Loyson S A, Rademakers L H, Joling P, Vroom T M, van den Tweel J G
Department of Pathology, University Hospital Utrecht, The Netherlands.
Histopathology. 1997 Nov;31(5):412-9. doi: 10.1046/j.1365-2559.1997.2980886.x.
To identify the stromal structures and haematopoietic cell lineages in normal bone marrow. The optimal conditions were studied for the reactivity of a panel of antibodies, applicable to paraffin sections of decalcified trephine biopsies using antigen retrieval methods.
Two methods of antigen retrieval (pepsin and acid citrate buffer) were tested. For the demonstration of most antigens and for reduction of background staining, heating in acid citrate buffer was preferred. In the case of elastase and von Willebrand Factor (factor VIIIrAg) pepsin pre-treatment was optimal, whereas Ulex europaeus agglutinin (UEA-1) and alpha-smooth muscle actin (alpha-SMA) required no pretreatment. Staining patterns obtained after 48 h EDTA decalcification and short electrolytic decalcification were identical. Both methods allowed recognition of HLA-A and HLA-B antigens, isolated CD34+ cells, mono-histiocytic cells (CD68+), myeloid cells (elastase and myeloperoxidase), erythroid cells (glycophorin C) and of megakaryocytic cells (Factor VIIIrAg). A relative simple lymphocyte-subset analysis was possible in decalcified paraffin sections allowing recognition of B-cells (CD20+) and T-cells (CD3+ and CD45RO+) in frequencies comparable to frozen sections. Suitable stromal cell staining was achieved by vimentin and desmin antibodies, whereas the bone marrow capillary network was visualized by CD34, factor VIIIrAg and UEA-1.
This immunohistochemical study indicates that all cellular components of the haematopoietic microenvironment of the bone marrow can be identified in decalcified paraffin sections using antigen retrieval methods and that the time of decalcification can be reduced to 1-1.5 h.
识别正常骨髓中的基质结构和造血细胞谱系。研究了一组抗体的反应性的最佳条件，这些抗体适用于使用抗原修复方法的脱钙环钻活检石蜡切片。
测试了两种抗原修复方法（胃蛋白酶和柠檬酸缓冲液）。对于大多数抗原的显示和背景染色的减少，柠檬酸缓冲液加热是首选。对于弹性蛋白酶和血管性血友病因子（因子VIIIrAg），胃蛋白酶预处理是最佳的，而荆豆凝集素（UEA-1）和α-平滑肌肌动蛋白（α-SMA）不需要预处理。48小时乙二胺四乙酸脱钙和短时间电解脱钙后获得的染色模式相同。两种方法都能识别HLA-A和HLA-B抗原、分离的CD34+细胞、单核-组织细胞（CD68+）、髓细胞（弹性蛋白酶和髓过氧化物酶）、红系细胞（血型糖蛋白C）和巨核细胞（因子VIIIrAg）。在脱钙石蜡切片中可以进行相对简单的淋巴细胞亚群分析，能够识别B细胞（CD20+）和T细胞（CD3+和CD45RO+），其频率与冰冻切片相当。波形蛋白和结蛋白抗体实现了合适的基质细胞染色，而骨髓毛细血管网络通过CD34、因子VIIIrAg和UEA-1可视化。
这项免疫组织化学研究表明，使用抗原修复方法可以在脱钙石蜡切片中识别骨髓造血微环境的所有细胞成分，并且脱钙时间可以缩短至1-1.5小时。
