40
Jacqui Calvin Addenbrooke’s Hospital Cambridge Thirty Years with Cystic Fibrosis

Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

  • Upload
    melora

  • View
    39

  • Download
    0

Embed Size (px)

DESCRIPTION

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. - PowerPoint PPT Presentation

Citation preview

Page 1: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

Jacqui CalvinAddenbrooke’s Hospital Cambridge

Thirty Years with

Cystic Fibrosis

Page 2: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

Screening area – Norfolk, Suffolk, Cambridgeshire

Page 3: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

Screening for CF since 1979 First year a pilot to set cut-offs

Routine screening - January 1980Various assays and protocols used

Page 4: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

Page 5: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

Page 6: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

Total babies screenedJan 1980 to December 2009

730,730Give or take a few!

Page 7: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

Trypsinogen-1(encoded by PRSS1 gene)cationic

Trypsinogen-2(encoded by PRSS2 gene)anionic

What are we measuring?

Page 8: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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)

Page 9: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

Trypsin(ogen) in blood1-antitrypsin

2-macroglobulin

Cationic trypsinogen

Anionic trypsinogen

trypsin

1-AATBinds 15% trypsin

2-macroglobulinBinds 85% trypsin

Page 10: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

Dhondt & Farriaux 1994Behring RIA kit

Samples from CF babies showed a bimodal decay curve suggesting a different mix of IRT species

Page 11: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

SorinRadioimmunoassayPolyclonal antibodies raised to inactivated cationic trypsin

Cross reactivity: trypsinogen - 98%

1AT bound trypsin 15% 2macroglobulin bound trypsin 1%

Page 12: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

AGEN

Two monoclonal antibodies raised tocationic trypsinogen

Enzyme immunoassay

?cross reactivity with other species

Page 13: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

Delfia/autodelfia

Dissociation-Enhanced Lanthanide Fluorescent Immunoassay

Monoclonals raised to ????

Antibodies chosen gave the bestdiscrimination between normal and CF bloodspots

Page 14: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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)

Page 15: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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.

Page 16: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

Page 17: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

Page 18: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

Page 19: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

Page 20: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

Page 21: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

Page 22: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

IRT pre and post treatment in galactosaemia

17/21 galactosaemics had raised IRT

Page 23: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

Page 24: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

           

Page 25: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

           

Page 26: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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.

Page 27: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

           

Page 28: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

           

Page 29: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

Page 30: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

Page 31: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

Page 32: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

Page 33: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

Page 34: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

Page 35: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

Page 36: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

p.Arg117HisExpression influenced by a polythymidine variant in intron 8

Affects splicing

T7 – neutralT5 – reduces protein synthesis

Page 37: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

Page 38: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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

Page 39: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

  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)

   

Page 40: Jacqui Calvin Addenbrooke ’ s Hospital Cambridge

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!