IRT

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Thirty Years
with
Cystic Fibrosis
Jacqui Calvin
Addenbrooke’s Hospital Cambridge
Screening area – Norfolk, Suffolk,
Cambridgeshire
Screening for CF since 1979
First year a pilot to set cut-offs
Routine screening - January 1980
Various assays and protocols used
First CF patient detected on screening
First CF child detected by IRT
screening – October 1980
Reported in ‘Current issues in neonatal screening for cystic fibrosis and
implications of the CF gene discovery’.
Farrell et al 1991 Ped Pulmonol
We have two earlier CFs –
dob June/July 1980
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
Total babies screened
Jan 1980 to December 2009
730,730
Give or take a few!
What are we measuring?
Trypsinogen-1
(encoded by PRSS1 gene)
cationic
Trypsinogen-2
(encoded by PRSS2 gene)
anionic
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 blood
a1-antitrypsin
Cationic
trypsinogen
a1-AAT
Binds 15% trypsin
a2-macroglobulin
a2-macroglobulin
trypsin
Binds 85% trypsin
Anionic trypsinogen
Dhondt & Farriaux 1994
Behring RIA kit
Samples from CF babies showed a
bimodal decay curve suggesting a
different mix of IRT species
Sorin
Radioimmunoassay
Polyclonal antibodies raised to inactivated
cationic trypsin
Cross reactivity:
trypsinogen - 98%
a1AT bound trypsin 15%
a2macroglobulin bound trypsin 1%
AGEN
Two monoclonal antibodies raised to
cationic trypsinogen
Enzyme immunoassay
?cross reactivity with other species
Delfia/autodelfia
Dissociation-Enhanced Lanthanide
Fluorescent Immunoassay
Monoclonals raised to ????
Antibodies chosen gave the best
discrimination between normal and
CF bloodspots
Time period
Kit
Protocol
1st action limit
2nd action limit
IRT mg/L
IRT mg/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)
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.
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
IRT-IRT
IRT-IRT
IRT-IRT
IRT-IRT
IRT-DNA-IRT
Kit
Sorin
Agen
Delfia
Autodelfia
Autodelfia
Total
234, 098
65,222
210,356
73,290
147,764
n
24
22
37
13
9
FP
%
0.01
0.03
0.02
0.02
0.006
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
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
False positives: IRT-IRT protocol
Clinical details
n
Term, well babies
43
Multi-organ failure and/or sepsis
Birth trauma, hypoxia
Extreme prematurity
31
6
1
Gastroschisis
Pyloric stenosis
Ileal atresia
3
1
1
Renal disease
Liver disease
3
2
Trisomy 13
Trisomy 18
2
2
Galactosaemia (symptomatic)
6
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
IRT pre and post treatment in galactosaemia
17/21 galactosaemics
had raised IRT
False positives IRT-DNA-IRT
First sample
Second sample
Age (days)
IRT (mg/L)
Age (days)
IRT (mg/L)
8
96
27
77
66
125
10
465
26
134
6
175
26
78
26
308
9
117
25
837
5
112
23
61
5
143
27
100
Comments
Well, thriving
HIE, abnormal LFTs
Multi-organ failure, died
Sepsis
Trisomy 13
26
65
Premature twin
Autoimmune enteropathy
Extreme prematurity
Congenital renal failure
False negatives IRT-IRT
Pancreatic insufficient
Age at
presentation
Clinical
details
First IRT
Second IRT
[mg/L]
(Cut-off)
[mg/L]
(Cut-off)
Mutation analysis
<60 (60)
Not done
p.Phe508del
homozygous
10.8m
RS
11w
RS +
sepsis
92 (60)
49 (50)
3m
RS + GI
54 (55)
6w
RS + GI
<60 (60)
3m
RS + GI +
FTT
11m
FTT
<1yr*
RS + GI
3m
RS + GI +
FTT
34 (35)
12 (35)
<70 (70)
110 (80)
Comments
Not done
Deceased:
diagnosed at pm
Not done
p.Phe508del
heterozygous
Abnormal sweat
test
Not done
Not done
Abnormal sweat
test
Not done
p.Phe508del
homozygous
Not done
p.Phe508del
heterozygous
Abnormal sweat
test
Not done
p.Phe508del/
c.3140-26A>G
Sib*
64 (70)
p.Phe508del/
621+1G>T
False negatives IRT-IRT
Pancreatic insufficient
Age at
presentation
Clinical
details
First IRT
Second IRT
[mg/L]
(Cut-off)
[mg/L]
(Cut-off)
Mutation analysis
<60 (60)
Not done
p.Phe508del homozygous
10.8m
RS
11w
RS +
sepsis
92 (60)
49 (50)
3m
RS + GI
54 (55)
6w
RS + GI
3m
RS + GI +
FTT
11m
FTT
<1yr*
RS + GI
3m
RS + GI +
FTT
Comments
Not done
Deceased:
diagnosed at pm
Not done
p.Phe508del heterozygous
Abnormal sweat
test
<60 (60)
Not done
Not done
Abnormal sweat
test
34 (35)
Not done
p.Phe508del homozygous
12 (35)
<70 (70)
110 (80)
Not done
p.Phe508del heterozygous
Abnormal sweat
test
Not done
p.Phe508del/
c.3140-26A>G
Sib*
64 (70)
p.Phe508del/
621+1G>T
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.
False negatives IRT-IRT
Pancreatic sufficient
Age at
presentation
Clinical
details
First IRT
Second IRT
[mg/L]
(Cut-off)
[mg/L]
(Cut-off)
Mutation analysis
Comments
Not done
Abnormal sweat test
3yr
Mild RS
88 (60)
63 (65)
14y
RS
69 (70)
Not done
p.Phe508del
heterozygous
Abnormal sweat test
7y
RS
40 (45)
Not done
p.Phe508del
heterozygous
Abnormal sweat test
1w
FTT, GI
57 (70)
Not done
p.Phe508del/
p.Arg117His
Echogenic bowel
prenatally
4w
Well sib
54 (70)
Not done
p.Phe508del/
2789+2insA
Currently
asymptomatic
False negatives IRT-IRT
Pancreatic sufficient
Age at
presentation
Clinical
details
First IRT
Second IRT
[mg/L]
(Cut-off)
[mg/L]
(Cut-off)
Mutation analysis
Comments
Not done
Abnormal sweat test
3yr
Mild RS
88 (60)
63 (65)
14y
RS
69 (70)
Not done
p.Phe508del
heterozygous
Abnormal sweat test
7y
RS
40 (45)
Not done
p.Phe508del
heterozygous
Abnormal sweat test
1w
FTT, GI
57 (70)
Not done
p.Phe508del/
p.Arg117His
Echogenic bowel
prenatally
4w
Well sib
54 (70)
Not done
p.Phe508del/
2789+2insA
Currently
asymptomatic
False negatives IRT-DNA-IRT
Pancreatic insufficient
Age at
presentation
Clinical
details
9m
RS + GI +
FTT
2m
RS + GI +
FTT
First IRT
Second IRT
[mg/L]
(Cut-off)
[mg/L]
(Cut-off)
Mutation
analysis
Not done
p.Phe508del
homozygous
Not done
p.Phe508del
homozygous
43 (70)
65 (70)
False negatives IRT-DNA-IRT
Pancreatic insufficient
Age at
presentation
Clinical
details
9m
RS + GI +
FTT
2m
RS + GI +
FTT
First IRT
Second IRT
[mg/L]
(Cut-off)
[mg/L]
(Cut-off)
Mutation
analysis
Not done
p.Phe508del
homozygous
Not done
p.Phe508del
homozygous
43 (70)
65 (70)
False negatives IRT-DNA-IRT
Pancreatic sufficient
Age at
presentation
3y
4m**
4m**
1m*
Clinical
details
RS
RS + GI
First IRT
Second IRT
[mg/L]
(Cut-off)
[mg/L]
(Cut-off)
82 (70)
32 (70)
Mutation analysis
Comments
<25 (60)
p.Phe508del/p.Pro750Leu
Designated a
carrier
Not done
p.Phe508del & p.Ile1027Thr
in cis /TG12T5
** Twins
** Twins
Sib of 7
RS + GI
38 (70)
Not done
p.Phe508del & p.Ile1027Thr
in cis /TG12T5
RS
64 (70)
Not done
p.Phe508del/c.3140-26A>G
False negatives IRT-DNA-IRT
Pancreatic sufficient
Age at
presentation
3y
4m**
4m**
1m*
Clinical
details
RS
RS + GI
First IRT
Second IRT
[mg/L]
(Cut-off)
[mg/L]
(Cut-off)
82 (70)
32 (70)
Mutation analysis
Comments
<25 (60)
p.Phe508del/p.Pro750Leu
Designated a
carrier
Not done
p.Phe508del & p.Ile1027Thr
in cis /TG12T5
** Twins
** Twins
Sib of 7
RS + GI
38 (70)
Not done
p.Phe508del & p.Ile1027Thr
in cis /TG12T5
RS
64 (70)
Not done
p.Phe508del/c.3140-26A>G
Sweat testing
5 ‘suspected’ mis-identified as carriers
Age
Chloride
Sodium
(months)
(mmol/L)
(mmol/L)
1
33
3
45
1.5
19
1.6
15
28
54
1.5
51
2.5
37
5
44
6
57
Non CF range
24
Conductivity
Osmolality
NaCl eq
(mmol/Kg)
42
99
p.Phe508del /p.Arg117His
p.Phe508del /p.Arg117His
56
30
76
p.Phe508del /p.Pro67Leu
62
Intermediate range
176
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
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
p.Arg117His
Expression influenced by a
polythymidine variant in intron 8
Affects splicing
T7 – neutral
T5 – reduces protein synthesis
Newborns screened France 2002-2006
61 p.Phe508del/p.Arg117His;T7
47
13
1
asymptomatic
respiratory symptoms
classical CF
Penetrance of classical CF 0.03%
Thavin-Robinet, Munck, Huet et al 2009
True negatives mis-identified
as false negatives
1. 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
Incidence
Combined protocols
(n=730,730)
Sensitivity
(%)
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)
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!
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