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Background: Formalin-fixed paraffin-embedded (FFPE) tissue archives in hospitals, biobanks, and others offer a vast collection of extensive, readily available specimens for molecular testing. Unfortunately, the use of tissue samples for molecular diagnostic applications is challenging; thus, the forensic pathology FFPE tissue archives in Africa have been a largely unexploited genetic resource, with the usability of DNA obtainable from these samples being unknown.Intervention: The study, conducted from January 2015 to August 2016, determined the usefulness of FFPE tissue as a reliable source of genetic material for successful post-mortem molecular applications and diagnostics. Formalin-fixed paraffin-embedded tissue samples were collected and archived from autopsies conducted over 13 years in the forensic medicine department of the University of Pretoria (Pretoria, South Africa). Deoxyribonucleic acid from FFPE tissue samples and control blood samples was amplified by high-resolution melt real-time polymerase chain reaction before sequencing. The procurement parameters and fixation times were compared with the quantity and quality of the extracted DNA and the efficiency of its subsequent molecular applications.Lessons learnt: This study has shown that FFPE samples are still usable in molecular forensics, despite inadequate sample preparation, and offer immense value to forensic molecular diagnostics.Recommendations: FFPE samples fixed in formalin for more than 24 h should still be used in molecular diagnostics or research, as long as the primer design targets amplicons not exceeding 300 base pairs.
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DNA , Resolutions , Paraffin , Archives , Autopsy , Tissues , Pain Measurement , Genetic Testing , Polymerase Chain Reaction , Pathology, Molecular , Molecular Docking SimulationABSTRACT
Resumen Las alteraciones en la metilación de dinucleótidos CpG en regiones promotoras es uno de los mecanismos epigenéticos implicados en cáncer que tiene uso potencial como biomarcador. Su evaluación, a partir de tejidos fijados en formalina y embebidos en parafina (FFPE), representa un gran desafío dadas la degradación parcial, el entrecruzamiento y las bajas cantidades del DNA obtenido. En esta nota técnica, describimos un protocolo para el estudio del estado de metilación del promotor distal del proto-oncogén K-RAS, a partir de varias muestras obtenidas de dos tejidos FFPE de cáncer colorrectal con antigüedad de 11 años. Se empleó un protocolo de conversión con bisulfito alternativo al usual; se usó una DNA polimerasa modificada y una PCR anidada y se optimizó la secuenciación directa del DNA convertido con bisulfito. Este protocolo podría ser aplicado para determinar estados de metilación en otros genes y tipos de cáncer en tejidos FFPE.
Abstract Alterations in the methylation of CpG dinucleotides in promoter regions is one of the epigenetic mechanisms involved in cancer that has potential use as a biomarker. Its evaluation from formalin-fixed and paraffin-embedded (FFPE) tissues represents a great challenge given the partial degradation, crosslinking, and low amounts of the obtained DNA. In this technical note we describe a protocol for the study of the methylation status of the distal promoter of the K-RAS proto-oncogene from several samples obtained from two 11-years old FFPE tissues of colorectal cancer. An alternative bisulfite conversion protocol to the usual one was used; a modified DNA polymerase and a nested PCR were used and the direct sequencing of the converted DNA with bisulfite was optimized. This protocol could be applied to determine methylation states in other genes and types of cancer.
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Humans , Paraffin , Colorectal Neoplasms , DNA Methylation , Biomarkers , Polymerase Chain Reaction , GenesABSTRACT
BACKGROUND: Single staining is commonly performed for practical pathologic diagnoses. However, this method is limited in its ability to specify cellular morphology and immunophenotype and often requires consumption of limited tissue. This study aimed to describe an optimized protocol for multiple in situ hybridization (ISH) and immunohistochemistry (IHC). METHODS: The quality of multistaining was evaluated by carefully changing each step of ISH and IHC in an angioimmunoblastic T-cell lymphoma (AITL) case on a Ventana BenchMark XT automated immunostainer. The optimized protocols were also performed using another immunostainer and in 15 cases of five Epstein-Barr virus (EBV)–associated malignancies using formalin-fixed paraffin-embedded tissue. RESULTS: The quality of various ISH-IHC staining protocols was semi-quantitatively evaluated. The best EBV-encoded RNA (EBER)-ISH/double IHC staining quality, equivalent to single staining, was obtained using the following considerations: initial EBER-ISH application, use of protease and antigen retrieval reagent (cell conditioning 1 [CC1] treatment time was minimized due to impact on tissue quality), additional baking/deparaffinization not needed, and reduced dilution ratio and increased reaction time for primary antibody compared with single immunostaining. Furthermore, shorter second CC1 treatment time yielded better results. Multiple staining was the best quality in another immunostainer and for different types of EBV-associated malignancies when it was performed in the same manner as for the Ventana BenchMark XT as determined for AITL. CONCLUSIONS: EBER-ISH and double IHC could be easily used in clinical practice with currently available automated immunostainers and adjustment of reagent treatment time, dilution ratio, and antibody reaction time.
Subject(s)
Benchmarking , Diagnosis , Herpesvirus 4, Human , Immunohistochemistry , In Situ Hybridization , Lymphoma, T-Cell , Methods , Reaction Time , RNAABSTRACT
Objective The purpose of this study was to evaluate the quality of DNA from the formalin-fixed paraffin-embedded (FFPE) specimens of non-small cell lung cancer (NSCLC) after immunohistochemical staining and investigate DNA extraction by immunohistochemical staining of the specimens in small in number or difficult to obtain and the feasibility of related molecular tests. Methods We randomly collected 50 FFPE biopsy specimens of NSCLC in our Department of Pathology from June 2017 to December 2017 and sliced each into 12 sections, of which, 6 were directly subjected to DNA extraction (the control group) and the other 6 to immunohistochemical Envision two-step staining for DNA extraction (the experimental group). Then, we detected the mutations of the epidermal growth factor receptor (EGFR), kirsten rat sarcoma viral oncogene (KRAS) and V-raf murine sarcoma viral oncogene homolog B (BRAF) in all the DNAs extracted. Results No statistically significant differences were observed between the experimental and control groups in the DNA concentration and purity in the 50 FFPE biopsy specimens of NSCLC (P>0.05). Of the 50 NSCLC FFPE specimens of the experimental group, 20 (40%) showed the mutation of EGFR, 8 (16%) exhibited that of KRAS, and 5 (10%) manifested that of BRAF. In the other 50 specimens of the control group, 33 showed the mutations of EGFR, KRAS and BRAF. A 100% consistency was found in the results of detection between the experimental and control groups (P=0.000, Kappa=1.000). Conclusion High-quality DNA can be extracted after immunohistochemical staining from NSCLC FFPE specimens, especially those small in number or difficult to obtain, and can be used for downstream molecular analysis of target genes, which is a good method for specimen recycling and provides a solution for subsequent molecular test of scarce or difficult-to-obtain clinical samples.
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Objective: To compare the quality of RNA extracted from fresh and formalin-fixed paraffin-embedded (FFPE) brain tissues and to explore the long non-coding RNA (lncRNA) expression level. Methods: FFPE samples stored under various conditions and paired frozen brain tissues were collected and total RNA qualities were then detected. Amplification efficiency (AE) and expression stability of each RNA marker were calculated and analyzed based on real-time quantitative PCR. After selecting reference biomarkers, normalized △ Ct values of candidate makers within different amplicon size were measured to assess the possibility of lncRNA quantification in FFPE tissues. Results: The purity of RNA extracted from FFPE was relatively high, but the RNA integrity was lower than fresh samples. All biomarkers were successfully amplified and amplification efficiencies of long-chain RNA markers were correlated with amplicon sizes, sample treatment and preservation conditions, namely temperature and storage time. 5S, miR-9 and miR-125b achieved optimal AE and showed quite stable expression in all specimens, therefore they were chosen as control markers. Compared with fresh samples, the △ Ct values of only 2 lncRNA (HAR1F and MALAT1-L, whose amplicon size were both higher than 200 bp, respectively) increased in the FFPE samples kept in 4 ℃, while in FFPE tissues kept in room temperature, increments of the △ Ct values were significant for most target genes except for short amplicon markers (<60 bp), which showed consistently stable expression in all brain specimens. Conclusion: RNA integrity is affected by sample treatment and preservation conditions, but lncRNA expression levels in FFPE tissues can be accurately quantificated by using optimal amplicon sizes and considerable reference markers.
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Objective·To compare the quality of RNA extracted from fresh and formalin-fixed paraffin-embedded (FFPE) brain tissues and to explore the long non-coding RNA (IncRNA) expression level.Methods·FFPE samples stored under various conditions and paired frozen brain tissues were collected and total RNA qualities were then detected.Amplification efficiency (AE) and expression stability of each RNA marker were calculated and analyzed based on real-time quantitative PCR.After selecting reference biomarkers,normalized △ Ct values of candidate makers within different amplicon size were measured to assess the possibility of lncRNA quantification in FFPE tissues.Results·The purity of RNA extracted from FFPE was relatively high,but the RNA integrity was lower than fresh samples.All biomarkers were successfully amplified and amplification efficiencies of long-chain RNA markers were correlated with amplicon sizes,sample treatment and preservation conditions,namely temperature and storage time.5S,miR-9 and miR 125b achieved optimal AE and showed quite stable expression in all specimens,therefore they were chosen as control markers.Compared with fresh samples,the △ Ct values of only 2 lncRNA (HAR1F and MALAT1-L,whose amplicon size were both higher than 200 bp,respectively) increased in the FFPE samples kept in 4 ℃,while in FFPE tissues kept in room temperature,increments of the △ Ct values were significant for most target genes except for short amplicon markers (＜60 bp),which showed consistently stable expression in all brain specimens.Conclusion·RNA integrity is affected by sample treatment and preservation conditions,but IncRNA expression levels in FFPE tissues can be accurately quantificated by using optimal amplicon sizes and considerable reference markers.
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Objective To analyze formalin-fixed and paraffin-embedded(FFPE) sample by next generation sequencing(NGS) technology for high throughput DNA sequencing and related bioinformatics analysis,and to explore the reliability and clinic significance of miRNAs in the detection of sample from FFPE gastric cancer tissue.Methods FFPE and fresh frozen samples of the same tissue were excised from 2 cases of gastric cancer in 2015 and 2016.RNA was extracted from the samples and the small RNA library was isolated and constructed,and high throughput DNA sequencing was conducted with Illumina sequencing platform.The DNA sequencing results were analyzed by quantifying the expression of known miRNAs families sequence,and the expression profiling of known miRNAs was constructed.Spearman correlation analysis was performed on the miRNAs of the samples to assess the difference in the expression of miRNAs.Results The total RNA extracted from the fresh frozen sample had higher quality and complete RNA coverage,and the results showed that the 28S was 1.3-1.4 times of 18S.The RNA extracted from the FFPE sample showed different degrees of degradation,and the peaks of 28S and 18S were not significant.The spearman correlation of miRNAs expression profiling in frozen and FFPE samples was 0.90 and 0.86 respectively,and the expression levels of miRNAs in two samples were consistent and had significant correlation.Conclusion miRNAs in FFPE sample can retain certain level of integrity and can be used in tumor test by NGS.
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A placa aural é uma dermatopatia associada à quatro Equus caballus papillomavirus (EcPVs). Até o momento, o DNA de EcPVs não foi identificado em amostras de placa aural fixadas em formalina e embebidas em parafina (FFPE). O objetivo deste estudo foi otimizar um método para a detecção dos quatro tipos de EcPVs em 21 amostras FFPE usando a PCR. O DNA dos EcPVs foram detectados em 11 amostras (52.4%). O DNA do EcPV4 foi detectado em 38.1% (8/21) e do EcPV3 em 4.8% (1/21) das amostras. Coinfecção foi identificada em duas amostras (9.5%); EcPV4 e 5 foram detectados simultaneamente em uma amostra, enquanto o DNA dos EcPV4 e 6 foi detectado em outra. A especificidade do DNA dos papilomavírus equinos foi avaliada por sequenciamento gênico direto, que confirmou a especificidade dos produtos. A metodologia de PCR proposta possibilita o diagnóstico dos EcPV3, 4, 5 e 6 em amostras FFPE de placa aural equina.(AU)
Subject(s)
Animals , Analytic Sample Preparation Methods/veterinary , Human Papillomavirus DNA Tests/veterinary , Horses/virology , Paraffin , Polymerase Chain Reaction/veterinaryABSTRACT
Strongyloides stercoralis can cause systemic infection, termed strongyloidiasis, and gastrointestinal ulcer disease in immunocompromised patients. However, to our knowledge, there are no reported cases of comorbid gastric adenocarcinoma and S. stercoralis infection. Here, we report a case of an 81-year-old Korean man who presented with S. stercoralis infection coexisting with early gastric adenocarcinoma (T1aN0M0). S. stercoralis eggs, rhabditiform larvae, and adult females were observed in normal gastric and duodenal crypts. They were also observed in atypical glands representative of adenocarcinoma and adenoma. Preliminary laboratory tests revealed mild neutrophilic and eosinophilic leukocytosis. A routine stool test failed to detect rhabditiform larvae in the patient's fecal sample; however, S. stercoralis was identified by PCR amplification and 18S rRNA sequencing using genomic DNA extracted from formalin-fixed paraffin-embedded tissues. Postoperatively, the patient had a persistent fever and was treated with albendazole for 7 days, which alleviated the fever. The patient was followed-up by monitoring and laboratory testing for 4 months postoperatively, and no abnormalities were observed thus far. The fact that S. stercoralis infection may be fatal in immunocompromised patients should be kept in mind when assessing high-risk patients.
Subject(s)
Aged, 80 and over , Animals , Female , Humans , Male , Adenocarcinoma/complications , Albendazole/therapeutic use , Anthelmintics/therapeutic use , DNA, Helminth/chemistry , DNA, Ribosomal/chemistry , Endoscopy, Digestive System , Histocytochemistry , Korea , RNA, Ribosomal, 18S/genetics , Sequence Analysis, DNA , Stomach Neoplasms/complications , Strongyloides stercoralis/isolation & purification , Strongyloidiasis/complications , Treatment OutcomeABSTRACT
Strongyloides stercoralis can cause systemic infection, termed strongyloidiasis, and gastrointestinal ulcer disease in immunocompromised patients. However, to our knowledge, there are no reported cases of comorbid gastric adenocarcinoma and S. stercoralis infection. Here, we report a case of an 81-year-old Korean man who presented with S. stercoralis infection coexisting with early gastric adenocarcinoma (T1aN0M0). S. stercoralis eggs, rhabditiform larvae, and adult females were observed in normal gastric and duodenal crypts. They were also observed in atypical glands representative of adenocarcinoma and adenoma. Preliminary laboratory tests revealed mild neutrophilic and eosinophilic leukocytosis. A routine stool test failed to detect rhabditiform larvae in the patient's fecal sample; however, S. stercoralis was identified by PCR amplification and 18S rRNA sequencing using genomic DNA extracted from formalin-fixed paraffin-embedded tissues. Postoperatively, the patient had a persistent fever and was treated with albendazole for 7 days, which alleviated the fever. The patient was followed-up by monitoring and laboratory testing for 4 months postoperatively, and no abnormalities were observed thus far. The fact that S. stercoralis infection may be fatal in immunocompromised patients should be kept in mind when assessing high-risk patients.
Subject(s)
Aged, 80 and over , Animals , Female , Humans , Male , Adenocarcinoma/complications , Albendazole/therapeutic use , Anthelmintics/therapeutic use , DNA, Helminth/chemistry , DNA, Ribosomal/chemistry , Endoscopy, Digestive System , Histocytochemistry , Korea , RNA, Ribosomal, 18S/genetics , Sequence Analysis, DNA , Stomach Neoplasms/complications , Strongyloides stercoralis/isolation & purification , Strongyloidiasis/complications , Treatment OutcomeABSTRACT
Purpose To investigate the comparison of miR-106a in fresh and paraffin-embedded tissues and its expression in precan-cerous 1esions of gastric cancer. Methods Human gastric cancer tissues inc1uding 30 coup1es of fresh and 40 coup1es of paraffin-em-bedded samp1es were co11ected,quantitative rea1-time PCR was used to detect the expression of miR-106a in these two samp1es. Anoth-er paraffin-embedded samp1es inc1uding 20 cases of precancerous 1esions and 40 cases of gastric adenocarcinoma were a1so co11ected,in situ hybridization was used to assess the expression of miR-106a in these stages. Results The re1ative expression of miR-106a in fresh tissues was 3. 25 ± 1. 99,in paraffin-embedded tissues was 3. 18 ± 2. 14,indicating that the expression of miR-106a in these two sam-p1es has no statistica1 difference(P>0. 05),corre1ation ana1ysis showed that there was a significant corre1ation of miR-106a expression in these two samp1es(rs =0. 998,P<0. 001). The expression of miR-106a was detected in the stage of precancerous 1esions with the positive rate was 70%. The positive signa1s were 1ocated in dysp1astic epithe1ia1 ce11s and appeared as dark b1ue fine granu1es. The positive rate of miR-106a in gastric cancer tissues was 87. 5% and the frequency and extent increased and enhanced compared with pre-cancerous 1esions. Conclusion miR-106a has significant corre1ation in gastric cancer fresh tissues and paraffin-embedded tissues. Detection of miR-106a with paraffin tissues and its ear1y changes in prema1ignant 1esions can provide va1uab1e information for the ear1y diagnosis of gastric cancer.
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Background: Granulomatous lesions occur in tuberculosis (TB), other infections, toxic, allergic, and autoimmune diseases among others. In absence of a an acid-fast bacilli (AFB) confirmation of TB is necessary. Objective: To assess the efficacy of PCR for TB detection and to correlate with granuloma histology and AFB staining. Methods: We analyzed 380 fixed paraffin-embedded tissues (PETs) of granulomas with and without caseous necrosis; suppurative; sarcoidal; or of chronic nonspecific nature. Nested PCR-IS6110 for Mycobacterium tuberculosis complex (MTB) and a nested pan-Mycobacterium for the hsp65 gene were used for Mycobacterium spp detection. Results: PCR was more sensitive than AFB staining for all five catageories of granulomas: G1: PCR 71%, AFB staining 28%. G2: PCR 37%, AFB 8%. G3: PCR 17%, AFB staining 7%. G4: PCR 8%, AFB staining 4%. G5: PCR 6%, AFB staining 0%. Conclusions: Molecular diagnosis of TB using PCR-based testing is a fast, efficacious and sensitive method that increased the accuracy of PET histological diagnosis associated with granulomatous lesions.
Introducción: Lesiones granulomatosas ocurren en tuberculosis (TBC), otras infecciones, condiciones tóxicas, alérgicas y autoinmunes, entre otras. Con baciloscopia negativa, es necesario confirmar el diagnóstico de TBC. Objetivo: Evaluar la eficacia de la RPC para detectar TBC comparado con baciloscopia en relación a la histología del granuloma. Métodos: Analisis de 380 tejidos fijados en formalina e incluidos en parafina (TFFP) con diferentes tipos de granulomas: con necrosis caseosa; sin necrosis caseosa; supurativo; sarcoidal; a cuerpo extraño/inespecífico. Utilizamos RPC anidada-IS6110 para detección del complejo Mycobacterium tuberculosis (MTB) y una pan-RPC anidada-hsp65 para Mycobacterium spp. Resultados: La detección de TBC mediante RPC fue significativamente superior a baciloscopia en los cinco tipos de granuloma: G1: RPC 71%, baciloscopia 28%; G2: RPC 37%, baciloscopia 8%; G3: RPC 17%, baciloscopia 7%; G4: RPC 8%, baciloscopia 4%; G5: RPC 6%, baciloscopia 0%. Conclusión: El diagnóstico de TBC por RPC es un método rápido, eficaz y de gran sensibilidad, que aumenta la precisión del diagnóstico diferencial de lesiones granulomatosas de TFFP procesados rutinariamente en histopatología.
Subject(s)
Adolescent , Adult , Child , Child, Preschool , Female , Humans , Infant , Male , Young Adult , DNA, Bacterial/genetics , Granuloma/microbiology , Mycobacterium tuberculosis/genetics , Tuberculosis/diagnosis , Diagnosis, Differential , Formaldehyde , Granuloma/diagnosis , Paraffin Embedding , Polymerase Chain Reaction , Predictive Value of Tests , Sensitivity and Specificity , Staining and Labeling , Tissue FixationABSTRACT
Background: Colorectal cancer is one of the most common causes of death in the world and third and fourth most common cancer among men and women in Iran respectively. Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that shows over expression in epithelial tumors and regulates important processes in tumorigenesis. Incidence and characteristics of colorectal cancer are based on the geographic region and race. Aim: In this research work, the over expression of EGFR in formalin fixed paraffin-embedded (FFPE) colorectal cancer tumor tissue of patients was studied. Materials and Methods: Fifteen FFPE colorectal cancer tumor tissues (10 women and 5 men; 25-65 years old and stage IV) and 15 non-patients (nine women and six men; 25-65 years old) that were collected during 2006-2012. EGFR gene expression level was analyzed by real-time quantitative reverse transcriptase polymerase chain reaction (PCR). All PCR reactions were performed in triplicate for both target gene and internal control (18s ribosomal ribonucleic acid) with the 2−ΔΔCT method. Gene expression differences in patients and controls were evaluated with t-test. Results: The results were showed EGFR gene over expression in 12 (80%) of 15 patients. There was a statistically significant difference in the prevalence of EGFR expression between patients and control (P < 0.05). Conclusion: Our results demonstrated EGFR gene over expression in colorectal cancer tumor tissue compared with controls.
Subject(s)
Colorectal Neoplasms/genetics , Fixatives , Formaldehyde , Gene Expression/genetics , Gene Expression Profiling , Humans , Male , Paraffin Embedding , ErbB Receptors/genetics , Reverse Transcriptase Polymerase Chain Reaction , Tissue FixationABSTRACT
Aims: To identify and differentiate mycobacterium spp. in archival formalin-fixed, paraffinembedded tissue by PCR to supply additional differential diagnostic method for Mycobacterium infections, where tuberculosis had been tested for by histopathological methods but without culturing. Place and Duration of Study: Department of Medical Laboratory Sciences, Department of Pathology and Microbiology (Faculty of Medicine) and King Abdulla University Hospital, between January 2004 and July 2010. Methodology: Fifty-six extra-pulmonary specimens of formalin-fixed, paraffin-embedded tissue obtained from patients showing granulomatus inflammation and/or other histopathologic features. Specimens were analyzed by a hemi-nested PCR assay targeting the gene encoding for 16S ribosomal RNA, which is common to all mycobacteria spp. The PCR positive specimens were amplified by a touch-down PCR targeting a fragment in the insertion sequence IS6110 specific to Mycobacterium tuberculosis complex. Results were compared to acid fast bacilli stain and with histopathology of each specimen. Results: M. tuberculosis complex DNA was detected in 27 (48.2%) specimens, and non-tuberculous mycobacteria in four specimens, compared to 10 (18%) specimens that were positive by acid fast staining. The positive cases were observed more in the lymph nodes, and pleural specimens. Conclusions: This study is among few studies to use the touch-down PCR assay as a promising auxiliary tool for the diagnosis of extra-pulmonary tuberculosis in archival tissue specimens. It could be used in conjunction with routine laboratory tests (e.g., cultures, acid fast staining) and clinical criteria of the patient to increase the accurate diagnosis of such cases. It is also recommended that culture be routinely done for all tuberculosis suspected cases.
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Objective To explore the expression and significance of hsa-miR-181a (miR-181a) in can-ceration progression of endometrial carcinoma. Methods A total of 75 formalin-fixed paraffin-embedded tissue specimens were studied in this study , of which , 13 were normal endometrium , 18 were endometrial hyperplasia , 44 were endometrial carcinoma. After total RNA had been extracted , real-time PCR was applied to detect the ex-pression level of miR-181a in endometrial tissue in each group. Results miR-181a expression in formalin-fixed paraffin-embedded tissue specimens can be detected. Expression of miR-181a in endometrial carcinoma was high-er than that in endometrial hyperplasia , its expression in endometrial hyperplasia was also higher than that in normal endometrium, and the difference was statistically significant (P < 0.05). The expression of miR-181a in endometrial carcinoma was associated with FIGO stages (P < 0.05). Conclusion The up-regulation of miR-181a expression in women with endometrial carcinoma may play the role of oncogenes. Abnormal expression of miR-181a is probably associated with the occurrence and development of endometrial carcinoma.
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Introducción: Helicobacter pylori es considerado uno de los principales agentes causales de gastritis crónica, úlcera péptica y neoplasias gástricas malignas en humanos. Objetivo: evaluar el uso de la reacción en cadena de la polimerasa para la identificación de H. pylori y sus genotipos en tejidos gástricos con neoplasias malignas embebidos en parafina. Material y Métodos: se estudiaron secciones de 5 bloques de parafina procedentes de 5 pacientes mexicanos con neoplasias gástricas malignas. Se realizaron coloraciones de rutina y especiales de anatomía patológica, así como la técnica de la reacción en cadena de la polimerasa para la detección del microorganismo y sus genotipos. Resultados: la técnica de la reacción en cadena de la polimerasa identificó a este agente infeccioso en todos los bloques analizados en correspondencia con su detección a través de las técnicas histológicas. Esta metodología permitió demostrar una variabilidad genética del patógeno en las muestras analizadas según los genotipos vacA y cagA. Conclusiones: la reacción en cadena de la polimerasa podría ser un método eficaz en la identificación del H. pylori en tejidos gástricos con neoplasias malignas embebidos en parafina. Esta se perfila como una estrategia atractiva para realizar estudios de epidemiología molecular y permitirá establecer posibles asociaciones de genotipos/subtipos del microorganismo con variables clínicas, epidemiológicas y de manejo del paciente.
Introduction: Helicobacter pylori is considered one of the main causal agents of chronic gastritis, peptic ulcer and gastric malignant neoplasms in humans. Objective: to evaluate polymerase chain reaction for identification of Helicobacter pylori and its genotypes in paraffin embedded gastric tissues with malignant neoplasms. Material and Methods: sections of five paraffin blocks from five patients with gastric malignant neoplasms were studied. They were analyzed through routine and special stains of pathological anatomy, as well as the polymerase chain reaction technic for microorganism and genotypes detection. Results: the infectious agent was identified in all of the analyzed blocks through the polymerase chain reaction technic in correspondence with its detection through histologic techniques. This methodology showed a genetic variability of the pathogen in the analyzed samples in respect to vacA and cagA genotypes. Conclusions: the polymerase chain reaction could be an efficacious method for the identification of H. pylori in paraffin embedded gastric tissues with malignant neoplasms. It is projected as an attractive strategy for performing studies of molecular epidemiology and the establishment of possible associations between genotypes/subtypes of the microorganism and clinic or epidemiologic variables, and patient handling.
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Objective: To optimize the process of making cell blocks by residual pleural and peritoneal effusions, and to explore its value in pathological diagnosis. Methods: Totally 150 residual pleural and peritoneal effusion specimens of thinprep cytologic test (TCT) were evenly divided into 3 groups according to cytopathological diagnosis. Specimens in the three groups were processed by direct centrifugal method, egg white as the bracket method and cell block test method. The detection rate of malignant cells, distribution status and morphological features of cells on the cellular sections were compared between the three different method s, and the immunohistochemical staining results were compared between the cell block and tissue block. Results: The TCT yielded a detection rate of malignant cells of 31. 3% (47/150), and examination of the cell block in this study yielded a detection rate of 40. 7% (61/150), with the rates of direct centrifugal method, egg white as the bracket method and cell block test method being 26. 0% (13/50), 46. 0% (23/50) and 50. 0% (25/50), respectively. The detection rates of malignant cells in egg white as the bracket method and cell block test method groups were significantly higher than that in the direct centrifugation method (P<0. 05); in addition, the former two groups also had better cell aggregation and distribution. The immunohistochemical staining results of cell blocks were also similar to those of tissue blocks. Conclusion: The cell blocks processed by egg white as the bracket method and cell block test method can improve the detection rate of malignant cells in residual pleural and peritoneal effusion, and the blocks can be used for immunocytochemistry staining.
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Objective To evaluate the value of paraffin-embedded sediments section combined with routine smear in diagnosis of malignant tumor from hydrothorax and hydroperitoneum.Methods 150 cases of hydrothorax and hydroperitoneum were analyzed by using the methods of paraffin-embedded section and routine smear respectively.Results Among 150 cases with hydrothorax and hydroperitoneum,120 cases were certified malignancy by clinical source and other methods.The positive rate was 62.5% (75/120) by routine smear and 84.2% (101/120) by paraffin-embedded sediments section respectively,while the positive rate was 94.2% (113/120) by the combined examination of the two and the difference was significant compared with routine smear and paraffin-embedded sediments section respectively. At the same time, It is easier for us to distinguish adenocarcinoma cell from mesothelial cell by means of detecting the expression of CEA,CK,CAL and VIM immunohistochemically.Conclusion The combined examination of the two methods along with immunohistochemistry can increase diagnosis rate significantly and worth of being generalized.
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Objective To establish a high quality genomic DNA preparation method from formalin fixed and paraffin embedded tissues (FFPET) by integrating previous DNA extraction methods. Methods We combined classical genomic DNA extraction methods and commercially available DNA affinity column, replaced the de-waxing by dimethylbenzene with water-bath, designed a fast and improved genomic DNA preparation method. We also extracted genomic DNA from paraffin embedded cervical cancer tissues, and checked the quality of DNA by agarose gel electrophoresis and polymerase chain reaction detection. Results The improved genomic DNA extraction method combined the advantages of the water-bath de-waxing and DNA affinity column, making it possible to get high quality genomic DNA from paraffin embedded cervical cancer tissues, and especially efficient to recover genomic DNA fragments larger than 20 kb. Conclusion The improved DNA extraction method is fast and convenient to recover high quality genomic DNA from paraffin embedded tissues.
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Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10 percent non-buffered or 10 percent buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10 percent non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.
O desenvolvimento e a padronização de métodos confiáveis para a detecção de Mycobacterium tuberculosis em amostras clínicas é um objetivo importante nos laboratórios de todo o mundo. Neste trabalho, fragmentos de pulmão e baço de paciente que morreu com o diagnóstico de tuberculose miliar foram usados para avaliar a influência do tipo de fixador e dos protocolos de fixação e inclusão em parafina na performance da PCR. Fragmentos de tecido foram fixados por quatro h a 48 h, usando formalina não tamponada a 10 por cento ou formalina tamponada a 10 por cento e incluídos em parafina pura ou misturada a cera de abelha. As amostras foram submetidas a PCR para amplificação do gene da beta-actina humana e, separadamente, para amplificação da sequência de inserção IS6110, específica do complexo M. tuberculosis. O resultado da amplificação do gene da beta-actina foi positivo em todas as amostras. Não foram gerados amplicons na PCR-IS6110 em amostras de tecido pulmonar fixadas usando formalina não tamponada a 10 por cento e incluídas em parafina com cera de abelha. Em conclusão, fatores inibitórios combinados interferiram na detecção de M. tuberculosis em material de arquivo. É importante controlar estes fatores inibitórios para poder implementar o diagnóstico molecular em laboratórios de patologia.