Download Review of The Impact of Water Quality on Reliable Laboratory

Review
Review of The Impact of Water Quality on
Reliable Laboratory Testing and Correlation
with Purification Techniques
Rana Nabulsi, PhD, MSc, BSc, CPHQ,1 Mousa A. Al-Abbadi, MD, FIAC, CPHQ2*
Lab Med Fall 2014;45:e159-e165
DOI: 10.1309/LMLXND0WNRJJ6U7X
ABSTRACT
In recent years, many rapid and automated instruments with
highly complex testing methodologies have been introduced to
our laboratories, almost all of which require the use of water.
Many investigators have discussed the impact of water quality
on the accuracy and reliability of clinical laboratory testing and
technologies. Evaluation of water quality and specifications according
The unique molecular properties of water, such as polarity
and reactivity, make it a nearly universal solvent for many
substances.1 Also, water is easily contaminated before
being distributed centrally in the laboratory and during
storage.1 Many laboratory scientists colloquially consider
Water quality to be one of the critical preanalytical factors
that influences laboratory testing. Many consider water
to be a laboratory reagent and use it to prepare buffers,
Abbreviations:
CLSI, Clinical and Laboratories Standards Institute; NCCLS, National
Committee for Clinical Laboratory Standards; ASTM, American Society
for Testing and Materials; CLRW, clinical laboratory reagent water;
TOC, total organic carbon; CFU, colony forming units; IFW, instrument
feed water; SRW, special reagent water; HPLC, high performance liquid
chromatography; LC-MS, liquid chromatography–mass spectrometry;
PCR, polymerase chain reaction; RT-PCR, reverse transcriptase; EIAs,
enzyme immunoassays; UV, ultraviolet; RO, reverse osmosis; EDI,
electrode deionization; IERs, ion-exchange resins; UF, ultrafiltration;
SNP, single nucleotide polymorphism; NS, not specified; GC, gas
chromatography; IVF, in-vitro fertilization; IVD, in-vitro diagnostic; AA,
atomic absorption; EU, endotoxin units
Proficiency Healthcare Diagnostics. Department of Pathology and
Laboratory Medicine, Abu Dhabi, United Arab Emirates
1
Department of Pathology and Laboratory Medicine, King Fahad
Specialist Hospital, Dammam, Saudi Arabia
2
*To whom correspondence should be addressed.
[email protected]
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to international standards and guidelines has helped laboratories
to judge water quality before using the water in question as part of
testing. In the past few years, dramatic introduction of different water
purification technologies has shown significant improvements in
laboratory testing accuracy. In this review, we delve into the intricate
factors that influence water quality.
Keywords: water quality, purification, contaminants
blanks, calibrators, controls, mobile phase, reaction
mixtures, and reagent reconstitution for many laboratory
assays. Therefore, the use of pure water is critical for
accurate laboratory testing.
Different contaminants may exist in water; these impurities
may inhibit and affect clinical-test results. Different
purification technologies are currently available to remove
these contaminants; each method has advantages and
limitations. Combined purification technologies are
required to produce different levels of purified water
that comply with the specifications defined for each
methodology.
The Clinical and Laboratories Standards Institute (CLSI;
formerly, the National Committee for Clinical Laboratory
Standards [NCCLS]) guidelines define specifications
for pure water; testing water purity usually requires
continuous measurement and monitoring the presence of
contaminants. Consequently, the CLSI classifies different
types of water according to specific and well-defined
purity standards that allow the suitable application of
test methodology in the laboratory. Recently, ASTM
International (formerly, the American Society for Testing
and Materials [ASTM]) established specifications
for types I, II, III, and IV reagent grade water. Also,
depending on endotoxin and bacterial content, further
classification for water was established as type A, type B
or type C.1
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Table 1. The 1998 NCCLS Guidelines for Classification of Water Typesa
Contaminants
Parameter and Measurement Unit
Type III
Type II
Type I
Ions
Organic Materials
pH
Particulates
Colloids
Bacteria
Resistivity, minimum (MΩ-cm) 25°C
TOC (ppb)
0.1
NS
NS
NS
1.0
NS
1.0
NS
NS
NS
0.1
<1000
10
Carbon filtration
5-8
0.22 μm filtration
0.05
<10
Particulates >0.22 µm (units/mL)
Silica (mg/L)
Bacteria (CFU/mL)
NCCLS, National Committee for Clinical Laboratory Standards; TOC, total organic carbon; NS, not specified; CFU, colony forming unit
a
The NCCLS is now known as the Clinical and Laboratory Standards Institute (CLSI).
Table 2. The 2006 CLSI Specification for Reagent Laboratory Water
Water Type
CLSI Specification(s)
Application (Example)
CLRW
Maximum microbial content (CFU/mL) <10
Minimum resistivity 10 MΩ-cm, 25°C
Free of particulates >0.22 µm
Organic materials (TOC) <500 ppb
Defined by lab for procedures that need different specifications
than CLRW
IVD measurement systems allows IVD instrument manufacturers
to clarify specifications for their particular methods
Most laboratory procedures; similar to types I and II
Defined as minimum quality for routine biochemistry assays
SRW
IFW
Immunoassay, molecular assays, HPLC, LC-MS, GC, microarray, IVF
Autoclave use and washing require
CLSI, Clinical and Laboratory Standards Institute; CLRW, clinical laboratory reagent water; CFU, colony-forming unit; TOC, total organic carbon; SRW, special reagent water;
HPLC, high performance liquid chromatography; LC-MS, liquid chromatography–mass spectrometry; GC, gas chromatography; IVF, in-vitro fertilization; IVD, in-vitro diagnostic;
IFW, instrument feed water
(removal of all particles >0.22 µm), as shown in Table 2.
Definition of Water Quality and
Related Contaminants
The original NCCLS guidelines recommended classification
of water depending on specific levels of purity for bacterial
content, ions, organic materials (hereafter, organics), pH,
silica, and particulates (Table 1).1 This original classification
system for water quality was a 3-tiered system (types I, II
and III).1,2
However, this system specified by NCCLS has since
been replaced with CLSI guidelines, which are different
and probably more meaningful given their descriptive
nomenclature for water purity types.3 Also, these guideline
recommend using pure water for optimal laboratory
testing.3 Clinical laboratory reagent water (CLRW) is
defined as the minimum quality water suitable for routine
biochemical testing. CLRW can replace types I and II
water for most applications. The criteria that define the
classification of pure water are based on measurements
of ionic purity (resistivity > 10 MΩ), organic purity (total
organic carbon [TOC] < 500 ppb), bacteria levels (> 10
colony-forming units [CFU]/mL), and particulate level
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Instrument feed water (IFW) can be used for autoclaving,
filling water baths, and washing glassware; this type
of water meets the type-III requirements from the
previous NCCLS classification (Table 3). The new waterquality definitions also include parameters that were
not previously specified. For example, special reagent
water (SRW) may be specified when CLRW purity is
unsatisfactory; additional parameters are needed to ensure
that the water quality is suitable for specific applications
(eg, high performance liquid chromatography [HPLC],
liquid chromatography–mass spectrometry [LC-MS], and
molecular biology based assays).3,4
IFW is considered colloquially to be the most basic
unpolished type of water according to default criteria.
Specifications for IFW can be defined by manufacturers in
determining the qualities of the water that will be suitable
and appropriate for use with their instruments. Also, IFW is
used for general rinsing and for filling incubators and water
baths.
The ASTM established specifications for type I, type
II, type III, and type IV reagent water (Table 4). Further
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Table 3. Former NCCLS Water-Types Classification and Its Applications
Water Type
Assay Methods
Type I
Type II
Type III
Ultrapure water applications such as HPLC, GC, MS, AA, molecular-biology applications, cell culture, and IVF
General laboratory applications such as biochemical assays, microbiological culture, media preparation, buffers, and pH solutions
Noncritical laboratory work, such as rinsing glassware, filling water baths, and autoclaving; used in filling or feeding type I lab watertreatment system
NCCLS, National Committee for Clinical Laboratory Standards; HPLC, high performance liquid chromatography; GC, gas chromatography; MS, mass spectrophotometry; AA,
atomic absorption; IVF, in-vitro fertilization
Table 4. ASTM Specifications for Reagent-Grade Water (Guideline D1193-6-2011)
Contaminants
Parameter and Measurement Unit
Type IV
Type III
Type II
Type I
Ions
Organic materials
pH
Chloride, maximum (µg/L)
Resistivity, minimum MΩ-cm (25°C)
TOC, max (µg/L)
pH units
0.2
NS
5-8
50
4.0
200
NS
10
1.0
50
NS
5
18
50
NS
1
50
NS
Type A
10
500
Type B
5
3
Type C
1
3
<0.03
0.25
NS
1
10
1000
Sodium, maximum (µg/L)
Colloids
Silica (µg/l)
Endotoxin (EU/mL)
Bacteria
Bacteria (CFU/100 mL)
ASTM, American Society for Testing and Materials; TOC, total organic carbon; NS, not specified; EU, endotoxin units; CFU, colony- orming unit
classification for water quality into types A, B, and C was
based on bacterial and endotoxin content.
can form bubbles that may block the optical sensors of any
machine and alter the numerical ability of counting, which is
detrimental to processes such as particulate counting and
spectrophotometric measurements.6,7
Water Contaminants
Oxygen and nitrogen do not ionize in water in normal
conditions. However, when the temperature rises to
37°C during testing procedures, more bubbles can form,
creating blockage problems.7
Many impurities can be present in water, which may
interfere with testing and lead to inaccurate results. Such
contaminants interfere with many laboratory assays; Table
5 summarizes these contaminants and the consequences
of their presence on laboratory testing and methodology.
Colloids and particulates are found in water; whether these
particulates are soft or hard, they usually interfere with
most laboratory assays.3,6 Bacteria or bacterial by-products
such as alkaline phosphatase, nucleases and pyrogens,
can interfere with many molecular biology tests, such as
polymerase chain reaction (PCR), reverse transcriptase
PCR (RT-PCR), microarrays, and cell culture because these
tests are highly sensitive to endotoxins and nucleases.4-6,9,10
Gases such as nitrogen, ammonia, oxygen, and carbon
dioxide can be detected in water in dissolved form. Ammonia
and CO2 can be ionized, forming weak acids and bases,
therefore changing the pH of the testing solutions. Changing
the pH is detrimental to many testing reactions.7 Nitrogen
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Organic contamination can be measured as TOC.
Organics may interfere with binding of substrate in enzyme
immunoassays (EIAs) and could inhibit the enzymes used
in these reactions.8 A high concentration of TOC interferes
mainly with spectrophotometric testing, particularly within
the ultraviolet (UV) absorbance range.4,7-8 Also, concentration
of organics in water should be low to avoid binding to
calcium when measured by the colorimetric method,
thereby reducing the actual concentration in the specimen.6
Organic contamination may increase the background in
chromatographic assays and may alter column performance
in HPLC and LC-MS.4,6-8 Moreover, in microarrays, some
organic contaminants may block hybridization and affect
washing steps. Consequently, improper washing may
increase the background of fluorescent assays, making
interpretation cumbersome.6-8,10 It is also well known that
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Table 5. Water Contaminants and Interference with Different Laboratory Assays
Contaminant
Interference with Laboratory Assays
Particles and colloids
Yes: most laboratory assays and methodologies
Particles could block nebulizer and prevent efficient spraying of specimen solution into flame of AA assay
Damage to HPLC pump and injectors and increase in system back-pressure
Bacteria and their endotoxins,
Endotoxin, RNAses, DNAses, and proteases catalyze the hydrolysis of DNA and RNA, making them unstable in PCR,
enzymes, and nucleases
RT-PCR, and microarrays
Endotoxin inhibits cell culture
High bacteria count increases level of calcium-binding proteins, resulting in decrease of actual level
Bacterial alkaline phosphates can lead to cross-reaction in EIA3
Bacteria and its byproducts change media pH and contaminate pure culture, prevent cell growth, and affect IVF
success8-12
Gases such as nitrogen, oxygen, Form bubbles that interfere with accurate particulate counting or spectrophotometric measurements and block optical
chlorine, or carbon dioxide
sensors or fluid lines6
Chlorine in water can bleach the H&E staining in histopathology slides13
Organic contaminants (TOC)
Interfere with spectrophotometric testing, especially in UV absorbance
Organic materials consume calcium and reduce actual calcium in specimen
Increase background in fluorescent assays and HPLC, drifting or noisy baselines, and distorted peak shapes (ghost peaks)
Deactivate enzymatic reactions and inhibit cell growth6,12
Organics can affect hybridization steps, washing, and analysis, and fluorescence detection in DNA microarrays
Inorganic ions
Ions act as catalysts
Contamination by metallic ions under investigation would lead to higher estimation and increase blank signal in AA
Heavy metals such as lead, mercury, and zinc are toxic to various cells in cell cultures
Some ions, such as nitrates, absorb in UV range
AA, atomic absorption; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase–PCR; EIA, enzyme immunoassay; IVF:
in-vitro fertilization; H&E, hematoxylin-eosin; TOC, total organic carbon; UV, ultraviolet
certain organic contaminants inhibit cell growth during the
performance of culture assays.6,7,13
Ions such as sodium, calcium, magnesium, zinc, bicarbonate
chloride, and sulfate may affect biochemical reactions by
acting as catalysts or by inhibiting enzymes in EIA.6,8,10 The
resistivity and conductivity of different types of laboratory
water depend on the amount of ions dissolved in water.
Therefore, interference can occur in many colorimetric and
enzymatic methods when ions are present in water.4,6,7 Also,
ions such as phosphate and many other divalent types can
bind to DNA or RNA and interfere with assay results.6,13
Methods of Water Purification
There are many different techniques for water purification
that help laboratories produce high quality purified
water suitable for laboratory testing procedures (Table
6). Although these techniques are numerous, none is
adequate to satisfy the CLSI guidelines. However, many
laboratories use a combination of these methods to
maximize the purification process and consequently to
obtain highly purified water.
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Distillation is one of the oldest methods used in the
laboratory for water purification and is of relatively low initial
cost. This process includes boiling the water followed by
condensing the resulting water vapor. This method removes
most contaminants, such as bacteria, ions, organic
materials, and dissolved gases.6,7 However, this technique
has a relatively high maintenance cost, with the associated
low-flow rate and the need for a storage reservoir. An
additional limitation of distillation is that it cannot remove
some contaminants, such as silica and sodium.
Reverse osmosis (RO) is considered to be an effective
method of removing most types of contaminants, such as
ions, organics, colloids, particulates, and silica.6,8 Water
is forced via hydraulic pressure through a membrane that
excludes materials with a molecular weight of 100 to 200
da.7 The most common disadvantages of this method
are limited flow rate due to the small pore size of the RO
membrane and RO membrane damage caused by scaling,
fouling, and piercing.
Electrode deionization (EDI) uses selective anion and
cation semipermeable membranes and ion-exchange
resins (IERs) that are regenerated with low electrical
current.8,10 Electrodes are used to ionize water molecules
and to separate dissolved ions from water by forming
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Table 6. Water-Purification Methods and the Benefits and Limitations of Each
Method
Distillation
RO
EDI
Ion exchange
Filtration
Ultrafiltration
UV photo-oxidation
Benefits
Limitations
Simple, removes many impurities such as bacteria, ions,
dissolved gases, and organic materials
Removes most types of contaminants (particles, ions,
organics, and bacteria) as first purification step
Minimal maintenance cost and easy-to-monitor operation
parameters
Efficient in removing ions; low maintenance
Softener technology reduces water hardness before RO
processing
Remove ions effectively, easy to use, and relatively
inexpensive
Removes all particles and microorganisms larger than pore
size (0.22 µm)
Remove small contaminants (25-30 kDa) that are not
removed by filtration
Removes endotoxins, RNase, DNase, ALP, and proteases
Reduces organic contamination
Limited energy needed
Polishing step decreases TOC level
High maintenance cost, low flow rates, and storage reservoir needed
Some contaminants such as silica and sodium are not removed
Limited flow rate
RO membrane damaged by scaling, fouling, or piercing
Requires high feed-water quality to prevent plugging (RO feed water)
Limited capacity if resin is occupied
Organics not removed; bacterial contamination possible
Clogging possible; nonregenerable
Clogging possible with large contaminants
CO2 produced may decrease water resistivity
UV light does not affect ions or particles
Of limited overall value
RO, reverse osmosis; EDI, electrode ionization; ALP, alkaline phosphatase; UV, ultraviolet; TOC, total organic carbon
channels.6 EDI is very efficient because it removes ions
and is requires little maintenance.
Ion exchange involves the removal of ions from water using
IER. Ions in water are exchanged for other ions fixed to
the beads. This method acts as a softening and polishing
step that reduces water hardness by removing calcium and
magnesium.7 Removing ions effectively will enhance the
purification process by permitting resistivity to be higher
than 18 MΩ-cm at 25°C. However, a major disadvantage of
this method is its limited capacity for ion exchange when
all ion-binding sites are occupied.
Filtration involves the separation of contaminants in the
water by using a porous material, such as cellulose or
activated carbon filters, in which all particles larger than
0.22 µm are retained.7 Bacterial organisms are removed
using this filter during the final step of the purification
system.7 The main limitations of the filtration method
are potential clogging and inability to remove ions and
organics.
Ultrafiltration (UF) is a method that eliminates other
contaminants not removed by regular filtration. UF
removes most particles, endotoxins, pyrogens, enzymes,
microorganisms, and colloids because the pores of
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UF are small, ranging from 25 to 30 kDa.6-8,10 UF is the
preferred method of removing RNAses, bacterial alkaline
phosphatase, and endotoxins.6,10 Frequent clogging
and passage of ions and organics are considered to be
limitations to UF purification.
Ultraviolet photooxidation using wavelengths 185
and 254 disrupt the DNA of living microorganisms
by breaking the bonds among carbon, nitrogen, and
hydrogen atoms.7 The use of photooxidation at these
wavelengths is considered a germicidal treatment
and disinfection system for water.7,10 Moreover,
photooxidation of organic compounds reduces the TOC
level below 5 ppb. However, an important limitation of
UV photo-oxidation is that it produces free radicals that
can increase the conductivity of water.
A combination of purification technologies can provide
laboratories with consistently high water quality and
reduced levels of contaminants in water (Figure 1). Typical
water purification technologies include general filtration
to reduce particle load and the method of RO. The latter
is considered a standard pretreatment technique to
decrease the amount of organics, ions, particles, and
colloids. To eliminate variations in the quality of tap water,
EDI is included in the pretreatment process.10 RO-EDI
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Tap water
Pre treatment
reverse osmosis
electro deionization
Pure water
types II, III
Polishing
ion exchange
activated carbon
Ultrafiltration
designed storage reservoir and in glassware that meets
certain specifications defined by the manufacturer.
Type III water typically is used for washing and rinsing of
glassware; there are no specifications for its storage, to
our knowledge. In most laboratories, this type of water
is virtually the same as filtered tap water. Proper storage
of water is critical to avoid additional environmental
contamination that may interfere with test procedures.1
Ultraviolet
photo oxidation
Conclusion
Ultrapure water
type I
Figure 1
Flowchart for water purification using a combination of technologies.
treatment yields types II and III water. To provide water
suitable for advanced techniques such as molecular
diagnostics, single nucleotide polymorphism (SNP)
analysis, HPLC, and LC-MS, further polishing steps are
needed, such as ion exchange, use of activated carbon,
UF, and UV photooxidation.4,6,7,10 These technologies
complete the water-purification process by removing ions,
organics, and bacterial by-products to trace or minimal
levels. Therefore, after completing these steps, the water,
as an end product, exhibits high resistivity, low TOC,
and nuclease-free and bacteria free characteristics, as
confirmed via molecular biology testing.
Water Storage
Water can be contaminated easily during storage by
ions, gases, bacteria, endotoxins, silica, and particulates
leaking from containers, inner liners, plasticizers,
and piping.1 Maintaining the quality of ultrapure water
requires using dedicated glassware. Laboratories using
type I water should dispense water immediately before
use.1 Type I water must not be stored in tanks because
the atmosphere and the water containers may contribute
to the development of ionic and organic contaminants.1
The specifications for type I water cannot be obtained
from commercial bottled water because storage often
introduces impurities.6,7 Therefore, type I water should
be stored in specific glassware determined by the
manufacturers to be effective for this purpose. Type
II water may be stored for short periods in a properly
e164 Lab Medicine Fall 2014 | Volume 45, Number 4
Water is widely considered to be a laboratory reagent
because it constitutes a high percentage of most
reagent solutions used in many laboratory technologies
and assays. The use of high-purity water is critical for
accurate, cost effective, reliable laboratory analysis. A
proper combination of specific purification technologies
is required to produce the ultrapure water that is suitable
for each laboratory application and technique. Inadequate
monitoring of contamination in purified water can cause
serious laboratory errors. CLSI and ASTM guidelines
recommend measuring certain parameters of purified
water used in clinical laboratory applications as a tool for
quality control to achieve the desired purity for specific
application. In closing, we hope that this review the impact
of water quality on reliable laboratory testing and the
purification methods will serve as a resource to novice and
veteran laboratory professionals alike.
Personal and Financial
Conflicts of Interest
None declared.
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