A laboratory guide to newborn blood spot screening for inherited metabolic diseases
Updated 6 November 2024
This handbook is for laboratories that provide an NHS newborn blood spot (NBS) screening service for inherited metabolic diseases (IMDs) in the UK. It defines a framework for the pre-analytical, analytical and post-analytical steps in the newborn screening process so as to:
- support newborn screening laboratories to provide the screening service
- improve consistency across the screening programme
- provide guidance on achieving good quality by application of standards and audit
Use this handbook alongside other NBS screening programme guidance.
Screening for IMDs
All babies across the UK are offered a blood test to identify IMDs as part of the NHS NBS screening programme. Screening aims to identify babies with an IMD before they develop symptoms, to enable the early initiation of high-quality care.
The six IMDs currently screened for as part of the NHS NBS screening programme are:
- phenylketonuria (PKU)
- medium-chain acyl-CoA dehydrogenase deficiency (MCADD)
- maple syrup urine disease (MSUD)
- isovaleric acidaemia (IVA)
- glutaric aciduria type 1 (GA1)
- homocystinuria (pyridoxine unresponsive) (HCU)
General organisation of NBS screening
IMD screening is fully integrated within the existing NBS screening programme and based on the same screening laboratory populations. The initial screening tests use blood collected on the standard newborn screening blood sample collection card. Quality assurance (QA) and performance management arrangements follow the same general principles as those for other newborn screening programmes.
With IMDs, as for other blood spot screening conditions, the screening laboratory is a major communication hub. Screening results are fed back to child health records departments (CHRDs) / child health information services (CHISs), with onward transmission of negative results to the parents. More than 99% of results are ‘IMD not suspected’ and generated promptly. However, in some cases, screening will not be completed until the baby is over a month old.
Some parents may decide that they do not want their baby to be screened for some or all of the conditions. Screening can be declined for congenital hypothyroidism, sickle cell disease and cystic fibrosis individually, but the 6 IMDs can only be declined as a group.
Complete the blood spot card to indicate which conditions have been accepted or declined, as outlined in the NBS screening sampling guidelines.
The screening protocols – common elements
Aims
The UK screening protocols for each IMD are intended to:
- maximise the early detection of IMDs so that pre-symptomatic treatment can be initiated to reduce the long-term morbidity associated with the conditions if untreated
- minimise the need for second heel pricks, and any other causes of diagnostic delay
The analytical cut-off
The screening protocols for each disorder are designed to maximise sensitivity while seeking to reduce the number of false positive results, and thus deliver an acceptable positive predictive value (PPV) for the test overall. In some cases, improved specificity is achieved by the use of secondary testing as part of the screening protocol (total homocysteine in the case of HCU and C10 in the case of MCADD).
In each IMD screening protocol, an analytical cut-off is set approximately 20% below the referral cut-off. Those samples which exceed the analytical cut-off are re-tested in duplicate on the same day. If the mean of the three results exceeds the referral cut-off then the patient is referred, or, in the case of MCADD and HCU, the C8:C10 ratio or total homocysteine is assessed respectively and the decision to refer is based upon these results.
New sibling testing
A new sibling born to parents who already have a child with PKU, MCADD, MSUD, IVA, GA1 or HCU has a 1 in 4 risk of having the same disorder. In these circumstances, ‘early’ screening (prior to day 5, counting day of birth as day 0) should be undertaken in addition to the routine screening test to avoid delays in diagnosis and to allay parental anxiety. The decision about when to carry out the additional ‘early’ screening test depends upon the condition suspected; see chapters 8 – 13 for specific guidance relating to each condition.
This additional ‘early’ screening test does not remove the need for routine screening. It is essential that the routine blood spot collection is undertaken on day 5 to screen for the other conditions tested for as part of the NBS programme.
Older sibling testing
Older siblings of newly diagnosed screen detected cases, when born to the same parents, have a 1 in 4 chance of having been born with the same disorder. The clinician responsible for managing the newly diagnosed baby is also responsible for considering the risks for older siblings in the family. Note that older sibling testing is undertaken by diagnostic laboratories.
Late testing
Screening should be offered to all eligible babies under a year of age (up to but not including their first birthday) without documented results (or declines) for all 9 conditions (note that CF can only be screened for up to 8 weeks of age). If any of the untested conditions are IMDs, and the parents accept screening, the screening laboratory will screen the baby for all 6 IMDs. This is regardless of whether the baby has documented results for some of the IMDs. If screening is accepted, the sample must be taken no later than 14 calendar days after the baby’s first birthday. Once the baby is above this age, then they are no longer eligible for screening. If the family or GP have any clinical concerns after this point, a referral for paediatric assessment would be appropriate. See Newborn blood spot screening: movers in with no available records for more information.
The standard national cut-off should be applied irrespective of the age of the baby at the time of screening. However, it is noted that rarely, older infants with MCADD may have C8 levels below the screening cut-off and there is a potential for false negative screening results (see section 10.3.1). These effects, linked in part to carnitine depletion, might also be expected to reduce the sensitivity of testing for IVA and GA1. In the case of MSUD, intermittent forms are known to occur, and similarly, GA1 patients with minimal excretion of key metabolites have been described. If any of the IMDs are suspected or there are any pre-existing clinical concerns, the infant should be referred to a clinician for diagnostic testing.
Receipt of ‘not required’ newborn screening cards
Occasionally laboratories may receive newborn screening cards which were not required and sent in error. Laboratories should only analyse and report the first valid day 5 sample received; the subsequent sample should be recorded on the system and a result code 0906 (not required, previous valid result) generated. However, laboratory processes may mean that analysis has commenced before the subsequent card is identified as a ‘not required’ sample. If analysis is performed and all the parameters are below the cut off for action, in accordance with the first valid sample, then it should be reported as 0906. If the results obtained require individual follow-up then this should be undertaken and reported appropriately. The samples should then be stored as usual in case further enquiries arise.
Genetic counselling
In most instances, questions about genetic inheritance and risk will be dealt with by the metabolic team. However, if there are any outstanding issues, further genetic counselling is available through the local Genetics Service.
General analytical aspects
Newborn screening for the IMDs should be provided using the nationally agreed screening protocols described in this document. No alternative method should be used. Any proposal to introduce new analytical methods needs careful collective consideration and approval by the NHS Newborn Blood Spot Screening Programme.
Specimen requirements
Blood spot sampling by heel prick should be undertaken according to the guidelines for newborn blood spot sampling. Specimens should be dispatched to the laboratory within 24 hours of taking the sample. They must be kept in a dry environment at room temperature or refrigerated at 4°C before analysis. Storage after analysis should follow guidelines in the ‘Code of Practice for the Retention and Storage of Residual Spots’.
Samples that do not meet the national screening requirements (for example red cell transfusion, poor blood spot quality, delayed in transit) and require a repeat sample must be analysed and positive results acted upon.
Venepuncture or venous/arterial sampling from an existing line is an alternative method to a heel prick sample is acceptable providing the sample is not contaminated with EDTA/heparin and the line is cleared of infusate. Anticoagulants may affect the assay. Capillary tubes (plain or heparinised) must not be used to collect blood samples.
Methodology
Newborn screening for IMD requires the analysis of phenylalanine (Phe), tyrosine (Tyr), leucine (Leu), methionine (Met), octanoylcarnitine (C8), decanoylcarnitine (C10), isovalerylcarnitine (C5) and glutarylcarnitine (C5-DC) in a blood spot disc which has been punched into a multi-well plate. Internal standard of stable isotopes of Phe, Tyr, Leu, Met, C8, (C10), C5 and C5DC in a suitable solvent (e.g. 80% methanol) is added and is used to elute the analytes of interest from the blood spot. Following extraction at room temperature with agitation, the solvent extracts are analysed using tandem mass spectrometry. Sampling may be direct from the original plate (with blood spots in situ) or the eluates may be transferred to a fresh plate before sampling. Transfer to a fresh plate may reduce blockage rates but has its own risks e.g. sample mix up / contamination. Multiple reaction monitoring (MRM) acquisition mode must be used and analysis restricted to the specified analytes. Results may be calculated by internal standard or external calibration; in the latter case in-house calibrators or commercial kits may be used.
Table 1. MRM ion transitions for amino acids and acylcarnitines
Potential for false negative results
False negative results may occur for a variety of reasons, those that are specific to a particular analyte are described in the condition specific sections below.
A blood spot missing from a well should result in total absence of analytes of interest and a several fold increase in internal standard abundances due to lack of ion suppression. Procedures should be in place to detect this, and should include a visual check that all designated wells contain a blood spot. Any suspicious results for example those with a very low Phe and/or Leu concentration should be investigated. Procedures should be in place to detect inappropriate addition of internal standard, for example samples with double the usual internal standard abundance, which will lead to a spuriously low analyte concentration.
Laboratory quality and performance
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 example United Kingdom Accreditation Service (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 for total homocysteine analysis where it is not available locally.
There should be documented local policies and standard operating procedures describing the whole screening process including pre-analytical, analytical and post-analytical processes. Where appropriate these will include reporting results, and referral and follow-up arrangements for presumptive positive cases and carriers, as specified in laboratory handbooks. Processes should be provided in line with relevant NBS guidance and should be reviewed periodically taking into account audit data, accumulating results, technical developments and local changes in healthcare provision.
Referral laboratory for total homocysteine analysis (where required)
In general, each screening laboratory should send samples to a single referral laboratory that:
- is capable of meeting the performance standards (specified below)
- uses ISO 15189 accredited processes
Overall performance and timeliness
The laboratory analytical service should be configured to enable PKU, MCADD, MSUD, GA1 and IVA screen positive results to be reported within 3 working days of receipt of an adequate sample. In addition, babies with screen positive results for MCADD, MSUD or IVA must be referred by the laboratory to the appropriate clinical team on the same day that abnormal results are identified. The NHSE service specification states that positive results for MCADD, MSUD and IVA must be reported within 24 hours of successful analysis; in practice this means that results must be checked on weekends and bank holidays (as required), but that if analysis is unsuccessful trouble-shooting may be undertaken on the next working day. If there is insufficient sample for repeat analysis to confirm an initial result raised above the analytical cut-off, referral to the designated specialist metabolic team should be instigated without delay for PKU, MCADD, MSUD, IVA and GA1. In the case of possible HCU (methionine above the analytical cut-off) a repeat sample should be requested if there is insufficient blood spot to confirm the initial result.
Follow-up diagnostic tests must be undertaken in line with the diagnostic protocol by UKAS accredited laboratories that must participate and demonstrate acceptable performance in the relevant, accredited EQA schemes.
Risk Management
There must be a documented risk management policy for the laboratory aspects of the IMD screening programme as part of an overall newborn screening risk management policy. This should describe the steps in the testing protocol where failures could occur and the procedures that have been implemented to minimise the risk of their occurrence. Reporting and learning from incidents and user feedback should be used as a means of continuous improvement.
Contingency planning
All screening laboratories should have a formalised and documented contingency plan for all aspects of the process. This must be invoked in the event of full or partial loss of the screening service (for example instrument downtime, staffing shortages) and take into account the reporting timelines required for reporting screen positive results, that is not more than 3 days.
Internal quality control and performance monitoring
Blood spot internal quality control (IQC) for each analyte measured should be performed with each plate of samples. IQC material should be used at concentrations which provide a clinically relevant challenge to the assay. Laboratories should consider, where possible, inclusion of IQC at concentrations near clinical decision limits
Laboratories should participate in audit at local, regional and national levels, to assess the effectiveness of the national screening programme. They should publish the results and performance of their newborn blood spot screening programme within an annual report.
External quality assurance of screening metabolites
All laboratories must participate in the UKNEQAS (https://birminghamquality.org.uk/ ) external quality assurance (EQA) scheme which includes Phe, Tyr, Met, Leu, C5, C5-DC, C8 and C10.
Following 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 safety of the programme on behalf of the public.
Where laboratories also undertake second tier testing for homocystinuria (HCU), participation in a blood spot scheme for homocysteine is also required, for example ERNDI Special assays in dried blood spots.
CDC also provide a newborn screening EQA programme.
Normal population data
Laboratories return quarterly population data for all screening analytes. Each laboratory’s data is displayed as box-whisker plots and are compared with other UK laboratories.
Performance monitoring and reporting
The results and performance of the IMD screening programme should be included within an annual report produced by the screening laboratory for circulation to local directors of public health (and others as required). Normally this will be a combined report covering all blood spot screens, dealing also with common issues such as specimen quality, timeliness, etc. There should also be periodic multidisciplinary review of local policies for IMD screening in the light of accumulated results, technical developments and any local changes in health care provision.
National Congenital Anomaly and Rare Disease Registration Service (NCARDRS) are involved in outcome studies and laboratories may be required to submit data to them.
Long term outcome studies are an important means of assessing the impact and effectiveness of newborn screening. The routes by which this data is gathered may vary by condition, but the newborn screening labs have a valuable role in submitting data on screen positive cases and their follow-up. When this is requested, it is important that access to the data complies with relevant data governance requirements such as, Section 251/254 of the National Health Service Act 2006, and the screening labs will be notified by NHSE if authorised returns are to be made and the route by which this data is to be collected.
Stability of acylcarnitines and amino acids in dried blood spots
At room temperature acylcarnitines in blood spotted on filter paper are slowly hydrolysed to free carnitine, and their corresponding fatty acids and amino acids may also degrade (acylcarnitine profile images are available at MetBioNet).
For the acylcarnitines, the rate of degradation depends on the carbon chain-length of the acyl group and the presence of functional groups. For saturated straight-chain acylcarnitines the stability of the carnitine esters in stored blood spots generally increases with the increasing chain-length of the acyl group. The decay appears to be biphasic with more rapid decay in the first few weeks at room temperature (Fingerhut et al, 2009). The stability of C5-DC (Johnson et al, 2004) appears particularly poor and at room temperature will degrade by at least 50% in six months. High humidity and ambient temperature will also increase the rate of degradation (Golbahar et al, 2014).
The amino acids Tyr, Leu, Phe and Met decrease at a rate of 1.7%, 3.1%, 5.7% and 7.3% per year respectively for the first five years in dried blood spots when stored at room temperature in a dry environment (Strnadova et al, 2007).
The stability of acylcarnitines is significantly improved if dried blood spots are stored in sealed bags at low temperature. At 4°C the decrease in the concentrations of C6 and C8 is about 4% per year. In specimens stored at -20°C the decrease in the concentration of acetylcarnitine is less than 10% per year and those of C6, C8 and C10 are less than 3%.
Clinical referral and follow up – common elements
The need for prompt and effective intervention in screen positive patients is important for all conditions that form part of the newborn screening programme. The inherited metabolic diseases we screen for have particularly complex needs and outcomes may be strongly influenced by referral and treatment pathways. Some of the conditions (MCAD, MSUD, IVA) may be associated with early decompensation and will need urgent referral and clinical review. It is therefore important that the referral of all IMD screen positive patients is undertaken in co-ordination with a clinical service that complies with the service specification for a paediatric IMD centre; this is largely aimed at ensuring a continuity of specialist care.
It is essential that each screening laboratory, in collaboration with commissioners and local implementation groups, makes detailed local arrangements for the follow-up of presumptive positive cases for all districts covered by their laboratory. These arrangements should be updated regularly to reflect personnel and organisational changes.
Reporting and communicating results
Reporting results 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 NBSFS using electronic messaging. Status codes should also be used for reporting to the NBSFS.
Newborn blood spot failsafe solution (NBSFS)
The NBSFS is an IT solution for England that reduces the risk of babies missing or having delayed NBS screening. It is in use by all maternity units across England. The system also records repeat requests and screening outcomes to support failsafe processes.
The NBSFS user guide and NBSFS operational level agreements provide more information about how to use the failsafe system.
Communicating results
‘Not suspected’ results should be communicated to the parents by 6 weeks of age. It is recommended that CHRDs/CHISs notify parents by letter.
‘Suspected’ results require follow-up / clinical referral. The laboratory should refer babies with positive screening results for an IMD the same or next working day following confirmation of a positive result. The referral notification should include a link to the IMD initial clinical referral guidelines. This initiates the clinical referral of screen positive cases.
See Newborn blood spot screening: reporting positive results from laboratories.
Template letters are available for the screening laboratory to notify:
- the designated clinician for a baby with a suspected IMD
- the baby’s GP of a ‘suspected’ result
- the baby’s GP of a ‘confirmed’ result
An appropriately trained healthcare professional should communicate the ‘suspected’ result as soon as possible to the parents/carers. Early contact enables them to start their baby’s treatment as soon as this can be arranged.
Reports of all screening results should have a generic disclaimer saying: ‘These tests are screening tests. No screening test is 100% reliable.’
Each screening laboratory should have an agreed arrangement for the follow-up and referral of all IMD suspected cases, which should be in line with the IMD initial clinical referral guidelines.
Programme monitoring and data collection
Programme standards
The NBS screening programme standards are used to assess the NBS screening process. The standards are a set of measures that have to be met to make sure screening is safe and effective. All health care professionals involved in the NBS screening pathway have a part to play in meeting these standards.
Laboratories should submit standards data to the NBS screening programme on an annual basis. The annual data collection template is shared with the screening laboratories via email each year, with instructions for completion and submission. Data submissions must be accurate, timely and complete. This enables performance monitoring and programme evaluation.
Laboratory standards
Laboratories screening for IMDs must be accredited by UKAS. There must be a member of staff at consultant level responsible for IMD screening with defined lines of accountability for all aspects of the service.
There should be local policies and standard operating procedures describing the whole screening process including pre-analytical, analytical and post-analytical processes; these include reporting normal and abnormal results, referral and follow-up arrangements for presumptive positive cases. Processes must be provided in line with relevant national standards and guidance and should be reviewed periodically taking in to account audit data, accumulating results, technical developments and local changes in healthcare provision.
Medical Laboratory Accreditation - ISO 15189 (ukas.com)
Data collection
Laboratories should request clinical information from metabolic paediatricians on each presumptive positive case. Data on each case notified to the clinical referral services (‘condition suspected’ and ‘other disorders follow-up’) should be collated and anonymised before submission to the NBS screening programme. It is the responsibility of the designated clinician to provide this information to the laboratory directors.
Laboratories should also provide data on screening performance to regional and local audit or quality management groups as required.
Babies diagnosed with an IMD not identified through newborn screening
If a screening laboratory director or clinical team is made aware of an IMD case that has not been detected via NBS screening, it is very important that the case is reported to the NBS screening programme.
The clinical team should gather the details in conjunction with the screening laboratory director using the ‘IMD notification of case not detected through screening’ form. The laboratory director should collate and anonymise the data and send it as soon as possible to the NBS screening programme at england.screeninghelpdesk@nhs.net. Data on false negative cases are collated on an annual basis as an important part of the audit and evaluation of the IMD screening programme.
There should be no patient identifiable data on the ‘IMD notification of case not detected through screening’ form.
If a laboratory director is made aware of a case that has been reported as ‘condition not suspected’, in some cases (PKU and MCADD) this should be reported as a serious incident to the Trust and regional SQAS team in accordance with the ‘managing safety incidents in NHS screening programmes’ guidance. Details must be collated by the clinical team in conjunction with the laboratory director and returned to the NBS screening programme. Some cases (MSUD, IVA, GA1 and HCU) are not always detected by newborn screening and while the circumstances should be investigated it would not be appropriate to automatically treat these as a serious incident.
Screening safety incidents
All safety concerns and incidents must be reported and managed in accordance with the ‘managing safety incidents in NHS screening programmes’ guidance. This guidance details the accountabilities for reporting, investigating and managing NHS screening programme safety incidents. It covers the management of screening programme:
- safety concerns
- safety incidents
- serious incidents
Phenylketonuria (PKU)
Scientific background
PKU is an autosomal recessively inherited disorder of amino acid metabolism caused by a deficiency of the enzyme phenylalanine hydroxylase (PAH). The average incidence across the UK is approximately 1 in 10,000- 15,000 births. PAH is required to metabolise phenylalanine to tyrosine. A deficiency of this enzyme results in an accumulation of phenylalanine and associated metabolites in blood and tissues. The infant brain is sensitive to high phenylalanine levels and if left untreated, patients with PKU develop severe mental retardation and microcephaly, and a proportion of patients develop epilepsy.
The clinical consequences of the metabolic defect are dependent on the degree of elevation of phenylalanine which is determined by the residual activity of PAH.
With early detection and monitoring, and a specialised diet, children diagnosed with PKU can lead normal lives.
Screening protocol
Figure 1. PKU newborn screening protocol
Pre-analytical aspects
9.3.1 Potential for false negatives
The potential exists for false negative results to arise for physiological reasons although this is in practice exceedingly rare for example in patients with mild mutations who are rapidly growing. There are no practical steps the laboratory can take to ensure detection of such cases but complete failure to detect a case of PKU is less likely in the UK due to the later sampling age at 5–8 days and the immediate referral of all cases with [Phe] ≥240 µmol/L.
Historically, using the Guthrie method for PKU newborn screening, there was a requirement for established milk feeding before sample collection to guarantee sufficient elevation of Phe levels for detection of abnormal cases. With MS/MS methodology and the UK protocols this is no longer necessary (UK Newborn Screening Programme Centre, 2005).
9.3.2 Potential for false positives
Increased blood phenylalanine is not specific for PKU and may occur in other clinical situations. In cases where the tyrosine is also increased, these are reported as “PKU is not suspected – other disorders follow-up. Urgent referral for investigation and management to an appropriate specialist clinician is essential. See Figure 1. PKU screening protocol
A false positive is defined as a phenylalanine result, which is confirmed on repeat analysis of the screening specimen (triplicate testing) as elevated (screen positive) but is not confirmed on follow-up i.e. confirmatory diagnostic testing result is normal. In practice, it may be impossible to differentiate an incorrect / artefactual result on the screening specimen from a genuine increase of phenylalanine that is transient and not present at diagnostic follow-up.
Possible causes of a ‘false positive’ include:
-1. Contamination of the sample –the artificial sweetener Aspartame (a methylester of phenylalanine / aspartic acid dipeptide) is a potential exogenous source of phenylalanine that may cause contamination if drinks containing it are spilt onto the screening card.
-1. Non-sample source contamination - contamination during the analytical process, for example from phenylalanine standards or with other compounds with a mass to charge ratio of 166.
Both of these situations (1 and 2) may manifest as discrepant results.
-1. Physiological reasons increased phenylalanine and, in some cases, tyrosine, may be seen in a range of clinical scenarios including organ failure, transient illness, prematurity (liver immaturity), , and patients on parenteral nutrition.
-1. Other disorder suspected – these are clinically significant situations which may be identified by screening for PKU and, as such, should be followed up for further investigation. Detection of these disorders is not the objective of the PKU screening programme and all cases will not be reliably detected.
Disorders of pterin metabolism
There are several different rare disorders of pterin synthesis / recycling which may be associated with an isolated increase in phenylalanine at screening and therefore detected by the PKU programme. They are disorders due to defects in the synthesis and recycling of the biopterin cofactor required for normal PAH activity, in particular deficiency of DHPR. These are very rare with a combined estimated incidence of 1–3% of all cases referred with hyperphenylalaninaemia.
It is not possible to exclude these disorders from the blood phenylalanine level and for this reason all babies with a PKU suspected screening result are tested for pterin disorders at follow-up (see section 9.5 – Diagnostic protocol).
Disorders associated with liver dysfunction
An increased blood phenylalanine and tyrosine may occur in babies with disorders associated with liver dysfunction.
Aetiologies include several causes of liver disease in the neonate for example hepatitis, biliary atresia, cytomegalovirus (CMV) and some inherited disorders, in particular galactosaemia and tyrosinaemia type 1. It is not possible to differentiate the cause of the liver dysfunction from the phenylalanine and tyrosine concentrations.
See British Inherited Metabolic Disease Group (BIMDG).
Clinical referral and follow up
Screen positive patients should be referred to the clinical liaison service promptly as patient will need to be contacted and reviewed in a timely manner. See PKU clinical management guidelines.
9.4.1 Follow up of presumptive/suspected positive cases
These are babies with mean of triplicate phenylalanine ≥240 µmol/L and tyrosine <240 µmol/L (Figure 1 – PKU newborn screening protocol):
All babies with a ‘PKU suspected’ screening result should be referred to the specialist clinical team (or designated local team) via the CLS (as per local arrangements) on the same working day that the ‘PKU suspected’ screening result is available. This referral with detailed screening results must be reported both verbally as well as in writing - a template is available.
The family will be instructed by the specialist or designated team to take the baby to an appropriate hospital where the first review appointment will take place on the same or next working day.
9.4.2 Other disorders follow up
These are babies with a mean triplicate phenylalanine ≥240 µmol/L and tyrosine ≥240 µmol/L also babies with a mean triplicate phenylalanine <240 µmol/L and tyrosine ≥240 µmol/L (when galactosaemia testing has been initiated because the first phenylalanine was ≥ 200) (Figure 1 – PKU newborn screening protocol):
Referral must be made to the metabolic or other appropriate specialist team (a template is available), and will depend on the specific local situation for example baby may already be under the care of a medical consultant. Refer to PKU not suspected, other disorder pathway available on BIMDG.
Diagnostic protocol:
Available on (BIMDG)
Figure 2. PKU diagnostic protocol
Medium-chain acyl-CoA dehydrogenase deficiency (MCADD)
Scientific background
MCADD is an autosomal recessively inherited defect of fatty acid oxidation caused by deficiency of the enzyme medium-chain acyl-CoA dehydrogenase. It is the commonest fatty acid oxidation defect, affecting between one in 10,000 and one in 20,000 babies born in the UK.
The metabolic defect is due to a block in the breakdown of medium-chain length fats (carbon chain lengths C6–C12). This can lead to a toxic build-up of medium-chain fatty acids, and octanoylcarnitine (C8). Clinical manifestations typically arise when affected individuals needs to break down fat quickly for example during periods of stress caused by an illness, situations associated with fasting and/or vomiting. Breast fed neonates are at particular risk during the first few days of life. A decompensated state develops which can result in serious life-threatening symptoms including seizures, brain damage and even death. Hypoglycaemia is a late finding.
With early detection and monitoring, and avoidance of fasts, children diagnosed with MCADD can lead normal lives.
Screening protocol
Figure 3. MCADD newborn screening protocol
1.If insufficient blood to re-test, but raised initial C8 (≥0.40) treat as MCADD suspected 2. To calculate C8:C10 ratio calculate the C8:C10 ratio on each spot that is analysed and then calculate the mean of the three C8:C10 ratios
Pre-analytical aspects
10.3.1 Potential for false negatives
Dextrose administration in a sick neonate with MCADD prior to blood collection may reduce octanoylcarnitine levels.
Carnitine depletion has resulted in C8 levels below the screening cut-off in older children with MCADD who have presented clinically. Carnitine stores in newborns generally reflect maternal levels and low carnitine is sufficiently rare for this to be an exceedingly low risk of false negative screening results. No cases were found during the pilot study. This theoretical risk should be borne in mind if testing is delayed beyond the normal postnatal time-frame.
10.3.2 Potential for false positives
Physiological stress in newborns can be associated with elevations of C8 above normal levels, particularly in heterozygote carriers of MCADD. False positives when screening at 5–8 days however are very rare when the C8:C10 ratio is included in the screening pathway.
Preterm neonates are often on special milk formulae with higher than normal medium chain triglyceride content. The majority of babies less than 32 weeks gestation will also require parenteral nutrition (PN) for a period of time. The usual PNlipid source is SMOF® (30% soybean oil, 30% medium triglyceride, 25% olive oil and 15% fish oil). Preterm formulae and PN with SMOF® will rarely lead to a false positive screening test for MCADD, with increased C8 and C8:10 ratio. These babies will require follow-up as a MCADD suspected case.
Clinical referral and follow up
Screen positive patients should be referred to the clinical liaison service immediately as patient will need to be contacted and reviewed urgently. See BIMDG MCADD clinical management guidelines.
10.4.1 Follow up of presumptive/suspected positive cases
These are babies with mean of triplicate C8 ≥0.5 µmol/L AND C8:C10 ratio ≥1.0 (Figure 3 – MCADD newborn screening protocol).
All babies with a ‘MCADD suspected’ screening result should be referred to the specialist or designated clinical team via the CLS (as per local arrangements) on the same day that the ‘MCADD suspected’ screening result is available. This referral with detailed screening results must be reported both verbally as well as in writing – a template is available.
The family will be instructed by the specialist or designated team to take the baby to an appropriate hospital (if not an inpatient already) where initial assessment will take place within 24 hours of the screening referral.
Diagnostic protocol
Figure 4. MCADD diagnostic protocol
FOLLOW-UP ANALYSES (at first review appointment)
- Blood acylcarnitines (dried blood spot or plasma)
- Qualitative urine organic acid (UOA) analysis
- Dried blood spot (or whole blood) for DNA
Results of acylcarnitines, urine organic acids and 985A>G variant analysis should be completed within 5 working days of receipt of the diagnostic samples into the laboratory.
Results of further follow-up diagnostic tests (Extended mutation testing (EMS) should be available within 15 working days from receipt of diagnostic sample
Maple syrup urine disease (MSUD)
Scientific background
Maple syrup urine disease (MSUD) is an autosomal recessive disorder caused by a deficiency of the branched chain alpha keto acid dehydrogenase complex. MSUD occurs in approximately 1 in 140,000 live births in the UK. The name of the condition derives from the maple syrup like odour of the urine sometimes produced by affected individuals.
The metabolic block leads to an increased concentration of the branched chain amino acids leucine, valine, isoleucine and alloisoleucine and their corresponding keto acids. These compounds accumulate in tissues, blood and urine resulting in a life-threatening metabolic decompensation in some affected individuals which without treatment can result in death or severe brain damage.
The classic form of the disorder presents a few days after birth with vomiting or difficulty feeding accompanied by lethargy and progressive neurological deterioration. Patients with the intermediate form may present with developmental delay although the characteristic elevation of branched chain amino acids is still present. The intermittent form of the disease may only manifest at times of stress or infection and branched chain amino acids may not be continuously elevated. A rarer E3 variant has been described which also affects the pyruvate dehydrogenase complex resulting in marked lactic acidosis. Newborn screening will detect patients with the classic form but may not detect the intermediate and intermittent forms.
With early detection and medical/dietetic treatment, a vast majority of patients avoid severe neurological sequelae.
Screening protocol
Figure 5. MSUD newborn screening protocol
Pre-analytical aspects
11.3.1 Potential for false negatives
Physiological reasons - Patients with intermediate and intermittent forms have intermediate enzyme activity and may not be detected by newborn screening.
11.3.2 Potential for false positives
- MS/MS analysis does not differentiate leucine from isoleucine or hydroxyproline. While elevation of leucine and isoleucine both result from MSUD, increased hydroxyproline may indicate the rare benign condition hydroxyprolinaemia
- Increased leucines can be observed in ketosis, severe liver disease (including galactosaemia) or in babies receiving parenteral nutrition.
Clinical referral and follow up
Screen positive patients should be referred to the clinical liaison service immediately as patient will need to be contacted and reviewed urgently. See MSUD clinical management guidelines
11.4.1 Follow up of presumptive/suspected positive cases
These are babies with mean of triplicate Leu ≥600 µmol/L (Figure 6 – MSUD newborn screening protocol):
All babies with a ‘MSUD suspected’ screening result should be referred to the specialist clinical team via the CLS (as per local arrangements) on the same day that the ‘MSUD suspected’ screening result is available. This referral with detailed screening results must be reported both verbally as well as in writing – a template is available.
The family will be instructed by the specialist team to take the baby to an appropriate hospital (if not an inpatient already) where initial assessment will take place. The baby will be transferred to the specialist IMD centre as soon as safely possible.
If transfer is not available, the specialist team will liaise with the local hospital to arrange diagnostic testing and supply of dietary supplements.
Diagnostic protocol
This applies to babies with a presumptive positive newborn screening test, i.e. leucine concentration ≥600 µmol/L that are reported as ‘MSUD suspected’.
Figure 6. MSUD diagnostic protocol
Isovaleric acidaemia (IVA)
Scientific background
Isovaleric acidaemia (IVA) is an autosomal recessive condition caused by a deficiency in isovaleryl-CoA dehydrogenase (IVD). The estimated incidence in the UK is around 1 in 140,000.
IVD is involved in the catabolism of leucine. Defective catabolism result in the toxic accumulation of isovaleric acid and its glycine and carnitine derivatives.
IVA has a spectrum of clinical phenotypes which might include acute neonatal presentations, acute presentations at a later age and chronic intermittent presentations. The acute neonatal presentation is within the first few days of life with vomiting and lethargy, progressing to coma. Patients may also present with similar symptoms from infancy to early childhood, usually precipitated by an infection. Other patients present with chronic symptoms – failure to thrive and/or developmental delay.
Although a firm phenotype/genotype correlation has not been identified, the 932C>T mutation in the IVD gene may be associated with a very mild phenotype and can be asymptomatic.
With early detection and medical/dietetic treatment, a vast majority of patients avoid neurological sequelae and lead normal lives.
Screening protocol
Figure 7. IVA newborn screening protocol
Pre-analytical aspects
12.3.1 Potential for false negatives
Physiological reasons – later onset IVA presenting with failure to thrive and developmental delay has been described. It is unclear whether all such cases would be detected by newborn screening.
12.3.2 Potential for false positives
Pivaloylcarnitine is isobaric with isovaleryl carnitine and can result in false positive results. Pivalate is found is some antibiotics and nipple creams. It is recommended that antibiotic and full drug history is taken from mother and baby, at the first appointment and blood spot C5 isobar testing is undertaken if pivalate suspected and is available from Guy’s and St Thomas’ Hospital.
Glutaric aciduria type 2 is often associated with an increase in C5, C8, C10 and C5-DC acylcarnitines, measured as part of the newborn screening programme. Screen positive results for a combination of these metabolites should prompt consideration of glutaric aciduria type 2.
2-methylbutyryl carnitine is elevated in short/branched chain acyl-CoA dehydrogenase deficiency (SBCAD) and is isobaric with isovalerylcarnitine and causes a positive screening result. SBCAD is a rare condition, probably harmless, but is known in the UK population.
Clinical referral and follow up
Screen positive patients should be referred to the clinical liaison service immediately as patient will need to be contacted and reviewed urgently. See BIMDG IVA clinical management guidelines.
12.4.1 Follow up of presumptive/suspected positive cases
Please refer to the screening protocol (Figure 8). These are babies with mean of triplicate C5 ≥2.0 μmol/L.
All babies with a ‘IVA suspected’ screening result should be referred to the specialist clinical team via the CLS (as per local arrangements) on the same day that the ‘IVA suspected’ screening result is available. This referral with detailed screening results must be reported both verbally as well as in writing – a template is available.
The family will be instructed by the specialist team to take the baby to an appropriate hospital (if not an inpatient already) where initial assessment will take place. The baby will be transferred to the specialist IMD centre as soon as safely possible.
If transfer is not available, the specialist team will liaise with the local hospital to arrange diagnostic testing and supply of dietary supplements.
Diagnostic protocol
Figure 8. IVA diagnostic protocol
Key
MADD | multiple acyl-CoA dehydrogenase deficiency |
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SBCAD | Short/branched chain acyl-CoA dehydrogenase deficiency |
Glutaric aciduria type 1 (GA1)
Scientific background
Glutaric aciduria type 1 (GA1) is an autosomal recessive condition caused by a deficiency of the enzyme glutaryl-CoA dehydrogenase (GCDH). The estimated incidence in the UK is around 1 in 100,000 live births.
GCDH is involved in the decarboxylation of glutaryl-CoA, which is an intermediate in the breakdown of the amino acids lysine, hydroxylysine and tryptophan. Defective catabolism causes the toxic accumulation of glutaric acid, 3-hydroxyglutaric acid, glutaconic acid, and glutaryl carnitine.
Patients are at risk of acute encephalopathic crisis typically within the first 3 years of life. These are usually precipitated by intercurrent illness which can lead to dystonia and dyskinesia as permanent sequelae but with relative preservation of the intellect. A minority of patients can develop insidious onset neurological features such as movement disorder.
With early detection and medical/dietetic treatment, a vast majority of patients avoid neurological sequelae and lead normal lives.
Screening protocol
Figure 9. GA1 newborn screening protocol
Pre-analytical aspects
13.3.1 Potential for false negatives
Physiological reasons – patients with glutaric aciduria type 1 who excrete a very low concentration of glutarate and related metabolites are well described. It is not clear whether these patients would be detected by measuring C5-DC acylcarnitine in the newborn period.
13.3.2 Potential for false positives
C6OH acylcarnitine is isobaric with C5-DC acylcarnitine. An elevated C6OH acylcarnitine is seen in association with ketosis in some patients and may produce a false positive result.
Glutaric aciduria type 2 is often associated with an increase in C5, C8, C10 and C5-DC acylcarnitines, measured as part of the newborn screening programme. Screen positive results for a combination of these metabolites should prompt consideration of glutaric aciduria type 2.
Glutarylcarnitine may be increased in renal impairment and cause a false positive screening result.
Clinical referral and follow up
Screen positive patients should be referred to the clinical liaison service immediately as patient will need to be contacted and reviewed urgently. See BIMDG GA1 clinical management guidelines.
13.4.1 Follow up of presumptive/suspected positive cases
These are babies with mean of triplicate C5-DC ≥0.70 μmol/L (Figure 10- GA1 newborn screening protocol).
All babies with a ‘GA1 suspected’ screening result should be referred to the specialist or designated clinical team via the CLS (as per local arrangements) on the same day that the ‘GA1 suspected’ screening result is available. This referral with detailed screening results must be reported both verbally as well as in writing- a template is available.
All presumptive positives should be referred to the specialist IMD team via the CLS (as per local arrangements) on the same day that the final screening result has become available. This must be reported both verbally as well as in writing – a template is available.
The specialist team will contact the family and make arrangements to review the baby the same day or the next working day.
Diagnostic protocol
Figure 10. GA1 diagnostic protocol
Homocystinuria (pyridoxine unresponsive) (HCU)
Scientific background
Classical homocystinuria is an autosomal recessively inherited defect in the enzyme cystathionine b-synthase (CBS) which requires pyridoxine (Vit B6) as a co-factor. The overall incidence in the UK is reported to be around 1 in 100,000 live births. Deficiency of CBS causes accumulation of homocysteine and increased methionine concentrations. The presentation of classical homocystinuria occurs over a period of years and can result in myopia followed by dislocation of the lens, osteoporosis, thinning and lengthening of the long bones, mental retardation and thromboembolism affecting large and small arteries and veins. Without treatment, the reported mortality is approximately 25% before the age of 30, usually as a result of thromboembolism. There is clinical heterogeneity, with some patients displaying all clinical symptoms whilst others display very few or none.
In the UK approximately 50% of patients with classical homocystinuria are classified as pyridoxine responsive; these patients usually have milder symptoms and slower disease and respond to oral pyridoxine (Vitamin B6) supplementation. They are very unlikely to be detected by newborn screening.
With early detection and treatment, patients can avoid long term complications of the condition.
Screening protocol
Figure 11. HCU newborn screening protocol
Key
THcy | Total homocysteine |
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Met | Methionine |
Pre-analytical aspects
14.3.1 Potential for false negatives
The blood spot screening programme for homocystinuria is not designed to detect pyridoxine responsive classical homocystinuria, defects of homocysteine remethylation (for example MTHFR deficiency or defects of vitamin B12 metabolism) or maternal B12 deficiency.
14.3.2 Potential for false positives
Liver disease (for example due to tyrosinaemia type I or galactosaemia), parenteral nutrition, and methionine adenosyl transferase (MAT) deficiency can give rise to an elevated methionine concentration in the newborn period.
Clinical referral and follow up
Screen positive patients should be referred to the clinical liaison service immediately as patient will need to be contacted and reviewed. See BIMDG HCU clinical management guidelines.
14.4.1 Follow up of presumptive/suspected positive cases
These are babies with mean of triplicate Met ≥50 µmol/L (Figure 12 – HCU newborn screening protocol) AND a THcy ≥15 µmol/L.
All babies with a ‘HCU suspected’ screening result should be referred to the specialist clinical team (or designated local team) via the CLS (as per local arrangements) on the same working day that the ‘HCU suspected’ screening result is available. This referral with detailed screening results must be reported both verbally as well as in writing – a template is available.
The specialist team will contact the family and make arrangements to review the baby the same day or the next working day.
Diagnostic protocol
This applies to babies with a presumptive positive newborn screening test, i.e. Methionine concentration ≥50 µmol/L AND THcy ≥15 µmol/L who are reported as ‘HCU suspected’.
Figure 12. HCU diagnostic protocol
Background to screening for IMDs
Newborn screening began in the late 1950s with locally organised screening initiatives for phenylketonuria (PKU). Universal screening for PKU using dried blood spots collected onto a filter paper card (the Guthrie card) at around one week of age was in place throughout the UK by the early 1970s.
In 2009, screening for MCADD was formally introduced in England (and in the devolved nations during 2009–2013). The technology needed to undertake screening for MCADD, MS/MS, simultaneously provided the potential to detect a range of rare metabolic conditions. In May 2014, the UK National Screening Committee recommended that MSUD, IVA, GA1 and HCU should be included as part of the national programme with effect from January 2015.
The programme is a service that seeks to balance the interests of families where a child is identified as having IMDs versus the interests of the large majority of families where children are unaffected.
Early detection of IMDs is a significant benefit and can be lifesaving. Whilst the majority of results represent a true positive, inconclusive results are a recognised outcome leading to uncertainty for families and health care professionals.
Scientific background to the screening protocol – common elements
The conditions included in this handbook share a common technological approach to their detection. MS/MS is used to detect all the key compounds of interest. The technique uses a triple quadrupole mass spectrometer. Ions produced by the source are selected by the first quadrupole to enter a second quadrupole which acts as a collision cell where molecular fragmentation takes place. The products, which are characteristic of the molecule, are then filtered by the final quadrupole before entering a detector. Complex mixtures can be resolved in this way, allowing untreated blood eluates to be analysed. The instrument can monitor a number of fixed transitions which restricts the compounds detected to a defined and pre-selected list including the amino acids and acylcarnitines of interest which are chosen as signature compounds related to the disorders to be detected. So, PKU may be detected by measuring phenylalanine (Phe), HCU by measuring methionine (Met), MSUD by measuring leucine (Leu), MCADD by measuring C8 acylcarnitine, GA1 by measuring C5-DC acylcarnitine and IVA by measuring C5 acylcarnitine. These analyses can be performed simultaneously in the same sample within two or three minutes and the process can be automated providing a useful basis for mass screening.
References
Fingerhut, R., Ensenauer, R., Röschinger, W., Arnecke, R., Olgemöller, B. and Roscher, A.A. (2009) ‘Stability of acylcarnitines and free carnitine in dried blood samples: Implications for retrospective diagnosis of inborn errors of metabolism and neonatal screening for carnitine transporter deficiency’, Anal Chem, 81, 3571-75.
Golbahar, J., Altayab, D.D. and Carreon, E. (2014) ‘Short-term stability of amino acids and acylcarnitines in the dried blood spots used to screen newborns for metabolic disorders’, J Med Screen, 21(1): 5-9.
Johnson, D.W. and Trinh, M.U. (2004) ‘Stability of malonylcarnitine and glutarylcarnitine in stored blood spots’, J Inher Metab Dis, 27(6), 789-90.
Strnadova, K.A., Holub, M., Muhl, A., Heinze, G., Ratschmann, R., Mascher, H., Stockler-Ipsiroglu, S., Waldhauser, F., Votava, F., Lebl, J. and Bodamer, O.A. (2007) ‘Endocrinology and Metabolism: Long-Term Stability of Amino Acids and Acylcarnitines in Dried Blood Spots’, Clinical Chemistry, 53(4), 717-22.
Tables and figures
Table 1. MRM ion transitions for amino acids and acylcarnitines 7
I
Figure 1. PKU newborn screening protocol 18
Figure 2. PKU diagnostic protocol 22
Figure 3. MCADD newborn screening protocol 24
Figure 4. MCADD diagnostic protocol 26
Figure 5. MSUD newborn screening protocol 28
Figure 6. MSUD diagnostic protocol 30
Figure 7. IVA newborn screening protocol 32
Figure 8. IVA diagnostic protocol 34
Figure 9. GA1 newborn screening protocol 36
Figure 10. GA1 diagnostic protocol 38
Figure 11. HCU newborn screening protocol 40
Figure 12. HCU diagnostic protocol 42
Abbreviations
C0 free carnitine
C2 acetylcarnitine
C5 isovalerylcarnitine
C5-DC glutarylcarnitine
C8 octanoylcarnitine
C10 decanoylcarnitine
CBS cystathionine b-synthase
CDC Centres for Disease Control and Prevention
CF cystic fibrosis
CHT congenital hypothyroidism
CLS clinical liaison service
DHPR dihydropteridine reductase
EQA external quality assessment
GA1 glutaric aciduria type 1
GA2 glutaric aciduria type 2
GAL-1-PUT galactose-1-phosphate-uridyl-transferase
GALP galactose-1-phosphate
GCDH glutaryl-CoA dehydrogenase
HCU homocystinuria
IMD inherited metabolic disease
IQC internal quality control
IS internal standards
IVA isovaleric acidaemia
IVD isovaleryl-CoA dehydrogenase
LCHADD long-chain hydroxyacyl-CoA dehydrogenase deficiency
LC/MS/MS or tandem mass spectrometer with liquid chromatography sample induction
UPLC-MS/MS
Leu leucine (when measured by MS/MS can include isoleucine and alloisoleucine)
MADD multiple acyl-CoA dehydrogenase deficiency
MAT methionine adenosyl transferase
MCADD medium-chain acyl-CoA dehydrogenase deficiency
Met methionine
MRM multiple reaction monitoring
MS/MS tandem mass spectrometry
MSUD maple syrup urine disease
PAH phenylalanine hydroxylase
Phe phenylalanine
PHE Public Health England
PKU phenylketonuria
PPV positive predictive value
SBCAD short/branched chain acyl-CoA dehydrogenase deficiency
SCD sickle cell disease
THcy total homocysteine
TSH thyroid stimulating hormone
Tyr tyrosine
UKCSNS-MCADD UK Collaborative Study of Newborn Screening for MCADD
UKNEQAS UK National External Quality Assessment Service
UK NSC UK National Screening Committee
Appendix 1: Newborn screening laboratory standards
Newborn screening laboratory standards | |
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NBS1 | Laboratories undertaking newborn blood spot screening, including molecular genetics laboratories, shall be accredited by the United Kingdom Accreditation Service (UKAS). |
NBS2 | Newborn blood spot screening shall be provided within the organisational structure of the newborn blood spot screening programme and undertaken by specialist newborn screening laboratories already providing screening programmes. |
NBS3 | There shall be documented local policies and standard operating procedures describing the whole screening process including pre-analytical, analytical and post-analytical processes. Where appropriate these shall include reporting results, referral and follow-up arrangements for presumptive positive cases and carriers, as specified in laboratory handbooks. |
NBS4 | Newborn blood spot screening tests shall be performed by a recommended method as defined in the screening programme laboratory handbook, capable of performing to the required sensitivity/specificity. |
NBS5 | Processes shall be provided in line with relevant national standards and guidance and screening specifications. |
NBS6 | Processes shall be reviewed periodically taking into account audit data, accumulating results, technical developments and local changes in healthcare provision. |
NBS7 | There shall be written and agreed procedures describing the working arrangements between the screening laboratory and any referral laboratory that is used. |
NBS8 | There must be a senior member of the laboratory staff at medical consultant or consultant clinical scientist level, evidenced by FRCPath or equivalent responsible for the newborn blood spot screening with defined lines of accountability for all laboratory aspects of the service. |
NBS9 | Laboratories undertaking newborn blood spot screening shall undertake internal quality control procedures for the screening test and demonstrate satisfactory performance in an approved external quality assurance scheme. |
NBS10 | There shall be a documented risk management policy for the laboratory aspects of the newborn screening programme. This should describe the steps in the testing protocol where failures could occur and the procedures that have been implemented to minimise the risk of their occurrence. |
NBS11 | Screening incidents shall be managed in accordance with the guidance on Managing Safety Incidents in NHS Screening Programmes. |
NBS12 | Laboratories shall participate in audit at local, regional and national level, to assess the effectiveness of the national screening programme. |
NBS13 | The laboratory must release reports on screening performance, including external quality assurance and UKAS assessments to any agency with a legitimate interest in the quality and safety of the programme on behalf of the public. |
NBS14 | Laboratories should publish the results and performance of their newborn blood spot screening programme within an annual report. |
NBS15 | Screening laboratories shall use the newborn screening results status codes and sub codes for acknowledging the receipt of specimens in the laboratory and when reporting results to the child health records departments. |
NBS16 | Clinical information should be requested from clinical referral centres on each presumptive positive case. Data on each case notified should be collated and anonymised before submission to the NHS Newborn Blood Spot Screening Programme. Cases presenting clinically should also be anonymised and reported to the NHS Newborn Blood Spot Screening Programme |