Neonatal blood spot screening

Abstract

This article describes the neonatal or newborn blood spot screening programme (NBSSP) in the United Kingdom. Babies are screened for nine diseases at 5 days of age by a heel prick test. Although midwifes are the primary health practitioner involved in NBSSP, GPs may be consulted antenatally or postnatally for advice. GPs can aid informed decision making for parents concerned about genetic testing, discuss the implications of positive screening, and provide support for families with confirmed diagnoses. This review of the newborn blood spot test will discuss the programme, its limitations, ethical considerations and the diseases screened.

Clinical case scenario

You have a telephone consultation booked with Emily, aged 29 years, to discuss her baby’s heel prick test. She had an emergency caesarean section 2 weeks ago; she was discharged well with no follow up required for the baby. Emily has no past medical history of note. She has been notified that her baby boy needs a repeat heel prick and that he may have cystic fibrosis. Emily is alarmed and upset as she cannot remember discussing this condition with her midwife. She wants to understand the risks of not undertaking the test, whether it can be delayed and whether she needs to inform the father of the child. She is living with her parents who are unaware of any relatives with cystic fibrosis. Her parents are reluctant to involve the father and have suggested she sees a private paediatrician.

Newborn blood spot screening programme

Neonatal screening began with urine testing for phenylketonuria in the 1950s, progressing to blood spot testing in the mid- to late-1960s. In 1968, Wilson and Junger developed their criteria for screening tests that local authorities used to implement their own programmes. East Anglia pioneered screening for congenital hypothyroidism (CHT) in 1979 and cystic fibrosis (CF) in 1980, with the city of Birmingham leading the way to screening for sickle cell disease (SCD) in 1979 (Downing and Pollitt, 2008). The diversity of public health screening strategies across the UK became more formalised and structured when the National Screening Committee (NSC) was established in 1996. The aims of the NSC were to assess the evidence and cost effectiveness of screening, working in collaboration with relevant stakeholders such as patient groups, charities and royal colleges such as the Royal College of General Practitioners. The NSC criteria for screening are fulfilled by the newborn blood spot (NBS) test programme. Table 1 displays the NSC criteria by condition, test, intervention, programme and evaluation, with corresponding information on how this is achieved by the NBS test. Since March 2020, all four home nations (England, Wales, Scotland and Northern Ireland) have adopted the same NBS tests. This uniformity in newborn screening has benefits for a mobile population with families no longer living their whole lives in one area of the UK.

Table 1.

UK National Screening Committee criteria for screening in relation to newborn blood spot.

	UK screening criteria	Newborn blood spot screening
Condition	Important public health problem, with epidemiology and risk factors understood	The conditions cause significant morbidity and mortality. Genetic and metabolic pathways are understood with effective treatment
	Cost-effective primary prevention implemented with understanding of natural history of carriers of genetic mutations	For SCD and CF screening detects carrier status which aids decision making when having offspring
Test	Safe, simple, precise and validated test with suitable cut offs defined	Simple test and laboratories have validated procedures for measurement and processing results
	Acceptable test	Heel prick is non-invasive and easy to perform
	Agreed policy on further diagnostic tests after positive screening	There are national laboratory standards for analysing screening tests and pathways for diagnosis
Intervention	Effective intervention for identified patients	Early management reduces morbidity and mortality
	Benefits compared to usual care	Usual care leads to poor outcomes
	Evidence-based policies for intervention	Specialist services use evidence-based medicine to manage patients
Programme	Acceptable to public and professionals	Informed consent with patient information used to maintain high uptake of screening
	Cost-effective and evidence based	Cost-effectiveness and evidence for benefit reviewed by NSC
	Benefit to individual outweighs harms with evidence to show reduced mortality and morbidity from screening	Evidence shows that early diagnosis reduces morbidity and mortality. Counselling, education and access to referral available for carriers
	Clinical management of the condition optimised, and all treatments considered	All conditions managed by Specialists services with guidelines on treatment
Evaluation	Plans for managing and monitoring quality assurance standards	Annual review of programme and audits of laboratory standards, uptake of screening, false positives and false negatives
	Adequate staffing and facilities	Professional training programmes and teaching resources for laboratory and healthcare staff
	Information on the purpose and consequences of screening, investigations and interventions available for the public	Regularly reviewed and updated patient information in various languages with print and online copies available

Adapted from Public Health England (2015) Criteria for appraising the viability, effectiveness and appropriateness of a screening programme.

Limitations and ethical considerations

For any screened condition, GPs can help patients understand the accuracy of screening tests in identifying individuals with disease. Sensitivity of a test is the probability of a positive test result given that the condition is present, otherwise known as a true positive. Specificity is the probability of a negative result given the condition is not present, a true negative. False positives lead to parental anxiety and use of expensive diagnostic resources. False negatives can provide false reassurance. Although these still occur, measures such as avoiding analysis of inadequate samples and repeat testing for premature babies attempt to decrease the rate.

Screening can also be described by the predictive value of the test. The positive predictive value estimates the chance that a patient with the disease will have a positive screen result. A negative predictive value is the chance that a patient without the disease will have a negative test result. These terms are related to the prevalence of the disease; with rarer diseases, the negative predictive value of a test increases, whereas the positive predictive value decreases. Figure 1 demonstrates how the sensitivity, specificity, positive and negative predictive value can be calculated based on test results and whether the disease is present.

Figure 1.

Measurement of the performance of a screening test.

Explaining the validity of tests to patients in a straightforward way that avoids jargon is essential for improving the uptake of screening. The difference between a screening test and diagnostic test allows understanding of false positives and false negatives. In all screening programmes there is self-selection bias, with those individuals who choose to take part having different characteristics from those who do not. Vulnerable groups, in particular, may miss out, such as travelling communities, the homeless, refugees and those with learning disabilities.

Informed consent and decision making is integral to NBS screening in the UK. Over the years, healthcare training and provision of educational material has improved. There is an acknowledgement of the anxiety and distress caused by screening, from false positives through to diagnosis of the condition or carrier status. As well as feelings of guilt from parents, there may be issues around paternity and stigma of diagnosis. A positive diagnosis may result in genetic testing of other family members and have implications for future pregnancies. Counselling of parents is important, as is early access to specialist services.

Consenting to the heel prick includes consent to all aspects of the programme, which includes storage of residual blood. The sample may be used for future research or part of quality appraisal of the screening programme. In England, positive results for SCD and the inherited metabolic diseases (IMDs) are sent to the National Congenital Anomaly and Rare Diseases Registration Service. Families have to opt-out of this if they do not want their details on the register.

The conditions

Information on the NBS test is given antenatally at the booking visit, in the third trimester and postnatally by the midwife. Ideally a heel prick will be performed on day 5 (counted from day of birth as day 0) though it can be taken up to day 8. If babies are missed at this stage the NBS test can be undertaken up to 1 year of age for all conditions except CF which needs to be completed by 8 weeks. Prematurity and blood transfusion can affect screening results. In the latter case, 3 days post transfusion, are required for the metabolites used in screening for CF, CHT and IMDs to return to normal.

The NBS programme screens for the nine conditions listed in Box 1. Parents can choose to decline testing for all or selected diseases. Four blood spots are taken by a heel prick, as shown in Figure 2. The six metabolic diseases are screened from the same blood spot so they cannot be differentially chosen. Results are fed back through the Child Health Record Department and are defined as:

Condition suspected - with immediate referral to the specialist team

Condition not suspected – health visitor and parents informed

Avoidable repeat test – insufficient blood, missing or inaccurately filled form

Inconclusive – borderline results which require repeat sampling

Carrier status- healthcare professional to discuss with parents and arrange follow up

Box 1.

The current NBS programme.

The current NBS programme includes the following diseases:

• Sickle cell disease

• Cystic fibrosis

• Congenital hypothyroidism

• Six inherited metabolic diseases: phenylketonuria,, medium-chain acyl-CoA dehydrogenase deficiency, maple syrup urine disease, isovaleric acidaemia, glutaric aciduria type 1, homocystinuria

Figure 2.

Neonatal blood spot (heel prick).

Sickle cell disease

Sickle cell anaemia has an autosomal recessive inheritance. Offspring of two carriers (sickle cell trait) have a 1-in-4 chance of not being affected or having the condition and a 2-in-4 chance of being a carrier. Figure 3, is the infographic produced by Public Health England explaining the inheritance pattern for autosomal recessive conditions. As well as sickle cell anaemia (homozygous sickle gene), SCD can occur with other genotypes described as compound heterozygotes. This occurs when the sickle haemoglobin (Hb S) interacts with haemoglobin variants (Hb V) such as Hb C or with beta (β) thalassaemia. SCD is thought to occur in 1 in 2000 babies born in the UK, commonly affecting people of Afro-Caribbean ethnicity. The clinical manifestations vary depending on the genotype. Infants with sickle cell anaemia can present with severe anaemia, splenomegaly, vaso-occlusive episodes (painful crises), bone infarcts, hepato-renal complications and bacterial infections. Babies who are compound heterozygotes can have mild-to-moderate clinical features, though if Hb S is inherited with β thalassaemia the condition can be as severe as sickle cell anaemia. Individuals born with sickle cell trait are asymptomatic, except at high altitude or under general anesthesia. Children with SCD are prescribed prophylactic antibiotics and are managed by specialist services.

Figure 3.

Inheritance of autosomal recessive conditions.

SCD is the only NBS condition that is included in antenatal screening. The family origin questionnaire is used in low prevalent areas to aid identification of women who may benefit from screening, whereas in high prevalent areas, the questionnaire provides additional information to the laboratory and all mothers are screened. Education and counselling are important, as mothers who are carriers need to inform the father of the child to enable testing of the father. In addition to paternity issues, if both parents are carriers counselling is required to discuss diagnostic tests such as Amniocentesis or Chorionic Villus Sampling.

Haemoglobin (Hb) fractions are used for screening. If abnormal fractions are detected using one method then results are validated using a second method. Fractions identified are fetal Hb (Hb F), adult Hb (Hb A), sickle Hb (Hb S) and Hb variants. Variants include Hb C, Hb D, Hb E, Hb O^Arab and Hb V encompassing unidentified variants. Unaffected infants will have Hb F as the major fraction with Hb A. Those with SCD can have either Hb S with Hb F, or Hb F and S in combination with Hb A (the quantity of Hb S is greater than Hb A) or Hb S, F and another Hb variant. Sickle cell carriers have Hb F, Hb A and Hb S, but the amount of Hb A will exceed Hb S. In addition to SCD, other haemoglobinopathies are identified that can be clinically significant (e.g. β thalassaemia) or benign conditions (e.g. homozygotes or compound heterozygotes of variant Hb) and carriers.

Like all screening methods there will be false positives and negatives. Most babies with β thalassaemia major have very low Hb A, so will be detected through screening, but those with Hb A above the cut off will not be detected. Neither are all thalassaemia or carriers of thalassaemia detected, or rare Hb variants. Furthermore, prematurity and blood transfusion can affect results. Procedures are in place in neonatal units to ensure a blood spot is taken at a suitable time, either on admission, before blood transfusion or 3 days after. Figure 4 is a flow diagram of SCD screening. Parents should receive information and counselling if their child has a benign condition or is a carrier, and newborns with suspected SCD or other significant condition are referred for genotyping and clinical review.

Figure 4.

Sickle cell screening outcomes based on analytical results and potential diagnoses.

Cystic fibrosis

CF occurs in 1-in-500 babies screened; this multisystem condition is autosomal recessive. As the inheritance is the same as sickle cell anaemia, it can be explained to patients using the diagram in Figure 1. Currently around 2000 gene mutations have been identified for the CF transmembrane conductance regulator which can cause a variety of phenotypes. The protein controls sodium and chloride transport with failure causing the features of the disease. Mucus plugging, impaired muco-ciliary clearance and reduced immunity affects the lungs with recurrent infections and bronchiectasis. Children can suffer from poor growth and malnutrition, due to pancreatic insufficiency. Abnormal liver function can progress into cirrhosis and portal hypertension, with many other long-term sequelae such as diabetes, arthropathy, osteoporosis and infertility.

The screening test is the immunoreactive trypsinogen (IRT) test. Increased levels are present in the first 8 weeks, so babies presenting after this time are unable to undergo testing. Figure 5 displays the flow diagram of possible results from screening. Several IRT cut off points are used before DNA testing for four common CF DNA mutations. If no mutation is identified the IRT level is assessed at the 99.9th centile. If one mutation is found, a search is performed for a further 29 to 31 DNA mutations. A second blood spot from the baby may be required at day 21. This step is undertaken to reduce the number of babies who have negative diagnostic sweat tests. CF is suspected after two mutations or from two raised IRT samples or one mutation and a raised second IRT sample. Carriers are suspected from one mutation and a second IRT below the cut off. If the baby is diagnosed as a carrier there is approximately a 1-in-200 chance of further offspring of the couple having CF. Parents receive counselling regarding the options of further genetic testing and review by CF specialists. The possibility of false positives can occur with prematurity, congenital infections and renal failure with meconium ileus and blood transfusions potentially leading to false negatives.

Figure 5.

CF screening test pathway.

Congenital hypothyroidism

Prior to the introduction of screening, congenital hypothyroidism was a common cause of developmental delay and learning disabilities. Around 1-in-3000 babies are diagnosed with CHT. Screening measures whole-blood thyroid stimulating hormone (TSH) levels. Regional laboratories have various ranges for diagnosis. Results from a screening test can be positive, negative or borderline. Borderline results are repeated 7 to 10 calendar days after initial sample, and if the result is not negative the baby is referred for further testing to a Paediatric endocrinologist. This requires a TSH level, free T4 level and may include a thyroid scan. Premature babies born less than 32 weeks have a second blood spot taken at day 28 or on discharge from the neonatal unit. CHT can be associated with other congenital malformations and babies may require lifelong thyroxine replacement.

Inherited metabolic diseases

Neonatal screening includes six autosomal recessive metabolic disorders. The most frequently diagnosed are phenylketonuria (PKU) and medium-chain acyl-CoA dehydrogenase deficiency (MCADD), which occur in 1-in-10 000 screened babies. Maple syrup urine disease (MSUD) and isovaleric acidaemia (IVA) occur in 1-in-150 000 babies. Glutaric aciduria type 1(GA1) and homocystinuria (HCU) are rarer, being found in 1-in-300,000 infants (Public Health England, 2018a). Each condition is due to specific enzyme deficiencies, which prevent breakdown of amino acids. The resulting accumulation of toxic metabolites can cause acute metabolic crises leading to coma and death. Some IMDs have long term sequelae of developmental delay and learning disability. Diagnosis requires both molecular genetic testing and analysis of plasma and urine amino acids. Currently the mainstay of treatment is dietary, with management involving a multidisciplinary team of specialists to monitor blood levels, growth and development of the child. Diets can be complicated, unpalatable and cause difficulties socially, however, new strategies such as gene therapy are being developed to improve management. It should be noted that many other inborn errors of metabolism are not currently screened for in the UK. The National Screening Committee reviewed evidence for inclusion of Gaucher’s disease in 2019, but it was declined as there was no clear benefit of treatment before diagnosis.

Phenylketonuria

Neonates with PKU have a deficiency of the phenylalanine hydroxylase enzyme that converts phenylalanine to tyrosine. High levels of phenylalanine are toxic to the brain, leading to developmental delay with learning disability, spasticity, epilepsy and behavioural problems. The disease is most common in Caucasians with characteristic appearance of being fairer than siblings. This is due to phenylalanine affecting the production of melanin. Breath and urine can have a distinctive musty smell. Dietary treatment requires essential amino acid supplements and restriction of phenylalanine while maintaining sufficient levels for normal growth and development.

Medium-chain acyl-CoA dehydrogenase deficiency

MCADD is the most common mitochondrial fatty acid oxidation disorder. During periods of fasting such as overnight and episodes of increased energy demand (e.g. trauma or febrile illness) neonates are unable to convert medium chain fatty acids to energy due to the enzyme deficiency. Thus glycogen stores are rapidly used up leading to hypoglycaemia. Babies may present with anorexia, vomiting, seizures and lethargy, which can progress to coma and death. In addition to risk of mortality recurrent episodes of decompensation are thought to lead to developmental delay (Gartner et al., 2015). Management is based on avoidance of fasting with guidance on recommended length of time between feeds changing with age of child, use of emergency glucose during episodes of illness and early medical intervention in metabolic crises.

Maple syrup urine disease

Historically, MSUD was diagnosed by the characteristic sweet smell of urine. The disorder in the metabolism of branched chained amino acids presents with high levels of leucine, isoleucine and valine, which can lead to a variety of clinical manifestations. As with MCADD babies may present acutely in a metabolic crisis during intercurrent infection with vomiting, irritability and lethargy. Neurological manifestations include seizures, abnormal movements and hypotonia. Some infants are diagnosed when presenting with failure to thrive, developmental delay and learning difficulties. Dieticians guide parents on restricting leucine, valine, isoleucine and alloisoleucine, but still maintaining sufficient levels in their diet for growth and development.

Isovaleric acidaemia

Babies with IVA have an acidaemia due to impaired breakdown of leucine, a branched chain amino acid. Lack of the enzyme, isovaleryl Co A dehydrogenase leads to increased levels of isovaleric acid. There can be differences in presentations from acute neonatal illness to a chronic intermittent form. Babies can present unwell with anorexia, vomiting, dehydration and lethargy progressing to metabolic acidosis, ketosis and lactic acidaemia. The resulting risk of coma and death can be avoided with early diagnosis and intervention. The chronic form can lead to learning difficulties. Dietary treatment involves restricting leucine, but preventing deficiency which requires parental education, prescription of appropriate medical foods and early intervention during illness.

Glutaric aciduria type 1

In this type of acidaemia, infants with GA1 lack the enzyme glutaryl CoA dehydrogenase, causing an accumulation of glutaric acid and its metabolites. Multiple genetic mutations have been identified for the condition and expression of the phenotype can vary. Infants may have macrocephaly and can present with encephalopathy during an illness or trauma. Other neurological symptoms include seizures, hypotonia, rigidity, loss of head control and dystonia. Similar to IVA dietary management involves the correct balance of amino acids and depends on the age of the infant. Medical foods are available and parents need intensive dietetic support.

Homocystinuria

HCU is a multisystem disorder caused by the deficiency of cystathionine beta-synthase, which is involved in methionine metabolism. Phenotypes and clinical characteristics can differ, with those severely affected presenting in infancy. Newborns may have high blood levels of homocysteine and methionine. These metabolites can affect the ocular, skeletal, central nervous and vascular systems. Dietary treatment aims to decrease levels of homocysteine and methionine using a protein restricted diet and use of vitamins B6, B12 and folate. Along with the other inherited metabolic disorders these patients will require lifelong monitoring and support.

KEY POINTS

NBS screening is a public health programme to identify babies who may have nine congenital disorders

The UK National Screening Committee regularly reviews the evidence and cost-effectiveness of screening for current and new conditions

GPs can help parents with decision making, discuss the implications of results and provide support for families with confirmed diagnoses

Screening is usually done at day 5 but can be undertaken up to 1 year for all conditions except CF

False positives and negatives can occur due to various factors such as prematurity, blood transfusions and if the baby has a rarer DNA mutation which is not screened for

When screening for SCD other significant and benign haemoglobinopathies and carrier types can be discovered

ORCID iD

Dr Thulani Ashcroft https://orcid.org/0000-0002-1829-6132

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