Confirmatory and differential diagnostic tests
after positive screening results
Péter Monostori, PhD
Neonatal Screening Laboratory,
Department of Pediatrics, University of Szeged
Primary screening tests
• Routine primary screening methods are designed to
identify as many abnormal infants as possible.
• Therefore, diagnostic sensitivity (=low number of falsenegative results) is more important than diagnostic
specificity (=to have few false-positive results)
• This approach exponentially increases the number of
false-positive test results as more disorders are included
in screening.
• Obtaining another blood sample to
confirm/exclude the disorder is a valid
choice but is not always necessary…
The problem with repeated sampling
• The newly obtained samples require additional work.
• The cost of the screening program is increased.
• Repeated sampling causes stress and anxiety in the
families:
– infants with false-positive screening results are more often
hospitalized than healthy children with normal screening
results
– families subjected to false-positive
newborn screening results are at
higher risk of developing dysfunctional
parent-child relationships
How to address this issue?
The aim is therefore to improve diagnostic
specificity (=decrease the number of false-positive
results) without reducing diagnostic sensitivity:
• Inclusion of secondary criteria, such as ratios
– C3/C2 for propionic/methylmalonic acidemia etc.
• Good interpretation of the results in view of the
clinical status (prematurity etc.), drugs, nutrition…
• Second-tier tests
Second-tier tests – What are these?
• More specific for the diagnostic compound than the
primary screening method and/or
• Measure additional metabolites
• Use the same sample (e.g. dried blood spot, DBS)
– there is no need to obtain a new sample
• Utility:
– support or exclude the diagnosis
suggested by the primary test
– differentiate
between
(differential diagnosis)
disorders
Second-tier tests
Then why don’t we use these as primary tests?
• Limitations:
– lower sample throughput
– greater complexity
– longer analysis time
– higher cost
Second-tier tests
• Biochemical tests (mainly MS/MS)
• Enzyme activity measurements
• Molecular genetic tests
Second-tier tests – Examples
1. Congenital adrenal hyperplasia (CAH)
2. Phenylketonuria (PKU)
3. Propionic acidemia (PA), methylmalonic acidemia
(MMA) with/without homocystinuria (HCYS)
4. Tyrosinemia type I (Tyr I)
5. Galactosemia
6. Isovaleric acidemia (IVA)
7. Maple syrup urine disease (MSUD)
1. Congenital adrenal hyperplasia (CAH)
First-tier tests for CAH
• First-tier screening tests for CAH use
immunoassays
to
measure
17-hydroxyprogesterone (17-OHP) levels in DBS:
– Dissociation-enhanced,
lanthanide
fluorescence
immunoassay (DELFIA®) is almost exclusively used
• However, the positive predictive value for
first-tier screening of CAH is generally about 1%.
Limitations of first-tier screening of CAH
• First, the antibodies used in the immunoassays cross-react with
other steroids, particularly 17-hydroxypregnenolone.
• Second, 17-OHP levels are normally high at birth and decrease
rapidly during the first few days. By contrast, 17-OHP levels
increase over time in newborn babies with CAH. Thus, diagnostic
accuracy is poor in the first 2 days.
• Third, newborn girls have lower 17-OHP levels than newborn boys
(sensitivity of screening for CAH in girls is lower).
• Fourth, premature, sick or stressed babies tend to have higher
levels of 17-OHP.
– most laboratories use a series of (birth weight- or) gestational ageadjusted threshold values
• Fifth, antenatal corticosteroids administered to
mothers at risk of preterm delivery might reduce
17-OHP levels (false-negative test results ↑).
• Finally, neonatal screening identifies only few
babies with mild, nonclassic CAH.
Second-tier tests for CAH –
Biochemical assays
• Direct analysis of steroid levels by LC-MS/MS
from DBS is used as second-tier tests.
• This 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.
• The run times for individual samples in most
LC-MS/MS assays are generally 6-12 min, which
would be too long for a first-tier screen.
Second-tier tests for CAH –
Biochemical assays
• Appropriately selected ratios of the steroids can further
improve the specificity of LC-MS/MS.
• The rationale for using ratios with cortisol:
– newborns under stress have high cortisol levels with secondary
accumulation of 17-OHP
– in CAH patients, cortisol levels are relatively low
• Ratio No. 1:
(17-OHP+androstenedione)/cortisol
– androstenedione is only secondarily increased in CAH (indirectly
due to the deficiency of 21-hydroxylase)
• Ratio No. 2:
(17-OHP+21-deoxycortisol)/cortisol
– 21-deoxycortisol is highly specific for
21-hydroxylase deficiency
Second-tier tests for CAH –
Molecular genetic assays
• CYP21A2 mutations can be detected in DNA
samples extracted from the same DBS used for
primary screening.
• However, this approach is not comprehensive
– CAH is a genetically heterogenous disorder
– not all mutations can be reliably detected in a
screening setting
– LC-MS/MS is less costly and time-consuming than
genotyping
A novel biochemical assay as a first-tier
test for CAH
• As shown earlier, the run times for individual
samples in most LC-MS/MS assays are
generally 6-12 min, which would be too long for
a first-tier screen.
• Exception: a US laboratory (Manitoba)
developed an LC-MS/MS assay using a modified
instrument for first-tier screening (turbo-flow
chromatography coupled to LC-MS/MS)
– rapid determination of 17-OHP, androstenedione and
cortisol
– no false-positives so far
Improvement of the specificity for CAH screening
(Mayo Clinic, USA)
2. Phenylketonuria (PKU)
GTP cyclohydrolase (GTPCH)
6-Pyruvoyl-tetrahydrobiopterin
synthase (PTPS)
Sepiapterin reductase (SR)
Dihydropteridine
reductase (DHPR)
q-Dihydrobiopterin
Pterin-4α-carbinolamine
dehydratase (PCD)
Phenylalanine
hydroxylase (PAH)
First-tier tests for PKU
• First-tier
screening
tests
for
PKU
determine
phenylalanine (Phe) levels in DBS. With MS/MS, tyrosine
levels and Phe/Tyr ratios are also obtained.
• A positive screening result is generally sufficient to conclude
that some form of hyperphenylalaninemia (PKU, transient
hyperphenylalaninemia or tetrahydrobiopterin (BH4)
deficiency) is present.
• For differential diagnosis:
– Phe and BH4 loading test,
– pterin profile analysis (from urine or DBS),
– dihydropteridine reductase (DHPR) activity
measurement (from DBS) should be performed
Differential diagnosis of BH4 deficiencies
• BH4 loading test
– useful in all forms of BH4 deficiency
– a 24 h Phe loading test is recommended previously, especially if
the basal Phe level is low (e.g. < 360 μM)
– single Phe dose plus a single BH4 dose 3 h later
– blood sampling: -3; 0; 4; 8; 12; 16; 24 h
• Pterin profile analysis (neopterin, biopterin and pterin)
– sample: DBS or urine (random urine specimen dried on filter
paper is better than liquid urine, as pterins are very unstable)
– HPLC plus fluorescent detection or MS/MS
• DHPR activity measurement from DBS
– spectrophotometry
Pterin levels and DHPR activity in variants of
BH4 deficiency
Phe
(plasma)
Biopterin
(urine)
Neopterin
(urine)
DHPR
activity
(blood)
Homovanillic
acid (HVA,
liquor)
5-hydroxyindoleacetic acid
(5-HIAA, liquor)
GTPCH1
(recessive)



N


GTPCH1
(dominant)
N
N ( in
liquor)
N ( in
liquor)
N

N/
PTPS



N


PCD


N/ 
primapterin
N
N
N
DHPR


N



SR
N
N ( in
liquor)
N ( liquor
sepiapterin)
N


3. Propionic acidemia (PA), methylmalonic acidemia
(MMA) with/without homocystinuria (HCYS)
First-tier tests for PA, MMA and MMA+HCYS
• First-tier screening tests for PA and MMA
measure propionylcarnitine (C3) levels and
C3/C2 ratios in DBS with MS/MS (in MMA,
C4DC may also be increased). For MMA+HCYS
(caused by defective cobalamine metabolism:
Cbl C, Cbl D), elevated C3 and C3/C2, plus
decreased methionine levels may be suggestive.
• C3 is frequently responsible for false-positive
results in newborn screening:
– dietary deficiency of vitamin B12
(newborn or mother), prematurity,
jaundice (hyperbilirubinemia)
– ratios and/or second-tier tests are used
Second-tier tests for PA, MMA and MMA+HCYS
• The simultaneous determination of methylmalonate,
methylcitrate and homocysteine in DBS by means of
LC-MS/MS allows confirmation and differential diagnosis of
the disorders (in some laboratories, 3-hydroxypropionate is
assayed instead of methylcitrate)
Initial screening
Second-tier test
C3
Met
methylmalonate
methylcitrate
homocysteine
PA
↑↑
N
N
↑↑
N
MMA
↑-↑↑
N
↑↑
↑
N
MMA+
HCYS
↑
↓
↑
N
↑
Testing algorithm for PA, MMA and MMA+HCYS
(Mayo Clinic, USA)
4. Tyrosinemia type I (Tyr I)
First-tier tests for Tyr I
• First-tier screening tests for tyrosinemias
detect tyrosine levels in DBS with MS/MS.
• Tyrosine elevation is not specific for Tyr I (in fact,
tyrosine is generally higher in Tyr II and III).
• Tyrosine levels in babies with Tyr I can be
relatively low during the first few days of life.
• Tyrosine elevation is most often associated with
benign transient tyrosinemia or an increased
protein uptake.
Second-tier test for Tyr I
• Succinylacetone (SA, SUAC) is a specific
marker for Tyr I.
• Succinylacetone in DBS can be measured in a
second-tier test or as a routine primary screen,
both by means of MS/MS.
Testing algorithm for Tyr I (Mayo Clinic, USA)
5. Galactosemia
Galactosemia
• First-tier screening tests for galactosemia
generally use assays to measure galactose plus
galactose-1-phosphate levels in DBS (enzymatic
test)
– the microbiological (Guthrie-)test measures galactose
only.
• However, a positive screening result can also be
caused by a portosystemic (liver) shunt or liver
dysfunction.
• Therefore, newborns with abnormal
screening results should be further
tested with confirmatory assays.
Second-tier tests for galactosemia
• Beutler-test: enzymatic assay for confirmation of
galactose-1-phosphate uridyltransferase deficiency
(GALT, classic galactosemia) in whole blood
• Multiplex enzyme assay using UPLC-MS/MS in
DBS: simultaneous determination of all three
enzymes in galactose degradation
6. Isovaleric acidemia (IVA)
First-tier tests for IVA
• First-tier
screening
tests
for
IVA
measure
isovalerylcarnitine (C5) levels in DBS with MS/MS.
• However, a positive screening result may also be caused
by increased 2-methylbutyrylcarnitine, valerylcarnitine
and pivaloylcarnitine levels (these are not determined
separately in routine MS/MS assays)
– 2-methylbutyrylcarnitine may be indicative for Short/branchedchain
acyl-CoA
dehydrogenase
(SBCAD)
deficiency
=2-methylbutyryl-CoA dehydrogenase deficiency
– pivaloylcarnitine may be derived from
pivalate-generating antibiotics (pivampicillin,
pivmecillinam, cefditoren pivoxil, cefcapene
pivoxil, cefteram pivoxil etc.) (pivalate
esterification is used to improve absorption
and oral bioavailability)
Second-tier tests for IVA
• LC-MS/MS-based assay for the quantitative
analysis of isovalerylglycine in DBS
– the number of false-positive results is reduced
7. Maple syrup urine disease (MSUD)
First-tier tests for MSUD
• Deficiency of the Branched-chain alfa-ketoacid
dehydrogenase complex results in elevated
levels of isoleucine (Ile), leucine (Leu), valine
(Val),
and
allo-isoleucine
(allo-Ile,
a
characteristic biomarker for MSUD).
• However, first-tier screening tests with MS/MS
cannot differentiate between the isomers of Leu,
Ile, allo-Ile and hydroxyproline (OH-Pro).
• Elevated levels may also be
caused by parenteral nutrition.
Second-tier tests for MSUD
• LC-MS/MS-based assay for the quantitative
analysis of Val, Leu, Ile, allo-Ile and OH-Pro
– the number of false-positive results is reduced
Testing algorithm for MSUD (Mayo Clinic, USA)
Summary
• Second-tier tests support or exclude the diagnosis
suggested by the primary screening test, and can
help to differentiate between disorders.
• They offer higher specificity than the primary test
without the need to obtain a new sample, which
– lowers the overall cost of the screening program and
– decreases stress and anxiety caused by repeated
sampling.
• Second-tier tests are expected to
become an essential part of the
routine screening procedure.
Thank you for your attention!
References
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