chromosome analysis protocols

&APTER 1 Mitotic Metaphase Chromosome Preparation from Peripheral Blood for High Resolution George Spowart 1. Introduction The advent of chromosome banding techniques some 20 years ago (1,2) allowed the unequivocal identification of every chromosome in the human metaphase and provided a mapping scheme along each chro- mosome. Subsequently, a great deal of research has centered on prepar- ing longer chromosomes with more bands visible. Chromosomes condense as they move through mitosis, and adjacent bands close up and appear to fuse. The earlier stages are longer with more bands recognized. It is not always possible to define the mitotic stage of a particular cell. International standards have been agreed for various numbers of bands in the haploid set. Thus we have 400-, 550-, and 850-band sets (3). Other workers report the use of even longer chromosomes (4,s). High-resolu- tion banding has undoubted advantages in many fields. As well as allow- ing greater accuracy in traditional karyotype analysis, there are many reports of microdeletions and other abnormalities detected only on extended chromosomes (6). Likewise, in situ hybridization and gene localization techniques are taking advantage of the improved resolution. The culture technique to prepare human chromosomes still follows the basic scheme laid down by Hungerford (7). Lymphocytes from peripheral blood are stimulated to divide in culture; cells are arrested in mitosis, swollen with hypotonic solution, fixed in an acid-alcohol fix, and spread on microscope slides by air-drying. From Methods m Molecular B/ology, Vol. 29 Chromosome Analysis Protocols Edlted by J R. Gosden Copynght 01994 Humana Press Inc , Totowa, NJ 2 Spowart Published methods on the preparation of elongated chromosomes are abundant. As with banding techniques, different laboratories have preferences for particular methods and have developed their own varia- tions. None of the methods is guaranteed to work with every speci- men, being the nature of biological material. There is no doubt that it is very difficult for one laboratory to reproduce exactly all the condi- tions in another, and it is likely that published methods need some experimentation to optimize them for local conditions. Methods of preparation fall into three general categories. An individual protocol may use one or more of these approaches. 1.1. Induction of Synchrony Stimulated peripheral blood lymphocytes in culture grow and divide asynchronously, and pass through the prometaphase and early metaphase stages relatively rapidly. Cells are blocked around S-phase (8) and, on release, will continue through mitosis in a wave of divisions, thus enhancing the potential yield of early stages. Methotrexate (9) and excess thymidine (10) are the most successful blocking agents. The timing of the interval between release of the block and harvest is the critical stage in the procedure and depends, in a complex way, on the various cul- ture conditions, This is the main reason that many workers have found it difficult to duplicate successfully published methods or to maintain a high level of success with a particular scheme. To release the cells from the block, the blocking agent is removed, or at least overcome, and cells are encouraged to enter mitosis. The choice of release agent may be determined by the banding technique to be used subsequently. 1.2. Use of Chemicals to Affect the Condensation of the Chromosomes Chromosomes progressively condense as the cell moves through mitosis. A number of chemicals have been found to counter this (12-14). Care must be taken to balance the reduction in contraction against lowering of mitotic index and/or induction of chromosome aberrations. 1.3. Alteration of Arrest and/or Hypotonic Treatments afZer Harvest (15-17) Colcemid is used in most chromosome preparation techniques to destroy spindle formation and arrest cells in metaphase. Wiley et al. (17) questioned the necessity for colcemid treatment, but most workers Mitotic Metaphase Chromosome Preparation 3 continue to use it in a variety of concentrations and exposure times. Hypotonic treatment with 0.075M KC1 still features in the majority of protocols, but many other formulations have been advocated, some with the specified aim of elongating chromosomes. There arevarious reasons why a particular method will be preferred for a line of research. However, in general, where time and material permit, it will be good practice to run tandem methods on each specimen. I will detail two protocols that have given good results. 2. Materials Z.l.Methodl 2.1.1. Supplemented Culture Medium This will usually be prepared in bulk and aliquoted to culture ves- sels just before cultures are set up. The number of specimens to be pro- cessed will determine batch size. A week’s supply is typical. 1. RPM1 1640 (Glbco, Galthersburg, MD), 340 mL. 2. Fetal bovine serum, 60 mL. 3. Phytohemagglutnun (HA15 Wellcome, Dartford, UK), 4 mL. 4. Penicillin and streptomycin (0.1 g and 100,000 U/mL) (Glaxo) mixed solution, 0.4 mL. Store at 4°C. 2.1.2. Blocking Agent Methotrexate injection (Lederle #4587-24) is obtained as 25 mg/mL (5 x lo-%) solution. A working solution ( lc5m is prepared by diluting 20 pL with 9.980 mL sterile distilled water. This can be stored at 4°C for several weeks. Methotrexate is a cytotoxic drug, and due care must be taken in handling. 2.1.3. Release Agents 1. Thymidine (Srgma [St. Louis, MO] #T-9250) (10m3jV) is prepared by dissolving 2.42 mg/mL rn sterile distilled water. Store at 4°C. 2. The alternative agent is 5bromo-2’-deoxyundine Bdu (Sigma #B-5002): Working solution (10e2M) 1s prepared by dissolving 3 mg/rnL in distilled water. Aliquots can be stored frozen for several months. Store vial in use at 4°C. Care must be taken in handling this teratogen and mutagen. 2.1.4. Arresting Agent Colcemid, 10 pg/rnL (Gibco). Store at 4°C. 4 Spowart 2.1.5. Hypotonic Solution KC1 (0.075M) (1.4 g in 250 mL deionized water): Make up fresh for each harvest, and heat to 37°C. 2.1.6. Fix Acetone-free methanol and glacial acetic acid are freshly mixed in the proportion 3: 1. 2.2. Method 2 2.2.1. Supplemented Culture Medium This will usually be prepared in bulk and aliquoted to culture ves- sels just before cultures are set up. The number of specimens to be processed will determine batch size. A week’s supply is typical. 1. RPM1 1640 (Gibco), 340 rnL. 2. Fetal bovine serum, 60 mL. 3. Phytohemagglutmin (HA15 Wellcome), 4 mL. 4. Penicrllm and streptomycin (0.1 g and 100,000 U/mL) (Glaxo) mixed solution, 0.4 mL Store at 4°C. 2.2.2. Inducing Agent Actinomycin D (Sigma #A- 1410) stock solution is prepared by dissolving 10 mg in 1 mL dimethylsulfoxide. Small aliquots are stored at -20°C. Working solution is 50 @n-J, made by diluting thawed stock 200-fold in distilled water. This can be stored at 4°C for up to 2 wk. Actinomycin is poisonous, a known carcinogen and teratogen, and due care must be taken to avoid all contact. 2.2.3. Arresting Agent Colcemid (Gibco) 10 pg/mL. Store at 4°C. 2.2.4. Hypotonic Solution KC1 (0.075M) 1.4 g in 250 mL deionized water: Make up fresh for each harvest, and heat to 37°C. 2.2.5. Fix Acetone-free methanol and glacial acetic acid are freshly mixed in the proportion 3: 1. Mitotic Metaphase Chromosome Preparation 5 3. Methods Aseptic laboratory procedures must be observed to avoid micro- bial contamination during the culture stages. All centrifugations are carried out in centrifuge with swing-out buckets in the rotor. 3.1. Method 1 1. Dispense 9.5 mL of supplemented RPM1 1640 medium (see Notes l-4) mto a sterrle culture vessel (see Note 5), and inoculate with 0.75 mL of whole blood (see Note 6). 2. Incubate at 37°C for 72 h. 3. Inject the culture with 100 pL methotrexate solution (see Note 7), and remcubate at 37°C. 4. After 17 h, carefully remove the supernatant above the cell layer, pref- erably with a tube from a suction pump followmg the meniscus. Pipeting can be used but 1s more laborious. 5. Flick the flask with the finger to distribute the cells, and resuspend in 9.5 mL supplemented medium. 6. Add 0.2 mL thymidine (see Note 8). Reincubate at 37°C for 3.75 h, or add 0.2 mL 5-bromo-2’-deoxyuridine (see Note 8). Reincubate at 37°C for 4.25 h. 7. Maintain the culture at 37’C while adding 60 pL colcemrd (see Note 9), and incubate for a further 10 min. 8. Gently shake the flask, and transfer the culture to a conical-based centrifuge tube. 9. Centrifuge at 15Og for 10 min. 10. Suck off the supernatant to around 3 mm above the cell pellet. 11. Flick the tube to distribute the cells and pipet m 10 mL of prewarmed KC1 (see Note 10). 12. Incubate at 37°C for 10 min. 13. Centrifuge at 15Og for 10 min. 14. Three layers should be visible, a red cell pellet at the bottom, a slightly opaque layer of white cells, and the supernatant. Suck off the superna- tant to within 3 mm of the white cell layer. 15. Flick the tube to loosen the cells. Use a vortex mrxer to stir the cells more thoroughly while carefully addmg a pipetful of frx dropwise to the middle of the vortex (see Note 11). Add two more pipetfuls of fix, and allow to stand for at least 30 mm. 16. Centrifuge at 15Og for 10 min, and remove most of the supernatant. Flick tube to resuspend cells, and add 2 pipetfuls of fix. 6 Spowart 17. Centrifuge at 150g for 10 min, and remove most of the supernatant. Flick tube to resuspend cells, and add 1 pipetful of fix. 18. Centrifuge at 150g for 10 mm, and remove supernatant to lust above cell pellet. Flick tube to resuspend cells, and add fix to give about 0.5 mL suspension. 19. Mix the suspension gently with a pipet and place a drop on a clean polished microscope slide (see Note 12). Allow to air-dry and examme under microscope to check cell density, spreading of chromosomes, and so forth. If cells are too densely packed, add more fix. If too sparse, spin down and reduce volume. Different methods of spreading may have to be adopted. 3.2. Method 2 1. Dispense 9.5 mL of supplemented RPM1 1640 medium (see Notes l-4) into a sterile culture vessel (see Note 5), and maculate with 0.75 mL of whole blood (see Note 6) 2. Incubate at 37OC for 68 h. 3. Inlect the culture with 100 pL actinomycm D solution (see Note 13), followed by 60 FL colcemid solution (see Note 9), and reincubate at 37°C for 4 h. 4. Transfer to 15 mL conical-based centrifuge tube, and spm at 150g for 10 min. 5. Suck off the supernatant to around 3 mm above the cell pellet. 6. Flick the tube to distribute the cells, and pipet in 10 mL of prewarmed KC1 (see Note 10). 7. Incubate at 37°C for 10 min. 8. Centrifuge at 150g for 10 min. 9. Three layers should be visible, a red cell pellet at the bottom, a slightly opaque layer of white cells, and the supernatant. Suck off the superna- tant to within 3 mm of the white cell layer. 10. Flick the tube to loosen the cells. Use a vortex mixer to stir the cells more thoroughly while carefully adding a pipetful of fix dropwise (see Note 11). Add two more pipetfuls of fix and allow to stand for at least 30 min. 11. Centrifuge at 150g for 10 mm and remove most of the supernatant. Flick tube to resuspend cells, and add 2 pipetfuls of fix. 12. Centrifuge at 150g for 10 mm and remove most of the supernatant. Flick tube to resuspend cells, and add 1 pipetful of fix. 13. Centrifuge at 150g for 10 min and remove supernatant to Just above the cell pellet. Flick tube to resuspend cells, and add fix to give about 0.5 mL suspension. Mitotic Metaphase Chromosome Preparation 7 14. Mix the suspension gently with a pipet and place a drop on a clean polished microscope slide (see Note 12). Allow to air-dry, and examine under microscope to check cell density, spreading of chromosomes, and so on. If cells are too dense, add more fix. If too sparse, spin down and reduce volume. Different methods of spreading may have to be adopted. 4. Notes 1, Good results have been obtained with a number of culture media, includ- ing RPM1 1640, RPM1 1603, TC 199, and McCoy’s 5A. I have chosen the readily available RPM1 1640 (Gibco). 2. Published methods suggest a wide range of supplements. Most con- sider bovine serum the appropriate source of necessary growth factors, but pooled human serum and even artificial supplements have their advo- cates. There is strong evidence that the serum chosen affects the timing of the mitotic cycle, and since rt is difficult to maintain a source of unchanging material, it must be anticipated that regular checks will have to be kept to optimize timings, especially of the interval between release and harvest 15% fetal bovine serum is chosen here. 3. Antibiotics are normally added to avoid microbial mfection. Pemcillm and streptomycm are the usual choice. The relatively short culture time means that mycoplasma infection is not a problem. 4. Phytohemagglutmm is unrivaled as mitogen to stimulate lymphocytes to divide. Lyophiltzed HA15 (Wellcome) is reconstitued with distilled water and added to the culture medium in the proportion 1: 100. With a few hematological conditions, it may be necessary to use pokeweed mitogen as well. 5. Heparinized peripheral venous blood is the most readily available and convenient material to produce chromosome preparations. Some work- ers prefer to enrich the proportion of leukocytes m the inoculum by centrifugation and taking plasma and white cell layers along with some of the red cell layer. I do not do this routinely, but if the medical history of the donor suggests high red cell or low white cell count, it could be advantageous. Specimens from patients on drug therapy or that have taken several days to reach the laboratory often benefit from havmg plasma replaced by pooled human serum, Blood from neonates nor- mally contams a very high number of leukocytes, and a smaller mocu- lum will suffice. 6. Several types of culture vessel give good results, and choice may be determined by budget and availability. There is a complex relationship among various dimenstons of culture vessel in whole blood culture. In general, glass or plastic can be used, although it is safer to use plasticware 8 Spowart that is designed for tissue culture. The ratio of cell volume of culture to area of base of the vessel is important, and so is the volume of the gas phase above the culture. Plastic or glass Universal contamers with a base area 4-5 sq cm are excellent for the recommended 10 mL culture. Tissue culture flasks with similar area of end wall can be used standmg on end. If available specimen volume demands smaller culture, then 15 mL glass McCartney bottles give good results with 5-mL cultures usmg 0.5 mL blood. Plastic Universals with conical base are not suitable. 7. Methotrexate is the most widely used reagent in blocking cells to encour- age synchronization to enrich the harvest of chromosomes in early metaphase. Most reliable is Methotrexate injection (Lederle) equiva- lent to 25 mg in 1 rnL. This ts diluted to a workmg solution of 10e5M and used to give a final concentratron of 10e7M. 8. Cells are released from the S-phase block by removing the blockmg agent and resuspending the cells in medium enriched in thymidine or its analog 5-bromo-2’-deoxyuridine. Some published methods require the blocking agent to be washed from the cells, but I prefer to minimize the handling of the culture, which may result in cell loss or damage and also requires more time and labor. The medium is stmply sucked from above the cells, and the cell are resuspended m fresh medium with the release agent. Thymidine is used as release agent for cultures that will be banded to show G-bands, whereas bromodeoxyuridine incorpora- tion is far better for staining of R-bands. Although different laborato- ries may prefer one type of banding, most agree that it is better to have preparations available from both for confirmatory analysis. I run two sets of cultures, one to be released with thymidine and the other with bromodeoxyuridine. 9. There is some debate over the role and efficacy of colcemid used to arrest cells. However, there ts no doubt that if it is not used, then the mitotic index drops dramatically, although the proportion of early stages is increased. On balance, I prefer to use 60 pL of 10 pg/mL for a 10 mL culture. 10. The hypotonic stage of the harvest is crucial. Many formulations are published, but the most popular is still 0.075M potassmm chloride. In my experience, it is better if this solution is made up m deionrzed rather than distilled water. 11. The first fixation stage is the most important. The red cells will fuse mto insoluble clumps entrapping the lymphocytes if the cells are not vortexed and the fix added dropwise to the middle of the vortex. Acetone-free methanol may require to be filtered before use. Three parts methanol and one part glacial acetic acid should be mixed in quantity required just before use. Mitotic Metaphase Chromosome Preparation 12. The spreading of cells on the slide depends on the size of the drop of cell suspension, cell density, slide surface, ambient temperature, humidity, and so on. If spreadmg is not satisfactory on a dry polished slide, the following can be tried: a. Breathing on the slide Just before placing the drop. b. Suspending slide above an open 60°C water bath. c. Chilling slide. d. Reducing or Increasing size of drop. e. Altering the height above the slide from which to drop the cells. 13. I agree with Wiley et al. (I 7) that actinomycin D and colcemld together give a high mitotlc Index. Although actmomycm alone gives a higher proportion of early stages, I prefer to have more cells of all stages to chose from. I agree also that lower concentration of actinomycm is supenor. References 1 Caspersson, T., Lomakka, G , and Zech, L (1971) The 24 fluorescence pat- terns of the human metaphase chromosomes-dlstmguishmg characters and varlablhty Hereditas 67,89-102 2. Sumner, A T., Evans, H. J , and Buckland, R. A. (1971) New technique for dlstmguishing between human chromosomes. Nature New Biol 232,3 l-32 3. ISCN, (1985) An International System for Human Chromosome Nomencla- ture (Harnden, D G. and Klinger, H P., eds.) S Karger, Base1 4 Yums, J J (198 1) Mid prophase human chromosomes; the attamment of 2000 bands Hum. Genet 56,295-298 5. Droum, R., Lemleux, N., and Richer, C -L. (1988) High-resolution R-banding at the 1250-band level. II Schematic representation and nomenclature of human RBG-banded chromosomes. Cytobios 56,425-439. 6. Schmzel, A. (1988) Microdeletion syndromes, balanced translocatlons, and gene mapping. J Med Genet 25,454-462 7 Hungerford, D A. (1965) Leukocytes cultured from small inocula of whole blood and the preparation of metaphase chromosomes by treatment with hypo- tonic KC1 Stain Technol. 40,333-338. 8. Camargo, M. and Cervenka, J (1980) Pattern of chromosomal replication m synchro- nised lymphocytes I. Evaluation of the methotrexate block Hum. Genet. 54,47-53 9. Yunis, J. J., (1976) High resolution of human chromosomes Science 191, 1268-1269. 10. Viegas-Peqmgnot, E and Dutrillaux, B. (1978) Une m&hode simple pour obtemr des prophases et des promttaphases Ann. G&w?. 21, 122-125. 11 Schwartz S , and Palmer, C G. (1984) High-resolution chromosome analysis. I. Apphcatlons and hmltations. Am J Med. Genet 19,291-299 12. Rybak, J., Tharapel, A., Robmett, S., Garcia, M., Ma&men, C , and Freeman, M. (1982) A simple reproducible method for prometaphase chromosome analy- sis. Hum Genet 60,328-333 10 Spowart 13 Matsubara, T. and Nakagone, Y. (1983) High-resolution banding by treating cells with acridine orange before fixation. Cytogenet Cell Genet 35, 148-15 1 14 Ikeuchi, T. (1984) Inhibitory effect of ethidium bromide on mitotlc chromosome condensation and its application to htgh-resolution chromosome banding Cytogenet. Cell Genet. 38,56-61. 15 Bigger, T. R L and Savage, J. R. K. (1975) Mapping G-bands on human prophase chromosomes Cytogenet Cell Genet 15, 112-121. 16 Ronne, M., Netlsen, K V , and Erlandsen, M (1979) Effect of controlled colcemid exposure on human metaphase chromosome structure. Heredltus 91, 49-52. 17. Wiley, J. E , Sargent, L M., Inhorn, S. L., and Meisner, L. F. (1984) Compari- son of prometaphase chromosome techniques wtth emphasis on the role of colcemid In Vitro 20,937-941 [...]... attention to detail From Methods fn Molecular Bology, Edited by J R Gosden Copyright Vol 29’ Chromosome Analysis Protocols 01994 Humana Press Inc., Totowa, NJ 11 12 Ross The main problems with attempting to prepare chromosomes from malignant tissue are the frequent low mitotic indices and the poor quality of the chromosomes A variety of conditioned media or potential mitogens have been employed in an... defined by ISCN (9) as two cells with the same rearrangement or additional chromosome, or three cells with loss of the same chromosome Care needs to be taken with the latter definition if many cells have been broken during preparation Thirty cells are ridiculously few in terms of marrow turnover, but the time involved in chromosome analysis usually precludes anything more Where patients are being followed... pachytene chromosomes, which can be used for mapping of individual bivalents by the “chromomere” patterns (14) Chromomere preparations have been used to aid the mapping of genes by in situ hybridization along the arms of human chromosomes (15), as an alternative to the use of banded somatic chromosomes In general, accessibility to the in situ hybridization procedures appears enhanced in meiotic chromosomes,... submicroscopic level The most notable example of this is in CML, where the same molecular rearrangement can be achieved by a visible translocation between chromosomes 9 and 22 or a submicroscopic insertion of part of the c-ABL gene from chromosome 9 into the BCR gene on chromosome 22 (7) Whether similar events will be shown to account for all the cases of acute leukemia where no abnormality can be detected (presently... DPX or Histomount 3.1.5 Chromosome Analysis Since it is usually necessary to analyze large numbers of cells to be sure of detecting clones that form only a small proportion of the dividing cells, it is not generally practicable to analyze fromphotokaryotypes With practice, it is possible to analyze most cases down the microscope, the exceptions usually being those with very high chromosome counts or very... success of their chromosome preparation; the acute myeloid leukemias are now relatively well understood and, in general, give fairly consistent results The acute lymphoid leukemias still have major problems with the quality of the abnormal chromosomes, and some diseases, such as Hodgkin’s disease, still do not have reliable methods to produce any abnormal metaphases The general standard of chromosome preparations... direct bone marrow chromosome technique for acute lymphoblastic leukaemia Cancer Genet Cytogenet 13,239-257 14 Macera, M J., Szabo, P , and Verma, R S (1989) A simple method for short term culturmg of bone marrow and unstimulated blood from acute leukemras Leuk Res 13,729-734 CHAPTER Meiotic Chromosome 3 Preparation Ann C Chundley, Robert M Speed, and Kun Ma 1 Introduction The study of chromosomes at meiosis... in particular (4) From Methods in Molecular B/ology, E&ted by J R Gosden Copynght Vol 29 Chromosome Analysa Protocols 01994 Humana Press Inc , Totowa, NJ 27 28 Chandley, Speed, and Ma In the 197Os,“microspreading,” which had originally been developed to examine the synaptonemal complexes formed between paired chromosomes at meiotic prophase in insect spermatocytes (5), was adapted by Moses et al (6)... collection of human oocytes in MI or MII, considerablepractical difficulties are encountered, and although a number of attempts to prepare chromosomes in these stages for analysis by air-drying have been made (II, 12), high-quality preparations that would allow accurate analysis of, for example, chiasma counts at MI have never really been produced For further information on this topic and the technical... Thus, although these cells are part of the abnormal clone, it makes sense to remove them before culturing the cells for chromosome analysis Fortunately, a large reduction in neutrophil numbers can be very simply achieved by standard lymphocyte separation techniques The quality of the chromosome preparations from such cultures is often better than in conventional CML cultures, and it is thought that this . B/ology, Vol. 29 Chromosome Analysis Protocols Edlted by J R. Gosden Copynght 01994 Humana Press Inc , Totowa, NJ 2 Spowart Published methods on the preparation of elongated chromosomes are. Bology, Vol 29’ Chromosome Analysis Protocols Edited by J. R Gosden Copyright 01994 Humana Press Inc., Totowa, NJ 11 12 Ross The main problems with attempting to prepare chromosomes from. a visible trans- location between chromosomes 9 and 22 or a submicroscopic inser- tion of part of the c-ABL gene from chromosome 9 into the BCR gene on chromosome 22 (7). Whether similar events