A new sensitive and specific combination of CD81/CD56/CD45 monoclonal antibodies for detecting circulating neuroblastoma cells in peripheral blood using flow cytometry

Purpose: Intensive chemoradiotherapy followed by peripheral blood stem cell transplantation has been introduced to treat children with advanced neurob lastoma (NBL). Detection of NBL cells in peripheral blood (PB) is important to prevent reinfusion of NBL cells. Several immunologic methods have been proposed for detecting NBL cells in hematologic samples. The development of a sensitive and specific combination of monoclonal antibodies (MoAbs) for detecting small numbers of NBL cells in PB using flow cytometry remains an important challenge. Methods: Twenty-one clinical samples from NBL tissues or smears containing NBL cells were examined for reactivity against CD81, CD56, and CD9 using an immunocytochemical technique. The expressions of CD81, CD56, CD9, and anti human disialoganglioside GD2 MoAb (GD2) in five NBL cell lines were assayed by flow cytometry. For the evaluation of sensitivity, five NBL cell lines were added to normal PB and the detection level of the combination of CD81/ CD56/CD45 MoAbs was compared with that of CD9/CD56/CD45 MoAbs (reported pre viously). One hundred thirty-three normal PB samples were examined to deter mine the sensitivity and specificity of this method. Results: All NBL cell lines showed strong positivity with CD81 and CD56 MoA b. However, CD9 MoAb was weakly positive against the five NBL cell Lines. G D2 MoAb reacted strongly with four NBL cell lines, although almost the enti re cell population of the SK-N-SH NBL line failed to bind the GD2 MoAb. in vitro experiments using NBL cell lines demonstrated that tumor cells added to normal PB cells could be detected by flow cytometry using CD81/CD56/CD45 MoAbs even at a concentration of 0.005%. Through comparative studies, the combination of CD81/CD56/CD45 MoAbs was found to be more sensitive and spec ific than that of CD9/CD56/CD45 MoAbs for detecting small numbers of NBL ce lls using the above cell lines. Conclusions: Triple-color flow cytometric analysis using CD81/CD56/CD45 MoA bs is useful for detecting NBL cells in PB, Further studies testing this ap proach using samples of PB with NBL contamination are needed to test this a pproach in patients.