CN104749086B - More reference channel pulse recognition method, apparatus and particle analyzer - Google Patents

Abstract

The present application discloses a multi-reference channel pulse identification method and device, and a particle analyzer. The identification method includes: for a single reference channel where a pulse is found, determining invalid data in the pending data of the single reference channel based on the found pulse to be identified Unify the start and end time of the invalid data area of multiple reference channels according to the invalid data area of each single reference channel; determine the valid data area of multiple reference channels based on the invalid data area after the unified start and end time; The pulses in the valid data area are pulse identified, and the pulses in the invalid data area are no longer pulse identified. In this application, by determining the invalid data area with uniform start and end times among multiple reference channels, the data that cannot or is difficult to obtain the correct area, amplitude and width information is excluded, and the pulse identification is not performed on them, thereby reducing the number of pulses The amount of data processing at the time of recognition.

Description

Multi-reference channel pulse identification method, device and particle analyzer

technical field

The present application relates to a medical instrument, in particular to a multi-reference channel pulse identification method and device in a cell analyzer.

Background technique

Flow cytometer is an instrument used to analyze the physical and chemical characteristics of cells or tiny particles (collectively referred to as particles, and cells are used as an example below). The principle is shown in Figure 1. Flow cytometers generally use sheath Flow technology, the cells in the sample are wrapped and sorted by the sheath fluid and flow through the flow chamber 1, the excitation light 2 is irradiated on the cells 3 one by one, so that the cells 3 generate scattered light or fluorescent signals, and the light signals are collected and converted by the photoelectric converter 5 After processing by the processing circuit 6, the electrical pulse signal 4 is formed, and the pulse identification module 7 performs pulse identification on the pulse signal to obtain parameters such as its amplitude (H), area (A), and width (W), and finally passes Graphically displayed for users to analyze. The H, A, W and other parameters of these scattered light or fluorescent signals can reflect the size, granularity, expression of antigen molecules, and the content of genetic material in cells, etc., and then obtain valuable reference from the perspective of clinical medicine or biology information. Therefore, it is of great significance to correctly identify the parameters such as the amplitude, area, and width of pulse signals formed by various excited cells.

There may be a variety of cells in the sample to be measured. After each cell is excited, it generally produces forward scattered light (FSC) and side scattered light (SSC). However, whether there is a signal in the fluorescence channel depends on the characteristics of the cell and the The presence or absence of fluorescein can be divided into the following situations: no, only one, and multiple fluorescent signals. By analyzing the H, A, W and other information of the signal generated by the cell in different channels, some characteristic information about the cell can be obtained. Since the sample may contain a variety of cells, and the user is only interested in some specific cells, at this time, a threshold is often set in a certain channel (called "reference channel"), so that pulses with peak values smaller than the threshold in this channel are ignored , The pulses greater than the threshold are collected and then analyzed to obtain H, A, W and other information. The process of analyzing and obtaining the H, A, W and other information of the pulse is called pulse recognition.

In some cases, the user is more interested in the signals of certain channels, then set these channels as reference channels, set thresholds in multiple "reference channels", and then perform combined logic on the threshold judgment results of these channels ("and "\"or") operation, judge whether to analyze the cell and give H, A, W and other information according to the obtained result.

Contents of the invention

The present application provides a multi-reference channel pulse identification method and device in a cell analyzer, which reduces the amount of data computation in the process of multi-reference channel pulse identification.

According to the first aspect of the application, the application provides a multi-reference channel pulse identification method, including:

Collecting signals output by a plurality of reference channels to obtain the data to be processed of each reference channel, the signals output by the reference channels are electrical signals converted from optical information emitted by the measured particles collected by the reference channels after they are excited by light;

Based on the data to be processed of each single reference channel and the characteristic threshold corresponding to the reference channel, it is judged whether to find a pulse, the characteristic threshold is the threshold of the characteristic of interest of the particle of interest, and the discovery of the pulse refers to the detected pulse peak value greater than the characteristic threshold pulse;

For the single reference channel where the pulse is found, determine the invalid data area in the data to be processed of the single reference channel based on the found pulse to be identified;

Unify the start and end times of the invalid data areas of multiple reference channels according to the invalid data areas of each single reference channel;

Determine the valid data areas of multiple reference channels based on the invalid data areas after the unified start and end times;

When performing pulse identification, perform pulse identification on the pulses in the valid data area, and no longer perform pulse identification on the pulses in the invalid data area.

According to the second aspect of the application, the application provides a multi-reference channel pulse identification device, comprising:

The data acquisition unit is used to collect the signals output by multiple reference channels to obtain the data to be processed by each reference channel. The signals output by the reference channels are converted from the light information emitted by the measured particles collected by the reference channels after they are excited by light. electric signal;

The pulse discovery unit is used to judge whether to find a pulse based on the data to be processed of each single reference channel and the feature threshold corresponding to the reference channel, the feature threshold is the threshold of the feature of interest of the particle of interest, and the found pulse refers to A pulse with a pulse peak value greater than the characteristic threshold is detected;

A single-channel invalid area determination unit is configured to determine an invalid data area in the data to be processed of the single reference channel based on the detected pulse to be identified for the single reference channel in which the pulse is found;

An invalid area unification unit is used to unify the start and end times of the invalid data areas of multiple reference channels according to the invalid data areas of each single reference channel;

a valid area determining unit, which determines the valid data areas of multiple reference channels based on the invalid data areas after the unified start and end times;

The pulse identification unit is used for performing pulse identification on the pulses in the valid data area during pulse identification, and no longer performing pulse identification on the pulses in the invalid data area.

According to a third aspect of the application, the application provides a particle analyzer, comprising:

an excitation light source, used to provide excitation light for irradiating the particles to be measured;

The flow chamber is used to provide a place where the measured particles are irradiated;

A light collection device, used to collect the light information emitted by the measured particles after being excited by light, the light collection device includes a plurality of reference channels; and

The above multi-reference channel pulse identification device.

In this application, by determining the invalid data area with unified start and end times among multiple reference channels, the data that cannot or is difficult to obtain the correct area, amplitude and width information is excluded, and the pulse identification is not performed on them, thereby reducing the number of pulses The amount of data processing at the time of recognition.

Description of drawings

Figure 1 is a schematic diagram of the working principle of the flow cytometer;

Fig. 2 is the structural representation of flow cytometer;

FIG. 3 is a schematic structural diagram of a multi-channel pulse identification device according to an embodiment of the present application;

Fig. 4 is a pulse shape characteristic analysis diagram;

FIG. 5 is a flow chart of multi-reference channel pulse identification in an embodiment of the present application;

Fig. 6 is a flowchart of determining an invalid data area for a single reference channel in an embodiment;

Fig. 7 is a flow chart of unifying the start and end times of invalid data areas of multiple reference channels in an embodiment;

Figure 8 is one of the schematic diagrams of vertical pollution and horizontal pollution;

Figure 9 is the second schematic diagram of vertical pollution and horizontal pollution;

FIG. 10 is a schematic structural diagram of an effective area determination unit according to an embodiment of the present application;

Fig. 11 is another schematic structural diagram of an effective area determination unit according to an embodiment of the present application;

FIG. 12 is a flow chart of valid area determination in an embodiment of the present application;

Fig. 13 is a schematic diagram of determining an invalid data area according to a similarity rule;

Fig. 14 is a schematic diagram of overlapping pulse windows in an embodiment of the present application.

Detailed ways

The present invention will be further described in detail below through specific embodiments in conjunction with the accompanying drawings.

Flow cytometer is a kind of particle analyzer. In the embodiment of the present application, flow cytometer is used to detect and analyze particles. The structure of flow cytometer is shown in Figure 2, including excitation light source 1025, flow chamber 1022, light collection Device and processor 1026, the excitation light source 1025 is used to provide excitation light for irradiating the measured particles, such as laser; the flow chamber 1022 is used to provide the place 1021 where the measured particles are irradiated; the light collection device is used to collect the measured particles excited by light The light information sent out last includes all reference channels and non-reference channels; each channel includes an optical component 1023 and a photoelectric conversion device 1024, the optical component 1023 is used to complete the collection and shaping of scattered light or fluorescence, and the photoelectric conversion device 1024 It is used to convert the collected optical information into electrical signals and output them to the processor 1026, and the processor 1026 is used to process the electrical signals.

To illustrate the analytical process, terms used in this application are explained.

Reference channel and non-reference channel: As shown in Figure 1, a particle can generate pulse signals in multiple channels. For a particle of interest, if the user sets a feature threshold related to the feature of interest of the particle of interest on n (n≥2) channels, these n channels are called "reference channels" , and the remaining channels are called "non-reference channels". If the user sets a feature threshold related to the feature of interest of the particle of interest in each channel, all the channels are reference channels and there is no non-reference channel.

Discovery pulse: The feature threshold set by the user in the reference channel is a reference value related to the feature of interest of the particle of interest. If data greater than the reference value is found in the channel, it is considered to be a pulse, which triggers the identification process. . If no data greater than this reference value is found, it is assumed that no pulse was found. There may be many "small pulses" in the reference channel, but they will not be detected because they are not larger than the threshold set by the user.

Invalid data area: The purpose of pulse identification is to calculate the A, H, W and other information of pulses existing in each channel. If it is impossible or difficult to obtain the correct A, H, and W information from the data in some areas, then for the convenience of processing, set this part of the data as invalid data, and do not identify and analyze it. This section of data area is the "invalid data area"; the purpose of this is to make the engineering implementation of the algorithm easier and feasible.

Valid data area: Corresponding to the invalid data area, the data that needs to participate in the pulse identification is valid data, and this section of the data area is the "valid data area".

Horizontal pollution and vertical pollution: Because a certain section of "invalid data area" causes another part of data to become "invalid data area" according to certain rules, the latter is said to be "polluted"; horizontal pollution refers to the data in the same reference channel Contamination carried out, longitudinal contamination refers to the contamination carried out between multiple reference channels.

Pulse combination: The pulses formed by the same particle in multiple channels have certain similarities, such as similar width, similar peak time, one-to-one correspondence, etc., and use this similarity to match the pulses of different reference channels one by one to the same effective particle The process of emitting pulses is called pulse combining.

In the embodiment of the present application, the invalid data area with a unified start and end time is first determined in the reference channel, and then the valid data area is determined. When performing pulse identification, only the data in the valid data area is subjected to pulse identification processing, thereby reducing the number of pulses. The amount of data processing in the identification process is reduced, and some data that cannot be obtained are discarded, which also improves the accuracy of various results after pulse identification.

Embodiment one:

Processor 1026 among Fig. 2 comprises multi-channel pulse identification device, is used for carrying out pulse identification to the multi-channel electric signal of input processor 1026, as shown in Figure 3, multi-channel pulse identification device 30 comprises data acquisition unit 31, pulse finds unit 32 , single channel invalid area determination unit 33 , invalid area unification unit 34 , valid area determination unit 35 and pulse identification unit 36 .

The data acquisition unit 31 is used to collect the signals output by multiple reference channels to obtain the data to be processed of each reference channel. The signal output by the reference channel is the electrical signal converted from the optical information emitted by the measured particles collected by the reference channel after being excited by light. .

The pulse discovery unit 32 is used to judge whether to find a pulse based on the data to be processed of each single reference channel and the feature threshold corresponding to the reference channel, the feature threshold is the threshold of the feature of interest of the particle of interest, and finding the pulse means detecting the peak value of the pulse Pulses greater than the characteristic threshold. As shown in FIG. 4 , the output waveform after circuit processing is maintained at a DC baseline voltage 41 when no particle emits light, and a pulse signal is generated when a particle passes by and generates a scattered light signal. A pulse is considered found when the pulse amplitude (the portion above the baseline) exceeds the set characteristic threshold 42 . The pulse area between the two intersection points (threshold points) before and after the pulse and the characteristic threshold is called the threshold window 43, and the front and rear threshold points extend forward and backward respectively to form a front expansion window 44 and a rear expansion window 45. The expansion windows 44\45 together with the threshold window 43 form the pulse window 46. The expansion range of the front and rear expansion windows is determined according to the characteristics of the test object and the user's usage habits. Generally speaking, it is necessary to ensure that the expanded range can cover the entire pulse waveform range.

The single-channel invalid area determination unit 33 is configured to determine an invalid data area in the data to be processed of the single reference channel based on the detected pulse to be identified for the single reference channel of the found pulse. During the identification process, if the pulse window of the pulse is found to overlap with the pulse windows of the previous and subsequent pulses, these pulses are considered to be invalid pulses, and this piece of data is discarded and no longer used. In addition, when the width of the pulse is found to be too wide or too narrow, it is also considered to be a false pulse and regarded as an invalid pulse. The time period covered by the invalid pulse window is determined as an invalid data area. Therefore, in a specific example, the single-channel invalid area determining unit 33 includes a pulse window determining subunit, a first judging subunit and a second judging subunit, the pulse window determining subunit is used to determine the pulse window of the pulse to be identified, The pulse window is the area where the pulse continues; the first judging subunit is used to judge whether the pulse window of the current pulse to be identified overlaps with the pulse windows of the adjacent pulses to be identified, and if so, the overlapping pulse window area is used as Invalid data area: the second judging subunit is used to judge whether the pulse width of the pulse to be identified is abnormal, and if yes, the abnormal pulse window area of the pulse to be identified is used as an invalid data area.

The invalid area unification unit 34 is used to unify the start and end times of the invalid data areas of multiple reference channels according to the invalid data areas of each single reference channel. In a specific example, the invalid area unified unit includes a vertical pollution subunit, a horizontal pollution subunit and a third judging subunit. The vertical pollution sub-unit is used to compare the single reference channel with invalid data area as the compared reference channel with other reference channels one by one, and make the start and end time of the invalid data area in the pending data area of other reference channels the same as that of the compared reference channel The period of time is used as an invalid data area; the horizontal pollution subunit is used to judge whether the invalid data area of each single reference channel overlaps with the adjacent pulse windows before and after, and if so, the overlapping pulse window area is also used as an invalid data area; The third judging subunit is used to judge whether the invalid data area of each single reference channel has a unified start and end time, and determine whether the vertical pollution subunit and the horizontal pollution subunit continue to work or end the cycle according to the judging result.

The valid area determining unit 35 determines the valid data areas of multiple reference channels based on the invalid data areas after the unified start and end times, for example, the data areas in the entire data area except the invalid data area are regarded as valid data areas.

The pulse identification unit 36 is used for performing pulse identification on the pulses in the valid data area when performing pulse identification, and no longer performing pulse identification on the pulses in the invalid data area.

Based on the multi-reference channel pulse identification device of this embodiment, when identifying multi-channel pulses, its processing flow is as shown in Figure 5, including the following flow:

Step 50, collect the signals output by multiple channels to obtain the data to be processed of each channel, and the signals output by each channel are electrical signals converted from the optical information emitted by the measured particles collected by each channel after being excited by light. Multiple channels, scattered light signals or fluorescent signals are collected through each channel, after photoelectric conversion and signal processing circuit processing in each channel, a time-continuous analog signal is formed, and the signal is sampled by an AD device with a certain sampling rate Conversion, which converts an analog signal into a discrete digital signal. Each sampling obtains the digital quantity of the analog voltage signal at that time, and each such sampling point is referred to as a "point" in this article. Due to the sampling conversion of the AD device, the continuous analog signal is converted into a series of digital "points". Since the "points" are very dense, the waveform of the analog signal can be basically restored after connection. The waveform data mentioned later in this application refers to the digital data after AD conversion. The waveform data is then analyzed and processed by means of software or logic circuits.

Step 51 , judging whether a pulse is found based on the data to be processed of each single reference channel and the feature threshold corresponding to the reference channel, where the feature threshold is the threshold of the feature of interest of the particle of interest. In this embodiment, at least two reference channels are set among the multiple channels, and firstly, the waveform data of the reference channels are processed to determine whether a pulse is found in each single reference channel. As shown in Figure 4, when the peak value of a certain pulse is detected to be greater than the characteristic threshold, it is considered that a pulse is found, and this pulse is called a pulse to be identified. If the peak value of a pulse is less than or equal to the characteristic threshold, the pulse is discarded.

Step 52, for the single reference channel where the pulse is found, determine the invalid data area in the data to be processed of the single reference channel based on the found pulse to be identified; if no pulse is found in a certain reference channel, it is not sure whether the single reference channel is There is an invalid data area. When determining the invalid data area of a single reference channel, if it is detected that the pulse windows overlap or the pulse width is abnormal, it is an invalid pulse, and the time period involved is the invalid data area. The specific processing flow is shown in Figure 6, including the following steps:

Step 60: Detect a threshold window of the pulse to be identified based on the characteristic threshold, and the threshold window is a pulse area between two threshold points where the pulse to be identified intersects a boundary formed by the characteristic threshold.

In step 61, the pulse window is obtained by extending the threshold window to a predetermined area forward and backward respectively. The pulse window is the pulse continuation area. In order to make the pulse window completely cover the pulse continuation area to prevent missing data, the threshold window is extended forward and backward by a predetermined area to obtain the pulse window. As shown in Figure 4, the threshold window 43 expands the predetermined area forward to obtain the front expansion window 44, and expands the predetermined area backward to obtain the rear expansion window 45. The time period of the pulse window 46 is equal to the front expansion window 44 plus the threshold window 43 plus the rear expansion window 45. The expansion has the following functions: 1. After the expansion, the pulse area can be integrated and calculated in the expansion window; 2. Through the expansion, the difference in the generation time of the pulse between channels can be redundant, and the area of each channel can be calculated within the expansion window. , it is guaranteed to cover the entire pulse. The setting of the front and rear expansion window width has a certain relationship with the setting of the characteristic threshold. If the characteristic threshold is set closer to the peak of the pulse, the larger the expansion is required to cover the entire pulse. The closer the threshold is to the bottom, the expansion can be covered without being too large. .

Step 62, judging whether the pulse window of the current pulse to be identified overlaps with the pulse windows of the adjacent pulses to be identified, if so, execute step 63, otherwise execute step 64.

In step 63, the overlapped pulse window area is regarded as an invalid data area. When the pulse windows overlap, the area of the pulse cannot be effectively calculated or the calculation of the area is inaccurate. Therefore, if the pulse windows overlap, the time period covered by the overlapping pulse windows before and after will be regarded as an invalid data area, and will not participate in subsequent pulses. identify.

Step 64, judging whether the pulse width of the pulse to be identified is abnormal, and if so, execute step 63, and use the abnormal pulse window area of the pulse to be identified as an invalid data area. Otherwise, execute step 65 and continue to detect the next pulse to be identified.

Usually, the pulse corresponding to the light generated by the excited particle has certain characteristics. Even if it is detected by different channels, the pulse shape of each channel is consistent and the generation time is the same. Therefore, it can be judged whether the pulse is generated by an effective particle according to the pulse shape generated by the measured particle. In this embodiment, the pulse width is used to reflect the pulse shape. For example, the half-peak width (that is, the pulse width at half of the peak value) or the equivalent width can be used as the pulse width. When the pulse width is too large or small, the pulse shape can be considered to be different If it matches, the pulse is likely to be an interference signal. Therefore, a pulse with an abnormal pulse width is considered an invalid pulse, and the time period involved in its pulse window is considered an invalid data area, which does not participate in subsequent pulse identification.

Step 53, unify the start and end times of the invalid data areas of multiple reference channels according to the invalid data areas of each single reference channel. By unifying the start and end times of the invalid data areas of each reference channel, data that is not expected or cannot be obtained with reasonable results is excluded, thereby reducing the amount of data processing during pulse identification. In a specific embodiment, the start and end times of the invalid data areas of multiple reference channels are unified through the following steps, as shown in FIG. 7 .

Step 70, the vertical pollution step. Compare the single reference channel with invalid data area as the compared reference channel with other reference channels one by one, and take the time period in the data area to be processed of other reference channels that has the same start and end time as the invalid data area of the compared reference channel as invalid data Area. The invalid data areas of multiple reference channels after longitudinal contamination have the same start time and end time.

Step 71, a horizontal contamination step. Determine whether the invalid data area of each single reference channel overlaps with the adjacent pulse windows after longitudinal pollution, and if so, the overlapping pulse window area is also used as the invalid data area. In each reference channel, due to the limited data buffer area, old data should be removed and new data should be added as the recognition proceeds. Therefore, in a preferred embodiment, only a small amount of data before the pulse identification time is retained, and the size of the data volume can ensure that the previous pulse data overlapping with the current pulse can be found in the buffer, but it cannot guarantee that more data before that can also be found . Therefore, the previous pulse window that overlaps with the invalid data area is only polluted once, and the subsequent pulse windows that overlap are continuously polluted. That is: when a section of data area becomes an invalid data area, if the pulse window that is earlier than the pulse generated by it overlaps or touches this section of invalid data, the pulse window area will be polluted into an invalid data area. The two are connected as one, and all become invalid data areas. If there is a pulse window in front of this piece of data, the pulse window will not be polluted, and the recognition result will be retained. If a piece of invalid data area overlaps or touches the pulse window generated later, the pulse window generated later will be polluted, and the two will be integrated. Invalid data area can continue to be polluted, which is called persistent pollution. The start and end times of the invalid data area of each reference channel may be different after the horizontal pollution is performed, so step 72 is performed.

Step 72, judging whether the start and end times of the invalid data areas of multiple reference channels are the same, if the start and end times of the invalid data areas of the reference channels are the same, then end the loop, otherwise turn to step 70.

This loops until the detection is completed and the "invalid data areas" of all reference channels have the same start time and end time. Vertical pollution and horizontal pollution are shown in Fig. 8, the pulse window T1-T2 shown by the solid line in reference channel 2 and the pulse window T3-T4 shown by the dotted line overlap, resulting in the pulse window overlap of all relevant pulses in channel 2 The data in the pulse window area (that is, the T1-T4 area in the figure) is invalid. According to the vertical pollution, the data area (T1-T4) in the same time period of channel 1 is also invalid, and then according to the horizontal pollution, the pulse window area of channel 1 T5-T6 also become invalid data, and then according to the longitudinal pollution, the areas T1-T6 of all reference channels (ie channel 1 and channel 2 shown in the figure) become invalid data areas. As shown in Figure 9, the pulse width of channel 1 is abnormal, the pulse is too wide, and the pulse window T3-T4 becomes an invalid data area. According to the vertical pollution, the T3-T4 area of channel 2 also becomes an invalid data area. The T3-T4 area overlaps with the pulse window T1-T2. According to the horizontal pollution, the pulse window T1-T2 becomes an invalid data area. According to the vertical pollution, the T1-T4 area of all reference channels eventually becomes an invalid data area.

Step 54: Determine the valid data areas of multiple reference channels based on the invalid data areas after the unified start and end times. The valid data area may be the data area of the entire data area excluding the invalid data area, or the valid data area may be determined by other methods. The valid data area should not overlap with the invalid data area.

Step 55, determine the logic value of each reference channel according to whether pulses are found in each reference channel. In each reference channel, the pulse validity Chx_Valid is assigned a value of "1" or "0" in each reference channel according to the rule of "1 if a pulse is found" and "0 if no pulse is found".

Step 56, perform logic operations based on the logic values and logic algorithms of each reference channel. On the UI operation interface, multiple channels can be selected to perform "AND" logic operation or "OR" logic operation. Based on different situations, the user may have different choices. According to the user's choice, the Chx_Valid of each reference channel is used for logical operation. Get the final validity result ChAll_Valid of multiple reference channels.

Step 57, according to the logic operation result, determine whether to perform pulse recognition on the data to be processed of all channels, if yes, execute step 58, otherwise execute step 59. For example, if the final result ChAll_Valid is "0", the valid data area of all channels is also set to "invalid data area", and pulse identification is no longer performed. If the final result ChAll_Valid is "1", continue to execute step 58 .

Step 58, perform pulse identification on the data to be processed in all channels, and only perform pulse identification on pulses in the valid data area during pulse identification, and do not perform pulse identification on pulses in the invalid data area. During pulse identification, calculate and output the A, H, W information of the pulse in the effective data area.

Step 59, end.

In this embodiment, after vertical and horizontal contamination, all the pulse window regions corresponding to pulses with abnormal width and overlapping pulse windows are removed, that is, the regions where effective calculation cannot be performed are defined as invalid data regions. When performing pulse identification, The pulse identification operation is not performed on the invalid data area, thereby reducing the amount of data operation. Because the pulses in the invalid data area are usually interference pulses or pulses that cannot be effectively calculated, the accuracy of the calculation results is also improved after removing these pulses.

Embodiment two:

According to the above-mentioned embodiment, after the invalid data area is determined, the valid data area is further determined, and the pulse identification operation is only performed on the data in the valid data area during subsequent pulse identification. The difference between this embodiment and the foregoing embodiments is that a better solution for determining a valid data area is provided.

In this embodiment, the effective area determination unit is used to combine the pulses of the data areas of the multiple reference channels except the invalid data area according to the similarity rule between the pulses formed by the same particle in multiple reference channels. When combining, the single reference channel where the pulse is found is used as the compared reference channel one by one, and the pulse window in the data area of the compared reference channel except the invalid data area is used as the compared time area. The effective data areas of multiple reference channels are determined according to the pulse combination results, and the effective data areas at least include the pulse window area where the pulse combination is successful.

In a specific example, as shown in FIG. 10 , the similarity rule includes: there is no more than one pulse in the corresponding pulse window of multiple reference channels. The valid area determination unit 351 includes a threshold point acquisition subunit 3510 , a fourth determination subunit 3511 , a fifth determination subunit 3512 and a sixth determination subunit 3513 . The threshold point acquisition subunit 3510 is used to acquire the threshold points in the compared time region of other reference channels; the fourth judging subunit 3511 is used to judge the single-compared time region (that is, a single compared time region) of other reference channels Is the number of threshold points equal to 0? If the number of threshold points is equal to 0, it means that there is no pulse in the compared time region of the reference channel. If the number of threshold points is not equal to 0, the number of threshold points may be 1 or 2 , 3 or even more. When the number of threshold points is equal to 0, it is directly considered that the pulse combination in the compared time zone is successful; the fifth judging subunit 3512 is used to judge that when the fourth judging subunit 3511 judges that the number of threshold points is not equal to 0, the judging single is compared Whether the number of threshold points in the time zone is equal to 2. If it is equal to 2, it means that there may be only one pulse in the compared time region of the reference channel. If it is not equal to 2, it means that there are more than one pulses in the compared time zone of the reference channel, such as 1.5 or 2. In this case, it is considered that the pulse combination in the compared time zone fails; the sixth The judging subunit 3513 is used to judge whether the time of the front threshold point of the two threshold points is earlier than the time of the rear threshold point when the fifth judging subunit 3512 judges that the number of threshold points in the single-comparison time region is equal to 2, If yes, it means that there is only one pulse in the compared time zone of the reference channel, and the pulse combination in the compared time zone is considered successful; otherwise, it is considered that the pulse combination in the compared time zone fails, and the In the time zone, it may be the back part of the previous pulse and the front part of the next pulse.

In another specific example, as shown in FIG. 11 , the similarity rule includes: there is no more than one pulse in the corresponding pulse window of multiple reference channels and the peak times are similar. The valid area determining unit 352 includes a threshold point obtaining subunit 3520 , a fourth judging subunit 3521 , a fifth judging subunit 3522 , a sixth judging subunit 3523 and a seventh judging subunit 3524 . The threshold point acquisition subunit 3520 is used to obtain threshold points in the compared time regions of other reference channels; the fourth judging subunit 3521 is used to judge whether the number of threshold points in the single compared time regions of other reference channels is equal to 0 , if yes, it is considered that the pulse combination in the compared time zone is successful; the fifth judging subunit 3522 is used to judge whether the number of threshold points in the single compared time zone is equal to 2, if not equal to 2, then it is considered that the The combination of pulses in the ratio time zone fails; the sixth judging subunit 3523 is used to determine the number of threshold points in the two threshold points when the fifth judging subunit judges that the number of threshold points in the ratio time zone is equal to 2 Whether the time is earlier than the time of the rear threshold point, if the time of the front threshold point is later than the time of the rear threshold point, then it is considered that the pulse combination in the compared time zone fails; the seventh judging subunit 3524 is used in the sixth judgment subunit When the unit judges that the time of the front threshold point is earlier than the time of the rear threshold point, it judges whether the difference between the pulse peak moments in the same compared time area of all reference channels is greater than the interval threshold, and if so, it is considered that the compared time The combination of pulses in the area fails, otherwise the combination of pulses in the compared time area is considered successful.

Because the pulses formed by the same particle in multiple channels have certain similarities, such as similar width, similar peak time, one-to-one correspondence (it is impossible for a particle to form more than 2 pulses in one channel, only 0 or 1 pulse ), etc., if it is found that the pulse between the reference channels does not meet these characteristics, it is considered that this is not a valid particle signal. Based on the effective area determination unit of this embodiment, the effective data area is determined by using the pulse similarity rule of the same particle at the same time. The flow of a preferred specific embodiment is shown in Figure 12, including the following steps:

Step 120, take the single reference channel that found the pulse as the compared reference channel one by one, and call the data area outside the invalid data area determined by the scheme of Embodiment 1 of the compared reference channel as the initial valid data area, and set the initial effective data area Each pulse window in the area is used as a compared time area, and the threshold points in the compared time areas of other reference channels are obtained.

Step 121 , judging whether the number of threshold points in the single-comparison time zone of other reference channels is equal to 0, if yes, it is considered that the pulse combination in the comparison time zone is successful, otherwise step 122 is executed.

Step 122, judging whether the number of threshold points in the single-compared time zone is equal to 2, if not equal to 2, it is considered that the pulse combination in the compared time zone fails, if it is equal to 2, then perform the following steps;

Step 123, judge whether the time of the front threshold point among the two threshold points in the single-compared time zone is later than the time of the rear threshold point, if yes, then consider that the pulse combination in the compared time zone fails, otherwise perform the following step;

Step 124, judge whether the difference between the pulse peak moments in the same compared time zone of all reference channels is greater than the interval threshold, if yes, then consider that the pulse combination in the compared time zone fails, otherwise consider the compared time zone The pulse combination in the zone was successful.

The pulse window area where the pulse combination is successful can be used as the effective data area, and the effective data area is the superposition of all the time areas corresponding to the pulse window where the pulse combination is successful. The area where the pulse combination fails can also be regarded as an invalid data area, and the newly determined invalid data area in this embodiment is removed from the initial valid data area, and the remaining area is a valid data area for pulse identification operations. E.g:

The above judgment steps are refined to identify other reference channels within the pulse window time period of any reference channel, and the following points are judged:

a) The pulses of other reference channels have only one threshold point, as in the case corresponding to P2 in channel 1 in Figure 13;

b) The pulses of other reference channels have more than 3 threshold points, as shown in the case corresponding to P1 in channel 1 in Figure 13;

c) Although other reference channel pulses have two threshold points, their "pre-threshold point" time is later than the "post-threshold point", as in the case corresponding to P3 in channel 1 in Figure 13;

If any of the above three situations is found, the invalid data area is determined according to "horizontal pollution" and "vertical pollution". If none of the above three situations occurs, that is, within the pulse window time period of any reference channel, other reference channels have only 2 front and rear threshold points in normal order (the time of the rear threshold point > the time of the front threshold point ) or 0 threshold points, it further includes the following steps:

Overlap all pulse windows of the active data area of the individual reference channels. Because a particle generates pulses in multiple channels, although the time is close, there are inevitably small differences, and the pulse windows on multiple channels are also inevitably different at the beginning and end of time. In this embodiment, by taking the "union" In this way, the earliest time T1 and the latest time T2 in all pulse windows are obtained to form a new time period, as shown in Figure 14, this time period covers all pulse windows in the effective data area of all reference channels, that is, "overlap" .

Compare the peak moments of pulses between multiple reference channels (the moment corresponding to the midpoint of the threshold window can be regarded as the peak moment), if the maximum deviation of the peak value is greater than a certain value (a value shown in Figure 14, a value can be set according to the actual situation), then all relevant pulse window areas are set as "invalid data area" according to the "horizontal pollution" and "vertical pollution" rules.

After the effective data area is determined, in the effective data area T1-T2 time period shown in Figure 14, in each reference channel, according to the rule of "if a pulse is found, it will be 1, and if a pulse is not found, it will be 0". Each reference channel assigns its pulse validity Chx_Valid as "1" or "0", and according to the "AND"/"OR" operation rules set by the user, perform logical operations on Chx_Valid of each reference channel to obtain the final validity of multiple reference channels Result ChAll_Valid. If the final result ChAll_Valid is "0", the data of all channels between T1-T2 is set as "invalid data area", otherwise further identification.

If ChAll_Valid is "1", the A, H, and W information of all reference channels and non-reference channels are identified and output within the T1-T2 time period shown in FIG. 14 . The general method is: scan the maximum value during the T1-T2 time period shown in Figure 14 as the pulse peak value H, the integration result obtained by adding all points is the pulse A, and the result of dividing A by H is the pulse W. In this way, the A, H, and W information of all channels are finally identified.

In this embodiment, after the invalid data area is determined, the similarity rule is used in the initial valid data area to further determine the valid data area corresponding to the valid pulse generated by the same particle, and further exclude some data that does not need to participate in pulse identification, thereby Further reduce the amount of data calculation and improve the accuracy of calculation results.

Those skilled in the art can understand that all or part of the steps of the various methods in the above embodiments can be completed by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium, and the storage medium can include: read-only memory, Random access memory, disk or CD, etc.

The above content is a further detailed description of the present invention in conjunction with specific embodiments, and it cannot be assumed that the specific implementation of the present invention is limited to these descriptions. Those of ordinary skill in the technical field to which the present invention belongs can also make some simple deduction or replacement without departing from the concept of the present invention.

Claims (21)

1. The multi-reference channel pulse identification method is characterized in that comprising: Collecting signals output by a plurality of reference channels to obtain the data to be processed of each reference channel, the signals output by the reference channels are electrical signals converted from optical information emitted by the measured particles collected by the reference channels after they are excited by light; Based on the data to be processed of each single reference channel and the characteristic threshold corresponding to the reference channel, it is judged whether to find a pulse, the characteristic threshold is the threshold of the characteristic of interest of the particle of interest, and the discovery of the pulse refers to the detected pulse peak value greater than the characteristic threshold pulse; For the single reference channel where the pulse is found, determine the invalid data area in the data to be processed of the single reference channel based on the found pulse to be identified; Unify the start and end times of the invalid data areas of multiple reference channels according to the invalid data areas of each single reference channel; Determine the valid data areas of multiple reference channels based on the invalid data areas after the unified start and end times; When performing pulse identification, perform pulse identification on the pulses in the valid data area, and no longer perform pulse identification on the pulses in the invalid data area. 2. The method according to claim 1, wherein determining the invalid data area in the data to be processed of the single reference channel based on the detected pulse to be identified comprises: Determine the pulse window of the pulse to be identified, where the pulse window is the area where the pulse continues; Judging whether the pulse window of the current pulse to be identified overlaps with the pulse windows of the adjacent pulses to be identified, and if so, takes the overlapping pulse window area as an invalid data area; It is judged whether the pulse width of the pulse to be identified is abnormal, and if yes, the pulse window area of the abnormal pulse to be identified is used as an invalid data area. 3. The method according to claim 2, wherein said determining the pulse window of the pulse to be identified comprises: Detecting a threshold window of the pulse to be identified based on the characteristic threshold, the threshold window is a pulse area between two threshold points where the pulse to be identified intersects a boundary formed by the characteristic threshold; The pulse window is obtained by expanding the threshold window to a predetermined area forward and backward respectively. 4. The method according to claim 2, characterized in that, unifying the start and end times of the invalid data areas of multiple reference channels according to the invalid data areas of each single reference channel comprises: Perform the following steps cyclically until the invalid data area of each single reference channel has a unified start and end time: Vertical pollution step, one by one, compare the single reference channel with invalid data area as the compared reference channel with other reference channels, and compare the start and end time of the invalid data area in the pending data area of other reference channels with the compared reference channel at the same time segment as an invalid data area; In the horizontal contamination step, it is judged whether the invalid data area of each single reference channel overlaps with the adjacent pulse windows, and if so, the overlapping pulse window area is also used as the invalid data area. 5. The method according to any one of claims 1 to 4, wherein determining the valid data zones of multiple reference channels based on the invalid data zone after the unified start and end time comprises: According to the similarity rules between the pulses formed by the same particle in multiple reference channels, the pulse combination is performed on the data areas of multiple reference channels except for the invalid data area, and the valid data areas of multiple reference channels are determined. The data area at least includes the pulse window area where the pulse combination is successful. 6. The method according to claim 5, wherein the similarity rule comprises: the pulses in the corresponding pulse windows of a plurality of reference channels are no more than 1, and the pulse combining step comprises: One by one, the single reference channel where the pulse is found is used as the compared reference channel, and the pulse window in the data area of the compared reference channel except the invalid data area is used as the compared time area, and the pulse window in the compared time area of other reference channels is obtained. threshold point; Judging whether the number of threshold points in the single-compared time zone of other reference channels is equal to 0, if yes, then consider that the pulse combination in the compared time zone is successful, otherwise perform the following steps; Judging whether the number of threshold points in the single-compared time zone is equal to 2, if it is not equal to 2, then it is considered that the pulse combination in the compared time zone fails, if it is equal to 2, then perform the following steps; Judging whether the time of the front threshold point of the two threshold points in the single-compared time zone is earlier than the time of the rear threshold point, if so, it is considered that the pulse combination in the compared time zone is successful, otherwise it is considered that the compared time The combination of pulses in the zone failed. 7. The method according to claim 5, wherein the similarity rule comprises: there are no more than one pulse in the corresponding pulse window of a plurality of reference channels and the peak times are similar, and the pulse combining step comprises: Take the single reference channel where the pulse is found as the compared reference channel one by one, use each pulse window in the data area of the compared reference channel except the invalid data area as a compared time area, and obtain the compared time of other reference channels Threshold points in the area; Judging whether the number of threshold points in the single-compared time zone of other reference channels is equal to 0, if yes, then consider that the pulse combination in the compared time zone is successful, otherwise perform the following steps; Judging whether the number of threshold points in the single-compared time zone is equal to 2, if it is not equal to 2, then it is considered that the pulse combination in the compared time zone fails, if it is equal to 2, then perform the following steps; Judging whether the time of the front threshold point in the two threshold points in the single compared time zone is later than the time of the rear threshold point, if yes, then consider that the pulse combination in the compared time zone fails, otherwise perform the following steps; Judging whether the difference between the pulse peak moments in the same compared time area of all reference channels is greater than the interval threshold, if yes, it is considered that the pulse combination in the compared time area has failed, otherwise it is considered that the pulse combination in the compared time area is Pulse combined successfully. 8 . The method according to claim 5 , wherein the effective data area is a superposition of time areas corresponding to all pulse windows in which the pulse combination is successful. 9. The method according to any one of claims 1 to 4, further comprising: after determining the valid data areas of a plurality of reference channels: Determine the logic value of each reference channel according to whether pulses are found in each reference channel; Perform logical operations based on the logical values and logical algorithms of each reference channel; Determine whether to perform pulse identification on the data to be processed of all channels according to the logic operation result. 10. The method according to claim 5, further comprising: after determining the valid data areas of a plurality of reference channels: Determine the logic value of each reference channel according to whether pulses are found in each reference channel; Perform logical operations based on the logical values and logical algorithms of each reference channel; Determine whether to perform pulse identification on the data to be processed of all channels according to the logic operation result. 11. The method according to claim 6, further comprising: after determining the valid data areas of multiple reference channels: Determine the logic value of each reference channel according to whether pulses are found in each reference channel; Perform logical operations based on the logical values and logical algorithms of each reference channel; Determine whether to perform pulse identification on the data to be processed of all channels according to the logic operation result. 12. The method according to claim 7, further comprising: after determining the valid data areas of a plurality of reference channels: Determine the logic value of each reference channel according to whether pulses are found in each reference channel; Perform logical operations based on the logical values and logical algorithms of each reference channel; Determine whether to perform pulse identification on the data to be processed of all channels according to the logic operation result. 13. The method according to claim 8, further comprising: after determining the valid data areas of a plurality of reference channels: Determine the logic value of each reference channel according to whether pulses are found in each reference channel; Perform logical operations based on the logical values and logical algorithms of each reference channel; Determine whether to perform pulse identification on the data to be processed of all channels according to the logic operation result. 14. A multi-reference channel pulse identification device, characterized in that it comprises: The data acquisition unit is used to collect the signals output by multiple reference channels to obtain the data to be processed by each reference channel. The signals output by the reference channels are converted from the light information emitted by the measured particles collected by the reference channels after they are excited by light. electric signal; The pulse discovery unit is used to judge whether to find a pulse based on the data to be processed of each single reference channel and the feature threshold corresponding to the reference channel, the feature threshold is the threshold of the feature of interest of the particle of interest, and the found pulse refers to A pulse with a pulse peak value greater than the characteristic threshold is detected; A single-channel invalid area determination unit is configured to determine an invalid data area in the data to be processed of the single reference channel based on the detected pulse to be identified for the single reference channel in which the pulse is found; An invalid area unification unit is used to unify the start and end times of the invalid data areas of multiple reference channels according to the invalid data areas of each single reference channel; a valid area determining unit, which determines the valid data areas of multiple reference channels based on the invalid data areas after the unified start and end times; The pulse identification unit is used for performing pulse identification on the pulses in the valid data area during pulse identification, and no longer performing pulse identification on the pulses in the invalid data area. 15. The device according to claim 14, wherein the single-channel invalid area determining unit comprises: The pulse window determination subunit is used to determine the pulse window of the pulse to be identified, and the pulse window is the area where the pulse continues; The first judging subunit is used to judge whether the pulse window of the current pulse to be identified overlaps with the pulse windows of the adjacent pulses to be identified, and if so, takes the overlapped pulse window area as an invalid data area; The second judging subunit is used for judging whether the pulse width of the pulse to be identified is abnormal, and if so, takes the abnormal pulse window area of the pulse to be identified as an invalid data area. 16. The device according to claim 15, wherein the invalid area unifying unit comprises: Vertical pollution subunit, used to compare the single reference channel with invalid data area as the compared reference channel with other reference channels one by one, and compare the start and end time of the data area to be processed in other reference channels and the invalid data area of the compared reference channel The same time period as the invalid data area; The horizontal pollution subunit is used to determine whether the invalid data area of each single reference channel overlaps with the adjacent pulse windows, and if so, the overlapping pulse window area is also used as an invalid data area; The third judging subunit is used to judge whether the invalid data area of each single reference channel has a unified start and end time, and determine whether the vertical pollution subunit and the horizontal pollution subunit continue to work or end the cycle according to the judging result. 17. The device according to any one of claims 14 to 16, wherein the effective region determining unit is used to, according to the similarity rule between the pulses formed by the same particle in multiple reference channels, by multiple reference Pulse combination is performed on the data area of the channel except the invalid data area, and valid data areas of multiple reference channels are determined according to the pulse combination result, and the valid data area at least includes the pulse window area where the pulse combination is successful. 18. The device according to claim 17, wherein the similarity rules include: there are no more than one pulse in the corresponding pulse window of multiple reference channels, and the single reference channel of the pulse will be found one by one when pulses are combined As the compared reference channel, the pulse window in the data area except the invalid data area of the compared reference channel is used as the compared time area, and the effective area determination unit includes: The threshold point acquisition subunit is used to acquire the threshold points in the compared time region of other reference channels; The fourth judging subunit is used to judge whether the number of threshold points in the single-compared time zone of other reference channels is equal to 0, and if so, it is considered that the pulse combination in the compared time zone is successful; The fifth judging subunit is used to judge whether the number of threshold points in the time zone of a single comparison is equal to 2 when the fourth judging subunit judges that the number of threshold points is not equal to 0, and if it is not equal to 2, it is considered that the compared Pulse combination failed in the time zone; The sixth judging subunit is used to judge whether the time of the front threshold point in the two threshold points is earlier than the time of the rear threshold point when the number of threshold points in the single ratio time zone is equal to 2, and if so, consider The pulse combination in the compared time zone is successful; otherwise, it is considered that the pulse combination in the compared time zone fails. 19. The device according to claim 17, wherein the similarity rules include: there is no more than one pulse in the corresponding pulse window of multiple reference channels and the peak time is similar, and the pulses will be found one by one when the pulses are combined The single reference channel is used as the compared reference channel, and the pulse window in the data area except the invalid data area of the compared reference channel is used as the compared time area, and the effective area determination unit includes: The threshold point acquisition subunit is used to acquire the threshold points in the compared time region of other reference channels; The fourth judging subunit is used to judge whether the number of threshold points in the single-compared time zone of other reference channels is equal to 0, and if so, it is considered that the pulse combination in the compared time zone is successful; The fifth judging subunit is used to judge whether the number of threshold points in the time zone of a single comparison is equal to 2 when the fourth judging subunit judges that the number of threshold points is not equal to 0, and if it is not equal to 2, it is considered that the compared Pulse combination failed in the time zone; The sixth judging subunit is used to judge whether the time of the front threshold point in the two threshold points is earlier than the time of the rear threshold point when the fifth judging subunit judges that the number of threshold points in the single ratio time region is equal to 2 , if the time of the front threshold point is later than the time of the rear threshold point, it is considered that the pulse combination in the compared time area fails; The seventh judging subunit is used to judge whether the difference between the pulse peak moments in the same compared time region of all reference channels is greater than the interval threshold, if yes, it is considered that the pulse combination in the compared time area fails, otherwise it is considered that the pulse combination in the compared time area succeeds. 20. The device according to claim 17, wherein the effective data area is a superposition of time areas corresponding to all pulse windows in which the pulse combination is successful. 21. A particle analyzer, characterized in that it comprises: an excitation light source, used to provide excitation light for irradiating the particles to be measured; The flow chamber is used to provide a place where the measured particles are irradiated; A light collection device, used to collect the light information emitted by the measured particles after being excited by light, the light collection device includes a plurality of reference channels; and A device as claimed in any one of claims 14-20.