DNA extraction from paraffin embedded colorectal carcinoma samples: A comparison study of manual vs automated methods,using four commercially kits

BACKGROUND Nucleic acid isolation from formalin-fixed, paraffin-embedded tissue(FFPET)samples is a daily routine in molecular pathology laboratories, but extraction from FFPET is not always easily achieved. Choosing the right extraction technique is key for further examinations.AIM To compare the performance of four commercially available kits used for DNA extraction in routine practice.METHODS DNA isolation was performed on 46 randomly selected formalin-fixed, paraffinembedded(FFPE) colorectal adenocarcinoma(CRC) surgical specimens. Four commercially available extraction kits were used: two for manual DNA extraction(the Pure Link Genomic DNA Mini Kit from Invitrogen and the High Pure FFPE DNA Isolation Kit from Roche) and two for automated DNA extraction(the i Prep Genomic DNA Kit from Invitrogen and the Magna Pure LC DNA Isolation Kit from Roche). The DNA concentration and quality(odds ratio) among the four systems were compared. The results were correlated with the clinicopathological aspects of CRC cases: age, gender, localization, macro-and microscopic features,lymph node metastases, and the lymph node ratio.RESULTS The highest DNA concentration was obtained using the manual kits: 157.24 ±62.99 ng/μL for the Pure Link Genomic DNA Mini Kit and 86.64 ng/μL± 43.84 for the High Pure FFPE DNA Isolation Kit(P < 0.0001). Lower concentrations were obtained with automated systems: 20.39 ± 21.19 ng/μL for the Magna Pure LC DNA Isolation Kit and 8.722 ± 6.408 ng/μL for the i Prep Genomic DNA Kit,with differences between the systems used(P < 0.0001). The comparison between age, gender, tumor localization, pT or pN stage and the lymph node ratio indicated no statistically significant difference in DNA concentration using any of the nucleic acid isolation kits. DNA concentration was influenced by the macroscopic features and grade of differentiation. A higher DNA concentration was obtained for well-differentiated polypoid colorectal adenocarcinomas(CRCs), compared with undifferentiated ulcero-infiltrative carcinomas,irrespective of the kit used.CONCLUSION For research or diagnosis that needs high DNA concentrations, manual methods of DNA isolation should be used. A higher amount of DNA can be obtained from polypoid-type differentiated CRCs. Automated systems confer comfort and a lower amount of DNA that is, however, sufficient for classic polymerase chain reaction(PCR) and real-time quantitative PCR molecular examinations. All four commercially available kits can be successfully used in daily practice.

Detection of BCL2-IGHrearrangement on paraffin-embedded tissue sections obtained from a small submucosal tumor of the rectum in a patient with recurrent follicular lymphoma

A 59-year-old woman was admitted to our hospital because of recurrent follicular lymphoma(FL).Colonoscopic examination revealed a rectal submucosal tumor(SMT)without any erosions and ulcers.In this patient,it was difficult to distinguish non-Hodgkin's lymphoma(NHL)invasion from other disorders of the colon including carcinoid tumor merely based on endoscopic findings.Histopathologic and immunohistochemical studies on biopsy specimens showed an infiltration of atypical lymphocytes that were positive for CD20 and BCL2 but negative for UCHL-1.Fluorescence in situ hybridization on paraffin-embedded tissue sections (T-FISH)identified a translocation of BCL2 with IGHgene. Based on these findings,the tumor was defined as an invasion of FL.T-FISH method is useful for the detection of a monoclonality of atypical lymphocytes in an SMT of the gastrointestinal tract,and particularly for the detection of chromosomal translocations specific to lymphoma subtypes.

DETECTION OF CYTOMEGALOVIRUS GENOME BY IN SITU HYBRIDIZATION IN PARAFFIN EMBEDDED ENDOMYOCARDIAL BIOPSY SPECIMENS OF VIRAL MYOCARDITIS

Cytomegalovirus (CMV) genes were detected by in situ hybridization in 25 Chinese patients with viral myocarditis (VMC). The positive hybridization signals werre found in cardiomyocytes (6 cases, 24%), capillary endothelial cells (4 cases, 16%) and interstitial cells (7 cases, 28%). The difference between VMC and control group (16 cases died of brain trauma and 10 cases of congenital heart diseases was statistically significant. There was no definite pathomorphological relationship between the detection of CMV genes and myocardial lesions. The results suggest that CMV infection may be one of the causes of myocarditis and chronic stimulation of the immune system induced by CMV may be a possible pathogenesis of this disease.

DNA Extraction from Formalin-fixed and Paraffin-embedded Tissues by Triton X-100 for Effective Amplification of EGFR Gene by Polymerase Chain Reaction

For first-line non-small-cell lung cancer（NSCLC） therapy,detecting mutation status of the epidermal growth factor receptor（EGFR） gene constitutes a prudent test to identify patients who are most likely to benefit from EGFR-tyrosine kinase inhibitor（TKI） therapy.Now,the material for detecting EGFR gene mutation status mainly comes from formalin-fixed and paraffin-embedded（FFPE） tissues.DNA extraction from FFPE and the amplification of EGFR gene by polymerase chain reaction（PCR） are two key steps for detecting EGFR gene mutation.We showed a simple method of DNA extraction from FFPE tissues for the effective amplification of EGFR gene.Extracting DNA from the FFPE tissues of NSCLC patients with 1% Triton X-100（pH=10.0） was performed by heating at 95 °C for 30 min.Meanwhile,a commercial kit was used to extract DNA from the same FFPE tissues of NSCLC patients for comparison.DNA extracted products were used as template for amplifying the exons 18,19,20 and 21 of EGFR by PCR for different amplified fragments.Results show that DNA fragment size extracted from FFPE tissues with 1% Triton X was about 250―500 base pairs（bp）.However,DNA fragment size extracted from FFPE tissues via commercial kit was about from several hundreds to several thousands bp.The DNA yield extracted from FFPE tissues with 1% Triton X was larger than that via commercial kit.For about 500 bp fragment,four exons of EGFR could not be amplified more efficiently from extracted DNA with 1% Triton X than with commercial kit.However,for about 200 bp fragment.This simple and non-laborious protocol could successfully be used to extract DNA from FFPE tissue for the amplification of EGFR gene by PCR,further screening of EGFR gene mutation and facilitating the molecular analysis of a large number of FFPE tissues from NSCLC patients.

Trefoil Factor 3 (TFF3) mRNA Expression Level in Follicular Thyroid Tumors Using Formalin-Fixed, Paraffin-Embedded (FFPE) Blocks

Background: Differential diagnosis of follicular thyroid carcinoma (FTC) from follicular thyroid adenoma (FTA) is often difficult since presence or absence of capsular/vascular invasion can not be determined by preoperative fine needle aspiration cytology, and may not be judged unanimously on permanent sections even among experienced pathologists. Determination of molecular-genetic factors such as trefoil factor 3 (TFF3) mRNA in the follicular thyroid tumors may be useful aid to improve the accuracy of diagnosis, though it is considered to be unstable and relatively low concentrated genetic substance. Purpose of our study is to investigate expression level of TFF3 mRNA of thyroid follicular tumors using formalin-fixed, paraffin-embedded (FFPE) tissue. Methods: Study population included FFPE sections from 19 FTC cases, 20 FTA cases, 11 adenomatous goiter (G) cases and 12 samples of normal thyroid tissue (N) adjacent to thyroid tumors. RNeasy FFPE kit was used for extraction of total RNA. Purification and concentration values were determined by spectrophotometer. Extracted RNA was used for cDNA synthesis in reverse transcription. Synthesized cDNA subsequently proceeded for relative quantification of TFF3 mRNA by RT-qPCR using TFF3 primers. Glyceroldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthin phosphorobosyltransferase1 (HPRT1) were used as control genes. The mean and standard deviation of TFF3 mRNA expression level were analyzed by software Multiplate RQ. Results: Extraction by the FFPE kit yielded high concentration of RNA in all cases. Purification values were 1.8 in average. Concentration values were significantly higher in FTC and FTA relative to G and N tissues, possibly due to high density of thyrocytes in the samples. Relative quantification of TFF3 mRNA expression level showed broad ranges both in FTC and FTA, while the analyses in G and N tissues indicated narrow ranges. Conclusion: FFPE tissues from thyroid follicular tumors can be used for measurement of unstable and low concentrated genetic substances such as TFF3 mRNA. Its diagnostic value yet remains to be determined.

围绕FFPE样本特性的DNA纯化纳米磁珠优化

目的 探讨围绕福尔马林固定石蜡包埋(formalin fixed paraffin embedded, FFPE)样本特性获取更高质量/得率的纳米磁珠核酸提取方案,改进分子病理技术。方法 合成4大类15个小类的备选磁珠,以FFPE样本为中心,筛选高质量/得率磁珠。模拟常规组织、粗针穿刺(肝脏)、纤维支气管镜样本(肺),装管相同张数连片。采用筛选的最佳磁珠与市场出售的常见磁珠试剂提取核酸,横向对比纯化总量、片段大小等质量参数。应用PCR和Sanger验证核酸的下游应用。结果 以FFPE样本DNA为中心筛选自制纳米磁珠,获得最佳性能纳米磁珠总回收率为58.5%±1.58%,5种市售商品化磁珠和3种国产磁珠总回收率为18.68%~40.71%。相同组织量(连片)提取的DNA总量,在模拟常规组织、粗针穿刺和纤维支气管镜样本核酸得率比市售试剂盒提高39.49%~181.72%(P<0.05)。模拟纤维支气管镜样本1张(4μm)总量可达100 ng以上、5张总量可达400 ng以上。结论 以FFPE样本DNA为中心筛选的DNA纯化纳米磁珠,与商品化磁珠相比有较大提升,为临床分子病理检测的质量保证、自动化检测和项目拓展提供空间。

石蜡包埋机评价指标体系的建立与应用

采用文献回顾、专家访谈、小组讨论等方法拟定石蜡包埋机评价指标体系草案及专家咨询问卷,采用德尔菲法确定评价指标,应用层次分析法构建判断矩阵得到评价指标权重,通过层次单排序及层次总排序一致性检验,形成石蜡包埋机评价指标体系,最后邀请临床医务人员根据已建立的评价指标体系开展石蜡包埋机评价。构建了一套石蜡包埋机评价指标体系,包含5个一级指标、26个二级指标,专家权威程度在满意范围之内;临床医务人员对国产某品牌石蜡包埋机进行试用并评价,从冷冻台和包埋机两个方面共计提出了18条改进意见建议。本研究所建立的石蜡包埋机评价指标体系,专家参与度高、积极性好、权威程度高,说明结果可参考。

电磁引导经会阴前列腺穿刺联合小标本快脱病理学检查在前列腺癌诊断中的应用价值

目的探讨电磁引导局麻经会阴前列腺mpMRI-TRUS多模态影像融合穿刺活检联合小标本快脱病理学检查在前列腺癌诊断中的应用价值。方法回顾性分析138例PI-RADS评分≥3分并行电磁引导局麻经会阴前列腺多模态影像融合穿刺活检患者的临床病历资料。使用AI技术将mpMRI与TRUS多模态影像进行融合,对可疑病灶靶向穿刺2针,然后行前列腺系统穿刺12针。靶向穿刺标本进行小标本快脱病理学检查,比较靶向穿刺、系统穿刺及联合穿刺csPCa检出率的差异。结果138例患者csPCa检出率为71.01%。靶向穿刺和系统穿刺csPCa检出率分别为62.32%和70.29%,差异无统计学意义(P=0.20)。联合穿刺csPCa检出率高于靶向穿刺和系统穿刺,但差异均无统计学意义(P>0.05)。前列腺PI-RADS评分3分患者靶向穿刺、系统穿刺和联合穿刺的csPCa检出率分别为30.95%、38.10%和40.48%;评分4分患者分别为52.94%、61.76%和61.76%;评分5分患者分别为90.32%、96.77%和96.77%,不同PI-RADS评分患者三种穿刺方法在csPCa检出率方面差异均无统计学意义(P>0.05)。前列腺PI-RADS评分3、4、5分患者,靶向穿刺csPCa漏诊率分别为23.53%、14.29%和6.67%。结论对于PI-RADS v2.1评分≥3分的可疑前列腺癌患者,电磁引导经会阴前列腺穿刺活检联合小标本快脱病理学检查可快速获取病理结果,且有较好的诊断准确性,但联合穿刺仍是目前诊断前列腺癌最合适的方法。

Detection of SYT-SSX fusion transcripts in paraffin-embedded tissues of synovial sarcoma by reverse transcription-polymerase chain reaction

OBJECTIVE: To assess the feasibility of detecting SYT-SSX fusion transcripts in paraffin-embedded tissues of synovial sarcoma by reverse transcription-polymerase chain reaction (RT-PCR). METHODS: RT-PCR was used to amplify the SYT-SSX fusion transcripts using archival formalin-fixed paraffin-embedded tumor specimens from a series of 37 synovial sarcoma cases. To investigate the specificity of the SYT-SSX fusion transcripts, a variety of non-synovial sarcoma tumors were included in the study as negative controls. The detected messages derived from fusion genes were confirmed by subsequent sequence analysis. RESULTS: SYT-SSX fusion transcripts were detected in 33 of 37 (89.2%) synovial sarcomas. None of the 34 cases of non-synovial sarcoma tumors showed amplified products of SYT-SSX fusion transcripts, although PBGD mRNA was detected in all specimens. Among 33 SYT-SSX-positive synovial sarcomas, 22 tumors had an SYT-SSX 1 fusion transcript, whereas 6 tumors had an SYT-SSX2 fusion transcript. Fusion types can not be distinguished in the remaining 5 cases. There was a significant relationship between SYT-SSX fusion type and histologic subtype. All 10 biphasic synovial sarcomas had the SYT-SSX1 fusion, whereas all tumors with SYT-SSX2 were of monophasic morphology (P

胸腹水液基细胞学检测联合胸腹水沉淀物琼脂石蜡双包埋切片法在病理检测的价值

目的探讨与分析胸腹水液基细胞学检测联合胸腹水沉淀物琼脂石蜡双包埋切片法在病理检测的价值。方法选取2019年3月—2022年9月在乌鲁木齐市中医医院诊治的92例胸腹水患者作为研究对象,所有患者都给予胸腹水液基细胞学检测联合胸腹水沉淀物琼脂石蜡双包埋切片法,都给予病理活检,判断诊断的价值。结果在92例患者中,胸腹水液基细胞学检测判断为恶性肿瘤54例,良性肿瘤38例,胸腹水液基细胞学检测诊断恶性肿瘤的敏感度与特异度分别为85.48%、96.67%。胸腹水沉淀物琼脂石蜡双包埋切片检测判断为恶性肿瘤55例,良性肿瘤37例,胸腹水沉淀物琼脂石蜡双包埋切片检测诊断恶性肿瘤的敏感性与特异性分别为88.71%、100.00%。胸腹水液基细胞学检测联合胸腹水沉淀物琼脂石蜡双包埋切片检测判断为恶性肿瘤61例,良性肿瘤31例,胸腹水液基细胞学检测联合胸腹水沉淀物琼脂石蜡双包埋切片检测诊断恶性肿瘤的敏感度与特异度分别为98.39%、100.00%。恶性肿瘤患者的人表皮生长因子受体2、突触素、嗜铬素A表达阳性率分别为40.32%、41.94%、43.55%,与良性肿瘤患者的10.00%、13.33%、10.00%相比有显著提高,差异有统计学意义(P<0.05)。胸腹水液基细胞学检测联合胸腹水沉淀物琼脂石蜡双包埋切片法对肿瘤良恶性的诊断敏感性高于单项检测,差异有统计学意义(P<0.05),不同检测方法的诊断特异性比较,差异无统计学意义(P>0.05)。ROC曲线分析显示,胸腹水液基细胞学检测联合胸腹水沉淀物琼脂石蜡双包埋切片法对肿瘤良恶性的诊断曲线下面积为0.883。结论胸腹水液基细胞学检测联合胸腹水沉淀物琼脂石蜡双包埋切片法在病理检测中的应用能提高诊断敏感度,还可保持非常高的诊断特异度,对患者的原发肿瘤良恶性判定具有很好的价值。

比较胸腹水液基细胞学剩余标本制作细胞块的3种方法

目的优化胸腹水液基细胞学剩余标本制作细胞块的程序,并探讨其在病理诊断中的应用价值。方法 150例胸腹水薄层液基细胞学技术(thinprep cytologic test,TCT)检测剩余标本,根据细胞学诊断结果分为3组,每组50例,分别采用直接离心法、蛋清液作为支架法和细胞块试剂盒法处理后制作石蜡细胞块,对比分析3种不同方法制成的细胞切片上的恶性细胞检出率、细胞分布状况和形态特征,并初步比较细胞块和组织块免疫化学染色效果。结果 150例胸腹水TCT检测出的恶性细胞率为31.3%(47/150),其剩余标本经细胞块法检测出的总恶性细胞检出率为40.7%(61/150),其中直接离心法、蛋清液作为支架法和细胞块试剂盒法的检出率分别为26.0%(13/50)、46.0%(23/50)和50.0%(25/50)。用蛋清液作为支架及细胞块试剂盒两种方法制作的细胞块,恶性细胞检出率高于直接离心法(P<0.05),细胞聚集度和细胞分布也优于直接离心法,细胞块和组织块免疫化学染色效果相似。结论用蛋清液作为支架及细胞块试剂盒两种方法制作的细胞块可提高胸腹水液基细胞学剩余标本的恶性细胞检出率,并可用于免疫细胞化学检测。

肺癌石蜡包埋组织及新鲜标本组织芯片制备方法学探讨

目的 :探讨和建立石蜡包埋组织和新鲜冷冻标本制备组织芯片的方法 ,以及在基因表达分析中的应用。方法 :选取肺癌石蜡包埋组织 90例 ,新鲜液氮冻存肺癌及癌旁肺组织 2 0例 ,常规切片HE染色下确定穿刺部位 ,石蜡包埋组织芯片制作按Kononem等方法 (1998) ,每例穿取 0 6mm组织柱 2条 ,制备 180点阵组织芯片 ;而新鲜组织芯片参考Fejzo等方法 (2 0 0 1) ,并略作改良 ,制备 5 0点阵组织芯片。所有组织芯片模块 ,经 4 μm切片、常规组织学染色和免疫组化染色 ,以评价其在形态学及基因表达分析中应用的可行性。结果 :180点阵石蜡包埋肺癌组织芯片 ,组织排列整齐 ,HE染色后组织形态可观察率在 98%以上 ;p5 3、c myc、bcl 2、bax和Ki 6 7等免疫组化染色后 ,肺癌组织可见不同程度的抗原表达 ,组织点阵的形态可观测率在 89%～ 95 %之间。新鲜组织芯片经HE染色后 ,形态可观测率和基因表达可分析率在 70 %左右。结论 :手工法组织芯片制备简便易行 ,使用组织少 ,不破坏原蜡块 ;实验均一性、可比性好 ,且节省试剂。与常规组织切片相比 ,2点 0 6mm组织可以获得原蜡块 95 %以上的信息 ,完全可以用于组织形态学和蛋白水平上的基因表达分析 ;新鲜组织芯片技术已经初步建立 ,但尚有待于进一步完善制备技术和mRNA分析方法。

普通甲醛固定石蜡包埋组织DNA提取方法的探讨

背景与目的:在国内,几乎所有的医院和科研机构均应用普通甲醛固定手术标本,但因为普通甲醛固定石蜡包埋组织中的DNA降解相对严重,从这些蜡块中提取高质量的DNA非常困难。本研究的目的是寻找从普通甲醛固定石蜡包埋组织中提取DNA的理想方法。方法:取我院2000年甲醛固定石蜡包埋的手术切除食管癌标本15例,分别以蛋白酶K消化和不同pH值下加热两种裂解方法裂解细胞,以酚氯仿抽提法提取其DNA;并在蛋白酶K消化法进行DNA提取的过程中,应用交叉设计对二戊烯或二甲苯脱蜡、37℃或56℃消化48h或72h、氯化钠盐析或酚氯仿抽提4种参数进行研究,所得DNA质量以电泳分析和PCR扩增结果判断。结果:蛋白酶K消化酚氯仿抽提法所得DNA产量(平均值为17.88μg)和质量均高于pH值为7～12条件下的加热提取法(P<0.05)。56℃消化所得的DNA质量明显优于37℃,而消化72h的DNA亦明显优于48h者。不同脱蜡方法和抽提方法对DNA的产量和质量影响不大,结论:以蛋白酶K消化氯化钠盐析法,从普通甲醛固定石蜡包埋组织中提取DNA质量较可靠。并且,应用蛋白酶K于56℃消化3天更能获得高质量和高产量的DNA。

甲醛固定石蜡包埋组织STR分型检测

目的评估10%甲醛固定石蜡包埋组织STR分型结果的影响因素。方法采用QIAGEN法、IQ法、Chelex法对2具新鲜尸体在尸检时常规制备的心、脑、肝、脾、肾、肺、胃、肠石蜡包埋组织进行DNA提取,用AmpFlSTR Identifiler试剂盒进行PCR扩增,在3100-Avant上完成片段分析。另外对15个案例中室温保存1～5年的心、肝、肺、肠存档石蜡包埋组织共56份采用同样的方法进行STR分型。以STR基因座检出率评估分型有效性。结果各种组织DNA片段均随着保存时间延长而持续降解,其中心、肺组织STR基因座检出率与保存时间存在线性相关。相同保存时间时,各种组织基因座检出率差异有统计学意义。其中以肺组织在不同保存时间中基因座检出率最高。结论在甲醛固定时间一定的条件下,存放时间、组织类型、DNA提取方法和PCR模板质量浓度是影响石蜡包埋组织STR分型的重要因素。

福尔马林固定石蜡包埋组织中DNA提取

由于甲醛介导的DNA损伤和石蜡对DNA提取的阻碍作用,使得常规DNA提取方法很难从福尔马林固定石蜡包埋组织(FFPET)中获取高质量的DNA。近年来,众多学者的研究表明,通过改良预处理方法,优化蛋白酶K消化作用,简化DNA提取步骤,纯化DNA提取物等,可以有效提高FFPET中提取的DNA质量,为FFPET的DNA分析奠定了基础。

石蜡包埋组织快速DNA提取方法在诊断病理PCR分析中的应用研究

目的改良石蜡组织DNA快速提取方法的流程,评价其提取效率,探讨其应用于病理诊断PCR分析的可行性。方法选取包括淋巴瘤及反应性病变的52例存档蜡块,以缓冲液煮沸方法快速提取DNA,并以传统酚-氯仿抽提法和试剂盒提取法作为对照,采用PCR反应扩增看家基因β-Globin以确定模板DNA质量,并通过测序确定结果的可靠性;同时根据临床病理诊断选择性的进行免疫球蛋白重链基因(IgH)或T细胞受体γ基因(TCRγ)重排检测。结果快速法提取DNA的浓度虽低于对照方法,但β-Globin扩增效率与对照方法相比差异没有统计学意义(P>0.05),对IgH和TCRγ重排的检出率与对照相比差异亦没有统计学意义(P均>0.05)。测序结果显示扩增片段与目的基因序列完全符合。DNA可在-20℃保存至少4周。结论快速DNA提取法可以从存档的石蜡包埋组织中提供足量的PCR反应模板,结果稳定可靠,且省时省力,在病理诊断PCR分析中具有应用价值。

从石蜡包埋肺癌组织提取RNA检测PML基因转录的研究

背景与目的甲醛固定石蜡包埋组织(FPE)包含大量临床病理信息,同时还含有大量的基因和蛋白物质可用来研究临床意义。本研究的目的是探讨从FPE中提取RNA的可能性,检测急性早幼粒细胞白血病基因(PML)在肺癌FPE中的转录表达。方法选取前期经免疫组织化学染色证实无PML蛋白表达的40例肺癌FPE,存档时间在60-85个月,5例新鲜冰冻肺腺癌组织作为对照;利用Trizol一步法提取RNA,经OD值检测验证RNA质量;RT-PCR检测PML的基因转录表达。结果自肺癌FPE和新鲜冰冻组织提取的RNA的OD比值在1.7-2.1。RT-PCR显示,40例FPE以及5例新鲜冰冻组织中,PML(295bp)以及β-actin(318bp)mRNA均有表达。结论利用Trizol一步法自FPE提取RNA进行RT-PCR,可有效扩增出300bp左右的产物;肺癌组织中PML蛋白存在转录后缺失。

聚羟基丙烯酸和Van-clear替代传统试剂在FISH法检测宫颈hTERC基因中的应用比较

目的:观察环保固定液(聚羟基丙烯酸、环保透明脱蜡液Van-clear单独或联合)替代传统固定液[4%(体积分数)中性缓冲甲醛、传统透明脱蜡液二甲苯]应用于荧光原位杂交(fluorescence in situ hybridization,FISH)法检测宫颈组织中人端粒酶核糖核酸组分(human telomerase RNA component,h TERC)基因扩增的差异。方法:收集2013年3月到2015年4月于中山市博爱医院妇科住院部送检的255例宫颈组织标本,同一病变部位切取4个样本,分为4组,命名为A、B、C、D组:A组采用4%中性缓冲甲醛固定、二甲苯透明脱蜡制作切片;B组采用聚羟基丙烯酸固定、二甲苯透明脱蜡制作切片;C组采用4%中性缓冲甲醛固定、Van-clear透明脱蜡制作切片;D组采用聚羟基丙烯酸固定、Van-clear透明脱蜡制作切片。采用FISH技术检测4组宫颈标本中h TERC基因。结果:在FISH法检测宫颈各级病变组织h TERC基因时,荧光显微镜下,A、B、C、D四组的组织轮廓和背景均清晰,探针定位准确,可见耀眼的红/绿荧光信号。B、C、D组与A组阳性率相比差异均无统计学意义(P>0.05),且FISH结果符合率高。结论:环保试剂聚羟基丙烯酸、Van-clear有潜在的可能单独或联合替代4%中性缓冲甲醛、二甲苯应用于FISH法检测宫颈h TERC基因。

miR-10b与乳腺癌侵袭及预后的相关性研究

背景与目的:miR-10b通过增加其下游转移基因RHOC的表达,影响肿瘤的转移和侵袭能力。本研究探讨miR-10b在乳腺癌石蜡组织中的表达及其与乳腺癌侵袭转移及预后的关系。方法:采用实时定量PCR(real-time qPCR)对60例乳腺癌患者原发肿瘤蜡块提取的RNA进行miR-10b表达水平的测定,MannWhitney-U非参数检验比较miR-10b表达水平和乳腺癌临床病理参数的关系,预后分析采用Kaplan-Meier生存分析和log-rank计算。结果:miR-10b表达水平与乳腺肿瘤的大小、淋巴结转移、组织学分级、激素受体状态、肿瘤病理分期、组织类型及是否复发等无明显关系(P>0.05)。Kaplan-Meier生存分析表明,miR-10b表达水平并不影响乳腺癌患者的生存期(P=0.485)。结论:miR-10b表达在乳腺癌侵袭转移中的作用有限,并不是影响预后的主要因素,尚不足以作为基于石蜡组织标本的乳腺癌预测预后指标。

大鼠眼部晶状体标本石蜡切片的改良制作方法

目的:探讨应用改良固定液和加压脱水法制作大鼠晶状体石蜡切片的效果。方法:将大鼠晶状体投入到由甲醛、冰醋酸和丙酮组成的改良固定液中固定后制成石蜡切片,经HE染色后在显微镜下观察。结果:晶状体组织结构完整清楚。结论:此改良固定液和加压脱水法优于其它方法,是适合大鼠眼部晶状体标本石蜡切片的一种有效方法。