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Bias of ionIzed CalcIum Results

from Blood

Gas Syringes

To the Editor:
The advent of whole-blood analyzers that measure blood gases and other an-

alytes has presented us with the problem of finding a blood-collection system that provides an acceptable sample for all analytes the tobe assayed. syringe A that works well for blood gases is not necessarily adequate for ionized calcium. Although it is generally known that heparin binds calcium and has the potential to interfere with the measurement of ionized calcium, little information is available on evaluation of different heparin-containing blood gas syringes for use in collecting samples

used in this study), both from Marquest MedicalProducts(Englewood, CO); and Pro-Vent (30 11.3 heparin, no. 4616) of and Pro-Vent, Low Heparin (15 113of heparin, no. 4318), both from Concord Portex, Keene, NH. The procedure for the study was in accordance with the ethical standards of our institutional Committee on Human Research. The approach was similar to that reported by Toffaletti et al. (1). Venous blood from each of 40 healthy subjects was drawn into a 12-mL syringe without any anticoagulant. A three-way plastic stopcock was then attached to the syringe so that 0.5 mL of blood was
added to each syringe and 3 mL to a Vacutainer Tube. Each container’s contents were mixed to distribute the hep-

Micro-ABG is probably the only one with clinically significant bias. Two syringes had statistically significant bias of the potassium results, but this was probably not clinically important. We speculate that the differences in sodium and potassium results may be

due to the use of different


counterions in the syringes; the countenon was specified only for the Mar-

quest products.

for measuring ionized calcium. An electrolyte-balanced heparinized (i.e., heparm-containing) syringehas been found
(1), but the syringe marketed with this anticoagulant was not acceptable to our hospital committee,

for reasons unrelated to the nature of the anticoagulant. Therefore, we tested for their effect on electrolyte results, especially ionized calcium, four 1-mL arterial blood-collecting syringes and a Vacutainer Tube (no. 6388, 3 mL, containing 45 11.3 f heparin; Becton Dicko inson,Rutherford, NJ). The syringes were Gas Lyte (2.811.3 Li heparin, no. of P9023RH) and Micro-ABG (5011.3 Li of heparin in the syringe plus 50111in the needle, no. P9023; the needle was not

arm uniformly. Blood gases and electrolytes were measured first in the sample without anticoagulant. The other syringe samples were then measured in random order. All assays were performed on the same Nova Stat Profile 5 blood gas analyzer (Nova Biomedical, Waltham, MA). We analyzed the results by one-way analysis of variance (ANOVA), using 0.05 as the level of statistical significance relative to the sample drawn without anticoagulant. Table 1 shows the mean, SD, and
statistical significance

for each group

of results. Only the Vacutainer Tube showed a bias in pH. Three containers showed statistically significant bias in the sodium results,ut the Marquest b

Table 1. Electrolyte measurements in whole blood collected different in blood gas syrInges.
Gas Lyle BD Hepauin Vacutalner

Concord Pro-Vent Low

Pro-Vent 60

Heparin, IU/mL blood




Heparin 30

Marquest Mlcro-ABG



Na, mmol/L Mean

7.363 0.026







1.7 4.33 0.26

141.68 1.6

The Marquest Lyte syringe was the only one that did not show bias of ionized calcium values relative to the blood drawn without anticoagulant. The other three syringes all had statistically significant bias, with mean ionized calcium values 0.05 mmo]JL lower than that in the unheparinized blood. This amount of bias is nearly one-half of the reference interval and must thus be considered clinically significant. The SD values of two of the groups were somewhat larger than the others. A variance homogeneity test showed that the differences were statistically insignificant at the 0.01 level; therefore, this factor does not invalidate the statistical significance of the ANOVA result. The calcium bias in the Vacutainer Tube sample is the least of the group that was statistically significant by ANOVA, the mean difference being 0.03 mmol/L. Thus even the low heparin concentration of 15 IU/mL in the Vacutainer Tube caused a statistically significant bias. This finding is consistent with the recommendations in the NCCLS Document C31-P (2), which was published after this Letter was submitted. That document recommends use of sodium or lithium heparin concentrations <10 IU/mL. The heparin concentrations of the blood samples shown in Table 1 were calculated from the heparin content of the syringes as provided by the manufacturers. Evidently, the bias of the ionized calcium results increases with
the heparin concentration. The syringe in this study with the greatest heparin content, the Marquest MicroABG, had the largest bias, and the syringe with least heparin, Marquest Gas Lyte, had no significant bias. The volume of blood drawn into the sy3). Syringes

SD K, mmol/L

4.31 0.28

4.29 0.27

4.268 0.27

4.31 0.25

Ionized Ca,








1.218c 0.021

1.1898 0.029



1.lola 0.053

Statistically significant byANOVA at 8P <0.001,

P = 0.006,



40 each. Becton BO, Dickinson.

ringe is also an important variable (1, that are filled only onehalf or one-quarter full will have blood heparin concentrations two- and fourfold, respectively, the concentration achieved when the syringe is filled (3).

CLINICAL CHEMISTRY, Vol. 40, No. 4, 1994

4. ad. Am J Clin Pathol 1979. participates in surveys. 2.Scand J Clin Lab Sci 1988. Haeckel R. Hyltoft Clin Biochem 1991. whereas biology is universal and thus should then be applicable over both locale and time. 3. as criteria for the acceptability of new analytical systems. References 1.5 SDb1OI ill w increase the rate by 194%. This strategy for bias is firmly based on the recommendations of the International Federation of Clinical Chemistry (8) regarding generation of reference values. a bias of +0.39:1514-8. in contrast to North American proficiency-testing schemes. 4. and in external quality-assessment schemes (which.30:811-7. In contrast. References 1. Sachs 5. laboratorians must be informed about the problem and must be involved in evaluating syringes for bias before the syringes are put into use in their hospitals. the model of Klee and our approach give identical results. It would be advantageous for the two regions to work together in the difficult task of generating and applying useful quality specifications for imprecision and bias. and employs welltrained laboratory technologists. However. rather than 2. Ann clinical strategies are often local and are modified over time. and handling proposed guideline. 48:757-64. 670 Vol. Harder M.1 SDbOI will increase the falsepositive rate by 26. Gowans EMS. Abrams B. 7.72:374-82. Approved recommendation (1987) on the theory of reference values. In Europe. Dry electrolyte-balanced heparinized syringes evaluated for determining ionized calcium and other electrolytes in whole blood. Solberg HE.Ionized calciumdeterminations: precollection variables. The European approach. When analytical imprecision is present. Assessing quality reM. Hyltoft Petersen P. Statistical treatment of collected Clin Biochem Petersen P. we must consider whether this assumption is relevant for clinical decision-making in situations when increased values are interpreted very differently from decreased values. Hunt P. based on biology. 1993. Part 5.medical staff. To prevent this error. Oregon Health Sci. No.95:161-309. Villanova. CLINICAL CHEMISTRY. we had no problems with sample clotting. Formally. 8.In this study the syringes were filled half full because this is the volume we realistically expect to receive. Blaabjerg 0. Kindermans C.g. Chaneac M. Analyticalgoals for the acceptance of common reference intervals laboratories throughout a geefor graphical area. Although the manufacturer may intend a syringe to be used only for blood gases. Statistical principles underlying analytic goal-setting in clinical chemistry. this can be achieved when the bias is less than or equal to onequarter of the group biological variation (4. and not with the total population outside the reference interval. Clin Chem 1991. and the widely used approach of Harris (7)that imprecision should be less than or equal to one-half of the biological variation-has been adopted. This study shows that the use of some commercially available syringes for collecting arterial blood will produce significant errors in ionized calcium results. Fraser CG. RicosC. 2. Gowans EMS. deVerdier C-H. the quality specification is that reference intervals should be transferrable over geography. Clin Chem 1993. Tolerance limits for shortterm analyticalias and analytical impreb cision derived from clinicalssay specifica ity. when bias is present. 3). NCCLS Document C31-P. 1994 . It is very difficult to combine approaches to the setting of quality specifications on clinical considerations and on biological bases. specimen choice. 1980. of Pathol. For example. National Committee for Clinical Laboratoiy Standards. Medical need for quality specifications in laboratorymedicine. Pk NCCLS. Upsala J Med Sci 1990. Ernst P. Proposed quality specifications for the imprecision and inaccuracy of analytical systems for clinical chemistry.and purchasing agents. Falch-Christiansen C. Helsinki: Finnish Govt. there appear to be many reasons to promote cooperation between North America and Europe.28: J Robert Swanson1 Carol Heeter Mark Limbocker Maureen Sullivan Dept. has different strategies for setting quality specifications for imprecision and bias. practices good instrument maintenance. similarly. Dechaux M. For bias. and appears to approximate to the percentage increase in the false-positive rates caused by both types of error. are educational and supportive rather than regulatory). In such situations the increased falsepositive rate caused by positive bias should be compared with the percentage of individuals whose results exceed the upper reference limit in the error-free situation. quirements in clinical chemistry.4% as suggested by Klee (1). collection. This is an error that can exist even though the laboratory has good daily quality control. however. Printing Center.37:1730-3. Rabouine F. Harris EK. Klee GG. when the model is applied to reference intervals that can be described as gaussian. This European perspective was presented at the Forum (9) and. Klee compares the increase in falsepositive rate due to increases in imprecision and bias with the two-tailed false-positive rate of the distribution in healthy individuals.5%. when the devices contain anticoagulant in solution) and unacceptably high concen- trations of anticoagulant ions (such as Na and K). 3. a bias of +0. considering the many efforts presented at that meeting. 167-73. this model appears to have merits. 1 or associated Author for correspondence. Portland. Univ. and thus links current European thinking to international guidelines. Imprecision should not increase the within-subject biological variation significantly. Eur J Clin Chem 6.” This is calculated as the sum of the squares of the imprecision and bias errors. the results are very different. including assessment of new analytical methods.8 IU of heparin per syringe. H#{248}rder ed. despite recommendations for full syringes. 5. Preanalytical errors in ionized calcium measurements induced by the use of liquid heparin. Hyltoft Petersen P. Blaabjerg 0. Herder M. Toffaletti J. OR 97201 Editor’s note: An experienced reviewer reminds us that blood-collection devices generally should be filled to their nominal capacities to avoid dilutional errors (e. 1992. and several examples have been described in detail in two Nordic publications (2. Analytical goals for the estimation of non-gaussian reference intervals. The main rationale for this use of two rather different but complementary approaches is that rim concentrations increase the risk of sample clotting. not 60. Clinical approaches are best applied in specific medical situations. this may be a subtlety that will escape laboratory staff. strategies based on biology (4-6) are best suited for laboratory management. and because no larger volume is required for assay.49:727-37. Scand J Clin Lab Invest 1989. Although low hepa- Analytical Quality Specifications in Clinical Chemistry To the Editor: Among the many interesting publications emanating from the 1992 Clinical Chemistry Forum (Accuracy and Precision Goals in Clinical Chemistry: Can They Be Defined by Medical Relevance?) is the proposal by Klee (1) for an “error budget. 5). even in the Mar- quest Gas Lyte syringe with 2. both strategies are applied-but in different situations. 40.0% by our proposal..