Guidance

NHS Cervical Screening Programme: laboratory quality control and assurance for human papillomavirus testing

Updated 25 July 2019

This document is for laboratories performing human papillomavirus (HPV) testing for the NHS Cervical Screening Programme (NHSCSP). It provides guidance on internal quality control (IQC) and internal quality assessment (IQA) procedures and their monitoring, plus external quality assessment (EQA).

Public Health England (PHE) commissioned the development of this guidance in response to queries received from English HPV laboratories that serve the cervical screening programme. We recognise that only laboratories accredited to ISO15189:2012 will provide testing to the programme.

This guidance reflects the standards implicit in ISO15189. Laboratories should refer to section 5.6.2 on quality control.

1. Internal Quality Control (IQC) Material

Some laboratories find in-house preparation of internal quality control material challenging. This is due to the volume of residual material available after routine processing and/or a lack of access to proxies of clinical material (for example, HPV containing cell lines).

This section provides guidance on IQC material and covers:

• sourcing
• preparation
• frequency of use
• monitoring
• expiry

1.1 IQC application, frequency and monitoring

The ISO standard 5.6.2.2 does not stipulate frequency of IQC inclusion. Variation in the use of IQC is to be expected. It will depend on factors such as:

• HPV test throughput of the laboratory
• platform constrictions or requirements
• local IQC protocols for molecular testing that may cover multiple targets (including HPV)

It is common practice to include an IQC positive and an IQC negative sample with each machine batch of samples in addition to any mandatory manufacturer controls. We note that as continuous loading platforms replace the batch type platforms, this way of operating may not be routinely feasible or relevant. Laboratories should include an IQC positive and negative sample every 24 hours as a minimum per instrument and with each new kit lot. This supports inter-laboratory analyser comparability and acceptance testing.

Laboratories should log and monitor IQC performance. Acceptability criteria for IQC pass or fail should be locally defined and the expected result should match the observed result at the qualitative level. A quantitative result is not currently relevant for management in the CSP. However, logging the numerical read-out of the assay (which will vary according to assay) using a Levey-Jennings chart is helpful in identifying assay-drift, and indicating any stability issues with the IQC.

Logging the numerical assay result can also help address the uncertainty of measurement of a particular assay. There are a number of specific software systems that can monitor IQC performance and support the identification of issues and outliers. Irrespective of system imposed for acceptability, laboratories should log any breach along with a description of the subsequent corrective actions.

Where possible, include the IQC material in the associated pre-analytical system so that the process is assessed in its entirety. We accept this will be operationally more challenging for some platforms. Recourse to the platform manufacturer to support with options is advised. Even if it is not routinely feasible to include IQC during the pre-analytical phase, laboratories should monitor the quality of the pre-analytical system in some way. This may be an intermittent testing of IQC samples.

Laboratories must label IQC material with the date it was made up and an expiry date which reflects the manufacturer’s product inserts for the LBC preservative and the HPV platforms.

1.2 IQC derivation or preparation: examples, pros and cons

Pooled material from residual clinical samples

Pooled, residual, anonymised cervical samples associated with positive or negative results can be used for IQC purposes.

1. Document associated results including date and type of testing.
2. Define and meet acceptability criteria before introducing into routine use. This may be aligned with local policy for molecular IQC.
3. Anonymise each pooled sample.

Pros: mimics a clinical sample.
Cons: some laboratories may have platforms that limit access to sufficient residual material so generating working pools is more challenging.

HPV containing cell line material

Laboratories can order HPV containing cell lines such as HeLa, Caski and SiHa from centralised accredited culture collections such as American Type Culture Collection (ATCC). HPV negative C33a cell lines are also available and laboratories may use these as negative controls.

Laboratories can use dilution of cell lines in cytological media for IQC material. However laboratories which are not co-located with culture facilities may not be able to store and propagate original material.

At the time of writing, the Scottish HPV Reference Library (SHPVRL) can provide pools of diluted cell-line material in preservative media to external laboratories. There is a charge for these samples to cover preparation and transit costs. Cell line material supplied by SHPVRL is linked to traceable National Institute for Biological Standards and Control (International Units) where possible.

Contact SHPVRL for further details and advice, for example on expiration and dilution.

Pros: ability to standardise input, more stable than clinical samples.
Cons: less of a proxy for a clinical sample.

Non NHS and commercial providers

Third party providers offer material that can be used for IQC purposes. The nature of the material ranges from lyophilised or freeze dried specimens that require reconstitution to buffered, to liquid samples representing separate HPV types. Relevance will depend on the chosen platform at testing site.

Pros: able to reference external existing validation.
Cons: additional costs (these will vary depending on the provider).

The matrix that some material is provided in may not reconcile with certain platforms. Laboratories must validate any manipulation of the commercially provided material outside the manufacturer’s instructions before the material is put into routine use.

2. Internal quality assessment

IQA is the selective retesting of a proportion of tested samples (approximately 0.5 to 1.0% of the total workload).

It is good laboratory practice to participate in IQA but not mandatory for IS015189. IQA can monitor all activities involved in the passage of specimens through the laboratory, starting from reception and ending in the dispatch of the final report. IQA schemes also assess the reproducibility of the laboratory sample processing and HPV testing techniques.

Laboratories may carry out IQA in ‘real-time’ before reporting results, or anonymise samples before resubmitting for testing. The latter approach avoids potential issues of discrepancies with previously authorised or reported results, which may cause anxiety in the women concerned.

When setting up an IQA scheme, take into account factors which may complicate analysis of results. These include:

• degradation of nucleic acid during storage
• inadequate specimen volume
• low frequency of positive specimens

Laboratories should record and regularly summarise discrepancies between results to identify any recurring problems or trends.

3. Inter-laboratory schemes

Inter-laboratory schemes involve (usually reciprocal) exchange of samples between willing laboratories. Laboratories must log, check and assess concordance between expected and observed results. They should fully investigate and record any discrepancies.

Inter-laboratory schemes are not mandatory for HPV testing if a laboratory already participates in an ISO accredited EQA scheme. However, they can complement existing procedures and can be helpful for troubleshooting.

4. External quality assessment

Laboratories providing HPV testing must participate in, and show adequate performance in, an ISO accredited EQA scheme.

EQA schemes which offer an HPV panel include Quality Control for Molecular Diagnostics (QCMD). A recent review of HPV EQA schemes is provided by Carozzi, F M and others (2015).

Laboratories should assess and document any performance issues with EQA and record any associated corrective actions – this is obligatory for accreditation purposes.

5. Validations

It is no longer obligatory for laboratories to test and report on a blinded external validation panel from SHPVRL if providing a ‘new’ service for HPV testing or if the laboratory is changing platform. Laboratories must use an HPV test that has been formally accepted by the NHSCSP (see guidance on acceptable HPV tests) and should perform validations in-house with the use of pre-annotated stored material and/or quality materials (external and, or internal).

Internal validation should include support from the commercial provider. It is anticipated that the kits required to support validation will be supplied gratis by the manufacturers.

Before live use:

• the commercial provider will determine the success of installation using analytical checks and processes relevant to the platform and provide documentation to confirm this
• an analytical validation panel of 16 samples in triplicate (n=48) with known/anticipated results should be tested over 3 independent runs by 3 separate operator(s)

Samples within the panel may include:

• standard positive and negative control material associated with the assay/platform
• residual material from HPV EQA schemes
• HPV containing cell line material, including that provided by NIBSC
• other independently sourced HPV control material

Additionally, SHPVRL can provide pre-annotated materials for quality control and validation purposes (where possible and where requested). These are managed on a case by case basis and cost-recovery for materials and courier costs are applied.

6. Annual QA data analysis

Laboratories must undertake yearly verification of HPV assays. Local templates for verification reports often incorporate a review of IQC and EQA performance as well as a summary of any operational or manufacture issues. Laboratories must clearly state the method of verification and criteria for success.

7. Other aspects of laboratory practice to ensure quality in HPV testing

7.1 Environmental swabbing

Laboratories should document results of environmental swabbing and record any associated issues or actions.

Some of the HPV techniques used in the laboratory amplify microbial targets present in screening samples. This amplification may produce billions of copies of these targets, which pose a contamination risk. Laboratories should reduce the risk of contamination with daily cleaning of benches, computer keyboards, equipment, door handles and so on, using DNA denaturing agent.

It is good laboratory practice to carry out monthly environmental swabbing of the testing areas to ensure quality of results pre- and post- decontamination procedures. Negative results from environmental samples provide reassurance that the laboratory is free from amplicon (which could cause false positive results).

7.2 Reagents, consumables and acceptance testing

Laboratories should verify each new reagent delivery prior to use, using a defined and documented procedure. There must be sufficient documentation explaining the criteria for acceptance. This ensures consistency of performance between batches, and that the change in reagent has had no impact on the quality of the examination.

7.3 Equipment calibration and metrological traceability

To meet the requirements of ISO15189 standard 5.3.1.4, laboratories must keep documented evidence of regular equipment servicing and calibration. The service report should be detailed enough to include tasks undertaken and results prior to handover, verifying the required measurement accuracy. If the supplier of the HPV platform maintains and services the equipment, laboratories should obtain calibration evidence from them.

Laboratories must regularly calibrate and service automated pipettes and pipetting instruments. They must have evidence of this, including a calibration certificate. Companies providing the calibrating and servicing must be compliant with ISO17024.

Laboratories should monitor refrigeration temperatures using a thermometer, calibrated regularly to United Kingdom Accreditation Standards (UKAS) standards. A data logger can be used to demonstrate a constant 24 hour temperature reading. This ensures monitoring of any peaks or falls out of the set range. Data loggers and appropriate software are commercially available.

7.4 Interfacing between HPV assay platforms and the laboratory information management system (LIMS)

ISO15189 standard 5.10.3 requires that the host laboratory verifies the electronic interfaces between the HPV platform and the LIMS. This ensures accurate transmission of results to service users. Further verification is required where a translation protocol or additional software (‘middleware’) is used to generate NHSCSP result codes from analyser HPV results as described in the implementation guide for primary HPV screening.

8. Staff training and competency

The supplier must provide training on a new HPV platform to an appropriate core of staff who can then cascade training to existing and new staff.

The Screening Quality Assurance Service (SQAS) must be provided with evidence that:

• training is provided on site
• all staff undertaking testing have completed their training
• staff have a certificate of completed training

All staff receiving cascade training should be provided with training logs. These should include:

• evidence of competency (an assemblage of observation, assessments, verbal assessments, panels of anonymised previously tested samples)
• signatures of assessors with dates
• review and future re-assessment dates

If a laboratory decides to adopt more than one HPV testing method, staff must be trained to the required level of competence in each of the tests used.