Considering the varied, and often critical, biochemical roles that calcium plays in human pathology and physiology, it is not surprising that its analysis is one of the most commonly performed tests in the clinical laboratory. The overwhelming majority of calcium, more than 99%, is found in the bones, while the remaining concentrations are involved in significant functions within the body. Calcium ions decrease neuromuscular excitability, are active in blood coagulation, and are necessary for the activation of certain enzymes. In addition, calcium and cyclic AMP play a role in the transfer of inorganic ions across cell membranes and in the release of neurotransmitters. Essentially all of the calcium found in blood is present in the plasma as two distinct forms: nondiffusible protein-bound calcium and the diffusible free calcium fraction. Of the two forms, the nondiffusible protein-bound fraction constitutes roughly 40% to 50% of the total extracellular calcium, while the diffusible free fraction can be further subdivided into ionized calcium (which is the physiologically active form) and complexed calcium (which is found bound to bicarbonate, citrate, phosphate, and sulfate).

The determination of calcium concentrations, therefore, is a vital and necessary component for the proper diagnosis of a number of disease states. An elevated calcium level, or hypercalcemia, is indicative of hyperparathyroidism, hypervitaminosis D, neoplastic bone disease, and numerous malignancies (including breast, lung, prostate, kidney, lower gastrointestinal tract, and multiple myeloma). Conversely, hypocalcemia, or decreased calcium concentrations, can be observed in hypoparathyroidism, pancreatitis, tetany, steatorrhea, nephritis, nephrosis, and as a side effect to certain medications.

A Traditional Problem
Despite the need for rapid and accurate test results, calcium determinations have been traditionally problematic for both diagnostic manufacturers and clinical laboratories. The classic reference methods—atomic absorption spectrophotometry and oxalate precipitation followed by titration—are hardly cost-efficient or convenient for routine use, given the demands of today’s laboratory environment. Consequently, the employment of biochemical reagents, using dye complexes read by spectrophotometric means (such as o-Cresolphthalein Complexone and Arsenazo III), are the most common and recognized methods now used. While these assays do yield accurate and precise results, they do, unfortunately, suffer from certain inherent problems as well. Calcium reagents have historically been prone to issues involving, among others, cross-contamination, water quality, reagent drift, calibration instability, and lot-to-lot variations, all of which, singly or combined, can contribute to erratic and less-than-optimum test results. This further necessitates additional instrument and reagent troubleshooting time required to identify root-cause problems.

Another significant issue facing the laboratory as a result of calcium testing stems from the use of certain toxic components and/or raw materials used in the manufacturing of these reagents. Due to environmental directives within certain geographical municipalities, proper waste disposal is a requirement, often resulting in monetary fines and legal obstacles for failure to comply. As a result, clinical analyzers performing the testing are modified, often at an additional expense, to accommodate separate drain and waste lines. Compounding this are the supplementary operational costs involved for the proper removal and disposal of this waste from the laboratory.

A New System
In direct response to these challenges and in keeping with prevailing laboratory industry trends, Diagnostic Chemicals Limited (DCL of Oxford, Conn) has recently developed its new single-component, fully liquid-stable Calcium L3K® reagent. This reagent system introduces a new and distinctive method for the measurement of calcium in clinical samples. The assay uses an innovative synthetic dye complex, Phosphonazo III, in a unique buffering system for the quantitative determination of calcium in serum, plasma, and urine. The test principle is relatively simple and straightforward. Calcium present in the test specimen reacts with the Phosphonazo III dye in a buffered and acidic environment, yielding a blue/violet calcium-dye complex. The resulting increase in absorbance caused by the formation of this complex is bichromatically measured in the area of 660/700 nm and is directly proportional to the calcium concentration of the sample. Noteworthy for the laboratory is that both the Phosphanazo III dye, as well as the buffer system used in this particular reagent, is classified as “environmentally friendly,” since no special disposal requirements are necessary.

As important as the environmental and disposal concerns are, the specific performance characteristics of this reagent system on an automated clinical platform are equivalent to, or in some cases superior to, the industry-accepted o-Cresolphthalein Complexone and Arsenazo III methods. For example, in recovery studies performed, calcium acetate was added to pooled human sera, plasma, and urine to increase calcium concentrations by 1.9 mg/dL and 4.8 mg/dL. Recovery of the added calcium averaged 99.6% in serum, 96.9% in plasma, and 104.4% in urine. Further to recovery, interference studies confirm no clinically significant interference from ascorbic acid, hemolysis, icterus, or lipemia. Reportable range studies, following NCCLS EP6-P protocols, demonstrated the linear limit of this procedure to be 20.0 mg/dL, while the lower limit of detection for the procedure was found to be 0.2 mg/dL. This data results in a reportable range of 0.2 to 20.0 mg/dL.

Continuing, accuracy studies following NCCLS EP9-P protocols demonstrated that the performance of Calcium L3K (y) compared well to the performance of commercially available o-Cresolphthalein Complexone and Arsenazo III Calcium methods (x), covering a wide range of values. The serum-comparison study, using the Arsenazo III methodology with 125 samples, yielded a correlation coefficient of 0.9979, with a resulting linear-regression equation of y = 1.02x – 0.18 mg/dL. The plasma-comparison study, using the o-Cresolphthalein method performed with 44 samples, yielded a correlation coefficient of 0.9994 and a linear-regression equation of y = 0.971x + 0.0 mg/dL. The urine study, using an Arsenazo III method, performed with 60 specimens, yielded a correlation coefficient of 0.9988 and a linear-regression equation of y = 0.957x – 0.36 mg/dL.

Finally, NCCLS EP5-T2 precision studies were also performed. Within-run precision was established by assaying each sample 20 times, while total precision was established by assaying two samples per run, two times per day, for a total of 20 days. Precision estimates in the serum study were obtained using two levels of commercially available serum-based control materials, with concentrations of approximately 9 mg/dL and 12 mg/dL. Within-run performance testing resulted in SDs of 0.05 mg/dL and 0.06 mg/dL, respectively, generating CV%s of 0.5 and 0.5. Estimates for total precision were also obtained in the same study. Here, the resulting SDs were 0.11 mg/dL and 0.17 mg/dL, with CV%s of 1.2 and 1.4. Precision estimates for plasma were determined using two levels of laboratory-prepared control materials with concentrations of approximately 9 mg/dL and 14 mg/dL. Within-run performance yielded SDs of 0.06 mg/dL and 0.12 mg/dL, respectively, resulting in CV%s of 0.6 and 0.8. Total precision-performance estimates resulted in SDs of 0.10 mg/dL and 0.15 mg/dL, respectively, with CV%s of 1.1 for both levels. Estimates for urine were obtained using two levels of commercially available urine-based control materials, with approximate concentrations of 5 mg/dL and 11 mg/dL. Within-run testing resulted in SDs of 0.07 mg/dL and 0.11 mg/dL, respectively, with CV%s of 1.3 and 1.0. Total precision-performance estimates displayed SDs of 0.08 mg/dL and 0.18 mg/dL respectively, yielding CV%s of 1.6 for both levels.

Benefits to the Lab
Aside from the obvious advantages associated with the reagent waste and disposal issue and the exceptional performance characteristics, DCL’s Calcium L3K reagent also offers clinical laboratories important additional benefits. It is supplied as a single-vial, liquid-stable reagent. Single-vial, liquid-stable technology eliminates the need for reagent-reconstitution procedures and reduces the incidence of procedural errors, while saving time, labor, and reagent waste. The assay features a minimal 3 µL sample-volume requirement, making it ideal for pediatric, geriatric, and veterinary testing. In addition, as the assay is fully validated to support a single-point calibration requirement, as well as the testing of serum, plasma, and urine specimens, this added testing flexibility allows for increased uptime and testing throughput. Finally, as each test requires only a maximum 3-minute reaction period, test turnaround time is significantly improved. DCL’s Calcium L3K reagent is currently available in two distinct operator-oriented packaging formats: a 4- x 125-mL kit configuration, as well as a 1- x 1,000-mL size. A comprehensive listing of instrument applications is also available for a range of chemistry analyzers, especially those most frequently encountered within the clinical laboratory.

Additional internal and external clinical evaluations of Calcium L3K are currently under way, following NCCLS guidelines for performance. Results of these evaluations will be reported during the upcoming American Association for Clinical Chemistry 2006 Annual Meeting and Clinical Lab Exposition in Chicago, July 25–27. DCL’s Calcium L3K reagent benefits the needs of medical laboratory professionals in physician’s office, hospital, and reference laboratories.

Robert Janetschek, MS, MT(ASCP), is director of business development for Diagnostic Chemicals Ltd.