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Ann Clin Biochem 1988; 25: 228-232 An isotonic potassium chloride liquid junction minimises the effects of ionic strength, protein and haematocrit on ionised calcium measurement RBPAYNE From the Department of Chemical Pathology, St James's University Hospital, Leeds LS9 7TF, UK When the reference electrode liquid junction of a Nova 2 analyser was changed to isotonic potassium chloride, increasing the ionic strength of aqueous solutions containing a constant total calcium concentration had a negligible effect on measured ionised calcium. In contrast, measurements using hypertonic potassium chloride, hypertonic sodium formate and isotonic sodium chloride liquid junctions showed significant sample ionic strength effects. Interferences by sample protein concentration and haematocrit were marked with hypertonic, but negligible with isotonic junctions. Ionised calcium values in samples containing 25 mmoVL acetate, bicarbonate, ~-hydroxybutyrate, lactate or pyruvate were all lower by fr.7% with an isotonic than a hypertonic potassium chloride junction. Thus, anions that replace bicarbonate during metabolic acidosis have a similar effect on residual liquid junction potential. The clinical usefulness of an isotonic potassium chloride liquid junction needs to be evaluated. SUMMARY. The clinically significant positive interference with the measurement of ionised calcium by sample protein concentration I. 2 was suggested by others to be a Donnan effect.? However, subsequent experiments showed that it disappeared when the reference electrodes liquid junction was changed from 2·0 moVL KCI or 4·6 moUKg HCOONa to 150 mmoVL NaCI; in addition, measurements became significantly more reproducible." Isotonic NaCI bridges are not used in clinical analysers because their residual liquid junction potential varies with the ionic strength of the sample and is said to vary with its anionic composition. Preliminary experiments suggested that variation in ionic strength had little effect when the brid~e was 150 mmoVL KCI rather than NaCI. The purpose of the work described here was to determine the effects of sample ionic strength, protein concentration, haematocrit and anion composition on ionised calcium measured using a range of reference electrode liquid junctions. Materials and methods Bicarbonate-free solutions with high and ,w protein concentrations were prepared trom reconstituted Qualify I human control serum lot QN21A (American Monitor UK Ltd, Storrington, Sussex, UK) with a total protein concentration of 62 giL. It was diluted with a solution : containing 1·0 mmoVL CaCI2 and 150 mmoVL NaCI to achieve a low total protein concentration of 32 giL. An aliquot was then ultrafiltered" to achieve a high total protein concentration of 82 giL in the retentate. Protein concentration was measured on : Monitor Parallel analyser by a biuret method with an imprecision of 1·3% at 70 giL. A bicarbonate-free 'whole blood' preparation with a high packed cell volume and its 'plasma' were prepared by washing the cells from 10 ml of heparinised blood three times with a solution containing 1·0 mmoVL CaCl2 and 150 mmoVL NaCI and then resuspending them in reconstituted Qualify I control serum; after centrifugation, an aliquot of the 'plasma' was removed, leaving a 'whole blood' preparation with an haematocrit of 75%. Bicarbonate-containing aqueous solutions were equilibrated with humidified 5% CO 2 at 37°C immediately after preparation to avoid the formation of carbonate complexes." Ionised calcium concentrations were mea- 228 Downloaded from acb.sagepub.com at PENNSYLVANIA STATE UNIV on May 11, 2016 Isotonic liquid junctions sured with a Nova 2 ionised calcium analyser. The reference electrode liquid junction geometry was T-shaped with the manufacturer's reference solution of 2·0 moVL KCI flowing from the side into an upward-moving sample stream; there was no liquid movement during measurement. The liquid junction solution was changed by removing the reference solution straw from the fluids pack, inserting it in the new solution and purging the machine twice. It was then recalibrated after the displayed potential (mY) had stabilised. A 4·6 mol/Kg HCOONa salt-bridge solution was purchased from Radiometer (Copenhagen, Denmark). The Nova's internal standards were ISO mmoVL NaCI containing 1·0 and 2·0 mmoVLCaCI2 • All measurements were made in duplicate after a single calibration except when otherwise stated. Results were recorded to three decimal places and are reported as means to two places. Results 229 1'04 ~ 0 1'02 .s 1'00 J! 8 0'98 ]c 0·96 e ~ .2 1 j 0·94 0'92 0'90 , , , , , , 100 110 120 130 140 150 Sample Noel concentration (mmoVLI FIG. I. Ionised calcium values in aqueous solutions containing 1·0 mmol/L CaCI2 with NaCI concentrations ranging from )(K) to 150 mmol/L, measured using reference electrode liquid junctions of 2·0 mol/L KCI (.). ISO mmol/L KCI (0) and ISO mmol/L NaCI (e). The data points using 4·6 mol/Kg HCOONa were close to those for 2·0 mol/L KCI and are not shown. IMPRECISION Coefficients of variation calculated from duplicate measurements made between calibrations were less than 0·5% when aqueous solutions, protein solutions or whole blood were measured with reference electrode liquid junctions of either ISO mmoVL NaCI or KCI, and also when aqueous solutions were measured with the manufacturer's 2·0 moVL KCI junction. However, within-calibration imprecisions were significantlygreater (P«HH) when a 2·0 moVL KCI bridge was used to measure either protein solutions (CY 0,77%) or whole blood (CY O·84'ra). SAMPLE IONIC STRENGTH EFFECTS The ionised calcium concentrations of aqueous solutions containing 1·0 mmoVL CaCI2 and having NaCIconcentrations ranging from to() to ISO mmol/L were measured using four different reference electrode liquid junctions (Fig. I). With the standard 2·0 moVL KCI bridge the values fell from 1·03 to HlO mmoVL (r=-O·994, P<()·OO5); with a 4·6 mol/Kg I1COONa bridge the values fell from 1·()4 to I·(K) mmoVL (r=-()'978, P«H)()5); with a ISO mmoVLNaCI bridge the values rose from ()·91 mmoVL to 1·00 mmoVL (r=()·996, P«HK)()5); and with a 150 mmoVL KCI bridge the values did not change significantly from I·()() mmoVL (r=0·594, P=()·2). The ionised calcium concentrations of three aqueous solutions each containing I·() mmoVL CaCh and either 100, 150 or 185 mmoVLNaCI were measured using reference liquid junctions of 2·0 mmol/L, 1·5 mol/L, 1·0 mol/L, 500 mmoVLand 150 mmol/L KCI. The calcium values of the solution containing 100 mmoVL NaCI fell significantly from 1·04 to ()·99mmollL as the junction molarity was reduced from 2·0 mollL to ISO mmoVL (r=-0·964, P<O·OOI). The calcium values in the solution containing ISO mmoVL NaCI did not change significantly across the range of bridge compositions from 0·99 mmoVL (r=-0·108, P=0·77). In the solution containing 185 mmoVL NaCl, the calcium values rose significantly from 0·96 mmoVL with the 2·0 mollL KCI bridge to 0·99 mmoVL with the 150 mmollL bridge (r=0'933, P<O·OOI). Thus, values that ranged from 0·96 in a hyperosmolar solution to 1·04 mmollL in a hypo-osmolar solution when measured using the conventional liquid junction all became 0·99 mmollL when an isotonic KCI junction was used. PROTEIN CONCENTRATION EFFECTS The ionised calcium concentrations of the control serum preparations with total protein concentrations ef 32 and 82 giL were 1·16 and 1·2() mmol/L, respectively, when measured with the 2·() mollL KCI liquid junction. The values became closer as the molarities of the bridge solutions were reduced, until both became 1·05 mmoVL with the isotonic KCI junction (Fig. 2). Downloaded from acb.sagepub.com at PENNSYLVANIA STATE UNIV on May 11, 2016 230 Payne 1'25 1'25 ~e .se $. "0 e .! e 1'20 .2 .~ ..\! ..\! 0 u 1.§ 0 u 1015 l 1015 .~ c: .2 "0 ; 2 ~ 1·20 "0 ..2 I! :> 1010 1010 ~ 1·05 1·05 2'0 1'5 1·0 0'5 2·0 0'15 Liquid junction KCI (mol/L) 1·5 1·0 0·5 0'15 Liquid junction KCI (mol/L) FIG. 2. Ionised calcium values in a diluted bicarbonate-free control serum and its own retentatc after Ultrafiltration. measured using a range of liquid junction KCI molarities. FIG. 3. Ionised calcium values in a bicarbonate-free control serum with and without the addition of red blood cells to give a hacrnatocrit of 75%. measured using a range of liquid Junction KCI molarities. HAEMATOCRIT EFFECTS for each anion heing lowered to a similar extent. When measured with the conventional bridge. .the ionised calcium of the whole blood preparation with an haematocrit of 75'Yo was 1·23 mmoUL while that of its plasma was 1·19 mrnol/L, Both values were 1·05 mmoVL when measurements were made using an isotonic KCI junction (Fig. 3). EFFECTS OF ANIONS The ionised calcium concentrations of isotonic aqueous solutions containing sodium 150 mmol/L, calcium 1·67 mmoVL, chloride 127 mmol/L and either acetate, bicarbonate, ~ hydroxybutrate, lactate or pyruvate 25 mmol/L were determined with 2·0 mol/L and 150 mmol/L KCI liquid junctions (Table I). In each case the values were significantly lower with the ISO mmol/L bridge (P<O·OOI), values Discussion Hypertonic reference electrode liquid junctions have practical disadvantages for ion-selective electrode measurements in patients' samples. A clinically significant positive effect of protein concentration has heen confirmed hy several workers. I. 2. 7-'} The interference is more apparent with ionised calcium and sodium measurements than with pH because the reference ranges for calcium and sodium arc only about 1·5 mY wide compared with 5-6 mY for pH.'} It has been shown previously that this interference, which is greater with a 4·6 mol/Kg HCOONa than with a 2·0 mol/L KCI bridge, is abolished by the use of an isotonic NaCI bridge" I. Ionised calcium concentrations in aqueous solutions containing ISO mmol/L sodium. 1·67 mmol/L calcium, 128 mmoIlL chloride and 25 mmoIlL acetate. bicarbonate. ~-hydroxybutyrate. lactate or pyruvate measured with different reference electrode liquid junctions TABLE Mean ionised calcium (mmoIlL) 2·0 moIlL KCI 150 mmoIlL KCL Difference Acetate Bicarbonate Butyrate Lactate Pyruvate 1·57 1-47 6·4% 1·50 1-41 1·54 1·43 7·I'Yc, 1·27 1·19 6·3% 1·17 I· J() 6-()% 6-()'}t,. Downloaded from acb.sagepub.com at PENNSYLVANIA STATE UNIV on May 11, 2016 Isotonic liquid junctions 231 2. Summary of the relationships between reference electrode liquid junction composition and interference by hacmatocrit. protein and ionic strength TABLE Liquid junction Interference 4·6 mol/Kg HCOONa _-_..- .. Haematocrit Protein Ionic strength ++ + 2·() mol/L KCI ().IS mol/L NaCI + + + ++ ..' - _.. presumably because it abolishes the precipitation of protein (and of red celIs) that is known to occur at hypertonic junctions.9-13 An additional advantage of an isotonic junction is that the instability of readings is reduced," 12 an observation confirmed here. The use of an isotonic NaCI bridge for pH measurements to lessen irreproducibility due to protein precipitation was proposed as long ago as 1959;14. 15 it was noted that it would have lowered normal blood pH values by about 0·1 units. 16 The proposal was not adopted because when an isotonic NaCI bridge was used the junction potential became more susceptible to the ionic strength of the sample. 12 This observation has been confirmed in the present work, but it was also found that even when 2·0 mol/L KCI or 4·6 moVKg HCOONa junctions were used the measured values changed with sample ionic strength (Fig. 1). In contrast, with isotonic KCI there was no measurable effect of changing ionic strength. It might be argued that the falling values with the hypertonic junctions are a reflection of the decreasing 'activity' of the calcium ion as ionic strength increases. If that were the case, the absence of the effect with isotonic KCI must be due to a fortuitous equivalence between faIling ionic activity and increasing residual liquid junction potential. With the hypertonic junction, the effect of ionic strength extended across the range of clinicalIy encountered sodium concentrations, from IOO to 180 mmol/L, and there were measurable differences in apparent ionised calcium at alI KCI junction molarities tested until a concentration of ISO mmoVL was reached. The present work has shown that interference by sample protein concentration is negligible with an isotonic KCI junction (just as it is with an isotonic NaCI junction") and that the haematocrit effect is also negligible (Fig. 2). The advantages of this junction are summarised in Table 2. 0·15 mol/L KCI The major problem with the adoption of an isotonic KCI junction for clinical use is that sample anions such as bicarbonate that diffuse more slowly than chloride alter the residual liquid junction potential, and therefore the measured values, and this effect is more marked the more dilute the liquid junction.'?' III However, the magnitude of the observed effect was similar for bicarbonate and for each of the organic anions tested (Table 1), so the effect may prove to be acceptable in practice because anions that replace bicarbonate during metabolic acidosis will have a similar effect on residual liquid junction potential. The clinical usefulness of an isotonic KCI junction needs to be evaluated to determine whether the disadvantage of any residual effect of anion composition is outweighed by the advantages of decreased imprecision, insensitivity to change in sample ionic strength and negligible interference by protein concentration and haematocrit. References 1 Payne RB. ClinieaIly significant effect of protein concentration on ion-selective electrode measurements of ionised calcium. Ann Clin Biochem 1982; 19: 233-7. 2 Butler SJ. Payne RB, Gunn IR et al. Correlation between serum ionised calcium and serum albumin in two hospital populations. Br Med J 1984; 289: 948-50. 3 Thode J. Fogh-Andersen N, Sigaard-Andersen O. Donnan effect or protein interference in ionised calcium measurements'! Ann Clin Biochem 1983; 20: 271-3. 4 Payne RB, Jones DP. Protein interferes with ionised calcium measurement at the reference electrode liquid junction. Ann Clin Biochem 1987; 24: 400-7. 5 Payne RB. Isotonic liquid junctions minimise protein interference with ionized calcium measurements. In: Mass AHJ. Buckley B, Marsoner H et al. cds. Methodology and Clinical Applications of Ion-Selective Electrodes. Proceedings of the 8th meeting of the wee European Downloaded from acb.sagepub.com at PENNSYLVANIA STATE UNIV on May 11, 2016 232 Payne Working Group on Ion-Selective Electrodes 1986 (ISBN 90-9001722-4). Copenhagen: IFCC Technical Secretariat, 1987; 287-8. 6 Boink FBTJ, Bijster P, Vink KI.J, Maas AHJ. Direct potentiometric determination of sodium ion in blood. III. Influence of (bi)carbonate. Clin Chem 1985; 31: 523-6. 7 Buckley BM, Smith SCH, Hcath DA, Bold AM. Clinical studies on ionized calcium using the Radiometer ICAI analyzer. Scan J Clin Lab Invest 1983; 43 (Suppl 165): 87-92. 8 Freaney R, Egan T, McKcnna MJ et al. Determination of ionised calcium is not independent of albumin concentration. Clin Chim Acta 1986; 158: 129-38. 9 Pedersen KO. The effect of protein concentration on potentiometric determination of ions in serum and blood. In: Maas AHJ, Boink FBTJ, Saris NEL et al., eds. Methodology and Clinical Applications of Ion-Selective Electrodes. Proceedings of the 7th meeting of the IFCC European Working Group on Ion-Selective Electrodes 1985 (ISBN 87-88138-10-0). Copenhagen: Radiometer, 1986; 77-98. 10 Siggaard-Anderscn O. Factors affecting thc liquid-junction potential in c1ectrometric blood pH measurement. Scan J Clin Lab Invest 1961; 13: 205-11. 11 Maas AHJ. pH determination in body Ilnids using a cell with an isotonic sodium chloride bridge. J Appl Physiol 1971; 30: 248-50. 12 Salling N, Siggaard-Andersen O. Liquid-junction potentials between plasma or erythrolysatc and KCI solutions. Scand J Clin Lab Invest 1971; 28: 33-40. 13 Siggaard-Anderscn O. Fogh-Anderscn N. Thode J. Christiansen TF. Elimination of the erythrocyte effect on the liquid junction potential in potentiometric measurements on whole blood using unconventional salt bridge solutions. c.g. sodium formate. In: Maas AHJ. Kolstad J. SiggaardAndersen 0, Kokholm G. cds. Ionized Calcium, Sodium and Potassium by Ion-selective Electrodes. Proceedings of the 5th meeting of the IFCC European Working Group on Ion-Selective Elcctrodes (ISBN 87-88 I38-()4-6). Copenhagen: Private Press. 1984; 149-51. 14 Wright MP. pH measurements with glass electrode. In: Woolmer RF, cd. A Symposium on pH and Blood Gas Measurements. London: Churchill, 1959; 5-15. 15 Mattock G. Electrochemical aspects of blood pH measurements. In: Wool mer RF, cd. A Symposium on pH and Blood Gas Measurements. London: Churchill, 1959; 19-29. 16 Semplc SGH. Observed pH differences of blood and plasma with different bridge solutions. J Appl Physiol 1961; 16: 576-7. 17 Czaban JD. Cormier AD, Lcgg KD. Establishing the direct-potentiometric 'normal' range for NalK: residual liquid junction potential and activity coefficient effects. Clin Chem 1982; 28: 1936-45. 18 Winkelman JW. Merritt C, Scott WJ et al. Effects of residual liquid junction potential in direct potentiometry of potassium. Clin Chem 1984; 30: 482-4. Accepted for publication 27 November 1987 Downloaded from acb.sagepub.com at PENNSYLVANIA STATE UNIV on May 11, 2016