title: Diagnosis and Monitoring of Cystinosis Using Immunomagnetically Purified Granulocytes.
abstract: Cystine determination is a critical biochemical test for the diagnosis and therapeutic monitoring of the lysosomal storage disease cystinosis. The classical mixed-leukocyte cystine assay requires prompt specialized recovery/isolation following blood drawing, providing cystine concentrations normalized to total protein from assorted types of white blood cells, each with varying cystine content.
  
  
  
  We present a new workflow for cystine determination using immunomagnetic granulocyte purification, and new reference ranges established from 47 patient and 27 obligate heterozygote samples assayed. Samples were collected in acid-citrate dextrose tubes and their stability was proven to allow for overnight shipping before analysis. Cystine was quantified by LC-MS/MS.
  
  
  
  The new method was reproducible (<15% root mean square error) and specific, assaying purified granulocytes from blood samples that no longer required immediate preparation and therefore allowing for up to 30 h before processing. There was a nearly a 2-fold increase in the therapeutic target (1.9 nmol half-cystine/mg protein) range, established using distributions of patient, obligate heterozygote, and control samples. The 2.5-97.5 percentile ranges (-2 SD to +2 SD around mean) for these cohorts were 0.67-6.05 nmol/mg protein for patients, 0.33-1.35 nmol/mg protein for obligate heterozygotes, and 0.09-0.35 nmol/mg protein for controls.
  
  
  
  The intracellular cystine determination method using immunopurified granulocytes followed by LC-MS/MS analysis improves the inherent variability of mixed leukocyte analysis and eliminates the need for immediate sample preparation following blood draw.
authors.affiliation: Biochemical Genetics and Metabolomics Laboratory, Department of Pediatrics, University of California, San Diego, La Jolla, CA.
authors.forename: Ilya
authors.initials: I
authors.lastname: Gertsman
authors.affiliation: Biochemical Genetics and Metabolomics Laboratory, Department of Pediatrics, University of California, San Diego, La Jolla, CA.
authors.forename: Wynonna S
authors.initials: WS
authors.lastname: Johnson
authors.affiliation: Biochemical Genetics and Metabolomics Laboratory, Department of Pediatrics, University of California, San Diego, La Jolla, CA.
authors.forename: Connor
authors.initials: C
authors.lastname: Nishikawa
authors.affiliation: Biochemical Genetics and Metabolomics Laboratory, Department of Pediatrics, University of California, San Diego, La Jolla, CA.
authors.forename: Jon A
authors.initials: JA
authors.lastname: Gangoiti
authors.affiliation: Biochemical Genetics and Metabolomics Laboratory, Department of Pediatrics, University of California, San Diego, La Jolla, CA.
authors.forename: Bonnie
authors.initials: B
authors.lastname: Holmes
authors.affiliation: Biochemical Genetics and Metabolomics Laboratory, Department of Pediatrics, University of California, San Diego, La Jolla, CA. bbarshop@ucsd.edu.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Barshop
mesh_terms: D000293:Adolescent; D000328:Adult; D002648:Child; D002675:Child, Preschool; D002853:Chromatography, Liquid; D003553:Cystine / Q000097:blood*; D003554:Cystinosis / Q000097:blood* / Q000175:diagnosis*; D005260:Female; D006098:Granulocytes / Q000473:pathology*; D006801:Humans; D018189:Immunomagnetic Separation; D008297:Male; D008875:Middle Aged; D053719:Tandem Mass Spectrometry; D055815:Young Adult
chemical_list: D003553:Cystine