石蜡包埋脱钙骨组织中结核杆菌DNA不同提取方法比较

目的 探讨石蜡包埋的10%硝酸脱钙骨组织中简易有效的结核杆菌DNA提取方法并进行验证.方法 以20例石蜡包埋、10%硝酸脱钙的坏死性肉芽肿炎疑为骨结核的组织标本为研究对象,常规法直接采用德国QIAGEN公司的QIAamp DNA FFPE Tissue Kit方法提取DNA,而改良法中加入QIAamp DNA Micro Kit试剂盒中的carrier RNA,以两种方法分别提取20例标本中结核杆菌DNA,通过荧光定量PCR,比较常规试剂盒提取法和改良提取法对结核杆菌检测结果的影响.结果 改良法提取的DNA[（35.32±3.48）ng/μl]比常规法[（6.68±3.72）ng/μl]提取的DNA显著增多（P＜0.05）,改良法提取的DNA经荧光定量PCR检测结核杆菌的阳性率（55%）明显高于常规法（15%,P＜0.01）.结论 改良法提取石蜡包埋脱钙骨组织中结核杆菌DNA具有高效简便的特点,是较为理想的方法,值得在puncture和极微小石蜡样本中推广.     

福尔马林固定的猴淋巴组织中HIV-1DNA的扩增

本文尝试从福尔马林固定的猴淋巴组织提取HIV-1CNA，并用PCR方法进行扩增和对扩增产物HIV-1DNA进行检测。结果认为，采取从福马林固定标本中提取PNA的方法是可行的。在提取DNA中，采用过量TE（ph9.0）长时间浸泡标本。并适当提高蛋白酶K（100μq/ml）和SDS（2%-3%）的浓度，可有助于DNA的提取，同时适当提高提取液的PH值（PH9.0），对DNA具有一定的保护作用。     

慢性乙肝患者石蜡包埋肝组织中HBV cccDNA定量检测方法的建立

目的建立慢性乙肝患者微量石蜡包埋肝组织共价闭合环状DNA(HBV cccDNA)定量检测方法。方法以37例慢性乙肝患者甲醛固定石蜡包埋肝组织为研究对象,提取肝组织HBV DNA,经不降解质粒的ATP依赖的DNA酶(PSAD)消化后,利用滚环扩增加跨缺口实时荧光PCR扩增技术检测肝组织中HBV cccDNA含量,以β-actin为内参照。通过已知浓度的模板DNA进行梯度稀释鉴定该方法的灵敏度,并对该方法进行批内和批间重复性检测。应用该方法分析37例慢性乙肝患者肝组织HBV cccDNA与血清总HBV DNA、肝组织总HBV DNA的关系,以及肝组织HBV cccDNA和HBV DNA与血清HBeAg表达之间的关系。结果成功建立了微量石蜡包埋肝组织HBV cccDNA的定量检测方法,该方法具有较好的特异性、灵敏度和稳定性。HBeAg阳性者肝组织中cccDNA含量明显高于HBeAg阴性者,HBV cccDNA水平与血清总HBV DNA水平(R2=0.48,P=0.042)及肝组织总HBV DNA水平(R2=0.63,P=0.001)呈正相关。结论该方法可特异灵敏地定量检测微量石蜡包埋组织切片中的HBV cccDNA。     

肺泡灌洗液联合肺组织PCR方法检测结核杆菌DNA对肺结核早期诊断的意义

目的探讨肺泡灌洗液(BALF)联合肺组织聚合酶链反应(PCR)方法检测结核杆菌DNA对肺结核早期诊断的意义。方法对104例结核杆菌痰阳性和确诊肺结核的痰菌阴性患者经纤维支气管镜采集BALF及活检肺组织进行石蜡包埋,对标本采用PCR方法检测结核杆菌DNA。结果 104例患者BALF中PCR检测结核杆菌DNA阳性92例,阳性率为88.46%;肺组织石蜡包埋检测结核杆菌DNA阳性98例,阳性率为94.23%。两组阳性率比较,差异无统计学意义(P>0.05)。结论 BALF联合肺组织PCR方法检测结核杆菌DNA有较高的敏感性及特异性,对肺结核病的早期诊断有重要临床意义。     

人胆管癌组织中CD44 mRNA的表达与胆管癌的关系

1　资料与方法1 1　一般资料　胆管癌组织标本 16例 ,均为腺癌 ;其中肝门部胆管癌 9例 ,中段胆管癌 3例 ,下段胆管癌 4例 ;高分化癌 10例 ,低分化癌 6例 ;伴有淋巴结转移者 5例。另取 10例胆管良性病变作为对照。所有标本均经病理证实。1 2　标本处理　标本在手术切除后即刻送到实验室 ,每份标本分成 2份 :1份立即置液氮中冷冻储存备用 ;另 1份用 4%的甲醛固定 ,石蜡包埋切片 ,常规组织学染色 ,光镜观察 ,病理诊断。1 3　方法及步骤　ＲＴ ＰＣＲ分析 :每份标本取 2 μｇ总ＲＮＡ先行逆转录反应 ,随机引物被用于ｃＤＮＡ第一条链的合成。Ｐ…     

P~(53)蛋白在胃癌中表达的意义

通过检测胃癌中Ｐ５３蛋白的异常表达情况,探讨Ｐ５３蛋白在胃癌诊断中的潜在价值及与淋巴结转移间的关系。１　材料和方法１－１　标本及处理　２１例胃癌中,７例早期胃癌,１４例进展期胃癌,均作胃切除术。男性１５例,女性６例,年龄３９～６８岁。全部标本都经１０％磷酸—福尔马林缓冲液固定,石蜡包埋,连续切片,片厚５μｍ。１－２　试剂　鼠抗人Ｐ５３单克隆抗体ＤＯ７及ＳＰ试剂盒（福州迈新公司）。１－３　染色、结果判断　按ＳＰ试剂盒说明ＳＰ法检测,其中Ｐ５３染色用微波抗原修复法处理。阴性对照以ＰＢＳ及鼠血…     

两种全血基因组DNA提取方法的比较

目的比较两种不同的DNA提取方法提取人全血基因组DNA,对DNA提取率、纯度以及PCR扩增的效果影响.方法分别用传统的酚、氯抽提法和改良法两种不同的方法,提取人全血基因组DNA.结果两种方法提取的DNA纯度用光吸收比值为指标检测A280/260值为:1.45和1.82,提取DNA总量分别为187μg/ml和260 μg/ml.用0.8%琼脂糖凝胶电泳鉴定改良法制备的DNA效果较好,PCR扩增效果有差异.结论用改良法提取的DNA总量明显较高,提取效率高,PGR扩增的效果好,是全血基因组DNA提取中首选考虑的方法.     

中性粒细胞的两种简易标记方法及在计量中的应用

本文在急性肺损伤中性粒细胞(PMN)计量的实验研究中,应用过碘酸雪夫氏(PAS)组织化学染色法标记石蜡切片肺组织中(PMN),取得了满意的效果,提高了PMN计量的准确性。并与粒细胞组合抗原(GAA)免疫组化染色作了比较,对二者的适用性进行了探讨。1 材料与方法 实验用Wistar大鼠内毒素(LPS,E.Coli0111 B4.USA)性急性肺损伤和急性阑尾炎标本,组织经10％福乐马林固定,石蜡包埋、切片(片厚5μm)。参照张贤良等的方法     

介绍痰标本做石蜡包埋制片法

多年来我们将痰标本制成切片后观察 ,取得了理想的结果 ,介绍如下。1　材料与方法　　选病人新鲜晨痰 ,最好在医生指导下咯深部痰。咯前先漱口 ,挑选带血丝的痰液。如痰液稀薄 ,可酌情置少许蛋清混合。痰用滤纸包埋 ,固定和常规脱水透明切片。　　混合固定液用 1 0 %福尔马林和 95 %的酒精各半。酒精脱水后经丙酮再行脱水 ,硬化标本 ,二甲苯I、II透明各 1 0min ,余同常规石蜡制片。2　讨论　　肺部疾病做痰常规细胞学检查 ,痰检多用涂片方法 ,由于涂片检查缺点多 ,对痰标本石蜡包埋制片检查切片薄 ,均匀一致 ,染色清晰 ,易观察 ,切片长期…     

粘膜活检病理分析纤维胃镜1073例

近年来,纤维胃镜活检已在临床上广泛应用,利用病理学诊断明确各种胃炎、胃溃疡、胃腺瘤等胃部疾病,特别是对胃癌的早期诊断具有相当重要的作用,现就1073例胃粘膜活检标本病理分析如下。1材料和方法1.1一般资料取1991年至2005年纤维胃镜活检标本1073例,组织采用10%福尔马林固定、石蜡包埋、常规HE染色、光镜观察。个别组织经网络纤维染色。由于条件有限,未做胃癌有关的免疫组化标记。病理检查结果:其中各种胃炎671例、非典型性增生53例、胃溃疡112例、胃腺瘤45例、胃癌192例。1.2方法从胃镜材料看,按文献分类法[1]。诊断慢性浅表性胃炎、胃…     

DNA identification of formalin-fixed organs is affected by fixation time and type of fixatives: using the AmpF l STR(R) Identifiler(R) PCR Amplification Kit

Personal identification using DNA typing of formalin-fixed tissue is very important in the forensic sciences. However, few studies have been conducted to determine the detection limit of DNA typing of formalin fixation time in samples using the AmpF?STR(?) Identifiler(?) PCR Amplification Kit (Identifiler Kit). We collected samples from five cadavers submitted for forensic autopsies, and fixed them either in a 10% formalin solution, or in a 10% neutral-buffered formalin solution. The amount of template DNA for polymerase chain reaction (PCR) amplification and the detection limit of DNA typing for the Identifiler Kit were determined. When tissues were fixed in 10% formalin, 10 ng of DNA template was required for successful genotyping even after three-hour fixation and 100 ng was required after one-week fixation for PCR amplification. However, when tissues were fixed in 10% neutral-buffered formalin, the required amount of DNA template was 1 ng for a fixation time of three hours to three days and 125 ng for three months. Fixation time in neutral-buffered formalin was longer for successful PCR than that in formalin solution. Dropout was more common with increasing formalin fixation time. These results suggest that neutral-buffered formalin is preferred to formalin for fixation of tissues if they are to be subjected to DNA typing and that tissues fixed with neutral-buffered formalin can be used for DNA typing using the Identifiler Kit unless the fixation time exceeds one month.     

A simple Duplex-PCR to evaluate the DNA quality of anthropological and forensic samples prior short tandem repeat typing

Typing of DNA from ancient or otherwise highly degraded material, e.g. formalin fixed tissues, can be difficult, time consuming and costly. Very often, genetic typing is not possible at all. We present an inexpensive and easy to use Duplex-PCR that amplifies a 164 bp fragment specific for nuclear DNA together with a 260 bp mitochondrial DNA fragment and that can be employed as a pretest prior to short tandem repeat (STR) typing. All together, we analyzed DNA from 20 ancient bones, 20 formalin fixed tissues and 20 other forensic samples in different concentrations. Each sample that failed in the presented Duplex-amplification was also negative for STR typing, while samples that showed strong and clear signals in the Duplex-PCR led to reproducible genetic profiles using the multiplex kits AmpFLSTR Identifiler and Powerplex ES. The Duplex-PCR worked as a reliable indicator of DNA quality in the sample.     

Detection of genetic variation in KCNQ1 gene by high-resolution melting analysis in a prospective-based series of postmortem negative sudden death: comparison of results obtained in fresh frozen and formalin-fixed paraffin-embedded tissues

High-resolution melting (HRM) analysis is a recently developed molecular technique proved to be applicable for detection of genetic variation, notably in sudden cardiac death. In certain circumstances, especially in postmortem genetic investigations, the formalin-fixed and paraffin-embedded (FFPE) tissues are the only DNA source available. The present study aimed to develop HRM assays, optimized for the analysis of FFPE tissues, to detect sequence variations in KCNQ1 exons in a prospective population-based series of postmortem negative sudden death and to compare the results between the paired freshly frozen and FFPE tissue samples simultaneously obtained from the same case. The analyses were conducted in each case of sudden death involving cases younger than 35 years with no significant morphological anomalies particularly with no cardiac structural disease and with negatives toxicological investigations. HRM analysis was successfully optimized for 13 of the 16 exons of the KCNQ1 gene. All mutated samples were correctly identified by HRM whatever the type of tissue tested. However, for FFPE samples, HRM indicated more positive samples than classical sequencing, used in parallel, due to the degradation of DNA by formalin fixation. This is the first postmortem study of KCNQ1 mutation detection with HRM on DNA extracted from FFPE samples with adapted protocol. Despite the false-positive detection, we concluded that the use of HRM as a screening method with FFPE samples to analyze KCNQ1 mutations can reduce the number of sequencing reactions and, thus, results in substantial time and cost savings.     

A fatal case of poisoning by lidocaine overdosage--analysis of lidocaine in formalin-fixed tissues: a case report

The death of a 76-year-old man with heart disease as a result of the injection of an excessive dose of lidocaine is presented. The patient was given 5 ml of 10% lidocaine hydrochloride (500 mg) intravenously instead of 2.5 ml of 2% lidocaine hydrochloride (50mg) in order to treat repeated paroxysmal ventricular arrhythmia. Immediately following the injection the patient had tonic clonic seizures and complete cardiopulmonary arrest followed. Although resuscitation attempts once successfully restarted his pulse and spontaneous respiration, the patient died on the eighth day after the injection. Toxicological examinations were carried out on the tissues obtained at the time of autopsy and which had been fixed in formalin solution for 40 days, and lidocaine was detected in each tissue examined. The concentrations were (ng/g or ml): parietal lobe, 308.0; occipital lobe, 208.7; temporal lobe, 318.0; frontal lobe, 223.2; cerebellum 200.9; pons 285.7; liver, 109.5; kidney 52.2; skeletal muscle 127.0; and formalin solution 8.4. In an experiment on rats we determined the concentration changes of lidocaine in formalin fixed tissues. The concentrations of lidocaine in these tissues significantly decreased to 1/3-1/4 from the original. This data shows that the cause of death was poisoning by lidocaine overdose.     

Determination of ABO genotypes with DNA extracted from formalin-fixed,paraffin-embedded tissues

Summary The gene encoding the specific glycosyltransferases which catalyze the conversion of the H antigen to A or B antigens shows a slight but distinct variation in its allelic nucleotide sequence and can be divided into 6 genotypes when digested with specific restriction enzymes. We extracted DNA from formalin-fixed, paraffin-embedded tissues using SDS/proteinase K treatment followed by phenol/chloroform extraction. The sequence of nucleotides for the A, B and O genes was amplified by the polymerase chain reaction (PCR). DNA fragments of 128 by and 200 by could be amplified in the second round of PCR, using an aliquot of the first round PCR product as template. Degraded DNA from paraffin blocks stored for up to 10.7 years could be successfully typed. The ABO genotype was deduced from the digestion patterns with an appropriate combination of restriction enzymes and was compatible with the phenotype obtained from the blood sample.     

皮瓣内微血管过氧化物酶组织化学染色方法的实验研究

本文选S—D大白鼠鼠背皮瓣为材料，对内源性过氧化物酶染色显示微血管的方法进行实验研究。结果以10％缓冲甲醛（PH7．2—7．4）在4℃固定6—12小时，冰冻切片40um，联苯胺液显示微血管效果最好，该方法不需灌注，对血管无扩张，破裂作用，无人为改变，可用于皮瓣微血管形态学研究和定量分析。     

Ultrasound-accelerated formalin fixation improves the preservation of nucleic acids extraction in histological sections

The aim of the present study was to examine an ultrasound-accelerated fixation technique that reduces the exposure time of the tissue to formaldehyde with respect to the analysis of nucleic acids. We extracted and analysed DNA and RNA from three series of autopsy specimens from five routine cases. Two series were shortly fixed in 4% buffered formalin (15 and 30 min, respectively) whilst being irradiated with high-frequency, high-intensity ultrasound. The last series (control) was routinely fixed in 4% buffered formalin for 24–48 h without irradiation. Although sufficient amounts of DNA of good quality could be extracted and amplified from all three series, the peak heights obtained from conventional fixation were smaller and allele dropout occurred more often, especially for the longer amplicons. RNA yield depended on the fixation procedure, i.e. the shortest fixation time led to the highest RNA yield and quality. No differences were observed with regard to the quality of the histological slides both with conventional and immunohistochemical staining methods. Keeping in mind the increasing need for molecular diagnosis, this fixation technique can be useful to ensure stable quality of nucleic acids in archived autopsy specimens.     

Assessment of microbial DNA extraction methods of cadaver soil samples for criminal investigations

Gravesoil beneath decomposing cadavers undergoes substantial biochemical changes that have the potential to aid in PMI estimation and identification of clandestine gravesites. The use of DNA extraction methods is necessary for culture-independent downstream molecular applications such as PCR and next-generation sequencing. In this study, a comparison of four methods was performed for cadaver-soil collected beneath the heads and feet of 11 cadavers decaying in a natural setting. The four methods isolated 3.6–263 ng/μl of genomic DNA as determined by optical density analysis. The purity of the extracted DNA according to A_260/280 and A_260/230 ratios was determined. The A_260/280 and A_260/230 ratios were 1.24–1.97 and 0.27–2.12, respectively. The optical density at 320 nm was measured for humic acid quantification of the lysates from the method that provided the most efficient removal of humic acid. The results demonstrated that this method provided a 98% reduction of humic acid. PCR of 16S rRNA genes followed by gel electrophoresis was performed. The statistical analysis revealed a significant correlation between the yields and days on/in the soil using a phenol-chloroform method for soil collected at the head and feet. No earlier published work has extensively elucidated the efficacy of DNA extraction methods for DNA obtained from cadaver-soil.     

Quantitative and qualitative analysis of DNA extracted from postmortem muscle tissues

Summary DNA extracted from 33 postmortem muscle specimens was analyzed using MZ 1.3, a hypervariable minisatellite probe, as well as locus-specific minisatellite probes (g3, MS1 and MS43). After storage at –25°C for 10 months, DNA from all the samples was partially (approximately 21% of total DNA) degraded even when autopsy was performed 1 day post mortem. However, more than 90% of DNA samples up to at least 3 days post mortem were suitable to obtain good restriction fragment length polymorphism (RFLP) patterns. When small strips of specimen were stored for 8 days at room temperature in moist chambers, approximately 42% of total DNA was degraded. Only 30% of these DNA samples still showed good RFLP patterns. However, no obvious relation between qualities of DNA analyzed by detection of RFLP and quantities of total and high-MW DNA became apparent. A case of familial relationship was ascertained by DNA fingerprints. Since DNA of good quality can be recovered from muscle tissues in large quantities, DNA extraction from muscle tissues and detection of RFLP patterns should be very useful for individual identification in autopsy cases.