Page 34 - Textbook of Pathology, 6th Edition

18 iv) Western blot is analogous to the previous two methods but cell suspensions are required for flow cytometry, its
is employed for protein fractionation; in this method applications are limited to flow assays e.g. leucocytes,
antibodies are used as probes. erythrocytes and their precursors; body fluids, and sometimes
Applications. In view of high degree of specificity and solid tissues homgenised to make into cell suspensions.
sensitivity of the molecular hybridisation techniques, these Applications. Flow cytometric analysis finds uses in clinical
techniques have widespread applications in diagnostic practice in the following ways:
pathology: 1. Immunophenotyping by detailed antigenic analysis of
i) In neoplasia, haematologic as well as non-haematologic. various haematopoietic neoplasias e.g. acute and chronic
ii) In infectious diseases for actual diagnosis of causative agent, leukaemias, lymphomas (Hodgkin’s and non-Hodgkin’s), and
SECTION I
epidemiologic studies and identification of newer infectious plasmacytic neoplasms.
agents. 2. Measurement of proliferation-associated antigens e.g. Ki67,
iii) In inherited genetic diseases for carrier testing, prenatal diag- PCNA.
nosis and direct diagnosis of the genetic disease. 3. Measurement of nucleic acid content e.g. measuring RNA
iv) In identity determination for tissue transplantation, forensic content of reticulocytes, quantifying DNA content and DNA
pathology, and parentage testing. ploidy counts in various types of cancers.

3. POLYMERASE CHAIN REACTION. Polymerase chain 4. Diagnosis and prognostication of immunodeficiency e.g. in
reaction (PCR) is a revolutionary technique for molecular AIDS by CD4 + T lymphocyte counts. Patients with CD4 + T
genetic purpose with widespread applications in diagnostics cell counts below 500/ml require antiviral treatment.
and research. The technique is based on the principle that a 5. To diagnose the cause of allograft rejection in renal trans-
single strand of DNA has limitless capacity to duplicate itself plantation in end-stage renal disease by CD3 + T cell counts.
to form millions of copies. In PCR, a single strand of DNA Patients with CD3 + T cells below 100-200/ml have lower
generates another by DNA polymerase using a short risk of graft rejection.
complementary DNA fragment; this is done using a primer 6. Diagnosis of autoantibodies in ITP, autoimmune neutro-
which acts as an initiating template. penia.
A cycle of PCR consists of three steps:
i) Heat denaturation of DNA (at 94°C for 60-90 seconds). METHODS FOR CELL PROLIFERATION ANALYSIS
ii) Annealing of the primers to their complementary Besides flow cytometry, the degree of proliferation of cells in
sequences (at 55°C for 30-120 seconds). tumours can be determined by various other methods. These
iii) Extension of the annealed primers with DNA polymerase include the following:
General Pathology and Basic Techniques
(at 72°C for 60-180 seconds).
Repeated cycling can be done in automated thermal cycler 1. Mitotic count. This is the oldest but still widely used
and yields large accumulation of the target sequence since method in routine diagnostic pathology work. The number
each newly generated product, in turn, acts as template in of cells in mitosis are counted per high power field e.g. in
the next cycle. categorising various types of smooth muscle tumours.
Applications. PCR analysis has the same applications as for 2. Radioautography. In this method, the proliferating cells
filter hybridisation techniques and has many advantages over are labelled in vitro with thymidine and then the tissue
them in being more rapid, can be automated by thermal processed for paraffin-embedding. Thymidine-labelled cells
cyclers and requires much lower amount of starting DNA. (corresponding to S-phase) are then counted per 2000 tumour
However, PCR suffers from the risk of contamination; thus cell nuclei and expressed as thymidine-labelling index. The
extreme caution is required in the laboratory during PCR method is employed as prognostic marker in breast
technique. carcinoma.

3. Microspectrophotometric analysis. The section is stained
OTHER MODERN AIDS IN
with Feulgen reaction which imparts staining to DNA content
DIAGNOSTIC PATHOLOGY of the cell and then DNA content is measured by
microspectrophotometer. The method is tedious and has
FLOW CYTOMETRY limited use.

Flow cytometry is a modern tool used for the study of pro- 4. Immunohistochemistry. The nuclear antigen specific for
perties of cells suspended in a single moving stream. Flow cell growth and division is stained by immunohistochemical
cytometry, thus, overcomes the problem of subjectivity method and then positive cells are counted under the
involved in microscopic examination of cells and tissues in microscope or by an image analyser. Such proliferation
histopathology and cytopathology. markers include Ki-67, PCNA, cyclins.
Flow cytometer has a laser-light source for fluorescence,
cell transportation system in a single stream, monochromatic 5. Nucleolar organiser region (NOR). Nucleolus contains
filters, lenses, mirrors and a computer for data analysis. Flow ribosomal components which are formed at chromosomal
cytometer acts like a cell sorter to physically sort out cells regions containing DNA called NORs. NORs have affinity
from liquid suspension flowing in a single-file. Since single- for silver. This property is made use in staining the section