No Family History of Cystic Fibrosis How Does Baby Have Presumptive Positive

This handbook is for laboratories that provide an NHS newborn blood spot (NHS NBS) screening service for cystic fibrosis (CF) in the UK. It defines a framework for the pre-analytical, belittling and post-belittling steps in the newborn screening process to:

support newborn screening laboratories to provide the screening service
improve consistency beyond the screening programme
provide guidance on achieving practiced quality past application of standards and audit

Use this handbook aslope other NBS screening programme guidance.

Screening for CF

All babies beyond the Britain are offered a blood test to identify CF equally part of the NHS NBS screening programme. Screening for CF aims to identify babies with a classical CF diagnosis earlier they develop symptoms, to enable the early initiation of high-quality care.

About CF

CF is a heterogeneous disorder with a large number of different factor variants affecting the putative gene (the CF Transmembrane Conductance Regulator (CFTR) gene) and has a range of different clinical phenotypes. For further information, see the Cystic Fibrosis Trust website and research on the hereafter of CF care.

Whilst babies with a clear CF diagnosis represent the majority of true positives, identification of babies with an inconclusive diagnosis after a positive NBS issue is an increasingly recognised outcome. This leads to uncertainty for both families and healthcare professionals.

The approach to the care of these babies is evolving with increased feel and reporting of outcomes. Babies with an unclear diagnosis later NBS screening are designated as 'CF Screen Positive, Inconclusive Diagnosis' (CFSPID). There is international guidance on the intendance of these babies. It is of import that strategies are based, whenever possible, on prove of benefit for the child and family. To default CFSPID babies to a full CF care pathway is non appropriate, but information technology is equally important that the potential risks that these babies confront are recognised and addressed.

General organisation of NBS screening

CF screening is fully integrated inside the existing NBS screening programme and based on the same screening laboratory populations. The initial screening exam (the assay of immunoreactive trypsinogen (IRT)) uses blood nerveless on the standard NBS screening sample collection carte (see section ix.ane). Quality assurance (QA) and performance management arrangements follow the same general principles equally those for other newborn screening programmes.

With CF, as for other blood spot screening conditions, the screening laboratory is a major communication hub. The laboratory sends screening results to child health records departments (CHRDs)/child health information services (CHISs), which send negative results to the parents/carers. Over 99% of results are 'CF not suspected' and are generated promptly. Yet, in some cases, screening may not be completed until the baby is over a month old.

The CF screening programme requires partnership working between the screening laboratory and molecular genetics laboratory.

Some parents/carers may decide that they do not want their baby to be screened for some or all of the conditions covered by NBS. Screening can be declined for CF, sickle prison cell illness and built hypothyroidism individually, only the 6 inherited metabolic disorders (IMDs) tin can only be declined equally a group. Details of these vi inherited metabolic disorders are available.

The sample taker completes the blood spot card to signal which conditions have been accepted or declined, as outlined in the NBS screening sampling guidelines.

The screening protocol

The Uk screening protocol is intended to:

maximise the early detection of CF across all populations so that pre-symptomatic handling tin can be initiated to reduce the long-term morbidity associated with the condition if untreated
minimise the demand for second heel pricks, and any other causes of diagnostic delay
minimise detection of unaffected heterozygotes
minimise recognition of babies with CFSPID
let for the fact that a clear diagnosis is not always possible

The standard protocol

IRT analysis is performed on a single spot from an initial dried claret sample followed by a one or two-stage genetic analysis of the CFTR gene on all samples with confirmed IRT concentrations in a higher place a defined cut-off. Subsequent activity depends on the results of the genetic assay.

This protocol is summarised diagrammatically in Figure i beneath. No alternative to this protocol is recommended. A text description of Figure one is available.

Figure 1: cystic fibrosis screening algorithm

Definition of the cut-off values

As of January 2021, the national cut-off values for the AutoDELFIA analyser are:

cut-off A = 52 µg/50
cut-off B = 62 µg/L

Equally of January 2021, the national cut-off values for the GSP analyser are:

cut-off A = 45 µg/L
cut-off B = 55 µg/L

These cutting-off values are bailiwick to regular review based upon continued data collection from the laboratories.

The value used for cutting-off A is based on selecting an average of one.five% of all results for echo IRT analysis. Information technology is set to 10µg/L below cutting-off B.

Cut-off B, used to select samples for further genetic analysis, has been defined based upon feel in East Anglia using a two-stage IRT-IRT screen over a 17 year period that showed a 99.5th centile cut-off for the initial sample gave an overall sensitivity of 97% (Heeley and others, 1999)^{[footnote ane]}.

For farther data about the rationale of the cutting-off values, see section 5.1 below.

The first screening specimen

Samples are initially assayed in singleton.

Consider babies in whom the IRT concentration is < cut-off A in the initial screening sample to accept a negative screening result for CF. Report them as 'CF non suspected'.

Re-assay samples with results ≥ cut-off A in indistinguishable from the aforementioned carte du jour simply on a different spot(s) to give a more definitive result. This is to minimise effects of volumetric variability of the punched discs, day-to-day variation in IRT assay scale, and to detect possible sample misidentification.

Accept the average of the 3 results.

Review the set of triplicate results for consistency as poor analytical performance tin can produce dissimilar results. Spuriously loftier results tin can occur with faecal contamination or blood spot layering and spuriously low results can occur if there is a missing spot or poor sample.

Farther action therefore requires an cess of agreement between results. The post-obit sections provide guidance merely and laboratories should use discretion for each individual set of results.

Review of triplicate mean results

For babies in whom the triplicate mean IRT concentration in the initial screening sample is < cut-off B, and provided in that location is no suspicion of a 'missing spot', effect a 'CF not suspected' event.

If the average of the 3 results is ≥ cut-off B *, assess the results for consistency before proceeding further.

The pathway for handling IRT results from the first screening specimen is summarised diagrammatically in Figure 2 below. No alternative pathway is recommended. A text description of Figure 2 is bachelor.

Figure 2: handling of IRT results from the get-go screening specimen

Regard a ready of three results as discrepant if they encounter any ane of the following criteria:

a coefficient of variation (standard deviation/hateful) ≥ 0.25
one or more than private results being ≥ xxx% higher up or beneath the average of all 3
the ratio of the highest event to the average of the other 2 results ≥ 1.five

For sets of 3 results considered to exist discrepant:

if at that place is a single loftier result (≥ cut-off B *) with the other two results < cutting-off B *, and provided that there is no uncertainty almost sample identification, ignore the single high issue and consequence a 'CF not suspected' report
if ii results are ≥ cut-off B * and the boilerplate of all 3 results ≥ cut-off B* but < 120 µg/50, proceed to genetic analysis every bit per the screening protocol
if the average of all three results is ≥ 120 µg/L, request a echo sample on the grounds that this is likely to be sample contamination (only if a repeat sample tin be taken earlier the babe is 21 days old, otherwise an immediate referral is indicated)

*If the sample was taken on twenty-four hours 21 or subsequently then employ cut-off A.

Assaying further samples from a menu with discrepant results is unlikely to completely resolve the issue.

Bereft sample: no re-assay possible

If it is not possible to dial whatsoever farther discs from the carte (for example, for a single result ≥ cut-off A), request that a repeat specimen is taken as before long as possible due to insufficient sample.

Only one re-assay possible

If but 2 IRT results tin be obtained (and so after the initial assay, only one further spot from the carte is possible) and then proceed as follows. If the average of both results is:

< cut-off B *, written report equally 'CF not suspected'
'≥ cutting-off B *, proceed to genetic assay; widely discrepant results may crave a repeat specimen taken as soon as possible– laboratory staff should use judgement on this.

*If the sample was taken on day 21 or after then apply cutting-off A.

The second specimen (repeat IRT exam)

Requests for 2nd claret samples for IRT testing are via locally agreed pathways defined as part of the newborn screening responsibilities. Laboratories should request that the repeat blood spot sample is collected on day 21 (by solar day 24; this allows for day 21 to fall on a weekend when a special visit is not warranted).

The 2nd dried claret spot specimen is for IRT testing only. Information technology is recommended practice not to repeat other screening tests on this specimen.

The sample taker should explicate to parents/carers that further tests need to be done for cystic fibrosis. An information canvas for parents about the repeat blood spot test for CF is available on GOV.United kingdom of great britain and northern ireland. The results of genetic analysis are not given out at this phase as this may issue in premature disclosure to the parents/carers earlier the definitive screening effect is known. Confirm the repeat asking in writing to the appropriate health professional person(due south).

If in that location is a delay and a second sample has not been taken past 8 weeks of age, it is too late for IRT testing to be carried out reliably (see section 2.3 below).

Actions to exist taken on the echo IRT test

The post-obit actions are undertaken on the repeat sample depending on the reason for its request.

If one CFTR variant is detected – assay the repeat sample for IRT in duplicate. Report babies with an average of results ≥ cutting-off A as 'CF suspected' and refer to a CF specialist. Report babies with an average of results < cut-off A equally 'CF carrier'. No referral is required. A health professional person contacts the parents/carers to discuss the screening results.

If no CFTR variants are detected and the initial IRT ≥ 120 µg/50 – assay the repeat sample for IRT in duplicate. Written report babies with an average event ≥ cut-off A as 'CF suspected' and refer to a CF specialist. Otherwise, result a 'CF non suspected' effect.

For information virtually the reporting and advice of results, see section 7.iii beneath.

Late sampling

Blood spot IRT concentration is increased in most babies with CF in the first few weeks of life. However, early inquiry on blood spot screening and on age-related decline in IRT indicates IRT becomes unreliable as an indicator of CF at around viii weeks. This reject in IRT has implications for the reliability of CF screening if samples are taken outside the specified time windows. It is therefore important that appropriate cut-off values are practical to the times at which samples are taken.

This means that CF screening cannot be undertaken or completed for babies who:

have arrived in the UK afterward 56 days (viii weeks) of age
have not, for any reason, had their commencement screening sample collected by 56 days (8 weeks) of age
require a second (day 21) blood spot sample for IRT assay but practise not have it taken at the correct time – for instance, because the sample drove was delayed or the sample was lost in transit

The following guidance applies in these situations.

Late outset samples

For samples taken before day 21, apply cut-off values equally for samples that have non been delayed.

For samples taken when the baby is anile between 21 days (3 weeks) and 56 days (eight weeks) inclusive, apply cutting-off A as the decision point for genetic assay.

Do not test any samples taken later on 56 days (8 weeks) for IRT. Procedures should exist in place for laboratories to place these samples.

If a first (routine screening) sample is taken after twenty-four hour period 56 (8 weeks) and is unavoidably tested, and then the laboratory staff should continue as follows. If the IRT is:

< cutting-off A, written report as 'CF – non screened (baby besides old >eight weeks age)'
≥ cut-off A (boilerplate result), written report as 'CF suspected' and refer the infant to a CF clinician; transport a claret spot sample at the same time for genetic analysis, and give communication to the family wellness visitor and/or midwife about the reason for referral so that this can be explained to the parents/carers

The pathway for handling late first IRT samples is summarised diagrammatically in Figure 3 below. No alternative pathway is recommended. A text description of Effigy 3 is available.

Effigy 3: handling of late first IRT samples

Belatedly or absent second samples

For second samples taken on or before day 56 (8 weeks) of age, employ cut-off A per the CF screening protocol.

Babies aged over 56 days (8 weeks) are too sometime for CF screening to be carried out reliably. Practise not collect the second sample.

If a second sample is non received because the infant aged over 56 days (8 weeks), study as 'CF suspected' and refer the baby to the CF clinical team for follow-up.

If a second sample is taken after 24-hour interval 56 (eight weeks) of age, do not analyse it for IRT. Written report as 'CF suspected' and refer the baby to the CF clinical team for follow-upward.

If a second sample is taken later twenty-four hour period 56 (8 weeks) of age and has been unavoidably analysed, study as 'CF suspected' and refer the baby to the CF clinical squad for follow-up regardless of the IRT concentration.

In these situations, laboratory staff should provide data to the CF clinical squad about the reason for referral and then an explanation tin be given to the parents/carers.

Report babies that take transferred out of the UK and are however awaiting the drove of a repeat first or second IRT sample as 'screening incomplete'. A 'cystic fibrosis screening not complete' template letter of the alphabet is available on www.GOV.Uk

Activity post-obit genetic analysis

Babies with 2 CFTR variants detected

Babies with CFTR variants detected in both copies of the gene (either a homozygote or compound heterozygote) have a presumptive positive diagnosis of CF. Study them as 'CF suspected' and refer to a CF paediatric service for firsthand evaluation (clinical cess and sweat exam).

Babies with one CFTR variant detected

About babies with but one CFTR variant detected will exist unaffected carriers simply at that place is a risk that they carry a second CFTR variant not detected past the panel of CFTR variants tested. They are therefore tested again (second IRT) on a repeat dried blood spot sample. Laboratories should asking that this second IRT sample is collected on day 21 (past day 24; this allows for twenty-four hours 21 to autumn on a weekend when a special visit is not warranted). For further information, meet department 2.2 in a higher place.

Babies with no detected CFTR variants

Babies with no detected CFTR variants (by the initial panel of 4 CFTR variants) are divided into 2 groups depending on the initial IRT effect:

those with first IRT < 120 µg/Fifty – written report as 'CF not suspected'
those with first IRT ≥ 120 µg/L – a second IRT test is undertaken on a repeat dried claret spot specimen; laboratories should request that this 2nd IRT sample is collected on twenty-four hour period 21 (by day 24, this allows for day 21 to fall on a weekend when a special visit is not warranted) – for further information encounter section two.two above

For more information on genetic analysis, see department 6 below.

Sibling testing

Older siblings (of a baby with confirmed CF diagnosed from newborn screening) may exist at take a chance of CF. Any testing is at the discretion of the clinician, with the family's consent.

For whatever subsequent siblings of the index newborn, the clinical team offers expedited genetic analysis of cord blood sample together with the mother's blood sample.

Newborn screening should be undertaken equally normal. 'Early on' screening for siblings (prior to solar day 5, counting day of birth as day 0) is not recommended. The blood spot sample should be nerveless on day 5 for all babies regardless of medical condition, milk feeding and prematurity. This is to enable detection of aberrant results and initiation of appropriate treatment.

Family unit history and other risk factors

Babies are regarded as high gamble if they:

nowadays with meconium ileus
take shown echogenic bowel in-utero
are known to be at chance due to family history

They should be investigated independently co-ordinate to clinical circumstances every bit well equally being screened in the normal way.

If a genetics centre has carried out genetic testing before routine screening, it is helpful if they propose the screening laboratory of the results. This may avoid unnecessary duplication of follow-up or diagnostic testing. It will besides help avoid any confusion arising from a 'CF not suspected' screening event (from IRT assay) which might cause business organisation for the family.

Unscreened babies

These include babies who were never role of the CF screening process, for example:

babies built-in away
babies not tested because screening was offered too late (> eight weeks)
babies for whom screening was declined (by the parents/carers)

For any subsequent requests for CF screening from the parents/carers, the family health visitor should explicate to the family why their baby has not been screened for CF.

Screening laboratories should inform the baby's GP if screening for CF is incomplete. A 'cystic fibrosis screening not consummate' template letter is bachelor.

The family health visitor should inform the GP of whatsoever subsequent requests for screening from the parents/carers.

Such babies should not routinely exist offered any testing and should only be referred on a clinical ground if required. If the family or GP has whatsoever concerns, a referral for assessment (which may include sweat testing) past the local designated CF team is advisable.

Previously screened babies where subsequent results differ

A raised IRT ≥ cut-off A on a sample taken after day 21 where there is a previous CF not suspected result should be dealt with equally described in the following situations.

Discrepant IRT result

Suspect contamination and asking a further repeat if there is sufficient time to obtain a specimen up to and including twenty-four hour period 56. Run across department 2.ane above.

Non-discrepant IRT consequence ≥ cut-off A with previous IRT < cut-off B and 'CF non suspected' reported

Send for genetic analysis. Subsequent action depends on the results of the genetic analysis. If:

ane or more than CFTR variant is detected, refer and report every bit 'CF suspected'
no CFTR variants are detected (sample taken at ≤ 56 days), request a repeat sample
no CFTR variants are detected (sample taken at > 56 days), refer and study equally 'CF suspected'

Non-discrepant IRT result ≥ cut-off A with previous IRT ≥ cut-off B, no CFTR variants detected, and 'CF not suspected' reported

This shows a persistently raised IRT. Refer and report equally 'CF suspected'.

Laboratory quality and operation

Screening laboratory

Laboratories undertaking NBS screening must use processes accredited in accordance with ISO 15189 for Medical Testing Laboratories by a competent accreditation testing service, for instance UKAS.

NBS screening is provided within the organisational structure of the NBS screening programme and undertaken by specialist newborn screening laboratories already providing screening programmes.

There should be written agreed procedures describing the working arrangements between the screening laboratory and their referral laboratory.

There should be documented local policies and standard operating procedures describing the whole screening process including pre-analytical, analytical and postal service-belittling processes. Where advisable these will include reporting results, and referral and follow-up arrangements for presumptive positive cases and carriers, as specified in laboratory handbooks. Provide processes in line with relevant NBS national standards and CF guidance. Processes should be reviewed periodically taking into business relationship audit data, accumulating results, technical developments and local changes in healthcare provision.

Molecular genetics laboratory

In general, each screening laboratory should send samples to a unmarried molecular genetics laboratory. Molecular genetics laboratories may receive samples from more than than i screening laboratory.

Screening laboratories may contract for this service any molecular genetics laboratory that:

is capable of meeting the performance standards (specified below)
uses ISO 15189 accredited processes
is part of one of the vii Genomic Laboratory Hubs (a nationally commissioned genomics laboratory in England)

Overall performance and timeliness

Laboratory services should be configured to enable CF newborn screening to be completed in time for all babies with positive screening results to have their first clinic appointment by:

day 28 for babies in whom 2 CFTR variants have been detected
solar day 35 for babies who have needed a second sample IRT measurement

Analysis for IRT should be performed frequently enough to generate a screening test result (including any retest results, where required, to be confirmed in indistinguishable), no later than 4 working days from receipt of an adequate sample. Definitive results from genetic analysis must be bachelor on or before the quaternary working mean solar day from receipt of the sample within the genetic laboratory.

Report CF suspected results to the appropriate clinical team within i working day of condign bachelor. Do non consequence intermediate reports (those showing increased IRT in the initial sample without follow-up results).

Ideally, parents/carers should non be informed of a positive screening result on a Fri or Saturday, to brand sure that the clinical cess and sweat test can exist arranged for the solar day later on the results.

Internal quality and performance monitoring

Laboratories should participate in audit at local, regional and national levels, to assess the effectiveness of the national screening program. They should publish the results and functioning of their newborn blood spot screening plan within an annual written report.

There must be a documented hazard management policy for the laboratory aspects of the CF screening programme as part of an overall pathway risk assessment. This should describe the steps in the testing protocol where failures could occur and the procedures that accept been implemented to minimise the chance of their occurrence.

External quality assessment

Laboratories should participate in an approved external quality assurance (EQA) scheme (for example The United Kingdom National External Quality Assessment Service (Uk NEQAS)). The EQA scheme assesses laboratories on the precision and accuracy of belittling steps.

Post-obit agreement from the NBS screening programme, the laboratory must release reports on screening performance (including external quality assurance and accreditation assessments) to agencies with a legitimate interest in the quality and rubber of the program on behalf of the public.

Pre-analytical factors

Specimen requirements

Claret spot sampling should exist undertaken co-ordinate to the Guidelines for newborn blood spot sampling. Specimens should be dispatched to the laboratory inside 24 hours of taking the sample. They must be kept in a dry surround at room temperature or refrigerated at 4°C before analysis. Storage after analysis should follow the guidelines provided by the code of practice for the retention and storage of remainder newborn blood spots.

Venepuncture or venous/arterial sampling from an existing line is an culling method to collect the blood spot sample. This is providing the sample is not contaminated with ethylenediaminetetraacetic acid (EDTA)/heparin and the line is cleared of infusate. Anticoagulants may affect the assay. Capillary tubes (patently or heparinised) must not be used to collect claret samples.

Blood spot stability

IRT was shown to exist relatively unstable in early on studies (Heeley and others, 1982; Kirby and others, 1981) and there are few published studies using modern methods of IRT analysis. In 2006, the Centres for Disease Control and Prevention (CDC) showed that IRT-enriched blood added to a filter newspaper matrix was stable when stored with desiccant for ane year at either -20°C or 4°C, and 30 days at ambient temperature. Only 75% of IRT remained in dried blood spots stored at ambience temperature for upwardly to one yr (Li and others, 2006). All the same, dried claret spots in this study were prepared by lysing blood before the addition of IRT and recovery studies demonstrated that IRT degrades more rapidly in whole blood. A further CDC study using whole blood with intact cells (thus more than closely mimicking newborn DBS samples) showed that samples stored at 37°C in low-humidity environments retained 83% of IRT for xxx days, whereas in a high humidity environment just 47% was retained (Adam and others, 2011).

Samples received in the laboratory more than 14 days afterward the sample was taken are therefore deemed unsuitable for testing. Asking a repeat sample as presently as possible in line with the blood sampling guidelines. Test the original sample following the standard algorithm and refer the infant if a screen positive result is obtained.

For CF, information technology is important to be aware that samples taken when the baby is anile over 56 days (viii weeks) should not exist analysed (see section 2.3 above).

Not-belittling factors affecting the screening consequence

Potential for fake negative results

Several factors (in addition to tardily testing – run into department 2.3 above) are known to lower blood IRT concentration in babies with CF, leading to simulated negative screening results.

Meconium ileus:

Some babies with CF and meconium ileus are likely to requite negative screening results when tested during the first week of life, though IRT concentrations may become clearly abnormal a calendar week or 2 later. It is unclear whether surgery itself is responsible or whether it is the concomitant lack of enteral feeding. In whatsoever example, CF should exist strongly suspected in whatsoever baby with meconium ileus irrespective of the screening event. Neonatal units should have appropriate investigation protocols in place (see Sathe and Houwen, 2017).

Blood transfusion:

The effects of blood transfusion on IRT are unclear but could result in a false negative IRT issue. A echo sample should therefore be taken afterwards a minimum of 72 hours has elapsed. As white cells are removed prior to claret transfusion, misleading results with genetic analysis are unlikely (meet Brauner and others, 1997).

Viral infection:

Viral infection leading to astute gastroenteritis or respiratory illness may too be associated with a false negative screening result (see Dunn and others, 2011).

Prematurity:

The performance of the IRT analysis for premature babies is less articulate and there is potential for both false positive and false negative results (meet Cortes and others, 2014; Bourguignon and others, 1986; Kirby and others, 1981; Heidendael and others, 2014).

It is not practicable to prefer culling diagnostic approaches routinely in these concluding 2 groups of babies. E'er bear in listen in mind that non all cases of CF will be detected on newborn screening and any kid showing advisable symptoms should exist investigated accordingly.

Potential for false positive results

Elevated IRT levels have been reported in association with congenital infections, renal failure, bowel atresias and nephrogenic diabetes insipidus. Imitation positives also occur in children who are very sick on paediatric intensive care unit (PICU) who do not specifically have whatever bowel or renal problems. High IRT levels can occur in babies with a variety of chromosomal abnormalities particularly trisomies 13 and eighteen (run into Castellani and others, 2009).

Other causes of raised IRT include ΔF508 carriers, hypoxic insult to pancreas, congenital heart disease, spina bifida, gastroschisis, viral infections and galactosaemia.

The standard screening protocol should be followed in all of these situations. If CFTR variants are not found, laboratory staff should explicate to the clinician looking after the baby that the raised IRT may be a secondary miracle and consider whether or not further investigation is required (for example, repeat IRT or sweat examination).

The IRT analysis

Assay IRT in the blood spot using a methodology that has been demonstrated to be fit-for-purpose and approved by the NBS screening program. Where possible, the reagents and instrumentation should be CE marked. The laboratory should follow the procedures detailed in the manufacturer's instructions.

Include internal quality control samples at 3 IRT levels with each analysis batch. Each laboratory should assign acceptable ranges for these samples.

Rationale for IRT cut-off values

The sensitivity and specificity of screening are crucially dependent on the operation of the IRT assay. In particular, if cut-off B is set too depression, too many samples will be sent for genetic analysis and there will be a disproportionate increase in the number of carriers detected; if it is besides high and then CF cases may exist missed.

Unfortunately, this is a especially difficult assay to control equally human blood contains a diversity of trypsinogen species which react differently with the various antibodies used for immunoassay. Additionally, the IRT species increased in neonates with CF has different properties from that present commonly in neonatal claret (meet Dhondt and Farriaux, 1994).

For these reasons, it is not possible to deliver the quality assurance required for newborn screening solely by ways of an EQA scheme with circulated claret spots, particularly as there are marked matrix effects.

The 99.5th centile for IRT is used to select samples for subsequent genetic analysis and consequently to help determine which patients will be referred for clinical investigation every bit a 'condition suspected' instance. Comparing data from several UK laboratories reveals meaning variation in the population distribution of IRT values with different kit lots (lot variation should non exceed +/-eight%) as well as systematic inter-laboratory variations due to differences in instrumentation (see Pollitt and Matthews, 2007) and possibly ethnicity. In order to determine the 99.fifth centile in a reliable way, information technology is estimated that >12,000 samples must be analysed, but even in laboratories with big workloads this is not possible in a reasonable fourth dimension frame.

National cut-off for IRT

Due to concerns about obtaining sufficient information and varying the cut-off ofttimes, a new approach came into result from 1 Apr 2020 to define a 'national' cut-off based on retrospective information assay from several laboratories over a longer period of time. It was agreed that this would be bailiwick to regular review based upon continued information collection from laboratories on a quarterly basis (for the electric current values, run into department 2.1 above). Note that these cut-offs may not apply in the aforementioned way in the Commonwealth of Ireland laboratory (Dublin) who screen at twenty-four hours three of life and in the Scottish laboratory (Glasgow) who screen at day 4.

Genetic analysis

Genetic analysis must be performed using an ISO 15189 accredited procedure.

Sample requirements, identity and ship

There is no requirement for a measured amount of blood, so the residue claret from a spot that has already had a disc punched out is likely to be sufficient.

At that place must exist a tracking system to ensure that stale blood spot samples sent for genetic analysis are identified unequivocally. The card should not ordinarily leave the screening laboratory.

Samples must exist deeply identified. The simplest method is to cut an irregularly shaped strip from the claret spot bill of fare (with the blood at one end), and on the bare section of the strip write patient identifiers including at to the lowest degree 2 from the infant's date of birth, surname and NHS number. The irregularly shaped strip can and then be matched with the card if subsequently required. With increasing automation and an electronic IT organisation, a bar-code sample identifier is desirable.

Appropriate timely transport arrangements to the genomics laboratory must exist organised and the laboratory made enlightened of the imminent inflow of a screening specimen(due south). Sample receipt by the genomic laboratory should be acknowledged dorsum to the screening laboratory.

2-stage analysis

Genetic analysis is carried out in two stages to minimise the detection of homozygotes for the 'milder' alleles (CFTR variants associated with developed onset CFTR-related disorders rather than classic CF) and the number of carriers detected.

The offset stage establishes whether the babe has any of the iv most common CFTR variants in the English population associated with severe affliction. Nomenclature complies with the Human Genome Variation Society Guidelines to ensure consistent and precise nomenclature. These iv variants are as detailed below.

cDNA	Poly peptide	Traditional
c.1521_1523delCTT	p.Phe508del	ΔF508 (run across Note 1)
c.1652G>A	p.Gly551Asp	G551D (see Annotation 2)
c.1624G>T	p.(Gly542Ter) or p.(Gly542*)	G542X
c.489+1G>T		621+1G>T

CFTR reference sequences: cDNA – LRG_663t1 (NM_000492.3); protein – LRG_663p1 (NP_000483.3)

Annotation 1: Depending on the technology used, the c.1519_1521delATC, p.(Ile507del), (ΔI507) variant may as well be detected.

Note 2: Depending on the technology used, the c.1657C>T, p.(Arg553Ter), (R553X) variant may also exist detected.

The beginning 3 variants are also described co-ordinate to the poly peptide change so p.Gly551Asp indicates that the glycine at amino acid residual 551 is predicted to change to aspartic acid. c.489+1G>T is an intronic variant in the approved splice site and thus has no directly associated amino acid alter. The numbering indicates the 1st nucleotide (+ane) after nucleotide 489 (which is at the last nucleotide of the exon).

This starting time stage detects over 80% of illness-causing variants in the United kingdom of great britain and northern ireland population.

There are several technologies, including a range of commercial kits, available for detection of these 4 CFTR variants. It is essential that in the starting time phase of genetic analysis, no variants other than the ones specified higher up are co-incidentally detected.

The second stage covers a wider range of CFTR variants and is applied, using the original sample, in all cases where a single variant is detected in the heterozygous state at the first phase.

The panels used are specified by the CF Screening Advisory Board and reviewed periodically in the light of technical developments and on-going evaluation of the plan.

Reporting results

Samples with no detected CFTR variants may be reported to the screening laboratory past list with appropriate specimen identifiers.

For any sample showing i or more CFTR variants, the genomic laboratory must supply the screening laboratory with a formal study, largely limited to a factual description of the findings (variants tested for and results). Standard advice on family studies, such as referring the babe to a CF heart for follow-up, is inappropriate in the screening context as these options are incorporated in the protocol. Send copies (not transcriptions) of the genetic assay report with the screening laboratory'southward study or referral letter to the baby's consultant or GP equally appropriate.

Next steps

Following the genetic analysis, the screening laboratory should go along according to the screening protocol (see section 2.4 above).

Reporting and communication of results

Reporting to CHRDs/CHISs

Screening laboratories and CHRDs/CHISs should use the national status codes and subcodes to record the outcomes of NBS screening. Ideally, the laboratory sends screening results to CHRDs/CHISs and the newborn claret spot failsafe solution (NBSFS) using electronic messaging. Status codes should also be used for reporting to the NBSFS.

Reports of all screening results should have a generic disclaimer fastened, stating:
These tests are screening tests; no screening test is 100% reliable.
Such a disclaimer is particularly relevant to CF because of the potential for falsely loftier or depression IRTs from other non-CF causes (see section 4.2 above).

Newborn blood spot failsafe solution (NBSFS)

The NBSFS is an IT system that identifies babies born in England who have missed NBS screening. It is in use by all maternity units across England. The system also records repeat requests and screening outcomes to back up failsafe processes.

The NBSFS user guide and NBSFS operational level agreements provide more than data most how to use the failsafe system.

Communicating results

CF not suspected

'CF not suspected' results should exist communicated to the parents/carers by CHRDs/CHISs by 6 weeks of age. If a 'CF not suspected' result is generated post-obit collection and assay of a repeat sample for a very high first IRT (> 99.ninth centile) then the family should be informed promptly (ideally inside 24 hours of the laboratory obtaining the second IRT result) as they are likely to take raised anxiety levels due to a repeat sample being needed.

Probable CF carrier, low likelihood of CF

Again, the family will likely have raised anxiety levels because a repeat blood spot sample was needed. The sample taker should have explained the reason for the echo and when to wait the result. The family should be informed by a healthcare professional of the 'probable CF carrier' result promptly, ideally within 24 hours of the laboratory obtaining the second IRT result. Where possible, the screening laboratory should have a screening nurse specialist contact the family health visitor (or preferably a designated wellness visitor if in that location is an appointed lead health visitor for giving screening results to families) by telephone. They should also ship confirmation of the result to the family in writing.

The laboratory should also inform the baby'south GP of the result in writing. A 'cystic fibrosis: probable carrier' template alphabetic character is available. The communication with the GP should include a link to the 'cystic fibrosis carrier' leaflet as well as data about which healthcare professional has been informed of the final result and will be communicating it to the parents/carers.

The designated wellness visitor or alternative healthcare professional (who must exist trained for the purpose) will visit the family to inform them of the upshot and give them the 'cystic fibrosis carrier' leaflet. The healthcare professional that gives the carrier issue to the family should consummate and return the 'cystic fibrosis screening: carrier of CF gene' follow-upwardly course to the newborn screening laboratory inside 24 hours of the visit.

Each laboratory should have an agreed arrangement for the advice and follow-up of 'probable CF carrier' results. This should be in line with the 'managing positive results from cystic fibrosis screening' guidelines.

The screening protocol is designed to choice up as few carrier babies as possible and therefore should non be regarded as a reliable means of detecting CF carrier babies.

CF suspected

'CF suspected' results require follow-up/clinical referral. The laboratory should refer babies with positive screening results for CF the same or next working twenty-four hours post-obit confirmation of a positive result. The screening laboratory should report the screen positive result past phone and in writing to the regional CF centre. For information on the regional CF centres, delight refer to the Cystic Fibrosis Trust website.

'Cystic fibrosis: presumed positive' template letters are available for the screening laboratory to notify the designated clinician and the baby's GP of the 'CF suspected' result. The regional CF centre will commonly be responsible for liaising with a more than local CF clinic (as required by local arrangements). The referral notification should include a link to the 'managing positive results from cystic fibrosis screening' guidelines. This initiates the clinical referral of screen positive cases.

An accordingly trained healthcare professional should communicate the 'CF suspected' result as soon as possible to the parents/carers. The 'cystic fibrosis suspected' leaflet tin support healthcare professionals to have this conversation. Early contact enables them to kickoff their baby's treatment as soon as this can be arranged.

It is imperative that the GP understands that the result has been communicated directly to the regional CF Center (who volition contact the parents/carers), and that the letter of the alphabet they (the GP) have received is for information only.

Each screening laboratory should take an agreed arrangement for the follow-upwardly and referral of all 'CF suspected' cases, which should be in line with the 'managing positive results from cystic fibrosis screening' guidelines.

Programme monitoring and data collection

Plan standards

The NBS screening programme standards are used to assess the NBS screening procedure. The standards are a set of measures that have to be met to make certain screening is safe and effective. All health care professionals involved in the NBS screening pathway accept a function to play in meeting these standards.

Laboratories should submit standards data to the NBS screening programme on an annual footing. The annual data drove template is shared with the screening laboratories via e-mail each year, with instructions for completion and submission. Data submissions must exist accurate, timely and complete. This enables operation monitoring and programme evaluation.

Monitoring clinical outcomes

Request clinical information from clinical referral centres on each presumptive positive case. Complete the presumptive positive follow-upwardly grade for each case and submit the form to the NBS screening programme. Information technology is the responsibility of the designated clinician at the clinical referral centres to ensure that these forms are completed and returned to their corresponding laboratory directors. The regional CF heart should besides be informed about diagnostic outcome to facilitate regional and national audit.

Follow-up information is also required on all cases reported as CF carriers. For each instance, send the CF carrier follow-up form to the relevant wellness professional (specialist health company/counsellor) for completion. The anonymised data from the form should incorporate part of the annual screening standards information submission by the screening laboratory to the NBS screening programme.

Follow-upward information is also required on all cases with an inconclusive exam effect. The newborn screening laboratory should complete the CF inconclusive test follow-up form and send it to the baby'due south midwife or health visitor (also ship a re-create to the baby's GP).

Babies diagnosed with CF not identified through newborn screening

If a screening laboratory director, clinical team or molecular laboratory director is made enlightened of a CF case (born later on 01 Apr 2007) that has not been detected via the CF screening programme, it is very important that information on the instance is reported to the NBS screening programme.

The clinical team should gather the details in conjunction with the screening laboratory director using the 'CF diagnosis not identified through screening' form. The laboratory director should collate and anonymise the data, and transport information technology as soon every bit possible to NBS screening program at phe.screeninghelpdesk@nhs.net. All course fields shaded in grey should exist removed prior to submitting the class.

There should exist no patient identifiable data on the 'CF diagnosis non identified through screening' form.

Data on these cases are collated on an annual ground every bit an important part of the audit and evaluation of the CF screening programme.

Screening safety incidents

All condom concerns and incidents must be reported and managed in accord with the 'managing safety incidents in NHS screening programmes' guidance. This guidance details the accountabilities for reporting, investigating and managing NHS screening programme prophylactic incidents. Information technology covers the management of screening programme:

safety concerns
rubber incidents
serious incidents

Background to screening for CF

Screening for CF was formally introduced as a national newborn screening programme in England in Apr 2001. Screening was already taking place in Wales, Northern Ireland and some parts of England. Screening started in Scotland in February 2003 and became universal across the UK in October 2007.

The programme seeks to rest the interests of families where a child is identified as having CF versus the interests of the big majority of families where children are unaffected. The programme was also designed to minimise the number of CF carriers unavoidably detected.

Scientific background to the screening protocol

Newborn screening for CF is founded on the work of Crossley and others (1979), who showed that IRT in blood is significantly increased in affected newborns. Screening programmes based on IRT measurement from a dried bloodspot sample were introduced in East Anglia in 1980 and subsequently elsewhere in the United kingdom. A raised IRT level is sensitive for identifying CF merely has poor positive predictive value (PPV) and therefore CF programmes require a second 'tier' test to improve PPV. In early programmes, this was traditionally IRT in a 2nd claret sample collected at 2 to 4 weeks of age. A 2-stage IRT procedure has the disadvantage of requiring a relatively high number of second samples, which increase the anxiety generated past screening, and the presumptive diagnosis is made relatively late.

One time the CFTR gene was identified in 1989, the selection of genetic analysis became possible to replace the second-tier IRT analysis. Initially, in 1990, but the most common CFTR variant, ΔF508 (now known as c.1521_1523delCTT p.Phe508del) was used, just more recently panels with a larger number of CFTR cistron variants have been used globally.

Over 2000 variants take been identified associated with CF and around 400 occur at sufficient frequency to determine their characterisation (run into Farrell and others, 2017; Sosnay and others, 2017).

For the United kingdom of great britain and northern ireland programme, the initial panel used for babies with a raised IRT measurement contains the 4 commonest CFTR variants in the UK population. If i variant is recognised, an expanded console is used. In cases where just one variant is recognised using both the initial and expanded console, a second IRT measurement is undertaken at day 21. If this is lower than a pre-defined cut-off the baby is classified as a probable carrier. Babies with a raised second IRT are referred for a clinical assessment and sweat testing. The express initial panel and repeated IRT measurement at day 21 are unique aspects of the UK program.

Because of the limited initial panel, a safe net is employed in the U.k. protocol. If the first IRT is extremely high (>99.9th centile), but in that location is no variant recognised on the limited 4-variant panel, then a 2d dried blood spot sample is collected at day 21. Babies with a raised second IRT are referred for clinical assessment and sweat testing. This aspect of the U.k. protocol recognises babies with CF who have unusual CFTR variants (disproportionately babies from a not-European background), but has a poor positive predictive value compared to other parts of the protocol (around 10 to xx babies are referred for sweat testing for each positive diagnosis).

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Source: https://www.gov.uk/government/publications/cystic-fibrosis-screening-laboratory-handbook/cystic-fibrosis-screening-laboratory-handbook