MSACL 2015 EU Abstract

Diagnosis of Inborn Metabolic Disorders via Absolute Enzymatic Activity
Zdenek Spacil
University of Washington

Zdenek Spacil(1), Arun Babu Kumar(1), Naveen Kumar Chennamaneni(1), Fan Yi(1), Sophia Masi(1), C. Ronald Scott(2), Frantisek Turecek(1), and Michael H. Gelb(1,3)
University of Washington Department of (1)Chemistry, (2)Pediatrics and (3)Biochemistry

Short Abstract

The newborn screening for lysosomal storage disorders, such as sphingolipidoses or mucopolysacharidoses is critical to detect affected individuals and to timely initiate treatment. Over 15 screening assays developed in our lab measure activity of lysosomal enzymes in dried blood samples (DBS) by tandem mass spectrometry technology (SRM). These assays greatly outperform other screening tests, such as fluorometric assays in terms of sensitivity and specificity, are easy to implement and allow for multiplexing, which was collectively demonstrated in numerous pilot studies worldwide. Besides the latest development and clinical data from pilot studies, we are planning to present on the absolute enzyme activity measurement in highly purified leukocyte populations as a second-tier diagnostic test, which is transforming the field.

Long Abstract


Lysosomal storage disorders (LSDs) comprises approximately 50 inherited defects in lysosomal function. They occur with an estimated collective prevalence of 1/7000-9000 in European populations. A continuum of severity ranging from infantile to adult-onset forms can manifest for each disorder depending on the character of genetic mutation (deletion, frameshift, point, etc.) posttranslational modifications and other mechanisms determining the residual enzyme activity. Relatively infrequent occurrence of LSDs and often lack of specific symptoms poses a real challenge for timely diagnosis. Specific therapies such as enzyme replacement (ERT), substrate reduction (SRT), bone marrow transplantation (BMT) or gene therapy were developed to treat LSDs. However, quick identification of affected individuals during asymptomatic stage is critical to initiate treatment preventing irreversible damage of somatic organs and central nervous system. Therefore screening methods for LSDs in dried blood samples (DBS) are highly desired. Enzyme activity assays based on tandem mass spectrometry (MS/MS) demonstrated their potential to reliably detect LSDs in numerous pilot studies worldwide and became the method of choice in LSDs screening. To date we have developed over 15 enzyme assays and currently we are exploring the potential to measure an absolute enzyme activity as second-tier diagnostic test. We are excited to present our first proof of concept results.


We have previously reported fully quantitative multiplex assays of lysosomal enzymes in DBS material, primarily used for newborn screening applications. The commercial 6 plex assay for Niemann-Pick-A/B, Pompe, Fabry, Gaucher, Krabbe and mucopolysacharidoses I (MPS-I) will be available since 2016 and reagents for 3-plex assay (MPS-II; MPS-IVA and MPS-VI) can be obtained through our lab. These multiplex assays were implemented in newborn screening and clinical labs worldwide. Last year, our partners in Newborn Screening Laboratory at Department of Health (Shoreline, WA) completed a 100 000 pilot study on Fabry, Pompe, MPS-I to demonstrate feasibility and stable performance. The published data indicated low false positive rates (0.005-0.012%) and offered previously unknown information on prevalence of LSDs (1/7500 combined). Lately, they adopted the 6-plex assay and up to date screened over 25 000 newborns, detecting 4 affected individuals at very low combined false positive rate. The NBS programs based on our MS/MS multiplex assays are underway e.g. in NYS Department of Health, Illinois Department of Public Health or National Taiwan University Hospital. Although advantageous for newborn screening, DBS material does not allow absolute enzymatic activity measurement necessary for diagnosis. To develop a second-tier diagnostic test we isolated specific leukocyte populations from the whole blood using magnetic microbeads and measured absolute enzyme activity in these populations with MS/MS assays. We assume the severity of a LSD manifestation corresponds to the residual enzyme activity in highly purified and characterized sample comparable within individuals and accounts for variances in hematocrit, white blood cell levels, leukocyte population diversity etc. The diagnostic test based on absolute enzyme activity is assumed to be more robust and have better predictive value than analysis of a specific metabolite.


Unlike other leukocyte based assays, we were able to isolate specific leukocyte populations from whole blood using magnetic microbeads technology and measure absolute enzyme activity in these leukocytes. The enrichment yields based on fluorescence-activated cell sorting were typically between 85-95% depending of cell type and the protocol used. We used the 9-plex enzyme assay to measure absolute activity and found up to 20-fold difference in enzyme activity across these populations. The finding demonstrates shortcomings of diagnostic tests, which uses an ensemble of white blood cells. Therefore assaying a highly purified specific population of leukocytes is essential to account for heterogeneity among individuals and to access the diagnostic information. Due to high sensitivity and wide analytical range, MS/MS assay requires only 10-15 × 10E3 leukocytes per assay, which allows for repeated measurements from the whole blood volumes as low as 250 µL.


We developed a second-tier diagnostic test based on absolute enzyme activity by MS/MS in specific leukocyte populations. This test allows for accurate prediction of LSD severity delivering an instant and unambiguous diagnosis for individuals with positive screening test in DBS.