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# Neonatal screening for cystic fibrosis using immunoreactive trypsinogen and direct gene analysis: four years' experience

BMJ 1994; 308 (Published 04 June 1994) Cite this as: BMJ 1994;308:1469
1. E Ranieri,
2. B D Lewis,
3. R L Gerace,
4. R G Ryall,
5. C P Morris,
6. P V Nelson,
7. W F Carey,
8. E F Robertson
1. Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, South Australia 5006, Australia
1. Correspondence to: Mr Ranieri.

## Abstract

Objective : To assess the performance and impact of a two tier neonatal screening programme for cystic fibrosis based on an initial estimation of immunoreactive trypsinogen followed by direct gene analysis.

>Design : Four year prospective study of two tier screening strategy. First tier: immunoreactive trypsinogen measured in dried blood spot samples from neonates aged 3-5 days. Second tier: direct gene analysis of cystic fibrosis mutations (δF508, δI506, G551D, G542X, and R553X) in samples with immunoreactive trypsinogen concentrations in highest 1% and in all neonates with meconium ileus20or family history of cystic fibrosis.

Setting : South Australian Neonatal Screening Programme, Adelaide.

Subjects : All 88 752 neonates born in South Australia between December 1989 and December 1993.

Interventions : Neonates with two identifiable mutations were referred directly for clinical assessment and confirmatory sweat test; infants with only one identifiable mutation were recalled for sweat test at age 3-4 weeks. Parents of neonates identified as carriers of cystic fibrosis mutation were counselled and offered genetic testing. Main outcome measures - Identification of20all children with cystic fibrosis in the screened population.

## Discussion

A two tier, neonatal screening strategy for cystic fibrosis, using immnoreactive trypsinogen quantification followed by direct gene analysis in blood spots from neonates aged 3-5 days, has been operating in South Australia since December 1989. Consistent with initial predictions,14 this two tier strategy has functioned at high sensitivity, achieved by the use of a lower immunoreactive trypsinogen decision level (highest 1% of the unpartitioned population), and high specificity, achieved by direct gene analysis in the second tier. In the first four years of its operation this strategy has been highly effective, requiring the recall of only 3.2 families for every neonate detected with cystic fibrosis. Of the total neonates screened, only 0.08% (69/88 752) were recalled for a sweat test, which is an improvement on 0.18-0.66% in those strategies recalling neonates on the basis of the estimation of immunoreactive trypsinogen alone.*RF 8,19- 22* This reduction minimises the extent of anxiety generated in the population screened.

## Two tier strategy

The incidence of cystic fibrosis within the screened population was 1 in 3060. During the four year screening period there were six prenatal diagnoses of cystic fibrosis (four singleton pregnancies and one twin pregnancy), which resulted in termination of the pregnancies. This gives a total incidence of cystic fibrosis of 1 in 2535, which compares well with a population incidence in Australia of 1:2500.

As most sweat tests are performed at one hospital and all children with cystic fibrosis are reviewed at some stage in one cystic fibrosis clinic, the outcome of all neonates screened could be monitored. We are not aware of any child with cystic fibrosis who was missed by the screening programme. However, from the results of other workers the expected theoretical false negative rate in the first tier of the screening strategy will be about 3-4% when immunoreactive trypsinogen alone is measured.4,5,8,21,23 For this reason we have incorporated clinical and familial information into our screening strategy. In such cases direct gene analysis is performed regardless of the immunoreactive trypsinogen result. One affected neonate, presenting with meconium ileus, was found through this branch of the strategy.

In the second tier of the screening strategy, direct gene analysis of the five common cystic fibrosis mutations within our population will theoretically fail to identify a further 5% of neonates with cystic fibrosis who carry mutations that cannot be identified at present. Overall, the two tier screening strategy operating in South Australia will fail to detect one child with cystic fibrosis every two years. However, if a fifth of children with cystic fibrosis do not present clinically until after the age of 5 years1,24,25 (presumably these may be carriers of rarer mutations with mild phenotype or patients with low immunoreactive trypsinogen) these children might not be seen for several more years. An extreme example of this may be congenital bilateral absence of the vas deferens, in which mutations in the cystic fibrosis gene cause male infertility.26,27

## Communicating results

The inclusion of four of the more common mutations, in addition to δF508, in our screening strategy allows additional cases of cystic fibrosis to be detected at minimal extra cost. Furthermore, the additional information gained determines the way in which the recall process is handled. In 23 neonates the diagnosis of cystic fibrosis was made because they were either homozygotes or compound heterozygotes for the mutations sought. In this group the referring medical officer was contacted by telephone and informed of the result of the direct gene analysis as soon as this was available. The medical officer contacted the family of the child and discussed the screening results with them. Many of these neonates had clinically important complications noted before the screening result was available. It is important therefore to communicate the results of the direct gene analysis rapidly.

Our cystic fibrosis clinic offers to assess these neonates and counsel the families within 48 hours of the initial contact by the referring medical officer. At the time of the assessment, arrangements are made for a sweat test to confirm the direct gene analysis result to exclude the small possibility of error (such as an incorrectly labelled initial blood spot sample). The clinic offers review of the neonate by a team including a paediatric pulmonologist, gastroenterologist, nutritionist, physiotherapist, and social worker, and genetic counsellor. The median age of the neonates detected with cystic fibrosis at the time of initial clinical assessment is 3 weeks.

## Further testing

For those neonates carrying only one identifiable mutation a sweat test is arranged at age 3-4 weeks. A telephone call is made to the referring medical officer stating that the child is at risk of cystic fibrosis. For this group it is emphasised that the risk is relatively low, with only 1 in 12 of such neonates recalled having cystic fibrosis. It has been found that the quality of information and its presentation to parents at this point is critical to establishing their favourable response to the resolution of the diagnosis in their infant. In an expert centre there are few problems associated with performing a sweat test on a 3-4 week old infant.

At the time of the sweat test the family is counselled for about 45 minutes; the genetics of cystic fibrosis inheritance, the implications of being a cystic fibrosis carrier, and the risks to future pregnancies, parental siblings, and for the infant in future years are discussed. The parents and referring medical officer are then contacted by telephone within 24 hours with the result of the sweat test. For most (91% in this study) the normal sweat test result confirms their child as a carrier of a cystic fibrosis mutation. These families are further contacted by telephone about two weeks later, when the results of the parents' genetic tests, if requested, are available. A summary of all the family results is sent to them in a letter that also reiterates what was discussed in the counselling session and offers further counselling and testing for themselves and other family members if desired.

The higher incidence of cystic fibrosis mutations (63/975, 1:15.5) among the population of neonates with increaed immunoreactive trypsinogen has been confirmed. This finding has been reported by several other workers28,29 and has meant that twice as many sweat tests than theoretically expected have been performed to exclude cystic fibrosis.

## Conclusion

The two tier screening strategy we implemented in 1989 has proved to be a highly sensitive and specific screen for cystic fibrosis. Deficiencies encountered in earlier strategies have been avoided.8,9,22 Our current false negative rate is less than the theoretical rate of about 8%; this is acceptable given that the definition of cystic fibrosis is now changing in the light of genotypephenotype studies.*RF 29-31* In practice the resources involved in counselling of recalled families are offset by the reduced number of neonates requiring follow up. For this reason alone the strategy used here is an improvement on other screening programmes, and the difficulties associated with the genetic information generated by the programme can be overcome as long as a standardised protocol for counselling and monitoring recalled families is followed.

We believe this screening strategy is an effective, practical, and specific approach for the neonatal screening of cystic fibrosis. If future clinical studies confirm the benefit of presymptomatic diagnosis of cystic fibrosis31,32 then this strategy will enhance neonatal screening for the disorder.

## Acknowledgments

We thank Dr E Edkins from the Princess Margaret Hospital for Children in Perth, Western Australia, for determining the extra 11 cystic fibrosis mutations; staff of the cystic fibrosis unit at the Women and Children's Hospital; and families, medical officers and other allied health care workers for their support of the programme.

## References

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