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From Cytogenetics to Cytogenomics-Top 5 Reasons to Automate FFPE Tissue FISH Processing

Cytogenetics has played a pivotal role in clinical and cancer diagnostics for over 60 years. Until now, not much has changed with sample processing. The use of genomic technologies such as array-based platforms and Next Generation Sequencing (NGS) speeds targeted genetic analysis, but how can high throughput automation in the cytogenetics laboratory broaden approaches? This article highlights the top 5 reasons to automate formalin-fixed paraffin embedded tissue fluorescent in situ hybridization (FFPE tissue FISH) in Cytogenomics.

Cytogenetics, then and now

In 1956, Tijo & Levan accurately determined the diploid human chromosome number to be 46.1 It was a big step for researchers and clinicians to understand the relationship among chromosome number, structure, organization, and disease states. Until recently, the experimental and clinical protocols to obtain chromosomes for analysis were low throughput, manual processes that have remained largely unchanged.

Genomic technologies such as array-based platforms and NGS speed targeted genetic analysis. New uses of high throughput automation in the cytogenomics laboratory broadens processing approaches, furthering the potential to impact the field of solid tumor diagnostics.

Solid Tumor Diagnostics and Formalin Fixed Paraffin Embedded Tissue

With the advent of molecular testing, such as fluorescent in situ hybridization (FISH), practitioners could visualize chromosomal rearrangements and numbers in both dividing and non-dividing cells. By "unzipping" DNA, brightly colored tags, called probes, are hybridized and inserted into specific chromosomal locations. When visualized, the colored patterns bound to the particular regions of interest provide insights into chromosomal changes that may indicate disease.

The ability to utilize FISH in non-dividing cell populations, or interphase FISH, enabled researchers and clinicians to use solid tumors as a diagnostic predictor of disease.

FISH: Then and Now

Hastings, et al. clarifies the importance of cytogenetics in the diagnostic pathway by stating, "In several diseases, tumour genetics correlate strongly with clinical risk; thus, cytogenetic information may help the Oncologist counsel the patient, choose a specific treatment, and/or modulate treatment intensity" (p.6).2 Because solid tumors are typically fixed in formalin and placed in paraffin blocks; these specimens are referred to as FFPE tissue. FFPE tissue adds longevity and far-reaching testing implications that allow clinicians to see disease progression over time. Cytogenomic laboratories perform interphase FISH on FFPE tissue specimens to determine clinical significance and treatment pathways.

Historically, FFPE tissue FISH was a highly labor-intensive process, with many steps to prepare the specimen for probe application. It was not an optimal process, requiring significant amounts of time; both technologists' hands-on time and prolonged wait time between stages. Further, these manual steps could be dangerous and have potential adverse effects on the sample. For example, accurate temperature regulation of the pre-treatment solutions in which sample slides become immersed, sometimes over 80-90°C, was a challenging yet essential requirement for quality processing. The glass Coplin jars containing these high-temperature solutions could crack during the process, causing precious samples to be lost and the protocol repeated.

Accuracy of enzyme digestion could also be a concern. The sample slide's pre-treatment plays a pivotal role in the FFPE tissue assay result's quality. Different tissue types and thicknesses have particular time and heat specifications for the pre-treatment, and in turn, an array of ancillary reagents making standardization a problematic task. Also, some laboratories check each sample's tissue digestion stage by using a blue DAPI counterstain to assess completeness. These protocols contain numerous opportunities for variability, as the inter-operational difference between person-to-person practice can be nearly impossible to eliminate, even with the best standard operating procedures.

Automating high throughput tissue processing offers many benefits, such as: make FFPE tissue FISH more user-friendly, add a high degree of consistency and reliability in results, and yield a potential impact on quality. Below are the top five reasons to automate FFPE tissue FISH in Cytogenomics.

Reason #1: Standardization

Leica Biosystems ThermoBrite Elite (TBE)* automates and standardizes FFPE tissue FISH slide preparation, including deparaffinization, pre-treatment, denaturation/hybridization, and post-hybridization wash steps. Application of probe, counterstain, and coverslipping are the only manual steps.4 The semi-automated solution's open system can add the flexibility of creating custom protocols and specimen specifications to suit user requirements.3

The ability to "load and go" highlights the automation's simplicity, allowing the user to load the slides into the instrument and walk away.3 The minimal hands-on time compared to the manual process allows for the reallocation of skilled technologists to perform the high-complexity testing rather than spending valuable time performing "dipping and dunking" tasks required for manual processes.4 The liquid handling system's automation can also monitor adequate fluidity volumes of reagents distributed to each slide using a low flow rate warning alarm.4

Reason #2: Simplification of Workflow

The TBE simplifies the workflow process. The TBE only requires 3 hands-on steps: loading slides, adding probe and coverslip, and removing the coverslip post-hybridization. Automation handles the stages of deparaffinization, pre-treatment/enzyme digestion, denaturation, hybridization, and post-washes. The TBE decreases the process's complexity from approximately 30 manual steps to 3 when preparing FFPE tissue FISH slides.4

Figure 1. TBE Minimization of Hands-on Time 4

Reason #3: Protocol Consistency

The TBE offers onboard processes that standardize and automate many manual steps that may cause protocol inconsistency.3 Regulating the time and temperatures are critical to maintaining standardization in sample processing that drives quality FFPE tissue FISH results.6

Reason #4: Reproducibility

The automation of TBE provides an opportunity to process samples with precision, showing little inter-run and inter-operator variability.5 It offers consistent, reliable, and trackable metrics for each step along the processing pathway.3

With accurate hybridization temperature as a part of the automated system, the instrument consistently monitors each sample for proper hybridization efficiency.3

Optimization and quality control of reagents, such as buffers and probes, offer reproducibility and reliability. Together with automation, this can offer an "end-to-end" solution for FFPE tissue FISH analytics.1

Reason #5: Traceability

Automation assists traceability of data and metrics.3 Samples processed in a cytogenomics laboratory often have many handwritten entries in logbooks to track patient identification, time and date processed, and type of probe used in the FISH assay.5

With the simplicity of the TBE's user interface, data points are captured and maintained in log files.3 Parameters of the analytic process, such as preloaded or user-tailored protocols containing times and temperatures, and FISH probes applied to the FFPE tissue slides for hybridization, can also be recorded.3

Onboard metrics can supplement and support the laboratory's Continuous Quality Improvement (CQI) program by real-time capturing of essential data, such as patient identifiers, reagents, probes used, temperatures, times, and dates. Logs are also maintained to increase traceability of problems and see trends in data for evaluating, tracking, and documenting potential error rates of specimen failures.3

Top 5 Reasons to Automate FFPE Tissue FISH

There are many reasons why a laboratory would want to automate their FFPE tissue FISH. The top 5 reasons to automate with the ThermoBrite Elite from Leica Biosystems are:

  1. TBE's semi-automated platform provides standardization, flexibility, and walk-away conveniences.4
  2.  TBE simplifies the manual workflow from approximately 30 steps to 3 steps.4
  3. TBE's automation temperature standardization and hybridization efficiency provide consistency to processing FFPE tissue FISH samples.5
  4. TBE reduces variation and offers reproducibility to sample processing.5
  5. TBE provides traceability of critical CQI data and metrics of sample processing during FFPE tissue FISH.3

References

  1. Tjio, J.H., Levan, A. (1956), The Chromosome Number Of Man. Hereditas, 42: 1-6. doi:10.1111/j.1601-5223.1956.tb03010.x
  2. Hastings, R. J., Bown, N., Tibiletti, M. G., Debiec-Rychter, M., Vanni, R., Espinet, B., van Roy, N., Roberts, P., van den Berg-de-Ruiter, E., Bernheim, A., Schoumans, J., Chatters, S., Zemanova, Z., Stevens-Kroef, M., Simons, A., Heim, S., Salido, M., Ylstra, B., Betts, D. R., Tumour Best Practice meeting, … Eurogentest (2016). Guidelines for cytogenetic investigations in tumours. European journal of human genetics: EJHG, 24(1), 6–13. https://doi.org/10.1038/ejhg.2015.35
  3. 3855_7500_501_B01_Thermobrite_Elite_User_Manual.pdf
  4. 200685_Rev_A_ThermoBrite_Elite_Flyer.pdf
  5. Tafe LJ, Allen SF, Steinmetz HB, et al. Automated processing of fluorescence in-situ hybridization slides for HER2 testing in breast and gastro-esophageal carcinomas. Exp Mol Pathol. 2014;97(1):116-119. doi:10.1016/j.yexmp.2014.06.003

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