Development of an Development of an automated method for automated - PowerPoint PPT Presentation

Development of an Development of an automated method for automated method for the measurement of the measurement of leucocyte cystine by leucocyte cystine by LCMS LCMS Dr. Amanda Lam Dr. Amanda Lam Clinical Scientist Clinical Scientist

Cystinosis Cystinosis Ø Rare autosomal recessive lysosomal Rare autosomal recessive lysosomal Ø storage disease storage disease Ø Defect in the lysosomal membrane protein Defect in the lysosomal membrane protein Ø cystinosin cystinosin Ø Infants may present with failure to thrive Infants may present with failure to thrive Ø and begins to show signs of Fanconi and begins to show signs of Fanconi syndrome syndrome Ø Treatment by cysteamine Treatment by cysteamine Ø

Current laboratory method- - Cystine Cystine Current laboratory method binding protein (CBP) method binding protein (CBP) method Sample 14 C - cystine Y 30 min incubation Y Y Y Y Filtration CPM Scintillation counting [cystine]

LC- -MS method (in brief) MS method (in brief) LC Ø Sample preparation Sample preparation Ø Ø 10 10µ µl of sample + 10 l of sample + 10µ µl d3 l d3- -cystine cystine Ø Ø 125 125 µ µl 0.2M borate buffer, pH 10.4, l 0.2M borate buffer, pH 10.4, Ø Ø 125 125 µ µl 6M derivative l 6M derivative Ø Ø HPLC HPLC- -MS/MS analyses using a C18 guard MS/MS analyses using a C18 guard Ø column, 5 min per sample. column, 5 min per sample.

Optimisation of method Optimisation of method 20µl sample homogenate 0.2M Borate buffer pH 10.4 20µl sample homogenate 0.1M Borate buffer pH 10.4 10µl sample homogenate 0.1M Borate buffer pH 10.4 10µl sample homogenate 0.2M Borate buffer pH 10.4

Linearity Linearity Cystine calibration curve 683.83>152.03 transition (3 replicates for each point) 0.9 0.8 0.7 0.6 d0-cystine/d4-cystine 0.5 ratio 0.4 0.3 0.2 0.1 0.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 µmol/l

Evaluation with patient samples Evaluation with patient samples Cystinosis patient WBC cystine Internal standard d4-cystine Control patient WBC cystine

Imprecision Imprecision Within batch Between batch Within batch Between batch %CV %CV %CV %CV Low QC Low QC 4.0 (n = 16) 5.8 (6 batches) 4.0 (n = 16) 5.8 (6 batches) (1.8 µ µmol / L) mol / L) (1.8 High QC High QC 6.2 (n = 19) 5.7 (6 batches) 6.2 (n = 19) 5.7 (6 batches) (3.1 µ µmol / L) mol / L) (3.1

Comparison of CBP and LCMS Comparison of CBP and LCMS methods methods 18 R 2 = 0.9181 16 LC-MS method (umol / L) 14 12 6 10 R 2 = 0.757 5 LCMS method (umol / L) 8 4 3 6 2 4 1 0 2 0 1 2 3 4 5 6 CBP method (umol / L) 0 0 2 4 6 8 10 12 14 16 18 CBP method (umol / L)

Bland Altman plot Bland Altman plot 3 Absolute difference (LCMS - CBP) 2.5 2 1.5 1 0.5 0 0 1 2 3 4 5 -0.5 Mean of two methods (umol / L)

Acknowledgments Acknowledgments Ø Dr. Kevin Mills Dr. Kevin Mills - - Biochemistry Research Biochemistry Research Ø Group, Institute of Child Health Group, Institute of Child Health Ø Furzana Malik Furzana Malik – – Enzyme Unit, GOSH Enzyme Unit, GOSH Ø