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.

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.

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.

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.

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.

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.