Nagai J, Ishida Y, Koga N, Tanaka Y, Ohnuma K, Toyoda Y, Katoh A, Hayabuchi Y, Kigasawa H
Division of Laboratory, Kanagawa Children's Medical Center, Yokohama, Japan.
J Pediatr Hematol Oncol. 2000 Jan-Feb;22(1):20-6. doi: 10.1097/00043426-200001000-00004.
Intensive chemoradiotherapy followed by peripheral blood stem cell transplantation has been introduced to treat children with advanced neuroblastoma (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.
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 antihuman 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 previously). One hundred thirty-three normal PB samples were examined to determine the sensitivity and specificity of this method.
All NBL cell lines showed strong positivity with CD81 and CD56 MoAb. However, CD9 MoAb was weakly positive against the five NBL cell lines. GD2 MoAb reacted strongly with four NBL cell lines, although almost the entire 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 specific than that of CD9/CD56/CD45 MoAbs for detecting small numbers of NBL cells using the above cell lines.
Triple-color flow cytometric analysis using CD81/CD56/CD45 MoAbs is useful for detecting NBL cells in PB. Further studies testing this approach using samples of PB with NBL contamination are needed to test this approach in patients.
强化放化疗后行外周血干细胞移植已被用于治疗晚期神经母细胞瘤（NBL）患儿。检测外周血（PB）中的NBL细胞对于防止NBL细胞回输至关重要。已经提出了几种免疫方法来检测血液样本中的NBL细胞。开发一种灵敏且特异的单克隆抗体（MoAb）组合，用于通过流式细胞术检测PB中少量的NBL细胞，仍然是一项重大挑战。
使用免疫细胞化学技术检测来自NBL组织或含有NBL细胞涂片的21份临床样本对CD81、CD56和CD9的反应性。通过流式细胞术检测5种NBL细胞系中CD81、CD56、CD9和抗人双唾液酸神经节苷脂GD2单克隆抗体（GD2）的表达。为评估敏感性，将5种NBL细胞系加入正常PB中，并将CD81/CD56/CD45单克隆抗体组合的检测水平与CD9/CD56/CD45单克隆抗体组合（先前报道）的检测水平进行比较。检测133份正常PB样本以确定该方法的敏感性和特异性。
所有NBL细胞系对CD81和CD56单克隆抗体均呈强阳性。然而，CD9单克隆抗体对5种NBL细胞系呈弱阳性。GD2单克隆抗体与4种NBL细胞系强烈反应，尽管SK-N-SH NBL细胞系几乎全部细胞群体均未与GD2单克隆抗体结合。使用NBL细胞系的体外实验表明，加入正常PB细胞中的肿瘤细胞即使在浓度为0.005%时也可通过使用CD81/CD56/CD45单克隆抗体的流式细胞术检测到。通过比较研究发现，对于使用上述细胞系检测少量NBL细胞，CD81/CD56/CD45单克隆抗体组合比CD9/CD56/CD45单克隆抗体组合更灵敏且特异。
使用CD81/CD56/CD45单克隆抗体的三色流式细胞术分析可用于检测PB中的NBL细胞。需要使用受NBL污染的PB样本进一步研究该方法，以在患者中测试该方法。
