Inherited Metabolic Disorders: Efficacy of Enzyme Assays on Dried Blood Spots for the Diagnosis of Lysosomal Storage Disorders

  • Jyotsna VermaEmail author
  • Divya C. Thomas
  • David C. Kasper
  • Sandeepika Sharma
  • Ratna D. Puri
  • Sunita Bijarnia-Mahay
  • Pramod K. Mistry
  • Ishwar C. Verma
Research Report
Part of the JIMD Reports book series (JIMD, volume 31)


High consanguinity rates, poor access to accurate diagnostic tests, and costly therapies are the main causes of increased burden of lysosomal storage disorders (LSDs) in developing countries. Therefore, there is a major unmet need for accurate and economical diagnostic tests to facilitate diagnosis and consideration of therapies before irreversible complications occur. In cross-country study, we utilized dried blood spots (DBS) of 1,033 patients clinically suspected to harbor LSDs for enzymatic diagnosis using modified fluorometric assays from March 2013 through May 2015. Results were validated by demonstrating reproducibility, testing in different sample types (leukocytes/plasma/skin fibroblast), mutation study, or measuring specific biomarkers. Thirty percent (307/1,033) were confirmed to have one of the LSDs tested. Reference intervals established unambiguously identified affected patients. Correlation of DBS results with other biological samples (n = 172) and mutation studies (n = 74) demonstrated 100% concordance in Gaucher, Fabry, Tay Sachs, Sandhoff, Niemann-Pick, GM1, Neuronal ceroid lipofuscinosis (NCL), Fucosidosis, Mannosidosis, Mucopolysaccharidosis (MPS) II, IIIb, IVa, VI, VII, and I-Cell diseases, and 91.4% and 88% concordance in Pompe and MPS-I, respectively. Gaucher and Pompe are the most common LSDs in India and Pakistan, followed by MPS-I in both India and Sri Lanka. Study demonstrates utility of DBS for reliable diagnosis of LSDs. Diagnostic accuracy (97.6%) confirms veracity of enzyme assays. Adoption of DBS will overcome significant hurdles in blood sample transportation from remote regions. DBS enzymatic and molecular diagnosis should become the standard of care for LSDs to make timely diagnosis, develop personalized treatment/monitoring plan, and facilitate genetic counseling.


Diagnostic accuracy Dried blood spots Enzymatic diagnosis Lysosomal enzymes Lysosomal storage disorders Molecular diagnosis 



Creatine phosphokinase


Dried blood spot


External Quality Assurance Scheme


European Research Network for evaluation and improvement of screening, diagnosis, and treatment of inborn errors of metabolism




Lysosomal storage disorders





We thank all patients and normal subjects whose blood was used as a control/reference material to generate the data for this study. We extend our thanks to Genzyme (India) who has chosen our center as a referral laboratory for lysosomal enzymes testing. We express our gratitude to doctors from India and neighboring countries for referring patients to our genetic center. We appreciate Genzyme (India), a Sanofi company for extending support to patients for testing and treatment. We also thank Chairman and Director Administration, Sir Ganga Ram Hospital, for their cooperation in setting up genetic testing facility.


  1. Bostick WD, Dlnsmore SR, Mrochek JE, Waalkes TP (1978) Separation and analysis of aryl sulfatase isoenzymes in body fluids of man. Clin Chem 24(8):1305–1316PubMedGoogle Scholar
  2. Burin M, Dutra-Filho C, Brum J, Mauricio T, Amorim M, Giugliani R (2000) Effect of collection, transport, processing and storage of blood specimens on the activity of lysosomal enzymes in plasma and leukocytes. Braz J Med Biol Res 33:1003–1013CrossRefPubMedGoogle Scholar
  3. Camelier MV, Burin MG, De Mari J, Vieira TA, Marasca G, Giugliani R (2011) Practical and reliable enzyme test for the detection of Mucopolysaccharidosis IVA (Morquio Syndrome type A) in dried blood samples. Clin Chim Acta 412:1805–1808CrossRefPubMedGoogle Scholar
  4. Ceci R, de Francesco PN, Mucci JM, Cancelarich LN, Fossati CA, Rozenfeld PA (2011) Reliability of enzyme assays in dried blood spots for diagnosis of 4 lysosomal storage disorders. Adv Biol Chem 1:58–64CrossRefGoogle Scholar
  5. Chace DH, Kalas TA, Naylor EW (2003) Use of tandem mass spectrometry for multianalyte screening of dried blood specimens from newborns. Clin Chem 49:1797–1817CrossRefPubMedGoogle Scholar
  6. Chamoles NA, Blanco M, Gaggioli D (2001) Fabry disease: enzymatic diagnosis in dried blood spots on filter paper. Clin Chim Acta 308:195–196CrossRefPubMedGoogle Scholar
  7. Chamoles NA, Blanco M, Gaggioli D, Casentini C (2002a) Gaucher and Niemann-Pick diseases- enzymatic diagnosis in dried blood spots on filter paper: retrospective diagnosis in newborn- screening cards. Clin Chim Acta 317:191–197CrossRefPubMedGoogle Scholar
  8. Chamoles NA, Blanco M, Gaggioli D, Casentini C (2002b) Tay-Sachs and Sandhoff diseases: enzymatic diagnosis in dried blood spots on filter paper: retrospective diagnosis in newborn-screening cards. Clin Chim Acta 318(1–2):133–137CrossRefPubMedGoogle Scholar
  9. Civallero G, Michelin K, de Mari J et al (2006) Twelve different enzyme assays on dried-blood filter paper samples for detection of patients with selected inherited lysosomal storage diseases. Clin Chim Acta 372:98–102CrossRefPubMedGoogle Scholar
  10. Cobos PN, Steqlich C, Santer R, Lukacs Z, Gal A (2015) Dried blood spots allow targeted screening to diagnose mucopolysaccharidosis and mucolipidosis. JIMD Rep 15:123–132PubMedGoogle Scholar
  11. Coelho J, Guigliani R (2000) Fibroblasts of skin fragments as a tool for the investigation of genetics diseases: technical recommendations. Genet Mol Biol 23:269–271CrossRefGoogle Scholar
  12. Filocamo M, Morrone A (2011) Lysosomal storage disorders: molecular basis and laboratory testing. Hum Genomics 5:156–169CrossRefPubMedPubMedCentralGoogle Scholar
  13. Hollak CE, Van Weely S, van Oers MH, Aerts JM (1994) Marked elevation of plasma chitotriosidase activity. A novel hallmark of Gaucher disease. J Clin Invest 93:1288–1292CrossRefPubMedPubMedCentralGoogle Scholar
  14. Hopwood JJ, Muller V, Smithson A, Baggett N (1979) A fluorometric assay using 4 methylumbelliferyl alpha-L-iduronide for the estimation of alpha-L-iduronidase activity and the detection of Hurler & Scheie syndromes. Clin Chim Acta 92:257–265CrossRefPubMedGoogle Scholar
  15. Kaminsky P, Lidove O (2014) Current therapeutic strategies in lysosomal disorders. Presse Med 43:1174–1184CrossRefPubMedGoogle Scholar
  16. Kelly S (1977) Biochemical methods in medical genetics, vol 1008, 18th edn, Harvard American lecture series. Thomas, SpringfieldGoogle Scholar
  17. Lukacs Z, Santavuori P, Keil A, Steinfeld R, Kohlschutter A (2003) Rapid and simple assay for the determination of tripeptidyl peptidase and palmitoyl protein thioesterase activities in dried blood spots. Clin Chem 49:509–511CrossRefPubMedGoogle Scholar
  18. Marsh J, Fensom AH (1985) 4 Methylumbelliferyl α-N-acetyl glucosaminidase activity for diagnosis of Sanfilippo B disease. Clin Genet 27:258–262CrossRefPubMedGoogle Scholar
  19. Mistri M, Oza N, Sheth F, Sheth J (2014) Prenatal diagnosis of lysosomal storage disorders: our experience in 120 cases. Mol Cytogenet 7(Suppl 1):P126CrossRefPubMedCentralGoogle Scholar
  20. Muller KB, Rodrigues MD, Pereira VG, Martins AM, D’Almeida V (2010) Reference values for lysosomal enzymes activities using dried blood spots samples- a Brazilian experience. Diagn Pathol 5:65–72CrossRefPubMedPubMedCentralGoogle Scholar
  21. Sheth J, Mistri M, Sheth F et al (2014) Burden of lysosomal storage disorders in India: experience of 387 affected children from a single diagnostic facility. JIMD Rep 12:51–63CrossRefPubMedGoogle Scholar
  22. van Diggelen OP, Keulemans JL, Winchester B et al (1999) A rapid fluorogenic palmitoyl-protein thioesterase assay: pre- and postnatal diagnosis of INCL. Mol Genet Metab 66:240–244CrossRefPubMedGoogle Scholar
  23. van Diggelen OP, Voznyi YV, Keulemans JLM et al (2005) A new fluorometric enzyme assay for the diagnosis of Niemann–Pick A/B, with specificity of natural sphingomyelinase substrate. J Inherit Metab Dis 28:733–741CrossRefPubMedGoogle Scholar
  24. Verma PK, Ranganath P, Dalal AB, Phadke SR (2012) Spectrum of lysosomal storage disorders at a medical genetics center in northern India. Indian Pediatr 49:799–804CrossRefPubMedGoogle Scholar
  25. Verma J, Thomas DC, Sharma S et al (2015) Inherited metabolic disorders: quality management for laboratory diagnosis. Clin Chim Acta 447:1–7CrossRefPubMedGoogle Scholar
  26. Voznyi YV, Keulemans JLM, van Diggelen OP (2001) A fluorimetric enzyme assay for the diagnosis of MPS II (Hunter disease). J Inherit Metab Dis 24:675–680CrossRefPubMedGoogle Scholar
  27. Willey AM, Carter TP, Kally S et al (1982) Clinical genetics: problems in diagnoses and counseling. Harcourt Brace/Jovanovich, New York/London, Page 41Google Scholar
  28. Winchester B, Bali D, Bodamer OA et al (2008) Methods for a prompt and reliable laboratory diagnosis of Pompe disease: report from an international consensus meeting. Mol Genet Metab 93:275–281CrossRefPubMedGoogle Scholar

Copyright information

© SSIEM and Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Jyotsna Verma
    • 1
    Email author
  • Divya C. Thomas
    • 1
  • David C. Kasper
    • 2
  • Sandeepika Sharma
    • 1
  • Ratna D. Puri
    • 1
  • Sunita Bijarnia-Mahay
    • 1
  • Pramod K. Mistry
    • 3
  • Ishwar C. Verma
    • 1
  1. 1.Biochemical Genetics, Centre of Medical Genetics, Sir Ganga Ram HospitalNew DelhiIndia
  2. 2.Clinical Institute of Laboratory MedicineMedical University of ViennaViennaAustria
  3. 3.Department of Internal Medicine (Digestive Diseases)Yale University School of MedicineNew HavenUSA

Personalised recommendations