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RISS 인기검색어
- 검색키워드
- 저자 : Elisa Legnini (검색결과 2 건)
- 무료
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Analysis of Acid Sphingomyelinase Activity in Dried Blood Spots Using Tandem Mass Spectrometry
Elisa Legnini,Joe J. Orsini,Adolf Mühl,Britt Johnson,Angela Dajnoki,Olaf A Bodamer 대한진단검사의학회 2012 Annals of Laboratory Medicine Vol.32 No.5
Background: Niemann Pick disease (NP) is a rare, lysosomal storage disorder due to deficiency of the intra-lysosomal enzyme acid sphingomyelinase (ASM) resulting in intracellular accumulation of sphingomyelin. We evaluated a tandem mass spectrometry (MS/MS)method to analyze ASM activity in dried blood spots (DBS) that may be suitable for laboratory diagnosis of NP including high throughput screening of at-risk populations and potentially for newborn screening. Methods: ASM activity was measured in 3.2 mm punches from DBS. The eluate was incubated with the ASM substrate (N-Hexanoyl-D-erythro-sphingosylphosphorylcholine [C6- sphingomyelin (C29H59N2O6P)]) and an internal standard (N-butyroyl-D-erythro-sphingosine [C4-ceramide (C22H43NO3)]). ASM product and IS were analyzed using MS/MS in multiple reaction monitoring mode for transitions m/z 370.6>264.3 (ASM internal standard) and m/z 398.6>264.3 (ASM product). Results: ASM activities were stable for up to 2 months at or below 4˚C. Position of the punch in the DBS and/or hematocrit of the DBS had a limited effect on ASM activities. Both intraand inter-assay variability were below 10%. There was no carry-over. The median ASM activity in 2,085 newborn infants was 9.5 μmol/h/L (mean 10.6) with a SD of 5.06 μmol/h/L. Six of 2,085 (0.3%) infants were found to have ASM activities below the cut-off of 2.5 μmol/h/L. ASM activities were below the cut-off level in all 10 previously diagnosed cases with NP (range: 0.16 to 2.08 μmol/h/L). Conclusions: This MS/MS method for the measurement of ASM activity in DBS is robust and suitable for laboratory diagnosis of NP. Background: Niemann Pick disease (NP) is a rare, lysosomal storage disorder due to deficiency of the intra-lysosomal enzyme acid sphingomyelinase (ASM) resulting in intracellular accumulation of sphingomyelin. We evaluated a tandem mass spectrometry (MS/MS)method to analyze ASM activity in dried blood spots (DBS) that may be suitable for laboratory diagnosis of NP including high throughput screening of at-risk populations and potentially for newborn screening. Methods: ASM activity was measured in 3.2 mm punches from DBS. The eluate was incubated with the ASM substrate (N-Hexanoyl-D-erythro-sphingosylphosphorylcholine [C6- sphingomyelin (C29H59N2O6P)]) and an internal standard (N-butyroyl-D-erythro-sphingosine [C4-ceramide (C22H43NO3)]). ASM product and IS were analyzed using MS/MS in multiple reaction monitoring mode for transitions m/z 370.6>264.3 (ASM internal standard) and m/z 398.6>264.3 (ASM product). Results: ASM activities were stable for up to 2 months at or below 4˚C. Position of the punch in the DBS and/or hematocrit of the DBS had a limited effect on ASM activities. Both intraand inter-assay variability were below 10%. There was no carry-over. The median ASM activity in 2,085 newborn infants was 9.5 μmol/h/L (mean 10.6) with a SD of 5.06 μmol/h/L. Six of 2,085 (0.3%) infants were found to have ASM activities below the cut-off of 2.5 μmol/h/L. ASM activities were below the cut-off level in all 10 previously diagnosed cases with NP (range: 0.16 to 2.08 μmol/h/L). Conclusions: This MS/MS method for the measurement of ASM activity in DBS is robust and suitable for laboratory diagnosis of NP.
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Analysis of Lyso-Globotriaosylsphingosine in Dried Blood Spots
Britt Johnson,Hermann Mascher,Daniel Mascher,Elisa Legnini,Christina Y Hung,Angela Dajnoki,Yin-Hsiu Chien,László Maródi,Wuh-Liang Hwu,Olaf A Bodamer 대한진단검사의학회 2013 Annals of Laboratory Medicine Vol.33 No.4
Recently, lyso-globotriaosylsphingosine (lyso-Gb3) was found to be elevated in plasma of treatment naive male patients and some female patients with Fabry Disease (FD). This study tested whether lyso-Gb3 could be analyzed in dried blood spots (DBS) from filter cards and whether concentrations are elevated in newborn infants with FD. Lyso-Gb3 concentrations were analyzed in DBS following extraction using a novel HPLC-mass spectrometry (MS)/MS method. Lyso-Gb3 levels in DBS were above the lower limit of quantitation (0.28 ng/mL) in 5/17 newborn FD infants (16 males; range: 1.02-8.81 ng/mL), but in none of the newborn controls, in all 13 patients (4 males) with classic FD (range: 2.06-54.1ng/mL), in 125/159 Taiwanese individuals with symptomatic or asymptomatic FD who carry the late onset α-galactosidase A (GLA) mutation c.936+919G>A (IVS4+919G>A) (3.75±0.69 ng/mL; range: 0.418-3.97 ng/mL) and in 20/29 healthy controls (0.77±0.24 ng/mL;range: 0.507-1.4 ng/mL). The HPLC-MS/MS method for analysis of lyso-Gb3 is robust and yields reproducible results in DBS in patients with FD. However, concentrations of lyso-Gb3 were below the limit of quantitation in most newborn infants with FD rendering this approach not suitable for newborn screening. In addition, most females with the late onset mutation have undetectable lyso-Gb3 concentrations.
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