Examining and

Analyzing
Chromosomes

Reported by:
Esporas, Jacqueline Anne
Galido, Leonel Jun Doven
Gerardo, Arianne Jean
Gerardo, Armie Joy
Macatuhay, Irene
 Basic Laboratory Procedure

 The study of chromosomes using traditional cytogenetic

techniques requires cells that are actively dividing.
 Specimens that contain spontaneously proliferating cells include
bone marrow, lymph nodes, solid tumors, and chorionic villi.
 SPECIMEN COLLECTION AND HANDLING

Peripheral Blood Specimen

Peripheral blood samples should be collected in sterile
syringes or vacuum tubes containing preservative-free sodium
heparin.

Bone Marrow Aspirates

The collection requirements for bone marrow samples are
essentially the same as for peripheral blood.

Amniotic Fluid Specimens

From 15 to 30 mL of amniotic fluid should be obtained under
sterile conditions and collected in a sterile container approved
for cell culture.
Solid Tissue Specimens
Solid tissue sources include skin biopsies, chorionic villi,
products of conception, and stillbirth biopsies.

Small samples should be collected and transported in

sterile culture vessels containing growth or tissue culture
medium (not formalin).
 CULTURE INITIATION
Growth Media
 All specimens for chromosome preparation are grown and
maintained in an aqueous growth medium.
L-Glutamine
 L-Glutamine is an amino acid essential for cell growth.
Serum
 Serum is essential for good cell growth.
Antibiotics
 This is a stopgap measure at best and should never be relied upon
to compensate for sloppy technique.
Mitotic Stimulants (Mitogens)
 Some cells, particularly mature lymphocytes, do not spontaneously
undergo cell division and must be stimulated to divide by the
addition of an appropriate mitogen to the cell culture.
Growth Factors
 A variety of additional growth factors are commercially
available and are used by some laboratories to achieve optimal
cell growth for different sample types.
Culture Vessels
 The choice of culture vessel depends in part on the growth
needs of the sample and in part on the individual preference of
the laboratory.
Flask Method
 Cells are grown on the inner surface of T-flasks until adequate
numbers of dividing cells are present. Cell growth is monitored
using an inverted microscope.
In Situ Method
 Amniotic fluid, chorionic villus (CVS) and other tissue
samples can be grown directly on cover slips in small Petri
dishes, in “flaskettes,” or in slide chambers.

Preparation of Specimens for Culture

 Amniotic fluid specimens, whole blood samples, and bone
marrow samples arrive in the laboratory as single cells in a
fluid environment.
CULTURE MAINTENANCE
 After cultures have been initiated, they are allowed to grow
under specific conditions of temperature, humidity, and pH
until adequate numbers of dividing cells are present.

 Open systems are those that allow the free exchange of

gases between the atmosphere inside the culture vessel
and the surrounding environment of the incubator.

 Closed systems are those in which the culture vessels are

tightly capped to prevent exchange of gases.
Culture Maintenance and Growth Interval
 Once the culture requirements are met, the cells must be
allowed time to grow and divide. The time in culture varies
depending on the cell type involved.
 Peripheral blood cultures require little maintenance once
the growth requirements have been met.
 Bone marrow cultures need little attention once the
culture has been initiated.
 Amniotic fluid and solid tissue specimens require longer
culture periods and do not grow at predictable rates.
 Amniotic fluid and solid tissue specimens cultured with
either the in situ or flask method become depleted of
required nutrients and additives during the culture period.
CELL HARVEST
 Harvest is the procedure of collecting the dividing cells at
metaphase, their subsequent hypotonic treatment and
fixation, and the placement of the chromosomes on glass
slides so they can be stained and microscopically
examined.

Mitotic Inhibitor
 A mitotic inhibitor must be used to obtain adequate
numbers of cells in the metaphase.
Hypotonic Solution
 A hypotonic solution is added to the cells after exposure to
Colcemid.
Fixative
 A solution of three parts absolute methanol to one part
glacial acetic acid is used to stop the action of the
hypotonic solution and to fix the cells in the swollen state.
Slide Preparation
 Fixed cells from suspension cultures are dropped onto
glass slides to allow for subsequent staining and analysis.
CHROMOSOME STAINING AND BANDING
 The chromosomes were classified according to their
overall length, centromere position, and the ratio of the
short arm to long arm.
 Methods that produce specific alternating bands along the
length of the chromosomes create unique patterns for
each individual chromosome pair.

Techniques That Create Bands Along the Length of the

Chromosomes
 As chromosomes condense during mitosis, sub-bands
begin to merge into larger landmarks along the
chromosome.
 G-Banding (Giemsa Banding)
 Q-Banding (Quinacrine Banding)
 R-Banding (Reverse Banding)

Techniques that Stain Selective Chromosome Regions

 C-Banding (Constitutive Heterochromatin Banding)
 T-Banding (Telomere Banding)
 Cd Staining (Centromeric dot or Kinetochore Staining)
 G-11 Banding (Giemsa at pH 11)
 NOR Staining (Silver Staining for Nucleolar Organizer Regions)
 DAPI/DA Staining (4,6-Diamino-2-Phenole-Indole/Distamycin A)
 Fluorescence In Situ Hybridization (FISH)
HIGH-RESOLUTION STUDIES
 Chromosomes are routinely examined during metaphase,
when they are at their most contracted state.
Cell Synchronization Techniques
 Randomly dividing cells can be synchronized with
knowledge of the average timing of the stages of the
human cell cycle.
 One method involves the addition of FUdR (5-
fluorodeoxyuridine) to peripheral blood cultures prior to
harvest.
Chemical Elongation
 Ethidium bromide (EB) can be added to cultures prior to
harvest to achieve longer chromosomes.

CULTURE FAILURE
 All culture failures must be investigated. The
circumstances of the failure should be recorded as a part
of an on-going quality assurance program
PRESERVATION OF CELLS
 Cells do not survive indefinitely in tissue
culture.
 Cultured cells can be kept alive by
cryopreservation, the storage of cell in liquid
nitrogen.
CHROMOSOME ANALYSIS
 Selection of the correct specimen for chromosome
analysis and additional tests is not always straightforward,
and the submission of an inappropriate sample to the
laboratory can create frustration for both patient and
clinician.
Procedure
 After all of the appropriate laboratory manipulations and
staining procedures have been performed, there are
several steps involved in the clinical analysis of
chromosomes.
 Once the appropriate number of mitotic cells
has been examined and analyzed, a
representative sample must be selected for
imaging and ultimate preparation of
karyotypes.
The Fundamentals of Microscopy

 Microscopy is an essential technique for

clinical cytogenetic analysis and is an integral
part of the cytogenetics laboratory.

BRIGHTFIELD MICROSCOPY
 The Transmitted Light Source and Power
Supply

 Microscope Filters for Brightfield Microscopy

 Köhler Illumination
 The Phase-Contrast Condenser

Immersion Oil

Microscope Slides, Cover Slips, and Mounting Media

The Microscope Stage and Coordinate Location

Magnification and Objective Lenses

 Plan Objective Lenses
 Achromat Objective Lenses
 Apochromat Objective Lenses
 Fluorite Objective Lenses
 Tube Length
 Cover-Slip Correction
 Non-Cover-Slip-Corrected Lenses
 Numerical Aperture
 Oil Immersion Lenses
 High-Dry Objective Lenses
 Infinity Correction
 Objective-Correction Collar
 Optivar Lenses and Magnification Changers
 Eyepieces
Microscope eyepieces increase the magnification of
the microscope image and position the image so that it can
be seen by each eye.
 Beam Splitter
A beam splitter is present on microscopes capable of
photomicrography or electronic image capture.
FLUORESCENCE MICROSCOPY
 Epifluorescence Lamp Housing and Microscope
Attachment
 Filters
 Fluorescence Filters and the Fluorescence Filter
Housing
 Viewing Fluorochromes at the Microscope
Fluorescence Objective Lenses
 Fluorescence objective lenses are made of
fluorite or quartz, not glass.

 Immersion Oil
 Beam Splitters
 The Brightfield Condenser