CN115777695B - Composite preservation solution suitable for cerebrospinal fluid cell morphology examination and preparation method thereof - Google Patents
Composite preservation solution suitable for cerebrospinal fluid cell morphology examination and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of clinical medicine, and discloses a compound preservation solution suitable for cerebral spinal fluid cell morphology examination and a preparation method thereof, wherein the compound preservation solution comprises a nutrient solution, a fixing solution and a protective solution, and the nutrient solution comprises sodium citrate, glucose, adenine, sodium dihydrogen phosphate, disodium hydrogen phosphate, naCl and albumin; the fixing liquid comprises alcohols, aldehydes and citric acid, and the protecting liquid 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 morphological and structural characteristics of the cerebrospinal fluid cells, fills the blank of the morphological protection of the cerebrospinal fluid cells in the prior art, and provides technical assurance for morphological examination of the cerebrospinal fluid.
Description
Technical Field
The invention relates to a compound preservation solution suitable for cerebrospinal fluid cell morphology examination and a preparation method thereof, in particular to a compound preservation solution capable of prolonging the service life of in-vitro cerebrospinal fluid cells and guaranteeing the stability of the morphology and the structure of the in-vitro 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 cells is an examination method for observing the cell morphology in cerebrospinal fluid by using a microscope, can provide help for diagnosis of nervous system diseases in clinical application, and brings great convenience in aspects of differential diagnosis, disease monitoring, curative effect evaluation and prognosis judgment, especially the types of the cerebrospinal fluid cells can be known by dyeing, whether the cerebrospinal fluid has abnormality or abnormal type and whether tumor cells and pathogenic microorganisms exist or not can be provided in the most intuitive mode, and the clinical doctor is provided with treatment basis. However, in the existing situation, the morphological examination of cerebrospinal fluid cells has a fatal defect that after the cells are isolated, the cells are quickly degenerated and have incomplete morphology due to the actions of intracellular autolyzing enzyme and the like. Thus, in clinical testing procedures, it is required that cerebrospinal fluid testing should be delivered immediately after receipt of the specimen, typically for no more than 1 hour.
At present, due to the defect of detection capability of part of hospitals, samples are required to be sent to a superior hospital or a third-party laboratory for detection, and long-term preservation and long-distance transportation of cerebrospinal fluid samples are required. There are studies showing that: after 2 hours of specimen collection, neutrophils have been reduced by about 50%, so it is very necessary to study a cell preservation solution to maintain the integrity of cells after they are isolated from the body and to provide technical support for morphological detection of cells.
In the prior art, the invention patent with the publication number of CN109644985A discloses a cell preservation solution and application thereof, 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) can avoid the loss of nucleic acid substances and improve the stability of maintaining cell morphology. Although this patent discloses that the cell preservation solution can be used for preservation of cerebrospinal fluid, it only describes that the cell preservation solution stores the stability of HPV viral nucleic acid and cervical cell morphology in cells at 25 ℃ respectively, and the effective preservation time can reach 45 days and 50 days respectively, however, the preservation stability of cerebrospinal fluid cells still needs to be verified.
In addition, the invention patent with publication number of CN111944877A also discloses a novel preservation solution of microbial nucleic acid in cerebrospinal fluid, wherein the pH value of the preservation solution is 7-8, and the novel preservation solution comprises the following components: 5-20 mmol/L nuclease inhibitor (ethylenediamine tetraacetic acid or ethylene glycol tetraacetic acid), 15-30 mmol/L guanidine hydrochloride buffer solution, 15-25 mmol/L protein denaturation inhibitor (sodium dodecyl sulfonate), 5-10 mmol/L trisodium citrate, 20-30 mmol/L bacteriostat (ethanol or potassium sorbate), 5-10 mmol/L stabilizer (glycine and/or sodium acetate) and 5-10% isopropyl alcohol. The preservation solution described in this patent is relatively stable in Ct value for cerebrospinal fluid of a patient to be preserved and does not differ much in preservation for 0 to 48 hours, but the patent also describes only the preservation of nucleic acid stability in cerebrospinal fluid cells, and the preservation effect of morphological and structural characteristics of cerebrospinal fluid cells is not clear.
Disclosure of Invention
The invention aims to provide the compound preservation solution suitable for the morphological examination of the cerebrospinal fluid cells, and the compound preservation solution adopts the nutrient solution, the fixing solution and the cell protection solution which are composed of specific formulas, so that the service life of the cells in vitro can be effectively prolonged, the morphological and structural characteristics of the cerebrospinal fluid cells are well protected, the blank of the morphological protection of the cerebrospinal fluid cells in the prior art is filled, and technical assurance is provided for the morphological examination of the cerebrospinal fluid. Therefore, the invention also provides a preparation method of the composite preservation solution.
The fixing liquid formula disclosed by 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 liquid, hydrothorax and ascites, joint fluid and the like through reasonable optimization of the formula and the proportion, so that the fixation of the cells is realized, the cell membrane keeps micro-permeability under the condition that the cell morphology and structure are kept unchanged, nutrient substances and dyes can permeate the membrane to enter the cell, the metabolism of the cells is maintained, and the cells can be stained.
The invention is realized by the following technical scheme: the compound preservation solution suitable for the cell morphology examination of the cerebrospinal fluid contains nutrient solution, fixing solution and protective solution,
the nutrient solution comprises the following components in concentration: sodium citrate 0.5-4.97 g/L, glucose 2.01-7.21 g/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 2.36.36-4.978 g/L and albumin 0.3-0.8 g/L;
the fixing liquid comprises the following components in concentration: 6.75-8.64 ml of alcohol substances, 0.1-0.75 ml of aldehyde substances and 10-26 g of citric acid;
the protective liquid comprises the following components in concentration: 3 to 8 milliliters of gelatin solution, 0.01 to 0.1g/L of trehalose, 0.1 to 0.2g/L of biological preservative, 0.1 to 0.2g/L of vitamin C and 2 to 4g/L of mannitol.
The pH value of the composite preservation solution is 7.0-7.5.
The osmotic pressure of the composite preservation solution is 280-320 mOsm/L.
In the fixing solution, the alcohol substance is at least one selected from propanol, isopropanol, benzyl alcohol and ethylene glycol.
In the fixing solution, aldehyde substances are selected from at least one of acetaldehyde, acrolein and glyoxal.
In the protective solution, the biological preservative is at least one selected from pinus koraiensis, chlorhexidine acetate, domiphen bromide and trisodium phosphate.
The preparation method of the compound preservation solution suitable for the cerebrospinal fluid cell morphology examination comprises the steps of respectively preparing a nutrient solution, a fixing solution and a protective solution according to the formula proportion, adding the nutrient solution into ion exchange water for dissolution, then sequentially adding the fixing solution and the protective solution, filtering by an ultrafiltration device of a 0.2um filter membrane after dissolution, and then adjusting pH and osmotic pressure to obtain the compound preservation solution.
Compared with the prior art, the invention has the following advantages:
(1) According to the invention, in the formula of the nutrient solution, the micro environment for cell survival is creatively simulated through the matching of the formula and the proportion of each component, necessary electrolyte, buffer solution, glucose and protein components are provided for living cells, and the life of the cells is prolonged as much as possible in vitro by providing proper pH, osmotic pressure and necessary nutrient components for living cells, so that the integrity of cell morphology and structure is maintained.
(2) According to the invention, in the formula of the fixing solution, substances such as alcohols, aldehydes and the like are adopted to form a compound fixing agent formula, through reasonable matching of the formula and the proportion of each component, the micro-fixation of cells can be realized, so that the cell membrane keeps micro-permeability under the condition that the cell morphology and structure are kept unchanged, nutrient substances and dyes can permeate the membrane to enter the cells, the metabolism of the cells is maintained, and the cells can be colored.
(3) In the formula of the protecting solution, gelatin is added as a special protecting agent of cerebrospinal fluid cells for the first time, plays an important role in protecting the morphology of leucocytes, belongs to natural proteins, has a complex molecular structure, and is proved by experiments: under the condition of 4 ℃, the gelatin can isolate the white blood cells from each other, reduce aggregation and achieve the effect of protecting the white blood cells.
(4) In the formula of the protective solution, trehalose is initially 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 effect of protecting biological tissues, cells and biological macromolecules, and can ensure the integrity of the cells at high temperature and high and cold, and particularly for a long time.
(5) The protective solution has the protective effect on cell membranes by adding mannitol in the formula of the protective solution, and 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, especially the pinus koraiensis, can kill various bacteria, viruses and fungi in combination with other preservatives for a long time, and is an internationally recognized safe and efficient broad-spectrum preservative.
(6) The invention does not adopt toxic substances such as trioxymethylene with unstable chemical property, does not relate to expensive instruments and freeze drying methods with complex procedures, adopts an integration mode, adopts a technology with complementary advantages of a plurality of methods to maintain cell morphology, achieves the aim of long-time preservation of cerebrospinal fluid cells, and provides technical assurance for morphological examination of the cerebrospinal fluid cells.
(7) After the formula and the proportion of the fixing liquid are reasonably adjusted, the fixing liquid can be independently applied to the protection and the storage of the exfoliated cells such as alveolar lavage liquid, hydrothorax and ascites, joint fluid and the like, and the fixing of the cells can be independently realized.
(8) The invention can creatively adopt the special sealing ring to seal the test tube and the annular sealing ring as the end face for sealing in the storage process, can isolate the air to the maximum extent and prevent the oxidation of oxygen in the air to cells.
(9) The invention adopts 4 ℃ low temperature to preserve cerebrospinal fluid cells, can reduce cell metabolism to the maximum extent, prolongs the cell life and maintains the cell morphological characteristics.
In summary, the invention provides the compound protective solution integrated by adopting the novel formula (namely, the nutrient solution, the fixing solution and the protective solution), which can be used for prolonging the service life of the in-vitro cerebrospinal fluid cells and protecting the morphology and structure of the in-vitro cerebrospinal fluid cells, solves the dilemma faced by the prior morphological examination of the cerebrospinal fluid cells, prolongs the preservation time of the cerebrospinal fluid cells to 72 hours at 4 ℃, and brings technical assurance for the detection of the cerebrospinal fluid cells.
Drawings
FIG. 1 is a cell morphology of experiment group A stored for 24 h.
FIG. 2 is a cell morphology of experiment group A stored for 48 h.
FIG. 3 is a cell morphology of experiment group A stored for 72h.
FIG. 4 is a cell morphology of experiment group B stored for 8 h.
FIG. 5 is a cell morphology of experiment group C stored for 8 h.
FIG. 6 is a diagram showing the morphology of cells preserved for 72 hours in different batches using the composite preservation solution I (one).
FIG. 7 is a diagram showing the morphology of cells preserved for 72 hours in different batches using the composite preservation solution I (second).
FIG. 8 is a graph of cell morphology (granulocytes) of experimental group A stored 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 of cell staining of experiment group A stored for 8 h.
FIG. 11 is a graph of cell staining of Experimental group A for 7 days.
FIG. 12 is a cell morphology of a cerebrospinal fluid sample 8h preserved with the preservation solution I of comparative example 1.
FIG. 13 is a cell morphology of a cerebrospinal fluid sample 8h preserved with preservation solution II of comparative example 2.
FIG. 14 is a cell morphology of a cerebrospinal fluid sample 8h preserved with preservation solution III of comparative example 3.
Description of the embodiments
The objects, technical solutions and advantageous effects of the present invention will be described in further detail below.
It is noted that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed, and unless otherwise indicated, 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 compound preservation solution for the cell morphology examination of cerebrospinal fluid, which solves the dilemma faced by the current cell morphology examination of cerebrospinal fluid, namely, the cell morphology examination is influenced by the cell damage caused by the long-term storage of a sample after the living cell is isolated. A composite fixer for micro-fixing cells to make cell membrane in micro-closed state is disclosed, which can make the extracellular substances (nutrients, dye, etc.) enter the cells via membrane holes for metabolism and color receiving of cells. 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 (pinus koraiensis) can inhibit microorganism proliferation and growth. Therefore, the invention can realize long-time in-vitro preservation of cerebrospinal fluid cells by reasonable collocation and proportion control of the three formula substances, and can effectively ensure the stability of cell morphology and structure.
Further, in the present invention, the nutrient solution comprises the following components in mass concentration: sodium citrate: 0.5-4.97 g/L, glucose: 2.01-7.21 g/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 fixing solution comprises the following components in percentage by volume (mass): alcohols: 6.75-8.64 ml), aldehyde substance: 0.1-0.75 ml), citric acid: 10-26 g percent. The protective liquid comprises the following components in percentage by volume: gelatin solution 3-8 ml%, 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 may 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 preservative may be selected from the group consisting of pinacolin (isothiazolinone), 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 one specific embodiment, the composite preservative fluid can be stored and preserved for cerebrospinal fluid cells at 4℃and the preservation time can be extended to 72 hours. In another embodiment, the protective fluid may be used alone, for example, after further optimization and adjustment of its formulation, to protect and store exfoliated cells such as alveolar lavage fluid, hydrothorax and ascites fluid, joint fluid, etc.
When preparing the composite preserving fluid, preparing the nutrient solution, the fixing solution and the protecting solution according to the above formula proportion, respectively, then selecting the nutrient solution, the fixing solution and the protecting solution with proper proportion, for example, in a specific embodiment, controlling the volume ratio of the nutrient solution, the fixing solution and the protecting solution to be 10:0.05:1, adding the nutrient solution into ion exchange water to dissolve, starting a stirring device, fully stirring until the nutrient solution is completely dissolved, then adding the fixing solution, fully stirring and uniformly mixing, and then adding the fixing solutionStirring to dissolve completely, adding ion exchange water to desired volume, stirring again, filtering (0.2 um membrane) with ultrafiltration device, and adding inorganic salt buffer solution (NaH 2 PO 4 /Na 2 HPO 4 ) And (3) regulating the pH value of the preservation solution to 7.0-7.5, regulating the osmotic pressure to 280-320 mOsm/L, and then obtaining the composite preservation solution, and subpackaging for preservation. All process flows do not require heating. Tested: under the condition of room temperature, the compound preservation solution can be stably preserved for 12 months through experimental verification for up to one year.
The following description of the invention will be given by way of example only, and the scope of the invention is not limited to the following examples. The reagent raw materials used in this example are all commercial products, for example:
trehalose: CAS number 99-20-7, guangzhou chemical Co., ltd;
gelatin: CAS number 9000-80-7, shandong Hengyuan Kangbio-technology Co., ltd;
vitamin C: CAS number 50-81-7, chengdu Kelong chemical reagent plant;
albumin: CAS number 9048-46-8, chengdu Kelong chemical reagent plant.
Example 1: composite preservation solution I
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.
Fixing solution: isopropanol 7.55 ml, acetaldehyde 0.45 ml, citric acid 15g%.
Protective solution: 3 ml of gelatin solution, 0.1g/L of trehalose, 0.15g/L of kathon, 0.12g/L of vitamin C and 3g/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 7.0, the osmotic pressure is 320mOsm/L, and the composite preservation solution I is obtained.
Example 2: composite preservation solution II
Nutrient solution: 1.59g/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; albumin 0.3g/L.
Fixing solution: 6.75 ml of propanol, 0.50 ml of acetaldehyde, 0.25 ml of acrolein and 26g of citric acid.
Protective solution: 8 ml of gelatin solution, 0.01g/L of trehalose, 0.1g/L of kathon, 0.1g/L of trisodium phosphate, 0.1g/L of vitamin C and 4g/L of mannitol.
The preparation is carried out according to the method of the invention, and after the preparation is finished, the pH7.4 is adjusted, the osmotic pressure is 280mOsm/L, and the composite preservation solution II is obtained.
Example 3: composite preservation solution 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 solution: 6.39 ml of propanol, 2.25 ml of glycol, 0.1 ml of acrolein and 10g of citric acid.
Protective solution: 6 ml of gelatin solution, 0.05g/L of trehalose, 0.1g/L of kathon, 0.2g/L of vitamin C and 2g/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 7.5, the osmotic pressure is 300mOsm/L, and the composite preservation solution III is obtained.
Example 4: composite preservation solution 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; albumin 0.3g/L.
Fixing solution: 6.75 ml of propanol, 0.50 ml of acetaldehyde, 0.25 ml of acrolein and 26g of citric acid.
Protective solution: 8 ml of gelatin solution, 0.01g/L of trehalose, 0.1g/L of kathon, 0.1g/L of trisodium phosphate, 0.1g/L of vitamin C and 4g/L of mannitol.
The preparation is carried out according to the method of the invention, and after the preparation is finished, the pH7.4 is adjusted, the osmotic pressure is 280mOsm/L, and the composite preservation solution IV is obtained.
Example 5: composite preservation solution V
Nutrient solution: sodium citrate 2.82g/L, glucose 5.20g/L, adenine 0.10g/L, sodium dihydrogen phosphate 0.65g/L, disodium hydrogen phosphate 6.4g/L, naCl3.72g/L, albumin 0.58g/L.
Fixing solution: 6.88 ml of propanol, 0.36 ml of acetaldehyde and 12g of citric acid.
Protective solution: 5.5 ml of gelatin solution, 0.02g/L of trehalose, 0.15g/L of kathon, 0.18g/L of vitamin C and 3g/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 7.2, the osmotic pressure is 290mOsm/L, and the composite preservation solution V is obtained.
Example 6: composite preservation solution VI
Nutrient solution: sodium citrate 0.5g/L, glucose 6.47g/L, adenine 0.01g/L, sodium dihydrogen phosphate 0.80g/L, disodium hydrogen phosphate 6.2g/L, naCl 4.56g/L, albumin 0.8g/L.
Fixing solution: 7.65 ml of ethylene glycol, 0.05 ml of acetaldehyde, 0.1 ml of acrolein and 23g of citric acid.
Protective solution: 5 ml of gelatin solution, 0.08g/L of trehalose, 0.1g/L of pinus koraiensis, 0.1g/L of chlorhexidine acetate, 0.1g/L of vitamin C and 3.2g/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, the osmotic pressure is 295mOsm/L, and the composite preservation solution 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 solution: isopropanol 7.23 ml, acetaldehyde 0.61 ml, citric acid 14g%.
Protective solution: 5.25 ml of gelatin solution, 0.1g/L of trehalose, 0.1g/L of kathon, 0.05g/L of domiphen bromide, 0.11g/L of vitamin C and 3.2g/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 7.5, the osmotic pressure is 300mOsm/L, and the composite preservation solution VII is obtained.
Example 8: composite 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 solution: 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.
Protective solution: 8 ml of gelatin solution, 0.01g/L of trehalose, 0.12g/L of kathon, 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, the osmotic pressure is 285mOsm/L, and the composite preservation solution VIII is obtained.
Examples
Experimental samples: blood samples of different individuals clinically were taken from cerebrospinal fluid.
Control samples: control 1 (normal saline), control 2 (nutrient only).
Taking 3 plastic test tubes, respectively numbering A, B and C, and adding 0.2ml of composite preservation solution (composite preservation solution I in optional selection example 1) into the group A to obtain an experimental group A; adding 0.2ml of physiological saline into the group B, namely the experimental group B (physiological saline control group); the experimental group C (nutrient control group) was obtained by adding 0.2ml of the nutrient solution of example 1 to group C.
Adding a blood sample cerebrospinal fluid sample according to the volume ratio of 1:9, immediately mixing, screwing a sealing test tube cover, and observing the preservation states of red blood cells and white blood cell morphology in different time periods at the temperature of 4 ℃.
Observation means and indexes: visual inspection, supernatant color and monoclonal hemoglobin colloidal gold reagent; the integrity of cell morphology and cell staining condition are directly observed under a microscope high-power microscope, whether the volume of red blood cells is large and saw-tooth-shaped is observed, whether the volume of white blood cells is changed and degenerated is observed, and the nuclei are dissolved.
Experimental effect:
the experimental group A (namely, the composite preservation solution I in the embodiment 1) has a very good protection effect on cerebrospinal fluid erythrocytes and leukocytes, the cell morphology in different time periods (24 h, 48h and 72 h) is perfect, the cells can be singly and uniformly distributed in the visual field, and the original morphology of the cells in the sample collection is maintained: red blood cells are yellowish and are in a discoid and biconcave state. White blood cells are slightly larger in volume than red blood cells, are off-white, round and have stronger refraction, and are shown in particular in fig. 1, 2 and 3.
When the test sample was stored for 8 hours in the test group B (physiological saline control group), a large number of jagged red blood cells (approximately 80% of the mature red blood cells were jagged) were observed, as shown in FIG. 4.
When the experimental group C (nutrient control group) is preserved for 8 hours, about 10% of red blood cells are saw-tooth-shaped and oval-shaped red blood cells, and the method is specifically shown in fig. 5.
Further, the experimental group a was used to store different batches of experimental samples (cerebrospinal fluid) for 72 hours, and the cells of different batches remained in their original state, as shown in fig. 6 and 7.
The experimental group A is stored for 72 hours, 2% glacial acetic acid is added into a test tube of the experimental group A, and the mixture is gently mixed, so that most of erythrocytes are completely dissolved within a few seconds (when white cells are counted, the erythrocytes are required to be destroyed, and interference is removed), and the cell membrane of a mononuclear cell (granulocyte) is complete, oval and clear, and the nucleus is shown in a specific view in fig. 8. In addition, the envelope of mononuclear cells (lymphocytes) is intact, oval, and the nucleus is clearly visible, see in particular FIG. 9.
The experimental group A was stored for 8 hours and was subjected to Ruiji staining, and it was found that both erythrocytes and lymphocytes were stained, as shown in FIG. 10. The experiment group A was stored for 7 days, and red blood cells were not uniform in size, but red blood cells and white blood cells were stained by Rajie staining, and nuclei were stained, as shown in FIG. 11.
Therefore, when the compound preservation solution is used for preserving cerebrospinal fluid cells, the morphology and the structure of the cells (red blood cells and white blood cells) can be effectively maintained by preserving the cerebrospinal fluid cells for 72 hours at 4 ℃, the compound preservation solution is suitable for morphological examination of the cerebrospinal fluid cells, and the white blood cells can stain cell nuclei by staining for 7 days at 4 ℃, so that technical assurance is brought to cerebrospinal fluid cell detection.
Comparative example 1: preservation solution I
The comparative example used only nutrient solutions (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 of example 1) as preservation solution I, which were slightly different in pH and osmotic adjustment, pH7.4, and osmotic pressure 302mOsm/L.
Comparative example 2: preservation solution II
In this comparative example, only a fixed solution (8.55 ml of isopropyl alcohol, 2.75 ml of acetaldehyde, 18g% of citric acid) was used as the preservation solution II, which was slightly different in pH from the osmotic pressure adjustment, pH 7.2, and osmotic pressure 315mOsm/L.
Comparative example 3: preservation solution III
This comparative example used only nutrient solution and fixative (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 isopropyl alcohol, 0.45 ml of acetaldehyde, 15g of citric acid, the same nutrient solution and fixative as in example 1, but without addition of a protective solution) as preservative solution III, which was slightly different in pH and osmotic adjustment, pH 7.25, osmotic pressure 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 experimental samples (cerebrospinal fluid samples of blood samples), and the cell morphology and structure of the experimental samples added to the complex preservation solution I of example 1 were sequentially observed at 4℃for 24 hours, 48 hours, and 72 hours, and the cell morphology and structure of the experimental samples added to 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 sequentially observed at 4℃for 8 hours, respectively.
The experimental results are shown in table 1 below.
TABLE 1 comparison of cell morphology for cerebrospinal fluid sample preservation
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent variation, etc. of the above embodiment according to the technical matter of the present invention fall within the scope of the present invention.
Claims (2)
1. Compound preservation solution suitable for cerebrospinal fluid cell morphology inspection, its characterized in that: the composite preservation solution contains a nutrient solution, a fixing solution and a protecting solution, wherein the pH value of the composite preservation solution is 7.0, the osmotic pressure of the composite preservation solution is 320mOsm/L,
the nutrient solution comprises the following components in concentration: 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 fixing solution comprises the following components in concentration: isopropanol 7.55% (v/v), acetaldehyde 0.45% (v/v), citric acid 15wt%;
the protective solution comprises the following components in concentration: gelatin solution 3% (v/v), trehalose 0.1g/L, kathon 0.15g/L, vitamin C0.12 g/L, mannitol 3g/L.
2. The preparation method of the composite preservation solution suitable for the morphological examination of the cerebrospinal fluid cells is characterized by comprising the following steps of: preparing nutrient solution, fixing solution and protective solution according to the formula proportion of claim 1, respectively adding the nutrient solution into ion exchange water for dissolution, then sequentially adding the fixing solution and the protective solution, filtering by an ultrafiltration device with a 0.2um filter membrane after dissolution, and then adjusting pH and osmotic pressure to obtain the composite preservation solution.
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| CN118330207B (en) * | 2024-06-12 | 2024-08-09 | 成都海默云因医学检验实验室有限公司 | Fixing agent for improving fluorescent background in fixed cell immunofluorescence method and method for fixing cells by using fixing agent |
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