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Dr. Krishnaja A.P. 05-09-11

Peripheral Blood lymphocytes - Cytogenetic Methods

The reason for these notes is my daughter Sukanya. A second year BSc student pursuing Life Sciences at St Xaviers College, Mumbai, an Honours programme requires her to study Human Lymphocyte Cultures and Heavy Metal Toxicity for two course credits. Her teachers asked her to go through the methodology for the same. So back home she came, brimming over with questions about the same. Sukanyas questions took me back 30 years, when I set up my first lymphocyte culture at PRL in K.E.M Hospital. Id just completed my PhD and met Prof Kumata, looking for a job. I dont believe in certificates, he categorically told me. You prove yourself. Come and set up the lab which is now closed due to contamination. My predecessor, Ricky Kolluri, had left a six-line note on how to set up cultures. Prof Kumata gave me a free rein. I took up the challenge, set up the lab and enjoyed working there. Those were the most wonderful three months of my professional life until then. I taught myself banding and karyotyping and was carried into the magical world of human chromosomes. I never got bored of it. When I joined B.A.R.C, I had to again start from scratch, set up another lab. But I totally loved working there for the first five years. The excitement of looking at beautiful, wellspread metaphase chromosomes continued for many years. Even when things changed, I was able to survive cantankerous bosses, the prevalent sycophancy and the stagnant atmosphere only because of the world of chromosomes. I hope that you also experience the same sense of enjoyment from setting up your own lymphocyte cultures, and that when you do look at the slide through your microscope, you will be as excited as I was to explore this intriguing world. A word of warning: Once you are hooked, there is no easy way out. Enter at your own risk

Peripheral blood lymphocytes for chromosome studies in humans.

Classic cytogenetic analysis depends on cells undergoing mitosis to obtain metaphase chromosome spreads. Peripheral blood lymphocytes are the material of choice for chromosome studies in humans. The advantage is that blood can be collected easily from subjects by veni-puncture. The T-lymphocytes from peripheral blood are stimulated by phytohaemagglutinin (PHA) to divide and a large number of metaphases will be accumulated at 72h after setting up cultures. When cultures are appropriately processed around this time, good metaphase spreads can be obtained for analysis. 1. Blood samples can be easily and repeatedly obtained from exposed person, easily transported and stored, only a few milliliters of blood needed for an analysis. 2. Most of the lymphocytes circulating in blood are in the resting stage (G0 or G1 phase of cell cycle) asynchronous population of cells. 3. In cultures, PBLs can be easily stimulated to divide and are an excellent source of mitosis. 4. PBLs have a relatively long life span an estimated average half-life of approximately 3 years enables the unrepaired damage to be preserved until the cells are stimulated to divide in vitro. 5. Blood distribution and circulation throughout the body permits the detection of the effects of partial-body exposures in the circulating lymphocytes. Method for the preparation of (a) metaphase chromosome spreads and (b) micronuclei in cytokinesis blocked binucleate cells. at a glance. Basics of Tissue culture Four primary elements are involved in a tissue culture set up. 1. Work environment 2. Culture-ware 3. Culture media and media ingredients 4. Tissue to be cultured Successful culturing requires well-balanced and stable environmental conditions especially with regard to basic parameters like pH, temperature and maintaining sterility. Cells in culture have no defense mechanism. The better way is to strictly adhere to an aseptic working technique.

Setting up cultures Growth medium F 10 / RPMI 1640 / MEM / Medium 199 etc. L-glutamine serves as an essential nutrient (energy source). Being unstable, added just before use. Fetal bovine serum Pencillin and Streptomycin ( No antibiotics were used in our laboratory ) Phytohaemagglutinin SCE - BrdU Micronuclei - Cytochalasin B Harvesting of cultures Colcemid addition at 69-70h metaphase spreads Slide preparation One of the most critical steps in obtaining quality chromosome spreads. Standard staining and mounting (GTG) Whole blood culture method to obtain Metaphase chromosome spreads / Micronuclei Collection of Blood samples- Blood collection vials Anticoagulent- Sodium Heparin 1000 IU/ml Setting up the whole blood cultures 4ml Culture Medium (F 10, RPMI 1640 etc) 0.5 ml Fetal Bovine Serum 0.1 ml reconstituted PHA 0.3 ml whole blood Antibiotics not used 1. Inoculate the appropriate amount of blood (0.3ml) into a blood culture vial with culture medium, Fetal Bovine Serum (FBS), Phytohaemagglutinin (PHA). 2. Incubate the culture at 37oC for 48 or 72h as the case may be. 3. For SCE 10 g/ml BrdU (5-Bromodeoxyuridine) added at the initiation of culture. 4. For metaphase chromosome spreads, 2-3h prior to harvesting, add colcemid solution (final concentration 0.02g/ml). or G- banding by Trypsin using Giemsa.

5. For MN 6 g/ml cytochalasin B added at 24h after the initiation of culture. 6. Cultures terminated at 48 or 72h as the case may be. Harvesting of cultures chromosome preparations. 1. Transfer the culture to a centrifuge tube and spin at 500 RPM for 5 min. 2. Remove the supernatant, mix thoroughly, add 9 ml of prewarmed 0.075 M KCl, Incubate at 370C for 8 min. 3. Add, drop by drop, 1 ml fresh cold fixative made up of 3: 1 Methanol-Acetic acid 4. Spin at 500 RPM for 5 min. 5. Remove the supernatant, mix thoroughly and add 10 ml cold fresh fixative. 6. Repeat steps 7 and 8 with 7 ml fixative twice more. 7. Spin at 500 RPM for 5 min. 8. Resuspend the cell pellet in a small volume 0.5 to 1 ml of fresh fixative (depending on the size of cell button) to get an opaque solution. 9. Prepare slides by air dry method. Drop on to a clean wet chilled microscope slide and allow to dry at 600C Technical variations in culture conditions and media have been adopted to 1. Obtain a high frequency of cell divisions 2. Achieve better chromosome morphology 3. Stimulate the cells to divide or enhance the rate of proliferation of cells that are otherwise dormant in vivo. Critical steps are Hypotonic treatment Fixation Slide preparation by standard air drying technique Slides thoroughly dried. Slide preparation is one of the most important critical steps in obtaining quality metaphase chromosome spreads. It is a good practice to examine the first slide or slides before making a large number of final slides. If preparations are unsatisfactory, there are several variations, that may eliminate an existing problem or at least improve the quality of the preparations.

Ambient temperature and relative humidity play a significant role in chromosome spreading. Staining: The prepared slides were stained conventionally or G -banded (GTG) by Trypsin / Giemsa. Conventional staining. Staining procedures which provide a uniform unbanded appearance to chromosomes are referred to as solid or conventional staining. Homogeneously stained. 1% Giemsa stain in Phosphate buffer (pH 6.8). Place the slides in the Giemsa stain for 20 min. Rinse the slides thrice in dist.water. Air dry.

Solid or conventional staining

G- banding

Under 20X this is how the metaphases appear.

A little more magnified - 40X. Metaphases in different stages of condensation

Solid or conventional staining

G- bands produced by Trypsin using Giemsa (GTG) Chromosomes stained by this protocol exhibit light and dark stained regions. (light and dark bands) along the length of the chromosomes. The G-band pattern produced by trypsin is grossly similar to that produced by quinacrine. Q-banding In general, slides for G-banding should be aged 3-5 days at room temperature or overnight (16-18h) at 55-600C for optimal results.

1. Phosphate buffered saline calcium and magnesium free. PBS-CMF. pH 7.0) NaCl 8g, KCl 0.2g, Na2HPO4 0.92g, KH2PO4, 0.2g, dist. water 1000 ml 2. Trypsin solution. 50 mg in 20 ml PBS 3. Normal saline solution (0.9%) 4. Working staining solution. 43 ml dist. water, 5.0 ml methanol, 2ml Giemsa stain Trypsin, prepare fresh and use for 3 to 4h. Discard the solution as soon as turbidity develops. The staining solution is made up fresh and used for 2 to 3 hours. 1. Incubate the slides for 10 min. in phosphate buffer (pH 6.8) at 600C 2. Treat the slides in trypsin solution.(20-40 sec, carried out in petri dishes)Trypsin time increases with increasing age of slides. The optimum time for trypsin treatment for each batch of slides has to be worked out. 3. Rinse the slides briefly in cold normal saline (2-50C kept in the refrigerator) 4. Stain the slides in Giemsa staining solution 10 min. The slides can be examined, without mounting, using dry objectives and later on mounted for use with oil immersion objectives. Trypsin and Giemsa staining solution should be changed periodically during the day to maintain optimum banding and staining conditions respectively. A Coplin jar filled with normal saline is kept in the refrigerator and is taken out briefly to rinse the slides after trypsin digestion. All other solutions are kept at room temperature. The critical steps again are: 1. Hypotonic treatment 2. Fixation 3. Slide preparation by standard air drying technique 4. Slides thoroughly dried (55-600C overnight) Results of Giemsa-banding techniques can be quite variable. If the chromosomes appear fuzzy, insufficient aging of slides is the most likely explanation. Over-trypsinized chromosomes appear puffy and pale with segments sometimes missing while undertrypsinized chromosomes are dark with poor distinction between light and dark bands.

Chromosome analysis - Applications: Clinical uses Determination of the constitutional karyotype of patients requiring genetic diagnosis or genetic counseling. Assessment of chromosome damage in individuals exposed to environmental hazards. Induced damage radiation, chemicals. Acquired abnormalities haematologic malignancies Metaphase FISH studies, gene mapping, microdissection and PCR, CGH, lymhoblast cell lines etc. Chromosome analysis: Karyotype is an orderly arrangement of chromosomes according to international conventions. With continued practice, microscope karyotyping becomes a useful skill. Banding - Uses the additional information provided by the banding pattern to identify each chromosome by number. Band level should be adequate for the diagnosis. At least 20 banded metaphses should be counted and examined. Mosaicism - 100 metaphases to be analysed. The International System for Human Cytogenetic Nomenclature (ISCN) to be followed for writing descriptive karyotypes. Conventionally-stained metaphase spreads were examined for unstable chromosome aberrations such as dicentrics and multiple forms, centric rings each with fragments Excess acentrics (interstitial and terminal deletions) consisting of fragments, minutes and acentric rings. Each metaphase had to contain 46 centromeres. Acentrics were counted as excess if they were not accounted for by a dicentric chromosome or centric ring. The dicentrics and rings seen without accompanying fragments were scored separately. Any other chromosome rearrangements such as (pericentric inversions, translocations, marker chromosomes) and chromatid breaks, gaps, chromatid exchanges, are also scored

In situ hybridization. Enzymatic detection with HRP/Anti-Digoxigenin and DAB

Painted chromosomes. Chromosome X

Painted chromosomes. Chromosome 16

45, XY,-13,-14,+t(13;14)

G- Banded Karyotype. 46,XX

Dicentric chromosomes

Ring chromosomes

Chromatid exchanges

Sister Chromatid Exchanges (SCE) assay in Human Lymphocytes.

Biomonitoring of human population. Baseline frequencies of SCE in human

population. In vitro- genotoxicity - Chemical exposures. SCE as a reflection of cellular DNA repair - agents that provoke DNA repair also induce SCE. In general, SCE reflect the amount of damage, which has occurred through the total period of DNA replication. SCE is recognized as a reciprocal switch of a newly replicated chromatid with its sister chromatid at apparently homologous loci, possibly originating from a double strand recombination of chromosomal DNA constrained by the polarity of the duplex.

Incorporation of 5-bromodeoxyuridine(BrdU) as a thymidine substitute during the last two successive cell cycles results in sister chromatid differentiation (SCD). After exposure to UV light, the sister chromatid with BrdU integrated into both DNA appears faintly stained while the other chromatid containing only one substituted DNA strand is dark when stained with Giemsa. Exchanges between sister chromatids result in patterned harlequin chromosomes where the label of one chromatid switches to the sister chromatid.

A modification of the FPG (Fluorescent plus Giemsa) method for SCEs staining. A modification of the FPG method (Perry and S. Wolff 1974, Nature, 251, 156-158. Morimoto et al. 1982, Mutation Research, 102, 183-192). Krishnaja A. P. and N. K. Sharma 1998, Environmental Mutagenesis and Carcinogenesis. Chauhan P. S. and P. M. Gopinath (Eds) Quest Publications, pp 77-90.

Procedure: Chromosome preparations aged two days were stained in Hoechst 33258 (100ug/ml in distilled water) for 20 min. Rinsed in tap water. Mounted in Sorensens buffer (M/15, pH 8.0 adjusted with 5% NaOH) under a coverslip and exposed to 360nm light from a Black ray lamp (distance 2cm, 20 J/m2/sec) for 12 min on a slide warmer at 60oC. (Sorensens buffer M/15(pH 8.0 adjusted with 5% NaOH) Na2HPO4 1.188g, KH2PO4 0.907g/dist.water 100ml. 5% NaOH solution for pH 8 adjustment. Rinsed in ice-cold Sorensens buffer pH 6.8. (Sorensens buffer pH 6.8(Na2HPO41.775g, KH2PO4 1.70g/dist.water 1000ml). Slides were again rinsed in tap water. Stained in 4% Giemsa in Sorensens buffer pH 6.8 for about 8min. SCE analysis: 1. 50 second division metaphases are analysed for SCE. 2. Every point of breakage and rejoining that is exchange is always counted as one exchange - the piece of chromatid exchange. 3. Intercalary exchanged piece including even a dot or minute is counted as two exchanges. 4..Any terminal exchange is counted as one exchange. 5. SCE at centromere is counted only if an obvious twisting at this point is ruled out. Lymphocyte proliferation kinetics: BrdU labelling of chromosomal DNA facilitated the analysis of lymphocyte proliferation kinetics. Harlequin staining of BrdU-substituted chromatids by the FPG staining allowed identification of chromosomes in the first/second/third and subsequent divisions. 1. First-division cells46 chromosomes with both sister chromatids stained uniformly darkly. 2. Second-division cells46 harlequin chromosomes with one chromatid darkly stained and its sister chromatid lightly stained.

3. Third-division cellssome harlequin chromosomes (1530 chromosomes) and other chromosomes with both sister chromatids stained uniformly lightly. 4. Fourth-division cellssome chromosomes show the light and dark pattern (713 chromosomes), and the rest with both sister chromatids stained uniformly lightly. The Proliferation rate index (PRI) calculated as M1+ 2M2 +3M3+ 4M4 / N. Where M1, M2, M3 and M4 represent I - IV metaphases and N is the total number of cells scored. SCE are indicators of genotoxic activity. SCE analysis has been used successfully for monitoring human exposure to potentially genotoxic agents. DNA damaging agents that are capable of inducing chromosome lesions at all stages of the cell cycle and that do not require an S phase for expression are relatively ineffective in inducing SCE. Agents that require a DNA synthesis period for inducing chromatid type damage are potent inducers of SCE. Micronuclei- Cytochalasin B method. Micronuclei originate from chromosome breakage and loss. Nucleoplasmic bridges are a marker of chromosome rearrangements. Micronucleus assay is a quick and reliable technique to assess genetic damage. 1. 6g/ml cytochalasin B addition at 24h after initiation o f cultures 2. Cultures terminated at 72h 3. Transfer the culture to a centrifuge tube and spin at 500 RPM for 5 min. 4. Remove the supernatant, mix thoroughly, add 9 ml of cold 0.8% KCl min. 5. Spin at 500 RPM for 5 min. 6. Remove the supernatant, mix thoroughly and add 10 ml cold fresh fixative. Keep for 10 min. 7. Repeat step 5, add fixative containing 1% formaldehyde, that enhances the preservation of cytoplasm. 8. Repeat step 5, add fresh fixative 3:1 Methanol-Acetic acid only. 9. Repeat step 5, resuspend the cell pellet in a small volume of fresh fixative for 5

10. Cells were gently dropped on wet chilled slides and allowed to dry at room temperature. 11. Cells were stained with Giemsa (Merck) 1 % in Sorensens buffer pH 6.8 for 20 min.

MN frequencies are determined in one thousand binucleated cells with well preserved cytoplasm. Nucleoplasmic bridges are also scored. Scoring criteria for BNC. 1. The two nuclei in the BNC should be of approximately equal size, staining pattern and staining intensity. 2. The two nuclei in a BNC should have intact nuclear membranes and be situated within the same cytoplasmic boundary. 3. The two nuclei within a BNC may be attached by a fine nucleoplasmic bridge which is no wider than one-third of the largest nuclear diameter. 4. The two main nuclei in a BNC may touch or overlap each other especially in preparations in which the cytoplasm has been preserved. A cell with two overlapping nuclei can be scored only if the nuclear boundaries of each nucleus are distinguishable. 5. The cytoplasmic boundary or membrane of a BNC should be intact and clearly distinguishable from the cytoplasmic boundary of adjacent cells. Scoring criteria for MN 1. The diameter of MN in human lymphocytes may vary between 1/16 and 1/3 of the diameter of the main nuclei. 2. MN are round or oval in shape.

3. MN are non-refractile and can be readily distinguished from artifacts such as staining particles. 4. MN usually have the same staining intensity as the main nuclei but occasionally staining may be more intense. 5. MN are not linked to the main nuclei but they may overlap the main nuclei. Nuclear division index (NDI) determined by scoring the number of mono, bi, tri ,tetra and more (polynucleate) cells in one thousand cells. Nuclear division index was calculated as NDI = (M1 + 2xM2 + 3xM3 + 4xM4) / N, where M1 - M4 represent the number of cells with 1 to 4 nuclei respectively and N is the total number of cells scored. In the routine laboratory environment chromosome and MN analysis are always concerned with light microscope studies. The aim of routine chromosome analysis is to extract information from the karyotype, conventional, G-banded, high resolution banded or FISH as to whether a chromosome aberration is present or not. How well this goal is achieved depends on the quality of the metaphases analysed and on the subjective judgement of the cytogeneticist. The former definitely influences the latter. Traditional Cytogenetics is labour intensive, requires a highly trained analyst. Banding analysis further requires a trained skilled analyst. A high degree of analytical skill at the microscope is necessary for clear cut interpretation to uncover translocations and other complicated chromosome aberrations. Scoring is an extremely important element in cytogenetic biomonitoring. Accurate dicentric and MN scoring may depend on the quality of the preparations and the skilled technical personnels subjective assessment of the relative quality ie scorability of the spreads. The highest yield of aberrations in culture is likely to be seen in first division metaphases, 48-52 h cultures. It is important to score cells after first division in the case of CA or MN and after second division in case of SCES in order to obtain the best estimate of damage induced in vitro. Asymmetrical chromosome type exchanges (dicentrics and rings) may arise either directly or from chromatid exchanges following replication in a subsequent cell cycle. Ideally, only first in vitro mitoses should be analysed for chromosomal aberrations because aberrations are lost or appear in altered form in subsequent cell divisions.

Human peripheral blood lymphocytes present a readily available source of cells to evaluate chromosome damage. However this requires careful attention to protocol design and good knowledge of the material, its reactions to different types of treatment, the spontaneous background frequencies of the endpoints studied, their variances as well as the factors that influence them. Cytogenetic biomonitoring studies are tedious and difficult in many respects, so careful planning and adherence to stringent laboratory protocols are very important. Rigorous study design is essential in all cytogenetic biomonitoring methods, since many inter individual factors not related to the specific exposures of study can affect the cytogenetic parameters of interest. Probably no two laboratories adopt precisely the same procedure. Nevertheless, all laboratories should follow simple standard protocols that work well in their set up. There is still a mysterious aspect to the procedure, and it is impossible to verbalize all of the necessary skills. One must simply practice until the intuitive part of the skill is mastered and there will always be some technologists with a better feel for it. The methodology may appear simple, but preparing good quality metaphase spreads is technically challenging. Careful attention to a number of variables certainly increases the chance of satisfactory results. Still, this aspect of cytogenetic methodology is something of an art. In that sense, preparation of good quality metaphase spreads and chromosome banding can be considered an art as well as a science.