A flow cytometer sort control system, sort method, and storage medium

Abstract

The application discloses a flow cytometer sorting control system, a sorting method and a storage medium, and belongs to the technical field of flow cytometer sorting. The flow cytometer sorting control system provided by the application comprises an interface for interacting with user software, an analog-to-digital conversion module, a data processing module, a sorting identification module, a sorting delay module and a sorting actuator. The application further provides a method for sorting cells by using the flow cytometer sorting control system provided by the application, wherein the method comprises a new sorting identification method and a new delay method. By using the control system and the sorting identification method provided by the application, the calculation time can be greatly shortened and the sorting speed can be improved when complex circle gates are used under the same calculation speed of hardware; meanwhile, the time difference between the start of liquid drop charging and the disconnection of the liquid drop can be fixed, the sorting stability and reliability can be ensured, the judgment of cell spacing for each cell is not needed, the judgment of whether each cell is a target cell is not needed, the processing process of cell signals is simplified, and the requirement for electronic performance is reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of flow cytometry sorting. Specifically, the present application relates to a flow cytometer sorting control system, a sorting method and a storage medium. BACKGROUND

[0002] Flow cytometry is a technology for multi-parameter analysis and sorting of single cells or other various micro-particles and their loads in a fast straight flow by using a flow cytometer. The technology has the characteristics of fast detection speed, multiple measurement parameters, large amount of collected information, comprehensive analysis and flexible method.

[0003] Generally, the sorting control process is as follows: when the sample passes through fluid focusing and the single cells pass through the laser spot one by one, the flow cytometer controls the photodetector to detect the scattered light and fluorescent light signals, and transmits the digital signals converted by AD to the data acquisition control system. The data acquisition control system processes the data and compares them with the target identification signals to determine whether the cells are the target cells to be sorted, and transmits the data to the user software. After the sorting signals are delayed, they are sent to the sorting execution module, so that the cells are sorted according to the identification results.

[0004] The common sorting identification method is to compare the cell data with the gate signals after the cell data are collected; if the cell is in the gate, the sorting flag bit is 1, and then the cell is determined whether to be sorted according to the sorting mode and the flag bits of the upstream and downstream cells of the cell. This method has a complex algorithm and a large real-time calculation amount, which results in a long sorting identification time and limits the sorting speed.

[0005] Meanwhile, the common sorting execution module controls the cell sorting by delaying the sorting signals. Since any execution mechanism has an execution process, if the sorting execution mechanism cannot frequently receive the sorting signals or makes an error during the process, the general fixed delay method will result in unstable sorting time and reduce the sorting purity and yield. SUMMARY

[0006] An object of the present application is to provide a flow cytometer sorting control system. The sorting control system provided by the present application can realize sorting identification of 500,000 cells per second, can realize rapid identification of target cells, and can improve the cell sorting throughput.

[0007] Another object of the present application is to provide a method for realizing cell sorting by using the flow cytometer sorting control system provided by the present application. The method provided by the present application can reduce cell loss and improve the yield.

[0008] Another object of the present application is to provide a computer storage medium storing the cell sorting method.

[0009] Another object of the present application is to provide a flow cytometer.

[0010] To achieve the above object, the present application provides the following technical solutions.

[0011] In the first aspect, the present application provides a flow cytometer sorting control system, which comprises:

[0012] an interface interacting with user software, for receiving user commands, sorting gate data or other data, and sending collected cell data to the user software;

[0013] an analog-to-digital conversion module, for converting the cell optical signal received by the photodetector into a digital signal;

[0014] a data processing module, for calculating the height, area, time or other cell parameters of the digital signal generated by the analog-to-digital conversion module, and then sending these parameters to the sorting identification module;

[0015] a sorting identification module, for comparing the channel data with the gate data according to the gate characteristics set by the user after receiving the real-time cell data, and sending the time data of the cell to the sorting delay module if the sorting condition is met;

[0016] a sorting delay module, for delaying the time data of the cell, generating a driving signal, driving the sorting actuator, and realizing cell sorting;

[0017] a sorting actuator.

[0018] Preferably, the interface interacting with the user software comprises USB, Gigabit Ethernet, PCle or other interfaces capable of realizing high-throughput and high-speed cell data transmission.

[0019] Preferably, the sorting identification module comprises m n×n memory blocks, each memory block corresponding to a data graph in the user software, and m being the number of sorting gates.

[0020] Preferably, the memory blocks are constructed according to the following method:

[0021] After receiving the sorting gate data and sorting mode data of the software, the position of the gate graph on the n×n two-dimensional graph is calculated according to the different coordinate axes and gate graph, and then the coordinates of the n×n points are compared with the position, if the point is within the gate, the value of the point is set to 1, otherwise it is set to 0;

[0022] The points passed by the door graph are adjusted according to the sorting mode, and are set to 0 if the sorting mode is the purity mode or the single-cell mode, and are set to 1 if the sorting mode is the enrichment mode.

[0023] After the calculation is completed, the n*n data is mapped to the corresponding memory, and then the calculation of the next door is performed until all the doors are calculated.

[0024] In a second aspect, the application provides a method for realizing cell sorting by using the flow cytometry sorting control system.

[0025] The analog-to-digital conversion module converts the cell optical signal received by the photodetector into a digital signal;

[0026] The data processing module compares the digital signal generated by the analog-to-digital conversion module with a preset threshold value; if the digital signal is greater than the threshold value, it is determined to be a cell signal, and then the calculation of cell parameters is performed, the cell parameters including height, area or time; different analog-to-digital conversions are converted into corresponding parameters, which are stored and sent to the sorting identification module in a certain order;

[0027] The sorting identification module receives the cell data, selects the parameters of the corresponding coordinate axes as the address, and performs addressing in the memory; if the read data is 1, it indicates that the cell is in the current door, and the next door comparison is continued until all door comparisons are completed; if the result is 1, it is a target cell and needs to be sorted; if the data read from the memory is 0 during a certain door comparison, it is a non-target cell and does not need to be sorted;

[0028] The sorting identification module sends the time data of the cells meeting the sorting conditions to the sorting delay module, and the sorting delay module performs delay processing on the time data of the cells to generate a driving signal, which drives the sorting actuator to realize cell sorting.

[0029] Preferably, the processing process of each door is as follows:

[0030] According to the n value, the cell data is converted into an address value; specifically, according to the n value, the data is high-bit truncated, and the number of truncated bits b is log2(n);

[0031] For example, if n=256, the high 8 bits are taken, if n=512, the high 9 bits are taken, and if n=65536, 16 bits are taken;

[0032] The corresponding address data is read from the memory, and m doors are calculated in turn to obtain the final sorting signal.

[0033] Preferably, the delay processing includes:

[0034] The delay time of detection sorting is divided into two parts, a first delay time and a second delay time, wherein the specific time of the first delay time is specified by a user and can be accurately adjusted, and must be greater than the calculation time of sorting identification; the time of the second delay time is an integer multiple of the vibration frequency Td;

[0035] With the vibration clock as a reference, the cell signal flow is cut into several small units, the cell number in the small unit and the target cell number are calculated, and the sorting flag is obtained according to the sorting mode, and output is performed at the rising edge of the next vibration clock, the charge loading circuit is driven, and the liquid drops are charged.

[0036] Preferably, in the calculation of the sorting flag,

[0037] If it is the enrichment mode, the target cell number (Count_target) > 0, and the sorting flag is 1;

[0038] If it is the purity mode, the total cell amount (Count_all) = target cell number (Count_target) > 0, and the sorting flag is 1;

[0039] If it is the single cell mode, the total cell amount (Count_all) = target cell number (Count_target) = 1, and the sorting flag is 1.

[0040] In a third aspect, the present application provides a computer storage medium, which stores a computer program, wherein the computer program is executed by a processor to implement the method of the present application.

[0041] In a fourth aspect, the present application provides a flow cytometer, which comprises the flow cytometer sorting control system of the present application and / or the computer storage medium of the present application.

[0042] In summary, the present application provides a flow cytometer sorting control system and a cell sorting method. The technical solution provided by the present application has the following advantages compared with the prior art:

[0043] 1. A new sorting identification method is adopted, which can greatly shorten the calculation time and improve the sorting speed under the same calculation speed of hardware when complex gating is performed;

[0044] 2. A new delay method is adopted to ensure that the time difference between the start of charging the liquid drops and the disconnection of the liquid drops is fixed, and the sorting is stable and reliable;

[0045] 3. The cell signal flow is divided into small units based on the vibration frequency, and the sorting flag is calculated, without the need to judge the cell spacing of each cell or whether each cell is a target cell, which simplifies the processing process of the cell signal and reduces the requirement for electronic performance. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 is a control block diagram for sorting.

[0047] Figure 2 is a block diagram of a sorting identification module.

[0048] Figure 3A Figure 3B and Figure 3C is a turnstile calculation.

[0049] Figure 4 is a sorting drive in different modes.

[0050] Figure 5 is a control system circuit implementation diagram. DETAILED DESCRIPTION

[0051] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application will be described in detail below, but it should not be understood as limiting the scope of the present application.

[0052] Throughout the specification, unless otherwise specifically indicated, the terms used herein are to be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application pertains. If there is a contradiction, the present specification takes precedence.

[0053] It should be noted that in the present application, the terms "comprise", "include" or any other variant thereof are intended to cover non-exclusive inclusion, so that a method or device comprising a series of elements not only includes the elements explicitly listed, but also includes other elements not explicitly listed, or includes elements inherent to the implementation of the method or device. Without more limitations, the element defined by the statement "comprises a" does not exclude the presence of another related element in the method or device comprising the element.

[0054] It should be noted that the terms "first", "second", "third" involved in the present application are only to distinguish similar objects, and do not represent the specific order of the objects. Understandably, "first", "second", "third" can be interchanged in specific order or sequence as allowed. It should be understood that the objects distinguished by "first", "second", "third" can be interchanged under appropriate circumstances, so that the present application described herein can be implemented in an order other than those illustrated or described herein.

[0055] It should be noted that "one" in the claims in the present application is used only for the convenience of description, and should not be understood as a quantitative limitation.

[0056] ​In the present invention, the term "enrichment mode" means all droplets with positive cells are sorted.

[0057] In the present invention, the term "purity mode" means all droplets with negative cells are discarded.

[0058] In the present invention, the term "single cell mode" means only one positive cell in a droplet is sorted.

[0059] In the present invention, the term "data plot" means a plot displayed on flow software, which can be a scatter plot, i.e. X and Y represent two cell parameters, or a histogram, i.e. X represents a cell parameter and Y represents the number. In a scatter plot, both X and Y are screened, while in a histogram, X is screened and Y is accepted.

[0060] In the present invention, "n" is the resolution of a plot displayed on flow software.

[0061] As shown in the accompanying drawings, the present invention provides a flow cytometer sorting control system, which comprises: Figure 1

[0062] an interface for interacting with user software, for receiving user commands, sorting identification gates or other data, and sending collected cell data to user software;

[0063] an analog-to-digital conversion module for converting cell optical signals received by a photodetector into digital signals;

[0064] a data processing module for calculating height, area, time or other cell parameters from digital signals generated by the analog-to-digital conversion module, and then sending these parameters to the sorting identification module;

[0065] a sorting identification module for comparing channel data with gate data according to gate characteristics set by the user after receiving real-time cell data; if the sorting condition is met, the time data of the cell is sent to the sorting delay module;

[0066] a sorting delay module for delaying the time data of the cell, generating a driving signal to drive the sorting actuator, and realizing cell sorting;

[0067] a sorting actuator.

[0068] In some specific embodiments, the interface for interacting with user software includes USB, Gigabit Ethernet, PCle or other interfaces that can realize high-throughput and high-speed cell data transmission.

[0069] ​In some specific embodiments, the sorting identification module comprises m n*n memory blocks, each of which corresponds to a data graph in the user software, and m is the number of the sorting ring gates.

[0070] In some specific embodiments, the memory blocks are constructed according to the following method:

[0071] After receiving the software sorting ring gate data and the sorting mode data, the position of the gate graph on the n*n two-dimensional graph is calculated according to the different coordinate axes and the gate graph, Figure 3A 、 Figure 3B 、 Figure 3C Then the coordinates of the n*n points are compared with the position, if the point is in the ring gate, the value of the point is set to 1, otherwise it is set to 0.

[0072] And the points passed by the gate graph are adjusted according to the sorting mode, if it is the purity mode and the single cell mode, it is set to 0; if it is the enrichment mode, it is set to 1.

[0073] After the calculation is completed, the n*n data is mapped to the corresponding memory, and then the calculation of the next gate is carried out, until all the gates are calculated.

[0074] Among them, the position calculation of the gate graph on the n*n two-dimensional graph is the binary graph filling of the memory according to the contour, which belongs to the public knowledge in the field of computer image recognition.

[0075] In some specific embodiments, the application also provides a method for realizing cell sorting by using the flow cytometry sorting control system, which comprises the following steps:

[0076] The analog-to-digital conversion module converts the cell optical signal received by the photodetector into a digital signal;

[0077] The data processing module compares the digital signal generated by the analog-to-digital conversion module with the preset threshold value; if it is greater than the threshold value, it is judged as a cell signal, and then the calculation of the cell parameters is carried out, the cell parameters include height, area or time; different analog-to-digital conversions are converted into corresponding parameters, stored and sent to the sorting identification module in a certain order;

[0078] The sorting identification module receives the cell data, selects the corresponding coordinate axis parameters as the address, and performs addressing in the memory; if the read data is 1, it means that the cell is in the current gate, and the next gate comparison is continued until all the gate comparisons are completed; if the result is 1, it is the target cell and needs to be sorted; if the data read from the memory is 0 when comparing a certain gate, it is a non-target cell and does not need to be sorted.

[0079] The sorting identification module sends the time data of the cells meeting the sorting condition to the sorting delay module, and the sorting delay module generates a driving signal after time delay processing of the time data of the cells, drives the sorting actuator, and realizes cell sorting (as shown in Figure 2

[0080] As an optional embodiment, the processing of each gate is as follows:

[0081] According to the value of n, the cell data is converted into an address value; specifically, the data is high-bit truncated according to the value of n, and the number of truncated bits is b = log2(n); for example, if n = 256, the high 8 bits are taken, if n = 512, the high 9 bits are taken, and if n = 65536, 16 bits are taken;

[0082] The corresponding address data is read from the memory, and the m-gate is calculated in sequence to obtain the final sorting signal.

[0083] In some more specific embodiments, according to the gate order and the number of gates set by the upper computer software, the identification is performed in sequence to determine whether sorting is needed; wherein the processing of each gate is as follows: according to the cell parameter selected by the gate, the corresponding data is read from the real-time cell data, and high-bit truncation is performed, if n = 256, the high 8 bits are taken, if n = 512, the high 9 bits are taken; the obtained data is used as an address, and the data in the corresponding address in the memory is read, if the data is 0, it indicates that the cell is not the target cell; if the data is 1, the processing of the next gate is performed. If the identification of the last gate is successfully performed and the read data is 1, it indicates that the current cell is the target cell.

[0084] Wherein, it takes one calculation period to read the corresponding address data from the memory; to calculate the m-gate, a total of 3xm+1 calculation periods are needed, and the final sorting signal can be obtained, m is the number of sorting gates, which is set by the person skilled in the art according to the target cell to be sorted. Generally, the value of m ranges from a natural number excluding zero, preferably 1-100, more preferably 1-50, and further preferably 1-20, so the entire calculation period is less than 50, if the FPGA is always 100MHz, one calculation period occupies 4 initial period, the entire calculation time is less than 2 microseconds, and the sorting identification of 500,000 cells per second can be realized. Therefore, this sorting identification process will not be a factor limiting the speed of cell sorting.

[0085] As an optional embodiment, the delay processing includes:

[0086] The delay of the detected sorting is divided into two parts, the first delay and the second delay, wherein the specific time of the first delay is specified by the user and can be accurately adjusted, and must be greater than the calculation time of the sorting identification; the time of the second delay is an integer multiple of the vibration frequency Td; ​

[0087] With the vibration clock as the reference, the cell signal flow is cut into several small units (for example, assuming that the frequency of cell data generation is 10MHz, and the vibration frequency is 1MHz, when the data flow is divided based on the vibration clock, there are 10 cell data in an average small unit. As long as the sorting process is ongoing, cell data is continuously generated, and the small unit is also continuously generated), the number of cells in the small unit is calculated, and the target cell number is combined with the sorting mode to obtain whether the sorting mark is needed, and output is performed at the next vibration clock rising edge, the charge loading circuit is driven, and the droplet is charged (as shown in Figure 4 ).

[0088] The delay processing method does not need to judge the cell spacing for each cell, and does not need to distinguish whether each cell is a target cell, simplifies the processing process of the cell signal, and reduces the requirement for electronic performance. Because the general processing method is to analyze a single cell as a target, if the current cell is a target cell, in the enrichment mode, it is not necessary to consider the state of the front and rear cells, and sorting is performed; in the purity mode, it is necessary to consider whether the front and rear cells are target cells, or the distance between the non-target cells in front and rear of the current cell is far from the current cell, which does not affect the separation of the current cell; in the single cell mode, it is necessary to consider that the front and rear cells are far away from the current cell. In this way, each target cell must be calculated once, and the method proposed in the present application considers that the droplet formation has a time interval, that is, the vibration period, and the frequency of droplet generation, so it is not necessary to calculate each cell, but to calculate the cell situation in each droplet, which reduces the calculation frequency and reduces the calculation amount.

[0089] As an optional embodiment, in the calculation of the sorting mark,

[0090] If it is the enrichment mode, the target cell number (Count_target) > 0, and the sorting mark is 1;

[0091] If it is the purity mode, the total cell amount (Count_all) = target cell number (Count_target) > 0, and the sorting mark is 1;

[0092] If it is the single cell mode, the total cell amount (Count_all) = target cell number (Count_target) = 1, and the sorting mark is 1.

[0093] In some specific embodiments, the present application provides a computer storage medium, which stores a computer program, wherein the computer program is executed by a processor to realize the method described in the present application.

[0094] As Figure 5As shown, the storage medium is executed by the processor (single-chip microcomputer, ARM, FPGA, etc.) to communicate with the host computer software; at the same time, the analog-to-digital converter (generally more than 12 bits, 16 bits, 24 bits) is controlled to convert the cell photoelectric signal into a digital signal and store it in the data buffer (which can be RAM, FIFO, etc.); the processor reads the data from the data buffer, calculates the characteristic data of the cell (such as height, area, width, etc.) through an algorithm, and uploads it to the host computer software. When setting the gate, the processor calculates the scatter plot into the RAM. When sorting, the processor converts the real-time cell signal into address data for RAM addressing and sends it to RAM, so that RAM outputs the value in the corresponding address; these values are input into the synchronous buffer to generate a cell sorting enable signal, and the synchronous buffer synchronizes this signal with the droplet generation clock and outputs it as a cell sorting signal to control the sorting actuator.

[0095] The above describes the preferred embodiments of the present application, but the present application is not limited thereto. Without departing from the spirit and essence of the present application, various technical features of the present application can be substituted, modified and combined, and these simple modifications and combinations should also be considered as disclosed by the present application, and all fall within the protection scope of the present application.

Claims

1. A flow cytometer sorting control system, comprising: An interface for interacting with user software, used to receive user commands, sort and identify cell gates, and send the collected cell data to the user software; An analog-to-digital converter module is used to convert the cell light signal received by the photodetector into a digital signal; A data processing module is used to calculate the height, area, and time from the digital signal generated by the analog-to-digital converter module, and then send these parameters to the sorting and identification module. The sorting and identification module is used to compare the channel data with the gate data according to the gate characteristics set by the user after receiving the real-time cell data. If the sorting conditions are met, the cell time data is sent to the sorting delay module. A sorting delay module is used to delay the cell time data, generate a drive signal, drive the sorting actuator, and realize cell sorting. One-way selector actuator.

2. The flow cytometer sorting control system according to claim 1, wherein, The interface for interacting with user software includes USB, Gigabit Ethernet, PCIe, or other interfaces capable of enabling high-throughput, high-speed cell data transmission.

3. The flow cytometer sorting control system according to claim 1, wherein, The sorting and identification module includes m n×n memory blocks, each memory block corresponding to a data graph in the user software, where m is the number of sorting gates.

4. The flow cytometer sorting control system according to claim 3, wherein, The memory block is constructed according to the following method: After receiving the sorting gate data and sorting mode data from the software, the position of the gate shape on the n×n two-dimensional graph is calculated according to different coordinate axis selections and gate shapes. Then, the coordinates of n×n points are compared with the position. If the point is inside the gate, the value of the point is set to 1; otherwise, it is set to 0. The points through which the gate pattern passes are adjusted according to the sorting mode: if it is the purity mode or the single-cell mode, it is set to 0; if it is the enrichment mode, it is set to 1. After the calculation is completed, the n×n data is mapped into the corresponding memory, and then the calculation of the next gate is performed, until all gates have been calculated.

5. A method for cell sorting using a flow cytometer sorting control system according to any one of claims 1-4, comprising the following steps: The analog-to-digital converter module converts the cell light signal received by the photodetector into a digital signal; The data processing module compares the digital signal generated by the analog-to-digital converter with a preset threshold; if it is greater than the threshold, it is determined to be a cell signal, and then cell parameters are calculated, including height, area or time; different analog-to-digital converters are converted into corresponding parameters, stored in a certain order and sent to the sorting and identification module; The sorting and identification module receives cell data, selects the parameter corresponding to the coordinate axis as the address, and performs addressing in memory. If the data read is 1, it means that the cell is in the current gate, and continues to the next gate comparison until all gate comparisons are completed. If the result is 1, it is the target cell and needs to be sorted; if the data read from memory during a certain gate comparison is 0, it is a non-target cell and does not need to be sorted. The sorting and identification module sends the time data of cells that meet the sorting conditions to the sorting delay module. The sorting delay module then delays the time data of the cells and generates a drive signal to drive the sorting actuator to achieve cell sorting.

6. The method according to claim 5, wherein, The processing procedure for each door is as follows: Based on the value of n, the cell data is converted into address values; specifically, based on the value of n, the data is truncated to the high-order bits, and the truncated bit value is b=log2(n). Read the data at the corresponding address from memory, calculate the m gates in sequence, and obtain the final sorting signal.

7. The method according to claim 5, wherein, The delay processing includes: The detection delay for sorting is divided into two parts: a first delay and a second delay. The specific time of the first delay is specified by the user and can be precisely adjusted, but it must be greater than the calculation time for sorting and identification. The time of the second delay is an integer multiple of the vibration frequency Td. Using the vibration clock as a reference, the cell signal stream is divided into several small units. The number of cells in each small unit and the number of target cells are calculated. Combined with the sorting mode, a sorting flag is determined. The flag is then output at the rising edge of the next vibration clock to drive the charge loading circuit and charge the droplet.

8. The method according to claim 7, wherein, When calculating sorting criteria, If it is enrichment mode, the number of target cells > 0, and the sorting flag is 1; If it is in purity mode, then the total number of cells = the number of target cells > 0, and the sorting flag is 1; If it is a single-cell mode, then the total number of cells = the number of target cells = 1, and the sorting flag is 1.

9. A computer storage medium having a computer program stored thereon, wherein, When the computer program is executed by a processor, it implements the method of any one of claims 5-8.

10. A flow cytometer sorting instrument, comprising a flow cytometer sorting control system according to any one of claims 1-4 or a computer storage medium according to claim 9.

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