Confirmatory and differential diagnostic tests after positive screening results

1
Confirmatory and differential diagnostic tests
after positive screening results

• Péter Monostori, PhD
• Neonatal Screening Laboratory, Department of
  Pediatrics, University of Szeged

2
Primary screening tests

• Routine primary screening methods are designed to
  identify as many abnormal infants as possible.
• Therefore, diagnostic sensitivity (low number of
  false-negative 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

3
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

4
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

5
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 disorders (differential
  diagnosis)

6
Second-tier tests

• Then why dont we use these as primary tests?
• Limitations
• lower sample throughput
• greater complexity
• longer analysis time
• higher cost

7
Second-tier tests

• Biochemical tests (mainly MS/MS)
• Enzyme activity measurements
• Molecular genetic tests

8
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)

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1. Congenital adrenal hyperplasia (CAH)
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First-tier tests for CAH

• First-tier screening tests for CAH use
  immunoassays to measure 17-hydroxy-progesterone
  (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.

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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 age-adjusted 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.

12
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 enzymes
  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.

13
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-OHPandrostenedione)/cortisol
• androstenedione is only secondarily increased in
  CAH (indirectly due to the deficiency of
  21-hydroxylase)
• Ratio No. 2
• (17-OHP21-deoxycortisol)/cortisol

• 21-deoxycortisol is highly specific for
  21-hydroxylase deficiency

14
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

15
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-?ow chromatography
  coupled to LC-MS/MS)
• rapid determination of 17-OHP, androstenedione
  and cortisol
• no false-positives so far

16
Improvement of the specificity for CAH screening
(Mayo Clinic, USA)
17
2. Phenylketonuria (PKU)
GTP cyclohydrolase (GTPCH)
6-Pyruvoyl-tetrahydrobiopterin synthase (PTPS)
Sepiapterin reductase (SR)
Dihydropteridine reductase (DHPR)
Phenylalanine hydroxylase (PAH)
q-Dihydrobiopterin
Pterin-4a-carbinolamine dehydratase (PCD)
18
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

19
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. lt 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

20
Pterin levels and DHPR activity in variants of
BH4 deficiency
Phe (plasma) Biopterin (urine) Neopterin (urine) DHPR activity (blood) Homovanillic acid (HVA, liquor) 5-hydroxy-indoleacetic 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 ? ?
21
3. Propionic acidemia (PA), methylmalonic
acidemia (MMA) with/without homocystinuria (HCYS)
22
First-tier tests for PA, MMA and MMAHCYS

• 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 MMAHCYS (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

23
Second-tier tests for PA, MMA and MMAHCYS

• 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 Initial screening Second-tier test Second-tier test Second-tier test
C3 Met methyl-malonate methyl-citrate homo-cysteine
PA ?? N N ?? N
MMA ?-?? N ?? ? N
MMAHCYS ? ? ? N ?
24
Testing algorithm for PA, MMA and MMAHCYS (Mayo
Clinic, USA)
25
4. Tyrosinemia type I (Tyr I)
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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.

27
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.

28
Testing algorithm for Tyr I (Mayo Clinic, USA)
29
5. Galactosemia
30
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.

31
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

32
6. Isovaleric acidemia (IVA)
33
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/branched-chain 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)

34
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

35
7. Maple syrup urine disease (MSUD)
36
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.

37
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

38
Testing algorithm for MSUD (Mayo Clinic, USA)
39
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.

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Thank you for your attention!
41
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