Whole blood platelet enrichment method

Abstract

The invention discloses a whole blood platelet enrichment method, which comprises the following steps: carrying out centrifugal treatment on a whole blood sample subjected to anticoagulation treatment to obtain upper plasma and a lower blood cell layer, and centrifuging the upper plasma layer again to obtain a precipitate, namely a first part of platelets, and a supernatant component, namely platelet-deficient plasma; mixing upper-layer platelet-deficient plasma separated after the second-time centrifugal treatment with a lower-layer blood cell layer separated after the first-time centrifugal treatment to prepare a whole blood sample again, and then carrying out centrifugal treatment on the newly prepared whole blood sample, and centrifuging the upper platelet-rich plasma layer obtained by separation again to obtain a precipitate, namely a second part of platelets, and mixing and collecting the first part of platelets and the second part of platelets to obtain all enriched platelets. The platelet enrichment method disclosed by the invention has the advantages of simplicity in operation, high platelet acquisition rate and high acquisition number of large platelets.

Description

Technical Field

[0001] The present invention relates to the technical field of platelet extraction, in particular to a whole blood platelet enrichment method. Background Art

[0002] In recent years, platelet transcriptome analysis, as a cancer liquid biopsy technique, has demonstrated significant advantages over other liquid biopsy methods. Platelets are readily available, contain abundant RNA, and specifically process RNA in response to external cues. Studies have shown that platelets play a central role in both systemic and local responses to tumor growth. Activation of platelet surface receptors and lipopolysaccharide (LPS) by external stimuli can induce specific splicing of pre-mRNA in circulating platelets. Platelets are not only the first responders to trauma but also the first responders to tumor development and metastasis, participating in tumor angiogenesis and metastasis. Platelets may undergo sequence-specific mRNA splicing in response to stimulation by tumor cells and stromal or immune cells within the tumor microenvironment. Combined with platelets' ability to perform specific splicing in response to external stimuli and their ability to directly uptake circulating mRNA, platelets, which contain a large number of dynamic mRNAs, may be useful in cancer diagnosis. Professor Wurdinger's team sequenced platelet RNA from 779 individuals and found that TEPs transcriptome data could distinguish between normal subjects and lung cancer, with diagnostic accuracy unaffected by factors such as age and tumor stage. In recent years, the results have been validated by multiple teams, and platelet transcriptome analysis has demonstrated promising diagnostic value not only in lung cancer, but also in breast cancer, ovarian cancer, and other cancers.

[0003] However, the key to analyzing the peripheral blood platelet transcriptome for cancer diagnosis lies in the enrichment and extraction of peripheral blood platelets. The current mainstream platelet extraction method developed by Professor Wurdinger's team involves centrifuging anticoagulated whole blood samples at 120g for 20 minutes to separate the upper platelet-rich plasma layer from the lower red blood cell layer. The upper platelet-rich plasma is then centrifuged at 360g for 20 minutes to separate the platelet-poor plasma and platelet pellet. This simple and widely accepted method, however, only yields approximately 30%-50% of platelets in the peripheral blood of cancer patients, leaving approximately 50%-70% unextracted. Sequencing revealed that these unextracted platelets may be involved in multiple mechanisms related to tumor progression, making enrichment of these platelets crucial for cancer research. Summary of the Invention

[0004] In response to the above problems, the present invention provides a whole blood platelet enrichment method, which has the advantages of simple operation, high platelet yield, and the ability to enrich platelets that are missed in traditional methods.

[0005] The technical solution adopted in the present invention is as follows:

[0006] A whole blood platelet enrichment method, characterized in that the extraction method is as follows: a whole blood sample after anticoagulation treatment is centrifuged to obtain an upper plasma layer and a lower blood cell layer, the upper plasma layer is centrifuged again to obtain a precipitate, namely a first portion of platelets, and the supernatant component is platelet-poor plasma; the upper platelet-poor plasma separated after the second centrifugation is mixed with the lower blood cell layer separated after the first centrifugation to prepare a new whole blood sample, and the newly prepared whole blood sample is centrifuged again, and the upper platelet-rich plasma layer obtained by separation is centrifuged again to obtain a precipitate, namely a second portion of platelets, and the first portion of platelets and the second portion of platelets are mixed and collected, i.e., all enriched platelets.

[0007] The working principle of the above technical solution is as follows:

[0008] The anticoagulated whole blood sample is centrifuged to obtain an upper plasma layer and a lower blood cell layer. The upper plasma layer is centrifuged again to obtain the precipitate (the first portion of platelets), and the supernatant component is the platelet-poor plasma. The platelet-poor plasma is cross-mixed with the lower blood cell layer obtained initially to prepare a new whole blood sample. The re-prepared whole blood sample is centrifuged again to extract platelets, and all extracted platelets are pooled and collected.

[0009] The platelets of tumor patients may be in an easily activated state due to the stimulation of tumor cells, so there are more large-volume and mutually adhered and aggregated platelets in the peripheral blood of tumor patients. In traditional extraction methods, these platelets will be centrifuged into the lower layer of liquid during the first centrifugation. The present invention can recover these large platelets by re-preparing the whole blood sample and centrifuging it again, which can effectively solve the technical problem of low platelet recovery rate in traditional extraction methods, in which the platelets sink into the blood cell layer during the first centrifugation of the whole blood sample, and cannot be extracted. The method of the present invention is helpful in collecting large-volume platelets, which is beneficial to related research on tumors and platelets.

[0010] In a further technical solution, the method of performing at least two stages of centrifugation on the anticoagulated blood sample is as follows:

[0011] A1. Place the anticoagulated whole blood sample in a hematology analyzer to detect and record the platelet count in the whole blood. Place the tested whole blood sample in a centrifuge, adjust the speed, and centrifuge for 20 minutes to separate the upper platelet-rich plasma layer and the lower blood cell layer.

[0012] A2. Pipette the upper platelet-rich plasma layer into another new EP tube, test again and record the platelet count, while leaving the lower blood cell layer in the original EP tube.

[0013] A3. Place the upper platelet-rich plasma layer in a centrifuge and adjust the speed for 20 minutes to separate the upper platelet-poor plasma layer and the lower platelet layer. Pipette the upper platelet-poor plasma layer into a new EP tube, retest the tube, and record the platelet count. The lower sediment is the first portion of enriched platelets.

[0014] In a further technical solution, a whole blood sample is re-prepared and then centrifuged as follows:

[0015] B1. Evenly mix the upper platelet-poor plasma layer obtained from the second centrifugation with the lower blood cell layer obtained from the first centrifugation to prepare a second whole blood sample;

[0016] B2. Place the second whole blood sample in a centrifuge, adjust the speed, and centrifuge for 20 minutes to separate the upper platelet-rich plasma layer and the lower precipitate layer, which is the second portion of enriched platelets.

[0017] B3. Mix the first portion and the second portion of platelets to obtain all the enriched platelets.

[0018] Furthermore, in step A1, the centrifugal speed is 120g.

[0019] Furthermore, in step A2, the centrifugal speed is 360g.

[0020] Furthermore, in step B2, the centrifugal speed is 120g.

[0021] The beneficial effects of the present invention are: by re-preparing the whole blood sample, the present invention can effectively solve the technical problem in the traditional extraction method that when the whole blood sample is centrifuged for the first time, large and aggregated platelets sink into the blood cell layer and cannot be extracted, resulting in a low platelet recovery rate. The present invention has the advantages of simple operation, high platelet acquisition rate, and high number of large platelets obtained. DETAILED DESCRIPTION

[0022] The embodiments of the present invention are described in detail below.

[0023] Example:

[0024] A whole blood platelet enrichment method comprises the following steps:

[0025] S1. Place the anticoagulated whole blood sample (P1) in a hematology analyzer to detect and record the number of platelets in the whole blood. Place the tested whole blood sample in a centrifuge at a speed of 120 g for 20 minutes to separate the upper platelet-rich plasma layer (PRP1) and the lower blood cell layer (B1).

[0026] S2. Pipette the upper platelet-rich plasma layer (PRP1) into another new EP tube, test again on the machine and record the platelet count;

[0027] S3. Place the tested upper platelet-rich plasma layer (PRP1) in a centrifuge at 360 g for 20 min to separate the upper platelet-poor plasma layer (PPP1) and the lower platelet layer (PLA1);

[0028] S4, mixing the upper platelet-poor plasma layer (PPP1) obtained after the test with the lower blood cell layer (B1) obtained by the first centrifugation to obtain a second whole blood sample (P2);

[0029] S5. Place the second whole blood sample (P2) in a centrifuge at 120 g for 20 minutes to separate the upper platelet-rich plasma layer (PRP2) and the lower blood cell layer (B2). Pipette the upper platelet-rich plasma layer (PRP2) into a new soft tube / EP tube, test again on the centrifuge, and record the platelet count.

[0030] S6. Place the tested upper platelet-rich plasma layer (PRP2) in a centrifuge, adjust the centrifugal speed to 360g, and centrifuge for 20 minutes to obtain an upper platelet-poor plasma layer (PPP2) and a lower platelet layer (PLA2). Pipette the two lower platelet layers (PLA1 and PLA2) into another new soft test tube / EP tube to collect all the enriched platelets.

[0031] The above extraction method was used to extract platelets from 6 groups of whole blood samples. Each group used 2 ml of whole blood sample. The data of the 6 groups are as follows:

[0032]

[0033] Calculate the recovery rates of the above 6 groups of tests, Among them, the HTC value is the hematocrit, V is the whole blood volume 4 ml, and the HTC values ​​of the six groups are 0.472, 0.409, 0.424, 0.39, 0.41, and 0.388, respectively. It can be clearly seen from the above two calculation formulas that the platelet recovery rate of the present invention is significantly higher than that of the traditional method. Moreover, more importantly, the present invention can effectively recover larger platelets that have settled in the lower layer liquid during the first centrifugation.

[0034] The results of the above 6 groups of tests are as follows:

[0035] The recovery rates of the six groups of platelets extracted by the extraction method of the present invention are 99.63%, 97.52%, 96.53%, 96.53%, 94.92%, and 93.36%, respectively, and the average platelet recovery rate is 96.41%.

[0036] The recovery rates of the six groups of platelets extracted using the traditional extraction method were 73.69%, 63.53%,

[0037] 67.85%, 65.36%, 66.38%, 65.88%, and the average platelet recovery rate was 67.11%;

[0038] Anticoagulated whole blood samples were centrifuged at 120g for 20 minutes to separate an upper platelet-rich plasma layer and a lower red blood cell layer. The upper platelet-rich plasma was then centrifuged at 360g for 20 minutes to separate platelet-poor plasma and a platelet pellet. The platelet-poor plasma and the lower red blood cell layer were then mixed and centrifuged at 120g for 20 minutes to separate an upper platelet-rich plasma layer and a lower red blood cell layer. The upper platelet-rich plasma was again centrifuged at 360g for 20 minutes to separate platelet-poor plasma and a platelet pellet. The platelets obtained from these two extractions were combined to form the enriched peripheral blood platelets. This method can recover nearly all platelets remaining in the lower red blood cell layer, achieving a platelet total of nearly 100% of the total peripheral blood platelet count. This provides technical support for TEPs research.

[0039] It can be seen that the platelet recovery rate using the extraction method of the present invention is significantly higher than that using the traditional extraction method.

[0040] The above-described embodiments merely represent specific implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A whole blood platelet enrichment method, characterized in that: The extraction method is as follows: the whole blood sample after anticoagulation treatment is centrifuged to obtain an upper plasma layer and a lower blood cell layer, the upper plasma layer is centrifuged again to obtain a precipitate, i.e., the first part of platelets, and the supernatant component is the platelet-poor plasma; the upper platelet-poor plasma separated after the second centrifugation is mixed with the lower blood cell layer separated after the first centrifugation, and the whole blood sample is re-prepared, and the newly prepared whole blood sample is centrifuged again, and the upper platelet-rich plasma layer separated is centrifuged again to obtain a precipitate, i.e., the second part of platelets, and the first part of platelets and the second part of platelets are mixed and collected, i.e., all the enriched platelets.

2. A whole blood platelet enrichment method according to claim 1, characterized in that: The method for processing anticoagulated blood samples is as follows: A1. Place the whole blood sample after anticoagulation treatment in a blood cell analyzer to detect and record the number of platelets in the whole blood. Place the whole blood sample after the test in a centrifuge, adjust the speed, and centrifuge for 20 minutes to separate the upper platelet-rich plasma layer and the lower blood cell layer; A2. Pipette the upper platelet-rich plasma layer into another new EP tube, test it again and record the platelet count, and leave the lower blood cell layer in the original EP tube; A3. Place the upper platelet-rich plasma layer after the test in a centrifuge, adjust the speed, centrifuge for 20 minutes, separate the upper platelet-poor plasma layer and the lower platelet layer, draw the upper platelet-poor plasma layer into another new EP tube, test it again on the machine and record the platelet count. The lower sediment is the first part of the enriched platelets.

3. A whole blood platelet enrichment method according to claim 2, characterized in that: The method for re-preparing a whole blood sample and centrifuging the newly prepared whole blood sample is as follows: B1. Evenly mix the upper platelet-poor plasma layer obtained by the second centrifugation with the lower blood cell layer obtained by the first centrifugation to prepare a second whole blood sample; B2. Place the second whole blood sample in a centrifuge, adjust the speed, and centrifuge for 20 minutes to separate the upper platelet-rich plasma layer and the lower precipitate, which is the second part of the enriched platelets. B3. Mix the first part and the second part of platelets to enrich all the platelets.

4. A whole blood platelet enrichment method according to claim 3, characterized in that: In step A1, the centrifugal speed is 120g.

5. A whole blood platelet enrichment method according to claim 3, characterized in that: In step A2, the centrifugal speed is 360g.

6. A whole blood platelet enrichment method according to claim 3, characterized in that: In step B2, the centrifugal speed is 120g.

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