Detection of hepatitis "C" virus in formalin-fixed liver tissue by nested polymerase chain reaction.

Interpretation of antibody to hepatitis C virus (HCV) in patients with liver disease is difficult due to false-positive reactivity in some conditions. To evaluate the feasibility of HCV in archival material, HCV was sought in formalin-fixed, paraffin-embedded liver biopsy specimens. Nested polymerase chain reaction was used to detect hepatitis C virus in formalin-fixed, paraffin-embedded liver biopsy specimens after total RNA was extracted from tissue by proteinase K digestion and phenol/chloroform purification. The relative efficiency of amplification of HCV RNA from formalin-fixed material was estimated semiquantitatively by serial dilution of cDNA synthesised from RNA extracted from fresh and formalin-fixed sections from the same liver. Although HCV RNA could be detected in formalin-fixed liver tissue by nested PCR in 5/5 cases in which HCV was detected in serum, amplification was approximately 5-fold less efficient than when HCV was amplified from fresh tissue. Nevertheless, nested PCR of HCV from formalin-fixed liver tissue represents a useful technique in addressing some important questions related to the pathogenesis of liver disease.     

Optimising restriction enzyme cleavage of DNA derived from archival histopathological samples: an improved HUMARA assay

AIMS: Our aim was to evaluate and optimise methylation-sensitive restriction enzyme assays for use in formalin-fixed, paraffin-embedded tissue (FFPET) samples, in order to improve the application of the HUMARA X-chromosome inactivation assay to FFPET samples. METHODS: We extracted DNA from normal male colon and thyroid FFPET. Several DNA clean-up procedures and restriction enzyme buffer compositions were tested for two methylation-sensitive enzymes, HhaI and HpaII and a non-methylation-sensitive isoschizomere, MspI. RESULTS: By including both a non-methylation-sensitive control enzyme and DNA from male archival specimens in our experiments, we were able to detect even subtle degrees of incomplete digestion. We showed that FFPET-derived DNA is a poor substrate for restriction enzymes, especially for methylation-sensitive restriction endonucleases. An optimised DNA clean-up protocol and restriction enzyme buffer-mix allowed us to achieve complete digestion. CONCLUSIONS: The combination of multiple, rigorous controls, DNA clean-up and restriction buffer optimisation increases the reliability of HUMARA-based X-chromosome inactivation analysis of FFPET samples. Analogous approaches are likely to allow optimisation of other restriction enzyme-based assays of FFPET samples.     

Real-time PCR analysis of RNA extracted from formalin-fixed and paraffin-embeded tissues: effects of the fixation on outcome reliability.

In many pathologic circumstances, quantitative mRNA expression levels are important for evaluation of possible genome mutations. The development of real-time polymerase chain reaction (RT-PCR) technology has facilitated the realization of nucleic acid quantification. Potentially, quantitative PCR offers a number of advantages over traditional methods because it permits the use of small amounts of genetic material. In the present study, we optimize a RNA purification technique on specimens that are formalin-fixed, paraffin-embedded and we examine prolonged formalin fixation effects on quantitative RT-PCR analysis. We compared RNA levels with 70 colic mucosa samples using the cyclooxygenase 2 gene as marker. The difference in amplification successes between formalin-fixed tissues and formalin-fixed, paraffin-embedded tissues was not statistically significant. Moreover, we compared the expression of formalin-fixed samples with the expression of each fresh tissue. Wilcoxon Mann-Whitney test shows that only the difference in the expression levels of 1- or 3-hour formalin-fixed samples is not statistically significant with respect to other fixation times. We found that the mRNA can be reliably extracted from formalin fixed, paraffin-embedded tissue sections but that prolonged formalin fixation produces different results in quantitative RT-PCR. It can be related to difference in RNA sequences length and the generation of secondary structures that are more susceptible to the prolonged formalin fixation. We suppose that the paraffin do not influence the RNA extraction yield because there are no statistical significant differences between amplification success of formalin-fixed tissues and paraffin-embedded tissues. Therefore, in relative expression quantization, we confirm that it is appropriate to use specimens with same protocols and time for formalin fixation.     

Feasibility of archival non-buffered formalin-fixed and paraffin-embedded tissues for PCR amplification: An analysis of resected gastric carcinoma

Although several factors affecting the sensitivity of potymer-ase chain reaction (PCR) amplification from formalin-fixed tissues have been Investigated mostly by experiments, the feasibility of archival formalln-flxed, paraffin-embedded tissue samples stored in pathology departments for PCR amplification has rarely been examined directly. Thus, the feasibility of 74 archival unbuffered 10% formalin-fixed, paraffin-embedded tissues for PCR amplification with primers producing a 190 b.p. DNA segment of p53 exon 5 was investigated. Fixation time was the critical factor influencing the sensitivity of PCR amplification. All (6/6) of the samples fixed for only 1 day, 44% (7/16) of the samples fixed for 2–3 days and 14% (4/28) of the samples fixed for 4–6 days showed successful amplification, while no amplification was obtained for the samples fixed for 7 days or more. The peak size of DNA extracted from the archival tissues decreased as the fixation time became longer. Experiments using xenografted tumor tissues fixed for various times showed longer permissible fixation time; up to 9 days of fixation, decreasing amounts of PCR products were obtained while no amplification was obtained for the samples fixed for 12 days or more. The time in paraffin seemed to be a minor factor for PCR amplification since all of the 1 day fixation samples, including those that had been embedded for up to 5 years, resulted in efficient amplification. The size of the amplified DNA segments, however, could be another factor influencing the sensitivity of amplification because even the 1 day fixation samples showed less amplification of 345 b.p. DNA compared with those of 167 and 262 b.p. DNA. Additionally, a point mutation was detected in the amplified pS3 products from archival tissues using a non-isotopic method, temperature gradient gel elec-trophoresis. In conclusion, archival tissue samples that had been fixed immediately for only up to 1 day were constantly available for PCR amplification of approximately 200 b.p. DNA segments, suggesting that surgical specimens should be subjected to cutting and paraffin embedding Just after 1 day or less fixation for subsequent use in PCR amplification.     

Improved PCR amplification for molecular analysis using DNA from long-term preserved formalin-fixed,paraffin-embedded lung cancer tissue specimens

Archival tissue specimens are valuable resources of materials for molecular biological analyses in retrospective studies, especially for rare diseases or those associated with exposure to uncommon environmental events. Although successful amplification with PCR is essential for analysis of DNA extracted from archival formalin-fixed, paraffin-embedded (FFPE) tissue specimens, we have often encountered problems with poor PCR amplification of target fragments. To overcome this, we examined whether heat treatment in alkaline solution could efficiently restore the PCR template activity of DNA that had already been extracted from FFPE lung cancer tissue specimens. The effect of the heat treatment was assessed by PCR for the TP53 gene and other lung cancer-related gene loci. The heat treatment of DNA samples in borate buffer resulted in successful PCR amplification of DNA fragments ranging from 91 to 152 bp. This technique for restoration of template activity of DNA for PCR amplification is very simple and economical, and requires no special apparatus, so it may be applicable for molecular analysis of DNA samples from FFPE tissue specimens at various laboratories.     

SCOMP is superior to degenerated oligonucleotide primed-polymerase chain reaction for global amplification of minute amounts of DNA from microdissected archival tissue samples

Global genome amplification from formalin-fixed tissues is still problematic when performed with low cell numbers. Here, we tested a recently developed method for whole genome amplification termed "SCOMP" (single cell comparative genomic hybridization) on archival tissues of different ages. We show that the method is very well suited for formalin-fixed paraffin-embedded samples obtained by nuclei extraction or laser microdissection. The polymerase chain reaction (PCR) products can be used for subsequent comparative genomic hybridization, loss of heterozygosity studies, and DNA sequencing. To control for PCR-induced artifacts we amplified genomic DNA isolated from 20 nuclei of archival formalin-fixed, paraffin-embedded nonpathological lymph nodes. Subsequent comparative genomic hybridization revealed the expected balanced profiles. For loss of heterozygosity analysis by microsatellite PCR 60 to 160 cells were sufficient. In comparative experiments the approach turned out to be superior to published degenerated oligonucleotide-primed-PCR protocols. The method provides a robust and valuable tool to study very small cell samples, such as the genomes of dysplastic cells or the clonal evolution within heterogeneous tumors.     

Quantitative gene expression analysis in microdissected archival tissue by real-time RT-PCR

Laser-assisted microdissection is a powerful tool for the analysis of morphologically defined cell populations. We report here that the combination of microdissection and real-time RT-PCR technologies together with an optimized RNA microscale extraction procedure allows the reproducible determination of gene expression levels in archival formalin-fixed, paraffin-embedded tissue samples.     

Molecular detection and characterization of yellow fever virus in blood and liver specimens of a non-vaccinated fatal human case

A yellow fever virus of a South American genotype was identified in the liver and blood samples of a non-vaccinated European patient after his return from Brazil. ELISA tests were negative for IgG and positive for IgM against yellow fever. Yellow fever proteins in the formalin-fixed and paraffin-embedded liver biopsy were detected by immunohistochemical procedures. Viral RNA extracted from the liver tissue was also detected using an RT-semi-nested PCR procedure and molecular hybridization. Alignment of the sequence obtained from a gene fragment amplified by RT-semi-nested PCR directly from a blood sample with those of African and South American yellow fever virus strains identified a Brazilian topotype as being responsible for the disease. RT-semi-nested PCR may be used advantageously for clinical specimens for rapid and specific diagnosis, and with archival biopsy material for retrospective studies. J. Med. Virol. 53:212–217, 1997. © 1997 Wiley-Liss, Inc.     

Quality control and sensitivity of polymerase chain reaction techniques for the assessment of immunoglobulin heavy chain gene rearrangements from fixed- and paraffin-embedded samples

Frozen tissue is considered the gold standard if DNA is to be extracted for polymerase chain reaction (PCR) analysis. In molecular studies from paraffin-embedded material, only positive results are usually taken into account. Our goal was to evaluate both the sensitivity and the specificity of PCR techniques for immunoglobulin heavy chain (IgH) gene rearrangement according to various lengths and types of fixative before paraffin embedding. One set of studies compared IgH rearrangement in a case of mantle cell lymphoma tissue that had been fixed in 14 different ways before paraffin embedding and frozen tissue. Formalin fixation was found not to be deleterious for DNA, amplification being possible up to 15 days after fixation with good sensitivity. In contrast, the performance of PCR decreased for samples fixed in Bouin's liquid for longer than 6 hours or after 48 hours of incubation in a vacuum infiltration processor (in which Bouin's liquid-fixed and formalin-fixed samples are mixed). In addition, we undertook a retrospective study of 20 routinely processed B-cell lymphomas, with frozen formalin-fixed and Bouin's liquid-fixed tissues for each case. Of the 14 positive cases on frozen material, 13 were also clonal from paraffin-embedded tissues. Whatever the IgH locus analyzed, each time the adapted control was positive, results from paraffin-embedded material were identical to results obtained from frozen tissue. In this study, we showed that the use of paraffin-embedded tissue is efficient for the study of IgH gene rearrangement. Whenever adapted controls are used, it is even possible to assess negative results.     

Assessment of genotype and molecular evolution of hepatitis C virus in formalin-fixed paraffin-embedded liver tissue from patients with chronic hepatitis C virus infection

Drawbacks of hepatitis C virus (HCV) RNA detection in paraffin-embedded liver tissue have satisfactorily been solved by RT-PCR amplification of the 5'non-coding region (5'NCR). However, detection of this highly conserved region does not provide information on epidemiological or pathogenetic aspects of HCV infection. This study explores whether other functionally important genetic regions of HCV, such as the hypervariable region 1 (HVR-1) and the interferon sensitivity-determining region (ISDR), can be retrieved from paraffin-embedded liver specimens by RT-PCR, and whether the amplified material is suitable for further molecular analyses. RT-PCR amplification of 5'NCR, HVR-1, and ISDR was assessed in RNA extracted from 50 formalin-fixed, paraffin-embedded liver specimens, including 23 needle liver biopsies (11 from patients with non-A, non-B chronic hepatitis diagnosed between 1971 and 1985, 8 from subjects with normal liver histology and 4 from sequential biopsies from a patient with HCV recurrence after liver transplantation), and 27 liver explants from patients undergoing transplantation between 1988 and 1996 (16 with HCV-related cirrhosis and 11 with other disorders). The 5'NCR was successfully amplified in 8 of 11 (73%) non-A, non-B chronic hepatitis biopsies and in all of the specimens from patients with serological documentation of HCV infection. There were no false-positive results. HCV genotype was identified by RFLP analysis of the 5'NCR in the 13 cases analyzed. HVR-1 and ISDR were amplified in 24 of 28 (86%) samples, which were positive for the 5'NCR. Efficient amplification was inversely related to the time of storage. The evolutionary changes of HVR-1 and ISDR were successfully analyzed by direct sequencing of amplificates from the explanted liver and from the sequential liver biopsies in a patient with HCV infection recurrence after transplantation. These observations indicate that paraffin-embedded liver tissue, even when stored for more than 20 years, is appropriate for advanced studies on the molecular biology of HCV.     

Development of a microarray platform for FFPET profiling: application to the classification of human tumors

Background

mRNA profiling has become an important tool for developing and validating prognostic assays predictive of disease treatment response and outcome. Archives of annotated formalin-fixed paraffin-embedded tissues (FFPET) are available as a potential source for retrospective studies. Methods are needed to profile these FFPET samples that are linked to clinical outcomes to generate hypotheses that could lead to classifiers for clinical applications.

Methods

We developed a two-color microarray-based profiling platform by optimizing target amplification, experimental design, quality control, and microarray content and applied it to the profiling of FFPET samples. We profiled a set of 50 fresh frozen (FF) breast cancer samples and assigned class labels according to the signature and method by van 't Veer et al [1] and then profiled 50 matched FFPET samples to test how well the FFPET data predicted the class labels. We also compared the sorting power of classifiers derived from FFPET sample data with classifiers derived from data from matched FF samples.

Results

When a classifier developed with matched FF samples was applied to FFPET data to assign samples to either "good" or "poor" outcome class labels, the classifier was able to assign the FFPET samples to the correct class label with an average error rate = 12% to 16%, respectively, with an Odds Ratio = 36.4 to 60.4, respectively. A classifier derived from FFPET data was able to predict the class label in FFPET samples (leave-one-out cross validation) with an error rate of ~14% (p-value = 3.7 × 10^-7). When applied to the matched FF samples, the FFPET-derived classifier was able to assign FF samples to the correct class labels with 96% accuracy. The single misclassification was attributed to poor sample quality, as measured by qPCR on total RNA, which emphasizes the need for sample quality control before profiling.

Conclusion

We have optimized a platform for expression analyses and have shown that our profiling platform is able to accurately sort FFPET samples into class labels derived from FF classifiers. Furthermore, using this platform, a classifier derived from FFPET samples can reliably provide the same sorting power as a classifier derived from matched FF samples. We anticipate that these techniques could be used to generate hypotheses from archives of FFPET samples, and thus may lead to prognostic and predictive classifiers that could be used, for example, to segregate patients for clinical trial enrollment or to guide patient treatment.     

A tissue fixative that protects macromolecules (DNA, RNA, and protein) and histomorphology in clinical samples

Preservation of macromolecules (DNA, RNA, and proteins) in tissue is traditionally achieved by immediate freezing of the sample. Although isolation of PCR-able RNA has been reported from formalin-fixed, paraffin-embedded tissues, the process has not been shown to be reproducible because high molecular weight RNA is usually degraded. We investigated the potential value of a new universal molecular fixative (UMFIX, Sakura Finetek USA, Inc., Torrance, California) in preservation of macromolecules in paraffin-embedded tissue. Mouse and human tissues were fixed in UMFIX from 1 hour to 8 weeks. They were then processed by a rapid tissue processing (RTP) system, embedded in paraffin, and evaluated for routine histology as well as for the quality and quantity of DNA, RNA, and proteins. Formalin-fixed tissues were processed by RTP and evaluated in a similar manner. Fresh-frozen samples were used as controls. The morphology of UMFIX-exposed tissue was comparable to that fixed in formalin. High molecular weight RNA was preserved in tissue that was immediately fixed in UMFIX and stored from 1 hour to 8 weeks at room temperature. There were no significant differences between UMFIX-exposed and frozen tissues on PCR, RT-PCR, real-time PCR, and expression microarrays. Similarly, physical and antigenic preservation of proteins in UMFIX tissue was similar to fresh state. Both RNA and proteins were substantially degraded in formalin-fixed and similarly processed specimens. We concluded that it is now possible to preserve histomorphology and intact macromolecules in the same archival paraffin-embedded tissue through the use of a novel fixative and a rapid processing system.