In Vivo Flow Cytometry of Extremely Rare Circulating Cells

Xuefei Tan,Judith M Runnels,Mark Niedre,Charles P Lin,Peter Bartosik,Roshani Patil

doi:10.1038/s41598-019-40143-2

Abstract

Circulating tumor cells (CTCs) are of great interest in cancer research, but methods for their enumeration remain far from optimal. We developed a new small animal research tool called “Diffuse in vivo Flow Cytometry” (DiFC) for detecting extremely rare fluorescently-labeled circulating cells directly in the bloodstream. The technique exploits near-infrared diffuse photons to detect and count cells flowing in large superficial arteries and veins without drawing blood samples. DiFC uses custom-designed, dual fiber optic probes that are placed in contact with the skin surface approximately above a major vascular bundle. In combination with a novel signal processing algorithm, DiFC allows counting of individual cells moving in arterial or venous directions, as well as measurement of their speed and depth. We show that DiFC allows sampling of the entire circulating blood volume of a mouse in under 10 minutes, while maintaining a false alarm rate of 0.014 per minute. In practice, this means that DiFC allows reliable detection of circulating cells below 1 cell per mL. Hence, the unique capabilities of DiFC are highly suited to biological applications involving very rare cell types such as the study of hematogenous cancer metastasis.

Highlights

There are many biological processes involving rare cells that circulate in the peripheral blood
There were a number of limitations which precluded its use in a real cancer metastasis studies, in particular, (i) it could not distinguish cells moving in arterial, venous, or capillary bed, making it susceptible to over-counting of cells on their return trip through the vasculature, (ii) it was susceptible to false-positive signals due to electronic noise or motion, and, (iii) the measured count rate was lower than predicted based on the cell concentration, which we later determined was primarily due to hypothermia and poor blood flow in the limb while the mouse was under anesthesia
Diffuse in vivo Flow Cytometry’ (DiFC) builds on our previous work, but introduces a number of critical advances

Summary

Introduction

There are many biological processes involving rare cells that circulate in the peripheral blood. Researchers have developed ‘in vivo flow cytometry’ (IVFC) methods to count cells directly in mice without having to draw blood samples. These are modified intravital fluorescence microscopes that operate in trans-illumination mode through a mouse ear, and optically sample blood flowing through a small arteriole[5,11,12,13,14,15]. For circulating cells at lower concentrations, mice usually must be euthanized and the entire peripheral blood volume drawn and analyzed In this case, experiments are terminal and individual animals cannot be followed longitudinally over time to track disease progression. We achieved better thermal control of the mouse tail while under anesthetic, avoiding hypothermia in the limbs and significantly increasing blood perfusion in the sampling volume versus our previous work

Methods

Findings

Conclusion