Factors affecting sedimentational separation of bacteria from blood

Abstract

Rapid diagnosis of blood infections requires fast and efficient separation of bacteria from blood. We have developed spinning hollow disks that separate bacteria from blood cells via the differences in sedimentation velocities of these particles. Factors affecting separation included the spinning speed and duration, and disk size. These factors were varied in dozens of experiments for which the volume of separated plasma, and the concentration of bacteria and red blood cells (RBCs) in separated plasma were measured. Data were correlated by a parameter of characteristic sedimentation length, which is the distance that an idealized RBC would travel during the entire spin. Results show that characteristic sedimentation length of 20 to 25 mm produces an optimal separation and collection of bacteria in plasma. This corresponds to spinning a 12-cm-diameter disk at 3,000 rpm for 13 s. Following the spin, a careful deceleration preserves the separation of cells from plasma and provides a bacterial recovery of about 61 ± 5%. K E Y W O R D Sbacterial bloodstream infection, bacterial separation, centrifugation, disk design, E. coli, human blood, sedimentation

Effect of dilution on sedimentational separation of bacteria from blood

“…In these experiments, disk trough volumes and V input were controlled such that the disk trough contained all the red cells upon deceleration and the red cell sediment thickness not exceed 3 mm 22 for red cells packed to 85%. 21 As a result, V flowdown $ (1 -ϕ 0 /0.85)V input , and the volume ratio Table S4 for regression details. The y-intercept at BR = 0.68 is consistent with previous experiments that measured BRs of 69%.…”

“…The y-intercept at BR = 0.68 is consistent with previous experiments that measured BRs of 69%. 21 5 | DISCUSSION…”

“…Our laboratory has recently developed a novel spinning disk that separates bacteria from RBCs and WBCs in less than 2 min using principles of sedimentation. [19][20][21] In our disk, centrifugal force pushes whole blood components toward an outer disk wall and leaves bacteria suspended in the supernatant plasma. Our current technology using a 12-cm-diameter disk can separate the RBCs from 7 ml of whole blood containing bacteria in about 20 s of spinning at 3,000 rpm, followed by 2 min of careful deceleration 22 during which time the red and white cells at the back wall slump down into a capture trough, and the bacteria-laden plasma flows into a collection space and is collected by hand pipet.…”

“…20 These experiments were done on disks of various sizes, spun at various velocity and time conditions. 21 In an effort to reduce the complexity of experimental factors, the data from hundreds of experiments were correlated by defining a characteristic length for each experiment, which is the distance that a hypothetical sphere (of 8 μm diameter and having the density of a RBC) would travel through plasma during the conditions of the spin, according to the physics of sedimentation of single spheres. Characteristic length described RBC and bacteria removal results well across all disk sizes and spin speeds and times, indicating that particle sedimentation was governing this separation process.…”

Analysis of Identification Method for Bacterial Species and Antibiotic Resistance Genes Using Optical Data From DNA Oligomers

“…Thus a 10 mL sample of blood would provide 100-fold more DNA than needed to place a 10-mer on each pyramid. Bacteria (and their DNA) can be collected from blood in minutes (Pitt et al, 2016;Alizadeh et al, 2017;Pitt et al, 2019), and the SERS analysis can commence immediately, followed by computations for identification of species and antibiotic resistance.…”