Supplementary Material for: Histamine skin prick tests: from established diagnostic technique to advanced experimental biomarker

Introduction Skin prick tests have a long history as diagnostic and pharmacodynamic biomarker. Besides visual assessments of the wheal and flare, objective blood flow measurements using laser Doppler imaging (LDI) and laser speckle contrast imaging (LSCI) have been reported. In light of these advancements, an up-to-date characterization of the histamine-evoked response is worthwhile. Methods A single-center study was completed in healthy males. Two parameters were addressed: (1) dermal blood flow (DBF) within a 7.65 mm ring encircling the skin prick site (DBFring), and (2) surface area of the flare (AREAflare). First, the dose response was assessed using placebo (0.9% sodium chloride) or histamine (histamine dihydrochloride 1, 3 or 10 mg/mL) skin pricks on the volar surface of subjects’ (n=12) forearm. The DBFring was measured by LDI, the AREAflare by LDI and by ruler. Secondly, the inter-arm and inter-period reproducibility of the DBFring and AREAflare, as evoked by histamine (10 mg/mL) and measured by LDI and LSCI, was examined (n=14). Lastly, the effect of aprepitant (125 mg), ketotifen (1 mg) and a single (5 mg) and fourfold (20 mg) dose of desloratadine and levocetirizine on the histamine-induced (10 mg/mL) DBFring and AREAflare was evaluated with LSCI (n=13 or 12). Results All three histamine doses induced a time-dependent vasodilation. Ruler recordings did not conclusively correlate with LDI assessments of the AREAflare. The DBFring and AREAflare were reasonably reproducible when measured by using LDI or LSCI, with negligible bias between arms and study periods and poor to moderate within-subject reproducibility (0.23≤ ICC≤ 0.71). While the fourfold dose of desloratadine (p=0.0041) and the single and fourfold dose of levocetirizine (p<0.0001) managed to reduce the AREAflare, only the fourfold dose of levocetirizine (p=0.0052) reduced the DBFring. Discussion/conclusion Caution is warranted when translating years of clinical experience with histamine skin prick tests to objective recordings of the associated changes in skin perfusion. Ruler and LDI assessments of the AREAflare do not consistently correlate, and the reproducibility and histamine-dependency of the measurements is not obvious. While 10 mg/mL histamine may be a good choice for qualitative diagnostic evaluations, a lower dose may be better suited to use as a quantitative biomarker.

引言 皮肤点刺试验（skin prick tests）作为诊断与药效学生物标志物已有悠久历史。除针对风团和潮红（wheal and flare）的视觉评估外，已有研究采用激光多普勒成像（laser Doppler imaging, LDI）与激光散斑对比成像（laser speckle contrast imaging, LSCI）开展客观血流测量的相关报道。鉴于上述技术进展，对组胺诱发的皮肤反应进行系统性最新表征颇具研究价值。

方法 本研究为单中心研究，纳入健康男性受试者。本次研究聚焦两项核心参数：(1) 皮肤点刺位点周围7.65 mm环形区域内的皮肤血流量（dermal blood flow, DBF），记为DBFring；(2) 潮红区域面积（AREAflare）。首先，针对12名受试者的前臂掌侧表面（volar surface），分别采用安慰剂（0.9%氯化钠）或不同浓度二盐酸组胺（histamine dihydrochloride，1、3或10 mg/mL）进行皮肤点刺，以评估剂量反应关系；其中采用LDI测量DBFring，采用LDI与直尺两种方式测量AREAflare。其次，针对14名受试者，采用10 mg/mL组胺诱发皮肤反应，分别通过LDI与LSCI测量DBFring与AREAflare，以此检验臂间及周期间的重现性。最后，采用LSCI分别针对13或12名受试者，评估阿瑞匹坦（125 mg）、酮替芬（1 mg）以及单次（5 mg）与四倍剂量（20 mg）的地氯雷他定、左西替利嗪对10 mg/mL组胺诱导的DBFring与AREAflare的影响。

结果 三种浓度的组胺均可诱发时间依赖性血管舒张。直尺测量的AREAflare结果与LDI评估结果未呈现明确相关性。采用LDI或LSCI测量得到的DBFring与AREAflare具有较好的重现性：双臂与各研究周期间的偏倚可忽略不计，但受试者内部的重现性为较差至中等水平（0.23≤组内相关系数（intraclass correlation coefficient, ICC）≤0.71）。尽管四倍剂量的地氯雷他定（p=0.0041）以及单次与四倍剂量的左西替利嗪（p<0.0001）均可有效降低AREAflare，但仅四倍剂量的左西替利嗪（p=0.0052）可显著降低DBFring。

讨论与结论 将多年来基于皮肤点刺试验的临床经验转化为皮肤灌注相关变化的客观记录时，需保持谨慎。直尺与LDI对AREAflare的评估结果并不始终具有相关性，且测量结果的重现性与组胺依赖性并不明确。尽管10 mg/mL的组胺或许可作为定性诊断评估的合适选择，但更低浓度的组胺或许更适合作为定量生物标志物。