Transcript Second-tier tests

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
1. Matern D, Tortorelli S, Oglesbee D, Gavrilov D, Rinaldo P. Reduction of the falsepositive rate in newborn screening by implementation of MS/MS-based second-tier
tests: the Mayo Clinic experience (2004-2007). J Inherit Metab Dis. 2007; 30: 585592.
2. Lehotay DC, Hall P, Lepage J, Eichhorst JC, Etter ML, Greenberg CR. LC-MS/MS
progress in newborn screening. Clin Biochem. 2011; 44: 21-31.
3. Chace DH, Hannon WH. Impact of second-tier testing on the effectiveness of
newborn screening. Clin Chem. 2010; 56: 1653-1655.
4. Ko DH, Jun SH, Park KU, Song SH, Kim JQ, Song J. Newborn screening for
galactosemia by a second-tier multiplex enzyme assay using UPLC-MS/MS in dried
blood spots. J Inherit Metab Dis. 2011; 34: 409-414.
5. Turgeon CT, Magera MJ, Cuthbert CD, Loken PR, Gavrilov DK, Tortorelli S, Raymond
KM, Oglesbee D, Rinaldo P, Matern D. Determination of total homocysteine,
methylmalonic acid, and 2-methylcitric acid in dried blood spots by tandem mass
spectrometry. Clin Chem. 2010; 56: 1686-1695.
6. Oglesbee D, Sanders KA, Lacey JM, Magera MJ, Casetta B, Strauss KA, Tortorelli S,
Rinaldo P, Matern D. Second-tier test for quantification of alloisoleucine and
branched-chain amino acids in dried blood spots to improve newborn screening for
maple syrup urine disease (MSUD). Clin Chem. 2008; 54: 542-549.
7. Shigematsu Y, Hata I, Tajima G. Useful second-tier tests in
expanded newborn screening of isovaleric acidemia and
methylmalonic aciduria. J Inherit Metab Dis. 2010; 33 (Suppl 2):
S283-288.