Interrogating Bronchoalveolar Lavage Samples via Exclusion-Based Analyte Extraction

Abstract

Although average survival rates for lung cancer have improved, earlier and better diagnosis remains a priority. One promising approach to assisting earlier and safer diagnosis of lung lesions is bronchoalveolar lavage (BAL), which provides a sample of lung tissue as well as proteins and immune cells from the vicinity of the lesion, yet diagnostic sensitivity remains a challenge. Reproducible isolation of lung epithelia and multianalyte extraction have the potential to improve diagnostic sensitivity and provide new information for developing personalized therapeutic approaches. We present the use of a recently developed exclusion-based, solid-phase-extraction technique called SLIDE (Sliding Lid for Immobilized Droplet Extraction) to facilitate analysis of BAL samples. We developed a SLIDE protocol for lung epithelial cell extraction and biomarker staining of patient BALs, testing both EpCAM and Trop2 as capture antigens. We characterized captured cells using TTF1 and p40 as immunostaining biomarkers of adenocarcinoma and squamous cell carcinoma, respectively. We achieved up to 90% (EpCAM) and 84% (Trop2) extraction efficiency of representative tumor cell lines. We then used the platform to process two patient BAL samples in parallel within the same sample plate to demonstrate feasibility and observed that Trop2-based extraction potentially extracts more target cells than EpCAM-based extraction.

Keywords: bronchoalveolar; clinical automation; lab on a chip; lavage; lung cancer; microtechnology; solid-phase extraction.

Figure 1

Bronchoalveolar lavage sampling (BAL) is used to sample lung lesions in a less invasive manner than traditional percutaneous needle biopsy method. (A) A bronchoscope is guided down the bronchial tree, where it is used to wash the lesion site with saline. The saline is collected and its contents are analyzed. (B) BAL sample after centrifugation in a 50 mL conical tube. BAL samples are very heterogenous, containing cells, proteins, and extracellular matrix, making it difficult to process and analyze.

Figure 2

SLIDE platform overview. (A) The robot deck elements include a waste bin, a tube rack for samples and buffers, a tip rack, and two extraction plates. Each extraction plate consists of an input (In), a wash (W), and an output well (Out). (B) Samples are extracted via bronchoalveolar lavage and preprocessed with buffers and paramagnetic particles (PMPs). Samples are then placed in the tube rack, where the robot protocol distributes them to an input well where they are allowed to bind for 30 min at room temperature. Afterward, the modified magnetic pipet head is moved into position at the input well with its magnets in the bottom position. The magnet head is then moved slowly along the length of the well, extracting PMPs along with any cells bound to them. After collecting PMPs from the input, the magnet head moves them to the wash well, which has magnets embedded just below it in the plate platform. The magnets in the modified pipet head are then raised into the top position, allowing the PMPs to be pulled down into the well by the now stronger magnetic force of the embedded magnets. The plate is slid until the output well is positioned above the embedded magnets and the wash well is briefly mixed. Next, the magnets in the pipette head are lowered back to the bottom position, and the magnet head is positioned at the wash well to collect the PMPs, which are then moved to the output well and mixed in the same fashion as previously used for the wash well. Further details of SLIDE processing are contained in ref. . Downstream processing is performed manually off chip.

Figure 3

Cell extraction via EpCAM. (A) Representative images (identically treated) of EpCAM expression in H358, A549, and H226 cells. (B) A probability density plot of the natural log of EpCAM fluorescence in each of the three cell lines. (C) Extraction efficiency of cell lines using EpCAM. The EpCAM-positive cell lines (H358 and A549) were spiked into separate fractions of a cell suspension of THP-1 monocytes for a total of eight technical replicates of each condition. Extraction efficiency was compared with that of the H226 cell line, which moderately expresses EpCAM. Extraction efficiency of cell clumps is also shown. (D) Comparison of extraction efficiency of H358 cells in phosphate-buffered saline using SLIDE versus standard tube-based paramagnetic particle extraction.

Figure 4

Cell extraction via Trop2. (A) Representative images (identically treated) of Trop2 expression in H358 and H226 cells. (B) A probability density plot of the natural log of Trop2 fluorescence in each of the two cell lines. (C) Extraction efficiency of the cell lines using Trop2.

Figure 5

Cancer diagnostic cell line controls. (A) Fluorescent immunohistochemistry images of H358 and H226 cells with channels split and merged. (B) The ratio of TTF1 and p40 fluorescent intensity was determined for each cell; the log of the ratio was then taken and plotted on a density curve.

Figure 6

Patient bronchoalveolar lavage cell extraction and staining. (A) An example image from a patient sample that has been processed with the automated SLIDE protocol and stained for TTF1, p40, CD45, and Hoechst. A cancer cell (TTF1 and p40 positive, white arrow) and a clump of leukocytes (CD45 positive, pink arrow) can be seen in this image. (B) Number of target cells extracted from each patient (Pt.) sample aliquot using EpCAM and Trop2 capture antigens. (C, D) Density histograms of log(p40:TTF1) for patient target cells and associated cell line controls. EpCAM capture for Pt. 1 produced too few cells to make a histogram. Instead, the seven cells are plotted as blue dots on the x-axis.