Role of Immunocytochemistry in body fluids

1. Role of immunocytochemistry in
body fluids
Moderator: Dr Nirmala M J
Presenter: Dr G Santhipriya G
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2. Contents
• Introduction
• Immunocytology Techniques
– 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
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3. INTRODUCTION
• The application of 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
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4. Immuncytology techniques
• 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 ICC6/3/2019 4Seminar PESIMSR

5. Specimen types
• Cytomorphology forms 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
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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
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7. Standardization issues
• Formalin fixative 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
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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
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9. Antigen retrieval
• High-temperature heating
• Antigen retrieval can be applied to these specimens for a
wide range of antibodies- cytology
• ADS>AFS
• Paraffin sections
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10. Fixation for Hormone Receptors
• 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
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11. Thin-Layer Technique
• Excellent immunostaining 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.
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12. Cell blocks
• Superior method 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.
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13. Papanicolaou-stained breast aspirate of tubular
carcinoma illustrating whole tubules en face,
tubular lumens
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14. Cell block of tubular carcinoma shows angular glands that
are negative for smooth muscle myosin heavy chain,
confirming the diagnostic impression
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15. 6/3/2019 15Seminar PESIMSR

16. H&E section of traditional cell block compared
with automated cell block.
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17. • RCB produces a 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
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18. Controls
• Positive and negative controls must be performed with
each test sample.
• The ideal control should be a comparably fixed cytology
sample.
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19. Specimens of Limited Quality
• 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
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20. Somatostatin negative HHF35 muscle actin positive
CAM5.2 negative
LCA positive
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21. Interpretation and Limitations of 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
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22. Standard IHC staining protocol
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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
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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)
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25. • Mesothelial markers
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26. Calretinin
• 29- kDa calcium 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
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27. Reactive mesothelial cells -calretinin postitive
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28. HBME1
• Antibody against cultured mesothelial cells and recognizes an
antigen on the microvillus surface.
• Mesothelial cells -thick bushy membrane pattern
• A thin membrane or cytoplasmic staining of -adenocarcinoma.
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29. Cytokeratin 5/6
• Family of 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
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30. Wilms’ Tumor Gene 1
• 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
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31. D2-40
• Lymphatic endothelium, as well as neoplastic mesothelial
cells
• Sensitive marker
• Malignant mesothelioma from pulmonary carcinoma in
effusion cytology
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32. GLUT1
• A member of 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
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33. XIAP
• X-linked inhibitor of apoptosis (XIAP)
• Is a monoclonal antibody
• Marker for distinguishing malignant from benign groups
of cells
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34. • Non – mesothelial (adenocarcinoma) markers
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35. MOC31
• Is a monoclonal 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
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36. MOC 31 positive adenocarcinoma, membranous
staining
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37. BG8
• Antibody against Lewis 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
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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
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39. CEA
• 180-kD glycoprotein
• Widely in effusion cytology
• CEA antibody in effusion cytology has a low sensitivity
[55%] and a high specificity [>90%]
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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%.
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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
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42. SITE-SPECIFIC MARKERS
• Thyroid transcription 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
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43. Metastatic pulmonary adenocarcinoma in 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
• Estrogen receptor
– 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
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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
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46. Breast cytology
• Gross cystic 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
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47. Breast cytology
• Mammaglobin
– Gene sequence fragment
– Primary and metastatic breast carcinomas
– Endometrial adenocarcinomas
– Salivary gland carcinomas
– Endocervical carcinomas in situ
Other organs
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48. Breast cytology
• E-CADHERIN AND P120 CATENIN
– Absence of E-cadherin, characteristic of lobular
neoplasms
– Diffuse signet ring carcinomas of stomach and rectum
may also show p120 cytoplasmic immunostaining
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49. Infiltrating ductal carcinoma demonstrating
strong membranous E-cadherin staining
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50. Metastatic breast carcinoma to bone- cell block Cyokeratin 7 positive
GCDFP-15 positive Mammaglobin positive
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51. Pleural effusion
Cell block preparation Gross cystic disease fluid
protein 15 positive
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52. Pleural effusion
Mammaglobin positive E- cadherin negative
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53. Pleomorphic lobular carcinoma in pleural
effusion
P120 cytoplasmic expression Calretinin- non specific staining
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54. Gynecological cytology
• P16 INK4a
– 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.
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55. Strong nuclear and cytoplasmic staining of p16
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56. p16 nuclear and cytoplasmic positive,
nonspecific staining in metaplastic cells
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57. Gynecological cytology
• P16 INK4a
– 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
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58. Gynecological cytology
• ProEx C
– 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
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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
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60. Ovarian Cytology
• Thyroid transcription 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
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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
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62. CARCINOMA OF UNKNOWN PRIMARY
• 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
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63. CK7
CK20
++
Lung
Breast
Gastric
Ovary
+ -
Small cell lung ca
- -
Poorly
differentiated
squamous cell
carcinoma
- -
Colorectal
Gastric
Pancreatic
+
colonic
TTF1
p63
CDX2
CK5/6
p63
+ +
Squamous cell carcinoma
PSA
CA125 WT1
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64. Non-epithelial Malignancies
Presenting as Tumors of Unknown Origin
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65. Sarcoma
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68. THERANOSTIC APPLICATIONS:
ICC FOR TARGETED THERAPIES
• CD117
• HER2/neu
• EGFR
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69. CONCLUSION
• In the fast-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.
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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
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71. • Thank you!!
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