Screening for hypotyreosis and
congenital adrenal hyperplasia
(CAH), second-tier testing –
laboratory methods
Péter Monostori
Definitions of positive/negative predictive
value, sensitivity and specificity
Sensitivity
 The proportion of affected subjects that have a positive test
result
Specificity
 The proportion of unaffected subjects that have a negative
test result
Positive predictive value
 The chance that a positive test result actually indicates an
affected individual
 The proportion of „real” positive samples within all positive
results
Negative predictive value
 The chance that a negative test result actually excludes the
disorder
 The proportion of „real” negative samples within all negative
results
Congenital hypothyreosis/hypothyroidism (CH)
 Primary hypothyroidism is caused a disorder of thyroid
hormone biosynthesis (the most frequent form of CH).
 Secondary/central/hypopituitary hypothyroidism results
from a deficiency of thyroid stimulating hormone (TSH,
thyrotropin).
 Peripheral hypothyroidism results from defects of thyroid
hormone transport, metabolism or action.
Screening strategies
Primary CH: TSH ↑, free T4 ↓
Hypopituitary CH: TSH ↓/normal, free T4 ↓
T4 concentrations have smaller physiological variations shortly
after birth than does TSH.
Therefore, the T4 method was more popular in the initial
screening strategy in newborn screening systems.
To improve screening sensitivity and specificity, initial T4
screening is usually accompanied by a second-tier TSH test.
However, elevation of TSH concentration is generally thought to
be a better disease predictor.
Screening methodology
T4 screening
 Radioimmunoassay (RIA):
radioactively labeled specific antibody and an
immobilized antibody are used
this method was associated with lower CH
incidences than the other techniques
is rarely used nowadays
 Enzyme immunometric assay (EIA): an enzyme is
connected to a specific antibody
 Fluoroimmunoassay (FIA): a fluorophore is connected to a
specific antibody
Primary T4+follow-up TSH test:
 Advantage: infants with hypopituitary hypothyroidism and
delayed TSH rise may also be detected.
 Disadvantage: lower sensitivity and specificity, higher
recall rates.
Screening methodology
TSH screening
 RIA:
this method was associated with
incidences than the other techniques
is rarely used nowadays
lower
CH
 Immunoradiometric assay (IRMA): „improved RIA”,
radioactively labeled specific antibody and an immobilized
antibody are used
 EIA
 FIA, incl. dissociation-enhanced, lanthanide fluorescence
immunoassay (DELFIA®)
Primary TSH test:
 Advantage: cases of mild subclinical hypothyroidism are
also detected, and lower recall rates are needed. If a
second routine sample is obtained, infants with delayed
TSH rise are also detected.
 Disadvantage: infants with hypopituitary hypothyroidism
cannot be detected.
DELFIA-assay for the measurement of TSH
This assay is based on a direct sandwich technique
where two monoclonal antibodies recognize separate
antigenic determinants on the hTSH molecule. The
second antigen is a europium-labeled anti-hTSH
immunglobuline.
The fluorescence signal is proportional to the
analyte concentration in the sample.
DELFIA-assay for hypothyreosis
Quantitative
Reproducible
Sensitivity: 2 μU/ml
Incubation time: 4 h, or overnight
EDTA (such as in violet-capped tubes)
disturbs
the
determination
(chelates
europium)
Screening methodology
Combined T4 and TSH screening:
 has the advantages of both previous methods
 disadvantage: more expensive and laborious,
higher recall rates
T4 ; T3 ; TSH levels on the first 6 days of life
T4
( μgdl )
18
15
T3
( ngdl )
1
8
1
8
400
TSH
( μUml )
80
T4
60
12
300
40
9
200
T3
6
20
3
0
100
TSH
0
0
1
2
3
4
5
6
The timing of blood sampling for CH screening
important, as T4, T3 and TSH levels decrease
with age
false-positives:
 in the first days of life, TSH may be transiently
elevated (normally lasting not more than 24 h)
 premature infants: physiological reduction in TSH
may occur
false-negative rate:
 is higher if T4 is primarily screened (this is
probably associated with the residual thyroid
hormone activity of the mother)
Prenatal diagnosis
 Genetic testing on fetal cells obtained by
amniocentesis is a more direct and safer
method of diagnosis than fetal cord blood
sampling.
 Measurement of amniotic fluid TSH or
thyroid hormone levels are not reliable,
sampling of fetal umbilical cord blood is
necessary to diagnose fetal hypothyroidism.
What is congenital adrenal hyperplasia (CAH)?
White, P. C. (2009) Neonatal screening for congenital adrenal hyperplasia Nat. Rev. Endocrinol. doi:10.1038/nrendo.2009.148
Screening methodology
First-tier screening tests for CAH employ
immunoassays to measure 17-OHP levels in dried
blood spots (DBSs).
The same filter paper cards are used as for other
neonatal screenings, such as the Guthrie test.
Three different methods currently exist:
 Radioimmunoassay (the first method to be
developed)
 Enzyme-linked immunoassays
 Dissociation-enhanced, lanthanide fluorescence
immunoassay (DELFIA®): almost exclusively used
DELFIA-assay for CAH
This assay is based on the competitive binding of
europium-labeled 17-OHP and 17-OHP in the sample to
17-OHP-specific antibodies.
The fluorescence signal is proportional to the analyte
concentration in the sample.
DELFIA-assay for CAH
Quantitative
Reproducible
Good sensitivity
No solvent-extraction step is required
Incubation time: 3 h, or overnight
EDTA (such as in violet-capped tubes)
disturbs
the
determination
(chelates
europium)
Limitations of first-tier screening of CAH
First, levels of 17-OHP are normally high at birth
and decrease rapidly during the first few postnatal
days.
By contrast, 17-OHP levels increase over time in
newborn babies who are affected with CAH.
Thus, diagnostic accuracy is poor in the first 2
days.
This can be a problem if newborn babies are
discharged from the hospital within this period.
Limitations of first-tier screening of CAH
Second, newborn girls have lower mean 17-OHP
levels than newborn boys, which slightly reduces
the sensitivity of neonatal screening for CAH in
girls.
As almost all girls with salt-wasting CAH are
virilized (i.e. the clinical symptoms help in the
diagnosis), neonatal screening is more necessary
for rapid detection of affected boys than affected
girls.
Limitations of first-tier screening of CAH
Third, premature, sick or stressed babies tend to
have higher levels of 17-OHP than healthy, term
babies do.
This problem seems to be particularly prominent
with DELFIA® assays (probably because these assays
are usually performed without an organic extraction
step that could remove cross-reacting substances).
Therefore, such babies generate many false-positive
test results (unless increased normal threshold values
are used).
Limitations of first-tier screening of CAH
No
universally accepted standards exist for
stratifying 17-OHP levels in newborn babies.
Most laboratories use a series of birth-weightadjusted threshold values (2 birth weight categories
- generally below and above 2500 g -; three, four or
five categories are also used).
The specificity of neonatal CAH screening can be
improved by stratifying test results according to the
babies’ actual gestational age, because 17-OHP
levels correlate much better with gestational age
than with birth weight.
In The Netherlands, adoption of gestational-age
criteria improved the positive predictive value of
CAH screening tests from 4.5% to 16%.
Limitations of first-tier screening of CAH
Fourth, antenatal corticosteroids administered to
mothers at risk of preterm delivery might reduce
17-OHP levels.
This may potentially increase the likelihood of falsenegative screening test results.
All babies who received such treatment and were
screened for CAH at birth should be tested again after
several days of life.
Limitations of first-tier screening of CAH
Finally, neonatal screening identifies only few
babies with mild, nonclassic CAH.
Incidence: 1:130000.
The true incidence of nonclassic CAH is predicted
to be approximately 1:2000 in most populations (as
indicated by the carrier frequency).
Predictive value, sensitivity and
specificity of neonatal CAH screening
White, P. C. (2009) Neonatal screening for congenital adrenal hyperplasia Nat. Rev. Endocrinol. doi:10.1038/nrendo.2009.148
The reason for a need for second-tier
screening with CAH
The positive predictive value for CAH screening is
generally about 1% (despite adjusting cutoff levels
for birth weight, gestational age, and stress factors).
 Organic solvent extraction before immunoassays
may be appropriate, but
direct biochemical analysis of steroid levels by
liquid chromatography-tandem mass spectrometry
(LC–MS/MS) addresses these issues more effectively.
The run times for individual samples in LC–MS/MS
assays are 6–12 min, which would be too long for a
first-tier screen, but are suitable for a second-tier
screen using the original DBSs.
Second-tier screening
Biochemical second screens
The LC-MS/MS assay does not only determine 17-
OHP as a direct substrate for 21-hydroxylase, but
also cortisol (a downstream product of this enzyme’s
reaction) and other steroids.
Appropriately selected ratios of the steroids can
further improve the specificity of LC–MS/MS.
The rationale for the using ratios with cortisol is
that newborns under stress will have high cortisol
levels with secondary accumulation of 17-OHP.
Measuring the level of 17-OHP only will yield
elevated results in these cases, which are not related
to CAH and do not require follow-up.
Biochemical second screens
Ratio No. 1:
(17-OHP+androstenedione)/cortisol
However, the accumulation of androstenedione is
only secondary in CAH (only indirect due to the
deficiency of 21-hydroxylase).
Biochemical second screens
Ratio No. 2:
(17-OHP+21-deoxycortisol)/cortisol
21-deoxycortisol is not normally secreted in large
amounts even in preterm babies.
Thus, elevated levels of this steroid are highly
specific for 21-hydroxylase deficiency.
In a primary screening program in Germany:
1609 samples (that tested positive out of a total of
242500 samples) were tested prospectively
all 16 affected children were identified and there
were no false-positive results (a positive predictive
value of 100%)
Second-tier screening
Molecular genetic second screens
Recently,
second-tier testing for CAH using
molecular genetic methods has been proposed to
confirm a diagnosis on the DNA level.
However, this approach is not comprehensive
CAH is a genetically heterogenous disorder
not all mutations can be reliably detected in a
screening setting
Therefore, molecular studies would not be enough
to exclude all unaffected subjects.
Molecular genetic second screens
CYP21A2 mutations can be detected in DNA
samples extracted from the same DBS used for
hormonal screening.
Detection
methods:
dot-blotting
protocols,
ligation-detection
assays,
real-time
PCR,
quantitative
PCR,
full
sequencing
and
minisequencing.
More than 90% of mutant alleles involve one or
more recombination events (deletion of CYP21A2,
or gene conversions that result in transfer of
deleterious mutations from the nearby CYP21A1P
pseudogene to CYP21A2).
Second-tier screening
Cost-effectiveness
LC–MS/MS is less costly and time-consuming
than genotyping per sample (even
equipment for LC–MS/MS is expensive).
if
the
Evaluation after positive tests
The
gold-standard
of
diagnosis of CAH is a
cosyntropin stimulation test.

This
test
employs
a
pharmacologic dose of 0.125–
0.25 mg cosyntropin, which
maximally
stimulates
the
adrenal cortex.

This diagnostic test is different
from the low-dose cosyntropin
stimulation
test,
used
to
evaluate the integrity of the
hypothalamic-pituitary-adrenal
axis.
Evaluation after positive tests/2
Measured
analytes
stimulation test:
during
the
cosyntropin

17-OHP, cortisol, deoxycorticosterone, 11-deoxycortisol
and 17-OH-pregnenolone at 0 min and 60 min of
stimulation

at least one measurement each of dehydroepiandrosterone
(DHEA) and androstenedione

in low-birth-weight babies who have a small blood volume,
only one sample is collected at 60 min

determination of precursor:product ratios is particularly
useful to distinguish between the different enzymatic
defects

preferable method: LC-MS/MS
Prenatal diagnosis
 Samples are obtained by means of chorionic
villous sampling at 10-12 weeks of pregnancy,
or by means of amniocentesis at 15-18 weeks of
pregnancy.
 HLA typing in combination with measurement of
17-OHP and androstenedione/21-deoxycortisol
in amniotic fluid may be used for prenatal
diagnosis.
Thank you for your attention!