免疫细胞化学染色及激光扫描共聚焦显微镜技术在脑膜癌病早期诊断中的应用

Early diagnosis of patients with meningeal carcinomatosis with carcino-embryonic antigen immunocytochemical stain examination and laser scanning confocal microscope

目的探讨免疫细胞化学染色及激光扫描共聚焦显微镜（LSCM）技术在脑膜癌病（meningeal carcinomatosis，MC）早期诊断中的应用价值。方法将所观察脑脊液标本分为MC组、非MC组（对照组），以Shandon Cytospin4离心沉淀仪收集脑脊液细胞。分别进行Giemsa染色（42例）、联合癌胚抗原（CEA）免疫细胞化学染色（29例，对照20名）、免疫荧光双标记染色（17例，对照20名）。CEA免疫细胞化学染色采用SP法，置于普通光学显微镜下观察；免疫荧光双标记染色以Cy5荧光二抗及4，6-联脒-2-苯基吲哚分别标记抗CEA单抗及细胞核DNA，置于LSCM下观察，进行定位、定性、定量分析。结果42例患者经脑脊液细胞学检查均发现异常细胞，首次脑脊液细胞学检查阳性率为85．7％（36／42），其中29例患者结合CEA免疫细胞化学染色，阳性率为79．3％（23／29），17例经免疫荧光双标记染色，13例阳性。MC组[CEA（＋）者1694．69±478．06，CEA（-）者1543．04±364．71]与对照组（603．72±178．04）比较胞核DNA荧光相对含量[CEA（＋）者t=21．386，CEA（-）者t=23．144，均P〈0．01]差异有统计学意义；CEA荧光相对含量CEA（＋）者（1407．04±275．30）与对照组（202．51±54．05）差异亦有统计学意义（f=42．934，P〈0．01）。结论免疫细胞化学染色是诊断MC的重要定性手段，结合LSCM可将MC的诊断提高到定性、定位与定量相结合的新水平。

Objective To explore the early diagnostic value of careino-embryonic antigen （CEA） immunocytochemistry examination combined with laser scanning confocal microscope （LSCM） in the patients with meningeal carcinomatosis （MC）. Methods The patients were divided into experimental group （patients with MC ） and control group （patients without MC ） . Thermo electron corporation shandon cytospin 4 centrifuge was used in the cytologic examination of cerebrospinal fluid （ CSF）, whose function was to produce a monolayer of cells onto a glass slide from CSF. Giemsa staining was used in 42 cases. The CEA immunocytochemistry staining was used in 29 cases and 20 controls. The double immunofluorescence staining was used in 17 cases and 20 controls. SP staining method was used in the CEA and the results were observed under the light microscope. Nuclear DNA and CEA were stained with fluorescent probe DAPI and Cy5 respectively and the results of double immunofluorescence staining were observed by the laser scanning confocal microscope. Results There was a high positive rate in cytologic examination of CSF, and malignant cells were found in all of 42 cases for repeated CSF testing. The positive rate of routine CSF cytologic examination and CEA immunocytochemistry examination was 85. 7% （36/42） and 79. 3% （23/29） respectively in the first CSF specimens . There were 17 cases using double immunofluorescence staining and observed by LSCM, and the positive rate was 13/17. Compared with experimental group and control group, fluorescent value both nuclear DNA （ CEA （ ＋ ） 1694. 04±478.06, CEA （ - ） 1543.04 ± 364. 71, control group 603.72 ＋ 178.04, t = 21. 386, 23. 144, both P 〈 0. 01 ） and CEA （ CEA （ ＋ ） 1407.04 ± 275.30, control group 202. 51 ± 54.05, t = 42. 934, P 〈 0. 01 ） were significantly different. Conclusion Immunocytochemistry examination of CSF is an important early qualitative diagnosis method for MC. LSCM improved the level of locating, qualitative and quantitative diagnosis of MC.