Neonatal Screening Tests for Inherited Metabolic Disorders using Tandem Mass Spectrometry: Experience of a Clinical Laboratory in Korea

Sung Eun Cho; Eun Jung Park; Dong Hee Seo; In Bum Lee; Hyun Ju Lee; Dae-Yeon Cho; Jung Min Oh

doi:10.3343/lmo.2015.5.4.196

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Cho, Park, Seo, Lee, Lee, Cho, and Oh: Neonatal Screening Tests for Inherited Metabolic Disorders using Tandem Mass Spectrometry: Experience of a Clinical Laboratory in Korea

Original Article

Clinical Chemistry

Laboratory Medicine Online 2015; 5(4): 196-203.

Published online: 1 October 2015

DOI: https://doi.org/10.3343/lmo.2015.5.4.196

Neonatal Screening Tests for Inherited Metabolic Disorders using Tandem Mass Spectrometry: Experience of a Clinical Laboratory in Korea

Sung Eun Cho, M.D.¹, Eun Jung Park, B.S.¹, Dong Hee Seo, M.D.^1,², In Bum Lee, M.S.², Hyun Ju Lee, M.T.¹, Dae-Yeon Cho, Ph.D.², Jung Min Oh, M.T.¹

¹LabGenomics Clinical Laboratories, Seongnam, Korea.

²LabGenomics Clinical Research Institute, Seongnam, Korea.

Corresponding author: Sung Eun Cho. LabGenomics Clinical Laboratories, B-6F, 700 Daewangpangyo-ro, Bundang-gu, Seongnam 13488, Korea. Tel: +82-31-628-0729, Fax: +82-31-628-0741, secho0824@gmail.com

Received 22 October 2014 Revised 3 April 2015 Accepted 9 April 2015

(open-access):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The purpose of this study is to investigate the positive rates of screening tests for inherited metabolic disorders, set cutoff values, and report the actual status of internal quality controls in LabGenomics Clinical Laboratories by using LC-MS/MS system.

Methods

We use Agilent 1260 Infinity HPLC System (Agilent Technologies, USA) for liquid chromatography, and API 2000 (AB Sciex, Canada) for MS/MS system. We set up screening tests for 55 diseases, which include metabolic disorders of 25 amino acids, 16 organic acids, and 14 fatty acids.

Results

We determined the analyte cutoff values as 99.9 or 0.1 percentiles in 15,000 newborn samples. The total number of samples tested from January 2012 to September 2014 was 119,948; of these, 6,681 were repeated. Of the repeated samples, 713 were presumed to be positive in the screening tests. Repeat screening with newly obtained dried blood spot specimens was recommended for these 713 samples and 600 specimens were obtained. Thus, the recall rate was 0.5% (600/119,948) for all samples and 84.2% (600/713) for the samples presumed to be positive in the screening tests. About 70 samples, that is, 0.06% of the total samples and 11.7% of the "reobtained" samples, again tested positive; we recommended confirmatory tests for these samples.

Conclusions

We have presented data on the status of neonatal screening tests for inherited metabolic disorders using LC-MS/MS, including positive rates and recall rates of screening tests, set up cutoff values and reported the actual status of internal quality controls in a clinical laboratory in Korea.

Keywords: Neonatal screening, Inherited metabolic disorders, Tandem mass spectrometry

Figures and Tables

Table 1

Target/internal standard mass (m/z) of parent ions of amino acids and acylcarnitines

	Mass (m/z) of the Target/Internal Standard
Amino acids
Phenylalanine	166/172
Leucine+Isoleucine	132/135
Methionine	150/153
Tyrosine	182/188
Valine	118/126
Arginine	175/180
Citrulline	176/178
Alanine	90/94
Glutamic acid	148/151
Glycine	76/78
Ornithine	133/135
Histidine	156/153
Lysine	147/178
Proline	116/135
Acylcarnitines
Free carnitine (C0)	162/171
Acetylcarnitine (C2)	204/207
Propionylcarnitine (C3)	218/221
Iso-/Butyrylcarnitine (C4)	232/235
Isovaleryl/2-CH3 Butyrylcarnitine (C5)	246/255
3-OH-Isovalerylcarnitine (C5OH)	262/255
Glutarylcarnitine (3-OH-C10) (C5DC)	276/291
Hexanoylcarnitine (C6)	260/255
Octanoylcarnitine (C8)	288/291
Decanoylcarnitine (C10)	316/291
Dodecanoylcarnitine (C12)	344/381
Tetradecanoylcarnitine (C14)	372/381
Hexadecanoylcarnitine (C16)	400/403
3-OH-Hexadecanoylcarnitine (C16OH)	416/403
Octadecanoylcarnitine (C18)	428/403
Malonylcarnitine (C3DC)+3-OH-Butyrylcarnitine (C4OH)	248/255
Tiglylcarnitine (C5:1)	244/255
Decenoylcarnitine (C10:1)	314/291
Decadienoylcarnitine (C10:2)	312/291
Tetradecenoylcarnitine (C14:1)	370/381
3-OH-Tetradecanoylcarnitine (C14OH)	388/381
Octadecenoylcarnitine (C18:1)	426/403
3-OH-Octadecanoylcarnitine (C18OH)	444/403
3-OH-Octadecenoylcarnitine (C18:1OH)	442/403

Table 2

Cutoff values for amino acids (µmol/L) extracted from blood spots of newborns

Amino acids	Cutoff values of our laboratory^*	Yoon et al.^†,‡	CDC cutoff^§	Zytkovicz et al.^∥	R4 99 percentile of normal population^¶
Phenylalanine	99	164^**	150	139^∥∥	97
Leucine+Isoleucine	236	402^††	300	373^¶¶
Methionine	60	87^‡‡	75	67^∥∥	44
Tyrosine	270	358^§§	400	442^§§	207
Valine	211	438^∥∥	300		212
Arginine	40	37	70	132^§§	32
Citrulline	40	75^§§	55	100^***	28
Alanine	694				507
Glutamic acid	850				551
Glycine	1,000	752			767
Ornithine	300	182		300^†††
Histidine	252
Lysine	850
Proline	354

^*99.9 percentile of newborns (n=15,000); ^†4 S.D. above the mean; ^‡Reference [4], The authors used PE Sciex micro-LC series 200, PE Sciex micro-Pump, and a Model PE Sciex autosampler in 79,719 Korean newborns; ^§Reference [6]; ^∥Reference [5], The authors used a series 1100 Hewlett Packard HPLC pump, a Model 215 Gilson autosampler and Micromass Quattro LC triple-quadrupole tandem mass spectrometer in more than 160,000 newborns in the USA, especially in Massachusetts, Maine, New Hampshire, Vermont, and Rhode Island; ^¶Reference [7], cumulative percentiles for amino acids in neonatal dried blood spots analyzed by tandem mass spectrometry by the participants of the Region 4 Stork collaborative project (as of December 1, 2010); ^**10 S.D. above the mean; ^††7 S.D. above the mean; ^‡‡8 S.D. above the mean; ^§§9 S.D. above the mean; ^∥∥6 S.D. above the mean; ^¶¶5 S.D. above the mean; ^***13 S.D. above the mean; ^†††11 S.D. above the mean.

Table 3

Cutoff values for acylcarnitines (µmol/L) extracted from blood spots of newborns

Acylcarnitines	Cutoff values of our laboratory^*	Yoon et al.^†,‡	CDC cutoff^§	Zytkovicz et al.^∥	R4 99 percentile of normal population^¶
C0	< 8.20^**, > 60.00	64.80 (High)	<8.60 (Low)		59.00 (High)
C2	49.56				52.00
C3	7.18	7.20^††	6.00	8.00^††	4.74
C4	0.70	1.47^‡‡	1.30	1.90^¶¶	0.75
C5	0.47	1.20^§§	0.70	1.20^§§	0.39
C5OH	0.60	0.38	0.90	0.80^†††	0.45 (U)
C5DC	0.30	0.22	0.35	0.21^††	0.25 (U)
C6	0.15	0.56	0.45		0.18
C8	0.50	0.52^∥∥	0.45	0.50^§§	0.21
C10	0.40	0.57^¶¶	0.45		0.26
C12	0.51	0.32			0.41
C14	0.61	1.05^¶¶	0.80		0.50
C16	5.57	8.47^***	7.50	12.00^‡‡‡	6.00
C16OH	0.15	0.08	0.15	0.10^‡‡‡	0.08
C18	2.00	1.66	2.50		1.70
C3DC+C4OH	0.57		0.45		0.33
C5:1	0.20	0.38	0.25	0.08^***
C10:1	0.22	0.23	0.30		0.18
C10:2	0.12		0.15		0.08
C14:1	0.40	0.21	0.60	0.90^†††	0.37
C14OH	0.14
C18:1	1.81	2.10	3.50		2.50
C18OH	0.08	0.05	0.10		0.06
C18:1OH	0.18	0.07			0.07

0.07For abbreviations of analytes, see Table 1.

^*99.9 percentile of newborns (n=15,000); ^†4 S.D. above the mean; ^‡Reference [4], The authors used PE Sciex micro-LC series 200, PE Sciex micro-Pump, and a Model PE Sciex autosampler in 79,719 Korean newborns; ^§Reference [6]; ^∥Reference [5], The authors used 1100 Hewlett Packard HPLC pump, a Model 215 Gilson autosampler, and Micromass Quattro LC triple-quadrupole tandem mass spectrometer in more than 160,000 newborns in the USA, especially in Massachusetts, Maine, New Hampshire, Vermont, and Rhode Island; ^¶Reference [7], acylcarnitine cumulative percentiles in neonatal dried blood spots analyzed by tandem mass spectrometry by the participants of the Region 4 Stork collaborative project (as of December 1, 2010); ^**0.1 percentile of newborns (n=15,000); ^††8 S.D. above the mean; ^‡‡17 S.D. above the mean; ^§§11 S.D. above the mean; ^∥∥15 S.D. above the mean; ^¶¶10 S.D. above the mean; ^***6 S.D. above the mean; ^†††12 S.D. above the mean; ^‡‡‡7 S.D. above the mean.

Abbreviation: U, underivatized.

Table 4

Positive cases of neonatal screening tests for inherited metabolic disorders that require confirmatory tests

	Increased analytes	Suspected disorders or false positive cases	Number of cases	Range of concentrations (median) (µmol/L)
Amino acid disorders	Phenylalanine	R/O Phenylketonuria	4	120-184 (155)
	Tyrosine	R/O Tyrosinemia	7	320-509 (397)
	Leucine+Isoleucine	R/O Maple syrup urine disease	1	740
	Citrulline	R/O Citrullinemia	6	45-62 (53)
	Ornithine	R/O Hyperornithinemia	14	325-429 (354)
	Arginine	R/O Argininemia	1	54
Organic acidurias	C3	R/O Propionic acidemia	1	10.92
	C5	R/O Isovaleric acidemia or R/O 2-Methylbutyryl CoA dehydrogenase deficiency or R/O 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency	21	0.57-4.00 (0.91)
	C5OH	R/O 3-OH-3 Methylglutaryl CoA lyase deficiency or R/O 3-Methylcrotonyl CoA carboxylase deficiency or R/O 3-Methylglutaconyl CoA hydratase deficiency	7	0.74-15.78 (1.92)
Fatty acid metabolic disorders	C0	R/O Carnitine palmitoyl transferase type I deficiency	1	77.65
	C4	R/O Short chain acyl CoA dehydrogenase deficiency	3	0.73-2.34 (1.22)
	C10:1	R/O Medium chain acyl CoA dehydrogenase deficiency	1	0.47
	C14OH	R/O Long chain hydroxy acyl CoA dehydrogenase deficiency	1	0.15
	C10:2	R/O 2,4-Dienoyl CoA reductase deficiency	1	0.34
Total number of cases			70

For abbreviations of analytes, see Table 1.

Notes

This article is available from http://www.labmedonline.org

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