Vol. 15 •Issue 8 • Page 83
Maintaining Water Quality in Clinical Chemistry
Water quality for clinical testing is essential for complying with the norms, in particular Clinical and Laboratory Standards Institute (CLSI), and for performing assays using analyzers that require purified water. The CLSI guideline was written as a suggestion for ensuring a minimum water quality, which is supported by recommendations made for levels of a few contaminants and the basic water purity to conduct clinical chemistry assays safely.
Water Quality Requirements
Assays performed routinely in clinical analyzers can be divided into several categories with varying sensitivity to contaminants that can be present in water. Water used for general chemistry assays should contain as few ions and bio-organic molecules (which are naturally present in tap water) as possible. Bacteria will also release all major ions found in blood and will be a major source of bio-organic molecules in water and release carbohydrates, aminoacids, lipids and proteins.
Many toxicology and therapeutic drug monitoring methods are immunoassays, which are sensitive to the presence of ions and high levels of organics. Sometimes coupled with mass spectrometry, methods based on chromatography (GC, HPLC) have been developed to monitor the presence and quantify these compounds, most of which are small organic molecules. In these cases, it is important to reduce the level of organics in the water that impacts chromatography.
Enzyme-immunoassays are sensitive to the presence of ions; for example, Mg and Zn are co-factors, and Pb and Cd would inhibit or inactivate the enzymes. Additionally, organics can not only interfere with the binding of substrates, but also act as inhibitors of the enzyme used in these reactions. Some high-level organics also would interfere with the UV/visible and fluorescence detection and enzymes released by microorganisms used in signal amplification cascades. Enzymes such as alkaline phosphotase (ALP) often are used as reporter enzymes and the level should be controlled in purified water.
Trace elements analysis finds transition metals (Fe, Cr, Cu, Co, Mn, Mb), heavy metals (Hg) and a few elements with critical levels (Se). Water with low ion levels is required for these analyses, which use atomic absorption or ICP-MS. Water must have a resistivity of 18.2 MWácm to ensure trace levels of the ions present. Avoid bacteria because they release the same ions as those being analyzed.
The quality of the water in the reaction is critical in emerging nucleic acid binding assays. To avoid interferences in the binding process, it is mandatory to eliminate DNA or RNA that could compete and ions and organic molecules such as acids and phosphates that could bind with the target or sample nucleic acids. Since nucleic acids are involved, it is important to reduce the levels of nucleases present in the water. Therefore, special water quality should be used for these experiments: 18.2 MWácm resistivity (guaranteeing low ionic contamination), low organic content (total organic carbon, or TOC, below 10 ppb) and nuclease-free water.
Purification technologies are combined to reduce the levels of contaminants and ensure a constant quality of water dispensed to the clinical analyzer. General filtration reduces the incoming particle load. Activated carbon eliminates the oxidative agents present in tap water to avoid microorganism development. Reverse Osmosis (RO), a membrane-based technology, has become a standard pretreatment filtration technique to decrease the load of ions, organics, colloids and particulates. RO, which also removes a large part of silica, rejects a percentage of the contaminants. Silica leaves deposits in the probes and modifies dispensed micro volumes.
To reach a more constant water quality and overcome daily and seasonal variations in tap water, electrodeionization (EDI) has been introduced to remove ions. This technology uses selective anionic and cationic semi-permeable membrane and ion exchange resins that are regenerated constantly with a small electrical current without maintenance. After RO-EDI treatment, water has a resistivity typically >10 MWácm, and a TOC level <50 ppb (off-line measurements). At this stage, water is stored temporarily in a reservoir. Depending on the assays, the clinical analyzer and laboratory, this water can be used to feed the analyzer directly or further purified to reach a CLSI Clinical Laboratory Reagent Water level.
At the outlet of the purification system, a 0.22 µm filter often is employed to avoid bacteria release from the purification system. Bacteria control has been achieved in water purification systems and clinical analyzers via screen membrane filtration (0.22 µm), germicidal UV254 and chemical sanitization. More recently, it has been recommended to use ultrafiltration for eliminating significant bacteria by-products.
For specialty chemistries, such as toxicology and nucleic acid based assays, additional purification technologies are available. Different water purification systems usually are selected based on experiment types. These systems may combine ion exchange resins (IEX), activated carbon, ultrafiltration and UV photooxidation (UV185/254).
Pre-analytical factors that influence analytic results are difficult to monitor because they occur outside of the laboratory. Reducing the number of tests failing for technical reasons and releasing erroneous results are indicators used to monitor the quality and improve the processes. The laboratorian can control the analytic factors, which primarily depend on instrumentation and reagents. Water is a major component used in the reactions and testing methods and should be a constant focus.
Stephane Mabic is worldwide applications support manager of the Bioscience Division, Millipore Corp.