3D pathology is intrinsically dependent on 3D microscopy, or the whole tissue imaging of patient tissue biopsies (TBs). Consequently, unsectioned needle specimens must be processed whole: a procedure which cannot necessarily be accomplished through manual methods, or by retasking automated pathology machines. Thus "millifluidic" devices (for millimeter-scale biopsies) are an ideal solution for tissue handling/preparation. TBs are large, messy and a solid-liquid mixture; they vary in material, geometry and structure based on the organ biopsied, the clinician skill and the needle type used. As a result, traditional microfluidic devices are insufficient to handle such mm-sized samples and their associated fabrication techniques are impractical and costly with respect to time/efficiency. Our research group has devised a simple, rapid fabrication process for millifluidic devices using jointed skeletal molds composed of machined, reusable metal rods, segmented rods and stranded wire as structural cores; these cores are surrounded by Teflon outer housing. We can therefore produce curving, circular-cross-section (CCCS) millifluidic channels in rapid fashion that cannot normally be achieved by microfabrication, micro-/CNC-machining, or 3D printing. The approach has several advantages. CLINICAL: round channels interface coring needles. PROCESSING: CCCS channels permit multi-layer device designs for additional (processing, monitoring, testing) stages. REUSABILITY: for a biopsy/needle diameter, molding (interchangeable) components may be produced one-time then reused for other designs. RAPID: structural cores can be quickly removed due to Teflon®'s ultra-low friction; housing may be released with ethanol; PDMS volumes cure faster since metal skeleton molds conduct additional heat from within the curing elastomer.
For cancer diagnoses, core biopsies (CBs) obtained from patients using coring needles (CNs) are traditionally visualized and assessed on microscope slides by pathologists after samples are processed and sectioned. A fundamental gain in optical information (i.e., diagnosis/staging) may be achieved when whole, unsectioned CBs (L = 5-20, D = 0.5-2.0 mm) are analyzed in 3D. This approach preserves CBs for traditional pathology and maximizes the diagnostic potential of patient samples. To bridge CNs/CBs with imaging, our group developed a microfluidic device that performs biospecimen preparation on unsectioned CBs for pathology. The ultimate goal is an automated and rapid point-of-care system that aids pathologists by processing tissue for advanced 3D imaging platforms. An inherent, but essential device feature is the microfluidic transport of CBs, which has not been previously investigated. Early experiments demonstrated proof-of-concept: pancreas CBs (D = 0.3-2.0 mm) of set lengths were transported in straight/curved microchannels, but dimensional tolerance and flow rates were variable, and preservation of CB integrity was uncontrolled. A second study used metal cylinder substitutes (L = 10, D = 1 mm) in microchannels to understand the transport mechanism. However, CBs are imperfectly shaped, rough, porous and viscoelastic. In this study, fresh/formalin-fixed porcine and human pancreas CBs were deposited into our device through a custom interface using clinical CNs. CB integrity (i.e., sample viability) may be assessed at every stage using an optomechanical metric: physical breaks were determined when specimen intensity profile data deviated beyond xavg + 2σ. Flow rates for human CBs were determined for several CNs, and microfluidic transport of fresh and formalin-fixed CBs was analyzed.
A minimally-invasive diagnosis of pancreatic cancer is accomplished by obtaining a fine needle aspirate and observing
the cell preparations under conventional optical microscopy. As an unavoidable artifact, native tissue architecture is lost,
making definite diagnosis of malignancy, or invasive neoplasm, impossible. One solution is the preparation of core
biopsies (CBs) within a microfluidic device that are subsequently imaged in 3D. In this paper, porcine pancreas CBs (L
= 1-2 cm, D = 0.4-2.0 mm) were formalin-fixed, stained and optically cleared (FocusClear®). In brightfield at 40x, light
transmission through the ordinarily opaque CBs was increased 5-15x, and internal islet structures were easily identified
250-300 μm beneath the tissue surface. Typically, specimen preparation is time intensive and requires precise handling
since CBs are delicate; thus, fixative, absorptive stain and FocusClear® diffusion were done slowly and manually. To
significantly speed up tissue processing, we developed a microfluidic device consisting of both a main channel (L = 12.5
cm, D = 1.415 mm) with a circular cross section used for fixing and transporting the CB and an intersecting U-channel
employed for staining. Space between the CB and channel wall provided a key feature not traditionally employed in
microfluidic devices, such that at low flow rates (5-10 mL/min) CBs were fixed and stained while the specimen
remained stationary. By switching quickly to higher flow rates (15-20 mL/min), we could precisely overcome adhesion
and transport the specimen within the channel towards the imaging platform for 3D pathology.
The pancreas is a deeply seated organ requiring endoscopically, or radiologically guided biopsies for tissue diagnosis. Current approaches include either fine needle aspiration biopsy (FNA) for cytologic evaluation, or core needle biopsies (CBs), which comprise of tissue cores (L = 1-2 cm, D = 0.4-2.0 mm) for examination by brightfield microscopy. Between procurement and visualization, biospecimens must be processed, sectioned and mounted on glass slides for 2D visualization. Optical information about the native tissue state can be lost with each procedural step and a pathologist cannot appreciate 3D organization from 2D observations of tissue sections 1-8 μm in thickness. Therefore, how might histological disease assessment improve if entire, intact CBs could be imaged in both brightfield and 3D? CBs are mechanically delicate; therefore, a simple device was made to cut intact, simulated CBs (L = 1-2 cm, D = 0.2-0.8 mm) from porcine pancreas. After CBs were laid flat in a chamber, z-stack images at 20x and 40x were acquired through the sample with and without the application of an optical clearing agent (FocusClear®). Intensity of transmitted light increased by 5-15x and islet structures unique to pancreas were clearly visualized 250-300 μm beneath the tissue surface. CBs were then placed in index matching square capillary tubes filled with FocusClear® and a standard optical clearing agent. Brightfield z-stack images were then acquired to present 3D visualization of the CB to the pathologist.
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