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Effect of time delay and storage temperature on blood gas and acid–base values of bovine venous blood
Research in Veterinary Science 76 (2004) 121–127 www.elsevier.com/locate/rvsc
Effect of time delay and storage temperature on blood gas and acid–base values of bovine venous blood Gurbuz Gokce a
a,*
, Mehmet Citil a, Vehbi Gunes a, Gultekin Atalan
b
Department of Internal Medicine, Faculty of Veterinary Medicine, University of Kafkas, Kars 36100, Turkey b Department of Surgery, Faculty of Veterinary Medicine, University of Kafkas, Kars 36100, Turkey Accepted 12 August 2003
Abstract 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 °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 °C (Group I, n ¼ 5), at a room temperature of about 22 °C (Group II, n ¼ 5) and in an incubator adjusted to 37 °C (Group III; n ¼ 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 pCO2 value for Group I was )0.72 kPa during the assessment, while for groups II and III, maximum alterations in pCO2 were detected as +2.68 and +4.16 kPa, respectively. Mean pO2 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 (HCO3 ) 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 °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. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: pO2 ; pCO2 ; Acid base parameters; Storage temperature time; Bovine
1. Introduction Various metabolic and respiratory diseases effect venous blood gas composition and acid–base values of cattle (Radostits et al., 1994; Carlson, 1996). Therefore, accurate measurement of these blood values is of great importance for veterinary clinicians and research workers. Human (Paerregaard et al., 1987; Muller-Plathe and Heyduck, 1992; Beaulieu et al., 1999) and cattle (Poulsen and Surynek, 1977; Szenci and Besser, 1990; Szenci et al., 1991) blood gas composition and acid–base values may change depending on the type of syringe used for sampling, delays in measurement time and variations in *
Corresponding author. Tel.: +90-474-242-6800; fax: +90-474-2426853. E-mail address: [email protected] (G. Gokce). 0034-5288/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2003.08.009
storage temperature. Continuous anaerobic and aerobic metabolism between the drawing of the blood sample and its analysis intervals may effect blood acid–base values in humans (Boink et al., 1991; Liss and Payne, 1993), cattle (Jagos et al., 1977; Krokavec et al., 1987; Szenci and Besser, 1990) and dogs (Haskins, 1977). In vitro blood metabolism includes both aerobic metabolism with production of carbon dioxide and anaerobic glycolysis with production of non-gaseous acids such as lactic acid. Oxygen consumption depends on the enzymatic activity of the citric acid and cytocrome systems in leucocytes and reticulocytes and will vary with the concentration of these cell types. Glycolysis is the predominant metabolic process in mature red blood cells. These processes are largely responsible for the rising of pCO2 in the stored blood of dogs (Haskins, 1977), and cattle (Poulsen and Surynek, 1977). These metabolic changes are temperature
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G. Gokce et al. / Research in Veterinary Science 76 (2004) 121–127
dependent (Liss and Payne, 1993). Additionally, severe leucocytosis (Schimidt and Plathe, 1992; Liss and Payne, 1993) and anaemia (Haskins, 1977) can lead to alterations in acid–base values. Since it is difficult to measure the acid–base and gas values of blood under field conditions in a short period of time its necessary to store blood (Szenci and Besser, 1990). Several authors have investigated the alterations in acid–base variables during the storage of blood samples from humans (Paerregaard et al., 1987; Schimidt and Plathe, 1992; Beaulieu et al., 1999), cattle (Jagos et al., 1977; Poulsen and Surynek, 1977; Krokavec et al., 1987; Szenci and Besser, 1990; Szenci et al., 1991, 1994) and dogs (Haskins, 1977). It would be useful to determine the extent to which changes in blood acid– base values and gas composition depend on the duration and temperature of storage. To our knowledge, there is no precise information available as to whether or not clinically significant changes occur when bovine venous blood is stored at 22 or at 37 °C for long periods. Therefore, this study was carried out to detect the changes in gas composition and acid–base values in bovine venous blood samples stored at different temperatures (+4, 22 and 37 °C) for up to 48 h.
2. Materials and methods
matically performed on a blood gas analyser (Chiron diagnostics, Rapidlab 248, UK) prior to the study. Blood samples from the five cattle were analysed for blood gas and acid base values four times each within 15 min to determine the imprecision of parameters, which emerged as follows: pH 0.01%; pCO2 1.9%; pO2 3.52%; ActHCO3 1.74%; StdHCO3 1.34%, BEact 1.98%, BEstd 1.02%, O2 SAT 0.39% and O2 CT 1.8% on the basis of variation co efficiency. Control of accuracy was carried out using a commercial accuracy control solution (Complete, Bayer, East Walpole, USA). Control values were within the normal range as described by the manufacturer. Calculated variables were automatically performed by blood gas analyser according to equations 1 programed to device by manufacterer (Chiron diagnostics, Rapidlab 248, UK) The values of pH, pCO2 and pO2 were automatically adjusted to the animalÕs rectal temperatures. 2.3. Haematological analyses For the initial haematological examinations, peripheral blood samples were collected from jugular vein into ethylenediaminetetraaceticacid (EDTA) treated tubes. These blood samples were used to manually establish total white blood cells (WBCs), total red blood cells (RBCs) and haemoglobin concentration (Hb), as described by Coles (1980).
2.1. Animals and sampling 2.4. Statistical analyses Five clinically healthy, 1–3 years old, crossbreed cattle were used in the study. Following rectal temperature measurement, three jugular venous blood samples were drawn from each animal into 10 ml plastic syringes the dead space (0.08 ml) of which was filled with heparin (LiquemineÒ -Roche, Istanbul, Turkey). The needle tips were closed with a rubber stopper after the removal of air bubbles from the samples. The samples were placed in a bed of crushed ice, taken immediately to the laboratory and analysed within 15 min. Following the first (0) hourÕs laboratory analysis, a total of 15 blood samples were collected from the animals, allocated into three groups and then stored, respectively, in a refrigerator set to +4 °C (Group I, n ¼ 5), at a room temperature of about 22 °C (Group II, n ¼ 5) and in an incubator adjusted to 37 °C (Group III, n ¼ 5), for up to 48 h. Blood gas and acid–base values were analysed at 0, 1, 2, 3, 4, 5, 6, 12, 24, 36 and 48 h of storage for all the groups. 2.2. Acid–base and blood gas analyses The measurement of pH, pO2 , pCO2 and the calculation of standard Base excess (BEstd), actual base excess (BEact), standard bicarbonate (stHCO3 ), actual bicarbonate (actHCO3 ) concentrations, oxygen saturation (O2 SAT) and oxygen content (O2 CT) were auto-
The effect of temperature on blood gas and acid base values was tested by analysis of variance using SPSS for Windows 6.0. The effects of time on the repeated measurements within each group were also examined using two-way variance analysis (Kirkwood, 1988). The data is presented as mean SE.
3. Results The mean initial haematological values of the animals used in the study were detected as 115 0.6 g/l for Hb, 5.9 0.5 1012 cell/l for RBC and 6.5 1.9 109 cell/l for WBC. The mean rectal temperature of the animals was 38.5 0.2 °C. 1
ActHCO3 ¼ pH þ logðpCO2 0:0307Þ 6:105; StdHCO3 ¼ 24:5þ 0:9A þ ðA 2:9Þ2 ð2:65 þ 0:31ctHbÞ=1000. A ¼ BEstd 0:2½ctHb ½100 O2 SAT=100; BEact ¼ cHCO3 24:8 þ 1:62ðpH 7:40Þ. BEstd ¼ ð1 0:014 ctHbÞ½ðcHCO3 24:8Þ þ ð1:43 ctHb þ 7:7Þ ðpH 7:40Þ. N 4 15N 3 þ 2045N 2 þ 2000N O2 SAT ¼ 4 100: N 15N 3 þ 2400N 2 31100N þ 2:4 106 N ¼ pO2 þ 10½0:48ðpH7:4Þ0:0013BEstd and BEstd is calculated assuming 100% O2 SAT; O2 CT ¼ O2 SAT 1:39 ctHb þ 0:00314 pO2 .
Table 1 Effect of storage time on pCO2 and pO2 of bovine venous blood samples stored at +4 °C (Group I), 22 °C (Group II) and 37 °C (Group III) (n ¼ 5) (mean SE) Parameter
Group
pCO2 (kPa)
I
Time (h)
II III
0
1
2
3
4
5
6
12
24
36
48
P1
6.59 0.11 ab 6.59 0.11 f 6.59 0.11 e
5.95 0.09 e 6.34 0.14 f 6.20 0.15 e
5.94 0.10 B,e 6.60 0.16 A,f 6.41 0.15 A,e
5.87 0.12 B,e 6.59 0.12 A,f 6.61 0.12 A,e
6.04 0.10 B,de 6.81 0.14 A,e 7.01 0.16 A,d
6.01 0.11 C,e 6.88 0.10 B,e 7.37 0.18 A,c
6.17 0.10 C,cde 7.34 0.12 B,c 9.01 0.26 A,b
5.86 0.11 C,e 7.07 0.13 B,d 8.88 0.29 A,b
6.34 0.12 C,bcd 8.06 0.16 B,b 10.75 0.32 A,a
6.40 0.12 B,bc 8.82 0.19 A,a 9.29 0.19 A,b
6.90 0.10 B,a 9.27 0.47 A,a 9.05 0.34 A,b
p < 0:001
p < 0:05
p < 0:01
p < 0:001
p < 0:001
p < 0:001
p < 0:001
p < 0:001
p < 0:001
p < 0:001
4.47 0.15 d 4.41 0.15 d 4.33 0.10 a
4.55 0.15 cd 4.64 0.11 c 4.38 0.12 a
4.54 0.16 d 4.57 0.10 d 4.32 0.11 a
4.67 0.17 bcd 4.56 0.11 d 4.31 0.12 a
4.77 0.16 bcd 4.70 0.11 c 4.31 0.15 a
4.77 0.18 A,bcd 4.91 0.11 A,b 4.08 0.15 B,a
5.24 0.24 A,ab 5.00 0.10 A,a 0.86 0.13 B,c
5.12 0.24 A,abc 4.98 0.12 A,a 1.43 0.41 B,b
5.42 0.24 A,a 5.06 0.07 A,a 0.69 0.04 B,c
p < 0:01
p < 0:001
p < 0:001
p < 0:001
P2 4.26 0.10 d 4.26 0.10 d 4.26 0.10 a
I II III
4.27 0.16 d 4.25 0.10 d 4.32 0.09 a
P2
p < 0:001
p < 0:001 p < 0:001 p < 0:001
P1, significant differences within group compared to baseline value (a,b,c,d,e,f), (p < 0:001); P2, in each column different letters (A, B, C) indicated significant between groups (p < 0:01), (p < 0:001); pCO2 , partial pressure of carbondioxide (kPa); pO2 , partial pressure of oxygen (kPa).
Table 2 Effect of storage time on O2 SAT and O2 ct of bovine venous blood samples stored at +4 °C (Group I), 22 °C (Group II) and 37 °C (Group III) (n ¼ 5) (mean SE) Parameter
O2 SAT
Group
I II III
Time (h) 0
1
2
3
4
5
6
12
24
36
48
P1
62.32 1.20 d 62.32 1.20
62.80 2.46 cd 61.82 1.02
63.28 2.16 cd 61.33 1.69
64.36 2.04 bcd 64.42 0.34
63.78 2.24 cd 62.44 1.01
61.10 1.42 b
60.16 1.55 b
59.30 1.70 b
57.48 1.82 b
66.62 2.09 A,abcd 62.76 1.18 A 51.82 2.30 B,c
66.56 2.27 A,abcd 65.60 1.32 A 45.54 2.71 B,d
71.52 2.72 A,ab 63.88 1.34 B 32.22 0.41 C,e
69.82 2.89 A,abc 60.70 1.88 B 8.17 3.38 C,f
72.78 2.00 A,a 61.75 0.35 B 1.85 0.05 C,f
p < 0:05
62.32 1.20 a
65.46 2.19 A,bcd 62.06 1.21 A 55.58 2.00 B,c p < 0:01
p < 0:001
p < 0:001
p < 0:001
p < 0:001
p < 0:001
10.66 0.34 A,c 10.12 0.35 AB 9.08 0.39 B,b
10.84 0.36 A,c 10.22 0.35 A 8.44 0.46 B,c
10.86 0.37 A,c 10.70 0.39 A 7.44 0.50 B,c
11.66 0.51 A,b 10.44 0.39 B 0.48 0.11 C,d
11.16 0.40 A,c 9.92 0.49 A 1.40 0.60 B,d
12.00 0.30 A,a 10.45 0.05 B 0.3 0.00 C,d
p < 0:05
p < 0:01
p < 0:001
p < 0:001
p < 0:001
p < 0:001
P2 O2 CT (mmol/l)
I II III
P2
10.16 0.30 c 10.16 0.30
10.50 0.44 c 10.08 0.32
10.30 0.36 c 10.15 0.42
10.46 0.35 c 10.50 0.39
10.38 0.38 c 10.18 0.33
10.16 0.30 a
9.92 0.24 a
9.80 0.38 a
9.66 0.33 a
9.38 0.42 b
p > 0:333 p < 0:001
G. Gokce et al. / Research in Veterinary Science 76 (2004) 121–127
pO2 (kPa)
p < 0:001
p < 0:05 p > 0:954 p < 0:001
123
P1, significant differences within group compared to baseline value (a,b,c,d,e,f), (p < 0:05, p < 0:001); P2, in each column different letters (A, B, C) indicated significant between groups (p < 0:05, p < 0:01, p < 0:001); O2 SAT, oxyhaemoglobin saturation (%); O2 CT, oxygen content (mmol/l).
124
Table 3 Effect of storage time on ActHCO3 and StdHCO3 of bovine venous blood samples stored at +4 °C (Group I), 22 °C (Group II) and 37 °C (Group III) (n ¼ 5) (mean SE) Parameter
Group
ActHCO3 (mmol/l)
I II III
Time (h) 0
1
2
3
4
5
6
12
24
36
48
P1
31.38 0.35 a 31.38 0.35
29.04 0.27 B,c 30.32 0.37 A 28.94 0.26 B,c
28.00 0.30 B,cde 30.38 0.30 A 28.88 0.32 B,c
27.62 0.40 B,de 29.94 0.49 A 28.48 0.39 B,d
28.12 0.34 B,cd 30.00 0.35 A 29.02 0.37 AB,c
27.88 0.33 C,cde 30.00 0.20 A 29.00 0.35 B,c
28.42 0.43 B,cd 30.88 0.63 A 31.44 0.48 A,a
26.90 0.31 e 29.44 0.40
28.74 0.31 B,cd 31.20 0.52 A 29.80 0.36 B,b
28.56 0.54 B,cd 30.24 0.29 A 27.57 0.15 B,e
30.20 0.29 A,b 30.55 0.35 B 26.85 0.85 B,e
p < 0:001
p < 0:05
p < 0:001
p < 0:01
p < 0:01
p < 0:01
p < 0:01
p < 0:001
p < 0:01
p < 0:01
26.42 0.97 cd 28.28 0.27 b 27.46 0.31 b
26.40 0.26 B,cd 28.15 0.15 A,b 26.68 0.28 B,c
26.32 0.27 B,cd 27.80 0.40 A,c 26.18 0.28 B,c
26.88 0.20 A,bc 27.54 0.26 A,c 26.08 0.31 B,c
26.40 0.22 B,cd 27.46 0.15 A,c 26.36 0.44 B,c
26.80 0.36 bcd 27.88 0.52 c 26.58 0.27 c
25.58 0.20 A,d 27.74 0.38 A,c 23.24 0.28 B,d
27.00 0.24 A,bc 27.36 0.51 A,c 22.60 0.20 B,e
27.30 0.20 A,bc 25.70 0.38 B,d 21.67 0.23 C,f
27.78 0.29 A,b 25.55 0.75 A,d 21.05 0.75 B,f
p < 0:01
p < 0:01
p < 0:01
p < 0:05
p < 0:001
p < 0:001
p < 0:001
p < 0:001
31.38 0.35 a
P2 I II III
29.24 0.29 a 29.24 0.29 a 29.24 0.29 a
P2
p < 0:001
p < 0:001 n.s p < 0:001
P1, significant differences within group compared to baseline value (a,b,c,d,e,f), (p < 0:05, p < 0:01, p < 0:001); P2, in each column different letters (A, B, C) indicated significant between groups (p < 0:05, p < 0:01, p < 0:001); ActHCO3 , actual bicarbonate concentrations (mmol/l); StdHCO3 , standard bicarbonate concentrations (mmol/l).
Table 4 Effect of storage time on BEact and BEstd of bovine venous blood samples stored at +4 °C (Group I), 22 °C (Group II) and 37 °C (Group III) (n ¼ 5) (mean SE) Parameter
BEact (mmol/l)
Group
I II III
Time (h) 0
1
2
3
4
5
6
12
24
36
48
P1
7.04 0.39 a 7.04 0.39 a 7.04 0.39 a
4.74 0.32 B,bc 5.90 0.36 A,b 4.44 0.22 B,c
3.58 0.35 B,cd 5.83 0.22 A,b 4.14 0.31 B,c
3.2 00.42 B,de 5.30 0.52 A,c 3.46 0.43 B,c
3.64 0.36 B,cd 5.14 0.35 A,c 3.68 0.36 B,c
3.32 0.35 B,de 5.04 0.23 A,c 3.30 0.33 B,c
3.86 0.49 cd 5.70 0.70 b 4.94 0.44 b
2.24 0.31 A,e 4.16 0.45 A,d 0.88 0.39 B,e
4.06 0.31 A,cd 5.34 0.61 A,c 1.64 0.39 B,d
3.76 0.57 A,cd 3.58 0.46 A,d )0.13 0.32 B,f
5.24 0.34 A,b 3.65 0.75 A,d )0.85 0.85 B,f
p < 0:001
p < 0:05
p < 0:01
p < 0:05
p < 0:05
p < 0:01
p < 0:001
p < 0:001
p < 0:01
p < 0:001
4.16 0.26 B,bc 5.10 0.31 A,b 3.86 0.16 B,b
3.24 0.25 B,bcd 4.95 0.14 A,b 3.42 0.30 B,c
2.84 0.34 B,de 4.50 0.45 A,c 2.78 0.36 B,d
3.16 0.31 B,cd 4.44 0.38 A,c 2.78 0.33 B,d
2.90 0.32 B,de 4.18 0.18 A,c 2.40 0.27 B,d
1.98 0.29 B,e 3.28 0.4 A,d )0.30 0.30 C,e
3.46 0.27 A,bcd 4.00 0.56 A,c )0.02 0.30 B,e
3.14 0.50 A,cd 2.14 0.44 A,d )1.58 0.35 B,f
4.28 0.32 A,b 2.05 0.85 B,d )1.95 0.85 C,f
p < 0:05
p < 0:01
p < 0:05
p < 0:05
p < 0:01
p < 0:001
p < 0:001
p < 0:001
p < 0:001
P2 BEstd (mmol/l)
I II III
P2
6.12 0.34 a 6.12 0.34 a 6.12 0.34 a
3.32 0.43 bcd 4.58 0.59 c 3.42 0.34 c
p < 0:001 p < 0:001
p < 0:001 p < 0:001 p < 0:001
P1, significant differences within group compared to baseline value (a,b,c,d,e,f), (p < 0:001); P2, in each column different letters (A, B, C) indicated significant between groups (p < 0:01, p < 0:05, p < 0:001); BEact, extracelluler base excess (mmol/l); BEstd, blood base excess (mmol/l).
G. Gokce et al. / Research in Veterinary Science 76 (2004) 121–127
StdHCO3 (mmol/l)
28.08 0.39 e
p > 0:056
G. Gokce et al. / Research in Veterinary Science 76 (2004) 121–127
Blood acid–base and blood gas values are summarised in Tables 1–4 and Fig. 1. The pH values of the samples kept at different temperatures tended to decrease significantly with time in all groups (p < 0:001) (Fig. 1). Compared to initial values, pH values were found to decrease significantly after 5, 3 and 2 h (p < 0:001) for the samples stored at +4, 22 and 37 °C, respectively. Alterations in pCO2 and pO2 values are summarised in Table 1. The mean pCO2 values of the samples stored at +4 °C decreased significantly between 1 and 24 h, but later increased again to their initial levels. Moreover, the pCO2 values of the samples kept at 22 and 37 °C increased significantly (p < 0:001) after the fourth hour and continued to increase until the end of the study (Table 1). The maximum mean alteration in pCO2 value for Group I was )0.72 kPa during the assessment, while for groups II and III maximum alterations for pCO2 were detected as +2.68 and +4.16 kPa, respectively. The pO2 values of the samples kept at +4 and 22 °C increased with time (p < 0:001). However, a significant decrease was recorded after 24 h for the samples kept at 37 °C (p < 0:001). Values of O2 SAT in group I increased (p < 0:05) after 24 h storage, on the other hand it was significantly decreased in group III after 5 h (p < 0:001) and there were no changes in group II (p > 0:333) (Table 2). The O2 CT values were significantly increased (p < 0:05) after 24 h of storage at +4 °C, but significantly decreased after 4 h of storage at 37 °C (p < 0:001) (Table 2). However, no significant alterations were observed in the O2 CT values of the samples stored at 22 °C. BE and HCO3 concentrations decreased signifi-
125
cantly for all three groups during the study compared to their baseline values. The most prominent decrease in BE and HCO3 concentrations was recorded for the samples kept at 37 °C (Tables 3 and 4).
4. Discussion The effects of storage time and temperature on blood gases and acid–base values in humans (Mahoney et al., 1991; Liss and Payne, 1993; Beaulieu et al., 1999; Lenfant and Aucutt, 1965) and in cattle (Poulsen and Surynek, 1977; Szenci and Besser, 1990; Szenci et al., 1991) have been investigated intensively. Previous studies have revealed that the blood gas and acid–base values of human blood samples usually stabilised between 15 min and 2 h (Paerregaard et al., 1987; Mahoney et al., 1991; Beaulieu et al., 1999). The effect of storage temperature and storage time on blood gas and acid–base values in cattle are disputed (Poulsen and Surynek, 1977; Szenci and Besser, 1990; Szenci et al., 1991). Szenci and Besser (1990) reported that cattle venous blood acid–base values could be used for diagnostic purposes up to 24 h. On the other hand, the pH values of blood samples taken from cattle and stored at +4 °C stabilised after for 5–6 h (Poulsen and Surynek, 1977). In the present study, the pH values of the blood samples stored at +4 °C were found to be suitable for clinical usage up to 48 h (Fig. 1). Furthermore, storage time and temperature were found to significantly alter the blood pH and bicarbonate values of all groups.
7,45
7,4 a a a
7,35
a ab ab
ab abc bc
pH
7,3
A,ab A,bc B,cd
A,abc B ,cd C,de
**
***
7,25
A,bc B,cde C,e A,bc B,de C,f
*** Group I
7,2
***
Group II Group III
A,bcd B,e C,g A,bcd B,f C,h
***
7,15
***
A.cd B,g C,h
A,d B,g C,h
***
***
*** *** ***
7,1
7,05 0
1
2
3
4
5
6
12
24
36
48
Fig. 1. Effects of storage time on the pH of bovine venous blood samples stored at +4 °C (Group I), 22 °C (Group II) and 37 °C (group III) (n ¼ 5) (mean SE). a,b,c,d,e,f,g,h: significant differences within group compared to baseline (0 h), ***(p < 0:001). A,B,C: significant differences between group **(p < 0:01), ***(p < 0:001).
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G. Gokce et al. / Research in Veterinary Science 76 (2004) 121–127
Particularly, rapid and significant decreases in blood pH (p < 0:001) were observed for those samples stored at 37 °C, compared to the other groups (Groups I and II) (Fig. 1). Metabolism of in vitro blood samples continues during storage. Oxygen consumption occurs due to anaerobic metabolism and CO2 generation in the tricarboxylic acid cycles (TCA). Additionally, lactic acid is accumulated via glycolysis depending on anaerobic metabolism. These metabolic activities cause a decrease in blood pH (Andersen, 1961; Szenci and Besser, 1990; Boink et al., 1991; Liss and Payne, 1993). Since sampling error was reduced to a minimal level in the present study the decrease in blood pH and HCO3 was attributed to the formation of lactic acid due to glycolysis and to an increase in the level of pCO2 . Moreover, the much greater decrease in the pH of the blood samples stored at 37 °C may be attributed to lactic acid generation due to rapid anaerobic metabolism as a result of the high storage temperature (Liss and Payne, 1993). In the present study, increases in the pCO2 values of the blood samples kept at 37 °C and at room temperature and a decrease in those of the samples stored at +4 °C were recorded as compared to their baseline values. Researchers have reported that in vitro blood pCO2 value rises depending on aerobic and anaerobic metabolism in blood cells (Sandhagen et al., 1988; Szenci and Besser, 1990; Beaulieu et al., 1999). Unlike O2 , CO2 does not influx through plastic syringe walls (Mahoney et al., 1991), and therefore the significant rises of pCO2 in groups II and III may be the result of high cell metabolic activities at +22 and 37 °C (Foster and Terry, 1967). Moreover the most prominent increase in pCO2 was observed in the sample stored at 37 °C. The significant decrease in the pCO2 values of the samples stored at +4 °C may be due to the blood being transferred to a lower than body temperature medium (Mahoney et al., 1991). In the case of blood samples stored over time, the type of syringe used for sampling (Mahoney et al., 1991; Beaulieu et al., 1999) and the aerobic metabolism of leucocytes (Poulsen and Surynek, 1977; Haskins, 1977) in the blood have been determined to influence alterations in the levels of pO2 . It has been revealed that pO2 in plastic syringes increases by the diffusion of oxygen through the plastic wall of the syringe (Paerregaard et al., 1987; Beaulieu et al., 1999). Leucocytes are responsible for most of the aerobic metabolism in the blood (Liss and Payne, 1993) and cause O2 consumption in blood samples stored under in vitro anaerobic conditions (Sandhagen et al., 1988; Szenci and Besser, 1990). A decrease in pO2 values has been found in blood samples with high leucocyte counts (Schimidt and Plathe, 1992) and in samples with anaemia (Haskins, 1977). Since the initial mean leucocyte and erythrocyte numbers and haemoglobin concentrations of the blood samples were within normal range in the present study,
the significant decrease (p < 0:001) in pO2 level found in the blood samples stored at 37 °C after 24 h may be attributed to an increase in aerobic metabolism and O2 consumption by leucocytes due to the high storage temperature (Liss and Payne, 1993). A significant increase in the level of pO2 was detected after 6 h in the samples stored at room temperature and in the refrigerated samples. This may have been due to the release of O2 from haemoglobin as a result of the reduction in blood pH (Szenci and Besser, 1990) and to the diffusion of O2 through the plastic syringe wall (Beaulieu et al., 1999; Mahoney et al., 1991) although oxygen consumption occurs during aerobic cell metabolism. Moreover, this finding reflects the fact that aerobic metabolism of leucocyte was lower at room temperature and under +4 °C in vitro conditions than at 37 °C. The SAT O2 value also decreased together with pH in the blood samples kept at 37 °C. This can be explained by the shifting of the haemoglobin–O2 dissociation curve to the right (Bohr effect) due to decrease in blood pH (Szenci and Besser, 1990; Carlson, 1996). The O2 CT and O2 SAT value displayed the least alteration within the parameters for the blood samples kept at room temperature and at +4 °C, which is consisted the findings of Beaulieu et al. (1999) in a study carried out on human blood. Therefore, we suggest that the O2 CT and O2 SAT value should be taken into consideration for blood samples stored at room temperature when assessing blood O2 . In conclusion, the acid–base values of the samples stored at 22 and 37 °C changed significantly when compared to the samples stored at +4 °C. However, the acid–base values of the samples stored at 22 and +4 °C were within normal range and could be used for clinical purposes for up to 12 and 48 h, respectively.
References Andersen, O.S., 1961. Sampling and storing of blood for determination of acid–base status. Scandinavian Journal of Clinical Laboratory Investigation 13, 196–204. Beaulieu, M., Lapointe, Y., Vinet, B., 1999. Stability of pO2 , pCO2 and pH in fresh blood samples stored in a plastic syringe with low heparin in relation to various blood-gas and hematological parameters. Clinical Biochemistry 32, 101–107. Boink, A.B.T.J., Buckley, B.M., Christiansen, T.F., Conington, A.K., Maas, A.H.J., Muller-Plathe, O., Andersen, O.S., 1991. Recomendation on sampling, transport and storage for the determination of the concentration of ionized calcium in whole blood, plasma and serum. Annual Biological Clinics 49, 234–438. Carlson, G.P., 1996. Clinical chemistry tests (acid–base imbalance). In: Smith, B.P. (Ed.), Large Animal Internal Medicine. Mosby-Year Book, London, pp. 456–460. Coles, E.H., 1980. Veterinary Clinical Pathology. third ed. London, pp. 15–122. Foster, J.M., Terry, M.L., 1967. Studies on the energy metabolism of human leucocytes. I. Oxidative phosphorylation by human leucocyte mitochondria. Blood 30, 168–175.
G. Gokce et al. / Research in Veterinary Science 76 (2004) 121–127 Haskins, S.C., 1977. Sampling and storage of blood for pH and blood gas analysis. Journal of American Veterinary Medical Association 170, 429–433. Jagos, P., Bouda, J., Priklylova, J., 1977. Dynamics of acid–base changes in the venous blood of cattle in vitro with time. Veterinary Medicine (Prague) 22, 257–262. Kirkwood, B.R., 1988. Essentials of Medical Statistics. Blackwell Scientific Publications, Oxford, USA. Krokavec, M., S_ımo, K., Mart_ınko, A., 1987. Changes in the acid–base equlibrium in cattle in relation to the time intervals between sampling and study under field conditions. Veterinary Medicine (Praha) 32 (3), 145–150. Lenfant, C., Aucutt, C., 1965. Oxygen uptake and change in carbondioxide tension in human blood stored at 37 °C. Journal Applied Phsiology 20, 503–508. Liss, H., Payne, C.P., 1993. Stability of blood gases in ice and at room temperature. Chest 103, 1120–1122. Mahoney, J.J., Harvey, J.A., Wong, R.J., Kessel, L.V., 1991. Changes in oxygen measurements when whole blood is stored in iced plastic or glass syringes. Clinical Chemistry 37, 1244–1248. Muller-Plathe, O., Heyduck, S., 1992. Stability of blood gases, electrolytes and haemoglobin in heparinized whole blood samples: Influence of the type of syringe. European Journal of Clinical Chemistry and Clinical Biochemistry 30, 349–355. Paerregaard, A., Nickelsen, C.N.A., Brandi, L., Andersen, G.E., 1987. The influence of sampling site and time upon umbilical cord blood
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acid–base status and pO2 in the newborn infant. Journal of Perinatal Medicine 15, 559–563. Poulsen, J.S.D., Surynek, J., 1977. Acid–base status of cattle blood. Nordisk Veterinaer Medicin 29, 271–283. Radostits, O.M., Blood, D.C., Gay, C.G., 1994. Veterinary Medicine, eighth ed. Bailliere Tindall, London. pp. 66–86. Sandhagen, C.F.H., Verdier, C.H., Eriksson, L., 1988. Distrubution of blood gases, glucose and lactate within stored blood units. Vox Sanguinis 55, 139–142. Schimidt, C., Plathe, O.M., 1992. Stability of pO2 , pCO2 and pH in heparinized whole blood samples: Influence of storage temperature with regard to leucocyte count and syringe material. European Journal of Clinical Chemistry and Clinical Biochemistry 30, 767–773. Szenci, O., Besser, T., 1990. Changes in blood gas and acid–base values of bovine venous blood during storage. Journal of American Veterinary Medical Association 197, 471–474. Szenci, O., Brydl, E., Bajcsy, C.A., 1991. Effect of storage on measurement of ionized calcium and acid–base variables in equine, bovine, ovine, and canine venous blood. Journal of American Veterinary Medical Association 199, 1167–1169. Szenci, O., Nemeth, F., Stollar, Z., Brydl, E., 1994. Effect of storage time and temperature on ionized calcium concentration in bovine and ovine blood, plasma and serum. Journal of American Veterinary Medical Association 204 (8), 1242– 1244.
Effect of time delay and storage temperature on blood gas and acid–base values of bovine venous blood Gurbuz Gokce a
a,*
, Mehmet Citil a, Vehbi Gunes a, Gultekin Atalan
b
Department of Internal Medicine, Faculty of Veterinary Medicine, University of Kafkas, Kars 36100, Turkey b Department of Surgery, Faculty of Veterinary Medicine, University of Kafkas, Kars 36100, Turkey Accepted 12 August 2003
Abstract 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 °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 °C (Group I, n ¼ 5), at a room temperature of about 22 °C (Group II, n ¼ 5) and in an incubator adjusted to 37 °C (Group III; n ¼ 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 pCO2 value for Group I was )0.72 kPa during the assessment, while for groups II and III, maximum alterations in pCO2 were detected as +2.68 and +4.16 kPa, respectively. Mean pO2 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 (HCO3 ) 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 °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. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: pO2 ; pCO2 ; Acid base parameters; Storage temperature time; Bovine
1. Introduction Various metabolic and respiratory diseases effect venous blood gas composition and acid–base values of cattle (Radostits et al., 1994; Carlson, 1996). Therefore, accurate measurement of these blood values is of great importance for veterinary clinicians and research workers. Human (Paerregaard et al., 1987; Muller-Plathe and Heyduck, 1992; Beaulieu et al., 1999) and cattle (Poulsen and Surynek, 1977; Szenci and Besser, 1990; Szenci et al., 1991) blood gas composition and acid–base values may change depending on the type of syringe used for sampling, delays in measurement time and variations in *
Corresponding author. Tel.: +90-474-242-6800; fax: +90-474-2426853. E-mail address: [email protected] (G. Gokce). 0034-5288/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2003.08.009
storage temperature. Continuous anaerobic and aerobic metabolism between the drawing of the blood sample and its analysis intervals may effect blood acid–base values in humans (Boink et al., 1991; Liss and Payne, 1993), cattle (Jagos et al., 1977; Krokavec et al., 1987; Szenci and Besser, 1990) and dogs (Haskins, 1977). In vitro blood metabolism includes both aerobic metabolism with production of carbon dioxide and anaerobic glycolysis with production of non-gaseous acids such as lactic acid. Oxygen consumption depends on the enzymatic activity of the citric acid and cytocrome systems in leucocytes and reticulocytes and will vary with the concentration of these cell types. Glycolysis is the predominant metabolic process in mature red blood cells. These processes are largely responsible for the rising of pCO2 in the stored blood of dogs (Haskins, 1977), and cattle (Poulsen and Surynek, 1977). These metabolic changes are temperature
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G. Gokce et al. / Research in Veterinary Science 76 (2004) 121–127
dependent (Liss and Payne, 1993). Additionally, severe leucocytosis (Schimidt and Plathe, 1992; Liss and Payne, 1993) and anaemia (Haskins, 1977) can lead to alterations in acid–base values. Since it is difficult to measure the acid–base and gas values of blood under field conditions in a short period of time its necessary to store blood (Szenci and Besser, 1990). Several authors have investigated the alterations in acid–base variables during the storage of blood samples from humans (Paerregaard et al., 1987; Schimidt and Plathe, 1992; Beaulieu et al., 1999), cattle (Jagos et al., 1977; Poulsen and Surynek, 1977; Krokavec et al., 1987; Szenci and Besser, 1990; Szenci et al., 1991, 1994) and dogs (Haskins, 1977). It would be useful to determine the extent to which changes in blood acid– base values and gas composition depend on the duration and temperature of storage. To our knowledge, there is no precise information available as to whether or not clinically significant changes occur when bovine venous blood is stored at 22 or at 37 °C for long periods. Therefore, this study was carried out to detect the changes in gas composition and acid–base values in bovine venous blood samples stored at different temperatures (+4, 22 and 37 °C) for up to 48 h.
2. Materials and methods
matically performed on a blood gas analyser (Chiron diagnostics, Rapidlab 248, UK) prior to the study. Blood samples from the five cattle were analysed for blood gas and acid base values four times each within 15 min to determine the imprecision of parameters, which emerged as follows: pH 0.01%; pCO2 1.9%; pO2 3.52%; ActHCO3 1.74%; StdHCO3 1.34%, BEact 1.98%, BEstd 1.02%, O2 SAT 0.39% and O2 CT 1.8% on the basis of variation co efficiency. Control of accuracy was carried out using a commercial accuracy control solution (Complete, Bayer, East Walpole, USA). Control values were within the normal range as described by the manufacturer. Calculated variables were automatically performed by blood gas analyser according to equations 1 programed to device by manufacterer (Chiron diagnostics, Rapidlab 248, UK) The values of pH, pCO2 and pO2 were automatically adjusted to the animalÕs rectal temperatures. 2.3. Haematological analyses For the initial haematological examinations, peripheral blood samples were collected from jugular vein into ethylenediaminetetraaceticacid (EDTA) treated tubes. These blood samples were used to manually establish total white blood cells (WBCs), total red blood cells (RBCs) and haemoglobin concentration (Hb), as described by Coles (1980).
2.1. Animals and sampling 2.4. Statistical analyses Five clinically healthy, 1–3 years old, crossbreed cattle were used in the study. Following rectal temperature measurement, three jugular venous blood samples were drawn from each animal into 10 ml plastic syringes the dead space (0.08 ml) of which was filled with heparin (LiquemineÒ -Roche, Istanbul, Turkey). The needle tips were closed with a rubber stopper after the removal of air bubbles from the samples. The samples were placed in a bed of crushed ice, taken immediately to the laboratory and analysed within 15 min. Following the first (0) hourÕs laboratory analysis, a total of 15 blood samples were collected from the animals, allocated into three groups and then stored, respectively, in a refrigerator set to +4 °C (Group I, n ¼ 5), at a room temperature of about 22 °C (Group II, n ¼ 5) and in an incubator adjusted to 37 °C (Group III, n ¼ 5), for up to 48 h. Blood gas and acid–base values were analysed at 0, 1, 2, 3, 4, 5, 6, 12, 24, 36 and 48 h of storage for all the groups. 2.2. Acid–base and blood gas analyses The measurement of pH, pO2 , pCO2 and the calculation of standard Base excess (BEstd), actual base excess (BEact), standard bicarbonate (stHCO3 ), actual bicarbonate (actHCO3 ) concentrations, oxygen saturation (O2 SAT) and oxygen content (O2 CT) were auto-
The effect of temperature on blood gas and acid base values was tested by analysis of variance using SPSS for Windows 6.0. The effects of time on the repeated measurements within each group were also examined using two-way variance analysis (Kirkwood, 1988). The data is presented as mean SE.
3. Results The mean initial haematological values of the animals used in the study were detected as 115 0.6 g/l for Hb, 5.9 0.5 1012 cell/l for RBC and 6.5 1.9 109 cell/l for WBC. The mean rectal temperature of the animals was 38.5 0.2 °C. 1
ActHCO3 ¼ pH þ logðpCO2 0:0307Þ 6:105; StdHCO3 ¼ 24:5þ 0:9A þ ðA 2:9Þ2 ð2:65 þ 0:31ctHbÞ=1000. A ¼ BEstd 0:2½ctHb ½100 O2 SAT=100; BEact ¼ cHCO3 24:8 þ 1:62ðpH 7:40Þ. BEstd ¼ ð1 0:014 ctHbÞ½ðcHCO3 24:8Þ þ ð1:43 ctHb þ 7:7Þ ðpH 7:40Þ. N 4 15N 3 þ 2045N 2 þ 2000N O2 SAT ¼ 4 100: N 15N 3 þ 2400N 2 31100N þ 2:4 106 N ¼ pO2 þ 10½0:48ðpH7:4Þ0:0013BEstd and BEstd is calculated assuming 100% O2 SAT; O2 CT ¼ O2 SAT 1:39 ctHb þ 0:00314 pO2 .
Table 1 Effect of storage time on pCO2 and pO2 of bovine venous blood samples stored at +4 °C (Group I), 22 °C (Group II) and 37 °C (Group III) (n ¼ 5) (mean SE) Parameter
Group
pCO2 (kPa)
I
Time (h)
II III
0
1
2
3
4
5
6
12
24
36
48
P1
6.59 0.11 ab 6.59 0.11 f 6.59 0.11 e
5.95 0.09 e 6.34 0.14 f 6.20 0.15 e
5.94 0.10 B,e 6.60 0.16 A,f 6.41 0.15 A,e
5.87 0.12 B,e 6.59 0.12 A,f 6.61 0.12 A,e
6.04 0.10 B,de 6.81 0.14 A,e 7.01 0.16 A,d
6.01 0.11 C,e 6.88 0.10 B,e 7.37 0.18 A,c
6.17 0.10 C,cde 7.34 0.12 B,c 9.01 0.26 A,b
5.86 0.11 C,e 7.07 0.13 B,d 8.88 0.29 A,b
6.34 0.12 C,bcd 8.06 0.16 B,b 10.75 0.32 A,a
6.40 0.12 B,bc 8.82 0.19 A,a 9.29 0.19 A,b
6.90 0.10 B,a 9.27 0.47 A,a 9.05 0.34 A,b
p < 0:001
p < 0:05
p < 0:01
p < 0:001
p < 0:001
p < 0:001
p < 0:001
p < 0:001
p < 0:001
p < 0:001
4.47 0.15 d 4.41 0.15 d 4.33 0.10 a
4.55 0.15 cd 4.64 0.11 c 4.38 0.12 a
4.54 0.16 d 4.57 0.10 d 4.32 0.11 a
4.67 0.17 bcd 4.56 0.11 d 4.31 0.12 a
4.77 0.16 bcd 4.70 0.11 c 4.31 0.15 a
4.77 0.18 A,bcd 4.91 0.11 A,b 4.08 0.15 B,a
5.24 0.24 A,ab 5.00 0.10 A,a 0.86 0.13 B,c
5.12 0.24 A,abc 4.98 0.12 A,a 1.43 0.41 B,b
5.42 0.24 A,a 5.06 0.07 A,a 0.69 0.04 B,c
p < 0:01
p < 0:001
p < 0:001
p < 0:001
P2 4.26 0.10 d 4.26 0.10 d 4.26 0.10 a
I II III
4.27 0.16 d 4.25 0.10 d 4.32 0.09 a
P2
p < 0:001
p < 0:001 p < 0:001 p < 0:001
P1, significant differences within group compared to baseline value (a,b,c,d,e,f), (p < 0:001); P2, in each column different letters (A, B, C) indicated significant between groups (p < 0:01), (p < 0:001); pCO2 , partial pressure of carbondioxide (kPa); pO2 , partial pressure of oxygen (kPa).
Table 2 Effect of storage time on O2 SAT and O2 ct of bovine venous blood samples stored at +4 °C (Group I), 22 °C (Group II) and 37 °C (Group III) (n ¼ 5) (mean SE) Parameter
O2 SAT
Group
I II III
Time (h) 0
1
2
3
4
5
6
12
24
36
48
P1
62.32 1.20 d 62.32 1.20
62.80 2.46 cd 61.82 1.02
63.28 2.16 cd 61.33 1.69
64.36 2.04 bcd 64.42 0.34
63.78 2.24 cd 62.44 1.01
61.10 1.42 b
60.16 1.55 b
59.30 1.70 b
57.48 1.82 b
66.62 2.09 A,abcd 62.76 1.18 A 51.82 2.30 B,c
66.56 2.27 A,abcd 65.60 1.32 A 45.54 2.71 B,d
71.52 2.72 A,ab 63.88 1.34 B 32.22 0.41 C,e
69.82 2.89 A,abc 60.70 1.88 B 8.17 3.38 C,f
72.78 2.00 A,a 61.75 0.35 B 1.85 0.05 C,f
p < 0:05
62.32 1.20 a
65.46 2.19 A,bcd 62.06 1.21 A 55.58 2.00 B,c p < 0:01
p < 0:001
p < 0:001
p < 0:001
p < 0:001
p < 0:001
10.66 0.34 A,c 10.12 0.35 AB 9.08 0.39 B,b
10.84 0.36 A,c 10.22 0.35 A 8.44 0.46 B,c
10.86 0.37 A,c 10.70 0.39 A 7.44 0.50 B,c
11.66 0.51 A,b 10.44 0.39 B 0.48 0.11 C,d
11.16 0.40 A,c 9.92 0.49 A 1.40 0.60 B,d
12.00 0.30 A,a 10.45 0.05 B 0.3 0.00 C,d
p < 0:05
p < 0:01
p < 0:001
p < 0:001
p < 0:001
p < 0:001
P2 O2 CT (mmol/l)
I II III
P2
10.16 0.30 c 10.16 0.30
10.50 0.44 c 10.08 0.32
10.30 0.36 c 10.15 0.42
10.46 0.35 c 10.50 0.39
10.38 0.38 c 10.18 0.33
10.16 0.30 a
9.92 0.24 a
9.80 0.38 a
9.66 0.33 a
9.38 0.42 b
p > 0:333 p < 0:001
G. Gokce et al. / Research in Veterinary Science 76 (2004) 121–127
pO2 (kPa)
p < 0:001
p < 0:05 p > 0:954 p < 0:001
123
P1, significant differences within group compared to baseline value (a,b,c,d,e,f), (p < 0:05, p < 0:001); P2, in each column different letters (A, B, C) indicated significant between groups (p < 0:05, p < 0:01, p < 0:001); O2 SAT, oxyhaemoglobin saturation (%); O2 CT, oxygen content (mmol/l).
124
Table 3 Effect of storage time on ActHCO3 and StdHCO3 of bovine venous blood samples stored at +4 °C (Group I), 22 °C (Group II) and 37 °C (Group III) (n ¼ 5) (mean SE) Parameter
Group
ActHCO3 (mmol/l)
I II III
Time (h) 0
1
2
3
4
5
6
12
24
36
48
P1
31.38 0.35 a 31.38 0.35
29.04 0.27 B,c 30.32 0.37 A 28.94 0.26 B,c
28.00 0.30 B,cde 30.38 0.30 A 28.88 0.32 B,c
27.62 0.40 B,de 29.94 0.49 A 28.48 0.39 B,d
28.12 0.34 B,cd 30.00 0.35 A 29.02 0.37 AB,c
27.88 0.33 C,cde 30.00 0.20 A 29.00 0.35 B,c
28.42 0.43 B,cd 30.88 0.63 A 31.44 0.48 A,a
26.90 0.31 e 29.44 0.40
28.74 0.31 B,cd 31.20 0.52 A 29.80 0.36 B,b
28.56 0.54 B,cd 30.24 0.29 A 27.57 0.15 B,e
30.20 0.29 A,b 30.55 0.35 B 26.85 0.85 B,e
p < 0:001
p < 0:05
p < 0:001
p < 0:01
p < 0:01
p < 0:01
p < 0:01
p < 0:001
p < 0:01
p < 0:01
26.42 0.97 cd 28.28 0.27 b 27.46 0.31 b
26.40 0.26 B,cd 28.15 0.15 A,b 26.68 0.28 B,c
26.32 0.27 B,cd 27.80 0.40 A,c 26.18 0.28 B,c
26.88 0.20 A,bc 27.54 0.26 A,c 26.08 0.31 B,c
26.40 0.22 B,cd 27.46 0.15 A,c 26.36 0.44 B,c
26.80 0.36 bcd 27.88 0.52 c 26.58 0.27 c
25.58 0.20 A,d 27.74 0.38 A,c 23.24 0.28 B,d
27.00 0.24 A,bc 27.36 0.51 A,c 22.60 0.20 B,e
27.30 0.20 A,bc 25.70 0.38 B,d 21.67 0.23 C,f
27.78 0.29 A,b 25.55 0.75 A,d 21.05 0.75 B,f
p < 0:01
p < 0:01
p < 0:01
p < 0:05
p < 0:001
p < 0:001
p < 0:001
p < 0:001
31.38 0.35 a
P2 I II III
29.24 0.29 a 29.24 0.29 a 29.24 0.29 a
P2
p < 0:001
p < 0:001 n.s p < 0:001
P1, significant differences within group compared to baseline value (a,b,c,d,e,f), (p < 0:05, p < 0:01, p < 0:001); P2, in each column different letters (A, B, C) indicated significant between groups (p < 0:05, p < 0:01, p < 0:001); ActHCO3 , actual bicarbonate concentrations (mmol/l); StdHCO3 , standard bicarbonate concentrations (mmol/l).
Table 4 Effect of storage time on BEact and BEstd of bovine venous blood samples stored at +4 °C (Group I), 22 °C (Group II) and 37 °C (Group III) (n ¼ 5) (mean SE) Parameter
BEact (mmol/l)
Group
I II III
Time (h) 0
1
2
3
4
5
6
12
24
36
48
P1
7.04 0.39 a 7.04 0.39 a 7.04 0.39 a
4.74 0.32 B,bc 5.90 0.36 A,b 4.44 0.22 B,c
3.58 0.35 B,cd 5.83 0.22 A,b 4.14 0.31 B,c
3.2 00.42 B,de 5.30 0.52 A,c 3.46 0.43 B,c
3.64 0.36 B,cd 5.14 0.35 A,c 3.68 0.36 B,c
3.32 0.35 B,de 5.04 0.23 A,c 3.30 0.33 B,c
3.86 0.49 cd 5.70 0.70 b 4.94 0.44 b
2.24 0.31 A,e 4.16 0.45 A,d 0.88 0.39 B,e
4.06 0.31 A,cd 5.34 0.61 A,c 1.64 0.39 B,d
3.76 0.57 A,cd 3.58 0.46 A,d )0.13 0.32 B,f
5.24 0.34 A,b 3.65 0.75 A,d )0.85 0.85 B,f
p < 0:001
p < 0:05
p < 0:01
p < 0:05
p < 0:05
p < 0:01
p < 0:001
p < 0:001
p < 0:01
p < 0:001
4.16 0.26 B,bc 5.10 0.31 A,b 3.86 0.16 B,b
3.24 0.25 B,bcd 4.95 0.14 A,b 3.42 0.30 B,c
2.84 0.34 B,de 4.50 0.45 A,c 2.78 0.36 B,d
3.16 0.31 B,cd 4.44 0.38 A,c 2.78 0.33 B,d
2.90 0.32 B,de 4.18 0.18 A,c 2.40 0.27 B,d
1.98 0.29 B,e 3.28 0.4 A,d )0.30 0.30 C,e
3.46 0.27 A,bcd 4.00 0.56 A,c )0.02 0.30 B,e
3.14 0.50 A,cd 2.14 0.44 A,d )1.58 0.35 B,f
4.28 0.32 A,b 2.05 0.85 B,d )1.95 0.85 C,f
p < 0:05
p < 0:01
p < 0:05
p < 0:05
p < 0:01
p < 0:001
p < 0:001
p < 0:001
p < 0:001
P2 BEstd (mmol/l)
I II III
P2
6.12 0.34 a 6.12 0.34 a 6.12 0.34 a
3.32 0.43 bcd 4.58 0.59 c 3.42 0.34 c
p < 0:001 p < 0:001
p < 0:001 p < 0:001 p < 0:001
P1, significant differences within group compared to baseline value (a,b,c,d,e,f), (p < 0:001); P2, in each column different letters (A, B, C) indicated significant between groups (p < 0:01, p < 0:05, p < 0:001); BEact, extracelluler base excess (mmol/l); BEstd, blood base excess (mmol/l).
G. Gokce et al. / Research in Veterinary Science 76 (2004) 121–127
StdHCO3 (mmol/l)
28.08 0.39 e
p > 0:056
G. Gokce et al. / Research in Veterinary Science 76 (2004) 121–127
Blood acid–base and blood gas values are summarised in Tables 1–4 and Fig. 1. The pH values of the samples kept at different temperatures tended to decrease significantly with time in all groups (p < 0:001) (Fig. 1). Compared to initial values, pH values were found to decrease significantly after 5, 3 and 2 h (p < 0:001) for the samples stored at +4, 22 and 37 °C, respectively. Alterations in pCO2 and pO2 values are summarised in Table 1. The mean pCO2 values of the samples stored at +4 °C decreased significantly between 1 and 24 h, but later increased again to their initial levels. Moreover, the pCO2 values of the samples kept at 22 and 37 °C increased significantly (p < 0:001) after the fourth hour and continued to increase until the end of the study (Table 1). The maximum mean alteration in pCO2 value for Group I was )0.72 kPa during the assessment, while for groups II and III maximum alterations for pCO2 were detected as +2.68 and +4.16 kPa, respectively. The pO2 values of the samples kept at +4 and 22 °C increased with time (p < 0:001). However, a significant decrease was recorded after 24 h for the samples kept at 37 °C (p < 0:001). Values of O2 SAT in group I increased (p < 0:05) after 24 h storage, on the other hand it was significantly decreased in group III after 5 h (p < 0:001) and there were no changes in group II (p > 0:333) (Table 2). The O2 CT values were significantly increased (p < 0:05) after 24 h of storage at +4 °C, but significantly decreased after 4 h of storage at 37 °C (p < 0:001) (Table 2). However, no significant alterations were observed in the O2 CT values of the samples stored at 22 °C. BE and HCO3 concentrations decreased signifi-
125
cantly for all three groups during the study compared to their baseline values. The most prominent decrease in BE and HCO3 concentrations was recorded for the samples kept at 37 °C (Tables 3 and 4).
4. Discussion The effects of storage time and temperature on blood gases and acid–base values in humans (Mahoney et al., 1991; Liss and Payne, 1993; Beaulieu et al., 1999; Lenfant and Aucutt, 1965) and in cattle (Poulsen and Surynek, 1977; Szenci and Besser, 1990; Szenci et al., 1991) have been investigated intensively. Previous studies have revealed that the blood gas and acid–base values of human blood samples usually stabilised between 15 min and 2 h (Paerregaard et al., 1987; Mahoney et al., 1991; Beaulieu et al., 1999). The effect of storage temperature and storage time on blood gas and acid–base values in cattle are disputed (Poulsen and Surynek, 1977; Szenci and Besser, 1990; Szenci et al., 1991). Szenci and Besser (1990) reported that cattle venous blood acid–base values could be used for diagnostic purposes up to 24 h. On the other hand, the pH values of blood samples taken from cattle and stored at +4 °C stabilised after for 5–6 h (Poulsen and Surynek, 1977). In the present study, the pH values of the blood samples stored at +4 °C were found to be suitable for clinical usage up to 48 h (Fig. 1). Furthermore, storage time and temperature were found to significantly alter the blood pH and bicarbonate values of all groups.
7,45
7,4 a a a
7,35
a ab ab
ab abc bc
pH
7,3
A,ab A,bc B,cd
A,abc B ,cd C,de
**
***
7,25
A,bc B,cde C,e A,bc B,de C,f
*** Group I
7,2
***
Group II Group III
A,bcd B,e C,g A,bcd B,f C,h
***
7,15
***
A.cd B,g C,h
A,d B,g C,h
***
***
*** *** ***
7,1
7,05 0
1
2
3
4
5
6
12
24
36
48
Fig. 1. Effects of storage time on the pH of bovine venous blood samples stored at +4 °C (Group I), 22 °C (Group II) and 37 °C (group III) (n ¼ 5) (mean SE). a,b,c,d,e,f,g,h: significant differences within group compared to baseline (0 h), ***(p < 0:001). A,B,C: significant differences between group **(p < 0:01), ***(p < 0:001).
126
G. Gokce et al. / Research in Veterinary Science 76 (2004) 121–127
Particularly, rapid and significant decreases in blood pH (p < 0:001) were observed for those samples stored at 37 °C, compared to the other groups (Groups I and II) (Fig. 1). Metabolism of in vitro blood samples continues during storage. Oxygen consumption occurs due to anaerobic metabolism and CO2 generation in the tricarboxylic acid cycles (TCA). Additionally, lactic acid is accumulated via glycolysis depending on anaerobic metabolism. These metabolic activities cause a decrease in blood pH (Andersen, 1961; Szenci and Besser, 1990; Boink et al., 1991; Liss and Payne, 1993). Since sampling error was reduced to a minimal level in the present study the decrease in blood pH and HCO3 was attributed to the formation of lactic acid due to glycolysis and to an increase in the level of pCO2 . Moreover, the much greater decrease in the pH of the blood samples stored at 37 °C may be attributed to lactic acid generation due to rapid anaerobic metabolism as a result of the high storage temperature (Liss and Payne, 1993). In the present study, increases in the pCO2 values of the blood samples kept at 37 °C and at room temperature and a decrease in those of the samples stored at +4 °C were recorded as compared to their baseline values. Researchers have reported that in vitro blood pCO2 value rises depending on aerobic and anaerobic metabolism in blood cells (Sandhagen et al., 1988; Szenci and Besser, 1990; Beaulieu et al., 1999). Unlike O2 , CO2 does not influx through plastic syringe walls (Mahoney et al., 1991), and therefore the significant rises of pCO2 in groups II and III may be the result of high cell metabolic activities at +22 and 37 °C (Foster and Terry, 1967). Moreover the most prominent increase in pCO2 was observed in the sample stored at 37 °C. The significant decrease in the pCO2 values of the samples stored at +4 °C may be due to the blood being transferred to a lower than body temperature medium (Mahoney et al., 1991). In the case of blood samples stored over time, the type of syringe used for sampling (Mahoney et al., 1991; Beaulieu et al., 1999) and the aerobic metabolism of leucocytes (Poulsen and Surynek, 1977; Haskins, 1977) in the blood have been determined to influence alterations in the levels of pO2 . It has been revealed that pO2 in plastic syringes increases by the diffusion of oxygen through the plastic wall of the syringe (Paerregaard et al., 1987; Beaulieu et al., 1999). Leucocytes are responsible for most of the aerobic metabolism in the blood (Liss and Payne, 1993) and cause O2 consumption in blood samples stored under in vitro anaerobic conditions (Sandhagen et al., 1988; Szenci and Besser, 1990). A decrease in pO2 values has been found in blood samples with high leucocyte counts (Schimidt and Plathe, 1992) and in samples with anaemia (Haskins, 1977). Since the initial mean leucocyte and erythrocyte numbers and haemoglobin concentrations of the blood samples were within normal range in the present study,
the significant decrease (p < 0:001) in pO2 level found in the blood samples stored at 37 °C after 24 h may be attributed to an increase in aerobic metabolism and O2 consumption by leucocytes due to the high storage temperature (Liss and Payne, 1993). A significant increase in the level of pO2 was detected after 6 h in the samples stored at room temperature and in the refrigerated samples. This may have been due to the release of O2 from haemoglobin as a result of the reduction in blood pH (Szenci and Besser, 1990) and to the diffusion of O2 through the plastic syringe wall (Beaulieu et al., 1999; Mahoney et al., 1991) although oxygen consumption occurs during aerobic cell metabolism. Moreover, this finding reflects the fact that aerobic metabolism of leucocyte was lower at room temperature and under +4 °C in vitro conditions than at 37 °C. The SAT O2 value also decreased together with pH in the blood samples kept at 37 °C. This can be explained by the shifting of the haemoglobin–O2 dissociation curve to the right (Bohr effect) due to decrease in blood pH (Szenci and Besser, 1990; Carlson, 1996). The O2 CT and O2 SAT value displayed the least alteration within the parameters for the blood samples kept at room temperature and at +4 °C, which is consisted the findings of Beaulieu et al. (1999) in a study carried out on human blood. Therefore, we suggest that the O2 CT and O2 SAT value should be taken into consideration for blood samples stored at room temperature when assessing blood O2 . In conclusion, the acid–base values of the samples stored at 22 and 37 °C changed significantly when compared to the samples stored at +4 °C. However, the acid–base values of the samples stored at 22 and +4 °C were within normal range and could be used for clinical purposes for up to 12 and 48 h, respectively.
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