C1.4.1The satisfactory application of nucleic acid detection techniques depends on the correct performance of all components of testing procedures, including patient preparation and consent (where appropriate), specimen collection, transportation, reagent preparation, nucleic acid isolation, amplification, product visualisation, data transcription, data interpretation, reporting, record keeping, sample storage and quality assurance.
Proficiency and competence
S1.4.1The laboratory shall maintain proficiency and competence by undertaking sufficient testing to maintain the knowledge, experience and expertise of staff.
C1.4.2Achieving and maintaining standards of practice and expertise requires an adequate number of samples to be tested with a given method (McGovern et al 1999).
G1.4.1The benefits of centralisation versus those of developing local expertise and autonomy should be carefully assessed by laboratories intending to establish a new nucleic acid detection test.
G1.4.2The performance of only a small number of tests with any given nucleic acid based method is discouraged.
G1.4.3Where small-volume testing is necessary, appropriate training, assessment and ongoing education procedures should be implemented to maintain and document the proficiency of staff in conducting these tests, and the number of quality assurance samples should be increased.
S1.4.2Trained personnel shall be available at the facility where the sample collection is performed. Specimens shall be collected in accordance with written specimen collection protocols.
S1.4.3Where patient-collected samples are used for diagnosis, clear instructions shall be provided to the patient, to reduce the likelihood of sample contamination.
C1.4.3To minimise the risk of contamination in nucleic acid amplification techniques, some special sample collection and preparation is needed, in addition to the usual requirements for pathology testing.
C1.4.4The precise method of sample collection, initial processing and transportation depends on the specimen concerned and the nucleic acid target (DNA or RNA). Specimens are to be collected according to the principles outlined in the guidelines below, because sample contamination can occur at any stage of specimen collection and processing.
C1.4.5The potential for false positive or false negative results to occur in genetic DNA testing, particularly for serious disorders, must be considered as part of the evaluation and setting up of diagnostic assays. One way in which this type of error can be minimised is to take two samples from a single patient at the clinical end, and process them at the laboratory end as if they were two distinct specimens. In some circumstances it may be appropriate to test a single sample by two methods, where two methods exist.
C1.4.6Patients may require that samples be collected using a deidentification protocol, through a trusted third party (TTP) intermediary such as a gene trustee (see Essentially Yours, ALRC Report 96, Vol. 1, Chapter 18, pp. 492-4). In such cases, patient and sample identification must use the coded identifiers provided by the TTP, and appropriate registration of the patient and sample identification numbers must be made with the TTP. To comply with the patient’s consent and with the TTP protocols, laboratories must not separately record any linkage between the actual physical identity of the patient and the identification codes provided by the TTP for the patient and any samples.
G1.4.4Wherever possible, nucleic acid detection tests should be performed on dedicated samples or on aliquots taken before other tests are performed. Where it is necessary to perform nucleic acid detection tests on samples that have already been used for other purposes and there is a significant risk of cross-contamination, the report should be annotated accordingly and the results confirmed on a dedicated sample (if one is available).
G1.4.5If samples are referred to another laboratory for testing, it is the responsibility of the referring laboratory to ensure that the sample conditions outlined above have been met, and to inform the receiving laboratory if they have not been met.
G1.4.6Single-use disposable collection equipment should be used.
G1.4.7Where nucleic acid detection tests are being performed for human genetic diseases that require written consent, additional procedures are strongly recommended to minimise the probability of errors through either:
a) the testing of two samples collected at different times, with both samples tested independently
b) where possible, splitting the samples on receipt in the laboratory and processing them in different batches.
G1.4.8However, in some cases, such as prenatal genetic testing, duplicate samples will not be available.
C1.4.7Guidelines for the appropriate transport of patient samples are contained in the NPAAC publication Information on the Transport of Pathology Specimens.
S1.4.4Where there are specific requirements for the transport and handling of samples for nucleic acid detection, these must be documented and must be available to referring practitioners and laboratories.
S1.4.5Where nucleic acid extraction is required, nucleic acids should be extracted and purified using standard methods. The procedures used for nucleic acid isolation from the full range of sample types, collection methods (eg patient versus staff collection) and the condition of specimens received by the laboratory must be validated, and procedures must be detailed in the laboratory methods manual.
C1.4.8The quality of nucleic acid prepared from a specimen has a major effect on the subsequent probability of successfully performing the test.
C1.4.9Care needs to be taken to ensure that DNA and RNA remain intact during sample storage, transport and preparation. If the number of target sequences in the sample is very small, a false negative may be obtained if degradation occurs. If the starting material for amplification is mRNA, the sample should be processed as rapidly as possible after collection to minimise RNA degradation by ribonucleases.
G1.4.9Specific instructions for handling samples to minimise nucleic acid degradation should be included in all relevant manuals and should be available to staff in collection centres.
G1.4.10Where samples of marginal quality, quantity or integrity have been received, the laboratory should notify the referring clinician and seek recollection. Where only a single sample is available, the test is essential and recollection is not possible, the report should be annotated accordingly.
C1.4.10Laboratory directors are responsible for ensuring the analytic validity of tests before they make them available for use in clinical practice. This is particularly important for nucleic acid detection techniques because of the range of tests, the high sensitivity of the methodology and the potential for variable specificity of amplification and hybridisation procedures.
C1.4.11The term ‘method’ includes kits, individual reagents, instruments, platforms and software. Elements of methods endorsed ‘research use only’ or ‘not for diagnostic use’ must be validated by the laboratory before use for diagnostic purposes, as outlined in the NPAAC publication Requirements for the Validation of In-House In-Vitro Diagnostic Devices (IVDs).
S1.4.6All test systems shall be appropriately validated before routine use. S1.4.7 Laboratories that have modified kit components or the manufacturer’s procedures must demonstrate equivalence or superiority of the modified procedure before putting the process into routine use. The modified procedure must be treated as an inhouse test for validation purposes.
C1.4.12Commercial kits have been developed for a number of nucleic acid detection tests for common human genetic disorders. A large number of nucleic acid detection techniques that are currently in use in Australia have been developed within individual laboratories, or adapted from published methods by laboratories.
G1.4.11Where a laboratory uses a commercial test kit in which the methodology and reagents are unchanged from the manufacturer’s instructions, the kit does not need to be independently revalidated in the testing laboratory.
G1.4.12Standards and guidelines for the validation of inhouse tests can be found in the NPAAC document Requirements for the Validation of In-House In-Vitro Diagnostic Devices (IVDs).
G1.4.13Both commercial kits and inhouse test systems should be regularly reviewed to ensure that the currency of results is maintained, taking into account new discoveries relevant to the field.
Validation of methods
S1.4.8In-house tests, or commercial tests endorsed by the manufacturer as ‘for research use only’ or ‘not for diagnostic use’, shall be validated in accordance with the NPAAC publication Requirements for the Validation of In-House In-Vitro Diagnostic Devices (IVDs).
S1.4.9If any modification is made to a commercial IVD, it must be treated as an in-house IVD and it must be fully validated in accordance with the NPAAC publication Requirements for the Validation of In-House In-Vitro Diagnostic Devices (IVDs).
S1.4.10Validation data shall be retained by the laboratory in sufficient detail to enable external review. The period for which laboratory records are to be retained is stipulated in the NPAAC publication Guidelines for the Retention of Laboratory Records and Diagnostic Material.
C1.4.13Laboratories shall only offer nucleic acid detection tests as routine tests if their technical validity has been established, either by the laboratory or the manufacturer.
G1.4.14Where a validated test is available, it should be used in preference to a nonvalidated test. If a nonvalidated test must be performed because of clinical necessity, the report should clearly indicate that the diagnostic validity of the test has not been established.
G1.4.15The procedures and methods used in nucleic acid detection techniques for diagnostic purposes should be validated for clinical use according to the following principles:
a) Evaluation should use known positive and negative samples.
b) Evaluation should be by comparison with proficiency test material, if available.
c) Evaluation should be by comparison with an existing validated method in the laboratory.
d) Evaluation should be with all specimen types and conditions that will be used to make laboratory diagnosis.
e) If a significant modification to an analytical procedure has been made, the modified procedures should be compared to the original using either identical samples or identical types of samples.
f ) Reproducibility should not be determined by repetitive analysis of the same sample.
g) The sensitivity of a test, or cutoff values, should be set at a level that is relevant to the diagnostic use of the test.
C1.4.14Nucleic acid detection techniques are usually designed to maximise sensitivity and are capable of detecting very small amounts of nucleic acid.
C1.4.15Contamination may occur:
a) during specimen collection or transport
b) during handling or testing in the testing or referring laboratory before nucleic acid detection
c) during extraction of nucleic acids from the sample
d) during amplification
e) during product detection
f ) by contamination from reagents used for the test. C1.4.16 The sources of potential contamination include:
a) positive samples (cross-contamination)
b) amplified nucleic acid (eg contamination of stock reagents or equipment, or in aerosol droplets)
c) operator-derived nucleic acid.
C1.4.17Mathematical methods can be used to assess the likelihood of contamination in the use of polymerase chain reaction (PCR)-based methods (Shapiro 1999).
Measures to control contamination
S1.4.11Laboratories shall retain records documenting contamination events, the identified source of the contamination and measures taken to reduce the risk of future similar contamination events.
G1.4.16The greatest protection a laboratory has against contamination arises from:
a) the competence of the staff in performing laboratory tasks
b) the design of the laboratory
c) the routine use of controls to detect contamination.
G1.4.17These issues are addressed in detail in the relevant sections of this document.
G1.4.18For single round PCRs, the contamination risk may be reduced by replacing thymidine with uracil. The amplified product can then be destroyed by uracil-N-glycosolase, which is added to new samples. This is not sufficient to deal with heavy contamination and is not a substitute for the other measures. Also, it cannot be used in nested PCR, a technique that poses additional contamination risks due to the large amounts of second-round product, which is smaller and more resistant to decontamination procedures. Probe amplification methods (eg branched-chain DNA) have low contamination potential and may be performed in routine laboratory areas, provided those areas are not used for specimen processing.
S1.4.12Spills shall be cleaned up promptly. Those containing amplified nucleic acids shall be covered with absorbent paper soaked in 2–10% sodium hypochlorite and left for 10 minutes. The absorbent paper shall then be discarded and the area wiped over with 2–10% sodium hypochlorite.
S1.4.13The level of laboratory hygiene shall be high. Staff shall be careful to avoid contamination of their gowns and gloves. These shall be changed promptly if they have potentially been contaminated or if they become soiled.
G1.4.19High standards of laboratory hygiene are required, including the measures below. All work areas, especially those used previously for other testing, should be cleaned thoroughly.
G1.4.20Gowns and gloves are to be worn by laboratory staff in the work areas. Gloves are to be discarded and hands washed before leaving the area. Gowns should be dedicated to each area. Gowns or gloves should be changed whenever there is evidence of soiling.
G1.4.21Tube or pack racks used for holding tubes or plates containing amplified nucleic acid should be decontaminated in 2–10% sodium hypochlorite (or similar reagent) for a minimum of four hours.
S1.4.14All equipment shall be maintained in working order and shall be subject to a regular schedule of maintenance in line with the manufacturer’s recommendations.
S1.4.15Centrifuges used for preparation of biological samples shall have either sealed buckets or a sealed rotor.
S1.4.16All assays shall include appropriate negative controls that are subject to the whole test process, including the extraction.
S1.4.17Maternal cell contamination controls shall be used for prenatal diagnosis of human genetic disease.
S1.4.18Where a negative result is obtained, there must be confirmation that DNA has been extracted.
S1.4.19In assays using mRNA as the starting material, control measures must be incorporated to detect the success of mRNA extraction and of reverse transcription on that particular sample.
C1.4.18The types of controls used in nucleic acid detection techniques will vary with specific assays.
C1.4.19The exact number of controls required for PCR-based systems depends on the number of samples in each run. A negative patient sample is essential, and laboratories may also include a ‘no nucleic acid’ sample (ie all reagents but no nucleic acid) to determine whether reagent contamination is the cause of reactions in the negative patient sample, should they occur. A negative control should be placed after the last patient samples.
G1.4.22Where test runs are expected to contain a large proportion of positive results, additional ‘no nucleic acid’ controls should be interspersed among the patient samples at an appropriate frequency, as validated by the particular laboratory.
G1.4.23Positive controls should be selected to verify that the test would detect significant variants.
G1.4.24Positive controls should be just above the limit of sensitivity of the test. G1.4.25 Assurance that successful mRNA extraction and reverse transcription has occurred can be achieved by testing the sample for a housekeeping gene such as beta-actin.
C1.4.21Laboratories performing nucleic acid amplification testing should be aware that erroneous results may occur for reasons other than contamination. These may occur through:
a) nonspecific primer binding and amplification of other sequences, which are then misidentified as the target sequence
b) amplification of similar or identical target sequences found in other portions of the eukaryotic genome
c) nonspecific, usually low-level, reactivity in the detection system
d) primer design and binding
e) limitations of the methodologies of detection
f ) presence of deletions.
C1.4.22The frequency and significance of these events will vary with the test technique, the target sequence chosen, the detection system used and the patient population.
G1.4.26It is strongly recommended that, where the implications of a positive result are substantial, and where the specificity of the test is known to be suboptimal or has not been established by extensive validation, one or more of the following methods be performed to verify positive results:
a) use of a nested PCR or other techniques that require the binding of more than one set of primers to generate a positive result
b) repeat testing of all positives using another set of primers directed at a different target sequence
c) identification of the product using methods such as specific probes, sequencing or restriction enzyme analysis.
G1.4.27It may be necessary to use several of these methods to achieve the desired specificity.
Nucleic acid sequencing
S1.4.20DNA sequencing used primarily to detect an unknown mutation must be undertaken with particular care. Efforts must be made to confirm that the sequencing methodology is appropriate (eg limited BLAST search or comparison with a known standard sequence, or a second independent assay).
C1.4.23DNA sequencing is now an alternative approach to the detection of DNA mutations or DNA changes in human genetic disorders. The benefits of DNA sequencing are that it is a robust technology that allows for high-throughput mutation detection and characterisation in a single assay. The major problem with the technology is that the signal from each base pair needs to be individually considered and compared with one or more controls. Software packages are being marketed that allow rapid screening of DNA sequence data, but in their current form are not sufficiently robust to allow fully automated analysis.
C1.4.24The reasons a laboratory may wish to sequence a PCR product may include:
a) mutation screening, which is the detection of an unknown mutation in a length of DNA
b) confirmatory testing
C1.4.25In some cases, the laboratory to which the sample is referred for DNA testing undertakes to have the sequencing carried out in a DNA sequencing facility. By their nature, centralised DNA sequencing facilities carry out a broad range of activities that, in the main, are research oriented. Diagnostic-quality DNA sequencing must be distinguished from research-based DNA sequencing, and must conform to standards which ensure that false positive and false negative results are minimised. Therefore, the DNA sequencing facility that is used to perform a DNA diagnostic test should be accredited by the National Association of Testing Authorities and the Royal College of Pathologists of Australasia or ISO certified.
C1.4.26The DNA sequencing facility may not always receive sufficient information to distinguish between research work and service diagnostic work. Therefore, the onus is on the referring laboratory to ensure that the DNA sequencing facility undertakes the necessary controls and standards that would normally be expected for a more traditional DNA laboratory. Included in this would be quality assurance, quality control, appropriate retention of records, standards with each run and published criteria on what is acceptable for the particular DNA sequencing run (including such criteria as peak intensity, baseline fluctuations and signal-to-noise ratio).
C1.4.27A valuable document for DNA sequencing of genetic disorders is the Clinical Molecular Genetics Society’s Best Practice Guidelines for DNA Sequencing Analysis and Interpretation.1
C1.4.28A guide to what standards would be expected in sequencing for human leukocyte antigen typing may be found in the American Society for Histocompatibility and Immunogenetics standard protocol for human leukocyte antigen typing. For new technologies, appropriate validation must be performed.
1 Available from http://www.cmgs.org