Pathological procedures to diagnose tumors include frozen sections of tissue during surgery, routine tissue sections examined microscopically using hematoxylin-eosin stain, and various types of biopsies. Additional diagnostic tools discussed are immunohistochemistry using monoclonal antibodies, DNA and RNA probes, electron microscopy, fluorescent microscopy, histochemical stains, blot assays extracting DNA and RNA from cells, and tumor markers. Modern techniques mentioned are flow cytometry, in situ hybridization, analysis of cell proliferation, image analysis and morphometry, and molecular diagnostic techniques including polymerase chain reaction and DNA microarrays.

1. LAB. Investigations / Diagnosis
Of
Tumours/ Cancers
• DR. U.S. PANDEY
• PROFESSOR & HOD,
• DEPTT OF PATHOLOGY,
• GOVT MEDICAL COLLEGE,
• BETTIAH-845438 (West Champaran

2. Pathological procedures that are used to diagnose tumors
include :
*1. Immediate Frozen Sections of tissue removed during
surgery: freeze the tissue, make thin sections on a
microtome, and stain in less than 3 minutes.
*2. Routine Tissue Sections (HISTO-pathology)-*usually use
hematoxylin-eosin stain.
* 3.Cytology (Needle Aspirations,Exfoliative Cyto), needle
biopsies, incisional biopsies (piece of the tumor),
excisional biopsies (entire lesion).
*4. Immunohistochemistry(IHC):
(use monoclonal antibodies against antigensin tissue
that can be identified as color changes in the tissue).

4. Histopathology of Biopsy Specimen

7. Common Immunohistochemical Stains For TumoursOf Uncertain Origin

8. Examples of IHC staining at different sites in the tumour cells. A, Membranous staining for
leucocyte common antigen (LCA) or CD45 in lymphomas. B, Cytoplasmic staining for
smooth muscle actin (SMA) in myoepithelium on breast acinus. C, Nuclear staining for
breast ER-PR receptor studies in breast Cancer

9. *5. D N A - Probes
• Probe is any searching device for examination
or investigation in order to get information &
exploration.
Mol.Probe is a sequence of D.N.A. used to
identify a complementary sequence.
Types of Mol. Probe : of different Source & Size: are
1. Genomic Probe
2. c DNA-Probe
3. Oligonucleotide Probe
4. Ribo;Probe
5.Radioactive Labelled Probe ( 52p, 125I )
6.Non-Radioactive Labelled Probe (Biotin,Digoxigenin)

10. * 6.Electron Microscopy :
Ultrastructural examination of tumour cells offers selective role
in diagnostic pathology. A few general features of malignant
tumour cells by EM examination can be appreciated:
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.

11. * 6.Electron Microscopy(Contd.,) is useful in identifying
the following tumors:
. Epithelial tumor---- Tonofilaments
. Angiosarcoma - - - demonstration of Weibel Palade
--bodies.
. Small cell carcinoma of the lung, neuroblastomas and
carcinoid tumors - -
- - neurosecretory granules
. Rhabdomyosarcomas - - - thick and thin myofilaments
. Histiocytosis X (malignant histiocytic tumor) - - -
----BirbeckGranulesinLangerhan’scells(Histiocytes)

12. 7.Fluorescent Microscopy
• Dye-Acridine Orange gives Polychromatic Picture &
Differentiates D.N.A. of Nucleus from R.N.A. of Cytoplasm.
• Malignant Cells stain Red or Orange & Normal Cells Yellow
or Green . Drugs Tetracycline concentrate more in cancer
cells.Smallest Pinpoint of yellow fluorescence is evidence of
cancers in 50% cases.
Thus Cytoplasmic R.N.A. is earlier sign of malignancy,
useful in Ut. Cancer,GIT,Respiratory & Urogenital
Cancers, esp.,Sputum & Bronchial Secretions.

13. Why use Fluorescence Microscopy?
Recently used for :

14. * 8..Histochemical Stains of importance include:
. Cytokeratin - - - separates a carcinoma (positive) from a
malignant lymphoma (negative) and a mesothelioma
(positive) from an adenocarcinoma (negative)
. S 100 - - -positive in malignant melanoma and
neuroblastomas
. Factor VIII related antigen - - - positive in vascular tumors.
. Carcinoembryonic antigen (CEA) - - - negative in
mesothelioma and positive in adenocarcinoma
. Desmin - - - positive in muscle sarcomas
* 9.Southern blot assays involve the extraction of DNA from
cells.
•10.Northern blot assays involve the extraction of RNA from
cells.
•11.Tumour Markers: e.g.,CEA,a-Fetoprotein,PAO,CA-125,CA-
19.9,PSA

16. Common Immunohistochemical Stains For TumoursOf Uncertain Origin

17. Examples of IHC staining at different sites in the tumour cells. A, Membranous staining for
leucocyte common antigen (LCA) or CD45 in lymphomas. B, Cytoplasmic staining for
smooth muscle actin (SMA) in myoepithelium on breast acinus. C, Nuclear staining for
breast ER-PR receptor studies in breast Cancer

18. 10.Tumour Markers
Tumor marker may be defined as substance of
varying nature which may be tumor antigen or
other tumor products such as enzyme ,
hormones, immunoglobulin or
chromosomes,the determination of which
from blood ,urine or other accessible parts of
the body of patients ,may help in suggesion,
confirmation ,prognosis or recurrence of any
human neoplasm.

19. Types of tumor marker
1. Oncofetal Antigens
2. Hormones
3. Enzymes
4. Specific proteins
5. Cancer associated proteins
6. Other macromolecules
7. Immunoglobulin
8. Intermediate filaments
9. Virus
10. New markers

26. 11.OTHER MODERN AIDS IN PATHOLOGIC DIAGNOSIS OF TUMOURS
Other modern diagnostic techniques have emerged for tumour diagnostic
pathology but their availability as well as applicability are limited.
i) Flow cytometry This 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.
ii) In situ hybridisation This 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.
iii) Cell proliferation analysis Besides flow cytometry, the degree of proliferation of cells in
tumours can be determined by various other methods e.g. Ki67
a) Mitotic count
b) Radioautography
c) Microspectrophotometric analysis
d) IHC proliferation markers
e) Nuclear Organiser Region ( NOR )
iv) Image analyzer and morphometry The system is used to perform measurement of
architectural, cellular and nuclear features of tumour cells.
v) 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. Molecular diagnostic techniques include : DNA analysis by
Southernblot,RNA analysis by Northern blot , & PCR(Polymerase Chain

27. Other Modern Aids in Pathological Diagnosis of Tumours :(Contd.)

29. Other Modern Aids in Pathological Diagnosis of Tumours :(Contd.)

31. Molecular diagnosis:
MICRO-ARRAYS Analy
DNA microarray analysis:
• Expression of thousands of
genes are studied.
• Different tissue has
different pattern of gene
expression.
• Powerful tool useful for
sub categorization of disease
e.g. Lymphoma
- confirmation of
morphologic diagnosis
- illustration of genes
involved in certain disease
& possible therapy.