Department of Dermatology, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.
Department of Dental Materials Science, Academic Centre for Dentistry Amsterdam, University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.
Clin Exp Allergy. 2020 Jun;50(6):722-732. doi: 10.1111/cea.13603. Epub 2020 Apr 14.
Skin patch testing is still seen as the gold standard for the diagnosis of allergic hypersensitivity. For several metals and for patients with a suspected adverse reaction to their medical device implant material, patch testing can be unreliable. The current alternative to metal allergy patch testing is the in vitro lymphocyte proliferation test (LPT) using tritiated thymidine. This method is well-established but requires handling of radioactive material, often uses heat-inactivated allogenic human pooled serum and cannot determine T cell subsets.
To develop a radioactive free LPT by using carboxyfluorescein succinimidyl ester (CFSE) and to evaluate the influence of serum source (heat-inactivated human pooled serum [HI HPS] vs autologous serum) on the sensitivity and specificity of the nickel-specific LPT.
Peripheral blood mononuclear cells derived from nickel-allergic patients and healthy controls were collected, labelled with CFSE and cultured in medium containing 10% HI HPS or 10% autologous serum with or without additional T cell skewing cytokine cocktails (Th1: IL-7/IL-12, Th2: IL-7/IL-4 or Th17: IL-7/IL-23/IL-1β) in the absence or presence of NiSO . The stimulation index (SI) was calculated as the ratio of divided cells, that is the percentage of CFSE CD3 CD4 T-lymphocytes upon nickel stimulation compared to the percentage of CFSE CD3 CD4 T-lymphocytes without antigen. These results were compared with the history of Ni allergy, patch test results and the MELISA test.
Autologous serum positively influenced Ni-specific proliferation while HI HPS negatively influenced Ni-specific proliferation. The test protocol analysing CD4 cells and autologous serum without skewing cytokines scored the best diagnostic values (sensitivity 95%; specificity 93%; and overall accuracy 94%) compared to the parallel test using HI HPS (accuracy 60%). Cytokine supplements did not further improve the test protocol which used autologous serum. The protocol using HI HPS could be further improved by addition of the cytokine skewing cocktails.
Here, we describe an optimized and highly accurate flow cytometric LPT which comprises of CFSE-labelled cells cultured in autologous serum (not heat inactivated) and without the presence of T cell skewing cytokines.
The sensitivity and specificity of LPT is enhanced, compared to HI HPS, when autologous serum without skewing cytokines is used.
皮肤贴片测试仍然被视为过敏反应诊断的金标准。对于某些金属和对其医疗器械植入材料有疑似不良反应的患者，贴片测试可能不可靠。目前，金属过敏贴片测试的替代方法是使用氚标记胸腺嘧啶的体外淋巴细胞增殖测试 (LPT)。该方法已得到很好的建立，但需要处理放射性物质，通常使用热失活同种异体人混合血清，并且无法确定 T 细胞亚群。
开发一种无放射性的 LPT，使用羧基荧光素琥珀酰亚胺酯 (CFSE)，并评估血清来源（热失活人混合血清 [HI HPS] 与自体血清）对镍特异性 LPT 敏感性和特异性的影响。
从镍过敏患者和健康对照者中采集外周血单核细胞，用 CFSE 标记，然后在含有 10%HI HPS 或 10%自体血清的培养基中培养，或在含有 T 细胞偏倚细胞因子鸡尾酒（Th1：IL-7/IL-12，Th2：IL-7/IL-4 或 Th17：IL-7/IL-23/IL-1β）的培养基中培养，在不存在或存在 NiSO4 的情况下。刺激指数 (SI) 计算为分裂细胞的比率，即镍刺激后 CFSE CD3 CD4 T 淋巴细胞的百分比与无抗原的 CFSE CD3 CD4 T 淋巴细胞的百分比。将这些结果与镍过敏史、贴片测试结果和 MELISA 测试进行比较。
自体血清可正向影响镍特异性增殖，而 HI HPS 则负向影响镍特异性增殖。分析 CD4 细胞和无偏倚细胞因子的自体血清的测试方案（敏感性 95%；特异性 93%；总准确性 94%）与使用 HI HPS 的平行测试相比（准确性 60%）得分最佳。细胞因子补充剂并未进一步提高使用自体血清的测试方案。添加偏倚细胞因子的 HI HPS 测试方案可以进一步改进。
本文描述了一种优化的、高度准确的流式细胞术 LPT，该方案包括用 CFSE 标记的细胞在自体血清（非热失活）中培养，且不存在 T 细胞偏倚细胞因子。
与使用 HI HPS 相比，当使用无偏倚细胞因子的自体血清时，LPT 的敏感性和特异性得到提高。
