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本文内容

  • 摘要
  • 摘要
  • 引言
  • 研究方案
  • 结果
  • 讨论
  • 披露声明
  • 致谢
  • 材料
  • 参考文献
  • 转载和许可

摘要

嗜碱性粒细胞活化试验是一种补充性体 外 诊断试验，用于评估 IgE 介导的过敏反应，该试验基于通过流式细胞术测量活化标志物在存在特定刺激的情况下检测嗜碱性粒细胞活化。

摘要

嗜碱性粒细胞活化试验 （BAT） 是一种补充性体外诊断试验，除了临床病史、皮肤试验 （ST） 和特异性 IgE （sIgE） 测定 外 ，还可用于评估 IgE 介导的对食物、昆虫毒液、药物以及某些形式的慢性荨麻疹的过敏反应。然而，该技术在诊断算法中的作用是高度可变的，并且尚未很好地确定。

BAT基于通过流式细胞术测量活化标志物（如CD63，CD203c）来确定嗜碱性粒细胞对变应原/药物交联IgE活化的反应。该测试可以成为避免受控挑战测试以确认过敏诊断的有用和补充工具，尤其是在经历严重危及生命的反应的受试者中。一般来说，如果 i） 变应原/药物在 ST 中产生假阳性结果;ii） 没有用于 ST 或 sIgE 测定的过敏原/药物来源;iii） 患者病史与 ST 或 sIgE 测定之间存在不一致;iv）症状提示ST可能导致全身反应;v） 在考虑进行 CCT 以确认罪魁祸首过敏原/药物之前。该测试的主要局限性与非最佳灵敏度有关，特别是在药物过敏中，需要在样品提取后不超过24小时进行测试，以及实验室之间在程序，浓度和细胞标志物方面缺乏标准化。

引言

IgE 介导的过敏诊断基于临床病史、皮肤试验 （ST）、血清特异性 IgE （sIgE） 的定量，以及对照激发试验 （CCT）（如果需要和指征）1，2，3，4，5，6。然而，临床病史可能不可靠，因为可能缺乏准确的信息，ST 和 CCT 不是无风险的程序，在经历严重危及生命的反应的受试者中是禁忌的 1，2，3，4，5，6.这些问题，加上通过经过验证和商业的荧光酶免疫测定测定sIgE仅适用于少数过敏原和药物的事实，突出了其他体外功能测定（如嗜碱性粒细胞活化试验（BAT））的重要作用。

嗜碱性粒细胞是参与 IgE 介导的过敏反应的关键效应细胞，在过敏原/药物暴露后，在细胞表面高亲和力受体 （FcεRI） 上结合的相邻 sIgE 交联时被激活。嗜碱性粒细胞活化触发细胞脱颗粒和细胞胞浆内分泌颗粒中所含的预形成和新合成的炎症介质的释放7，8，9。BAT是一种体外方法，它试图在刺激（过敏原或药物）存在的情况下模拟嗜碱性粒细胞活化，并通过流式细胞术7，10确定嗜碱性粒细胞活化标志物表达的变化。有不同的策略来鉴定嗜碱性粒细胞（IgE+、CCR3+、CRTH2+、CD203c+）和使用荧光染料标记抗体的组合来测量细胞活化（主要是 CD63 和 CD203c 的上调7，10）。CD63是临床验证的最佳活化标志物11，12，13，14，是一种膜蛋白，锚定在含有组胺的分泌颗粒上，在细胞活化和颗粒与膜融合后，在嗜碱性粒细胞表面表达15，16，17，18，19，20，21.CD203c是一种表面标志物，在嗜碱性粒细胞上形成型表达，并在FcεRI刺激后上调，在BAT15，22，23，24，25中也显示出可靠的结果。此外，它似乎与CD6326共同表达。

在过去的几十年中，BAT已被证明可用于诊断由药物，食物或吸入剂等不同触发因素引起的IgE介导的过敏反应，以及某些形式的慢性荨麻疹，如下所述。然而，该技术在诊断算法中的位置变化很大，并且尚未很好地确定。

药物超敏反应
BAT已被证明可作为特定药物和患者的补充测试，特别是对于那些由于ST的诊断价值尚未很好地确定而出现严重反应的患者，因为它们对有限数量的药物进行了验证和标准化27，28，29，30。此外，sIgE 的定量仅适用于有限数量的药物，灵敏度低于 ST27，28，29，30，31，32。因此，药物超敏反应的诊断通常依赖于药物激发试验，这可能在经历严重危及生命的反应的受试者中禁忌33。

据报道，在选定的患者中使用BAT有希望的结果，这些患者报告对β内酰胺类（BLs）的即时超敏反应20，34，35，36，37，38，39，神经肌肉阻滞剂（NMBAs）19，22，40，41，42，43，44，45，氟喹诺酮类（FQ）46，47，48，49，吡唑啉酮类50，51，52，放射造影剂（RCM）53，54，55，56和铂化合物57，58，59.据报道，BAT的敏感性和特异性分别为51.7-66.9%和89.2-97.8%;阳性和阴性预测值分别在 93.4% 和 66.3% 之间27，31。此外，BAT已被提议作为铂化合物脱敏过程中突破性反应的预测生物标志物，因为在药物脱敏期间不良反应风险高的患者中，CD203c表达与CD63相比增加57。

值得注意的是，BAT仅在反应涉及嗜碱性粒细胞脱颗粒时才对药物超敏反应有用;因此，它在由酶促抑制环加氧酶 142引起的反应中没有用。

食物过敏
BAT已成为食物过敏的潜在诊断工具，因为测定血清sIgE对整个过敏原提取物或单一过敏原通常是模棱两可的，需要口服食物激发试验来确认诊断，这与药物超敏反应类似，是一种昂贵且并非无风险的过程60。多项研究显示了牛奶 61，62、鸡蛋 61，63、小麦64、65、66、67、68、花生 63、69、70、71、72、榛子73、74、75，76，77，贝类78，桃子79，80，81，苹果21，芹菜和胡萝卜82，83。

与血清中的STs和sIgE相比，BAT在食物过敏诊断中的主要附加值是它显示出更高的特异性和相似的敏感性。因此，BAT是区分临床过敏患者与具有高特异性（75-100%）和敏感性（77-98%）的致敏但耐受的受试者的有用工具63，69，84。敏感性和特异性值取决于变应原和其他因素，如表型（例如口腔过敏综合征与全身过敏反应）、年龄和地理相关的致敏模式63，85。

使用单一过敏原成分的BAT可以潜在地提高某些食物过敏原的诊断准确性61，80。有使用种子储存蛋白的研究（例如，来自花生的Ara h 1，Ara h 2，Ara h 3和Ara h 6）86;脂质转移蛋白（例如，来自桃子的Pru p 3和来自花生的Ara h 9）80，86;和 Bet v 1 同系物（例如，来自花生的 Ara h 8）87。其他潜在的效用与在花粉食物过敏综合征21，87，88，对红肉过敏89或食物依赖性运动诱导的过敏反应66的情况下识别罪魁祸首过敏原有关。

有趣的是，BAT可以提供有关过敏反应的严重程度和阈值的信息，因为反应更严重的患者表现出更大比例的活化嗜碱性粒细胞，如花生和牛奶过敏患者的研究中观察到的那样84，90，91;对微量过敏原有反应的患者表现出更大的嗜碱性粒细胞敏感性84，90，92。这些数据表明，BAT可能有助于识别需要更密切随访和更强化教育的高风险过敏患者93。此外，据报道，BAT可以预测食物挑战反应70，91，92，94和反应性阈值90，95，以帮助确定何时可以安全地（重新）引入食物84。然而，这些发现在一些研究中存在争议63，96，需要更多的研究。

另一方面，随着时间的推移，BAT已被用于监测食物过敏的缓解情况，无论是自然的还是在免疫调节治疗下的，到目前为止，仅通过口服食物激发进行评估，具有相关的风险和成本84，97，98，99，100，101，102，103，104，105，106，107，108。此外，它还被用于监测奥马珠单抗在食物过敏中的作用，因为在奥马珠单抗治疗期间嗜碱性粒细胞活化降低，但在停止治疗后增加109。

吸入剂过敏
BAT很少对吸入剂过敏有益，因为可以通过sIgE定量和ST常规确定诊断。然而，在局部过敏性鼻炎病例（sIgE 水平检测不到和鼻激发试验阳性的 ST 阴性）病例中，BAT 允许诊断 50% 的病例110.进一步报道了嗜碱性粒细胞敏感性与鼻/支气管激发试验反应之间的相关性，以及哮喘严重程度与奥马珠单抗 111，112 治疗效果之间的相关性。

BAT也被用于监测屋尘螨和花粉的变应原免疫治疗，因为在免疫治疗期间嗜碱性粒细胞敏感性降低，可能是由于阻断IgG抗体113，114，115，116，117的干扰。

膜翅目毒液过敏
膜翅目毒液过敏的诊断通常基于 ST 和血清 sIgE。BAT显示出高敏感性（85-100%）和特异性（83-100%），据报道，对于结果模棱两可的病例或有提示性毒液过敏临床病史但sIgE检测不到且ST 118，119阴性的患者，BAT是有用的。然而，BAT似乎不能预测这些反应的严重程度120，121。

高达 60% 的患者对黄蜂和蜂毒均表现出 sIgE，鉴定显性变应原对于充分的免疫治疗至关重要。据报道，在这些情况下，BAT可用于鉴定主要过敏原119，122，123，124。尽管对蜜蜂和黄蜂毒液的主要过敏原的sIgE可能会降低BAT在两种毒液双重阳性患者中的效用，但它主要在sIgE测定结果为阴性的受试者中提供有用的信息123。

一些研究表明，在毒液免疫治疗的积累阶段，BAT可能可作为副作用的预测生物标志物，因为据报道这种治疗选择会降低嗜碱性粒细胞的敏感性。然而，反应性并没有降低，这个BAT实用程序现在有争议的是13，120，125，126，127，128，129，130。

荨麻疹和血管性水肿
一部分慢性荨麻疹患者具有自身免疫病理生理学，这是由于针对自身过敏原的 IgE 自身抗体和靶向肥大细胞表面存在的 FcεRI 或 IgE-FcεRI 复合物的 IgG 自身抗体 131，132。在临床实践中，这种类型的慢性荨麻疹的诊断依赖于自体血清ST阳性，该血清ST具有意外感染的风险。BAT已被提议作为诊断和监测疑似慢性荨麻疹患者的体外检查。据报道，在慢性荨麻疹患者的血清刺激后，嗜碱性粒细胞表面的CD63和CD203c表达均增加，显示检测到活性自身抗体133，134，135，136，137。最近，据报道，与BAT138阴性的患者相比，BAT阳性患者通常经历最活跃的疾病状态，通过荨麻疹活动评分评估，并且需要更高剂量的抗组胺药联合三线治疗（环孢素A或奥马珠单抗）。

研究方案

协议执行是根据赫尔辛基原则宣言进行的，并得到当地道德委员会（西班牙马拉加省调查委员会）的批准。所有受试者都口头告知了这项研究，并签署了相应的知情同意书。

注意:本协议详细介绍了作者每天使用的BAT程序。然而，这不是一种标准化的方法，并且与其他作者发表的程序存在差异。主要的方案修改与刺激缓冲液中IL-3的使用，刺激物的孵育时间，停止嗜碱性粒细胞脱颗粒的方法以及流式细胞术策略有关。此外，不同的市售BAT试剂盒包括制造商推荐的特定方案。

1. 样品制备

  1. 在 9 mL 肝素化管中收集外周血，并将样品保持在室温 （RT） 在转子中，直到实验方案需要为止。
  2. 标记 5 mL 细胞仪管，用于阴性对照（2 管）、阳性对照（2 管）和不同浓度的过敏原/药物（每个测试的过敏原/药物浓度 1 个管）。将试管放在试管完美贴合而不会滑落的架子上。
  3. 在含有 2% （v/v） HEPES、78 mg/L NaCl、3.7 mg/L KCl、7.8 mg/L CaCl 2、3.3 mg/L MgCl2、1 g/L HSA 的双蒸水中制备刺激缓冲液。将 pH 值调节至 7.4，并以 2 ng/mL 的浓度添加 IL-3。通常准备100mL并分成2.5mL等分试样在-20°C冷冻。
  4. 在PBS-吐温-20中准备阳性对照0.05%（v / v）（PBS-T）:阳性对照1，N-甲酰甲硫酰基亮酰基苯丙氨酸（fMLP）（4μM），以确认嗜碱性粒细胞的质量;阳性对照2，抗IgE（0.05mg / mL）作为IgE介导的阳性对照。
  5. 在PBS-T中以所需最终浓度的2倍制备过敏原/药物。
    注意:必须事先通过使用各种浓度，剂量反应曲线以及遵循相同方案步骤139的细胞毒性研究来确定要使用的最佳过敏原/药物浓度。

2. 染色混合物制备

  1. 按照制造商推荐的抗体浓度或先前的抗体滴定，将用荧光染料标记的单克隆抗体添加到刺激缓冲液中。在该协议中，我们添加 1 μL 每种抗体（CCR3-APC 和 CD203c-PE 用于嗜碱性粒细胞鉴定;CD63-FITC用于嗜碱性粒细胞活化）每20μL刺激缓冲液140 。
    注意:保护染色混合物制剂免受光照。
  2. 向每个试管中加入 23 μL 染色混合物。

3. 血液刺激

  1. 向试管 1 和 2（阴性对照）中加入 100 μL PBS-T，向试管 3 中加入 100 μL fMLP，向试管 4 中加入 100 μL 抗 IgE，向以下试管中加入 100 μL 不同过敏原/药物浓度。在恒温浴中在37°C下孵育10分钟，培养基搅拌以预热试剂。
  2. 向每个试管中轻轻添加 100 μL 血液以避免溶血。轻轻涡旋管并在37°C下在恒温浴中孵育25分钟，培养基搅拌。
  3. 停止脱颗粒，将管保持在4°C至少5分钟。
    注意:如果需要，可以在4°C下暂停协议30-45分钟141，142，143。

4.红细胞裂解

  1. 向每个试管中加入 2 mL 的 1x 裂解缓冲液以裂解红细胞。涡旋每个管并在室温下孵育5分钟。
    注意:在此步骤中，由于缓冲液中含有固定剂（甲醛），细胞被固定。
  2. 在4°C下以300× g 离心5分钟。倒出上清液，将架子翻转成水槽。细胞保留在管的底部。
  3. 向每个试管中加入 3 mL PBS-T 以洗涤细胞。涡旋每个管子。
  4. 在4°C下以300× g 离心5分钟。倒出上清液，将架子翻转成水槽。
    注意:将样品保持在4°C，避光直至流式细胞仪采集。

5. 流式细胞术采集

  1. 通过流式细胞仪（例如BD FACSCalibur流式细胞仪）采集样品。将流式细胞仪连接到计算机软件，等待细胞仪准备就绪。加载模板和仪器设置（表1）。
  2. 开始样品采集。
  3. 使用以下细胞仪策略选择活化的嗜碱性粒细胞139。
    1. 从侧向散射 （SSC） - 前向散射 （FSC） 图中门控淋巴细胞。
    2. 将淋巴细胞群中的嗜碱性粒细胞作为CCR3 + CD203c +细胞。每管至少采集 500 个嗜碱性粒细胞。
    3. 显示 CCR3 - CD63 图以使用 CD63 作为激活标记来分析激活。使用阴性对照管将CD63阴性阈值设置为约2.5%。
    4. 获取所有样品。

结果

使用变应原或药物进行的 BAT 可以检查 IgE 依赖性超敏反应。嗜碱性粒细胞反应性应在至少两个最佳浓度下测量，以获得最佳结果34 ，并通过细胞表面CD63的上调来观察活化。此外，在过敏原的情况下，为了确认嗜碱性粒细胞反应性，应通过测量多个降低的过敏原浓度的反应性来分析嗜碱性粒细胞敏感性114。该测量允许确定诱导50%嗜碱性粒细胞（EC50）反应的过敏?...

讨论

BAT是一种补充体 外 诊断试验，用于评估IgE介导的过敏反应，已被证明可用于诊断由药物，食物或吸入剂等不同触发因素引起的反应，以及某些形式的慢性荨麻疹。一般来说，如果 i） 变应原/药物在 ST 中产生假阳性结果;ii） 过敏原/药物不能用于 ST 或 sIgE 定量;iii） 临床病史与 ST 或 sIgE 测定之间存在不一致;iv）症状表明ST段可能诱发全身反应;v） 在 CCT 之前，用于致病原原/药物确认

披露声明

作者没有什么可透露的。

致谢

我们感谢克劳迪娅·科拉扎（Claudia Corazza）宝贵的英语语言支持。这项工作得到了MIENCO卫生研究所"Carlos III"（ISCIII）的支持（由ERDF共同资助的赠款:"Una manera de hacer Europa";授权号PI20/01715;PI18/00095;PI17/01410;PI17/01318;PI17/01237 和 RETIC ARADYAL RD16/0006/0001;安达卢西亚地区卫生部（批准号PI-0127-2020，PIO-0176-2018;PE-0172-2018;PE-0039-2018;PC-0098-2017;PI-0075-2017;PI-0241-2016）。ID是一名临床研究者（B-0001-2017），AA持有高级博士后合同（RH-0099-2020），两者都由安达卢西亚地区卫生部支持（由ESF共同资助:"Andalucía se mueve con Europa"）。

材料

NameCompanyCatalog NumberComments
5 mL Round Bottom Polystyrene Test Tube, without Cap, NonsterileCorning352008
APC anti-human CD193 (CCR3) AntibodyBioLegend310708
BD FACSCalibur Flow CytometerBD Biosciences
Calcium chlorideSigma-AldrichC1016
FITC anti-human CD63 AntibodyBioLegend353006
HEPES (1 M)Thermo-Fisher15630106
Lysing Solution 10x concentratedBD Biosciences349202
Magnesium chlorideSigma-AldrichM8266
N-Formyl-Met-Leu-PheSigma-AldrichF3506
PE anti-human CD203c (E-NPP3) AntibodyBioLegend324606
Potassium chlorideSigma-AldrichP9541
Purified Mouse Anti-Human IgEBD Biosciences555857
Recombinant Human IL-3R&D Systems203-IL
Sheath FluidBD Biosciences342003
Sodium chlorideSigma-AldrichS3014
TUBE 9 mL LH Lithium HeparinGreiner Bio-One455084
Tween 20Sigma-AldrichP1379

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