title: Characterization of a Blood Spot Creatine Kinase Skeletal Muscle Isoform Immunoassay for High-Throughput Newborn Screening of Duchenne Muscular Dystrophy.
abstract: Duchenne muscular dystrophy (DMD) is a progressive, lethal X-linked neuromuscular disorder with an average worldwide incidence of 1:5000. Blood spot creatine kinase (CK) enzyme assays previously used in newborn screening programs for DMD are nonspecific because measured CK enzyme activity is attributable to 3 isoenzyme forms of CK (CK-MM, CK-MB, and CK-BB) and it is the CK-MM isoform that is found predominantly in skeletal muscle. CK-MM is increased in boys with DMD owing to muscle damage. We describe a sensitive and specific automated immunoassay for CK-MM to screen for DMD in blood spots.
  
  
  
  The prototype assay was developed on the PerkinElmer GSP®analyzer to enable high-throughput screening. CK-MM was assayed using a solid phase, 2-site immunofluorometric system. Purified human CK-MM was used to create calibrators and controls.
  
  
  
  The limit of blank (LOB), detection (LOD), and quantification (LOQ) values were <1, 3, and 8 ng/mL, respectively. The analytical measurement range was 4-8840 ng/mL. Interassay (n = 40) imprecision was <7% across the analytical range. Cross-reactivity was <5% for CK-MB and 0% for CK-BB. The mean recovery of CK-MM was 101% (range 87%-111%). Blood spots from newborn infants (n = 277) had a mean CK-MM concentration of 155 ng/mL and a 99th centile of 563 ng/mL. The mean blood spot CK-MM concentration from 10 cases of DMD was 5458 ng/mL (range 1217-9917 ng/mL).
  
  
  
  CK-MM can be reliably quantified in blood spots. The development of this CK-MM assay on a commercial immunoassay analyzer would enable standardized and high-throughput newborn blood spot screening of DMD.
authors.affiliation: Wales Newborn Screening Laboratory, Department of Medical Biochemistry, Immunology and Toxicology, University Hospital of Wales, Cardiff, Wales, UK; stuart.moat@wales.nhs.uk.|School of Medicine, Cardiff University, Cardiff, Wales, UK.
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Moat
authors.affiliation: PerkinElmer, Wallac Oy, Turku, Finland.
authors.forename: Teemu
authors.initials: T
authors.lastname: Korpimäki
authors.affiliation: PerkinElmer, Wallac Oy, Turku, Finland.
authors.forename: Petra
authors.initials: P
authors.lastname: Furu
authors.affiliation: PerkinElmer, Wallac Oy, Turku, Finland.
authors.forename: Harri
authors.initials: H
authors.lastname: Hakala
authors.affiliation: PerkinElmer, Wallac Oy, Turku, Finland.
authors.forename: Hanna
authors.initials: H
authors.lastname: Polari
authors.affiliation: PerkinElmer, Wallac Oy, Turku, Finland.
authors.forename: Liisa
authors.initials: L
authors.lastname: Meriö
authors.affiliation: PerkinElmer, Wallac Oy, Turku, Finland.
authors.forename: Pauliina
authors.initials: P
authors.lastname: Mäkinen
authors.affiliation: School of Medicine, Cardiff University, Cardiff, Wales, UK.
authors.forename: Ian
authors.initials: I
authors.lastname: Weeks
mesh_terms: D000328:Adult; D003402:Creatine Kinase / Q000097:blood* / Q000378:metabolism; D005260:Female; D057166:High-Throughput Screening Assays; D006801:Humans; D007118:Immunoassay; D007223:Infant; D007231:Infant, Newborn; D007527:Isoenzymes / Q000097:blood / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D018482:Muscle, Skeletal / Q000201:enzymology*; D020388:Muscular Dystrophy, Duchenne / Q000097:blood / Q000175:diagnosis*
chemical_list: D007527:Isoenzymes; D003402:Creatine Kinase