Rapid tissue processing using a temperature-controlled collection device to preserve tumor biomarkers.

Precision tissue diagnostics rely on high quality input specimens so that assay results are not affected by artifact, but advances in collection and processing of tissue specimens have lagged behind innovations in diagnostic assay development. Therefore, we have designed and evaluated a novel surgical tissue collection device that maintains and monitors sample temperature and motion throughout transport so that the major preanalytical variable of tissue temperature can be controlled and measured. This device, in combination with an improved cold-hot tissue fixation protocol affords optimal biomarker preservation in less overall time, thereby simultaneously improving diagnostic quality and turnaround time. We collected 50 primary and metastatic liver tumors using a novel transport device. Tissue was fixed using a rapid cold-hot fixation protocol and immunohistochemical assays were used to assess the performance of the device, in comparison to control tissue preserved using standard clinical fixation protocol. Two pathologists evaluated the IHC studies in a blinded fashion to determine the immunophenotype of each tumor. The observed IHC staining intensities and the clinical impressions of the immunophenotypes did not differ between tissue collected with the novel device and control tissue, while improvements in processing time were achieved. The novel cold transport device and rapid fixation protocol can be successfully and safely combined and used to monitor specimen conditions, thus preserving the diagnostic utility of specimens and improving the overall turn-around time of the diagnostic process.

Two-temperature formalin fixation preserves activation states efficiently.

Modern pathology is built around the principle of preserving tissues such that the in vivo molecular status is maintained at levels representative of the disease state. Tissues are immersed in a solution of fixative which slowly inactivates biological activities, thus preserving the sample. Further processing ultimately allows the tissue to be embedded into wax for thin sectioning and staining for interpretation microscopically. Every year, around 7 billion tissue samples are submitted for processing in the United States alone. With this huge workload, histology laboratories are looking for faster methods of performing fixation, which currently require from several hours to days to complete. Ideally, this procedure could be standardized and would be quicker with better preservation over a wide range of biologically relevant molecules.

Assessing Tissue Fixation Time and Quality with Label-free Mid Infrared Spectroscopy and Machine Learning.

Objectives: This work investigates whether changes in a biospecimen's molecular composition from formaldehyde fixation drive changes in the mid infrared (MID-IR) spectrum. Our ultimate goal was to develop an analytical metrology that could be used to accurately determine the fixation time of a tissue sample as a surrogate to overall tissue quality. Methods: Multiple unstained formalin-fixed paraffin-embedded tissue samples were scanned with an MID-IR microscope to identify a molecular fingerprint of formaldehyde fixation. The fixation specific patterns were then mined to develop a predictive model. A multiple tissue experiment using greater than 100 samples was designed to train the algorithm and validate the accuracy of predicting fixation status. Results: We present data that formaldehyde crosslinking results in alterations to multiple bands of the MID-IR spectra. The impact was most dramatic in the Amide I band, which is sensitive to the conformational state of proteins. The spectroscopic fixation signature was used to train a machine-learning model that could predict fixation time of unknown tissues with an average accuracy of 1.4 hours. Results were validated by histological stain quality for bcl-2, FOXP3, and ki-67. Further, two-dimensional imaging was used to visualize the spatial dependence of fixation, as demonstrated by multiple features in the tissue's vibrational spectra. Conclusions: This work demonstrates that it is possible to predict the fixation status of tissues for which the preanalytics are unknown. This novel capability could help standardize clinical tissue diagnostics and ensure every patient gets the absolutely best treatment based on the highest quality tissue sample.

Bioprinted Human Lung Cancer-Mimics for Tissue Diagnostics Applications.

Developing a reproducible and secure supply of customizable control tissues that standardizes for the cell type, tissue architecture, and preanalytics of interest for usage in applications including diagnostic, prognostic, and predictive assays, is critical for improving our patient care and welfare. The conventionally adopted control tissues directly obtained from patients are not ideal because they oftentimes have different amounts of normal and neoplastic elements, differing cellularity, differing architecture, and unknown preanalytics, in addition to the limited supply availability and thus associated high costs. In this study, we demonstrated a strategy to stably produce tissue-mimics for diagnostics purposes by taking advantage of the three-dimensional (3D) bioprinting technology. Specifically, we take anaplastic lymphoma kinase-positive (Alk+) lung cancer as an example, where a micropore-forming bioink laden with tumor cells was combined with digital light processing-based bioprinting for developing native-like Alk+ lung cancer tissue-mimics with both structural and functional relevancy. It is anticipated that our proposed methodology will pave new avenues for both fields of tissue diagnostics and 3D bioprinting significantly expanding their capacities, scope, and sustainability.

Making a science out of preanalytics: An analytical method to determine optimal tissue fixation in real-time.

Modern histopathology is built on the cornerstone principle of tissue fixation, however there are currently no analytical methods of detecting fixation and as a result, in clinical practice fixation is highly variable and a persistent source of error. We have previously shown that immersion in cold formalin followed by heated formalin is beneficial for preservation of histomorphology and have combined two-temperature fixation with ultra-sensitive acoustic monitoring technology that can actively detect formalin diffusing into a tissue. Here we expand on our previous work by developing a predictive statistical model to determine when a tissue is properly diffused based on the real-time acoustic signal. We trained the model based on the morphology and characteristic diffusion curves of 30 tonsil cores. To test our model, a set of 87 different tonsil samples were fixed with four different protocols: dynamic fixation according to our predictive algorithm (C/H:Dynamic, N = 18), gold-standard 24 hour room temperature (RT:24hr, N = 24), 6 hours in cold formalin followed by 1 hour in heated formalin (C/H:6+1, N = 21), and 2 hours in cold formalin followed by 1 hour in heated formalin (C/H:2+1, N = 24). Digital pathology analysis revealed that the C/H:Dynamic samples had FOXP3 staining that was spatially uniform and statistically equivalent to RT:24hr and C/H:6+1 fixation protocols. For comparison, the intentionally underfixed C/H:2+1 samples had significantly suppressed FOXP3 staining (p<0.002). Furthermore, our dynamic fixation protocol produced bcl-2 staining concordant with standard fixation techniques. The dynamically fixed samples were on average only submerged in cold formalin for 4.2 hours, representing a significant workflow improvement. We have successfully demonstrated a first-of-its-kind analytical method to assess the quality of fixation in real-time and have confirmed its performance with quantitative analysis of downstream staining. This innovative technology could be used to ensure high-quality and standardized staining as part of an expedited and fully documented preanalytical workflow.

Effect of immediate cold formalin fixation on phosphoprotein IHC tumor biomarker signal in liver tumors using a cold transport device.

Phosphoproteins are the key indicators of signaling network pathway activation. Many disease treatment therapies are designed to inhibit these pathways and effective diagnostics are required to evaluate the efficacy of these treatments. Phosphoprotein IHC have been impractical for diagnostics due to inconsistent results occurring from technical limitations. We have designed and tested a novel cold transport device and rapid cold plus warm formalin fixation protocol using phosphoproteins IHC. We collected 50 liver tumors that were split into two experimental conditions: 2 + 2 rapid fixation (2 hours cold then 2 hour warm formalin) or 4 hour room-temperature formalin. We analyzed primary hepatocellular carcinoma (n = 10) and metastatic gastrointestinal tumors (n = 28) for phosphoprotein IHC markers pAKT, pERK, pSRC, pSTAT3, and pSMAD2 and compared them to slides obtained from the clinical blocks. Expression of pERK and pSRC, present in the metastatic colorectal carcinoma, were better preserved with the rapid processing protocol while pSTAT3 expression was detected in hepatocellular carcinoma. Differences in pSMAD2 expression were difficult to detect due to the ubiquitous nature of protein expression. There were only 3 cases expressing pAKT and all exhibited a dramatic loss of signal for the standard clinical workflow. The rapid cold preservation shows improvement in phosphoprotein preservation.

Site-directed cleavage of DNA by protein-Fe(II) EDTA conjugates within model chromatin complexes.

The nucleosome and other chromatin complexes are examples of complicated protein-DNA assemblies that are not easily studied by traditional structural methods. Site-directed cleavage of DNA is a method for mapping the location of interaction of a specific site in a protein such as a linker histone within a large complex such as the nucleosome. In this chapter we describe the application of the site-directed cleavage method, employing linker histones site-specifically modified with the chemical cleavage reagent Fe(II)(EDTA-2-aminoethyl) 2-pyridyl disulfide (ebr). Addition of hydrogen peroxide and a reducing agent to the complex containing the modified protein leads to the production of hydroxyl radicals from the iron center, resulting in cleavage of DNA backbones in the vicinity of the modified residue. The cleavages can then be mapped and ascribed to a particular location within the nucleosome, allowing the binding site of the protein within this structure to be determined.

Monitoring Dehydration and Clearing in Tissue Processing for High-Quality Clinical Pathology.

The development of precision testing for disease diagnosis has advanced medicine by specifically matching patients with drugs to treat specific diseases. High-quality diagnostics start with high-quality tissue specimens. The development and optimization of tissue handling and processing have lagged behind bioassay development. Ultrasound time-of-flight (TOF) technology has been successfully used to monitor the critical processing step of tissue fixation with formalin. In this study, we expand the use of this technology to monitor tissue dehydration and clearing by analyzing TOF signals from 270 different specimens, representing 13 different tissue types obtained through surgical resections. We determined the time constant τ90 for each tissue type for the following tissue processing solvents: 70% ethanol, 90% ethanol, 100% ethanol, and xylene. The TOF signals were correlated with tissue morphology to ensure that high-quality tissue was produced. Tissues can be grouped into those exhibiting fast and slow reagent diffusion. We monitored incomplete dehydration of tissue by skipping a key processing step, dehydration in absolute ethanol, and then correlated the τ90 with poor histomorphology, demonstrating that the technique can detect significant processing errors. Ultrasound TOF technology can therefore be used to monitor all phases of tissue processing cycle and yields an important preanalytical quality metric.

A New Paradigm for Tissue Diagnostics: Tools and Techniques to Standardize Tissue Collection, Transport, and Fixation.

PURPOSE OF REVIEW: Studying and developing preanalytical tools and technologies for the purpose of obtaining high-quality samples for histological assays is a growing field. Currently, there does not exist a standard practice for collecting, fixing, and monitoring these precious samples. There has been some advancement in standardizing collection for the highest profile tumor types, such as breast, where HER2 testing drives therapeutic decisions. This review examines the area of tissue collection, transport, and monitoring of formalin diffusion and details a prototype system that could be used to help standardize tissue collection efforts. RECENT FINDINGS: We have surveyed recent primary literature sources and conducted several site visits to understand the most error-prone processes in histology laboratories. This effort identified errors that resulted from sample collection techniques and subsequent transport delays from the operating room (OR) to the histology laboratories. We have therefore devised a prototype sample collection and transport concept. The system consists of a custom data logger and cold transport box and takes advantage of a novel cold + warm (named 2 + 2) fixation method. SUMMARY: This review highlights the beneficial aspects of standardizing tissue collection, fixation, and monitoring. In addition, a prototype system is introduced that could help standardize these processes and is compatible with use directly in the OR and from remote sites.

Precision Medicine Starts With Preanalytics: Real-Time Assessment of Tissue Fixation Quality by Ultrasound Time-of-Flight Analysis.

Personalized medicine promises diagnosis and treatment of disease at the individual level and relies heavily on clinical specimen integrity and diagnostic assay quality. Preanalytics, the collection and handling steps of a clinical specimen before immunohistochemistry or other clinical assay, are critically important to enable the correct diagnosis of disease. However, the effects of preanalytics are often overlooked due to a lack of standardization and limited assessment tools to quantify their variation. Here, we report a novel real-time ultrasound time-of-flight instrument that is capable of monitoring and imaging the critical step in formalin fixation, diffusion of the fixative into tissue, which provides a quantifiable quality metric for tissue fixation in the clinical laboratory ensuring consistent downstream molecular assay results. We analyzed hundreds of tissue specimens from 34 distinct human tissue types and 12 clinically relevant diseased tissues for diffusion and fixation metrics. Our measurements can be converted into tissue diffusivity constants that correlate with the apparent diffusion constant calculated using magnetic resonance imaging (R=0.83), despite the differences in the approaches, indicating that our approach is biophysically plausible. Using data collected from time-of-flight analysis of many tissues, we have therefore developed a novel rapid fixation program that could ensure high-quality downstream assay results for a broad range of human tissue types.

Active monitoring of formaldehyde diffusion into histological tissues with digital acoustic interferometry.

The preservation of certain labile cancer biomarkers with formaldehyde-based fixatives can be considerably affected by preanalytical factors such as quality of fixation. Currently, there are no technologies capable of quantifying a fixative's concentration or the formation of cross-links in tissue specimens. This work examined the ability to detect formalin diffusion into a histological specimen in real time. As formaldehyde passively diffused into tissue, an ultrasound time-of-flight (TOF) shift of several nanoseconds was generated due to the distinct sound velocities of formalin and exchangeable fluid within the tissue. This signal was resolved with a developed digital acoustic interferometry algorithm, which compared the phase differential between signals and computed the absolute TOF with subnanosecond precision. The TOF was measured repeatedly across the tissue sample for several hours until diffusive equilibrium was realized. The change in TOF from 6-mm thick ex vivo human tonsil fit a single-exponential decay ([Formula: see text]) with rate constants that varied drastically spatially between 2 and 10 h ([Formula: see text]) due to substantial heterogeneity. This technology may prove essential to personalized cancer diagnostics by documenting and tracking biospecimen preanalytical fixation, guaranteeing their suitability for diagnostic assays, and speeding the workflow in clinical histopathology laboratories.

Immunohistochemistry of colorectal cancer biomarker phosphorylation requires controlled tissue fixation.

Phosphorylated signaling molecules are biomarkers of cancer pathophysiology and resistance to therapy, but because phosphoprotein analytes are often labile, poorly controlled clinical laboratory practices could prevent translation of research findings in this area from the bench to the bedside. We therefore compared multiple biomarker and phosphoprotein immunohistochemistry (IHC) results in 23 clinical colorectal carcinoma samples after either a novel, rapid tissue fixation protocol or a standard tissue fixation protocol employed by clinical laboratories, and we also investigated the effect of a defined post-operative "cold" ischemia period on these IHC results. We found that a one-hour cold ischemia interval, allowed by ASCO/CAP guidelines for certain cancer biomarker assays, is highly deleterious to certain phosphoprotein analytes, specifically the phosphorylated epidermal growth factor receptor (pEGFR), but shorter ischemic intervals (less than 17 minutes) facilitate preservation of phosphoproteins. Second, we found that a rapid 4-hour, two temperature, formalin fixation yielded superior staining in several cases with select markers (pEGFR, pBAD, pAKT) compared to a standard overnight room temperature fixation protocol, despite taking less time. These findings indicate that the future research and clinical utilities of phosphoprotein IHC for assessing colorectal carcinoma pathophysiology absolutely depend upon attention to preanalytical factors and rigorously controlled tissue fixation protocols.

Rapid two-temperature formalin fixation.

Formalin fixation is a mainstay of modern histopathologic analysis, yet the practice is poorly standardized and a significant potential source of preanalytical errors. Concerns of workflow and turnaround time drive interest in developing shorter fixation protocols, but rapid protocols can lead to poor histomorphology or inadequate downstream assay results. Additionally, assays such as immunohistochemistry for phosphorylated epitopes have historically been challenging in the context of formalin-fixed tissue, indicating that there may be room for improvement in this process that is fundamental to the practice of anatomic pathology. With these issues in mind, we studied basic formalin biochemistry to develop a novel formalin fixation protocol that involves a pre-incubation in subambient temperature formalin prior to a brief exposure to heated formalin. This new protocol is more rapid than standard protocols yet preserves histomorphology and yields tissue that is compatible with an expanded set of downstream clinical and research assays, including immunohistochemistry for phosphorylated epitopes.

DNA ligase I competes with FEN1 to expand repetitive DNA sequences in vitro.

Repeat sequences in various genomes undergo expansion by poorly understood mechanisms. By using an oligonucleotide system containing such repeats, we recapitulated the last steps in Okazaki fragment processing, which have been implicated in sequence expansion. A template containing either triplet or tandem repeats was annealed to a downstream primer containing complementary repeats at its 5'-end. Overlapping upstream primers, designed to strand-displace varying numbers of repeats in the downstream primer, were annealed. Human DNA ligase I joined overlapping segments of repeats generating an expansion product from the primer strands. Joining efficiency decreased with repeat length. Flap endonuclease 1 (FEN1) cleaved the displaced downstream strand and together with DNA ligase I produced non-expanded products. However, both expanded and non-expanded products formed irrespective of relative nuclease and ligase concentrations tested or enzyme addition order, suggesting the pre-existence and persistence of intermediates leading to both outcomes. FEN1 activity decreased with the length of repeat segment displaced presumably because the flap forms structures that inhibit cleavage. Increased MgCl(2) disfavored ligation of substrate intermediates that result in expansion products. Examination of expansion in vitro enables dissection of substrate and replication enzyme dynamics on repeat sequences.

Flap endonuclease 1 efficiently cleaves base excision repair and DNA replication intermediates assembled into nucleosomes.

Flap Endonuclease 1 (FEN1) plays important roles both in DNA replication and in base excision repair (BER). However, in both processes FEN1 substrates are likely to be assembled into chromatin. In order to examine how FEN1 is able to work within chromatin, we prepared model nucleosome substrates containing FEN1-cleavable DNA flaps. We find that human FEN1 binds and cleaves such substrates with efficiencies similar to that displayed with naked DNA. Moreover, we demonstrate that both FEN1 and human DNA ligase I can operate successively on DNA within the same nucleosome. These results suggest that some BER steps may not require nucleosome remodeling in vivo and that FEN 1 activity during Okazaki fragment processing can occur on nucleosomal substrates.