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Standardization of the Immunocytochemical Detection of Neuroblastoma Cells in Bone Marrow

Katrien Swerts, Peter F. Ambros, Chantal Brouzes, José M. Fernandez Navarro, Nicole Gross, Dyanne Rampling, Roswitha Schumacher-Kuckelkorn, Angela R. Sementa, Ruth Ladenstein and Klaus Beiske

Department of Pediatric Hematology and Oncology, Ghent University Hospital, Ghent, Belgium (KS); Children's Cancer Research Institute, St. Anna Children's Hospital, Vienna, Austria (PFA,RL); Laboratory of Hematology, Institut Gustave Roussy, Villejuif, France (CB); Unidad de Oncología Pediátrica, Hospital Infantil La Fe, Valencia, Spain (JMFN); Pediatric Oncology Research, Pediatrics, University Hospital CHUV, Lausanne, Switzerland (NG); Histopathology Department, Great Ormond Street Hospital for Children, London, United Kingdom (DR); University of Cologne, Universitätskinderklinik, Cologne, Germany (RS-K); Servizio di Anatomia Patologica, Istituto G. Gaslini, Genoa, Italy (ARS); and Department of Pathology, Rikshospitalet, Oslo, Norway (KB)

Correspondence to: Katrien Swerts, Lab of Hematology (1P8 West), Ghent University Hospital, De Pintelaan 185, B-9000 Gent, Belgium. E-mail: Katrien.Swerts{at}UGent.be

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 10⁶ cells were analyzed to reach a 95% probability of detecting one tumor cell in 1 x 10⁶ 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 10⁶. 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.

(J Histochem Cytochem 53:1433–1440, 2005)

Key Words: immunocytochemistry • minimal residual disease • neuroblastoma • bone marrow

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