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

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. Diagnostic Instruments
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. Diagnostic Instruments
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
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
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
Diagnostic Instruments
Quality Control
Medical Technologist
Patient Results

9. Troubleshooting procedures
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.
Diagnostic Instruments
Quality Control
Medical Technologist
Patient Results

10. Diagnostic Instruments
Next Steps
Diagnostic Instruments
Water Quality
Medical Technologist NO
YES NO
YES
Outside Source
YES
WHY ??
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 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 Instruments
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. Diagnostic Instruments
Biofilm Formation
Diagnostic Instruments
Water Quality
Organic
Particles
Surface
Bacteria
A biofilm is an agglomeration of particulates, organic substances and bacteria on the inner surface of the reservoir.
As indicated earlier, bacteria do not “like” to grow alone, in the middle of water . They rather tend to stick to a support . This is what happens ususally inside reservoirs : some partciulates and organic substances bind to the surface; bacteria attach themselves to the same point, and a biofilm grows and becomes thicker with time, releasing from time to time bacteria in the stored water.
NB : just to give an order of magnitude about what represents 1 cfu/ml. For a simple calculation, let’s consider that a bacteria occupies 0.1 um 3 (it usually occupies a smaller volume : Pseudomonas Diminuta volume is about 0.01 um3- this is just to make the demonstration easier).A 0.1 um3 volume corresponds to 0.1 x (10*-6m)3 = 10*-19 m3. Therefore, the volume occupied by 1 billion bacteria would be 10*-19 x 10*9 = 10*-10 m3 = 10*-7L = 10*-4 ml= ml . This means that 1ppb (1 part per billion ) bacteria corresponds to 1 bacteria per ml , or 10,000 bacteria per ml.
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
Bacteria Strain
(identification by 16S rDNA sequencing)
Bacteria level
(x 106 cfu/mL)
ALP concentration (mUnit/mL)
Sphingomonas paucimobilis
Caulobacter crescentus
Ralstonia pickettii
29.2
9.7
29.5
6.22
9.95
8.29

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

17. CLSI Water Quality Standards
Quality Control
Water Quality
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 Water: H2O …. and some other things
Presence of contaminants
Particles
Gases
Microorganisms
Ions
Organics
Purification technologies

19. Protecting the Water Purification Unit: Pretreatment cartridge
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.
Example of a pretreatment cartridge

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

21. Technology Insight: Electro-Deionization
EDI module
- Ion selective membranes
- Ion exchange resins
- Continuous current
A - Anionic Membrane
C - Cationic Membrane A C
Cathode
Na+ H+
OH-
Cl-
Product +
Reverse Osmosis Water
mS/cm -
Anode
Reject
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.