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 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…

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 disorders (differential diagnosis)

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-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%.

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.

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)

Pterin-4α-carbinolamine dehydratase (PCD)

q-Dihydrobiopterin

6-Pyruvoyl-tetrahydrobiopterin synthase (PTPS)

Sepiapterin reductase (SR)

GTP cyclohydrolase (GTPCH)

Phenylalanine hydroxylase (PAH)

Dihydropteridine reductase (DHPR)

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-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

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 methyl-malonate

methyl-citrate

homo-cysteine

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/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)

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!

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positive 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: 585-592.

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