PDF(Pubmed)
Abstract：
BACKGROUND: Calcitonin gene-related peptide (CGRP) is the most promising candidate to become the first migraine biomarker. However, literature shows clashing results and suggests a methodological source for such discrepancies. We aimed to investigate some of these methodological factors to evaluate the actual role of CGRP as biomarker.
METHODS: Previous to the experimental part, we performed a literature review of articles measuring CGRP in migraine patients. Using our 399 bio-bank sera samples, we performed a series of experiments to test the validity of different ELISA kits employed, time of sample processing, long-term storage, sampling in rest or after moderate exercise. Analysis of in-house data was performed to analyse average levels of the peptide and the effect of sex and age.
RESULTS: Literature review shows the high variability in terms of study design, determination methods, results and conclusions obtained by studies including CGRP determinations in migraine patients. CGRP measurements depends on the method and specific kit employed, also on the isoform detected, showing completely different ranges of concentrations. Alpha-CGRP and beta-CGRP had median with IQR levels of 37.5 (28.2-54.4) and 4.6 (2.4-6.4)pg/mL, respectively. CGRP content is preserved in serum within the 24 first hours when samples are stored at 4°C after clotting and immediate centrifugation. Storages at -80°C of more than 6 months result in a decrease in CGRP levels. Moderate exercise prior to blood extraction does not modulate the concentration of the peptide. Age positively correlates with beta-CGRP content and men have higher alpha-CGRP levels than women.
CONCLUSIONS: We present valuable information for CGRP measurements in serum. ELISA kit suitability should be tested prior to the experiments. Alpha and beta-CGRP levels should be analysed separately as they can show different behaviours even within the same condition. Samples can be processed in a 24-h window if they have been kept in 4°C and should not be stored for more than 6 months at -80°C before assayed. Patients do not need to rest before the blood extraction unless they have performed a high-endurance exercise. For comparative studies, sex and age should be accounted for as these parameters can impact CGRP concentrations.
METHODS: Previous to the experimental part, we performed a literature review of articles measuring CGRP in migraine patients. Using our 399 bio-bank sera samples, we performed a series of experiments to test the validity of different ELISA kits employed, time of sample processing, long-term storage, sampling in rest or after moderate exercise. Analysis of in-house data was performed to analyse average levels of the peptide and the effect of sex and age.
RESULTS: Literature review shows the high variability in terms of study design, determination methods, results and conclusions obtained by studies including CGRP determinations in migraine patients. CGRP measurements depends on the method and specific kit employed, also on the isoform detected, showing completely different ranges of concentrations. Alpha-CGRP and beta-CGRP had median with IQR levels of 37.5 (28.2-54.4) and 4.6 (2.4-6.4)pg/mL, respectively. CGRP content is preserved in serum within the 24 first hours when samples are stored at 4°C after clotting and immediate centrifugation. Storages at -80°C of more than 6 months result in a decrease in CGRP levels. Moderate exercise prior to blood extraction does not modulate the concentration of the peptide. Age positively correlates with beta-CGRP content and men have higher alpha-CGRP levels than women.
CONCLUSIONS: We present valuable information for CGRP measurements in serum. ELISA kit suitability should be tested prior to the experiments. Alpha and beta-CGRP levels should be analysed separately as they can show different behaviours even within the same condition. Samples can be processed in a 24-h window if they have been kept in 4°C and should not be stored for more than 6 months at -80°C before assayed. Patients do not need to rest before the blood extraction unless they have performed a high-endurance exercise. For comparative studies, sex and age should be accounted for as these parameters can impact CGRP concentrations.
摘要：
背景：降钙素基因相关肽（CGRP）是最有希望成为第一个偏头痛生物标志物的候选物。然而,文献显示了冲突的结果，并提出了这种差异的方法学来源。我们旨在研究这些方法学因素中的一些，以评估CGRP作为生物标志物的实际作用。
方法：在实验部分之前，我们对偏头痛患者CGRP测定的文献进行了综述.使用我们的399个生物银行血清样本，我们进行了一系列实验，以测试所使用的不同ELISA试剂盒的有效性，样品处理时间，长期储存,在休息或适度运动后取样。进行内部数据的分析以分析肽的平均水平以及性别和年龄的影响。
结果：文献综述显示研究设计方面的高度可变性，测定方法,通过包括偏头痛患者CGRP测定在内的研究获得的结果和结论。CGRP测量取决于所用的方法和特定试剂盒，也在检测到的亚型上，显示完全不同的浓度范围。α-CGRP和β-CGRP的IQR水平中位数为37.5（28.2-54.4）和4.6（2.4-6.4）pg/mL，分别。当样品在凝固和立即离心后在4°C下储存时,CGRP含量在最初24小时内保存在血清中。在-80°C下储存超过6个月会导致CGRP水平降低。在血液提取之前的适度运动不调节肽的浓度。年龄与β-CGRP含量呈正相关，男性的α-CGRP水平高于女性。
结论：我们为血清CGRP测量提供了有价值的信息。ELISA试剂盒的适用性应在实验前进行测试。应分别分析α和β-CGRP水平，因为即使在相同条件下，它们也可能表现出不同的行为。如果将样品保存在4°C中，则可以在24小时窗口中处理样品，并且在测定之前不应在-80°C下储存超过6个月。患者在抽血前不需要休息，除非他们进行了高耐力运动。对于比较研究，应考虑性别和年龄,因为这些参数会影响CGRP浓度.
方法：在实验部分之前，我们对偏头痛患者CGRP测定的文献进行了综述.使用我们的399个生物银行血清样本，我们进行了一系列实验，以测试所使用的不同ELISA试剂盒的有效性，样品处理时间，长期储存,在休息或适度运动后取样。进行内部数据的分析以分析肽的平均水平以及性别和年龄的影响。
结果：文献综述显示研究设计方面的高度可变性，测定方法,通过包括偏头痛患者CGRP测定在内的研究获得的结果和结论。CGRP测量取决于所用的方法和特定试剂盒，也在检测到的亚型上，显示完全不同的浓度范围。α-CGRP和β-CGRP的IQR水平中位数为37.5（28.2-54.4）和4.6（2.4-6.4）pg/mL，分别。当样品在凝固和立即离心后在4°C下储存时,CGRP含量在最初24小时内保存在血清中。在-80°C下储存超过6个月会导致CGRP水平降低。在血液提取之前的适度运动不调节肽的浓度。年龄与β-CGRP含量呈正相关，男性的α-CGRP水平高于女性。
结论：我们为血清CGRP测量提供了有价值的信息。ELISA试剂盒的适用性应在实验前进行测试。应分别分析α和β-CGRP水平，因为即使在相同条件下，它们也可能表现出不同的行为。如果将样品保存在4°C中，则可以在24小时窗口中处理样品，并且在测定之前不应在-80°C下储存超过6个月。患者在抽血前不需要休息，除非他们进行了高耐力运动。对于比较研究，应考虑性别和年龄,因为这些参数会影响CGRP浓度.
