title: Frequency, interobserver reproducibility and clinical significance of equivocal peaks in PCR clonality testing using Euroclonality/BIOMED-2 primers.
abstract: OBJECTIVE
  PCR studies for lymphoid clonality are now widely employed, especially using Euroclonality/BIOMED-2 primers. Criteria for interpretation as a clonal result, however, have proven controversial. This study examines the frequency and clinical significance of equivocal amplification patterns and measures the interobserver reproducibility of clonality interpretations.
  
  
  METHODS
  At our institution, results of each primer set are first classified as clonal, non-clonal or abnormal (equivocal peak on polyclonal background). Final results for all primer sets are then collectively reported as positive (≥1 clonal result), negative (non-clonal results) or indeterminate (≥1 abnormal result) for a clonal population. Results of 274 consecutive clonality cases were reviewed, and the interobserver reproducibility of individual primer set reactions and final results was determined in a subset of 30 cases.
  
  
  RESULTS
  44/161 (27%) B-cell and 50/163 (31%) T-cell cases contained at least one abnormal peak. Of these, 29 (64%) and 31 (62%), respectively, showed clonal results in another primer set. Interobserver reproducibility was excellent for most primer sets and for final interpretations, but only fair to good for IGK V-J and TCRB D-J1+2 primer sets. A definitive diagnosis of lymphoma was rendered in 93%, 20% and 6% of B-cell cases and 90%, 42%, and 14% of T-cell cases positive, indeterminate or negative for a clonal population, respectively.
  
  
  CONCLUSIONS
  Using a subjective approach, abnormal (equivocal) peaks are frequently observed in routine practice. However, most cases with abnormal peaks contain clonal rearrangements in other primer sets, facilitating overall interpretation of final results with excellent interobserver reproducibility.
authors.affiliation: Department of Laboratory Medicine, Ajou University School of Medicine, Suwon, Republic of Korea.
authors.forename: Il Joong
authors.identifier: http://orcid.org/0000-0003-2419-8165
authors.initials: IJ
authors.lastname: Park
authors.affiliation: Department of Quantitative Health Sciences, Cleveland Clinic, Cleveland, Ohio, USA.
authors.forename: James
authors.initials: J
authors.lastname: Bena
authors.affiliation: Department of Laboratory Medicine, Cleveland Clinic, Cleveland, Ohio, USA.
authors.forename: Claudiu V
authors.initials: CV
authors.lastname: Cotta
authors.affiliation: Department of Laboratory Medicine, Cleveland Clinic, Cleveland, Ohio, USA.
authors.forename: Eric D
authors.initials: ED
authors.lastname: Hsi
authors.affiliation: Department of Laboratory Medicine, Cleveland Clinic, Cleveland, Ohio, USA.
authors.forename: Armin G
authors.initials: AG
authors.lastname: Jegalian
authors.affiliation: Department of Laboratory Medicine, Cleveland Clinic, Cleveland, Ohio, USA.
authors.forename: Heesun J
authors.initials: HJ
authors.lastname: Rogers
authors.affiliation: Department of Laboratory Medicine, Cleveland Clinic, Cleveland, Ohio, USA.
authors.forename: Raymond R
authors.initials: RR
authors.lastname: Tubbs
authors.affiliation: Department of Quantitative Health Sciences, Cleveland Clinic, Cleveland, Ohio, USA.
authors.forename: Lu
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Laboratory Medicine, Cleveland Clinic, Cleveland, Ohio, USA.
authors.forename: James R
authors.initials: JR
authors.lastname: Cook
mesh_terms: D002999:Clone Cells; D017931:DNA Primers; D006801:Humans; D008223:Lymphoma / Q000175:diagnosis*; D008495:Medical Oncology / Q000379:methods / Q000592:standards*; D015588:Observer Variation; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers
keywords: HEMATOPATHOLOGY; LYMPHOMA; MOLECULAR PATHOLOGY; PCR