1 Water in the Clinical Laboratory Mikael Cleverstam WW Clinical Business Manager Role of water in Clinical Diagnostic Purification technologies basics.

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Water in the Clinical Laboratory

Mikael Cleverstam

WW Clinical Business Manager

Role of water in Clinical Diagnostic Purification technologies basics Delivering water to the clinical analyzer

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Putting it all together

Patient Patient ResultsResults

Water Water QualityQuality

Diagnostic Diagnostic InstrumentsInstruments

Medical Medical TechnologistTechnologist

Quality ControlQuality Control

Diagnostic instruments

Assay developmentPatient results

Troubleshooting Your analyzer

Water quality as part of the quality process

CLSI New standards

Water purification

Quality system

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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

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Water for Clinical Analysers

Cuvette washing Tubing and probe rinsing Reagent and buffer reconstitution Dilution Water Baths

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Clinical Analyzers

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


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Assay Development

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


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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


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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



Quality ControlQuality ControlDiagnostic Diagnostic

InstrumentsInstruments

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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.



Quality ControlQuality ControlDiagnostic Diagnostic

InstrumentsInstruments

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Next Steps

YES

YES

YES

NO

NO

Assay

Mechanical

Decontamination

Problem solved

Problem

WHY ??

Outside Source

Water Quality




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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


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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


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Diagnostic Dilemma

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



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Biofilm Formation

Time

Surface

Particles

Organic

Bacteria

Biofilm may shed bacteria, pyrogens etc



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Demonstration of ALP release from bacteria

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 (Unit/L)

Sphingomonas paucimobilis

Caulobacter crescentus

Ralstonia pickettii

29.2

9.7

29.5

6.22

9.95

8.29



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Detection methods

ALP

+

Substrate-Phosphate

Pi

UV-Visible pNPP

Fluorescence Attophos Starbright MUP ELF

Chemiluminescence CDP-Star (dioxetane) CSPD (dioxetane) Lumigen PPD AMPPD



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CLSI Water Quality Standards

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


Quality ControlQuality Control

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Water Contaminants

Water: H2O …. and some other things

Purification technologies

H

H

H

H

Presence of contaminants

Particles

Gases

Microorganisms

Ions

Organics

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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

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What is reverse osmosis ?

P P

Feed Water

PermeateReject

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Technology Insight: Electro-Deionization

Resistivity: > 10 M.cmTOC: < 30 ppbNo need for regeneration

A - Anionic MembraneC - Cationic Membrane

A C A C

CathodeCathode

Na+

Na+

H+

H+

OH-

Cl- Na+

ProductProduct

+ Cl-

Cl-

Cl-

Cl-

Reverse Osmosis WaterReverse Osmosis Water10 - 20 10 - 20 S/cmS/cm

Na+

-AnodeAnode

RejectReject

Na+

OH-

EDI module- Ion selective membranes- Ion exchange resins- Continuous current

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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.

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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

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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

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Water Purification Unit

Feed water

To analyzer

Pretreatment cartridge

Pump

Reverse Osmosis cartridge

Electrodeionization module

Drain

UV Germicidal

Ion exchange resins

Tank

Simplified flow schematic combining purification technologies

The electrodeionization module is not present in some purification units

Resistivity cell

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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.

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Conclusions

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

1 Water in the Clinical Laboratory Mikael Cleverstam WW Clinical Business Manager Role of water in Clinical Diagnostic Purification technologies basics.

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