Purification: A Valuable Preparation Step in Disease Screening
AcroPrep Advanced filter plates aid rapid sample purification in research study on screening newborns for congenital disorders
April 22, 2021
A collaborative research group from the U.S. and Japan is interested in improving diagnostic screening for newborns at risk of having inherited enzyme deficiency disorders that cause severe degenerative disease. Treatment measures are more effective when they are initiated before clinical symptoms begin to appear, so early detection is critical.
Mucopolysaccharidoses (MPS) are a family of disorders that cause deficiency of the lysosomal enzymes responsible for the natural breakdown of long carbohydrate chains. Buildup of these carbohydrates in the body can lead to multiple problems, including organ damage, developmental issues, and neurological disorders. There is an unmet need for reliable early screening methods that will help identify MPS disorders in newborns within the timeframe that will allow treatment to be most effective.
The authors of this publication investigated the effectiveness of two different assay strategies for MPS screening. Detection of MPS disorders was based on measuring either enzyme activity levels or the concentration of specific glycosaminoglycans (long-chain sugar groups, or GAGs) in the blood of newborns. In a pilot study, the group had developed cutoff values for establishing normal versus abnormal levels for both indicators.
To carry out their study, the authors screened 18,222 dried blood spot samples collected from newborns at least three years prior during routine health exams. MPS clinical disease symptoms nearly always appear within the first three years of a child’s life, so this strategy would allow results to be matched post-study with confirmed occurrences of MPS disease.
Sample preparation required a purification step to separate the MPS indicators from other blood solution components. This was accomplished using Pall’s AcroPrep™ Advance 96-well filter plates with a 10 K Omega™ MWCO (molecular weight cutoff) membrane. Pall provides AcroPrep plates to accommodate a wide variety of sample volume formats, membrane types, and MWCOs.
AcroPrep Advance filter plates provide rapid, efficient separation. Intrinsic plate and membrane properties prevent target molecules from binding to the plate, resulting in typical recovery rates of >90% of target biomolecules.
Following purification, newborn blood samples were analyzed via mass spectrometry. 300 samples containing elevated levels of more than one MPS disease-specific GAG were flagged and rescreened. Only one sample was confirmed as being true positive after the secondary screening, establishing a predicted false-positive rate of 1.64%.
For the second half of the study, lysosomal enzyme activity for two MPS-associated enzymes was measured via a fluorescence assay. The false positive rate for enzyme assay measurements were 0.21% and 0.18% for each enzyme, respectively.
Most importantly, when the authors used a combined two-tier screening strategy measuring both disease-specific enzyme activity levels and disease-specific GAG concentrations, the false positive rate dropped to 0%. Results were confirmed when the samples were unmasked and matched to real-world data. The authors believe the new two-tier strategy should become the new gold standard for screening newborns for MPS.
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