jacqui calvin addenbrooke ’ s hospital cambridge
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Jacqui CalvinAddenbrooke’s Hospital Cambridge
Thirty Years with
Cystic Fibrosis
Screening area – Norfolk, Suffolk, Cambridgeshire
Screening for CF since 1979 First year a pilot to set cut-offs
Routine screening - January 1980Various assays and protocols used
First CF patient detected on screening
Reported in ‘Current issues in neonatal screening for cystic fibrosis andimplications 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
ProtocolsJan 1980 – April 2004 IRT–IRT protocolBased on prolonged hypertrypsinaemiaseen in the majority of babies with CF
May 2004 onwards IRT-DNA-IRTp.Phe508del followed by 29 panelJan 09 changed to 4 mutation panel
CFTR
Total babies screenedJan 1980 to December 2009
730,730Give or take a few!
Trypsinogen-1(encoded by PRSS1 gene)cationic
Trypsinogen-2(encoded by PRSS2 gene)anionic
What are we measuring?
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)
Trypsin(ogen) in blood1-antitrypsin
2-macroglobulin
Cationic trypsinogen
Anionic trypsinogen
trypsin
1-AATBinds 15% trypsin
2-macroglobulinBinds 85% trypsin
Dhondt & Farriaux 1994Behring RIA kit
Samples from CF babies showed a bimodal decay curve suggesting a different mix of IRT species
SorinRadioimmunoassayPolyclonal antibodies raised to inactivated cationic trypsin
Cross reactivity: trypsinogen - 98%
1AT bound trypsin 15% 2macroglobulin bound trypsin 1%
AGEN
Two monoclonal antibodies raised tocationic trypsinogen
Enzyme immunoassay
?cross reactivity with other species
Delfia/autodelfia
Dissociation-Enhanced Lanthanide Fluorescent Immunoassay
Monoclonals raised to ????
Antibodies chosen gave the bestdiscrimination between normal and CF bloodspots
Time period Kit Protocol1st action limit 2nd action limit
IRT g/L IRT g/L
Jan 80 – March 90 Sorin IRT-IRT 60 50
April 90 – Sept 92 Agen IRT-IRT 35 35
Oct 92 – March 01 Delfia IRT-IRT 70 60
April 01 – April 04 Autodelfia IRT-IRT 70 60
May 04 – Dec 09 Autodelfia IRT-DNA-IRT 70 60
1st samples collected day 6-10 (Jan 1980 to March 2000), day 5 - 8 (April 2000 onwards)
DefinitionsProtocol intended to maximise diagnosis ofCFTR defects producing preventable/treatabledisease in infancy or childhood.
To minimise diagnosis of very mild forms ofCFTR defects producing late-onset,essentially unpreventable disease.
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
Numbers screened and false positives
Protocol Kit Total FP n %
IRT-IRT Sorin 234, 098 24 0.01IRT-IRT Agen 65,222 22 0.03IRT-IRT Delfia 210,356 37 0.02IRT-IRT Autodelfia 73,290 13 0.02IRT-DNA-IRTAutodelfia 147,764 9 0.006
Babies with CFTotal number known to screening lab: 325 Positive screen: 296 Includes53 with meconium ileus13 sibs or diagnosed prior to screening
29 ‘Not suspected’Including 10 meconium ileus
Meconium ileus53 cases of MI9 ‘Not suspected’1 ‘Probable carrier’
Well known that MI causes problems – up to 25% have negative screening results
Traditionally excluded from screening stats
False positives: IRT-IRT protocolClinical details n
Term, well babies 43Multi-organ failure and/or sepsis 31Birth trauma, hypoxia 6Extreme prematurity 1
Galactosaemia (symptomatic) 6
Gastroschisis 3Pyloric stenosis 1Ileal atresia 1Renal disease 3Liver disease 2
Trisomy 13 2Trisomy 18 2
GALACTOSAEMIA17/21 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
IRT pre and post treatment in galactosaemia
17/21 galactosaemics had raised IRT
False positives IRT-DNA-IRT
First sample Second sample
CommentsAge (days) IRT (g/L) Age (days) IRT (g/L) 8 96 27 77 Well, thriving
66 125 HIE, abnormal LFTs10 465 26 134 Multi-organ failure, died6 175 26 78 Sepsis
26 308 Trisomy 139 117 26 65 Premature twin
25 837 Autoimmune enteropathy 5 112 23 61 Extreme prematurity 5 143 27 100 Congenital renal failure
False negatives IRT-IRTPancreatic insufficient
Age at presentation
Clinical details
First IRT Second IRT
Mutation analysis Comments[g/L]
(Cut-off)[g/L]
(Cut-off)
10.8m RS <60 (60) Not donep.Phe508del homozygous
11wRS + sepsis 92 (60) 49 (50) Not done
Deceased: diagnosed at pm
3m RS + GI 54 (55) Not donep.Phe508del heterozygous
Abnormal sweat test
6w RS + GI <60 (60) Not done Not doneAbnormal sweat test
3mRS + GI + FTT 34 (35) Not done
p.Phe508del homozygous
11m FTT 12 (35) Not donep.Phe508del heterozygous
Abnormal sweat test
<1yr* RS + GI <70 (70) Not donep.Phe508del/c.3140-26A>G Sib*
3mRS + GI + FTT 110 (80) 64 (70)
p.Phe508del/621+1G>T
False negatives IRT-IRTPancreatic insufficient
Age at presentation
Clinical details
First IRT Second IRT
Mutation analysis Comments[g/L]
(Cut-off)[g/L]
(Cut-off)
10.8m RS <60 (60) Not done p.Phe508del homozygous
11wRS + sepsis 92 (60) 49 (50) Not done
Deceased: diagnosed at pm
3m RS + GI 54 (55) Not donep.Phe508del heterozygous
Abnormal sweat test
6w RS + GI <60 (60) Not done Not doneAbnormal sweat test
3mRS + GI + FTT 34 (35) Not done p.Phe508del homozygous
11m FTT 12 (35) Not donep.Phe508del heterozygous
Abnormal sweat test
<1yr* RS + GI <70 (70) Not donep.Phe508del/c.3140-26A>G Sib*
3mRS + GI + FTT 110 (80) 64 (70)
p.Phe508del/621+1G>T
King et al 1986
Small bowel, liver, respiratory and pancreaticchanges, as described in CF,
a common finding at autopsy in prematurebabies receiving prolonged intensive care.
False negatives IRT-IRTPancreatic sufficient
Age at presentation
Clinical details
First IRT Second IRT
Mutation analysis Comments[g/L]
(Cut-off)[g/L]
(Cut-off)
3yr Mild RS 88 (60) 63 (65) Not done Abnormal sweat test
14y RS 69 (70) Not donep.Phe508delheterozygous Abnormal sweat test
7y RS 40 (45) Not donep.Phe508delheterozygous Abnormal sweat test
1w FTT, GI 57 (70) Not donep.Phe508del/p.Arg117His
Echogenic bowelprenatally
4w Well sib 54 (70) Not donep.Phe508del/2789+2insA
Currently asymptomatic
False negatives IRT-IRTPancreatic sufficient
Age at presentation
Clinical details
First IRT Second IRT
Mutation analysis Comments[g/L]
(Cut-off)[g/L]
(Cut-off)
3yr Mild RS 88 (60) 63 (65) Not done Abnormal sweat test
14y RS 69 (70) Not donep.Phe508delheterozygous Abnormal sweat test
7y RS 40 (45) Not donep.Phe508delheterozygous Abnormal sweat test
1w FTT, GI 57 (70) Not donep.Phe508del/p.Arg117His
Echogenic bowelprenatally
4w Well sib 54 (70) Not donep.Phe508del/2789+2insA
Currently asymptomatic
False negatives IRT-DNA-IRTPancreatic insufficient
Age at presentation
Clinical details
First IRT Second IRT
Mutation analysis
[g/L] (Cut-off)
[g/L] (Cut-off)
9mRS + GI + FTT 43 (70) Not done
p.Phe508del homozygous
2mRS + GI + FTT 65 (70) Not done
p.Phe508del homozygous
False negatives IRT-DNA-IRTPancreatic insufficient
Age at presentation
Clinical details
First IRT Second IRT
Mutation analysis
[g/L] (Cut-off)
[g/L] (Cut-off)
9mRS + GI + FTT 43 (70) Not done
p.Phe508del homozygous
2mRS + GI + FTT 65 (70) Not done
p.Phe508del homozygous
False negatives IRT-DNA-IRTPancreatic sufficient
Age at presentation
Clinical details
First IRT Second IRT
Mutation analysis Comments[g/L]
(Cut-off)[g/L]
(Cut-off)
3y RS 82 (70) <25 (60) p.Phe508del/p.Pro750Leu Designated a carrier
4m** RS + GI 32 (70) Not donep.Phe508del & p.Ile1027Thr in cis /TG12T5 ** Twins
4m** RS + GI 38 (70) Not donep.Phe508del & p.Ile1027Thr in cis /TG12T5 ** Twins
1m* RS 64 (70) Not done p.Phe508del/c.3140-26A>G Sib of 7
False negatives IRT-DNA-IRTPancreatic sufficient
Age at presentation
Clinical details
First IRT Second IRT
Mutation analysis Comments[g/L]
(Cut-off)[g/L]
(Cut-off)
3y RS 82 (70) <25 (60) p.Phe508del/p.Pro750Leu Designated a carrier
4m** RS + GI 32 (70) Not donep.Phe508del & p.Ile1027Thr in cis /TG12T5 ** Twins
4m** RS + GI 38 (70) Not donep.Phe508del & p.Ile1027Thr in cis /TG12T5 ** Twins
1m* RS 64 (70) Not done p.Phe508del/c.3140-26A>G Sib of 7
Age Chloride Sodium Conductivity Osmolality(months) (mmol/L) (mmol/L) NaCl eq (mmol/Kg)
1 33
3 45 24 42 99
1.5 19
1.6 15
28 54 56 76 176
1.5 51
2.5 37 30
5 44
6 57 62
Non CF range Intermediate range
p.Phe508del /p.Arg117His
p.Phe508del /p.Arg117His
p.Phe508del /p.Pro67Leu
Sweat testing 5 ‘suspected’ mis-identified as carriers
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/LUp to 6 months of age
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
p.Arg117HisExpression influenced by a polythymidine variant in intron 8
Affects splicing
T7 – neutralT5 – reduces protein synthesis
Newborns screened France 2002-200661 p.Phe508del/p.Arg117His;T7
47 asymptomatic13 respiratory symptoms1 classical CF
Penetrance of classical CF 0.03%
Thavin-Robinet, Munck, Huet et al 2009
True negatives mis-identified as false negatives
1. MaleIRT less than 25 g/Lrectal prolapse, sweat Cl 45, 47 mmol/Lno mutations identified
2. & 3. Male twins8yr steatorrhoea and respiratory problems abnormal sweat test in one child no mutations identifiedmultiple sweat Cl – normalnasal potential difference - normalpancreatic function tests – normal10yr mild asthma
Incidence Sensitivity (%)
Combined protocols (n=730,730) 1 in 2248
Excluding MI 93.0 (89.1 - 95.6)
Unexpected cases, <3yr 95.3 (91.8 - 97.4)
IRT-IRT protocol (n=582,966) 1 in 2286
Excluding MI 93.8 (89.4 - 96.5)
Unexpected cases, <3yr 96.0 (92.0 - 98.1)
IRT-DNA-IRT protocol (n=147,764) 1 in 2111
Excluding MI 90.2 (79.1 - 95.9)
Unexpected cases, <3yr 93.0 (82.1 - 97.7)
Conclusion95% 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!
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