Malin M J, Hwang D R, Ben-David D
Anal Biochem. 1986 Jul;156(1):1-10. doi: 10.1016/0003-2697(86)90145-4.
Within the past year, it has become apparent, in connection with its use on automatic flow cytometers, that the quality of commercially available Alcian Blue has significantly declined. A homologous series of alkylated (C1-C7) Astra Blue quaternary ammonium halides was prepared, characterized, and evaluated for the detection of basophils in whole blood. On the Technicon H6000 flow cytometer, the resolution of the basophil cluster from the main population of unstained white blood cells was found to depend on the chain length of the quaternizing alkyl group. Optimal basophil resolution was observed for the n-propyl derivative. Correlation of the new method vs Alcian Blue as the reference on the H6000 was expressed as follows: %Baso (Astra Blue) = 0.89% Baso (Alcian Blue) + 0.12% for 180 fresh whole blood samples. Within-run precision at a basophil differential count of 0.73% was characterized by SD = 0.11, identical to that obtained for Alcian Blue. Aqueous solutions of n-propyl Astra Blue iodide, in contrast to Alcian Blue, are thermally stable. Heating the reagent for 1 h at 100 degrees C did not alter solubility or cytochemical behavior. In contrast, parallel treatment of Alcian Blue yielded insoluble material by hydrolysis of the isothiouronium groups. The reagent for basophil detection comprises n-propyl Astra Blue iodide, lanthanum chloride, sodium chloride, Tween 20, and cetylpyridinium chloride. The Astra Blue derivatives were characterized by uv-vis, ir, percentage halide, paper chromatography, and 13C NMR.
在过去一年里，与在自动流式细胞仪上的使用相关，市售阿尔新蓝的质量显著下降已变得明显。制备了一系列烷基化（C1 - C7）阿斯特拉蓝季铵卤化物的同系物，对其进行了表征，并评估了其在全血中检测嗜碱性粒细胞的性能。在Technicon H6000流式细胞仪上，发现嗜碱性粒细胞簇与未染色白细胞主要群体的分辨率取决于季铵化烷基的链长。对于正丙基衍生物观察到了最佳的嗜碱性粒细胞分辨率。在H6000上，新方法与以阿尔新蓝为参比的相关性表示如下：对于180份新鲜全血样本，阿斯特拉蓝嗜碱性粒细胞百分比（%Baso）= 0.89×阿尔新蓝嗜碱性粒细胞百分比（%Baso）+ 0.12%。在嗜碱性粒细胞分类计数为0.73%时，批内精密度的特征为标准差（SD）= 0.11，与阿尔新蓝获得的结果相同。与阿尔新蓝相反，正丙基阿斯特拉蓝碘化物的水溶液具有热稳定性。将该试剂在100℃加热1小时不会改变其溶解度或细胞化学行为。相比之下，对阿尔新蓝进行平行处理会因异硫脲鎓基团的水解产生不溶性物质。用于检测嗜碱性粒细胞的试剂包含正丙基阿斯特拉蓝碘化物、氯化镧、氯化钠、吐温20和十六烷基氯化吡啶。阿斯特拉蓝衍生物通过紫外 - 可见光谱、红外光谱、卤化物百分比、纸色谱法和13C核磁共振进行表征。
