Cell Wash

The core needle biopsy is today’s standard procedure for the laboratory testing of tissue samples when malignancy is suspected. Sampling in this manner continues to provide invaluable insights into the morphological features of mass lesions in the body. Through morphological analysis, pathologists can discern whether the mass is benign or cancerous and – if the latter – gather insights into its stage, level of aggressiveness, and metastatic potential.

However, there resides in these core needle biopsy specimens much more potential than the delivery of conventional morphology. Herein is presented a simple method that opens the doors for pathology labs to improve upon the recovery and uncovering of the secrets behind the microscopic image.

Formalin-fixation: a double-edged sword?
 

The handling of core needle biopsies is critical to the reliability and reproducibility of results. At many facilities, the collected sample has to be transferred to the pathology laboratory, which demands formalin fixation. The resulting formalin-fixed, paraffin-embedded tissues are stained and analyzed with immunohistochemistry or immunofluorescence methods. Thus, formalin fixation enables morphological analysis by allowing phenotypic evaluation, visualization between normal and cancerous cells and protein detection by immunohistochemistry.

Despite these benefits, core needle biopsies have limitations that make further sample analysis cumbersome. In today’s era of precision medicine, there exists a strong demand for molecular analysis on this fixed material, as the underlying genomic content in these cancer cells can reveal critical mutations that contribute to specific altered pathways. Such information is instrumental in biomarker discovery and drug development, with rapidly emerging critical implications for patient treatment. 

Nevertheless, the nature of the core needle biopsy specimen, and the subsequent tissue fixation it routinely undergoes, are in direct conflict with molecular analysis. To begin with, the tissue obtained via core needles is quantitatively minute, thus limiting the ability to perform multiple modality testing on the same sample. More importantly, formalin is a chemical that creates crosslinks between macromolecules making it a challenging proposition to collect high-quality genetic content for biochemical applications such as PCR and next-generation sequencing, both vitally important tools in today’s burgeoning field of precision medicine.

Cell harvest for molecular analysis
 

The key to a comprehensive sample analysis lies in the ability to extract sufficient amounts of cells and use a fraction solely for molecular testing. This can be achieved by an additional washing step during sample preparation. When a core needle is used for collecting a tissue sample, a number of cells are dislodged and adhere to the inner wall of the cylinder. These residual cells can be recovered and retained for molecular testing by submerging the needle in a phosphate buffer saline (PBS) solution. The non-toxic and isotonic nature of the PBS buffer allows these dislodged cells to go into solution and be recoverable while protecting the cells’ integrity and preventing the loss of their precious genetic content. With this minor modification step in specimen processing, the pathologist gains access to two samples, one for morphological analysis and the other for molecular testing.

The role of DNA recovery in taking full advantage of cell washing cannot be overstated. The primary objective is to recover sufficient amounts of high-quality DNA from these dislodged cells that originated from the already diminutive core needle biopsy tissue specimen. The phrase “high quality” here refers to DNA integrity and purity – crucial features for reliable biochemistry assay outputs. To that end, the solid phase reversible immobilization (SPRI) technique has repeatedly shown value in genomic DNA purification. Through manipulation with a magnetic field, SPRI paramagnetic beads enable seamless isolation of genomic DNA by reversibly binding nucleic acids and separating them from the rest of the cell lysate solution.

Wilfrido D. Mojica, Chief of Pathology at the Niagara Falls Memorial Medical Center, developed a cell wash method to harvest these dislodged cells that concurrently enables tissue recovery. Mojica’s lab demonstrated the applicability of the method using a variety of biopsy specimens. “The recovery of these cells and the rapid stabilization of their nucleic acids helped us freeze and preserve the DNA, giving us flexibility as to when to perform the molecular testing,” Mojica explains. “We successfully obtained high-quality DNA with the desired purity and integrity for demanding molecular testing applications. More importantly, integrating a commercially available reagent kit into the workflow significantly accelerated DNA recovery by allowing rapid separation and high-throughput implementation, as these kits are often amenable to automated liquid handling.”

Future implications of cell washing and DNA recovery
 

Cell wash with DNA recovery is gaining more recognition as an easy and valuable addition to core needle biopsies. Unlike extracting nucleic acids from fixed tissue, this process is easy to adopt without high-end instrumentation or advanced expertise, making it highly accessible to pathology labs with limited resources. In addition, it broadens the scope of research applications for diagnostics by enabling morphological and molecular analysis simultaneously from the same tissue sample. Even in the event that immediate molecular analysis is not desired, the liquid aliquot containing the dislodged diagnostic tumor cells can be preserved should testing be sought at a later timepoint.

Cell washing of core needle biopsies is in its infancy, performed mainly for research use in small-scale laboratories, but has the potential to grow in popularity as laboratories continue to examine new methods and see the benefits firsthand. As envisioned by Mojica, “It is the wise and prescient pathologist who begins to optimally process these specimens to take full advantage of this unrealized – but clinically valuable asset – so as to improve upon not only small tissue biopsy management but also patient care.”