CN108956431B

CN108956431B - Method for detecting residual cells in platelet product obtained by apheresis

Info

Publication number: CN108956431B
Application number: CN201810904639.0A
Authority: CN
Inventors: 胡佳
Original assignee: Suzhou Diedai Biotechnology Co ltd
Current assignee: Suzhou Diedai Biotechnology Co ltd
Priority date: 2018-08-09
Filing date: 2018-08-09
Publication date: 2021-04-30
Anticipated expiration: 2038-08-09
Also published as: CN108956431A

Abstract

The scheme relates to a method for detecting residual cells in a platelet product obtained by apheresis, which comprises the following steps: step 1) taking a cell sample, and adding a staining antibody reagent to finish cell specificity marking; step 2) after the cell sample is tiled, observing and counting through a fluorescence microscope; wherein, the staining antibody reagent comprises: to achieve staining of platelets

Labeled CD41 and CD 61; to achieve staining of leukocytes

Labeled CD 45; and nuclear dyes that effect staining of white cell nuclei. The scheme can realize the simultaneous detection of residual white blood cells, red blood cells and platelets by adopting a means of combining cell staining markers with fluorescence imaging; the fluorescent microscope and software calculation can be adapted at the later stage, and automatic detection and counting are realized; the operation is simple and the efficiency is high.

Description

Method for detecting residual cells in platelet product obtained by apheresis

Technical Field

The invention relates to the field of platelet product detection, in particular to a method for detecting residual cells in an apheresis platelet product.

Background

Platelet transfusion is currently one of the most major therapeutic modalities for thrombocytopenia and many diseases, and has evolved to be the second clinical major component transfusion after red blood cell transfusion. However, platelet transfusion ineffectiveness events still occur frequently, blood patients generally need to transfuse platelets for multiple times, transfusion ineffectiveness is easy to occur after the platelets are transfused for multiple times, allogenic antibodies are mainly generated, and researches show that HLA antibodies, HPA antibodies and ABH antibodies exist in most platelet transfusion ineffectiveness patients, and HLA antibodies exist in 80% of the patients. The main cause of antibody production is probably due to the stimulation of the patient's immune system by leukocytes and erythrocytes remaining in platelets supplied from many and different donors.

Therefore, the quality of the platelet product is improved from the source, the platelet product is strictly monitored, especially, the leukocyte residual quantity in the platelet is strictly detected, the blood transfusion ineffectiveness caused by the leukocyte and erythrocyte residual quantity is avoided, and the safety of the platelet product is ensured, so that the method becomes the first problem to be clinically urgently solved.

The quality control of the platelet product has clear requirements on international and domestic standards according to the standardsThe quality requirements of quasi-GB 18469-2012 whole blood and component blood are that the mixing amount of white blood cells in the white blood cell removing single blood collecting platelet is lower than 5 x10⁶Per bag, the mixing amount of red blood cells is less than 8X 10⁹One/bag, which is one of the daily quality control items of the blood station.

The existing detection method for mixed cells in the platelet product mainly refers to the standard WST 550 + 2017 whole blood and component blood quality monitoring guidelines and the requirements of national clinical examination operating procedures. The mixed red blood cell count is still performed using classical microscopy, while the white blood cells can likewise be counted using classical blood cell counting plates or using a blood cell analyzer. In recent years, although some blood stations use more accurate flow cytometry analysis, the flow technology is complex in sample preparation, high in requirement on operators, and high in price, so that the method is not generally applicable.

The existing residual cell detection method has more problems and larger limitations. The manual counting mode based on the large-capacity Nageotte blood cell counting disc has the advantages of large error, poor repeatability and no original record, and does not meet the modern requirements on record storage and evidence chain.

The conventional blood cell counting instrument on the market is difficult to accurately count related indexes, particularly residual white blood cells and red blood cells, in an apheresis platelet product. The detection limit of the conventional cell counting method (such as a hemocytometer, a flow cytometer and the like) is generally 10⁴About one/mL, residual cells in the leukoreduced blood platelet cannot be detected quickly and effectively.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a method for detecting residual cells in a platelet product by single collection, which aims to realize the simultaneous and accurate detection of various cells by a method combining cell staining labeling and fluorescence imaging, and can realize the one-time high-speed and accurate interpretation of the content of residual white blood cells, residual red blood cells and platelets in the platelet product if cell imaging and data processing are combined.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a method for detecting residual cells in an apheresis platelet product comprising:

step 1) taking a cell sample, and adding a staining antibody reagent to finish cell specificity marking;

step 2) after the cell sample is tiled, observing and counting through a fluorescence microscope;

wherein, the staining antibody reagent comprises:

alexa to achieve staining of platelets

488-labeled CD41 and CD 61;

staining of leukocytes with Alexa

594 labeled CD 45; and

the cell nucleus dye for staining white cell nucleus is realized.

Preferably, the detection method is carried out in a concentration ratio of CD41 to CD61 of 1: 6.7-6.9.

Preferably, the detection method is carried out in which the concentration ratio of CD41 to CD61 is 1: 6.8.

Preferably, in the detection method, the structural formula of the nuclear dye is:

preferably, the detection method, wherein in the nuclear dye, R is₁Is COOEt or CH₃Or H.

Preferably, the detection method, wherein in the nuclear dye, R is₂Is CH₃Or CH₂CH₃Or H.

Preferably, the detection method, wherein in the nuclear dye, R is₃Is CH₃Or CH₂CH₃Or H.

Preferably, the detection method, whereinIn the nuclear dye, R₁Is COOEt; r₂Is CH₃；R₃Is CH₂CH₃。

The invention has the beneficial effects that: the scheme can realize the simultaneous detection of residual white blood cells, red blood cells and platelets by adopting a means of combining cell staining markers with fluorescence imaging; the fluorescent microscope and software calculation can be adapted at the later stage, and automatic detection and counting are realized; the operation is simple and the efficiency is high.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic diagram of a microporous carrier chip for fluorescence detection.

FIG. 2 is a graph of cell size for example 1.

Fig. 3 is a photograph showing fluorescence imaging of platelets in example 1.

FIG. 4 is a photograph of a fluorescent image of leukocytes obtained in example 1.

FIG. 5 is a photograph of fluorescence images of white cell nuclei of example 1.

FIG. 6 is a photograph of a fluorescent image of white cell nuclei of comparative example 1.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Examples

Materials (I) and (II)

1. A cell sample;

2. fluorescent dye:

CD41(GeneTex Inc., Cat. GTX113758), CD61 (Wuhan Boohot, Cat. orb86360), CD41 and 61 were all Alexa

488(ThermoFisher corporation) tag;

CD45 (Shanghai research, cat # 11-0451-82) in Alexa

594(ThermoFisher Co., Ltd.);

nuclear dye:

R₁is COOEt or CH₃Or H; r₂Is CH₃Or CH₂CH₃Or H; r₃Is CH₃Or CH₂CH₃Or H.

The synthesis method is as follows (with R)₁Is COOEt; r₂Is CH₃；R₃Is CH₂CH₃For example):

the total yield is as follows: 17.5 percent.¹H NMR (400MHz, deuterated DMSO) < delta > 8.41(s,1H, ArH),7.91(s,1H, ArH),7.59(s,1H, ArH),7.23(s,2H, ArH),7.21(s,2H, ArH),4.38(m,2H, NHCH)₂),4.30(m,2H,COOCH₂CH₃),4.01(s,1H,NHCH₂),3.90(m,1H,C＝NH),3.76(m,1H,HCl),3.35(m,2H,NCH₂CH₃),3.01(s,3H,NCH₃),1.29(s,3H,COOCH₂CH₃),1.12(s,3H,NCH₂CH₃)。

3. Cell fixing solution:

4% PFA: 4% paraformaldehyde (PBS dissolved): it is a cell fixative used to prevent lysis. Or using glutaraldehyde or formaldehyde or glacial ethanol (precooled at-20 ℃) PBS solution for substitution;

4. cell membrane permeabilizing agent: 0.1-0.5% Triton X100 to facilitate the entry of nuclear dyes into cells;

5.5% calf serum, used to block antibody non-specific binding sites;

glycerol: 1-10% of cell membrane stabilizer;

6. cell wetting agent: 1% BSA (bovine serum albumin) solution (PBS dissolved).

Second, the experimental procedure

1. Cell samples were centrifuged at 600rpm and washed 2 times with PBS.

2. 1ml of cell fixative was added to the cell pellet and incubated at 37 ℃ for 10 min. Centrifuging at 600rpm for 5min, and removing supernatant.

3. The cell pellet was resuspended in 1ml of 0.1% Triton X100(PBS lysis) and incubated for 5-15min at room temperature. Centrifuging at 600rpm for 5min, and removing supernatant.

4. The cell pellet was sealed with 1ml of 5% calf serum plus 1% glycerol for 30min at room temperature. Centrifuging at 600rpm for 5min, and removing supernatant.

5. The cell pellet was resuspended in 0.5ml PBS and 5ul Alexa at 0.5mg/ml was added

488-CD41/61 antibody (concentration ratio of CD41 to CD61 is 1: 6.7-6.9) and Alexa

594-CD45 antibody, applied at room temperature for 15-30 min.

6. Adding cell nucleus dye to the solution to a final concentration of 0.1ug/ml, and allowing the solution to act at room temperature for 15-30 min. PBS was washed 2 times.

7. The cell pellet was dissolved with 1% BSA solution (PBS) to prevent cell aggregation.

8. The structure of the microporous carrier chip is shown in figure 1, the diameter of a sample feeding pool 1 is about 2mm, the depth of an observation pool 2 is 100 mu m, the length and the width of the observation pool 2 are about 5-10mm, and the inner diameter of a connecting channel between the sample feeding pool 1 and the observation pool 2 is about 1 mm. The sample enters the observation pool 2 through the siphoning effect, and the observation pool 2 structure leads the cells to be paved in the observation pool 2, thereby facilitating the observation. And adding the marked cell sample into a sample introduction pool 1, introducing the cell sample into an observation pool 2 through siphonage, and observing and counting the cells through a fluorescence microscope after the cells are tiled. The photo can be taken and counted by the automatic counting software.

The basis of cell identification in the scheme is as follows: cell differentiation is achieved based on the fluorescent color of the cell label and the cell size. CD41 and CD61 are platelet common antigens that can be used to label assay platelet numbers; CD45 is a leukocyte common antigen, and can mark leukocytes; the designed nuclear dye is a nuclear specific marker dye, white blood cells have nuclei, and platelets and red blood cells have no nuclei.

Dye Alexa for CD41 and CD61 antibodies

488 label, platelet staining can be realized, and fluorescence microscopic imaging is green. Alexa for CD45 antibody

594 to achieve white blood cell staining and fluorescence imaging to red. The cell nucleus dye designed by the scheme is a specific cell nucleus dye, and the fluorescence imaging is blue, so that white cell nucleus dyeing can be realized.

Platelet size (human platelets mean diameter about 2-4 microns): significantly smaller than red blood cells (6-9 microns) and white blood cells (7-20 microns). Based on the size difference of three blood cells, the combined cell nucleus dye can be used for analyzing the number of red blood cells.

Therefore, based on the fluorescence imaging and white light imaging analysis of the markers, accurate imaging counting of the number of the residual white blood cells, red blood cells and platelets can be realized, and the result is shown in table 1.

TABLE 1

Although in the prior art DAPI (4', 6-diamidino-2-phenylindole) has been usedIs a nuclear dye, but it has the following disadvantages: 1) strong carcinogens, high risk to operators; 2) increased non-specific binding when used in combination with CD41, CD61, and CD 45; 3) and Alexa

488 and Alexa

594, the fluorescence color generated by DAPI is disturbed, the color is bluish black, close to the background color, and is not easy to observe and count. Therefore, the structural formula of the nuclear dye is redesigned in the scheme:

right side of the chemical formula

Partially inserted into the minor groove of the DNA double strand and freely rotated in space by-NH-CH linked thereto₂The chain, which can be externally connected with a pi-electron-rich group, can form steric hindrance effect to effectively improve the specific binding with the nucleus, and the left side

But also can increase the fluorescence wavelength, make the color thereof bright blue, and be easy to observe and count.

To further illustrate, the compounds designed in this case were compared to the use of DAPI as a nuclear dye.

Preparing a cell standard solution:

	blood platelet	White blood cell	Red blood cell
				Concentration of	2×10⁶mu.L/u	2.5×10³mu.L/u	3.5×10⁴mu.L/u

Using the above examples as templates, respectively, DAPI and the compounds of the present case were used as nuclear dyes:

the test results were as follows:

	nuclear dye	Blood platelet	White blood cell	Red blood cell
					Example 1	Compound (I)	1.95×10⁶mu.L/u	2.45×10³mu.L/u	3.40×10⁴mu.L/u
Example 2	Compound (II)	1.83×10⁶mu.L/u	2.40×10³mu.L/u	3.22×10⁴mu.L/u
					Example 3	Compound (III)	1.85×10⁶mu.L/u	2.42×10³mu.L/u	3.15×10⁴mu.L/u
Comparative example 1	DAPI	1.82×10⁶mu.L/u	1.22×10³mu.L/u	3.12×10⁴mu.L/u

FIG. 2 is a graph of the cell size of example 1 (white light 10X imaging) with the result that platelets are distinguishable by cell size, calculated to be about 1.91X 10⁶μ L, but cannot accurately distinguish between white blood cell and red blood cell numbers.

FIG. 3 is a fluorescence image of platelets (488nm wavelength, 10 Ximage) obtained from example 1, which can be used to accurately count the number of platelets by fluorescence spectroscopy, and the result is 1.95X 10⁶One/mu L, which is basically consistent with the quantity of the cell standard solution; FIG. 4 is a photograph showing fluorescence imaging of leukocytes (590nm wavelength, 10X imaging) in example 1, and the number of leukocytes was 2.46X 10³mu/L; FIG. 5 is a photograph of fluorescence imaging of white cell nuclei (350nm wavelength, 10) of example 1X imaging) with the result of 2.45 × 10³The number of the white blood cells is 2.45 multiplied by 10 which can be accurately determined by the number of the white blood cells/mu L and the fluorescence imaging result of the white blood cells in the graph 4³μ L (slightly higher numbers of leukocytes, possibly with a slight amount of non-specific binding); FIG. 6 is a photograph showing fluorescence imaging of white blood cell nucleus (350nm wavelength, 10X imaging) of comparative example 1, and the result of white blood cell count thereof was 1.22X 10³The count of counts/. mu.L, which is significantly less than the count of the nuclear dye of the present invention, combined with the results of FIG. 4, is judged to be a serious distortion of the white blood cell count, while the comparison of FIGS. 5 and 6 shows that there are more fluorescence spots in FIG. 6, but the final count is lower, indicating that DAPI causes an increase in nonspecific binding, and the formed fluorescence spots are darker in color and not easily observed when combined with the background. The result obtained by adopting the nuclear dye designed by the scheme is closer to the true value, less in nonspecific binding, clear and bright in fluorescence point and easy to observe and count.

The counting method of the red blood cells comprises the following steps: by white light imaging of cells, platelets, red blood cells and white blood cells can be distinguished obviously according to the sizes of the cells, but the number of the white blood cells and the number of the red blood cells can not be calculated accurately, and the total number of the red blood cells and the white blood cells can be calculated. FIG. 2 is a graph of the cell size of example 1 (white light 10X imaging) combined with the platelet count results of FIG. 3 to accurately calculate the total number of red blood cells and white blood cells as 3.647X 10⁴mu/L; the number of leukocytes was accurately counted by comparing fluorescence imaging of leukocytes with nuclear imaging, and FIGS. 4 and 5 are the results of counting leukocytes of example 1 in an amount of 2.45X 10³mu/L; further, the number of red blood cells was calculated to be 3.402X 10⁴One/. mu.L, which is substantially identical to the amount of the cell standard solution.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

Claims

1. A method for detecting residual cells in an apheresis platelet product comprising:

wherein, the staining antibody reagent comprises:

alexa to achieve staining of platelets

488-labeled CD41 and CD 61;

staining of leukocytes with Alexa

594 labeled CD 45; and

a nuclear dye that effects staining of white nuclei;

the structural formula of the cell nucleus dye is as follows:

wherein R is₁Is COOEt; r₂Is CH₃；R₃Is CH₂CH₃。

2. The detection method according to claim 1, wherein the concentration ratio of CD41 to CD61 is 1: 6.7-6.9.

3. The detection method according to claim 2, wherein the concentration ratio of CD41 to CD61 is 1: 6.8.