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The distance from the sensing zone to the break off point is controlled by a microscope and held constant. The delay setting is fixed during sorting and in general the break off distance is kept constant by the operator. If the velocity of the liquid jet is constant during sorting the sorting works fine, but in practice this is not always the case.
Small changes of sheath pressure for example due to partial clogging of the sheath filter can alter jet velocity during sorting. Timothy Petersen and Gerrit van den Engh have examined the problem and showed how little variations of sheath pressure can disturb the sorting process and how the operator can handle it Toralf Kaiser examined how temperature changes of sheath fluid alters sorting performance and gives a solution for stabilizing sheath fluid temperature From a technical point of view a flow cytometer is a light detection device capable of detecting photons of different wavelengths over a high dynamic range.
In order to achieve a high dynamic range, the optics, signal detection, and processing units must be carefully designed. In flow cytometers, lenses are used to collect light emitted from the cell of interest, i. Furthermore, they are used to make the collected light parallel in order to direct it through the optical bench to the detectors. A flow cytometer employs collection and collimation lenses. Collection lenses convex lenses are used to focus the light from the interrogation point either to the end of an optical fiber or directly to a collimation lens e.
Some instruments use optical fibers to route the detected light to detectors which are installed in an octagon. In this case a collimation lens is installed at the other end of the fiber to ensure that all light is routed parallel through the octagon. Inside the octagon another collimation lens is placed in front of each detector to focus the parallel light onto the photocathode. In instruments without fiber optics the parallel light is routed through the optical bench and then focused onto the photocathode by a collimation lens. The photodetectors used in flow cytometers are spectrally broadband and therefore unable to generate a signal exclusively from specific wavelengths and thus specific markers.
To add specificity, optical filters and dichroic mirrors are used in a well defined manner to route the light to the detectors. Optical filters are designed as band pass BP , long pass LP , or short pass SP filters and are mostly installed in front of the light detectors. The common property of the filters is that they transmit light only within a spectral range.
A BP filter transmits light in a certain range. For example, a LP of nm will transmit all light above nm. Due to aging, quality of coating, and contamination, the actual parameter of an optical filter can differ from the technical description. Therefore, it is recommended to check the transmission spectra of new filters provided by the manufacturer and always keep filters dust free.
Sometimes mirrors usually silver mirrors are used in the optical bench of a flow cytometer in order to deflect light for geometrical or constructive reasons. The effect of the dichroic is dependent on the operating angle. Recently, commercial cytometers have become available which use spatially dispersing elements instead of or in combination with optical filters in order to deflect light wavelength specific to a detector array. The rationale behind this is the measurement of the entire emission spectra of a cell see Section I. A dispersing element can be a dispersive prism or a grating.
Prisms have a higher light efficiency over gratings and they are not sensitive for polarized light. This maybe the reason why they are employed in the spectral flow cytometer from Sony. Such lasers have a small footprint and a typical output power range from 20 to mW. Lasers are coherent light sources which allow a high photon density at the illumination point, and therefore an efficient energy transfer to the fluorochrome. Modern cytometers are equipped with up to seven different lasers in a typical laser line ranging from to nm.
This gives high flexibility in choosing the fluorophores. As a flow cytometer measures the biological information of a particle e. In this section, the main components of cytometer electronics are briefly described. From a technical point of view, the detection of cell related light is difficult due to i the low light level, ii the high analysis rate, and iii the high dynamic range of the light level.
Photomultiplier tubes PMTs meet these requirements and are therefore employed in almost all flow cytometers. PMTs are vacuum tubes containing a photocathode, electron focusing electrodes, and a series of dynodes for electron multiplication. The photocathode converts photons to photoelectrons which are then multiplied by a series of dynodes driven by a high voltage Fig.
In many applications, PMTs are increasingly being replaced, e. Amplifiers in a flow cytometer can be grouped as pre and main amplifiers. All amplifiers in a cytometer are analogue hardware devices which must be very well designed for optimal signal to noise ratios SNRs. In modern cytometers, the conversion of the continuous analog voltage signal into discrete digital values is done by ADCs which are defined by their sampling frequency and sample resolution. The required dynamic detection range DNR of a flow cytometer can be defined as the intensity range of stained and unstained cells, for example.
In practice, the effective number of bits of an ADC is, due to noise and distortion of the circuit, some decibels below the theoretical value e. This limits the dynamic range to less than 4 decades and, more importantly, shrinks the resolution of dim signals. In the digital domain the signals are processed by filters, baseline restorer, pulse height, pulse width algorithms, and trigger see Section I. The resulting signal consists of an unwanted DC part due to laser scatter light and electronic noise among others and a specific AC part. The baseline restorer attempts to keep the baseline at zero.
In practise however, baseline restoring is not perfect and can lead to negative values on the histogram axis or introduce a slight distortion of low signals and therefore to a increased CV of dim signals. Taken together, the analogue and digital components of a flow cytometer in combination with the baseline and pulse shaping algorithms need to be well adjusted in order to maximize SNR and DNR.
Since the invention of the first prototype of a Fluorescence Activated Cell Sorter in at Stanford University, the technology has become a powerful tool to analyze and sort individual cells based on their functional status. Moreover, flow cytometry provides a robust statistic of thousands of individual cells and can detect rare events at a frequency below 10 —4 cells.
In a typical cytometer, the sensitivity decreases with increasing flow rate due to the increasing diameter of the cell stream within the flow cell. Typically these markers are fluorescently tagged antibodies, molecular sensors, as well as genetically encoded reporters. In practice, this high number of parameters is not achievable because at the moment the range of appropriate fluorescent dyes is limited.
Technical limitations regarding the maximum number of detectable markers are also given by the overlap of the emission spectra of the different fluorescent tags, since each fluorescence detection channel is correlated to a biological marker. To overcome this, fluorescent tags became available which have different excitation wavelengths. Currently, up to seven lasers with emission wavelengths from to nm are used in order to achieve a high flexibility in the choice of the fluorescent tags.
Furthermore, tunable lasers are used for special applications like fluorescent life time measurements FLIMs. Flow cytometers use either photomultipliers PMTs or avalanche diodes to convert the emitted or scattered light into amplified electrical pulses which are processed by appropriate electronics to extract information like pulse height, area, length, and time. The electronics of the cytometer consist basically of a preamp circuit, baseline restoration circuit, and an analog to digital converter ADC.
All components together must have a low noise level i. Within this instrument, the emitted fluorescence light is divided by a wavelength division multiplexer WDM through a series of band pass filters and integrated optics, onto an array of avalanche diodes which enables a high sensitivity in the detection of e. Avalanche diodes or PMTs itself are light detectors which are unsuitable for wavelength detection, hence the fluorescent light needs to be filtered by optical filters and mirrors.
These filters must be carefully chosen because a multiparameter experiment, i. Conventional flow cytometers circumvent this problem by compensation see Section III. Following this, the data are analyzed in a multivariate fashion in combination with a hierarchical gating strategy see Section VI. It is essential to adapt the combination of fluorescent tags to the given optical, laser, and electronic setup of the instrument to minimize spillover, increase Q, and lower B signals.
For instance, by choosing the right concentration of a certain reagent see Section IV. This can help to increase the separation the distance between the means between a blank and a fluorescent population which is a function of Q and B. Thus, it requires the characterization of Q and B of the used instrument. Mostly polystyrene particles beads are used for this purpose in combination with software based protocols implemented in the instruments e. Scale calibration is an especially useful approach to measure absolute values e.
Beside beads, scale calibration can also be achieved by using LED light pulses. Furthermore, by using this tool, instruments can be compared within or between labs regarding their Q and B values. Up to this point, analytical cytometers have been described but cells can, in addition, be sorted based on specific marker expression for downstream analysis molecular biology, sequencing, etc.
After excitation A in Fig.
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Gratings are susceptible for polarized light. As polarization occurs frequently in flow cytometry 22 , the total efficiency of a grating may be reduced. In fact, prisms are better suited for spectral light dispersion because they have a better light transmission and are also stable for polarized light. Unfortunately, the dispersion of a prism is not linear with regard to the wavelength, which makes it difficult to use linear detector arrays such as multianode PMTs On the other hand, this in combination with high spectral resolution allows the spectral detection of Raman scattering which is a characteristic spectrum of molecular vibrations, much narrower than fluorescence spectra.
This allows the application of new biological markers, such as surface enhanced Raman scattering tags or near infrared fluorescent dyes 24 , More recently, Robinson et al. This spectrograph was implemented in the optical pathway of a conventional flow cytometer and was able to take spectra of single cells and microspheres as well as to discriminate free versus bound propidium iodide.
The first commercially available spectral flow cytometer, the SP, was developed by Sony Moreover, the instrument is equipped with 3 lasers , , and nm , which allows for full spectral detection of the resulting emission spectra. The measured spectra from single cells are subsequently unmixed by using reference spectra of all used dyes and the autofluorescence spectrum.
Least Square Fitting algorithms are used to calculate the most accurate fit for all reference spectra, leading to an accurate determination of which dyes are present on each cell and at which intensity. Using this method, a complete fluorescence emission is used instead of only a small portion of emitted light entering a dedicated detector through a specific set of mirrors and optical filters. This is a major advantage over conventional flow cytometry, in which light that is lost outside of the optical filters also contaminates other channels with unwanted light which has to be corrected by a subtractive method see Section III.
Since dyes frequently used in flow cytometry have rather broad emission spectra and large spectral overlaps, spectral unmixing can help mitigate this problem. Moreover, control of reagents especially tandem dyes is paramount with the increased need for standardization. Given that spectral flow cytometry shows full spectrum unbiased data, quality control is more or less integrated.
In this fashion, spectral flow cytometers are designed to measure the biological information across multiple detection channels, where the optical configuration can be fixed for all experiments, giving the added benefit of instrument stability, sensitivity 33 , and easier standardization across instruments, aided by the lack of individual PMTs and individual optical filters and mirrors. Imaging flow cytometers combine conventional flow cytometry with the additional benefit of imaging each individual cell.
By utilizing the speed and phenotyping ability of flow cytometry with the imagery of microscopy, it allows a broad range of applications to be studied that would be impossible using either technique alone. Each generation has become faster with higher resolution, and the addition of a benchtop model has made imaging flow cytometry more accessible to researchers. Both capture 12 images of each cell, of which 10 can be fluorescent.
The high throughput cell imaging of these instruments allows cellular functions, which are often only otherwise measurable by microscopy, to be investigated. It is very time consuming and user biased to analyze large number of cells by microscopy, and near impossible for rare cell types. An antibody panel appropriate for the biological question should be chosen and selection of the fluorochrome conjugates should take into account the expression level of the molecules while avoiding excessive compensation. Web based software can aid in the panel design, such as BD fluorescence spectrum viewer and Biolegend fluorescence spectra analyzer.
Since the laser powers frequently differ from conventional flow cytometers, even antibodies, which provide optimal cell detection in conventional flow cytometry require titration. The imaging component helps to determine the appropriate concentration and ensures that the protein is detected in the expected cell compartment. As for conventional flow cytometry, correct controls positive and negative need to be included, i. Positive experimental controls are also vital to assist in the generation of the best analysis strategy. After acquisition, the machines return unused sample, and this could be useful when setting up a new assay allowing direct comparison of imaging flow cytometer data to an established technique i.
Therefore, when performing titration experiments, it is important to test antibodies from the same panel at the same laser power. This prevents saturation of bright stains when they are used in combination with dim stains. Data quality is enhanced when the brightness levels of all probes excited off a single laser are balanced within one log scale of fluorescence intensity. Due to long acquisition times and the lack of temperature control of the machines, fixation of cells is recommended for further information see Section IV.
File sizes which are generated after acquisition can be very large, for example MB for a 10 event file. To investigate rare cell populations several s of cells may need to be acquired. Here it would be beneficial to collect data only from the cells of interest. Thus, the file size becomes manageable and the analysis is sped up, as it needs to be remembered that the software is slow when handling large data files.
FCS files and the associated images, in. The FCS files alone can also be exported into other data analysis software for flow cytometry, but would only provide information about fluorescence intensity and not imaging. Analysis of a new experiment can be very time consuming, but once optimized, for example the optimal mask and feature have been determined, it can be quickly applied to future experiments.
IDEAS has many features to aid new users with analysis, as well as user defined features for advanced users. The first step is compensation. IDEAS guides the user through the process automatically, selecting what it considers as positive events for each channel. This can be inaccurate, and therefore it is important to check that the correct population has been selected by clicking on the values in the compensation matrix and if necessary adjusting the gating in the compensation graphs. These guide the user step by step through the analysis. If no analysis wizard exists, the feature finder wizard is a useful tool to determine the best feature to use.
Once an analysis method has been developed, samples can be batch analysed. One should be aware that each sample might require a different gating. A treatment or activation may change the properties of the cell e. Therefore, the analysis should be checked ensuring the gating is still valid for each treatment and adjust if necessary.
Following analysis, a statistics report can be then generated of the parameters of interest. The brightness and contrast can be manipulated for each channel and any background staining removed. Importantly, changing the way the images are viewed does not alter the raw data or analysis. However, slow running and long complicated analysis should be taken into consideration when opting for this technique over conventional flow cytometry.
The mass cytometer combines a cell introduction system with a mass spectrometer consisting of three basic components: the ion source, the ion analyzer, and the ion detector. Essential parts and steps of the measurement are summarized in Fig. Using argon as a carrier gas, the nebulizer creates an aerosol that is guided into the ion source. The ion source of the CyTOF instrument is an inductively coupled argon plasma. At a plasma temperature of approx. Thus, each cell generates an ion cloud that expands by diffusion and enters the vacuum.
Ions are accelerated by an electric field of a known strength, resulting in ions receiving the same energy. Since the ions all have the same charge, the ions can be separated by their mass difference. The velocity of lighter ions is higher and they reach the detector first, followed by heavier and slower ions, in the sequence of increasing ion mass.
The ion cloud of a given cell is measured in small portions, termed pushes.
Since the CyTOF technology focuses on metal isotopes with high atomic mass, only the segment of the spectrum corresponding to atomic masses higher than 80 Da is taken in consideration. Typically, a single ion cloud is captured by approximately 10—40 spectra. An electron multiplier is used for ion detection and consists of a series of dynodes maintained at increasing potentials, resulting in serial amplification of the original signal. The spectra are then analyzed by two successive integration steps, to obtain information about the amount of metal associated with each ion cloud corresponding to a single event.
The first integration is an area under curve calculated over an around 19—26 nanosecond interval according to the region of a given mass spectrum and represents the intensity of the peak for a given isotope. The region used for the first integration is determined during the instrument setup procedure termed mass calibration, using a tuning solution.
The second integration summarizes consecutive positive peaks corresponding to a single cell event. Finally, the integrated signal intensities obtained for one cell in the different mass channels are converted into flow cytometry standard FCS 3. Thus, mass cytometric data can be viewed and analyzed manually using standard flow cytometry software packages.
An important point to consider is that data analyses of a given study more and more often employ several algorithms organized in an analysis pipeline, very similar to an experimental procedure that needs to be described and annotated in appropriate detail At present, Fluidigm Corp. Similar data can be generated using an alternative approach i.
However, it is advisable to have the instrument maintained and managed by an expert operator. While the advantages of mass cytometry are striking for various applications, it should be noted that due to the destruction of the cells in the argon plasma, CyTOF instruments cannot recover the original cell sample for subsequent experiments. Instrument sensitivity, cell throughput and recovery should be taken in consideration when planning a study involving mass cytometry. The variability in sensitivity for the detection of different reporters is lower in mass cytometry compared with that in flow cytometry.
In theory, sensitivity could be improved by hardware design, allowing for the detection of more of the injected target ions, and by the use of probes that carry more metal per specific probe, such as heavy metal nanoparticles 64 - Mass cytometers need to be set up and tuned daily procedure detailed in The experimental workflow for preparing mass cytometry assays is typically very similar to that for conventional flow cytometry, except for the strict requirement of cell fixation and their resuspension in water prior to acquisition on the CyTOF instrument.
Briefly, cells are subjected to cell surface staining and optional dead cell label incubation, fixed, usually using formaldehyde , permeabilized, stained for intracellular antigens and DNA content, and finally resuspended in water optionally supplemented with normalization beads for injection into the mass cytometer. Mass cytometers do not measure the light scatter parameters usually employed in flow cytometry for detection of cell events and separation of cell aggregates.
In mass cytometry, cells are solely detected by the metal associated with them. A typical gating strategy is provided in Fig. The design of mass cytometry panels is generally easier as compared to flow cytometric panels of similar marker capacity, since signal spillover and sensitivity differences are comparably minor issues However, the mere number of parameters and the implementation of quality control for antibodies 74 both make panel design a significant effort. Panel design includes optimizing the pairing of specific probes with unique heavy metal isotopes considering instrument sensitivity for that particular isotope mass, target antigen abundance, and additionally potential signal spillover.
A careful panel design, an optimally tuned instrument and highly pure reagents, however, can minimize these spillovers to very low levels that are orders of magnitude lower than fluorescent spectral overlaps. However, the sole fact that, in mass cytometry, typical panels include approximately 40 antibodies renders the routine and consistent realization of these controls quite complicated, and often unfeasible. Isotope controls require the use of an antibody with a matching isotype and the same amount of metal per antibody as the reagent that is to be controlled, and are presently not commercially available.
However, both strategies deliver only limited control information. Here, the expression of a given marker is evaluated in the same sample on different cell populations, or by comparing samples from untreated versus treated conditions. Counterstaining for multiple cell lineage markers in antibody conjugate evaluation experiments enables the identification of reference cell populations serving as positive and negative controls for a given antibody conjugate in the multitude of populations identifiable by a 40 parameter panel.
Therefore, sample banking and assay automation are actively pursued research areas in the mass cytometry field. Mass cytometry is a new hybrid technology employing principles of flow cytometry and mass spectrometry. There is great diversity amongst biological cells. Studying the function of different cell types and subsets often requires the isolation of many cells of a specific population with a high degree of purity or the isolation of single cells for a better understanding of the heterogeneity of cells within a subset.
Parallel cell sorting also called bulk cell sorting is useful when either simple physical parameters, e. In particular, magnetic cell sorting techniques see Section II. As detailed in Sections II. With some methods more than 10 11 cells can be processed in less than 1 h. Serial cell sorting technologies use rapid measurements at the single cell level. This allows the isolation of even very rare cells from complicated mixtures. Serial cell sorting discerns cell subsets by staining with combinations of fluorescently labeled antibodies.
Analytical methods for rapid electrostatic serial cell sorting have been refined to use multiple lasers and more than 18 optical parameters derived from the reaction of cells with fluorescently labelled affinity reagents providing diverse excitation and emission signatures to define very specific subsets with many applications in immunology see Section II. The combination of many serial cell sorters in a microfluidic chip promises very high sorting rates see Section II.
Present serial cell sorters process cells at rates from a few cells per hour to 10 5 cells per second depending on the diverse range of applications being done and the specific cell sorter configuration being used. Parallel sorting uses parameters like cell size, density, magnetic, or electrical properties. Affinity binding reagents e. General considerations : Bulk cell sorting from a cell mixture can be done by many methods, each one having different advantages and challenges.
The main variable parameters to be considered are specificity, yield, purity, viability, functionality. Moreover, speed, cost, and consumables for equipment must be also taken into account Fig. The importance of the different functional parameters will depend on the specific experimental goals, e. Instrumentation features depend on the specific needs and the experience of the user s.
Figure 10 illustrates the various parameters needed in deciding on a sorting strategy or method. Not always can all parameters be set at optimal levels simultaneously. First, because it reduces time of the cell sort, and second because it helps to improve gating quality by eliminating potential fluorescence overlap between stained and unstained cells Fig.
An overview of cell sorting technologies and applications can be found in Keeping track of cell numbers, viability, and analyzing the sorted cells before, during and after any separation is good routine in order to determine cell yield and cell purity, and to detect any unreasonable cell losses or damages. To quantitatively evaluate sorting performance, several calculations can be performed.
The purity, i. This provides a helpful metric when optimizing a sorting technology. Another approach for the evaluation of bulk sorting performance is described in 84 , where it only uses fractions of cells in the original and positive fraction and does not need information about the yield of the positive wanted population. At lower yields there are small differences between the two metrics. Table 1 provides an example showing that final purity values alone are not a good measure for sorting performance rows 4 and 5 in Table 1 , even though it may be the important measure for biological activity.
Physical properties of cells can be changed by the reaction with specially tagged affinity reagents like antibody conjugates with magnetic particles. In this way specific subsets can be isolated with bulk sorting methods. This technique uses the force of magnetism to sort out cells according to specific cell surface markers. Several commercial systems are available, which use either inorganic superparamagnetic or ferromagnetic materials embedded in polystyrene beads or in a matrix such as dextran, or coated with graphene Beads in sizes from tens of nanometers up to several times the size of a typical mammalian cell are available for bulk cell sorting.
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Nanometer sized beads require high field strength and field gradients, generally achieved in columns or microfluidic channels with optimized ferromagnetic structures. Unwanted cells are poured off or eluted. In negative selection strategies, all unwanted cells are labeled, leaving the wanted ones untouched for downstream applications or a second round of selection by another surface marker.
Several bead or affinity reagent chemistries allow the detachment from the cells if needed. The bulk sorting method hinges on the quality of the antibodies used, and the density of the surface markers on the cells. Cells with a low density surface marker expression may be more difficult to sort. Bulk sorting with beads, especially with large beads, cannot distinguish between high and low expression of a given antigen on the cells. Selection of a good antibody is crucial for successful sorting, as is the concentration of beads in the labeling step. Nowadays, many kits for sorting a range of cell types in various species are commercially available.
Note : the sort quality must always be analyzed to detect possible cell losses and impurities. Specificity is achieved by the antibodies and, again, the quality of the antibodies is important.
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As beads vary in size, several cell subsets can be sorted out of a mixture by using different sized beads for different antibodies. A potential advantage is that the size of the beads may prevent phagocytic uptake. Beads can be detached by a special buffer, and sequential sorting is possible. Temperature and duration for binding must be considered in the context of phagocytosis, decreasing possibility of unspecific binding, capping, or efficient binding kinetics.
Cells, organelles, parasites etc. Efficient removal of dead cells from a mixture is possible as well note of caution: this procedure is stressful for the living cells. When separating blood, the upper fraction contains both lymphocytes and other mononuclear cells. They thus are not based on a polysaccharide net Pitfalls : Density for similar cells between species can differ, e. Centrifugation must be done at room temperature and without brakes.
Loss of cells and recontamination when harvesting them from the gradient surface is possible. Cell activation can be an issue, e. Manufacturers: gelifesciences. A second density separation medium is Percoll, made from colloidal nanosized silica particles coated with polyvinylpyrrolidone Cells of differing densities collect at the different interfaces and can be taken off. Size differences of cells of interest, e.
The pore size enables larger cells to be retained on the membrane and smaller cells to pass through. However classical filter membranes do not have homogeneous and precisely controlled pore sizes, so the resolving power of this separation is limited and, due to the material of the filter, the recovery of white blood cells may be inefficient. Another separation method based on cell size that targets red blood cells and platelets specifically uses microfibrated silicon chips.
These feature homogeneously etched slots of a certain size designed to let erythrocytes pass through under a certain pressure whilst retaining leukocytes on the surface of the chip. The leukocytes can then be recovered by elution. Early evaluation of this technology has demonstrated Pitfalls : Throughput of the filters is limited by surface area and overload may result in reduced purity and recovery of leukocytes. So far the commercial devices can only handle up to 2 mL of whole blood which is sufficient for some cell analysis assays but not enough for blood transplantation and cell therapy applications.
The recovery of leukocytes is sensitive to the pressure applied—pushing with higher pressure and higher flow rate may result in decreased recovery. A method of bulk sorting currently under development is based on cell size. There are several publications reporting a microfluidic device that separates particles and cells with high resolution 97 and is able to not only fractionate whole blood components by their sizes 98 but to also isolate CTCsfrom whole blood Recent work describes improvements for the routine use of the technology Multiple sections of an obstacle matrix with varying gap sizes can be built in one device so that multiple sized particles can be isolated because each sized particle will follow its own determined path flowing through the device.
In theory, there should be no throughput limitation of the technology as it is a continuous flow system; however, some surface treatment of the device may be needed to avoid cell adhesion. Particles exposed to an acoustic field are known to move in response to an applied acoustic radiation force. Numerous researchers have investigated the effect of acoustic waves on cells and particles in aqueous solution.
Thus, acoustic focusing can be used to separate and position particles based on size, density, and deformability. The ultrasonic standing wave is generated by a piezoelectric transducer and resonance vibration of the microfluidic device made in silicon or glass. The acoustic pressure pushes leukocytes to the pressure node located at the center of the channel and leaves platelets at the side stream going to a waste outlet.
Size is a dominant parameter for acoustic cell sorting but not the only parameter as shown in the equation above. For example, separation of leukocytes from erythrocytes in whole blood is not easily done on an acoustic device as erythrocytes, though having a smaller diameter, move to the acoustic energy node along with leukocytes as the erythrocytes have a higher density.
Pitfalls : The cell moving trajectory in the flow channel is determined by both the acoustic pressure and the shear pressure so the flow rate and channel configuration need to be well controlled otherwise the separation efficiency will suffer. No commercial product is available yet. Enucleated erythrocytes are more susceptible to hypotonic shock than nucleated cells.
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