Ionized calcium in serum exposed to air

Robin Mirdell; Ganan Solika

doi:10.1515/cclm-2025-1183

Article Open Access

Ionized calcium in serum exposed to air

and

Published/Copyright: October 9, 2025

Published by

Become an author with De Gruyter Brill

Submit Manuscript Author Information Explore this Subject

From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 64 Issue 2

Keywords: ionized calcium; air exposure; serum; ABL

To the Editor,

One of the preanalytical errors that has received a lot of attention when it comes to ionized calcium (iCa²⁺) is air exposure. When exposing a sample to air, the partial pressure of CO₂ will decrease [1]. This will cause the pH to increase, which increases albumin’s affinity for iCa²⁺, decreasing the concentration of iCa²⁺ [2]. As the partial pressure of CO₂ decreases in the blood, carbonic anhydrase will convert HC0₃⁻ + H⁺ into CO₂ and H₂O, allowing even more CO₂ to escape and further increasing the pH of the sample [1]. Air exposure can potentially occur for two different reasons: air already present in the collection tube and when uncapping the sample. For this reason, the draw volume has to be adequate to ensure the intended ratio of blood to air in the sample from the start.

With advances within the field of robotics, it has become easier to half-automate analysis on instruments that are intended for human users. These robots are often not equipped with critical thinking and may sometimes fail to analyze individual samples. This means that certain samples may have been uncapped and then recapped without obtaining a result. If this issue is identified promptly, can the samples still be analyzed?

The aim of this study was therefore to investigate how the measured level of iCa²⁺ in serum samples is affected by air exposure near the time of analysis.

Specimens were selected from routine iCa²⁺ samples sent to Linköping University Hospital between the dates: 2024-01-29 and 2024-01-31. Sixty serum specimens were included in the study. Serum samples were collected in vacutainer tubes with a silica coagulation activator and without a gel separator, BD Vacutainer CAT 5/4 mL (Becton Dickinson, Franklin Lakes, NJ, USA). To qualify for inclusion, the sample first had to meet the standard local criteria for measurement of iCa²⁺. These criteria include the following: well-filled vial, never uncapped, and stored at 2–8 °C unless measurement can be done within 4 h from sampling. Additionally, visually hemolyzed samples (fHb ≳0.5 g/L) were avoided. All samples were centrifuged for 10 min at 2500×g.

After patient results had been reported and the specimen was destined for disposal, each sample was anonymized before any additional measurements were made. The study was conducted in accordance with the revised declaration of Helsinki from 2013. According to the guidelines of the regional ethics review board, no ethical permit is required for research on discarded blood samples where no traceable personal data remains.

Ionized calcium was measured with the ABL800 Flex instrument (Radiometer, Copenhagen, Denmark). The same instrument was used for all measurements. In addition to iCa²⁺, the pH of the sample was also determined during each measurement. QC-measurements on the instrument during January and February 2024 showed a CV for iCa²⁺ <0.5 %.

All statistical analysis was done with the aid of Excel (Microsoft, Redmond, WA, USA) and SPSS (IBM, Armonk, NY, USA). Normal distribution of data was investigated using the Kolmogorov-Smirnov test. ANOVA and Dunnett’s test for multiple comparisons was used to test if differences over time were significant. The correlation of a change in pH and a change in iCa²⁺ was determined using Pearson’s correlation coefficient. An alpha value of 0.05 was regarded as significant.

The median time to analysis was 4.46 h (range: 0.92–23.60 h) and the average initial value of iCa²⁺ in all samples was 1.23 mmol/L (range: 1.01–1.40 mmol/L). All iCa²⁺, pH-values, and differences compared to original values were normally distributed. Initial iCa²⁺ values and the difference to the new measurement can be seen in Table 1.

Table 1:

Average change of ionized calcium (iCa²⁺) in serum samples without separator gel exposed to air.

Air exposure and, time to new measurement	Initial iCa²⁺, mmol/L	New measurement	Average iCa²⁺ change	Range of discrepancy	n
1 min, after 1 h	1.22 (±0.06)	1.20 (±0.06)	−1.5 % (±0.8 %)^a	−0.03 to +0.01	20
1 min, after 4 h	1.23 (±0.07)	1.21 (±0.07)	−1.8 % (±1.2 %)^a	−0.05 to +0.01	20
Continuously, 15 min	1.24 (±0.08)	1.21 (±0.08)	−2.2 % (±0.8 %)^a	−0.04 to −0.01	20
Continuously, 30 min	1.24 (±0.08)	1.18 (±0.08)	−4.4 % (±1.4 %)^a	−0.10 to −0.03	20
Continuously, 60 min	1.24 (±0.08)	1.16 (±0.08)	−6.2 % (±2.2 %)^a	−0.13 to −0.03	20
Continuously, 120 min	1.27 (±0.07)	1.12 (±0.06)	−11.3 % (±2.0 %)^a	−0.20 to −0.09	12
Continuously, 180 min	1.27 (±0.07)	1.06 (±0.07)	−16.0 % (±2.6 %)^a	−0.25 to −0.14	12
Continuously, 240 min	1.27 (±0.07)	1.03 (±0.06)	−18.4 % (±2.7 %)^a	−0.28 to −0.16	12

All values are expressed in mmol/L. A total of 60 samples were used for the measurements. For 40 of these, air exposure lasted for 1 min and a new measurement was done after 1 or 4 h. The remaining samples were continuously exposed to air and additional measurements were done up to 4 h after the initial measurement. The iCa²⁺ values are expressed as mean (±SD). The range of individual discrepancies from the initial iCa²⁺ is also shown. ^aSignificantly different compared to the initial iCa²⁺ value (p<0.0001).

In addition to iCa²⁺, pH was also measured. For the recapped samples, the initial pH was 7.35 (SD: ±0.04) and it significantly (p<0.0001) increased to 7.40 (SD: ±0.04) after 1 h. For the samples that were reanalyzed after 4 h, the initial pH was 7.37 (SD: ±0.03), which then increased significantly (p<0.0001) to 7.43 (SD: ±0.03). In comparison, the samples that were never capped again showed a dramatic (p<0.0001) increase in pH from 7.35 (SD: ±0.06) to 7.82 (SD: ±0.07) after 4 h of continuous exposure to air. Change in pH and change in iCa²⁺ were inversely correlated (Figure 1) and showed a strong relationship with an R²-value of 0.89.

Figure 1:

Correlation using Pearson’s correlation coefficient between change in pH and change in ionized calcium (iCa²⁺) in 20 serum samples without separator gel continuously exposed to air. Change in iCa²⁺ from the initial measurement is shown on the y-axis and the change in pH on the x-axis. The same 20 samples were measured again three times during the first 60 min, and 12 of these samples had additional measurements done at 120, 180, and 240 min, generating a total of 96 data points. A strong correlation could be seen between the change in pH and iCa²⁺.

Our findings are similar to what has been previously reported [3], [4], [5], [6], [7]. There however seems to be an entrenched belief that air exposure may have a significant effect on iCa²⁺. Several studies have indeed shown large effects on the measured level of iCa²⁺ when pH is altered by artificial means such as adding acid or base, or by controlling the concentration of CO₂ [3], [4], [5], [6]. However, these studies never actually investigated how fast pH change occurs when serum, plasma, or whole blood is exposed to air. Nonetheless, these results have been used as the rationale behind the perceived lability of iCa²⁺ when exposed to air, prompting many laboratories to discard samples if not analyzed right away after uncapping.

The findings in this study suggest that a brief duration of air exposure has a very limited effect on pH and in turn the level of iCa²⁺. This however only applies to well-filled tubes, as incompletely filled samples may be more sensitive to air exposure. Our data also suggest that there is a slight delay before the pH change from the air exposure occurs. This likely explains why the pH increase in samples exposed to air for only 1 min and measured after 1 or 4 h was larger, 0.06 pH, than for samples exposed to air for 15 min and measured directly, 0.03 pH (p<0.0001).

There are several limitations in this study. Convenience sampling was used and as such, there was no true randomization of the samples selected for inclusion. Another limitation is that we did not investigate the effect of air exposure on plasma and whole blood samples or if serum vials with gel separators are less sensitive to air exposure than those without. There was also no clinical information available, which means that we do not know anything about any underlying conditions or how the small differences identified might have affected clinical interpretation of the value.

While it is important to minimize errors in sampling handling, it is also important to minimize the number of samples that are needlessly discarded. Although our findings add to the knowledge of this subject, there is still a further need to investigate the effects of air exposure in serum and plasma, with and without gel separator, and in whole blood. Additional knowledge could help facilitate automated solutions for iCa²⁺ without the need for cap penetration. An acceptable solution, for example, could be to uncap serum samples a few minutes before analysis in an ISE-module with a calcium ion selective electrode. The limited air exposure in such a scenario would only have a minimal impact on the measured level of iCa²⁺.

Air exposure for 1 min causes minimal changes in both iCa²⁺ (−1.6 %) and pH (from 7.35 to 7.40) if a new measurement is done within 4 h. Continuous exposure to air for 15 min followed by immediate measurement showed a similar decrease in levels of iCa²⁺ (2.2 %) and an increase in pH (from 7.35 to 7.38). Based on these findings, we recommend that serum samples should not be discarded when exposed to air for a duration of <15 min.

Corresponding author: Robin Mirdell, PhD, MD, Department of Biomedical and Clinical Sciences, Linköping University, 58185, Linköping, Sweden; and Clinical Department of Clinical Chemistry, Region Östergötland, Linköping, Sweden, E-mail: robin.mirdell@regionostergotland.se

Acknowledgments

We would like to extend our gratitude to Mohamed Elsayed for making all the measurements included in this study. We could not have done it without you.

Research ethics: Not applicable.
Informed consent: Not applicable.
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Use of Large Language Models, AI and Machine Learning Tools: None declared.
Conflict of interest: The authors state no conflict of interest.
Research funding: None declared.
Data availability: Data available in Supplementary Materials.

References

1. Boron, WF. Evaluating the role of carbonic anhydrases in the transport of HCO3--related species. Biochim Biophys Acta 2010;1804:410–21. https://doi.org/10.1016/j.bbapap.2009.10.021.Search in Google Scholar PubMed PubMed Central

2. Baird, GS. Ionized calcium. Clin Chim Acta 2011;412:696–701. https://doi.org/10.1016/j.cca.2011.01.004.Search in Google Scholar PubMed

3. Fogh-Andersen, N. Ionized calcium analyzer with a built-in pH correction. Clin Chem 1981;27:1264–7. https://doi.org/10.1093/clinchem/27.7.1264.Search in Google Scholar

4. Wybenga, DR, Ibbott, FA, Cannon, DC. Determination of ionized calcium in serum that has been exposed to air. Clin Chem 1976;22:1009–11. https://doi.org/10.1093/clinchem/22.7.1009.Search in Google Scholar

5. Wang, S, McDonnell, EH, Sedor, FA, Toffaletti, JG. pH effects on measurements of ionized calcium and ionized magnesium in blood. Arch Pathol Lab Med 2002;126:947–50. https://doi.org/10.5858/2002-126-0947-peomoi.Search in Google Scholar PubMed

6. Moore, EW. Ionized calcium in normal serum, ultrafiltrates, and whole blood determined by ion-exchange electrodes. J Clin Investig 1970;49:318–34. https://doi.org/10.1172/jci106241.Search in Google Scholar

7. Kirschbaum, B. Loss of carbon dioxide from serum samples exposed to air. Effect on blood gas parameters and strong ions. Clin Chim Acta 2003;334:241–4. https://doi.org/10.1016/s0009-8981(03)00242-0.Search in Google Scholar PubMed

Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/cclm-2025-1183).

Received: 2025-09-04

Accepted: 2025-09-30

Published Online: 2025-10-09

Published in Print: 2026-01-29

This work is licensed under the Creative Commons Attribution 4.0 International License.

Supplementary Material

Articles in the same Issue

https://doi.org/10.1515/cclm-2025-1183

Keywords for this article

ionized calcium; air exposure; serum; ABL

Creative Commons

BY 4.0