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 ...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.展开更多
BACKGROUND Mycobacterium mucogenicum(M.mucogenicum)belongs to the group of rapidly growing Nontuberculous mycobacteria.This microorganism is associated with a wide spectrum of infectious diseases.Due to a low detectio...BACKGROUND Mycobacterium mucogenicum(M.mucogenicum)belongs to the group of rapidly growing Nontuberculous mycobacteria.This microorganism is associated with a wide spectrum of infectious diseases.Due to a low detection rate or the time required for conventional culture methodology,a rapid and broad-spectrum method is necessary to identify rare pathogens.CASE SUMMARY A 12-year-old immunocompetent girl presented with painful masses for five months.The first mass was found in the right upper quadrant of the abdomen,and was about 1 cm×1.5 cm in size,tough but pliable in texture,with an irregular margin and tenderness.An abscess gradually formed and ulcerated with suppuration of the mass.Three new masses appeared on the back one by one.Chest computed tomography showed patchy and streaky cloudy opacities in both lungs.Needle aspiration of the abscess was performed,but the smear and conventional culture were negative,and the pathological examination showed no pathogens.We then performed next-generation sequencing using a formalinfixed,paraffin-embedded specimen to identify the pathogen.A significantly high abundance of M.mucogenicum was detected.The patient’s abscesses gradually decreased in size,while inflammation in both lungs improved following 12-wk of treatment.No recurrence was observed four months after the end of the one-year treatment period.CONCLUSION Next-generation sequencing is a promising tool for the rapid and accurate diagnosis of rare pathogens,even when using a formalin-fixed,paraffin-embedded specimen.展开更多
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 preope...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.展开更多
Stains are applied to impart contrast to the tissue and identify particular features of interest. However, the use of synthetic dyes as staining reagents has been associated with significant human health challenges an...Stains are applied to impart contrast to the tissue and identify particular features of interest. However, the use of synthetic dyes as staining reagents has been associated with significant human health challenges and pollution of the ecosystem. These developments have necessitated a shift towards using natural dyes that are eco-friendlier and readily available. We investigated the staining reaction patterns of teak tree leaves (Tectona grandis) dye extracts and explored their suitability as a cytoplasmic stain in micromorphological assessments. Dye extracts were prepared using acetone, methanol, and ethanol as solvents from air-dried (under shade) teak tree young leaves. The dye extracts were applied as a counterstain and evaluated against eosin in formalin-fixed paraffin-embedded (FFPE) bovine tissue sections at varying concentrations and different staining times. Teak tree leaves (Tectona grandis) dye extracts produced relatively varying staining intensities of reddish-brown cytoplasmic coloration when used on bovine tissue at different concentrations and staining times comparable to eosin and with blue-purple hematoxylin nuclear stain. The present study showed that Tectona grandis leaf dye extracts provide an excellent cytoplasmic staining pattern and can be used as an alternative counterstain in routine H&E staining techniques.展开更多
目的探讨实时荧光定量PCR(qPCR)在结核病理诊断中的应用价值。方法回顾性分析1323例疑似结核病的石蜡包埋组织qPCR结果,并与临床诊断结果、抗酸染色结果进行对比分析。结果1323例病例中1073例有明确的诊断结果,其中结核阳性率为68.22%(7...目的探讨实时荧光定量PCR(qPCR)在结核病理诊断中的应用价值。方法回顾性分析1323例疑似结核病的石蜡包埋组织qPCR结果,并与临床诊断结果、抗酸染色结果进行对比分析。结果1323例病例中1073例有明确的诊断结果,其中结核阳性率为68.22%(732/1073)。1073例病例中,qPCR阳性率为45.39%、敏感性为0.663、特异性为0.994,肺、胸膜、骨、淋巴结、肠中qPCR敏感性分别为0.706、0.676、0.437、0.678、0.625,特异性分别为0.993、1.000、1.000、1.000、1.000,手术组织阳性率和敏感性分别为48.42%、0.692,均高于活检组织,且差异有统计学意义(P<0.05)。755例同时具有qPCR结果和抗酸染色结果的病例中,qPCR与抗酸染色法符合率75.36%(95%CI 72.17~78.30),qPCR阳性率(43.44%,328/755)高于抗酸染色的阳性率(21.5%,162/755)(P<0.05),qPCR的AUC(95%CI)=0.837(0.817~0.859)高于抗酸染色AUC(95%CI)=0.656(0.634~0.678)(P<0.05),敏感性高于抗酸染色(0.677 vs 0.327)(P<0.05)。结论qPCR在结核病的病理诊断中具有良好的应用价值,联合抗酸染色可以更准确地进行鉴别诊断。展开更多
本研究以10例甲醛固定石蜡包埋组织(FFPET)作为疑难检材,将其制成蜡膜,分别以二甲苯-Chelex100法、脱蜡液-Chelex100法、脱蜡液-Vazyme FastPure FFPE DNA Isolation试剂盒(简称FP试剂盒)、脱蜡液-北京天根石蜡包埋组织基因组提取试剂盒...本研究以10例甲醛固定石蜡包埋组织(FFPET)作为疑难检材,将其制成蜡膜,分别以二甲苯-Chelex100法、脱蜡液-Chelex100法、脱蜡液-Vazyme FastPure FFPE DNA Isolation试剂盒(简称FP试剂盒)、脱蜡液-北京天根石蜡包埋组织基因组提取试剂盒法(简称天根试剂盒)提取DNA,进行紫外分光光度计测定(浓度、纯度)及短串联重复序列(STR)分型检测(23个常染色体),通过比较分析FFPET在4种不同提取方法中的提取质量、STR分型质量和检出率,从而寻求一种高效简便、经济实用、方便快捷的甲醛固定石蜡包埋组织中DNA提取方法。结果表明:应用4种方法提取的石蜡包埋组织蜡膜DNA经紫外分光光度计测定后显示,应用2种Chelex100法提取的DNA浓度高于2款试剂盒,试剂盒提取的DNA的纯度优于另外2种方法,以上差异均具有统计学意义(P<0.05);FP试剂盒提取DNA的平均浓度和纯度均优于天根试剂盒,无显著性差异(P>0.05);STR分型普遍存在不同基因座和同一基因座不同等位基因之间的扩增不均衡,呈现前高后低状态;STR平均检出率为FP试剂盒>天根试剂盒>Chelex100法,2种Chelex100法STR基因座检出率比较无显著差异(P>0.05),但分别和另外2种方法试剂盒方法相比有显著差异(P<0.05),FP试剂盒和天根试剂盒法相比有显著差异(P<0.05)。因此,在涉及甲醛固定石蜡包埋组织这类疑难检材的法医DNA鉴定案件时,脱蜡液-Vazyme FastPure FFPE DNA Isolation试剂盒法优于其余3种提取DNA方法。展开更多
基金Supported by the Jilin Science & Technology Development Plan,China(No.201201060)the Scientific Research Foundation of Jilin Province,China(No.20100942)+1 种基金the Fund of Developing and Reforming Community of Jilin Province,China(No.2010-1928)the Health Scientific Research Foundation of Jilin Province,China(Nos.2009z081,2010Z083)
文摘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.
基金Supported by the Clinical Research Foundation of the Third Affiliated Hospital of Sun Yat-Sen University,No.YHJH201904National Science and Technology Major Project,No.2018ZX10302204.
文摘BACKGROUND Mycobacterium mucogenicum(M.mucogenicum)belongs to the group of rapidly growing Nontuberculous mycobacteria.This microorganism is associated with a wide spectrum of infectious diseases.Due to a low detection rate or the time required for conventional culture methodology,a rapid and broad-spectrum method is necessary to identify rare pathogens.CASE SUMMARY A 12-year-old immunocompetent girl presented with painful masses for five months.The first mass was found in the right upper quadrant of the abdomen,and was about 1 cm×1.5 cm in size,tough but pliable in texture,with an irregular margin and tenderness.An abscess gradually formed and ulcerated with suppuration of the mass.Three new masses appeared on the back one by one.Chest computed tomography showed patchy and streaky cloudy opacities in both lungs.Needle aspiration of the abscess was performed,but the smear and conventional culture were negative,and the pathological examination showed no pathogens.We then performed next-generation sequencing using a formalinfixed,paraffin-embedded specimen to identify the pathogen.A significantly high abundance of M.mucogenicum was detected.The patient’s abscesses gradually decreased in size,while inflammation in both lungs improved following 12-wk of treatment.No recurrence was observed four months after the end of the one-year treatment period.CONCLUSION Next-generation sequencing is a promising tool for the rapid and accurate diagnosis of rare pathogens,even when using a formalin-fixed,paraffin-embedded specimen.
文摘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.
文摘Stains are applied to impart contrast to the tissue and identify particular features of interest. However, the use of synthetic dyes as staining reagents has been associated with significant human health challenges and pollution of the ecosystem. These developments have necessitated a shift towards using natural dyes that are eco-friendlier and readily available. We investigated the staining reaction patterns of teak tree leaves (Tectona grandis) dye extracts and explored their suitability as a cytoplasmic stain in micromorphological assessments. Dye extracts were prepared using acetone, methanol, and ethanol as solvents from air-dried (under shade) teak tree young leaves. The dye extracts were applied as a counterstain and evaluated against eosin in formalin-fixed paraffin-embedded (FFPE) bovine tissue sections at varying concentrations and different staining times. Teak tree leaves (Tectona grandis) dye extracts produced relatively varying staining intensities of reddish-brown cytoplasmic coloration when used on bovine tissue at different concentrations and staining times comparable to eosin and with blue-purple hematoxylin nuclear stain. The present study showed that Tectona grandis leaf dye extracts provide an excellent cytoplasmic staining pattern and can be used as an alternative counterstain in routine H&E staining techniques.
文摘目的探讨实时荧光定量PCR(qPCR)在结核病理诊断中的应用价值。方法回顾性分析1323例疑似结核病的石蜡包埋组织qPCR结果,并与临床诊断结果、抗酸染色结果进行对比分析。结果1323例病例中1073例有明确的诊断结果,其中结核阳性率为68.22%(732/1073)。1073例病例中,qPCR阳性率为45.39%、敏感性为0.663、特异性为0.994,肺、胸膜、骨、淋巴结、肠中qPCR敏感性分别为0.706、0.676、0.437、0.678、0.625,特异性分别为0.993、1.000、1.000、1.000、1.000,手术组织阳性率和敏感性分别为48.42%、0.692,均高于活检组织,且差异有统计学意义(P<0.05)。755例同时具有qPCR结果和抗酸染色结果的病例中,qPCR与抗酸染色法符合率75.36%(95%CI 72.17~78.30),qPCR阳性率(43.44%,328/755)高于抗酸染色的阳性率(21.5%,162/755)(P<0.05),qPCR的AUC(95%CI)=0.837(0.817~0.859)高于抗酸染色AUC(95%CI)=0.656(0.634~0.678)(P<0.05),敏感性高于抗酸染色(0.677 vs 0.327)(P<0.05)。结论qPCR在结核病的病理诊断中具有良好的应用价值,联合抗酸染色可以更准确地进行鉴别诊断。
文摘本研究以10例甲醛固定石蜡包埋组织(FFPET)作为疑难检材,将其制成蜡膜,分别以二甲苯-Chelex100法、脱蜡液-Chelex100法、脱蜡液-Vazyme FastPure FFPE DNA Isolation试剂盒(简称FP试剂盒)、脱蜡液-北京天根石蜡包埋组织基因组提取试剂盒法(简称天根试剂盒)提取DNA,进行紫外分光光度计测定(浓度、纯度)及短串联重复序列(STR)分型检测(23个常染色体),通过比较分析FFPET在4种不同提取方法中的提取质量、STR分型质量和检出率,从而寻求一种高效简便、经济实用、方便快捷的甲醛固定石蜡包埋组织中DNA提取方法。结果表明:应用4种方法提取的石蜡包埋组织蜡膜DNA经紫外分光光度计测定后显示,应用2种Chelex100法提取的DNA浓度高于2款试剂盒,试剂盒提取的DNA的纯度优于另外2种方法,以上差异均具有统计学意义(P<0.05);FP试剂盒提取DNA的平均浓度和纯度均优于天根试剂盒,无显著性差异(P>0.05);STR分型普遍存在不同基因座和同一基因座不同等位基因之间的扩增不均衡,呈现前高后低状态;STR平均检出率为FP试剂盒>天根试剂盒>Chelex100法,2种Chelex100法STR基因座检出率比较无显著差异(P>0.05),但分别和另外2种方法试剂盒方法相比有显著差异(P<0.05),FP试剂盒和天根试剂盒法相比有显著差异(P<0.05)。因此,在涉及甲醛固定石蜡包埋组织这类疑难检材的法医DNA鉴定案件时,脱蜡液-Vazyme FastPure FFPE DNA Isolation试剂盒法优于其余3种提取DNA方法。
