By Jan Holinsky, Robert Janetschek, Lee Lipski, and Tracey Jewells-Treige

 Over the past decade, there has been a steady evolution away from traditional dry-fill and lyophilized reagent platforms to full liquid-stable formats. Aside from the obvious conveniences that a liquid-stable format features, the inherent benefits this reagent design offers include a savings in time and labor by the removal of routine reagent reconstitution procedures, a savings in cost by the elimination of possible reconstitution errors and, more often than not, improved test turnaround time and efficiencies due to increased instrument uptime. All of these enhancements combine to result in increased overall laboratory productivity. However, regardless of the conveniences offered by a liquid-stable format, the challenging problematic issues related to reagent shelf life, reagent drift, and onboard and calibration stability still remain.

As a direct result of these challenges and keeping in line with prevailing laboratory industry trends, Diagnostic Chemicals Limited (Oxford, Conn; Mexico City, Mexico; and Charlottetown, Prince Edward Island, Canada) has introduced the L3K® reagent line of chemistry assays, a significantly improved and unique line of single-vial, liquid-stable reagents. The core technology behind the majority of the assays comprising the L3K reagent line is the incorporation of a proprietary manufacturing process that utilizes patented synthetic enzymatic cofactor analogs, primarily for NADH. Outside of an alkaline environment, NADH rapidly degrades to NAD+ (nicotinamide adenine dinucleotide) greatly reducing both reagent onboard and calibration stability. However, use of a synthetic NADH analog not only demonstrates a superior stability at a neutral pH, but also allows for an optimal pH that ensures the stability of other components within the reagent. This, in turn, allows for the manufacture and use of a single-vial reagent assay system. As an added benefit, the use of a synthetic analog also positively impacts assay reaction kinetics, available wavelength selection, and molar absorptivity.

As a prime example to demonstrate the key advantages of the L3K technology, one just needs to review the Carbon Dioxide L3K (CO2 L3K) reagent. One of the most frequently ordered tests within the laboratory to assist in the detection of electrolyte imbalance as a result of either respiratory acidosis or alkalosis, traditional methods for the determination of serum/plasma CO2 concentrations have incorporated either ion selective electrode (ISE) technology or enzymatic/colorimetric reagent systems. Not surprisingly, the most common enzymatic/colorimetric reagent systems for CO2 utilize NADH as the primary cofactor. Unfortunately, as explained earlier, NADH is only truly stable in an alkaline environment and degrades rather quickly outside of this pH range, negatively impacting overall reagent stability. Additionally, as NADH peaks at an absorbance of 340 nm and then rapidly drops off, it leaves only a very narrow band of wavelengths (340 nm–380 nm) accessible for accurate photometric readings by the vast majority of clinical chemistry analyzers currently available.

 Conversely, the utilization of a synthetic NADH analog improves overall performance in the CO2 L3K reagent system for a number of key reasons. First, the synthetic analog used demonstrates superior stability at a neutral pH. A neutral pH greatly reduces reagent CO2 absorption from the air, prolonging both onboard and calibration stability, with no observable reagent drift. Second, a neutral pH ensures the stability of other vital enzymatic components within the reagent, again extending overall reagent stability. Lastly, the use of this patented synthetic analog results in a reaction absorbance curve, which permits expanded readings from 340 nm to 415 nm, thereby allowing increased flexibility for photometric readings.

The current L3K reagent assays available, along with their appropriate test methodologies, include alanine aminotransferase (ALT)—kinetic (IFCC); bile acids (veterinary use only)—enzymatic rate (3-a-HSD); carbon dioxide (CO2)—enzymatic rate (PEPC); ethanol—enzymatic rate (ADH); and urea nitrogen (BUN)—enzymatic rate (urease). Additional assays are presently in active development.

L3K reagents are available in a variety of operator-oriented packaging configurations ranging in size from 13.5 mL to 1,200 mL, offer an average shelf life stability of 13 months, and feature exceptional onboard and calibration stability, while displaying superior performance characteristics with regard to accuracy, precision, and linearity. All L3K reagents are suitable for use on fully automated analyzers, with a comprehensive number of applications currently available for a broad range of chemistry instruments, including those most frequently encountered within the clinical laboratory.

In summary, all of the inherent attributes the L3K reagent technology offers combine to result in a reagent system demonstrating unparalleled onboard stability, calibration stability, shelf life, and operator convenience. Now actively producing reliable and accurate results in some of the industry’s most demanding facilities, the availability of L3K reagents will serve to benefit the needs of physician office, hospital, and reference laboratories alike. Convenience, simplicity, and enhanced reagent performance characteristics for clinical chemistry testing can be achieved without additional expense to the clinical laboratory.

All of the authors are affiliated with Diagnostic Chemicals Limited. Inquiries can be sent to the attention of Robert Janetschek c/o Diagnostic Chemicals Limited, 160 Christian St, Oxford, CT 06478; (203) 881-2020; fax: (203) 888-1143; email: [email protected].