Guidance

Referral of influenza samples to RVU, UKHSA Colindale, 2025 to 2026

Published 3 November 2025

Main messages

1. Samples to refer to Respiratory Virus Unit (RVU) during the 2025 to 2026 flu season

UK Health Security Agency (UKHSA) Clinical Network Laboratories (CNLs) should refer a maximum of 25 influenza-positive samples per week to the UKHSA National Reference Laboratory RVU, Colindale, prioritising samples as in Table 1.

Designated NHS collaborating laboratories should refer a maximum of 10 influenza positive samples per week to RVU, prioritising samples as in Table 1.

Clinical NHS laboratories can subtype locally; those that do not should refer a locally-agreed proportion of samples to their regional UKHSA CNL for influenza A subtyping.

2. Severe influenza

All influenza positive samples from individuals with severe influenza A (ITU/HDU/ECMO/fatal cases) should have subtyping attempted from an influenza A positive sample. Subtyping can be performed locally, if validated testing platforms are available, or by a UKHSA laboratory.

3. Unsubtypeable influenza A

There are a number of reasons why samples may fail H1pdm09/H3 subtyping attempts, including most commonly if the sample has low viral load, or due to recent viral evolution affecting assay performance, but also if the sample contains a zoonotic influenza:

• any unsubtypeable samples where clinical risk assessment indicates potential zoonotic exposure should be discussed with a CNL virologist and referred urgently to the appropriate CNL for A(H5) testing
• other unsubtypeable samples should be forwarded to UKHSA RVU Colindale.
• unsubtypeable samples once known to result from an established commercial diagnostic assay performance issue do not need to be forwarded for routine surveillance purposes but should be reported by the laboratory using the assay via the MHRA yellow card scheme

4. Suitability of materials

Please send ≥400 microlitres (µL) primary sample in viral transport medium, not in lysis buffer, for routine surveillance purposes.

Background

General principles

This guidance focuses on the sample flow for samples from individuals infected with seasonal influenza. For suspected zoonotic influenza, please see Guidance on the diagnosis and management of avian influenza.

The purposes of influenza surveillance are:

• to determine UK epidemiology to inform the World Health Organization vaccine program
• to assure influenza diagnostics performance and strategy
• to support patient care
• to contribute to pandemic signalling

Influenza strain surveillance informs the global vaccination programme and provides information for empirical antiviral choice, as well as pandemic early warning systems. Surveillance of influenza depends on accurate and timely virological information indicating which types of influenza viruses (Influenza A or B) and which subtypes of influenza A (H1N1pdm09 and H3N2) or lineages of influenza B (B/Victoria and B/Yamagata) are circulating and how closely related these are to seasonal influenza vaccine components. Influenza type classification is dependent on the characteristics of the internal genes of the segmented genome (usually NP, M or NS1 gene) whereas influenza subtyping is dependent on the characteristics of the genes coding for the highly variable viral surface protein haemagglutinin (HA).

Typically, most commercial assays for influenza detection provide influenza type information (differentiate influenza A and B), but do not provide influenza A subtyping or influenza B lineage assignment. Therefore, the majority of influenza test results determined in a clinical diagnostic laboratory setting will not provide the necessary strain information as needed for public health surveillance purposes or be able to differentiate a novel or zoonotic influenza A. Confirming the disappearance of B/Yamagata lineage since 2020 also remains critical for global vaccination strategy.

UKHSA National RVU is the WHO-designated National Influenza Centre (NIC) for England. There is a statutory requirement for UK laboratories to participate in national virological surveillance for influenza as part of UK’s responsibilities agreed at the World Health Assembly. It is for this reason that both positive and negative influenza testing results must be captured in the second-generation surveillance system (SGSS). Detailed data on the virological characterisation of influenza viruses is reported by the RVU to WHO on a weekly basis.

Comparison is made between virus strains circulating in the community, causing milder illness, and those strains associated with more severe illness requiring hospitalisation, and the degree of genetic and antigenic match between circulating strains to those included in the national vaccine programmes, informing tracking of disease burden and effectiveness of national vaccine programmes.

Surveillance periods

All clinical sample contributions are very valuable and boost the overall national picture of surveillance. UK national virological surveillance data is regarded as Official National Statistics output with some of the highest viewing figures of content on the government website. Influenza positive samples should be submitted regularly to ensure virological data is available for accurate weekly reporting (both nationally and internationally). All test results (both positive and negative) for influenza from all test centres must be reported to SGSS through electronic reporting from laboratory information systems.

Surveillance is divided into 2 periods yearly:

• ‘out of season’: the period of low influenza incidence
• ‘epidemic period’ or ‘in season’: the period of high influenza incidence, defined each year by observed influenza rates, and usually occurring somewhere between epidemiological week 40 of one year to week 19 of the next (approximately October to the following May) — a Chief Medical Officer alert notifies the start of the influenza epidemic season each year based on surveillance indicators

An example of the changing incidence of the influenza season can be seen in Figure 1. This shows the number of samples received by the RVU for influenza testing in a previous winter.

Figure 1. Influenza positive samples referred to RVU in a previous winter

Note: grey shading indicates epidemiological week 40 to week 20. The epidemic period usually falls somewhere within this and can vary from year to year.

Samples to send ‘out of season’

Out of season, when influenza incidence is low, UKHSA CNLs and NHS Collaborating Laboratories should refer all influenza A and B positive samples to RVU.

Other NHS laboratories can also refer influenza A and B positive samples directly to RVU out of season, up to a maximum of 10 samples per week (not subtyped or subtyped).

Samples from returning travellers must be sent with details of travel history, as these out of season or early season travel associated influenza strains are of particular interest for surveillance purposes.

Samples to send out of season are summarised in Figure 2.

Figure 2. Referral of samples ‘out of season’

See Guidance on the diagnosis and management of avian influenza for more information on suspected H5 samples. All suspected H5 samples should be discussed with a receiving virologist prior to referral.

Samples to send ‘in season’

During the epidemic period a proportion of representative positive samples should be referred from both CNLs and NHS laboratories.

UKHSA CNLs should send a pre-determined number of samples each week to the UKHSA RVU at Colindale. This includes representative, unusual and special interest samples that have already had subtyping performed. The maximum number of samples to be sent each week is determined yearly.

In the 2025 to 2026 season, 25 samples per week should be sent from CNLs.

Early and late in the season, there may be fewer than 25 appropriate samples, and therefore all appropriate samples may be referred in order to reach 25. Mid-epidemic period, when there are more than 25 appropriate samples, a maximum of 25 should be referred, selected using the priorities indicated in Table 1.

Designated NHS Collaborating Laboratories should send a maximum of 10 samples per week that have already had subtyping performed to RVU, with similar considerations.

Other NHS laboratories that do not perform their own subtyping should refer samples to UKHSA CNLs for subtyping (see Surveillance samples from NHS laboratories 2025 to 2026). Numbers of samples should be agreed locally. UKHSA CNLs will not charge for subtyping of influenza A positive samples performed for surveillance purposes.

Referral pathways are summarised in Figure 3.

Figure 3. Sample testing and referral pathways in England during the epidemic period (mid-season)

Table 1a. Priority level 1 [note 1]

Category	Samples
A	ALL Influenza A positive samples which cannot be subtyped as H3/H1pdm09 (or H5 where performed) that is, unsubtypeable.
B	Influenza A positive samples that have an unusual or unexpected PCR profile (not resulting from a known assay issue), or higher than usual Ct value difference between typing and subtyping assays (‘Ct mismatch’).
C	Patients admitted to hospital with severe influenza [note 2]. All ITU/HDU/ECMO and fatal cases should be included.
D	Influenza A samples associated with suspected outbreaks. Please ensure the samples are clearly marked as from an outbreak, with details of the outbreak setting name or location and type.
E	Samples from patients with suspected antiviral resistance.

Table 1b. Priority level 2

Category	Samples
F	Influenza positive samples from influenza only co-infections (that is, H1N1pdm09/H3N2 or influenza A and B), or influenza and SARS CoV-2 co-infections, with adequate viral load (for example, PCR Ct value ≤31, >400µL, in VTM).
G	Influenza positive samples from all individuals with a vaccination history for current season, with adequate viral load (for example, PCR Ct value ≤31, >400µL, in VTM)

Table 1c. Priority level 3

Category	Samples
H	Samples positive for influenza B [note 3], A (H3N2), or A (H1N1)pdm09, with adequate viral load (for example, PCR Ct value ≤31, >400µL, in VTM)

Note 1: for priority 1 samples, specimens with higher Ct values or below the usual volume requirement may be accepted where necessary – please indicate clearly on request form to avoid samples being inadvertently excluded. In these cases please also acquire and refer adequate samples where possible. Unsubtypeable samples resulting from an established commercial diagnostic assay performance issue do not need to be forwarded once the root case is identified, but should be reported by the laboratory using the assay via the MHRA yellow card scheme.

Note 2: severe influenza: Influenza requiring hospital admission with symptoms and signs of lower respiratory tract infection (hypoxaemia, dyspnoea, lung infiltrate), central nervous system involvement and/or a significant exacerbation of an underlying medical condition.

Note 3: lineage assignment for influenza B positives is necessary to confirm the observed disappearance of B/Yamagata lineage since 2020.

Surveillance samples from UKHSA Clinical Network Laboratories and NHS Collaborating Laboratories, 2025 to 2026

Priority samples for referral ‘in season’

Laboratories referring samples to RVU (25 per week for CNLs, 10 per week for NHS Collaborating Laboratories) should select samples according to the priority list given in Table 1.

Include all influenza positive samples from categories A to E (priority level 1) and F to G (priority level 2) and complete the batch with samples randomly selected from category H.

If a laboratory has more than 25 positive samples per week in categories A to G, please ensure that the 25 samples referred includes all those in categories A to E.

If more than 25 samples in categories A to E are identified in one week, please select the most useful samples for surveillance purposes, noting Acceptable referred samples. Unsubtypeable samples resulting from an established assay performance issue or due to low viral load (high Ct value) do not need to be prioritised. If more than 25 samples in categories A or B are noted within a week, consider local investigation of the underlying cause (such as an assay performance issue).

Process for referral of samples to RVU

UKHSA RVU Colindale requests influenza referrals from regional UKHSA CNLs and NHS Collaborating Laboratories, for surveillance purposes. No charge is made for processing these.

Primary samples (minimum 400µL) in viral transport medium (VTM) (not in lysis buffer) should be selected for referral to the reference laboratory for surveillance purposes, with subtyping assay results included. See the Acceptable referred samples section and the printable checklist in Appendix 1 to aid the sample referral process in laboratories.

Please use the current version of the E3 request form. The form collects information on detection and any subtyping assays used in the sender’s laboratory, which can help identify trends in assay performance including assay failures and is therefore an important piece of information for the Reference laboratory to fulfil its national advisory role. It is vital that the requested information – mainly through tick boxes - is completed as best as possible to support this function.

It is recommended to send samples in regular (weekly or biweekly) batches, if possible, except for those in categories A to E, which should be sent promptly to RVU for investigation and with the reason for sending, for example ECMO patient, clearly stated on the E3 request form. Please do not refer samples with low viral load unless in category A to E. Please do not send nucleic acid extracts or inactivated samples in lysis buffer. Samples (including those in lysis buffer) stored at room temperature for extended periods are frequently unsuitable for virus characterisation. Extracts and samples referred in lysis buffer or of inadequate volume may be rejected without testing unless in category A to E.

Surveillance samples from NHS laboratories 2025 to 2026

Severe cases

All influenza positive samples from individuals with severe influenza who require treatment in ITU/HDU/ECMO and fatal cases must have subtyping attempted from an influenza positive sample. Subtyping can be performed locally if validated testing platforms available or be referred to a UKHSA laboratory. Locally subtyped samples from ITU/HDU/ECMO/fatal cases should be sent on to RVU for further characterisation. Clinical information should be clearly stated on the current version of the typing of influenza strains E3 request form to avoid the sample being inadvertently excluded.

Subtyping for surveillance

During the winter epidemic period, NHS laboratories should not refer routine samples directly to the RVU for influenza diagnosis (for primary testing or for subtyping). Instead, NHS laboratories normally contribute to influenza surveillance through forwarding influenza positive samples to their regional UKHSA CNL (see Table 2).

During the winter epidemic period, NHS laboratories that perform their own subtyping do not need to send their samples to their regional UKHSA CNL for surveillance purposes. These laboratories can send up to 5 samples per week (following priority categories listed in Table 1) to RVU for further characterisation after subtyping. Please note Acceptable referred samples.

Unsubtypeable samples

NHS laboratories that perform their own H1pdm09/H3 subtyping and identify an unsubtypeable influenza A, should assess for the possibility of a non-seasonal subtype and forward clinically suspect samples urgently to their regional CNL (Table 1) for H5 subtyping, after discussion with a CNL virologist. Please also refer to Guidance on the diagnosis and management of avian influenza.

Unsubtypeable samples resulting from an established assay performance issue should not be sent to RVU after the root cause has been identified (see the Influenza A subtyping and referral of unsubtypeable or Ct mismatch samples section). Concerns with diagnostic assay performance should be reported by the laboratory using the assay via the MHRA yellow card scheme.

Table 2. Services provided by UKHSA CNLs and NHS Collaborating Laboratories

Region/HPT	Laboratory for seasonal influenza subtyping and avian H5 subtyping
Midlands	Birmingham
South West	Bristol
South East	Southampton
East of England, London	Cambridge
North West	Manchester
North East	Newcastle
Yorkshire and the Humber	Leeds

Note: the duty CNL virologist must be contacted to agree the sample referral pathway before sending any samples for avian influenza subtyping.

Guidance on specific sample types

Influenza A subtyping and referral of unsubtypeable or Ct mismatch samples

Influenza A detections may fall into one of several categories as far as subtyping information is concerned.

Either:

• subtyped — influenza A subtyping performed and subtype successfully allocated
• not subtyped — influenza A subtyping not performed and subtype unknown
• unsubtypeable — influenza A subtyping performed, but no subtype determined because the assay has failed to detect seasonal influenza H1N1pdm09 or H3N2
• Ct mismatch — influenza A subtyping performed, subtype Ct differs widely to influenza A Ct from the established norm

The category ‘unsubtypeable’ is different from not subtyped (that is, subtyping not attempted), which have separate tick-boxes on the E3 request form.

Causes of unsubtypeable and Ct mismatch samples

1. Non-seasonal influenza A (rare, high consequence)

Unsubtypeable results can arise if the sample contains non-seasonal influenza A. It is important to consider this possibility especially in cases of severe illness particularly if there is a history of travel or unusual zoonotic exposures and considering the ongoing worldwide epizootic of H5N1. In clinically suspect cases, H5 specific subtyping assays should be urgently performed in the appropriate UKHSA CNL or collaborating NHS laboratory (see Table 2) (see Guidance on the diagnosis and management of avian influenza). Suspected H5 samples should be discussed with a virologist at the receiving laboratory prior to referral.

It is unusual to see a Ct differential ≥5 between typing and subtyping assays where the same or a similar assay/platform is used for both. Samples with unexpected discrepancies observed between typing and subtyping assays (a higher than usual Ct difference when analysed on different platforms) could also indicate the presence of unusual viruses (that is, non seasonal/zoonotic H1/H3 viruses). Unexpected Ct mismatch samples where clinical risk assessment indicates potential zoonotic exposure should be urgently referred for further characterisation. The priority and suspected diagnosis should be clearly indicated on the request form, and relevant clinical information included.

2. Low viral load (frequent)

A plausible technical explanation for failure to subtype includes a low viral load, where subtyping assays may have a lower sensitivity compared to the generic detection assay. This can be observed where higher sample volume was used in the detection assay. A sample with Ct ≤30 in a generic influenza A assay should subtype.

3. Sequence variations affecting assay performance (frequent, with potential clinical significance)

Rapid virus evolution means that continued assurance for accuracy of influenza diagnostics for clinical use is required. For many commercial subtyping assays, the gene target and primer/probe sequences are not disclosed. In some influenza seasons, influenza A samples are classified as unsubtypeable due to minor virus strain sequence variations affecting performance of subtyping assays. Such variation can reflect recent in-season virus evolution. Samples should be forwarded for initial investigation together with information on the assay/manufacturer combination used for detection by completing the relevant sections in the E3 request form, until further investigation has identified the root cause of the issue.

Once the issue has been identified, please consider an update to the subtyping assay or use of an alternative assay. Further samples do not need to be sent to the reference laboratory.

Viral mutations can also affect pan-influenza typing assays targeting the internal genes with loss of sensitivity compared with subtyping results, as evidenced by higher-than-expected Ct values. This may lead to false negative results and/or loss of sensitivity of surveillance and clinical detections. Such discrepant results can usually only be detected by laboratories that perform multiple assays and observe an unusual or discordant pattern of reactivity – please forward any affected samples together with details of assay used for detection for initial investigation. Concerns with diagnostic assay performance should be reported by the laboratory using the assay via the MHRA yellow card scheme.

Sample processing and reporting at the RVU during the 2025 to 2026 season

Sample analysis at the RVU

In the 2025 to 2026 season, influenza positive samples received at the national reference laboratory will be selected for detailed analysis using influenza whole genome sequencing (WGS), subject to technical qualification, typically by ensuring samples have a high enough viral load and adequate volume. Samples that are collected in lysis buffer may be unsuitable for producing good quality influenza WGS data and preclude antigenic characterisation by culture. Primary material collected in VTM (>400µL) should be referred where available. Please do not send inactivated samples collected into lysis buffer. Details can be found in the Acceptable referred samples section.

Antigenic (phenotypic) characterisation is performed on a subset of viruses which are selected for virus isolation in cell culture. Both genotyping and phenotyping pathways have stringent requirements for clinical sample quality to increase chances for completed full analysis. Hence, selection for further characterisation beyond subtype classification will be based primarily on the viral load and availability of non-lysed material, with exemption for samples associated with severe illness or death, suspected exposure to non-seasonal influenza, and those with unusual testing profile/ greater than usual Ct mismatch, as this pattern of results could indicate the presence of unusual viruses or performance issues with individual assay platforms. Good completion of the questionnaire on the Typing of influenza strains request form; E3 especially for these exemption samples is crucial to prevent them from being inadvertently excluded based on Ct value.

Outbreak-related samples

RVU will further investigate subtyped samples for which characterisation is required for an outbreak investigation. Subtyped samples from localised or unusual outbreaks should always be submitted and will be subject to enhanced testing including sequencing. There is no PCR Ct value cut-off for referring influenza samples from outbreaks. Please ensure the samples are clearly marked as from an outbreak, with details of the outbreak setting name or location and type, and the CIMS reference if known, using the E3 request form.

Reporting of results from the RVU during the 2025 to 2026 season

The information obtained from each referred sample is an important component of the UK influenza virological surveillance. For typical influenza positive samples, referring laboratories will receive a standard report, without further sample specific results, with a link to the aggregated characterisation data provided in the weekly national influenza report. The data will be reported in the weekly national joint flu and COVID reports.

Reporting of results of genome sequencing, virus isolation, antigenic typing, or genomic typing for antiviral susceptibility markers of influenza virus to referring laboratories will be performed only for those samples where a specific request for characterisation has been made for patient management or epidemiological purposes, for example as part of an outbreak investigation. Reports to referring laboratories will also be issued if clinical or epidemiological follow up is required during surveillance testing, such as, if antiviral resistance is suspected.

Testing to inform patient management

To support clinical diagnosis and management, RVU can receive the following samples.

Influenza positive samples for antiviral susceptibility testing for clinical purposes

RVU analyses influenza whole genomes for genetic markers that have theoretically or clinically been linked with antiviral resistance. Laboratories with genuine suspicion of antiviral resistance must contact the RVU laboratory before sending samples for sequencing with the explicit purpose of antiviral genotypic testing. The routine turnaround time for results on these samples is such that the results are unlikely to be able to impact on patient management, unless specific arrangements are made.

AV genotypic testing should be considered when there is clinical concern about AV treatment failure. The greatest risk of oseltamivir resistance is currently in immunocompromised patients with influenza A(H1N1)pdm09. Further recommendations can be found in the guidance on use of antiviral agents for the treatment and prophylaxis of seasonal influenza.

The RVU Colindale laboratory can be contacted on:

• 020 8327 7125
• 020 8327 7002

Clinical queries can be directed to Dr Anika Singanayagam (Consultant Medical Virologist) via the Virus Reference Department enquiries line:

• 020 8327 7887

Acceptable referred samples

a. Volume and material

Reference laboratory work requires primary clinical material, minimum 400µL. Please do not send nucleic acid extracts or samples in lysis buffer to RVU.

Extracts and samples referred in lysis buffer in categories F to H may be rejected without testing if received. This is because a range of different analytical approaches that are part of the surveillance workflow, are incompatible with diverse lysis buffers. Samples (including those in lysis buffer) stored at room temperature for extended periods are frequently unsuitable for analysis. To prepare virus isolates and to obtain good quality virus whole genome sequence for surveillance purposes, it is necessary to use original non-lysed material, with minimum volume of 400µL.

b. Viral load

Please do not refer samples with low viral load (defined locally, for example Ct values over 31), unless priority 1 (see Table 1). Samples with low viral load are unlikely to be successfully sequenced or cultured.

c. Local assay used

Please do not refer samples which have been tested with an assay that does not generate PCR Ct/Cn values, unless in categories A to E (see Table 1).

A printable checklist to aid sample referral is provided in Appendix 1.

It is recommended to send samples in regular batches (weekly or biweekly), if possible, except for those in categories A to E, which should be sent rapidly to RVU for investigation. Samples greater than 2 weeks old may be unsuitable for virological analysis. Samples (including those in lysis buffer) stored at room temperature for extended periods may also be unsuitable for virological analysis. The current version of the typing of influenza request form (E3) should be used.