WO2021238015A1 - 一种细胞病理学样本的多重染色制片方法 - Google Patents
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Definitions
- the invention relates to a method for multiple staining of cells in the field of cytopathology.
- This method can simultaneously display detailed cell morphological characteristics and the expression information of one or more specific biomarkers on cell samples.
- the staining results are suitable for observation under an ordinary optical microscope.
- the expression of one or more specific biomarkers on the same cell can be observed at the same time.
- the present invention also relates to the cells obtained by the method and the pathological section carrying the cells.
- Cytopathology is mainly based on the abnormal conditions within the cells to study the causes and pathogenesis of diseases, as well as the changes in the physiological functions of cells in the process of disease occurrence, so as to propose the basis for diagnosis and prevention of diseases.
- Clinical samples cover exfoliative cytology, fine needle aspiration cytology, blood circulating tumor cells, and other cytology (cytology during surgery, bone marrow, peripheral blood cytology, AIDS cytology, etc.).
- Staining results can show the characteristics of cell membranes, such as cell membrane folds and bulges; cytoplasmic mucus, fat granules, neuroendocrine granules; the characteristics of cell nuclei, including the shape and size of the nucleus, the number and shape of nucleoli, the number of chromosomes, and chromatin
- the texture provides cell pathologists with diagnostic information on cell morphology.
- cytopathology The workflow of cytopathology is to make pathological smears of cell samples, perform routine staining, and then perform cytopathological morphological diagnosis.
- cytopathologists can perform immunocytochemical staining or nucleic acid chromogenic in situ hybridization staining on protein or nucleic acid markers of cells to further help diagnosis, judge prognosis, and select treatment.
- current cytopathological samples cannot be further stained by cellular immunohistochemistry or nucleic acid chromogenic in situ hybridization after routine morphological staining. This clinical disadvantage is caused by the following reasons:
- the invention relates to a cell multiple staining method in the field of cytopathology, which can simultaneously display detailed diagnosable cell morphological characteristics and expression information of one or more specific biomarkers on the same sample cell.
- the staining results are suitable for observation under a common optical microscope. Under the premise of meeting the pathological diagnosis requirements of pathologists, the expression of one or more specific biomarkers on the same cell can be observed at the same time.
- the staining process does not lose cells and is suitable for multiple staining of samples with a small number of cells.
- the invention solves the technical difficulties of conventional cytopathological staining and multiple staining of biomarkers. After the cells are subjected to immunohistochemistry or nucleic acid chromogenic in situ hybridization staining, the same cell is then subjected to cytopathological conventional staining. Using these methods, it is possible to obtain diagnostic-level cell morphology characteristics and information of multiple biomarkers on the same cell under an ordinary optical microscope.
- the staining results can show the morphological characteristics required for cytopathological diagnosis, including but not limited to: cell membrane morphology such as cell membrane folds and overall cell characteristics and size; cytoplasmic mucus, fat granules, neuroendocrine granules; cell nucleus characteristics, Including the shape and size of the nucleus, the number and shape of nucleoli, and the texture of chromatin.
- This method clearly provides cytopathologists with basic information about cell morphological diagnosis, and at the same time combines the expression information of one or more biomarkers on the same cell, which helps improve the accuracy of cytopathological diagnosis.
- the present invention also relates to the innovation of cell immunohistochemistry and nucleic acid chromogenic in situ hybridization staining.
- cell nucleic acid markers on conventional pathological slides is observed by the method of chromogenic in situ hybridization staining of nucleic acid.
- chromogenic in situ hybridization staining of nucleic acid By adding specific nucleic acid fragments to the samples on the pathological slides as the original nucleic acid probes, and then adding special reagents that can recognize the original probes labeled with chromogenic enzymes, and then adding the corresponding chromogenic enzyme substrates, after the chromogenic enzymes Catalyze the reaction, and finally produce the precipitation of the color product, which shows the expression signal of a specific nucleic acid in the cell on the pathological slide.
- the core of a cytopathological sample is cells, and each cell is individually adsorbed or placed on a pathological glass slide by methods such as smears to form a so-called cell smear.
- the cell smear sample is in an open environment during the staining process. During the staining process, the staining liquid must be constantly replaced, washed, and then discarded. With the continuous replacement and disposal of the staining solution and washing solution, some cell samples on the pathological slides will inevitably fall off and be lost, reducing the sensitivity and accuracy of the staining results, and prone to false negatives, especially when the sample contains a small number of cells. in the case of.
- the chromogenic reaction of immunohistochemistry and chromogenic in situ hybridization is carried out on pathological glass slides.
- the catalyzed reaction produces optically visible chromogenic products, which are concentrated and precipitated near the chromogenic enzyme at the cellular level, representing targeted biomarkers Expression signal.
- the precipitated chromogenic product is opaque. If the deposition is too dense on the cells, the chromogenic product will cover the characteristics of cell morphology and affect the pathomorphological diagnosis of cells when observed under an ordinary optical microscope.
- the inventor adopted a new and improved staining method of cellular immunohistochemistry and nucleic acid chromogenic in situ hybridization.
- the pathological slide is not used as a carrier for staining, but the sample cells are placed in a container and stained in the form of cell suspension. After the cells are stained, the smear is fixed and observed under a microscope. The order in which the sample cells are fixed is opposite to the conventional method.
- the inventor places the cells in a container, and completes the incubation of the first antibody or nucleic acid probe in the cell suspension, and the color reaction and other dyeing processes.
- centrifugal precipitation of cell samples is used to eliminate target cells in the discarded supernatant rinsing solution, which greatly reduces the loss of clinical cell samples during the staining process.
- spread the stained cells on the pathological glass slide to ensure the stability and reliability of the results.
- the inventors did not use pathological slides as a carrier during the staining and chemical development process, and always allowed the cells to be fully immersed and contacted in a suspension state.
- the staining reagents there are no staining blind spots and dead corners, which can improve the staining effect of cells, which is obviously helpful for improving the sensitivity of staining and reducing the false negative rate of staining.
- the improved chemical color development method makes the color products evenly distributed.
- Conventional immunohistochemical staining and nucleic acid chromogenic in situ hybridization are carried out on pathological glass slides.
- the color reaction is carried out on fixed tissues.
- the catalyzed chromogenic precipitation products are easy to accumulate locally because they are opaque and can be covered.
- the morphological characteristics of the microstructure of cells affect the morphological diagnosis of cytopathology.
- the inventor continuously oscillates the cells suspended in the liquid to uniformly disperse the color development precipitate products produced by the catalysis, and does not accumulate concentratedly, which affects the smoothness of the optical path.
- the chromogenic products produced by the catalysis can be reasonably distributed on the cells, which can display the expression information of the biomarkers without hindering the diagnosis of cell morphology, and greatly improve the accuracy of the diagnosis of cell morphology by pathologists.
- the present invention can fully wash a single cell in the cell suspension in all directions without blind areas and dead corners. At the same time, because there are no adverse effects such as non-specific deposition and edge effects on the pathological slides, it can greatly reduce the pathological slides. Non-specific background staining noise. It is obviously helpful to improve the staining effect of cells, increase the sensitivity of staining, and reduce the false positive rate of staining results.
- the present invention relates to the following technical solutions:
- the present invention relates to a multiple staining method for immunohistochemical staining and/or nucleic acid chromogenic in situ hybridization staining of cell samples and cell morphological staining that can be used for pathological diagnosis, including:
- the first antibody and/or first nucleic acid probe that specifically binds to the antigen and/or nucleic acid marker on the cell is added to the cell suspension to complete the first antibody and/or first nucleic acid The specific binding of the probe to the antigen and/or nucleic acid marker;
- c) Perform cell morphological staining for pathological diagnosis on the cells in the container, and then coat the multiple stained cells on a pathological glass slide; or, perform immunohistochemical staining and/or nucleic acid chromogen The cells stained by position hybridization are spread on the pathological glass slide, and the cell morphology staining can be used for pathological diagnosis.
- step a) includes centrifuging the cell sample with the cell fixation solution added, and then preparing a cell suspension in a container, and then adding a reagent that increases the permeability of the cell membrane to facilitate staining of the nucleus and cytoplasm.
- steps ii), iv) and vi) the unbound first antibody and/or the first nucleic acid probe are removed, and the unbound second antibody conjugated with the chromogenic enzyme is removed And/or the second nucleic acid probe labeled with a chromogenic enzyme and removing excess chromogenic substrate go through the following steps:
- the cell morphological staining that can be used for pathological diagnosis is selected from Diff-Quik staining, Papanicolaou, Wright-Giemsa staining, and hematoxylin/eosin ( H&E) dyeing, or its derivative or modified form.
- the cell morphological staining that can be used for pathological diagnosis is Diff-Quik staining.
- the cell sample is selected from a cytopathology fine needle aspiration (FNA) sample, a tumor cell sample, a cervical scrape cell sample, and a urine exfoliated cell sample, or the cell sample is obtained from a patient by dilution Obtained from the cell precipitate of the sample from the patient, the sample from the patient is selected from body fluids, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, pleural fluid , Ascites, lung lavage fluid, cerebrospinal fluid, stool and tissue samples.
- FNA cytopathology fine needle aspiration
- the first antibody and/or first probe is a combination of multiple antibodies and/or probes, which respectively stain the cell membrane, cytoplasm, and/or cell nucleus.
- the present invention relates to multi-stained cells in vitro, which have both morphological staining and cell biomarker staining that can be used for pathological diagnosis.
- the cells are from a cell sample selected from the group consisting of: cytopathology fine needle aspiration (FNA) sample, blood circulating tumor cell sample, cervical smear cell sample, urine exfoliated cell sample, body fluid, blood , Serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, pleural fluid, ascites, lung lavage fluid, cerebrospinal fluid, stool and tissue samples.
- FNA cytopathology fine needle aspiration
- the cell is a tumor cell, which has both morphological staining and tumor marker staining that can be used for pathological diagnosis.
- the cell is obtained by the method of the invention.
- the present invention relates to a pathological section, which contains multiple stained ex vivo cells as described above.
- the present invention also relates to a multiple staining kit for immunohistochemical staining and/or nucleic acid chromogenic in situ hybridization staining of cell samples and cell morphological staining that can be used for pathological diagnosis.
- a multiple staining kit for immunohistochemical staining and/or nucleic acid chromogenic in situ hybridization staining of cell samples and cell morphological staining that can be used for pathological diagnosis.
- a multiple staining kit for immunohistochemical staining and/or nucleic acid chromogenic in situ hybridization staining of cell samples and cell morphological staining that can be used for pathological diagnosis.
- a multiple staining kit for immunohistochemical staining and/or nucleic acid chromogenic in situ hybridization staining of cell samples and cell morphological staining that can be used for pathological diagnosis.
- the reagents for immunohistochemical staining and/or nucleic acid chromogenic in situ hybridization staining of cell samples include a first antibody and/or a first nucleic acid probe, and a second chromogenic enzyme conjugated reagent.
- the reagents used for cell morphological staining are selected from reagents used for Diff-Quik staining, Pap, Wright-Giemsa staining, and H&E staining, or derivatives or modified forms thereof.
- Figure 1 shows the exfoliated urinary epithelial cells in urine, which were simultaneously stained by Diff-Quik and liquid immunohistochemistry. All exfoliated urinary epithelial cells are stained blue by Diff-Quik, which meets the requirements of pathologists for cell morphological diagnosis. Two of the morphologically atypical urinary epithelial cells were stained brown by liquid immunohistochemistry. The brown signal shown in the figure is the expression of cytokeratin 20 (CK20) in two exfoliated urinary epithelial cells.
- CK20 cytokeratin 20
- Figure 2 shows the exfoliated urinary epithelial cells in urine, which are simultaneously stained by Diff-Quik and liquid-phase nucleic acid color in situ hybridization. All exfoliated urinary epithelial cells are stained blue by Diff-Quik, which meets the requirements of pathologists for cell morphological diagnosis.
- Four of the cells morphologically diagnosed as urinary epithelial carcinoma were stained brown by nucleic acid chromogenic in situ hybridization.
- the brown signal shown in the figure corresponds to the expression of human transcription factor GATA-3 in exfoliated urinary epithelial cancer cells.
- Figure 3 shows the completion of cellular immunohistochemical staining and Diff-Quik staining after smear.
- Two tumor cells in the field of view were stained brown.
- Three normal urine exfoliated epithelial cells were stained dark blue and light pink by Diff-Quik. All cell staining can be used for cytopathological diagnosis.
- chromogenic reagent Peroxidase-Liquid DAB+ (source Dako), place the cell suspension sample on a shaker, shake at 600-2500 rpm, and incubate for 10 minutes.
- DIFF Fast Stain No. 2 source Fisher Scientific, product code #22750012
- DIFF Fast Stain No. 1 source Fisher Scientific, product code #22750012
- Diff Fast Stain No. 1 source Fisher Scientific, product code #22750012
- nucleic acid chromogenic in situ hybridization after smearing, and complete Diff rapid staining in the form of pathological slides.
- chromogenic reagent Peroxidase-Liquid DAB+ (source Dako), place the cell suspension sample on the shaker, shake at 600-2500 rpm, and incubate for 5 minutes.
- the above-mentioned cell suspension is manually prepared with an ultra-thin Pap smear (ThinPrep), which means that the cells are transferred and adsorbed on the pathological glass slide.
- ThinPrep ultra-thin Pap smear
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Abstract
一种细胞病理学样本的多重染色制片方法。涉及对细胞样本进行液相免疫组化染色和/或核酸免疫原位杂交染色以及常规的细胞病理学染色的多重染色方法。还涉及由制片方法得到的离体细胞，载有细胞的病理切片，以及用于制片方法的试剂盒。
Description
本发明涉及细胞病理学领域的一种细胞多重染色制片方法。该方法可以在细胞样本上，同时显示详细的细胞形态学特点，和一个或者多个特定生物标记物的表达信息。染色结果适合在普通光学显微镜下观察，在符合病理学家对细胞病理形态学诊断要求的前提下，可以同时观察一个或者多个特定生物标记物在同一细胞上的表达。本发明还涉及所述方法得到的细胞和载有所述细胞的病理切片。
细胞病理学主要是根据细胞内异常状况，研究疾病发生的原因、发病原理，以及疾病发生过程中，细胞的生理功能发生的改变，从而提出诊断和防治疾病的依据。临床样本涵盖脱落细胞学、细针吸取细胞学、血液循环肿瘤细胞，其他细胞学(手术中的细胞学，骨髓、外周血细胞学，艾滋病细胞学等)。
早期的细胞病理学常规染色方法包括巴氏(Papanicolaou)染色、瑞氏-吉姆萨(Wright-Giemsa)染色等。目前最常用的是迪夫快速染色(Diff-Quik)，其染液是采用世界卫生组织(WHO)推荐的快速染色方法而配制，与瑞氏-吉姆萨类似都是利用巴氏染色技术原理改良而来的。染色结果可以显示细胞膜的特点，比如细胞膜皱褶和凸起；细胞质的粘液、脂肪颗粒、神经内分泌颗粒；细胞核的特点，包括细胞核的形态和大小、核仁的数目和形状、染色体数目、染色质的质地，为细胞病理学家提供细胞形态学诊断信息。
细胞病理学的工作流程是先对细胞样本制作病理涂片，进行常规染色，然后再进行细胞病理形态学诊断。随着科学和技术的发展，细胞病理学家可以对细胞的蛋白或者核酸标志物进行免疫细胞化学染色或者核酸显色原位杂交染色，来进一步帮助诊断、判断预后，和选择治疗。但是，目前细胞病理学样本经过常规形态学染色以后，无法再进一步进行细胞免疫组化或者核酸显色原位杂交染色。这个临床不利点是由以下原因造成的：
1)常规细胞病理学染色方法的染料会阻碍第一抗体或者第一探针 和相关靶点的结合，因此需要除去这些染料，才能实现生物标记物的免疫组化染色。2)除去染料后，常常需要与常规相比更高浓度的蛋白抗体或者核酸探针来进行染色，容易造成假阳性，误导临床诊断、治疗和判断预后。3)玻片上的细胞涂片在染色过程中处在一个开放的环境，在染色过程中染色液体要不断更换，冲洗，随后丢弃。随着对染色液和冲洗液的不断更换和丢弃，会造成病理玻片上细胞样本脱落而丢失，影响结果的准确性。4)即使个别情况下不需要去除常规染色，免疫组化染色或者显色原位杂交染色过程可导致常规染料脱落，导致常规染色下的细胞形态学特点观察效果不佳。5)通常的免疫组织化学或者核酸显色原位杂交染色，为了提高染色的敏感性，会尽量提升化学染色的过程，会造成显色产物过度集中堆积而覆盖细胞，影响观察形态学的诊断。
尽管免疫组化和显色原位杂交染色成为组织病理学上已经成为不可或缺的辅助染色，因为以上的技术阻碍，细胞病理学上免疫组化和显色原位杂交染色的应用还是非常规的，没有广泛临床应用。因此，本领域还需要新的染色方法以克服上述现有技术的缺点。
发明内容
本发明涉及细胞病理学领域的一种细胞多重染色方法，可以在同一个样本细胞上，同时显示详细的可诊断的细胞形态学特点，和一个或者多个特定生物标记物的表达信息。染色结果适合在普通光学显微镜下观察，在符合病理学家对细胞病理形态学诊断要求的前提下，可以同时观察一个或者多个特定生物标志物在同一细胞上的表达。染色过程不丢失细胞，适合具有少量细胞的样本的多重染色。
本发明解决了细胞病理常规染色和生物标志物多重染色的技术困难，对细胞进行免疫组化或者核酸显色原位杂交染色以后，再对同一细胞进行细胞病理学常规染色。采用这些方法，可以在同一细胞上，在普通光学显微镜下，同时得到诊断级别的细胞形态学特点，和多种生物标志物的信息。染色结果可以显示细胞病理学诊断所需要的形态学特点，包括但不局限于：细胞膜的形态比如细胞膜皱褶和总体细胞性状和大小；细胞质的粘液、脂肪颗粒、神经内分泌颗粒；细胞核的特点，包括细胞核的形态和大小、核仁的数目和形状、染色质的质地。 该方法清晰地为细胞病理学家提供细胞形态学诊断的基本信息，同时结合了一个或者多个生物标记物在同一个细胞上的表达信息，有助提高细胞病理学诊断的准确性。
本发明还涉及到对细胞免疫组化和核酸显色原位杂交染色的创新。
常规的免疫组化染色都是以病理玻片为载体来进行的，常规病理玻片上细胞蛋白标记物的表达是通过免疫组织化学的方法来实现的。通过对病理玻片上的样本加入第一抗体，能够特异性识别第一抗体的显色酶标记的第二抗体，再加入相应的显色酶底物，经过显色酶的催化反应，最终通过产生的显色产物沉淀，在病理玻片显示某种特异性生物标志物在细胞中的表达信号。
常规病理玻片上细胞核酸标志物表达是通过核酸显色原位杂交染色的方法来观察的。通过对病理玻片上的样本加入特异性的核酸片段作为原始核酸探针，再加入能够识别原始探针的显色酶标记的特殊试剂，随后加入相应的显色酶底物，经过显色酶的催化反应，最终产生显色产物沉淀，在病理玻片上显示某种特异性核酸在细胞中的表达信号。
以上两种方法的最大区别是一个使用抗体，一个使用核酸探针进行初始标记，随后的显色反应原理一致。经过病理制片以后都可以在普通光学显微镜下观察，来帮助病理医生做进一步的诊断。
现有的免疫组化和显色原位杂交染色技术对细胞病理学样本来说有以下缺点：
1)细胞病理学样本的核心是细胞，经涂片等方法将每一个细胞单独吸附或者放置在病理玻片上，形成所谓的细胞涂片。细胞涂片样本在染色过程中处在一个开放的环境，在染色过程中染色液体要不断更换，冲洗，随后丢弃。随着对染色液和冲洗液的不断更换和丢弃，不可避免会造成病理玻片上一些细胞样本脱落而丢失，降低染色结果的敏感性和准确性，容易产生假阴性，尤其在样本包含少量细胞数目的情况下。
2)常规细胞病理学免疫组化染色和核酸显色原位杂交以病理玻片为细胞样本的载体，要求细胞样本和病理玻片两者直接紧密接触，之间不能有间隙而导致细胞从玻片上脱落。但染色液因此也无法到达细胞和玻片接触的位置，在细胞和玻片的接触部位形成染色盲区，从 而不能把细胞全方位充分染色，留有染色死角，影响染色结果的敏感性和准确性。
3)免疫组化和显色原位杂交的显色反应在病理玻片上进行，经催化反应产生光学可见的显色产物，在细胞水平上集中沉淀在显色酶附近，代表靶向生物标志物的表达信号。沉淀的显色产物不透光，如果在细胞上沉积过多过密，在普通光学显微镜下观察时，显色产物会覆盖细胞相态学的特点，影响细胞的病理形态学诊断。
发明人在免疫组化和显色原位杂交的染色过程中，采用了新型改良的细胞免疫组化和核酸显色原位杂交的染色方法。
1)改变的染色顺序和流程：采用对细胞样本先染色，再涂片的方法。传统的细胞免疫组化和核酸显色原位杂交染色是以病理玻片为载体，细胞样本先涂片固定，再对固定在病理玻片上的细胞进行染色。
本发明不用病理玻片为载体染色，而是把样本细胞放置在一个容器中，以细胞悬液的形式进行染色，染好细胞以后再进行涂片固定，显微镜下观察。在样本细胞固定的顺序上，和常规的方法相反。
2)改良的换液和冲洗手段，避免细胞样本的丢失。常规的免疫组化染色或者核酸显色原位杂交都是在病理玻片上进行，细胞涂片在染色过程中处在一个开放的环境，在染色过程中染色液体要不断更换，冲洗，随后丢弃。随着对染色液和冲洗液的不断更换和丢弃，会造成病理玻片上细胞样本脱落而丢失，不可避免会影响样本的最终诊断。
在本发明中，发明人把细胞放置于一个容器中，在细胞悬液中完成第一抗体或者核酸探针的孵育，和显色反应等染色过程。在多次更换染色试剂和冲洗液的过程中，通过离心沉淀细胞样本等手段，使丢弃的上清冲洗液中没有靶向细胞，极大幅度地减少了临床细胞样本在染色过程中的流失。完成全部染色以后，再把染好的细胞涂布在病理玻片上，保证结果的稳定可靠。
3)改变的染色环境避免传统染色方法细胞上的染色盲区。传统的免疫组化和核酸显色原位杂交，是在病理玻片上进行染色的。染色试剂无法到达细胞和玻片的接触部位，形成染色盲区和死角。
为使样本细胞能够充分染色，避免细胞上的染色盲区和死角，发明人在染色和化学显色过程中，不用病理玻片为载体，始终让细胞在悬液状态下，充分浸泡和接触在不同的染色试剂中，没有染色盲区和 死角，提高细胞的染色效果，对提高染色的敏感性，降低染色的假阴性率，有显而易见的帮助。
4)改良的化学显色方法使显色产物分布均匀。常规的免疫组化染色和核酸显色原位杂交是在病理玻片上进行的，显色反应在固定的组织上进行，催化产生的显色沉淀产物容易在局部集中堆积，因为其不透明，可以遮盖细胞细微结构的形态学特点，影响细胞病理学的形态学诊断。
发明人在显色过程中，不断震荡悬浮在液体中的细胞，使催化产生的显色沉淀产物均匀散开，不集中堆积而影响光学路径的通畅。使催化产生的显色产物在细胞上分布合理，既能够显示生物标记物的表达信息，又不妨碍细胞形态学的诊断，大幅提高病理学家对细胞形态学诊断的准确性。
5)改进的更有效的消除染色背景噪音的方法。常规的免疫组化染色和核酸显色原位杂交是在病理玻片上进行的，显色反应在固定的组织上进行，各种染色试剂会非特异性沉淀吸附在病理玻片或者细胞组织上。细胞和玻片接触的部位也可以产生染色过程中所谓的边缘效应，造成染色成分的非特异性吸附和沉淀。需要在病理玻片上多次彻底冲洗，才能消除非特异性的试剂吸附和沉淀，也增加细胞从病理玻片脱落而丢失的可能性。
本发明在完成染色反应后，在细胞悬液中对单个细胞全方位无盲区无死角充分冲洗，同时因为不存在病理玻片上的非特异性沉积和边缘效应这些不良效果，能够大幅降低病理玻片上的非特异性背景染色噪音。对提高细胞的染色效果，提高染色的敏感性，降低染色结果的假阳性率，有显而易见的帮助。
具体地，本发明涉及以下技术方案：
一方面，本发明涉及对细胞样本进行免疫组化染色和/或核酸显色原位杂交染色以及可用来病理学诊断的细胞形态学染色的多重染色方法，包括：
a)在容器中制备细胞样本的细胞悬液；
b)在所述容器中的所述细胞悬液中，对所述细胞进行免疫组化染色和/或核酸显色原位杂交染色，包括：
i)在所述细胞悬液中加入特异性结合所述细胞上的抗原和/或 核酸标志物的第一抗体和/或第一核酸探针，完成所述第一抗体和/或第一核酸探针与所述抗原和/或核酸标志物的特异性结合；
ii)去除未结合的所述第一抗体和/或第一核酸探针，然后重新制备细胞悬液，
iii)在所述细胞悬液中加入缀合了显色酶的第二抗体和/或显色酶标记的第二核酸探针，使其特异性结合第一抗体和/或第一核酸探针；
iv)去除未结合的所述缀合了显色酶的第二抗体和/或显色酶标记的第二核酸探针，然后重新制备细胞悬液，
v)在所述细胞悬液中加入显色底物进行显色，在显色过程中，不断震荡悬浮在细胞悬液中的细胞，使催化产生的显色沉淀产物均匀散开而避免集中堆积；以及
vi)去除多余的显色底物，然后重新制备细胞悬液；以及
c)在所述容器中对所述细胞进行可用来病理诊断的细胞形态学染色，然后将多重染色的细胞涂布到病理玻片上；或者，将经过免疫组化染色和/或核酸显色原位杂交染色的细胞涂布到病理玻片上，进行可用来病理诊断的细胞形态学染色。
在一些实施方案中，步骤a)包括离心沉淀加入了细胞固定液的细胞样本，然后在容器中制备细胞悬液，然后加入增加细胞膜通透性的试剂，有利于细胞核和细胞质的染色。
在一些实施方案中，步骤ii)、iv)和vi)中去除未结合的所述第一抗体和/或第一核酸探针、去除未结合的所述缀合了显色酶的第二抗体和/或显色酶标记的第二核酸探针和去除多余的显色底物通过以下步骤：
i)清洗细胞；
ii)离心沉淀细胞；和
iii)丢弃上清液。
在一些实施方案中，所述可用来病理诊断的细胞形态学染色选自Diff-Quik染色、巴氏(Papanicolaou)、瑞氏-吉姆萨(Wright-Giemsa)染色，和苏木精/曙红(H&E)染色，或其衍生或修饰形式。
在一些实施方案中，所述可用来病理诊断的细胞形态学染色是Diff-Quik染色。
在一些实施方案中，所述细胞样本选自细胞病理学细针穿刺(FNA) 样本、肿瘤细胞样本、宫颈刮片细胞样本，和尿液脱落细胞样本，或者所述细胞样本是通过稀释来自患者的样本的细胞沉淀物而得到的，所述来自患者的样本选自体液、血液、血清、血浆、尿、唾液、汗液、痰、精液、粘液、泪液、淋巴液、羊水、间质液、胸水、腹水、肺灌洗液、脑脊液、粪便和组织样本。
在一些实施方案中，所述第一抗体和/或第一探针是多重抗体和/或探针组合，分别染色细胞膜、细胞质，和/或细胞核。
另一方面，本发明涉及经过多重染色的离体细胞，所述细胞同时具备可用来病理诊断的形态学染色和细胞生物标志物染色。
在一些实施方案中，所述细胞来自选自下组的细胞样本：细胞病理学细针穿刺(FNA)样本、血液循环肿瘤细胞样本、宫颈刮片细胞样本、尿液脱落细胞样本、体液、血液、血清、血浆、尿、唾液、汗液、痰、精液、粘液、泪液、淋巴液、羊水、间质液、胸水、腹水、肺灌洗液、脑脊液、粪便和组织样本。
在一些实施方案中，所述细胞是肿瘤细胞，其同时具备可用来病理诊断的形态学染色和肿瘤标志物染色。
在一些实施方案中，所述细胞是通过本发明的方法得到的。
另一方面，本发明涉及病理切片，其载有上文所述的经过多重染色的离体细胞。
另一方面，本发明还涉及用于对细胞样本进行免疫组化染色和/或核酸显色原位杂交染色以及可用来病理学诊断的细胞形态学染色的多重染色的试剂盒，所述试剂盒包含用于对细胞样本进行免疫组化染色和/或核酸显色原位杂交染色的试剂，以及用于细胞形态学染色的试剂。
在一些实施方案中，用于对细胞样本进行免疫组化染色和/或核酸显色原位杂交染色的试剂包括第一抗体和/或第一核酸探针、缀合了显色酶的第二抗体和/或显色酶标记的第二核酸探针和显示底物。
在一些实施方案中，用于细胞形态学染色的试剂选自用于Diff-Quik染色、巴氏、瑞氏-吉姆萨染色，和H&E染色，或其衍生或修改形式的试剂。
图1显示尿液中的脱落泌尿上皮细胞，同时被Diff-Quik和液相免 疫组化多重染色。所有脱落泌尿上皮细胞被Diff-Quik染成蓝色，满足病理医生对细胞形态学诊断的要求。其中两个形态学上不典型的泌尿上皮细胞，被液相免疫组化染成棕色。图中显示的棕色信号是细胞角蛋白20(CK20)在两个脱落泌尿上皮细胞中的表达。
图2显示尿液中的脱落泌尿上皮细胞，同时被Diff-Quik和液相核酸显色原位杂交多重染色。所有脱落泌尿上皮细胞被Diff-Quik染成蓝色，满足病理医生对细胞形态学诊断的要求。其中四个形态学上诊断为泌尿上皮癌的细胞，被核酸显色原位杂交染成棕色。图中显示的棕色信号对应于人类转录因子GATA-3在脱落泌尿上皮癌细胞中的表达。
图3显示完成细胞免疫组化染色，涂片后再进行Diff-Quik染色。视野中两个肿瘤细胞被染成棕色。三个正常尿液脱落上皮细胞被Diff-Quik染成深蓝色和淡粉色。所有细胞染色可以用来进行细胞病理形态学诊断。
实施例1
基于新型改良的细胞免疫化学蛋白标记物染色方法和迪夫快速的细胞多重染色
(所有离心都是以1500r/min离心5min，去上清液，然后将沉淀物重新悬浮在PBS中)
1. 25毫升含有4％多聚甲醛细胞固定液的膀胱癌患者尿液，离心沉淀细胞，丢弃上清液，然后稀释沉淀物并且重新悬浮在1毫升的PBS(pH7.4)缓冲液中，放置到10毫升塑料试管中。
2.加入20微升Triton-100(增加细胞膜通透性，利于细胞质、细胞核染色)。
3.加热细胞悬液92摄氏度5分钟进行抗原修复。
4.在悬浮液中加入100微升Dual Endogenous Enzyme Block(Dako，Carpinteria，Ca)，并且孵育5分钟，以抑制细胞内源性的过氧化物酶(peroxidase)和碱性磷酸酶(alkline phosphatase)的活性。
5.离心后去除上清液和多余的Dual Endogenous Enzyme Block，然后稀释沉淀物并且重新悬浮在1毫升的PBS(pH7.4)缓冲液中。
6.加入200微升Serum-Free Block(Dako)，孵育5分钟(填充非 特异性蛋白结合点)。
7.加入第一抗体，即，200微升CK20小鼠抗体，室温下孵育2小时。
8.加入1毫升PBS缓冲液，混匀后离心，去掉上清液。重复一到三次，冲洗掉未结合的第一抗体。
9.加入200微升羊抗鼠的、多聚体酶标结合的过氧化物酶的第二抗体(EnVision，Dako)；室温下孵育30分钟。
10.用1毫升PBS缓冲液离心冲洗细胞样本三次，去掉未结合的第二抗体。
11.加入显色剂：过氧化物酶-Liquid DAB+(来源Dako)，细胞悬液样本放置振荡器上，震荡速度600-2500rpm，孵育10分钟。
12.加入1毫升PBS缓冲液，然后离心沉淀细胞，去除多余显色剂，
13.用1毫升PBS缓冲液洗涤冲洗染好的细胞样本，然后离心沉淀细胞，丢弃上清液，重复三次，有效消除非特异性的染色背景噪音。
14.加入1毫升迪夫快速染色剂2号(来源Fisher Scientific，产品编号#22750012)，孵育1分钟然后离心，去除多余显色剂；
14.加入1毫升迪夫快速染色剂1号(来源Fisher Scientific，产品编号#22750012)，孵育1分钟然后离心，去除多余显色剂；
16.加入200微升PBS缓冲液，涂片。
在显微镜下观察细胞病理涂片，如图1所示。
也可以完成免疫组化以后涂片，以病理玻片的形式完成迪夫快速染色。
实施例2
基于新型改良的细胞核酸显色原位杂交的染色方法和迪夫快速细胞的多重染色：
(所有离心都是以1500r/min离心5min，去上清液，然后将沉淀物重新悬浮在PBS中)
1. 25毫升含有4％多聚甲醛细胞固定液的膀胱癌患者尿液，离心沉淀细胞，丢弃上清液，然后稀释沉淀物并且重新悬浮在1毫升的PBS(pH7.4)缓冲液中，放置到10毫升塑料试管中。
2.加入20微升Triton-100(增加细胞膜通透性，利于细胞质、细 胞核染色)。
3.用
2.5HD Reagent Kit(ACD Bio，美国加州)免疫组化核酸原位杂交试剂盒按照厂家推荐的实验步骤进行核酸标记物染色。加入人类转录因子GATA-3的RNA核酸探针(Hs-GATA3，ACD Bio，美国加州)，40摄氏度孵育2小时。
4.离心细胞悬液，弃掉上清液，加入1毫升厂家提供的冲洗缓冲液，离心5分钟，弃掉上清液。加入冲洗缓冲液再重复冲洗一次。
5.加入4滴杂交液AMP1，40摄氏度孵育30分钟。
6.离心细胞悬液，弃掉上清液，加入1毫升冲洗缓冲液，离心5分钟，弃掉冲洗上清液。再重复冲洗一次。
7.加入4滴杂交液AMP2，40摄氏度孵育30分钟。
8.离心细胞悬液，弃掉上清液，加入1毫升冲洗缓冲液，离心5分钟，弃掉冲洗上清液。再重复冲洗一次。
9.加入4滴杂交液AMP3，40摄氏度孵育30分钟。
10.离心细胞悬液，弃掉上清液，加入1毫升冲洗缓冲液，离心5分钟，弃掉冲洗上清液。再重复冲洗一次。
11.加入4滴杂交液AMP4，40摄氏度孵育15分钟。
12.离心细胞悬液，弃掉上清液，加入1毫升冲洗缓冲液，离心5分钟，弃掉冲洗上清液。再重复冲洗一次。
13.加入4滴杂交液AMP5，40摄氏度孵育30分钟。
14.离心细胞悬液，弃掉上清液，加入1毫升冲洗缓冲液，离心5分钟，弃掉冲洗上清液。再重复冲洗一次。
15.加入4滴杂交液AMP6，40摄氏度孵育15分钟。
16.离心细胞悬液，弃掉上清液，加入1毫升冲洗缓冲液，离心5分钟，弃掉冲洗上清液。再重复冲洗一次。
17.加入120微升事先混匀的DAB-A和DAB-B显色液，细胞悬液样本放置振荡器上，震荡速度600-2500rpm，孵育10分钟。离心细胞悬液，弃掉上清液。
18.用1毫升1X冲洗液洗涤冲洗染好的细胞样本，然后离心沉淀细胞，丢弃上清液，重复三次，有效消除非特异性的染色背景噪音。
19.加入1毫升迪夫快速染色剂2号(来源Fisher Scientific，产品编号#22750012)，孵育1分钟然后离心，去除多余显色剂。
20.加入1毫升迪夫快速染色剂1号(来源Fisher Scientific，产品编号#22750012)，孵育1分钟然后离心，去除多余显色剂。
21.加入200微升PBS缓冲液，涂片。在显微镜下观察细胞病理涂片，如图2所示。
22.也可以完成核酸显色原位杂交以后涂片，以病理玻片的形式完成迪夫快速染色。
实施例3
基于新型改良的细胞免疫化学蛋白标记物染色方法和迪夫快速细胞多重染色
(所有离心都是以1500r/min离心5min，去上清液，然后将沉淀物重新悬浮在PBS中)
1. 25毫升含有4％多聚甲醛细胞固定液的膀胱癌患者尿液，离心沉淀细胞，丢弃上清液，然后稀释沉淀物并且重新悬浮在1毫升的PBS(pH7.4)缓冲液中，放置到10毫升塑料试管中。
2.加入20微升Tween 20(增加细胞膜通透性，利于细胞质、细胞核染色)。
3.加热细胞悬液92摄氏度5分钟进行抗原修复。
4.在悬浮液中加入100微升Dual Endogenous Enzyme Block(Dako，Carpinteria，Ca)，并且孵育5分钟，以抑制细胞内源性的过氧化物酶(peroxidase)和碱性磷酸酶(alkline phosphatase)的活性。
5.离心后去除上清液和多余的Dual Endogenous Enzyme Block，然后稀释沉淀物并且重新悬浮在1毫升的PBS(pH7.4)缓冲液中。
6.加入200微升Serum-Free Block(Dako)，孵育5分钟(填充非特异性蛋白结合点)。
7.加入第一抗体，即，200微升CK20小鼠抗体，室温下孵育2小时。
8.加入1毫升PBS缓冲液，混匀后离心，去掉上清液。重复一次，冲洗掉未结合的第一抗体。
9.加入200微升羊抗鼠的、多聚体酶标结合的过氧化物酶的第二抗体(EnVision，Dako)；室温下孵育30分钟。
10.用1毫升PBS缓冲液离心冲洗细胞样本三次，去掉未结合的第二抗体。
11.加入显色剂：过氧化物酶-Liquid DAB+(来源Dako)，细胞悬液样本放置振荡器上，震荡速度600-2500rpm，孵育5分钟。
12.加入1毫升PBS缓冲液，然后离心沉淀细胞，去除多余显色剂，
13.用1毫升PBS缓冲液洗涤冲洗染好的细胞样本，然后离心沉淀细胞，丢弃上清液，重复三次，有效消除非特异性的染色背景噪音。重新制备200微升细胞悬液。
14.将上述细胞悬液用手工方法制备超薄柏氏抹片(ThinPrep)，既将细胞转移吸附在病理玻片上。
15.加入被PBS缓冲液稀释成5％的1毫升迪夫快速染色剂2号(来源Fisher Scientific，产品编号#22750012)，孵育30秒，然后用1毫升PBS缓冲液冲洗去除多余显色剂；
16.加入被PBS缓冲液稀释成5％的1毫升迪夫快速染色剂1号(来源Fisher Scientific，产品编号#22750012)，孵育30秒，然后用1毫升PBS缓冲液冲洗去除多余显色剂；
17.加盖玻片然后封片。
在显微镜下观察细胞病理涂片，如图3所示。
Claims (16)
- 对细胞样本进行免疫组化染色和/或核酸显色原位杂交染色以及可用来病理学诊断的细胞形态学染色的多重染色方法，包括：a)在容器中制备细胞样本的细胞悬液；b)在所述容器中的所述细胞悬液中，对所述细胞进行免疫组化染色和/或核酸显色原位杂交染色，包括：i)在所述细胞悬液中加入特异性结合所述细胞上的抗原和/或核酸标志物的第一抗体和/或第一核酸探针，完成所述第一抗体和/或第一核酸探针与所述抗原和/或核酸标志物的特异性结合；ii)去除未结合的所述第一抗体和/或第一核酸探针，然后重新制备细胞悬液，iii)在所述细胞悬液中加入缀合了显色酶的第二抗体和/或显色酶标记的第二核酸探针，使其特异性结合第一抗体和/或第一核酸探针；iv)去除未结合的所述缀合了显色酶的第二抗体和/或显色酶标记的第二核酸探针，然后重新制备细胞悬液，v)在所述细胞悬液中加入显色底物进行显色，在显色过程中，不断震荡悬浮在液体中的细胞，使催化产生的显色沉淀产物均匀散开而避免集中堆积；以及vi)去除多余的显色底物，然后重新制备细胞悬液；以及c)在所述容器中对所述细胞进行可用来病理诊断的细胞形态学染色，然后将多重染色的细胞涂布到病理玻片上；或者，将经过免疫组化染色和/或核酸显色原位杂交染色的细胞涂布到病理玻片上，进行可用来病理诊断的细胞形态学染色。
- 权利要求1所述的方法，其中步骤ii)、iv)和vi)中去除未结合的所述第一抗体和/或第一核酸探针、去除未结合的所述缀合了显色酶的第二抗体和/或显色酶标记的第二核酸探针和去除多余的显色底物通过以下步骤：i)清洗细胞；ii)离心沉淀细胞；和iii)丢弃上清液。
- 权利要求1或2所述的方法，其中所述可用来病理学诊断的细胞形态学染色选自Diff-Quik染色、巴氏(Papanicolaou)、瑞氏-吉姆萨(Wright-Giemsa)染色，和苏木精/曙红(H&E)染色，或其衍生或修改形式。
- 权利要求3所述的方法，其中所述可用来病理学诊断的细胞形态学染色是Diff-Quik染色。
- 权利要求1-3的任一项所述的方法，其中所述细胞样本选自细胞病理学细针穿刺(FNA)样本、血液循环肿瘤细胞样本、宫颈刮片细胞样本，和尿液脱落细胞样本，或者所述细胞样本是通过稀释来自患者的样本的细胞沉淀物而得到的，所述来自患者的样本选自体液、血液、血清、血浆、尿、唾液、汗液、痰、精液、粘液、泪液、淋巴液、羊水、间质液、胸水、腹水、肺灌洗液、脑脊液、粪便和组织样本。
- 权利要求1-5的任一项所述的方法，其中所述第一抗体和/或第一探针是多重抗体和/或探针组合，分别染色细胞膜、细胞质，和/或细胞核。
- 经过多重染色的离体细胞，所述细胞同时具备可用来病理诊断的形态学染色和细胞生物标志物染色。
- 权利要求7的离体细胞，其中所述细胞来自选自下组的细胞样本：细胞病理学细针穿刺(FNA)样本、血液循环肿瘤细胞样本、宫颈刮片细胞样本、尿液脱落细胞样本、体液、血液、血清、血浆、尿、唾液、汗液、痰、精液、粘液、泪液、淋巴液、羊水、间质液、胸水、腹水、肺灌洗液、脑脊液、粪便和组织样本。
- 权利要求7的离体细胞，其中所述细胞是肿瘤细胞，其同时具备可用来病理诊断的形态学染色和肿瘤标志物染色。
- 权利要求7-10的任一项的离体细胞，其是通过权利要求1-5的任一项的方法得到的。
- 病理切片，其载有权利要求7-10的任一项的细胞。
- 用于对细胞样本进行免疫组化染色和/或核酸显色原位杂交染色以及可用来病理学诊断的细胞形态学染色的多重染色的试剂盒，所述试剂盒包含用于对细胞样本进行免疫组化染色和/或核酸显色原位杂交染色的试剂，以及用于细胞形态学染色的试剂。
- 权利要求12所述的试剂盒，其中用于对细胞样本进行免疫组化染色和/或核酸显色原位杂交染色的试剂包括第一抗体和/或第一核酸探针、缀合了显色酶的第二抗体和/或显色酶标记的第二核酸探针和显示底物。
- 权利要求12或13所述的试剂盒，其中所述用于细胞形态学染色的试剂选自用于Diff-Quik染色、巴氏、瑞氏-吉姆萨染色，和H&E染色，或其衍生或修改形式的试剂。
- 权利要求13所述的试剂盒，其中所述第一抗体和/或第一探针是多重抗体和/或探针组合，分别染色细胞膜、细胞质，和/或细胞核。
- 权利要求12-15的任一项所述的试剂盒，其中所述细胞样本选自细胞病理学细针穿刺(FNA)样本、血液循环肿瘤细胞样本、宫颈刮片细胞样本，和尿液脱落细胞样本，或者所述细胞样本是通过稀释来自患者的样本的细胞沉淀物而得到的，所述来自患者的样本选自体液、血液、血清、血浆、尿、唾液、汗液、痰、精液、粘液、泪液、淋巴液、羊水、间质液、胸水、腹水、肺灌洗液、脑脊液、粪便和组织样本。
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| CN118091127A (zh) * | 2023-12-07 | 2024-05-28 | 深圳市森盈生物科技有限公司 | 一种乳腺细胞免疫化学染色试剂盒及检测方法 |
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| CN115372107A (zh) * | 2021-05-21 | 2022-11-22 | 深圳安侣医学科技有限公司 | 前处理试剂及制备方法和细胞染色方法及前处理方法 |
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| WO2018208122A1 (ko) * | 2017-05-12 | 2018-11-15 | 주식회사 온코태그디아그노스틱 | 담도 세포에서 메티오닐-타알엔에이 합성효소를 이용한 담도암 진단 방법 |
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| CN118091127A (zh) * | 2023-12-07 | 2024-05-28 | 深圳市森盈生物科技有限公司 | 一种乳腺细胞免疫化学染色试剂盒及检测方法 |
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