Comparative analysis of immunofluorescence double staining for foamy macrophages and Mycobacterium tuberculosis in paraffin-embedded tissue of clinical tuberculous wound
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摘要:
目的 观察免疫荧光双重染色对临床结核性创面石蜡组织中泡沫细胞及结核分枝杆菌(MTB)的显示效果，并与3种常规染色方法进行比较。 方法 采用实验研究方法。2019年4月—2020年5月，厦门大学附属翔安医院烧伤整形与创面修复科收治10例符合入选标准的结核性创面患者(男女各5例，年龄28～77岁)，取其扩大清创术中切取并于该院病理科保存的创面石蜡组织进行研究。将每例患者创面石蜡组织制成40张切片，分别进行苏木精-伊红(HE)染色、免疫组织化学染色、抗酸-免疫组织化学染色、免疫荧光双重染色，每种方法10张。计算4种染色方法切片剔除率；观察4种染色方法的创面肉芽肿组织识别检出情况并计数，观察4种染色方法的创面组织泡沫细胞识别检出情况；比较4种染色方法的创面肉芽肿组织及非肉芽肿组织中MTB检出情况；比较抗酸-免疫组织化学染色和免疫荧光双重染色的创面肉芽肿组织及非肉芽肿组织中泡沫细胞的亚型分型及其分布、MTB检出率，创面组织中泡沫细胞形态清晰度以及MTB与泡沫细胞的非特异性着色率和阳性反应丢失率。对数据行Fisher确切概率法检验、单因素方差分析、独立样本
t 检验、Wilcoxon符号秩检验。 结果 HE染色、免疫组织化学染色、抗酸-免疫组织化学染色、免疫荧光双重染色切片剔除率分别为3%(3/100)、1%(1/100)、6%(6/100)、2%(2/100)，总体比较差异无统计学意义(
P ＝0.26)。4种染色方法均能识别创面肉芽肿组织，肉芽肿结构数相近(
F ＝1.284，
P ＝0.28)。4种染色方法均能识别创面组织泡沫细胞，每张切片均检出。HE染色和免疫组织化学染色创面肉芽肿组织与非肉芽肿组织中均未见MTB；抗酸-免疫组织化学染色和免疫荧光双重染色均显示MTB在创面肉芽肿组织及非肉芽肿组织中均有分布，多数MTB分布于创面肉芽肿组织中。抗酸-免疫组织化学染色不能区分泡沫细胞是否吞噬MTB；免疫荧光双重染色显示吞噬了MTB的泡沫细胞多分布在创面肉芽肿组织中，未吞噬MTB的泡沫细胞多分布在创面非肉芽肿组织中。免疫荧光双重染色的创面肉芽肿组织及非肉芽肿组织MTB检出率分别为(89.00±0.08)%、(82.67±0.05)%，明显高于抗酸-免疫组织化学染色的(54.56±0.14)%、(44.44±0.13)%(
t ＝-12.495、-7.961，
P <0.01)。相较于抗酸-免疫组织化学染色，免疫荧光双重染色的创面组织泡沫细胞清晰度更佳(
Z ＝-3.162，
P <0.01)。免疫荧光双重染色的创面组织MTB与泡沫细胞非特异性着色率和阳性反应丢失率分别为(9.11±0.07)%、(9.22±0.07)%，明显低于抗酸-免疫组织化学染色的(20.67±0.06)%、(44.00±0.12)%(
t ＝4.569、15.519，
P <0.01)。 结论 相较于HE染色、免疫组织化学染色、抗酸-免疫组织化学染色，免疫荧光双重染色呈现图像清晰直观、操作简便，能够精确实现MTB与泡沫细胞在临床结核性创面石蜡组织中的共定位显像。
Abstract:Objective To observe the effect of immunofluorescence double staining for foamy macrophages and
Mycobacterium tuberculosis (MTB) in paraffin-embedded tissue of clinical tuberculous wound, in comparison with three routine staining methods. Methods The experimental method was used. From April 2019 to May 2020, 10 patients with tuberculous wound (5 males and 5 females, aged 28-77 years) meeting the inclusion criteria were treated in the Department of Burns and Plastic & Wound Repair Surgery of Xiang′an Hospital of Xiamen University. The paraffin-embedded wound tissue were collected during extended debridement and preserved in the Department of Pathology of this hospital. Forty paraffin sections were made from the wound tissue of each patient. Hematoxylin-eosin (HE) staining, immunohistochemical staining, Ziehl-Neelsen and immunohistochemical double staining, immunofluorescence double staining were performed respectively, with 10 sections in each method. The section rejection rate of four staining methods were calculated. The recognition and detection of wound granuloma tissue in the four staining methods were observed and counted, and the recognition and detection of foamy macrophages in the wound tissue stained with four methods were observed. The MTB detection in the wound granuloma tissue and non-granuloma tissue in the four staining methods were compared. The subtyping and distribution of foamy macrophages and detection rate of MTB in the wound granuloma tissue and non-granuloma tissue, the morphologic clarity of foamy macrophages, as well as the non-specific staining rate and the loss rate of positive reaction of MTB and foamy macrophages by Ziehl-Neelsen and immunohistochemical double staining were compared with those of immunofluorescence double staining. Data were statistically analyzed with Fisher′s exact probability test, one-way analysis of variance, independent sample
t test and Wilcoxon signed rank test. Results The section rejection rate of HE staining, immunohistochemical staining, Ziehl-Neelsen and immunohistochemical double staining, and immunofluorescence double staining were 3% (3/100), 1% (1/100), 6% (6/100), and 2% (2/100), respectively. There was no statistically significant difference among the four groups (
P =0.26). All the four staining methods could identify granuloma tissue, and the number of granuloma structures was similar (
F =1.284,
P =0.28). All the four staining methods were able to identify foamy macrophages in the wound tissue, which was detected in each section. No MTB was observed in the wound granuloma tissue or non-granuloma tissue by HE staining or immunohistochemical staining. MTB was observed distributing in the wound granuloma tissue and non-granuloma tissue by Ziehl-Neelsen and immunohistochemical double staining and immunofluorescence double staining, and most MTB distributed in the wound granuloma tissue. Ziehl-Neelsen and immunohistochemical double staining could not distinguish foamy macrophages engulfed MTB from that non-engulfed MTB. Immunofluorescence double staining showed that foamy macrophages engulfed MTB mostly distributed in the wound granuloma tissue, and the foamy macrophages non-engulfed MTB mostly distributed in the wound non-granuloma tissue. The detection rates of MTB in wound granuloma and non-granuloma tissue in immunofluorescence double staining were (89.00±0.08)% and (82.67±0.05)%, respectively, which were significantly higher than (54.56±0.14)% and (44.44±0.13)% in Ziehl-Neelsen and immunohistochemical double staining (
t =-12.495, -7.961,
P <0.01). Compared with that of Ziehl-Neelsen and immunohistochemical double staining, immunofluorescence double staining showed better foamy macrophages clarity in wound tissue (
Z =-3.162,
P <0.01). The nonspecific staining rate and positive reaction loss rate of MTB and foamy macrophages in wound tissue of immunofluorescence double staining were (9.11±0.07)% and (9.22±0.07)%, respectively, which were significantly lower than (20.67±0.06)% and (44.00±0.12)% of Ziehl-Neelsen and immunohistochemical double staining (
t =4.569, 15.519,
P <0.01). Conclusions Compared with HE staining, immunohistochemical staining, and Ziehl-Neelsen and immunohistochemical double staining, the immunofluorescence double staining is easy to operate, giving clear and intuitive images. It allows accurate imaging co-localization of MTB and foamy macrophages in paraffin-embedded tissue of clinical tuberculous wound.
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