Yi Yan, Zhang Guang-sen
Department of Hematology, Institute of Molecular Hematology, Second Xiangya Hospital, Central South University, Changsha 410011, China.
Zhonghua Yi Xue Za Zhi. 2003 Aug 10;83(15):1313-6.
To observe the localization of inclusion and expression of nonmuscle myosin heavy chain-A (NMMHC-A) in cytoplasm of neutrophils of May-Hegglin anomaly (MHA) patients, and elucidate and identify the property of the inclusions in constitutional elements.
Peripheral blood was drawn from the MHA proband, the proband's father, and a healthy control. White blood cells and platelets were isolated and smeared. Indirect immunofluorescence technique combined with propidium iodide (PI) nuclei staining technology was used to detect the inclusion and nonmuscle myosin in cytoplasm of neutrophils and platelet. Neutrophils were isolated. Protein in the neutrophils was extracted and underwent Western blot assay to examine the expression of NMMHC-A.
Spindle-like inclusions with yellow fluorescence were clearly displayed in the neutrophils of the MHA patient and her father, that matched very well in shape, size and localization with the inclusions, revealed by Wright-Giemsa's stain. In normal control, except a diffusive distribution of fluorescent spot in neutrophils cytoplasm, not any inclusion was detected. As for NMMHC-A expression, Western blot assay showed that NMMHC-A was upregulated in the neutrophils of the MHA patient (60.9) and her father (58.9).
A new method to display MHA inclusions and identify the major component of inclusions in the neutrophils, which was originated from a mutant of nonmuscle myosin, of MHA was set up. Immunofluorescence analysis is more sensitive than Wright-Giemsa's staining in detecting inclusions of MHA.
观察May-Hegglin异常（MHA）患者中性粒细胞胞质中包涵体的定位及非肌肉肌球蛋白重链A（NMMHC-A）的表达，阐明并鉴定构成成分中包涵体的性质。
采集MHA先证者、先证者父亲及健康对照者的外周血。分离白细胞和血小板并涂片。采用间接免疫荧光技术结合碘化丙啶（PI）核染色技术检测中性粒细胞和血小板胞质中的包涵体及非肌肉肌球蛋白。分离中性粒细胞。提取中性粒细胞中的蛋白质，进行蛋白质印迹分析以检测NMMHC-A的表达。
MHA患者及其父亲的中性粒细胞中清晰显示出呈黄色荧光的纺锤样包涵体，其形状、大小和定位与瑞氏-吉姆萨染色显示的包涵体非常匹配。在正常对照中，除中性粒细胞胞质中有弥散分布的荧光点外，未检测到任何包涵体。关于NMMHC-A的表达，蛋白质印迹分析显示MHA患者（60.9）及其父亲（58.9）的中性粒细胞中NMMHC-A上调。
建立了一种显示MHA包涵体并鉴定MHA中性粒细胞中包涵体主要成分的新方法，该成分源自非肌肉肌球蛋白的突变体。免疫荧光分析在检测MHA包涵体方面比瑞氏-吉姆萨染色更敏感。
