Summary
Using slit-scan flow cytometry, the shape of human metaphase chromosomes, as expressed in their centromeric index (CI), and the DNA content of the chromosomes have been used as parameters in bivariate flow karyotyping. The resolution of the DNA vs CI flow karyogram of the larger chromosomes up to chromosome 13 is much higher than the resolution obtained in the DNA-based monovariate flow karyogram. Chromosome length appears to be an important factor in the resolution of the DNA vs CI-based flow karyogram. A method has been developed to obtain chromosomes in suspension that are long enough for adequate analysis. Several chromosomes that cannot be distinguished or are difficult to discriminate in the DNA-based karyogram can now be distinguished as individual peaks, e.g., chromosomes 1 and 2. The peak of chromosomes 9–12 can be separated into two peaks formed by chromosomes 9 and 11, and 10 and 12, respectively. The advantage of the system applied in this study is that the DNA vs CI analysis is performed on-line, allowing chromosomes to be sorted on the bases of their CI. Pulse shapes of the selected chromosomes can be recorded simultaneously with the transmission of the sorting command. The purity of the sorted fraction can be estimated from the offline inspection of these pulse shapes. Fractions of chromosome 1 have been sorted out on the basis of the CI information, centrifuged on slides, fixed and subsequently banded with trypsin and Giemsa or hybridized with the chromosome 1 specific probe, pUC 1.77. The observed purity under the selected conditions ranges from 80%–99% and is in accordance with the estimates of the purities made on the basis of the simultaneously recorded pulse shapes. Fixation of the chromosome suspension prior to flow cytometric analysis and sorting appears to be essential for the preservation of their morphology and has no adverse influence on the resolution of Giemsa banding or on the quality of in situ hybridization.
Similar content being viewed by others
References
Aten JA, Kipp JBA, Barendsen GW (1980) Flow-cytofluorometric determination of damage to chromosomes from X-irradiated Chinese hamster cells. Flow Cytometry IV, Proceedings of the 5th International Symposium on Flow Cytometry. Universitetsforlaget, Oslo, pp 287–292
Aten JA, Buys CHCM, Veen AY van der, Mesa JR, Yu LC, Gray JW, Osinga J, Stap J (1987) Stabilization of chromosomes by DNA intercalators for flow karyotyping and identification by banding of isolated chromosomes. Histochemistry 87:359–366
Aten JA, Manders E, Oven C van, Rens W, Stap J (1988) Detection of chromosome aberrations by flow cytometry as a test for radiation sensitivity. Cytometry, Suppl 2:23
Berg FM van den, Jiwa M, Rook R, Geelen JLMC (1988) Analysis and isolation of cytomegalovirus DNA by field inversion gel electrophoresis. J Gen Virol 69:699–704
Buys CHCM, Koerts T, Veen AY van der (1982) Well identifiable human chromosomes isolated from mitotic fibroblasts by a new method. Hum Genet 61:157–159
Buys CHCM, Aten JA, Koerts T, Osinga J, Veen AY van der (1984) Isolated metaphase chromosomes stabilized by DNA intercalation or polyamine addition: a comparison. Cell Biol Int Rep 8:273
Carrano AV, Gray JW, Langlois RG, Burghart-Schultz KJ, Van Dilla MA (1979) Measurement and purification of human chromosomes by flow cytometry and sorting. Proc Natl Acad Sci USA 76:1382–1384
Cooke HJ, Hindley J (1979) Cloning of human satellite DNA: different components are on different chromosomes. Nucleic Acids Res 6:3177–3197
Cotter FE, Nasipuri S, Lam G, Young BD (1989) Rapid mapping of chromosome 11q22 alterations by enzymatic amplification from small numbers of flow sorted chromosomes (abstract). In: Buys CHCM (ed) Proceedings of the 21st European Society of Human Genetics Symposium. Imedex, Groningen, p 33
Cremer C, Rappold G, Gray JW, Muller CR, Ropers H-H (1984) Preparative dual-beam sorting of the human Y-chromosome and in situ hybridization of cloned DNA-probes. Cytometry 5:572–579
Cremer C, Hausmann M, Zuse P, Aten JA, Barths J, Bühring HJ (1989) Flow cytometry of chromosomes: principles and applications in medicine and molecular biology. Optik 82:9–18
Cremer T, Lichter P, Borden J, Ward DC, Manuelidis L (1988) Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes. Hum Genet 80:235–246
Davies KE, Young BD, Ellis RG, Hill ME, Williamson R (1981) Cloning of a representative genomic library of the human X-chromosome after sorting by flow cytometry. Nature 293:374–377
Dean PN, Pinkel D (1978) High resolution dual laser flow cytometry. J Histochem Cytochem 26:622–627
Engh G van den, Trask B, Cram S, Bartholdi M (1984) Preparation of chromosome suspensions for flow cytometry. Cytometry 5:108–123
Gooderham K, Jeppeson P (1983) Chinese hamster metaphase chromosomes isolated under physiological conditions. A partial characterization of associated non-histone proteins and protein cores. Exp Cell Res 144:1–14
Gray JW, Langlois RG, Carrano AV, Burkhart-Schultz K, Van Dilla MA (1979) High resolution chromosome analysis: one and two parameter flowcytometry. Chromosoma 73:9–27
Gusella JF, Keys C, Varsanyi-Breiner A, Kao F-T, Jones C, Puck TT, Hausman D (1980) Isolation and localization of DNA segments from specific human chromosomes. Proc Natl Acad Sci USA 77:2829–2833
Hausmann M, Dudin G, Aten JA, Bühring HJ, Diaz E, Dölle J, Bier F, Cremer C (1989) Flow cytometric detection of isolated chromosomes following fluorescence hybridization. (Unpublished)
Hopman AHN, Ramaekers FCS, Raap AK, Devilee P, Van der Ploeg M, Vooijs GP (1988) In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumours. Histochemistry 89:307–316
Hutter KJ, Stoehr M (1985) Detection and separation of the submetacentric marker chromosome of the Walker (W-256) carcinoma using flowcytometry and sorting. Histochemistry 82:469–475
Kunkel LM, Tantravahi U, Kurnit DM, Eisenhard M, Bruns GP, Latt SA (1983) Identification and isolation of transcribed human X chromosome DNA sequences. Nucleic Acids Res 11:7961–7979
Langlois RG, Yu L-C, Gray JW, Carrano AV (1982) Quantitative karyotyping of human chromosomes by dual beam flowcytometry. Proc Natl Acad Sci USA 79:7876–7880
Lebo RV (1982) Chromosome sorting and DNA sequence localization. Cytometry 3:145–154
Lebo RV, Gorin F, Fletterick RY, Kao FT, Cheung MC, Bruce BD, Kan YW (1984) High resolution chromosome sorting and DNA spot-blot analysis assign McArdle's syndrome to chromosome 11. Science 225:57–59
Lebo RV, Cheung MC, Bruce BD, Riccardi VM, Kao FT, Kan YW (1985) Mapping parathyroid hormone, beta-globin, insulin, and LDH-A genes within the human chromosome 11 short arm by spot-blotting sorted chromosomes. Hum Genet 69:316–320
Lebo RV, Golbus MS, Cheung MC (1986) Detecting abnormal chromosome constitutions by dual laser flow cytogenetics. Am J Med Genet 25:519–529
Lichter P, Cremer T, Tang CC, Watkins PC, Manuelidis L, Ward DC (1988a) Rapid detection of human chromosome 21 aberrations by in situ hybridization. Proc Natl Acad Sci USA 85:9664–9668
Lichter P, Cremer T, Borden J, Manuelidis L, Ward DC (1988b) Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization suing recombinant DNA libraries. Hum Genet 80:224–234
Lucas JN, Gray JW (1987) Centromeric index versus DNA content flowkaryotypes of human chromosomes measured by means of slit-scan flowcytometry. Cytometry 8:273–279
Lucas JN, Gray JW, Peters DC, Van Dilla MA (1983) Centromeric index measurement by slit-scan flowcytometry. Cytometry 4:109–116
Lüdecke HJ, Senger G, Claussen U, Horsthemke B (1989) Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification. Nature 338:348–350
Mayall BH, Carrano AV, Moore DH, Ashworth LK, Bennet DE, Mendelsohn ML (1984) The DNA-based human karyotype. Cytometry 5:376–385
Mullikin J, Norgren R, Lucas J, Gray J (1988) Fringe-scan flow-cytometry. Cytometry 9:111–120
Oven C van, Aten JA (1990) An instrument for real-time pulse-shape analysis of slit-scan flow cytometry signals. Cytometry 9 (in press)
Paris Conference (1972) Standardization in human cytogenetics. National Foundation — March of Dimes. Birth Defects, Original Article Series VIII, vol 7
Peters D, Branscomb E, Dean P, Merril T, Pinkel D, Van Dilla, Gray JW (1985) The LLNL high-speed sorter: design features, operational characteristics, and biological utility. Cytometry 6:290–301
Pinkel D, Landegent J, Collins C, Fuscoe J, Segraves R, Lucas J, Gray J (1988) Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4. Proc Natl Acad Sci USA 85:9138–9142
Shapiro HM (1985) Practical flow cytometry. Liss, New York
Sillar R, Young BD (1981) A new method for the preparation of metaphase chromosomes for flow analysis. J Histochem Cytochem 29:74–78
Tantravahi U, Stewart GE, Van Keuren M, McNeil G, Roy S, Patterson D, Drabkin H, Lalande M, Kurnit D, Latt SA (1988) Isolation of DNA sequences on human chromosome 21 by application of a recombination-based assay to DNA from flowsorted chromosomes. Hum Genet 79:196–202
White TJ, Arnheim N, Erlich HA (1989) The polymerase chain reaction. Trends Genet 5:185–189
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Boschman, G.A., Rens, W., Manders, E. et al. On-line sorting of human chromosomes by centromeric index, and identification of sorted populations by GTG-banding and fluorescent in situ hybridization. Hum Genet 85, 41–48 (1990). https://doi.org/10.1007/BF00276324
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00276324