title: Standardization of the immunocytochemical detection of neuroblastoma cells in bone marrow.
abstract: Standard cytomorphological examination of bone marrow (BM) aspirates does not appear to be sensitive enough to detect single neuroblastoma cells. The SIOPEN Neuroblastoma Bone Marrow Committee developed a sensitive and reproducible anti-GD2 immunocytochemical assay and introduced morphological and immunocytological criteria for the interpretation of results. Fixed cytospins were incubated with a commercially available anti-GD2 monoclonal antibody and an APAAP kit. Cells fulfilling all morphological and immunocytological criteria were called criteria-positive cells (CPCs). Not convincingly interpretable cells fulfilled some, but not all, criteria, and negative cells displayed only exclusion criteria. The genetic profile of doubtful cells was checked by fluorescence in situ hybridization. Ideally, 3 x 10(6) cells were analyzed to reach a 95% probability of detecting one tumor cell in 1 x 10(6) mononuclear cells. Four quality control rounds were organized to validate the method. A total of 111 quality control samples were analyzed. Two main improvements were achieved: in discordant cases, the range between the lowest and highest reported result was reduced by half, and discordant results were only found in samples with less than 10 CPCs per 1 x 10(6). This article describes the first internationally standardized protocol to detect and quantify rare neuroblastoma cells by immunocytochemistry. This method is an indispensable tool for multicenter studies evaluating the clinical significance of minimal residual disease in neuroblastoma.
authors.affiliation: Department of Pediatric Hematology and Oncology, Ghent University Hospital, Belgium. Katrien.Swerts@UGent.be
authors.forename: Katrien
authors.initials: K
authors.lastname: Swerts
authors.forename: Peter F
authors.initials: PF
authors.lastname: Ambros
authors.forename: Chantal
authors.initials: C
authors.lastname: Brouzes
authors.forename: José M Fernandez
authors.initials: JM
authors.lastname: Navarro
authors.forename: Nicole
authors.initials: N
authors.lastname: Gross
authors.forename: Dyanne
authors.initials: D
authors.lastname: Rampling
authors.forename: Roswitha
authors.initials: R
authors.lastname: Schumacher-Kuckelkorn
authors.forename: Angela R
authors.initials: AR
authors.lastname: Sementa
authors.forename: Ruth
authors.initials: R
authors.lastname: Ladenstein
authors.forename: Klaus
authors.initials: K
authors.lastname: Beiske
mesh_terms: D001853:Bone Marrow / Q000473:pathology*; D001854:Bone Marrow Cells / Q000473:pathology; D002460:Cell Line; D005189:False Positive Reactions; D006801:Humans; D007150:Immunohistochemistry / Q000592:standards*; D017404:In Situ Hybridization, Fluorescence / Q000592:standards; D018365:Neoplasm, Residual; D009447:Neuroblastoma / Q000473:pathology*; D011786:Quality Control; D012680:Sensitivity and Specificity