Use of SYTOX in detecting red blood cell maturity in peripheral blood and test reagents and methods

Abstract

The application belongs to the technical field of cell detection, and relates to application of SYTOX in detection of maturity of peripheral blood red blood cells and a detection kit method. The application provides application of SYTOX in detection of maturity of peripheral blood red blood cells. SYTOX staining is used for distinguishing red blood cells at different maturity stages of peripheral blood, and has high sensitivity, high accuracy and high consistency, and is efficient, simple and easy to popularize.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cell detection, and particularly relates to application of SYTOX in detection of maturity of peripheral blood red blood cells and a detection kit method. BACKGROUND

[0002] Reticulocytes are a stage between late erythroblasts and mature red blood cells, the late erythroblasts form reticulocytes by de-nucleating in the bone marrow, and the reticulocytes become mature red blood cells by further removing ribosomes and mitochondria in the cytoplasm after entering the periphery. Currently, the following three methods are mainly used to evaluate the maturity of peripheral blood red blood cells: (1) manual microscopic examination: blood smears are dyed by using Giemsa, new base blue, brilliant toluene blue and the like, and then reticulocytes, nucleated red blood cells and the like are observed and counted under a microscope, which is tedious and low in counting efficiency; (2) flow cytometry: a flow cytometer uses the principle that thiazole orange and the like are combined with RNA in reticulocytes and the size of cells to distinguish red blood cells of different maturity, which is commonly used in large hospital laboratories and is high in automation, but the RNA in reticulocytes is distributed in a net or cluster, the fluorescence signal is scattered after being combined with the fluorescent dye, and it is difficult to distinguish single cells according to the fluorescence signal when the cells pass through the detector, and repeated detection can easily cause cross contamination between samples, which is not suitable for wide use; (3) in general laboratories, CD71, CD44 and the like are commonly used to stain red blood cell surface molecules to distinguish the maturity of red blood cells, which is high in cost. The method provided in the present application is a method for distinguishing the maturity of red blood cells by using DNA dye SYTOX, which is higher in efficiency than manual microscopic examination; is simpler in operation and lower in cost than the flow cytometer commonly used in clinical laboratories, and is more economical than conventional CD71 or CD44 staining. Therefore, it is crucial to find a detection method which is higher in efficiency and easy to popularize. SUMMARY

[0003] The present application provides application of SYTOX in detection of maturity of peripheral blood red blood cells and a detection kit method.

[0004] The present application provides application of SYTOX in detection of maturity of peripheral blood red blood cells.

[0005] The present application also provides a reagent for detecting the maturity of peripheral blood red blood cells, which comprises SYTOX and a red blood cell antibody.

[0006] Preferably, the red blood cell antibody comprises Ter119 or CD235a.

[0007] The application further provides a method for detecting the maturity of red blood cells in peripheral blood for non-diagnostic purposes, comprising the following steps:

[0008] The sample to be tested is mixed with the reagent described in the above technical solution for staining, flow detection and analysis, the cell population is determined using SS and FS, the red blood cell population is determined using the red blood cell antibody channel, and the red blood cells of different maturity are marked using the SYTOX fluorescence signal gate, with SYTOX negative representing mature red blood cells and SYTOX positive representing erythroid precursor cells.

[0009] Preferably, the source of the sample to be tested comprises peripheral blood, and the cell concentration of the sample to be tested is 1x10 7 ~ 5x10 7 / mL.

[0010] Preferably, when the source of the sample to be tested is mouse peripheral blood, the red blood cell antibody in the reagent is Ter119, and when the source of the sample to be tested is human peripheral blood, the red blood cell antibody in the reagent is CD235a.

[0011] Preferably, the staining time is 15-30 min.

[0012] The application further provides the use of SYTOX or the reagent described in the above technical solution in the preparation of a tumor or blood system disease diagnostic kit or a bone marrow hematopoietic function evaluation kit.

[0013] Preferably, the blood system disease comprises anemia.

[0014] The application provides the use of SYTOX in the detection of the maturity of red blood cells in peripheral blood. SYTOX, as a nucleic acid dye that is not easy to penetrate the cell membrane of living cells, can be used to stain dead cells, and the staining of nucleated dead cells is strongly positive. Peripheral blood red blood cells mainly include mature red blood cells and erythroid precursor cells (mainly IV stage reticulocytes), which are both anucleated cells. When SYTOX is stained at an appropriate ratio, it can penetrate the cell membrane of reticulocytes to stain mitochondria, thereby distinguishing mature red blood cells from immature erythroid precursor cells. The application first discovers that SYTOX staining can also be used to distinguish red blood cells at different maturity stages in peripheral blood. SYTOX staining for distinguishing red blood cells at different maturity stages in peripheral blood has high sensitivity, accuracy and consistency, and is efficient, simple and easy to promote. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0016] Figure 1 A flow cytometry analysis of tumor-related anemia mouse peripheral blood red cell maturity result chart provided by the present application;

[0017] Figure 2 A normal mouse and anemic mouse peripheral blood red cell maturity comparison result chart provided by the present application;

[0018] Figure 3 A normal human and tumor patient peripheral blood red cell maturity comparison result chart provided by the present application;

[0019] Figure 4 A tumor patient peripheral blood SYTOX pos Red cell and anemia relationship result chart;

[0020] Figure 5 A normal mouse and anemic mouse Mito tracker, CD71, SYTOX staining detection peripheral red cell maturity result chart provided by the present application;

[0021] Figure 6 A normal mouse and anemic mouse CD71, SYTOX staining detection peripheral red cell maturity comparison result chart provided by the present application;

[0022] Figure 7 A normal human and tumor patient CD71, SYTOX staining detection peripheral blood red cell maturity comparison result chart provided by the present application. DETAILED DESCRIPTION

[0023] The present application provides the application of SYTOX in detecting peripheral blood red cell maturity. SYTOX, as a nucleic acid dye which is not easy to penetrate the cell membrane of living cells, can be used to stain dead cells, and the staining of nucleated dead cells is strongly positive. The present application first discovers that SYTOX staining can also be used to distinguish red cells at different maturity stages in peripheral blood. The reticulocyte is continuously cleared of residual cytoplasmic mitochondria, and the staining is continuously moderate to low intensity fluorescence, and the mature red cell is negative. SYTOX staining is used to distinguish red cells at different maturity stages in peripheral blood, and the sensitivity is high. The fluorescence intensity of red cell SYTOX staining is consistent with Mito tracker and CD71 staining, which indicates that the use of SYTOX staining to detect red cell maturity has high accuracy and consistency. The present application is more accurate in evaluating peripheral blood red cell maturity by detecting mitochondrial DNA. The present application can realize the distinction of red cell maturity through simple SYTOX staining, which is more conducive to promotion. The present application provides a new strategy for reticulocyte staining counting, and has certain clinical application value.

[0024] The application also provides a reagent for detecting the maturity of red blood cells in peripheral blood, which comprises SYTOX and a red blood cell antibody. In the application, the red blood cell antibody preferably comprises Ter119 or CD235a. The application does not have special limitations on the source of SYTOX and the red blood cell antibody, and the conventional commercially available products of SYTOX, Ter119 and CD235a known to those skilled in the art can be used. In the application, the Ter119 is preferably a Pacific Blue anti-mouse Ter119 antibody (manufacturer: Beijing Dakewei Biotechnology Co., Ltd.; product number: 116232). In the application, the CD235a is preferably an APC anti-human CD235a (manufacturer: Beijing Dakewei Biotechnology Co., Ltd.; product number: 349113). In the application, the SYTOX is preferably a FITC-SYTOX (manufacturer: Thermo Fisher Scientific (China) Co., Ltd.; product number: S7020).

[0025] The application also provides a method for detecting the maturity of red blood cells in peripheral blood for non-diagnostic purposes, comprising the following steps:

[0026] The test sample is mixed with the reagent described in the above technical solution for staining, flow detection and analysis, the cell population is determined using SS and FS, the red blood cell population is determined using the red blood cell antibody channel setting, and the red blood cells of different maturity are marked using the SYTOX fluorescence signal setting, the SYTOX negative represents mature red blood cells, and the SYTOX positive represents erythroid precursor cells.

[0027] The application mixes the test sample with the reagent described in the above technical solution for staining. In the application, the source of the test sample preferably comprises peripheral blood; the cell concentration of the test sample is preferably 1×10 7 ~ 5×10 7 / mL, and more preferably 5×10 7 / mL. In the application, the test sample is more preferably peripheral blood cells. The application does not have special limitations on the method for obtaining peripheral blood cells, and a conventional method can be used. In the application, the method for obtaining peripheral blood cells preferably comprises the following steps: mixing a blood sample with PBS (phosphate buffered saline), resuspending the blood cells, and adjusting the cell concentration to 1×10 7 ~ 5×10 7After obtaining the cell pellet, preferably 200 μL is pipetted into a 96-well plate, centrifuged at 1800 rpm for 2 min, the supernatant is discarded, the sample is washed once with PBS, and centrifuged at 1800 rpm for 2 min to obtain the cell pellet. In this invention, the blood sample preferably includes mouse tail vein peripheral blood or human peripheral blood. In this invention, when the sample is derived from mouse peripheral blood, the erythrocyte antibody in the reagent is preferably Ter119; when the sample is derived from human peripheral blood, the erythrocyte antibody in the reagent is preferably CD235a. In this invention, the Ter119 is preferably a 1:100 diluted Pacific Blue anti-mouse Ter119 antibody (manufacturer: Beijing Dachuang Biotechnology Co., Ltd.; catalog number: 116232). In this invention, the CD235a is preferably a 1:100 diluted APC anti-human CD235a (manufacturer: Beijing Dachuang Biotechnology Co., Ltd.; catalog number: 349113). In this invention, the SYTOX is preferably FITC-SYTOX (manufacturer: Thermo Fisher Scientific (China) Co., Ltd.; product number: S7020), and the FITC-SYTOX is preferably diluted 1:1000 for use. In this invention, the staining time is preferably 15–30 min, more preferably 20 min. In this invention, the staining is preferably performed in DMEM complete medium, which is preferably purchased from Thermo Fisher Scientific (China) Co., Ltd., product number: C11995500BT. In this invention, after staining, the sample is preferably washed 1–3 times with PBS, more preferably 2 times.

[0028] After staining, this invention performs flow cytometry detection and analysis. SS and FS are used to determine the cell population. Red blood cell populations are determined using erythrocyte antibody (Ter119 or CD235a) channel gating. SYTOX fluorescence signals are used to gating and label red blood cells at different maturity levels. SYTOX-negative (SYTOX...) neg ) represents mature red blood cells, SYTOX positive (SYTOX) pos ) represents erythroid precursor cells.

[0029] This invention also provides the application of SYTOX or the reagents described in the above-described technical solutions in the preparation of diagnostic kits for tumors or hematological diseases or bone marrow hematopoietic function assessment kits. In this invention, the hematological diseases preferably include anemia.

[0030] To further illustrate the present invention, the application of SYTOX in detecting the maturity of peripheral blood erythrocytes and the detection kit method provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1

[0032] 1. Obtain peripheral blood cells: Take 10 μL of mouse tail vein blood or patient peripheral blood in PBS (phosphate buffered saline), resuspend the blood cells, adjust the cell concentration to 5 x 10 7 / ml, take 200 μL in a 96-well plate, centrifuge at 1800 r for 2 min, discard the supernatant, wash once with PBS, centrifuge at 1800 r for 2 min to obtain cell pellets;

[0033] 2. Flow cytometry staining: Prepare flow cytometry antibodies according to the sample species for staining the cells obtained in step 1, according to the sample species - mouse samples use antibody Pacific Blue anti-mouse Ter119 1:100 (manufacturer: Beijing Dakewei Biotechnology Co., Ltd.; item number: 116232), human samples use antibody APC anti-human CD235a 1:100 (manufacturer: Beijing Dakewei Biotechnology Co., Ltd.; item number: 349113), respectively, with FITC-SYTOX 1:1000 (manufacturer: Thermo Fisher Scientific (China) Co., Ltd.; item number: S7020) in DMEM complete medium (DMEM - manufacturer: Thermo Fisher Scientific (China) Co., Ltd., item number: C11995500BT; serum - manufacturer: Thermo Fisher Scientific (China) Co., Ltd., item number: 10091148), 100 μL per well, room temperature staining for 20 min, after staining, wash twice with PBS, and flow cytometry detection.

[0034] 3. Flow cytometry analysis steps are as follows: (1) use SS and FS to determine cell populations, exclude cell debris and adhesion; (2) use Ter119 or CD235a channel to determine red blood cell population; (3) use SYTOX fluorescence signal to mark red blood cells of different maturity.

[0035] 4. Peripheral blood red blood cell maturity markers are as follows: SYTOX neg represent mature red blood cells (neg: negative); SYTOX pos represent erythroid precursor cells (pos: positive).

[0036] Example 2

[0037] 1. Flow cytometry analysis of peripheral blood red blood cell maturity in tumor-related anemia mice:

[0038] The peripheral blood red blood cell maturity of tumor-related anemia mice was detected by the method of Example 1. Construction of a mouse tumor-related anemia model: 1 x 10 6Three weeks after tumor-bearing mice, female C57BL / 6 mice were subcutaneously inoculated with LLC (Lewis lung cancer cell). Blood samples were collected from both the tumor-bearing mice and control mice (normal mice that had not undergone any treatment) for flow cytometry analysis.

[0039] The results are as follows Figure 1 As shown, peripheral blood erythrocytes from mice with tumor-associated anemia can be divided into two groups based on the intensity of SYTOX fluorescence signal: SYTOX... neg Represents mature red blood cells; SYTOX pos Represents erythroid precursor cells.

[0040] 2. Maturity of peripheral blood erythrocytes in normal mice and anemic mice:

[0041] The mouse tumor-associated anemia model was constructed as described in Example 1. For the hemolytic anemia model: on day one, normal mice were intraperitoneally injected with 200 μL of phenylhydrazine, and hemolytic anemia was induced 5 days later. Blood samples were collected from tumor-bearing mice 3 weeks after tumor induction, mice 5 days after phenylhydrazine induction, and control mice for flow cytometry analysis. Specific experimental procedures were as described in Example 1, and the results were as follows: Figure 2 As shown, the results indicated that the peripheral blood of healthy control mice mainly contained SYTOX. neg Mature red blood cells and a small amount of SYTOX pos Erythroid precursor cells; mice with tumor-associated anemia and hemolytic anemia exhibited active compensatory hematopoiesis and abundant SYTOX in the peripheral blood. pos Erythroid precursor cells.

[0042] 3. Peripheral blood erythrocyte maturity in normal individuals and cancer patients (lung cancer):

[0043] The experimental method is as described in Example 1. The results are as follows: Figure 3 As shown, peripheral blood of cancer patients contains more SYTOX than that of healthy individuals due to compensatory hematopoiesis. pos Erythroid precursor cells.

[0044] 4. SYTOX in peripheral blood of cancer patients pos The relationship between red blood cells and anemia:

[0045] The number of erythroid precursor cells reflects the body's compensatory hematopoietic function and is generally negatively correlated with the degree of anemia in cancer patients. Using the method described in Example 1, 44 cancer patients (n=44) with varying degrees of anemia were tested, and the results are as follows: Figure 4 As shown, the results indicate that peripheral blood SYTOX... pos The red blood cell ratio is significantly negatively correlated with the patient's own hemoglobin content, indirectly confirming the effects of SYTOX. pos Accuracy of labeling red blood cell maturity.

[0046] The above illustrates that SYTOX and red blood cell antibody staining can distinguish peripheral blood red blood cell maturity, and can be used in experimental mice and human peripheral blood detection, without species specificity, simple operation, good repeatability, and potential application value of erythroid precursor cell marker and counting.

[0047] Comparative Example 1

[0048] 1. Normal mice and anemic mice Mito tracker staining, CD71 staining and SYTOX staining for detecting peripheral red blood cell maturity of red blood cells:

[0049] 1) The tumor-related anemia model was constructed according to the method described in Example 2, and APC-Mitotracker 1:5000 (manufacturer: Thermo Fisher Scientific (China) Co., Ltd.; item number: M22426) and PE-Cyanine7 anti-mouse CD71 (manufacturer: Beijing Dakewe Biotechnology Co., Ltd.; item number: 113812) were added in the flow staining step, and the staining method was the same as that in Example 1. According to the flow cytometry analysis step in Example 1, the SYTOX neg and SYTOX pos two groups of cells were circled, and the expression of CD71 and Mito tracker in the two groups of cells was observed, respectively. The analysis is shown in the following figure:

[0050] The results are shown in Figure 5 , the SYTOX staining of the two groups of cells is consistent with the same color represented Mito tracker and CD71 subgroups, which confirms that the method established in the application is reliable.

[0051] 2) The tumor-related anemia and hemolytic anemia mouse models were constructed according to the method described in Example 2, and the staining and analysis were performed according to the method described in Example 1, wherein PE-Cyanine7 anti-mouse CD71 (manufacturer: Beijing Dakewe Biotechnology Co., Ltd.; item number: 113812) was added at the same time when the original antibodies (Pacific Blue anti-mouse Ter119 and FITC-SYTOX) were added in the staining step, and the staining method was the same as that in Example 1. According to the flow cytometry analysis step in Example 1, the red blood cells were circled, and the co-expression of CD71 and SYTOX in the red blood cells was detected.

[0052] The results are shown in Figure 6 , the SYTOX staining of the two groups of cells is consistent with the same color represented Mito tracker and CD71 subgroups, which confirms that the method established in the application is reliable. pos pos The red blood cells of the healthy control group were basically CD71 pos erythroid precursor cells; the red blood cells of the tumor-related anemia group were also CD71 pos , and a small part of CD71 pos ​SYTOX neg Red blood cells, the possible reason is that in the case of active compensatory hematopoiesis, the degradation of ribosomes, mitochondria and membrane molecule CD71 in the process of red blood cell maturation is inconsistent, and such cells have more mature red blood cells with no mitochondria; Another hemolytic anemia mouse appears a large number of CD71 neg SYTOX pos Erythroid precursor cells (the possible reason is that CD71 membrane molecules are shed in the case of red blood cell membrane damage). Therefore, CD71 antibody staining alone cannot effectively distinguish the maturation of peripheral red blood cells, and SYTOX staining is more accurate.

[0053] 2. Comparison of CD71 staining and SYTOX staining of normal people and tumor patients

[0054] Peripheral blood of normal people and tumor patients was obtained, and staining and analysis were performed according to the method of Example 1, wherein Brilliant Violet 421 anti-human CD71 1:100 (manufacturer: Beijing Dakewei Biotechnology Co., Ltd.; item number: 334121) was added at the same time as the original antibody (APC anti-human CD235a and FITC-SYTOX) was added, and the staining method was the same as in Example 1. According to the flow cytometry analysis steps in Example 1, red blood cells were circled, and the co-expression of red blood cell CD71 and SYTOX was detected.

[0055] The results are shown in Figure 7 CD71 pos Erythroid precursor cells, tumor patients can see SYTOX pos Red blood cells of CD71 pos Erythroid precursor cells, SYTOX staining of red blood cell maturity is more accurate.

[0056] The above comparative experiment proves that the fluorescence intensity of red blood cell SYTOX staining is consistent with Mito tracker and CD71 staining, indicating that its accuracy and consistency are high; and in the detection of erythroid precursor cells in the peripheral blood of hemolytic anemia mice, it has a more precise advantage.

[0057] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which are within the scope of protection of the present application.

Claims

1. Use of SYTOX for detecting the maturity of red blood cells in peripheral blood for non-diagnostic purposes.

2. A method for detecting the maturity of red blood cells in peripheral blood for non-diagnostic purposes, comprising the following steps: mixing the sample to be tested with SYTOX and red blood cell antibodies for staining, flow detection and analysis, using SS and FS to determine cell groups, using red blood cell antibody channels to set gates to determine red blood cell groups, using SYTOX fluorescence signals to set gates to mark red blood cells of different maturity, SYTOX negative representing mature red blood cells, and SYTOX positive representing erythroid precursor cells; the red blood cell antibodies include Ter119 or CD235a.

3. The method of claim 2, wherein, The source of the sample to be tested includes peripheral blood; the cell concentration of the sample to be tested is 1 x 10 7 5 x 10 7 per mL.

4. The method of claim 2, wherein, When the sample to be tested is derived from mouse peripheral blood, the red blood cell antibody is Ter119; when the sample to be tested is derived from human peripheral blood, the red blood cell antibody is CD235a.

5. The method of claim 2, wherein, The staining time is 15-30 min.

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