A bone marrow cell paraffin block, its preparation method and application

Abstract

This invention discloses a bone marrow cell paraffin block, its preparation method, and its application, belonging to the field of pathological detection technology. The preparation method includes the following steps: Red blood cell lysis: Bone marrow aspirate is taken, and a precipitate containing cells and sediment is separated. A red blood cell lysis agent is added to lyse the red blood cells, and a lysed precipitate containing unlysed cells and sediment is separated. Formation: The lysed precipitate is resuspended in formalin solution for fixation, the supernatant is removed, and the precipitate is resuspended in agar solution. The agar is cooled and solidified, and then fixed again in neutral formalin. Dehydration: The solidified agar block is dehydrated. Embedding: The dehydrated agar block is embedded in paraffin to form a paraffin block. The cell paraffin block prepared by this method can supplement the diagnosis when bone marrow smears or bone marrow biopsy samples are not ideally prepared.

Description

Technical Field

[0001] This invention relates to pathological testing techniques, and in particular to a bone marrow cell paraffin block, its preparation method, and its application. Background Technology

[0002] Bone marrow aspiration is an important diagnostic tool for hematological diseases. It mainly relies on a combination of bone marrow smear examination and bone marrow biopsy. Currently, the detection technologies have different focuses in diagnosis, and they complement each other, which is of great significance in the diagnosis, differential diagnosis, treatment selection, efficacy evaluation and prognosis of hematological diseases.

[0003] Bone marrow aspiration smears are used to observe cell morphology and proportion, and are one of the important examination items for patients with hematological diseases and clinically difficult cases. They are of great significance for diagnosing hematological diseases, their treatment efficacy, prognosis, and the assessment of the recovery of hematopoietic function after bone marrow transplantation. Bone marrow aspiration biopsy can preserve the complete bone marrow tissue structure, which can compensate for the shortcomings of bone marrow smears. It can reveal the composition of bone marrow cells and the distribution of primitive cells, and observe tissue structure, degree of proliferation, cell distribution, cell infiltration, fibrosis, and interstitial changes, which facilitates pathological diagnosis.

[0004] Currently, bone marrow aspiration in clinical practice combines bone marrow smear examination and bone marrow biopsy for diagnosis. However, both examinations have their limitations. For example, in many primary care hospitals, due to limitations in materials and technology, bone marrow aspiration can only perform bone marrow smear examinations and not bone marrow biopsies. Even hospitals with the necessary resources may encounter cases of "dry aspiration," where no bone marrow tissue can be extracted, thus hindering the formation of a complementary diagnostic framework. For these patients, clinical diagnosis relies solely on traditional bone marrow smears or blood clots. Furthermore, the clinician's procedure involves only one puncture point, which can be affected by puncture technique and excessive aspiration force, leading to blood mixing and affecting accurate judgment.

[0005] Therefore, there is an urgent need for a bone marrow pathology testing protocol for cases where bone marrow biopsy is not performed due to various reasons, or where bone marrow biopsy cannot be performed in primary hospitals and only bone marrow fluid is sent for flow cytometry analysis, and the clinical manifestations are suspected to be malignant, requiring further clarification of the disease. Summary of the Invention

[0006] To address the above problems, this invention provides a method for preparing bone marrow cell paraffin blocks. The cell paraffin blocks prepared by this method can be used for supplementary diagnosis when the preparation of bone marrow smears or bone marrow biopsy samples is not ideal.

[0007] On one hand, this invention discloses a method for preparing bone marrow cell paraffin blocks, comprising the following steps:

[0008] Red blood cell lysis: Take bone marrow aspirate, separate the precipitate containing cells and sediment, add red blood cell lysis agent to lyse the red blood cells, and separate the lysed precipitate containing unlysed cells and sediment.

[0009] Molding: The lysed precipitate is resuspended in formalin solution for fixation, the supernatant is removed, and then agar solution is added for resuspending. The agar is cooled and solidified, and then placed in formalin for fixation again.

[0010] Dehydration: Dehydrating the solidified agar blocks;

[0011] Embedding: The agar block that has undergone the above dehydration treatment is embedded in paraffin wax to make a wax block.

[0012] Considering that the reason why bone marrow pathological testing cannot be performed is the lack of samples of suitable morphology, this invention utilizes bone marrow aspirate obtained by bone marrow puncture (either the remaining specimen after collecting samples for flow cytometry and preparing blood smears, or the remaining specimen after flow cytometry testing) to prepare a new type of bone marrow cell wax block by adding techniques such as breaking down red blood cells and shaping the bone marrow aspirate, based on the traditional cell wax block preparation method. This new type of bone marrow cell wax block can be used as a supplementary diagnostic basis in pathological testing.

[0013] Meanwhile, during preliminary research, the inventors discovered that in the pathological slide preparation process, routine bone marrow biopsy tissue undergoes decalcification and acid treatment before paraffin embedding, which significantly affects cell morphology. The inventors verified that DNA and RNA expression results are affected when using acid-treated specimens for molecular detection. This prevents further molecular diagnostic testing in difficult cases. While plastic-embedded pathological slides are relatively simple to examine, do not require decalcification, and maintain good cell morphology, the large amount of heat generated during plastic embedding can cause antigen denaturation, making immunohistochemical examination impossible. Furthermore, basophils are difficult to identify due to loss of basophilic granules during slide preparation, and certain late erythroblasts and lymphocytes are difficult to distinguish in the slides. Therefore, the bone marrow cell blocks prepared by the method described in this invention are neither acid-treated nor heat-treated, and can be used for HE staining, special staining, immunohistochemical staining, and molecular detection, all with good results.

[0014] In some embodiments, a pretreatment step is included before the redness-breaking step. This pretreatment step involves: when bone marrow biopsy tissue cannot be obtained during clinical bone marrow aspiration, aspirating 2-3 ml of bone marrow aspirate and injecting it into an anticoagulant tube for later use; when bone marrow biopsy tissue can be obtained during clinical bone marrow aspiration, aspirating ≥1 ml of bone marrow aspirate and injecting it into a bone marrow cell fixative for later use; the bone marrow cell fixative is a PBS buffer containing 40%-60% methanol (by volume) and 15-22 mg / 100 ml dipotassium ethylenediaminetetraacetate (EDTA-D); preferably, the methanol concentration is 50% by volume. Preserving the bone marrow aspirate with the above-mentioned bone marrow cell fixative maintains good cell morphology.

[0015] In some embodiments, when the bone marrow aspirate is stored in an anticoagulant tube, it is stored at 2-8°C, and the bone marrow cell block is prepared within 72 hours of cell ex vivo in the bone marrow aspirate, preferably stored at 4-6°C and the bone marrow cell block is prepared within 8-48 hours.

[0016] When the bone marrow aspirate is stored in bone marrow cell fixative, it should be kept at 2-40°C, and the bone marrow cell block should be prepared within 72 hours of cells being removed from the bone marrow aspirate, preferably at 20-30°C, with the bone marrow cell block preparation completed within 8-48 hours. It is understood that when the bone marrow aspirate is stored in bone marrow cell fixative, it can be refrigerated or stored at room temperature without affecting its fixation effect, but room temperature storage expands its application prospects.

[0017] In some embodiments, the pH of the PBS buffer is 7.0-7.4; preferably 7.2-7.4.

[0018] In some embodiments, the ratio of the bone marrow cell fixative to the bone marrow aspirate is 2-4:1, preferably 3:1.

[0019] In some embodiments, the anticoagulant is dipotassium ethylenediaminetetraacetate or sodium heparin, preferably dipotassium ethylenediaminetetraacetate.

[0020] In some embodiments, the erythrocyte lysis agent is selected from: erythrocyte lysis buffer R1010 from Beijing Solarbio Science & Technology Co., Ltd., erythrocyte lysis buffer RT122-02 from Tiangen Biotech Co., Ltd., erythrocyte lysis buffer 100-302-100 from Albatross Biotechnology Co., Ltd., and preferably erythrocyte lysis buffer RT122-02 from Tiangen Biotech Co., Ltd.

[0021] In some embodiments, the volume ratio of the erythrocyte lysis agent to the precipitate is 2-4:1, preferably 3:1.

[0022] In some embodiments, the time for erythrocyte lysis by adding the erythrocyte lysis agent is 3-10 minutes, preferably 4-6 minutes.

[0023] In some embodiments, the erythrocyte lysis agent is added to induce erythrocyte lysis at a temperature of 20°C-35°C, preferably 25-30°C.

[0024] In some embodiments, during the red-breaking step, the precipitate is separated by centrifugation at 1300±300 rpm for 5±3 min.

[0025] In some embodiments, the lysis of red blood cells is accelerated by inverting the reagent container.

[0026] In some embodiments, during the red-breaking step, the pyrolysis precipitate is separated by centrifugation at 3000±300 rpm for 5±2 min.

[0027] In some embodiments, during the red blood cell lysis step, if the lysed precipitate is found to contain red blood cells, the red blood cell lysis step is repeated; furthermore, if the lysed precipitate is observed to be red, it is determined that it contains red blood cells.

[0028] In some embodiments, a washing step is included between the red lysis step and the shaping step. The washing step is as follows: resuspending the lysed precipitate in PBS buffer, centrifuging, and removing the supernatant; further, the amount of PBS buffer is 5-8 times the volume of the lysed precipitate, and / or, centrifuging at 1500±300 rpm for 2-3 min and then removing the supernatant, preferably at 2-6°C, more preferably at 4°C; even further, the washing step is repeated 1-3 times.

[0029] In some embodiments, during the molding step, the volume percentage concentration of the formalin solution is 4 ± 1%, preferably 4%.

[0030] In some embodiments, during the molding step, the volume of formalin solution added is 5-10 times the volume of the sample. For example, when resuspending lysed precipitates in formalin solution for fixation, the volume of formalin solution is 5-10 times the volume of the lysed precipitates; similarly, when placing agar blocks that have cooled and solidified into agar blocks in formalin for fixation, the volume of formalin solution is 5-10 times the volume of the agar blocks.

[0031] In some embodiments, the fixing time in the molding step is 5-10 minutes.

[0032] In some embodiments, during the molding step, the supernatant is removed after centrifugation at 1500±300 rpm for 2-3 minutes.

[0033] In some embodiments, the agar solution is added dropwise during the molding step, and the temperature of the agar solution is 45-50°C.

[0034] In some embodiments, during the molding step, the agar solution has a mass-volume percentage concentration of 4-10%, preferably 5%.

[0035] In some embodiments, during the molding step, the amount of agar solution added is 0.1-0.5 ml, preferably 0.1 ml.

[0036] In some embodiments, the solidified agar block is wrapped in filter paper or gauze and placed in an embedding cassette for dehydration by an automated tissue dehydrator.

[0037] In some embodiments, the largest surface of a whole block of agar containing tissue cells is embedded.

[0038] On the other hand, the present invention also discloses bone marrow cell wax blocks prepared by the above preparation method.

[0039] On the other hand, the present invention also discloses the application of the above-mentioned bone marrow cell blocks in the study of hematological diseases for non-diagnostic and therapeutic purposes.

[0040] For example, the bone marrow cell paraffin blocks of the present invention can be used in the following scenarios:

[0041] 1. Cases where there are no conditions to perform bone marrow biopsy or where bone marrow biopsy samples are always of poor quality for various reasons.

[0042] 2. Cases where bone marrow biopsy cannot be performed at primary hospitals, and only bone marrow fluid is sent for flow cytometry analysis, with clinical manifestations suggesting malignancy, requiring further clarification.

[0043] 3. When bone marrow aspirate comes into contact with blood, the diluted bone marrow aspirate may affect the results of the bone marrow smear.

[0044] 4. When bone marrow biopsy tissue fails to decalcify completely or the acid treatment exceeds the time limit, thus affecting the results of subsequent experiments.

[0045] 5. When the number of hematopoietic stem cells of each lineage in the bone marrow biopsy tissue is too low.

[0046] 6. Bone marrow cell blocks that have not undergone acidification and decalcification treatment can be used to retain tissue cell antigens, DNA, and RNA, enabling molecular pathology and genetic testing.

[0047] In some of these embodiments, the bone marrow cell paraffin block is used for immunohistochemical staining.

[0048] For example, it can be used for the following immunohistochemical detection items:

[0049] Screening for rare diseases: CD34, CD117, CD61, CD71, CD3, CD20, CD56, CD30, CK, CD138.

[0050] Multiple myeloma package: CD38, CD138, Kappa, Lambda, CD3, CD20, CD19, CD117, CD56, Ki-67.

[0051] Small B-cell lymphoma: CD3, CD5, CD10, CD20, CD22, CD23, Cyclin-D1, CD138, CD103, Ki-67.

[0052] Large B-cell lymphoma: CD3, CD5, CD10, CD20, CD22, Cyclin-D1, CD30, BCL-2, C-myc, Ki-67.

[0053] T-cell lymphoma: CD3, CD4, CD8, CD5, CD7, ALK, CD30, CD56, CD57, Ki-67.

[0054] Acute myeloid leukemia: CD34, CD117, MPO, CD33, CD13, CD3, CD22, CD14, CD68R, CD123.

[0055] Acute lymphoblastic leukemia: CD34, TdT, CD117, CD2, CD3, CD7, CD19, CD20, CD22, CD10.

[0056] Bone marrow metastases: CK, CK7, CK20, PSA, PSAP, P504s, TTF-1, Napsin A, CDX-2, villin (male).

[0057] Bone marrow metastases: CK, CK7, CK20, ER, PR, HER2, AR, TTF-1, Napsin A, villin (female).

[0058] Langerhans cells: CD1a, S-100, CD68, Langerin, CD34, CD117, CD3, CD20, CD56, CD30.

[0059] Neuroendocrine tumors: Syn, CgA, NSE, S-100, MelanA, HMB45, TdT, CD3, CD20, CD22.

[0060] blastoid plasmacytic dendritic tumors: CD34, TdT, MPO, CD4, CD43, CD56, CD123, TCL-1, CD3, CD22.

[0061] Immunohistochemical staining involves binding a primary antibody to the target protein antigen in the tissue being tested, followed by binding of the primary antibody to a secondary antibody labeled with HRP or similar substances. Finally, the reaction with a DAB chromogenic agent confirms the localization and semi-quantification of the target protein antigen. Immunohistochemistry is primarily significant for determining the localization of the target protein. The antibodies involved in the validation process of this protocol include, but are not limited to, CD20 and / or CD138. The illustrations in the examples are for illustrative purposes only; it is understood that the bone marrow cell blocks of this invention can also be applied to other immunohistochemical detection projects.

[0062] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0063] The reagents and raw materials used in this invention are all commercially available.

[0064] The positive and progressive effects of this invention are as follows:

[0065] The method for preparing bone marrow cell paraffin blocks of the present invention allows for bone marrow pathological examination when bone marrow tissue is unavailable. This method is simple to operate, requiring only anticoagulated bone marrow aspirate. After red blood cell lysis, the prepared bone marrow cell paraffin blocks can be stored long-term. Because conventional bone marrow biopsy tissue contains a small amount of bone tissue, acidification and decalcification are required before preparing paraffin blocks. However, the preparation method of the present invention does not involve acidification or decalcification. Therefore, the staining results obtained from subsequent immunohistochemical staining are significantly more stable and reliable than those obtained from immunohistochemical staining using bone marrow tissue. Furthermore, it meets the pretreatment requirements for specimens in molecular biology and genetic testing, eliminating the possibility that acid treatment might affect experimental results. Additionally, molecular tests such as FISH, multicolor immunohistochemistry for observing the tumor microenvironment, and gene sequencing can be performed according to diagnostic needs.

[0066] Meanwhile, compared with bone marrow smears, bone marrow cell blocks prepared by this method have the potential for sustainable sectioning and repeated experiments, while bone marrow smears can only be used for a single experiment and cannot be stored for a long time or for further experiments.

[0067] Furthermore, this invention optimizes the preparation method, resulting in bone marrow cell blocks free of erythrocytes. The cell membrane exhibits high sensitivity to antigen-antibody binding. When performing immunohistochemical staining experiments, there are sufficient cells available for diagnosis under a 20X field of view, with no erythrocyte interference in diagnosis. This reduces the impact of nonspecific staining on the results and demonstrates good practical effectiveness. Attached Figure Description

[0068] Figure 1 The results of immunohistochemical staining (CD20) of bone marrow cell paraffin blocks in Example 2 are shown.

[0069] Figure 2 The result of immunohistochemical staining of bone marrow smear (CD20) in Example 2.

[0070] Figure 3 The results of immunohistochemical staining for CD138 in group A of Example 3 are shown.

[0071] Figure 4 The results of immunohistochemical staining for CD138 in group B of Example 3 are shown.

[0072] Figure 5 The results of immunohistochemical staining for CD138 in group C of Example 3 are shown.

[0073] Figure 6 The results of immunohistochemical staining for CD138 in group D of Example 3 are shown.

[0074] Figure 7 The results of the first group of immunohistochemical staining for CD138 in Example 4 are shown.

[0075] Figure 8 The results of the second group of immunohistochemical staining for CD138 in Example 4 are shown.

[0076] Figure 9 The results of the third group of immunohistochemical staining for CD138 in Example 4 are shown.

[0077] Figure 10 The results of the fourth group of immunohistochemical staining for CD138 in Example 4 are shown.

[0078] Figure 11 The results of the fifth group of immunohistochemical staining for CD138 in Example 4 are shown.

[0079] Figure 12 The results of the first group of immunohistochemical staining for CD138 in Example 5 are shown.

[0080] Figure 13 The results of the second group of immunohistochemical staining for CD138 in Example 5 are shown. Detailed Implementation

[0081] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0082] Example 1

[0083] A bone marrow cell wax block is prepared by the following method.

[0084] 1. Pre-processing

[0085] During a bone marrow aspiration, bone marrow fluid is aspirated to obtain the aspirated bone marrow solution. The specific steps are as follows:

[0086] 1) When bone marrow biopsy tissue cannot be obtained smoothly

[0087] In clinical bone marrow aspiration, if bone marrow biopsy tissue cannot be obtained smoothly, 2-3 ml of bone marrow aspirate can be aspirated. After preparing several bone marrow smears at the bedside, the remaining bone marrow aspirate can be injected into an EDTA-K2 anticoagulant blood collection tube. Store and transport at 2-8℃, preferably 4-6℃.

[0088] 2) When bone marrow biopsy tissue is successfully obtained

[0089] When bone marrow biopsy tissue is successfully obtained during clinical bone marrow aspiration, ≥1ml of bone marrow aspirate is aspirated. Several bone marrow smears are prepared at the bedside. The remaining bone marrow aspirate is then injected into 2-4 times the volume of bone marrow cell fixative, with repeated aspiration and respiration, preferably 3 times the volume of bone marrow cell fixative. It is ensured that all remaining bone marrow aspirate in the syringe is injected into the bone marrow cell fixative to maintain cell morphology. The tissue is transported at room temperature, such as 10-40℃ or 20-30℃.

[0090] The formula for the bone marrow cell fixation solution is shown in the table below.

[0091] Table 1. Bone marrow cell fixation solution formulation

[0092]

[0093] 2. Break the red

[0094] Take the bone marrow aspirate after the above pretreatment, separate the precipitate containing cells and sediment, add red blood cell lysis agent to lyse the red blood cells, and separate the lysed precipitate containing unlysed cells and sediment.

[0095] The specific operations in this embodiment are as follows:

[0096] 1) Take the above bone marrow aspirate, centrifuge at 1500 rpm for 5 minutes, discard the supernatant after centrifugation, and obtain a precipitate containing cells and sediment.

[0097] 2) Add 3 times the volume of erythrocyte lysis buffer to the precipitate containing cells and sediment obtained above (e.g., add 900 μl of erythrocyte lysis buffer to 300 μl of precipitate), mix by inversion, and incubate at room temperature (about 20-30℃) for 5 min, and invert and mix 6 more times during this period (if the precipitate is less than 300 μl, add 1 ml of erythrocyte lysis buffer).

[0098] The erythrocyte lysis buffer was selected from the erythrocyte lysis buffer with product number RT122-02 from Tiangen Biotech Co., Ltd.

[0099] 3) Centrifuge at 3000 rpm for 5 min, remove the supernatant, and leave the precipitate, which is the lysis precipitate.

[0100] 4) A small amount of red blood cells will not affect subsequent tests. The red blood cells can be visually assessed to determine whether they have been completely lysed. If the precipitate is dark red, it is determined that the red blood cells have not been completely lysed. That is, if the precipitate is red after centrifugation, the red blood cell lysis step can be repeated until the precipitate is no longer red.

[0101] 3. Washing

[0102] The lysed precipitate was resuspended in PBS buffer, centrifuged, and the supernatant was removed to obtain the precipitate.

[0103] The specific operation in this embodiment is as follows: Add 5-8 times the volume of the lysed precipitate in PBS with a pH of 7.2-7.4 to the above precipitate, resuspend the precipitate, centrifuge at 1500 rpm for 2-3 seconds, and discard the supernatant. Centrifugation at 4°C yields better results. Wash twice.

[0104] 4. Molding

[0105] The lysed precipitate was resuspended in formalin solution for fixation, the supernatant was removed, and then agar solution was added for resuspending to solidify the agar.

[0106] The specific operation in this embodiment is as follows: Add 5-10 times the volume of the precipitate and 4% neutral formalin for fixation, resuspend the precipitate for 5-10 minutes for fixation, centrifuge at 1500 rpm for 2-3 minutes, discard the supernatant, add agar solution at 45-50℃, and after the agar cools and solidifies, place it again in 5-10 times the volume of neutral formalin for fixation.

[0107] 5. Dehydration

[0108] The solidified agar blocks are dehydrated. In this embodiment, the solidified agar blocks are wrapped in filter paper or gauze and placed in an embedding cassette, where an automated tissue dehydrator performs dehydration according to a conventional procedure.

[0109] 6. Embedding

[0110] The dehydrated agar blocks were embedded in paraffin wax, with the largest surface being embedded to form cell wax blocks, thus obtaining bone marrow cell wax blocks.

[0111] The above procedures, from the aspiration of bone marrow aspirate to obtaining the bone marrow cell block, must be completed within 72 hours.

[0112] Example 2

[0113] In this embodiment, the bone marrow cell paraffin blocks prepared in Example 1 were used for immunohistochemical staining experiments, and the results were compared with those obtained from bone marrow smears.

[0114] 1. Method

[0115] Immunohistochemical staining was performed on bone marrow cell blocks and bone marrow smears prepared in Example 1, respectively, using the following specific methods:

[0116] 1) Immunohistochemical staining method for bone marrow cell paraffin blocks:

[0117] Following standard procedures, the primary antibody used was the CD20 antibody produced by Dako, and the blocking agent and secondary antibody were the RQ7600 immunochromatographic reagent from Zhongshan Aoquan Medical Technology Co., Ltd. The bone marrow cell blocks obtained in Example 1 were subjected to immunohistochemical staining according to the following procedure: dewaxing, antigen retrieval, blocking, primary antibody, secondary antibody, and immunochromatographic staining.

[0118] 2) Immunohistochemical staining method for bone marrow smears:

[0119] Following standard procedures, CD42b antibody from GeneTech (Shanghai) Co., Ltd. was used as the primary antibody and the blocking agent. The Leica RE7280-K polymer detection system was used as the secondary antibody. Immunohistochemical staining of bone marrow smears was performed according to the following procedure: blocking, primary antibody, secondary antibody, and immunochromatography.

[0120] 2. Results

[0121] The results of immunohistochemical staining of the bone marrow cell paraffin block prepared in Example 1 are as follows: Figure 1 As shown, Figure 1 The result of immunohistochemical staining of CD20 cell membranes is positive. The result of immunohistochemical staining of bone marrow smears is as follows: Figure 2 As shown, Figure 2 The result is positive for CD42b megakaryocytes after immunohistochemical staining.

[0122] from Figure 1 As can be seen, immunohistochemical staining with the above-mentioned bone marrow cell paraffin blocks improves the sensitivity of antigen-antibody binding, provides a sufficient number of cells for diagnosis under 20X field of view, eliminates erythrocyte interference in diagnosis, and reduces the impact of nonspecific staining on the results.

[0123] And from Figure 2 As can be seen, under 20X vision, the number of nucleated cells is sparse, and the stroma is filled with a large number of red blood cells, resulting in a limited number of cells that can be effectively used for diagnosis. Furthermore, because the smear contains a large number of red blood cells, the reaction between the red blood cells and the immunohistochemical chromogenic agent DAB produces a brownish-yellow background, which interferes with interpretation.

[0124] Example 3

[0125] This embodiment screens commercially available erythrocyte lysis buffer.

[0126] 1. Method

[0127] Following the method in Example 1, different commercially available erythrocyte lysis buffers were selected and designated as Group A, Group B, and Group C, respectively. Group D (without erythrocyte lysis buffer) was set up as a control group. Different bone marrow cell blocks were prepared according to the method in Example 1, and then immunohistochemical staining was performed according to the method in Example 2. The antibody used for testing was CD138.

[0128] Group A: Beijing Solarbio Science & Technology Co., Ltd., Product Name: Red Blood Cell Lysis Buffer, Product No.: R1010, Specification: 100ml.

[0129] Group B: Tiangen Biotech (Beijing) Co., Ltd., Product Name: Red Blood Cell Lysis Buffer, Product No.: RT122-02, Specification: 250ml.

[0130] Group C: Albatross Biotechnology (Guangzhou) Co., Ltd., Product Name: No-wash flow cytometry erythrocyte lysis buffer, Product No.: 100-302-100, Specification: 100ml.

[0131] Group D: The red blood cell lysis buffer treatment step was not performed; after the fixation step, the red blood cell lysis step was skipped and the washing step was performed.

[0132] 2. Results

[0133] Group A staining results are as follows: Figure 3 As shown, Figure 3 The results of immunohistochemical staining for CD138 in group A (20×) are shown in the figure. The red arrows in the figure point to plasma cells, which should be positive for CD138 expression. However, it can be seen from the figure that the staining results show no positive expression of CD138 in the target cells, which reduces the binding force between the antigen and the antibody.

[0134] The staining results for group B are as follows: Figure 4 As shown, Figure 4 The results of immunohistochemical staining for CD138 in group B (20×) are shown in the figure. As can be seen from the figure, the immunohistochemical detection results accurately assess the positive localization of CD138 target cells, with strong staining, suggesting that the red blood cell lysis buffer in group B can lyse red blood cells while having minimal impact on white blood cells.

[0135] Group C staining results are as follows Figure 5 As shown, Figure 5 The results of immunohistochemical staining for CD138 in group C (20×) are shown in the figure. The red arrows point to plasma cells, which should be positive for CD138 expression. However, it can be seen from the figure that the target cells for CD138 in immunohistochemical staining are accurately located, but the positive expression intensity is reduced, resulting in weak staining and poor observation effect, showing a decrease in the binding force between antigen and antibody.

[0136] The staining results of group D are as follows: Figure 6 As shown, Figure 6 The image shows the results of CD138 detection by immunohistochemical staining in group D (20×). As can be seen from the image, the antigen-antibody binding affinity of the sample without erythrocyte lysis buffer is still acceptable. However, the fibrinogen and erythrocytes in the bone marrow aspirate show a large area of ​​adsorption of the chromogenic agent DAB, which is consistent with the positive result of the CD138 target cells being observed, creating significant interference and making accurate interpretation difficult.

[0137] The above experimental results show that group B, which uses erythrocyte lysis buffer from Tiangen Biotech (Beijing) Co., Ltd., exhibits the lowest cell integrity and antigen expression loss, resulting in the best staining effect.

[0138] Example 4

[0139] This embodiment verifies the stability of the method in Example 1, and verifies the effect of different storage temperatures and durations on the experimental results.

[0140] 1. Method

[0141] Following the method of Example 1, different storage temperatures and durations were selected to set up different experimental groups. Different bone marrow cell paraffin blocks were prepared according to the method of Example 1, and then immunohistochemical staining was performed according to the method of Example 2 to test the antibody CD138.

[0142] Group 1: Bone marrow aspirate was injected into the bone marrow cell fixation solution in Table 1 of Example 1 and stored and transported at room temperature (approximately 20-35°C) for 8-48 hours.

[0143] Group 2: Bone marrow aspirate was injected into commercially available EDTA.K2 anticoagulant tubes for preservation and testing. It was stored and transported at refrigerated temperatures of 2-8°C for 8-48 hours.

[0144] Group 3: Bone marrow aspirate was injected into the bone marrow cell fixation solution in Table 1 of Example 1, and stored and transported at room temperature for 48-72 hours.

[0145] Group 4: The bone marrow aspirate was injected into commercially available EDTA.K2 anticoagulant tubes for preservation and testing. It was stored and transported at refrigerated temperatures of 2-8°C for 48-72 hours.

[0146] Group 5: Without injecting the bone marrow aspirate into the bone marrow cell fixative, store and transport it directly at room temperature for 48-72 hours.

[0147] 2. Results

[0148] The staining results of the first and second groups are as follows: Figure 7-8 As shown, Figure 7-8 The results of immunohistochemical staining for CD138 in the first and second groups are shown (20×). The results showed that there was no difference in cell morphology and immunohistochemical positivity between the two groups, indicating good staining effect.

[0149] The staining results for groups three and four are as follows: Figure 9-10 As shown, Figure 9-10 The results of immunohistochemical staining for CD138 in groups 3 and 4 (20×) are shown. The results showed no significant difference in cell morphology between the two groups, but the positivity for CD138 in immunohistochemical staining was reduced. Furthermore, the results indicated that storage at room temperature for more than 48 hours affected erythrocyte lysis; erythrocyte fragments (indicated by the red arrows in the figure) reacted with DAB in the tissue cells, appearing as a brownish-yellow interference background.

[0150] The results of the fifth group of staining are as follows Figure 11 As shown, Figure 11 The results of the fifth group of immunohistochemical staining for CD138 (20×) show that after 48-72 hours of storage at room temperature without the addition of bone marrow cell fixative, the cells self-healed without structure, and the antigens on the cells could not bind to the antibodies, resulting in experimental failure.

[0151] The above experimental results show that treatment with commercially available EDTA.K2 anticoagulant tubes or self-prepared bone marrow cell fixative has little impact on tissue cells. However, EDTA.K2 anticoagulant tubes need to be stored and transported at refrigeration at 2-8℃, while self-prepared bone marrow cell fixative can be stored and transported at room temperature. For both storage and transport methods, the optimal fixation time for specimens is within 8 to 48 hours.

[0152] Example 5

[0153] This embodiment compares the number of times the red nucleus is washed in the method of Example 1.

[0154] 1. Method

[0155] Following the method in Example 1, different numbers of erythrocyte lysis and washing were selected to set up different experimental groups. Different bone marrow cell paraffin blocks were prepared according to the method in Example 1, and then immunohistochemical staining was performed according to the method in Example 2 to test the antibody CD138.

[0156] Group 1: Repeat the red-breaking step 4-6 times, and repeat the washing step 4-6 times.

[0157] Group 2: Repeat the red-breaking step 1-3 times, and repeat the washing step 1-3 times.

[0158] 2. Results

[0159] The staining results of the first and second groups are as follows: Figure 12-13 As shown, Figure 12-13 The results of immunohistochemical staining for CD138 in the first and second groups are shown (20×). The results showed that with the increase of the number of erythrocyte lysis and washing cycles, the impact on cell staining increased, such as changes in cell morphology, unclear nuclear membrane, and diffuse cell structure.

[0160] The above experiments show that the number of red blood cell lysis cycles and washing cycles should ideally be 1-3 times.

Claims

1. A method for preparing bone marrow cell paraffin blocks, characterized in that, Includes the following steps: Pretreatment: When bone marrow biopsy tissue cannot be obtained during clinical bone marrow aspiration, aspirate 2-3 ml of bone marrow aspirate and inject it into an anticoagulant tube containing anticoagulant for later use; when bone marrow biopsy tissue can be obtained during clinical bone marrow aspiration, aspirate ≥1 ml of bone marrow aspirate and inject it into bone marrow cell fixation solution for later use. Red blood cell lysis: Take bone marrow aspirate, separate the precipitate containing cells and sediment, add red blood cell lysis agent to lyse the red blood cells, and separate the lysed precipitate containing unlysed cells and sediment. Molding: The lysed precipitate is resuspended in formalin solution for fixation, the supernatant is removed, and then agar solution is added for resuspending. The agar is cooled and solidified, and then placed in formalin for fixation again. Dehydration: Dehydrating the solidified agar blocks; Embedding: The agar block that has undergone the above dehydration treatment is embedded in paraffin wax to make a wax block, which is the result; The bone marrow cell fixative is a PBS buffer containing 40%-60% methanol (by volume) and 15-22 mg / 100 ml dipotassium ethylenediaminetetraacetate.

2. The preparation method according to claim 1, characterized in that, The volume percentage concentration of methanol is 50%.

3. The preparation method according to claim 1, characterized in that, When the bone marrow aspirate is stored in an anticoagulant tube, it is stored at 2-8°C, and the bone marrow cell wax block is prepared within 72 hours of cells being ex vivo in the bone marrow aspirate. When the bone marrow aspirate is stored in bone marrow cell fixative, it is stored at 2-40°C, and the bone marrow cell block is prepared within 72 hours after the cells are removed from the bone marrow aspirate.

4. The preparation method according to claim 3, characterized in that, When the bone marrow aspirate is stored in an anticoagulant tube, it should be stored at 4-6°C, and the bone marrow cell wax block should be prepared within 8-48 hours after the cells in the bone marrow aspirate are removed from the body. When the bone marrow aspirate is stored in bone marrow cell fixative, it is stored at 20-30°C, and the bone marrow cell block is prepared within 8-48 hours after the cells are removed from the bone marrow aspirate.

5. The preparation method according to claim 1, characterized in that, The pH value of the PBS buffer is 7.0-7.4; And / or, the ratio of the amount of bone marrow cell fixative to the amount of bone marrow aspirate is 2-4:1; And / or, the anticoagulant is dipotassium ethylenediaminetetraacetate or sodium heparin.

6. The preparation method according to claim 5, characterized in that, The pH value of the PBS buffer is 7.2-7.4; And / or, the ratio of the amount of bone marrow cell fixative to the amount of bone marrow aspirate is 3:1; And / or, the anticoagulant is dipotassium ethylenediaminetetraacetate.

7. The preparation method according to claim 1, characterized in that, The erythrocyte lysis agent is selected from any one of the following erythrocyte lysis buffers: erythrocyte lysis buffer R1010 from Beijing Solarbio Science & Technology Co., Ltd., erythrocyte lysis buffer RT122-02 from Tiangen Biotech Co., Ltd., and erythrocyte lysis buffer 100-302-100 from Albatross Biotechnology Co., Ltd.

8. The preparation method according to claim 1, characterized in that, The erythrocyte lysis agent is the erythrocyte lysis buffer with product number RT122-02 from Tiangen Biotech Co., Ltd.

9. The preparation method according to claim 1 or 4, characterized in that, The volume ratio of the erythrocyte lysis agent to the precipitate is 2-4:1; And / or, the time for erythrocyte lysis after adding the erythrocyte lysis agent is 3-10 min; And / or, the erythrocyte lysis agent is added to bring the erythrocyte lysis temperature to 20°C-35°C.

10. The preparation method according to claim 1 or 4, characterized in that, The volume ratio of the erythrocyte lysis agent to the precipitate is 3:1; And / or, the red blood cell lysis time is 4-6 minutes after adding the red blood cell lysis agent; And / or, the erythrocyte lysis agent is added to bring the erythrocyte lysis temperature to 25-30°C.

11. The preparation method according to claim 1, characterized in that, One or more of the following conditions must be met: (1) In the red breaking step, the precipitate is obtained by centrifugation at 1300±300rpm for 5±3min; (2) In the red blood cell lysis step, the red blood cell lysis is accelerated by inverting the reagent container. (3) In the red-breaking step, the pyrolysis precipitate is obtained by centrifugation at 3000±300rpm for 5±2min; (4) In the red blood cell breaking step, if it is observed that the lysed precipitate still contains red blood cells, the red blood cell breaking step is repeated; (5) Between the red rupture step and the molding step, there is also a washing step, which is: resuspending the lysed precipitate in PBS buffer, centrifuging, and removing the supernatant; (6) In the molding step, the volume percentage concentration of the formalin solution is 4±1%; (7) In the molding step, the volume of formalin solution added is 5-10 times the volume of the sample; (8) In the molding step, the fixing time is 5-10 min; (9) In the molding step, the supernatant is removed after centrifugation at 1500±300 rpm for 2-3 minutes; (10) In the molding step, the agar solution is added dropwise, and the temperature of the agar solution is 45-50℃; (11) In the molding step, the mass-volume percentage concentration of the agar solution is 4-10%; (12) In the molding step, the amount of agar solution added is 0.1-0.5 ml; (13) In the dehydration step, the solidified agar block is wrapped with filter paper or gauze and placed in the embedding box for dehydration by an automatic tissue dehydrator; (14) In the embedding step, the largest surface of the whole piece of agar containing tissue cells is embedded.

12. The preparation method according to claim 11, characterized in that, One or more of the following conditions must be met: (1) In the red-breaking step, when the lysed precipitate is observed to be red, it is determined that it contains red blood cells, and the red-breaking step is repeated; (2) Between the red rupture step and the molding step, there is also a washing step, which is: resuspending the lysed precipitate in 5-8 times the volume of the lysed precipitate in PBS buffer, centrifuging, and removing the supernatant; (3) Between the red rupture step and the molding step, there is also a washing step, which is: resuspending the lysed precipitate in PBS buffer, centrifuging at 1500±300 rpm for 2-3 min and then removing the supernatant; (4) In the molding step, the volume percentage concentration of the formalin solution is 4%; (5) In the molding step, the mass-volume percentage concentration of the agar solution is 5%; (6) In the molding step, the amount of agar solution added is 0.1 ml.

13. The preparation method according to claim 12, characterized in that, Between the red lysis step and the molding step, there is also a washing step, which is to resuspend the lysed precipitate in PBS buffer, centrifuge at 2-6°C, and remove the supernatant; the washing step is repeated 1-3 times.

14. Bone marrow cell paraffin blocks prepared by the preparation method according to any one of claims 1-13.

15. The use of the bone marrow cell block of claim 14 in research on hematologic diseases for non-diagnostic and therapeutic purposes.

16. The application as described in claim 15, characterized in that, The bone marrow cell paraffin blocks were used for immunohistochemical staining.

17. The application as described in claim 16, characterized in that, The immunohistochemical staining was used to detect CD20 and / or CD138.

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