For Doctors

Disorders included in newborn screening are genetic disorders imposing huge burden on health care infrastructure though many of these are treatable conditions.

Depending on the technique used to perform these tests, they belong to three broad categories:

  • Biochemical Assays- congenital hypothyroidism, congenital adrenal hyperplasia, galactosemia, glucose-6-phosphate dehydrogenase deficiency, biotinidase deficiency, cystic fibrosis
  • Tandem Mass Spectrometry (TMS) based assays- Amino acid, fatty acid and organic acid disorders
  • Hemoglobinopathies – Disorders of haemoglobin structure and function, detected mostly by Isoelectric Focusing.

New Born Screening- Biochemical Assays

  • Galactosemia (GALT)- Inability of body to breakdown galactose.
  • Congenital Hypothyroidism (CH)- Defect in synthesis of thyroid hormones.
  • Congenital Adrenal Hyperplasia (CAH)- Defect in adrenal cortical hormone synthesis.
  • Glucose-6-Phoshate Dehydrogenase Deficiency (G6PDH)- Defective glucose-6-phosphate dehydrogenase enzyme (Pentose Phosphate Pathway).
  • Biotinidase Deficiency(BIOT)- Defective recycling of biotin due to defective biotinidase enzyme.
  • Cystic Fibrosis (CF)- defective Cl- channel, build up of thick mucus in lungs and intestines and leading to infections and nutritional deficiencies.
  • Phenylketonuria (PKU)- Inability of body to breakdown phenylalanine.

Tandem Mass Spectrometry Assays

Amino Acid Disorders (AA): Inability of body to breakdown a particular amino acid (16 disorders).

Fatty Acid Oxidation Disorders (FAO): Inability of body to use fatty acids for energy (12 disorders).

Organic acid disorders: Defective metabolic enzymes resulting in build-up of toxic organic acids in the body (16 disorders).

Amino acid disorders and their primary biomarkers

Disorder Primary Biomarker
Argininemia (ARG) Arginine
Argininosuccinic Aciduria (ASA Lyase) Citrulline
Carbamoylphosphate Synthase Deficiency (CPS) Citrulline
Citrullinemia (CIT-I) Citrulline
Homocystinuria (HCY) Methionine
Hypermethioninemia (MET) Methionine
Hyperammonemia, Hyperornithinemia, Homocitrullinemia (HHH Syndrome) Ornithine and homocitrulline
Hyperornithinemia with Gyrate Atrophy (HOGA) Ornithine
Maple Syrup Urine Disease (MSUD) Leucine plus isoleucine and/or valine
Liver Disease Various biomarkers
Phenylketonuria (PKU)– Classical Hyperphenylalaninemia

– Biopterin Cofactor Deficiencies

Tyrosinemia-Transient Neonatal Tyrosinemia

– Tyrosinemia Type I

– Tyrosinemia Type II

– Tyrosinemia Type III


Fatty acid oxidation disorders and their primary biomarkers

Disorder Primary Biomarker (Acylcarnitines)
2,4-Dienoyl-CoA Reductase deficiency (DE-RED) C 10:2
Carnitine/Acylcarnitine Translocase Deficiency (CACT) C16
Carnitine Palmitoyl Transferase Deficiency Type I (CPT-I) C16
Carnitine Uptake Deficiency (CUD) C0 (Free Carnitine)
Long Chain Hydroxy Acyl-CoA Dehydrogenase Deficiency (LCHAD) C16-OH and/or C18:1-OH
Medium Chain Acyl-CoA Dehydrogenase Deficiency (MCAD) C8
Multiple Acyl-CoA Dehydrogenase Deficiency (MADD or GA-2) C4-C18 saturated and unsaturated species
Neonatal Carnitine Palmitoyl Transferase Deficiency Type-II (CPT-II) C16
Short-chain Acyl-CoA Dehydrogenase Deficiency (SCAD) C4
Short-Chain Hydroxyacyl-CoA Dehydrogenase Deficiency (SCHAD) C4-OH
Trifunctional Protein Deficiency (TFP) C16-OH and/or C18:1-OH
Very Long Chain Acyl-CoA Dehydrogenase Deficiency (VLCAD) C14:1
Organic acid disorders and their primary biomarkers

Disorder Primary Biomarker (Acylcarnitines)
2-Methyl-3-hydroxybutyryl-CoA Dehydrogenase Deficiency (2M3HBA) C5-OH
2-Methylbutyryl-CoA Dehydrogenase Deficiency (2MBG) C5
3-Hydroxy-3-methylglutaryl-CoA Lyase Deficiency (HMG) C5-OH
3-Methylcrotonyl-CoA Carboxylase Deficiency (3MCC) C5-OH
3-Methylglutaconyl-CoA Hydratase Deficiency (3MGA) C5-OH
Glutaric Acidemia Type 1 (GA-1) C5DC
Isovaleric Acidemia (IVA) C5
Isobutyryl-CoA Dehydrogenase Deficiency (IBG) C4
Malonic Aciduria (MAL) C3DC
Mitochondrial Acetoacetyl-CoA Thiolase Deficiency (βKT) C5:1 and/or C5-OH
Methylmalonic Acidemias (MMA)– Methylmalonyl-CoA Mutase Deficiency (MUT)

– Some Adenosylcobalamin Synthesis Defects (CBL A, B/CBL C, D)

– Maternal Vitamin B12 Deficiency

Multiple CoA Carboxylase Deficiency (MCD) C5-OH and/or C3
Propionic Acidemia (PROP) C3

Hemoglobinopathies (4 disorders)

Sickle cell disease (Hb SS)

Defective hemoglobin leads to change in shape of RBC from “doughnut” to “sickle” causing complications such as tissue and organ damage, hemolytic anemia, spleen infections, etc.

Hemoglobin SC disease

Defective hemoglobin due to presence of one defective sickle (S) gene and another defective C gene. Same symptoms as above bu generally milder than Hb SS disease.

Sickle ß Thalassemia (Hb S/ß thal)

Defective hemoglobin due to one sickle (S) gene and one gene causing reduced production of beta chain of hemoglobin. Symptoms include mild anaemia, tiredness, etc.

Other hemoglobin variants

Do not cause severe symptoms but may be picked up during the screening.

Tandem Mass Spectrometry(MS/MS) is widely used in the Inborn Errors of Metabolism (IEM) screening protocol, for the analysis of amino acids (AA) and acylcarnitines (AC). Various amino acids and acylcarnitines and some specific ratios of these compounds can be used for the assessment of various inborn errors of metabolism.

The mass spectrometer is a device that separates and quantifies ions based on their mass/charge (m/z) ratios.The modern tandem mass spectrometer usually consists of two quadrupole mass spectrometers separated by a reaction chamber or collision cell; the latter is often another quadrupole. The mixture to be analyzed is subjected to a soft ionization procedure (e.g., fast atom bombardment or electrospray) to create quasimolecular ions, and is injected into the first quadrupole, which separates these parent ions from each other. These ions then pass (in order of m/z ratio) into the reaction chamber, where they are fragmented; the m/z ratios of the fragments are then analyzed in the second quadrupole. Because separation of compounds in the mixture is by mass spectrometry instead of chromatography, the entire process, from ionization and sample injection to data acquisition by computer, takes only seconds.


As a key technology in future clinical endocrinology, MSMS brings the following benefits:

  • Due to its sensitivity MS/MS is widely regarded as the ”Gold Standard” among assay technologies.
  • Permits very rapid, sensitive and accurate measurement of many different types of metabolites including biomarkers for amino acidemias, organic acidemias, and disorders of fatty acid oxidation.
  • Results for multiple analytes in a single assay
  • High throughput, cost effectiveness and reproducibility
  • Multiple sample matrices can be used

tech-5Time Resolved Fluorometry

Perkin-Elmer time resolved fluorometry is used for detection of seven disorders- congenital hypothyroidism, congenital adrenal hyperplasia, galactosemia, glucose-6-phosphate dehydrogenase deficiency, biotinidase deficiency, cystic fibrosis and phenylketonuria.

The time-resolved detection relies on two important properties of the fluorophore, which contribute to the sensitivity of the assay. The fluorphore has a long decay time and it has a wide Stokes’ shift (difference between the excitation and emission wavelength). Measurement can then be made at a time and wavelength at which background is minimal.Measurement takes place by repeatedly exciting the fluorophore, and after each excitation, measuring emitted fluorescence for a period that starts after an interval.

Newborn screening required dried blood spot specimen. A few drops of blood from specific area of infant’s heel are collected on a specially designed filter paper. The blood is allowed to dry for 3-4 hours at room temperature and then the screening card with the blood on the filter paper and containing the details such as date of birth of the baby, sample collection date, any medications currently being administered, contact information of parents and the doctor etc.

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