POSSIBLE SOURCES OF ERROR IN THE DETERMINATION OF ARTERIAL CARBON DIOXIDE TENSION BY AN INTERPOLATION TECHNIQUE

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1 Brit. J. Anaesth. (1963), 35, 338 POSSIBLE SOURCES OF ERROR IN THE DETERMINATION OF ARTERIAL CARBON DIOXIDE TENSION BY AN INTERPOLATION TECHNIQUE BY F. A. NEEMATALLAH,* LINDA M. CHAPMAN AND M. K. SYKES Department of Anaesthesia, Postgraduate Medical School, London SUMMARY There are two main sources of error in the determination of Pa C o 3 by an interpolation technique which utilizes bubble equilibrations. These are: (1) The occurrence of metabolic acidosis during the process of storage and equilibration. (2) Incomplete equilibration of the blood samples with the known carbon dioxide and oxygen mixture. Incomplete equilibration is particularly likely to occur if the blood has a high packed cell volume: in these circumstances there may be an underestimate of Pco 2 of as much as 20 mm Hg. It is suggested that a Pco 2 electrode is both more accurate and more convenient than the interpolation technique described. However, this too is subject to error and its accuracy should be repeatedly checked by tonometry. In 1961 a series of acid-base studies were performed on patients undergoing open-heart surgery under total body perfusion. Although strict precautions were taken to obtain a steady state during arterial sampling, the arterial carbon dioxide tensions (Pa 002 ) in pre- and postoperative samples were lower than would have been expected. Furthermore, the pre-operative standard bicarbonate values were lower than the normal value of 22.9 ±1.5 m.equiv/1. quoted by Astrup et al. (1960). This suggested the possibility of technical error. The investigations which were undertaken to determine the possible sources of this error are described in this paper. METHODS Arterial blood samples were collected in 10-ml glass syringes lubricated with silicone oil. The deadspace of the syringes was filled with heparin (5,000 units/ml). After collection of the sample, the syringe was capped, the blood was mixed by rotating the syringe rapidly, and the syringe was then stored in a vacuum flask containing ice and water. * Lecturer in Anaesthesia, University of Alexandria, Egypt. This work was supported by a grant from the Medical Research Council. The initial whole blood was always determined within 30 minutes of sampling, using a combined glass and calomel electrode (GK 2641f) and a PHM4 meterf as described by Astrup and Schr0der (1956). All values quoted in this paper were the mean of two duplicate determinations. The meter was standardized with two phosphate buffers ( and at 37 C) of the National Bureau of Standards Scale (Bates and Acree, 1945; Semple, Mattock and Uncles, 1962). The electrode system, buffers and wash solution were maintained at 37 C±0.rC. INTERPOLATION TECHNIQUE After determination of the whole blood, the blood was equilibrated with two carbon dioxide and oxygen mixtures of known composition by bubbling the gases through 2-ml blood samples contained in small tubes standing in a water bath at 37 C. The gas mixtures commonly used were 3 per cent and 7 per cent carbon dioxide in oxygen. Each gas mixture was analyzed on a standard Haldane gas analysis apparatus before use. (The Haldane apparatus was calibrated with mercury and checked by the analysis of total tradiometer Ltd., Copenhagen. 338

2 DETERMINATION OF ARTERIAL CARBON DIOXIDE TENSION 339 absorbable gas in air.) The equilibrating gases were humidified by passing them over the surface of 0.1 N lactic acid contained in a 2-litre bottle lying on its side in the water bath. The gases were then bubbled into the blood samples through polyethylene tubes (size 48, Portland Plastics Ltd.). The rate of bubbling was maintained at about five bubbles per second, one drop of silicone emulsion RD (Midland Silicones Ltd.) being added to prevent foaming. Equilibration was continued for 7 minutes. The of the samples was then determined and the results plotted on a nomogram (Siggaard Andersen and Engel, 1960). Tonometry. Fifteen to 20 ml of heparinized venous or arterial blood was placed in a glass tonometer 4.5 cm in diameter and 28 cm long. This was rotated in a water bath for 40 minutes at 37 C. The equilibrating gas was humidified by passing it over the surface of 0.1 normal lactic acid at 37 C and was then allowed to flow through the tonometer at a rate of 2 litres per minute. The haematocrit was checked before and after each run to guard against haemoconcentration or dilution. The blood sample was withdrawn through a polyethylene tube into a syringe. Both syringe and sampling tube were flushed with the gas used for tonometry before the sample was withdrawn. The syringe was held slightly lower than the tonometer when the sample was being taken. Packed cell volume. Haematocrit values were determined by a micromethod (Hawksley Ltd.). Percentage haemolysis. One ml of blood was withdrawn from the tonometer and mixed thoroughly; 0.02 ml of this blood was then diluted with 4 ml of 0.04 per cent ammonium hydroxide (NH 4 0H). This was taken as the standard for 100 per cent haemolysis. The remainder of the sample was centrifuged at 2,500 r.p.m. for 5 minutes; 0.02 ml of the resultant plasma was diluted with 4 ml of the ammonium hydroxide solution and this was taken as the standard for 0 per cent haemolysis. The percentage haemolysis produced by the process of equilibration was determined by spinning a sample of the equilibrated blood (approximately 0.5 ml) at 2,500 r.p.m. for 5 minutes and then diluting 0.02 ml of the resulting plasma with 4 ml of the ammonium hydroxide solution as above. The optical density of this sample was then compared with the 100 per cent and 0 per cent samples on the Unicam SP600 spectrophotometer. Readings were taken at 560 m^. Total carbon dioxide content. One-ml samples of whole blood were analyzed in duplicate on a standard Van Slyke apparatus using the technique of Peters and Van Slyke (1932). Duplicates were not accepted unless they agreed within 0.1 m.equiv/1. ACCURACY OF THE INTERPOLATION METHODS The accuracy of the method described was checked by a series of tonometer experiments before the method was used clinically. The results are shown in table I. There was a mean from the tonometer gas Pco 2 of mm Hg (SD 1.53 mm Hg). It was concluded from these results that the accuracy of the determination was Comparison of determined by Tonometer gas Pco TABLE I tonometered blood samples with Pco a the whole blood equilibration technique. Pco Mean= 0-34 mm Hg SD 1-53 Blood-gas sufficient for the clinical research proposed. However, when the technique was applied to a large number of cases, results were obtained which were unexpectedly low. A further series of tonometer experiments was, therefore, performed to check the accuracy of the Pco 2 determinations. The results are shown in table II. In this series, the mean of Pco 2 from tonometer gas was mm Hg (SD 2.99 mm Hg). The range of s was also greater than had been obtained before. When Pco 2 was calculated from the whole blood and from the total carbon

3 340 BRITISH JOURNAL OF ANAESTHESIA TABLE II Comparison between blood tonometered with gas of known Pco 2 and Pco 2 determined by interpolation technique {Column I) and with Pco 2 determined by Singer-Hastings nomogram using and total carbon dioxide content {Column II) Tonometer gas Pco Pco 2 (interpolation) Blood-gas (interpolation) I Mean 2-45 mm Hg SD 2-99 SD Blood-gas Pco 2 nomogram II (nomogram) ^ = +011 mmhg 3-35 dioxide content of the tonometer blood sample, using the Singer-Hastings nomogram (Singer and Hastings, 1948), the Pco 2 had a mean from the tonometer gas Pco 2 of mm Hg (SD 3.35 mm Hg). Examination of the nomogram (Siggaard Andersen and Engel, 1960) suggested that the most likely explanation of the low Pco 2 values obtained was that the buffer line, in which the original blood was interpolated, was shifted to the left. Two possible causes were postulated: a reduction in total base content resulting from cellular metabolism during storage and equilibration, and incomplete equilibration of blood samples with the low carbon dioxide and oxygen mixture. The latter supposition was supported by the abnormally steep slope of some of the buffer lines plotted during clinical studies. PLAN OF INVESTIGATION Effect of storing blood samples in iced water. The and carbon dioxide content of the blood samples were measured immediately after sampling and again after storage in capped syringes placed in iced water for 2 hours. Effect of storing blood samples at 37 C. The of blood samples was measured before and after storage in capped syringes at 37 C for 30, 60 and 90 minutes. Other samples were stored in iced water for 2 hours and then at 37 C for 30 minutes. (This was to simulate conditions experienced on a number of occasions during clinical studies when the number of samples exceeded the capacity of the laboratory. In these circumstances the initial readings were made immediately and the equilibrations were performed later on blood which had been kept iced for up to 2 hours.) Optimal time for equilibration. A number of blood samples were divided into five 2-ml aliquots. These aliquots were then equilibrated with the same gas mixture for different periods of time. The after varying periods of equilibration was then compared with the initial of the blood sample. The same procedure was carried out on blood samples with a high packed cell volume. These samples were taken from patients with the Tetralogy of Fallot, since it was in these patients that the greatest discrepancies in Pco 2 determination had been observed to occur. A further check on the completeness of equilibration was obtained by measuring the Pco 2 after equilibration with a known gas mixture by means of the Pco 2 electrode (Severinghaus and Bradley, 1958). This was carried out on blood samples having both normal and high packed cell volumes. Other effects of the equilibration process. On total base content. Blood was tonometered with gas of a known Pco 2 and the was determined. Aliquots of this blood were then equilibrated with the same gas for 7, 10 and 15 minutes and the was again determined. Similar measurements were made on blood which had been stored in iced water for 2 hours after tonometry. In addition, total carbon dioxide content was measured on tonometered blood samples before and after 10 minutes equilibration with the same gas. All equilibrations and determinations were performed at 37 C. On haemolysis, (i) The percentage haemolysis produced by 10 minutes equilibration was determined on normal blood and on blood taken from a pump-oxygenator after total body perfusion.

4 DETERMINATION OF ARTERIAL CARBON DIOXIDE TENSION 341 (ii) The and percentage haemolysis were determined on tonometered blood samples. Each sample was then divided into three parts and equilibrations were performed with the same gas using 1, 2 or 3 drops of antifoam in each of the tubes. The change in and percentage haemolysis was noted. (iii) Tonometered blood samples were divided into three aliquots. Each of these was then equilibrated with the same gas for 10 minutes using slow, normal and fast rates of bubbling. and percentage haemolysis were compared before and after this procedure. (iv) The and percentage haemolysis levels were determined on samples of tonometered blood which had been subjected to periods of 10, 20 and 30 minutes equilibration with the same gas. (v) Similar observations were made on tonometered blood samples which had been stored in iced water for 1 hour and then equilibrated with the same gas for 10 minutes. Comparison of Pco 2 determined by the interpolation technique with that determined by the Pco 2 electrode. In one series of comparisons tonometered normal blood was used. In a second series the determinations were made on blood taken from patients with the Tetralogy of Fallot in whom the packed cell volume was more than 55 per cent. In this second series the blood was not subjected to tonometry. In this group of experiments, the period of equilibration used in the interpolation technique was 10 minutes if the blood had a normal packed cell volume and 20 minutes if the haematocrit reading was above 55 per cent. RESULTS Effect of storage at 0 C. In every case but one there was a fall in in blood samples stored for 2 hours in iced water (table in). The average fall in was units. There was no change in the total carbon dioxide content (table IV). Effect of storage at 37 C. There was a fall in in all the blood samples stored at 37 C. This increased with duration of storage. The reduction in averaged 0.023, and units after 30, 60 and 90 TABLE III of blood samples before and after 2 hours storage in iced water. Initial after 2 hours storage in ice Average fall=0-008 units change after storage in ice O TABLE IV Total carbon dioxide content of tonometered blood before and after 2 hours storage in a capped syringe in iced water. ( Van Slyke duplicate analyses agreed within 0 1 m.equiv/l.} Total CO 2 content after tonometry (m.equiv/l.) Total CO 2 content after 2 hours storage in ice (m.equiv/l.) Difference (m.equiv/l.) ( Mean=0 0 minutes storage respectively. When samples were stored at 0 C for 2 hours and then at 37 C for 30 minutes, there was an average fall in of units. The average fall during the period of storage at 0 C in these samples was units (table V). When each of these samples was equilibrated with two gases of known composition, the buffer lines obtained were shifted to the left. Figure 1 shows the results from one of these experiments (Case No. 1, table V) plotted on a nomogram. The lines reveal a progressive loss of base amounting to 1.2 m.equiv/l. in the first 30 minutes. After 1 hour's storage at 37 C the base loss may amount to 3 m.equiv/l.

5 Standard Bicarbonate meq/l units. FIG. 1 Effect of storing blood at 0 and 37 C. Buffer lines plotted on nomogram of Siggaard Andersen and Engel. Numbers on buffer lines refer to numbered samples of Case No. 1 (table V). TABLE V Effect of storage on of blood samples. Blood samples Casel PH Case 2 Case 3 Case 4 PH Case 5 Case 6 Average change in PH (1) Immediately after sampling (2) After 30 min at 37 C (3) After 60 min at 37 C (4) After 90 min at 37 C (5) After 2 hours at 0 C (6) After 2 hours at 0 C and 30 min at 37 C

6 DETERMINATION OF ARTERIAL CARBON DIOXIDE TENSION 343 TABLE VI Normal blood. Comparison ofpco 2 of tonometer gas {determined by Haldane analysis) with Pco 2 determined by electrode and with Pco 2 of tonometered blood determined by electrode. Tonometer gas Pco 2 3% (Haldane) Tonometered blood Blood-gas Pco Mean= 0 2 mm Hg Tonometer gas Electrode gas-haldane gas Pco Mean=+0-29 mm Hg Tonometer gas Pco 2 5% (Haldane) Tonometer blood Pco a (electrode) Blood-gas Pco Mean= 0-5 mmhg Tonometer gas Electrode gas-haldane gas Pco Mean = 0 mm Hg Tonometer gas Pco 2 10% (Haldane) Tonometer blood Blood-gas Pco Mean=3 mmhg Tonometer gas Electrode gas-haldane gas Pco Mean=+0-2 mmhg Optimal time of equilibration. Blood of normal packed cell volume. The change in with increasing periods of equilibration was caused by two processes the change in Pco 2 and the change in base content due to cellular metabolism. Consequently a true plateau was not obtained. Equilibration of blood samples with low carbon dioxide and oxygen mixtures caused the to rise initially and then to fall again (fig. 2). The decline was probably due to the second of the two processes. From the curves it may be concluded that equilibration was complete in 10 minutes. To allow for the fall due to metabolism the downslope can be extrapolated back to zero time. The average correction factor so obtained was units. Equilibration of blood samples with a high carbon dioxide and oxygen mixture yielded a curve which declined steeply at first, and then more gradually (fig. 3). Equilibration was complete in 10 minutes. Extrapolation to zero time yielded an average correction of units. Blood with a high packed cell volume. Although equilibration was complete by 20 minutes in most cases, in some cases an even longer period was required (fig. 4). When the Pco 2 of samples of normal blood equilibrated with a 3 per cent carbon dioxide and oxygen mixture for 10 minutes was checked with the Pco 2 electrode, the mean from the equilibrating gas Pco 2 was mm Hg (SD 1.4 mm Hg). When a 10 per cent carbon dioxide and oxygen mixture was used the mean was +1 mm Hg (SD 0.25 mm Hg).

7 344 BRITISH JOURNAL OF ANAESTHESIA O 10 c 7-45 X Q. 74O IO 15 2O 25 Time in minutes. FIG. 2 Equilibration of six normal blood samples with 3 per cent carbon dioxide and oxygen gas mixture O O units O 715 o 5 IO Time in minutes. FIG. 3 Equilibration of six normal blood samples with 10 per cent carbon dioxide and oxygen gas mixture. 7 IO 15 Time in minutes FIG. 4 2O 25 Equilibration of five blood samples having packed cell volumes greater than 55 per cent with 3 per cent and 10 per cent carbon dioxide and oxygen mixtures-

8 DETERMINATION OF ARTERIAL CARBON DIOXIDE TENSION 345 TABLE VII High haematocrit blood. Comparison ofpco 2 of tonometer gas (determined by Haldane analysis) with Pco% determined by electrode and with Pco 2 of tonometered blood determined by electrode. Tonometer gas Pco 2 3% (Haldane) Tonometered blood Blood/gas Pco Mean= 0-35 mmhg Tonometer gas Electrode gas/actual gas Pco Mean=+0-21mmHg Tonometer gas Pco a 5% (Haldane) Tonometered blood Blood/gas Pco 2 Tonometer gas Electrode gas/actual gas Pco Mean= 0-93 mmhg Mean=+0-25 mmhg Tonometer gas Pco 2 10% (Haldane) Tonometered blood Blood/gas Pco 2 Tonometer gas Electrode gas/actual gas Pco Mean= 2 8 mm Hg Mean=+0-52 mmhg With high haematocrit blood samples the s were +3 mm Hg (SD 1.3 mm Hg) and - 3 mm Hg (SD 2.31 mm Hg) respectively. This confirmed that equilibration was not complete in 10 minutes when the blood sample had a high packed cell volume. To exclude electrode error as the cause of these s, a series of tonometry experiments were performed concurrently. These revealed that with normal blood samples the mean between the Pco 2 determined by the electrode and the tonometer gas was small (table VI). With blood of a high packed cell volume, the gas-electrode was small with 3 per cent and with 5 per cent carbon dioxide and oxygen mixtures, but was greater with 10 per cent carbon dioxide and oxygen mixtures (table VII). It was thought possible that these s might be due either to the failure to reach equilibration during tonometry or to failure to reach equilibrium between the blood and bicarbonate solution within the electrode. Further experiments suggested that both these factors may have contributed to the errors. TABLE VIII of blood after 7, 10 and 15 minutes equilibration with the same gas that was used for tonometry. Posttonometer Average fall in after 7 minutes equilibration units after 10 minutes equilibration units after 15 minutes equilibration units

9 346 BRITISH JOURNAL OF ANAESTHESIA TABLE IX Change in during 10 minutes equilibration with tonometer gas. Equilibrations performed immediately after tonometry and after 2 hours storage in ice. Post-tonometer after 10 minutes equilibration Average change in Change in units after 2 hours at0 C after 10 minutes equilibration after storage for 2 hours at0 C units Change in When tonometry was prolonged for over 1 hour and two successive samples of blood were pushed through the electrode before reading, the mean between tonometer gas and blood Pco 3 determined by the electrode was - 2 mm Hg. A 3 per cent carbon dioxide and oxygen mixture was used for calibrating the carbon dioxide electrode in all these experiments. If a gas mixture with a Pco 2 closer to that of the blood was used, then the blood-gas with blood of high packed cell volume was reduced still further. Other effects of the equilibration procedure. On total base content. When tonometered blood was removed anaerobically and transferred to the equilibrating chamber with the same gas for 7, 10 and 15 minutes at 37 C there was a fall in during the process of equilibration. This amounted to an average of units after 7 minutes, units after 10 minutes, and units after 15 minutes equilibration (table VIII). When tonometered blood samples were stored in capped syringes in ice for 2 hours and then equilibrated with the same gas, the fall in was more marked. In this group of samples the average fall in after 10 minutes equilibration was units in samples equilibrated immediately after tonometry and units in those samples stored in ice for 2 hours and then equilibrated. The average drop in after 2 hours storage in ice alone was again units (table IX). There was a slight diminution in total carbon dioxide content in the tonometered blood samples after equilibration with the same gas for 10 minutes. The mean reduction in total carbon TABLE X Total carbon dioxide content of blood after tonometry and after 10 minutes equilibration {measured by Van Slyke analysis). Duplicates agreed within 01 m.equiv/l. Total CO 2 content after tonometry (m.equiv/l.) Total CO 2 content after 10 minutes equilibration (m.equiv/l.) TABLE XI Total CO 2 content (m.equiv/l.) O Mean= m.equiv/l. SDO-6 Percentage haemolysis in post-perfusion pump blood and normal blood after equilibration with a gas mixture for 10 minutes. {Duplicate readings taken for every case.) Percentage haemolysis of post-perfusion pump blood after 10 minutes equilibration Mean=6-45 Percentage haemolysis of normal blood after 10 minutes equilibration Mean=0-9

10 DETERMINATION OF ARTERIAL CARBON DIOXIDE TENSION 347 TABLE XII Tonometered blood. Effect of using 1, 2 and 3 drops antifoam on and percentage haemolysis of blood equilibrated for 10 minutes. Posttonometer drop antifoam in = units % haemolysis haemolysis = drops antifoam in = units % haemolysis haemolysis = drops antifoam in= units % haemolysis haemolysis = 5-87 TABLE XIII Tonometered blood. Effect of 10 minutes equilibration {using slow, normal and fast rates of bubbling) on and percentage haemolysis. Posttonometer norma I bubbling in = % haemolysis haemolysis= 4-87 slow bubbling in = % haemolysis haemolysis= 3-67 fastiwbbling % haemolysis in = haemolysis= dioxide content was 0.4 m.equiv/1. (SD 0.6 m.equiv/1.) (table X). On haemolysis. The percentage haemolysis produced by 10 minutes equilibration of normal blood was small the mean in seven samples being 0.9 per cent. In blood that had been used for total body perfusion, the mean increase in haemolysis during the equilibration procedure was 6.45 per cent (table XI). The effect of adding 1, 2 or 3 drops of antifoam is shown in table XII. There was little either in the amount of haemolysis produced or in the degree of fall noted. However, as would be expected, a fast rate of bubbling greatly increased the percentage haemolysis and also the mean fall in (table XIII). In addition, prolonging the period of equilibration produced a slight increase in haemolysis (table XIV). Preliminary storage in ice for 1 hour produced a slight increase in haemolysis and fall when compared

11 348 BRITISH JOURNAL OF ANAESTHESIA TABLE XIV Tonometered blood samples. Effect of varying periods of equilibration on and percentage haemolysis. Control samples of blood (in brackets) were standing at 37 C for similar periods without equilibration. Posttonometer PH % haemolysis (7-220) (7-212) (7-359) (7-110) (7-144) (7-220) (7-069) (7-370) (7-409) (7-395) in = (0 013) (0-6) 2-3 (0-6) (3-8) (0-75) haemolysis= 4-87 (0-66) 20 min equilibration % haemolysis 7175 (7-218) (7-192) (7-330) (7-100) (7-135) 7162 (7-218) (7-059) (7-361) (7-393) (7-382) in = (0 025) TABLE XV Effect of 10 minutes equilibration (after storage at 0 Cfor 1 hour) on and percentage haemolysis. Posttonometer PH min equilibration PH % haemolysis Mean drop in = haemolysis =3-64 after 1 hour storage ato C % haemolysis Mean drop in = Mean% haemolysis = (0-7) 1-8 (0-4) (3-95) 1-8 (1-8) 1 (0-83) 4-5 (1-99) haemolysis= 5-27 (0-96) 30 min equilibration % haemolysis (7-217) (7-180) (7-310) (7-096) (7-124) 7150 (7-199) (7-045) (7-349) (7-388) (7-380) in = (0 030) (2-8) 2-2 (0-5) 8-2 (0 8) (6-4) 2-2 (0-55) 2-99 d-3) 50 (3-5) haemolysis= 6-92 (1-76) with control samples of the same blood which had been equilibrated immediately after tonometry (table XV). Comparison with Pco 2 determined by Pco 2 electrode. Despite the longer periods of equilibration used in these experiments, there was still a mean between tonometer gas and the Pco 2 determined by the interpolation technique of mm Hg (SD 0.89 mm Hg). The electrode comparisons of this series of samples revealed a mean of mm Hg (SD 1.04 mm Hg) (table XVI). In the five blood samples having a packed cell volume in excess of 55 per cent there was a mean of mm Hg between the Pco 2 determined by the electrode and the Pco 3 determined by the interpolation technique, using 10-minute periods of equilibration.

12 DETERMINATION OF ARTERIAL CARBON DIOXIDE TENSION 349 TABLE XVI Tonometered blood samples. Comparison ofpco^ determinations by electrode and interpolation techniques. Tonometer gas Pco 2 Electrode PcOj Electrode-gas Pco 2 Interpolation Pco 2 Interpolation-gas Pco j « ^ Mean= 0 59 mm Hg SD 104 Mean= 2 03 mm Hg SD 0-89 TABLE XVII Comparison ofpco 2 determinations on blood samples of high PCV using Pco 2 electrode and revised interpolation technique with 10 and 20 minute equilibrations. Electrode Pco ' Interpolation Pco 2 10 min equilib Difference 10 min equilib Mean= 5 13 mm Hg Interpolation Pco 2 20 min equilib Difference 20 min equilib Mean= 3 03 mm Hg When 20-minute periods of equilibration were used, the mean was still mm Hg (table XVII). Since it has already been shown that the electrode tends to underestimate Pco 2 when used with blood of high haematocrit value (table VII), it is likely that the error noted with the interpolation technique is even greater than these figures suggest. DISCUSSION In the last decade, rapid advances have been made in the design of electrode systems and meters. These have greatly facilitated the accurate measurement of. There are, however, many pitfalls for the inexperienced (Nunn, 1962) and the greatest care is necessary in order to avoid errors of technique. If blood can be determined to within units, the error in the estimation of Pa M2 from the Singer-Hastings nomogram should not exceed 2 mm Hg in the mm Hg range. (This assumes that the total carbon dioxide content of plasma bicarbonate can be determined to within 0.2 m.equiv/1. by chemical analysis.) If the interpolation technique is used the error should be no greater (Robinson and Utting, 1961). In addition, small errors in the assumed buffer values should make no to the determination of Pa OT2 as long as all three determinations are referred to the same buffer. However, it has been shown in this paper that there are two main

13 350 BRITISH JOURNAL OF ANAESTHESIA sources of error in the interpolation technique described. The first is a loss of base during the processes of storage and equilibration. This loss of base may be due to cellular metabolism or to the haemolysis of red cells. Cellular metabolism is not completely inhibited by sodium fluoride (Nunn, 1959) but is greatly inhibited by storage at 0 C (Siggaard Andersen, 1961). From the experiments reported in this paper, it would appear that the loss of base during storage and equilibration with the equilibration technique described would not amount to more than 0.4 m.equiv/1. This, in itself, is insufficient to account for the error in determination of Pa C02. The degree of haemolysis produced by the equilibration process in normal blood samples is small. In blood samples which have been taken from patients after total body perfusion, the degree of haemolysis produced is greater. There will, of course, be a raised plasma haemoglobin level resulting from the perfusion itself, but any lowering of from this cause will be applied to the initial reading as well as to of the equilibrated blood samples. J0rgensen and Astrup (1957) point out that the of plasma falls about 0.01 unit for each gramme of haemoglobin which has been liberated by haemolysis in 100 ml of plasma. It is apparent that the degree of haemolysis occurring under normal conditions of equilibration is insufficient to account for a marked reduction in the Pa c02. It would seem, therefore, that the second source of error, incomplete equilibration, is the more important. It has been shown that in normal blood samples equilibration is not complete until bubbling has been continued for at least 10 minutes, whilst in blood samples of a high packed cell volume equilibration may not be complete in 20 minutes. This may be related to the viscosity which increases markedly with high packed cell volumes (Reemtsma and Greech, 1962). If the initial Pa CO2 is high, and equilibrations are performed with low carbon dioxide and oxygen mixtures, equilibrium will be delayed even more. This probably explains why, in two cases of the Tetralogy of Fallot, both with haematocrit values above 60 per cent die between the Pa c02 determined by the Pco 2 electrode and that determined by the interpolation technique was over 20 mm Hg! For these reasons the interpolation technique described above has now been abandoned by the present authors in favour of a direct determination by the Pco 2 electrode. However, this too has been known to read inaccurately without any obvious sign of malfunction (Nunn, unpublished observations). In addition it has been shown that Pa c02 determinations by the electrode on blood of a high packed cell volume may be somewhat in error if the between blood and calibrating gas Pco 2 is too great. To guard against these possibilities, a small sample of the patient's blood, tonometered with a gas of known Pco 2, is always run with each batch of patient samples. In addition, a second electrode system is maintained to check the first. Experience over the past five years has convinced us that such precautions are vital if trustworthy results are to be obtained. CONCLUSIONS There are two probable sources of error in the determination of Pa 002 using the interpolation technique described in this paper. These are: (i) The! occurrence of a metabolic acidosis during the processes of storage and equilibration. This probably amounts to no more than 0.4 m.equiv/1. in normal circumstances and would result in an underestimate of Pa c02 of about 1 mm Hg in the normal range. (ii) Incomplete equilibration of the blood samples with the known carbon dioxide and oxygen mixture. This is particularly likely to occur if the blood has a high packed cell volume. It is concluded that the technique described may produce erroneous results in some circumstances. These criticisms are unlikely to apply to the micro-astrup technique where equilibration of a small quantity of blood is achieved with great rapidity. For the above reasons Pa c02 is now determined by the Pco 2 electrode and concurrent checks are made with tonometered blood samples. If further information concerning acid-base balance is required, and haematocrit or haemoglobin concentration are determined. The variables may then be plotted on a suitable nomogram to yield a graphic demonstration of the acid-base balance.

14 DETERMINATION OF ARTERIAL CARBON DIOXIDE TENSION 351 REFERENCES Astrup, P., and Schroder, S. (1956). Apparatus for anaerobic determination of the of the blood at 38 C. Scand. J. din. Lab. Invest., 8, 30. J0rgensen, K., Siggaard Andersen, O-, and Engel, K. (1960). Acid-base metabolism. Lancet, 1, Bates, R. G., and Acree, S. F. (1945). of aqueous mixtures of potassium dihydrogen phosphate and disodium hydrogen phosphate at 0 C to 60 C. /. Res. nat. Bur. Stand., 34, 373. J0rgensen, K., and Astrup, P. (1957). Standard bicarbonate, its clinical significance and a new method for its determination. Scand. J. din. Lab. Invest., 9, 122- Nunn, J. F. (1959). The accuracy of the measurement of blood Pco 2 by interpolation methods. In A Symposium of and Blood Gas Measurement (ed. Woolmer, R. F.), p. 60. London: Churchill. (1962). The undressing of. Lancet, 1, 803. Peters, J. P., and van Slyke, D. D. (1932). Quantitative Clinical Chemistry, Vol. II Methods, p London: Bailliere, Tyndall and Cox. Reemtsma, K., and Greech, O. (1962). Viscosity studies of blood plasma and plasma substitutes. J. thorac. cardiovasc. Surg., 44, 674. Robinson, J. S., and Utting, J. E. (1961). A simple interpolation method for the estimation of Pco 2 in whole blood. Brit. J. Anaesth., 33, 963. Semple, S. J. G., Mattock, G., and Uncles, R. (1962). A buffer standard for blood measurement. /. biol. Chem., 237, 963. Severinghaus, J. W., and Bradley, A. F. (1958). Electrodes for blood Po 2 and Pco 2 determination. /. appl. Physiol, 13, 515. Siggaard Andersen, O. (1961). Sampling and storing of blood for determination of acid-base status. Scand. J. din. Lab. Invest., 13, 196. Engel, K. (1960). A new acid-base nomogram. Scand. J. din. Lab. Invest., 12, 177. Singer, R. B., and Hastings, A. B. (1948). An improved clinical method for the estimation of disturbances of the acid-base balance of human blood. Medicine, 27, 223. SOURCES POSSIBLES D'ERREUR DANS LE DOSAGE DE LA TENSION DE DIOXYDE DE CARBONE PAR INTERPOLATION TECHNIQUE SOMMAIRE Deux sources d'erreurs existent dans le rosage du Paco 2 par interpolation, technique d'equilibrage par bulles. Ce sont: (1 ) L'acidose metabolique survenant pendant la mise en reserve et l'equilibrage. (2 ) L'etablissement incomplet d'equilibre des echantillons de sang par le melange CO 2 O 2 connu. Un e'tablissement incomplet d'equilibre se produit surtout lorsque les cellules du sang sont etroitement "empilees", dans ces circonstances une sous-estimation du Pco 2 jusqu' a 20 m/m Hg est possible. Les auteurs proposent l'electrode de Pco 2 comme plus precise et plus facilement maniable que la technique d'interpolation decrite. Toutefois l'^lectrode est elle aussi susceptible de provoquer des erreurs et on devrait verifier sa precision par tonome'trie a plusieurs reprises. MOGLICHE FEHLERQUELLEN BEI DER BESTIMMUNG DER ARTERIELLEN KOHLEN- DIOXYDSPANNUNG BEI EINER INTERPOLA- TIONSMETHODE ZUSAMMENFASSUNG Es gibt zwei Hauptfeblerquellen bei der Bestimmung des CO 2 -Partialdruckes durch eine Interpolationsmethode, die eine Blasenwaage benutzt. Diese sind: 1. Das Auftreten von metabolischer Azidose wahrend des Stehenlassens und der Einbalanzierung. 2. Unvollstandige Einbalanzierung der Blutproben auf die bekannte CO 2 O 2 Mischung. Unvollstandige Einbalanzierung tritt besonders leicht bei Blut mit hohem Zellvolumen auf, unter diesen Umstanden kann der CO 2 -Partialdruck bis um 20 mm/hg unterschatzt werden. Es wird behauptet, dafi die Bestimmung des CO 2 -Partialdruckes mittels einer Elektrode sowohl genauer als auch bequemer als die beschriebene Interpolationsmethode ist. Es konnen jedoch auch dabei Fehler auftreten, weshalb die Genauigkeit haufig durch Tonometrie uberpriift werden soil.