Quantitative Diagnosis of Malignant Pleural Effusions by Single-Cell Mechanophenotyping

Abstract

Biophysical characteristics of cells are attractive as potential diagnostic markers for cancer. Transformation of cell state or phenotype and the accompanying epigenetic, nuclear, and cytoplasmic modifications lead to measureable changes in cellular architecture. We recently introduced a technique called deformability cytometry (DC) that enables rapid mechanophenotyping of single cells in suspension at rates of 1000 cells/s-a throughput that is comparable to traditional flow cytometry. We applied this technique to diagnose malignant pleural effusions, in which disseminated tumor cells can be difficult to accurately identify by traditional cytology. An algorithmic diagnostic scoring system was developed on the basis of quantitative features of two-dimensional distributions of single-cell mechanophenotypes from 119 samples. The DC scoring system classified 63% of the samples into two high-confidence regimes with 100% positive predictive value or 100% negative predictive value, and achieved an area under the curve of 0.86. This performance is suitable for a prescreening role to focus cytopathologist analysis time on a smaller fraction of difficult samples. Diagnosis of samples that present a challenge to cytology was also improved. Samples labeled as "atypical cells," which require additional time and follow-up, were classified in high-confidence regimes in 8 of 15 cases. Further, 10 of 17 cytology-negative samples corresponding to patients with concurrent cancer were correctly classified as malignant or negative, in agreement with 6-month outcomes. This study lays the groundwork for broader validation of label-free quantitative biophysical markers for clinical diagnoses of cancer and inflammation, which could help to reduce laboratory workload and improve clinical decision-making.

Measuring Cell Viscoelastic Properties Using a Microfluidic Extensional Flow Device

“…To address these issues, microfluidic tools have recently been explored as a strategy to measure cellular structural and mechanical properties with a rapidity that may be better suited to drug discovery and clinical application (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). Although these approaches have indeed massively improved measurement throughput and reduced operator skill/bias issues relative to traditional measurements, the extraction of cell mechanical properties (e.g., elastic modulus) remains challenging, primarily due to complex viscous forces that severely complicate analysis of deformations.…”

“…In an attempt to achieve high-throughput mechanical measurements within a simpler geometry, Di Carlo and colleagues developed higher-Reynolds-number (Re > 40) microfluidic systems that measure cell deformability with throughput ranging from 1000 cells/s (14) to 65,000 cells/s (15). By elongating cells at the stagnation point of extensional flow or pinching cells with two sheathing flows, they successfully developed population ''signatures'' based on distributions of cell deformability versus size.…”

“…By elongating cells at the stagnation point of extensional flow or pinching cells with two sheathing flows, they successfully developed population ''signatures'' based on distributions of cell deformability versus size. These population signatures responded in expected ways to cytoskeletal drugs in the pinched-flow sheathing device for which strain rates and imposed cell strains were not too large (15) (the expected effects of cytoskeletal depolymerization drugs were not detected in the high-strain-rate, highstrain-extensional-flow device (13)) and enabled prediction of disease state from clinical samples (13,14). Nonetheless, this work did not present an analytical route for extracting cell constitutive model parameters, instead requiring numerical solutions due to the high inertial component of the flow.…”

High-throughput physical phenotyping of cell differentiation

“…Using a standard inverted microscope setup equipped with a high-speed camera, the technology successfully monitored cell size and deformation at a throughput of~2000 cells per second. The developed technology, with cell size and deformability alone, successfully discriminated activated/non-activated leukocytes and identified malignancy in pleural effusion samples 21 ; however, it was also noted that there were potentially other easily extracted parameters (e.g., timedependent deformation and cell morphology) that might provide additional physical phenotypic information about cell type/status. As such, we hypothesized that expanding the analysis to additional physical properties may help to distinguish a spectrum of changes that occur as stem cells differentiate.…”

High‐throughput linear optical stretcher for mechanical characterization of blood cells

“…One approach is the microfluidic cell deformability cytometer which are inexpensive to prototype, use small sample volumes (nanoliters), and employ laminar flow characteristics (24) allowing for predictable and controllable flow. For example, physical constrictions (25)(26)(27)(28) or inertial focusing flow (29)(30)(31) have been used to create contact or shear forces (> 1 nN (30)) capable of significantly deforming flowing cells. Large strains (>10% deformation) however, can damage cells and should be avoided when cell isolation and viability post-analysis are of interest.…”