Survivin expression as a prognostic marker for breast cancer

SUMMARY OBJECTIVE: Due to the speed of development observed in breast cancer, several studies aimed at discovering new biomarkers have been carried out in order to arrive at an early diagnosis. As survivin plays a fundamental role in the evasion of apoptosis in tumor cells, the aim of this study was to verify the expression profile of the survivin gene in paraffin-embedded breast tumor samples and associate it with the clinical characteristics of the patients. METHODS: This is a cross-sectional study, for which 100 tumor samples were obtained from cancer patients treated throughout the year 2019 at Instituto de Mama do Cariri (Juazeiro do Norte, in the state of Ceará). This study included women over 30 years old who had confirmed breast cancer through anatomopathological examination but excluded those with non-neoplastic breast comorbidities, other neoplasms, or chronic diseases. Survivin gene expression was assessed by quantitative polymerase chain reaction. RESULTS: The expression of survivin is associated with the lack of expression of estrogen (p=0.027) and progesterone (p>0.0005) receptors. It means that survivin expression is higher in patients in which labeling was absent for estrogen receptor and progesterone receptor. CONCLUSION: Our data reinforce that survivin expression is higher in estrogen receptor-patients, thus representing an additional prognostic tool.

N-butanol combined with turpentine instead of xylene for paraffin embeddin / 解剖学报

Objective To look for a tissue paraffin embedding method that can replace xylene transparent agent. Methods The mixture of N-butanol and turpentine was used to replace the role of anhydrous ethanol and xylene in the process of conventional paraffin embedding. The brain, kidney, stomach, liver and duodenum of multiple cerebral infarction model rats were embedded in paraffin. Finally, the new dehydration procedure was evaluated according to the effect of paraffin section, HE staining and immunohistochemical result . Results The mixture of N-butanol and turpentine could replace not only the dehydration effect of anhydrous ethanol but also the transparency effect of xylene in the conventional paraffin embedding process. The tissue sections treated with the mixture of N-butanol and turpentine were smooth, and the tissue did not become brittle or hard; After HE staining, the nucleus and cytoplasm of the new dehydrated tissue were distinct, and the chromaticity, color and transparency of the tissue were not different from those of the conventional dehydration procedure; Immunohistochemical staining was performed on different tissues of rats, and the comparison result were no different from conventional embedded tissue immunohistochemical staining. Conclusion N-butanol combined with turpentine for tissue dehydration can not only avoid the toxic effect of xylene on human, but also reduce the tissue damage caused by excessive dehydration of anhydrous alcohol.

Quantitative Expression of RNA from Frozen Organs and Formaldehyde-fixed and Paraffin-embedded Tissues / 法医学杂志

Objective Quantitative analysis and comparison of the expression of ribonucleic acid （RNA） from frozen organs and formaldehyde-fixed and paraffin-embedded （FFPE） tissues. Methods Frozen specimens of human brain, myocardium and liver tissues as well as FFPE samples at different postmortem intervals were collected and mass concentration of RNA was extracted and detected. Reverse transcription-quantitative polymerase chain reaction （RT-qPCR） technology was used to analyze the amplification efficiency and relative expression of each RNA marker. Results The mass concentration and integrity of RNA extracted from FFPE samples were relatively low compared with frozen specimens. The amplification efficiency of RNA markers was related with RNA species and the length of amplification products. Among them, glyceraldehyde-3-phosphate dehydrogenase （GAPDH） and β-actin （ACTB） with relatively long amplification products failed to achieve optimal amplification efficiency, whereas 5S ribosomal RNA （5S rRNA） achieved ideal amplification efficiency and showed quite stable expression across various tissues, therefore it was chosen as internal reference marker. The expression quantity of GAPDH and ACTB in frozen specimens with longer postmortem intervals and in FFPE samples with relatively long amplification products was decreased. The expressions of tissue-specific microRNAs （miRNAs）, GAPDH and ACTB with relatively short amplification products had consistency in the same tissues and FFPE samples. Conclusion Through standardizing the RT-qPCR experiment, selecting the appropriate RNA marker and designing primers of appropriate product length, RNA expression levels of FFPE samples can be accurately quantified.

Comparison between poly hydroxy acrylic acid and Van-clear replacing the tradi-tional reagents to detect the cervical hTERC genes by adopting FISH technique / 北京大学学报(医学版)

Objective:To observe the difference of the human telomeres RNA component (hTERC) genes’amplification in the cervical tissue by applying the environment-friendly fixative poly hydroxy acrylic acid and the transparent dewaxing solution Van-clear separately or jointly to replace the traditional fixative 4% (volume fraction)neutral buffered formalin and the conventional transparent dewaxing solu-tion xylene in the use of fluorescence in situ hybridization (FISH)for detection.Methods:In the study, 255 cases of cervical tissue specimens submitted by the Department of Gynecology in Zhongshan Boai Ho-sipital were collected from Mar.2013 to Apr.2015.Four samples were taken from the same lesion site. All the cases were divided into 4 groups and named group A,B,C,and D.Group A used 4% neutral buffered formalin fixed and xylene dewaxing to make slices.Group B used poly hydroxy acrylic fixed and xylene dewaxing to make slices.Group C used 4% neutral buffered formalin fixed and Van-clear trans-parent to make slices.Group D used poly hydroxy acrylic fixed and Van-clear transparent dewaxing to make slices.The amplification of hTERC genes in the four groups of cervical specimens was also detected by FISH technique.Results:When the hTERC genes were detected by FISH method under the fluore-scence microscope,it was obvious that the tissue profile and the background of group A,B,C and D were all clear.The probe was fixed in the accurate position so that the bright red or green fluorescence signals were easily found in these four groups.Compared with the positive rate of group A,there was no statistical significance in that of group B,C and D (P>0.05).At the same time,the coincidence rate of the FISH results was high,which showed that the new environment-friendly reagent had no significant difference in the detection of cervical hTERC genes by FISH technique.Conclusion:It is possible for the environment-friendly reagent poly hydroxy acrylic acid and Van-clear to replace 4%neutral buffered for-malin and xylene separately or jointly to detect the cervical hTERC genes by adopting FISH technique.

Sentinel lymph node biopsy in rats. Comparison between paraffin and frozen section analysis

PURPOSE: To simulate a lymph node metastasis in an animal model using activated carbon, assess their identification in frozen section analysis and compare with histopathological examination in paraffin. METHODS: Thirty two adult female rats were used. They received the carbon injection on its hind legs. Half of the rats was sacrificed on day one, and the other half after 21 days. Thus, 64 lymph nodes were dissected and split longitudinally. One half of the lymph node was sent immediately to frozen section analysis. The other half was fixed in 10% formaldehyde to be cut in paraffin. Slides were divided into quadrants and classified by the presence of carbon in these four quadrants_ They were also classified by the carbon staining intensity. RESULTS: Comparing the slides obtained in the first day and 21 days, there was a tendency of carbon to spread over time, but without statistical significance. The intensity did not alter over time. CONCLUSION: There was no concordance between the two methods of pathological analysis, however the actived carbon was seen in all lymph nodes. .

The application value of HPV E6/E7 mRNA detection in paraffin-embedded samples for cervical cancer screening / 肿瘤研究与临床

Objective To explore the application value of detecting HPV E6/E7 mRNA in cervical cancer screening and prognosis.Methods bDNA technology was used to detect the HPV E6/E7 mRNA levels in 301 cases of paraffin-embedded cervical samples that were grouped based on pathological diagnosis.Statistic differece between multiple groups were assessed by chi-square segmentation method.Results The positive detection rate of HPV E6/E7 mRNA is higher in cervical cancer group than that in CIN1-3 groups and cervicitis group,which were 94.29 ％ (66/70),30.30 ％ (20/66),50.00 ％ (19/38),59.46 ％ (44/74),5.66 ％ (3/50),respectively (all P ＜ 0.004 5).The HPV E6/E7-positive rates in all three CIN groups were higher than that in cervicitis group (P ＜ 0.004 5).The positive rates in CIN2 and CIN3 group were higher than that in CIN1 group (P ＜ 0.004 5).HPV E6/E7 mRNA levels in cervical cancer group were higher than those in CIN groups and cervicitis group (16 508.730±8 741.402,929:337±460.880,3 239.681±1 447.399,5 286.224±2 933.445,4.941±2.263,respectively,all P ＜ 0.05).CIN groups had higher copy numbers of HPV E6/E7 mRNA than cervicitis group (P ＜ 0.05).More copies of HPV E6/E7 mRNA were detected in CIN3 group than those in CIN1 group (P ＜ 0.05).Conclusions The positive rate and expression levels of HPV E6/ E7 mRNA was related to the severity of cervical diseases.HPV E6/E7 mRNA detection in paraffin-embedded samples have a high clinical application value in predicting the risk of cervical cancer.It' s an effective measure for cervical cancer screening and follow-up.

Preparation of Compact Agarose Cell Blocks from the Residues of Liquid-Based Cytology Samples

BACKGROUND: Inevitable loss of diagnostic material should be minimized during cell block preparation. We introduce a modified agarose cell block technique that enables the synthesis of compact cell blocks by using the entirety of a cell pellet without the loss of diagnostic material during cell block preparations. The feasibility of this technique is illustrated by high-throughput immunocytochemistry using high-density cell block microarray (CMA). METHODS: The cell pellets of Sure- Path residues were pre-embedded in ultra-low gelling temperature agarose gel and re-embedded in standard agarose gel. They were fixed, processed, and embedded in paraffin using the same method as tissue sample processing. The resulting agarose cell blocks were trimmed and represented on a CMA for high-throughput analysis using immunocytochemical staining. RESULTS: The SurePath residues were effectively and entirely incorporated into compact agarose cell buttons and embedded in paraffin. Sections of the agarose cell blocks revealed cellularities that correlated well with corresponding SurePath smears and had immunocytochemical features that were sufficient for diagnosis of difficult cases. CONCLUSIONS: This agarose-based compact cell block technique enables preparation of high-quality cell blocks by using up the residual SurePath samples without loss of diagnostic material during cell block preparation.

Comparación de métodos de extracción de ADN en tejidos parafinados y utilidad para amplificación por PCR / Comparison of DNA extraction methods from formalin-fixed paraffin sections for RCP amplification

Los tejidos fijados en formol e incluidos en parafina son una fuente de material para hallazgos moleculares en el ámbito clínico y científico, demostrándose que el ADN extraído de éstos, es adecuado para amplificación a través de la reacción en cadena de la polimerasa (RCP). En este estudio, se ensayaron tres métodos de extracción de ADN en tejidos incluidos en parafina, con el objetivo de comparar la eficiencia de estos para obtener ADN adecuado, además se analizó su utilidad en amplificación por RCP. Se emplearon tres muestras, correspondientes a una biopsia de pulmón, legrado endometrial y ganglio linfático, todas fijadas en formaldehido al 10% e incluidas en parafina. Utilizándose tres métodos diferentes de extracción de ADN (extracción por salting out, método modificado de Sambrook y kit comercial) El ADN obtenido se cuantificó por espectrofotometría, además se realizó electroforesis en gel de agarosa al 1%, para comprobar si el ADN era de buena calidad y se realizó RCP para el exón 3 del gen caveolina 1. Todos los métodos dieron como resultado una buen producto de ADN genómico, observándose mayor cantidad y pureza en los métodos de salting out y kit comercial, asimismo se obtuvo amplificación del producto esperado por estos dos métodos, no hubo buenos resultados con el ADN extraído por el método modificado "Preparation of Genomic DNA from Mouse Tails y Other Small samples, según Sambrook". El ADN obtenido a partir de tejidos FFIP puede ser amplificado por varios métodos, entre estos, la extracción por salting out es útil y con poca toxicidad, permite obtener ADN de buena calidad para amplificación por RCP.

Formalin-fixed and paraffin-embedded tissues are a source of important molecular findings in clinical and scientific, demonstrating that the DNA extracted from these is suitable for amplification by polymerase chain reaction (PCR). In this study, we tested three methods of DNA extraction, in order to compare the efficiency of these DNA for RCP amplification. Three samples were used, corresponding to a lung biopsy, endometrial curettage and lymph node, all fixed in 10% formaldehyde and embedded in paraffin. Three different methods were used for DNA extraction (extraction by salting out, modified Sambrook method and commercial kit) The DNA obtained was analyzed by spectrophotometry, and gel electrophoresis was performed in 1% agarose to check if the DNA was amplifiable. PCR was performed for exon 3 of caveolin-1 gene. All methods resulted in a good product of genomic DNA, obtaining more quality and purity in the salting out and commercial kit methods. Also, we obtained amplification of the product by these two methods, without favorable results with the DNA extracted by the modified "Preparation of Genomic DNA from Mouse Tails and Other Small samples, according to Sambrook et al." The DNA obtained from FFPE can be amplified by several methods, among them, salting out extraction is an easy, effective and low toxicity for obtaining good quality DNA for PCR amplification.

Detection of Varicella-Zoster Virus (VZV) by Nested Polymerase Chain Reaction / 임상검사와정도관리

BACKGROUND: Polymerase chain reaction (PCR) is known as a sensitive and specific method for the detection of varicella-zoster virus (VZV). Nested PCR is reliably used than conventional PCR to increase the sensitivity and specificity, especially in cases of small sized tissue samples. METHODS: We detected VZV infection in tissues from 111 patients using conventional PCR and nested PCR. Ninety-one cases of fresh tissues and twenty cases of formalin-fixed paraffin-embedded (FFPE) tissues were evaluated. The column method or home made lysis buffer method was used for the DNA extraction of fresh tissues and FFPE tissues. RESULTS: Among total 111 cases, VZV were detected in 62 (55.9%) cases by conventional PCR and 79 (71.2%) cases by nested PCR. The detection rate of nested PCR was higher than conventional PCR (1.27 folds). In 91 cases of fresh tissues, 56 (61.5%) were positive by conventional PCR and 68 (74.7%) by nested PCR. In 20 cases of FFPE tissues, 6 (30%) were positive by conventional PCR and 11 (55%) by nested PCR. The detection rate of VZV was increased by nested PCR both in fresh tissues (1.21 folds) and FFPE tissues (1.83 folds). CONCLUSIONS: Nested PCR is the more sensitive method than conventional PCR for the detection of VZV infection in tissues regardless of DNA extraction methods, especially for the small sized FFPE tissues.

Application of pathological diagnosis by rapid paraffin sections for biopsy in the diagnosis and treatment of cervical diseases / 中华妇产科杂志

Objective To evaluate the application of pathological diagnosis by rapid paraffin sections in the diagnosis and treatment of cervical diseases. Methods A total of 176 cases from our hospital between September 2009 and January 2010 with abnormal cervical cancer screening (including abnormal cytology result and high-risk HPV continuous positive) were randomly divided into 2 groups. Eighty-seven cases of them whose biopsy were got by Belinson forceps under the direction of colposcopy with rapid paraffin sections by ultrasonic histopathological rapid processor and BT transparent agents were selected as group A, while 89 cases with conventional paraffin sections were selected as group B. The production time and quality for paraffin sections were analyzed in the two groups. Those diagnosed as cervical intraepithelial neoplasia (CIN)Ⅱ or even worse and some special patients with CIN Ⅰ in the two groups received surgery, including loop electrosurgical procedure (LEEP) ,cold knife conization (CKC),.hysterectomy or radical hysterectomy.Tissue obtained after surgery was sent for routine pathological examination. If the results of postoperative routine pathological examination were inconsistent with the rapid or routine biopsy pathological examination,the heavier results were regard as the final diagnoses. The pathological results and diagnose accordance rates were recorded and compared between group A and group B. Results The quality of sections in two groups were all satisfied or basically satisfied to meet the diagnostic requirements. There were statistically significant difference in average production time between group A and B (40 minutes vs 24 hours, P 0. 05). Conclusion Bapid paraffin sections technology is safe, accurate and economical for rapid pathological diagnosis of cervical diseases, which is worthy for being widely used in hospitals.