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Journal of Applied Sciences Research, 5(10): 1425-1435, 2009
© 2009, INSInet Publication
Preliminary Results of Egypt Experience for Use of Tandem Mass Spectrometry for
Expanded Metabolic Screening
1
Fayza Hassan M.D. 1Fatma El Mougy M.D, 1 Iman Mandour M.D, 2Sawsan Abdel Hady MD,
Laila 2 Selim M.D, 1 Sahar Abdel Atty M.D, 1Marian Morgan M.D, 2 Fadia Salem 2 M.D, Azza
Oraby M.D 1Dina Mehaney MSc, 1 Mohammed Abdel Monem MSc, 3Ibrahim El Nekhely,
MSc and 3Nadia Moharam MD
1
From the clinical and chemical pathology and 2pediatrics departments,
3
Faculty of Medicine, Cairo University and Ministry of Health and Population
Abstract: It is suspected that metabolic disorder rates among Egyptian newborn has high incidence due
to the prevalence of consanguinity (38%), where NBS for metabolic disorders is neither mandatory nor
available. This pilot study is the first attempt to elicit prevalence of metabolic disorders in Egypt by
tandem mass spectrometry (MS/MS). Subjects and Methods: 16000 neonates (3-7 days) and 550 children
(3 months to 15 years) presenting to the neuro-metabolic outpatient (OP) clinic, Cairo University Children
Hospitals (CUCH) underwent expanded metabolic screening by MS/MS. Dried blood spots samples –were
extracted, derivatized and submitted to MS/MS analysis. Amino acids & acylcarnitine values were
calculated. Method validation was achieved through participation in CDC NBSQA program and our own
QC samples. Results: From the 16000 screened neonates, 10 cases were detected (1/1600). Four PKU
(1/4000); 1case MSUD (1/16000), 5 cases with abnormal acylcarnitine profile approximately (1/3000).
Accordingly, the first Egyptian reference values for expanded screening by MS/MS were calculated. From
550 patients presenting to the neurometabolic clinic, 24 patients with metabolic disorders were detected
approximately (1/23). Fourteen PKU cases (1/39), 2 MSUD (1/275), 1 patients with citullinemia(1/550)
, 2 homocystinuria (1/275) and 5 cases with abnormal acylcarnitine profile (1/110). Conclusion: MS/MS
technology was effective in identifying several metabolic disorders. Overall incidence was found to be high
particularly among disabled children, who could have been saved from disabilities if expanded NBS was
offered. The study provided the first Egyptian reference values for amino acids and acylcarnitines
measured by MS/MS for newborns.
Key words: TMS, newborn screening, amino acids, acylcarnitines, metabolic disorders, pilot study.
INTRODUCTION
Newborn screening, for the detection of inborn
errors of metabolism (IEM), has long been recognized
as an essential, life-saving, and effective preventive
public health service.
In many cases, detecting these disorders spells the
difference between life and death for these babies; in
other instances, identifying newborns with a disorder
means that they can be treated and thus not face lifelong disability or cognitive impairment. Now, with the
advent of this new screening technology, babies can be
tested-and treated- for many more disorders than was
previously possible.
Newborn screening clearly is “more than a test”
because it involves follow-up care, counseling, and
other important services. Yet a critical piece of any
comprehensive newborn screening program begins with
the test itself and with its reliability. Laboratories,
pediatricians and parents must be confident that test
results are accurate and that disorders are not
missed [1].
A breakthrough in technology particularly the use
of MS/MS have allowed for the development of fast
and inexpensive protocols to quantify amino, organic
and fatty acid disorders from a single dried blood
spot[2].
Developing of a comprehensive neonatal screening
program was one of the major objectives of the
program for prevention, early detection and early
intervention in Egyptian children with genetic disability
and children at risk, a project financed by the
European commission (EC).
Corresponding Author: Iman Mandour M.D., From the clinical and chemical pathology
Iman Mandour; (+2)-0123-954-879;
E-mail: [email protected]
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J. Appl. Sci. Res., 5(10): 1425-1435, 2009
An increasing number of reports on the use of the
new technology; tandem mass spectrometry (MS/MS),
as a technique for newborn screening[3] have stimulated
national interest in adding MS/MS to the current
newborn screening program [4]. This technology, which
uses a tiny amount of blood, can rapidly identify and
measure the concentrations of a large number of amino
acids, fatty acids, and organic acids. This method has
the potential to detect >30 rare genetic metabolic
disorders [5].
This Pilot Study Aims At:
1- Setting the first Egyptian reference intervals for
NBS from dried blood spots by MS/MS and the first
database registry for inborn errors of metabolism (IEM)
in Egypt.
2- Early detection, via newborn screening, of treatable
and preventable heritable disorder s to ensur e
appropriate and timely intervention.
3- Finding the prevalence of IEM detectable by
MS/MS screening among children presenting with
different types of disabilities (high risk group)
4- Gaining practical experience in using the new
technology and to evaluate the overall effectiveness of
the new test.
Subjects and Methods: The sample of this study was
composed of sixteen thousand neonates aged 3- 7 days
and 550 infants and childr en attending the
neurometabolic clinic of Cairo University Children
Hospital (CUCH).
Blood samples were taken by heel prick and
spotted on Whatman filter paper cards and sent to the
laboratory for screening by MS/MS. The blood spots
were analyzed for acylcarnitines and amino acids by
triple-quadruple tandem mass spectrometer (Quattro LC;
Micromass, Mancheste r, UK) with a positive
electrospray ionization probe. Using a BSD 400
automated Puncher, a single 3-mm disk (3 μl blood)
was punched in the center of the dried blood spot,
placed in flat bottomed 96-well polypropylene
microtitre plate for extraction of the blood with 200 μl
of methanol, containing known concentrations of a
mixture of isotopically labeled internal standards of
acylcarnitines and amino acids, provided by Cambridge
Isotope Laboratories (Woburn,Mass., USA). The
concentrations of the deuterium labeledworking internal
standards per liter of methanol were as follows: 2.5
μmol/L ([2H3]-Leu); [2H3]-phenylalanine ([2H5]-Phe)
; [2H3]-methionine ([2H3]-Met); [2H4]-tyrosine ([2H4]Tyr); [2H6]-ornithine ([2H6]-Orn); [2H2]- citrulline
([2H2]-L-Cit); [2H4]-[13C1]-arginine ([2H4]-[13C1]-LArg)and 12.5 μmol/L for 15N2 glycine (15N2Gly);
[2H9]-carnitine( [2H9 )C0), 0.76 μmol/L; [2H3 ]1426
acetylcarnitine 2H3C2, 0.19 μmol/L. And 0.04 μmol/L
[2H3]- pro pio nylcarnitine ([2H3]-C3 ); [2H3]octanoylcarnitine
([2H3]-C8);
([2H 3]myristoylca rnitine ([2H 3]C14
while[ 2H3] palmitoylcarnitine ([2H3]-C16), 0.08 μmol/L.
After 20 min of shaking, the solvent was
transferred to U bottom microtiter plate, gently
evaporated under a stream of nitrogen and the dried
residue was derivatized with 80 μl of butanolic HCl
and heated at 65°C for 15 min. The solvent was
evaporated again and the residue was finally
reco nstituted in 80 μl of the mobile phase
(acetonitrile/water, 80/20 v/v). A 20μl sample was
directly injected into the MS at a flow rate of 0.2
ml/min. A Waters 2695 HPLC with an autosampler
(Waters, Milford, Mass., USA) was used for automatic
injection. The run cycle time for each sample was 2
min from injection to injection.
Principle of MS/MS:
A mass spectrometer is a device that ionizes
molecules and separates the charged products, or ions
according to their mass-to-charge ratios (m/z). The
instrument has two mass analyzers separated by a
reaction cell. The latter contains an inert gas (argon),
whose molecules collide with ions entering the cell
from the first analyzer. These collisions cause
fragmentation of ions. The second mass analyzer in
turn separates and detects the charged products
according to the chemistry of the metabolite.
Electrospray ionization used is a soft ionization method
which simplifies the analysis – by reducing sample
preparation steps and eliminating matrix interference –
as it produces predominantly intact molecular ions from
the mixture [6].
ESI-MS/MS was applied to the analysis of amino
acids after extraction from bloo d spots and
derivatization to butyl esters.
The butylated AA species give a common neutral
loss (NL) of 102D upon fragmentation. This
corresponds to loss of butylformate from their
protonated molecular ions. It is noteworthy that glycine
produces a very weak signal which can be easily
missed. Six AA including asparagine, glutamine,
arginine, citrulline, lysine and ornithine lose ammonia
first followed by butyl formate, resulting in a total NL
of 119 D [7] . Therefore to get more complete picture of
AA in the sample, four constant NL scan functions
were used at 102 116 119 and 56 generating a separate
profile for each scan function. Normal profile at 102
and at 119 NL is shown in figures 3 and 5.
Multiple reaction monitoring (MRM) experiment
was performed for Phenyalanine and Tyrosine (Fig. 1).
As for acylcarnitines, they were chemically
J. Appl. Sci. Res., 5(10): 1425-1435, 2009
converted to their butyl esters. Electrospray ionization
produces mostly intact molecular ions that undergo
fragmentation in the reaction cell to produce a common
fragment of m/z 85. Fixing the second mass analyzer
to transmit only ions of m/z 85 and scanning the
output of the first analyzer over the mass range 200500, which includes the molecular weights of all the
acylcarnitines, allows a computer to generate a special
type of mass spectrum that consists of the molecular
masses of individual acylcarnitines and their relative
abundance (Fig. 7).
Data interpretation by the system used was
facilitated by the use of the provided Neolynx program,
where interpretation process was an objective efficient
and automated process. This program allowed the
introduction of m/z ratio for each analyte and its
labeled form, and concentrations of internal standards
added during sample preparation, whereby metabolite
concentrations are automatically calculated. If the
analyte is not available in the labeled form, the nearest
homologue was used as a substitute for ratio
calculations.
The cutoff levels were first selected according to
values reported by several published data (1 and 8).
All sample results together with newborn or infant
demographic data were stored in a database. Analytes
exceeding the assigned cut off values were flagged.
Butanolic HCl was obtained from Regis
Technologies (Morton Grove,Ill.,USA). HPLC-grade
methanol and acetonitrile were purchased from Merck.
W ater wa s pur ified b y M illi-Q d evice
(Millipore,Bedford, Mass.,USA). Whatmann filter paper
cards (S and S 903; Schleicher and Schull, Dassel,
Germany) were used to collect blood samples.
RESULTS AND DISCUSSION
Discussion: Inborn errors of metabolism are common
in Egypt, presumably because of the high rates of
consanguinity (38%) (10). This is the first study to be
carried out to estimate the exact prevalence of IEM
among neonates in 3 governorates in Egypt.
The establishment of metabolic screening unit to
diagnose and treat IEM was supported by a project
funded by the EC. The introduction of ESI/MS/MS as
a diagnostic tool in our metabolic unit has allowed
diagnosis of aminoacidopathies and organic acidemias
among both neonates and sick children referred to the
metabolic clinic.
ESI/MS/MS allowed high sample throughput and
efficient neonatal screening method. About 360 samples
were run daily. Automation of the whole process was
facilitated by the use of 96-well plates, automated
punching and automated data processing by Neolynx
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software ; however, more efficient performance could
have been achieved by the introduction of barcode
readers and computerized microplate mapping.
At the start of our work reference ranges and
cutoff values used were those used by CDC and from
the literature [8,11,12,13]. One of our goals was to establish
our own population-based cutoff values from a large
number of normal samples recruited from an ongoing
neonatal screening program.
The values obtained in the present study from
16000 normal neonates are presented in table (1 and
2). Values for amino acids and acylcarnitines are very
similar to those reported by Sander et al. [14] and in
partial agreement with Rashed et al., [11]. Minor
elevations of acylcarnitines especially calculated ratios
were responsible for >50% of our flagged results. In
reality these increases are of no pathological
significance and fine adjustments of the cutoff values
to be based on our population would minimize these
false flags [11]. Proper interpretation of results was
achieved by examination of the reason of flagged
results and visually inspecting the spectra, thus
allowing proper data analysis.
For some of the flagged results retesting was done
if it seemed appropriate and for abnormal profiles
denoting disease. Samples were reanalyzed in the next
batch to assure reproducibility of initial results.
Among 16000 neonates examined through this
screening program 10 cases of IEM were detected
(1/1600), 5cases with aminoacidopathies (1/3200) and
5 cases with organic acidemias (1/3200), this frequency
is higher than that reported by Applegarth et al., 2000
(1/4160) in Caucasian population as well as Schulze
and coworkers, 2003 (1/3800) and Torres-Sepúlveda
and coworkers, 2008 (1/5000) and Hostetler, 1999
(1/2500) [15,8,16,17]. This can be explained by the ethnic
differences between populations studied. However our
prevalence of IEM was less than that reported by
Rashed and coworkers, 1997 (1/740) [11]. This may be
due to the higher rate of consanguinity among the
Saudi population (56%) [18] as compared to that of
Egypt.
Among 550 OP cases presenting to the
neurometabolic clinic, 24 cases with IEM were detected
(1/ 23; 4.3%) 19 cases with aminoacid disorders (1/28)
and 5 cases with organic acidemias (1/110). Similar
results were reported by Kumta, 2005 in India who
detected 5.75% IEM among mentally retarded children
[19]
. Slightly higher values were reported by Xie and
coworkers, 20 08 in China (11%) [20]. In Omani
population, Joshi et al., 2002 detected 32% IEM by
MS/MS in the metabolic unit [21]. The authors reported
that parental consanguinity was twice as frequent in the
study patients as compared to the general population.
J. Appl. Sci. Res., 5(10): 1425-1435, 2009
Table 1: Egyptian Reference values for AA by MS/MS
MEDIAN
99th percentile
5th percentile
------------------------------------------------------------------------------------------------------------Aminoacid (µmol/L)
Tyrosine (TYR)
97.89
396.3
45.4
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Phenylalanine (PHE)
45.03
84.7
30.7
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------phe/tyr
0.45
1.32
0.18
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Leucine/Isoleucine (LEU/Ile)
159.03
305.5
96.9
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Valine (VAL)
152.2
321.2
94.8
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Methionine (MET)
15.9
34.16
9.6
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Citrulline (CIT)
11.9
40.7
3.3
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Alanine (ALA)
253.1
511.8
160.4
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Glycine (GLY)
71.14
236.4
38.4
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Proline (PRO)
7.99
18.54
4.6
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Glutamine (GLU)
225
727
101.8
Table 2: Egyptian Reference Values for AC by MS/MS
MEDIAN
99th percentile
5th percentile
------------------------------------------------------------------------------------------------------------Acylcarnitine (µmol/L)
Free carnitine (C0)
24.38
59.22
13.76
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Acetylcarnitine (C2)
19.55
62.5
10.3
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Propionylcarnitine C3
1.94
7.02
0.84
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C3/C2
0.1
0.39
0.05
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Malonylcarnitine (C3DC)
0.06
0.16
0.02
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Butyrylcarnitine (C4)
0.29
0.86
0.12
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Isovalerylcarnitine (C5)
0.3
0.88
0.16
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Methylcrotonylcarnitine (C5:1)
0.04
0.17
0.01
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Hydroxyisovalerylcarnitine (C5:OH)
0.25
0.89
0.11
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Glutarylcarnitine (C5DC)
0.04
0.16
0
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Hexanoylcarnitine (C6)
0.08
0.37
0.04
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Octanoylcarnitine (C8)
0.07
0.21
0.03
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Decanoylcarnitine (C10)
0.1
0.31
0.04
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C3DC/C10
0.57
2.69
0.12
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Decenoylcarnitine (C10:1)
0.08
0.22
0.03
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Tetradecanoylcarnitine ( C14)
0.2
0.49
0.04
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Tetradecenoylcarnitine (C14:1)
0.07
0.25
0.03
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Hexadecanoylcarnitine (C16)
2.67
6.55
1.0
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Hydroxyhexadecanoylcarnitine (C16:OH)
0.05
0.13
0.01
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Octadecanoylcarnitine (C18)
0.96
2.3
0.39
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Octadecenoylcarnitine (C18:1)
1.14
2.47
0.39
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J. Appl. Sci. Res., 5(10): 1425-1435, 2009
Fig. 1: Normal MRM scan
Fig. 2: A case of PKU as seen in a MRM scan mode
Fig. 3: Normal AA profile
Fig. 4: A case of MSUD
Fig. 5: Normal Scan in NL 119
Fig. 6: A case of Citrulinemia
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J. Appl. Sci. Res., 5(10): 1425-1435, 2009
Fig. 7: Normal AC profile
Fig. 8: A case of MMA showing high C3 (46.4 µmol/L)
Fig. 9: A case of IVA
Fig. 10: A case with elevated C5OH
Fig. 11: Urinary Argininosuccinic Acid Profile by MS/MS
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J. Appl. Sci. Res., 5(10): 1425-1435, 2009
Fig. 12: Urinary Acylcarnitine Profile by MS/MS showing high methylmalonyl carnitine m/z 374.5
Cases of aminoacidopaties detected in the present
study were 10 (NBS) and 19 (OP). Diseases detected
were PKU, MSUD, Citrulinemia and Homocystinuria
(table 3)
The profile in Fig. 2 corresponds to a case of
PKU where a prominent ion at m/z 222 corresponding
to phenylalanine was observed, in the MRM mode, and
phenylalanine/tyrosine ratio was elevated. These results
are clearly consistent with the diagnosis of PKU.
The profile in Fig. 4 shows a prominent ion at m/z
188 corresponding to leucine/isoleucine, a finding
strongly diagnostic of maple syrup urine disease MSUD
Fig. (13) shows the initial Leucine/isoleucine and valine
levels among the three cases of MSUD detected. Fig.
6 shows an abnormal profile for a patient suspected of
having a urea cycle disorder. The profile o f NL of
119Dshowed an abundant ion at m/z ratio 232
corresponding to butyl ester of citrulline .The level of
citrulline in this patient was 646 µmol/L a finding
strongly suggestive of citrullinemia. Diagnosis of
citrulinemia was confirmed by argininosuccinic acid in
urine (Fig. 11). An elevated methionine level of 339
µmol/L was detected in one case in the NL 102 profile
with a prominent ion at m/z 206, a finding diagnostic
of homocystinuria.
As for the acylcarnitine profile 10 cases from the
two studied groups were detected. The disorders
detected were MMA, PA, isovaleric acidemia and
methylcrotonylglycinuria. The distribution of diagnosed
cases among the two groups is presented in table 3.
The acylcarnitine profile presented in Fig. 9 is for a
case of isovaleric acidemia affected with disorders of
1431
branched chain aminoacid catabolism, the profile
reveals a prominent ion at m/z ratio 302 corresponding
to isovalerylcarnitine with elevated C5 7.46µmol/L and
C5/C2 ratio 0.67. The decision criteria for positive
testing for isovaleric acidemia where C5> 2 and
C5/C2> 0.06 according to Schulze et al., [8]. This
preliminary diagnosis was further confirmed by urine
organic acid analysis (GC/MS) where highly elevated
isovalerylglycine (mono and di-derivatives) were
detected.
Propionylcarnitine (C3), with a prominent ion at
m/z 274.5, was elevated in 6 cases, 3 of them were
found to have methylmalonic acidemia after
confirmation by urinary organic acid analysis by
GC/MS. Urinary acylcarnitine profile with very high
methylmalonylcarnitine at m/z 374.5 is shown in figure
12. One case was found to have propionic acidemia,
confirmed by urinary acylcarnitine (m/z 274.5) by
MS/MS and two cases from NBS were not confirmed.
This was due to failure of recall of these cases for
further testing. Two cases with elevated C5OH at m/z
318.2 (Fig. 10) were found to have elevated urinary 3
methyl crotonyl glycine by GC/MS, diagnosed as 3
methylcrotonyl Co-A carboxylase deficiency.
It is noteworthy that no cases of fatty acid
oxidation (FAO) defects in the form of MCAD, LCAD,
VLCAD and MADD were found. This stands in
contrast with other population based studies in-UK,
Germany and USA [22,23,24] and indicate the rarity of
these disorders in our country, or possibly late
presentation of these disorders.
J. Appl. Sci. Res., 5(10): 1425-1435, 2009
Table 3: Cases detected among screened newborns (n=16000) and outpatient clinic patients (n=550) from January to August 2008
Detected disorders
Elevated Marker Range (µmol/L)
Screened Newborns OP ClinicClinical presentation
Amino Acids
PKU
Phenylalanine
(458- 2047)
4
14
Microcephaly, MR, seizures,
Phe/tyr
(6.3-39.5)
fair complexion
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------MSUD
Leu/Ileu
(1945- 3930)
1
2
Poor feeding, vomiting,
Valine
(460-900)
muscle hypertonicity, seizures
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Citrulinemia
Citrulline
(678)
1
Hyperammonemia, vomiting,
diarrhea, lethargy, seizures
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Homocystinuria
Methionine
(149- 339)
2
Skeletal and ocular problems, MR
Organic Acidemia
MMA and PA
C3
(10.7- 46.4)
4
2
Failure to thrive, MR, hyperammonemia,
lethargy, vomiting
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Isovaleric Acidemia
C5
(7.46- 12.89)
1
1
Poor feeding, acidosis, seizures
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Methylcrotonylglycinuria C5OH
(9.59- 60.1)
2
Hypoglycemia, severe metabolic acidosis
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Amino Acidemias Total
5
19
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Organic Acidemias Total
5
5
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Total Screened
16000
550
Table 4: Precision from 23 replicates of one QC CDC sample (AA and AC)
Analyte
CDC mean
Mean (SD) mg/dl
CV%
Aminoacids (mg/dl)
Tyrosine
13.8
11.12 (0.74)
8.6
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Phenylalanine
11.3
10.65(0.93)
8.7
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Valine
10.2
11.3(1.0)
11.5
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Leu/Ileu
14.6
11.9 (0.68)
9.3
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Methionine
5.7
4.77
11.7
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Citruline
4.8
4.2 (0.69)
17.3
Acylcarnitines (µmol/L)
Free carnitine C0
251
200 (13.6)
6.8
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Acetylcarnitine C2
97.5
111.2 (12.3)
11.0
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Propionylcarnitine C3
14.25
18.6 (2.5)
13.5
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Isovalerylcarnitine C5
3.55
3.2 (0.14)
11.87
Fig. 13: MSUD Cases Detected (2 OP and 1 NBS)
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J. Appl. Sci. Res., 5(10): 1425-1435, 2009
Fig. 14: Participation of Egypt, represented by our lab, in the Laboratory Quality Improvement of Newborn
Screening by MS/MS (Quoted from Rinaldo, 2008) (9)
Fatty acid oxidation and organic acid disorders
represent a group of inborn errors of mitochondrial ?
oxidation and inborn errors of carnitine cycle which
were not detected by conventional screening methods
[8]
. It was reported that the number and favorable
outcome of these newborns provide the strongest
argument for improved outcomes with expanded
screening by MS/MS.
To assure and validate the results obtained in our
study participation in the Center for Disease Control
(CDC) newborn screening quality assurance program
(NBSQAP) where both quality control (QC) and
proficiency testing (PT) dried blood spots samples were
delivered to our lab and results submitted before the
deadline. Replicates for intra and inter-assay precision
were analyzed from the same spots of CDC QC and
PT samples (table 4).
CDC evaluation for our lab PT samples indicated
100% satisfactory results with no misclassification
Furthermore, spiked blood samples from normal
subjects with aminoacid standards phenylalanine,
tyrosine, leucine and valine and samples from
confirmed PKU patients were prepared and used as
internal quality control samples.
Our laboratory also participated in the Laboratory
Quality Improvement collaborative project of Newborn
Screening By MS/MS among 100 participants labs
worldwide [9]. This project collects data from all
participant labs to implement universal screening and
confirmatory testing of newborns for inborn errors of
metabolism of aminoacids, organic acids and fatty acid
oxidation defects.
Effective management of detected cases is
established through referral of the cases to the
metabolic clinic for assessment and treatment.
Due to unavailability of GC/MS in our laboratory,
contacts with several metabolic centers in Kuwait and
USA were established to perform the confirmatory tests
required for some of the abnormal acylcarnitine
profiles.
1433
Conclusions and Recommendations: Difficulties
encountered during the implementation of this work
included the delay in DBS card receipt. This may be
solved by assigning separate DBS cards for the
metabolic lab to be sent promptly. PKU, a condition
that silently causes neurological devastation but is
amenable to early detection and effective prevention
with diet control, needs raising public awareness for
hazards of non compliance especially in rural areas and
among low socioeconomic individuals.
The high rejection rate for poor blood spots
encountered (30%), calls for the necessity of training
personnel on proper sample collection.
It is to be noted that due to incomparable value of
expanded screening by MS/MS for detection of a wide
range of metabolic disorders with a high degree of
reliability and high throughput and the high prevalence
detected in our newborns and patients with mental and
physical disabilities expanded metabolic screening by
MS/MS is highly recommended and every effort should
be done to implement it in our country. A back up
plan for instrument downtimes should be established.
The plan should include ready access to additional
instruments such as MS/MS and GC/MS.
ACKNOWLEDGEMENT
We express our deepest gratitude to Prof. Dr.
Mohammed Abdel Hamid, Faculty of Pharmacy Kuwait
University; Prof. Dr. Mohamed Rashed, King Faisal
Hospital, Riyadh; and Prof. Dr. Johannes Sander,
Hannover, Germany for their continuous support and
help throughout our work by offering their experiences
and collaboration. We also would like to thank Prof.
Dr. Piero Rinaldo, Mayo Clinic, USA for sincere help
with establishing our reference ranges and always
offering a helping hand. Our deepest gratitude also
goes to Prof. Dr. Ibrahim El Nekhely and his team at
the MOHP for facilitating supply of DSB samples for
neonates included in this work. We are extremely
J. Appl. Sci. Res., 5(10): 1425-1435, 2009
grateful to our poor affected patients who have been
the greatest motive for our efforts to implement this
work. Last but not least we are grateful for the
administr ati on, clinicia ns, and nurse s of our
participating hospitals and for our colleagues and
chemists without whom this work would have not
existed.
8.
9.
Footnotes: Nonstandard abbreviations: MS/MS, tandem
mass spectrometry; PKU, phenylketonuria; MCAD,
medium chain acyl-co A dehydrogenase; QC, quality
control; CDC, centre of disease control; C3,
propionylcarnitine; C5, isovalerylcarnitine ; C5OH,
3-hydroxydecanoylcarnitine; PA, propionic acidemia;
MMA, methylmalonic academia; FAO, fatty acid
oxidation; MSUD, maple syrup urine disease; MADD,
multiple acyl-coA dehydrogenase deficiency; LCAD,
long chain acyl-coA dehydrogenase;; GC/MS, gas
chromatography/mass spectrometr y; ESI/MS/MS,
electrospray ionization/tandem mass spectrometry;
MOHP, ministry of health and population; CH,
congenital hypothyroidism; IEM, inborn errors of
metabolism; EC, European Commision, CUCH, Cairo
University Children Hospital; NBSQAP, newborn
screening quality assurance program.
10.
11.
12.
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