Laboratory Guidelines For

Screening And Detection Of

Inborn Errors Of Metabolism

Dr. Fayza Abdel-Hamid Hassan

Prof. Clinical and Chemical pathology
Faculty of Medicine ,Cairo University
The first few days of a newborn’s life
can be critical to his future well-being.
Clinical manifestations of IEM are non
specific & overlap with common illnesses
 Frequently remain
undiagnosed until late
 Delay in recognition & ttt

tragic consequences
-5 IQ units lost /month
 20% of infants presenting
with a “sepsis” picture in
absence of risk factors
(prematurity,
chorioamnionitis …. Etc)
have IEM
 Two types of presentations :
• Apparently healthy at birth after symptom-free interval .
Placental protection: placenta provides an effective dialysis
system for removal of toxic metabolites, most babies with IEM
are born in good condition & normal birth wt .
 exceptions
Lactic acidosis
Glutaric aciduria II
Non ketotic hyperglycinuria

2. Neonate with over whelming neurological illness with

unconsciousness, convulsions, apnea with no apparent
symptom-free interval.
:Pathogenesis of IEM
Enzyme or structural protein deficiency .1
. accumulation of toxic metabolites
2. Defect in membrane transport
cystinuria.
. Deficiency of co-factors or other substrates .3

E
Substrate Product
Alt activity
ern eg: albinism
ati
PKU ve Collagen defects
me
MSUD t. pat
hw
ay
Toxic metabolites
galactosemia
 Classical
Classical Phenylketonuria (PKU)
Phenylketonuria

O Hydroxylase O
Phenylalanine

NH2
OH
X
Hydroxylase
NH2
OH

HO

[Phenylalanine] [Tyrosine]
 Detection of IEM

 Mass neonatal screening.

 Screening the at risk.
 Detection of suspected IEM.
 Requirements for newborn screening
 Rapid and accurate analysis and reporting.
Screen a large number of newborns with:

* Minimum false positives

* Eliminates false negatives
 Cost effective.
* Multiple tests from one sample for many disorders.
* Treatable disorders included will reduce cost of
special care for mentally and physically disabled.

 Testing should be doable utilizing existing personnel.

* Result reporting must be straightforward & not
cumbersome.
tives/False Negatives
 There are two possible scenarios when the sample, the analysis, or the
interpretation of the data give erroneous results:
 False Positives
 A laboratory test detects an abnormally high metabolite
 The sample is retested and is still high
 A new sample is taken from the child
 The new sample is normal

 Think of the parental anxiety when the new sample is taken!

 False Negatives
 A laboratory test DOES NOT detect an abnormally high
metabolite
 The sample is reported as being normal
 Nothing is known until the child presents clinically

 The screening has failed!

 RETURN
 History of NBS
 1957 - Dr. Centerwall (father of a child
with mental retardation (Ferric chloride)

 1961 - Dr. Guthrie (BIA)

 1963 - MA and OR implement

legislation
Early screening methods
 Bacterial inhibition assays.
Single test for single disorder (BIA)
 Paper and thin layer chromatography.
few disorders of single type
These methods are:
 Tedious
 Time consuming
 False positive and negative rates (high)
 Few disorders ,difficult interpretation (TL,&PC)
 Technical challenges have limited their applications
PKU by Bacterial Inhibition
Assay
 Recent use of the tandem mass
spectrometer has allowed for changes in
traditional newborn screening services
leading to expansion and improvement of
testing
 History of NBS

 1997 - NC begins piloting MS/MS

 1999- NC and MA begin newborn

screening with MS/MS

 2000 - WI begins MS/MS NBS

 Itrequires a very small sample size to screen
for

a large number of rare

disorders
in

a very quick amount of time, about 2

minutes
 Expanded neonatal
screening by MS/MS

Although individually rare their

cumulative effect can be
devastating
USA 1 in 3500
Germany 1 in 4000

KSA 1 in 750
 ?What tests and when to order
 Lab abnormalities can be transient.
 Tests need to be repeated during an episode of
acute illness,or require provocation in a
specialized center.
 Most IEMs with acute life threatening episodes
can be categorized based on initial lab findings
as one of the following:
 Metabolic acidosis
 Hypoglycemia
 Hyper-ammonemia
Investigations of suspected hypoglycemia
in children
Obtain critical blood sample
12h fast if< 6m
24h fast if>12m

Measure blood
glucose

glucose Normal glucose

No hepatomegaly
Measure:
Insulin, GH, Cortisol,
Lactate, B(OH)B

Normal
No in B(OH)B GH Or
Ketotic insulin lactate
FAO defects Cortisol
hypoglycemia
All Mass Spectrometers

• Must generate ions

• separate ions
• detect ions
• compute ion intensities
• interpret Data
 1.Quadrupole Mass Analyzer

Resonant Ion
Detector
Nonresonant Ion

rf and -dc

dc and Rf voltages
Source

rf and +dc
 What is a tandem mass
spectrometer
 Two mass spectrometers in series connected by a
chamber that breaks a molecule into pieces
(collision cell).
 A sample is sorted and weighed in the first MS,
then broken into pieces in the collision cell& a
piece or pieces sorted and weighed in the second
MS.
 Why tandem mass
Can improve laboratory analysis because:
 Very specific in identification of compounds
 Very accurate and sensitive.
 More than one compound simultaneously in a single
two minute analysis.
 Reduces false positive rates by more than ten fold.
Ion Fragmentation
 Disorders Screened Using MS
 Amino Acid Disorders
 Fatty Acid Oxidation Disorders
 Organic Acid Disorders
d -Leu 3
100
d5-Phe Normal
Le
d4-Ala Phe
Al u
d6-Tyr
% a Ty
d3-Met r
Me
0 t
Phe
100
Elevated Phenylalanine PKU
%

0 m /z
140 160 180 200 220 240 260 280
 Selective Screening by Tandem
MS
Amino Acids Disorders:

 1.Phenylketonuria (PKU)/ Hyperphenylalaninemia

 2. Maple syrup urine disease (MSUD)
 3. Homocystinuria/ Hypermethioninemia
 4. Hypemethioninemia associated with abnormal B12 metabolism
 5. Pyroglutamic aciduria
 6. Citrullinemia
 7. Argininosuccinase Def. (Argininosuccinic Aciduria)
 8. Tyrosinemia type 1
 9.Non-ketotic Hyperglycinemia
 10. Argininemia
 :Organic Acids Disorders

 1. Methylmalonic Acidemia (MMA; different types)

 2. Propionic Acidemia (PA)
 3. Isovaleric Acidemia (IVA)
 4. Glutaric Acidemia type I (GA-I)
 5. Multiple CoA Carboxylase def. (MCD)
 6. 3-Hydroxy-3-methylglutaryl- CoA Lyase def. (HMG)
 7. 3-ketothiolase def. (BKT)
 . Methylcrotonyl-CoA carboxylase def. (MCC)
 .Malonic acidemia
 :Fatty Acid Oxidation Defects
 . Short-Chain acyl-CoA dehydrogenase def. (SCAD)
 . Medium-Chain Acyl-CoA Dehydrogenase Deficiency
(MCAD)
 . Very long-Chain Acyl-CoA Dehydrogenase Deficiency
(VLCAD)
 Hydroxy-long-Chain Acyl-CoA Dehydrogenase
Deficiency (LCHAD)
 . Glutaric acidemia Type-II (GA-II)
 .Carnitine transport defect (CTD)
 . Carnitine palmitoyltransferase def. type I (CPTI)
 . Carnitine-acylcarnitine translocase def.
 Decision Criteria, Interpretation,
and Newborn Screening
Protocol
1. Technical interpretation of acquired
data.
2. Clinical interpretation and decision-
making.
 Test Limitations & Interfering
:Substances
MS/MS screening and other screening tests should be
done at the appropriate time after birth, which is
24-72 hours.
Collection of sample before 24 hours may lead to false-
negative results.
The results of this test do not include values for
acylcarnitines.
Antibiotics that are used as pivalate esters give interfering
signal with that for the diagnosis of isovaleric acidemia
In this case, confirmation by urine GC/MS analysis for
organic acids is necessary.
 Reporting of results

 The interpretation is by pattern recognition.

 Most newborn screening laboratories are used to
provide quantitative results without interpretation.
 A decision limit (cutoff) for each analyte or analyte
ratio should be set on the 99.5th (0.05th) percentile,
based on
 Data collected and analyzed from a large number of
healthy babies.
 Samples, flagged for one or more analyte must be
repeated from the same blood spot.
frequencies

Metabolite level
frequencies

Metabolite level
Laboratories can elect to establish two levels of abnormal
.results
Flagged indicative of a particular disorder for immediate referral to the clinical
.management team for follow-up

Borderline that require re-sampling and retesting.

:Decision to use two-level system might be dependent on

.the availability of follow-up resources
 Reference ranges
 Phe: 37 - 85 μmol/L
 Leu/Ile: 107 - 286 μmol/L
 Met: 12 - 43 μmol/L
 Tyr: 51 - 275 μmol/L
 Phe/Tyr ratio: 0.20 - 1.00

(From 3000 normal neonates)

AIM
A pilot prospective neonatal screening using MS/MS will be initiated with a view
to subsequent introduction into general use &/or high risk groups

:Screening will be directed to a limited range of clearly defined diseases with

 Adequate specificity
no need for repeat sampling
 Available confirmatory tests

:Aims
Technical validation of the method in Egypt-
:Collection of data on-
Reference ranges
Cut off values in different pediatric age
PPV of different metabolites groups
development of EQAS-
Important points for sampling for
:screening of IEM

- Obtain samples before giving supportive therapy

to avoid false negative tests .

- For amino acid disorders take samples after 2 – 3

feedings with protein meal.
to allow abnormal a a to accumulate to avoid
false negative results.
 Special problems of obtaining proper
specimens from newborns
 Skin puncture → least hazardous
 Depth < 2,4 mm
 Respect Site allowed for heel puncture
 Venepuncture → avoid prolonged stasis
 Arterial puncture → best for BG
Lactate
Ammonia
 Umbilical Artery
 Venous catheters → avoid using first 2 drops of
blood
Sample size:
 Smallest possible for neonates but sufficient for reliable
results
Sampling

 Time:
3rd -7th day after 4-5 milk feeding to

allow accumulation of metabolites.

 Site:
Heel prick – free flowing blood on
filter paper. (Air dry-send by mail)
Dried blood spot samples
(Guthrie Card)

A punch is taken out of the filter card.

In most laboratories a 3mm punch is used, equal to

approximately 3µL of whole blood
 Lab request form for suspected IEM
 NAME:
 HOSP NO:
 DOB:
 SEX:
 WARD:
 HOSPITAL:
 CONSULTANT:
 Tests required:
 Date of specimen
 Time of specimen
 Date & time of admission/acute symptoms
 History
 Family history:
 Consanguinous yes/No
 Previous sibling death or affected case?
 Nutrition
 Feeding: Breast fed
Formula fed
Normal diet
TPN
Confirm protein 2 g/kg yes / no
Any supplements
 Follow-up
Positive MS/MS NBS Result

 PMD called and faxed results

 Consultant names provided
 Medical management recommended
 F-up by PMD or Genetic Center
 For the critically ill
Important samples to be taken before giving any supportive
treatment

 2-3 ml heparinized & EDTA blood .

 Dried blood spots on screening cards.
 5-10 ml urine in sterile container save &
freeze all urine passed for future analysis.
 For lactate & ammonia, arterial samples are
preferable, contact lab for precautions.
 Special precautions for certain
analytes for the critically ill
Lactate
 Arterial samples are preferable, best during attack do not
use tourniquet.
 Sample after exercise or struggling during vene-puncture
(false increase)
 Anticoagulant : Fluoride / oxalate.
Ammonia:
 Arterial sample is better, (avoid smoking in the sampling
area) .
 Fill tube (no air bubbles).
 Place sample tube immediately on ice centrifuge at -4oC
rapidly .
 Prolonged crying
 ↑ Lactate
↑ glucose
use smallest syringe

completely sealed → no air send immediately

 Pyruvate

 Same precautions as lactate except :

 Pyruvate is extremely unstable, 2 ml blood
immediately added to 4.0 ml perchloric
acid (8%) kept on ice & delivered to lab
for rapid separation.
When death is inevitable establishing
a diagnosis is very important
 genetic counseling
For 
Later prenatal diagnosis

Collect relevant specimens & biopsy

before or shortly after death
 Blood spots on screening cards
 Lithium heparin & EDTA blood
 All possible urine
 Skin, liver biopsy
Close co-operation between
attending clinician & lab
supervisors is important to
avoid unnecessary and
expensive lab investigations
 Newborn
Neonatal Screening
Screening Summary
Summary

Reproduced with permission from American Scientist vol. 90 (2002)

tives/False Negatives
 There are two possible scenarios when the sample, the analysis, or the
interpretation of the data give erroneous results:
 False Positives
 A laboratory test detects an abnormally high metabolite
 The sample is retested and is still high
 A new sample is taken from the child
 The new sample is normal

 Think of the parental anxiety when the new sample is taken!

 False Negatives
 A laboratory test DOES NOT detect an abnormally high
metabolite
 The sample is reported as being normal
 Nothing is known until the child presents clinically

 The screening has failed!

 RETURN