Umbilical cord blood source platelet separation method

Abstract

The invention provides an umbilical cord blood source platelet separation method which comprises the following steps: firstly, removing red blood cells in waste umbilical cord blood by using inverted heavy centrifugation, connecting a blood collection bag after red blood removal with a second plasma separation bag, and performing upright light centrifugation to obtain a plasma mixture; connecting the plasma mixture of the second plasma separation bag with the residues of the blood collection bag, carrying out upright heavy centrifugation, separating pure plasma at the upper part of the second plasma separation bag, backwashing the pure plasma into the residues of the blood collection bag, and uniformly mixing the pure plasma and the residues of the blood collection bag; the blood collection bag and a second plasma separation bag from which part of plasma is separated are positively arranged together for light centrifugation, then plasma on the albuginea layer is separated into the second plasma separation bag, and plasma contents in the second plasma separation bag are evenly mixed; and finally, connecting the second plasma separation bag containing the plasma contents with a third plasma separation bag, and carrying out upright heavy centrifugation to slowly separate out upper-layer plasma, so that the remainder is the concentrated platelets. The problem that the content of platelets in umbilical cord blood is low is solved, and the risk that other cell debris is introduced into the product to increase the use of the platelets is avoided.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a method for separating platelets from umbilical cord blood. Background Art

[0002] Platelets (PLT) have the functions of promoting hemostasis, accelerating coagulation and maintaining the integrity of blood vessel walls. When thrombocytopenia occurs, minor trauma or just increased blood pressure can cause petechiae or even large bruises, which can even be life-threatening in severe cases. Thrombocytopenia is one of the important problems encountered in the clinical treatment of malignant blood diseases and tumors. When tumor patients receive concentrated chemotherapy and radiotherapy, the bone marrow will be suppressed to varying degrees. Bone marrow suppression and its complications of anemia, granulocytopenia and thrombocytopenia are the main factors causing patient death, and are also the main obstacles to limiting the dose of radiotherapy and chemotherapy and improving the efficacy in clinical practice. At present, recombinant EPO, G-CSF, and GM-CSF have been used clinically to improve anemia and granulocytopenia caused by chemotherapy. However, there is currently no drug that can effectively improve thrombocytopenia after radiotherapy and chemotherapy. Platelet transfusion plays an important role in preventing spontaneous bleeding in patients and ensuring that anticancer drugs and chemotherapy drugs are used up according to the prescribed course of treatment. Platelet transfusion can reduce the risk of severe bleeding, but its clinical application is limited by many factors, such as fever, disseminated intravascular coagulation, hypersplenism, previous BMT, and human leukocyte antigen (HLA) antibodies, etc. These factors can affect the effect of platelet transfusion, and platelet transfusion may also be complicated by infection and immune complications.

[0003] Most of the blood products used in clinical practice, including platelets, are therapeutic products made by separating and purifying the plasma of healthy people or specific immune people. Therefore, they have a certain scarcity of raw materials and a very short shelf life for clinical use, which is only 5 days. At present, plasma can be collected in two ways: single plasma collection and recycled plasma. All raw plasma in China comes from single plasma collection, while the proportion of recycled plasma in the United States accounts for 10%, and the proportion in some European countries even reaches 30%. The gap between supply and demand of blood products in my country is as high as 50%. Sufficient plasma raw materials are the prerequisite for the rapid development of the blood products industry. my country's upstream resources for blood products are limited, but downstream demand remains unchanged. Due to China's large population base, the intensification of the aging trend, and the continuous increase in corresponding indications that stimulate the continuous growth of demand for blood products, there are currently few or almost no completely alternative products, resulting in a huge gap in blood products in my country, including platelets, which restricts the development of the industry, but also fully proves that the market prospects of blood products such as platelets are broad.

[0004] Therefore, how to effectively separate and purify platelets from umbilical cord blood is a problem that needs to be solved. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for separating platelets from umbilical cord blood, which organically combines the process of separating and preparing umbilical cord blood platelets and the process of platelet purification while expanding the source of platelets, thereby solving the problem of low platelet content in umbilical cord blood and not introducing other cell fragments into the product to increase the risk of platelet use.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] The present invention provides a method for separating platelets from umbilical cord blood, using discarded umbilical cord blood as a raw material to separate and purify platelets therefrom, comprising the following steps:

[0008] Step 1, connecting the discarded umbilical cord blood collection bag to the first plasma separation bag, and separating the red blood cells in the discarded umbilical cord blood into the first plasma separation bag by inverted re-centrifugation;

[0009] Step 2, connecting the blood collection bag after the red blood cells are removed to a new second plasma separation bag, placing it upright and lightly centrifuging it to separate the plasma into the second plasma separation bag to obtain a plasma mixture;

[0010] Step 3, connecting the plasma mixture in the second plasma separation bag with the remainder of the blood collection bag, centrifuging in an upright position, separating the pure plasma in the upper part of the second plasma separation bag, and backwashing it into the remainder of the blood collection bag and mixing;

[0011] Step 4, placing the blood collection bag and the second plasma separation bag for separating part of the plasma upright and gently centrifuging, controlling the flow rate, and then separating the plasma on the buffy coat into the second plasma separation bag, and mixing the plasma content in the second plasma separation bag;

[0012] Step 5, connect the second plasma separation bag containing the plasma content obtained in step 4 to a new third plasma separation bag, place it upright and re-centrifuge it, slowly separate the upper plasma, and the remainder is the concentrated platelets.

[0013] Furthermore, in step 2, the separation flow rate is controlled when approaching the buffy coat layer, and the separation is performed slowly. When a small amount of buffy coat layer enters the plasma line, the separation is stopped immediately.

[0014] Furthermore, in steps 1, 3 and 5, the re-centrifugation conditions are: 2000G, 5min.

[0015] Furthermore, in steps 2 and 4, the light centrifugation conditions are: 500G, 3min.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The method for separating platelets from umbilical cord blood of the present invention uses discarded umbilical cord blood as raw material to separate and purify platelets therefrom, and organically combines the process of separating and preparing umbilical cord blood platelets with the process of purifying and freezing platelets, thereby solving the problem of low platelet content in umbilical cord blood and preventing the introduction of other cell fragments into the product to increase the risk of platelet use;

[0018] 2. Existing platelet preparation usually adopts density centrifugation. Red blood cells are the main factor affecting the platelet separation effect. Light centrifugation allows platelets to be suspended in plasma; heavy centrifugation allows red blood cells to settle quickly. The present invention uses heavy centrifugation to separate red blood cells while reducing the centrifugation time to 5 minutes, thereby reducing the sedimentation of platelets as much as possible and keeping them suspended in plasma. The existing conventional gravity centrifugation method for directly separating platelets has a platelet recovery rate of 50%-70%. After separating the red blood cells, the present invention uses the plasma in the umbilical cord blood to backwash the separated red blood cells, which can increase the recovery of platelets by 13.22%±1.56%, so that the average platelet recovery rate after preparation by the method of the present invention reaches 84.83%±4.65%, the average white blood cell removal rate is 96.18%, and the average red blood cell removal rate is 99.42%;

[0019] 3. The umbilical cord blood-derived platelets isolated by the method of the present invention are activated and proliferated in vitro. On the basis of treating the primary disease of the patient, they can play their hematopoietic support treatment function in the patient's body, provide good support for improving the patient's quality of life and promoting the treatment of the primary disease. They also have very broad and important application value in the support treatment of various anemic diseases, infectious diseases, hereditary diseases and autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0021] Figure 1 This is a schematic diagram of the recovery rate of plasma and suspended layer PLT in Comparative Example 1;

[0022] Figure 2 This is a schematic diagram of the results of the residual amount of PLT after separation of the plasma and the suspension layer in Comparative Example 1;

[0023] Figure 3 This is a schematic diagram comparing the recovery rates of PLT separated by different preparation methods in comparative example 2. DETAILED DESCRIPTION

[0024] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0025] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0026] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention in detail, and are not used to limit the present invention.

[0027] Example 1

[0028] This embodiment provides a method for separating platelets from umbilical cord blood, using discarded umbilical cord blood as a raw material to separate and purify platelets therefrom, comprising the following steps:

[0029] Step 1: The discarded cord blood collection bag is connected to the first plasma separation bag, and the red blood cells in the discarded cord blood are separated into the first plasma separation bag by inverted gravity centrifugation at 2000G for 5 minutes, thereby removing most of the red blood cells in the cord blood. The red blood cell removal accounts for about 60%-75%, and the red blood cell removal flow rate needs to be very slow to prevent the formation of a funnel vortex and cause platelet loss.

[0030] Step 2, connect the blood collection bag after removing red blood cells to a new second plasma separation bag, centrifuge lightly at 500G for 3 minutes, separate the plasma into the second plasma separation bag, control the separation flow rate when approaching the white film layer, and separate slowly. When a small amount of white film layer enters the plasma pipeline, stop immediately to obtain a plasma mixture.

[0031] Step 3, connect the plasma mixture in the second separation bag with the remainder of the blood collection bag, centrifuge at 2000G for 5 minutes, separate the pure plasma in the upper part of the second separation bag, and backwash it into the remainder of the blood collection bag and mix well.

[0032] Step 4, place the blood collection bag and the second plasma separation bag for separating part of the plasma upright and centrifuge lightly at 500G for 3 minutes to control the flow rate, then separate the plasma on the buffy coat into the second plasma separation bag, and mix the plasma contents in the second plasma separation bag.

[0033] Step 5, connect the second plasma separation bag containing the plasma content obtained in step 4 to a new third plasma separation bag, centrifuge upright at 2000G for 5 minutes, slowly separate the upper 2 / 3 of the plasma, and the remainder is the concentrated platelets.

[0034] Before and after the above separation treatment, the volume of plasma was measured and the cells were counted. The counting results of whole blood and concentrated samples were used to calculate the recovery rate.

[0035] The calculation formula is as follows:

[0036]

[0037] Table 1 PLT preparation and concentration recovery rate according to the embodiment method

[0038]

[0039] Comparative Example 1 - Density Gradient Centrifugation to Separate Platelets

[0040] This comparative example provides a method for separating platelets using a traditional density gradient centrifugation method, the specific steps of which are:

[0041] A cord blood sample was subjected to a complete blood count. After a large number of red blood cells were removed by inverted re-centrifugation (2000G5min), the plasma mixture was evenly divided into 5ml samples per tube for gradient centrifugation. The centrifugal force was selected from 100G to 800G for 5 minutes. The centrifugal force was increased by 50G every time. Two samples were prepared at the same centrifugal force.

[0042] After centrifugation, extract the red blood cell layer, plasma layer and suspension layer of each sample.

[0043] The volume of each cell layer was calculated and the cells were counted. The recovery rate was calculated based on the counting results of whole blood and concentrated samples. The recovery rate curves of plasma and suspension layer PLT under different centrifugal forces were obtained.

[0044] The calculation formula is as follows:

[0045]

[0046] Table 2 Preparation conditions and PLT recovery rate of comparative example 1

[0047]

[0048]

[0049] Figure 1 Figure 2 is the PLT recovery curve of plasma and suspension layer. Figure 2 is the PLT recovery rate curve of the residual in the red blood cells. Figure 1 , Figure 2 Combined with the analysis, the following conclusions are drawn:

[0050] 1. When the centrifugal force ranges from 100G to 250G, the PLT recovery rate is at an extremely low stage. This indicates that the centrifugal force at this stage is too small to effectively separate platelets from other components. A large number of platelets are lost.

[0051] 2. The PLT recovery rate increased significantly when the centrifugal force was increased from 300G. The overall PLT recovery rate was high and the experimental results were stable in the centrifugal force range of 500G-750G.

[0052] 3. After heavy centrifugation at 2000G to remove red blood cells, the overall centrifugal force is relatively large. Finally, the light centrifugation method of 500G was selected to shorten the overall preparation time.

[0053] In summary, the recovery rate of PLT in the plasma suspension layer is higher in the centrifugal force range of 500G-750G, while the recovery rate of PLT in the residual amount after separation in this range is lower.

[0054] Comparative Example 2

[0055] This comparative example adopts a method of directly separating platelet-rich plasma by single centrifugal force and single centrifugation. Table 3 below shows some specific experimental data.

[0056] Table 3 Partial experimental results of direct separation of platelet-rich plasma by single centrifugal force

[0057]

[0058] According to the analysis of the data results in Table 3 above, a single centrifugal force cannot efficiently prepare platelet-rich plasma. The PLT recovery rate is basically 50-70%, and a large number of platelets are lost during the preparation process. Further adjustment of the centrifugal force still cannot improve the recovery rate. It is necessary to achieve the purpose of preparing platelet-rich plasma by combining heavy and light centrifugation. At the same time, there are few residual white blood cells and a high red blood cell removal rate.

[0059] Furthermore, in order to explore the recovery of PLT in umbilical cord plasma after heavy centrifugation to remove a large number of red blood cells under different low-speed centrifugal forces, the specific results are as follows Figure 3 shown.

[0060] Figure 3 There are three different experimental steps, among which, blue-single centrifugal force is a method of directly separating platelet-rich plasma with only one centrifugation. Red-red-removed and de-plasma is a method of separating platelets with only one heavy centrifugation and one light centrifugation, without a backwashing step. Gray-backwash preparation experimental method is to add a backwashing step after the red-removed and de-plasma preparation method. Gray-backwash preparation data is the final experimental method data (Example 1 of the present application).

[0061] After a large number of experiments, it was found that red blood cells are the main factor affecting the platelet separation effect. Therefore, this application attempts to change the method of directly separating platelets from a single centrifugation to a multi-step centrifugation, first using heavy centrifugation to remove a large number of red blood cells. After experiments, the centrifugation conditions were gradually increased from 200G to 2000G. It was finally determined that the PLT residue of red blood cells removed at 2000G for 5 minutes was the lowest. And after a large number of repetitive experiments, it was learned that after heavy centrifugation to remove a large number of red blood cells, the PLT recovery rate in the separated platelet-rich plasma was increased to 60-70%.

[0062] After consulting relevant literature, it was found that the separation of platelets is directly related to the hematocrit in umbilical cord blood. After repeated experiments, it was determined that adding the operation step of backwashing with pure plasma after the first separation of plasma can further reduce the hematocrit of the initial platelet-rich plasma. After a large number of experimental explorations, it was found that the overall operation steps of heavy centrifugation-light centrifugation-backwashing-plasma separation described in Example 1 can ultimately increase the recovery rate of PLT to more than 80%. Thereby greatly improving the recovery effect of platelet preparation.

[0063] According to the data comparison between the comparative example and the method described in the embodiment of the present application. The comparative example only carried out a single centrifugal force and a single centrifugation. The results of multiple data showed that the PLT recovery rate was less than 60% and the PLT loss was large. After multiple experiments, the experimental method was improved. The PLT recovery rate increased from 50-60% to 80-90%. And multiple repetitive experiments can meet the higher recovery rate. It shows that the experimental repeatability of the experimental conditions described in the embodiment is good.

[0064] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred arrangement scheme, a person skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for separating platelets from umbilical cord blood, characterized in that: Using discarded umbilical cord blood as raw material, separating and purifying platelets therefrom, the method comprises the following steps: Step 1, connecting the discarded umbilical cord blood collection bag to the first plasma separation bag, and separating the red blood cells in the discarded umbilical cord blood into the first plasma separation bag by inverted re-centrifugation; Step 2, connecting the blood collection bag after the red blood cells are removed to a new second plasma separation bag, placing it upright and lightly centrifuging it to separate the plasma into the second plasma separation bag to obtain a plasma mixture; Step 3, connecting the plasma mixture in the second plasma separation bag with the remainder of the blood collection bag, centrifuging in an upright position, separating the pure plasma in the upper part of the second plasma separation bag, and backwashing it into the remainder of the blood collection bag and mixing; Step 4, placing the blood collection bag and the second plasma separation bag for separating part of the plasma upright and gently centrifuging, controlling the flow rate, and then separating the plasma on the buffy coat into the second plasma separation bag, and mixing the plasma content in the second plasma separation bag; Step 5, connect the second plasma separation bag containing the plasma content obtained in step 4 to a new third plasma separation bag, place it upright and re-centrifuge it, slowly separate the upper plasma, and the remainder is the concentrated platelets.

2. The method according to claim 1, characterized in that: In step 2, the separation flow rate is controlled when approaching the buffy coat layer, and the separation is slow. When a small amount of buffy coat layer enters the plasma line, the separation is stopped immediately.

3. The method according to claim 1, characterized in that: In steps 1, 3 and 5, the re-centrifugation conditions are: 2000G, 5min.

4. The method according to claim 1, characterized in that: In steps 2 and 4, the light centrifugation conditions are: 500G, 3min.

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