scholarly journals Evaluation of PCR in Detection of Mycobacterium tuberculosis from Formalin-Fixed, Paraffin-Embedded Tissues: Comparison of Four Amplification Assays

1998 ◽  
Vol 36 (6) ◽  
pp. 1512-1517 ◽  
Author(s):  
Giulia Marchetti ◽  
Andrea Gori ◽  
Lidia Catozzi ◽  
Luca Vago ◽  
Manuela Nebuloni ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
False Negative ◽  
Pcr Amplification ◽  
Positive Culture ◽  
Single Copy ◽  
High Rate ◽  
Antigen Gene ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

We compared the sensitivities and specificities of four nested PCR assays for the detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues. Thirty-seven autopsy samples from human immunodeficiency virus-positive patients were analyzed: 15 were M. tuberculosis positive, 11 served as negative controls, and 11 were Ziehl-Neelsen positive without cultural confirmation of M. tuberculosis. Three genomic sequences (mtp40, 65-kDa antigen gene, and IS6110) with different molecular masses and numbers of repetitions within the M. tuberculosis genome were targeted. On the IS6110 sequence, two fragments of different sizes (106 and 123 bp, respectively) were amplified with two separate pairs of primers. The highest sensitivity rates were obtained by amplifying the highly repetitive IS6110 insertion sequence, and the different primers tested showed a sensitivity ranging from 80 to 87%. Amplification of the large 223-bp fragment of themtp40 sequence present in a single copy in the M. tuberculosis genome yielded a high rate of false-negative results, ranging from 66 to 80%. A poor sensitivity (from 47 to 60%) was also shown by PCR amplification of the 142-bp 65-kDa antigen gene. All the PCRs except that for the 65-kDa antigen gene showed a specificity of 100%. Moreover, different results were obtained with different dilutions of DNA, and DNA concentrations of 1 and 3 μg yielded the highest sensitivities depending upon which protocol was used. Application of the PCRs to the Ziehl-Neelsen-positive, culture-negative samples confirmed the sensitivities of the PCRs obtained with the control samples. In conclusion, PCR can successfully be used to detect M. tuberculosis from paraffin-embedded tissues and can be particularly useful in the validation of a diagnosis of tuberculosis in clinical settings in which the diagnosis is uncertain. However, the efficacy of PCR strictly depends on several amplification parameters such as DNA concentration, target DNA size, and the repetitiveness of the amplified sequence.

Inter-laboratory validation of PCR-based detection of Mycobacterium tuberculosis in formalin-fixed, paraffin-embedded tissues

2005 ◽  
Vol 447 (3) ◽  
pp. 573-585 ◽  
Author(s):  
Christiane Schewe ◽  
Torsten Goldmann ◽  
Marianne Grosser ◽  
Albert Zink ◽  
Karsten Schlüns ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

scholarly journals Selection of endogenous control genes for normalising gene expression data derived from formalin-fixed paraffin-embedded tumour tissue

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Tim A. D. Smith ◽  
Omneya A. AbdelKarem ◽  
Joely J. Irlam-Jones ◽  
Brian Lane ◽  
Helen Valentine ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Candidate Genes ◽  
Pcr Amplification ◽  
The Cancer Genome Atlas ◽  
Analysis Tool ◽  
Endogenous Control ◽  
Formalin Fixed Paraffin ◽  
Stability Ranking ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Abstract Quantitative real time polymerase chain reaction (qPCR) data are normalised using endogenous control genes. We aimed to: (1) demonstrate a pathway to identify endogenous control genes for qPCR analysis of formalin-fixed paraffin-embedded (FFPE) tissue using bladder cancer as an exemplar; and (2) examine the influence of probe length and sample age on PCR amplification and co-expression of candidate genes on apparent expression stability. RNA was extracted from prospective and retrospective samples and subject to qPCR using TaqMan human endogenous control arrays or single tube assays. Gene stability ranking was assessed using coefficient of variation (CoV), GeNorm and NormFinder. Co-expressed genes were identified from The Cancer Genome Atlas (TCGA) using the on-line gene regression analysis tool GRACE. Cycle threshold (Ct) values were lower for prospective (19.49 ± 2.53) vs retrospective (23.8 ± 3.32) tissues (p < 0.001) and shorter vs longer probes. Co-expressed genes ranked as the most stable genes in the TCGA cohort by GeNorm when analysed together but ranked lower when analysed individually omitting co-expressed genes indicating bias. Stability values were < 1.5 for the 20 candidate genes in the prospective cohort. As they consistently ranked in the top ten by CoV, GeNorm and Normfinder, UBC, RPLP0, HMBS, GUSB, and TBP are the most suitable endogenous control genes for bladder cancer qPCR.

scholarly journals Improved RT-PCR Amplification for Molecular Analyses with Long-term Preserved Formalin-fixed, Paraffin-embedded Tissue Specimens

2006 ◽  
Vol 54 (7) ◽  
pp. 773-780 ◽  
Author(s):  
Kiyohiro Hamatani ◽  
Hidetaka Eguchi ◽  
Keiko Takahashi ◽  
Kazuaki Koyama ◽  
Mayumi Mukai ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Rt Pcr ◽  
Molecular Analyses ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Tissue Specimens ◽  
Formalin Fixed

scholarly journals BCL-1 Gene Rearrangements in Iranian Non-Hodgkin Lymphoma Patients

2015 ◽  
Vol 8 (8) ◽  
pp. 108
Author(s):  
Manoush Tohidirad ◽  
Mehrdad Asghari Estiar ◽  
Azim Rezamand ◽  
Saeid Ghorbian ◽  
Sasan Andalib ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
Pcr Amplification ◽  
B Cell Lymphoma ◽  
Gene Rearrangements ◽  
Non Hodgkin Lymphoma ◽  
Formalin Fixed Paraffin ◽  
Mantle Cell ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

<p>In the present study, our aim was to assess the incidence of BCL-1 gene rearrangements in formalin-fixed paraffin embedded (FFPE) tissue in patients with non-Hodgkin lymphomas (NHL). The BIOMED-2 protocol was applied to assess the BCL-1 gene rearrangements in NHL patients. PCR amplification was carried out on FFPE in 100 patients with B-cell lymphoma including 89 cases with diffused large B-cell lymphoma (DLBCL) (15 cases under 18 years old) and 11 cases with mantle cell lymphoma (MCL). Out of the 100 patients, 19 cases (19%) were identified to have concurrent translocation involving BCL-1. The significant association was seen between BCL-1 gene rearrangements and the lymphomas in patients older than 55 years (P&lt;0.05). Out of 100 cases, 80 cases were positive and 20 cases were negative regarding CD20. No significant association was found between DLBCL lymphoma in patients under 18 years old and BCL-1 gene rearrangements (P&gt;0.05). In addition, the positive and negative expressions of LCA/CD45 marker were 76% (76/100) and 26% (26/100), respectively. Our findings revealed that BCL-1 gene rearrangement assays using BIOMED-2 protocol can be considered as a valuable approach in detection of the lymphomas.</p>

scholarly journals KIT Somatic Mutations and Immunohistochemical Expression in Canine Oral Melanoma

Animals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2370
Author(s):  
Ginevra Brocca ◽  
Beatrice Poncina ◽  
Alessandro Sammarco ◽  
Laura Cavicchioli ◽  
Massimo Castagnaro
Keyword(s):  
Pcr Amplification ◽  
Mean Value ◽  
Single Nucleotide ◽  
Oral Melanoma ◽  
Pathway Activation ◽  
Formalin Fixed Paraffin ◽  
Frequent Event ◽  
Formalin Fixed Paraffin Embedded ◽  
Exon 11 ◽  
Formalin Fixed

Canine oral melanoma (COM) is an aggressive neoplasm with a low response to therapies, sharing similarities with human mucosal melanomas. In the latter, significant alterations of the proto-oncogene KIT have been shown, while in COMs only its exon 11 has been adequately investigated. In this study, 14 formalin-fixed, paraffin-embedded COMs were selected considering the following inclusion criteria: unequivocal diagnosis, presence of healthy tissue, and a known amplification status of the gene KIT (seven samples affected and seven non-affected by amplification). The DNA was extracted and KIT target exons 13, 17, and 18 were amplified by PCR and sequenced. Immunohistochemistry (IHC) for KIT and Ki67 was performed, and a quantitative index was calculated for each protein. PCR amplification and sequencing was successful in 97.62% of cases, and no single nucleotide polymorphism (SNP) was detected in any of the exons examined, similarly to exon 11 in other studies. The immunolabeling of KIT was positive in 84.6% of the samples with a mean value of 3.1 cells in positive cases, yet there was no correlation with aberration status. Our findings confirm the hypothesis that SNPs are not a frequent event in KIT activation in COMs, with the pathway activation relying mainly on amplification.

Rapid real-time PCR for detection of Mycobacterium tuberculosis complex DNA in formalin-fixed paraffin embedded tissues: 16% of histological ‘sarcoid’ may contain such DNA

2014 ◽  
Vol 67 (12) ◽  
pp. 1084-1087 ◽  
Author(s):  
Güzin Surat ◽  
William A Wallace ◽  
Ian F Laurenson ◽  
Amie-Louise Seagar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Extraction Methods ◽  
Ffpe Tissue ◽  
Negative Results ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Dna Extraction Methods ◽  
Formalin Fixed

AimsTo investigate the diagnostic accuracy of IS6110 real-time PCR for detection of Mycobacterium tuberculosis complex (MTBC) in DNA extracted from formalin-fixed paraffin embedded (FFPE) tissues using two different methods. In the absence of material submitted for tuberculosis (TB) culture, MTBC detection in FFPE tissue can be an important aid to diagnosis.MethodsWe collected 144 FFPE tissue blocks (lung and lymph node) for IS6110 real-time PCR. Two DNA extraction methods (QIAamp FFPE tissue kit and NucliSENS easyMAG) were assessed within a general laboratory setting. PCR results were compared with histology and culture.ResultsIn the histological MTBC and culture MTBC (TB-positive) groups, 72.4% were IS6110-positive and 27.6% negative. IS6110-negative results were obtained from 98%, 61.5% and 84% of the histologically MTBC-negative (TB-negative) group, histologically TB/no culture group and sarcoidosis group, respectively. Review of 19 IS6110-positive patients in the latter three groups showed that 15 had clinical TB. Thirteen of 15 (86.7%) IS6110-positive patients in the histological TB/no culture group and 2 of 4 (50%) IS6110-positive patients in the sarcoidosis group were clinically diagnosed with TB which highlights the difficulty of a pathological diagnosis.ConclusionsIS6110 real-time PCR using easyMAG extracted DNA is a moderately sensitive, specific and rapid method for MTBC detection in FFPE material, but must be interpreted in the overall clinical context. PCR results can be available in around 5 h from FFPE specimen receipt, with minimal hands-on time.

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