流式细胞术(FC)分析红细胞形态及相关生物力学特性,比如渗透脆弱性,还没有从研究工具转向常规的临床试验。主要原因是现有证据表明,各种预分析因素会影响对所获得数据的数学解释。为了促进FC在人类红细胞形状评估中的标准化和拓宽其用途,将从健康供体中分离的新鲜制备的外周血红细胞在等渗和低渗溶液中孵育(纯盐水,含钾和钙的盐水,和磷酸盐缓冲盐水),并通过FC使用前向散射(FSC)和侧向散射(SSC)的值进行检查。峰度,偏斜度,皮尔逊不对称第二偏度系数(PCD),和球形指数,根据FSC分布计算,用于红细胞形状评价。在所有等渗介质中,FSC分布和基于FSC的形态参数显示出巨大的个体间和介质间变化。随着渗透压降低,在所有介质和样品中,红细胞的大小增加,肿胀指数和峰度下降。然而,偏度和PCD的变化受所用培养基和测试样品的影响。与FSC相比,等渗的SSC信号及其在低渗介质中的变化显示出较低的个体间差异,并且不受介质类型的影响。我们提出球形指数和峰度作为基于FSC的红细胞形状指标。由于对预分析处理的影响更具抵抗力,在评估红细胞形状时,SSC数据似乎被不公平地忽略了,与通常采用的FSC数据相比。
Flow cytometry (FC) analysis of erythrocyte shape and related biomechanical properties, such as osmotic fragility, have not moved from a research tool to regular clinical testing. The main reason is existing evidence that various pre-analytical factors influence the mathematical interpretation of the data obtained. With an aim to contribute to the standardization and broaden the use of FC for human erythrocyte shape assessment, freshly prepared peripheral blood erythrocytes isolated from healthy donors were incubated in iso and hypo-osmotic solutions (pure saline, saline with potassium and calcium, and phosphate buffered saline) and examined by FC using values of forward scatter (FSC) and side scatter (SSC). Kurtosis, skewness, Pearson\'s second skewness coefficient of dissymmetry (PCD), and spherical index, calculated from FSC distributions, were used for the erythrocyte shape evaluation. In all isotonic media FSC distribution and FSC-based morphology parameters showed huge inter-individual and inter-medium variation. With decreasing osmolality, in all media and samples, the size of the erythrocytes increased, and swelling index and kurtosis decreased. However, changes in skewness and PCD were influenced by the medium used and the sample tested. Compared to FSC, SSC signal in isotonic and its change in hypotonic media showed lower inter-individual variation and was not influenced by the type of medium. We propose a spherical index and kurtosis as FSC-based indicators of erythrocyte shape. As more resistant to the influence of the preanalytical treatment, SSC data appeared to be unfairly neglected for the assessment of erythrocyte shape, in comparison to the usually employed FSC data.