CN109517792B

CN109517792B - A kind of separation medium of new and old red blood cells and preparation method and application thereof

Info

Publication number: CN109517792B
Application number: CN201811474007.1A
Authority: CN
Inventors: 桂嵘; 刘凤霞; 纪宏文; 周明; 黄雪原; 曾娇辉
Original assignee: Third Xiangya Hospital of Central South University
Current assignee: Hunan Zhonghui Information Technology Co ltd
Priority date: 2018-12-04
Filing date: 2018-12-04
Publication date: 2021-03-16
Anticipated expiration: 2038-12-04
Also published as: CN109517792A

Abstract

The invention discloses a separation medium for new and old red blood cells and a preparation method and application thereof. The separation medium is composed of dimethyl phthalate and dibutyl phthalate, wherein the dimethyl phthalate and the The volume ratio of dibutyl phthalate is (0.95～1.05):(1.95～2.05). The present invention utilizes that the density of reticulum or new red blood cells is 1.085, and the density of old red blood cells is 1.114. The present invention utilizes the characteristics that dimethyl phthalate and dibutyl phthalate are incompatible with red blood cells and have no damage to red blood cells. , configure it into a solution with a density between the old and new red blood cells, so in the process of high-speed centrifugation, it will be clearly divided into four layers in the capillary, completely separate the old and new red blood cells, ensure the accuracy of the test results, and enhance the accuracy of the test results. precision. The consumables of the separation medium of the present invention are simple, the separation medium of the present invention is used to separate old and new red blood cells, the required time is short, the blood consumption is small, the results are intuitive, and the error is small, and can be widely used in routine separation and detection.

Description

Separation medium for new and old red blood cells and preparation method and application thereof

Technical Field

The invention belongs to the technical field of cell separation, and particularly relates to a separation medium for new and old red blood cells, and a preparation method and application thereof.

Background

In the detection of accurate blood type identification, clinical transfusion adverse reaction investigation and the like, new and old red blood cells need to be separated, and the blood type is identified by obtaining the quantity of certain new and old red blood cells through the specific gravity difference of the new and old red blood cells. At present, the separation of new and old red blood cells is mainly carried out by adopting a capillary ultracentrifugation separation method. The capillary ultracentrifugation method mainly utilizes the difference of the density of new and old red blood cells caused by the difference of the nuclear-to-cytoplasmic ratio of fresh cells and old cells, so that the sedimentation velocity is different in the ultracentrifugation process, and the cells with different densities are subjected to hierarchical separation, thereby achieving the purpose of separating the new and old red blood cells. At present, the separation of new and old red blood cells only depends on visual inspection to take the separated red blood cells at the proximal end as reticulocytes or newborn red blood cells of a patient, and the red blood cells at the distal end as old cells or input red blood cells of a blood donor, but the boundary of the separated new and old red blood cells is not obvious, the reticulocytes or newborn red blood cells of different patients can be distinguished, and the error which is easy to exist through the subjective judgment of naked eyes is larger, so that the separated detection result is inaccurate, and the transfusion treatment of the patient is delayed.

Disclosure of Invention

The invention aims to provide a separating medium for new and old red blood cells and a preparation method and application thereof, and solves the problems that the new and old red blood cells are not obviously limited and the separation and detection results are inaccurate in the ultracentrifugation separation process.

The medium for separating the new and old red blood cells consists of dimethyl phthalate (relative density is 1.190-1.193) and dibutyl phthalate (relative density is 1.0463-1.0475), wherein the volume ratio of the dimethyl phthalate to the dibutyl phthalate is (0.95-1.05): 1.95-2.05).

Preferably, the volume ratio of dimethyl phthalate to dibutyl phthalate is 1: 2.

The preparation method of the separation medium for the old and new red blood cells comprises the following steps: mixing the two materials according to the set volume ratio of dimethyl phthalate to dibutyl phthalate, uniformly mixing, and testing the density of the mixture to obtain the separation medium for new and old red blood cells.

The density of the separating medium for the old red blood cells and the new red blood cells is 1.094-1.096.

The method for separating the old red blood cells from the new red blood cells by adopting the separation medium of the old red blood cells comprises the following steps: firstly filling packed red blood cells washed by a patient, then filling a separation medium into a capillary tube, then filling 4-5mm sealing mud into the end of the red blood cells, sealing, then placing the capillary tube into a high-speed centrifuge for separation, and after separation, dividing the capillary tube into four layers: the proximal end is a small amount of plasma layer, reticulocyte or newborn erythrocyte layer, the middle layer separation medium layer and the distal end are old erythrocyte layers; cutting out the reticulocyte or new erythrocyte layer at the proximal end by using a grinding wheel for testing; the amount of reticulocyte or neoerythrocyte layer will vary from patient to patient.

The filling amount of the packed red blood cells is 65-67 mm, the filling amount of the new and old red blood cell separation medium is 2.5-3.5 uL, and the length is 2.5-3.5 mm.

The centrifugal rotating speed is 10000-12000 r; the centrifugation time is 20-25 min.

The invention has the beneficial effects that: the density of reticulocytes or new red blood cells is 1.085, the density of old red blood cells is 1.114, and the invention utilizes the characteristics that dimethyl phthalate and dibutyl phthalate are incompatible with red blood cells and do not damage the red blood cells, and prepares the old red blood cells and the new red blood cells into solution with the density between that of the old red blood cells, so that the old red blood cells and the new red blood cells can be obviously divided into four layers in a capillary tube in the high-speed centrifugal separation process, the new red blood cells and the old red blood cells are completely separated, the accuracy of a test result is ensured, and the precision of the test result is enhanced. The separation medium has simple material consumption, needs short time for separating new and old red blood cells, uses less blood, has intuitive result and small error, and can be widely applied to conventional separation detection.

Drawings

FIG. 1 is a schematic diagram of the process of the present invention after separating old and new red blood cells.

FIG. 2 is a photograph of the capillary tube after separating old and new red blood cells in example 1.

FIG. 3 is a graph showing the results of Rh typing of the novel erythrocytes in example 1 and comparative example 1; wherein: a is example 1 and B is comparative example 1.

Detailed Description

In the present invention, after separating old and new red blood cells by using a separation medium, the condition of the capillary after separation is shown in fig. 1 according to the principle.

Example 1

1) Configuration of the separation medium: weighing 48 microliters of dimethyl phthalate, 96 microliters of dibutyl ester, uniformly stirring, and testing the density, wherein the relative density is 1.095 +/-0.001.

2) Separation of old and new red blood cells: taking a capillary tube, firstly filling 66mm packed red blood cells, then filling 3uL of separation medium into the capillary tube, wherein the length of the separation medium is about 3mm, finally filling 5mm of sealing mud into the end of the red blood cells for sealing, then placing the capillary tube in an ultracentrifuge, centrifuging for 20-25min at 11000r, and after the centrifugation is finished, dividing the capillary tube into four layers, as shown in figure 2, wherein the separation condition of the new and old red blood cells in figure 2 is completely consistent with that in figure 1, which shows that the separation medium of the invention can well separate the new and old red blood cells, cutting the reticulocyte or the new and old red blood cells at the near heart end by a grinding wheel, testing the Rh parting result of the reticulocyte or the new and old red blood cells, and testing the Rh parting result of the reticulocyte or the new and old red blood cells, wherein the Rh parting result of the reticulocyte or the new and old red blood cells is.

Comparative example 1

Filling 69mm packed red blood cells into a capillary tube, filling 4mm sealing mud into the capillary tube for sealing, then placing the capillary tube in an ultracentrifuge, centrifuging for 20-25min at 11000r (please supplement the rotating speed and time), after the centrifugation is finished, carefully observing the boundary, intercepting reticulocyte or new red blood cells at the near-core end by a grinding wheel, and testing the Rh parting result of the reticulocyte or new red blood cells, wherein the parting result is CCDee, and the result is shown in figure 3B.

The final typing results of fig. 3A in example 1 and fig. 3B in comparative example 1 are not different, but the typing results in fig. 3A are more obvious from the picture, while a small amount of old red blood cells may exist in fig. 3B, and a phenomenon which is not obvious on the picture of the typing results is not obvious; the separation medium of the invention can better separate old and new red blood cells and reduce detection errors.

Claims

1. A separation medium for new and old red blood cells is characterized by comprising dimethyl phthalate and dibutyl phthalate, wherein the volume ratio of the dimethyl phthalate to the dibutyl phthalate is (0.95-1.05) to (1.95-2.05).

2. Separation medium according to claim 1, wherein the volume ratio of dimethyl phthalate to dibutyl phthalate is 1: 2.

3. A method for preparing a medium for separating old and new red blood cells according to claim 1 or 2, comprising the steps of: mixing the two materials according to the set volume ratio of dimethyl phthalate to dibutyl phthalate, uniformly mixing, and testing the density of the mixture to obtain the separation medium for the new and old red blood cells.

4. The method according to claim 3, wherein the density of the medium for separating old and new red blood cells is 1.094 to 1.096.

5. Method for separating old and new red blood cells using a separation medium for old and new red blood cells according to claim 1 or 2, comprising the steps of: firstly, filling packed red blood cells, then filling a separation medium into a capillary, then filling 4mm sealing mud into the red blood cell end, sealing, then placing the capillary in a high-speed centrifuge for separation, and after separation, dividing the capillary into four layers, wherein the proximal end is a small amount of plasma layer, a reticulocyte or new erythroblast layer, a middle layer separation medium layer and the distal end is an old erythroblast layer; cutting out the reticulocyte or new erythrocyte layer at the proximal end by using a grinding wheel for testing; the amount of reticulocyte or neoerythrocyte layer will vary from patient to patient.

6. The method for separating old and new red blood cells according to claim 5, wherein the packed volume of red blood cells is 65-67 mm, the packed volume of the old and new red blood cell separation medium is 2.5-3.5 μ L, and the packed volume of the old and new red blood cell separation medium is 2.5-3.5 mm.

7. The method for separating old and new red blood cells according to claim 5, wherein the centrifugation speed is 10000-12000 r; the centrifugation time is 20-25 min.