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Quantifying Podocytes and Parietal Epithelial Cells in Human Urine Using Liquid-based Cytology and WT1 Immunoenzyme Staining
使用基于液体的细胞学检查和WT1免疫酶染色定量人尿中的足细胞和壁层上皮细胞   

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Di Wang Di Wang
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参见作者原研究论文

本实验方案简略版
Cytopathology
Dec 2017

Abstract

In glomerular disease, podocytes and parietal epithelial cells (PECs) are shed in the urine. Therefore, urinary podocytes and PECs are noninvasive biomarkers of glomerular disease. The purpose of this protocol is to employ immunocytochemistry to detect podocytes and PECs, using the WT1 antibody on liquid-based cytology slides.

Keywords: Podocytes (足细胞), Parietal epithelial cells (壁层上皮细胞), Liquid-based cytology (基于液体的细胞学检查), WT1 (WT1), Immunocytochemistry (免疫组化), Glomerular disease (肾小球疾病), Crescent formation (新月体形成), Urine (尿液)

Background

Podocytes line the exterior of glomerular capillaries and thus face the Bowman’s capsule and primary urine. In glomerular injury, podocytes may detach from the glomerular basement membrane. The detachment of podocytes and their shedding in the urine have been implicated in the progression of glomerular diseases and crescent formation. Parietal epithelial cells (PECs) cover the inner aspect of the Bowman’s capsule. Similar to podocytes, it has been reported that PECs positive for WT1, proliferate and are shed in the urine during active glomerular disease (Zhang et al., 2012; Fujita et al., 2017).

Previous studies have shown a relationship between the number of urinary podocytes and PECs and various types of glomerular disease (Hara et al., 1998; Nakamura et al., 2000; Achenbach et al., 2008). The studies cited above used conventional methods (direct smears and cytospin) to prepare the urine samples. However, these conventional methods have common problems, including an air-drying effect and the possibility of significant cell loss during the cytocentrifugation and fixation processes. In addition, standardization and quantitative evaluation are difficult with the conventional methods. Although most previous studies have used immunofluorescence staining with the podocalyxin antibody, this method is difficult to perform in small- and medium-sized hospitals because of the specialty antigen and the requirement for a fluorescence microscope.

A useful urinary biomarker test should be easy to perform and have minimal requirements for standardized sample preparation and immunocytochemistry. Therefore, we have devised a method for detecting podocytes and PECs by combining liquid-based cytology, the WT1 antibody, and immunoenzyme staining. SurePathTM liquid-based cytology was developed as a replacement for the conventional sample preparation method because of its better cell preservation and higher cell recovery rate. SurePathTM allows the possibility of standardization and quantitative evaluation, and this system can be operated manually without machines. Furthermore, immunoenzyme staining using the WT1 antibody is performed in most pathological laboratories. Therefore, our method can be an inexpensive, simple, and internationally standardized method for the detection of urinary podocytes and PECs.

Materials and Reagents

  1. Saline-soaked gauze balls
  2. Medicine spoon
  3. Conical centrifuge tube (Corning, Falcon®, catalog number: 352097 ) or equivalent
  4. Kimwipes (KCWW, Kimberly-Clark, catalog number: 34133 ) or equivalent
  5. PAP pen (Abcam, catalog number: ab2601 ) or equivalent
  6. BD TotalysTM Slide Prep Slide Rack (BD, catalog number: 491294 )
  7. Coverslip (Matsunami Glass, catalog number: C024321 ) or equivalent
  8. BD SurePathTM Manual Method Kit (including settling chamber and positively charged slide) (BD, catalog number: 491266 )
  9. BD CytoRichTM Red Preservative Fluid (BD, catalog number: 491336 )
  10. Ethanol (Wako Pure Chemical Industries, catalog number: 059-06957 ) or equivalent
  11. Methanol (Wako Pure Chemical Industries, catalog number: 136-09475 ) or equivalent
  12. Hydrogen peroxide (Wako Pure Chemical Industries, catalog number: 086-07445 ) or equivalent
  13. Phosphate-buffered saline (PBS) (Nichirei Biosciences, catalog number: 415223 ) or equivalent
  14. WT1 antibody (clone 6F-H2) (Agilent Technologies, catalog number: M3561 )
  15. Antibody diluent (Agilent Technologies, catalog number: S3022 ) or equivalent
  16. N-Histofine® Simple StainTM MAX PO (MULTI) (Nichirei Biosciences, catalog number: 414151F )
  17. N-Histofine® DAB-3S Kit (Nichirei Biosciences, catalog number: 415192F )
  18. Mayer’s hematoxylin (Muto Pure Chemicals, catalog number: 30002 ) or equivalent
  19. Xylene (Wako Pure Chemical Industries, catalog number: 245-00717 ) or equivalent
  20. Malinol (mounting medium) (Muto Pure Chemicals, catalog number: 20093 ) or equivalent

Equipment

  1. Pipette (Nichiryo, model: Nichipet EXII ) or equivalent
  2. Centrifuge (Kubota, model: M4000 ) or equivalent
  3. Vortex mixer (Scientific Industries, model: Vortex-Genie 2 ) or equivalent
  4. Microscope (Olympus, model: BX43 ) or equivalent

Procedure

  1. Urine collection
    1. First-morning mid-stream-voided urine specimens are the most suitable for our method.
    2. Although spot mid-stream-voided urine is also likely usable, we have not verified such evidence.
    3. In catheter urine, WT1-positive cells are numerically diluted by mechanically detached urothelial cells.
    4. In the case of female samples, prior cleaning of the urethral meatus using saline soaked gauze balls before urine collection is preferable to prevent contamination by cells from the vulva. Additionally, samples collected during menstruation are not suitable for our method.

  2. Cytology specimen preparation (SurePathTM liquid-based cytology)
    The principle underlying the SurePathTM system is gravity sedimentation and electrical adhesion. In brief, the settling chamber is clamped to a positively charged slide, and the cell suspension is added. Gravity then settles the cells onto the slide, and the positively charged slide is combined with negatively charged cells (Figures 1 and 2).


    Figure 1. SurePathTM system manual method kit. A. Slide rack and manual method kit. B. A positively charged slide and a settling chamber set in a rack.


    Figure 2. The principle underlying the SurePathTM system. A. Transfer the specimen to a settling chamber and mount on a positively charged slide. B. Negatively charged cells sediment by gravity and electrically combine with positively charged slides. C. Excess cells are removed together with the supernatant. D. SurePathTM slides consist of a 13 mm circular monolayer smear.

    1. Stir the whole amount of urine with a medicine spoon to make it homogeneous.
    2. Distribute 10 ml of the homogeneous urine into a none-sterilized 15 ml conical test tube.
    3. Spin at 800 x g for 5 min in a swing-type centrifuge.
      Note: The centrifuge should be swing-type, not angle-type.
    4. Decant the supernatant with an aspirator or 5 ml pipette.
    5. Resuspend the sediment in 10 ml of CytoRich RedTM.
      Notes:
      1. In the case of macroscopic hematuria, collect and fix the buffy coat only after removal of the supernatant.
      2. The urine sediment needs to be fixed by CytoRich RedTM within 3 h after urine collection.
      3. Cells are stable for 3 days in CytoRich RedTM, they should not be stored longer than that.
    6. Fix the sample for at least 30 min.
    7. Centrifuge the specimens at 800 x g for 5 min and remove the supernatant.
    8. Add 6 ml of distilled water to the sediment and resuspend it for 10 sec with a vortex mixer.
    9. Centrifuge the specimen at 800 x g for 5 min and remove the supernatant.
    10. Add another 300 μl of distilled water to the sediment and resuspend it for 10 sec with a vortex mixer.
    11. After resuspension, transfer the entire amount of the specimen to a settling chamber and mount on a slide for 10 min.
    12. Invert the slide rack and discard the supernatant.
    13. Rinse the inside of the settling chamber with 95% ethanol.
      Note: Do not apply 95% ethanol directly to smears (Figure 3).
    14. Invert the slide rack and discard the 95% ethanol.
      Note: Take care not to dry the smear.
    15. Repeat once more Steps B13 and B14.
    16. Remove the settling chamber and immediately soak the slide in 95% ethanol.
      Notes:
      1. Immunostaining is carried out after fixation with 95% ethanol for 30 min or more.
      2. Cells are stable for 3 days in the 95% ethanol, they should not be stored longer than that.


    Figure 3. Schematic diagram of Step B13

  3. Immunocytochemistry (immunoenzyme staining)
    1. After fixing with 95% ethanol, incubate the slides in methanol containing 0.3% hydrogen peroxide to block endogenous enzyme activity (for 15 min).
      Notes:
      1. Hydrogen peroxide solution needs to be prepared freshly each time.
      2. In any process, when moving the slides through the solutions, mix the solutions and slides adequately by dipping with the slide holder several times.
    2. Hydrate the slides in a series of ethanol solutions of decreasing concentration (70% ethanol, 50% ethanol, and distilled water, for 1 min each).
    3. Remove the slide and use Kimwipes to wipe away the distilled water surrounding the cell smear.
    4. Enclose the area around the smear with a PAP pen.
      Notes:
      1. Use the PAP pen on the part wiped dry of the distilled water.
      2. Move to the next step only after the PAP pen line has dried (for 10-20 sec).
    5. Rinse the slide with PBS (2 times, for 1 min each time).
      Note: If stored at 4 °C, PBS can be used for several months.
    6. Incubate the cell smear with 100 µl of diluted (1:1,000) WT1 antibody in a humidified chamber (for 1 h at room temperature).
    7. Rinse the slide with PBS (3 times, for 1 min each time).
    8. Incubate the cell smear with 3 drops of N-Histofine Simple Stain MAX PO (MULTI) in a humidified chamber (for 30 min at room temperature).
    9. Rinse the slide with PBS (3 times, for 1 min each time).
    10. Incubate the cell smear with 100 µl of DAB reagent (for 1 min).
      Note: Prepare the DAB reagent immediately before use.
    11. Rinse the slide with distilled water (3 times, for 1 min each time).
    12. Counterstain the slide with Mayer’s hematoxylin (for 30 sec).
    13. Rinse the slide under running tap water (for 5 min).
    14. Dehydrate the slide with 100% ethanol (4 times, for 5 min each time).
    15. Move the slide from the ethanol into the xylene (4 times, for 5 min each time).
    16. Mount the coverslip onto the slide using mounting medium.

Data analysis

Count the WT1-positive cell numbers in a total field of smear under a light microscope (objective 20x or 40x).
Representative data:

  1. Urinary WT1-positive cells
    Urinary WT1-positive cells exhibited moderate to strongly positive staining of the cytoplasm and were negative for the nuclei. The morphology of these cells was observed to be round to polygon-shaped, with a diameter of 20-50 μm and a clear or vacuolated cytoplasm (Figures 4A). The WT1-positive cells showed a variety of morphologies, ranging from anucleated to multinucleated cells. Occasionally, cast encasement of WT1-positive cells was present (Figures 4B) (Ohsaki et al., 2016).
    Previous study has reported that WT1 exist not only at the nucleus, but also at cytoplasm (Niksic et al., 2004). The reason why urinary WT1-positive cells showed positive staining of the cytoplasm and were negative for nuclear staining in our method is that we employed an alcohol-based fixative (CytoRichTM Red). Since alcohol-based fixatives yield less reproducible staining for antibodies to nuclear epitopes (Gong et al., 2004), nuclear expression of WT1 is only detectable in formalin-fixed material (Skoog and Tani, 2011).


    Figure 4. Urinary WT1-positive cells. WT1 immunocytochemistry (400x). A. WT1-positive cell surrounded by neutrophils and squamous cells. B. Cast encasement of a WT1-positive cell.

  2. Correlation between urinary WT1-positive cells and glomerular disease
    In patients with glomerular disease, WT1-positive cells were found in 33 samples (50.0%, 33/66). No WT1-positive cells were found in patients with benign lower urinary tract disease (0%, 0/45) and in healthy volunteers (0%, 0/30). The frequency of WT1-positive cells in glomerular disease was significantly higher than that in benign lower urinary tract disease (P < 0.001) and the healthy volunteer samples (P < 0.001) (Ohsaki et al., 2016).

  3. Correlation between urinary WT1-positive cells and crescent formation
    The number of urinary WT1-positive cells in patients with crescent formation was significantly higher than that in patients without crescent formation (P = 0.007) (Fujita et al., 2017).

  4. Cutoff value of urinary WT1-positive cells
    The best cutoff value of urinary WT1-positive cells to differentiate patients with crescentic lesion from those without the lesion was 5 cells/10 ml (sensitivity 73.3%, specificity 64.9%). Urinary WT1-positive cells produced an AUC of 0.735 (Fujita et al., 2017).

Recipes

  1. PBS
    9.55 g of PBS powder
    Bring the volume to 1,000 ml with distilled water
  2. 0.3% hydrogen peroxide
    1 ml 30% hydrogen peroxide
    Bring the volume to 99 ml with methanol
  3. WT1 antibody
    1 μl WT1 antibody
    Bring the volume to 999 μl with antibody diluent

Acknowledgments

Dr. Ohsaki was supported by a grant from The Kidney Foundation, Japan (Grant number: JKF12-3) and JSPS KAKENHI (Grant number: JP15K08382). The authors declare no conflict of interest.

References

  1. Achenbach, J., Mengel, M., Tossidou, I., Peters, I., Park, J. K., Haubitz, M., Ehrich, J. H., Haller, H. and Schiffer, M. (2008). Parietal epithelia cells in the urine as a marker of disease activity in glomerular diseases. Nephrol Dial Transplant 23(10): 3138-3145.
  2. Fujita, T., Sofue, T., Moritoki, M., Nishijima, Y., Tokuhara, Y., Wakisaka, H., Kushida, Y., Haba, R. and Ohsaki, H. (2017). Urinary WT1-positive cells as a non-invasive biomarker of crescent formation. Cytopathology 28(6): 524-530.
  3. Gong, Y., Symmans, W. F., Krishnamurthy, S., Patel, S. and Sneige, N. (2004). Optimal fixation conditions for immunocytochemical analysis of estrogen receptor in cytologic specimens of breast carcinoma. Cancer 102(1): 34-40.
  4. Hara, M., Yanagihara, T., Takada, T., Itoh, M., Matsuno, M., Yamamoto, T. and Kihara, I. (1998). Urinary excretion of podocytes reflects disease activity in children with glomerulonephritis. Am J Nephrol 18(1): 35-41.
  5. Nakamura, T., Ushiyama, C., Suzuki, S., Hara, M., Shimada, N., Ebihara, I. and Koide, H. (2000). Urinary excretion of podocytes in patients with diabetic nephropathy. Nephrol Dial Transplant 15(9): 1379-1383.
  6. Niksic, M., Slight, J., Sanford, J. R., Caceres, J. F. and Hastie, N. D. (2004). The Wilms’ tumour protein (WT1) shuttles between nucleus and cytoplasm and is present in functional polysomes. Hum Mol Genet 13 (4): 463-471.
  7. Ohsaki, H., Sofue, T., Kawakami, K., Nishijima, Y., Hara, T., Matsunaga, T., Kushida, Y., Haba, R., Shigematsu, Y., Irino, S. and Norimatsu, Y. (2016). WT1 immunoenzyme staining using SurePathTM processed urine cytology helps to detect kidney disease. Cytopathology 27(1): 43-49.
  8. Skoog, L. and Tani, E. (2011). Immunocytochemistry: an indispensable technique in routine cytology. Cytopathology 22(4): 215-229.
  9. Zhang. J., Hansen, K. M., Pippin, J. W., Chang, A. M., Taniguchi, Y., Krofft, R. D., Pickering, S. G., Liu, Z. H., Abrass, C. K. and Shankland, S. J. (2012). De novo expression of podocyte proteins in parietal epithelial cells in experimental aging nephropathy. Am J Physiol Renal Physiol 302(5): 571-580.

简介

在肾小球疾病中,足细胞和顶壁上皮细胞(PEC)在尿中脱落。 因此,尿足细胞和PECs是肾小球疾病的非侵入性生物标志物。 该协议的目的是使用免疫细胞化学来检测足细胞和PEC,在液基细胞学载玻片上使用WT1抗体。

【背景】足细胞排列在肾小球毛细血管的外部,因此面对鲍曼氏囊和初级尿。在肾小球损伤中,足细胞可能从肾小球基底膜脱落。足细胞的脱落及其在尿液中的脱落与肾小球疾病和月牙形成的进展有关。顶壁上皮细胞(PECs)覆盖鲍曼囊的内部。与足细胞类似,已经报道了在活动性肾小球疾病期间PEC对WT1呈阳性,增殖并在尿中脱落(Zhang等人,2012; Fujita等人 >,2017)。

先前的研究已经显示了尿足细胞和PECs的数量与各种类型的肾小球疾病之间的关系(Hara等人,1998; Nakamura等人,2000; Achenbach ,2008)。上述引用的研究使用常规方法(直接涂片和细胞离心涂片)来制备尿样。然而,这些常规方法具有常见问题,包括风干效应和在细胞离心和固定过程期间显着细胞损失的可能性。另外,传统方法的标准化和定量评估是困难的。虽然以前大多数研究使用免疫荧光染色与podocalyxin抗体,但由于特异性抗原和荧光显微镜的要求,这种方法在中小医院很难实现。

有用的尿生物标志物检测应该容易实施,并且对标准化样品制备和免疫细胞化学的要求最低。因此,我们设计了一种通过结合液基细胞学,WT1抗体和免疫酶染色来检测足细胞和PEC的方法。由于其更好的细胞保存和更高的细胞回收率,SurePath TM基于液体的细胞学被开发作为常规样品制备方法的替代品。 SurePath TM 允许标准化和定量评估的可能性,并且该系统可以在没有机器的情况下手动操作。此外,使用WT1抗体的免疫酶染色在大多数病理学实验室中进行。因此,我们的方法可以成为检测尿足细胞和PECs的廉价,简单和国际标准化的方法。

关键字:足细胞, 壁层上皮细胞, 基于液体的细胞学检查, WT1, 免疫组化, 肾小球疾病, 新月体形成, 尿液

材料和试剂

  1. 盐渍纱布球
  2. 药勺
  3. 锥形离心管(Corning,Falcon ,目录号:352097)或同等产品
  4. Kimwipes(KCWW,Kimberly-Clark,产品目录号:34133)或同等产品
  5. PAP笔(Abcam,产品目录号:ab2601)或同等产品
  6. BD Totalys Slide Slide Rack(BD,产品目录号:491294)
  7. Coverslip(Matsunami Glass,产品目录号:C024321)或同等产品
  8. BD SurePath TM手动方法试剂盒(包括沉降室和带正电荷的载玻片)(BD,目录号:491266)
  9. BD CytoRich TM红色防腐液(BD,目录号:491336)
  10. 乙醇(Wako Pure Chemical Industries,产品目录号:059-06957)或同等产品
  11. 甲醇(Wako Pure Chemical Industries,产品目录号:136-09475)或同等产品
  12. 过氧化氢(Wako Pure Chemical Industries,目录号:086-07445)或同等产品
  13. 磷酸盐缓冲盐水(PBS)(Nichirei Biosciences,产品目录号:415223)或同等产品
  14. WT1抗体(克隆6F-H2)(Agilent Technologies,目录号:M3561)
  15. 抗体稀释液(安捷伦科技公司,产品目录号:S3022)或等同物
  16. N-Histofine®Simple Stain™MAX PO(MULTI)(Nichirei Biosciences,目录号:414151F)
  17. N-Histofine DAB-3S试剂盒(Nichirei Biosciences,目录号:415192F)
  18. Mayer's苏木精(Muto Pure Chemicals,产品目录号:30002)或同等产品
  19. 二甲苯(Wako Pure Chemical Industries,产品目录号:245-00717)或同等产品
  20. 马林醇(固定介质)(Muto Pure Chemicals,目录编号:20093)或同等产品

设备

  1. 移液器(Nichiryo,型号:Nichipet EXII)或同等产品
  2. 离心机(久保田,型号:M4000)或同等产品
  3. 涡旋混合器(Scientific Industries,型号:Vortex-Genie 2)或同等产品
  4. 显微镜(奥林巴斯,型号:BX43)或同等产品

程序

  1. 尿液收集
    1. 第一天早上中流尿液标本最适合我们的方法。
    2. 虽然现场中流尿液也可能有用,但我们还没有证实这种证据。
    3. 在导管尿液中,WT1阳性细胞通过机械分离的尿道上皮细胞进行数字稀释。
    4. 在女性样品的情况下,优选在收集尿液之前使用盐水浸泡的纱布球清洁尿道口以防止来自外阴的细胞污染。此外,在月经期间收集的样本不适合我们的方法。

  2. 细胞学标本制备(SurePathTM液基细胞学)
    SurePathTM系统的基本原理是重力沉降和电气附着。简而言之,将沉降室夹紧到带正电荷的载玻片上,并加入细胞悬液。然后重力将细胞沉降到载玻片上,带正电荷的载玻片与带负电的细胞结合(图1和2)。


    图1. SurePath™系统手动方法套件。 A.滑动机架和手动方法套件。 B.一个带正电荷的滑块和一个放置在机架中的沉降室。


    图2. SurePath的基本原理 TM 系统 A.将样品转移到沉降室并安装在一张带正电的幻灯片上。 B.带负电的细胞通过重力沉降并与带正电荷的载玻片电结合。 C.过量的细胞与上清液一起被除去。 D. SurePath TM载玻片由13mm圆形单层涂片组成。


    1. 用药匙搅拌全部尿液,使其变得均匀。

    2. 将10毫升均匀的尿液分配到未经消毒的15毫升锥形试管中。

    3. 在旋转式离心机中以800gxg旋转5分钟 注意:离心机应该是摆动式的,而不是角型的。
    4. 用吸气器或5毫升移液器滗析上清液。

    5. 10毫升CytoRich Red™TM重新悬浮沉淀物。
      注意:
      1. 在肉眼血尿的情况下,仅在去除上清液后收集并固定血沉棕黄层。
      2. 尿液沉积物需要在尿液收集后3小时内由CytoRich Red TM TM 固定。
      3. 细胞在CytoRich Red中稳定3天 TM ,它们的存储时间不应超过此长度。 />
    6. 将样品固定至少30分钟。

    7. 在800×g的条件下离心5分钟,取出上清液。

    8. 加入6毫升蒸馏水到沉淀物中,并用旋涡混合器重悬10秒。

    9. 在800×g的条件下离心5分钟,取出上清液。
    10. 向沉淀物中加入300μl蒸馏水,用旋涡混合器重悬10秒钟。
    11. 重新悬浮后,将全部样本转移至沉降室并在载玻片上放置10分钟。
    12. 倒置滑动架并丢弃上清液。

    13. 用95%乙醇冲洗沉降室内部 注意:不要将95%的乙醇直接涂抹在涂片上(图3)。
    14. 倒置幻灯片架并丢弃95%的乙醇。
      注意:小心不要擦干污迹。
    15. 再重复一次步骤B13和B14。
    16. 16.取下沉降室,立即将玻片浸泡在95%乙醇中。
      注意:
      1. 用95%乙醇固定30分钟或更长时间后进行免疫染色。
      2. 细胞在95%乙醇中稳定3天,它们不应该长于此时间。


    图3.步骤B13的示意图

  3. 免疫细胞化学(免疫酶染色)
    1. 用95%乙醇固定后,将载玻片在含有0.3%过氧化氢的甲醇中孵育以阻断内源性酶活性(15分钟)。
      备注:
      1. 每次都需要新鲜制备过氧化氢溶液。
      2. 在任何过程中,当将载玻片移动通过溶液时,通过将载玻片浸入载玻片几次,充分混合溶液和载玻片。
    2. 在一系列浓度降低的乙醇溶液(70%乙醇,50%乙醇和蒸馏水,每次1分钟)中水化幻灯片。

    3. 删除幻灯片并使用Kimwipes擦拭细胞涂片周围的蒸馏水。

    4. 用PAP笔围住涂片周围的区域。
      备注:
      1. 在擦干蒸馏水的部分使用PAP笔。
      2. 只有在PAP笔管线干燥后(10-20秒),才能进入下一步。
    5. 用PBS冲洗载玻片(2次,每次1分钟)。
      注意:如果储存在4°C,PBS可以使用几个月。
    6. 孵育细胞涂片与100μl稀释(1:1,000)WT1抗体在潮湿的室(在室温1小时)。
    7. 用PBS冲洗载玻片(3次,每次1分钟)。
    8. 孵育细胞涂片与3滴N-Histofine Simple Stain MAX PO(MULTI)在加湿室(在室温下30分钟)。
    9. 用PBS冲洗载玻片(3次,每次1分钟)。
    10. 用100μlDAB试剂孵育细胞涂片(1分钟)。
      注意:使用前立即准备DAB试剂。
    11. 用蒸馏水冲洗载玻片(3次,每次1分钟)。
    12. 用Mayer的苏木精制作幻灯片(持续30秒)。

    13. 在流动的自来水下冲洗载玻片(5分钟)。

    14. 用100%乙醇将玻片脱水(4次,每次5分钟)。
    15. 将乙醇中的载玻片移入二甲苯(4次,每次5分钟)。
    16. 使用安装介质将盖玻片安装到载玻片上。

数据分析


在光学显微镜下(目标为20x或40x),在总涂片领域中计数WT1阳性细胞数量。
代表性数据

  1. 尿WT1阳性细胞
    尿WT1阳性细胞呈现中度到强烈的胞质阳性染色,并且对细胞核呈阴性。观察到这些细胞的形态为圆形至多边形,直径为20-50μm,细胞质澄清或空泡(图4A)。 WT1阳性细胞显示多种形态,从无核细胞到多核细胞。有时,存在WT1阳性细胞的包膜(图4B)(Ohsaki等人,2016)。
    先前的研究已经报道WT1不仅存在于细胞核中,而且存在于细胞质中(Niksic等人,2004)。在我们的方法中,尿WT1阳性细胞显示细胞质阳性染色和核染色阴性的原因是我们使用醇基固定剂(CytoRich TM TM Red)。由于醇基固定剂对核表位的抗体产生较少的可重复染色(Gong等人,2004),所以WT1的核表达仅在福尔马林固定的材料中才能检测到(Skoog和Tani,2011)。


    图4.尿WT1阳性细胞WT1免疫细胞化学(400x)。 A. WT1阳性细胞被嗜中性粒细胞和鳞状细胞包围。 B.铸造一个WT1阳性细胞。

  2. 尿WT1阳性细胞与肾小球疾病的相关性
    在患有肾小球疾病的患者中,33个样品中发现WT1阳性细胞(50.0%,33/66)。在良性下尿路疾病患者(0%,0/45)和健康志愿者(0%,0/30)中未发现WT1阳性细胞。肾小球疾病中WT1阳性细胞的频率显着高于良性下尿路疾病( <0.001>)和健康志愿者样品( p <0.001>)的频率。 0.001)(Ohsaki et al。,2016)。

  3. 尿WT1阳性细胞与新月体形成之间的相关性
    新月体形成患者的尿WT1阳性细胞数量明显高于无新月体形成的患者( P = 0.007)(Fujita等,2017) 。

  4. 尿WT1阳性细胞的临界值
    尿WT1阳性细胞将新月体病变与无病变病灶区分的最佳临界值为5个细胞/ 10 ml(敏感性73.3%,特异性64.9%)。尿WT1阳性细胞产生0.735的AUC(Fujita等人,2017)。

食谱

  1. PBS

    9.55克PBS粉末
    用蒸馏水将容量加至1000毫升
  2. 0.3%过氧化氢
    1毫升30%过氧化氢
    用甲醇将体积加至99 ml
  3. WT1抗体
    1μlWT1抗体

    用抗体稀释液将体积加至999μl

致谢

Ohsaki博士获得日本肾脏基金会(基金编号:JKF12-3)和JSPS KAKENHI(基金编号:JP15K08382)的资助。作者宣称没有利益冲突。

参考

  1. Achenbach,J.,Mengel,M.,Tossidou,I.,Peters,I.,Park,J.K。,Haubitz,M.,Ehrich,J.H。,Haller,H.and Schiffer,M.(2008)。 尿中壁层上皮细胞作为肾小球疾病疾病活动的标志 Nephrol拨号移植 23(10):3138-3145。
  2. Fujita,T.,Sofue,T.,Moritoki,M.,Nishijima,Y.,Tokuhara,Y.,Wakisaka,H.,Kushida,Y.,Haba,R.和Ohsaki,H。(2017)。 尿WT1阳性细胞作为新月体形成的非侵入性生物标志物 细胞病理学 28(6):524-530。
  3. Gong,Y.,Symmans,W.F.,Krishnamurthy,S.,Patel,S。和Sneige,N。(2004)。 乳腺癌细胞学标本中雌激素受体免疫细胞化学分析的最佳固定条件 癌症 102(1):34-40。
  4. Hara,M.,Yanagihara,T.,Takada,T.,Itoh,M.,Matsuno,M.,Yamamoto,T。和Kihara,I。(1998)。 足细胞尿排泄反映肾小球肾炎患儿的疾病活动 Am J Nephrol 18(1):35-41。
  5. Nakamura,T.,Ushiyama,C.,Suzuki,S.,Hara,M.,Shimada,N.,Ebihara,I。和Koide,H。(2000)。 糖尿病肾病患者的足细胞排泄 肾移植< / em> 15(9):1379-1383。
  6. Niksic,M.,Slight,J.,Sanford,J.R。,Caceres,J.F。和Hastie,N.D。(2004)。 维尔姆斯肿瘤蛋白(WT1)穿梭于细胞核和细胞质之间,存在于功能性多核糖体中。 / Hum> Mol Genet 13(4):463-471。
  7. Ohsaki,H.,Sofue,T.,Kawakami,K.,Nishijima,Y.,Hara,T.,Matsunaga,T.,Kushida,Y.,Haba,R.,Shigematsu,Y.,Irino,S。和Norimatsu,Y.(2016)。 使用SurePath TM处理的尿液细胞学检测WT1免疫酶染色有助于检测肾脏疾病。 细胞病理学 27(1):43-49。
  8. Skoog,L。和Tani,E。(2011)。 免疫细胞化学:常规细胞学中不可或缺的技术。 细胞病理学 22(4):215-229。
  9. 张。 J.,Hansen,K.M.,Pippin,J.W.,Chang,A.M.,Taniguchi,Y.,Krofft,R.D.,Pickering,S.G.,Liu,Z.H.,Abrass,C.K。和Shankland,S.J。(2012)。 实验性老年性肾病中顶体上皮细胞中足细胞蛋白的从头表达 Am J Physiol Renal Physiol 302(5):571-580。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ohsaki, H., Matsunaga, T., Fujita, T., Tokuhara, Y., Kamoshida, S. and Sofue, T. (2018). Quantifying Podocytes and Parietal Epithelial Cells in Human Urine Using Liquid-based Cytology and WT1 Immunoenzyme Staining. Bio-protocol 8(9): e2827. DOI: 10.21769/BioProtoc.2827.
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