White blood cell differential counting device

Abstract

The present invention provides a device for classifying and counting white blood cells, comprising: an upper magnet and a lower magnet, with a sample channel disposed between the upper and lower magnets; a sample inlet and a buffer inlet disposed at one end of the sample channel; a white blood cell output port and a red blood cell output port disposed at the other end of the sample channel, the white blood cell output port being connected to the input end of a white blood cell shunt tube, the output end of the white blood cell shunt tube being connected to a sorting chamber; and a plurality of sensors mounted at the output end of the sorting chamber for classifying and counting purified samples. The device provided by the present invention is compact, portable, and provides accurate measurement results. It also avoids the drawbacks of traditional methods, such as the need for high-precision camera acquisition components, the reduced reliability due to compromised image quality, the generation of significant noise due to the need to lyse red blood cells prior to detection, and the resulting lack of data support for diagnosis of related diseases due to red blood cell lysis.

Description

Technical Field

[0001] The present invention relates to the technical field of blood analysis, and in particular to a white blood cell classification and counting device. Background Art

[0002] White blood cells are the immune system's frontline defense mechanism. Their composition changes dynamically in response to foreign bodies, infection, inflammation, or other malignant events occurring within the body. Monitoring the composition of white blood cells, known as the leukocyte differential, is an important, regular test used to diagnose infection or assess a person's vulnerability to health abnormalities. Measuring the proportions of white blood cell subtypes in the blood is a routine test performed in clinics to identify blood disorders, potential infections, or specific vulnerabilities. For example, neutropenia, a deficiency of neutrophils, makes the body highly susceptible to infection and must be monitored while patients are taking broad-spectrum antibiotics. Similarly, CD4+ lymphocytopenia is observed in autoimmune diseases such as HIV infection. In addition to deficiencies, diseases and disorders may also manifest as abnormal abundance of cells, such as mononucleosis, which is often caused by tuberculosis, subacute bacterial endocarditis, or malaria. White blood cells, also known as leukocytes (WBCs), are blood cells involved in the body's immune response. Each leukocyte subtype—lymphocytes, granulocytes, and monocytes—performs a specific role in the immune system. Lymphocytes are involved in recognizing foreign substances and producing antibodies as a defense mechanism, granulocytes respond to bacterial or fungal infections, allergic reactions, and inflammation, and monocytes engage in endocytosis. These cells can be distinguished from each other based on their biophysical and biochemical properties, such as their antigen expression, cytoplasmic composition, and the number and size of their nuclei.

[0003] Currently, differential white blood cell analysis is performed using hematology analyzers or flow cytometers, both of which are bulky instruments that require trained and certified technicians to operate in centralized laboratories, limiting access in resource-poor settings. Furthermore, to analyze white blood cells, these instruments lyse red blood cells beforehand and measure the cells' impedance. Based on the impedance and device-specific parameters, the white blood cell types are classified. Summary of the Invention

[0004] The present invention provides a leukocyte classification and counting device, which is used to solve the defect in the prior art that leukocyte classification and counting needs to be performed using bulky instruments.

[0005] A leukocyte differential counting device comprises: an upper magnet and a lower magnet, wherein a sample channel is provided between the upper magnet and the lower magnet;

[0006] A sample inlet and a buffer inlet are provided at one end of the sample channel; the sample liquid injected from the sample inlet and the buffer injected from the buffer inlet merge at one end of the sample channel and flow to the other end;

[0007] A white blood cell output port and a red blood cell output port are provided at the other end of the sample channel, the white blood cell output port is connected to the input end of the white blood cell shunt tube, and the output end of the white blood cell shunt tube is connected to the sorting chamber;

[0008] A plurality of sensors are installed at the output end of the sorting chamber, and the purified samples injected into the sorting chamber are classified and counted by the sensors; the sensors are arranged in parallel according to the direction of the magnetic field, so that different sensors correspond to different magnetic field gradients;

[0009] The sorting chamber and the plurality of sensors are both arranged between the upper magnet and the lower magnet.

[0010] Furthermore, the white blood cell classification and counting device as described above further includes a base plate disposed between the upper magnet and the lower magnet, and the sample channel and the sorting chamber are both mounted on the base plate.

[0011] Furthermore, the leukocyte differential counting device as described above comprises a left purification channel and a right purification channel arranged on the left and right sides of the sample channel; the left purification channel and the right purification channel are both mounted on the substrate;

[0012] The red blood cell liquid flowing out of the red blood cell output port is divided into two branches, one branch flows into the left purification channel through the left red blood cell shunt tube, and the other branch flows into the right purification channel through the right red blood cell shunt tube;

[0013] A left buffer input port is provided at the input end of the left purification channel, and a right buffer input port is provided at the input end of the right purification channel;

[0014] A left white blood cell liquid outlet and a left red blood cell liquid outlet are respectively provided at the output end of the left purification channel; a right white blood cell liquid outlet and a right red blood cell liquid outlet are respectively provided at the output end of the right purification channel;

[0015] The left leukocyte liquid output port is fed into the leukocyte shunt tube via the left leukocyte inlet tube; the right leukocyte liquid output port is fed into the leukocyte shunt tube via the right leukocyte inlet tube.

[0016] Furthermore, in the above-mentioned leukocyte classification and counting device, the sorting chamber comprises: a left sorting chamber and a right sorting chamber; the left sorting chamber and the right sorting chamber are both mounted on the substrate;

[0017] The output end of the leukocyte shunt tube is divided into two left and right branches, namely a left leukocyte branch and a right leukocyte branch; the left leukocyte branch is connected to the left sorting chamber; the right leukocyte branch is connected to the right sorting chamber;

[0018] Several sensors are respectively installed at the output ends of the left sorting chamber and the right sorting chamber.

[0019] Furthermore, in the white blood cell classification and counting device as described above, a white blood cell serpentine channel is provided between the output end of the white blood cell shunt tube and the left and right branches. The white blood cell liquid flowing out of the white blood cell shunt tube passes through the white blood cell serpentine channel and then flows into the left white blood cell branch and the right white blood cell branch respectively.

[0020] Furthermore, in the above-mentioned leukocyte differential counting device, a left buffer inlet is provided at one end of the left sorting chamber, and a left sample outlet is provided at the other end thereof; the left buffer inlet and the output end of the left leukocyte branch are provided on the same side of the left sorting chamber;

[0021] One end of the right sorting chamber is provided with a right buffer inlet, and the other end thereof is provided with a right sample outlet; the right buffer inlet and the output end of the right leukocyte branch are arranged on the same side of the right sorting chamber.

[0022] Furthermore, in the white blood cell differential counting device as described above, the sensor is a code-multiplexed Coulter sensor, and each sensor is assigned a unique 31-bit digital code from a set of orthogonal golden sequences.

[0023] Furthermore, in the white blood cell classification and counting device as described above, the sensor installed at the output end of the left sorting chamber is electrically connected to the left electrode, and the sensor installed at the output end of the right sorting chamber is electrically connected to the right electrode. The left electrode and the right electrode are respectively electrically connected to the input ends of two transimpedance amplifiers, and the output ends of the two transimpedance amplifiers are respectively connected to the input ends of the phase-locked amplifier, and the output end of the phase-locked amplifier is electrically connected to the processor.

[0024] Furthermore, in the leukocyte differential counting device as described above, the buffer inlet merges the buffer at one end of the sample channel through two branch pipes.

[0025] The white blood cell classification and counting device provided by the present invention achieves binary separation of a whole blood sample through a sample channel disposed between upper and lower magnets, obtaining a purified white blood cell liquid. A sensor disposed at the output end of the sorting chamber then performs classification and counting on the purified white blood cell liquid. The device is compact, portable, and provides accurate measurement results. Furthermore, it avoids the drawbacks of traditional methods, such as the need for high-precision camera acquisition components and the resulting reduction in reliability due to image quality degradation. It also avoids the drawbacks of traditional methods, such as the need to lyse red blood cells before testing, which results in significant noise generation and the loss of relevant red blood cell indicator information due to the lysis of red blood cells, resulting in a lack of data support for relevant disease diagnoses. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 Schematic diagram of the structure of the white blood cell classification and counting device provided by the present invention Figure 1 ;

[0028] Figure 2 for Figure 1 Top view after removing the magnet;

[0029] Figure 3 A flow chart for classifying white blood cells using the device provided in this application;

[0030] Figure 4 This is a schematic diagram of the principle of using sensors to classify and count white blood cells in the sorting chamber;

[0031] Figure 5 Schematic diagram of the sensor provided for this application;

[0032] Figure 6 This is an overall schematic diagram of the white blood cell differential counting device provided by the present invention;

[0033] Figure 7 A schematic diagram of the principle of the white blood cell differential counting device provided by the present invention;

[0034] Figure 8 Set up density scatter plots corresponding to healthy blood sample analysis and immune magnetic load gating;

[0035] Figure 9 Figure 1 shows the results of a benchmark test using healthy blood samples against conventional methods.

[0036] Figure 10 This is the result of analyzing the blood sample after processing the white blood cell components;

[0037] Reference numerals:

[0038] 1-sample channel; 2-sorting chamber; 21-left sorting chamber; 211-left sample outlet; 22-right sorting chamber; 222-right sample outlet; 23-left purification channel; 24-right purification channel, 3-leukocyte shunt tube; 31-right leukocyte branch tube; 32-left leukocyte branch tube; 33-left leukocyte inlet tube; 34-right leukocyte inlet tube; 35-leukocyte serpentine channel; 36-left red blood cell shunt tube; 37-right red blood cell shunt tube; 4-sample inlet; 5-sensor; 51-left sensor; 52-right sensor; 6-buffer inlet; 61-first left buffer tube; 62-first right buffer tube; 63-second left buffer tube; 64-second right buffer tube; 65-total buffer inlet; 71-left electrode; 72-right electrode; 8-substrate. DETAILED DESCRIPTION

[0039] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0040] Since the expression of the membrane protein CD33 varies among leukocyte subsets, with monocytes expressing the highest level of CD33, granulocytes having intermediate expression, and lymphocytes having the lowest expression, this application, in order to analyze the CD33 expression of leukocytes, mixes magnetic microbeads with a whole blood sample and performs leukocyte classification analysis on the peripheral whole blood sample by analyzing the CD33 expression of the leukocytes (it should be noted here that this application only uses monocytes, granulocytes, and lymphocytes as examples for three classifications. This application can also use more cells that need to be classified with different CD33 expressions to distinguish more types of cells). Since the magnetic load on leukocytes corresponds to the density of the target surface antigen (i.e., each type of cell can carry a different number of magnetic microbeads), this application places the whole blood sample carrying magnetic microbeads in an environment with a magnetic field. The different types of leukocytes can be distinguished by the strength of the magnetic field and the number of magnetic beads carried by each type of leukocyte.

[0041] The device provided in this application goes through the following four stages when classifying and counting white blood cells: Figure 3 The flow chart of the device provided in this application for classifying white blood cells; Figure 3As shown, first, white blood cells are labeled with anti-CD33-coupled magnetic microbeads. Second, the immunomagnetic load (magnetic microbeads) on the white blood cells is used to perform binary separation of red blood cells and white blood cells to obtain a purified sample. Third, the purified sample enters the characterization stage, in which the white blood cells are differentially magnetically classified based on their CD33 surface expression. Finally, a barcoded electronic sensor is used to quantify the white blood cells in the separated and purified sample.

[0042] The device provided by the present invention is introduced below:

[0043] Figure 1 Schematic diagram of the structure of the white blood cell classification and counting device provided by the present invention Figure 1 ; Figure 2 for Figure 1 Top view after removing the magnet. Figure 1 、 Figure 2 As shown, the device includes: an upper magnet 91 and a lower magnet 92, with a sample channel 1 provided between the upper magnet 91 and the lower magnet 92;

[0044] A sample inlet 4 and a buffer inlet 6 are provided at one end of the sample channel 1; the sample liquid injected from the sample inlet 4 and the buffer solution injected from the buffer inlet 6 merge at one end of the sample channel 1 and flow to the other end;

[0045] A white blood cell output port and a red blood cell output port are provided at the other end of the sample channel 1. The white blood cell output port is connected to the input end of the white blood cell shunt tube 3, and the output end of the white blood cell shunt tube 3 is connected to the input end of the sorting chamber 2.

[0046] A plurality of sensors 5 are installed at the output end of the sorting chamber 2, and the white blood cell liquid injected into the sorting chamber 2 is classified and counted by the sensors 5; the sensors are arranged in parallel according to the direction of the magnetic field, so that different sensors correspond to different magnetic field gradients;

[0047] The sorting chamber and the plurality of sensors are arranged between the upper magnet 91 and the lower magnet 92 .

[0048] Specifically, if Figure 1As shown, because upper magnet 91 comprises two magnets, and the magnetic poles of these two magnets are opposite on the side facing sample channel 1, and similarly, the polarities of the two magnets of lower magnet 92 are also opposite on the side facing sample channel 1, and the polarities of the two opposing magnets are the same, the area of ​​strongest magnetism within the space formed by upper magnet 91 and lower magnet 92 is concentrated in the central region between upper magnet 91 and lower magnet 92. Consequently, when a whole blood sample is placed within sample channel 1 and within the magnetic field formed by upper magnet 91 and lower magnet 92, white blood cells, labeled with magnetic microbeads, are attracted to the center of sample channel 1 by the magnetic field, while unlabeled red blood cells are distributed near the walls of sample channel 1. Consequently, driven by the buffer solution, the whole blood sample flushes the liquid carrying red blood cells out of both sides of sample channel 1, while the liquid carrying white blood cells flows out of the center of sample channel 1.

[0049] The working process of the device is as follows: First, the whole blood sample to be tested is injected from the sample inlet 4 (at a rate of 1.5 mL / h), and the 1-fold diluted phosphate buffered saline is injected from the buffer inlet 6 (at a rate of 4.9 mL / h) to realize the sample addition operation. Due to the adsorption of the magnetic field to the paramagnetic beads, when the whole blood sample is driven by the buffer to flow into the distribution area with the magnetic field strength, the white blood cells labeled with the magnetic microbeads in the whole blood sample are attracted to the position with stronger field strength, that is, they are attracted to the center of the sample channel 1 and flow (see Figure 1 The remaining unlabeled liquid carrying red blood cells flows in the direction indicated by the arrow in the middle of the sample channel 1, while the remaining unlabeled liquid carrying red blood cells has no attraction to the magnetic field and flows along the upper and lower sides of sample channel 1. Therefore, the liquid carrying white blood cells is flushed out of the outlet of sample channel 1 and eventually flows into the sorting chamber, where the white blood cells are classified and counted by the sensor installed at the output end of the sorting chamber.

[0050] The following describes how to use sensors to classify and count white blood cells in the sorting chamber:

[0051] Figure 4 The schematic diagram of using sensors to classify and count white blood cells in the sorting chamber is as follows: Figure 4 As shown, a row of parallel sensors are set up at the output end of the sorting chamber, and each sensor is under a different magnetic field gradient. After the previous red blood cell binary classification step, the labeled white blood cell liquid will flow to different sensor channel outlets under the action of the magnetic field gradient. The white blood cells are classified according to the outlet position, and the sensor counts the white blood cells according to the number of white blood cells passing through each sensor channel (displayed in the form of a waveform). Among them, the classification and counting of white blood cells are specifically affected by the following factors:

[0052] First, when it comes to white blood cell classification, the amount of magnetic microbeads affects the degree of deflection of white blood cells under the influence of a magnetic field. Each type of white blood cell carries a different number of magnetic microbeads. Therefore, different white blood cells can be driven to corresponding sensor channels based on the strength of the magnetic field and the amount of magnetic microbeads. Second, cell size also has a certain impact on deflection. Because the Stokes drag force on white blood cells offsets part of the magnetic force of the magnetic field, larger white blood cells with the same magnetic load will ultimately experience less deflection. Furthermore, the flow rate of the liquid also affects the degree of white blood cell deflection. The faster the white blood cell flows, the shorter the deflection time, resulting in smaller deflection. In summary, the sensor collects signals corresponding to white blood cell position, size, and flow rate, and samples these signals to a processor. The processor decodes the collected signals and recovers the various parameter information of individual cells based on the received signals. This information is then used to ultimately achieve white blood cell classification and counting.

[0053] In order to quantify white blood cells and capture the specific information of each cell, this application uses a code-multiplexed Coulter sensor. Figure 5 The sensor diagram provided for this application is as follows: Figure 5 As shown, each sensor is assigned a unique 31-bit digital code from a set of orthogonal golden sequences. The orthogonality of the different sensor codes ensures that the signals collected by the sensors can be reliably decoded with minimal interference. The decoded signals are then compared with correlation-matched signals from a template library to distinguish white blood cells. The uniqueness of the code is achieved by varying the spatial arrangement of the positive and negative electrode fingers of the excitation electrodes that power the circuit. When a cell interacts with the sensor, the digital code generated by the sensor reflects three parameters (cell size, velocity, and position): the power of the code signal represents the cell size, the duration of the code signal provides the cell's velocity, and the code itself provides the cell's position within the sorting chamber. Finally, the processor decodes the height, velocity, and position information based on the orthogonality between the sensor's golden sequences and the different coding sequences for different position channels, thereby achieving highly accurate classification of white blood cells.

[0054] The device provided by the present invention utilizes a code-multiplexed Coulter sensor, enabling the device to combine magnetic immunity and code division multiple access technologies to achieve the purpose of cell counting and classification using barcodes. The sensor can alleviate the common interference factors in encoding and decoding. At the same time, it utilizes the orthogonality between each golden sequence and different coding sequences in different position channels to embed information such as height, speed, and position. A specific decoding method is used to perform highly accurate classification of white blood cells, thereby achieving highly accurate classification and counting of white blood cells.

[0055] Furthermore, if Figure 1 As shown, the device further includes a substrate 8 disposed between the upper magnet 91 and the lower magnet 92 . The sample channel 1 and the sorting chamber are both mounted on the substrate 8 , and the substrate 8 is located at the center between the upper magnet 91 and the lower magnet 92 .

[0056] Furthermore, the device also includes a left purification channel 23 and a right purification channel 24 respectively arranged on the left and right sides of the sample channel 1; the left purification channel 23 and the right purification channel 24 are both installed on the substrate 8; the red blood cell liquid flowing out of the red blood cell output port is divided into two branches, one branch flows into the left purification channel 23 through the left red blood cell shunt tube 36, and the other branch flows into the right purification channel 24 through the right red blood cell shunt tube 37; a left buffer input port is provided at the input end of the left purification channel 23, and the left buffer input port is connected to the second left buffer tube 63; a right buffer input port is provided at the input end of the right purification channel 24, The right buffer input port is connected to the first right buffer tube 62; a left white blood cell liquid output port and a left red blood cell liquid output port are respectively provided at the output end of the left purification channel 23, and the left white blood cell liquid output port is connected to the left white blood cell inlet tube 33, and the white blood cell liquid purified by the left purification channel 23 is finally discharged into the white blood cell shunt tube 3; a right white blood cell liquid output port and a right red blood cell liquid output port are respectively provided at the output end of the right purification channel 24, and the right white blood cell liquid output port is connected to the right white blood cell inlet tube 34, and the white blood cell liquid purified by the right purification channel 24 is finally discharged into the white blood cell shunt tube 3 through the right white blood cell inlet tube 34.

[0057] Specifically, in order to avoid errors in subsequent quantitative analysis caused by some white blood cells that have not been completely separated and purified being mixed in the red blood cells, it is necessary to continue the second round of cell purification. The specific purification method is: the white blood cells output from the white blood cell output port of the sample channel 1 pass smoothly through the white blood cell shunt tube 3 into the subsequent sorting chamber, while the red blood cell liquid mixed with white blood cells enters the left purification channel 23 (0.5 mm wide, 25 mm long) and the right purification channel 24 along the arc channel from the upper and lower sides of the sample channel 1; under the action of the second magnetic field, the labeled white blood cells will flow into the white blood cell shunt tube 3 along the outlets of the left purification channel 23 and the right purification channel 24, and finally enter the sorting chamber, while the red blood cells flow out through the outlets set on the left purification channel 23 and the right purification channel 24.

[0058] In summary, the present invention improves the accuracy of leukocyte classification and counting by designing the left purification channel 23 and the right purification channel 24 to recover leukocytes that have not been captured before.

[0059] Furthermore, the sorting chamber includes: a left sorting chamber 21 and a right sorting chamber 22; the left sorting chamber 21 and the right sorting chamber 22 are both installed on the substrate 8; the output end of the leukocyte shunt tube 3 is divided into two left and right branches, namely the left leukocyte branch 31 and the right leukocyte branch 32; the left leukocyte branch 31 is connected to the left sorting chamber 21; the right leukocyte branch 32 is connected to the right sorting chamber 22; a number of sensors are respectively installed at the output ends of the left sorting chamber 21 and the right sorting chamber 22.

[0060] The purpose of dividing the sorting chamber into two parts is to: divert the sample liquid for parallel detection, thereby improving the sorting rate and capture rate of white blood cells; at the same time, reducing the overlap caused by the large sample volume, which ultimately affects the signal passing through the sensor, thereby avoiding the defect of reducing the detection accuracy of the sensor.

[0061] Furthermore, a leukocyte serpentine channel 35 is provided between the output end of the leukocyte shunt tube 3 and the left and right branches. The leukocyte liquid flowing out of the leukocyte shunt tube 3 passes through the leukocyte serpentine channel 35 and flows into the left leukocyte branch 31 and the right leukocyte branch 32 respectively.

[0062] The device provided by the present invention controls and buffers the flow rate of the liquid by using the leukocyte serpentine channel 35 to act as a fluid dynamic resistor, thereby ensuring the correct direction of fluid flow and preventing backflow, further improving the accuracy of leukocyte classification and counting.

[0063] Furthermore, a left buffer inlet is provided at one end of the left sorting chamber 21, communicating with the first left buffer tube 61; a left sample outlet 211 is provided at the other end of the left sorting chamber 21; the left buffer inlet and the output end of the left leukocyte branch tube 32 are located on the same side of the left sorting chamber 21; a right buffer inlet is provided at one end of the right sorting chamber 22, communicating with the first right buffer tube 62; a right sample outlet 222 is provided at the other end of the right sorting chamber 22; the right buffer inlet and the output end of the right leukocyte branch tube 31 are located on the same side of the right sorting chamber 22. Buffer is diverted from the main buffer inlet 65 through the first left buffer tube 61, the first right buffer tube 62, the second left buffer tube 63, and the second right buffer tube 64.

[0064] The device provided by the present invention realizes buffer diversion through the total buffer inlet 65, so that the buffer pressures entering the left purification channel 23, the right purification channel 24, the left sorting chamber 21, and the right sorting chamber 22 are the same, thereby further improving the accuracy of white blood cell classification and counting.

[0065] Specifically, in order to enable the finally purified and collected liquid carrying leukocytes to flow smoothly from the output end of the sorting chamber to each sensor channel, it is also necessary to drive the liquid carrying leukocytes out of the sorting chamber through a buffer solution.

[0066] Figure 6 This is an overall schematic diagram of the white blood cell classification and counting device provided by the present invention. Figure 7 The schematic diagram of the principle of the white blood cell classification and counting device provided by the present invention is as follows: Figure 6 、 Figure 7 As shown, the sensor installed at the output end of the left sorting chamber 21 is electrically connected to the left electrode 71, and the sensor installed at the output end of the right sorting chamber 22 is electrically connected to the right electrode 72. The left electrode 71 and the right electrode 72 are electrically connected to the input ends of two transimpedance amplifiers respectively, and the output ends of the two transimpedance amplifiers are respectively connected to the input ends of the phase-locked amplifier, and the output end of the phase-locked amplifier is electrically connected to the processor.

[0067] Specifically, the present invention uses a 1.5V 500kHz sine wave to drive the excitation electrodes, measures the current from a pair of sensing electrodes, and combines the measured signals into a bipolar waveform using a differential amplifier. The waveform of each cell is then compared with a database waveform to determine the specific exit point to which the cell was classified. This waveform is then decoded using a specific program to estimate the cell trajectory. Specifically, the sensing electrodes (two positive and two negative electrodes) are first connected to a transimpedance amplifier, which converts the current measured by the sensor into a voltage signal. These voltage signals are then sampled at a frequency of 57.6kHz using a lock-in amplifier. The data stream is saved as a local file on a computer, and the sensor information (cell position, size, and velocity) is subsequently extracted for processing and analysis, and the results are ultimately output. When processing and analyzing the electrical signals acquired from the sensors, the correlation between the recorded signal and the template library signal is first determined. The orthogonality of the code sequences generated by the sensors ensures minimal crosstalk between different sensors, and matching sensor identifiers are identified from autocorrelation peaks. For situations where multiple cells interact with different sensors simultaneously, an iterative continuous interference cancellation algorithm is employed, and the interfering signals are subtracted until no residual signal remains in the waveform.

[0068] Furthermore, the buffer inlet 6 merges the buffer at one end of the sample channel 1 through two branch pipes.

[0069] The device provided by the present invention combines the buffer solution at the buffer solution inlet 6 through two branch tubes, which can better dilute the blood sample, thereby further improving the accuracy of white blood cell classification and counting.

[0070] Experimental example:

[0071] Complete cell analysis is inseparable from the pre-preparation of samples. In this example, venous blood samples were collected using vacuum blood collection tubes, and each blood sample was then divided into four equal parts for testing and three independent validation studies. In order to label leukocytes, biotin-conjugated anti-CD33 antibodies were introduced into the test aliquots at a concentration of 45fg antibody per leukocyte and incubated at room temperature for 30 minutes. 1μm streptavidin-conjugated magnetic beads were then washed, precipitated, and added to the blood sample at a concentration of 100 magnetic beads per leukocyte. The mixture was incubated for another 30 minutes at room temperature without any magnetic field interference to ensure that the beads did not aggregate.

[0072] To facilitate subsequent control experiments, a granulocyte-rich blood sample was also prepared. The blood sample was placed in a separation tube supplemented with lymphocyte culture medium for a period of time and then centrifuged at 1200 grpm for 10 minutes to separate granulocytes and red blood cells from plasma and monocytes. Centrifugation produced three suspensions containing: (1) granulocytes and red blood cells, (2) lymphocytes and monocytes, and (3) plasma. The granulocyte-red blood cell suspension was mixed with plasma in a 1:1 ratio and diluted to an appropriate hematocrit level, and aliquoted for subsequent testing and validation. For the test samples, the same immunomagnetic labeling protocol was followed. The present invention determined the quantitative gating parameters for distinguishing white blood cells based on the immunomagnetic load, processed blood from different healthy samples, and calibrated the data of this embodiment using the white blood cell difference obtained from a commercial blood analyzer using matched samples. Based on this calibration process, two immunomagnetic gate thresholds were set at different numbers of beads for determination to distinguish white blood cell subpopulations. Next, to verify these set gates, this embodiment also analyzed blood samples from three different healthy individuals. The blood sample was driven through the device provided by the present application at 1500 μL / h, and at least 4000 events were recorded in less than 5 minutes. First, the density scatter plot of the acquired data was analyzed to determine the location of the subpopulation ( Figure 8 (a) In all cases, three distinct cell populations can be qualitatively identified based on the contrast between cell size and immunomagnetic loading. Given that lymphocytes are expected to have the lowest CD33 expression among other leukocyte subsets, leukocytes with an immunomagnetic loading below the first threshold are scored as lymphocytes. At the other end of the spectrum, monocytes have the highest CD33 expression and are larger, so leukocytes carrying beads larger than this threshold are scored as monocytes. Finally, the largest fraction of leukocytes falls between these two threshold gates; these are labeled granulocytes, consistent with the fact that the average CD33 expression of granulocytes falls between that of lymphocytes and monocytes.

[0073] The set immunomagnetic loading gate was applied to the cell sorting data of this example, and then the frequencies of different leukocyte subsets of each sample were calculated ( Figure 8 The corresponding b in (a). The lymphocyte frequencies of sample #1, sample #2, and sample #3 were measured to be 34.46%, 27.54%, and 36.38%, respectively. Similarly, granulocytes were measured to account for 54.32%, 63.24%, and 55.32% of all white blood cells in samples #1, #2, and #3, respectively, and monocytes were measured to account for 10.22%, 9.22%, and 8.30% of all white blood cells, respectively. To test the accuracy of the measurements of this example, three independent analyses were performed on validation aliquots of each sample (Table 1). First, the samples were fluorescently stained for CD33, and the distribution was analyzed using flow cytometry for direct comparison with the CD33 measurements of this example ( Figure 8 (c) Fluorescence-based measurement of CD33 expression resulted in estimated concentrations of 34.2%, 29.3%, and 35.3% lymphocytes; granulocytes were 55.0%, 62.1%, and 54.4% for samples #1, #2, and #3, respectively, and monocytes were 10.8%, 8.6%, and 10.3%, respectively. Assuming the CD33-based flow cytometry results as ground truth, the close match between the two assays confirms the validity of the present invention's device. Nevertheless, the present invention also analyzed the samples using a standard hematology analyzer, as well as another set of flow cytometric measurements, this time using CD45 staining, and using the traditional side scatter and CD45 expression method to quantify the different fractions of leukocyte subsets.

[0074] Table 1 Comparison of experimental results of three samples with independent verification methods

[0075]

[0076] In order to quantitatively compare the measurement results of the device of the present application with those of the traditional method, the absolute percentile error of the estimated subpopulation frequency was calculated ( Figure 9 The corresponding a) and relative percentile error ( Figure 9 Corresponding to b). For all leukocyte subtypes, the absolute percentile error between the frequency measurements of the microchip device designed by the present application and the validation method was always less than ±2.5% on average. In terms of relative percentile error, the lymphocyte and granulocyte measurement results obtained using the device of the present application were accurate compared to the blood analyzer results, with average relative percentile errors of 3.8% (STD = 2.5) and 0.4% (STD = 1.69), respectively. On the other hand, monocyte measurements were consistently underestimated by the device of the present application, showing a negative average absolute percentile error (-1.3%) and a higher relative percentile error, with an average of -15.6% (STD = 8.28). This is because monocytes are scarce among other leukocytes, which amplifies small differences in absolute frequency measurements when expressed as relative percentile errors.

[0077] In order to test the consistency of the present application's device and traditional methods, linear regression analysis was used. Specifically, the present application has regressed the subpopulation frequency (table 2) of the same measurement performed relative to other technologies. For granulocytes and lymphocytes, regression analysis demonstrates better fitting (R2>0.83), particularly about CD45 flow cytometry measurement (R2>0.92). The R2 measurement of monocytes is not too accurate and is between 0.56 and 0.74, most compatible with CD45 flow cytometry data. In addition, the present application calculates these transition points of lymphocytes, granulocytes and monocytes and is respectively 25%, 60% and 10%. About the flow cytometry measurement of CD33 and CD45 expression, the regression analysis of the present application produces a slope close to 1 to lymphocytes and granulocytes, shows that these results are very consistent with the result discussed before, and also in line with expectation.

[0078] Table 2 Linear regression analysis of the measurement results of the traditional method

[0079]

[0080] To simulate hematological conditions with abnormal leukocyte composition, the present invention prepared blood samples that were depleted of monocytes (i.e., only lymphocytes and granulocytes), resulting in an abnormally high frequency of granulocytes among the leukocytes. These samples were analyzed in the present invention's device at 1500 μL / h, and at least 4000 leukocytes were analyzed per run. Consistent with the manipulated leukocyte composition, the assay reported abnormally high levels of granulocytes, with samples #1, #2, and #3 reporting 78.48%, 72.30%, and 84.53%, respectively ( Figure 10 These measurements were compared with those of matched samples processed using a hematology analyzer and CD33 and CD45 fluorescence flow cytometry. Figure 10 The corresponding b) and CD45-based flow cytometry data ( Figure 10The corresponding c) is very consistent. For sample #1, the measured granulocyte frequency was 78.48%, which is very consistent with the CD33-based measurement value of 81.1% and the CD45-based flow cytometry measurement value of 79.2% (<3% difference). Similarly, the lymphocyte measurement results also had only <2% deviation. For sample #1, the biggest mismatch was observed by the measured monocyte frequency. The measured monocyte fraction of 9.26% was higher than the CD33-based and CD45-based flow cytometry, which were 5.3% and 5.73%, respectively. In sample #2, the granulocyte measurement value of 72.30% was slightly lower than the granulocyte measurement concentrations of 79.3% and 82.5% based on CD33 and CD45 flow cytometry. For sample #3, the analysis correctly identified the sample as the sample with the highest granulocyte frequency among the test samples, with a measured granulocyte frequency of 84.53%. These measurements further verified that the white blood cells falling between the two threshold immunomagnetic gates set by this application are granulocytes. Overall, the measurement results of the device of the present application are in good agreement with the results of independent analysis.

[0081] Finally, to further validate the immunomagnetic gate threshold, the present application also processed residual samples containing only monocytes and plasma, which are easily obtained as a by-product of preparing granulocyte-rich blood samples. These samples are almost devoid of granulocytes because these samples are depleted along with red blood cells to prepare the primary suspension previously analyzed. In fact, the present application's assay detected abnormally low (≈1.5%) levels of white blood cells with a magnetic load of 20-60 magnetic beads. In contrast, a high proportion of white blood cells carrying magnetic beads less than the first magnetic gate threshold (i.e., lymphocytes) or white blood cells carrying magnetic beads greater than the second magnetic gate threshold (i.e., monocytes) were observed in these samples, and these results were very consistent with fluorescence-based flow cytometry of CD33 and CD45 expression from these control samples.

[0082] In summary, these results validate the applicability of the immunomagnetic gate of the present application in leukocyte identification based on CD33 expression.

[0083] The device of the present invention first separates immunomagnetically labeled white blood cells from other blood cells and then differentially classifies them according to their immunomagnetic load to separate these white blood cells into subpopulations. Because the results are readily available as electrical signals, the provided platform is ideally suited to expand white blood cell differential analysis to virtually any setting, point of care, bedside, or at home, for easy and rapid monitoring of immune system disorders and emergencies, potentially eliminating the cumbersomeness of regular hospital visits for immunocompromised patients.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A leukocyte differential counting device, characterized in that: include: an upper magnet (91) and a lower magnet (92), wherein a sample channel (1) is provided between the upper magnet (91) and the lower magnet (92); A sample inlet (4) and a buffer inlet (6) are provided at one end of the sample channel (1); the sample liquid injected from the sample inlet (4) and the buffer injected from the buffer inlet (6) merge at one end of the sample channel (1) and flow to the other end; A white blood cell output port and a red blood cell output port are provided at the other end of the sample channel (1), the white blood cell output port is connected to the input end of the white blood cell shunt tube (3), and the output end of the white blood cell shunt tube (3) is connected to the input end of the sorting chamber (2); A plurality of sensors (5) are installed at the output end of the sorting chamber (2), and the white blood cell liquid injected into the sorting chamber (2) is classified and counted by the plurality of sensors (5); the plurality of sensors (5) are arranged in parallel according to the direction of the magnetic field, so that different sensors correspond to different magnetic field gradients; The sorting chamber (5) and the plurality of sensors (2) are both arranged between the upper magnet (91) and the lower magnet (92); It also includes a base plate (8) disposed between the upper magnet (91) and the lower magnet (92), and the sample channel (1) and the sorting chamber (2) are both mounted on the base plate (8); It comprises a left purification channel (23) and a right purification channel (24) arranged on the left and right sides of the sample channel (1); the left purification channel (23) and the right purification channel (24) are both mounted on the substrate (8); The red blood cell liquid flowing out of the red blood cell output port is divided into two branches, one branch flows into the left purification channel (23) through the left red blood cell shunt tube (36), and the other branch flows into the right purification channel (24) through the right red blood cell shunt tube (37); A left buffer input port is provided at the input end of the left purification channel (23), and a right buffer input port is provided at the input end of the right purification channel (24); A left white blood cell liquid output port and a left red blood cell liquid output port are respectively provided at the output end of the left purification channel (23); a right white blood cell liquid output port and a right red blood cell liquid output port are respectively provided at the output end of the right purification channel (24); The left leukocyte liquid output port is fed into the leukocyte shunt tube (3) through the left leukocyte inlet tube (33); the right leukocyte liquid output port is fed into the leukocyte shunt tube (3) through the right leukocyte inlet tube (34); The sorting chamber (2) comprises: a left sorting chamber (21) and a right sorting chamber (22); the left sorting chamber (21) and the right sorting chamber (22) are both mounted on the base plate (8); The output end of the leukocyte shunt tube (3) is divided into two left and right branches, namely a left leukocyte branch (32) and a right leukocyte branch (31); the left leukocyte branch (32) is connected to the left sorting chamber (21); the right leukocyte branch (31) is connected to the right sorting chamber (22); The plurality of sensors are respectively installed at the output ends of the left sorting chamber (21) and the right sorting chamber (22); A leukocyte serpentine channel (35) is provided between the output end of the leukocyte shunt tube (3) and the left and right branches. The leukocyte liquid flowing out of the leukocyte shunt tube (3) passes through the leukocyte serpentine channel (35) and then flows into the left leukocyte branch tube (32) and the right leukocyte branch tube (31) respectively.

2. The leukocyte differential counting device according to claim 1, characterized in that: A left buffer inlet is provided at one end of the left sorting chamber (21), and a left sample outlet (211) is provided at the other end; the left buffer inlet and the output end of the left leukocyte branch (32) are provided on the same side of the left sorting chamber (21); One end of the right sorting chamber (22) is provided with a right buffer inlet, and the other end thereof is provided with a right sample outlet (222); the right buffer inlet and the output end of the right leukocyte branch (31) are provided on the same side of the right sorting chamber (22).

3. The leukocyte differential counting device according to claim 1, characterized in that: The sensors are code-multiplexed Coulter sensors, and each sensor is assigned a unique 31-bit digital code from a set of orthogonal golden sequences.

4. The leukocyte differential counting device according to claim 1, characterized in that: The sensor installed at the output end of the left sorting chamber (21) is electrically connected to the left electrode (71), and the sensor installed at the output end of the right sorting chamber (22) is electrically connected to the right electrode (72). The left electrode (71) and the right electrode (72) are respectively electrically connected to the input ends of two transimpedance amplifiers. The output ends of the two transimpedance amplifiers are respectively connected to the input ends of the lock-in amplifier, and the output end of the lock-in amplifier is electrically connected to the processor.

5. The leukocyte differential counting device according to claim 1, characterized in that: The buffer inlet (6) merges the buffer at one end of the sample channel (1) through two branch pipes.

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