LABORATORY METHODS:
CHROMOSOME ABERRATION ANALYSISFOR BIOLOGICAL DOSE ASSESSMENTRadiation Biology Center, Kyoto University,Yoshida-konoecho, Sakyo-ku, Kyoto 606-8501, Japan

Chromosome Aberration Analysis in Peripheral Blood Lymphocytes
OutlineBlood Sampling and the Establishment of CultureTransportation of the Remote Samples“Cold” or “Chilled” Stimulation Method

Leibovitz’s L-15 medium is essential.
(Amino acid-buffered medium)Mitotic Progression of T-lymphocytes and Culture Time

Cell Harvest and Fixation

Chromosome Preparation and StainingChromosome Preparation for Bone Marrow Cellsand Leukemia Cells in Peripheral Blood
Usually, bone marrow cells and leukemia cells in circulating blood are progressing
mitosis. Chromosome preparations may be made directly with these mitotic cells.

Procedure(1) Bone marrow samples: Mix about 0.5ml BM aspirate with 5ml complete
medium, and incubate 2~4 hrs at 37oC. Hypotonic treatment, fixation, andslide preparation my depend on chromosome analysis method concerened.
(2) Usually leukemic cells are dividing in thecirculating blood. Mix the cells in leukocyterich buffy layer with complete medium, andproceed similarly to the method for BM cells.
* The cells in mitosis are scarce due to time
lapse after sampling, the cells can be cultured
in culture medium (without colcemid) andharvested as is the case for culture cells.

CML cells in bone marrow:
Normal Giemsa staining (upper)
and G-banding (lower)

Cell Culture from Tiny Skin Sample
Procedure

Minute skin sampling and cell culture. Sampling is painless and does
not require anesthesia.
(1) Disinfect all apparatus including skin of concern with alcohol. Pick up
the skin with sharp hooked forceps, and cut 1-2mm part beneath theforceps with surgical knife or razer blade. (Dissection does not resultin bleeding).
(2) Sample (1~2mm) should be put into culture medium, or placed on
gauze wet with physiological saline. (Prevent drying)(3) Prepare 5cm Petri dish with 5ml culture medium.
(4) Pick up the skin fragment on a tip of a fine needle. And, thus float theskin fragment on the surface of culture medium with the skin surface
side up. If not successful, try again until floating with the skin surface
side up and dermis side down.
(5) Withdraw culture medium leaving approximately 1ml, which is smallenough not to allow moving but provides thin layer to provide nutrientto the dermis side. (Leave in CO2 incubator).
(6) After 2-3 day incubation, keratinocytes will first migrate out. At thisstage, the culture dishes can be fed with fresh medium.

Fibroblast outgrowth(7) Approximate 1 w after, outgrowth of fibroblasts is seen. The culture isthen available for chromosome analysis or or further propagation bycell transfer.

Chromosome Preparation for Meiotic Cells (Testis)

Testis samples are usually very small (needle biopsy). An, moreover, thecells are easily lost during preparation due to their sticky nature to theapparatuses. To avoid the cell loss, handling apparatuses should be coatdwith serum before handling (Petri dishes, scissors, pipette, centrifuge tubes.

Procedure(1) Mince the biopsy sample (usually 1-2mm) with fine-point scissors
together with serum.
* In the case of mouse testis sample, the cells may be released byneedles under dissection microscope. But, it can not be done forhuman testis samples.
(2) Add 1~2ml 75mM KCl, and gently pipette in a centrifuge tube. (tubeand pipette should be coated with serum in advance).
(3) Leave for 20 mins at room temperature.
(4) Overlay freshly prepared Carnoy fixative (3ml), gently mix, and leave
for 10 min.
(5) Change the fixative by centrifugal sedimentation, and leave for at least
30 min before making slide preparation.

Human male meiotic metaphase I

Technical Modifications for Aberration Analysis(1) G-band Analysis

Technical Modifications for Aberration Analysis(2) C-band Analysis
C-banding is a simple Giemsa staining method for identification of pericentromeric heterochromaticregions of chromosomes. The method is not routinely used for the chromosome aberration analysis in
human radiation cytogenetics. However, the method is highly efficient in identifying centromere position,
namely identification of dicentric aberrations in some mammalian cells (e.g., mouse cells). In human cells,
the identification of the centromeric regions are currently possible by the use of FISH method applied tolabelling with DNA or peptide probes specific to the centromeric regions.

ProcedureC-banding
1. Use fresh slide
2. 1.0 N HCl 5 min
3. 0.2 N HCl Briefly immerse and air dry
4. 5% Ba(OH)2 6 min
5. 0.2 N HCl Immerse
6. 2×SSC 60oC, 30 min
7. Rinse in phosphate buffer (pH 6.8)
8. Stain in Giemsa (6%, pH 6.8), 30 min

Human (unirradiated)

Mouse (irradiated)Technical Modifications for Aberration Analysis(3) Q-band (R-band) Analysis

Technical Modifications for Aberration Analysis(4) Analysis of Replication Pattern
In the G-banding, G-positive (dark) regions are gene poor regions and replicate in the later stages of S-phase.
Conversely, G-negative (light) regions are gene rich and replicate in the earlier stages of S-phase. When BrdU isincorporated into DNA only in the late stages of S-phase, the chromosomes are differentially marked (stained)
after appropriate treatment. The banging pattern is called replication banding, but is reverse of the G-band. The
followings are culture procedures using peripheral blood lymphocytes.

Procedure

TdR

BrdU

-24h-8h

Colcemid Harvest-2h0h(1) Establish PB lymphocyte culture to be harvested at 72nd hrs.
(2) Add thymidine (TdR, 300μg/ml) at 24hs before harvest.
(3) At -8h, wash the cells with TdR-free medium, and continue
culture in medium with bromodeoxyuridine (BrdU, 25μg/ml).
(4) Add colcemid (0.05μg/ml) 2h before harvest.
(5) Harvest (0h) and make chromsome preparation by air-drying
method.
(6) Expose the preparations to black light and treat with 2x SSC
at 62oC.
(7) Stain either with Giemsa, acridine orange, or propidiumiodide.
If the TdR and BrdU treatments are reverted, inverse images maybe obtained.

Technical Modifications for Aberration Analysis(5) Chromosome Painting (FISH) Analysis

Technical Modifications for Aberration Analysis(6) Sister Chromatid Differentiation for SCE Analysis

Number Cells to be ScoredScoring of Chromosome Aberration in Score SheetScoring of Chromosome Aberration by Binary Gigits

International System for Human Cytogenetic Nomenclature
Historical perspectives(Discovery of normal human chromosomes, Tjio JH and Levan A: Hereditas, 42:1-16, 1956.)
1. Denver Report: A proposed standard system of nomenclature of human mitotic chromosomes. Ann Hum Genet, 24:319-322. 1960.
2. London Conference: London Conference on the Normal Human Karyotype. Cytogenet Cell Genet, 2:264-268, 1963.
3. Chicago Conference: Standardization in Human Cytogenetics. Birth Defects Original Article Series II(2), National Foundation,New York, 1966.
4. Paris Conference (1971): Standardization in Human Cytogenetics. Cytogenet Cell Genet, 11:313-362, 1972.
(G-, Q-, R- and C-banding)
5. Paris Conference (1971), Supplement (1975): Standardization in Human Cytogenetics. Cytogenet Cell Genet, 5:17-21, 1975.
(Banding homology among human, chimpanzee, gorilla, and orangutan)
6. ISCN (1978): An International System or Human Cytogenetic Nomenclature (1978). Cytogenet Cell Genet, 21:309-404, 1978.
(Nomenclature for acquired aberrations)
7. ISCN (1981): An International System or Human Cytogenetic Nomenclature. High-resolution banding (1981). (1978). Cytogenet
Cell Genet, 31:1-23, 1981.
(High resolution banding: 400-, 550- and 850-band level)
8. ISCN (1985): An International System or Human Cytogenetic Nomenclature (1985). Birth Defects Original Article Series XXI(1).
National Foundation, New York, 1985.
(Nomenclature for human meiotic chromosomes)
9. ISCN (1991): Guidelines for Cancer Cytogenetics. Supplement to An International System or Human Cytogenetic Nomenclature.
F. Mitelman (ed). S. Karger, Basel, 1991.
(Proposed nomenclature for chromosomes in cancer)
10. ISCN (1995): An International System or Human Cytogenetic Nomenclature. F. Mitelma (ed). S. Karger, Basel, 1995.
11. ISCN (2005): An International System or Human Cytogenetic Nomenclature (2005). LG Shaffer and N Tommerup (eds).
Karger, Basel, 2005.
(……): Newly highlighted issues

END

Compiled by M. S. Sasaki, D. Sc.
Professor Emeritus, Kyoto University