METHODS AND TECHNIQUES IN

PATHOLOGY … 2
Other techniques in surgical
pathology
 Frozen Section:
 It is a rapid intraoperative diagnostic procedure,
which is used to confirm/ rule out malignancies
and to determine if the surgical plane of
resection is free of the tumor. In this technique,
afresh piece of tissue is rapidly frozen, which
leads to freezing of water within the tissues,
which is covered to ice, thus hardening it and
making it suitable for sectioning.
 Decalcification:
 Calcified tissues like bone and teeth are very
hard and therefore are difficult to section with a
microtome knife. Such tissues need to be
treated with chemicals(formic acid, nitric acid,
hydrochloric acid) to remove the calcium
deposits prior to embedding to allow easy
section.
 II AUTOPSY PATHOLOGY

 Autopsy is a careful postmortem

examination of the human body, which
enlightens the clinicians about the
pathogenesis of diseases, reveals hazardous
effects of therapy administered and is the
final verdict in settlings the discrepancies
between ante- and post- mortem findings.
Therefore autopsy is carried out for
confirming the cause of death and
establishing the final diagnosis.
 III MICROSCOPY

 They are used to view magnified images of

minute objects.
 The most widely used type of microscope is a
light microscope that uses visible light as its
light source. Light microscope is of two types:
 Simple microscope has a simple hand magnifying
lens.
 Compound microscope has a series of sequential
lenses, which are capable of producing highly
magnified images.
 IV IMMUNOFLUORESCENCE

 Immunofluorescence is a technique, which identifies

antigenic molecules at subcellular levels in tissues.
 This is achieved by use of antibodies which are
irreversibly bound to fluorescent labels.
 There are two types of fluorescence technique
performed on cryostat sections-direct (the antibody
is directly conjugated against antigen and then
examined under fluorescence microscope) and
indirect (the antigen antibody interacts, followed by
step of washing and then addition of fluorochrome
for reaction.
 The main applications of immunofluorescence
are as follows:
 Detection of autoantibodies in serum, e.g.
antinuclear antibody.
 For determination of deposits of immunoglobulins
in renal disease.
 For detection of immunoglobulin deposits in skin
disorders.
 Study of cell surface markers.
 Diagnosis of infective disorders, e.g. viral hepatitis.
 V IMMUNOHISTOCHEMISTRY

 To detect the lineage of tumor cells by the

presence or absence of tissue specific
lineage markers.
 IHC employs the use of specific antibodies,
which are visualized by a chromogen like
diaminobenzidine tetrachloride (DAB),
which gives a brown color.
 The antibodies used in IHC are produced by
monoclonal or polyclonal techniques.
 The two most commonly employed techniques
of IHC are:
 Avidin-biotin conjugate immunoenzymatic method
and
 Peroxidase-antiperoxidase method.
 Immunohistochemical staining should always
be done with appropriate positive controls.
 The positivity of the antibodies should be
interpreted depending upon the localization of
the antigen within the cell.
 For example, nuclear staining for estrogen and
progesterone receptors, membranous positivity
for HER-2/ neu and cytoplasmic staining for
cluster of differentiation (CD) markers.
 The main applications of
immunochemistry are as follows:
 Detection of lineage of tumor cells.
 Diagnosis of tumors of uncertain origin.
 Prognostication of tumors by identification of
enzymes, tumor specific antigens and oncogenes.
 To predict therapeutic response in certain types of
tumors. For example, carcinoma of breast and
prostate.
 For quick and precise identification of infectious
agent in tissues by use of specific antibodies
against microbial deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA).
 VI CYTOGENETICS

 Cytogenetics is the branch of genetics that

deals with the study of chromosomal
abnormalities.
 The most commonly used technique is
karyotyping, which is a photograpic
representation of stained pairs of
chromosomes.
 The main application of the cytogentics is for
the detection of abnormalities in number and
structure of chromosomes.
 VII MOLECULAR PATHOLOGY
 Molecular pathology detects abnormalities at the
level of the genetic makeup of cells (DNA/RNA).
 The following molecular techniques are most widely
used.
 Polymerase chain reaction: it is revolutionary technique
works on the principle that a single strand of DNA has
limitless capacity to duplicate itself to form millions of
copies.
 In situ hybridisation: It allows localization of the nucleic
acid sequence directly in the intact cell without DNA
extraction.
 Used in viral infections, human tumors and chromosomal
disorder.
 VIII FLOW CYTOMETRY

 Flow cytometry identifies hemopoietric

neoplasms by the presence or absence of
cellular antigen expression.
 It is particularly suited for the
immunophenotypic analysis of blood, bone
marrow aspirates and lymphnode suspensions.
 It simultaneously allows the quantative and
qualitative analysis of several characteristics of
cells as they flow in single line through a beam
of light.
Applications are as follows for
flow cytometery.
 Diagnosis of hematolymphoid neoplasms ,e.g.
acute and chronic leukemias.
 For exact categorization/ classification on the
hemopoietic neoplasms.
 CD4+ T cell counts for the diagnosis and
prognosis of patients with human
immunodeficiency virus (HIV) infection.
 To diagnose the cause of allograft rejection by
CD3 + T cell counts.
 DNA ploidy studies in various cancers.