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A Human Erythrocyte-based Haemolysis Assay for the Evaluation of Human Complement Activity
Alternatives to Laboratory Animals ( IF 2.4 ) Pub Date : 2020-05-01 , DOI: 10.1177/0261192920953170
Ruby Anne N. King 1 , Fresthel Monica M. Climacosa 1, 2 , Bobbie Marie M. Santos 1, 3 , Salvador Eugenio C. Caoili 1
Affiliation  

The complement system consists of at least 50 proteins that serve as one of the first lines of defence against foreign, or damaged, cells and invading microorganisms. Its dysregulation underlies the pathophysiology of many different diseases, which makes functional assays of complement activity crucial; they are, however, underutilised. Standard haemolysis assays for the analysis of complement function employ sensitised non-human erythrocytes (e.g. from the sheep, guinea-pig or rabbit), the use of which raises animal welfare concerns. To provide an alternative to the use of such animal-derived products for complement function assays, we developed a method that employs modified human erythrocytes to evaluate the activity of complement pathways. Human erythrocytes were subjected to various chemical and/or proteolytic treatments involving 2,4,6-trinitrobenzene sulphonate (TNBS) and pancreatin. Haemolysis assays demonstrated that sequential treatment with TNBS and pancreatin resulted in significantly greater complement-mediated haemolysis, as compared to TNBS or pancreatin treatment alone. Evidence that lysis of the modified erythrocytes was complement-mediated was provided by the chelation and subsequent restoration of calcium in the plasma. Thus, such modified human erythrocytes could be used as an alternative to animal-derived erythrocytes in haemolysis assays, in order to evaluate complement activity in human plasma during, for example, the screening of patients for complement deficiencies and other abnormalities in a clinical setting.

中文翻译:

用于评估人类补体活性的基于人类红细胞的溶血测定

补体系统由至少 50 种蛋白质组成,它们是抵御外来或受损细胞和入侵微生物的第一道防线。它的失调是许多不同疾病病理生理学的基础,这使得补体活性的功能测定变得至关重要;然而,它们没有得到充分利用。用于分析补体功能的标准溶血试验使用致敏的非人类红细胞(例如来自绵羊、豚鼠或兔),其使用会引起动物福利问题。为了提供一种替代使用此类动物源性产品进行补体功能测定的方法,我们开发了一种方法,该方法采用改良的人类红细胞来评估补体途径的活性。人类红细胞经过各种化学和/或蛋白水解处理,包括 2,4、6-三硝基苯磺酸盐 (TNBS) 和胰酶。溶血试验表明,与单独的 TNBS 或胰酶治疗相比,用 TNBS 和胰酶进行序贯治疗导致补体介导的溶血显着增加。血浆中钙的螯合和随后的恢复提供了修饰红细胞裂解是补体介导的证据。因此,此类修饰的人红细胞可在溶血试验中用作动物来源红细胞的替代物,以评估人血浆中的补体活性,例如在临床环境中筛查患者的补体缺乏和其他异常情况。溶血试验表明,与单独的 TNBS 或胰酶治疗相比,用 TNBS 和胰酶进行序贯治疗导致补体介导的溶血显着增加。血浆中钙的螯合和随后的恢复提供了修饰红细胞裂解是补体介导的证据。因此,这种修饰的人红细胞可在溶血试验中用作动物源性红细胞的替代物,以评估人血浆中的补体活性,例如在临床环境中筛查患者的补体缺乏和其他异常。溶血试验表明,与单独的 TNBS 或胰酶治疗相比,用 TNBS 和胰酶进行序贯治疗导致补体介导的溶血显着增加。血浆中钙的螯合和随后的恢复提供了修饰红细胞裂解是补体介导的证据。因此,此类修饰的人红细胞可在溶血试验中用作动物来源红细胞的替代物,以评估人血浆中的补体活性,例如在临床环境中筛查患者的补体缺乏和其他异常情况。血浆中钙的螯合和随后的恢复提供了修饰红细胞裂解是补体介导的证据。因此,此类修饰的人红细胞可在溶血试验中用作动物来源红细胞的替代物,以评估人血浆中的补体活性,例如在临床环境中筛查患者的补体缺乏和其他异常情况。血浆中钙的螯合和随后的恢复提供了修饰红细胞裂解是补体介导的证据。因此,此类修饰的人红细胞可在溶血试验中用作动物来源红细胞的替代物,以评估人血浆中的补体活性,例如在临床环境中对患者进行补体缺乏和其他异常的筛查。
更新日期:2020-05-01
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