Water in the Clinical Laboratory

1. Water in the Clinical Laboratory • Role of water in Clinical Diagnostic • Purification technologies basics • Delivering water to the clinical analyzer Mikael Cleverstam WW Clinical Business Manager

2. Putting it all together Patient Results Water Quality Medical Technologist Diagnostic Instruments Diagnostic instruments Troubleshooting Your analyzer Assay development Patient results CLSI New standards Water quality as part of the quality process Water purification Quality system Quality Control

3. Normal seen Problems • Frequent Calibrations • High CV% • Fluctuation in quality results over the day/week/month • Interfered assays • Calcium Interfered by rocks, leaves, geology • ALP Interfered by biofilm, detergent, rocks • CK Interfered by water treatment • Amylase Interfered by citrus fruit, detergents, leaves • LD Interfered by effluent, leaves, H2O2 • Phosphorus Interfered by citrus fruit, leaves • Iron Interfered by rocks, leaves, detergents • Magnesium Interfered by citrus fruit • Triglycerides Interfered by plastics, chemicals • Urea Interfered by citrus fruit, water treatment • Troponin I Interfered by biofilm

4. Water for Clinical Analysers • Cuvette washing • Tubing and probe rinsing • Reagent and buffer reconstitution • Dilution • Water Baths

5. Clinical Analyzers Diagnostic Instruments • Features and benefits of automation • Precision optical systems for accuracy in testing • Automatic sampling and dilutions modes • Real time alerts to patient and QC failures • Improved software alerts end user to mechanical failures • Cost benefits • Workflow efficiency and high speed through put • Instrumentation targeted to reduce operating cost with more efficient technology • Reduced operator interface

6. Assay Development Diagnostic Instruments • Measuring chemical changes in the body for diagnosis, therapy and prognosis has resulted in new assay development • Multiple method testing on a single analyzer • Current research methodologies for infectious disease and tumor marker’s are moving from research labs in universities to the clinical laboratory • Complex methodologies are being fully automated for more routine use

7. Unique Challenges for Medical Technologist Medical Technologist • Verification of final clinical results to be accurate and precise are determined by Medical Technologist • Clinical decisions are not solely made on the test result, but in conjunction with the patient’s history and symptoms • Software alerts, QC reviews, calibration must all be within stated limits before results are released • Troubleshooting instrument problems result in production delays, are costly and non-productive activities that must be performed and documented ➙ Try to avoid diagnostic instrument service because it is expensive

8. Reviewing Patient Results Patient Results Diagnostic Instruments Medical Technologist • All analytical and pre-analytical factors must be reviewed and documented • Medical Technologist must review all test results • If results are flagged, troubleshooting the cause is necessary Quality Control

9. Troubleshooting procedures Patient Results Diagnostic Instruments Medical Technologist • Sample handling procedures confirmed • Quality control must be reviewed • Shifts and trends • Peer group • Previous data • Assay • Reagent issue • Calibrator stability • Mechanical • Instrument malfunction • Error codes • If above solutions do not correct the erroneous result, further troubleshooting must identify cause before results can be released to physicians ➙ Delayed patient treatment. Quality Control

10. Next Steps Diagnostic Instruments Water Quality Medical Technologist WHY ?? NO YES NO YES Outside Source YES Water Quality

11. Water Quality Water Quality • Quality results are dependent upon reliable instrumentation and known water quality • Analytical factors need to be controlled and optimized to reduce the number of test failures, failed calibrations, and high blanks that can contribute to erroneous patient results • Maintenance of high purity water system is essential to reliable results

12. Understanding Water Quality and Methodology Water Quality • Water should be considered a bulk reagent on any analyzer • The high purity water system is a separate unit, not monitored by diagnostic software on the clinical analyzer • The unique properties of water if not processed and monitored can produce subtle changes in assay methods • These changes in water quality can lead to erratic and inconsistent results • The quality of water required or its impact on the testing method is often not considered until the purchase is complete

13. Diagnostic Dilemma Diagnostic Instruments Water Quality • Smaller sample size and reaction vessel are subjected to harsher environment • Inevitable build-up of biofilm in instruments, manifolds and tubing require more frequent decontaminations but Less and less time available for maintenance of the instruments • Some sensitive assays can become contaminated with bacteria and ions • Bacteria release enzymes and ions whose behavior is similar to the enzymes targeted in the assay method ➙ Increased need to monitor water quality as closely as any other instrument malfunction

14. Biofilm Formation Diagnostic Instruments Water Quality Organic Particles Surface Bacteria Time Biofilm may shed bacteria, pyrogens etc

15. Demonstration of ALP release from bacteria Diagnostic Instruments Water Quality • Correlation between bacteria concentrations and levels of ALP in water

16. Detection methods Diagnostic Instruments Water Quality • UV-Visible • pNPP • Fluorescence • Attophos • Starbright • MUP • ELF • Chemiluminescence • CDP-Star (dioxetane) • CSPD (dioxetane) • Lumigen PPD • AMPPD Substrate-Phosphate ALP + Pi

17. CLSI Water Quality Standards Water Quality Quality Control • New Standards released July 2006 (C3-A4 Vol. 26 No. 22) • Nomenclature Type I,II,III has been replaced with purity types that provide more meaningful parameters • CLRW (Clinical Laboratory reagent Water) replaces Type I,II for most applications • IFW (Instrument Feed Water) allows instrument manufacturers to clarify specifications for their particular methods • SRW ( Special Reagent Water) may be specified for specific applications when additional parameter are needed to insure water quality • Autoclave and wash water will meet the requirements of previously classified Type III • Complete review of the document should be done when considering new applications to insure the contaminants found in the source water do not become an issue

18. Water Contaminants Presence of contaminants Particles Gases Microorganisms Ions Organics Purification technologies Water: H2O …. and some other things

19. Due to the difference in water quality around the world, additional pretreatment cartridges are required. The cartridges provide protection and insure good performance of the reverse osmosis membrane The pretreatment packs include 0. 5 micron filter (1) to remove particles and activated carbon (2) to remove chlorine The activated carbon is impregnated with a small level of silver to prevent bacterial growth. Protecting the Water Purification Unit: Pretreatment cartridge Example of a pretreatment cartridge

20. What is reverse osmosis ? P P Feed Water Permeate Reject

21. Technology Insight: Electro-Deionization Reverse Osmosis Water 10 - 20 mS/cm A C A C Na+ Cl- H+ OH- Cl- - Na+ + Cathode Anode Cl- Na+ OH- H+ Na+ Cl- Cl- Na+ Reject Product A - Anionic Membrane C - Cationic Membrane EDI module - Ion selective membranes - Ion exchange resins - Continuous current Resistivity: > 10 MW.cm TOC: < 30 ppb No need for regeneration

22. Filters – Bacteria Removal • Screen 0.2 µm filters • Designed for the removal of particles and microorganisms from liquids and gases. • Use of PVDF membranes, provide high flow rates and throughputs, low extractables, broad chemical compatibility and the lowest protein binding of any membrane available.

23. Ultrafiltration • Cut-off: 5 KDa to 20 KDa • Removes bacterial by-products such as most proteins and macromolecules (e.g. endotoxins) • Utilized for immunochemistry assays • Immunoenzyme assays based on reporter enzymes (alkaline phosphatase, ALP) are sensitive to ALP released by bacteria • Also filters bacteria

24. Storage • CLRW water with a resistivity >10 megohm-cm cannot be stored because ionic and organic contamination will leach from the atmosphere and container materials in which it is stored. • CLRW water should be used as it is produced • Stored water is never as pure as when it is made • Storage of water enhances bacterial contamination • Containers need to be cleaned thoroughly between refilling. • Carboys, tanks, bottles • Notorious source of contamination since we often refill them without thoroughly cleaning them when they are emptied • Some plastic materials out-gas polymers and plasticizers, and these end up in the water

25. Water Purification Unit Simplified flow schematic combining purification technologies Tank Pretreatment cartridge Reverse Osmosis cartridge Electrodeionization module Resistivity cell Feed water Pump To analyzer UV Germicidal Ion exchange resins Drain The electrodeionization module is not present in some purification units

26. Connecting the Water Purification Unit to the Clinical Analyzer • Water is delivered in its purified state to a harsh environment within the chemistry analyzer bottle • Water bottles inside analyzer are not frequently decontaminated • Electronics, mechanical hardware, pumps all create heat within the analyzer cabinet, thus raising the interior temperature of the water bottle. • Increased temperatures enhance the growth of bacteria and biofilm within the instruments manifolds and tubings.

27. Conclusions • Water is a reagent. • The quality of water has an impact on the testing method.