CN115777695A - Compound preservation solution suitable for morphological examination of cerebrospinal fluid cells and preparation method thereof - Google Patents
Compound preservation solution suitable for morphological examination of cerebrospinal fluid cells and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of clinical medicine, and discloses a composite preservation solution suitable for morphological examination of cerebrospinal fluid cells and a preparation method thereof, wherein the composite preservation solution comprises a nutrient solution, a fixing solution and a protective solution, wherein the nutrient solution comprises sodium citrate, glucose, adenine, sodium dihydrogen phosphate, disodium hydrogen phosphate, naCl and albumin; the fixing solution comprises alcohol substances, aldehyde substances and citric acid, and the protective solution comprises gelatin solution, trehalose, biological preservative, vitamin C and mannitol. The compound preservation solution can be used for preserving and storing cerebrospinal fluid cells, can well protect the morphology and structural characteristics of the cerebrospinal fluid cells, fills the gap of morphological protection of the cerebrospinal fluid cells in the prior art, and provides technical guarantee for cerebrospinal fluid morphological examination.
Description
Technical Field
The invention relates to a compound preservation solution suitable for morphological examination of cerebrospinal fluid cells and a preparation method thereof, in particular to a compound preservation solution capable of prolonging the service life of the cerebrospinal fluid cells in vitro and ensuring the morphological and structural stability of the cerebrospinal fluid cells, and a preparation method of the compound preservation solution, and belongs to the technical field of clinical medicine.
Background
The morphological examination of cerebrospinal fluid is an examination method for observing the cell morphology in cerebrospinal fluid by using a microscope, can provide help for the diagnosis of nervous system diseases in clinical application, brings great convenience in the aspects of differential diagnosis, disease monitoring, curative effect evaluation and prognosis judgment, particularly, the type of cerebrospinal fluid cells can be known by dyeing the cerebrospinal fluid cells, and the basis for judging whether the cerebrospinal fluid is abnormal or abnormal and whether tumor cells and pathogenic microorganisms exist or not and providing treatment basis for a clinician is provided in a most intuitive mode. However, the morphological examination of cerebrospinal fluid cells has a fatal defect that after the cells are separated from the body, the cells are rapidly degenerated due to the action of intracellular autolytic enzyme and the like, and the morphology is incomplete. Therefore, in clinical practice, it is required that the cerebrospinal fluid examination should be sent immediately after the specimen is received, typically not more than 1 hour.
At present, due to the insufficient detection capability of part of hospitals, samples need to be sent to a superior hospital or a third-party laboratory for detection, so that the cerebrospinal fluid samples need to be stored for a long time and transported for a long distance. Research shows that: after the sample is collected for 2 hours, the neutrophil is reduced by about 50 percent, so that a cell preservation solution is researched, the integrity of the cells can be maintained after the cells are separated from the body, and the technical support is provided for cytomorphology detection.
In the prior art, an invention patent with publication number CN109644985A discloses a cell preservation solution and its application, wherein the cell preservation solution comprises the following components: 0.03-0.07M Tris buffer solution with pH of 7.5-8.0 45-55% (v/v), 95-97% ethanol 45-55% (v/v), calcium salt 8-12% (w/w) and magnesium salt 8-12% (w/w), thus avoiding the loss of nucleic acid substances and improving the stability of maintaining cell morphology. Although the patent discloses that the cell preservation solution can be used for preserving cerebrospinal fluid, the patent only describes that the cell preservation solution can respectively preserve the stability of HPV virus nucleic acid and cervical cell morphology in cells at 25 ℃, and the effective preservation time can respectively reach 45 days and 50 days, but the preservation stability of the cerebrospinal fluid cells is still to be verified.
In addition, the invention patent with publication number CN111944877A discloses a novel preservation solution for microbial nucleic acid in cerebrospinal fluid, wherein the pH of the preservation solution is 7-8, and the preservation solution comprises the following components: 5-20 mmol/L nuclease inhibitor (ethylene diamine tetraacetic acid or ethylene glycol tetraacetic acid), 15-30 mmol/L guanidine hydrochloride buffer solution, 15-25 mmol/L protein denaturation inhibitor (sodium dodecyl sulfate), 5-10 mmol/L trisodium citrate, 20-30 mmol/L bacteriostatic agent (ethanol or potassium sorbate), 5-10 mmol/L stabilizer (glycine and/or sodium acetate) and 5-10% isopropanol. The storage solution described in this patent is relatively stable in Ct value of cerebrospinal fluid of a patient to be stored, and does not greatly differ from the case of storing for 0 to 48 hours, but the patent also only describes protection of stability of nucleic acid in cerebrospinal fluid cells, and does not clearly show the effect of storing morphological and structural characteristics of cerebrospinal fluid cells.
Disclosure of Invention
The invention aims to provide a compound preservation solution suitable for morphological examination of cerebrospinal fluid cells, which adopts a nutrient solution, a fixing solution and a cell protection solution which are composed of specific formulas, can effectively prolong the service life of in vitro cells, well protects the morphology and structural characteristics of the cerebrospinal fluid cells, fills the blank of morphological protection of the cerebrospinal fluid cells in the prior art, and provides technical guarantee for morphological examination of the cerebrospinal fluid. Therefore, the invention also provides a preparation method of the compound preservation solution.
The fixing liquid formula described in the invention is formed by combining alcohol substances, aldehyde substances, citric acid and the like, and can be independently applied to the protection and storage of exfoliated cells such as alveolar lavage fluid, pleural effusion, joint fluid and the like through reasonable optimization of the formula and the proportion to realize the fixation of the cells, so that cell membranes keep micro permeability under the state that the cell morphology and the structure are not changed, nutrient substances and dye can permeate the membranes to enter the cells, the metabolism of the cells is maintained, and the cells can be colored.
The invention is realized by the following technical scheme: the compound preservation solution suitable for morphological examination of cerebrospinal fluid cells comprises nutrient solution, stationary solution and protective solution,
the nutrient solution comprises the following components in concentration: 0.5-4.97 g/L of sodium citrate, 2.01-7.21 g/L of glucose, 0.01-0.14 g/L of adenine, 0.45-0.94 g/L of sodium dihydrogen phosphate, 4.0-8.2 g/L of disodium hydrogen phosphate, 2.36-4.978 g/L of NaCl and 0.3-0.8 g/L of albumin;
the fixing liquid comprises the following components in concentration: 6.75-8.64 ml of alcohol substance, 0.1-0.75 ml of aldehyde substance and 10-26 g of citric acid;
the protective solution comprises the following components in concentration: 3-8 ml of gelatin solution, 0.01-0.1 g/L of trehalose, 0.1-0.2 g/L of biological preservative, 0.1-0.2 g/L of vitamin C and 2-4 g/L of mannitol.
The pH value of the compound preservation solution is 7.0-7.5.
The osmotic pressure of the composite preserving fluid is 280-320 mOsm/L.
In the stationary liquid, the alcohol substance is at least one selected from propanol, isopropanol, benzyl alcohol and ethylene glycol.
In the fixing solution, the aldehyde substance is at least one selected from acetaldehyde, acrolein and glyoxal.
In the protective solution, the biological preservative is at least one selected from the group consisting of cason, chlorhexidine acetate, domiphen bromide and trisodium phosphate.
The preparation method of the composite preservation solution suitable for the morphological examination of the cerebrospinal fluid comprises the steps of preparing a nutrient solution, a stationary solution and a protective solution according to the formula proportion, adding the nutrient solution into ion exchange water for dissolving, then sequentially adding the stationary solution and the protective solution, filtering by using an ultrafiltration device with a 0.2-micron filter membrane after dissolving, and then adjusting the pH value and the osmotic pressure to obtain the composite preservation solution.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) According to the invention, in the formula of the nutrient solution, through the matching of the formula and the proportion of each component, the microenvironment for cell survival is creatively simulated, the electrolyte, the buffer solution, the glucose and the protein components which are necessary for living cells are provided, and through providing the pH value and the osmotic pressure which are suitable for the cell survival and the nutritional components which are necessary for the living cells, the service life of the cells in vitro is prolonged as much as possible, and the integrity of the cell morphology and the structure is maintained.
(2) In the formula of the fixing solution, substances such as alcohols, aldehydes and the like are adopted to form a compound fixing agent formula, and through reasonable matching of the formula and the proportion of the components, micro-fixation on cells can be realized, so that cell membranes keep micro-permeability under the condition that the cell morphology and the structure are kept unchanged, nutrient substances and dyes can permeate the membranes to enter the cells, the metabolism of the cells is maintained, and the cells can be colored.
(3) In the formula of the protective liquid, the gelatin is firstly adopted as a special protective agent for cerebrospinal fluid cells and plays an important role in protecting the morphology of leucocytes, the gelatin belongs to natural protein and has a complex molecular structure, and experiments prove that: under the condition of 4 ℃, the gelatin can isolate the leucocytes from each other, reduce aggregation and achieve the effect of protecting the leucocytes.
(4) In the formula of the protective solution, trehalose is creatively added as a special protective agent, plays an important role in protecting the cell morphology, is a non-specific natural protective agent, can form a unique protective film on the cell surface, has the function of protecting biological tissues, cells and biological macromolecules, and can ensure the integrity of the cells at high temperature and high cold, particularly in a longer time.
(5) In the formula of the protective solution, the mannitol is added to protect cell membranes, and the protective solution can be applied to the preservation of stem cells in actual work; vitamin C has antioxidant effect; the biological preservative can inhibit the propagation and growth of microorganisms, particularly cason, is used as a new generation of biological preservative, can kill various bacteria, viruses and fungi for a long time when being combined with other preservatives, and is an internationally recognized safe and efficient broad-spectrum preservative.
(6) According to the invention, toxic and unstable chemical properties of trioxymethylene and other substances are not adopted, expensive instruments and freeze drying methods with complex procedures are not involved, an integration mode is adopted, and a technology with complementary advantages of multiple methods is adopted to maintain the cell morphology, so that the purpose of long-time preservation of cerebrospinal fluid cells is achieved, and technical guarantee is provided for morphological examination of the cerebrospinal fluid cells.
(7) After the formula and the proportion of the fixing solution are reasonably adjusted, the fixing solution can be independently applied to the protection and the storage of exfoliated cells such as alveolar lavage fluid, pleural effusion, ascites, joint fluid and the like, and can independently realize the fixation of the cells.
(8) The invention can creatively adopt the special sealing ring to seal the test tube and the annular sealing ring as end face sealing in the preservation process, can isolate the air to the maximum extent and prevent the oxidation of oxygen in the air to cells.
(9) The invention adopts the low temperature of 4 ℃ to store the cerebrospinal fluid cells, can reduce the cell metabolism to the maximum extent, prolong the cell life and keep the morphological characteristics of the cells.
In conclusion, the invention provides the composite protective solution integrated by adopting a novel formula (namely, the nutrient solution, the fixing solution and the protective solution), which can be used for prolonging the service life of cerebrospinal fluid cells in vitro and protecting the morphology and the structure of the cerebrospinal fluid cells, solves the dilemma faced by the conventional morphological examination of the cerebrospinal fluid cells, prolongs the storage time of the cerebrospinal fluid cells to 72 hours at 4 ℃, and brings technical guarantee for the detection of the cerebrospinal fluid cells.
Drawings
FIG. 1 is a cell morphology map of experiment group A stored for 24 h.
FIG. 2 is a cell morphology map of experiment group A stored for 48 h.
FIG. 3 is a cell morphology map of experiment group A stored for 72h.
FIG. 4 is a morphological diagram of cells preserved for 8h in Experimental group B.
FIG. 5 is a morphological diagram of cells preserved for 8h in Experimental group C.
FIG. 6 is a cell morphology chart (I) of different batches of samples preserved for 72h by using the compound preservation solution I.
FIG. 7 is a cell morphology chart of different batches of samples preserved for 72h by using the compound preservation solution I (II).
FIG. 8 is a cell morphology (granulocytes) of experiment group A preserved for 72h (2% glacial acetic acid added).
FIG. 9 is a cell morphology (lymphocytes) of experiment group A stored for 72h (2% glacial acetic acid added).
FIG. 10 is a graph showing staining of cells stored for 8h in Experimental group A.
FIG. 11 is a graph showing staining of cells stored for 7 days in Experimental group A.
FIG. 12 is a cell morphology map of cerebrospinal fluid samples stored for 8h using the storage solution I of comparative example 1.
FIG. 13 is a cytomorphology map of cerebrospinal fluid samples preserved for 8h using the preservation solution II of comparative example 2.
FIG. 14 is a cytomorphology chart of cerebrospinal fluid samples preserved for 8h using the preservation solution III of comparative example 3.
Detailed description of the preferred embodiments
The objects, technical solutions and advantageous effects of the present invention will be described in further detail below.
It is to be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention claimed, unless otherwise specified, and all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The invention aims to provide a composite preservation solution for morphological examination of cerebrospinal fluid, which aims to solve the dilemma faced by the morphological examination of cerebrospinal fluid at present, namely after living cells are isolated, a sample is kept for a long time to cause cell damage so as to influence the morphological examination of the cerebrospinal fluid. One is fixing liquid, which is compounded with composite fixing agent to fix cell in micro closed state and to make extracellular matter capable of entering cell through the pores for metabolism and color reception. One is a protective solution, which adopts special protective agents (gelatin and trehalose) to protect cells, mannitol, trehalose and gelatin all have protective effect on cells, vitamin C has antioxidant effect, and biological preservative (Kathon) can inhibit the reproduction and growth of microorganisms. Therefore, the invention can realize the long-time preservation of the cerebrospinal fluid cells in vitro by reasonably matching and controlling the proportion of the three formula substances, and can effectively ensure the stability of the cell morphology and structure.
Further, in the invention, the nutrient solution comprises the following components in mass concentration: sodium citrate: 0.5-4.97 g/L, glucose: 2.01 to 7.21g/L, adenine: 0.01-0.14 g/L, sodium dihydrogen phosphate: 0.45-0.94 g/L, disodium hydrogen phosphate: 4.0-8.2 g/L, naCl: 2.36-4.978 g/L, albumin: 0.3-0.8 g/L. The stationary liquid comprises the following components in percentage concentration by volume (mass): alcohol substances: 6.75-8.64 ml%, aldehyde substance: 0.1-0.75 ml%, citric acid: 10 to 26g percent. The protective solution comprises the following components in mass concentration (volume percentage concentration): 3-8 ml of gelatin solution, trehalose: 0.01-0.1 g/L, biological preservative: 0.1-0.2 g/L, vitamin C: 0.1-0.2 g/L, mannitol: 2-4 g/L.
In a specific embodiment, the alcohol can be selected from propanol, isopropanol, benzyl alcohol, ethylene glycol, or any combination thereof; the aldehyde substance can be selected from acetaldehyde, acrolein, glyoxal or any combination thereof; the biological antiseptic can be selected from casone (isothiazolinone compounds), chlorhexidine acetate (1, 1' -hexylbis [5- (p-chlorophenyl) biguanide ] diacetate), domiphen bromide (dodecyl-dimethyl-2-phenoxy-ethyl ammonium bromide), trisodium phosphate, or any combination thereof.
In a specific embodiment, the compound preservation solution can be used for preserving and storing cerebrospinal fluid cells at 4 ℃, and the preservation time can be prolonged to 72 hours. In another embodiment, the protective solution can be used alone, for example, after further optimization and adjustment of its formulation, for the protection and storage of exfoliated cells in alveolar lavage fluid, pleural effusion, joint fluid, and the like.
In preparing the composite preservation solution, the nutrient solution, the fixative solution and the protective solution are prepared according to the formula proportion, and then the nutrient solution, the fixative solution and the protective solution are selected according to the proper proportion, for example, in a specific embodiment, the volume ratio of the nutrient solution, the fixative solution and the protective solution is controlled to be 10: 0.05: 1, the nutrient solution is added into ion exchange water for dissolving, a stirring device is started, the mixture is fully stirred until the nutrient solution is fully dissolved, the fixative solution is added after fully stirring and uniformly mixing, the ion exchange water is added until the fixative solution is fully dissolved, the required volume is determined by volume after stirring until the ion exchange water is added, the mixture is filtered by an ultrafiltration device (0.2 um filter membrane) after being stirred again and uniformly mixing, and inorganic salt buffer solution (NaH) is adopted 2 PO 4 /Na 2 HPO 4 ) Adjusting the pH value of the preservation solution to 7.0-7.5, adjusting the osmotic pressure to 280-320 mOsm/L at the same time to obtain the composite preservation solution, and subpackaging and preserving. All process flows do not require heating. After testing: the compound preservation solution can be stably preserved for 12 months under the condition of room temperature through experimental verification for up to one year.
The following examples are provided to illustrate specific embodiments of the present invention, and it is understood that the scope of the present invention is not limited to the following examples. The reagent raw materials used in the present example are all commercial products, for example:
trehalose: CAS number 99-20-7, guangzhou Hao Kaihai chemical Co., ltd;
gelatin: CAS number 9000-80-7, shandong Hengyuan kang Biotech Ltd;
vitamin C: CAS number 50-81-7, chengdu Kelong chemical reagent factory;
albumin: CAS number 9048-46-8, chengdu Kelong chemical industry reagent works.
Example 1: compound preserving fluid I
Nutrient solution: 2.85g/L of sodium citrate, 7.21g/L of glucose, 0.08g/L of adenine, 0.55g/L of sodium dihydrogen phosphate, 4.0g/L of disodium hydrogen phosphate, 3.88g/L of NaCl and 0.5g/L of albumin.
Fixing liquid: 7.55 ml of isopropanol, 0.45 ml of acetaldehyde and 15g of citric acid.
Protection solution: 3 ml of gelatin solution, 0.1g/L of trehalose, 0.15g/L of cason, 0.12g/L of vitamin C and 3g/L of mannitol.
The compound preservation solution I is prepared according to the method, after the preparation is finished, the pH value is adjusted to 7.0, and the osmotic pressure is adjusted to 320mOsm/L, so that the compound preservation solution I is obtained.
Example 2: compound preserving fluid II
Nutrient solution: 1.59g/L sodium citrate, 5.25g/L glucose, 0.01g/L adenine, 0.45g/L sodium dihydrogen phosphate, 8.2g/L disodium hydrogen phosphate and 4.978g/L NaCl; 0.3g/L albumin.
Fixing liquid: 6.75 ml of propanol, 0.50 ml of acetaldehyde, 0.25 ml of acrolein and 26g of citric acid.
Protection solution: 8 ml of gelatin solution, 0.01g/L of trehalose, 0.1g/L of cason, 0.1g/L of trisodium phosphate, 0.1g/L of vitamin C and 4g/L of mannitol.
The compound preservation solution II is prepared according to the method, after the preparation is finished, the pH value is adjusted to 7.4, and the osmotic pressure is 280mOsm/L, so that the compound preservation solution II is obtained.
Example 3: compound preserving fluid III
Nutrient solution: 3.42g/L of sodium citrate, 7.21g/L of glucose, 0.14g/L of adenine, 0.94g/L of sodium dihydrogen phosphate, 4.0g/L of disodium hydrogen phosphate, 2.36g/L of NaCl and 0.8g/L of albumin.
Fixing liquid: 6.39 ml of propanol, 2.25 ml of ethylene glycol, 0.1 ml of acrolein and 10g of citric acid.
Protection solution: 6 ml of gelatin solution, 0.05g/L of trehalose, 0.1g/L of cason, 0.2g/L of vitamin C and 2g/L of mannitol.
The compound preservation solution III is prepared according to the method, after the preparation is finished, the pH value is adjusted to 7.5, and the osmotic pressure is adjusted to 300mOsm/L, so that the compound preservation solution III is obtained.
Example 4: compound preserving fluid IV
Nutrient solution: 4.36g/L of sodium citrate, 5.25g/L of glucose, 0.01g/L of adenine, 0.45g/L of sodium dihydrogen phosphate, 8.2g/L of disodium hydrogen phosphate and 4.978g/L of NaCl; 0.3g/L albumin.
Fixing liquid: 6.75 ml of propanol, 0.50 ml of acetaldehyde, 0.25 ml of acrolein and 26g of citric acid.
Protection solution: 8 ml of gelatin solution, 0.01g/L of trehalose, 0.1g/L of cason, 0.1g/L of trisodium phosphate, 0.1g/L of vitamin C and 4g/L of mannitol.
The compound preservation solution IV is prepared according to the method, after the preparation is finished, the pH value is adjusted to 7.4, and the osmotic pressure is 280mOsm/L, so that the compound preservation solution IV is obtained.
Example 5: compound preserving fluid V
Nutrient solution: 2.82g/L of sodium citrate, 5.20g/L of glucose, 0.10g/L of adenine, 0.65g/L of sodium dihydrogen phosphate, 6.4g/L of disodium hydrogen phosphate, 3.72g/L of NaCl3 and 0.58g/L of albumin.
Fixing liquid: 6.88 ml of propanol, 0.36 ml of acetaldehyde and 12g of citric acid.
Protection solution: 5.5 ml of gelatin solution, 0.02g/L of trehalose, 0.15g/L of kasons, 0.18g/L of vitamin C and 3g/L of mannitol.
The compound preservation solution V is prepared according to the method, after the preparation is finished, the pH value is adjusted to 7.2, and the osmotic pressure is 290mOsm/L, so that the compound preservation solution V is obtained.
Example 6: compound preserving fluid VI
Nutrient solution: 0.5g/L of sodium citrate, 6.47g/L of glucose, 0.01g/L of adenine, 0.80g/L of sodium dihydrogen phosphate, 6.2g/L of disodium hydrogen phosphate, 4.56g/L of NaCl and 0.8g/L of albumin.
Fixing liquid: 7.65 ml of ethylene glycol, 0.05 ml of acetaldehyde, 0.1 ml of acrolein and 23g of citric acid.
Protection solution: 5 ml of gelatin solution, 0.08g/L of trehalose, 0.1g/L of cason, 0.1g/L of chlorhexidine acetate, 0.1g/L of vitamin C and 3.2g/L of mannitol.
The compound preservative fluid VI is prepared according to the method, after the preparation is finished, the pH value is adjusted to 7.4, and the osmotic pressure is adjusted to 295mOsm/L, so that the compound preservative fluid VI is obtained.
Example 7: composite preservation solution VII
Nutrient solution: 4.97g/L of sodium citrate, 5.02g/L of glucose, 0.12g/L of adenine, 0.65g/L of sodium dihydrogen phosphate, 6.0g/L of disodium hydrogen phosphate, 2.64g/L of NaCl and 0.47g/L of albumin.
Fixing liquid: 7.23 ml of isopropanol, 0.61 ml of acetaldehyde and 14g of citric acid.
Protection solution: 5.25 ml of gelatin solution, 0.1g/L of trehalose, 0.1g/L of cason, 0.05g/L of domiphen bromide, 0.11g/L of vitamin C and 3.2g/L of mannitol.
The compound preservation solution VII is prepared according to the method, after the preparation is finished, the pH value is adjusted to 7.5, and the osmotic pressure is adjusted to 300mOsm/L, so that the compound preservation solution VII is obtained.
Example 8: compound preservation solution VIII
Nutrient solution: 3.56g/L of sodium citrate, 2.58g/L of glucose, 0.14g/L of adenine, 0.68g/L of sodium dihydrogen phosphate, 5.6g/L of disodium hydrogen phosphate, 2.36g/L of NaCl and 0.8g/L of albumin.
Fixing liquid: 5.25 ml of propanol, 2.08 ml of isopropanol, 0.05 ml of acetaldehyde, 0.05 ml of acrolein and 13g of citric acid.
Protection solution: 8 ml of gelatin solution, 0.01g/L of trehalose, 0.12g/L of cason, 0.18g/L of vitamin C and 2.36g/L of mannitol.
The preparation is carried out according to the method of the invention, after the preparation is finished, the pH is adjusted to 7.4, and the osmotic pressure is 285mOsm/L, thus obtaining the composite preserving fluid VIII.
Examples
Experimental samples: cerebrospinal fluid is sampled from different clinical individuals.
Control samples: control 1 (saline), control 2 (containing only nutrients).
Taking 3 plastic test tubes, respectively numbering A, B and C, and adding 0.2ml of compound preservation solution (the compound preservation solution I in the example 1 is arbitrarily selected) into the group A to obtain an experimental group A; adding 0.2ml of normal saline into group B to obtain experimental group B (normal saline control group); 0.2ml of the nutrient solution of example 1 was added to group C, which was the test group C (nutrient control group).
Adding the blood sample and the cerebrospinal fluid sample according to the volume ratio of 1: 9, immediately mixing uniformly, screwing and sealing a test tube cover, placing at 4 ℃, and observing the preservation states of the shapes of the red blood cells and the white blood cells in different time periods.
Observation means and indices: visual inspection of the color of the supernatant and a monoclonal hemoglobin colloidal gold reagent; the integrity of cell morphology and cell staining condition are directly observed under a microscope high power microscope, and the presence or absence of volume growth and sawtooth shape of red blood cells, volume change and degeneration of white blood cells and nucleolysis are observed.
The experimental effect is as follows:
the experimental group a (i.e. using the complex preservation solution i of example 1) had very good protective effect on cerebrospinal fluid erythrocytes and leukocytes, the morphology of the cells was intact at different time periods (24 h, 48h and 72 h), the cells could be uniformly and uniformly distributed in the visual field, and the original morphology of the cells at the time of sample collection was maintained: the red blood cells are light yellow, disc-shaped, biconcave. White blood cells are slightly larger than red blood cells, are gray white, round, and have stronger refractive index, as shown in fig. 1, 2, and 3.
When the experimental sample is stored for 8 hours in experimental group B (normal saline control group), a large number of indented red blood cells (about 80% of mature red blood cells are indented) are observed, as shown in FIG. 4.
When the experimental group C (nutrient substance control group) stored the experimental sample for 8 hours, about 10% of the red blood cells appeared to be jagged and oval red blood cells, as shown in fig. 5.
Further, different batches of the experimental sample (cerebrospinal fluid) were preserved for 72h using experimental group a, and most of the cells of the different batches still remained in the original state, as shown in fig. 6 and 7.
The experimental group a was stored for 72 hours, 2% glacial acetic acid was added to the test tube of the experimental group a, and the mixture was gently mixed to dissolve most of the erythrocytes within a few seconds (during white blood cell counting, erythrocytes were destroyed and interference was removed), and it was found that the cell membrane of mononuclear cells (granulocytes) was intact, oval, and clear nuclei were visible, as shown in fig. 8. In addition, the cell membrane of the mononuclear cell (lymphocyte) is intact, oval, and the nucleus is clearly visible, as shown in fig. 9.
The experimental group A was stored for 8h and stained with Rieger, which shows that both red blood cells and lymphocytes can be stained, as shown in FIG. 10. The test group A was stored for 7 days, and it was found that the red blood cells were not uniform in size, but the red blood cells and white blood cells were stained by Ruegy staining, as shown in FIG. 11.
Therefore, when the compound preservation solution is used for preserving the cerebrospinal fluid cells, the shape and the structure of the cells (red blood cells and white blood cells) can be effectively maintained after 72 hours of preservation at 4 ℃, the compound preservation solution is suitable for morphological examination of the cerebrospinal fluid cells, and the white blood cells can be stained by staining after being preserved for 7 days at 4 ℃, so that technical guarantee is brought to cerebrospinal fluid cell detection.
Comparative example 1: preserving fluid I
In the comparative example, only the nutrient solution (sodium citrate 2.85g/L, glucose 7.21g/L, adenine 0.08g/L, sodium dihydrogen phosphate 0.55g/L, disodium hydrogen phosphate 4.0g/L, naCl 3.88g/L, albumin 0.5g/L, the same as the nutrient solution in example 1) was used as the preservation solution I, and the pH and the osmotic pressure were slightly different, and the pH value was 7.4, and the osmotic pressure was 302mOsm/L.
Comparative example 2: preservation solution II
The comparative example only adopts the stationary liquid (8.55 ml of isopropanol, 2.75 ml of acetaldehyde and 18g of citric acid) as the preservation liquid II, and the pH and the osmotic pressure are slightly different, wherein the pH is 7.2 and the osmotic pressure is 315mOsm/L.
Comparative example 3: preservative fluid III
In the comparative example, only a nutrient solution and a stationary liquid (2.85 g/L of sodium citrate, 7.21g/L of glucose, 0.08g/L of adenine, 0.55g/L of sodium dihydrogen phosphate, 4.0g/L of disodium hydrogen phosphate, 3.88g/L of NaCl, 0.5g/L of albumin, 7.55 ml of isopropanol, 0.45 ml of acetaldehyde and 15g of citric acid which are the same as the nutrient solution and the stationary liquid in example 1 but are not added with a protective liquid) are used as a preservation solution III, the pH and osmotic pressure are slightly different, the pH is 7.25, and the osmotic pressure is 310mOsm/L.
Appropriate amounts of the complex preservation solution I of example 1, the preservation solution I of comparative example 1, the preservation solution II of comparative example 2 and the preservation solution III of comparative example 3 were added to the test sample (blood sample cerebrospinal fluid sample), and the cell morphology and structure of the test sample to which the complex preservation solution I of example 1 was added, which was preserved at 4 ℃ for 24h, 48h and 72h, and the cell morphology and structure of the test sample to which the preservation solution I of comparative example 1, the preservation solution II of comparative example 2 and the preservation solution III of comparative example 3 was added, which was preserved at 4 ℃ for 8h, were observed in this order.
The results of the experiments are shown in table 1 below.
TABLE 1 comparative Table of cell morphology preserved in cerebrospinal fluid samples
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and all simple modifications and equivalent variations of the above embodiments according to the technical spirit of the present invention are included in the scope of the present invention.
Claims (7)
1. The compound preservation solution applicable to morphological examination of cerebrospinal fluid cells is characterized in that: contains nutrient solution, stationary liquid and protective solution,
the nutrient solution comprises the following components in concentration: 0.5-4.97 g/L of sodium citrate, 2.01-7.21 g/L of glucose, 0.01-0.14 g/L of adenine, 0.45-0.94 g/L of sodium dihydrogen phosphate, 4.0-8.2 g/L of disodium hydrogen phosphate, 2.36-4.978 g/L of NaCl and 0.3-0.8 g/L of albumin;
the fixing liquid comprises the following components in concentration: 6.75-8.64 ml of alcohol substance, 0.1-0.75 ml of aldehyde substance and 10-26 g of citric acid;
the protective solution comprises the following components in concentration: 3-8 ml of gelatin solution, 0.01-0.1 g/L of trehalose, 0.1-0.2 g/L of biological preservative, 0.1-0.2 g/L of vitamin C and 2-4 g/L of mannitol.
2. The composite preservation solution according to claim 1, characterized in that: the pH value of the compound preservation solution is 7.0-7.5.
3. The composite preservation solution according to claim 1, characterized in that: the osmotic pressure of the composite preserving fluid is 280-320 mOsm/L.
4. The composite preservation solution according to claim 1, characterized in that: in the stationary liquid, the alcohol substance is at least one selected from propanol, isopropanol, benzyl alcohol and ethylene glycol.
5. The composite preservation solution according to claim 1, characterized in that: in the fixing solution, the aldehyde substance is at least one selected from acetaldehyde, acrolein and glyoxal.
6. The composite preservation solution according to claim 1, characterized in that: in the protection solution, the biological preservative is at least one selected from cason, chlorhexidine acetate, domiphen bromide and trisodium phosphate.
7. The preparation method of the composite preservation solution suitable for the morphological examination of cerebrospinal fluid cells is characterized by comprising the following steps of: preparing nutrient solution, fixative solution and protective solution according to the formula proportion of claim 1, dissolving the nutrient solution in ion exchange water, adding fixative solution and protective solution in turn, filtering with ultrafiltration device with 0.2um filter membrane, and adjusting pH and osmotic pressure to obtain the composite preservative solution.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117546837A (en) * | 2023-11-22 | 2024-02-13 | 深圳泽医细胞治疗集团有限公司 | Blood preservation solution suitable for NK cell culture and preparation method and application thereof |
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