Acoustic impedance matched buffers enable separation of bacteria from blood cells at high cell concentrations

Abstract

Sepsis is a common and often deadly systemic response to an infection, usually caused by bacteria. The gold standard for finding the causing pathogen in a blood sample is blood culture, which may take hours to days. Shortening the time to diagnosis would significantly reduce mortality. To replace the time-consuming blood culture we are developing a method to directly separate bacteria from red and white blood cells to enable faster bacteria identification. The blood cells are moved from the sample flow into a parallel stream using acoustophoresis. Due to their smaller size, the bacteria are not affected by the acoustic field and therefore remain in the blood plasma flow and can be directed to a separate outlet. When optimizing for sample throughput, 1 ml of undiluted whole blood equivalent can be processed within 12.5 min, while maintaining the bacteria recovery at 90% and the blood cell removal above 99%. That makes this the fastest label-free microfluidic continuous flow method per channel to separate bacteria from blood with high bacteria recovery (>80%). The high throughput was achieved by matching the acoustic impedance of the parallel stream to that of the blood sample, to avoid that acoustic forces relocate the fluid streams.

Gradient acoustic focusing of sub-micron particles for separation of bacteria from blood lysate

“…Many label-free microfluidic methods for bacteremic blood sample preparation rely on direct size-based separation without prior lysis 26,27,45,[63][64][65][66][67][68][69] . Besides the reduction of blood cells of larger or similar diameter than the targeted bacteria and the potential release of phagocytosed but viable bacterial cells 38 , other benefits might arise from selective blood cell lysis as a first step in sample preparation.…”

“…Purification of small particles, e.g. bacteria and platelets [25][26][27][28] has been achieved by acoustically pushing away the larger particles from the smaller particles, but this approach does not enable isolation of sub-micron-sized biological particles from a complex background of molecules, particles or debris that are of smaller size, which is essential for purification protocols. Recently sub-micron particles were pushed away from 100-nm-particles by surface acoustic waves 29,30 and the same approach was applied to separate red blood cells and microvesicles from exosomes in a two-stage process 31 .We recently found that, for bulk acoustic waves, acoustic streaming can be greatly reduced by introducing a gradient in acoustic impedance (mass density times speed of sound) in the acoustic cavity by standard gradient centrifugation media 32,33 .…”

Factors affecting sedimentational separation of bacteria from blood

“…Successful techniques for bacterial separation from blood include microfluidics [17][18][19] and capture on magnetic beads. [20][21][22][23] These techniques have been reviewed recently 24 and are summarized in Table S2.…”

A portable microfluidic platform for rapid determination of microbial load and somatic cell count in milk

“…The device was capable of identifying not only the internal structure of the bacteria such as Gram positive or negative but also measuring the size of individual bacteria at the same time. Very recently, two microfluidic platforms have been proposed for isolation of bacteria from blood cells at high cell concentrations employing acoustophoresis in a silicon (Ohlsson et al 2018) and plastic microfluidic chips (Dow et al 2018) at a sample flow rate of 50-400 μL/min which leads to high enough throughput for many clinical applications (Cetin et al 2014). However, both platforms rely on bulky hardware for flow control and acoustic actuation, and lack of a rapid detection systems.…”