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Quality Control in a Virology Laboratory
Part 1; General Principles of Quality Control
The validity of diagnostic test results produced in each laboratory is entirely dependent on the
measures employed before, during, and after each assay. Consistency in the production of good
results requires an overall program that includes quality assurance, quality control, and quality
assessment.
Definitions
Quality Control - QC refers to the measures that must be included during each assay to verify
that the test is working properly.
Quality Assurance - QA is defined as the overall program that ensures that the final results
reported by the laboratory are correct
Quality Assessment - quality assessment (also known as proficiency testing) is a means to
determine the quality of the results generated by the laboratory. It is usually an external evaluation
of the laboratory's performance. Internal quality assessment programs can also be instituted.
Quality assessment is a challenge to the effectiveness of the QA and QC programs.
"The aim of quality control is simply to ensure that the results generated by the test are correct.
However, quality assurance is concerned with much more: that the right test is carried out on the
right specimen, and that the right result and right interpretation is delivered to the right person at
the right time".
Many variables can affect the quality of results
1. The educational background and training of the laboratory personnel
2. The condition of the specimens
3. The controls used in the test runs
4. The interpretation of the results
5. The transcription of results

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6. The reporting of results
Quality Assurance
QA is an ongoing process that requires daily attention by all laboratory staff. Some fundamental
issues in QA related to specimens include;
1. Inspection of all specimens upon receipt and before testing to ensure that they are suitable,
Lipaemic, haemolysed or contaminated samples should not be used as these may interfere
with assay performance.
2. Patient information should be available on the tube
3. If the serum sample is frozen before testing, the sample must be well mixed after thawing
and before testing.
4. All test results, controls and records must be checked and rechecked to ensure that the
proper specimen was resulted before reporting.
QA also includes such factors as;
1. Reporting results in a timely manner
2. Being sure that the results are reported to the appropriate individual
3. Making sure that the laboratory is functioning in the most efficient way
4. Including a continuing education program for laboratory workers
5. Evaluation of laboratory personnel to identify areas for improvement
6. Using the most reliable tests
7. Reviewing transcriptional measures
8. Verifying final reports
The following sections indicate components that must be continually monitored and represent
fundamental aspects of a good QA program

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A. Record Keeping
An efficient laboratory will be able to monitor the records of specimens from the time the samples
arrive until the time that results are released. Logbooks are an essential step in the recording of
laboratory specimens and should be kept confidential. Any specimen that is determined to be
inadequate for testing or that does not contain the essential information e.g. for HIV testing, should
not be tested and a note should be entered in the logbook. A worksheet must accompany each test
run in the laboratory. The worksheet serves as a guide when placing samples on the run. QC
records are important in validating laboratory results. A standard operating procedure manual
should be kept in the laboratory at all times and should be reviewed and updated frequently.
B. Monitoring Laboratory Staff
Laboratory managers may wish to periodically monitor the performance of their laboratory staff.
Samples with known results may be resubmitted discreetly along with the routine workload.
C. Vigilance in the Laboratory
Vigilance refers to the watchfulness and consists of always
1. Watching that the identification on the specimen matches that of the requisition slip.
2. noting the condition of specimens as they are received
3. reviewing QC charts everyday
4. observing co-workers
5. taking care that the laboratory remains a safe place to work
6. rechecking paperwork and worksheets before reporting a result
D. Verification of True Positive and Negatives
A positive HIV result is a serious concern and each laboratory must be absolutely certain that each
positive specimen result is correct. Once a sample is found to be positive by a screening test, an
aliquot from the initial specimen tube should be retested, wherever possible, a second specimen
should be collected from the individual and rested to eliminate any possible handling, labeling, or
clerical errors.

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E. Parallel Testing of Resubmitted specimens
Parallel testing of resubmitted specimens is important in those patients whose specimens have
yielded indeterminate results.
F. Reviewing Transcriptional measures
Transcriptional or clerical errors include mistakes made during the transfer of information from
the test readout to the worksheet, and from the worksheet to the computer or report form. These
type of errors probably account for the majority of errors in the laboratory. Possible mechanisms
include having a second technologist to check the final result and the supervisor to check the results
before releasing.
G. Reporting of Results
In the case of HIV testing, the handling of the results must be controlled so that the confidentiality
of all persons tested is protected. A policy decision on the handling of HIV test results must be
established and uniformly enforced in the laboratory. When an indeterminate result is reported,
care should be taken to ensure that the clinician understands the significance of such a result and
the importance of a follow-up specimen.
H. Interaction with Physicians
The relationship between laboratory personnel and physicians should be one of mutual trust and
respect.
I. Storage of specimens for follow-up testing
Once a specimen has been tested, it should be stored at -20o
C in the appropriate vial. An organized
serum or other specimen bank should be available
J. Laboratory Efficiency
The aim is to provide turnaround time for submitted samples. Certain items must be periodically
evaluated to increase efficiency in the laboratory.

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1. are the best available and economical tests being used?
2. are newer tests being continually evaluated?
3. Is the laboratory budget properly divided and utilized?
K. Total Quality Management
TQM refers to a comprehensive organizational approach that is focused on continually improving
the quality and efficiency with which the laboratory operates. QA is a defined program that is
focused on maximizing detection of laboratory error, while TQM aims to assist in this process by
maximizing efficiency. TQM is not only concerned with the monitoring of the QC/QA program,
but should also include other technical or administrative considerations that may indirectly
influence the quality and efficiency of the laboratory operation. This includes the evaluation of the
laboratory staff and continuing education.
Quality Control: Monitoring the Testing Process
As mentioned previously, QC refers to those measures that must be included during each assay in
order to verify that the test is working properly. The following items are essential elements of
quality control that must be performed during every assay:
1. Each run must include one full set of controls
2. The controls for each test run must yield results within the limits of the manufacturer's
criteria for acceptability and validity of the run.
3. All test kits must be used before the expiration date to ensure valid results
4. Physical parameters of the test such as incubation time and temperature must be followed
to ensure proper performance.
Ordinarily, each test kit has a set of positive and negative control that is to be included in each test
run. These controls are considered to be internal controls, while any other controls included in the
run are referred to as external controls. Internal controls are essential for QC measures for each
run and are intended for use only with the lot number of the corresponding test kit. External
controls can be included on a run to monitor consistent performance, lot to lot variation between
kits, and to serve as an indicator of assay performance on samples that are borderline reactors.

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External controls, or otherwise known as internal quality control (IQC) specimens are used in
internal quality control programs, whereby IQC samples are included in serological assays. The
IQC samples are then evaluated against Westgard rules, whereby the IQC values are plotted in a
Shewhart type chart (this may be in terms of arbitrary units or IQC o.d./Cut-off o.d.). Westgard
rules define specific performance limits and are designed to detect both random and systematic
errors. Of the six commonly used Westgard rules, three are warning rules and the other three are
mandatory rules. The latter, if broken should result in the rejection of the test run.
Warning Rules
1. The control value exceeds the mean by 2 s.d. (p<.005)
2. Two consecutive values exceed the target value on the same side of the mean by 2 s.d. This
may indicate the presence of systematic errors.
3. Four consecutive control values exceed the same limit. This again indicates the possible
presence of systematic errors and may indicate the need to perform instrument maintenance
or equipment calibration.

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Mandatory rules
1. The control value exceeds the target value by 3.s.d. (p<0.01:99.7% of values should lie
within 3 s.d.) This indicates that the assay run is out of control
2. Where the IQC sample is tested in duplicate, the difference in SD between the duplicates
should not exceed 4 SD
3. 10 consecutive values are on the same side of the mean or target value. This detects
systematic errors. This may happen when a new test batch or introduced or changes in the
calibration of equipment.

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Quality Assessment
Quality assessment is a means to determine the quality of results. It is usually an external
evaluation of a laboratory's performance that relies on incorporating proficiency panels of well-
characterized sera into the testing routine. External quality assessment (EQA) is now recognized
as an essential component of quality assurance and is the only means to give the laboratory
manager an independent means of ensuring that his routine quality control is adequate and
effective. The National External Quality Assessment Scheme (NEQAS) is administered by the
PHLS
The following are distributed under the NEQAS scheme:
1. Hepatitis B serology
2. HIV serology
3. Rubella serology (IgM and IgG)
4. General virus serology
5. Virus isolation

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6. Electron microscopy
It is important for the participating laboratory to treat NEQAS specimens in the same manner as
normal routine specimens. Rubella IgM and HIV currently lack national reference preparations, as
opposed to rubella IgG and HBsAg. Occasionally, more difficult specimens than usual are
distributed as "education" material, such as those containing very low levels of HBsAg or anti-
HIV. Scores are not normally allocated for this type of specimen, but participants may find it useful
to compare their results with those of their peers.
The scoring system for NEQAS is as follows
2 fully correct report
1 partially correct report e.g. partial identification of a virus or an equivocal serological result
0 erroneous result which would not have serious clinical consequences e.g. false negative rubella
IgG
-1 erroneous result which would have serious clinical consequences, e.g. false negative HBsAg
or anti-HIV
It is highly desirable for the laboratory itself to have an internal quality assessment program,
whereby anonymous clinical samples are submitted to the laboratory. An internal quality
assessment scheme can be used to monitor the quality of the work more frequently and accurately
than EQA schemes, since EQA samples are usually received infrequently and they are usually
treated differently from the routine specimens. Experience at laboratories that have an internal
quality assessment scheme has generally been that internal schemes are much better at identifying
quality problems in the laboratory than external schemes.

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Part 2; Quality Control in Clinical Virology Quality Control in Clinical Virology
Quality control in the clinical laboratory consists of a set of procedures designed to help ensure
delivery to the medical staff of laboratory results that are consistent and accurate. These results
must be supplied in a timely fashion while the data is still clinically relevant. A clinical virology
laboratory should be designed in a manner so that biohazard risks to the laboratory personnel and
the general public is minimized and that cultures are protected from environmental contamination.
A facility designed specifically for clinical virology should;-
1. Be physically separate from the microbiology laboratory and not share common air returns
or equipment such as hoods and incubators.
2. The environment should be controlled so that the ambient temperature is 22 - 26o
C and the
relative humidity 30 -50%.
3. The facility should be under negative pressure with respect to the rest of the laboratory
area.
4. Internally, the laboratory can be divided into positive and negative air pressure areas; the
positive for tissue culture and media preparation, the negative for viral isolation or serology
because they deal with viable pathogens.
5. All surfaces should be composed of materials that can be decontaminated easily.
6. Good standard microbiology measures should be observed such as daily decontamination
of all work surfaces, proper laboratory attire, use of safe pipetting devices, and to minimize
aerosol generation.
7. Biological safety hoods should be available for tissue culture and viral isolation. Hood
rooms should not have common air ducting and the exhaust from hoods in which pathogens
are handled be externally vented.
8. The facility must be properly maintained; biohazard wastes properly disposed, floors
disinfected periodically, air pressure balance checked.

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Written Procedures
Two sets of written procedures are important. One is for use by medical staff and the other for
laboratory use as procedure manuals. The procedures for medical staff should include;-
1. Purpose and limitations of the test
2. Hours the test is performed
3. Test turnaround times
4. Types and quantity of specimen required
5. Specimen transport and holding instructions
The procedure manual used in the laboratory should be complete enough in detail so that an
inexperienced technologist can perform the procedure without additional information. One copy
of the manual should be readily available to bench personnel, another copy should be stored
separately in case of accidents.
Specimen Transport
Specimens for viral isolation are often held for long periods of time before it reaches the laboratory.
Enveloped viruses such as RSV and CMV are extremely liable to room temperature and freeze-
thaw cycles whereas non-enveloped viruses such as enteroviruses tolerate these conditions well.
As a general rule, viral specimens held for short periods should be refrigerated, while those for
longer periods may be frozen at -20 or -70o
C.
Transport Media - the composition and type of viral transport media can affect viral isolation
rates. In general, the media should be a balanced isotonic solution at physiological pH. It should
contain a substance that will stabilize the virus such as gelatin, fetal calf serum or bovine serum
albumin, and antibiotics against bacteria and fungi. The swab should be made of a material that is
non-toxic to viruses, such as dacron or rayon.
Smears - smears are becoming increasingly popular because of the rapidity of staining techniques.
The smear should contain a reasonable number of cells, be of a reasonable size and not over-
contaminated by blood or pus, as the latter may lead to nonspecific staining.

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Specimens for Serology - Excessively haemolysed, lipaemic, bacterially contaminated, or leaking
specimens should be rejected. Sera should be heat-inactivated depending on the tests to be
performed. In the event of a specimen being rejected, the ward must be informed, preferably by an
oral report followed by a written one. Extenuating circumstances may warrant the acceptance of a
substandard specimen.
Tissue Culture and Media
Tissue culture remain the mainstay of non-serologically viral diagnosis. Therefore, adequate
quality control for commercially purchased or for in-house preparation of tissue culture cells is of
great importance. Within a given cell line, there may be significant variations in sensitivity to virus
isolation which may depend on the particular cell subline or clone and the passage number.
Information on a particular cell line should be recorded pertinently including the source, type,
passage number, confluency and cell condition. The loss of a cell line routinely passaged for use
can lead to a severe disruption of workflow and therefore, provisions must be made for back-up
cells in the event of contamination or laboratory accident. These back-up systems include;
1. Freezing and storage of low passage cells at -70o
C
2. Use of paired stock flasks. When the flasks reach confluency, only one is passaged, while
the second flask is held as a back up until the new flask displays good growth.
3. Carrying of a parallel set of stock flasks using a separate set of tissue culture reagents and
glassware.
Cells purchased from a commercial company should be certified to be free from mycoplasma,
fungal, and bacterial contamination and examined for contamination on receipt. Tissue culture
lines carried in-house should be subjected to;
1. Daily checks for growth rate and contamination
2. Mycoplasma contamination monitored monthly by Hoechst stain.
3. The sensitivity to viral isolation monitored by periodic TCID50 experiments with stocks of
reference virus.

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Other quality control procedures that may aid in minimizing the risk of contamination include the
exclusion of laboratory with infectious diseases from handling tissue culture, separate laboratory
apparel, reagents and glassware for tissue culture. Cell lines should be handled separately and the
cabinet be decontaminated in between.
Media - following filter sterilization, aliquots of the media should be taken and checked for
bacteriological or fungal investigation. These samples should be examined daily for 5 days and
should be free from contamination before that lot of media is released for use. Aliquots of all other
medium components such as fetal calf serum and L-glutamine should also be checked. New lots
of medium and fetal calf serum that have passed the sterility check should be monitored for their
ability to support cell growth.
Reagents and Kits
Reagents and kits should be ordered from reputable manufacturers or dealers with reliable
transportation systems. Upon receipt, the reagents should be checked for obvious breakage or
contamination. The quantity, source, lot number and date of receipt should be entered in a logbook
and the reagents stored according to the manufacturer’s storage specifications. When new lots of
any reagent are opened, the date should be noted on the container. Caution must be exercised in
the case of kits as different components of a kit may have different storage conditions and
expiration dates.
Instruments
Laboratory instruments should be subjected to routine preventive maintenance and checked and
calibrated on a regular basis. Some of these checks can be performed by laboratory staff and
entered into a logbook. The following are some recommendations for routine laboratory
maintenance and performance checks on instruments.
1. Incubators - daily temperature, CO2 and humidity checks. Weekly decontamination of
interior

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2. Safety cabinets - daily air pressure check and cleaning of UV lamp. Work surface should
be decontaminated after each use. Annual checks for air velocity and filter integrity and
paraldehyde decontamination as applicable.
3. Microscopes - daily cleaning of objectives and stage, log of lamp usage and annual
overhaul
4. Refrigerators and freezers - daily temperature check; annual check of compressor and
refrigerant levels
5. Water baths - daily temperature checks, weekly decontamination
6. Centrifuges - weekly decontamination, quarterly speed calibrations with tachometer,
annual inspection of motor and drive system
7. Autoclaves - daily temperature check and spore strip testing
8. pH meters - single reference buffer check before each use, multiple point check monthly
9. Pipetting devices - gravimetric volume check monthly; annual overhaul