The Impact of Water on a Calcium Assay

By: Stephane Mabic, Application Manager and Worldwide Training Manager, Millipore S.A.S, Lab Water, Saint-Quentin-en-Yvelines, France


This paper discusses the impact of water on the Arsenazo calcium assay and the fact that water purity may influence the accuracy of assay results. Bacteria appear as the most common source of problems for the calcium assay, due to the release of proteins and small organic acids that can bind to calcium. Two other sources of interferences, namely the effects of calcium leakage and large organic acids on the calcium concentration, may be due to water quality when ion-exchange resins alone (service deionization - SDI) are used to purify water. Solutions for reducing bacterial counts in clinical analyzers and water purification systems that use combined technologies are briefly discussed.


The Arsenazo calcium assay is a very standard and fundamental parameter in the biochemical panel of blood and serum analysis. This assay, used on clinical analyzers proposed by several manufacturers, is sensitive to the conditions of the reagents and the analyzer. In particular, deviations from quality control (QC) standards and calibration curves, high blanks, and shifts of mean patient values are recurrent issues.

Effect of calcium leakage on calcium concentration

When water feeding the clinical analyzer is purified by deionization tanks (so-called “service deionization”, or SDI), there can be a drop in the ionic purity of the water. Indeed, ion-exchange resins used in SDI equipment have a defined and limited capacity. When these resins are saturated, they cannot retain ions anymore, and the ions present in the feed water can leach into the “purified water”. This situation, although temporary until the exchange of saturated resins for new ones, can lead to a severe increase of calcium concentration in the water delivered to the analyzer and utilized in the assay, resulting in interferences.

Effect of large organic acids on calcium concentration

It is well known that calcium can bind readily to organic acids and proteins (via their amines and carboxylic functions). One of the drawbacks of the ion-exchange resins is their limited capacity to retain organic acids, in particular the humic and fulvic acids often present in tap water, which are a result of the degradation of leaves. These large acidic and phenolic molecules bind the calcium dosed in the serum and can modify its concentration, leading to erroneous values.

Therefore, the two issues discussed above can arise when simple SDI water is used to feed clinical analyzers:

  • Leakage of calcium, which will interfere with the blood calcium concentration
  • Presence of large organic acids that can modify the concentration of blood calcium by binding

Issues may still arise, however, where other types of water purification processes are used. More sophisticated water purification processes include a combination of reverse osmosis (RO), and electrodeionization (EDI), usually supported by a small amount of virgin ion-exchange resin. The combination of RO-EDI ensures a constant water quality, without the drop in ionic purity of the water that occurs when SDI water is used. In addition, RO is very efficient at removing large organic acids (humic and fulvic acids), reducing the binding of calcium by these molecules. The resins used ensure very high purity of the water, and the purification systems are equipped with alerts that recommend exchanging the resins before the drop in ionic purity.

Role of bacteria as a contaminant

In those purification units using combined technologies, the ionic or organic purity of the water is not the root cause of the issues with the calcium assay; therefore, another type of contaminant must be considered: bacteria.1,2

Bacteria have the ability to grow and develop very rapidly under the right conditions. These conditions are typically found in clinical analyzers: heat (30-40°C inside the analyzer), nutrients (reagents) and plenty of places to grow (valves, needles, tubing, pumps, etc.). Bacterial counts up to 105 to 106 CFU/mL are not rare on the reagent needles and sample probes. At such high levels, the proteins and the small organic acids (i.e., oxalic, citric) released by the bacteria can bind to calcium and modify the concentration dosed.

It is noteworthy that in many cases the solution to calcium assay problems is a sanitization or decontamination of the analyzer, which fully supports the role of bacteria in calcium assay errors. The problems due to bacteria may also arise when SDI source water is used.

It is important to ensure a low bacterial count at the outlet of the purification unit by selecting the correct purified water solution. This solution, however, is not sufficient, and it is critical to avoid contamination of the analyzer by checking the microbiological levels in the analyzer on a regular basis. Most of the time, the source of the issue is the on-board reservoir that has not been drained and cleaned often enough, thus leading to biofilm formation.

Calcium complex with oxalate

Calcium complex with oxalate

Summary of common sources of interferences with the Arsenazo calcium assay

Summary of common sources of interferences with the Arsenazo calcium assay

SDI: service deionization, RO: reverse osmosis,
EDI: electrodeionization, DI: deionization

In order to support the selection of the optimum solution for water in the clinical laboratory, the Clinical and Laboratory Standards Institute® (CLSI®) C3-A4 Guideline3 defines water suitable for clinical analyzers based on three main parameters: ionic purity, organic purity, and bacteria levels. The final filters (screen filter or ultrafilter)4 included as the final purification step on purification units provide a low bacterial count and also ensure low particle content in water feeding the analyzer. A maximum bacterial count of 10 CFU/mL is recommended in the guideline.


The selection of a purified water solution combining RO, EDI, germicidal UV, ion-exchange and end filter (AFS® range of systems) ensures good ionic purity (resistivity > 10 MΩ·cm) and organic purity (TOC < 50 ppb) of the water, and can reduce the risk of bacterial contamination in the analyzer (consistent bacterial level < 10 CFU/mL), hence leading to a more stable Arsenazo calcium assay.





  1. J Shephard; B Baldwin. Interference in Measurement of Potassium Caused by Bacterial Contamination of an Analyzer. Clin.Chem. 50, No. 12, 2463-2464, 2004.
  2. K Kamiyama et al. Sources of bacterial contamination of autoanalyzers and counter measures. JJCLA, Vol 27, 5, 684- 689, 2002.
  3. CLSI® – Clinical and Laboratory Standards Institute®. Preparation and testing of reagent water in the clinical laboratory.Approved guideline. 4th ed. CLSI® document C3-A4 [ISBN 1-56238-610-7], 2006.
  4. J Bole; S Mabic. Utilizing ultrafiltration to remove alkaline phosphatase from clinical analyzer water. Clin Chem Lab Med, v. 44, n. 5, p. 603-8, 2006.


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