The effects of diseases on the human body were first
documented by ancient Egyptians however, the concept of
organ-specific disease and anatomic pathology have begun
to evolve only in the last few centuries, and the alterations
at the tissue and cellular level have gained attention
following the invention of the microscope
1. The
molecular pathology era has begun with the integration
of molecular tests into pathology practice, especially in
diagnosis of solid tumors and hematological malignancies,
as a part of the improvements in molecular sciences that
had been led by the completion of the Human Genome
Project in early 2000s. Pathologists now have a critical role
of morphomolecular assessment in this new generation
medicine era
2 and therefore have critical impact on the
preanalytical phase of molecular testing
3. Given that
pathologists combine molecular tests with conventional
pathological evaluation methods, pathology laboratories
should be designed and operated in accordance with the
requirements of molecular testing procedures.
Adequate space, appropriate equipment and qualified
personnel are required to establish a molecular pathology
laboratory. While the specifics of the requirements may
vary depending on the spectrum of the tests that will be
performed, there are several basic criteria that need to
be fulfilled for standardization. In this paper, the criteria
required to establish a molecular pathology laboratory will
be reviewed.
Required Physical Conditions
One of the most important points that should be taken
into consideration while designing a molecular pathology
laboratory is to create a plan to prevent contamination.
Polymerase chain reaction (PCR)-based methods are
especially susceptible to contamination 4. To obtain
a large number of copies from a very small amount of
the target sequence with the PCR method provides an
important diagnostic advantage, but this ability also leads to
false results in case of contamination. False positive results
may occur due to contamination from sample to sample,
transport of amplicons from the previous amplification
of the same target, cross-contamination of different
reactions prepared simultaneously, and contamination
of the reagents with DNA templates 5. In real-time
PCR methods; when the PCR reactions are finished with
fluorescence-based detection techniques, the analysis is
also completed at the same time. Since the PCR products
do not need to be reprocessed, the reaction tubes or closed
plates are not opened and the amplicon transport does not
occur 4. The laboratories using real-time PCR methods
therefore have less risk of contamination 5.
Main procedures performed in a molecular pathology
laboratory using PCR-based methods are pre-PCR
procedures (sample preparation, PCR preparation) and
post-PCR procedures (performing PCR and post-PCR
analysis) 4. It is critical to perform these operations in
areas separated as “clean” and “dirty”. The “clean” area represents the area where all pre-PCR procedures (such as
microdissection, DNA/RNA extraction, PCR preparation)
are performed, and the “dirty” area represents the area
where all post-PCR products (amplicons) are processed.
The staff and researchers should keep all reagents,
materials and equipment used in these areas separate at
all times and never move them back from the dirty area to
the clean area 6. Contamination is significantly reduced
by physical separation of the clean and dirty areas and by
doing pre-PCR and post-PCR activities in separate rooms.
Therefore, planning at least two separate rooms is essential
while designing a molecular pathology laboratory. It is
recommended to perform sample preparation steps such
as nucleic acid isolation in the pre-PCR laboratory, and to
perform PCR reactions and other post-PCR procedures in
the post-PCR laboratory 4. However, if there is sufficient
space, four separate rooms are recommended for the
preparation of reagents, sample preparation, PCR step
and post-PCR steps for an ideal molecular pathology
laboratory. Each room must have its own equipment,
protective clothing and consumables, and there should be
no material/equipment transport between the rooms 7.
The requirements for laboratory design may vary according
to the method used. For example, as mentioned previously,
3 rooms may be ideal in a laboratory where a real-time PCR
method is applied, since post-PCR analysis is not necessary
in the real-time PCR method 5.
Reagent preparation room is the room where reagent
stocks are prepared and then divided into a certain number
of small usable parts (aliquoted), and the reaction mixes
are prepared. This room should be free of any biological
materials such as DNA/RNA extracts, PCR products, etc.
The sample separation room is where the nucleic acid
isolation is performed and the isolated samples are added
to the PCR reaction mixes 5. This room is also called a
“low copy” room, as the number of copies has not yet been
amplified by PCR 8. Ideally, it is recommended to perform
the steps of nucleic acid isolation and addition of isolated
samples to the PCR reaction mixes in separate rooms, but
these two steps are usually performed in the same room but
in different areas/compartments since most laboratories
do not have sufficient space 5. Preparation of the PCR
reactions in a laminar flow biosafety cabinet ensures that
the area remains clean 9. The amplification (PCR) room
is where PCR devices are located and the amplification
steps are performed, and the post-PCR room is where the
analysis of PCR products by gel electrophoresis, sequencing,
nested-PCR, etc., methods are carried out. These two rooms
constitute contaminated-dirty rooms and no equipment
or materials used in these rooms should be used in other rooms 5. These rooms are also called “high copy” rooms
8. In the PCR applications such as real-time PCR where
single-stage PCR reactions are sufficient and tubes with
PCR products are not required to be opened, PCR devices
can be placed in the post-PCR room. However, in the
laboratories using PCR applications such as nested PCR,
etc., where multiple PCR reaction steps are required and
the tubes must be opened, PCR devices should be placed
in a separate room/area 9. In the amplification phase,
the primary and secondary PCR steps (if any), should be
separated according to the physical state of the laboratory,
preferably in separate rooms. If this is not possible, they
should be performed in separate compartments and on
separate PCR devices 7. Next-generation sequencing
applications also include one or more PCR amplification
steps, which are similarly recommended to be performed
in separate rooms/areas 5. Various recommendations
about minimum room sizes can be found in international
guidelines. For example, according to the field planning
criteria of the United States of America Military Health
System Pathology and Clinical Laboratories guide, the
reagent preparation room should be at least 120 sq ft
(approx. 33.5 m2) and the amplification room should be
240 sq ft (approx. 22.3 m2) in size 10. The relevant guide
published by the Republic of Turkey Ministry of Health is
detailed below.
Workflow
The workflow in the molecular pathology laboratory must
be unidirectional from the clean area to the dirty area 7.
When laboratory personnel and researchers are required to
move from dirty rooms to clean rooms, laboratory coats,
gloves and all kinds of protective equipment should be
changed and hands should be washed. No material should
be carried from the dirty room to the clean room 5. To
prevent the passage of personnel from the dirty room to
the clean room, it is appropriate to have separate personnel
working in each room or to perform pre-PCR and post-
PCR procedures on different days 9. There are automated
molecular pathology platform systems that provide
automatic one-way workflow and isolate nucleic acid from
the sample, combine the isolated DNA with amplification
reagents, and perform the analysis, and their use is becoming
increasingly common 5,11). The rooms and workflow
that should be present in an ideal molecular pathology
laboratory are shown in Figure 1. If all operations have to
be performed in a singl room, separate compartments/
benches are required for the reagent preparation, sample
preparation, PCR stage and post-PCR stages. The rule of
unidirectional workflow from the clean compartments
to the dirty compartments must be followed. If possible, sample preparation should be carried out in a laminar flow
biosafety cabinet including UV light. In the absence of
separate rooms, a timetable should be established in which
the pre-PCR and post-PCR steps are performed at different
times of the day 4.
Click Here to Zoom |
Figure 1: The Design and Workflow of an Ideal Molecular
Pathology Laboratory (Modified from references 4 and 8). |
Ventilation
Circulating air between pre- and post-PCR laboratories is
an important source of contamination in laboratories where
techniques detecting very small amounts of DNA/RNA are
used. Each laboratory should be ventilated separately and
the air pressure must be adjusted separately. At positive pressure, the air pressure inside the room is higher than
the air pressure outside the room, preventing the transport
of unwanted substances from outside. Negative pressure,
on the other hand, allows air to enter into the room and
prevents the migration of the air to the surrounding rooms/
laboratories. The doors must be kept closed to maintain
the negative pressure. There should be slight positive
pressure in the pre-PCR laboratory to prevent the entrance
of contaminated air from outside, while the post-PCR
laboratory should have a slight negative pressure to keep
the air in and thus to prevent the escape of amplicons from
the completed PCR samples. The ventilation of pre-PCR
and post-PCR laboratories should be opened to different
air channels and opened out from different locations 4.
Ultra-Violet (UV) Irradiation
UV rays that cause DNA damage are useful for eliminating
the contaminated DNA that may occur during addition
of the DNA template. UV light can therefore be used to
sterilize the pre-PCR laboratory. Since this method is
based on cross-linking with thymidine residues, the base
sequence of the target region plays a role in its success. In
addition, the hydration status of the DNA has a significant
effect on the UV resistance of the DNA. As dry-state DNA
is more resistant to UV light, UV light is less effective in
preventing contamination on dry laboratory surfaces
9. If UV light is going to be used on master mixes for
decontamination, care must be taken to ensure that no
dNTPs and enzymes are damaged in the UV light 8. The
UV light source can be placed on the laboratory ceiling or
bench and can be activated by a device on the exit door
as the last person leaving the laboratory closes the outer
door. If UV lights are used, UV-induced ozone must be
removed by ventilation. Accumulation of deposits due
to the precipitation of oxidation products on the glass
of the bulb during radiation occurs and this reduces the
effectiveness of the UV system. These deposits should be
removed monthly and the performance of the UV bulbs
must be strictly monitored 4.
The physical conditions required in molecular laboratories
in Turkey are defined by the “Guideline for Physical
Infrastructural Standards of Medical Laboratories applying
Molecular Tests” published by the Republic of Turkey
Ministry of Health 12. According to this guideline;
• Molecular diagnostic laboratories should have at least
two, preferably three rooms, each with a minimum
area of 15 square meters, physically separated from
each other to allow unidirectional workflow (from
preamplification to postamplification) and preferably
with separate ventilation systems.
• These rooms are defined in parallel with those
recommended in the literature and international
guidelines, as a ‘pre-amplification laboratory’ where
sample acceptance and nucleic acid extraction are
performed, an ‘amplification laboratory’ where target
amplification methods are applied, and a ‘postamplification
laboratory’ where analysis methods such
as electrophoresis, DNA sequence analysis etc. take
place after amplification.
• In order to prevent contamination, the issues of
providing a clean and preferably separate airflow,
preparation and storage of all reagents and chemicals in
their own areas, use of separate devices and materials for
each laboratory area (freezers, refrigerators, cabinets,
centrifuges, water baths, vortex mixers, pipettes, pencils,
timers, all kinds of consumables, etc.) are pointed out.
• It is emphasized that if only two rooms can be reserved
for the molecular diagnosis laboratory, preamplification
procedures and amplification / postamplification
analyses should be performed in separate rooms.
• For each laboratory; the issues of the presence of single
piece floor covering without pores, the presence of
hand washing sinks, the provision of temperature
and humidity monitoring, the use of UV irradiation
systems (on counter tops and / or room ceilings) during
non-working hours, the presence of sufficient storage
space, the presence of a sufficient number of grounded
electrical outlets and uninterruptible power supplies
(UPS, generator, etc.) for the laboratory devices,
and the placement of laboratory equipment to allow
unidirectional workflow are pointed out.
What to Do to Avoid Intermixing and Contamination of
Samples
Molecular laboratory tests are generally quite sensitive and
specific, providing very precise results 6. Even so, false
positive or false negative results may sometimes occur.
Control mechanisms that include the verification of the
primer and probe sequences, checking and confirming
whether the test conditions are optimal, and the use of
negative controls 9 should be employed to reduce false
results.
While amplification is generally a part of the molecular
diagnostic method, current nucleic acid amplification
methods are very sensitive with the capability of detecting
even a single molecule. Although this seems to be an
advantage, it should be kept in mind that the contaminated
DNA molecule may also be amplified, causing false positive results. Therefore, prevention of contamination must be a
priority in molecular pathology laboratories 9.
Cross-contamination is one of the sources of error and
contamination in the pathology laboratory and may occur
at any stage of the tissue processing process, such as during
macroscopic and/or microscopic evaluation, or during
DNA extraction, and may cause false positive or negative
results. Microorganisms (viruses, bacteria, etc.) may also be
transferred from one case to another during these processes.
Immunohistochemical staining with ABH blood group
antibodies, microdissection, and microsatellite instability
analysis can be performed to prevent and detect crosscontamination
2,13. As processing of the samples from
different patients occurs in the same area with recurrent
use of several instruments (e.g., microtome blade, water
bath) in pathology laboratories, precautions such as using
a new blade for each sample, washing the blade with DNA
decontamination solution, and/or sectioning of an empty
paraffin block between samples (called the “sandwich
model”) are used by various laboratories to reduce the risk of
cross-contamination 13. However, cross-contamination
rates have been reported to be around 3% (0-8.8 %) despite
these precautions 14.
The amplified DNA from the positive reactions in the
previous test, when the reaction tubes are opened after
amplification, is the source of contamination for the
subsequent tests 9. Also, amplification reactions are
exposed to contamination from other patients’ samples
and the target-containing plasmid 15. Samples may
contaminate the laboratory environment while pipetting
and the risk of contamination increases if multiple samples
are run together. Positive controls studied in the test are
risk sources for contamination as well. Clothing, laboratory
waste and/or uncleaned tables may contain contaminating
nucleic acids 9.
In order to prevent and control contamination, the
appropriate physical conditions, architectural structure
and design, meticulous application of laboratory
techniques and environmental control protocols, and
the workflow plan are essential. Using separate areas or
rooms for pre-amplification, amplification and postamplification
stages with separate ventilation systems is
an efficient way to prevent contamination, as discussed
in detail in the “Physical Conditions” section above. The
risk of contamination is lower in closed system devices.
Only personnel in charge should be present in the test
area. Every area/room should have its own equipment
including laboratory coats and pipettes 4,6,15-17. Minimum aerosolization while opening tubes is also
necessary to prevent transport between samples 15.
Cleaning before and after each procedure should be carried
out by using nucleic acid removers. For example, washing
with 10% bleach that is freshly prepared and rinsing with
70% ethanol can be performed 16. Thus, both biologically
hazardous substances and nucleic acids that may be
sources of contamination can be removed 15. Adding
enzymes such as uracil DNA glycosylase, which break
down DNA, into the amplified DNA to exchange some or
all of the thymidine with uracil in the reaction products
can prevent contamination biochemically 18. In addition,
contamination can be prevented or reduced by discarding
unopened tubes in the last stage, using pipettes with
positive pressure displacements, not talking during tests
such as PCR, and regular exposure of laboratory devices
to UV radiation 4,6. Aliquoting the reagents for each run
is another precaution for the prevention of contamination.
Patient samples and positive controls should be the last to
be put into reaction tubes to reduce the risk of transport of
the nucleic acids. If positive controls are going to be used,
the lowest dilutions should be preferred 15.
Water or DNA-free buffers can be used as negative controls
to detect and monitor contamination. The control tube
should contain all materials for all stages of the test, like
any other sample. A positive result detected in the negative
control indicates the possibility of contamination 4,9.
The adequacy control of chemical sterilization (such as
uracil glycosylase protocols) can be done by incorporating
a small amount of amplicon into a negative control 15.
Surface and equipment contamination can be checked by
swab samples from the laboratory surfaces where the test is
carried out with damp filter papers, and a positive result in
the swab sample indicates the presence of contamination.
In addition, more than expected positivity rates of a given
test may indicate contamination 4,9.
As RNA is more reactive than DNA and is vulnerable to
RNAses that are present in all cells, prevention of RNAse
contamination is very important in RNA-based molecular
tests. RNases are resistant to metal chelating agents and can
persist even after prolonged boiling or autoclaving. The
most common sources of exogenous RNAse contamination
are contaminated buffers and automatic pipettors. Also,
all laboratory surfaces and glassware can be contaminated
with RNAse from the laboratory personnel’s skin, hair, etc.
Wearing gloves and changing them frequently during all
stages of the test, use of separate laboratory equipment for
RNA based tests, aliquoting small amounts of buffers, use
of RNAse inhibitors (DEPC, etc.), and use of RNAase-free solutions and tubes are among the laboratory precautions
to prevent RNAase contamination 19.
Preparation of a documented action plan in case of
contamination is recommended. Most laboratories
quarantine and/or destroy all contaminated reagents and
consumables 15.
Equipment
Equipment and appliances may slightly differ between
molecular pathology laboratories based on their testing
profile. On the other hand, often a detailed inventory
list (Table I) is required to set up a molecular pathology
laboratory and provide standard testing. It should also be
noted here that equipment and consumables used in the
routine pathology setting must be available in a molecular
pathology laboratory, as well. It is also important to budget
for service contracts for maintenance and repair 6.
Unidirectional workflow should be taken into consideration
while organizing/placing the equipment (Figure 2). The list
of the appliances and equipment required in a molecular
pathology laboratory provided by Republic of Turkey
Ministry of Health is also a useful guide (Table II) 12.
Click Here to Zoom |
Table I: The list of equipment and appliances required in a molecular pathology laboratory. |
Click Here to Zoom |
Table II: The list of appliances and equipment required in a molecular laboratory as set by the Republic of Turkey
Ministry of Health 12. |
Click Here to Zoom |
Figure 2: Equipment required in a two-room PCR laboratory (Modified from reference no. 4); *: must be kept on different benches/ in
different areas. |
Instructions for calibration and maintenance should be
kept in the laboratory as written guidelines. Calibration
guidelines must include the schedule for calibration (e.g.,
daily, monthly etc.), instructions describing the steps of the
calibration procedure, calibration material specifications,
preparation and storage conditions, troubleshooting and
documentation methods, maintenance guidelines, the
schedule for maintenance, instructions for performing
maintenance, and troubleshooting guidelines 20.
Quality Assessment
Quality management is essential in all steps of pathology
evaluation (i.e., pre-analytical, analytical and postanalytical)
and a very important component in molecular
pathology practice. However, as the details of quality
management are beyond the scope of this review, only the
basic principles will be mentioned here.
There are several regulatory guidelines, including standard
operating procedure manuals, to be followed to set-up and/
or manage a molecular pathology laboratory 12,17,20.
The presence of errors affecting the accuracy of the results
in a molecular pathology laboratory should be checked
regularly under the supervision of the pathologist (“quality
control; QC”) to prevent or minimize erroneous reports
and provide the confidence that quality requirements will
be fulfilled (“quality assurance; QA) 21.
Turn-around time and test result statistics should be
checked and validated by using standard validation studies
22. For internal quality assessment (IQA), the use of
control materials is recommended (see previous sections
for details).
In addition to the internal precautions mentioned in the
previous sections, external quality assessment (EQA) must
also be performed at given intervals for specific types of
tests, as it is the most critical stage of quality management.
EQA, a measure of laboratory performance 23, has been
shown to be helpful to improve molecular pathology
laboratories 24, and EQA programs are the key elements
of a laboratory’s QA framework 25. Regular participation
in EQA is needed to verify and improve the quality of
testing, as molecular pathology EQA schemes score the
report and the test result 26,27. As a part of an EQA
scheme, participants receive test samples and their results
are then reviewed to check for errors.
The reports are scored and the participant laboratories
gain the opportunity to improve their service. Laboratories
across Europe are also required to have accreditation
26. Accreditation is a process in which an authorized
independent body officially recognizes that the laboratory
is competent to perform certain tasks, and may be
considered the most effective system for QA as compliance
with ISO standards is checked by accreditation bodies. Both
accreditation and participation in EQA are recognized as
effective and important tools to improve the accuracy and
reliability of molecular testing 28.
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