title: A multiplex immunoassay using the Guthrie specimen to detect T-cell deficiencies including severe combined immunodeficiency disease.
abstract: BACKGROUND
  Severe combined immunodeficiency (SCID) fulfills many of the requirements for addition to a newborn screening panel. Two newborn screening SCID pilot studies are now underway using the T-cell receptor excision circle (TREC) assay, a molecular technique. Here we describe an immunoassay with CD3 as a marker for T cells and CD45 as a marker for total leukocytes that can be used with the Guthrie specimen.
  
  
  METHODS
  The multiplexing capabilities of the Luminex platform were used. Antibody pairs were used to capture and detect CD3 and CD45 from a single 3-mm punch of the Guthrie specimen. The assay for each biomarker was developed separately in identical buffers and then combined to create a multiplex assay.
  
  
  RESULTS
  Using calibrators made from known amounts of leukocytes, a detection limit of 0.25 x 10(6) cells/mL for CD3 and 0.125 x 10(6) cells/mL for CD45 was obtained. Affinity tests showed no cross-reactivity between the antibodies to CD3 and CD45. The multiplex assay was validated against 8 coded specimens of known clinical status and linked to results from the TREC assay that had identified them. All were correctly identified by the CD345 assay.
  
  
  CONCLUSIONS
  The performance parameters of the CD345 assay met the performance characteristics generally accepted for immunoassays. Our assay classifications of positive specimens concur with previous TREC results. This CD345 assay warrants evaluation as a viable alternative or complement to the TREC assay as a primary screening tool for detecting T-cell immunodeficiencies, including SCID, in Guthrie specimens.
authors.affiliation: Biggs Laboratory, Wadsworth Center, Department of Health, NYS, Albany, NY 12201-0509, USA.
authors.forename: David K
authors.initials: DK
authors.lastname: Janik
authors.forename: Barbara
authors.initials: B
authors.lastname: Lindau-Shepard
authors.forename: Anne Marie
authors.initials: AM
authors.lastname: Comeau
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Pass
mesh_terms: D001800:Blood Specimen Collection; D017252:CD3 Complex / Q000097:blood; D002138:Calibration; D005240:Feasibility Studies; D006801:Humans; D007118:Immunoassay; D007153:Immunologic Deficiency Syndromes / Q000097:blood / Q000175:diagnosis*; D007230:Infant, Low Birth Weight; D007231:Infant, Newborn; D017493:Leukocyte Common Antigens / Q000097:blood; D007962:Leukocytes / Q000473:pathology; D016511:Severe Combined Immunodeficiency / Q000097:blood / Q000175:diagnosis; D013601:T-Lymphocytes / Q000473:pathology*
chemical_list: D017252:CD3 Complex; D017493:Leukocyte Common Antigens