Wahrmann Markus, Exner Markus, Regele Heinz, Derfler Kurt, Körmöczi Günther F, Lhotta Karl, Zlabinger Gerhard J, Böhmig Georg A
Division of Nephrology and Dialysis, Department of Internal Medicine III, University of Vienna, Währinger Gürtel 18-20, A-1090, Vienna, Austria
J Immunol Methods. 2003 Apr 1;275(1-2):149-60. doi: 10.1016/s0022-1759(03)00012-7.
Complement-dependent cytotoxicity (CDC) panel reactive antibody (PRA) testing is used to assess recipient presensitization and post-transplant alloantibody formation in transplant recipients. However, CDC test results can be affected by false-positive reactions brought about by autoantibodies or antilymphocyte reagents. As an alternative to the CDC-PRA assay, detection of HLA alloantibodies using HLA antigen-coated microbeads (FlowPRA test) was recently established. FlowPRA testing, however, does not distinguish between (presumably more harmful) complement-fixing and noncomplement-fixing alloantibodies. In this study, we established a novel assay allowing flow cytometric detection of HLA alloantibody dependent classical complement activation using the FlowPRA test. For the detection of complement activation, FlowPRA beads were incubated with sera from highly sensitized dialysis patients (CDC-PRA reactivity >60%) and then stained for C4 (C4d, C4c) and C3 (C3d, C3c) fragments, as well as C1q deposition using indirect immunofluorescence. We demonstrate alloantibody induced induction of C4 fragment, and in parallel C1q deposition to HLA class I or class II beads. As shown by immunoblotting, C4 staining was not due to the presence of preformed C4 fragment-IgG/M complexes. Indeed, C4 fragment deposition in our in vitro system was demonstrated to result from de novo complement activation. First, inactivation of C4 by treatment of sera with methylamine, which inhibits cleavage of the internal thioester, completely abolished C4 fragment deposition. Second, C4 fragment deposition was not observed in the evaluation of C4-free immunoadsorption eluates obtained from highly sensitized dialysis patients. After supplementation with complement, however, eluates induced C4 deposition. Deposition of C4 split products and C1q was temperature-dependent with maximum binding after incubation at 4 degrees C for 60 min. In contrast, maximum C3 fragment deposition was found at 37 degrees C. At this temperature, C3 deposition occurred in an alloantibody and C4-independent fashion, presumably as a result of alternative complement activation. In summary, we describe a novel cell-independent and easy-to-perform PRA test that permits flow cytometry based detection of alloantibody induced classical complement activation. Future studies will have to evaluate its possible relevance as an alternative to CDC-PRA testing in clinical transplantation.
补体依赖细胞毒性（CDC）群体反应性抗体（PRA）检测用于评估移植受者的预致敏状态以及移植后同种异体抗体的形成。然而，CDC检测结果可能会受到自身抗体或抗淋巴细胞试剂导致的假阳性反应的影响。作为CDC - PRA检测的替代方法，最近建立了使用HLA抗原包被微珠检测HLA同种异体抗体的方法（FlowPRA检测）。然而，FlowPRA检测无法区分（可能更具危害性的）补体结合性和非补体结合性同种异体抗体。在本研究中，我们建立了一种新的检测方法，可利用FlowPRA检测通过流式细胞术检测HLA同种异体抗体依赖性经典补体激活。为了检测补体激活，将FlowPRA微珠与高度致敏透析患者的血清（CDC - PRA反应性>60%）孵育，然后使用间接免疫荧光法对C4（C4d、C4c）和C3（C3d、C3c）片段以及C1q沉积进行染色。我们证明了同种异体抗体可诱导C4片段的产生，同时C1q沉积于HLA I类或II类微珠。免疫印迹显示，C4染色并非由于预先形成的C4片段 - IgG/M复合物的存在。实际上，在我们的体外系统中，C4片段沉积是由补体的从头激活导致的。首先，用甲胺处理血清使C4失活，甲胺可抑制内部硫酯的裂解，这完全消除了C4片段的沉积。其次，在对从高度致敏透析患者获得的无C4免疫吸附洗脱液进行评估时，未观察到C4片段沉积。然而，补充补体后，洗脱液可诱导C4沉积。C4裂解产物和C1q的沉积具有温度依赖性，在4℃孵育60分钟后结合量最大。相比之下，C3片段的最大沉积在37℃时出现。在此温度下，C3沉积以同种异体抗体和C4非依赖性方式发生，可能是替代补体激活的结果。总之，我们描述了一种新的非细胞依赖且易于操作的PRA检测方法，该方法允许基于流式细胞术检测同种异体抗体诱导的经典补体激活。未来的研究将不得不评估其作为临床移植中CDC - PRA检测替代方法的潜在相关性。
