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![CEA
• 180-kD glycoprotein
• Widely in effusion cytology
• CEA antibody in effusion cytology has a low sensitivity
[55%] and a high specificity [>90%]
6/3/2019 43Seminar PESIMSR](https://image.slidesharecdn.com/13-200915110854/85/13-role-of-icc-in-body-fluids-39-320.jpg)
![CEA
• 180-kD glycoprotein
• Widely in effusion cytology
• CEA antibody in effusion cytology has a low sensitivity
[55%] and a high specificity [>90%]
6/3/2019 43Seminar PESIMSR](https://image.slidesharecdn.com/13-200915110854/75/13-role-of-icc-in-body-fluids-39-2048.jpg)
Uploaded byDr SANTHIPRIYA GOPASANA
13. role of icc in body fluids
This document discusses the role of immunocytochemistry in body fluids. It begins by introducing immunocytochemistry techniques for body fluids and their importance in diagnostic cytopathology. It then covers topics like specimen types, fixation methods, standardization issues, antigen retrieval, controls, and the interpretation and limitations of immunocytochemistry. The document also discusses the application of immunocytochemistry to specific organ cytology like effusion cytology and breast cytology. It notes important site-specific markers and their role in determining the primary site of carcinomas of unknown primary. In conclusion, the document emphasizes that immunocytochemistry continues to play an important role as an adjuvant tool in diagnostic cytopathology and
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13. role of icc in body fluids
- 1. Role of immunocytochemistryin body fluids Moderator: Dr Nirmala M J Presenter: Dr G Santhipriya G 6/3/2019 1Seminar PESIMSR
- 2. Contents • Introduction • ImmunocytologyTechniques – Specimen types – Fixation – Standardization issues – Rehydration and storage – Antigen retrieval – Fixation for hormone receptors – Thin – layer technique – Cell blocks – Controls – Specimens of limited quality – Interpretation and limitations of ICC – Standard IHC staining protocol • Specific Organ Cytology – Effusion cytology – Site specific markers – Breast cytology – Gynecological cytology – Ovarian cytology • Carcinoma of Unknown Primary – Epithelial malignancies – Non epithelial malignancies • Theranostic Applications: ICC for Targeted Therapies • Conclusion • References 6/3/2019 2Seminar PESIMSR
- 3. INTRODUCTION • The applicationof immunohistochemistry (IHC) in diagnostic cytopathology. • With the use of automation, there has been a great deal of quality improvement in recent years. • IHC continues to play an important role in diagnostic cytopathology, and it is evolving as an important adjuvant tool in targeted therapies 6/3/2019 3Seminar PESIMSR
- 4. Immuncytology techniques • Specimentypes • Fixation • Standardization issues • Rehydration and storage • Antigen retrieval • Fixation for hormone receptors • Thin – layer technique • Cell blocks • Controls • Specimens of limited quality • Interpretation and limitations of ICC6/3/2019 4Seminar PESIMSR
- 5. Specimen types • Cytomorphologyforms the basis • Conventional Romanowsky or Papanicolaou stains have been examined, that a differential diagnosis is generated, IHC generated. – Exfoliative cell preparations – Effusions – Direct imprints – Fine-needle aspirates – Thin-layer collection samples • Air drying or immediate fixation in alcohol • Cytocentrifuge or cell block preparations 6/3/2019 5Seminar PESIMSR
- 6. Fixation • Important prerequisites –Well-spread film of cells on a glass slide – Adequate fixation – Removal of blood and proteinaceous material • Wet fixation in alcohol (WFA) • Air-dried smears (ADS) • Cold acetone and 95% alcohol are common fixatives • B5 may be used for lymphoid markers and neuroendocrine antibodies 6/3/2019 6Seminar PESIMSR
- 7. Standardization issues • Formalinfixative as the standard • A minimum of 8 hours fixative time –ER, PR, and HER2/ neu • Alcohol fixatives can be used for other antibodies • Appropriate alcohol-based controls if alcohol fixation of cytologic specimens is used 6/3/2019 7Seminar PESIMSR
- 8. Rehydration and storage •Air-dried slides -rehydrated in normal saline (<1 min) • Air-dried slides -1 week at room temperature • Slides for IHC, whether air-dried or fixed, can be stored at –70°C for at least 1 month and still maintain immunoreactivity 6/3/2019 8Seminar PESIMSR
- 9. Antigen retrieval • High-temperatureheating • Antigen retrieval can be applied to these specimens for a wide range of antibodies- cytology • ADS>AFS • Paraffin sections 6/3/2019 9Seminar PESIMSR
- 10. Fixation for HormoneReceptors • As per the CAP-ASCO guidelines, the recommended minimum fixation in – 10% neutral buffered formalin for HER2/neu IHC is 6 hours, and – Hormone receptors it is 8 hours. • PreservCyt: 56 days of storage • Formalin-fixed cell blocks are the venue of choice for ER/PR and HER2/neu 6/3/2019 10Seminar PESIMSR
- 11. Thin-Layer Technique • Excellentimmunostaining results • Proprietary solutions Cytolyt and PreservCyt • Lower antibody concentrations – Background is cleaner – Immunostaining is crisp • Immunoreactivity is stable even with long-term storage in PreservCyt. • PreservCyt fixed controls can be used. 6/3/2019 11Seminar PESIMSR
- 12. Cell blocks • Superiormethod for IHC for cytologic specimens. – Suspensions or bloody specimens may be fixed in formol-saline to lyse red cells, or – The specimen may be collected in RPMI salt solution, treated with a commercial thrombin-plasma agent to organize a clot • Then fixed in 10% formalin and processed like a surgical specimen • The main disadvantage of this method is availability of enough material. 6/3/2019 12Seminar PESIMSR
- 13. Papanicolaou-stained breast aspirateof tubular carcinoma illustrating whole tubules en face, tubular lumens 6/3/2019 13Seminar PESIMSR
- 14. Cell block oftubular carcinoma shows angular glands that are negative for smooth muscle myosin heavy chain, confirming the diagnostic impression 6/3/2019 14Seminar PESIMSR
- 15.
- 16. H&E section oftraditional cell block compared with automated cell block. 6/3/2019 16Seminar PESIMSR
- 17. • RCB producesa cell block in 15 minutes from residual Thin-prep vials or other specimens and can be used for a variety of gynecological and respiratory tissues, FNA biopsies, body fluids, and other materials. • Cytoscrape cell blocks (SCB) – Decoverslip – Destain – Scrap – 3 % mottled agar – wrapped in Whatman filter paper No. 1 and put in a tissue cassette – Processes 6/3/2019 17Seminar PESIMSR
- 18. Controls • Positive andnegative controls must be performed with each test sample. • The ideal control should be a comparably fixed cytology sample. 6/3/2019 18Seminar PESIMSR
- 19. Specimens of LimitedQuality • Immunohistochemistry can be hampered by limited quantity of specimen • A double labelling method to address the problem of limited material when more than one antibody is required to make a diagnosis • Cytology slides that were subjected to an immunoperoxidase test and produced a negative result can be subjected to another immunoperoxidase test using a different antibody 6/3/2019 19Seminar PESIMSR
- 20. Somatostatin negative HHF35muscle actin positive CAM5.2 negative LCA positive 6/3/2019 20Seminar PESIMSR
- 21. Interpretation and Limitationsof ICC • A patient workup • Heterogeneity of immunostaining is the rule rather than the exception – The pattern – Cell localization – Distribution of positive and negative immunostaining relative to normal cells • False positive • False negative 6/3/2019 Seminar PESIMSR 22
- 22. Standard IHC stainingprotocol 6/3/2019 Seminar PESIMSR 24
- 23. Specific organ cytology •Effusion cytology: – IHC serves as a valuable adjunct tool in definitive interpretation. – Reactive mesothelial cells versus adenocarcinoma versus mesothelioma • Various cytology preparation 6/3/2019 27Seminar PESIMSR
- 24. Mesothelial markers • Calretinin •HBME1 • Cytokeratin 5/6 • Wilms’ Tumor Gene 1 • D2-40 • GLUT1 • XIAP Non mesothelial (adenocarcinoma) markers • MOC31 • BG8 • Ber-EP4 • Monoclonal CEA • TAG-72.3 • CD15(Leu M1) 6/3/2019 28Seminar PESIMSR
- 25. • Mesothelial markers 6/3/201929Seminar PESIMSR
- 26. Calretinin • 29- kDacalcium binding protein • Member of EF proteins • Role in cell cycle • The sensitivity of calretinin to distinguish reactive mesothelial cells from adenocarcinoma cells is 100%, and the specificity is up to 80%. • Strong nuclear and cytoplasmic staining patter 6/3/2019 30Seminar PESIMSR
- 27. Reactive mesothelial cells-calretinin postitive 6/3/2019 31Seminar PESIMSR
- 28. HBME1 • Antibody againstcultured mesothelial cells and recognizes an antigen on the microvillus surface. • Mesothelial cells -thick bushy membrane pattern • A thin membrane or cytoplasmic staining of -adenocarcinoma. 6/3/2019 32Seminar PESIMSR
- 29. Cytokeratin 5/6 • Familyof water insoluble intracellular fibrous proteins present in almost all epithelia • Marker for epithelial differentiation • The sensitivity and specificity in distinguishing malignant mesothelioma from adenocarcinoma in pleural effusions is 90% to 100% • No value in differentiating malignant mesothelioma from metastatic pulmonary squamous cell carcinoma. • Breast carcinoma 6/3/2019 33Seminar PESIMSR
- 30. Wilms’ Tumor Gene1 • Tumor suppressor gene • Chromosome 11 • Epithelioid mesotheliomas • Sarcomatoid mesotheliomas(rare) • Desmoplastic round cell tumors • Wilms’ tumor vs pulmonary adenocarcinoma • Metastatic carcinoma- ovarian serous type- strong nuclear positivity 6/3/2019 34Seminar PESIMSR
- 31. D2-40 • Lymphatic endothelium,as well as neoplastic mesothelial cells • Sensitive marker • Malignant mesothelioma from pulmonary carcinoma in effusion cytology 6/3/2019 35Seminar PESIMSR
- 32. GLUT1 • A memberof the family of glucose transporter isoforms (GLUT) • Facilitates the entry of glucose into cells and is expressed in a variety of malignancies • Reactive mesothelial cells from malignant mesothelioma • Cannot, discriminate malignant mesothelioma and lung carcinoma 6/3/2019 36Seminar PESIMSR
- 33. XIAP • X-linked inhibitorof apoptosis (XIAP) • Is a monoclonal antibody • Marker for distinguishing malignant from benign groups of cells 6/3/2019 37Seminar PESIMSR
- 34. • Non –mesothelial (adenocarcinoma) markers 6/3/2019 38Seminar PESIMSR
- 35. MOC31 • Is amonoclonal antibody • An epithelial-associated transmembrane glycoprotein of 40 kD • Squamous cell carcinomas, adenocarcinomas, and small cell carcinomas show a membrane staining pattern • Highly specific non-mesothelial marker in addition to BG8 for distinguishing epithelioid mesothelioma from adenocarcinoma 6/3/2019 39Seminar PESIMSR
- 36. MOC 31 positiveadenocarcinoma, membranous staining 6/3/2019 40Seminar PESIMSR
- 37. BG8 • Antibody againstLewis antigen • ABH blood antigens – Tumor metastasis – Solid tumors • Sensitivity and specificity of 86% and 90%, differentiating adenocarcinoma from epithelioid mesothelioma. • Shows a membrane and cytoplasmic staining pattern of adenocarcinoma cells 6/3/2019 41Seminar PESIMSR
- 38. Ber-EP4 • Monoclonal antibody •Reacts with two glycoproteins on the surface as well as in the cytoplasm of epithelial cells • Does not react with mesothelial cells to a significant degree • Characteristic membranous pattern, and lack of cross- reaction with background inflammatory cells Strong membrane staining of Ber-EP4 in adenocarcinoma 6/3/2019 42Seminar PESIMSR
- 39. CEA • 180-kD glycoprotein •Widely in effusion cytology • CEA antibody in effusion cytology has a low sensitivity [55%] and a high specificity [>90%] 6/3/2019 43Seminar PESIMSR
- 40. TAG-72.3 • mAb B72.3,which is directed against a tumor-associated antigen (TAG-72) • A combination of B72.3 and Ber-EP4 has high sensitivity and specificity, up to 98%. 6/3/2019 44Seminar PESIMSR
- 41. CD15 (Leu M1) •CD15 or Lewis X antigen can be identified with the LeuM1 antibody. • Leu-M1 (CD15 granulocyte antigen) • BMA/070 (CD16 natural killer antigen) • Did not react with mesothelial cells, although they stained carcinoma cells. Monoclonal Abs 6/3/2019 45Seminar PESIMSR
- 42. SITE-SPECIFIC MARKERS • Thyroidtranscription factor-1 (TTF-1) – Pulmonary adenocarcinomas – Thyroid tumors – Small cell carcinomas (pulmonary and extrapulmonary) • Anti–TTF-1 : adenocarcinoma of pulmonary origin in patients presenting with metastatic adenocarcinoma in serous fluid(s) with an unknown primary site 6/3/2019 46Seminar PESIMSR
- 43. Metastatic pulmonary adenocarcinomain pleural effusion Cell block demonstrates malignant groups with marked nuclear pleomorphism x40 TTF-1 shows a diffuse strong nuclear staining, confirming the lung origin of the adenocarcinoma.6/3/2019 47Seminar PESIMSR
- 44. SITE-SPECIFIC MARKERS • Estrogenreceptor – Antibodies- identify metastatic breast carcinoma in effusions from patients without solid tissue metastasis – A positive ER result can be useful in indicating a breast or gynecological origin 6/3/2019 48Seminar PESIMSR
- 45. SITE-SPECIFIC MARKERS • CDX2 –Is a homeobox domain–containing transcription factor – Is important in the development and differentiation of the intestine and is expressed in colorectal carcinoma. – Gastrointestinal and pancreatic malignancies in ascites cytologic samples and to differentiate them from reactive mesothelial cells – Mucinous tumors with gastrointestinal differentiation that originate in the lung or ovary – Marker of neuroendocrine tumors of midgut origin 6/3/2019 49Seminar PESIMSR
- 46. Breast cytology • Grosscystic fluid protein 15 (GCDFP-15)/BRST-2 – Breast cyst fluid – Plasma of invasive mammary carcinoma – prostate – salivary – sweat glands – central (bronchial) lung carcinomas other organs 6/3/2019 50Seminar PESIMSR
- 47. Breast cytology • Mammaglobin –Gene sequence fragment – Primary and metastatic breast carcinomas – Endometrial adenocarcinomas – Salivary gland carcinomas – Endocervical carcinomas in situ Other organs 6/3/2019 51Seminar PESIMSR
- 48. Breast cytology • E-CADHERINAND P120 CATENIN – Absence of E-cadherin, characteristic of lobular neoplasms – Diffuse signet ring carcinomas of stomach and rectum may also show p120 cytoplasmic immunostaining 6/3/2019 52Seminar PESIMSR
- 49. Infiltrating ductal carcinomademonstrating strong membranous E-cadherin staining 6/3/2019 53Seminar PESIMSR
- 50. Metastatic breast carcinomato bone- cell block Cyokeratin 7 positive GCDFP-15 positive Mammaglobin positive 6/3/2019 54Seminar PESIMSR
- 51. Pleural effusion Cell blockpreparation Gross cystic disease fluid protein 15 positive 6/3/2019 55Seminar PESIMSR
- 52. Pleural effusion Mammaglobin positiveE- cadherin negative 6/3/2019 56Seminar PESIMSR
- 53. Pleomorphic lobular carcinomain pleural effusion P120 cytoplasmic expression Calretinin- non specific staining 6/3/2019 57Seminar PESIMSR
- 54. Gynecological cytology • P16INK4a – Overexpression of p16INK4a has been strongly linked to high-risk HPV infection and is expressed in dysplastic squamous cells. – Discriminates in situ and invasive cervical adenocarcinomas from benign endocervical cells – Thin-prep and Surepath slides as well as cell blocks. 6/3/2019 58Seminar PESIMSR
- 55. Strong nuclear andcytoplasmic staining of p16 6/3/2019 59Seminar PESIMSR
- 56. p16 nuclear andcytoplasmic positive, nonspecific staining in metaplastic cells 6/3/2019 60Seminar PESIMSR
- 57. Gynecological cytology • P16INK4a – A score of more than 10 cells showing predominantly nuclear as well as cytoplasmic staining is considered positive – Metaplastic cells – Trichomonas vaginalis – Endometrial cell nuclei (LBC) Non-specific staining 6/3/2019 61Seminar PESIMSR
- 58. Gynecological cytology • ProExC – Cocktail of monoclonal antibodies directed against proteins associated with aberrant S-phase cell-cycle induction – 100% positivity for HSIL • MIB1 (Ki-67) – Is complementary surrogate biomarkers for HPV- related pre-invasive squamous cervical disease 6/3/2019 62Seminar PESIMSR
- 59. Ovarian Cytology • Wilms’tumor gene product(WT1) – Serous carcinomas of ovarian surface epithelial origin (both ovarian and extra-ovarian) – Mucinous and micropapillary breast carcinomas Serous carcinoma in malignant effusions 6/3/2019 63Seminar PESIMSR
- 60. Ovarian Cytology • Thyroidtranscription factor-1(TTF-1) – Mixed serous and endometrioid carcinoma and pure highgrade serous carcinoma, showed strong nuclear stain – Marker for non–small cell carcinoma of primary pulmonary origin 6/3/2019 64Seminar PESIMSR
- 61. Ovarian Cytology • SOX9 –Sertoli cell tumors – Endometrioid borderline tumors – Well differentiated endometrioid carcinomas – Sertoliform endometrioid carcinomas – Carcinoid tumors • Hepatocyte nuclear factor-1β (HNF-1β) – Clear cell from adenocarcinomas 6/3/2019 65Seminar PESIMSR
- 62. CARCINOMA OF UNKNOWNPRIMARY • Body effusions -common presentation of metastasis CUP • Serous effusions -commonly • Adenocarcinoma • Squamous cell carcinoma • Non–small cell carcinoma • Small cell carcinoma • Identification of the organ of origin -therapeutic significance • Additional clinical history Possible to recognize four different tumor types cytologically in effusions 6/3/2019 66Seminar PESIMSR
- 63. CK7 CK20 ++ Lung Breast Gastric Ovary + - Small celllung ca - - Poorly differentiated squamous cell carcinoma - - Colorectal Gastric Pancreatic + colonic TTF1 p63 CDX2 CK5/6 p63 + + Squamous cell carcinoma PSA CA125 WT1 6/3/2019 67Seminar PESIMSR
- 64. Non-epithelial Malignancies Presenting asTumors of Unknown Origin 6/3/2019 68Seminar PESIMSR
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- 68. THERANOSTIC APPLICATIONS: ICC FORTARGETED THERAPIES • CD117 • HER2/neu • EGFR 6/3/2019 72Seminar PESIMSR
- 69. CONCLUSION • In thefast-growing era of technology, diagnostic cytopathology has managed to adopt and incorporate modern ancillary techniques such as ICC to aid in diagnosis. • The role of ICC continues to grow not only in arriving at diagnosis but also for targeted therapies. • However, the major challenge that remains to be addressed is standardization of immunoreactions within and across laboratories. 6/3/2019 73Seminar PESIMSR
- 70. REFERENCES • Mamatha c,Dabbs D J. Immunocytology .In Diagnostic immunohistochemistry. 3rd Ed. Elsevier. p890-918 • Ronald A D, Rana S H. Immunochemistry and molecular biology in cytological diagnosis. In Koss’ diagnostic cytology and its histologic bases. 5th ed. Lippicott. p1636- 80 • Prabab D.Special stains and immunocytochemistry. In Diagnostic cytology. 2nd ed. Jaypee. p237-48 • Ramdas N. Immunohistochemistry. In Histopathology techniques and its management. Jaypee. p267-90 6/3/2019 74Seminar PESIMSR
- 71. • Thank you!! 6/3/201975Seminar PESIMSR