The Pre-analytical Phase of Pathology Research and Diagnostics

markdownabstract__Abstract__ For the development of 'personalized medicine' it is necessary to perform translational research on patient tissue samples. It is of crucial importance that the pre-analytical phase does not influence the desired results. In this thesis a number of experiments on many levels (e.g. gene expression, immunohistochemistry) are described, which shed light on the adverse effects of ischemia and formalin fixation on gene expression and antigenicity. Some of the main conclusions are that surgery can cause gene up or down regulation, smaller tissue samples decay faster than larger ones and that the adverse effects of formalin fixation can be undone by fine tuning the analytical phase, rather than standardizing the pre-analytical phase. These results will become part of new ISO guidelines and protocols in the near future and will lead to less variabilty in down stream techniques

A reference image-based method for optimization of clinical immunohistochemistry

AimsCold ischaemic and formalin fixation time (CIT and FFT) are considered to be crucial parameters for intralaboratory variation in immunohistochemistry (IHC). Here we describe a new method to optimize IHC, by using control tissue blocks with known pre-analytical history and comparing the IHC outcome with digitized reference slides. Methods and resultsTissue specimens (two per tissue type) were divided into eight samples, which were subjected to different CIT and FFT. Immunohistochemistry was performed with 34 routinely used antibodies, following standard operating procedures. Relative staining intensity of four sections per slide was scored. Of the antibodies studied, seven were influenced by CIT, 13 by FFT and five by both parameters. IHC protocols were adapted until most sections on the slide showed the same intensity. Changing the antibody dilution for 10 protocols and the antigen retrieval method for six protocols improved the consistency of the IHC staining. Nine protocols could not be optimized. The optimized staining results were compared to reference slides and were found to be of adequate quality. ConclusionsIt was possible to optimize most IHC protocols by adapting the analytical, rather than the pre-analytical, phase. If global references can be established, this method could decrease interlaboratory variation, preceding standardization of the pre-analytical workflow

Fit for Purpose Frozen Tissue Collections by RNA Integrity Number-Based Quality Control Assurance at the Erasmus MC Tissue Bank

About 5000 frozen tissue samples are collected each year by the Erasmus Medical Center tissue bank. Two percent of these samples are randomly selected annually for RNA isolation and RNA Integrity Number (RIN) measurement. A similar quality assessment was conducted during centralization of a 20-year-old tissue collection from the cancer institute, a 15-year-old liver sample archive (-80 degrees C), and a 13-year-old clinical pathology frozen biopsy archive (Liquid Nitrogen). Samples were divided into either high-quality (RIN >= 6.5) or low-quality overall categories, or into four "fit-for-purpose" quality groups: RIN = 8: suitable for all downstream techniques. In general, low RIN values were correlated with fatty, fibrous, pancreatic, or necrotic tissue. When the percentage of samples with RIN >= 6.5 is higher than 90%, the tissue bank performance is adequate. The annual 2011 quality control assessment showed that 90.3% (n=93) of all samples had acceptable RIN values; 97.4% (n=39) of the cancer institute collection had RIN values above 6.5; and 88.6% (n=123) of samples from the liver sample archive collection had RIN values higher than 6.5. As the clinical pathology biopsy collection contained only 58.8% (n=24) acceptable samples, the procurement protocols used for these samples needed immediate evaluation. When the distribution of RIN values of the different collections were compared, no significant differences were found, despite differences in average storage time and temperature. According to the principle of "fit-for-purpose" distribution, the vast majority of samples are considered good enough for most downstream techniques. In conclusion, an annual tissue bank quality control procedure provides useful information on tissue sample quality and sheds light on where and if improvements need to be made

The Influence of Tissue Procurement Procedures on RNA Integrity, Gene Expression, and Morphology in Porcine and Human Liver Tissue

The advent of molecular characterization of tissues has brought an increasing emphasis on the quality of biospecimens, starting with the tissue procurement process. RNA levels are particularly affected by factors in the collection process, but the influence of different pre-analytical factors is not well understood. Here we present the influence of tissue specimen size, as well as the transport and freezing protocols, on RNA quality. Large, medium, and smaller porcine liver samples were stored either dry, on moist gauze, or in salt solution for various times, and then frozen in either liquid nitrogen or in pre-cooled isopentane. Large and small human liver samples were frozen in pre-cooled isopentane either immediately or after one hour at room temperature. The small samples were stored dry, on moist gauze, or in salt solution. RNA was isolated and RIN values were measured. The RNA for six standard reference genes from human liver was analyzed by RT-qPCR, and tissue morphology was assessed for artifacts of freezing. Experiments using porcine liver samples showed that RNA derived from smaller samples was more degraded after one hour of cold ischemia, and that cooled transport is preferable. Human liver samples showed significant RNA degradation after 1h of cold ischemia, which was more pronounced in smaller samples. RNA integrity was not significantly influenced by the transport or freezing method, but changes in gene expression were observed in samples either transported on gauze or in salt solution. Based on observations in liver samples, smaller samples are more subject to gene expression variability introduced by post-excision sample handling than are larger samples. Small biopsies should be transported on ice and snap frozen as soon as possible after acquisition from the patient

Fit for Purpose Frozen Tissue Collections by RNA Integrity Number-Based Quality Control Assurance at the Erasmus MC Tissue Bank

About 5000 frozen tissue samples are collected each year by the Erasmus Medical Center tissue bank. Two percent of these samples are randomly selected annually for RNA isolation and RNA Integrity Number (RIN) measurement. A similar quality assessment was conducted during centralization of a 20-year-old tissue collection from the cancer institute, a 15-year-old liver sample archive (-80 degrees C), and a 13-year-old clinical pathology frozen biopsy archive (Liquid Nitrogen). Samples were divided into either high-quality (RIN >= 6.5) or low-quality overall categories, or into four "fit-for-purpose" quality groups: RIN = 8: suitable for all downstream techniques. In general, low RIN values were correlated with fatty, fibrous, pancreatic, or necrotic tissue. When the percentage of samples with RIN >= 6.5 is higher than 90%, the tissue bank performance is adequate. The annual 2011 quality control assessment showed that 90.3% (n=93) of all samples had acceptable RIN values; 97.4% (n=39) of the cancer institute collection had RIN values above 6.5; and 88.6% (n=123) of samples from the liver sample archive collection had RIN values higher than 6.5. As the clinical pathology biopsy collection contained only 58.8% (n=24) acceptable samples, the procurement protocols used for these samples needed immediate evaluation. When the distribution of RIN values of the different collections were compared, no significant differences were found, despite differences in average storage time and temperature. According to the principle of "fit-for-purpose" distribution, the vast majority of samples are considered good enough for most downstream techniques. In conclusion, an annual tissue bank quality control procedure provides useful information on tissue sample quality and sheds light on where and if improvements need to be made

Inactivation of Influenza A virus, Adenovirus, and Cytomegalovirus with PAXgene Tissue Fixative and Formalin

Formalin fixation is known to inactivate most viruses in a vaccine production context, but nothing is published about virus activity in tissues treated with alternative, non-crosslinking fixatives. We used a model assay based on cell culture to test formalin and PAXgene Tissue fixative for their virus-inactivating abilities. MDCK, A549, and MRC-5 cells were infected with Influenza A virus, Adenovirus, and Cytomegalovirus, respectively. When 75% of the cells showed a cytopathic effect (CPE), the cells were harvested and incubated for 15min, or 1, 3, 6, or 24 hours, with PBS (positive control), 4% formalin, or PAXgene Tissue Fix. The cells were disrupted and the released virus was used to infect fresh MDCK, A549, and MRC-5 cells cultured on cover slips in 24-well plates. The viral cultures were monitored for CPE and by immunocytochemistry (ICC) to record viral replication and infectivity. Inactivation of Adenovirus by formalin occurred after 3h, while Influenza A virus as well as Cytomegalovirus were inactivated by formalin after 15min. All three virus strains were inactivated by PAXgene Tissue fixative after 15min. We conclude that PAXgene Tissue fixative is at least as effective as formalin in inactivating infectivity of Influenza A virus, Adenovirus, and Cytomegalovirus