Method, device and kit for analyzing immune cells for companion animals

Abstract

The present invention relates to a technique in which leukocytes are isolated from whole blood of a companion animal, and then are separated with high purity into polymorphonuclear leukocytes (PMNs) and cytotoxic lymphocytes (CTLs) according to the presence or absence of expression of a CD4 surface antigen, and each cell group is treated with a different activation composition (PMN: PMA, CTL: PHA·PMA·IL-2) so that functional activation of immune cells is induced. For the activated cells, a diffraction pattern is acquired through a lens-free shadow imaging (LSIT) device, and a combined shadow parameter (CSP) defined as a product of a peak-to-peak distance (PPD) and a pattern width standard deviation (WSM_SD) is calculated, and thus innate immunity and acquired immune activities can be simultaneously and quantitatively evaluated.

Description

Immunocyte analysis method, device, and kit for companion animals

[0001] The present invention relates to a method for analyzing immune cells, an analysis apparatus, an analysis kit for the same, and an immune cell activation composition for quantitatively evaluating the immune function of companion animals.

[0002] More specifically, the present invention relates to a technology capable of simultaneously diagnosing the defense ability against bacterial and viral infections in companion animals by selectively separating polymorphonuclear cells (PMN) and cytotoxic lymphocytes (CTL) from whole blood samples of companion animals, including dogs, using the expression characteristics of CD4 surface antigens, treating each cell group with an activation-inducing substance (PMA or ASC-K9 composition) optimized for the characteristics of each cell group, and then analyzing changes in the diffraction patterns of the cells using lens-free shadow imaging technology (LSIT).

[0003] The above device may include an LED, a CMOS image sensor, a disposable multi-channel slide, and a drive motor for slide transport, and may be configured to sequentially measure multiple samples on the slide with a single sensor combination.

[0004] The immune system of companion animals is a complex defense system of cells and organs organized to defend the body against external pathogens such as viruses, bacteria, and parasites, playing a key role in maintaining homeostasis and preventing disease. Among these, polymorphonuclear cells (PMNs) are key cells of innate immunity that respond first in the early stages of pathogen invasion to suppress the spread of infection, while cytotoxic lymphocytes (including CTLs, CD8+ T cells, and NK cells) directly eliminate virus-infected cells or tumor cells or perform anticancer immune surveillance functions.

[0005] With the recent increase in the pet-owning population, the aging of pets, and the rising incidence of cancer and infectious diseases, there is a rapidly increasing need to accurately diagnose the immune status of pets and perform preventive health care. Currently, in the field of clinical veterinary medicine, the assessment of pet immunity mainly relies on checking white blood cell counts through CBC (Complete Blood Count) tests or measuring the concentration of specific cytokines using ELISA (Enzyme-Linked ImmunoSorbent Assay).

[0006] However, CBC tests only provide quantitative information such as the number or distribution ratio of cells in the blood, and have limitations in evaluating the actual status of whether immune cells can perform an appropriate immune response. In addition, precision testing methods such as ELISA analysis and flow cytometry require expensive equipment, reagents, and skilled professionals, and because it takes a long time to obtain results and the pre-processing is complex, there are limitations to their use as Point-of-Care Testing (POCT) in veterinary clinics.

[0007] Furthermore, some companion animals, including canines, possess unique immunological characteristics distinct from humans. For instance, while CD4 surface antigens are primarily expressed on helper T cells in humans, there is a species-specific difference in dogs where CD4 antigens are also expressed in the PMN population. Due to these differences, applying existing immunoassay platforms or reagents developed for humans directly to companion animals presents a problem in that accurate cell isolation and activity evaluation are difficult.

[0008] Accordingly, there is a growing need for technology capable of effectively isolating immune cells from the whole blood of companion animals while considering species-specific characteristics, and rapidly quantifying their activity in a label-free manner. In particular, Lens-Free Shadow Imaging (LSIT) technology is attracting attention as a next-generation immunodiagnostic technology because it can acquire high-resolution diffraction patterns without the need for separate complex optical lenses, offering advantages such as device miniaturization and suitability for automated analysis.

[0009] The present invention aims to overcome the limitations of existing CBC-based blood tests or ELISA analysis methods, which provide only the number of immune cells or the concentration of specific proteins and lack information reflecting the activation state or responsiveness of immune cells, and to provide a technology that can directly evaluate the immune function of companion animals quickly and economically without expensive equipment or complex fluorescent labeling processes.

[0010] This invention reflects the species-specific immunological characteristic that, unlike humans, polymorphonuclear leukocytes (PMNs) in certain companion animals, including canines, express CD4 surface antigens, thereby overcoming the limitations of existing human-based immunoassay platforms and analyzing CD4-positive PMN-containing cell populations and CD4-negative cytotoxic lymphocytes (CTLs, CD8+ The purpose is to provide an optimized method that can selectively isolate cell populations containing T cells and NK cells.

[0011] The present invention aims to provide an immune cell analysis device and method capable of acquiring cell diffraction patterns in a label-free manner by applying lens-free shadow imaging (LSIT) technology, and visualizing and quantifying changes in cell activity in an automated manner through individual parameters or combinations of individual parameters capable of quantifying the patterns.

[0012] The present invention aims to precisely diagnose the immune activity of companion animals through the above technology and to establish a standardized range of immune activity criteria that is not affected by breed or weight variables, thereby ensuring diagnostic reliability in clinical settings.

[0013] To solve the above problem, the present invention obtains leukocytes by removing red blood cells from a whole blood sample of a companion animal, and separates the obtained leukocytes based on the presence or absence of CD4 surface antigen expression to form a cell group containing CD4-positive polymorphonuclear leukocytes (PMN) and CD4-negative cytotoxic lymphocytes (CTL, CD8 +A method is provided for accommodating cell populations (including T cells and NK cells) in separate reaction vessels. The CD4-positive cell population is treated with a PMA monotherapy, and the CD4-negative cell population is treated with a composite composition (ASC-K9) containing PHA, PMA, and IL-2 to induce activation under different conditions, and a diffraction pattern image of each activated cell population is obtained using a lens-free shadow imaging device. Subsequently, eight individual parameters (CMV, PPD, MMD, MMD_SD, WCM, WCM_SD, WSM, WSM_SD) or combinations of individual parameters, such as the inter-peak distance (PPD) and the standard deviation of the fringe width (WSM_SD) of the cell diffraction pattern, are calculated from the obtained image, and a combined shadow parameter (CSP = PPD Х WSM_SD), defined as the product of these parameters, is derived to simultaneously determine the cell activity of the CD4-positive cell population and the CD4-negative cell population.

[0014] In addition, the present invention provides a technology for separating innate immune cell populations and adaptive immune cell populations with high purity based solely on CD4 surface antigens rather than physical morphology, by utilizing the species-specific immune characteristic that CD4-positive cell populations in certain companion animal species, including dogs, are composed mainly of polymorphonuclear leukocytes (PMNs) unlike humans. Furthermore, the invention induces degranulation and morphological changes of neutrophils by culturing CD4-positive cell populations at a PMA concentration range of 1 to 100 nM for 10 to 60 minutes, and induces the activation of CTLs by culturing CD4-negative cell populations with an ASC-K9 composition consisting of PHA 1 to 20 μg / ml, PMA 1 to 50 nM, and IL-2 100 to 2000 U / ml for 1 to 24 hours.

[0015] In addition, to acquire a diffraction pattern image, a blue LED light source in the 400–500 nm band is passed through a pinhole to form a point light source, and this is irradiated onto a reaction vessel containing cells to acquire a shadow image projected onto a CMOS image sensor without a lens. The calculation of CSP is performed by generating a radial brightness profile from the center of an individual cell image, calculating the distance between the central maximum brightness and the peripheral peak as PPD, calculating the standard deviation of the diffraction fringe width as WSM_SD, and then calculating the product of these two values ​​as CSP.

[0016] In addition, in the activity determination step, the difference between the average CSP value of the control group not treated with the activating substance and the average CSP value of the experimental group treated with the activating substance is calculated to determine the final immune activity value, and normality can be evaluated by applying a single standard range regardless of breed and body weight.

[0017] Furthermore, the present invention includes a method for providing immune information to a user by comparing the decrease in bacterial / viral infection and anticancer immune surveillance function with a reference value of the increase in CSP, and also provides an activation composition comprising PHA, PMA, and IL-2 to induce activation of CD4-negative lymphocytes.

[0018] In addition, the device of the present invention includes a fluid control module for performing pretreatment of a pet blood sample, an optical module for acquiring an image of the pretreated sample, and a processor for analyzing the acquired image to calculate a CSP value. The optical module includes an LED, a pinhole, a sample chamber having a transparent bottom surface, and an image sensor, and is configured to maintain a distance of 0.5 to 2 mm between the sample chamber and the image sensor so that a holographic pattern of cells is formed. The device may include an LED, a CMOS image sensor, a disposable multi-channel slide, and a drive motor for slide transport, and may be configured to sequentially measure multiple samples on the slide using a single sensor combination.

[0019] In addition, the immunodiagnostic kit of the present invention comprises an RBC Lysis Buffer, an anti-CD4 antibody attached to a PE for capturing CD4-positive cells, an antibody attached to the PE, a magnetic bead attached to the antibody, a first reagent for PMN activation, and a second reagent for CTL activation, and may be configured in the form of an assay slide that can be linked with an LSIT device.

[0020] According to the present invention, by lysing red blood cells from whole blood of companion animals to separate leukocytes and separating PMNs and CTLs with high purity based on the expression of CD4 surface antigens, core cell populations performing innate and adaptive immune functions can be independently secured. Furthermore, by applying different activation conditions, such as PMA alone to PMNs and PHA, PMA, and IL-2 combined treatment to CTLs, the intrinsic physiological response of the cells can be effectively induced.

[0021] In addition, using lens-free shadow imaging technology, changes in diffraction patterns resulting from cell activation can be rapidly observed in a label-free manner, and the activity of immune cells can be quantified by deriving CSP parameters based on eight individual parameters or combinations of individual parameters such as PPD and WSM_SD. This analytical method is faster and more direct than conventional CBC or ELISA-based evaluations, and offers high economic efficiency and potential for automation as it does not require expensive fluorescent labels or antibodies.

[0022] Furthermore, since CSP-based activity measurement enables the establishment of a single reference range unaffected by breed or body weight, it allows for the creation of a standardized immunity assessment system for all companion animals. This enables the comprehensive evaluation of defense capabilities against bacterial infections, responsiveness to viral infections, and anticancer immune surveillance functions, and can be utilized in clinical diagnosis, preventive medicine, vaccine efficacy verification, and the evaluation of immunomodulators.

[0023] Figure 1a is the result data obtained by confirming the purity of each separated cell group based on whether CD4 surface antigen is expressed, after removing red blood cells from canine whole blood according to one embodiment of the present invention, using a flow cytometer.

[0024] Figure 1b is a dot plot showing the cell composition ratios of polymorphonuclear leukocytes (PMN), monocytes, lymphocytes, etc. within each fraction by analyzing forward scatter (FSC) and side scatter (SSC) signals for the separated CD4-positive and CD4-negative cell groups.

[0025] Figure 2 is a flow cytometry result demonstrating that when activation is induced in a group of CD4-positive cells (PMN) isolated according to the method of the present invention by treating them with PMA, an activating substance, the FSC and SSC values ​​of the cells increase compared to the control group due to morphological changes resulting from the activation.

[0026] Figure 3 is a flow cytometry result showing that when activation is induced in CD4-negative cell populations (CTLs) isolated according to the method of the present invention by treating them with the activating composition ASC-K9 (containing PHA, PMA and IL-2), the average FSC and SSC values ​​of the activated cells are significantly increased compared to the control group.

[0027] Figure 4 is a graph showing that the change in diffraction pattern according to the activation status of PMN and CTL was measured using the lens-free shadow imaging (LSIT) system of the present invention, and that the combined shadow parameter (CSP) value, which is quantified therefrom, increased significantly compared to the control group.

[0028] Figure 5 is a graph comparing PMN activity and CTL activity between a large dog group (over 25 kg) and a small dog group (under 10 kg) by applying the analysis method of the present invention, showing that there is no statistically significant difference according to breed or body weight, and thus a unified diagnostic standard can be applied.

[0029] Figure 6 is a statistical distribution graph showing the standard reference range for PMN and CTL activity established by performing the analysis of the present invention on 40 clinically healthy normal dogs.

[0030] FIGS. 7A, 7B, and 7C are drawings illustrating a multi-sample automatic measuring device of a motor-based slide transfer method according to an embodiment of the present invention.

[0031] Hereinafter, specific details for implementing the present invention are described in detail with reference to preferred embodiments of the present invention. However, these embodiments are merely examples to aid in understanding the present invention and do not limit the present invention; the invention may be implemented in various modified forms without departing from the technical spirit and scope of the present invention.

[0032] Example 1: Immune cell isolation and purity verification

[0033] In this embodiment, as the first step of the analysis of companion animal immune cells according to the present invention, a specific cell population was isolated from the whole blood of a canine based on the CD4 surface antigen, and its purity was verified.

[0034]

[0035] 1. Cell separation process

[0036]

[0037] First, peripheral whole blood collected from experimental dogs was treated with RBC Lysis Buffer diluted at a 1:10 ratio to selectively remove red blood cells and obtain a leukocyte pellet. The obtained leukocyte population was treated with an anti-canine CD4 antibody conjugated with phycoerythrin (PE) fluorescence to label the expression of CD4 antigens on the cell surface. Subsequently, the CD4-positive cell group (CD4+) and the CD4-negative cell group (CD4-) were physically separated using magnetic force with a PE Positive Selection Kit (magnetic particle-based).

[0038]

[0039] 2. Analysis of results for Fig. 1a

[0040]

[0041] To verify the purity of each isolated cell population, the cell distribution according to fluorescence intensity was analyzed using a flow cytometer.

[0042] Figure 1a is the result data obtained by confirming the purity of each separated cell group based on whether CD4 surface antigen is expressed, after removing red blood cells from canine whole blood according to one embodiment of the present invention, using a flow cytometer.

[0043] Referring to Fig. 1a,

[0044] CD4 Positive Fraction: It was confirmed that more than 90% of the cells are concentrated in the region where the CD4-PE fluorescence signal is strongly detected (right peak). This means that the target cells of the present invention, polymorphonuclear leukocytes (PMN), were captured with high purity.

[0045] CD4 Negative Fraction: It was confirmed that more than 90% of the cells were distributed in the region where CD4-PE fluorescence signals were barely detected (left peak). This indicates that cytotoxic lymphocytes that do not express CD4 (including CTLs, CD8+ T cells, and NK cells) successfully remained.

[0046] Of particular note is that the results in Fig. 1a experimentally demonstrate the species-specific immunological characteristics of canines. Unlike human CD4-positive cells, which are typically helper T cells, this experiment reconfirmed that the CD4-positive cell population in canines is predominantly composed of granulocyte-type **polymorphonuclear leukocytes (PMNs)**. In other words, the high separation purity in Fig. 1a suggests that, beyond simply distinguishing between the presence or absence of CD4 antigens, it is possible to effectively separate PMN populations and CTL populations using only a single marker (CD4).

[0047] Therefore, the data in FIG. 1a proves that the analysis system of the present invention has secured a reliable input sample, and serves as basic data supporting that the activation experiment (PMA alone or ASC-K9 combined treatment) and LSIT analysis results performed in the embodiments to be described later accurately reflect the unique characteristics of each immune cell group.

[0048]

[0049] Example 2: Analysis of Cytological Characteristics and Composition Ratios of Isolated Cell Groups

[0050]

[0051] Figure 1b is a dot plot showing the cell composition ratios of polymorphonuclear leukocytes (PMN), monocytes, lymphocytes, etc. within each fraction by analyzing forward scatter (FSC) and side scatter (SSC) signals for the separated CD4-positive and CD4-negative cell groups.

[0052] In a flow cytometer, FSC is used as a signal reflecting cell size, and SSC is a signal indicating internal granularity or internal structural complexity. In one embodiment of the present invention, the two signals were utilized together to quantitatively compare the cell composition of CD4-positive cell groups and CD4-negative cell groups.

[0053] As shown in Figure 1b, the CD4-positive cell population was found to consist of, on average, about 76–85% polymorphonuclear cells (PMNs), i.e., neutrophils. This experimentally confirms the species-specific immunological characteristic of PMNs expressing CD4 surface antigens in canines, and stands in stark contrast to the fact that CD4-positive cells in humans are mainly composed of helper T cells.

[0054] In addition, the CD4-positive cell population contains a small amount of CD4 T cells in addition to PMN, and in this example, it was observed to be approximately 15% on average. This result is consistent with previous reports that PMN and specific lymphocyte subtypes express CD4 together in dogs.

[0055] In contrast, among the CD4-negative cell population, the lymphocyte population accounted for the largest proportion at 48–68%, suggesting that cytotoxic lymphocytes (CTLs), namely CD8 T cells and NK cells, are the main components. Additionally, monocytes were observed at a level of approximately 12–15% in the CD4-negative cell population. Although PMNs were present in some parts of the CD4-negative cell population, their proportion was significantly lower compared to the CD4-positive cell population (around 15%).

[0056] The results of this comparison of cell composition support the fact that the CD4-based separation technology of the present invention can separate PMNs and CTLs with high separation efficiency. Furthermore, it enhances the reliability of the results by clarifying the identity of the cell population used in the morphological changes (FSC / SSC changes) and lens-free shadow imaging (LSIT)-based activity analysis following PMA alone treatment (PMN activation) or ASC-K9 complex composition treatment (CTL activation) presented in subsequent examples.

[0057] Therefore, FIG. 1b serves as key data proving that the CD4 antigen-based separation method proposed in the present invention separates the innate immune cell population (PMN) and adaptive immune cell population (CTL) of companion animals with high purity and accuracy, respectively, and demonstrates that the prerequisites for the subsequent immune cell activation step and LSIT-based quantitative analysis step are correctly met. In other words, the results of FIG. 1b demonstrate that the CD4-based separation process of the present invention can effectively compartmentalize the PMN population (CD4-positive group) and the CTL population (CD4-negative group) so that they do not mix with each other. This high separation efficiency serves as the basis for independently applying a stimulus specialized for PMN (PMA alone) and a stimulus specialized for CTL (ASC-K9 complex composition) in the activation step described later, and ultimately guarantees the reliability that the activity measured through the LSIT device accurately represents the function of a specific cell group.

[0058] FIG. 1b visually and quantitatively supports the fact that the present invention is an optimal platform for diagnosing the immune system of companion animals, going beyond simply isolating cells.

[0059]

[0060] Example 3: Induction and Verification of Activation of CD4-Positive Cell Populations (PMN)

[0061]

[0062] Figure 2 is a flow cytometry result demonstrating that when activation is induced in a group of CD4-positive cells (PMN) isolated according to the method of the present invention by treating them with PMA, an activating substance, the FSC and SSC values ​​of the cells increase compared to the control group due to morphological changes resulting from the activation.

[0063] First, leukocytes were isolated from whole blood of dogs using RBC lysis buffer in the same manner as described in Fig. 1, and then labeled with PE-conjugated anti-CD4 antibody, and CD4-positive cell populations were isolated using a PE Positive Selection kit. The CD4 cell populations obtained in this way consisted mainly of PMNs and were used in the PMN activation experiment of the present invention.

[0064] Separated CD4 + PMN cells were stabilized in RPMI-1640 medium supplemented with 10% FBS (Fetal Bovine Serum) and 1% Penicillin / Streptomycin at 37°C in a 5% CO2 environment. Subsequently, the experimental group was treated with PMA at a concentration of 50 nM and cultured for 30 minutes, while the control group was treated with the same solvent but without PMA.

[0065] Observing the changes in FSC and SSC values ​​obtained via flow cytometry, cells activated by PMA showed the following characteristic changes compared to the control group.

[0066] FSC increase:

[0067] FSC is an indicator reflecting cell size, and it was found that cell size increased in PMA-treated PMNs. This suggests that changes in cell morphology, such as granule migration, cell expansion, or membrane activation within the cell, occurred due to PMA stimulation.

[0068] SSC increase:

[0069] SSC is a signal indicating the density or complexity of intracellular granules. The increase in SSC values ​​in the PMA-treated group is interpreted as being due to degranulation, internal structural rearrangement, or changes in the refractive index of granules, which are characteristic of the PMN activation process.

[0070] As shown in the graph in Figure 2, the PMA-treated group showed a statistically significant increase in both FSC and SSC compared to the control group. Accordingly, when cells are gated on the FSC-SSC plane, more cells in the PMA-treated group are located in the upper region corresponding to the activated state than in the lower region corresponding to the non-activated state, which is represented by an increase in the proportion belonging to the activated gate in Figure 2.

[0071] Specifically, in Figure 2, the proportion of cells included in the activation gate upon PMA treatment increased significantly compared to the control group, which is an important indicator confirming that PMA rapidly induces morphological and functional activation of PMN.

[0072] In addition, PMA is known to be a potent activator of protein kinase C (PKC), and in PMNs, it induces various activation responses through stimulation of the PKC pathway, such as increased interaction with the extracellular matrix, promotion of oxygen radical production (ROS generation), cell membrane activation, and granule release. The FSC / SSC increase pattern in Fig. 2 is a result of these physiological phenomena being reflected as optical scattering signals, and provides the basis for selecting PMA as the sole composition for PMN activation in the present invention.

[0073] Therefore, Figure 2 shows that the PMA-based PMN activation method of the present invention induces a distinct morphological change, which is important data supporting the correlation with the change in diffraction pattern through lens-free shadow imaging (LSIT) presented in Example 4.

[0074]

[0075] Example 4: Induction and Verification of Activation of CD4-Negative Cell Population (CTLs)

[0076]

[0077] Figure 3 is a flow cytometry result showing that when activation is induced in CD4-negative cell populations (CTLs) isolated according to the method of the present invention by treating them with the activating composition ASC-K9 (containing PHA, PMA and IL-2), the average FSC and SSC values ​​of the activated cells are significantly increased compared to the control group.

[0078] As previously described in Figure 1, leukocytes were isolated from whole blood of dogs after red blood cell removal (RBC lysis), and CD4-negative cell populations were isolated using the PE Positive Selection method after being fluorescently labeled with an anti-CD4 antibody conjugated with PE (Phycoerythrin) fluorescence. This CD4-negative population is a CTL population composed mainly of CD8 T cells and NK cells, and is a key cell population for evaluating the adaptive immune activity of the present invention.

[0079] The isolated CTL cell population was suspended in RPMI-1640 medium (10% FBS, 1% Penicillin / Streptomycin) and stabilized at 37°C under 5% CO2 conditions. Subsequently, the ASC-K9 composition, i.e.

[0080] Phytohemagglutinin (PHA) 10 μg / ml,

[0081] PMA (Phorbol 12-myristate 13-acetate) 10 nM,

[0082] IL-2 (Interleukin-2) 1000 U / ml

[0083] Cells were treated with a mixture containing [the substance] and cultured for a total of 18 hours to induce activation.

[0084] As a result of measuring FSC and SSC signals in a flow cytometer after activation, as can be seen in Figure 3, the experimental group treated with ASC-K9 showed a significant increase in both the average FSC and SSC values ​​compared to the control group (vehicle).

[0085] FSC increase (increase in cell size)

[0086] During the activation process, CTLs undergo changes such as cytoplasmic expansion, increased membrane protein expression, and rearrangement of internal granules. These structural changes lead to an increase in cell size, which is observed as an increase in FSC signaling.

[0087] In Figure 3, the average FSC value of the ASC-K9 treatment group increased significantly compared to the control group, showing that the CTL reached a distinctly activated state.

[0088] Increase in SSC (increase in internal granules and complexity)

[0089] SSC is an indicator that reflects the degree of granularity and optical complexity inside the cell.

[0090] When CTL is activated, the rearrangement and release preparation process of granules containing perforin and granzyme is promoted, which is manifested as an increase in SSC.

[0091] The significant increase in SSC values ​​compared to the control group in Figure 3 indicates that the CTL activating composition (ASC-K9) effectively induced functional activation of immune cells.

[0092] In particular, the graph in Figure 3 quantitatively presents the average value of each signal, clearly showing that CTL activation is not a simple trend but a statistically significant change.

[0093] The average FSC value of the control group was approximately 323.1 and the average SSC value was 158.5, whereas in the ASC-K9 treated group, FSC increased to 357.6 and SSC to 184.5. This magnitude of change is experimental evidence that reliably demonstrates that CTLs were activated by ASC-K9.

[0094] In addition, the ASC-K9 composition is characterized by inducing multi-pathway activation by simultaneously stimulating key signaling pathways (IL-2R / JAK-STAT, PKC pathway, lectin-mediated activation, etc.) for T cell and NK cell activation. This very effectively enhances the cytotoxic function of CTLs and is directly linked to the increase in FSC / SSC shown in Figure 3.

[0095] In conclusion, Figure 3 is a diagram that experimentally confirms that the CD4-negative cell population isolation and ASC-K9-based activation method proposed in the present invention can rapidly and stably induce functional activation of CTLs, and provides key basic data for changes in diffraction patterns (increase in CSP) through lens-free shadow imaging (LSIT) presented in subsequent examples.

[0096]

[0097] Example 5: Diffraction pattern analysis and CSP calculation using lens-free shadow imaging (LSIT)

[0098] Figure 4 is a graph showing that the change in diffraction pattern according to the activation status of PMN and CTL was measured using the lens-free shadow imaging (LSIT) system of the present invention, and that the combined shadow parameter (CSP) value, which quantifies this, increased significantly compared to the control group.

[0099] In this embodiment, PMN and CTL cell populations, whose activation was induced by PMA or ASC-K9 in FIG. 2 and FIG. 3, respectively, were injected into an LSIT device to acquire diffraction images. The LSIT system uses a blue light-emitting diode (LED) with a central wavelength of approximately 470 nm as a light source and passes the LED light through a micro pinhole to form a point light source. The light emitted from the point light source passes through a cell chip or sample chamber in which cells are placed, and then creates a shadow pattern in the form of a hologram of the cells on a CMOS image sensor without a lens.

[0100] The acquired LSIT images show clear structural differences depending on the activation state of the cells. Cells in an inactive state form a relatively uniform and low-contrast diffraction pattern, whereas activated cells show changes in the shape of the central maximum, the position of the rings, and the width and contrast of the secondary maxima due to increased cell size, internal granule rearrangement, and changes in refractive index.

[0101] In this invention, two parameters were calculated to quantitatively evaluate these diffraction pattern differences.

[0102] 1) PPD (Peak-to-Peak Distance)

[0103] It is a value measuring the distance between the central brightness peak and the subsequent peripheral peak (secondary maximum) in an individual cell image.

[0104] When PMNs or CTLs are activated, cell size increases and internal structure changes, causing the diffraction pattern to expand; consequently, PPD values ​​tend to increase.

[0105]

[0106] 2) WSM_SD(Standard Deviation of Width of Secondary Maxima)

[0107] It is a value calculated as the standard deviation of the variation in the width of secondary maxima (WSM) of the peripheral peaks.

[0108] In activated cells, the width of the peripheral diffraction pattern becomes irregular due to increased internal granule density and increased structural complexity, resulting in an increase in WSM_SD.

[0109]

[0110] 3) CSP(CSP = PPD Х WSM_SD)

[0111] The Combined Shadow Parameter, defined by multiplying PPD and WSM_SD, is an indicator that comprehensively reflects the effect of size and internal structure changes caused by cell activation on the diffraction pattern.

[0112]

[0113] As shown in Fig. 4,

[0114] In both PMN (PMA treatment group) and CTL (ASC-K9 treatment group), CSP values ​​significantly increased compared to the control group (vehicle).

[0115] In particular, CSPs showed very high sensitivity in distinguishing whether they were activated in both PMNs and CTLs, which demonstrates the technical feasibility of LSIT-based diffraction pattern analysis technology being able to quantify immune cell activity without fluorescent labels or antibody-based procedures (label-free).

[0116] In addition, although PMNs and CTLs have different physiological activation pathways, LSIT-based analysis confirmed that both cell populations exhibit the same pattern of increased CSP upon activation. This leads to the significant advantage that the comprehensive immune cell activity analysis of companion animals, including dogs, can be evaluated on a single technology platform according to the present invention.

[0117] Figure 4 is a key figure demonstrating that the LSIT-based activity measurement technology of the present invention can quantitatively capture optical changes resulting from morphological changes in PMNs and CTLs, and serves as the basis for establishing variety- and body weight-independent activity indicators and normal reference ranges presented in Examples 5 and 6.

[0118]

[0119] Example 6. Motor-based slide-transfer type multi-sample automatic measuring device

[0120]

[0121] FIGS. 7A, 7B, and 7C are drawings illustrating a multi-sample automatic measurement device of a motor-based slide transport method according to an embodiment of the present invention. As shown in FIGS. 7A, 7B, and 7C, a device according to another embodiment of the present invention is configured to sequentially pass in front of a single LED light source and a single CMOS image sensor using a driving motor that fixes a multi-channel slide including a plurality of sample chambers and transports the slide.

[0122] For example, an integrated slide with four sample channels arranged at regular intervals can be mounted, and each channel can be driven via a precision stepper motor to be accurately aligned to a measurement position (light source and sensor alignment position). When each position is reached, the slide is stopped to acquire a holographic image, and then the slide is moved to the next channel.

[0123] This configuration allows for the automatic processing of multiple samples while minimizing the number of sensors and light sources, thereby reducing power consumption and achieving device miniaturization and weight reduction. Accordingly, the device of this embodiment can be connected to mobile devices (e.g., tablets, laptops) via USB or wireless communication and has the advantage of being able to operate on a battery basis without an external power source.

[0124] In addition, this configuration is particularly suitable as a point-of-care (POC) device for analyzing immune cells in companion animals, and has the advantage of enabling continuous measurement of samples under various stimulation conditions (e.g., vehicles, ASC-K9, PMA-treated samples, etc.) in a single device.

[0125]

[0126] Example 7: Analysis of the correlation between immune cell activity according to pet breed and body weight

[0127]

[0128] Figure 5 is a graph comparing PMN activity and CTL activity between a large dog group (over 25 kg) and a small dog group (under 10 kg) by applying the analysis method of the present invention, showing that there is no statistically significant difference according to breed or body weight, and thus a unified diagnostic standard can be applied.

[0129] In this embodiment, a comparative experiment was conducted to determine whether the immune cell analysis method and CSP indicator according to the present invention are affected by the body size or breed of the pet, and to verify whether they can be utilized as universal diagnostic criteria.

[0130] Experimental Design: A total of 40 clinically healthy dogs were recruited and divided into two experimental groups based on body weight as follows.

[0131] Small Breed Group: 20 individuals weighing less than 10 kg (e.g., Maltese, Toy Poodle, Chihuahua, etc.)

[0132] Large Breed Group: 20 individuals weighing over 25 kg (e.g., Retrievers, German Shepherds, etc.)

[0133] For peripheral whole blood collected from each individual, the process of [erythrocyte hemolysis] -> [CD4-based cell separation] -> [activation induction (PMN: PMA treatment / CTL: ASC-K9 treatment)] -> [LSIT diffraction pattern analysis] was performed in the same manner as described in Figures 1 to 4 above, and finally, the CSP (Combined Shadow Parameter) values ​​of each cell group were calculated and compared.

[0134] Figure 5 is a graph comparing PMN activity and CTL activity between a large dog group (over 25 kg) and a small dog group (under 10 kg) by applying the analysis method of the present invention, showing that there is no statistically significant difference according to breed or body weight, and thus a unified diagnostic standard can be applied.

[0135] PMN Activity: Analysis of changes in PMN activity induced by PMA treatment showed that both the small dog and large dog groups exhibited a similar high increase in CSP compared to the control group. The difference in average activity between the two groups was not statistically significant (p > 0.05), indicating that there is almost no difference in neutrophil responsiveness due to body size differences.

[0136] CTL activity: No significant difference was observed between the two groups in the change in CTL activity following ASC-K9 treatment. This demonstrates that micro-optical changes accompanying cell activation, such as physical size increase (PPD) or internal granule rearrangement (WSM_SD), are unique physiological phenomena at the cellular level independent of the individual's body weight.

[0137] The experimental results of Figure 5 suggest that the technology of the present invention has the following differentiated advantages compared to existing diagnostic methods.

[0138] First, there is the independence and objectivity of the activity indicator. The normal reference range for white blood cell counts in a general blood test (CBC) varies depending on age, breed, and body weight, requiring a complex correction process. However, since the CSP value of the present invention has been confirmed to be an independent indicator not affected by body weight or breed variables, it is possible to objectively evaluate immunity without being constrained by individual characteristics.

[0139] Second is the possibility of establishing a standardized Universal Reference Range. The absence of variation between breeds means that it is possible to set a 'single standard reference value' applicable to all companion dogs. This presents a significant technical advantage, enabling the provision of standardized protocols without complex considerations when performing tasks such as evaluating immunity formation after vaccination, screening for immune decline in senior dogs, or assessing immune surveillance functions during anticancer treatment at veterinary clinics and diagnostic sites.

[0140] Figure 5 is key data proving that the immune cell analysis technology of the present invention is a universal platform technology capable of overcoming breed specificity and providing standardized immune diagnostic indicators in the pet medical market where various breeds coexist.

[0141]

[0142] Example 8: Setting the Reference Range for Immune Activity Using a Population of Normal Dogs

[0143]

[0144] In this embodiment, to verify whether the immune cell analysis method of the present invention can be utilized as a diagnostic indicator in actual clinical settings, PMN and CTL activities were measured on a statistically significant number of normal pet dogs, and a standardized normal reference range was established based on this.

[0145]

[0146] Subjects and Selection Criteria: A total of 40 companion dogs with verified clinical health status were selected for the experimental group. All subjects were limited to those aged 2 to 8 years, with no medical history, and included in the experimental group only if they showed normal findings in physical examination and basic blood tests (CBC and Chem10 serum chemistry tests).

[0147]

[0148] After collecting peripheral whole blood from each of the 40 selected individuals, the standard operating procedure established in Examples 1 to 5 was applied in the same way. That is, the process of [erythrocyte hemolysis and leukocyte acquisition] -> [CD4-based PMN / CTL separation] -> [activation induction (PMN: PMA / CTL: ASC-K9)] -> [LSIT-based CSP calculation] was followed to derive the change in PMN and CTL activity (△CSP = Activated CSP / Control CSP) for each individual.

[0149]

[0150] Figure 6 is a statistical distribution graph showing the standard reference range for PMN and CTL activity established by performing the analysis of the present invention on 40 clinically healthy normal dogs.

[0151] PMN activity: Analysis results confirmed that the increase in CSP following PMA treatment was concentrated within a relatively narrow distribution range in all 40 individuals. The interquartile range (IQR) based on the median was narrow, suggesting that the basal activity level of the innate immune response remains constant among healthy individuals without significant variation.

[0152] CTL activity: CTL activity also showed a stable distribution pattern without extreme outliers. Although lymphocyte activity is generally known to be highly variable due to age or environmental factors, the LSIT-based CSP analysis method of the present invention directly measures minute physical changes (size and internal structure) of cells, and thus it was confirmed to be robust against biological noise between individuals compared to existing cytokine concentration measurement methods.

[0153] Based on the data in Fig. 6, the present invention applied statistical techniques (including a median-based 95% confidence interval) to establish a cut-off value for determining whether the pet's immunity is 'normal'.

[0154] Clinical Utility: The established reference range can be used as a single standard that does not require correction based on breed or body weight. In clinical practice, if a patient's CSP measurement falls below the lower limit of this range, an immediate diagnosis can be made as 'impaired ability to defend against bacterial infection (PMN dysfunction)' or 'impaired ability to monitor viral / tumor immunity (CTL dysfunction),' respectively.

[0155] The results demonstrate that the analysis platform of the present invention secures high reproducibility and objectivity required of a clinical diagnostic device, even though it is performed in a label-free manner without fluorescent labels or expensive reagents.

[0156]

[0157] Although the embodiments have been described by means of limited embodiments and drawings, those skilled in the art can make various modifications and variations from the above description. For example, suitable results may be achieved even if the described techniques are performed in a different order than described, and / or the components of the described system, structure, device, circuit, etc. are combined or assembled in a form different from the described method, or are replaced or substituted by other components or equivalents.

[0158] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims set forth below.

Claims

1. A step of obtaining white blood cells by removing red blood cells from a whole blood sample of a companion animal; A step of separating the above-mentioned leukocytes based on the presence or absence of CD4 (Cluster of Differentiation 4) surface antigen expression, dividing them into a group of CD4-positive (CD4+) polymorphonuclear leukocytes (PMN) and a group of CD4-negative (CD4-) cytotoxic lymphocytes (CTL), and placing each into a separate reaction vessel; A step of inducing activation under different conditions by treating the CD4-positive cell group with a PMA (Phorbol 12-myristate 13-acetate) monotherapy and treating the CD4-negative cell group with a composite composition (ASC-K9) containing PHA (Phytohemagglutinin), PMA, and IL-2 (Interleukin-2); A step of acquiring a diffraction pattern image of each activated cell group using a Lens-free Shadow Imaging Technology (LSIT) device; and a step of calculating the inter-peak distance (PPD) and the standard deviation of the fringe width (WSM_SD) of the cell diffraction fringe from the acquired image, and deriving the combined shadow parameter (CSP = PPD × WSM_SD), which is the product thereof, to simultaneously determine innate immune activity through the CD4-positive cell group and adaptive immune activity through the CD4-negative cell group; A method for analyzing animal immune cells including 2. In Paragraph 1, The above separation step utilizes the species-specific characteristic that, unlike human CD4-positive cells which are primarily helper T cells, CD4-positive cells in certain companion animals, including canines, are predominantly PMNs, thereby separating the CD4-positive PMN-containing cell group from the CD4-negative cytotoxic lymphocytes (CTLs, CD8 + A method for analyzing immune cells in companion animals, characterized by selectively isolating cell populations containing T cells and NK cells.

3. In Paragraph 1, A method for analyzing animal immune cells, characterized in that the step of inducing activation of the above CD4 positive cell population (PMN) involves treating with PMA at a concentration of 1 to 100 nM and culturing for 10 to 60 minutes to induce degranulation and morphological changes of neutrophils.

4. A method for analyzing animal immune cells according to claim 1, wherein the composite composition (ASC-K9) comprises 1 to 20 ug / ml of PHA, 1 to 50 nM of PMA, and 100 to 2000 U / ml of IL-2, and wherein the CD4 negative cell population (CTL) is cultured with the composite composition for 1 to 24 hours.

5. In Paragraph 1, The step of acquiring the above diffraction pattern image is, A method for analyzing immune cells in companion animals, characterized by passing a blue LED light source in the wavelength band of 400nm to 500nm through a pinhole to form a point source, and irradiating the point source onto a reaction vessel containing the cell group to acquire a shadow image of the cells projected onto a CMOS image sensor without a lens.

6. In Paragraph 1, The step of deriving the combined shadow parameter (CSP) above comprises the step of generating a radial profile from the center of an individual cell image; A step of calculating PPD by measuring the distance between the central maximum brightness point and the secondary maximum appearing after the first dark ring in the above profile; a step of calculating WSM_SD by calculating the standard deviation for the diffraction fringe width of the above profile; and A step of calculating a CSP value proportional to cell activity by multiplying the above PPD value and WSM_SD value; A method for analyzing animal immune cells including 7. In Paragraph 1, The step of determining the above activity level is, A method for analyzing immune cells in a pet, characterized by calculating the difference between the average CSP value of a control group (Vehicle) not treated with an activating substance and the average CSP value of an experimental group treated with an activating substance, and determining this as the final immune activity value of the pet.

8. In Paragraph 7, A method for analyzing immune cells in a pet, characterized in that the above-mentioned final immune activity value is determined to be normal within a single reference range without correction based on the pet's weight or breed.

9. A method for providing information using activity measured according to the method of claim 1, wherein if the increase in CSP of the CD4 positive cell group is below a reference value, it is determined that the defense ability against bacterial infection is reduced, and if it exceeds the reference value, it is determined that the state is hyperactive against bacterial infection or acute inflammatory response; and if the increase in CSP of the CD4 negative cell group is below a reference value, it is determined that the defense ability against viral infection or anticancer immune surveillance ability is reduced, and if it exceeds the reference value, it is determined that the cytotoxic immune response to viral stimulation or tumor-associated antigen stimulation is increased, and the immune information of the pet according to the determination result is output to the user.

10. An immune cell activation composition for inducing activation of lymphocytes derived from companion animals, comprising phytohemagglutinin (PHA), phorbol myristate acetate (PMA), and interleukin-2 (IL-2) as active ingredients, and inducing specific activation of CD4-negative lymphocytes (CD4- Lymphocytes) of companion animals to promote cell morphological changes and internal granule migration.

11. An optical module for acquiring an image of a pre-processed sample; and A processor that analyzes acquired images; A pet immune cell analysis device comprising: an optical module that acquires diffraction images of cells within each chamber using a lens-free shadow imaging (LSIT) method; and a processor configured to calculate a Combined Shadow Parameter (CSP) according to the method described in claim 1.

12. In Paragraph 11, The above optical module includes an LED that emits light; A pinhole positioned on the light path of the above LED to filter light; A sample chamber having a transparent bottom surface for accommodating cells; and An image sensor positioned in close contact with the bottom of the sample chamber; A pet immune cell analysis device comprising, wherein the distance between the sample chamber and the image sensor is maintained at 0.5 mm to 2 mm to form a holographic pattern of cells.

13. In Paragraph 12, It includes an integrated slide comprising a plurality of sample channels, a driving motor for transporting the slide, and a single light source and a single image sensor for sequentially acquiring images corresponding to each sample channel of the slide. A pet immune cell analysis device for measuring the immune cell activity of pets.

14. A kit for diagnosing immune activity in companion animals, comprising: magnetic beads or well plates to which CD4 antibodies are attached to capture CD4-positive cells in the whole blood of companion animals; a first reagent for activating polymorphonuclear leukocytes (PMNs) (containing PMA); and a second reagent for activating cytotoxic lymphocytes (CTLs) (containing PHA, PMA, and IL-2).

15. In Paragraph 14, A pet immunity diagnostic kit comprising a tube or cartridge form applicable to a lens-free shadow imaging device, and further including an instruction manual instructing to determine immunity using the CSP (Combined Shadow Parameter) value, which is the amount of change in the diffraction pattern of cells generated after treatment with the first and second reagents.

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