DEVICE OR METHOD FOR DETECTION OF LEUKOCYTE IN DISEASE STATE OR FOR DIAGNOSIS OF LEUKOCYTE-RELATED DISEASE

Abstract

Provided is a device or method for detection of leukocytes in a disease state or leukocytes in an abnormal state, or diagnosing a leukocyte-related disease, according to the device or method according to an aspect, it is possible to detect leukocytes in a disease state or leukocytes in an abnormal state at an early stage using a small amount of sample isolated from a subject, and thus, there is an effect that allows diagnosis of a leukocyte-related disease, for example, inflammation, an infectious disease, an immune disease, a metabolic disease, or cancer, etc.

Claims

1. A method of detecting leukocytes in a disease state, comprising: contacting an isolated biological sample including leukocytes, or leukocytes isolated from the biological sample with leukocyte extravasation factors to capture leukocytes in a disease state in the sample by the leukocyte extravasation factors; and detecting the captured leucocytes.

2. The method of claim 1, wherein the leukocyte extravasation factors are immobilized on a wall of a channel, a surface of a particle, at least a portion of a vessel, or at least a portion of a well.

3. The method of claim 1 or 2, comprising counting a total number of leukocytes per unit sample volume in an isolated biological sample including the leukocytes, or isolating the leukocytes from the isolated biological sample including the leukocytes and counting the isolated leukocytes.

4. The method of claim 3, wherein the detecting comprises analyzing a ratio of the number of leukocytes captured by the leukocyte extravasation factors to the total number of counted leukocytes per unit sample volume, or analyzing the number of leukocytes captured by the leukocyte extravasation factors among the number of the isolated leukocytes.

5. The method of claim 1, wherein the leukocyte extravasation factors are at least one factor selected from the group consisting of selectins, CD34, intercellular adhesion molecule-1 (ICAM-1), soluble ICAM-1, ICAM-2, soluble ICAM-2, glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1), mucosal vascular addressin cell adhesion molecule 1 (MadCAM-1), platelet/endothelial-cell-adhesion molecule (PECAM-1), junctional adhesion molecule A (JAM-A), JAM-B, JAM-C, endothelial cell-selective adhesion molecule (ESAM), vascular cell-adhesion molecule 1 (VCAM-1), cluster of differentiation 99 (CD99), integrins, extracellular domains thereof, cells including the same, membranes of cells including the same, and combinations thereof.

6. The method of claim 5, wherein the selectins are P-selectin, E-selectin, or a combination thereof.

7. The method of claim 2, wherein the leukocyte extravasation factors are immobilized to the channel, particle, vessel, or well by an immobilizing compound or a linker.

8. The method of claim 1, wherein the leukocytes in a disease state or a cell population of the leukocytes in a disease state have increased or decreased binding capacity with leukocyte extravasation factors, compared to leukocytes in a normal state or a cell population of the leukocytes in a normal state.

9. The method of claim 8, wherein the leukocytes in a disease state have increased or decreased expression or activity of factors capable of binding to leukocyte extravasation factors, compared to leukocytes in a normal state, or the cell population of the leukocytes in a disease state have increased or decreased expression or activity of factors capable of binding to leukocyte extravasation factors, compared to a cell population of the leukocytes in a normal state.

10. The method of claim 9, wherein the factors capable of binding to the leukocyte extravasation factors are at least one selected from the group consisting of sialylated carbohydrates, L-selectin, P-selectin glycoprotein ligand 1 (PSGL-1), and leukocyte function-associated antigen 1 (LFA-1), macrophage-1 antigen (Mac-1; integrin alpha M), VLA-4, CD24, CD44, and E-selectin ligand 1 (ESL-1).

11. The method of claim 1, wherein the disease state is inflammation, an infectious disease, an immune disease, cancer, or cancer metastasis.

12. The method of claim 11, wherein the infectious disease is systemic or local infections of viruses, bacteria, mold, or fungi, or sepsis, bacteremia, or viremia.

13. The method of claim 1, wherein the detecting is detecting by imaging the captured leukocytes, detecting by fluorescence staining the captured leukocytes, measuring isolated leukocyte lysates by lysing the captured leukocytes, or detecting by attaching a detectable label to the leukocytes or the leukocyte extravasation factors.

14. A method of providing information on diagnosis of disease related to leukocyte in the disease state, comprising: contacting an isolated biological sample including leukocytes, or leukocytes isolated from the biological sample with leukocyte extravasation factors to capture leukocytes in a disease state in the sample by the leukocyte extravasation factors; and detecting the captured leukocytes.

15. The method of claim 14, wherein the leukocyte extravasation factors are immobilized on a wall of a channel, a surface of a particle, at least a portion of a vessel, or at least a portion of a well.

16. The method of claim 14, wherein the leukocyte extravasation factors are at least one factor selected from the group consisting of selectins, CD34, intercellular adhesion molecule-1 (ICAM-1), soluble ICAM-1, ICAM-2, soluble ICAM-2, glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1), mucosal vascular addressin cell adhesion molecule 1 (MadCAM-1), platelet/endothelial-cell-adhesion molecule (PECAM-1), junctional adhesion molecule A (JAM-A), JAM-B, JAM-C, endothelial cell-selective adhesion molecule (ESAM), vascular cell-adhesion molecule 1 (VCAM-1), cluster of differentiation 99 (CD99), integrins, extracellular domains thereof, cells the same, membranes of cells including the same, and combinations thereof.

17. The method of claim 16, wherein the selectins are P-selectin, E-selectin, or a combination thereof.

18. The method of claim 14, wherein the disease related to leukocyte is inflammation, an infectious disease, an immune disease, cancer, or cancer metastasis.

19. The method of claim 18, wherein the infectious disease is systemic or local infections of viruses, bacteria, mold, or fungi, or sepsis, bacteremia, or viremia.

20. A device for detecting leukocytes in a disease state, comprising a detector for detecting leukocytes in a disease state, comprising a channel, a particle, a vessel, or a well, in which leukocyte extravasation factors are immobilized on a wall of the channel, a surface of the particle, at least a portion of the vessel, or at least a portion of the well, wherein leukocytes in a disease state in an isolated biological sample are captured by the leukocyte extravasation factors and detected.

21. The device of claim 20, wherein the leukocyte extravasation factors are at least one factor selected from the group consisting of selectins, CD34, intercellular adhesion molecule-1 (ICAM-1), soluble ICAM-1, ICAM-2, soluble ICAM-2, glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1), mucosal vascular addressin cell adhesion molecule 1 (MadCAM-1), platelet/endothelial-cell-adhesion molecule (PECAM-1), junctional adhesion molecule A (JAM-A), JAM-B, JAM-C, endothelial cell-selective adhesion molecule (ESAM), vascular cell-adhesion molecule 1 (VCAM-1), cluster of differentiation 99 (CD99), integrins, cells including at least one thereof and membranes of the cells, extracellular domains thereof, and combinations thereof.

22. The device of claim 21, wherein the selectins are P-selectin, E-selectin, or a combination thereof.

23. The device of claim 20, wherein the leukocyte extravasation factors are immobilized to the channel, particle, vessel, or well by an immobilizing compound or a linker.

24. The device of claim 20, wherein the leukocytes in a disease state or a cell population of the leukocytes in a disease state have increased or decreased binding capacity with leukocyte extravasation factors, compared to leukocytes in a normal state or a cell population of the leukocytes in a normal state.

25. The device of claim 24, wherein the leukocytes in a disease state have increased or decreased expression or activity of factors capable of binding to leukocyte extravasation factors, compared to leukocytes in a normal state, or the cell population of the leukocytes in a disease state have increased or decreased expression or activity of factors capable of binding to leukocyte extravasation factors, compared to a cell population of leukocytes in a normal state.

26. The device of claim 25, wherein the factors capable of binding to the leukocyte extravasation factors are at least one selected from the group consisting of sialylated carbohydrates, L-selectin, P-selectin glycoprotein ligand 1 (PSGL-1), and leukocyte function-associated antigen 1 (LFA-1), macrophage-1 antigen (Mac-1; integrin alpha M), VLA-4, CD24, CD44, and E-selectin ligand 1 (ESL-1).

27. The device of claim 20, wherein the disease state is inflammation, an infectious disease, an immune disease, cancer, or cancer metastasis.

28. The device of claim 27, wherein the infectious disease is systemic or local infections of viruses, bacteria, mold, or fungi, or sepsis, bacteremia, or viremia.

29. The device of claim 20, wherein the detection is detecting by imaging the captured leukocytes, detecting by fluorescence staining the captured leukocytes, measuring isolated leukocyte lysates by lysing the captured leukocytes, or detecting by attaching a detectable label to the leukocytes or leukocyte extravasation factors.

30. A device for diagnosing a leukocyte-related disease, comprising a detector for detecting leukocytes in a disease state, comprising a channel, a particle, a vessel, or a well, in which leukocyte extravasation factors are immobilized on a wall of the channel, a surface of the particle, at least a portion of the vessel, or at least a portion of the well, wherein the leukocytes in a disease state in an isolated biological sample are captured by the leukocyte extravasation factors and detected.

31. The device of claim 30, wherein the leukocyte extravasation factors are at least one factor selected from the group consisting of selectins, CD34, intercellular adhesion molecule-1 (ICAM-1), soluble ICAM-1, ICAM-2, soluble ICAM-2, glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1), mucosal vascular addressin cell adhesion molecule 1 (MadCAM-1), platelet/endothelial-cell-adhesion molecule (PECAM-1), junctional adhesion molecule A (JAM-A), JAM-B, JAM-C, endothelial cell-selective adhesion molecule (ESAM), vascular cell-adhesion molecule 1 (VCAM-1), cluster of differentiation 99 (CD99), integrins, cells including at least one thereof and membranes of the cells, extracellular domains thereof, and combinations thereof.

32. The device of claim 31, wherein the selectins are P-selectin, E-selectin, or a combination thereof.

33. The device of claim 30, wherein the leukocyte-related disease is inflammation, an infectious disease, an immune disease, a metabolic disease, cancer, or cancer metastasis.

34. The device of claim 33, wherein the infectious disease is systemic or local infections of viruses, bacteria, mold, or fungi, or sepsis, bacteremia, or viremia.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0069] FIG. 1 shows a diagram schematically illustrating a difference between a surface receptor of a leukocyte in a normal state and a surface receptor of a leukocyte in a disease state, according to an embodiment.

[0070] FIG. 2 shows a diagram schematically showing a principle of a method and a device for detecting leukocytes in a disease state, according to an embodiment.

[0071] FIG. 3 shows a graph showing degrees of adhesion of leukocytes in a normal state and leukocytes in a sepsis state to a channel, by using a leukocyte extravasation factor, according to an embodiment.

[0072] FIG. 4 shows an image showing the results of fluorescence staining of leukocytes in a normal state or a sepsis state, attached to a channel by using leukocyte extravasation factors, according to an embodiment.

[0073] FIG. 5 shows a graph showing average expression levels of PSGL-1 protein of leukocytes in a normal state or a sepsis state, according to an embodiment.

[0074] FIG. 6 shows a graph showing (number of leukocytes attached to the channel)/(total number of leukocytes in the sample) % of leukocytes in a normal state or a sepsis state, according to an embodiment.

[0075] FIG. 7 shows results showing (number of leukocytes attached to the channel)/(total number of leukocytes in the sample) % of leukocytes in a normal state or a sepsis state, according to an embodiment.

[0076] FIG. 8 shows an image showing results of fluorescence staining of leukocytes in a normal state or a cancer state, which are attached to a channel by using leukocyte extravasation factors, according to an embodiment.

[0077] FIG. 9 shows results showing (number of leukocytes attached to the channel)/(total number of leukocytes in the sample) % of leukocytes in a normal state or a cancer state, according to an embodiment.

[0078] FIG. 10A shows results showing the time-dependent change of tumor size after inoculation of cancer cells in a mouse tumor model.

[0079] FIG. 10B shows results showing the time-dependent change of tumor weight after inoculation of cancer cells in a mouse tumor model.

[0080] FIG.11 shows results of counting leukocytes in a normal state or a cancer state, which are attached to a channel by using leukocyte extravasation factors, according to an embodiment.

[0081] FIG. 12 shows results showing the proportion of neutrophils among leukocytes attached to a channel by using leukocyte extravasation factors, according to an embodiment.

[0082] FIG. 13A shows a photograph of leukocytes attached to a channel by using leukocyte extravasation factors, imaged with a fluorescence microscope, according to an embodiment.

[0083] FIG. 13B shows a photograph of leukocytes attached to the same channel as in FIG. 13A, imaged under a microscope on a Bright field (BF).

[0084] FIG. 13C is a photograph (ImageJ, USA), in which the number of cells was automatically counted by using the image taken under a microscope on the BF of FIG. 13B.

[0085] FIG. 13D shows a graph showing the difference between counting leukocytes attached to a channel by using leukocyte extravasation factors in a fluorescence image (CT) and a Bright field (BF) image, according to an embodiment.

MODE OF DISCLOSURE

[0086] Hereinafter, the present disclosure will be described in more detail through examples. However, these examples are intended to illustrate the present disclosure, and the scope of the present disclosure is not limited to these examples.

EXAMPLE 1

Preparation of Microfluidic Chip Including Microfluidic Channel for Detecting Leukocytes in a Disease State

[0087] In order to detect leukocytes in a disease state, a microfluidic chip including a microfluidic channel coated with leukocyte extravasation factors was prepared.

[0088] Specifically, polydimethylsiloxane (PDMS) including a surface having a pattern of a channel (width: about 400 μm, height: about 100 μm, length: about 27 mm) was prepared. The surface of the PDMS having the pattern of a channel was treated with air plasma and treated with about 10% 3-aminopropyltriethoxysilane (APTES) dissolved in about 99.9% ethanol. Thereafter, the surface of PDMS treated with APTES was bonded to a glass slide (LumiNano, Korea), in which aldehyde groups were activated, and reacted at about 37° C. for about 5 hours to prepare a microfluidic chip including microfluidic channels.

[0089] Then, the inside of the microfluidic channel was coated with leukocyte extravasation factors. Specifically, ICAM-1+E-selectin; and 1×PBS (pH 7.4) including ICAM-1+E-selectin+P-selectin (each about 5 μg/ml) were injected through the inlet to the microfluidic channel at a rate of about 10 μL/min for about 2 to 4 minutes. In this state, after stopping the operation of the micropump controlling the flow of the fluid, the liquid injected into the microfluidic channel was left for about 30 minutes at room temperature to induce the proteins to attach to the aldehyde group. After that, 1×PBS (pH 7.4) including about 3% bovine serum albumin was injected into the channel at a flow rate of about 10 μL/min for about 4 minutes, and after stopping the micropump, the liquid was left in the channel for about 1 hour, to undergo a blocking process to prevent non-specific reactions, and 1×PBS (pH 7.4) was injected into the channel at a flow rate of about 10 μL/min for about 4 minutes to wash.

[0090] As a result, a microfluidic chip including a microfluidic channel coated with leukocyte extravasation factors was obtained. Treatment of all samples were processed through the inlet and outlet provided in the microfluidic chip.

EXPERIMENTAL EXAMPLE 1

Detection of Leukocytes in Sepsis State Using Leukocyte Extravasation Factors

[0091] In order to detect leukocytes in a sepsis state by using leukocyte extravasation factors, the following experiment was performed.

[0092] First, sepsis was induced by intraperitoneally injecting E. coli K12 (about 10.sup.8 CFU/1 mL physiological saline) into 9-week-old Wistar male rats.

[0093] Then, about 50 μL of the blood of prepared sepsis-induced rat or normal rat was collected, mixed with ACK lysis buffer at a ratio of about 1:20, and reacted at room temperature for about 5 minutes, and then centrifuged to isolate the leukocytes. The isolated leukocytes were washed with 1×PBS, and then diluted in about 100 μL of 1×PBS to prepare a biological sample including leukocytes. In the same manner as in Example 1, a microfluidic chip including a microfluidic channel in which rat ICAM-1, rat P-selectin, and rat E-selectin, etc. were immobilized was prepared (each using a solution at a concentration of about 5 μg/ml), and after injecting and flowing the biological sample into the inlet of the prepared channel for about 10 minutes at a rate of about 8 μl/min, the channel was washed for about 4 minutes by injecting 1×PBS into the channel at a flow rate of about 8 μl/min, and this was repeated about two more times, in order to remove the leukocytes not attached to the channel. Then, in order to stain the leukocytes attached or trapped in the microfluidic channel, the inside of the channel was filled with dyes such as Hoechst and Cell tracker, and incubated at room temperature for about 20 minutes, and the channel was washed for about 4 minutes by injecting 1×PBS into the channel at a flow rate of about 8 μl/min. After that, the inside of each channel was photographed with a fluorescence microscope, and images inside each channel to which leukocytes were attached were captured, and the total number of leukocytes captured in the channel was counted.

[0094] About 50 μL of blood was separately collected from sepsis-induced or normal rats prepared for total leukocyte count analysis of the sample, and leukocytes were isolated in the same manner as described above, and the isolated leukocytes were diluted in about 100 μL of 1×PBS to prepare a biological sample, and the total number of leukocytes was counted by using a hemocytometer after staining with CellTracker, DAPI, or Hoechst.

[0095] Thereafter, ratios of leukocytes captured in each channel of the control group (normal rat) and the sepsis group were compared using (leukocytes attached to the channel)/(number of total leukocytes in the sample) (%), and the results are shown in FIG. 3.

[0096] As shown in FIG. 3, as a result of immobilizing leukocyte extravasation factors according to an embodiment to each channel, and capturing leukocytes in a sepsis state (leukocytes of a sepsis rat) and leukocytes in a normal state (leukocytes of a normal rat) in each channel, more leukocytes in a sepsis state were attached to the channel than the leukocytes in a normal state.

[0097] In addition, the cells stained with CellTracker in a channel in which ICAM-1+E-selectin+P-selectin, etc were immobilized were photographed with a fluorescence microscope, and the results are shown in FIG. 4.

[0098] As shown in FIG. 4, as a result of staining the leukocytes attached to each channel with CellTracker, more leukocytes in a sepsis state were attached to the channels than leukocytes in the normal control group.

[0099] These results indicate that, leukocytes in a disease state or leukocytes in an abnormal state have increased expression of factors related to leukocyte extravasation (for example, factors capable of binding to leukocyte extravasation factors), or in the subject having a disease, the number of leukocytes with an increased expression level of the factor increases. And these results indicate that, when the expression of the factors related to leukocyte extravasation are increased, the leukocytes in a disease state or leukocytes in an abnormal state become a state capable of binding more strongly to the leukocyte extravasation factors compared with normal leukocytes, and using such characteristics of leukocytes, by contacting leukocyte extravasation factors immobilized on the channel, particle, vessel, or well, with the leukocytes isolated form the subject, leukocytes in a disease state or leukocytes in an abnormal state may be detected.

EXPERIMENTAL EXAMPLE 2

Identification of Expression Factors of Leukocytes in Disease State

[0100] It was confirmed whether the expression of proteins interacting with leukocyte extravasation factors was actually increased in leukocytes in a disease state.

[0101] Specifically, in the same manner as in Experimental Example 1, leukocytes were isolated from sepsis-induced rats and normal rats, and the leukocytes were fluorescence stained. Average expression levels of PSGL-1 of the isolated leukocytes were compared by measuring the fluorescence intensity at the single cell level, and the results are shown in FIG. 5.

[0102] As shown in FIG. 5, it was confirmed that the expression of PSGL-1 in leukocytes of sepsis-induced rats was increased by about two times compared to leukocytes of normal rats.

[0103] These results indicate that, expression of factors interacting with leukocyte extravasation factors increases in leukocytes in a disease state or leukocytes in an abnormal state, and by using the leukocyte extravasation factors according to an embodiment, it is possible to diagnose a disease that increases expression of factors related to the surface of a leukocyte, such as sepsis.

EXPERIMENTAL EXAMPLE 3

Early Detection of Sepsis by Using Leukocyte Extravasation Factors

[0104] It was confirmed whether sepsis may be diagnosed early by using leukocyte extravasation factors.

[0105] Specifically, in the same manner as in Experimental Example 1, (number of leukocytes attached to the channel)/(total number of leukocytes in the sample) % was measured over time after bacterial inoculation, and the results are shown in FIG. 6.

[0106] As shown in FIG. 6, it was found that the numerical values of the sepsis group and the control group were significantly different even after about 1 hour after bacterial inoculation.

[0107] These results indicate that sepsis may be detected early even with a very small sample by using the leukocyte extravasation factors according to an embodiment.

EXPERIMENTAL EXAMPLE 4

Diagnosis of Infectious Disease or Inflammation Induced by Various Infectious Agents Using Leukocyte Extravasation Factors

[0108] It was confirmed whether an infectious disease or inflammation caused by various infectious agents may be diagnosed by using leukocyte extravasation factors.

[0109] First, 1 mL of physiological saline (sham control), E. coli K12 (about 10.sup.8 CFU/1 mL physiological saline), lipopolysaccharide (LPS; about 5 mg/kg), and methicillin-resistant Staphylococcus aureus (MRSA; about 10.sup.8 CFU/1 mL physiological saline) were respectively intraperitoneally injected into 8-week-old male Wistar rats, and the rats were bred for about 4 hours to prepare rats with infectious diseases or inflammation induced by various sepsis-inducing substances and bacteria.

[0110] In the same manner as in Example 1, a microfluidic chip including a microfluidic channel in which rat ICAM-1, rat P-selectin, and rat E-selectin, etc. were immobilized was prepared (each using a solution at a concentration of about 5 μg/ml), and in the same manner as in Experimental Example 1, after injecting the leukocytes isolated from the rat into the microfluidic channel, (number of leukocytes attached to the channel)/(total number of leukocytes in the sample) % was measured, and the results are shown in FIG. 7.

[0111] As shown in FIG. 7, as a result of using the leukocytes of the sham control group, and the leukocytes in a sepsis state caused by various causes, and the leukocytes in an inflammatory state, in the channel injected with leukocytes of the sham control group (rats in which only physiological saline was injected intraperitoneally) and the control group (normal rats), there was no difference in the percentage of leukocytes attached to each channel, but it was found that the number of leukocytes attached to the channel was remarkably increased in the channel into which the leukocytes of the experimental group with infectious diseases or inflammation induced by various infectious agents such as E. coli, MRSA, and LPS were injected.

[0112] These results indicate that by detecting leukocytes in a state with various infectious diseases or inflammation, by using leukocyte extravasation factors according to an embodiment, it is possible to diagnose a corresponding disease.

EXPERIMENTAL EXAMPLE 5

Early Detection of Cancer by Using Leukocyte Extravasation Factors

[0113] The following experiment was conducted to confirm whether cancer may be diagnosed early by using leukocyte extravasation factors.

[0114] First, 4T1 cancer cells (breast cancer cells) were cultured in RPMI 1640 medium containing about 10% fetal bovine serum (FBS) and about 1% antibiotics in an incubator at about 37° C. and under the condition of 5% CO.sub.2. The medium was changed about once every 2 days to 3 days, and the cells were subcultured using a trypsin/EDTA solution of about 0.25% when the flask was about 80% full with the cells.

[0115] Then, the cultured 4T1 cancer cells were injected into the mammary fat pad of 8-week-old female BALB/C mice at a concentration of about 3×10.sup.6 cells/0.1 mL 1×PBS (pH 7.4). After injection, they were bred in cages for one week.

[0116] In the same manner as in Example 1, a microfluidic chip including a microfluidic channel in which mouse ICAM-1, mouse P-selectin, and mouse E-selectin, etc. were immobilized was prepared (each using a solution at a concentration of about 5 μg/ml), and about 50 μL of the blood of the prepared cancer-induced mice or non-cancerous mice was collected, mixed with ACK lysis buffer in a ratio of about 1:20, and reacted at room temperature for about 5 minutes, and then the leukocytes were isolated by using centrifugation. The isolated leukocytes were washed with 1×PBS, and then diluted in about 100 μL of 1×PBS to prepare a biological sample including leukocytes. After injecting a sample including leukocytes isolated from the mice into the microfluidic channel, the cells captured in the channel were photographed with fluorescence staining, and (number of leukocytes attached to the channel)/(number of total leukocytes in the sample) % was measured, and the results are shown in FIGS. 8 and 9, respectively.

[0117] As shown in FIGS. 8 and 9, when leukocytes of cancer-induced mice were injected into the channel, it was found that significantly more leukocytes were attached to the channel and detected than when leukocytes of a control mouse were injected into the channel, and it was found that cancer may be diagnosed at an early stage in a subject with cancer, even when it was only about a week after the onset of cancer.

[0118] These results indicate that in leukocytes in a disease state or in leukocytes in an abnormal state, expression of factors that interact with leukocyte extravasation factors increases or a number of leukocytes with increased expression of factors that interact with leukocyte extravasation factors increases, and it is possible to diagnose a disease that increases expression of surface-related factors in a leukocyte, such as cancer, by using leukocyte extravasation factors according to an embodiment. In particular, these results mean that cancer may be diagnosed at an early stage with a very small sample using the leukocyte extravasation factor according to an embodiment.

EXPERIMENTAL EXAMPLE 6

Detection of Cancer by Using Leukocyte Extravasation Factors

[0119] In order to confirm whether cancer may be diagnosed using leukocyte extravasation factors, the following experiment was conducted.

[0120] First, 4T1 cancer cells were cultured in the same manner as in Experimental Example 5.

[0121] Then, the cultured 4T1 cancer cells were injected into the mammary fat pad of 8-week-old female BALB/C mice at a concentration of about 3×10.sup.6 cells/0.1 mL 1×PBS (pH 7.4). After the injection, the cells were subdivided into 4 groups (week-1, week-2, week-3, and week-4) of intervals of about one week according to the cancer progression period (4 weeks in total). In addition, a healthy mouse model that was not injected with anything was subdivided into 4 groups like the tumor models, and used as a control group. The sham control group, in which about 0.1 mL of 1×PBS (pH 7.4) was injected into the mammary fat pad of the mouse, was also subdivided into 4 groups like the tumor models and used. In addition, in order to confirm that the tumor models were well prepared, the tumor size and weight of the mouse tumor models were measured at intervals of about 1 week until 4 weeks after injection of 4T1 cancer cells, and the results are shown in FIG. 10. Specifically, the tumor diameter (cm) was measured after surgically isolating the tumor from the mouse tumor models (the diameter was measured at least twice in the direction perpendicular to each other over the largest part of the tumor),and after measuring the tumor diameter, the weight (mg) was measured by using an electronic scale. The average diameter of the tumor was calculated by using the following formula:

Formula=√(d1×d2) (d1 and d2 are the longest diameters of the tumor, which are diameters perpendicular to each other.)

[0122] As shown in FIG. 10, it was confirmed that both tumor size and weight were significantly increased from week 1 to week 4 after injection of 4T1 cancer cells. Therefore, it was confirmed that cancer was successfully formed in the mouse tumor model.

[0123] In the same manner as in Example 1, a microfluidic chip including a microfluidic channel in which mouse ICAM-1, mouse P-selectin, and mouse E-selectin, etc. were immobilized (each using a solution at a concentration of about 5 μg/ml) was prepared. After isolating leukocytes from the mice and washing the isolated leukocytes with 1×PBS, some of them were isolated separately, stained with Hoechst, and counted by using a hemocytometer and a fluorescence microscope, and the remaining leukocytes were diluted with 1×PBS to a final concentration (about 10.sup.6 cells/ml), and a biological sample including the leukocytes was prepared. The biological sample was injected into the microfluidic channel at a flow rate of about 8 μL/min for about 10 minutes, in order that about 80,000 leukocytes were injected into each channel. After the biological sample including the leukocytes was injected into the channel and flowed, the channel was washed for about 4 minutes by injecting 1×PBS into the channel at a flow rate of about 8 μl/min to remove the leukocytes that were not attached to the channel, and this was repeated about two more times. Then, in order to stain the leukocytes attached or trapped in the microfluidic channel, the inside of the channel was filled with dyes such as Hoechst and Cell tracker, and incubated at room temperature for about 20 minutes, and the channel was washed for about 4 minutes by injecting 1×PBS into the channel at a flow rate of about 8 μl/min. After that, the inside of each channel was photographed with a fluorescence microscope, and images inside each channel to which leukocytes were attached were captured, and the total number of leukocytes captured in the channel was counted, and the results are shown in FIG. 11.

[0124] As shown in FIG. 11, it may be seen that when leukocytes of cancer-induced mice were injected into the channel, significantly more leukocytes were attached to the channel and detected than when leukocytes of a control mouse or a sham control mouse were injected into the channel. Specifically, it was confirmed that the number of leukocytes attached to the channel was significantly increased in the case of all groups of mouse tumor models from week 1 to week 4 after injection of 4T1 cancer cells, compared to the mouse models in which tumor was not induced. In particular, it was confirmed that the tumor size gradually increased over the course of 4 weeks after injection of 4T1 cancer cells into mice (see FIG. 10), and the amount of leukocytes attached to the channel and detected also increased, among the leukocytes isolated from cancer-induced mice, as the tumor size of the cancer-induced mice increased.

[0125] These results indicate that in leukocytes in a disease state or in leukocytes in an abnormal state, expression of factors that interact with leukocyte extravasation factors increases or a number of leukocytes with increased expression of factors that interact with leukocyte extravasation factors increases, and it is possible to effectively diagnose a disease that increases expression of surface-related factors in a leukocyte, such as cancer, by using leukocyte extravasation factors, according to an embodiment. In addition, these results indicate that by using the leukocyte extravasation factors according to an embodiment, it is possible to quantitatively evaluate the progress of a disease, such as cancer, etc., in a subject with a disease that increase the expression of the surface-related factors of leukocytes, such as cancer, etc., or to make diagnosis on progression of a disease, such as cancer.

EXPERIMENTAL EXAMPLE 7

Confirmation of Proportion of Neutrophils Among the Detected Leukocytes

[0126] The proportion of neutrophils among the detected leukocytes was analyzed by using the leukocyte extravasation factors according to an embodiment.

[0127] Specifically, leukocytes isolated from the mouse tumor model of Experimental Example 6 (experimental group) and the mouse models in which tumor was not induced (control group, sham control group) were injected into the microfluidic channel prepared as in Experimental Example 6, and then the leukocytes captured in each channel were analyzed as a target. More specifically, each microfluidic channel in which the leukocytes were captured was reacted with about 4% paraformaldehyde solution at room temperature for about 10 minutes to fix the captured leukocytes in the channel. After fixation, the channel was washed with 1×PBS, and 0.5% Triton-X solution was injected into the channel for about 10 minutes to increase reagent permeability, and then the channel was washed with 1×PBS. Afterwards, about 3% BSA was injected and reacted at room temperature for about 1 hour to block non-specific binding. Next, antibodies to which FITC fluorescence was attached targeting neutrophil myeloperoxidase were injected into the channel to identify neutrophils. In addition, in order to visualize all the leukocytes captured in the channel, the cells in the channel were stained with Hoechst reagent at about 4° C. for about 24 hours. After washing the channels with 1×PBS, the total number of leukocytes and the number of neutrophils captured in each channel were counted by using a fluorescence microscope, and the proportion of neutrophils in the captured leukocytes was calculated, and the results are shown in FIG. 12.

[0128] As shown in FIG. 12, it was confirmed that neutrophils were present in the highest proportion among the leukocytes captured in the microfluidic channel coated with leukocyte extravasation factors in all the mouse models of the experimental group, the control group, and the sham control group.

[0129] These results may mean that the majority of leukocytes interacting with the leukocyte extravasation factors according to an embodiment are neutrophils.

EXPERIMENTAL EXAMPLE 8

Detection and Analysis of Leukocytes with Bright Field

[0130] If captured leukocyte may be counted with a Bright field (BF) as well as with fluorescence images, analysis may be performed with a regular camera, etc., and the present disclosure may be more useful at the point-of-care, and thus, in this experimental example, detection and analysis of leukocytes by using a BF was performed.

[0131] Specifically, in the same manner as in Experimental Example 1, blood of rats was collected about 12 hours after bacterial inoculation to isolate leukocytes, and the isolated leukocytes were injected into a microfluidic chip prepared in the same manner as in Example 1, including a microfluidic channel (each using a solution having a concentration of about 5 μg/ml), in which rat ICAM-1, rat P-selectin, and rat E-selectin were immobilized. Thereafter, in the same manner as in Experimental Example 1, after staining the cells captured in each channel using CellTracker, the cells were imaged with a fluorescence microscope, and imaged under a microscope in a BF mode, respectively, and (number of leukocytes attached to the channel)/(total number of leukocytes in the sample) % was measured in each image above, and the results are shown in FIG. 13.

[0132] As shown in FIG. 13, it was found that there was no statistical difference between the results of an analysis through the fluorescence image and an analysis with a BF, and it was found that a number of cells may be counted by imaging on the BF as by using fluorescence images. These results indicate that the method and device according to an embodiment allow on-site diagnosis of diseases or immune conditions without expensive and difficult-to-carry equipments.

[0133] Thus far, specific parts of the present disclosure are described in detail, and it will be apparent for those of ordinary skill in the art, that this specific description is only for preferred embodiments, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present disclosure will be defined by the appended claims and their equivalents.