NEWBORN SCREENING

Newborn screening for cystic fibrosis:
Techniques and strategies

Bridget Wilcken

Received: 13 February 2007 / Submitted in revised form: 29 March 2007 / Accepted: 4 April 2007 / Published online: 12 May 2007

#

SSIEM and Springer 2007

Summary Newborn screening for cystic fibrosis has been
carried out for over 25 years, and clinical and cost benefits
have been documented. There is still much variation in the
methods and strategies adopted. All current screening
programmes use a measurement of immunoreactive
trypsin as a primary screening test, and in most, a second
tier test involves analysing DNA mutations. The choice of
DNA mutations depends on the genetic background in
the region, and considerations of cost. Using DNA
analysis as part of a screening procedure has introduced
unwanted carrier detection, and protocols have now been
devised in an attempt to avoid this. There are at least
seven distinct protocols in use, all of which have different
advantages and disadvantages, and no method or strategy
will suit every region. Further careful study of perfor-
mance and costs of various strategies is needed.

Abbreviations
CF

cystic fibrosis

CFTR

cystic fibrosis transmembrane
conductance regulator

DGGE

denaturing gradient gel electrophoresis

DNA

deoxyribonucleic acid

IRT

immunoreactive trypsin

PAP

pancreatic-associated protein

Introduction

Newborn screening for cystic fibrosis has been carried
out for over a quarter of a century, and some benefits
were quickly established. Only recently has it become
less controversial as clinical and cost benefits have
become clearer, the range of benefits seen has extended,
harms have been found to be minimal, and benefits are
seen to outweigh any harms. Now, as Farrell has so
cogently written, the key question is no longer

Fshould

we screen

_ but Fhow should we screen?_ (Farrell

2004

).

Regions where screening has long been established, such
as north-western France, East Anglia (UK), Australasia,
areas of Italy, and Colorado and Wisconsin (USA), still
use a variety of strategies, and as screening is becoming
more widespread, and is being planned in many regions,
information about the advantages and drawbacks of
different screening strategies is very important.

Screening for cystic fibrosis (CF) is currently

carried out universally in Australia and New Zealand,
in 27 of 51 states in the USA, with partial screening or
planned screening in a further 7 (as of February 2007,

http://genes-r-us.uthscsa.edu/nbsdisorders.pdf

), and in

26 regional or state programmes in Europe (Southern
et al

2007

). There is interest in the Middle East (Nazer

1992

). It is planned for screening to become universal

in England this year (

http://www.newbornscreening-

bloodspot.org.uk/

, accessed February 2007) (it is

already carried out in Scotland, Wales and Northern
Ireland); two Canadian provinces will begin screening

J Inherit Metab Dis (2007) 30:537–543
DOI 10.1007/s10545-007-0584-0

Communicating editor: Rodney Pollitt

Competing interests: None declared

B. Wilcken (*)
Biochemical Genetics and Newborn Screening,
The Children

_s Hospital at Westmead, Hawkesbury Road,

Westmead, NSW 2145, Australia
e-mail: [e-mail]

B. Wilcken
The University of Sydney, Sydney, NSW, Australia

this year; and it is likely to be taken up by many more
states in the USA. We reviewed CF screening methods
in 2003, (Wilcken and Wiley

2003

) and a synopsis of

that review is presented here. There was a similar
review in the same year dealing with strategies
(Southern and Littlewood

2003

). Since that time there

have been proposals of new strategies, as well as studies
comparing different strategies, and these are discussed.

Dried blood spot immunoreactive trypsin
as the primary screening test

Newborn screening for CF was being discussed in the
1960s and 1970s, when the only method applicable to mass
screening was the measurement of meconium albumin, a
test with many drawbacks including poor sensitivity and
specificity. A test-strip for screening was developed in
1975 (Stephan et al

1975

). However, the discovery in

1979 of elevated blood immunoreactive trypsin (IRT)
in babies with CF (a surprise initially, as low levels had
been predicted) immediately led to the possibility of
effective mass newborn screening (Crossley et al

1979

).

All CF screening strategies now use dried blood

spot IRT as the primary screening test. Crossley

_s

original test used a polyclonal antibody and radioim-
munoassay. This was a successful approach from the
point of view of case finding, but it was time-
consuming. Initial modifications included the use of a
monoclonal antibody-based enzyme immunoassay us-
ing a 96-well microtitre plate ELISA technique
(Bowling et al 1987). Further improvements led to
the widespread, and now near-universal, use of an
automated dissociated enhanced lanthanide fluoroim-
munoassay (autoDELFIA, Perkin Elmer Life and
Analytical Sciences, Wallac Oy, Turku, Finland) (Soini
and Kojola

1983

).

The problem with a primary IRT assay is one of poor

specificity in the first few days of life. The positive
predictive value at 2–5 days of age is about 3–10%
(Travert

1988

). The initial strategy using IRT was a two-

stage assay, IRT/IRT: an initial elevation of IRT led to
a repeat test at around 2–4 weeks, when the positive
predictive value was approximately 50%, a much more
manageable proportion. The Wisconsin group reported
that perinatal stress factors accounted for some 25% of
false-positive results in the first IRT test, with other
causes including renal failure, congenital infection,
bowel atresia and some aneuploidies (trisomies 13 and
18) (Rock et al

1990

). A sweat-test was required for

babies with two positive IRT test results, to confirm or
exclude cystic fibrosis. Although the specificity of an
initial positive IRT test was not good, the sensitivity

proved to be high—we found a sensitivity of 98.1% for
the first IRT test after screening over 1 million babies,
if a cut-off of 1% was used (Wilcken et al

1995

).

Adding DNA testing

Mutation testing can readily be performed on dried
blood spots. The identification of the cystic fibrosis
transmembrane conductance regulator (CFTR) gene
in 1989 and the discovery of a common mutation,
D

F508, immediately made it possible to screen babies

for CF using a single sample, carrying out mutation
testing on samples with an elevated IRT level, without
the immediate need for requesting a second sample
(Seltzer et al

1991

). In areas where the common

mutation was particularly frequent—among popula-
tions derived mainly from Northern Europe—it was
possible to screen using only that mutation. Babies
with two copies had cystic fibrosis and were referred
directly to a CF clinic, while babies with only one copy
needed a sweat test to differentiate affected babies
from carriers. In regions where the common mutation
was less frequent, more mutations could be tested for
in the initial blood spot, depending on the CF mutation
background in the population (e.g. Scotet et al

2000

;

Spence et al

1993

). Cost was one major consideration.

Initially, adding new mutations was very costly, and
this is to some extent still the case. This type of
strategy is referred to as IRT/DNA.

Problems associated with DNA testing

The advantages of using a DNA test as second-tier
testing are clear. Not only was there good sensitivity
with little increase in cost but there was of course no
need to recall babies for a second test, abolishing
parental anxiety for many. The main disadvantage was
the detection of carriers of CF—an outcome not
included in the aim of screening. The population of
newborn babies with elevated IRT levels is enriched
with carriers (Castellani et al

2005

; Parsons et al

2003

).

In several studies, about one carrier is detected for
every CF patient identified. This still represents only a
small proportion of all carriers—about 1%. While
some people see this as a possible advantage, it has
also been seen as undesirable, causing anxiety and
possible harm to the developing mother–baby rela-
tionship. (There is in fact no evidence to support the
latter assertion; e.g. Parsons et al

2003

).

Several strategies have been suggested to avoid

some of the problems of carrier detection, most

538

J Inherit Metab Dis (2007) 30:537–543

notably the IRT/DNA/IRT strategy (Pollitt et al

1997

). Babies with an elevated level of IRT and one

copy of the common mutation proceeded to a repeat
IRT test. Only if this remained elevated was a sweat
test requested. There was a 92% reduction in the
number of second tests requested compared to an
IRT/IRT strategy with no DNA component, and an
80% reduction in sweat tests. Thus very few carriers
were formally detected; parents of babies with one
copy of a CFTR mutation were told that cystic
fibrosis was very unlikely, and were offered referral
for genetic counselling. This approach has proved
popular (e.g. Corbetta et al

2002

), and other variations

have melded IRT/IRT and IRT/DNA/IRT approaches
by requesting a second sample from all babies. This
latter protocol surrenders the advantages of a reduc-
tion in the number of second samples requested
(Littlewood et al

1995

).

Another problem has been a legal one: in France,

bioethics laws mean that it is not permitted to perform
any DNA testing without written, informed consent.
To fulfil these obligations, the Ethics and Genetics
committee of the French Association for Neonatal
Screening recommended that informed consent should
be obtained for all neonates at birth by having the
parents sign directly on the sampling paper. Using this
approach, the refusal rate was low, and declined from
0.8% at the start of the program to 0.2% at the end of
the first year of screening (Dhondt

2005

). Specific

consent for screening for CF was introduced in
Massachusetts, with a similar fall in refusals over the
first year of screening.

Which DNA mutations?

A further difficulty in choosing a screening protocol
is the realization that (again as in all screening) mild
cases will be detected. This seems particularly so
when a panel of DNA mutations is used. A consid-
eration of the different DNA mutations chosen for
inclusion in a screening strategy is of course depen-
dent on the local population, and on the funding
available, and is beyond the scope of this review. An
example of a region where using just the common
mutation might seem justified is the Hunter region of
New South Wales, Australia, where it was shown that
some 98% of babies with CF carried at least one
copy of the common mutation, DF508, which com-
prised 80% of the mutations occurring (Henry et al

1996

). In New South Wales as a whole, the occur-

rence of the common mutation is also high, compris-
ing about 75% of all mutations. In some contrast, a

study in north-eastern Italy found that it was neces-
sary to use the 16 most common CFTR mutations to
cover 86.6% of the mutations occurring in the CF
patients—the

Fcommon_ mutation being much less

common in this region (Bombieri and Pignatti

2001

).

There has been a move to avoid inclusion of mild
mutations, such as the fairly common R117H, from
panels even although this mutation can be quite
severe, depending upon the haplotype background
(O

_Sullivan et al

2006

).

Alternative methods

Other approaches suggested in areas with high allelic
heterogeneity include use of meconium lactase as an
extra test to improve an IRT/DNA strategy (Castellani
et al

1997

), and to avoid the use of DNA altogether by

the use of pancreatic-associated protein (PAP) as an
initial test, with a follow-up IRT test for samples with an
elevated PAP (Sarles et al

2005

). Neither of these

approaches has been used outside the region where they
were developed, although both seem to be effective.

Strategies for CF screening

The main strategies in use have already been
mentioned: IRT/IRT, IRT/DNA, either with single
or multiple mutations, and an extension of this, IRT/
DNA/IRT, in which a second blood spot for IRT is
requested when the initial IRT/DNA testing has
revealed only one mutation. Further interesting
variations on these have also been used. In Massa-
chusetts, a strategy with IRT/DNA (using multiple
CFTR mutation testing) also employs a third
Ffailsafe_ step of referring for sweat testing not only
those with two mutations or one mutation detected
but also those with an extremely elevated IRT
(Comeau et al

2004

). This differs from the IRT/

DNA/IRT strategy proposed by Pollitt, in that the
second IRT is requested for patients without any
mutation detected but with a very high initial IRT.
On a different tack, the randomized controlled trial
of screening in Wisconsin first used a strategy in
which an elevated IRT with rather a high cut-off led
immediately to a sweat test (IRT strategy, Rock et al

1990

). Another proposed strategy employs an IRT/

multiple DNA test, with the addition of an extended
mutation analysis for samples where only one muta-
tion has been found (Merelle et al

2006

). The apparent

benefits and drawbacks of these different approaches
are shown in Table

1

.

J Inherit Metab Dis (2007) 30:537–543

539

Table

1

Comp

arison

of

dif

ferent

CF

sc

reening

strategies:

actua

l

ben

efits

and

drawba

cks

will

dep

end

on

cut-off

points

adopte

d

for

the

IR

T

assay,

local

geneti

c

variati

on,

and

man

y

other

factors

Strate

gy

Step

s

Like

ly

benefi

ts

Likely

dra

wbacks

IRT/IRT

1.

IRT

on

initi

al

blood

spot.

If

elevated

:

G

ood

spec

ificity

and

sen

sitivity

second

test

Poor

spe

cificity

first

test:

thus,

more

famil

ies

with

anxie

ty

2.

Re

sample

:

IRT

on

seco

nd

bl

ood

spo

t.

If

ele

vated:

No

carri

ers

dete

cted

3.

Swea

t

te

st

IRT

1.

IRT

on

initi

al

blood

spot.

If

elevated

:

G

ood

spec

ificity

Either

poor

sensiti

vity

or

greatly

inc

reased

false-

positive

rate.

2.

Swea

t

te

st

N

o

carri

ers

dete

cted

High

sweat

te

st

rat

e

IRT/DN

A

Si

ngle

(c

ommon

)

m

utation

1.

IRT

on

initi

al

blood

spot.

If

elevated

:

G

ood

sensiti

vity

in

some

commu

nities

Detectio

n

o

f

some

carriers

2.

DNA

on

same

blood

spo

t.

Si

ngle

(comm

on

)

mutati

on:

Will

have

increas

ed

chanc

e

o

f

missing

CF

for

cert

ain

et

hnic

gro

ups

3.

If

one

copy

of

mutat

ion:

sw

eat

test.

If

no

copy

of

m

utation:

CF

not

indicat

ed

IRT/DN

A

Mul

tiple

m

utations

1.

IRT

on

initi

al

blood

spot.

If

elevated

:

B

etter

sensitivi

ty

compare

d

w

ith

single

mutati

on

test

Increased

cost

2.

DNA

on

same

blood

spo

t.

M

ultiple

mutat

ions,

chosen

for

genetic

backgro

und:

Lower

specificity:

inc

reased

nu

mber

of

ca

rriers

identifie

d

3.

If

on

ly

one

cop

y

o

f

any

mutati

on:

sweat

te

st.

If

no

copy

of

any

mutati

on:

CF

not

indicated

IRT/DN

A/IRT

1.

IRT

on

initi

al

blood

spot.

If

elevated

:

Incre

ased

spe

cificity

compare

d

w

ith

IRT/DN

A

Repe

at

bl

ood

samp

le

for

a

small

number

of

babies

2.

DNA

on

same

blood

spo

t.

Si

ngle

or

m

ultiple

mutati

ons:

Re

duced

nu

mber

of

sw

eat

test

s

False-n

egative

result

possibl

e

if

n

o

m

utations

dete

cted,

or

if

seco

nd

IRT

is

normal

(2

severe

mutati

ons,

on

e

unde

tected

3.

If

one

m

utation

only

dete

cte

d:

new

samp

le

for

IR

T

No

carri

ers

hav

e

auto

matic

sw

eat

test

Some

carriers

are

identifie

d,

and

offere

d

genetic

counse

lling.

4.

If

IRT

ele

vated:

sweat

test

If

IRT

no

t

ele

vated:

CF

not

indicat

ed

Some

of

these

cou

ld

have

another

mutati

on

IRT/DN

A/

fa

ilsafe

st

ep

1.

IRT

on

initi

al

blood

spot.

If

elevated

:

Incre

ased

sen

sitivity

comp

ared

with

IR

T/DNA

or

IR

T/DNA

/IRT

strategies

Increased

n

u

mber

of

sw

eat

test

s

2.

DNA

on

same

blood

spo

t.

Si

ngle

or

mult

iple

mutati

ons:

3.

If

one

m

utation

dete

cted

or

if

no

mutati

on,

but

extrem

ely

high

IR

T:

sweat

test

IRT/DN

A/DNA

(exp

erimen

tal

st

rategy)

1.

IRT

on

initi

al

blood

spot.

If

elevated

:

Incre

ased

spe

cificity

Increased

cost

comp

ared

with

IRT/

IRT

2.

DNA

on

same

blood

spo

t.

Si

ngle

or

m

ultiple

mutati

ons:

B

etter

sensitivi

ty

with

mult

iple

mutati

ons

in

seco

nd

tier:

only

pati

ents

with

two

rare

mutati

ons

missed

A

fe

w

new

DNA

vari

ants

of

unkno

wn

pathog

enicity

dete

cted:

need

for

a

sweat

test

3.

If

on

e

mutati

on

only

dete

cted:

exten

ded

mutati

on

analy

sis

CF

id

entifie

d

if

two

mutati

ons

dete

cted

Carriers

are

iden

tified

within

the

laborato

ry

but

are

not

reporte

d

to

parents

4.

Uncom

monly

,

DNA

variant

s

o

f

unkno

wn

pathog

enicity

dete

cte

d:

sweat

test

For

refere

nces

see

te

xt.

540

J Inherit Metab Dis (2007) 30:537–543

Comparison of strategies

Several studies have been conducted to compare
strategies, and a selection of these are summarized in
Table

2

. It is clear that there is a balancing act (as

always in screening) between, on the one hand,
adopting protocols with the highest sensitivity and, on
the other, keeping the

Fnoise_ of false-positive results

as low as possible. (An ideal approach might be an
IRT test at 2–3 weeks, when it has high discrimination,
followed by a sweat test at, say, 4 weeks on all babies
with a positive IRT test; but alas this would be
impracticable and expensive.)

Comparison of costs

Costing health care is very complex. There have been a
few small studies examining costs of screening diagno-
sis versus clinical diagnosis (e.g. Lee et al

2003

, more

recently extended by Rosenberg and Farrell

2005

). An

interesting study modelled the costs of four screening
strategies and assessed these in relation to health
effects (Van den Akker-van Marle et al

2006

). This

type of economic-modelling exercise requires a num-
ber of assumptions to be made, and in this case it was

assumed that there would be a gain of 40 life-years
for every early non-meconium-ileus CF death pre-
vented. The authors compared an IRT/IRT strategy
with IRT/DNA, IRT/DNA/IRT, and IRT/DNA/
DGGE strategies. The DGGE step was extended muta-
tion analysis by denaturing gradient gel electrophoresis
(see Table

1

). They concluded that the most favourable

cost–effectiveness ratio was achieved with IRT/IRT
and that IRT/DNA/DGGE achieved more health
effects at a lower cost than IRT/DNA/IRT. These
costs will of course vary greatly from country to
country and will depend heavily on the performance
of the tests employed (the sensitivity and specificity,
which can to some extent be manipulated by cut-off
points) and the health-care system in the country, but
these authors do conclude that screening is a good
economic option.

An alternative to newborn screening

Offering an effective prenatal carrier-screening
programme is considered by some as an alternative
strategy to newborn screening. A programme in the
East Lothian area of Scotland was in place for many
years. Different approaches to carrier testing under

Table 2 Studies comparing different strategies

Comparison

Region

Finding

Reference

IRT vs IRT/DNA

Wisconsin, USA

Fewer sweat tests and fewer

false-positive subjects contacted using
IRT/DNA

Gregg et al (

1993

)

IRT/IRT vs IRT/DNA

New South Wales, Australia

Similar sensitivity. Some carrier

detection but no recall samples
using IRT/DNA

Wilcken et al (

1995

)

IRT vs IRT/DNA

Wisconsin, USA

Improved positive predictive

value, fewer false-positives,
quicker diagnosis, no recall
specimens using IRT/DNA

Gregg et al (

1997

)

IRT/DNA vs

IRT/DNA/IRT

Trent region, UK

92% reduction in need for second

blood sample, 80% reduction in
sweat tests, using IRT/DNA/IRT;
similar (very slightly lower)
detection rate

Pollitt et al (

1997

)

Different mutation

strategies

Veneto and Trentino Alto

Adige regions, Italy

Complete gene screening

detected 90% mutations vs
86.6% using 16 most common
mutations, in one area

Bombieri and Pignatti (

2001

)

IRT/IRT vs

IRT/DNA/IRT

Lazio region, Italy

Increased sensitivity using

IRT/DNA/IRT

Narzi et al (

2002

)

IRT/multiple

DNA/failsafe vs
IRT/single DNA/failsafe

Massachusetts, USA

Increased sensitivity;

increased carrier detection

Comeau et al (

2004

)

J Inherit Metab Dis (2007) 30:537–543

541

these circumstances include screening one member of a
couple, normally the pregnant woman; if she is found to
carry the common mutation, her partner is offered
screening. Another approach is couple testing; both
members of the couple are tested, but the result is given
only as

Fcouple at risk_ if both are carriers or Fcouple at

low risk

_ for other couples. In the United Kingdom and

in subjects originating in Northern Europe, in a couple
with one member carrying the common mutation and
the other member not a carrier of this, the risk of CF in a
baby is about 1:400. Currently the programme in E.
Lothian has a very low uptake (A. Mehta, personal
communication 2007) and a similar proposal in
Denmark (Schwartz et al

1993

) was not implemented

(F. Skovby, personal communication 2007).

Aims of screening, methods and strategies

When considering the way forward for cystic fibrosis
screening, it is important to keep in mind the general
aims of newborn screening: to provide a benefit to an
affected baby from early diagnosis, together with
minimal harm to the baby and the community.
Documenting outcomes is vital, so that benefits can
be measured. Happily, good studies of CF outcome
have been undertaken, as documented in the accom-
panying article (McKay

2007

). The possible harms of

screening are few, and include the financial costs borne
by the community and the psychological and other
costs that stem from false-positive results. Both of
these are very dependent on the screening method and
the strategy adopted. No method or strategy will suit
every region. However, it is to be hoped that carefully
designed studies will provide guidance, and that both
established programmes and developing ones will
benefit from the knowledge emerging about the results
of different approaches to cystic fibrosis screening.

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