Rapid separation of bacteria from blood-review and outlook

Abstract

The high morbidity and mortality rate of bloodstream infections involving antibiotic-resistant bacteria necessitate a rapid identification of the infectious organism and its resistance profile. Traditional methods based on culturing the blood typically require at least 24 h, and genetic amplification by PCR in the presence of blood components has been problematic. The rapid separation of bacteria from blood would facilitate their genetic identification by PCR or other methods so that the proper antibiotic regimen can quickly be selected for the septic patient. Microfluidic systems that separate bacteria from whole blood have been developed, but these are designed to process only microliter quantities of whole blood or only highly diluted blood. However, symptoms of clinical blood infections can be manifest with bacterial burdens perhaps as low as 10 CFU/mL, and thus milliliter quantities of blood must be processed to collect enough bacteria for reliable genetic analysis. This review considers the advantages and shortcomings of various methods to separate bacteria from blood, with emphasis on techniques that can be done in less than 10 min on milliliter-quantities of whole blood. These techniques include filtration, screening, centrifugation, sedimentation, hydrodynamic focusing, chemical capture on surfaces or beads, field-flow fractionation, and dielectrophoresis. Techniques with the most promise include screening, sedimentation, and magnetic bead capture, as they allow large quantities of blood to be processed quickly. Some microfluidic techniques can be scaled up. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:823-839, 2016.

Keywords: bacterial bloodstream infection; centrifugation; chemical binding; filtration; hydrodynamic focusing; rapid identification; sedimentation.

Figure 1

Centrifugal separation device adapted from Ref. , with permission from Royal Society of Chemistry. Left: schematic representation of microfluidic channels on a rotating disk. Right: photograph of blood cell pellet and separated plasma after rotation has stopped, and plasma is being drained through a capillary duct.

Figure 2

Centrifugal separation device adapted from Ref. , with permission from Bioanalysis as agreed by Future Science Ltd. This compact disk device has a separation chamber and a capillary connection to a plasma collection chamber (bottom left of panels E and F) that drains the plasma when centrifugation is stopped.

Figure 3

(A) Drawing (to scale) of rotating hollow disk for sedimentation-based separation of bacteria from blood. (B) Schematic showing that initially 7 mL of bacteria-spiked blood are placed in the disk. Upon rotation at 3,000 rpm, the blood is spun to the inside surface of the wall of the hollow disk and the blood components start to sediment toward the wall. (C) After about 1 min there is a layer of clear plasma which still contains the bacteria because they sediment much more slowly than RBCs and WBCs. (D) When the rotation stops, the packed cells slough down and are trapped in the well at the base of the channel, while the plasma containing bacteria drains off and is collected.

Figure 4

Amount of RBCs (log₁₀ of RBC/mL) from porcine blood remaining in the plasma recovered from the spinning hollow disk apparatus. Spinning was done at 3,000 rpm. Error bars indicate the standard deviation of the mean of repeated experiments (n = 3). Where no error bars are evident, the range of standard deviation is less than the symbol size.

Figure 5

Preliminary results of recovery of E. coli spiked into fresh anticoagulant-treated human blood. Blood was collected from human volunteers into vacutainer tubes containing anticoagulants of either citrate, EDTA, or heparin. Error bars represent the standard error of the mean from repeated experiments, n ≥ 4.

Figure 6

Illustration of the three-stage separation device by which bacteria were separated from RBCs in whole mouse blood (above left). The bacteria and plasma are skimmed from the cell-free layer near the wall (above right), and the core of red cells is returned to the mouse. Taken from Ref. , with permission from Royal Society of Chemistry.

Figure 7

Illustration of the concept of inertial microfluidic focusing by which bacteria were separated from RBCs in highly diluted blood. The bacteria and red cells are deflected by the acting flow and protected by the sheath flow, and finally focused at a particular location in the exit channel. Reproduced from Ref. , with permission from Royal Society of Chemistry.

Figure 8

Illustration of the concept of Dean flow focusing by which bacteria and platelets were separated from RBCs in slightly diluted blood. The Dean flow circulation cycles the bacteria and platelets from the outer wall to the inner wall and back to the outer wall while the RBCs get trapped near the inner wall of the curved channel. Reproduced from Ref. , with permission from Royal Society of Chemistry.