Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

1. Thirty Years with Cystic Fibrosis Jacqui Calvin Addenbrooke’s Hospital Cambridge

2. Screening area – Norfolk, Suffolk, Cambridgeshire

3. Screening for CF since 1979 First year a pilot to set cut-offs Routine screening - January 1980 Various assays and protocols used

4. First CF patient detected on screening Reported in ‘Current issues in neonatal screening for cystic fibrosis and implications of the CF gene discovery’. Farrell et al 1991 Ped Pulmonol First CF child detected by IRT screening – October 1980 We have two earlier CFs – dob June/July 1980

5. Protocols Jan 1980 – April 2004 IRT–IRT protocol Based on prolonged hypertrypsinaemia seen in the majority of babies with CF May 2004 onwards IRT-DNA-IRT p.Phe508del followed by 29 panel Jan 09 changed to 4 mutation panel CFTR

6. Total babies screenedJan 1980 to December 2009 730,730 Give or take a few!

7. What are we measuring? Trypsinogen-1 (encoded by PRSS1 gene) cationic Trypsinogen-2 (encoded by PRSS2 gene) anionic

8. In health cationic > anionic Pancreatitis anionic >> cationic 19 CF blood spots anionic:cationic ratio = 0.49-3.5 15/19 anionic > cationic (Lindau-Shepard and Pass 2010)

9. Trypsin(ogen) in blood Cationic trypsinogen a1-antitrypsin a1-AAT Binds 15% trypsin a2-macroglobulin trypsin a2-macroglobulin Binds 85% trypsin Anionic trypsinogen

10. Dhondt & Farriaux 1994 Behring RIA kit Samples from CF babies showed a bimodal decay curve suggesting a different mix of IRT species

11. Sorin Radioimmunoassay Polyclonal antibodies raised to inactivated cationic trypsin Cross reactivity: trypsinogen - 98% a1AT bound trypsin 15% a2macroglobulin bound trypsin 1%

12. AGEN Two monoclonal antibodies raised to cationic trypsinogen Enzyme immunoassay ?cross reactivity with other species

13. Delfia/autodelfia Dissociation-Enhanced Lanthanide Fluorescent Immunoassay Monoclonals raised to ???? Antibodies chosen gave the best discrimination between normal and CF bloodspots

15. Definitions Protocol intended to maximise diagnosis of CFTR defects producing preventable/treatable disease in infancy or childhood. To minimise diagnosis of very mild forms of CFTR defects producing late-onset, essentially unpreventable disease.

16. False negative: A ‘not suspected’ result in a child presenting before the age of 16 years with signs and symptoms of CF, subsequently diagnosed clinically False positive: A ‘suspected’ result in a child not diagnosed with CF

17. Numbers screened and false positives Protocol Kit Total FP n % IRT-IRT Sorin 234, 098 24 0.01 IRT-IRT Agen 65,222 22 0.03 IRT-IRT Delfia 210,356 37 0.02 IRT-IRT Autodelfia 73,290 13 0.02 IRT-DNA-IRT Autodelfia 147,764 9 0.006

18. Babies with CF Total number known to screening lab: 325 Positive screen: 296 Includes 53 with meconium ileus 13 sibs or diagnosed prior to screening 29 ‘Not suspected’ Including 10 meconium ileus

19. Meconium ileus 53 cases of MI 9 ‘Not suspected’ 1 ‘Probable carrier’ Well known that MI causes problems – up to 25% have negative screening results Traditionally excluded from screening stats

20. False positives: IRT-IRT protocol Clinical details n Term, well babies 43 Multi-organ failure and/or sepsis 31 Birth trauma, hypoxia 6 Extreme prematurity 1 Gastroschisis 3 Pyloric stenosis 1 Ileal atresia 1 Renal disease 3 Liver disease 2 Trisomy 13 2 Trisomy 18 2 Galactosaemia (symptomatic) 6

21. GALACTOSAEMIA 17/21 galactosaemics had raised IRT at time of diagnosis (Anthony & Mary Heeley and Karen Poyser) IRT concentrations in untreated galactosaemia are comparable to those seen in CF

22. IRT pre and post treatment in galactosaemia 17/21 galactosaemics had raised IRT

23. False positives IRT-DNA-IRT

24. False negatives IRT-IRTPancreatic insufficient

25. False negatives IRT-IRTPancreatic insufficient

26. King et al 1986 Small bowel, liver, respiratory and pancreatic changes, as described in CF, a common finding at autopsy in premature babies receiving prolonged intensive care.

27. False negatives IRT-IRTPancreatic sufficient

28. False negatives IRT-IRTPancreatic sufficient

29. False negatives IRT-DNA-IRTPancreatic insufficient

30. False negatives IRT-DNA-IRTPancreatic insufficient

31. False negatives IRT-DNA-IRTPancreatic sufficient

32. False negatives IRT-DNA-IRTPancreatic sufficient

33. Sweat testing 5 ‘suspected’ mis-identified as carriers p.Phe508del /p.Arg117His p.Phe508del /p.Arg117His p.Phe508del /p.Pro67Leu

34. Sweat chloride Over 60 mmol/L - supports the diagnosis of CF Under 40 mmol/L - normal, low probability of CF 40-60 mmol/L - intermediate, suggestive but NOT diagnostic of CF

35. Sweat chloride Over 60 mmol/L - supports the diagnosis of CF Under 40 mmol/L - normal, low probability of CF 40-60 mmol/L – - intermediate, suggestive but NOT diagnostic of CF Sweat chloride ULN = <30 mmol/L Up to 6 months of age

36. p.Arg117His Expression influenced by a polythymidine variant in intron 8 Affects splicing T7 – neutral T5 – reduces protein synthesis

37. Newborns screened France 2002-2006 61 p.Phe508del/p.Arg117His;T7 47 asymptomatic 13 respiratory symptoms 1 classical CF Penetrance of classical CF 0.03% Thavin-Robinet, Munck, Huet et al 2009

38. True negatives mis-identified as false negatives • Male IRT less than 25 mg/L rectal prolapse, sweat Cl 45, 47 mmol/L no mutations identified 2. & 3. Male twins 8yr steatorrhoea and respiratory problems abnormal sweat test in one child no mutations identified multiple sweat Cl – normal nasal potential difference - normal pancreatic function tests – normal 10yr mild asthma

40. Conclusion 95% of unexpected cases CF detected 15/19 False negatives missed on first IRT Specificity IRT-DNA-IRT >> IRT-IRT Outcome data very difficult to collate! Interpret normal and intermediate sweat tests with caution!