Since the 1980s, the world's manufacturers of flow cytometers have changed, BC & BD is still there, new players are constantly entering, mergers and acquisitions are required, they produce a variety of different performance and function of the flow Cytometer. Overall, the following trends are emerging:

1. Application object from cell to particle  

In general, the subject detected by flow cytometry is a cell, and is a cell suspended in a liquid in a separate state, that is, a single cell suspension. Cells in tissues and organs must be prepared as single cell suspensions in a variety of ways for detection.

Bacteria, plankton, etc. can also be analyzed by flow cytometry.

Some individual particles that are not cells, such as viruses, nuclei, chromosomes, protoplasts, etc., are also subject to flow cytometry. In fact, the use of "particles" rather than "cells" to define the subject of flow cytometry is clearly more representative.

Because flow cytometry can treat "particles" as "cells", coated with antigens, antibodies or nucleic acid probes on special microspheres, and microspheres as carriers to detect various soluble proteins and cytokines. , autoantibodies, specific nucleic acid sequences, etc., so that the detection object of the flow cytometer is extended to the molecular range.

Luminex 's multiplex flow detection platform is capable of simultaneously detecting 50, 100 or even up to 500 analytes from a single microreactor, making it the gold standard for this technology. The official website is listed at https:// Com/zh/partners/ Nearly 100 partners.

Domestic, Weigong Technology and other manufacturers are also developing and producing streamed multi-detection platforms based on coded microspheres.

Weigong Technology EasyCell Streaming and Liquid Phase Chip Platform

2. Detecting particle size is greatly broadened

Conventional flow cytometry detects particles in the range of 0.5-50 μm.

Charge sorting flow cytometry detects the particle range and nozzle diameter, etc., up to 1/3-1/2 of the nozzle diameter, and the current maximum diameter nozzle is 200μm.

Union Biometrica 's airflow sorting platforms COPAS FP and BioSorter are capable of accommodating particles up to 1500 μm and can be used to sort eggs and larvae of animal models; model plant seeds and pollen; large volume cells and cell clusters. CytoBus from CytoBuoy can also go to 1500μm.

Union Biometrica Airflow Sorting Flow Schematic

Through optical path optimization, multi-angle light scattering, special fluorescent probes, and even purple lasers to collect scattered light signals, some flow cytometers can identify 0.2μm particles and noise signals.

Focusing on microparticle analysis, Apogee's A50-Micro plus scattered light detection is limited to 80nm and can be used for circulating microparticles, exosomes and specific microbial assays.

Apogee A50-Micro plus

3. Commercialization of various flow technologies

Most flow cytometers are based on the fluid dynamics mode of the sheath fluid.

Because the negative pressure injection eliminates the huge compressed gas source and can flexibly use various sample tubes, it has become the choice of almost all new instruments.

It is generally believed that flow cytometry uses a capillary rather than a sheath fluid, which is easily blocked by large cells or aggregated cells, and it is difficult for the flow cells to be accurately focused, and the flow rate is difficult to maintain constant.

The Guava platform easyCyte (now acquired by Luminex ) uses a high pressure syringe pump and PEEK tubing to create a self-stabilizing microcapillary system through negative pressure injection. It is said to solve the above problems, and the benefit is that no sheath fluid is needed. There is also less waste.

Guava easyCyte (Luminex)

Thermo 's sonic flow cytometer, the Attune NxT , uses ultrasound to concentrate cells on the central axis of the sample stream. When the injection speed is 1 mL/min, the so-called axial flow broadening and cell dispersion based on the sheath liquid flow can also be avoided. The biggest advantage of this technique is rapid detection, and the analysis of rare cells is advantageous.

Thermo Attune NxT

4. From relative count to direct absolute count

In order to meet the statistical requirements, the flow cytometer must collect a certain number of cells, and the instrument is set to stop the condition with the number of cells. The traditional flow cytometry test results are relative counts, that is, the ratio of target cells in a certain total system.

As the application deepens, people find that the ratio changes or does not change, and the magnitude of the change does not reflect the true change of the cell. The absolute count, that is, the number of cells per unit volume, proves to be more practical.

The dual platform method was first used to solve the above problem, that is, using a blood cell analyzer to obtain the concentration of white blood cells, lymphocytes, etc., and then calculating the absolute count value of the measured cells by the percentage measured by flow cytometry. Because the method requires the use of two instruments, the operation steps are large, the coefficient of variation is large, and the difference between different laboratories is also large, so the application is limited.

Later, people used the microsphere method, that is, using the known concentration of microspheres as a reference, and the absolute counting was performed by the proportional relationship between the measured cells and the microspheres. However, since the counting microspheres were expensive, the practical application was limited.

In recent years, the manufacturer's consensus has returned to the idea of ​​measuring the injection volume, instead of the microsphere method to directly count the absolute, to obtain more accurate results. The difference lies in the difference in the specific measurement techniques of different products.

Peristaltic pump: Accuri C6 Plus, CytoFlex

Syringe pump: easyCyte, NovoCyte, CytoFlow

Flow sensor: FACSVerse, Bricyte E6

Sysmex CytoFlow Series TVAC Volumetric Principle

5. Optical path optimization and application of new optical components

Large water-cooled or air-cooled lasers are gradually being replaced by miniaturized solid-state lasers.

Fiber optic transmission applications are becoming more common.

Fiber coupling makes optical energy transfer more efficient and not proprietary to the company.

New detectors such as avalanche photodiodes offer new options. Please see the expert analysis:

How does the flow cytometer "see" the light?

And the god-level combination of optical components gives sensitivity to new heights.

MESF is a measure of instrument sensitivity based on microspheres based on sheath fluid.

Different companies have no customary rules, not so easy to compare, but for the same company products:

BD FACSCelesta FITC 25 PE 15

Beckman Coulter CytoFlex FITC 30 PE 10

6. Rapid development of new optical technologies with multicolor analysis as the core

The main application of early flow cytometry is the determination of cellular DNA content. This technique is not very demanding on equipment. It is usually sufficient to use a flow cytometer with scattered light and a fluorescent signal. With the continuous development of new fluorescent probes and the gradual updating of instrument software and hardware, multi-color fluorescence analysis of flow cytometry has been rapidly developed, including two aspects:

Single laser multi-color, such as 488nm laser simultaneously exciting 7 colors, purple laser simultaneously exciting 10 colors...

Multiple lasers: such as 10 laser machines.

Also included are two new optical design models:

Spectral flow mode, using a special receiving device to collect the full emission spectrum in a certain range, without compensation, to distinguish the fluorescent dye pairs with high emission spectral coincidence. There are currently Sony SP6800Z and SA3800, Aurora from Cytek.

Mass spectrometry, a flow technique for multi-parameter detection of single cells using mass spectrometry. The platform, called CyTOF, was originally developed by DVS Sciences and later acquired by Fluidigm. The instrument uses an isotope-labeled antibody to label or recognize the signal molecules on the surface and inside the cell, and separates individual cells according to the principle of flow cytometry, and then observes the atomic mass spectrum of individual cells by inductively coupled plasma mass spectrometry (ICP-MS), and finally the atomic mass. The spectral data is converted to signal molecular data on the cell surface and inside. The Helios launched in 2015 reached 135 detection channels with almost no signal overlap or background noise.

From the CyTek website, it does not represent my concept

It must be noted that multicolor analysis provides a correlation map of various cellular properties to more precisely define a subpopulation of cells to better classify different cells. However, the more fluorescent dyes are detected at the same time, the need to accurately adjust the compensation between the channels, the technical difficulty is greater, because the amount of information contained therein is very large, the probability of mis-doping of the error signal is correspondingly increased, so the data analysis needs to be exceptional Note that it is generally necessary to use multiple labeling schemes to verify each other multiple times in order to draw important conclusions. The appearance of spectral flow and mass flow streaming overcomes the compensation problem.

However, fluorescent dye matching remains unresolved. After all, not every laser can be designed to excite 6 colors, 7 colors to 10 colors, or even 16 or 32 colors as desired by the spectral flow. Therefore, for multi-color analysis instruments of more than 20 colors, the nominal is often the theory, at least at present, the designer's imagination.

7. Data processing capabilities continue to improve

With the introduction of digital technology and the upgrade of data processing systems, the data processing capabilities of flow cytometry have been greatly improved.

Data processing speed: reflects the data scanning frequency, expressed in Hz.

Processing accuracy: Signal acquisition accuracy, expressed in bits, reflecting how many acquisition channels. At present, the instrument is generally above 20 bits (2 to 20 powers, 1 million channels) and up to 32 bits (4.3 billion channels).

Linear range: currently generally above 10 ⁵ , up to 10 ⁷ .

Single file storage capability: Reflects file storage capabilities and is beneficial for rare cell analysis.

The increase in data capabilities directly affects the instrument's analysis and sorting speed.

The speed increase is the result of the optimization of the optical path, the flow path and the data acquisition system.

The traditional flow cytometer has an analysis speed of no more than 10,000 events/s, and the newly launched products basically reach 25,000 events/s (note: not cells/s). For example, Beckman Coulter 's CytoFlex is 30000 events/s, Cytek 's Aurora is 35,000 events/s, BD 's LSRFortessa is 40,000 events/s with beads, and Bio-Rad 's ZE5 is 100,000 events/s.

Bio-Rad ZE5

The mechanical sorting of BD FACSCalibur is only 300/sec. It is difficult to collect a small amount of cells. It needs to be equipped with a concentration system for cell recovery. The movement and concentration system of the capture tube has great mechanical damage to the cells, which affects the functional characteristics of the cells. Because the movement of the trap inevitably affects the stability of the flow path, causing uncertainty; the mechanical device is difficult to thoroughly clean and disinfect. Mechanical sorting has been completely replaced by faster charge sorting.

Such as BD FACSAria III (70 psi /90 kHz) and InFlux (60 psi /100 kHz) four-way sorting, purity >98%, yield >80%, sorting speed 25000 events / s, continue to improve sorting speed The purity is not affected and the yield will decrease according to the Poisson distribution.

Beckman Coulter 's MoFlo XDP and MoFlo Astrios EQ are said to have an effective sorting speed of 70,000 cells/s.

8. The instrument continues to be miniaturized to microfluidic

The pursuit of miniaturization is almost the consensus of all streaming manufacturers to develop new products.

Look at a set of numbers: host W x D x H

BD Accuri C6 plus / FACSVia 37.5 x 41.9 x 27.9 cm

BD FACSVerse / FACSlyric 63.2 x 57.9 x 57.9 cm

BD FACSCelesta 58 x 61 x 59 cm

Beckman Coulter CytoFlex 42.5 x 42.5 x 34 cm

Miltenyi Biotec MACSQuant 60 x 35 x 40 cm

Thermo Attune NxT 58 x 43 x 40 cm

Cytek Aurora 54 x 52 x 52 cm

Mindray BriCyte E6 50 x 55 x 50 cm

(There is a kind of waist that is bigger than 70 and does not dare to go out.)

“Small” means space saving, easy to install and transfer. “Small” is more desirable to reduce sample volume, reduce reagent consumption, increase detection speed, and reduce the photoelectric and liquid path improvements caused by space.

Microfluidic technology emerged in the 1990s, as the name suggests, using microchannels (tens to hundreds of microns in size) to handle or manipulate tiny fluids.

On-chip launched the world's first microfluidic chip streaming On-Chip Sort in 2012 to sort cells at low pressure, reducing damage to cells; simplifying experimental operations; and easily achieving system sterility; Use disposable exchange modules to reduce cross-contamination between samples; no need to clean the flow path. Miltenyi Biotec 's MACSQuant Tyro and Namocell 's Namocell have also introduced similar microfluidic flow sorting products.

On-chip company's on-chip sort

The two core technologies of Sony 's sorting platform are the so-called CoreFinder fully automatic calibration technology and the liquid path design of the replaceable microfluidic chip. The latest model MA900 , fully automatic optical path and flow calibration; three-dimensional double spot four laser excitation, up to 12-color fluorescence detection at the same time; four-way sorting.

Sony MA900

Handyem , a manufacturer specializing in the production of analytical microfluidic flow, has introduced HPC-150 (domestic brand Shenzhen Core Kerry) in 2015 for the problem that microfluidic devices are prone to cell adhesion and clogging. The syringe pump replaces the peristaltic pump or the single-injection pump, and it is said to overcome problems such as the blocked flow deposition that is easy to see in the previous generation.

9. Fusion microscopy imaging and flow cytometry

The two major technology platforms of cell biology are based on microscope and flow, respectively. The former is excellent in morphological analysis and can provide detailed image information of the cell, but it is subjective and time-consuming. The latter is statistically long but lacks imaging ability. Understand subcellular localization.

Image flow cytometry with fusion microscopy and flow cytometry, represented by the Amnis platform (now acquired by Luminex ), the latest model ImageStreamX Mark II , linearly split from the cell through the flow cell Photography, the cold CCD uses time-delay integration to perform signal acquisition, and each sample analysis generates fluorescence intensity measurement parameters and image database of all cells in the sample, including bright field, dark field and fluorescent image of “per cell” .

For the weak fluorescence signal, the mask can be set by the bright field cell image, and the cell fluorescence signal under the mask can be specifically enhanced to capture and analyze the weak fluorescence image.

The system can analyze more than 500 quantitative parameters for each cell, including the scattered light and fluorescence signal intensity of the cell as a whole, as well as the analysis of cell morphology, cell structure and subcellular signal distribution.

Image flow cytometry does not require loss of morphological information in order to obtain cell population statistics, nor does it require loss of statistical function in order to obtain cellular details.

Amnis Image Flow Cytometry ImageStreamX Mark II

Sysmex recently introduced the MI-1000 , which automatically detects image fluorescence up to 10,000 cells.

Union Biomet rica 's air separation system COPAS VISION also added brightfield images.

10. Specialized and clinical instruments are available

CytoBuoy manufactures a variety of flow cytometers for microbiological analysis of water bodies, such as CytoBuoy, which is placed on a buoy; CytoSub has a special pressure-resistant device and an internal sheath fluid circulation device that does not require external sheath fluids. Use under 200 meters. Bentley Instruments and Delta Instruments specialize in providing specialized testing equipment for dairy farms.

The clinical market is the most promising development direction for commodity flow cytometry. However, the development and documentation of diagnostic flow reagents, as well as the development of instruments that conform to the testing process, do not match the growing demand for clinical applications.

Some companies introduced a special small flow cytometry for rapid counting of CD4 cells, such as FACSCount, CyFlow Counter like.

Mindray introduces BriCyte E6 for clinical user workflows and scenarios. For clinical routine analysis such as lymphocyte subsets, it can achieve the results of one-click test instruments, eliminating complicated voltage and compensation adjustments, and two-way communication of LIS. Data management is in line with the expectations of clinical diagnosis. A similar idea was also reflected in the later-listed BD FACSlyric .

BD FACSlyric six-color TBNK automatic analysis and two-way LIS

In the fifth year after the acquisition of Partec, the old blood cell manufacturer Sysmex introduced the PS-10 flow sample pretreatment system, in conjunction with the new flow XF-1600 , to achieve a fully automated process for sample processing, centrifugation and inspection. For details, please see:

The arrival of the era of automatic flow detection!

It can be expected that the flow cytometer will be integrated into the blood cell analysis pipeline as an important component in the future. In this system, the blood cell analyzer provides blood routine results and abnormal alarm information, and the flow cytometer is based on these. Information is used to fine-tune different strategies for abnormal cells.