MLS 007 (Human Cytogenetics)

BS MEDICAL TECHNOLOGY / SECOND YEAR

Session # 5-7
Ruchie Ann P. Baraquil
BSMLS-2C

08-17-2021

Activity 1 (Session 5)

A. BRIEF DISCUSSION. Give short, straight-to-the-point answers to the following questions:

1. What are the requirements in performing the following:

1.1. Cell culture initiation Controlled temperature, substrate for cell attachment, appropriate growth medium and incubator
that maintains correct pH and osmolality.
1.2. Cell culture maintenance

This fluorescent staining method, uses quinacrine to spot individual chromosomes and their structural
anomalies, given the resulting banding pattern. The characteristic banding pattern can be used to spot each
chromosome accurately

1.3. Cell harvest

Performed by separating the cell culture from the growing medium, several techniques are used to
perform this delicate operation.

2. What are the proper handling requirements for the following samples used in chromosomal
studies:

2.1. Peripheral Blood specimens

 Volume: 2 – 5 ml peripheral blood
 Container: Sodium Heparin blood collection tube; invert tube 4 – 8 times to prevent clots
 If Microarray is requested the preferred container is purple-top EDTA tube
 Do Not Use Lithium
 Invert tube 4 – 8 time to prevent clots
 Storage Conditions: Room temperature, 20 – 22°C (68 - 72°F) or refrigerated temperature, 2 - 8°C
(35.6 –
46.4°F)
Do Not Freeze

2.2. BM aspirates

 Volume: 2 – 5 ml of bone marrow; from the first or, at least, the second tap
 Container: Sodium Heparin blood collection tube
If Microarray is requested the preferred container is a purple top EDTA tube.
Do Not Use Lithium Heparin
Invert tube 4 – 8 time to prevent clots
 Storage Conditions: Room temperature, 20 - 22°C (68 - 72°F) or refrigerated temperature, 2-8°C (35.6 –
46.4°F).
Do Not Freeze Specimen

2.3. Amniotic fluid

 Volume: 10 – 20 ml; discard the first ml of fluid or use for other testing
 Container: Sterile centrifuge tube or container
  Storage Conditions: Room temperature, 20 - 22°C (68 - 72°F)
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2.4. Solid tissue specimens

 Volume: 3mm3 tissue biopsy
 Container: Sterile specimen cup containing sterile transport media
Sterile transport media provided by GAI upon request, sterile media such as RPMI, or
balanced salt solution may be used.
Do Not Use Formalin
 Storage Conditions: Room temperature, 20 - 22°C (68 - 72°F) or refrigerated temperature, 2 - 8°C
(35.6 – 46.4°F)
Do Not Freeze

Activity 2
(Session 6)

1. Compare and contrast the following techniques that create bands along the length of
chromosomes : 1.1. G-banding

G banding or Giemsa banding is a technique used in cytogenetics to produce a visible karyotype by staining
condensed chromosomes.

1.2. Q-banding

This is fluorescent staining method that uses quinacrine to spot individual chromosomes and their structural
anomalies, given the resulting banding pattern. The characteristic banding pattern can be used to identify each
chromosome accurately

1.3. R-banding

A cytogenetics technique that produces the reverse of the G-band stain on chromosomes. Resulting
chromosome patterns show darkly stained R bands, the complement to G-bands
_________________________________________________________________________________________________

2. Differentiate the following techniques that stain selective chromosome regions:

2.1. C-banding

Used for identifying heterochromatin by denaturing chromosomes in a saturated alkaline solution followed by
Giemsa staining.

2.2. T-banding

A modified reverse chromosomal banding technique wherein the telomeres are strongly stained as the faint
R-banding still occurs over the rest of the chromosome

2.3. Cd staining or Kinetochore staining

The type of chromosomal damage induced can be distinguished by using the kinetochore or pancentromeric
staining using molecular probes that label the centromeric regions of chromosomes allowing the
determination of aneugenic (chromosome loss) or clastogenic (chromosome breakage) agents.

2.4. G-11 banding

Is used to delineate these heterochromatin polymorphisms, it also has research applications. Used to
differentiate between human and rodent chromosomes in hybrid cells. The human chromosomes stain pale
blue, whereas the rodent chromosomes stain magenta.
 2.5. NOR Ag staining

Stains a specific protein associated with the activity of ribosomal cistrons during the preceding interphase by
counting the number of chromosomes with darkly stained nucleolus organizer regions (NORs), the number of
active NORs per metaphase can be determined.

2.6. DAPI/DA staining

Added to fix human chromosomes. The fluoresence of certain chromosome regions containing constitutive
heterochromatin is highlighted.

2.7. FISH
A laboratory technique used for detecting and locating a specific DNA sequence on a chromosome. The
technique relies on exposing chromosomes to a small DNA sequence called a probe that has a fluorescent
molecule attached to it.

Activity 3 (Session 7)

1. In high resolution chromosomal studies, cell synchronization &chemical elongation techniques are employed.
Give the principle behind each of the two techniques.

Cell synchrony is a vital process in the study of cells progressing through the cell cycle as it allows
population-wide data to be collected rather than relying solely on single-cell experiments. The types of
synchronization is broadly categorized into two groups; physical fractionization and chemical blockade.

Cell elongation requires lateral extension of the murein sacculus by intercalation of new glycan strands
and crosslinking of peptide subunits into the pre-existing shell.

2. What contributes to culture failure?

It will result to improper specimen collection or transport, improper laboratory technique, or the
condition of the sample

3. How is cell preservation intended for chromosomal analyses done?

Cultured cells can be kept alive by cryopreservation, the storage of cell in liquid nitrogen.

4. Outline the general procedure in doing chromosome analysis.

Chromosome analysis is usually done on a blood sample. Sometimes amniotic fluid (fluid from inside the
womb) or tissue (like skin) is tested. A laboratory (lab) will first grow the cells in special chemicals.