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Chromosome preparations of leukocytes cultured from human peripheral blood
Chromosome coiling early telophase was inhibited; prophase contraction occurred but the metaphase chromosomes were fused into a single mass. These results are evidence that polyamines are capable of upsetting the normal processes of chromosome coiling and uncoiling. REFERENCES AMES, B. N., DUBIN, L). T. and ROSENTHAL, S. M., Science 127, 814 (1958). ANDERSON, N. G., Quart. Reu. Hiof. 31, 169 (1956). ANDERSON, N. G. and NORRIS, C. B., Ezpfl. Cell Research. In press. DAVIDSON, D., Chromosoma 9, 39 (1957). FISHER, %. D., AXDERSO~~, N. G. and WILBUR, I<. 31., Ezpfl. Cd/ Research 18, 100 (1959). GUGGENHEIM, M., Die biogenen Amine. S. Karger, New York, 1951. Kurxx, W., HARGITAY, B.: KATCHALSKY, A. and EISENBERG, II., Sature 165, 514 (1950). 8. RfARQUARDT, H., &perienfia 5, 401 (1949). 9. HIEGEH, R. and MICHAELIS, A., Xonafsber. deut. Akad. Wissenschaffen Berlin 1, 51 (1959).
1. 2. 3. 4. 5. 6. 7.
CHROMOSOME PREPARATIONS OF LEUKOCYTES FROM HUMAN PERIPHERAL BLOOD1 P. S. MOORHEAD,
P. C.NOWELL,3 W. J. MELLMAN,4 and D.A. HUNGERFORD
CULTURED
D. M. BATTIPS,
Received May 18, 1960
s- I~CE t h’tdt’ e m ro UC ion of the hypotonic pretreatment of mitotic cells by Hsu and Pomerat [ 11 and by Hughes [2] mammalian chromosomes from tissue-cultured material have been extensively studied. Published studies concerning the human karyotype and somatic chromosome anomalies are increasing in frequency. Most of the work on humans has depended upon bone-marrow puncture or skin biopsy for material, and upon classic squashing techniques. The techniques described here represent the successful attempt to combine the convenience of Nowell’s [3] method for the cultivation of peripheral blood leukocytes with the advantages inherent in the air-drying method of Rothfels and Siminovitch [4]. Air-drying of metaphase cells cultured on glass provides well-spread chromosomes in one focal plane with a minimum of overlay 1 This investigation was supported in part by the United States Public Health Service Grants C-4534, C-4659 (National Cancer Institute), SF-4 (Senior Research Fellowship), A-2231 (National Institute of Arthritis and Metabolic Diseases), CF-11,168 (Pre-Doctoral Fellowship, National Cancer Institute), and by an Institutional Grant from the American Cancer Society. 2 Wistar Institute of Anatomy and Biology, Philadelphia. 3 Department of Pathology, School of Medicine, University of Pennsylvania, Philadelphia. * Department of Pediatrics, Hospital of the University of Pennsylvania and School of Medicine, University of Pennsylvania, Philadelphia. 5 Institute for Cancer Research, Philadelphia. Experimenful
Cell Research 20
614
P. 5’. Moorhead
et al.
and scattering. However, very few leukocytes adhered to cover glasses inserted in these cultures. Attempts to obtain good-quality air-dried preparations from unattached leukocytes handled in suspension failed consistently prior to the introduction of the modifications described here. Material and methods.-Separation of leukocytes. Draw 10 ml venous blood into a sterile syringe wet with commercial heparin. Add Bacto-Phytohemagglutinin (Difco Laboratories, Detroit l), 0.2 ml/10 ml blood. Mix and allow to stand 30-60 minutes in an ice bath. Centrifuge at 300-350 rpm (approximately 25 G) for 5-10 minutes at 5°C to sediment most erythrocytes. Remove supernatant plasma containing leukocytes in suspension. Count leukocytes. Culture of leukocytes. Plant cells in 60 x 22 mm screw-top bottle(s) in medium consisting of (a) 30-40 per cent fresh autologous or homologous plasma; (b) 60-70 per cent TC-199 (Difco); (c) penicillin and streptomycin. Plant S-10 ml culture(s) with 1.0-1.2 x lo6 cells/ml. Ajust pH to 7.0-7.2 initially and maintain in this range by daily adjustments with 0.1 N HCl or CO, gas. Incubate undisturbed at 37°C with room air as gas phase and screw-top tightened. Chromosome cytology. After 3 days (65-70 hours) add colchicine to the culture(s) in a final concentration of 0.5-1.0 x 1O-6 M. After 6 hours’ exposure to colchicine (9-10 hours yields over-contraction of the chromosomes) sacrifice culture(s) as follows. Loosen cells from bottom of bottle by agitation with pipette and centrifuge at 800 rpm for 5 minutes, using siliconized Pasteur pipette and 12-ml conical centrifuge tube, graduated. Remove most of supernatant medium and gently resuspend cells in 556 ml of warm Hanks’ or Earle’s balanced salt solution (BSS) at pH 7.0. Centrifuge as before. Remove supernatant, leaving some known volume in tube (such as 0.5 ml). Carefully resuspend cells in this small volume. With continual mixing, slowly add warm distilled water, adding 3 times original volume (such as 1.5 ml added to 0.5 ml). Let stand in incubator at 37°C for 3-5 minutes. Centrifuge at 600 rpm for 5 minutes. Total time in hypotonic BSS is 8-10 minutes. Make fresh fixative (1: 3 of glacial acetic acid and methyl alcohol, absolute). Remove all supernatant and add 334 ml of fixative without disrupting the “button” of cells. Let cells stand in fixative for at least 30 minutes. Break up button and cell clumps as completely as possible by careful and repeated pipetting. Centrifuge at 600 rpm for 5 minutes and replace supernatant fixative with more of the freshly made fixative. Add about one-half ml fixative to obtain a fairly dense cell suspension. Dip a pre-cleaned slide into chilled distilled water, shake off excess, and then pipette 223 drops of the cell suspension onto the wet slide. Immediately tilt slide and draw off all excess fluid by holding edge of slide on blotter. Drying should be completed in 30-60 seconds by fanning or by gently warming slide over a spirit flame. The quality of the slide can be checked immediately under phase contrast optics. Inadequate spreading may often be corrected by an additional change
Fig. l.-Air-dried metaphase of peripheral blood leukocyte from human female (count 45). Note satellites on certain acrocentrics (arrows) and a translocation metacentric (broad arrow). The cytology of this case will be presented elsewhere. (Bar in lower left corner represents IO micra.) Fig. 2.-Air-dried somes, including as Fig. 1.)
Experimental
metaphase of DBA mouse lymphoma cell, cell line L-5178-Y. Piate 44 chromo4 minutes and one distinctive chromosome (broad arrow). (Same magnification
Cell Research
20
Chromosome preparations of leukocytes
Experimental
615
Cell Research 20
P. S. Moorhead et al.
616
of fixative, even after long storage in the refrigerator. A routine staining dish passage of the slides from freshly filtered stain (1 per cent natural orcein, Gurr Ltd., in 60 “/b acetic acid) through a dehydration schedule and permanent mounting in Diaphane or Permount complete the preparation. Accessory notes. Peripheral blood from leukemic patients which contains immature forms may be treated exactly as above, except that colchicine is added after 2 days (40-45 hours) in culture. Aspirated bone marrow, normal or leukemic, may also be treated exactly as above, except that colchicine is added after one day (20-25 hours). In general, a shorter time in colchicine is desirable for bone marrow material. Phytohemagglutinin is essential for mitotic activity in normal peripheral blood, but not essential in leukemic blood or in bone marrow (normal or leukemic). Do not centrifuge excessively during initial separation of leukocytes, as a few red cells will not interfere with these short-term cultures. Normal peripheral blood usually yields a minimum of lo6 leukocytes/ml blood. The number of analyzable spreads varies from slide to slide, but usually a single culture from 10 ml of original blood is more than adequate for a chromosome characterization. Although most of the details of the cytological protocol are not considered critical, consistently well-spread metaphase plates appear to be dependent upon the surfacetension forces involved during drying on the wet slide. Consequently, successive changes of fresh fixative, well-cleaned slides, and the speed of drying are important in achieving good flattening and attachment of the cell membrane to the glass surface (Fig. 1). The protocol for chromosome cytology is also quite satisfactory for other cells which do not adhere to a glass surface in culture, such as the DBA mouse lymphoma, L-5178Y (Fig. 2.). Summary.-A combination of cytological and leukocyte culture techniques is described which constitutes a convenient, reliable approach for chromosome studies of humans and yields the following advantages: 1. Relative ease of obtaining blood and small volume required. 2. Adequate mitotic yield from the short-term culture of leukocytes. 3. High numbers of “exact count” quality metaphase spreads, permitting a critical analysis of chromosome morphology. REFERENCES 1. Hsu, T. C. and POMERAT, C. &I., J. Heredity 44, 23 (1953). 2. HUGHES, A., Quart. J. Microscop. Sci. 93, 207 (1952). 3. NOWELL, P. C., Cancer Research. In press. 4. ROTHFELS, K. H. and SIYI~L’OVITCH, L., Stain l’echnol. 33, 73 (1958).
Experimental
Cell Research 20
1. 2. 3. 4. 5. 6. 7.
CHROMOSOME PREPARATIONS OF LEUKOCYTES FROM HUMAN PERIPHERAL BLOOD1 P. S. MOORHEAD,
P. C.NOWELL,3 W. J. MELLMAN,4 and D.A. HUNGERFORD
CULTURED
D. M. BATTIPS,
Received May 18, 1960
s- I~CE t h’tdt’ e m ro UC ion of the hypotonic pretreatment of mitotic cells by Hsu and Pomerat [ 11 and by Hughes [2] mammalian chromosomes from tissue-cultured material have been extensively studied. Published studies concerning the human karyotype and somatic chromosome anomalies are increasing in frequency. Most of the work on humans has depended upon bone-marrow puncture or skin biopsy for material, and upon classic squashing techniques. The techniques described here represent the successful attempt to combine the convenience of Nowell’s [3] method for the cultivation of peripheral blood leukocytes with the advantages inherent in the air-drying method of Rothfels and Siminovitch [4]. Air-drying of metaphase cells cultured on glass provides well-spread chromosomes in one focal plane with a minimum of overlay 1 This investigation was supported in part by the United States Public Health Service Grants C-4534, C-4659 (National Cancer Institute), SF-4 (Senior Research Fellowship), A-2231 (National Institute of Arthritis and Metabolic Diseases), CF-11,168 (Pre-Doctoral Fellowship, National Cancer Institute), and by an Institutional Grant from the American Cancer Society. 2 Wistar Institute of Anatomy and Biology, Philadelphia. 3 Department of Pathology, School of Medicine, University of Pennsylvania, Philadelphia. * Department of Pediatrics, Hospital of the University of Pennsylvania and School of Medicine, University of Pennsylvania, Philadelphia. 5 Institute for Cancer Research, Philadelphia. Experimenful
Cell Research 20
614
P. 5’. Moorhead
et al.
and scattering. However, very few leukocytes adhered to cover glasses inserted in these cultures. Attempts to obtain good-quality air-dried preparations from unattached leukocytes handled in suspension failed consistently prior to the introduction of the modifications described here. Material and methods.-Separation of leukocytes. Draw 10 ml venous blood into a sterile syringe wet with commercial heparin. Add Bacto-Phytohemagglutinin (Difco Laboratories, Detroit l), 0.2 ml/10 ml blood. Mix and allow to stand 30-60 minutes in an ice bath. Centrifuge at 300-350 rpm (approximately 25 G) for 5-10 minutes at 5°C to sediment most erythrocytes. Remove supernatant plasma containing leukocytes in suspension. Count leukocytes. Culture of leukocytes. Plant cells in 60 x 22 mm screw-top bottle(s) in medium consisting of (a) 30-40 per cent fresh autologous or homologous plasma; (b) 60-70 per cent TC-199 (Difco); (c) penicillin and streptomycin. Plant S-10 ml culture(s) with 1.0-1.2 x lo6 cells/ml. Ajust pH to 7.0-7.2 initially and maintain in this range by daily adjustments with 0.1 N HCl or CO, gas. Incubate undisturbed at 37°C with room air as gas phase and screw-top tightened. Chromosome cytology. After 3 days (65-70 hours) add colchicine to the culture(s) in a final concentration of 0.5-1.0 x 1O-6 M. After 6 hours’ exposure to colchicine (9-10 hours yields over-contraction of the chromosomes) sacrifice culture(s) as follows. Loosen cells from bottom of bottle by agitation with pipette and centrifuge at 800 rpm for 5 minutes, using siliconized Pasteur pipette and 12-ml conical centrifuge tube, graduated. Remove most of supernatant medium and gently resuspend cells in 556 ml of warm Hanks’ or Earle’s balanced salt solution (BSS) at pH 7.0. Centrifuge as before. Remove supernatant, leaving some known volume in tube (such as 0.5 ml). Carefully resuspend cells in this small volume. With continual mixing, slowly add warm distilled water, adding 3 times original volume (such as 1.5 ml added to 0.5 ml). Let stand in incubator at 37°C for 3-5 minutes. Centrifuge at 600 rpm for 5 minutes. Total time in hypotonic BSS is 8-10 minutes. Make fresh fixative (1: 3 of glacial acetic acid and methyl alcohol, absolute). Remove all supernatant and add 334 ml of fixative without disrupting the “button” of cells. Let cells stand in fixative for at least 30 minutes. Break up button and cell clumps as completely as possible by careful and repeated pipetting. Centrifuge at 600 rpm for 5 minutes and replace supernatant fixative with more of the freshly made fixative. Add about one-half ml fixative to obtain a fairly dense cell suspension. Dip a pre-cleaned slide into chilled distilled water, shake off excess, and then pipette 223 drops of the cell suspension onto the wet slide. Immediately tilt slide and draw off all excess fluid by holding edge of slide on blotter. Drying should be completed in 30-60 seconds by fanning or by gently warming slide over a spirit flame. The quality of the slide can be checked immediately under phase contrast optics. Inadequate spreading may often be corrected by an additional change
Fig. l.-Air-dried metaphase of peripheral blood leukocyte from human female (count 45). Note satellites on certain acrocentrics (arrows) and a translocation metacentric (broad arrow). The cytology of this case will be presented elsewhere. (Bar in lower left corner represents IO micra.) Fig. 2.-Air-dried somes, including as Fig. 1.)
Experimental
metaphase of DBA mouse lymphoma cell, cell line L-5178-Y. Piate 44 chromo4 minutes and one distinctive chromosome (broad arrow). (Same magnification
Cell Research
20
Chromosome preparations of leukocytes
Experimental
615
Cell Research 20
P. S. Moorhead et al.
616
of fixative, even after long storage in the refrigerator. A routine staining dish passage of the slides from freshly filtered stain (1 per cent natural orcein, Gurr Ltd., in 60 “/b acetic acid) through a dehydration schedule and permanent mounting in Diaphane or Permount complete the preparation. Accessory notes. Peripheral blood from leukemic patients which contains immature forms may be treated exactly as above, except that colchicine is added after 2 days (40-45 hours) in culture. Aspirated bone marrow, normal or leukemic, may also be treated exactly as above, except that colchicine is added after one day (20-25 hours). In general, a shorter time in colchicine is desirable for bone marrow material. Phytohemagglutinin is essential for mitotic activity in normal peripheral blood, but not essential in leukemic blood or in bone marrow (normal or leukemic). Do not centrifuge excessively during initial separation of leukocytes, as a few red cells will not interfere with these short-term cultures. Normal peripheral blood usually yields a minimum of lo6 leukocytes/ml blood. The number of analyzable spreads varies from slide to slide, but usually a single culture from 10 ml of original blood is more than adequate for a chromosome characterization. Although most of the details of the cytological protocol are not considered critical, consistently well-spread metaphase plates appear to be dependent upon the surfacetension forces involved during drying on the wet slide. Consequently, successive changes of fresh fixative, well-cleaned slides, and the speed of drying are important in achieving good flattening and attachment of the cell membrane to the glass surface (Fig. 1). The protocol for chromosome cytology is also quite satisfactory for other cells which do not adhere to a glass surface in culture, such as the DBA mouse lymphoma, L-5178Y (Fig. 2.). Summary.-A combination of cytological and leukocyte culture techniques is described which constitutes a convenient, reliable approach for chromosome studies of humans and yields the following advantages: 1. Relative ease of obtaining blood and small volume required. 2. Adequate mitotic yield from the short-term culture of leukocytes. 3. High numbers of “exact count” quality metaphase spreads, permitting a critical analysis of chromosome morphology. REFERENCES 1. Hsu, T. C. and POMERAT, C. &I., J. Heredity 44, 23 (1953). 2. HUGHES, A., Quart. J. Microscop. Sci. 93, 207 (1952). 3. NOWELL, P. C., Cancer Research. In press. 4. ROTHFELS, K. H. and SIYI~L’OVITCH, L., Stain l’echnol. 33, 73 (1958).
Experimental
Cell Research 20