Establishment of Age Specific Reference Interval for Aminoacids and Acylcarnitine in Dried Blood Spot by Tandem Mass Spectrometry.

The Extended Screening for Inborn Errors of Metabolism is done for aminoacidopathies, fatty acid oxidation disorders and organic acid disorders. In a single dried blood spot, the tandem mass spectrometry is capable of measuring multiple analytes like amino acids, acylcarnitines, nucleosides, succinylacetone and lysophosphatidylcholines. This study was proposed to establish age specific reference internal for aminoacids and acylcartinitine in dried blood spot by tandem mass spectrometry. A total of 480 apparently healthy children were enrolled for the study and sub classified into four groups as follows: Group A: 0–1 month, Group B: 1 month–1 year, Group C: 1–5 year and Group D: 5–12 years each having 120 participants. Sample size were calculated as per CLSI approved guidelines. Tables 1 and 2 presents the age-specific percentile distribution of aminoacids and acylcarnitines established from healthy subjects as per rank-based method recommended by the IFCC and CLSI. Tables 3, 4 and 5 presents the cut-off values of primary and secondary marker/ratios for screening of aminoacidopathies, fatty acid oxidation disorders and organic acid disorders respectively. As a general principle, the interpretation of extended newborn screening results should be based on age specific cut-off established by the laboratory for primary analyte concentration and secondary analyte concentration/ ratios. This study was useful in establishing age specific cut-off values for various amino acids and acylcarnitines in South Indian population. Table 1 Age-specific percentile distribution of aminoacids Study groups Group A (μmol/L) Group B (μmol/L) Group C (μmol/L) Group D (μmol/L) Percentile 1 2.5 50 97.5 99 1 2.5 50 97.5 99 1 2.5 50 97.5 99 1 2.5 50 97.5 99 Glycine 44.00 50.00 361.85 745.00 840.00 76.00 81.00 351.41 705.00 820.00 88.00 92.00 383.00 620.00 785.00 109.00 115.00 397.74 600.00 790.00 Alanine 66.00 72.00 325.95 570.00 620.00 66.00 72.00 325.82 580.00 623.00 91.00 99.00 323.40 480.00 514.00 112.00 134.00 355.43 510.00 536.00 ASA 0.00 0.02 0.87 1.20 1.43 0.00 0.00 0.50 0.82 1.15 0.00 0.01 0.55 0.64 0.93 0.00 0.01 0.57 0.72 0.98 Leucine 24.00 33.52 120.84 270.00 300.00 23.00 33.78 113.42 264.00 300.00 44.00 52.01 110.43 223.00 250.00 42.00 52.76 118.91 224.00 250.00 Methionine 0.00 1.00 18.13 37.79 42.00 0.00 1.16 19.10 38.68 44.00 2.21 6.93 18.86 36.79 39.00 2.56 5.36 17.37 35.56 39.00 Ornithine 19.57 21.34 121.49 239.00 257.00 19.00 18.27 125.46 235.00 260.00 18.00 21.91 121.49 220.00 248.00 15.16 27.66 116.84 226.00 244.00 Phenylalanine 15.00 17.00 59.12 134.00 140.00 12.51 15.31 58.66 124.00 136.00 20.00 29.09 52.19 120.00 132.00 22.50 27.69 51.23 125.00 150.00 Proline 30.00 32.00 160.13 326.30 348.00 45.00 52.32 149.49 317.47 330.00 34.00 38.89 141.74 269.47 300.00 49.00 56.38 149.65 324.44 360.00 Tyrosine 15.34 16.38 116.28 274.00 300.00 17.00 20.59 115.67 260.00 290.00 16.50 17.12 94.94 180.00 220.00 24.00 29.14 91.50 190.00 220.00 Valine 29.00 31.86 111.86 265.03 285.00 28.00 32.65 102.24 252.76 260.00 41.00 58.80 113.00 250.80 264.00 54.00 63.99 130.96 242.74 260.00 Arginine 1.12 1.76 24.39 57.35 65.00 1.23 1.84 27.41 60.00 68.00 1.56 2.16 29.20 72.00 77.00 1.68 2.24 31.12 78.00 82.00 Citrulline 4.00 4.62 18.11 48.67 51.00 3.50 3.79 19.57 47.11 50.00 5.80 6.54 17.37 33.92 37.00 8.80 9.35 18.37 35.45 38.00 Table 2 Age-specific percentile distribution of acylcarnitines Study groups Group A (μmol/L) Group B (μmol/L) Group C (μmol/L) Group D (μmol/L) Percentile 1 2.5 50 97.5 99 1 2.5 50 97.5 99 1 2.5 50 97.5 99 1 2.5 50 97.5 99 Free carnitine (C0) 6.00 7.12 68.33 113.26 119.23 7.20 9.54 40.43 90.69 96.80 7.10 8.00 37.40 84.10 86.70 8.78 10.43 44.13 79.00 87.90 Acetylcarnitine (C2) 1.80 2.00 26.24 60.23 62.00 2.10 2.30 25.25 58.76 60.58 2.10 2.50 24.93 55.72 57.68 2.60 3.00 24.79 54.00 56.70 Propionylcarnitine (C3) 0.01 0.02 2.65 6.81 7.00 0.12 0.17 2.38 6.70 6.89 0.38 0.57 2.35 6.65 6.70 0.39 0.50 2.38 6.50 6.70 3-OH-butrylcarnitine (C3DC/C4OH) 0.00 0.01 0.23 0.68 0.69 0.01 0.02 0.22 0.65 0.67 0.02 0.04 0.21 0.47 0.50 0.01 0.04 0.21 0.48 0.50 Butrylcarnitine (C4) 0.06 0.09 0.59 1.20 1.30 0.06 0.07 0.52 1.20 1.27 0.06 0.09 0.51 1.00 1.16 0.08 0.10 0.50 1.00 1.13 3-OH-isovalerylcarnitine (C4DC/C5-OH) 0.02 0.04 0.38 0.82 0.87 0.04 0.06 0.37 0.79 0.81 0.07 0.09 0.34 0.69 0.70 0.07 0.08 0.33 0.67 0.68 Isovalerl/2-methylbutrylcarnitine (C5) 0.02 0.02 0.28 0.62 0.63 0.02 0.03 0.27 0.63 0.65 0.02 0.03 0.27 0.60 0.61 0.02 0.03 0.26 0.61 0.62 Tiglycarnitine (C5:1) 0.00 0.00 0.03 0.09 0.09 0.00 0.00 0.02 0.07 0.08 0.00 0.00 0.02 0.06 0.07 0.00 0.00 0.02 0.06 0.07 Glutarylcarnitine (C5DC/C6OH) 0.00 0.02 0.14 0.35 0.37 0.00 0.01 0.15 0.37 0.37 0.01 0.04 0.18 0.38 0.40 0.01 0.04 0.19 0.41 0.42 Hexanoylcarnitine (C6) 0.00 0.02 0.13 0.30 0.31 0.00 0.02 0.14 0.29 0.30 0.00 0.02 0.11 0.23 0.25 0.00 0.02 0.12 0.25 0.26 Adipylcarnitine (C6DC) 0.00 0.02 0.27 0.58 0.60 0.00 0.03 0.27 0.60 0.61 0.00 0.03 0.16 0.34 0.35 0.00 0.02 0.16 0.36 0.37 Octanoylcarnitine (C8) 0.00 0.00 0.15 0.34 0.35 0.00 0.00 0.16 0.34 0.35 0.00 0.01 0.11 0.25 0.26 0.00 0.01 0.12 0.25 0.26 Octenoylcarnitine (C8:1) 0.00 0.00 0.01 0.30 0.32 0.00 0.00 0.02 0.31 0.32 0.00 0.01 0.01 0.31 0.32 0.00 0.01 0.01 0.31 0.32 Decanoylcarnitine (C10) 0.00 0.02 0.20 0.44 0.45 0.00 0.02 0.20 0.41 0.42 0.00 0.03 0.21 0.53 0.54 0.00 0.03 0.22 0.54 0.55 Decenoylcarnitine (C10:1) 0.00 0.01 0.11 0.24 0.25 0.00 0.02 0.10 0.23 0.24 0.00 0.01 0.14 0.30 0.32 0.00 0.02 0.16 0.33 0.34 Dodecanoylcarnitine (C10:2) 0.00 0.00 0.02 0.06 0.07 0.00 0.00 0.02 0.06 0.07 0.00 0.00 0.02 0.06 0.07 0.00 0.00 0.02 0.06 0.07 Dodecanoylcarnitine (C12) 0.00 0.01 0.17 0.41 0.42 0.00 0.02 0.16 0.41 0.42 0.00 0.02 0.14 0.41 0.42 0.00 0.01 0.14 0.40 0.41 Docecenoylcarnitine (C12:1) 1.00 0.01 0.03 0.38 0.39 0.00 0.01 0.02 0.36 0.37 0.00 0.00 0.02 0.36 0.37 0.00 0.00 0.02 0.36 0.37 Tetradecanoylcarnitine (C14) 0.01 0.04 0.18 0.40 0.41 0.02 0.04 0.18 0.38 0.40 0.02 0.04 0.16 0.32 0.33 0.01 0.03 0.16 0.31 0.32 Tetradecenoylcarnitine (C14:1) 0.00 0.01 0.13 0.28 0.29 0.00 0.01 0.14 0.27 0.29 0.00 0.01 0.12 0.24 0.25 0.00 0.01 0.11 0.23 0.25 Tetradecadienoylcarnitine (C14:2) 0.00 0.00 0.01 0.03 0.04 0.00 0.00 0.01 0.03 0.04 0.00 0.00 0.01 0.03 0.04 0.00 0.01 0.01 0.03 0.04 3-OH-tetradecenoylcarnitine (C14-OH) 0.00 0.00 0.03 0.07 0.08 0.00 0.00 0.03 0.07 0.08 0.00 0.00 0.02 0.07 0.07 0.00 0.00 0.02 0.07 0.07 Hexadecanoylcarnitine (C16) 0.20 0.30 4.17 7.44 7.50 0.30 0.50 3.15 7.44 7.50 0.65 0.71 3.90 6.50 6.50 0.70 0.71 3.90 6.50 6.50 Hexadecenoylcarnitine (C16:1) 0.03 0.04 0.31 0.70 0.71 0.03 0.04 0.28 0.65 0.68 0.03 0.04 0.22 0.47 0.48 0.02 0.04 0.22 0.48 0.50 3-OH-hexadecanoylcarnitine (C16-OH) 0.00 0.00 0.03 0.09 0.10 0.00 0.00 0.03 0.08 0.09 0.00 0.00 0.03 0.07 0.08 0.00 0.00 0.03 0.08 0.09 3-Ohhexadecenoyl/heptadecanoylcarnitine (C16:1-OH/C17) 0.00 0.01 0.04 0.11 0.12 0.00 0.01 0.05 0.11 0.12 0.00 0.01 0.05 0.12 0.13 0.00 0.01 0.05 0.12 0.13 Stearoylcarnitine (C18) 0.09 0.09 1.10 2.20 2.30 0.09 0.09 1.10 2.20 2.30 0.23 0.24 0.90 1.70 1.80 0.23 0.24 0.89 1.70 1.80 Oleylcarnitine (C18:1) 0.11 0.12 1.70 3.00 3.04 0.12 0.13 1.51 3.00 3.10 0.32 0.34 1.13 2.30 2.40 0.31 0.33 1.24 2.40 2.50 3-OH-octadecanoylcarnitine (C18-OH) 0.00 0.00 0.01 0.08 0.09 0.00 0.00 0.00 0.08 0.08 0.00 0.00 0.00 0.07 0.07 0.00 0.00 0.00 0.07 0.08 Linoleylcarnitine (C18:2) 0.00 0.02 0.38 0.84 0.86 0.00 0.02 0.37 0.83 0.85 0.00 0.02 0.38 0.85 0.88 0.00 0.02 0.36 0.85 0.88 3-OH-oleylcarnitine (C18:1-OH) 0.00 0.01 0.09 0.17 0.18 0.00 0.01 0.09 0.17 0.18 0.00 0.01 0.07 0.15 0.16 0.00 0.01 0.07 0.15 0.16 Adenosine (ADO) 0.00 0.01 1.40 3.19 3.20 0.00 0.00 1.54 3.19 3.20 0.00 0.01 1.52 3.10 3.12 0.00 0.01 1.49 3.11 3.12 2-Deoxyadenosine (dADO) 0.00 0.01 0.09 0.14 0.15 0.00 0.01 0.06 0.13 0.14 0.00 0.01 0.05 0.11 0.12 0.00 0.01 0.06 0.11 0.12 Table 3 The cut-off values of primary and secondary marker/ratios for screening of aminoacidopathies RUSP core condition (Aminoacidopathies) Primary marker secondary marker Cut-offs Group A (μmol/L) Cut-offs Group B (μmol/L) Cut-offs Group C (μmol/L) Cut-offs Group D (μmol/L) Phenylketonuria (PKU) Phe 140 136 132 150 Phe/Tyr 1.5 1.4 1.4 1.6 Phe/(Leu + Ile) 1.2 1.2 1.2 1.3 Maple syrup urine disease (MSUD) (Leu + Ile) 300 300 250 250 Val 285 260 264 260 (Leu + Ile)/Phe 6.1 6.2 4.8 4.9 (Leu + Ile)/Ala 0.92 0.92 0.77 0.70 Homocystinuria (CBS) Met 42 44 39 39 Met/(Leu + Ile) 0.35 0.39 0.35 0.33 Citullinemia type I (CIT I) Cit 51 50 37 38 Cit/Arg 2.1 1.8 1.3 1.2 Arginosuccinic acidemia (ASA) ASA 1.43 1.15 0.93 0.98 Cit 51 50 37 38 Cit/Phe 0.86 0.85 0.70 0.74 Tyrosinemia type I (TYR) Tyr 300 290 220 220 Tyr/Phe 5.07 4.94 4.21 4.29 Argininemia (ARG) Arg 65 68 77 82 Arg/Orn 0.53 0.54 0.63 0.70 Table 4 The cut-off values of primary and secondary marker/ ratios for screening of fatty acid oxidation disorders RUSP core condition (fatty acid oxidation disorders) Primary marker secondary marker Cut-offs Group A (μmol/L) Cut-offs Group B (μmol/L) Cut-offs Group C (μmol/L) Cut-offs Group D (μmol/L) Medium-chain acyl-CoA dehydrogenase deficiency (MCAD) C8 0.35 0.35 0.26 0.26 C6 0.31 0.30 0.25 0.26 C10:1 0.25 0.24 0.32 0.34 C8/C2 0.01 0.01 0.01 0.01 Very long chain acyl-CoA dehydrogenase deficiency (VLCAD) C14:1 0.29 0.29 0.25 0.25 C14 0.41 0.40 0.33 0.32 C14:2 0.04 0.04 0.04 0.04 C12 0.42 0.42 0.42 0.41 C14:1/C2 0.01 0.01 0.01 0.01 Long chain l-3hydroxy acyl CoA dehydrogenase deficiency (LCHAD) C18-OH 0.09 0.08 0.07 0.08 C16-OH 0.10 0.09 0.08 0.09 C18:1-OH 0.18 0.18 0.16 0.16 C18-OH/C18 0.08 0.07 0.07 0.08 Carnitine uptake defect/carnitine transport defect (CUD) C0 (low) 6.00 7.20 7.10 8.78 C2 (low) 1.80 2.10 2.10 2.60 C3 (low) 0.12 0.12 0.38 0.39 C16 (low) 0.20 0.30 0.65 0.70 C18 (low) 0.09 0.09 0.23 0.23 (C0 + C2 + C3 + C16 + C18:1 + C18)/CIT 0.45 0.51 0.62 0.71 Trifunctional protein deficiency (TFP) C18-OH 0.09 0.08 0.07 0.08 C16-OH 0.10 0.09 0.08 0.09 C18:1-OH 0.18 0.18 0.16 0.16 C14-OH 0.08 0.08 0.07 0.07 C18-OH/C18 0.08 0.07 0.07 0.08 Table 5 The cut-off values of primary and secondary marker/ ratios for screening of organic acid disorders RUSP core condition (organic acid disorders) Primary marker secondary marker Cut-offs Group A (μmol/L) Cut-offs Group B (μmol/L) Cut-offs Group C (μmol/L) Cut-offs Group D (μmol/L) Propionic acidemia/methylmalonic acidemia (PROP/MUT) C3 7.00 6.89 6.70 6.70 C3/C2 0.27 0.27 0.27 0.27 C3/C16 1.68 2.19 1.72 1.72 C16:1OH/C17 0.12 0.12 0.13 0.13 Isovaleric acidemia (IVA) C5 0.63 0.65 0.61 0.61 C5/C0 0.01 0.01 0.01 0.01 C5/C2 0.02 0.02 0.02 0.02 Glutaric acidemia type I (GA I) C5-DC 0.37 0.37 0.40 0.42 C5-DC/C5-OH 0.97 1.00 1.17 1.27 C5-DC/C8 2.46 2.31 3.63 3.50 3-Methylcrotonyl-CoA carboxylase deficiency (3MCC) C5-OH 0.87 0.81 0.70 0.68 C5-OH/C8 5.8 5.06 6.36 5.66 3-Hydroxy-3-methyglutaric aciduria (HMG) C5-OH 0.87 0.81 0.70 0.68 C6-DC 0.60 0.61 0.35 0.37 ß-Ketothiolase deficiency (BKT) C5:1 0.09 0.08 0.07 0.07 C5-OH 0.87 0.81 0.70 0.68 C4-OH 0.69 0.67 0.50 0.50 Malonic acidemia (MAL) C3-DC 0.69 0.67 0.50 0.50 C3-DC/C10 3.45 3.35 2.38 2.27 [ABSTRACT FROM AUTHOR]