盐裂皮肤-间接免疫荧光方法优化及在大疱性类天疱疮抗体检测中的应用

Optimization of indirect immunofluorescence on salt-split skin and its application in detection of bullous pemphigoid antibodies

目的优化盐裂皮肤-间接免疫荧光方法(IIF-SSS),评价其在大疱性类天疱疮(BP)抗体检测中的应用。方法通过调整试验条件,利用正常人包皮或非包皮皮肤制备盐裂底物,分为3组:传统组在4℃旋转皮肤48~72 h;低温浸泡组在4℃浸泡皮肤48~72 h;室温浸泡组在室温25℃(23℃~27℃)浸泡皮肤24 h。收集2019年8月至2020年8月中国医学科学院皮肤病医院确诊的20例BP患者血清,采用倍比稀释法,利用上述3种试验条件制备的盐裂底物、未盐裂底物进行IIF检测。采用配对样本t检验进行两配对样本的比较。结果低温浸泡组在48~72 h未出现真表皮分裂;室温浸泡组非包皮盐裂底物与传统组一样,真表皮分裂界面位于透明板下部。20例BP患者血清,在室温浸泡组盐裂非包皮皮肤、盐裂包皮及传统组盐裂非包皮皮肤这3种底物上,抗体滴度的倒数M(Q1,Q3)分别为5120(2560,17920)、1280(640,2560)、1280(640,2560)。室温浸泡组制备的盐裂皮肤效果优于传统组,其中19例(95%)BP患者的滴定抗体滴度比传统盐裂方法高出1~5个倍比稀释度,差异有统计学意义(t=8.04,P<0.001);而室温浸泡组制备的盐裂包皮与传统组相比,抗体滴度差异无统计学意义(t<0.001,P>0.05)。在以正常未行盐裂的皮肤及包皮为底物的常规IIF中,抗体滴度的倒数分别为320(160,640)、480(160,1120);室温浸泡组盐裂包皮、非包皮皮肤及传统组盐裂非包皮皮肤这3种底物上IIF检测的抗体滴度与常规IIF检测的抗体滴度一致或高出1~7个倍比稀释度,差异有统计学意义(t=6.47、14.83、5.26,均P<0.001)。结论采用室温25℃(23℃~27℃)浸泡法来制备盐裂底物,不仅耗时短,流程简单,而且检测BP抗体的敏感性等于甚至优于传统盐裂方法。

Objective To optimize indirect immunofluorescence on salt-split skin(IIF-SSS),and to evaluate its performance in detection of bullous pemphigoid(BP)antibodies.Methods Normal human foreskin and non-foreskin skin tissues were used to prepare salt-split substrates under 3 different experimental conditions:traditional group rotated at 4℃for 48-72 hours,low-temperature immersion group soaked at 4℃for 48-72 hours,room-temperature immersion group soaked at 25℃(range:23-27℃)for 24 hours.Serum samples were obtained from 20 patients with bullous pemphigoid(BP)in Hospital of Dermatology,Chinese Academy of Medical Sciences between August 2019 and August 2020,and subjected to IIF on the intact skin or salt-split substrates by using a multiple dilution method.Paired-sample t test was used for comparisons of means between two paired samples.Results No dermal-epidermal separation was observed in the substrates prepared in the low-temperature immersion group at 48-72 hours,while dermal-epidermal separation occurred in the lower lamina lucida of the foreskin and non-foreskin substrates in the room-temperature immersion group and the traditional group.For the 20 patients with BP,the reciprocal end-point titers(M[Q1,Q3])detected with the salt-split non-foreskin skin and salt-split foreskin in the room-temperature immersion group,and with the salt-split non-foreskin skin in the traditional group were 5120(2560,17920),1280(640,2560),1280(640,2560),respectively.Moreover,19(95%)patients with BP showed that the reciprocal end-point titers detected with the substrates in the room-temperature immersion group were 1-5 times those in the traditional group(t=8.04,P<0.001),suggesting that the performance of salt-split skin in the room-temperature immersion group was superior to that in the traditional group in the detection of BP antibodies;however,there was no significant difference in the reciprocal end-point titers of BP antibodies between the salt-split foreskin in the room-temperature immersion group and salt-split non-foreskin skin in the traditional group(t<0.001,P>0.05).The reciprocal end-point titers in 20 BP sera detected by conventional IIF on the intact non-foreskin skin and foreskin were 320(160,640)and 480(160,1120),respectively;the reciprocal end-point titers detected by IIF on the salt-split foreskin and non-foreskin skin in the room-temperature immersion group,as well as on the salt-split non-foreskin skin in the traditional group,were all consistent with or 1-7 times higher than those detected by conventional IIF(t=6.47,14.83,5.26,respectively,all P<0.001).Conclusion The soaking method at room temperature 25℃(23-27℃)for preparing salt-split substrates has advantages of short duration and simple procedure,and the sensitivity of IIF-SSS using the substrates prepared by this method is equal or superior to the traditional salt-split method for detecting BP antibodies.