CN107858325B - Method for diagnosing and prognosis monitoring of blood system diseases by using marrow small granulocyte suspension centrifugal enrichment method - Google Patents
Method for diagnosing and prognosis monitoring of blood system diseases by using marrow small granulocyte suspension centrifugal enrichment method Download PDFInfo
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Abstract
The invention relates to the technical field of diagnosis and prognosis monitoring of blood system diseases, in particular to a method for diagnosing and prognosis monitoring of blood system diseases by applying a marrow small granulocyte suspension centrifugal enrichment method.
Description
Technical Field
The invention relates to the technical field of blood system disease diagnosis and prognosis monitoring, in particular to a method for applying a marrow granulocyte suspension centrifugal enrichment method to blood system disease diagnosis and prognosis monitoring.
Background
Although the cytology examination of bone marrow smears is the most important test for diagnosing hematopathy and for prognosis monitoring of hematopathy, the dilution of blood (venous blood) in blood sinuses cannot be avoided by the bone marrow smears of patients with successful puncture are shown to be diluted by (20+/-8)% of blood sinuses. Thus, the positive rate of cells such as primordial cells, megakaryocytes, tumor cells, plasma cells, macrophages, parasites, etc. can be reduced. Causing false negative and missing diagnosis. The patient is burdened by the complete diluter who is required to puncture again, and potential medical disputes exist. The trouble of re-pumping and the risk of medical alarm disputes are brought to doctors.
Bone marrow biopsy is also an important test for the diagnosis of hematological disorders. The inspection method preserves the original tissue structure of bone marrow tissue, avoids dilution of blood in blood sinus, and is an essential structure reflection of bone marrow tissue structural components. However, since the cells are not developed, the observation of the cells is insufficient, and the judgment of the cell-stage properties is affected. The manufacturing process is also complicated, corresponding instruments and equipment are expensive, the requirement is high, and the reporting period is long (the result is obtained about one week), so that the diagnosis and treatment of the patient are delayed. There is also a potential medical alarm dispute. At present, only a few three-dimensional hospitals and part of third-party inspection institutions are developed in China. It is inconvenient for the patient to check.
The bone marrow particles are small groups of bone marrow tissues and also preserve the essential structures of the bone marrow tissues, and the bone marrow particle pull tab is formed by collecting a plurality of bone marrow particles of a patient, placing the bone marrow particles on one end of a glass slide, extruding the bone marrow particles by using another glass slide, pushing the bone marrow particles in parallel, and performing microscopic examination after Rayleigh staining. Its advantages are no dilution of blood in blood sinus, full cell expansion, and easy observation of fine structure. Is also an essential structural reflection of bone marrow tissue. And the manufacturing process is simple and easy to implement. The primary hospitals can operate. The disadvantage is that the cells are spread out and distributed uniformly, and the cells are damaged. Affecting the observation.
All three detection methods are limited to morphological detection, and cannot be detected in the aspects of accurate detection such as immunology, molecular biology and other methodologies.
Disclosure of Invention
The invention aims to provide a method for diagnosing and prognosis monitoring diseases of a blood system by using a marrow small granulocyte suspension centrifugal enrichment method, which separates hematopoietic cells from tissues by using a physical method, can avoid dilution of blood in blood sinuses, ensures that the cells are fully expanded, facilitates observation of microstructure, makes accurate and clear judgment on original structural components of marrow tissues, can be applied to the aspects of accurate inspections such as immunology, molecular biology and the like, and the detected data value is more similar to the data value which is supposed to be of the marrow tissues per se.
In order to solve the technical problems, the invention is solved by the following technical scheme:
a method for diagnosing a disease of the blood system by applying a marrow granulocyte suspension centrifugation enrichment method, comprising the following steps:
a. extracting about 0.1-0.3ml of bone marrow liquid of a patient as bone marrow smear, then pumping about 2-6ml of bone marrow liquid into an anticoagulation tube of EDTA-K2, and uniformly mixing;
b. placing the anticoagulation tube into a horizontal throwing type medical centrifuge for 500-1000 revolutions per minute, and centrifuging for 3-8 minutes to enrich a bone marrow small particle layer;
c. taking about 1ml of the mixed solution of the bone marrow small particle floating layer at the uppermost layer and the plasma of a patient into a centrifuge tube by using a disposable plastic straw;
d. sucking the mixed solution of the bone marrow pellets and the blood plasma in the centrifuge tube by using a medical injector with the specification of 2 milliliters, injecting the mixed solution back into the centrifuge tube, and repeating the steps for 3 times to prepare bone marrow cell suspension;
e. replacing a medical injector with the specification of 1 milliliter to suck the mixed solution of the bone marrow pellets and the blood plasma in the centrifuge tube into a syringe, and then injecting the mixed solution back into the centrifuge tube, and repeating the steps for 3 times to prepare bone marrow cell suspension;
f. putting the centrifugal tube into a horizontal throwing type medical centrifugal machine, and centrifuging for 3-8 minutes under the centrifugal force of 300-1000g relative to the centrifugal force;
g. sucking the floating layer and a small amount of plasma by a disposable plastic suction tube, and uniformly mixing most of plasma with the hematopoietic cell enrichment layer at the bottom of the tube and filtering;
h. the hematopoietic cell mixed suspension is filtered by a 300 mesh filter gauze to remove fiber or other large particle impurities, and the marrow granulocyte suspension is prepared.
It is further preferred that in step b, the preparation of the bone marrow granulocyte suspension should be performed as soon as possible after the extraction of the patient's bone marrow into the anticoagulant tube, otherwise it should be refrigerated in a refrigerator at 4-8 ℃ and not more than 24 hours.
Further preferably, the step e operation may be performed directly without the step d operation for a patient with a small amount of bone marrow; if the resistance is encountered during the operation of the step d, the operation of the step d is performed after the medical injector with the specification larger than 2 milliliters is replaced by one step.
Further preferably, step e is based on the degree of proliferation of the bone marrow smear, wherein the degree of myeloproliferation is greater than or equal to that required by the hyperplastic active person, and the degree of proliferation is that required by the hyperplastic active person to replace the stainless steel needle with smaller inner diameter, then the mixed solution of the bone marrow pellets and the blood plasma in the centrifuge tube is pumped into the syringe, and then the mixed solution is injected back into the centrifuge tube, and the steps are repeated for 3 times to prepare the bone marrow cell suspension.
Further preferred is for diagnosis and prognosis monitoring at morphological time: 2-3ul bone marrow cell suspension is placed at one end of a glass slide, cell smear is carried out by a push plate, and after drying, the dried bone marrow cell suspension is subjected to microscopic examination by a Ruigy/Ruigy stain, so that the mixed suspension of hematopoietic cells can be used morphologically without filtering, and metastatic tumors can be prevented from being filtered.
Further preferred is for diagnostic and prognostic monitoring at the time of immunology: taking a plurality of filtered bone marrow cell suspensions, and detecting the relevant markers on the flow cytometer.
Further preferred is for diagnostic and prognostic monitoring in molecular biology:
(1) polymerase chain reaction experiment (PCR): and taking a plurality of filtered bone marrow cell suspensions to extract DNA or RNA for PCR detection.
(2) Fluorescence in situ hybridization assay (FISH): taking a plurality of filtered marrow cell suspensions, extracting DNA or RNA and detecting by using related FISH probes.
The invention has the beneficial effects that: the invention respectively prepares a bone marrow smear by extracting bone marrow liquid of a patient, uniformly mixing the bone marrow liquid in an anticoagulation tube, enriching the anticoagulation tube into a bone marrow granule layer by a medical centrifuge, then placing the bone marrow granule floating layer and the plasma mixed liquid of the patient into a centrifuge tube, sucking the bone marrow granule and the plasma in the centrifuge tube by a medical injector, mixing the bone marrow granule and the plasma in a syringe, injecting the mixture back into the centrifuge tube, placing the centrifuge tube into a horizontal throwing type medical centrifuge, uniformly mixing most of the plasma without impurities with the hematopoietic cell enrichment layer at the bottom of the tube, and finally filtering the hematopoietic cell mixed suspension by a filter gauze to prepare the bone marrow granule cell suspension; therefore, the hematopoietic cells are separated from the tissue by a physical method, so that the dilution of blood in the blood sinus can be avoided, the cells are fully unfolded, the observation of microstructure is facilitated, the accurate and clear judgment on the original structural components of the bone marrow tissue is made, the bone marrow granulocyte suspension is used for diagnosis and monitoring in morphology, immunology and molecular biology, and the detected data value is more similar to the data value which is required by the bone marrow tissue itself.
Drawings
FIG. 1 shows the distribution of megakaryocytes under a high power microscope (1000 times) when applied to morphology; microscopic observations after sufficient cell expansion following rayleigh staining.
FIG. 2 shows the distribution of megakaryocytes under high magnification (1000 times) when applied to morphology; hematopoietic cells were sufficiently expanded and post-rayleigh staining microscopic images were taken avoiding sinus blood cell dilution.
FIG. 3 is a view of a microscope after Rayleigh staining, showing that cells are uniformly distributed on a low power microscope (100 times) when applied to morphology, and avoiding the sheet making defects of smear and pull-tab.
FIG. 4 is a schematic diagram showing a patient using enrichment versus conventional methods when applied to immunology.
FIG. 5 is a schematic diagram showing the enrichment method compared with the conventional method for the second patient when the method is applied to immunology.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Example 1
A method for diagnosing a disease of the blood system by applying a marrow granulocyte suspension centrifugation enrichment method, comprising the following steps:
a. after about 0.1ml of bone marrow fluid of a patient is extracted to be used as a bone marrow smear, about 4ml of bone marrow fluid is pumped into an anticoagulation tube of EDTA-K2 and is uniformly mixed;
b. placing the anticoagulation tube into a horizontal throwing type medical centrifuge for 500 revolutions per minute, and centrifuging for 8 minutes to enrich a bone marrow small particle layer; after centrifugation of some patients, the upper layer of the anticoagulation tube is not provided with a bone marrow granule layer, and most of the cases are related to marrow material dilution. Part of which is related to the patient itself (dry pumping). After the bone marrow of the patient is extracted into an anticoagulant tube, the preparation of the bone marrow granulocyte suspension should be carried out as soon as possible, otherwise, the bone marrow granulocyte suspension should be put into a refrigerator with the temperature of 4-8 ℃ for refrigeration, and the time is not more than 24 hours.
c. Taking the uppermost bone marrow small particle floating layer and the mixed solution of the plasma of a patient into a 1.5 ml specification centrifuge tube by using a disposable plastic straw;
d. pumping the mixed solution of the bone marrow pellets and the blood plasma in the centrifuge tube into a syringe by using a medical injector with 2 milliliters of specifications (the specification of a domestic needle is 6# and the specification of an international needle is 23G), injecting the mixed solution into the centrifuge tube again, and repeating the steps for 3 times to prepare bone marrow cell suspension;
e. replacing a medical injector (the domestic needle specification is 4.5# and the international needle specification is 26G) with 1ml of specification, sucking the mixed solution of the bone marrow pellets and the blood plasma in the centrifuge tube into a syringe, injecting the mixed solution back into the centrifuge tube, and repeating the steps for 3 times to prepare bone marrow cell suspension; according to the proliferation degree of the bone marrow smear, the proliferation degree is more than or equal to that of the proliferation-active person, the proliferation degree is that the proliferation-active person requires to replace a stainless steel needle with smaller inner diameter, then the mixed solution of bone marrow granules and blood plasma in the centrifuge tube is pumped into a syringe, and then the mixed solution is injected back into the centrifuge tube, and the bone marrow cell suspension is prepared by repeating the steps for 3 times.
For patients with small bone marrow size, the step e operation can be directly performed without the step d operation; if resistance is encountered during the operation of the step d, the step d operation is performed after the medical injector with the size larger than 2 milliliters is needed to be replaced for one time;
f. the centrifuge tube was placed in a flat-flighted medical centrifuge and centrifuged at a centrifugal force of 400g (rotational speed about 1500 rpm) relative to the centrifugal force (RCF) for 8 minutes; the rotational speed is generally selected to be proportional to the degree of myeloproliferation;
g. sucking the floating layer and a small amount of plasma by a disposable plastic suction tube, uniformly mixing most of the plasma with the hematopoietic cell enrichment layer (the ratio is about 1:1) at the bottom of the tube, and filtering to prepare marrow cell suspension, wherein the process is easy to operate gently;
h. the hematopoietic cell mixed suspension is filtered by a 300 mesh filter gauze to remove fiber or other large particle impurities, and the marrow granulocyte suspension is prepared.
This example can be used to perform experiments with bone marrow biopsy tissue pieces, since the bone marrow pellet itself is a small mass of bone marrow tissue. The properties are almost the same as those of bone marrow pellets.
Diagnostic and prognostic monitoring for morphological applications: 2ul of bone marrow cell suspension is placed at one end of a glass slide, cell smear is carried out by using a push plate, and after drying, the cell smear is microscopically inspected after Rayleigh/Rayleigh staining.
For diagnostic and prognostic monitoring in immunology: taking a plurality of filtered bone marrow cell suspensions, and detecting the relevant markers on the flow cytometer.
Diagnostic and prognostic monitoring in molecular biology: (1) polymerase chain reaction experiment (PCR): and taking a plurality of filtered bone marrow cell suspensions to extract DNA or RNA for PCR detection.
(2) Fluorescence in situ hybridization assay (FISH): taking a plurality of filtered marrow cell suspensions, extracting DNA or RNA and detecting by using related FISH probes.
Example two
The marrow granulocyte suspension centrifugal enrichment method is applied to diagnosis and prognosis monitoring methods of blood system diseases, and is prepared from the following raw materials in parts by weight:
a method for diagnosing a disease of the blood system by applying a marrow granulocyte suspension centrifugation enrichment method, comprising the following steps:
a. extracting about 0.3ml of bone marrow liquid of a patient as a bone marrow smear, then pumping about 2ml of bone marrow liquid into an anticoagulation tube of EDTA-K2, and uniformly mixing;
b. placing the anticoagulation tube into a horizontal throwing type medical centrifuge for 1000 revolutions per minute, and centrifuging for 3 minutes to enrich a bone marrow small particle layer; after centrifugation of some patients, the upper layer of the anticoagulation tube is not provided with a bone marrow granule layer, and most of the cases are related to marrow material dilution. Part of which is related to the patient itself (dry pumping). After the bone marrow of the patient is extracted into an anticoagulant tube, the preparation of the bone marrow granulocyte suspension should be carried out as soon as possible, otherwise, the bone marrow granulocyte suspension should be put into a refrigerator with the temperature of 4-8 ℃ for refrigeration, and the time is not more than 24 hours.
c. Taking the uppermost bone marrow small particle floating layer and the mixed solution of the plasma of a patient into a 1.5 ml specification centrifuge tube by using a disposable plastic straw;
d. pumping the mixed solution of the bone marrow pellets and the blood plasma in the centrifuge tube into a syringe by using a medical injector with 2 milliliters of specifications (the specification of a domestic needle is 6# and the specification of an international needle is 23G), injecting the mixed solution into the centrifuge tube again, and repeating the steps for 3 times to prepare bone marrow cell suspension;
e. replacing a medical injector (the domestic needle specification is 4.5# and the international needle specification is 26G) with 1ml of specification, sucking the mixed solution of the bone marrow pellets and the blood plasma in the centrifuge tube into a syringe, injecting the mixed solution back into the centrifuge tube, and repeating the steps for 3 times to prepare bone marrow cell suspension; according to the proliferation degree of the bone marrow smear, the proliferation degree is more than or equal to that of the proliferation-active person, the proliferation degree is that the proliferation-active person requires to replace a stainless steel needle with smaller inner diameter, then the mixed solution of bone marrow granules and blood plasma in the centrifuge tube is pumped into a syringe, and then the mixed solution is injected back into the centrifuge tube, and the bone marrow cell suspension is prepared by repeating the steps for 3 times.
For patients with small bone marrow size, the step e operation can be directly performed without the step d operation; if resistance is encountered during the operation of the step d, the step d operation is performed after the medical injector with the size larger than 2 milliliters is needed to be replaced for one time;
f. the centrifuge tube was placed in a flat-flighted medical centrifuge and centrifuged for 3 minutes at a centrifugal force of 700g (rotational speed about 2000 rpm) relative to the centrifugal force (RCF); the rotational speed is generally selected to be proportional to the degree of myeloproliferation;
g. sucking the floating layer and a small amount of plasma by a disposable plastic suction tube, uniformly mixing most of the plasma with the hematopoietic cell enrichment layer (the ratio is about 1:1) at the bottom of the tube, and filtering to prepare marrow cell suspension, wherein the process is easy to operate gently;
h. the hematopoietic cell mixed suspension is filtered by a 300 mesh filter gauze to remove fiber or other large particle impurities, and the marrow granulocyte suspension is prepared.
This example can be used to perform experiments with bone marrow biopsy tissue pieces, since the bone marrow pellet itself is a small mass of bone marrow tissue. The properties are almost the same as those of bone marrow pellets.
The marrow granulocyte suspension centrifugal enrichment method is applied to a prognosis monitoring method of blood system diseases, and is used for diagnosis and prognosis monitoring in morphology: 3ul of bone marrow cell suspension is placed at one end of a glass slide, cell smear is carried out by using a push plate, and after drying, the dried bone marrow cell suspension is subjected to microscopic examination by using a Ruigi/Ruigi stain, so that the mixed suspension of hematopoietic cells can be used in morphology without filtering, and metastatic tumors can be prevented from being filtered.
Immunology (flow method)
For diagnostic and prognostic monitoring in immunology: taking a plurality of filtered bone marrow cell suspensions, and detecting the relevant markers on the flow cytometer.
Diagnostic and prognostic monitoring in molecular biology: (1) polymerase chain reaction experiment (PCR): and taking a plurality of filtered bone marrow cell suspensions to extract DNA or RNA for PCR detection.
(2) Fluorescence in situ hybridization assay (FISH): taking a plurality of filtered marrow cell suspensions, extracting DNA or RNA and detecting by using related FISH probes.
Example III
A method for diagnosing a disease of the blood system by applying a marrow granulocyte suspension centrifugation enrichment method, comprising the following steps:
a. extracting about 0.2ml of bone marrow fluid of a patient as a bone marrow smear, then pumping about 3-4ml of bone marrow fluid into an anticoagulation tube of EDTA-K2, and uniformly mixing;
b. placing the anticoagulation tube into a horizontal throwing type medical centrifuge for 800 revolutions per minute, and centrifuging for 5 minutes to enrich a bone marrow small particle layer; after centrifugation of some patients, the upper layer of the anticoagulation tube is not provided with a bone marrow granule layer, and most of the cases are related to marrow material dilution. Part of which is related to the patient itself (dry pumping). After the bone marrow of the patient is extracted into an anticoagulant tube, the preparation of the bone marrow granulocyte suspension should be carried out as soon as possible, otherwise, the bone marrow granulocyte suspension should be put into a refrigerator with the temperature of 4-8 ℃ for refrigeration, and the time is not more than 24 hours.
c. Taking the uppermost bone marrow small particle floating layer and the mixed solution of the plasma of a patient into a 1.5 ml specification centrifuge tube by using a disposable plastic straw;
d. pumping the mixed solution of the bone marrow pellets and the blood plasma in the centrifuge tube into a syringe by using a medical injector with 2 milliliters of specifications (the specification of a domestic needle is 6# and the specification of an international needle is 23G), injecting the mixed solution into the centrifuge tube again, and repeating the steps for 3 times to prepare bone marrow cell suspension;
e. replacing a medical injector (the domestic needle specification is 4.5# and the international needle specification is 26G) with 1ml of specification, sucking the mixed solution of the bone marrow pellets and the blood plasma in the centrifuge tube into a syringe, injecting the mixed solution back into the centrifuge tube, and repeating the steps for 3 times to prepare bone marrow cell suspension; according to the proliferation degree of the bone marrow smear, the proliferation degree is more than or equal to that of the proliferation-active person, the proliferation degree is that the proliferation-active person requires to replace a stainless steel needle with smaller inner diameter, then the mixed solution of bone marrow granules and blood plasma in the centrifuge tube is pumped into a syringe, and then the mixed solution is injected back into the centrifuge tube, and the bone marrow cell suspension is prepared by repeating the steps for 3 times.
For patients with small bone marrow size, the step e operation can be directly performed without the step d operation; if resistance is encountered during the operation of the step d, the step d operation is performed after the medical injector with the size larger than 2 milliliters is needed to be replaced for one time;
f. placing the centrifuge tube into a horizontal centrifugal type medical centrifuge, and centrifuging for 5 minutes under the centrifugal force of 500g (the rotating speed is about 1500-2000 rpm) relative to the centrifugal force (RCF); the rotational speed is generally selected to be proportional to the degree of myeloproliferation;
g. sucking the floating layer and a small amount of plasma by a disposable plastic suction tube, uniformly mixing most of the plasma with the hematopoietic cell enrichment layer (the ratio is about 1:1) at the bottom of the tube, and filtering to prepare marrow cell suspension, wherein the process is easy to operate gently;
h. the hematopoietic cell mixed suspension is filtered by a 300 mesh filter gauze to remove fiber or other large particle impurities, and the marrow granulocyte suspension is prepared.
This example can be used to perform experiments with bone marrow biopsy tissue pieces, since the bone marrow pellet itself is a small mass of bone marrow tissue. The properties are almost the same as those of bone marrow pellets.
The marrow granulocyte suspension centrifugal enrichment method is applied to a prognosis monitoring method of blood system diseases, and is used for diagnosis and prognosis monitoring in morphology: 2-3ul bone marrow cell suspension is placed at one end of a glass slide, cell smear is carried out by a push plate, and after drying, the dried bone marrow cell suspension is subjected to microscopic examination (see figures 1, 2 and 3 for details), so that the mixed suspension of hematopoietic cells can be used morphologically without filtering to prevent metastatic tumor from being filtered out.
Cell morphology test application verifies that:
for diagnostic and prognostic monitoring in immunology: taking a plurality of filtered bone marrow cell suspensions and detecting the uplink related markers of the flow cytometer; the prepared marrow liquid of the same patient is taken for a plurality of flow cytometry examinations, and meanwhile, compared with the traditional method, the flow cytometry examinations have the comparison experiment data difference (see the figures 4 and 5 for details).
Immunology (flow method)
Diagnostic and prognostic monitoring in molecular biology: (1) polymerase chain reaction experiment (PCR): and taking a plurality of filtered bone marrow cell suspensions to extract DNA or RNA for PCR detection.
(2) Fluorescence in situ hybridization assay (FISH): taking a plurality of filtered marrow cell suspensions, extracting DNA or RNA and detecting by using related FISH probes.
Molecular biology (PCR)
The foregoing is merely exemplary of the present invention, and those skilled in the art should not be considered as limiting the invention, since modifications may be made in the specific embodiments and application scope of the invention in light of the teachings of the present invention.
Claims (2)
1. A method for preparing a marrow small granule single cell suspension, which is characterized by comprising the following steps:
a. sucking 2-6ml bone marrow fluid into EDTA-K2 anticoagulant tube, and mixing;
b. placing the anticoagulation tube into a horizontal throwing type medical centrifuge for 500-1000 revolutions per minute, and centrifuging for 3-8 minutes to enrich a bone marrow small particle layer;
c. taking 1ml of the mixed solution of the bone marrow small particle floating layer and the blood plasma at the uppermost layer by using a disposable plastic straw, and putting the mixed solution into a centrifuge tube;
d. sucking the mixed solution of the bone marrow pellets and the blood plasma in the centrifuge tube by using a medical injector with the specification of 2 milliliters, injecting the mixed solution back into the centrifuge tube, and repeating the steps for 3 times to prepare bone marrow cell suspension;
e. replacing a medical injector with the specification of 1 milliliter to suck the mixed solution of the bone marrow pellets and the blood plasma in the centrifuge tube into a syringe, and then injecting the mixed solution back into the centrifuge tube, and repeating the steps for 3 times to prepare bone marrow cell suspension;
f. putting the centrifugal tube into a horizontal throwing type medical centrifugal machine, and centrifuging for 3-8 minutes under the centrifugal force of 300-1000g relative to the centrifugal force;
g. sucking the floating layer and a small amount of plasma by a disposable plastic suction tube, and uniformly mixing most of plasma with the hematopoietic cell enrichment layer at the bottom of the tube and filtering;
h. the hematopoietic cell mixed suspension is filtered by a 300 mesh filter gauze to remove fiber or other large particle impurities, and the marrow granulocyte suspension is prepared.
2. The method for preparing a bone marrow small granulocyte suspension according to claim 1, wherein: in the step b, the preparation of the bone marrow granulocyte suspension should be carried out as soon as possible after the bone marrow is sucked into an anticoagulant tube, otherwise, the bone marrow granulocyte suspension should be put into a refrigerator with the temperature of 4-8 ℃ for refrigeration, and the time is not more than 24 hours.
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