ABC-ELISA法诊断肠螨病的研究

目的：探讨生物素-亲和素酶联免疫吸附试验（ABC—ELISA）在肠螨病诊断中的应用价值。方法：采集48例肠螨病患者血清，用ABC—ELISA法检测血清螨特异性抗体IgG，并与葡萄球菌A-蛋白酶联免疫吸附试验（SPA—ELISA）进行比较。结果：用ABC—ELISA法和SPA—ELISA法检测48例肠螨病患者血清螨特异性抗体IgG阳性率分别为89.58％（43／48）和56.25％（27／48），两者比较，差异具显著性（χ^2=13．50，P〈0．01）。结论：ABC—ELISA法比SPA—ELISA法更有效诊断肠螨病。     

二种快速ELISA用于检测囊虫病患者血清特异性抗体的实验观察

用快速ＩｇＧ－ＥＬＩＳＡ和快速ＳＰＡ－ＥＬＩＳＡ检测囊虫病人血清抗体，其阳性率分别为９０．００％和９４．２９％，两虫之间无显著性差异（Ｐ＞０．０５）。两法假阴性率分别为１．４３％和２．８６％，对包虫病人血清出现较强交叉反应，对肝吸虫、肺吸虫病人血清出现一定程度的交叉反应，而对血吸虫病人、绦虫病人血清未出现交叉反应。若两种方法同时使用可提高敏感性。两种方法均较简便，在包虫病非流行区可作为较好的流行病学调查筛检方法，亦可用于临床     

一步法实时荧光定量PCR检测大鼠胰腺组织中Insulin mRNA和PDX-1 mRNA的表达

目的通过检测大鼠胰腺组织中Insulin mRNA和PDX-1 mRNA的表达探讨EGF/Gastrin联用促进胰岛PDX-1表达增强可能的作用机制。方法采用一步法实时荧光定量PCR检测各组大鼠胰腺组织中Insulin基因和PDX-1基因在转录水平的表达。结果大鼠胰腺组织中Insulin mRNA在转录水平的表达情况正常对照组最高;糖尿病组最低,与正常对照组相比相差2.4倍（P〈0.05）;EGF/Gastrin组是糖尿病组的2.1倍（P〈0.05）。大鼠胰腺组织中PDX-1mRNA在转录水平的表达情况正常对照组最高,糖尿病组最低,与正常对照组相比相差2.8倍（P〈0.05）,EGF/Gastrin组是糖尿病组的2.2倍（P〈0.05）。结论 EGF/Gastrin联用促进胰岛PDX-1表达增强的作用机制可能通过其改善胰岛β细胞功能、降低血糖进而减弱糖毒性对PDX-1表达的不良影响,间接地使PDX-1的表达增强。而EGF/Gastrin联用促进胰岛新生、改善胰岛β细胞功能又有可能是通过提高PDX-1的表达而实现的。     

A differential intra-operative molecular biological test for the detection of sentinel lymph node metastases in breast carcinoma. An extended experience from the first UK centre routinely offering the service in clinical practice

Introduction

One-Step Nucleic acid Amplification (OSNA) is a molecular biological assay of cytokeratin-19 (a breast epithelial marker) mRNA. It can be employed intra-operatively for detection of lymph node metastases in breast carcinoma. Patients with positive sentinel nodes may proceed to axillary lymph node dissection (ALND) level I or higher dependent upon the OSNA quantitative result, during the same surgical procedure, avoiding a second operation and eliminating the technical difficulties possibly associated with delayed ALND.

Aims

Our Breast Unit was the first in the UK to implement this novel technique in routine practice. This study reviews our first 44-month data following introduction of OSNA “live” on whole sentinel nodes following an extensive validation study (Snook et al.).⁹

Methods

Data was collected prospectively from the period of introduction 01/12/2008 to 30/08/2012. All patients eligible for sentinel node biopsy were offered OSNA and operations were performed by five consultant breast surgeons. On detection of macro-metastasis a level II/III and for a micro-metastasis a level I ALND was performed.

Results

A total of 859 patients (1709 sentinel lymph nodes) were analysed. All except one were females. The majority underwent wide local excision (73.4%, n = 631) or mastectomy 25% (n = 215) and 1.6% (13) underwent SLN biopsy alone. IDC was seen in 79% (n = 680) of the patients and 53.5% (n = 460) had grade II tumours. One-third (30.8%, n = 265) had positive sentinel nodes and had further axillary surgery at the time of SLN biopsy. Of these, 47% (n = 125/265) had macro-metastases, 38% (n = 101/265) had micro-metastases and 14.7% (n = 39/265) had “positive but inhibited” results. Positive non-sentinel lymph nodes (NSLN) were seen in 35% (44/125) of those with macro-metastases; 17.8% (18/101) of the patients with micro-metastases and 10.2% (4/39) of the “positive but inhibited” group.

Conclusion

In our series over a third of our patients had positive lymph nodes detected with OSNA allowing them to proceed directly to axillary surgery at the same operation. This technique eliminates the need for a second operation in sentinel lymph node positive patients and avoids the anxiety waiting for histological results.     

SPA-ELISA检测卡氏肺孢子虫抗原的研究

目的 探讨SPAK-ELISA检测卡氏肺孢子虫抗原对诊断卡氏肺孢子虫肺炎的价值。 方法 采用SPA-ELISA检测免疫抑制大鼠模型在不同时期肺泡灌洗液(BALF)和血清中肺孢子虫(PC)抗原,以及造血干细胞移植并发肺炎患者痰液和血清抗原,并与病原学检查结果比较。 结果 感染大鼠于用药后第4周,BALF中PC抗原,以及肺组织印片和BALF涂片包囊检查开始出现阳性,但前者阳性率(60％)高于后者(20％和40％),而且全部阳性达到的时间(8周)也早于后者(10周)。第10周有2只鼠血清抗原阳性,但此后又转阴。30例肺炎患者痰液抗原阳性8例,阳性率为26.67％,8例抗原阳性患者中只有3例发现包囊,符合率为37.5％。血清抗原检测全部阴性。 结论 SPA-ELISA检测痰液和肺泡灌洗液抗原比病原学检查敏感性高,而检测血清抗原不适用于诊断卡氏肺孢子虫肺炎。     

弓形虫病诊断试剂盒阴性血清保存方法的研究

目的：研究SPA-ELISA弓形虫病诊断试剂盒不同稀释液及灭活处理对阴性血清的保存效果。方法：正常人阴性血清（n=5）分别用0．01MPBS缓冲液、1％胎牛血清、15％甘油1：100进行稀释后保存于4℃，分别于稀释后1d、1周、2周、4周、2个月、3个月用SPA-ELISA检测阴性血清OD410比较其变化情况：同时观察灭活对阴性血清的影响。结果：用15％甘油稀释的阴性血清可以保存至3个月，用0．01M PBS缓冲液和1％胎牛血清稀释的阴性对照一个月之后保存效果较差。结论：15％甘油稀释的阴性血清可以比较长期稳定的保存．保证了SPA-ELISA弓形虫病诊断试剂盒的稳定性。     

Intraoperative assessment of sentinel lymph node by one-step nucleic acid amplification in breast cancer patients after neoadjuvant treatment reduces the need for a second surgery for axillary lymph node dissection

PurposeSentinel lymph node (SLN) biopsy has been shown to be both accurate and feasible for women who receive neoadjuvant chemotherapy (NAC). Intraoperative assessment of SLN by frozen sections can produce false negative results. The aim of this study was to compare two different techniques of intraoperative assessment of SLN in breast cancer patients treated with NAC: frozen section (FS) and molecular assay (OSNA).MethodsA multicenter cohort of 320 consecutive breast cancer patients treated with NAC between 2010 and 2014 was analyzed. FS was performed intraoperatively in 166 patients (H&E cohort) and OSNA in 154 patients (OSNA cohort).ResultsA mean of 2.15 SLNs by FS and 1.22 SLNs by OSNA was assessed (p = 0.03). SLN metastasis was found in 44 patients (26.5%) by FS and in 48 (31.2%) by OSNA (p = 0.4). There was no statistical significance in rates of macrometastasis (75%), micrometastasis (20.5%) or ITCs (4.5%) when assessed by FS compared to OSNA (52.3%, 36.3% and 11.4%, respectively) (p = 0.06). There were 10 patients in the H&E cohort with positive-SLN in the definitive pathology assessment with negative intraoperative FS. When OSNA and definitive pathology were compared, there were no differences in rates of macrometastasis (61.1%), micrometastasis (33.3%) nor ITCs (5.6%) (p = 0.5). Fifty-four patients in the H&E cohort and 44 in the OSNA cohort had ALND after positive-SLNs. ALND was performed in a second surgery in 10 patients (18.5%) in the H&E cohort for intraoperative FS false negative results, 90% being micrometastasis. 42 out of 44 patients (95.5%) in the OSNA cohort had an ALND in the same surgery (p = 0.03).ConclusionsOSNA assay detects SLNs metastases as accurately as conventional pathology in the NAC setting. Intraoperative definitive assessment of the SLN by OSNA reduces the need for a second surgery for ALND in 18.5% of breast cancer patients with a positive-SLN after NAC.     

二种滤纸血滴片SPA-ELISA诊断囊虫病的应用

用快速SPA－ELISA和SPA－ELISA一步法检测囊虫病人的滤纸血滴片和血清，前者阳性率分别为83．05％和86.44％．后者分别为96．67％和94.91％，均无统计学差异（P＞0．05）；两法的假阳性率均为3．33％。实验条件的研究表明：血滤纸4℃浸泡过夜和室温浸泡2h的阳性率为83．05％和84．38％，直径1．2cm和0．6cm阳性率为83．05％和85．29％，亦均无显著性差异（P＞0．05）。说明滤纸血滴片可以代替血清样本，具有敏感、安全、简便以及非常实用等优点，血滴片SPA－ELISA在囊虫病的诊断及流行病学调查中有较好的应用价值。     

乳腺癌前哨淋巴结转移术中检测进展：一步法核酸扩增技术

乳腺癌已成为女性最常见的恶性肿瘤之一,腋窝淋巴结（ALN）转移是乳腺癌最主要的转移方式,其是判断预后和指导术后辅助治疗方案决策最重要的指标。腋窝淋巴结清扫（ALND）是评估ALN状态最准确的方法,亦是造成上肢淋巴水肿、疼痛、感觉障碍等并发症的主要原因。随着早期乳腺癌的增多,ALN阴性乳腺癌已占据一半以上,如对所有乳腺癌患者行ALND,将只有少部分患者受益,大部分患者接受了过度治疗。乳腺癌前哨淋巴结（SLN）是乳腺癌患者肿瘤细胞经淋巴结转移的首道屏障。历经数十年发展,前哨淋巴结活检（SLNB）已成为乳腺癌患者ALN状态分期的标准程序,常用于确定乳腺癌治疗方式的选择。准确、快速的SLN术中诊断可以使SLN阴性的乳腺癌患者避免ALND,SLN阳性患者通过一次手术完成ALND,避免二次手术的费用负担和手术风险。术中检测SLN是否转移常用的病理学方法是冷冻切片和印片细胞学检查,这两种常规病理检测方法仅检测SLN的代表性切片,均存在敏感度较低、主观性、非标准化、检测的组织量少（远远低于5%）、没有统一诊断标准等缺点,因此临床上迫切需要一种结果准确,操作简便的新型检测方法。近年来分子诊断技术迅速发展,一步法核酸扩增（OSNA）是通过逆转录环介导的靶向细胞角蛋白19mRNA等温扩增法精确检测术中乳腺癌淋巴结转移的分子诊断方法。OSNA检测运作时间约30~40 min,在SLN检测分析中,OSNA的准确率、敏感度均优于常规病理学检测方法。术中快速定量可区分宏转移和微转移,指导手术方案,并且在检测SLN微转移时,OSNA也更胜一筹。笔者对OSNA检测在乳腺癌SLN转移中的进展及应用前景做一综述。     

检测弓形虫抗体SPA—ELISA

为了制备ＳＰＡ－ＥＬＩＳＡ弓形虫抗体诊断试剂盒，优选了最佳的实验条件，包括反应时间、ＳＰＡ－ＨＲＰ浓度及待测血清标本浓度。观察了封闭对实验结果的影响及酶保护剂对ＳＰＡ－ＨＲＰ活性的影响，并对该方法的敏感性、特异性、重复性及破坏性进行了实验研究。结果本试剂盒方法简便快捷，敏感性高，特异性强，重复性好，可４℃保存３个月，－２０℃保存半年，其活性不变。检测了５０份孕妇及可疑病例务清或脑脊液，阳性主继４％