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 Ø Rare autosomal recessive lysosomal

Rare autosomal recessive lysosomal storage disease storage disease

Ø Ø Defect in the lysosomal membrane protein

Defect in the lysosomal membrane protein cystinosin cystinosin

Ø Ø Infants may present with failure to thrive

Infants may present with failure to thrive and begins to show signs of Fanconi and begins to show signs of Fanconi syndrome syndrome

Ø Ø Treatment by cysteamine

Treatment by cysteamine

Current laboratory method Current laboratory method-

• Cystine

Cystine binding protein (CBP) method binding protein (CBP) method Y

Sample

14C - cystine

Y Y Y Y

30 min incubation Filtration CPM [cystine] Scintillation counting

LC LC-

• MS method (in brief)

MS method (in brief)

Ø 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 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)

0.0 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 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

µmol/l ratio d0-cystine/d4-cystine

Evaluation with patient samples Evaluation with patient samples

Internal standard d4-cystine Cystinosis patient WBC cystine Control patient WBC cystine

Imprecision Imprecision

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

Comparison of CBP and LCMS Comparison of CBP and LCMS methods methods

R2 = 0.9181 2 4 6 8 10 12 14 16 18 2 4 6 8 10 12 14 16 18 CBP method (umol / L) LC-MS method (umol / L)

R2 = 0.757 1 2 3 4 5 6 1 2 3 4 5 6 CBP method (umol / L) LCMS method (umol / L)

Bland Altman plot Bland Altman plot

• 0.5

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

Acknowledgments Acknowledgments

Ø Ø Dr. Kevin Mills

• Dr. Kevin Mills -
• Biochemistry Research

Biochemistry Research Group, Institute of Child Health Group, Institute of Child Health

Ø Ø Furzana Malik

Furzana Malik – – Enzyme Unit, GOSH Enzyme Unit, GOSH