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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

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.

Figures and Tables

Table 1

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

lmo-5-196-i001
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

lmo-5-196-i002
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

lmo-5-196-i003
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

lmo-5-196-i004
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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