNS28
12VenousPolypropylene (10 mL)23–25NS0.9NS28
Caprine14VenousPolypropylene (10 mL)0–4NS1.01.629
14VenousPolypropylene (10 mL)23–25−0.0022.50.629
Porcine3ArterialPolypropylene (2 mL)4NSNS3.533
3ArterialPolypropylene (2 mL)20−0.0080.6−14.633
7VenousPolypropylene (5 mL)0–4NSNSND31
15VenousPolypropylene (10 mL)4NSNDND32
2VenousPolypropylene (10 mL)0–4−0.0020.1NS30
2VenousPolypropylene (10 mL)21–24−0.0100.6NS30
Equine10ArterialGlass (2 mL)0–5NS1.14.537
6Tonometer-equilibratedGlass (3 mL)0NSNSNS39
8Tonometer-equilibratedGlass (3 mL)0NS0.4NSNA
10ArterialGlass (2 mL)22–25−0.0051.9−2.937
6Tonometer-equilibratedGlass (3 mL)22NS0.6−2.539
8Tonometer-equilibratedGlass (3 mL)22NSNS−7.3NA
10ArterialPolypropylene (1 mL)0–5−0.005NS16.037
10ArterialPolypropylene (2 mL)0–5NSNS24.537
8Tonometer-equilibratedPolypropylene (3 mL)0NS0.710.0NA
8Tonometer-equilibratedPolypropylene (3 mL)0NS0.65.5NA
10ArterialPolypropylene (10 mL)0–4−0.003NS0.735
10ArterialPolypropylene (1 mL)22–25−0.0101.07.537
10ArterialPolypropylene (2 mL)22–25−0.015NS8.037
8Tonometer-equilibratedPolypropylene (3 mL)22−0.012NS−1.1NA
8Tonometer-equilibratedPolypropylene (3 mL)22−0.0081.0NSNA
10ArterialPolypropylene (10 mL)21–24−0.0111.1−4.135
10VenousPolypropylene (10 mL)0–4−0.0051.2NS35
15VenousPolypropylene (10 mL)4−0.002NSNS27
10VenousPolypropylene (10 mL)21–24−0.0132.20.335

Change is calculated from that reported for the first 0.5 to 3 hours of storage and assuming a linear change. The Po2 in tonometer-equilibrated samples approximated that of arterial blood for animals when breathing room air.

NA = Not applicable. ND = Not determined. NS = Change from baseline value was not significant (P ≥ 0.05).

Discussion

Preanalytic errors in blood gas analysis can be caused by the presence of air bubbles in the sample, incorrect storage temperature, prolonged storage before analysis, nonhomogeneous samples, dilution of the sample by liquid heparin in the syringe or saline (0.9% NaCl) solution in catheter lines, arterial-venous admixture during sample collection, incorrect positioning (such as lateral or dorsal recumbency) of an animal during sample collection, and an unsteady state of ventilation.1,2,18,41 The use of tonometer-equilibrated blood in the study reported here, coupled with the rapid elimination of air bubbles from the syringe and application of multivariable regression analysis with linear and curvilinear (quadratic) equations, permitted accurate characterization of the influence of syringe characteristics, storage temperature, and storage time on preanalytic errors in blood gas analysis.

Analysis of results of the study reported here indicated that syringe characteristics, storage temperature, and storage time resulted in preanalytic changes in equine blood gas variables, with the most important clinical effect on Po2. A linear decrease in Po2 of 7.3 mm Hg/h was detected when equine blood was stored in a GS at 22°C; this decrease was slightly higher than that reported36,39 for equine arterial blood stored in a GS at room temperature (ΔPo2/Δt = −6.6 and −2.5 mm Hg/h, respectively) and greater than that reported3,19,20,22 for human blood stored in a GS at 22° to 24°C (ΔPo2/Δt = −1.4 to −4.5 mm Hg/h). Aerobic metabolism by leukocytes and thrombocytes is responsible for the decrease in Po2 when blood is stored in a GS at room temperature, but cellular metabolism is markedly slowed at 0°C.3,4 Equine blood Po2 remained stable when blood was stored for at least 2 hours via the reference method (GS at 0°C); this finding was consistent with other studies36,37,39 conducted with equine blood.

Important findings of the study reported here were that Po2 increased in a curvilinear manner when blood was stored at 0° or 22°C in a GPPS. Regression analysis indicated that the difference in Po2 when blood was stored for a 2-hour period in a GPPS at 22°C versus 0°C (16.6 mm Hg) was numerically similar to the difference in Po2 when blood was stored for a 2-hour period in a GS at 22°C versus 0°C (14.5 mm Hg). This result indicated that the rate of oxygen metabolism was similar in a GPPS and GS when blood samples were stored under similar conditions. Similarly, regression analysis indicated that the difference in Po2 when blood was stored in a PSBGA for a 2-hour period at 22°C versus 0°C (12.2 mm Hg) was numerically similar to the difference in Po2 when blood was stored in a GS at 22°C versus 0°C (14.5 mm Hg). This result indicated that the rate of oxygen metabolism was similar in PSBGAs and GSs when blood samples were stored under similar conditions. Therefore, the difference in the Po2-time relationship for the polypropylene syringes reflected differences in gas permeability of the syringes, with the GPPS being more permeable to oxygen than was the PSBGA when blood was stored at room temperature or in ice water, possibly because of differences in pore size and density40,44,i as well as thickness of the syringe wall and fit of the plunger and stopper within the syringe barrel.1 Analysis of our findings suggested that polypropylene syringes developed specifically for blood gas and acid-base analysis (such as those used in the present study) would be superior to GPPSs; moreover, polypropylene syringes that contain lyophilized lithium heparin as the anticoagulant avoid the potential effect of liquid heparin on measured values of pH, Pco2, and Po2.5–7 The main apparent disadvantage with the routine use of a PSBGA in veterinary medicine is that its cost exceeds that of a GPPS.

Small increases in Pco2 were detected for all 3 syringe types when blood samples were stored at 0° and 22°C, with a significant increase at 0°C for all 3 syringe types and at 22°C for PSBGAs. The increase in Pco2 was most likely attributable to aerobic metabolism by leukocytes and thrombocytes.3,4 Carbon dioxide is more soluble in polypropylene than is oxygen45; consequently, some of the CO2 may have remained within the wall of the plastic syringes rather than escaping from the sample into the atmosphere. This mechanism, in conjunction with the fact that carbon dioxide is approximately 25 times as soluble as oxygen in plasma, may have limited efflux of carbon dioxide from polypropylene syringes.12

Equine blood pH remained stable when blood was stored for at least 2 hours via the reference method (GS at 0°C) or in polypropylene syringes (Figure 3; Table 1); this finding was consistent with results in some studies37,39 conducted with equine blood, whereas ΔpH/Δt was −0.003 to −0.005 U/h in 1 study35 and −0.002 U/h in another study.27 As expected, the magnitude of the value for ΔpH/Δt was increased for blood samples stored in propylene syringes at 22°C (ΔpH/Δt = −0.008 to −0.012 U/h) and was accompanied by an increase in Pco2 because of aerobic metabolism by leukocytes and thrombocytes.3,4 Although we did not detect a significant decrease in pH or increase in Pco2 when blood was stored in GSs at 22°C, the value for ΔpH/Δt (−0.007 U/h) was numerically similar to that obtained when blood was stored in polypropylene syringes. Other investigators reported values for the ΔpH/Δt of equine blood stored at room temperature of −0.005 to −0.013 U/h; the rate of decrease of pH in equine blood during storage at room temperature was lower than that in human blood (ΔpH/Δt = −0.021 to −0.040)3,5,14,20 but higher than that in cattle blood (ΔpH/Δt = −0.001 to −0.005)23,25,26 or goat blood (ΔpH/Δt = −0.002).29 Presumably, species differences in ΔpH/Δt reflect differences in experimental methods, the number of leukocytes per milliliter of blood,46 or the magnitude of leukocyte activation during storage.

Blood gas and acid-base analysis is used in some racing jurisdictions to detect prerace administration of alkalinizing agents that may improve the athletic performance of horses. Compared with blood gas analyzers that are traditionally located in a laboratory some distance from these animals, point-of-care units are more expensive on a per-test basis and are not as accurate for the measurement of Po2 and Pco2.47,48 Therefore, there is a need to collect and transport samples for blood gas and acid-base analysis. This fact raises concerns about errors in the concentration of HCO3 and Tco2 that may arise because of preanalytic changes resulting from syringe or storage differences. Analysis of the results of the study reported here indicated that the calculated values for the concentration of HCO3, Tco2, and BEecf did not change for at least 2 hours when blood samples were stored in a GS or polypropylene syringe at room temperature. In contrast, the concentration of HCO3 and Tco2 in plasma increased linearly when blood was collected in a GS or GPPS and stored for 2 hours in ice water. The most likely reason for the increase in plasma concentration of HCO3 and Tco2 was the higher value for the solubility of carbon dioxide at 0°C relative to that at 22°C49; an increase in the solubility of carbon dioxide results in an increase in the concentration of HCO3 because of the equation HCO3 concentration = S × Pco2 × 10(pH–pK1′) despite the concurrent temperature-induced increase in the value for pK1′.39,49 Although the blood gas analyzer warmed blood samples to 37°C before analysis, the analytic process required < 90 seconds. Because plasma lacks carbonic anhydrase50 and blood gas analysis measures Pco2 in plasma, it is possible that blood gas analysis was completed before equilibrium had been reached for the following reaction after the sample was rewarmed back to 37°C: CO2 + H2O ⟷ H2CO3 ⟷ HCO3 + H+. Therefore, it is possible (but unproven) that the value for Pco2 measured with the blood gas analyzer at 37°C in blood samples stored in ice water was slightly higher than the true value because the blood samples did not have sufficient time for equilibration. This explanation is consistent with our finding that Pco2 was significantly (P = 0.012) increased when blood was stored at 0°C in a GS (ΔPco2/Δt = 0.41 mm Hg/h), but Po2 was not significantly (P = 0.69) increased. This explanation is also consistent with the finding in a study39 recently conducted by our research group that the concentration of HCO3 in equine blood typically was increased (but not significantly [P = 0.072]) when blood was stored at 0°C, compared with when it was stored at 22°C. However, this potential mechanism does not explain the fact that the concentration of HCO3 and Tco2 were not changed when blood was stored at 0°C in a PSBGA.

For the study reported here, we concluded that blood samples for blood gas and acid-base analysis should be collected in a GS and stored at 0°C or collected in a PSBGA and stored at room temperature. Equine blood samples collected and stored in this manner should be analyzed within 2 hours after collection. A review of the results of other studies indicates that blood samples collected in polypropylene syringes for blood gas and acid-base analysis should be collected in the largest syringe possible; for horses, this would suggest 3-mL polypropylene syringes are preferable to 1- or 2-mL polypropylene syringes. Additional studies appear warranted to determine whether equine blood samples are more stable during storage when collected into 6- or 12-mL polypropylene syringes.

ABBREVIATIONS

BEecf

Extracellular fluid base excess

ΔBEecf/Δt

Change in extracellular fluid base excess per unit of time

ΔHCO3/Δt

Change in concentration of HCO3 in plasma per unit of time

ΔPco2/Δt

Change in Pco2 per unit of time

ΔpH/Δt

Change in pH per unit of time

ΔPo2/Δt

Change in Po2 per unit of time

ΔTco2/Δt

Change in concentration of total CO2 in plasma per unit of time

GPPS

General-purpose polypropylene syringe

GS

Glass syringe

PSBGA

Polypropylene syringe designed for blood gas analysis

Tco2

Concentration of total CO2 in plasma

a.

Vacutainer blood collection needles, Becton-Dickinson, Franklin Lakes, NJ.

b.

Vacutainer Plus plastic plasma tube, Becton-Dickinson, Franklin Lakes, NJ.

c.

IL 237, Instrumentation Laboratory Inc, Lexington, Mass.

d.

Scott Specialty Gases Inc, Plumsteadville, Pa.

e.

Excel, Microsoft Corp, Seattle, Wash.

f.

Luer-tip glass syringe, Becton-Dickinson, Franklin Lakes, NJ.

g.

Monoject syringe, Tyco Healthcare Group LP, Mansfield, Mass.

h.

Portex syringe, Smiths Medical ASD Inc, Keene, NH.

i.

Zuokamore PA, Malachy OC, McDowell G, et al. The effect of temperature on the stability of blood gas samples using five different blood gas syringes (abstr). Anesthesiology 2003;99:A546.

j.

Kendall Monoject needle, Tyco Healthcare Group LP, Mansfield, Mass.

k.

ABL5, Radiometer, Copenhagen, Denmark.

l.

Qualicheck, Radiometer America, Westlake, Ohio.

m.

Tyco Healthcare Group LP, Mansfield, Mass.

n.

PROC REG, SAS, version 9.2, SAS Institute Inc, Cary, NC.

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Contributor Notes

Ms. Kennedy's present address is Department of Biological Sciences, College of Science, University of Notre Dame, Notre Dame, IN 46556.

Supported in part by a grant from the Indiana Horse Racing Commission.

Ms. Kennedy was supported in part by the Purdue University College of Veterinary Medicine 2010 Veterinary Scholars Summer Research Program.

Dr. Sen was supported in part by The Scientific and Technical Research Council of Turkey.

The authors thank Donna Griffey for technical assistance.

Address correspondence to Dr. Constable (constabl@purdue.edu).