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.

Proteins and antibodies in serum, plasma, and whole blood—size characterization using asymmetrical flow field-flow fractionation (AF4)

“…Due to the complex nature and the large size range of components in blood, such samples are difficult to analyze and extensive sample pre-treatment is generally included. Typical sample preparation steps involve centrifugation, extraction, and filtration [9, 10]. This pre-treatment can, however, cause unintended artifacts such as unwanted loss of components, contamination, and protein aggregation.…”

Rapid separation of bacteria from blood – Chemical aspects

“…The most practical (for large volumes of whole blood) of these methods generally fall into the broad categories of 1) binding bacteria by selective ligands and subsequent removal, 2) centrifugation, and 3) hydrodynamic separation. These separation techniques have recently been reviewed [10], and only a few will be mentioned herein as background.…”

“…Thus centrifugation will separate bacteria from plasma, but will not separate bacteria from RBCs. However, centrifugation for a short period of time can transiently separate the bacteria from the RBCs because the sedimentation velocity of smaller bacteria is about 30-fold less than the sedimentation velocity of larger RBCs [10]. For horizontal centrifugation of ideal spherical particles that do not interact with each other, the sedimentation velocity, v s , of the particles relative to the fluid is given by $${\nu }_s=\frac{D_p^2({\rho }_p-{\rho }_f){\left(R^2{\omega }^4+g^2\right)}^{1/2}}{18\mu }$$ where D p is the particle diameter, ρ p is the particle density, ρ f is the fluid density, R is the rotational radius, ω is the rotational angular velocity, g is the gravitational constant, and μ is the fluid viscosity [22].…”

Rapid separation of very low concentrations of bacteria from blood

“…Chemical and magnetic capture techniques have shown promising results with high bacterial concentrations (~10 3 to 10 4 CFU/ml) and high removal efficiencies (12–14). Drawbacks of these capture methods include that the binding agents must be specific to the bacteria, there must be a large number of beads to capture the low numbers of bacteria found in septic blood, and the process is often time-consuming (15). Few of these techniques have been demonstrated on blood with colony forming units (CFUs) in the range of 10–100 per milliliter.…”

“…Few of these techniques have been demonstrated on blood with colony forming units (CFUs) in the range of 10–100 per milliliter. In addition, the materials required in magnetic and chemical separation of bacteria from blood are often associated with a high cost (15). Microfluidic devices have also received attention in the literature for successfully separating red blood cells (RBCs) from bacteria or other smaller particles using red cell migration and particle focusing (16, 17).…”