# Technique Evolutions for Microorganism Detection in Complex Samples: A Review
## 3 Developments in Innovative Detection Methods
### Improvements and Developments in Analytical Methods Requiring Sampling
#### 3.2.2 Microfluidics
By matching the acoustic impedance of the central stream to that of the blood sample, they could process 1 mL of undiluted blood within 12.5 min, with a bacteria recovery rate of 90% and a blood cell removal rate above 99%. As shown in Table 1, flow rate and dilution factor can be tuned differently to optimize either the bacteria recovery rate (up to 99.7%) or the blood cell removal (up to 99.99%). \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Flow Rate** & **Dilution Factor** & **Time to Process 1 mL** & **Red Blood Cells Removal** & **Bacteria Recovery** & **Optimized for** \\ \hline
400 \(\mu\)L/min & 100 & 4 h & \textgreater{}99.9\% & **99.7\%** & Bacteria recovery \\
100 \(\mu\)L/min & 5 & 50 min & **99.99\%** & 75\% & Blood cell removal \\
400 \(\mu\)L/min & 5 & **12.5 min** & \textgreater{}99\% & 90\% & Throughput \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Comparison of label-free microfluidic continuous flow methods for separation of bacteria from red blood cells. All numbers are per channel, adapted from [81], licensed under CC BY 4.0. Figure 4: Schematic description of acoustic separation principle. The acoustic field separates blood cells by pushing them in the central stream while bacteria stay in the side streams. [PERSON] et al. combined a similar acoustophoresis system with a bacteriophage-based luminescence assay [91]. Tested on _Pseudomonas aeruginosa_, _E. coli_, and _Staphylococcus aureus_, they demonstrated an LOD of six bacteria from blood with initial concentration of \(10^{2}\) CFU/mL. Acoustophoresis greatly improved the LOD compared with unpurified samples. In conclusion, acoustophoresis can be used to separate bacteria from blood cells with excellent bacterial recovery. The high throughput enables the processing of clinical samples in a short period of time. It can also allow real-time monitoring without the need to collect samples during cell therapy production. Rate flow can further be increased through the use of higher blood cell concentrations, larger channels, or multiple parallel separation channels to monitor all types of ATMPs. In combination with downstream bacterial detection, this separation method paves the way for the potential development of fast detection methods. More detection methods should be tested with acoustophoresis in order to find the most suited and reduce the LOD and the time of response. This method can also be used to remove bacteria from a sample for sterilization purposes and not just for detection. The manufacturing of cell or gene therapies can be facilitated. As for previous methods, acoustophoresis should be validated on a larger number of bacterial species and strains. Other individual bacteria should behave in a similar way during acoustic separation, because the size difference with blood cells is always large. However, pathogenic bacteria are known to form clumps, to reside within white blood cells, or to adhere to blood cells or platelets. The risk is therefore that bacteria are sorted out with the large cells. One solution could be to chemically prevent the adhesion of bacteria to blood cells during the assay with the risk of denaturing the ATMP sample. Microdroplets and 3D Particle Counter  
Droplet microfluidics is another microfluidic method that can improve the detection of pathogens. The idea is to analyze a solution by decomposing it into a microdroplet surrounded by oil. By doing so, every microliter of the solution is screened individually, allowing a finer detection. By sorting the droplets of interest, it is as if the initial solution was concentrated, or enriched, by a factor of several hundred [92]. While droplet microfluidics can improve detection sensitivity and specificity, initial flow rates limited its application to sample with a volume in the microliter range. However, recent developments resulted in a 3D particle counter able to process, within minutes, samples of several milliliters, as usually required for clinical relevance. [PERSON] et al.