RAPID IMMUNOHISTOCHEMISTRY MICROFLUIDIC PROTOCOL ALLOWS THE

DETECTION OF CANCER CELLS AT THE MARGINS OF SURGICAL CUTS

Diego G. Dupouy*,

**, Sahar Ghiasikhou*, Ata Tuna Ciftlik**, Maryse Fiche***, Laurence de

Leval***, and Martin A. M. Gijs*

*Laboratory of Microsystems, École Polytechnique Féderale de Lausanne (EPFL), Switzerland

**Lunaphore Technologies SA, EPFL Innovation Park - Building C, CH-1015, Lausanne, Switzerland

*** Institute of Pathology, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland.

ABSTRACT

The assessment of the margin of the surgical cuts is crucial for the success of oncoplastic surgeries.

We show a rapid approach to assess the presence of tumoral cells at the margin of tumor resections using

a microfluidic tissue processor. We optimize the staining protocol to reduce the staining time to 8 minutes.

KEYWORDS: Rapid immunohistochemistry, Microfluidics, Intra-operative, Tumor resection margins

INTRODUCTION

During oncoplastic surgery of breast carcinomas, the location of cancer cells at the borders of the

excision is indicative of probable recurrence. Several intra-operative margin assessment techniques have

been described in literature, which make use of magnetic resonance [1], ultrasound [2] or impedance

spectroscopy [3] to create an image or detect a signal due to the presence of cancer cells. Staining of cryo-

fixated surgical specimens using hematoxylin and eosin remains the most common technique due to the

simplicity and speed (5 minutes) of the assay. The major common drawback these techniques share is the

lack of cancer cell-specificity, preventing the pathologist to spot a small number of infiltrating tumor cells

and eventually resulting in late positives [4]. A tool that helps the surgeons assess the proximity of

tumoral cells to the cut within surgical times is therefore highly desirable. In a previous study, we have

showed that our MTP can perform fast fluorescence immunostaining of tissue samples of Her2 antibodies

[5]. However, fast staining using chromogenic techniques using cytokeratins as markers of epithelial cells

was never demonstrated. We developed a microfluidic protocol that allows performing rapid IHC assays

on the surface of tumorectomy samples and, therefore, help localize cancer cells at the margins of the cut.

We optimized a chromogenic protocol that allowed us to perform an IHC staining in 8 minutes, compared

to the 70 minutes required with classical methods.

EXPERIMENTAL

Breast carcinoma samples of blocks located in close proximity to the surgical margins of the

tumorectomy were obtained from the bio-bank of the Institute of Pathology. The sample preparation

protocol, was done off-chip following the guidelines given by the pathology laboratory. Once the samples

were ready to be stained, they were loaded on the microfluidic setup and the protocol was launched.

Figure 1A is a cross-section schematic of the microfluidic device. By clamping a histological glass slide

to the MTP via an elastomer gasket, a thin (100 μm) chamber is formed that allows fast delivery and

washing of the reagents across the tissue section. A schematic of the reaction chamber is depicted in

Figure 1B, where the IHC steps are shown: (i) washing, (ii) primary antibody incubation, (iii) secondary

antibody incubation and (iv) chromogen substrate incubation. Figure 1C shows the microfluidic setup

used to interface the sample-containing slide, the MTP and the delivery system. Upon finalization of the

staining protocol, the histological glass slides are unclamped and mounted using a coverslip to be imaged

by bright-field microscopy.

2089978-0-9798064-8-3/µTAS 2015/$20©15CBMS-0001 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 25-29, 2015, Gyeongju, KOREA

Figure 1. A) Schematic cross-section of the microfluidic tissue processor (MTP) with the microfluidic chamber

formed at the interface with the histological glass slide. B) Zoom of the microfluidic chamber showing the 4 main

steps of the immunohistochemical (IHC) staining: (i) washing, (ii) primary antibody incubation, (iii) secondary

antibody incubation, and (iv) chromogen substrate incubation. C) Picture of the setup used to clamp the MTP to the

slide and deliver the reagents.

RESULTS AND DISCUSSION

We optimized the chromogenic step by performing a flush of the substrate during a multiple of fixed

short incubation times of 1 minute (figure 2A). On a further optimization step we increased the delivery

flow rate from 10 μL/s to 25 μL/s, showing an increased uniformity in the final staining across the

chamber (figure 2B). In figure 2C we show a typical breast carcinoma tissue section stained with anti-

cytokeratin antibodies, where cancer cells are found in close proximity to the surgical margin. The arrows

show the margins of the surgical cut, which is marked with a special ink administrated to the sample.. In

brown, the specific staining of epithelial cells can be observed. Pathologists can recognize the presence of

tumoral cells at the margins of the resection, which determines whether further excision is necessary.

Figure 2. Study of the chromogen incubation time given as t=2n-1

, with n the number of times the chromogen is

flushed in the chamber and incubated for 1 min. A) Brightfield images composed of microscope images of adjacent

slides for increasing chromogen incubation time. Scale bar: 500 μm. B) Comparison between chromogen flush done

at 10μL/s (blue) and 25μL/ (red). C) Brightfield microscopy image of human breast carcinoma sample stained using

anti-cytokeratin (AE1/AE3) antibodies in chromogenic staining. Epithelial cells are highlighted in brown. Blue ink

is applied prior to sectioning in order to identify the margins of the cut. Scale bar: 1 mm.

CONCLUSION

Fast IHC staining enabled by the MTP has the potential to increase the quality of intra-operative

margin assessment by providing cancer cell-specific information, reducing in this manner the rate of re-

excision.

REFERENCES

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[5] A. T. Ciftlik, H.-A. Lehr, and M. A. M. Gijs, “Microfluidic processor allows rapid HER2 immunohistochemistry of

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Mar. 2013.

CONTACT

* Diego G. Dupouy: +41 (21) 693 89 64/ diego.dupouy@epfl.ch

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