Calidad del agua en el Laboratorio Clínico

1. 1
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

2. 2
Putting it all together
PatientPatient
ResultsResults
WaterWater
QualityQuality
DiagnosticDiagnostic
InstrumentsInstruments
MedicalMedical
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

3. 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. 4
Water for Clinical Analysers
 Cuvette washing
 Tubing and probe rinsing
 Reagent and buffer reconstitution
 Dilution
 Water Baths

5. 5
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
DiagnosticDiagnostic
InstrumentsInstruments

6. 6
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
DiagnosticDiagnostic
InstrumentsInstruments

7. 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
MedicalMedical
TechnologistTechnologist

8. 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
MedicalMedical
TechnologistTechnologist
PatientPatient
ResultsResults
Quality ControlQuality Control
DiagnosticDiagnostic
InstrumentsInstruments

9. 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.
MedicalMedical
TechnologistTechnologist
PatientPatient
ResultsResults
Quality ControlQuality Control
DiagnosticDiagnostic
InstrumentsInstruments

10. 10
Next Steps
YES
YES
YES
NO
NO
WHY ??
Outside Source
Water Quality
MedicalMedical
TechnologistTechnologist
DiagnosticDiagnostic
InstrumentsInstruments
WaterWater
QualityQuality

11. 11
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
WaterWater
QualityQuality

12. 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
WaterWater
QualityQuality

13. 13
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
DiagnosticDiagnostic
InstrumentsInstruments
WaterWater
QualityQuality

14. 14
Biofilm Formation
Time
Surface
Particles
Organic
Bacteria
Biofilm may shed bacteria, pyrogens etc
DiagnosticDiagnostic
InstrumentsInstruments
WaterWater
QualityQuality

15. 15
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
(µUnit/µL)
Sphingomonas paucimobilis
Caulobacter crescentus
Ralstonia pickettii
29.2
9.7
29.5
6.22
9.95
8.29
DiagnosticDiagnostic
InstrumentsInstruments
WaterWater
QualityQuality

16. 16
Detection methods
ALP
+
Substrate-Phosphate
Pi
UV-Visible
 pNPP
Fluorescence
 Attophos
 Starbright
 MUP
 ELF
Chemiluminescence
 CDP-Star (dioxetane)
 CSPD (dioxetane)
 Lumigen PPD
 AMPPD
DiagnosticDiagnostic
InstrumentsInstruments
WaterWater
QualityQuality

17. 17
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
WaterWater
QualityQuality
Quality ControlQuality Control

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

19. 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. 20
What is reverse osmosis ?
P P
Feed Water
Permeate
Reject

21. 21
Technology Insight: Electro-Deionization
Resistivity: > 10 MΩ.cm
TOC: < 30 ppb
No need for regeneration
A - Anionic Membrane
C - Cationic Membrane
A C A C
CathodeCathode
Na+
Na+
H+
H+
OH-
Cl-
Na+
ProductProduct
+ Cl-
Cl-
Cl-
Cl-
Reverse Osmosis WaterReverse Osmosis Water
10 - 2010 - 20 µµS/cmS/cm
Na+
-AnodAnod
ee
RejectReject
Na+
OH-
EDI module
- Ion selective membranes
- Ion exchange resins
- Continuous current

22. 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. 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. 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. 25
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

26. 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. 27
Conclusions
 Water is a reagent.
 The quality of water has an impact on the testing method.