This power point presentation is intended for II MBBS student

1. Dr Sunita B. Patil
Assoc Prof
Dept. of Pathology
D.Y.Patil Medical College, Kolhapur

2. 1. Histological Methods (Biopsy)
2. Cytological Methods
i) Exfoliative cytology
ii) Fine needle aspiration cytology (FNAC)
3. Histochemistry and Cytochemistry
4. Immunohistochemistry
5. Electron Microscopy
6. Tumour Markers (Biochemical Assays)
7. Other Modern Aids in Pathologic Diagnosis of Tumours

3.  microscopic examination of properly fixed tissue (excised tumour mass or
open/needle biopsy from the mass), supported with complete clinical and
investigative data.
 These methods are most valuable in arriving at the accurate diagnosis.
 The tissue must be fixed in
i) 10% formalin for light microscopic examination and
ii) glutaraldehyde for electron microscopic studies, while
iii) quick frozen section and hormonal analysis are carried out on
fresh unfixed tissues.

4.  The histological diagnosis by either of these methods is based on the
morphological features
 benign tumours resemble those of normal tissue and are unable to invade and
metastasise,
 while malignant tumours are identified by lack of differentiation in cancer cells
termed ‘anaplasia’ or ‘cellular atypia’ and may invade as well as metastasise.

5. Cytological methods for diagnosis consist of
 study of cells shed off into body cavities (exfoliative cytology) and
 Study of cells by putting a fine needle introduced under vacuum into the lesion
(fine needle aspiration cytology, FNAC).

6.  Cytologic smear (Papanicolaou or Pap smear) method employed for detecting
dysplasia, carcinoma in situ and invasive carcinoma of the uterine cervix.
 examination of sputum and bronchial washings; pleural, peritoneal and
pericardial
effusions; urine, gastric secretions, and CSF.
 The method is based on microscopic identification of the characteristics of
malignant cells which are incohesive and loose and are thus shed off or ‘exfoliated’
into the lumen.
 However, a ‘negative diagnosis’ does not altogether rule out malignancy due to
possibility of sampling error.

7. A: Normal smear of the uterine cervix
B: Abnormal smear containing sheet of malignant
cells showing large Hyperchromatic and
pleomorphic nuclei with the presence of mitosis
in one cell

8.  study of materials obtained from superficial and deep-seated lesions in the body which
do not shed off cells freely.
 This method consists of study of cells obtained by a fine needle introduced under
vacuum into the lesion, so called fine needle aspiration cytology (FNAC).
 The superficial masses can be aspirated under direct vision
 while deep-seated masses such as intra-abdominal, pelvic organs and retroperitoneum are
frequently investigated by ultrasound (US) or computed tomography (CT)-guided fine needle
aspirations.
 The smears are fixed in 95% ethanol by wet fixation, or may be air-dried unfixed.
 Papanicolaou method of staining is routinely employed for wet fixed smears, and H & E due to
similarity
in staining characteristics in the sections obtained by paraffin embedding.
 Air-dried smears are stained by May-Grunwald-Giemsa or Leishman stain.
 FNAC has a diagnostic reliability between 80-97%

10.  These are additional diagnostic tools which help in identifying the chemical
composition of cells, their constituents and their products by special staining
methods.
 Though immunohistochemical techniques are more useful for tumour diagnosis,
histochemical and cytochemical methods are still employed for this purpose.

11. Substance Stain
1. Basement membrane/
Collagen
• Periodic acid-Schiff (PAS)
• Reticulin
• Van Gieson
• Masson’s trichrome
2. Glycogen • PAS with diastase loss
3. Glycoproteins, glycolipids,
glycomucins (epithelial origin)
• PAS with diastase
persistence
4. Acid mucin (mesenchymal origin) • Alcian blue
5. Mucin (in general) • Combined Alcian blue-PAS
6. Argyrophilic/ argentaffin granules • Silver stains
7. Cross striations • PTAH stain
8. Enzymes • Myeloperoxidase
• Acid phosphatase
• Alkaline phosphatase
Nucleolar organiser regions (NORs) • Colloidal silver stain

12.  An immunological method of recognizing a cell by one or more of its specific
components in the cell membrane, cytoplasm or nucleus.
 These cell components (called antigens) combine with specific antibodies on the
formalin-fixed paraffin sections or cytological smears.
 The complex of antigen-antibody on slide is made visible for light microscopic
identification by either fluorescent dyes (‘fluorochromes’) or by enzyme system (‘chromogens’).
 The specific antibody against a particular cellular antigen is obtained by hybridoma technique
for monoclonal antibody production.
 These monoclonal antibodies are not only antigen specific , are highly sensitive in detection of
antigenic component, and, therefore, impart objectivity to the subjective tumour diagnosis
made by the surgical pathologist.

15.  Categorization of undifferentiated tumours e.g.,Leukemias/ Lymphomas
 Site of origin
 Receptors, e.g., ER, PR
Gastric adenocarcinoma is positive for cytokeratin, with brown-red
reaction product in the neoplastic cell cytoplasm . This is a typical
staining reaction for carcinomas and helps to distinguish carcinomas
from sarcomas and lymphomas.

16. This sarcoma is positive for vimentin by
immunohistochemical staining. This is a typical
immunohistochemical staining reaction for sarcomas.

17. Intermediate Filament Tumour
1. Keratins Carcinomas, mesotheliomas,
some germ cells tumours
2. Vimentin Sarcomas, melanomas, lymphomas
3. Desmin Myogenic tumours
4. Neurofilaments (NF) Neural tumours
5. Glial fibrillary acidic protein (GFAP) Glial tumours
an important group of antibody stains directed against various classes of intermediate filaments,
useful in classification of poorly-differentiated tumours of epithelial or mesenchymal origin

18.  Ultrastructural examination of tumour cells offers selective role in diagnostic
pathology.
 EM examination may be helpful in confirming or substantiating a tumour diagnosis
arrived at by light microscopy and immunohistochemistry.
 A few general features of malignant tumour cells by EM examination can be
appreciated:

19. i) Cell junctions, their presence and type.
ii) Cell surface, e.g. presence of microvilli.
iii) Cell shape and cytoplasmic extensions.
iv) Shape of the nucleus and features of nuclear membrane.
v) Nucleoli, their size and density.
vi) Cytoplasmic organelles—their number is generally reduced.
vii) Dense bodies in the cytoplasm.
viii) Any other secretory product in the cytoplasm e.g. melanosomes in melanoma
and membrane-bound granules in endocrine tumours.

20.  Tumour markers are biochemical assays of products elaborated by the tumour
cells in blood or other body fluids.
 many of the products are produced by normal body cells too, and thus the
biochemical estimation of the product in blood or other fluid reflects the total
substance and not by the tumour cells alone.
 These methods, therefore, lack sensitivity as well as specificity and can only be
employed for the following:
i) as an adjunct to the pathologic diagnosis arrived at by other methods and not
for primary diagnosis of cancer.
ii) it can be used for prognostic and therapeutic purposes.

21. Marker Cancer
1. ONCOFOETAL ANTIGENS:
i. Alpha-foetoprotein (AFP) Hepatocellular carcinoma, non-seminomatous
germ cell tumours of testis
ii. Carcinoembryonic antigen (CEA) Cancer of bowel, pancreas, breast
2. ENZYMES
i. Prostate acid phosphatase (PAP) Prostatic carcinoma
ii. Neuron-specific enolase (NSE) Neuroblastoma, oat cell carcinoma lung
iii. Lactic dehydrogenase (LDH) Lymphoma, Ewing’s sarcoma

22. 3. HORMONES:
i. Human chorionic gonadotropin (hCG) Trophoblastic tumours, non-seminomatous
germ cell tumours of testis
ii. Calcitonin Medullary carcinoma thyroid
iii. Catecholamines and vanillylmandelic acid
(VMA)
Neuroblastoma, pheochromocytoma
iv. Ectopic hormone production Paraneoplastic syndromes
4. CANCER ASSOCIATED PROTEINS:
i. CA-125 Ovary
ii. CA 15-3 Breast
iii. CA 19-9 Colon, pancreas, breast
iv. CD30 Hodgkin’s disease, anaplastic large cell
lymphoma (ALCL)
v. CD25 Hairy cell leukaemia (HCL), adult T cell
leukaemia lymphoma (ATLL)
vi. Monoclonal immunoglobulins Multiple myeloma, other gammopathies
vii. Prostate specific antigen (PSA) Prostate carcinoma

24. i)Flow cytometry - is a computerised technique by which the detailed
characteristics of individual tumour cells are recognised and quantified and the
data can be stored for subsequent comparison too.
 Single cell suspensions are required to ‘flow’ through the ‘cytometer’, it can be
employed on blood cells and their precursors in bone marrow aspirates and body
fluids, and sometimes on fresh-frozen unfixed tissue.
 The method is employed for identification of
-cell surface antigen (e.g. in classification of leukaemias and lymphomas), or
- DNA content analysis (e.g. aneuploidy in various cancers).

26. ii) In situ hybridization - is a molecular technique by which nucleic acid sequences
(cellular/viral DNA and RNA) can be localised by specifically-labelled nucleic acid
probe directly in the intact cell (in situ) rather than by DNA extraction.
 A modification of in situ hybridization technique is fluorescence in situ
hybridisation (FISH) in which fluorescence dyes applied and is used to detect
microdeletions, subtelomere deletions and to look for alterations in chromosomal
numbers.
 In situ hybridisation used for analysis of certain human tumours by the study of
oncogenes aside from its use in diagnosis of viral infection.

28. iii) Molecular diagnostic techniques. –
 The group of molecular biologic methods in the tumour diagnostic laboratory are a
variety of DNA/RNA-based molecular techniques in which the DNA/RNA are extracted
(compared from in situ above) from the cell and then analysed.
 These techniques are highly sensitive, specific and rapid and have revolutionised diagnostic
pathology in neoplastic as well as non-neoplastic conditions (e.g. in infectious and inherited
disorders, and in identity diagnosis).
 Molecular diagnostic techniques include:
- DNA analysis by Southern blot,
- RNA analysis by northern blot, and
- polymerase chain reaction (PCR).
 The following techniques of molecular methods in tumour diagnosis have applications in
haematologic as well as malignancies:
- Analysis of molecular cytogenetic abnormalities
- Mutational analysis
- Antigen receptor gene rearrangement
- Study of oncogenic viruses at molecular level.

30. Besides tumour diagnosis newer molecular techniques are being applied for –
- predicting prognosis,
- biologic behaviour of tumour,
- detection of minimal residual disease and
- for hereditary predisposition of other family members to develop a particular
cancer.

31. iv) DNA microarray analysis of tumours - molecular profiling of a tumour by use
of gene chip technology which allows measurement of levels of expression of
several thousand genes (up-regulation or down-regulation) simultaneously.
- Fluorescent labels are used to code the cDNA synthesised by trigger from
mRNA.
- The conventional DNA probes are substituted by silicon chip which contains the
entire range of genes and high resolution scanners are used for the
measurement.

32. THANK YOU