Page 33 - Textbook of Pathology, 6th Edition

1. Cell selection. Cells capable of growth and division are DIAGNOSTIC MOLECULAR PATHOLOGY 17
selected for cytogenetic analysis. These include: cells from
amniotic fluid, chorionic villus (CVS) sampling, peripheral During the last quarter of 20th Century, rapid strides have
blood lymphocytes, bone marrow, lymph node, solid tumours been made in the field of molecular biology. As a result,
etc. molecular techniques which were earlier employed for
research purposes only have now been made available for
2. Cell culture. The sample so obtained is cultured in mito- diagnostic purposes. These techniques detect abnormalities CHAPTER 2
gen media. A mitogen is a substance which induces mitosis in at the level of DNA or RNA of the cell.
the cells e.g. PPD, phytohaemagglutinin (PHA), pokeweed Broadly speaking, all the DNA/RNA-based molecular
mitogen (PWM), phorbol ester etc. The dividing cells are techniques employ hybridization (meaning joining together)
then arrested in metaphase by the addition of colchicine or technique based on recombinant technology. Specific region
colcemid, both of which are inhibitory to microtubule
formation. Subsequently, the cells are lysed by adding of DNA or RNA is detected by labelling it with a probe (Probe
is a chain of nucleotides consisting of certain number of known
hypotonic solution. base pairs). Probes are of different sizes and sources as under:
The metaphase cells are then fixed in methanol-glacial
acetic acid mixture. 1. Genomic probes derived from a region of DNA of cells.
2. cDNA probe derived from RNA by reverse transcription.
3. Staining/banding. When stained, chromosomes have the 3. Oligonucleotide probe is a synthetic probe contrary to
property of forming alternating dark and light bands. For this genomic DNA and cDNA probe both of which are deri-
purpose, fixed metaphase preparation is stained by one of ved from cellular material.
the following banding techniques: 4. Riboprobe is prepared by in vitro transcription system. Techniques for the Study of Pathology
a) Giemsa banding or G-banding, the most commonly used.
b) Quinacrine banding or Q-banding used to demonstrate MOLECULAR METHODS
bands along chromosomes. Following is a brief account of various molecular techniques
c) Constitutive banding or C-banding is used to demonstrate available as diagnostic tool in surgical pathology:
constitutive heterochromatin.
d) Reverse staining Giemsa banding (or R-banding) gives pattern 1. IN SITU HYBRIDISATION. In situ hybridisation (ISH) is
opposite to those obtained by G-banding. a molecular hybridisation technique which allows localisation
of nucleic acid sequence directly in the intact cell (i.e. in situ)
4. Microscopic analysis. Chromosomes are then photo- without DNA extraction unlike other hybridisation-based
graphed by examining the preparation under the microscope. methods described below. ISH involves specific hybridisation
From the photograph, chromosomes are cut and then of a single strand of a labelled nucleic acid probe to a single
arranged according to their size, centromeric location and strand of complementary target DNA or RNA in the tissue.
banding patterns. The pairs of chromosomes are identified The end-product of hybridisation is visualised by radioactive-
by the arm length of chromosomes. The centromere divides labelled probe ( P, 125 I), or non-radioactive-labelled probe
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the chromosome into a short upper arm called p arm (p for (e.g. biotin, digoxigenin).
petit in French meaning ‘short’) and a long lower arm called q Applications. ISH is used for the following:
arm (letter q next to p). i) In viral infections e.g. HPV, EBV, HIV, CMV, HCV etc.
Currently, molecular cytogenetic analysis and charac-
terisation of chromosomes is possible by the revolutionary ii) In human tumours for detection of gene expression and
oncogenes.
technique of multicolour fluorescence in situ hybridization iii) In chromosomal disorders, particularly by use of fluorescent
(FISH) (vide infra under Molecular Pathology).
in situ hybridisation (FISH).
Applications 2. FILTER HYBRIDISATION. In this method, target DNA
or RNA is extracted from the tissue, which may either be fresh,
The field of cytogenetics has widespread applications in frozen and unfixed tissue, or formalin-fixed paraffin-
diagnostic pathology (Chapter 10). In brief, karyotyping is embedded tissue. Extracted target DNA or RNA is then
employed for the following purposes: immobilised on nitrocellulose filter or nylon. Hybridisation
i) Chromosomal numerical abnormalities e.g. Down’s syndrome of the target DNA is then done with labelled probe. DNA
(trisomy 21 involving autosome 21), Klinefelter’s syndrome analysis by filter hybridisation includes various methods as
(trisomy 46), Turner’s syndrome (monosomy 45, XO), under:
spontaneous abortions. i) Slot and dot blots in which the DNA sample is directly
ii) Chromosome structural abnormalities include translocations bound to the filter without fractionation of nucleic acid size.
{e.g. Philadelphia chromosome t(9;22), cri-du-chat (5p) ii) Southern blot which is similar to dot-blot but differs in
syndrome, repeated spontaneous miscarriages}, deletions, performing prior DNA-size fractionation by gel electro-
insertions, isochromosome, and ring chromosome formation. phoresis (E.M. Southern is the name of scientist who described
iii) Cancer is characterised by multiple and complex chromo- Southern blot technique).
somal abnormalities which include deletions, amplifications, iii) Northern blot is similar to Southern blot but involves size
inversions and translocations, especially in leukaemias and fractionation of RNA (Northern is, however, opposite
lymphomas, germ cell tumours, some sarcomas. direction of southern and not someone’s name).