Introduction

Single antigen bead testing (SAB) for HLA-specific antibody enables efficient organ allocation and aids in the diagnosis of antibody mediated rejection. In this retrospective cohort study, a population of kidney transplant recipients possessing HLA Class I antibodies was used to evaluate the best method for resolving complement interference, the so called “prozone” effect. The aim was to compare the use of EDTA versus a Biotin-Streptavidin Complex as methodological approaches for abating the prozone effect using a fixed 1 in 10 dilution as validation.

Methods

One hundred and seventeen patients transplanted in our centre between 2009 and 2014 were identified as having class I HLA-specific antibody(−ies) using a Labscreen® Mixed assay. Positive sera underwent class I HLA-specific SAB testing; for comparison a standard SAB with and without EDTA, BSC and dilution (1 in 10) modifications were utilised. Samples were processed on the Luminex platform generating 11,349 bead reactions for analysis.

Results

We identified sera from 23 patients giving rise to 170 bead reactions showing complement interference. Using linear modelling, we observed slightly higher MFIs on average in both EDTA and BSC modifications when compared to the standard assay, allowing the nominal threshold MFI of 2000 in the standard assay to be adjusted to 2097 and 2033 in the EDTA and BSC assays respectively. We calculated 99% prediction intervals to establish outlier bead reactions for each assay. The 1 in 10 dilution was used as a crosscheck for determining which prozone reactions were overcome by EDTA and BSC. Using ROC curve analysis, EDTA was found to be ~90% sensitive and 100% specific compared to BSC which was ~60% sensitive and 100% specific in ameliorating prozone positive reactions at the thresholds defined by linear models.

Discussion

Our data indicates that both EDTA and BSC are suitable assays in overcoming CMI. We recommend that all clinical laboratories adopt a validated assay designed specifically to abrogate CMI for all potential renal transplant recipients, as the standard assay is inhibited in nearly 20% of a post-transplant cohort.

中文翻译：

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肾移植后调查I类HLA特异性抗体的补体介导干扰。

介绍

针对HLA特异性抗体的单抗原珠测试（SAB）可实现有效的器官分配，并有助于诊断抗体介导的排斥反应。在这项回顾性队列研究中，使用一群拥有HLA I类抗体的肾移植受者评估了解决补体干扰的最佳方法，即所谓的“ prozone”效应。目的是比较使用EDTA与生物素-链霉亲和素复合物作为方法方法的效果，以固定的十分之一的稀释度作为验证方法，以减轻前带效应。

方法

2009年至2014年间，在我们的中心移植的一百一十七名患者通过Labscreen®Mixed assay被鉴定为具有I类HLA特异性抗体。阳性血清经过I类HLA特异性SAB测试；为了进行比较，使用具有和不具有EDTA，BSC和稀释度（十分之一）的标准SAB。在Luminex平台上处理样品，产生11,349个珠反应，以进行分析。

结果

我们从23例患者中鉴定出血清，这些血清引起170例珠粒反应，显示出补体干扰。使用线性建模，我们观察到与标准测定法相比，EDTA和BSC修饰的平均MFI均稍高，从而将标准测定法中2000的标称阈值MFI分别调整为EDTA和BSC测定法中的2097和2033。我们计算了99％的预测间隔，以建立每种测定的异常珠反应。十分之一的稀释度用作交叉检查，以确定EDTA和BSC克服了哪些前区反应。使用ROC曲线分析，发现EDTA与BSC相比具有〜90％的敏感性和100％的特异性，而BSC在线性模型定义的阈值下改善前区阳性反应时具有〜60％的敏感性和100％的特异性。

讨论区

我们的数据表明，EDTA和BSC都是克服CMI的合适方法。我们建议所有临床实验室均采用经过验证的分析方法，该方法专为所有潜在的肾脏移植接受者取消CMI而设计，因为标准分析法在移植后队列中几乎有20％被抑制。