Silent Time-Bomb, Mesothelioma

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1 Silent Time-Bomb, Mesothelioma ーClinical Pathology Shotaro Maeda Tama-Nagayama Hospita,lNippon Medical School JAPAN

2 Mesothelioma 1 General remarks 2 Pathology 3 Cytology 4 6 cases of the mesothelioma diagnosed by cytological materials The utility of cell transfer method and cell block method for immunochemical staining

3 Mesothelioma 1 General remarks 2 Pathology 3 Cytology 4 6 cases of the mesothelioma diagnosed by cytological materials The utility of cell transfer method and cell block method for immunochemical staining

4 A quiet time bomb Mesothelioma More than 80 to 90% cases of mesothelioma occur because of exposure to asbestos(amocyte and crocidolite are considerably more potent than chrysotile,, and crocidolite is more dangerous than amosite.) In Japan a large quantity of asbestos was imported in 1970 to 1990 as well as in Thailand. It is considered that patient shows symptom of mesothelioma after about 40 years being exposed to asbestos. So, mesothelioma will be considered to occur in high frequency in 2010 to I worry that the same situation will occur in Thailand as well.

5 The volume of asbestos import in Japan ERCA The number of death due to mesothelioma The number of accidents authorizaed by government

6 Mesothelioma Lung cancer The number of asbestos related mesothelioma and lung cancer authorized by government 独 立 法 人 環 境 再 生 保 全 機 構

7 Mesothelioma The first case of mesothelioma Klemperer,P.&Robin,C.B Robin,C.B.:.: Primary neoplasms of the pleura. A report of five cases. Arch.Pathol.11: , 412, The first case report of mesothelioma by asbestos exposure Wagner JC, Sleggs CA, Marchand P: Diffuse pleural mesothelioma and asbestos exposure in the North-Western Cape province. Br J Indust Med 17: , 271, 1960 A A report of 28 cases of mesothelioma by asbestos exposure in an asbestos mine of South Africa --- At that time Wagner was critisized--- At that time Wagner was bitterly

8 Mesothelioma A malignant tumor to occur to a body cavity (pleura, peritoneum, peicardium) and tunica vaginalis testis. Pleura Peritoneum Pericardium Tunica vaginalis testis 80~90% 10~20% about 1~ 5% 0.1~ 2% about 80 about 10 about about 0.1 Frequency: man78%>woman >woman22% (Occupation characteristics) Average age: about 60 years old

9 Benign Adenomatoid tumor Mesothelioma (Japan Lung Cancer Society 2003; 003; WHO 1999) Malignant mesothelioma Epi thelioid mesothelioma 50~60 60% Sarcmatoid mesothelioma 10~20 ~20% Desmoplastic mesothelioma very difficult Biphasic mesothelioma 20~30% ~30% Others

10 Benign Mesothelioma (Japan Lung Cancer Society 2003; 003; WHO 2004) Adenomatoid tumor Malignant mesothelioma Epi thelioid mesothelioma Sarcmatoid mesothelioma Desmoplastic mesothelioma Biphasic mesothelioma Others

11 In mesothelioma, body fluid accumulates in early stages because it is derived from the serous membrane different from other malignant tumors. Therfore,, body fluid cytology is very important for early detection and early treatment of mesothelioma. The prognosis of mesothelioma in early stages(stage Ia to Ib) has become better recently because of pleuropulmonary resection Stage 1a: parietal pleura Stage 1b: parietal pleura and visceral pleura Therefore, misdiagnosis for mesothelioma by body fluid cytology in early stages means that the chance of survivals of a patient is lost.

12 Clinical Clinical findings Diagnosis & Therapy (Japan) Asbestos exposure career, a symptom, an image, hyaluronic acid values of effusion 100,000ng/ml ;highly suspicious of mesothelioma Asbestos exposure career: about 40% ( occupation career) Living environment of asbestos exposure should be examined more. Asbestos exposure career: about 40% ( Diagnosis rate by cytology and histology Cytology (body fluid): about 30 to 40% Core Needle biopsy: about 20~80 80% Thoracoscopic pleural biopsy: 98% Cytologists(MD.CT) should make an effort to improve the diagnosis rate by cytology. TherapyTherapy Chemotherapy: Radiotherapy: Operation: 3 years survival rate of pleuroneumonectomy: : about 50% An An early diagnosis and an early treatment of mesothelioma are very important. So, we should study cytology of mesothelioma more seriously!!!

13

14 1 General remarks 2 Pathology 3 Cytology Mesothelioma 4 Six cases of the mesothelioma diagnosed only by cytological materials The utility of cell transfer method and cell block method for immunocytochemical staining

15 Benign Mesothelioma (Japan Lung Cancer Society 2003; 003; WHO 2004) Adenomatoid tumor Malignant mesothelioma Epi thelioid mesothelioma Sarcmatoid mesothelioma Desmoplastic mesothelioma Biphasic mesothelioma Others

16 Calretinin Epithelioid mesothelioma (tubulopapillary)

17 Calretinin Epithelioid mesothelioma (papillary)

18 Epithelioid mesothelioma Calretinin (tubular) mesothelioma (tubular)

19 Calretinin Epithelioid mesothelioma(solid)

20 Calretinin Sarcomatoid mesothelioma

21 Desmoplastic mesothelioma(who2004 WHO2004) Calretinin

22 Biphasic mesothelioma CK7 Calretinin

23

24 1 General remarks 2 Pathology 3 Cytology Mesothelioma 4 Six cases of the mesothelioma diagnosed only by cytological materials The utility of cell transfer method and cell block method for immunocytochemical staining

25 Differential Diagnosis Mesothelioma(Epithelial) VS Adenocarcinoma Reactive mesothelial cell

26 We love cytology

27 STUDY DESIGN About 2 years ago, we studied 10 cases of mesothelioma that were diagnosed by body fluid cytology in Nippon Medical School. Pleura: 6 cases Peritoneum: 2 cases Pericardium: 1 case Tunica vaginalis testis: 1 case

28 Thickened pleura Binucleated mesothelioma cells

29 70 -year-old womnan Pericadial mesothelioma

30 40-year-old male Mesothelioma of the tunica vaginalis testis

31 Characteristic cytological findings of mesothelioma numerous mesothelioma cells Variety of the cells; Single or groups consisting of many cells

32 Numerous, large to small clusters Scattered cells

33 Mesothelioma Variety of the cells; Large to small clusters (Papillary ~spherical) or s or single of numerous cells Reactive mesothelial cells Uniformity of the cells

34 Binucleated cells Prominent nucleoli

35 multinucleated cells with prominent nucleoli

36 Pair cells(cell mutual inclusions)

37 pair cells (cell mutual inclusions) with hump-like protruded cytoplasm

38 Differential Diagnosis Mesothelioma(Epithelial) VS Reactive mesothelial cells Adenocarcinoma

39 Mesothelioma (epithelial) Reactive mesothelial cells Adenocarcinoma nucleus nucleoli cytoplasm round to oval centrally located prominent dense cell border: unclear round to oval centrally located not prominent dense cell border: unclear irregular eccentrically centrically located clear~den dense cell border :clear PAS Alcian blue +(intracytoplasm intracytoplasm) digested in diastase +(cell surface intracytoplasm intracytoplasm) digested by hyaluronidase ±(inracytoplasm) digested by diastase +~±(intracytoplasm intracytoplasm:droplet~diffuse)not not digested in diastase +(intracytoplasm intracytoplasm) Not digested by hyaluronidase Useful antibody Calretinin Thrombomodulin HBME1 Mesothelin CK 5/6 AE1/AE3(pan-cytokeratin) WT-1 D2-40 ~ ± + ± CK7.CK20 CEA Ber-EP4 MOC31 31 CAM5.2 34βE12 & 35βH11H11 Electron microscope Numerous, long microvilli Tonofilament Glycogen A few, scattered short microbill Tonofilament Glycogen A few, short microvilli Tonofilament Glycogen

40 mesothelioma Reactive mesothelialcells Adenocarcinoma

41 Mesothelioma (epithelial) Reactive mesothelial cells Adenocarcinoma nucleus cytoplasm PAS Round to oval Centrally located Cell border: unclear + (intracytoplasm) digested by diastase a a few small clucters Binucleated~multnuleated cells pair cells (cell mutual inclusion) with hump-like protruded cytoplasm Prominent nucleoli ± (intracytoplasm) digested by diastase irregular Excentrically located Cell border: clear +~± (intracytoplasm) droplet~diffuse diffuse) not digested by diastase Alcian blue + (cell surface intracytoplasm) digested by hyaluronidase + (cell surface) digested by hyaluronidase + (intracytoplasm) not digested by hyaluronidase Useful antibody Calretinin Thrombomodulin HBME1 Mesothelin CK 5/6 AE1/AE3(pan (pan-cytokeratin) WT-1 D2-40 ~ ± + ± CK7.CK20 CEA Ber-EP4 MOC31 CAM5.2 34βE12 & 35βH11 Electron microscope Numerous, long microvilli Tonofilament Glycogen A few, scattered short microbill Tonofilament Glycogen A few, short microvilli Tonofilament Glycogen

42 Mesothelioma Reactive mesothelial cell Adenocarcinoma PAS

43 Mesothelioma (epithelial) Reactive mesothelial cells Adenocarcinoma nucleus cytoplasm Round to oval Centrally located Cell border: unclear a a few small clucters Binucleated~multnuleated cells pair cells (cell mutual inclusion) with hump-like protruded cytoplasm Prominent nucleoli irregular Excentrically located Cell border: clear PAS + (intracytoplasm) digested by diastased ± (inracytoplasm) digested by diastase +~ +~±(intracytoplasm) droplet ~diffuse) not digested by diastase Alcian blue + (cell surface intracytoplasm) digested by hyaluronidase + (cell surface) digested by hyaluronidase + (intracytoplasm) not digested by hyaluronidase Useful antibody Calretinin Thrombomodulin HBME1 Mesothelin CK 5/6 AE1/AE3(pan (pan-cytokeratin) WT-1 D2-40 ~ ± + ± CK7.CK20 CEA Ber-EP4 MOC31 CAM5.2 34βE12 & 35βH11 Electron microscope Numerous, long microvilli Tonofilament Glycogen A few, scattered short microbill Tonofilament Glycogen A few, short microvilli Tonofilament Glycogen

44 Alcian blue Mesothelioma Mesothelial cell Adenocarcinoma

45 Mesothelioma (epithelial) Reactive mesothelial cells Adenocarcinoma nucleus cytoplasm Round to oval Centrally located Cell border: unclear a a few small clucters Binucleated~multnuleated pair cells (cell mutual inclusion) with hump-like protruded cytoplasm Prominent nucleoli uniformity cells irregular Excentrically located Cell border: clear PAS Alcian blue Useful antibody +(intracytoplasm intracytoplasm) digested in diastase +(cell surface intracytoplasm intracytoplasm) digested by hyaluronidase Calretinin (+) CK 5/6 (+) D2-40 (+) (+) Thrombomodulin (+) Mesothelin(+) EMA (+) E-cadherin(+) P53(+) ±(inracytoplasm) digested in diastase Calretinin (+) CK 5/6 (+) D2-40 (+) (+) Thrombomodulin (+) Mesothelin (+) +) EMA(ー ~±) E-cadherin(ー ~±) P53(ー ~±) +~±(intracytoplasm intracytoplasm:droplet~diffuse) not digested in diastase +(intracytoplasm intracytoplasm) Not digested by hyaluronidase CEA(+) MOC31 31(+) Ber-EP4 EP4(+) Electron microscope Numerous, long microvilli Tonofilament Glycogen A few, scattered short microbill Tonofilament Glycogen A few, short microvilli Tonofilament Glycogen

46 The results of immunocytochemical staining for reactive mesothelial cells(13 cases) frequency of positive cases p53 2/13 ( 15 % ): only for a few cells EMA 3/13 ( 23 % ): weakly positive E-cadherin 0/13 ( 0 % )

47 How we can perform the immunochemical staining for several antibodies using body fluid.

48 Cell block method 1. Body fluid 2. Centrifuge at 1500rpm for 5minutes 3.Fixtation 4. Cell block 5.Immunochemical staining

49 Cell transfer method 1. Taking off the cover glass 2.Pasting the mount 3.Softening the mount 4.Taking off the mount &Trimming 5.Transfer

50 Cell block method 1. Body fluid 2. Centrifuge at 1500rpm for 5minutes 3.Fixtation 4. Cell block 5.Immunochemical staining 1.Ask clinical department to submit as much body cavity fluid as possible. 2.Centrifuge the body cavity fluid (1500 rpm for 5 minutes). 3.Decant the supernatant into another container, add 1-2 ml of 20% formalin to the supernatant, and fix overnight. 4.Embed the pellets in paraffin to prepare cell blocks. 5.Carry out immunochemical staining.

51 Cell transfer method 1. Taking off the cover glass 2.Pasting the mount 3.Softening the mount 4.Taking off the mount &Trimming 5.Transfer 1. Removal of the cover glass: Remove the cover glass with xylene. 2. Pasting the mount: Dilute malinol with xylene two times and apply it to the cell smear surface. Fix the mounted specimen by placing the glass slide on a hot plate at 75 C. Fix the specimen for 30 minutes to allow the malinol membrane to harden. 3. Softening the mount: Place the glass slide in a water bath at approximately 50 C for about 15 minutes to soften the mounting medium. 4. Removal of the mount and trimming: Take out the glass slide from the water bath and trim the softened malinol membrane using a pair of tweezers and a razor. 5. Cell transfer: Mount the trimmed softened malinol membrane to other glass slides. Dry the glass slides on a 75 C hot plate for about 30 minutes. After removing the malinol with xylene, carry out immunocytochemical staining.

52 アユタヤ

53 CEA Immunohistochemical staining using cell tranfer method in a case of mesothelioma Calretinin mesothelin l

54 An estimate of histology and primary site by a combination of CK 7 & CK20 CK7 + CK20 + CK20 - Transitional cell carcinoma Mucinous adenocarcinoma(ovary) Adenocarcinoma ( pancreas) Mesothelioma Adenocarcinoma(Lung,breast) Serous adenocarcinoma(ovary) Endometrioid adenocarcinoma(uterus) CK7 - Adenocarcinoma(stomach,, colon) Squamous cell carcinoma Renal cell carcinoma Hepatocellular carcinoma Adenocarcinoma(prostate)

55 CEA CK20 Immunohistochemical staining using cell tranfer method in a case of mesothelioma Calretinin mesothelin l CK7

56 EMA E-Cadherin p53 A case of mesothelioma Immunocytochemical staining using cell transfer technique

57 EM sample procedure on Cell block Smear sample It takes 2 to 3 days --- cytocentrifuge glutaraldehyde fixation wash osmium tetroxyde post- fixation dyhydration epoxy resin electronmicroscope semi section staind with uranyl acetate and lead citrate ultrathin section Embedded in quetol 812 ( 60 )

58 Mesothelioma (epithelial) Reactive mesothelial cells Adenocarcinoma nucleus cytoplasm Round to oval Centrally located Cell border: unclear a a few small clucters Binucleated~multnuleated pair cells (cell mutual inclusion) with hump-like protruded cytoplasm Prominent nucleoli uniformity cells irregular Cell border:nclear PAS Alcian blue +(intracytoplasm intracytoplasm) digested in diastase +(cell surface intracytoplasm) digested by hyaluronidase ±(inracytoplasm) digested in diastase +~±(intracytoplasm intracytoplasm:droplet~diffuse)not digested in diastase +(intracytoplasm intracytoplasm) Not digested by hyaluronidase Useful antibody Calretinin Thrombomodulin HBME1 Mesothelin CK 5/6 AE1/AE3(pan (pan-cytokeratin) WT-1 D2-40 ~ ± ~ ± ~ ± CK7.CK20 CEA Ber-EP4 MOC31 31 CAM5.2 34βE12 & 35βE11E11 Electron microscope Numerous, long microvilli Tonofilament Glycogen A few, scattered short icrovilli Tonofilament Glycogen A few, short microvilli Tonofilament Glycogen

59 Mesothelioma 電 顕 写 真 Mesothelial cell Adenocarcinoma

60 Why immunochemical staining is necessary to make a definitive diagnosis of mesothelioma?

61 Case 54-year year-old female Ascites

62

63 CEA CK 7 CEA CK 20 CEA EMA EMA Calretinin 組 織 像

64 Case 54-year year-old female Ascites SSPC(Serous surface papillary carcinoma) NSOCS(normal-sized ovary carcinoma syndrome)

65 Relationship between cytology and histology in the diagnosis of mesothelioma Histology by cell block method using body fluid Body fluid cytology (cell transfer method) Biopsy

66

67 Mesothelioma 1 General remarks 2 Pathology 3 Cytology 4 6 cases of the mesothelioma diagnosed by cytological materials The utility of cell transfer method and cell block method for immunochemical staining

68 Six cases of mesothelioma within 2 years diagnosed in our department were diffecult to detect only by cytological findings. However, the definite diagnosis of mesothelioma was possible by immunochemical staining for several antibodies using cell transfer method and/or cell block method as well as by electron- microscopical examination. So, I would like to emphasize that the definitive diagnosis of mesothelioma is possible from only aspirated body fluid.

69 Case Age sex 60 male 72 male 85 female 84 female 79 male 51 male Asbestos exposure unknown + unknown unknown unknown unkown Clinical diagnosis ascites pleural fluid pleural fluid pleural fluid pleural fluid pleural fluid Hyaluronic acid (ng/ml) 1,110,000 39, ,000 20,700 ND 920,000 Judgement (Class) Ⅱ Ⅴ Ⅲ Ⅳ Ⅲb Ⅳ Ⅲb Ⅳ Ⅳ Immunochemical Staining cell ransfer method cell transfer method cell transfer method cell block method cell transfer Method & cell block mrethod cell ransfer method Electroscopic materials ascites pleural fluid Biopsy material ND Ppleural fluid Pleural fluid Cytological diagnosis mesothelioma mesothelioma mesothelioma mesothelioma mesothelioma mesothelioma Histological findings ND Desmoplastic Mesothelioma Biphasic mesothelioma Epithelioid mesothlioma ND? Follow up (January.2007) Survival (18 momths) Survival (12months) Died (10months) Survival (12months) Dead (6months) Survival (2months)

70 Asbestos Case 1 Age sex exposure Clinical diagnosis Hyaluronic acid (ng/ml) Judgement Immunochemical staining Electroscopic materials Cytological diagnosis Histological findings Follow up (January.2007) 60 male - ascites 1,110,000 ClassⅤ cell transfer method ascites mesothelioma ND Survival (18 momths)

71 Binucleated cells PAS Multinucleated cells Multinucleated cells cell mutual inclusions hump-like protruded cytoplasm

72 Frequency of binucleated and multinucleated cells Mononucleated cell 77% 4% Multinucleated cells 19% Binucleated cells Reactive mesothelial cells binucleated cells<10% multimucleated cells<2%

73 calretinin EMA E-cadherin p53

74 Electron-microscopic findings cell mutual inclusions

75 Case 2 Age sex Asbestos exposure Clinical diagnosis Hyaluronic acid (ng/ml) Judgement Immunochemical staining Electroscopic materials Cytological diagnosis Histological findings Follow up (January.2007) 72 male + pleural fluid 39,900 ClassⅢ cell transfer method Pleural fluid mesothelioma Desmoplastic Mesothelioma Survival (12months)

76 l Binucleated cells Multinucleated cells cell mutual inclusions with hump-like protruded cytoplasm Cell mutual inclusions

77 Frequency of binucleated and multinucleated cells イケナガ Mononucleated cell 70% 24% Binucleated cells 6% Multinucleated cells Reactive mesothelial cells binucleated cells<10% multimucleated cells<2%

78 E-cadherin CK20 Calretinin mesothelin l CK7 CEA CK7

79 Case 3 Age sex Asbestos exposure Clinical diagnosis Hyaluronic acid (ng/ml) Judgement Immunochemical staining Electroscopic materials Cytological diagnosis Histological findings Follow up (January.2007) 85 female - pleural fluid 426,000 Class Ⅳ cell transfer method Tumor tissue mesothelioma Biphasic mesothelioma Died (10months)

80 Binucleated cells Multinucleated cells Pair cells Binucleated cells Cell mutual inclusions

81 Frequency of binucleated and multinucleated cells フジイ Mononucleated cell 66.0% 10% Multinucleated cells 24.0% Binucleated cells Reactive mesothelial cells binucleated cells<10% multimucleated cells<2%

82 EMA Calretinin EMA EMA CK 7 CK20

83 Calretinin CK7 CK20 HE Calretinin

84

85 Case 4 Age sex Asbestos exposure Clinical diagnosis Hyaluronic acid (ng/ml) Judgement Immunochemical staining Electroscopic materials Cytological diagnosis 84 female unknown pleural fluid 20,700 ClassⅣ cell block method ND mesothelioma Histological findings Follow up (January.2007). Epithelioid mesothelioma Alive (12months)

86 binucleated cells CK7 Binucleated cells & Binucleated cells Multinucleated cells

87 Frequency of binucleated and multinucleated cells Mononucleated cell 77% 9% Multinucleated cells 14% Binucleated cells Reactive mesothelial cells binucleated cells<10% multimucleated cells<2%

88 Cell block method HE Calretinin EMA Biopsy CK7 CK20 HE

89 Case 5 Age sex Asbestos exposure 79 male unknown Clinical diagnosis Hyaluronic acid (ng/ml) Judgement Immunochemical Staining Electroscopic materials Cytological diagnosis Histological findingsf Follow up (January.2007). pleural luid ND ClassⅣ Cell transfer Method & cell block method Pleural fluid mesothelioma ND Dead (6months)

90 Multinucleated cells Binucleated cells ell cell mutual inclusion Cell mutual inclusion

91 Frequency of binucleated and multinucleated cells Mononucleated cell 74% 2% Multinucleated cells 24% Binucleated cells Reactive mesothelial cells binucleated cells<10% <10% multimucleated mucleated cells<2% <2%

92 P-53 Calretinin E-cadherin E-カドヘリン

93 Case 6 Age sex Asbestos exposure Clinical diagnosis 51 male unkown pleural fluid Hyaluronic acid (ng/ml) Judgement (Class) Immunochemical Staining Electroscopic materials Cytological diagnosis 920,000 Ⅳ cell ransfer method Pleural fluid mesothelioma Histological findings Follow up (January.2007) Epithelial mesotheliom Survival (2months)

94 Cell mutual inclusion Cell mutual inclusion Binucleated cell

95 Reactive mesothelial cells binucleated cells<10% multimucleated cells<2% Frequency of binucleated and multinucleated cells 単 核 57% 2 核 30% 多 核 (3 核 以 上 ) 13%

96 Immunocytochemical staining by cell tansfer method Calretinin CK5/6 EMA

97 Immunohistochemical staining by cell block method HE Calretinin mesothelin CK5/6 thrombomodulin EMA E-cadherin CEA

98 Immunohistochemical staining by cell block method HE Calretinin mesothelin CK5/6 thrombomodulin EMA E-cadherin CEA

99

100 Thoracoscopic findings Numerous small nodules in the peritoneum Hyaluronic acid 920,000ng/ml

101 Thoracoscopic pleural biopsy HE Calretinin mesothelin CK5/6 C K 5 / 6 D2- HBME-1 EMA CEA

102 Stage 1

103 Stage 1 :Surgical margin(-)

104 Making a definitive diagnosis of mesothelioma by effusion cytology Use of cell transfer method or cell block method for immunochemical staining Maeda Shotaro1, Hosone Masaru1, Katayama Hironori1, Iwase Hiromi1, Kawano Kiyoko1, Yanagida Yumi1, Isobe Hiroaki1, Atarashi Hirotsugu2, Tanimura Shigeo3, and Naito Zenya4 1Department of Pathology, Tama-Nagayama Hospital, Nippon Medical School 2Department of Internal Medicine, Tama-Nagayama Hospital, Nippon Medical School 3Department of Respiratory Surgery, Tama-Nagayama Hospital, Nippon Medical School 4Department of Pathology, Nippon Medical School J.Jpn.Soc.Clin.Cytol.. 47: in press 2008(2)

105 Conclusion 1 1 In diagnosis of the mesothelioma, the information of asbestos exposure, clinical diagnosis( imaging etc.) and the value of hyaluronic acid in the body fluid are important. 2 The early detection of mesothelioma by body fluid cytology y is very important for the improvement of survival rate by pleuropulmonary resection The following cytological finding are important for the diagnosis of mesothelioma. 1The variety of the cytological findings, such as papillary and spherical, large and small clusters, and individual cells. 2The frequency of binucleated and multinucleated cells with large nucleoli. 3pair cells (cell mutual inclusions) with hump-like protruded cytoplasm

106 Conclusion 2 3 Immunostainig for the several antibodies is important for the 4 definite diagnosis of mesothelioma Calretinin CK5/6 CK5/6 D2-40 Thrombomodulin Mesothelin CEA MOC31 MOC31 P53 EMA EMA E-cadherin) Cell transfer method and/or cell block method is useful for the immunocytochemical staining. 5 In cytology of body fluid, the possibility of the mesothelioma should be always kept k in mild. The diagnosis of mesothelioma should be generally performed from the clinical, cytological, immunochemical and electron-microscopical findings.

107 The 1 announcement of the JSCC congress

108

109

110 Case 1 Calretinin p53 Multinucleated cells cell mutual inclusions

111 Case2 l Calretinin E-cadherin Multinucleated cells cell mutual inclusions with hump-like protruded cytoplasm Cell mutual inclusions Binucleated cells

112 Case 3 Calretinin EMA Cell mutual inclusions Cell mutual inclusions

113 Case 4 calretinin EMA

114 Case 5 Cell mutual inclusions Calretinin E-cadherin

115 Case6 Calretinin Acrobat ถ CK5/6 Cell mutual inclusions Cell mutual inclusions

116 Fig 3: Appearance frequencies of mononucleated, binucleated and multinucleated cells as determined by body fluid cavity cytology 100% 80% 4.1% 18.6% 2.3% 24.0% 5.6% 23.6% 10.0% 24.0% 8.7% 14.5% 12.5% 29.7% 60% 40% 77.3% 73.6% 70.8% 66.0% 76.8% 57.8% 20% 0% C ase1 C ase2 C ase3 C ase4 C ase5 C ase6 Multinucleated ce ls 4.1% 2.3% 5.6% 10.0% 8.7% 12.5% Binucleated ce ls 18.6% 24.0% 23.6% 24.0% 14.5% 29.7% M ononucleated cells 77.3% 73.6% 70.8% 66.0% 76.8% 57.8%

117 Multinucleated cells 4 Dense cytoplasm Prominent nucleoli

118 binucleated cells

119 Introduction(general remarks) of mesothelioma Asbestos body Binucleated mesothelioma cell

120 Mesothelioma Lung cancer The number of asbestos related mesothelioma and lung cancer authorized by government 独 立 法 人 環 境 再 生 保 全 機 構

121 Cytology of mesothelioma Binucleated mesothelioma cell

122 Cell transfer method It takes 1 and half hours----- Taking off the cover glass Pasting the mount Softening the mount Transfer 3X6=18 Taking off the mount Trimming

123 患 者 の 救 済 申 請 独 立 行 政 法 人 環 境 再 生 保 全 機 構 (2007 年 ) 3 救 済 給 付 の 種 類 ( 2 頁 ) 現 在 療 養 中 の 方 への 給 付 医 療 費 ( 本 人 が 請 求 ) 自 己 負 担 分 療 養 手 当 ( 本 人 が 請 求 ) ,870 円 / 月 認 定 後 療 養 中 の 方 がお 亡 くなりになった 場 合 の 給 付 葬 祭 料 ,999 円 救 済 給 付 調 整 金 個 別 に 算 定 7 労 災 補 償 制 度 又 は 特 別 遺 族 給 付 金 について( 頁 ) 医 学 的 判 定 に 係 わる 資 料 に 関 する 留 意 事 項 (3) 病 理 組 織 学 的 検 査 記 録 がない 場 合 について (ア) 細 胞 診 については パパニコロウ 染 色 による 形 態 的 特 徴 及 び 免 疫 染 色 の 結 果 については 詳 細 に 記 載 すること (イ) 陽 性 となる 抗 体 としてcalretinin calretinin 陰 性 となる 抗 体 としてCEA CEAを を 用 いた 免 疫 染 色 の 結 果 を 添 付 すること が 強 く 推 奨 されること 上 記 以 外 の 陽 性 となる 抗 体 としてcytokeratin cytokeratin5/6 thrombomodulin D2-40 などが 陰 性 となる 抗 体 として Ber Ber-EP4 MOC-31などがあり こららの 抗 体 を 用 いた 免 疫 染 色 の 結 果 も 参 考 になる 場 合 があること

124 Body fluid for cell block method

125 Fig.. 2 Cell block method 1. Body fluid 2. Centrifuge at 1500rpm for 5minutes 3.Fixtation 4. Cell block 5.Immunochemical staining 1.Ask clinical department to submit as much body cavity fluid as possible. 2.Centrifuge the body cavity fluid (1500 rpm for 5 minutes). 3.Decant the supernatant into another container, add 1-2 ml of 20% formalin to the supernatant, and fix overnight. 4.Embed the pellets in paraffin to prepare cell blocks. 5.Carry out immunochemical staining.

126 Useful antibodies for diagnosis of mesothelioma (The journal of JSCC:1970~2006) 1985 keratin 1988 EMA vimentin 1996 thrombomodulin 1997 mesothelin 2000 calretinin 2001 CK5/ WT D2-40

127 E-cadherin CEA CK20 Immunohistochemical staining using cell tranfer method in a case of mesothelioma Calretinin mesothelin l CK7

128 中 皮 腫 の 確 定 診 断 のために 何 故 免 疫 染 色 が 必 要 か!! 診 断 の 確 認!!

129 Body fluid for cell block method

130

131 Mesothelioma cells VS Reactive mesothelial cells 写 真 追 加 Variety Multinucleated cells Binucleated cells Reactive mesothelial cells Uniformity

132 Multinucleated cells 4 Dense cytoplasm Prominent nucleoli

133

134

135 Making a definitive diagnosis of mesothelioma by effusion cytology The utility of cell transfer method and cell block method for immunocytochemical staining Maeda Shotaro1, Katayama Hironori1,Naito Zenya2 1Department of Pathology, Tama-Nagayama Hospita,, Nippon Medical School 2Department of Pathology, Nippon Medical School

136 70 year old male

137

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