Annals of Clinical & Laboratory Science 37:85-88 (2007)
© 2007 Association of Clinical Scientists
Fluorescence In Situ Hybridization (FISH) Using Snap Frozen Buffy Coat
Hun Jung Min1,4,5,
Jinkyoung Lee2,
Jung Eun Choi1,
Su Shin3 and
Dong Soon Lee1,4,5
1 Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Korea2 Department of Laboratory Medicine, Korea Cancer Center Hospital Seoul, Korea3 Department of Laboratory Medicine, Boramae Hospital, Seoul, Korea4 Cancer Reseach Institute, Seoul National University College of Medicine, Seoul, Korea5 National Research Laboratory for Molecular Cell Imaging, Seoul, Korea
Address correspondence to Dong Soon Lee, M.D., Department of Laboratory Medicine, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul, 110-744, Korea; tel 82 2 2072 3986; fax 82 2 745 6653; e-mail: soonlee{at}plaza.snu.ac.kr or leukemia{at}plaza.snu.ac.kr.
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Abstract
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We report the successful application of interphase fluorescent in situ hybridization (FISH) using extracted nuclei from snap frozen buffy coat stored for the purpose of molecular analysis. Included in this study were 30 frozen buffy coat specimens, ranging from <1 day to 3 yr old. Thawing of the buffy coat at 37°C was followed by lysing erythrocytes with lysing solution (LYSE S III DIFF, Coulter Corp.). Cells were fixed and slides were prepared according to manufacturer’s FISH protocol. All specimens from the 30 frozen-thawed buffy coats were suitable for FISH evaluation, and signal intensities were not significantly related to the age of specimens. In conclusion, interphase FISH is feasible using frozen-thawed buffy coat; the technique will be useful for retrospective molecular cytogenetic analysis of hematologic malignancies.
Keywords: cytogenetics, chromosome probes, buffy coat, FISH analysis
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Introduction
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Fluorescence in situ hybridization (FISH) is a validated and reproducible method to detect chromosomal aberrations [1,2]. Interphase FISH is usually performed with mononuclear cells from bone marrow, but it is often applied to fresh cytologic materials such as bone marrow aspiration smears or touch prints [3]. For the purpose of molecular analysis, frozen buffy coat specimens from peripheral blood or bone marrow with no cryoprotectant has been used for several decades [4], but the procedure is unsuitable for molecular cytogenetic analysis such as FISH. Freezing-thawing causes injury to the plasma membrane and use of a cryoprotectant may prevent the membrane damage [5]. Nevertheless, it is unclear whether freezing-thawing causes nuclear membrane damage or chromosomal fragmentation.
We hypothesized that rettrospective chromosome analysis using FISH could be feasible if the nuclear membrane is not severely damaged by cell freezing-thawing stress. Supposing that the freeze-thawed buffy coat could be a useful source of interphase nuclei for FISH, we performed a FISH study with 30 frozen buffy coat specimens.
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Materials and Methods
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This study used 30 frozen buffy coat specimens that had been prepared from peripheral blood and stored for extraction of nucleic acids for molecular studies. The storage periods ranged from <1 day to 3 yr.
Freezing-thawing of buffy coats.
Peripheral blood specimens anticoagulated with EDTA or sodium citrate were centrifuged at 2500 rpm for 15 min and the leukocyte layers were collected by pipetting. The leukocytes were stored at –70°C until future testing. Thawing was pereformed in a 37°C water bath until the ice was all melted.
Lysing erythrocytes and preparation of nuclei.
The thawed cells were transferred to a 15 ml conical tube. After addition of 5 mL of erythrocyte lysing solution (LYSE® III DIFF, Coulter Corp., Miami, FL, USA), the cells were thoroughly mixed with lysing solution by pipetting up and down 10–15 times and separated by centrifugation at 1600 rpm for 5 min. The cells were resuspended in hypotonic solution (0.075 M KCl) at 37°C for 10 min and then fixed with fixative solution (methanol: glacial acetic acid, 3:1, v/v). Fixed nuclei were spread onto positively charged slides for the FISH procedure. Wright staining of the cells was performed simultaneously to evaluate the proportion of intact nuclei among the extracted nuclei. The absolute number of extracted nuclei was determined using a Neubauer counting chamber.
FISH analysis.
The FISH probe used in this study was LSI AML1/ETO dual color, dual fusion DNA probe, which hybridizes to chromosome 8q22 (ETO Spectrum Orange) and chromosome 21q22 (AML1 Spectrum Green) (Vysis, Inc., Downers Grove, IL, USA). Microscope slides were prepared were by placing 8 µl of the nuclei suspension on a positively charged slide to enhance cell adhesion to the surface. The FISH procedure was then performed according to the manufacturer’s instructions.
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Results and Discussion
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Individual cell nuclei were successfully extracted and clear hybridization signals (2 orange and 2 green signals) were observed in all 30 specimens. The fluorescence intensity was unaffected by the duration of storage of the frozen buffy coat specimens (Fig. 1
). In one specimen, extracted nuclei were not individually separated and the hybridization signals were ambiguous on the first attempt. However, we achieved prominent signals after protease K treatment (37°C, 10 min) on the slide (Fig. 2). Although such proteolytic enzyme treatment might have been helpful in separation of each nuclei and hybridization of probe in interphase chromosomes, it was not required for all specimens, but optional only for microclotted specimens.
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Fig. 1. Examples of interphase FISH signals according to the age of specimens. With LSI AML1/ETO dual color, dual fusion DNA probe, all the cells tested showed 2 orange (8q22) and 2 green (21q22) signals. Each nucleus in this figure was extracted from frozen buffy coat stored for 1 day (A), 1 week (B), or 1 yr (C).
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Fig. 2. Effect of protease K treatment on a slide of poorly separated nuclei with ambiguous FISH signals. Extracted nuclei were not individually separated and the signals were ambiguous at the first attempt (A). After protease K treatment, the signals became prominent (B).
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The absolute counts of extracted nuclei and the percentages of intact nuclei in each specimen are listed in Table 1. The average number of extracted nuclei per specimen was 665 nuclei/µl, which was sufficient for FISH evaluation. On average, 79% of the nuclei maintained intact round morphology. As we did not count the number of cells before freezing the buffy coat, the recovery rate could not be calculated. As expected, the percentage of intact nuclei was higher in younger specimens (Table 1).
The plasma membrane of cells is known to be damaged by freezing [5], which may possibly aid in permeabilization of the FISH probe through the plasma membrane into nuclei in the process of FISH staining. In the present study, by use of commercially available red blood cell (RBC) lysing solution, we preserved the nuclear membrane of white blood cells without disrupting nuclear DNA. By this protocol, individual nuclei of hematologic cells can be isolated from archived frozen buffy coat specimens without any cryoprotectant, which facilitates the retrospective application of interphase FISH in clinical practice.
In conclusion, interphase FISH is possible with frozen-thawed buffy coat, which will be useful for retrospective molecular cytogenetic analysis of hematologic malignancies and constitutional anomalies including microdeletion syndromes.
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Acknowledgement
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This study was supported by the National Research Laboratory Program of the Korean Ministry of Science and Technology (M1-0302-00-0112).
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References
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- Jenkins RB, Le Beau MM, Kraker WJ, Borell TJ, Stalboerger PG, Davis EM, Penland L, Fernald A, Espinosa R 3rd, Schaid DJ. Fluorescence in situ hybridization: a sensitive method for trisomy 8 detection in bone marrow specimens. Blood 1992;15:3307–3315.
- Chen Z, Morgan R, Berger CS, Sandberg AA. Application of fluorescence in situ hybridization in hematological disorders. Cancer Genet Cytogenet 1992; 63:62–69.[Medline]
- Heerema NA, Argyropoulos G, Weetman R, Tricot G, Secker-Walker LM. Interphase in situ hybridization reveals minimal residual disease in early remission and return of the diagnostic clone in karyotypically normal relapse of acute lymphoblastic leukemia. Leukemia 1993;7:537–543.[Medline]
- Farkas DH, Drevon AM, Kiechle FL, DiCarlo RG, Heath EM, Crisan D. Specimen stability for DNA-based diagnostic testing. Diagn Mol Pathol 1997;5:227–235.
- McGann LE, Yang HY, Walterson M. Manifestations of cell damage after freezing and thawing. Cryobiology 1988;25:178–185.[Medline]
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Abstract
Introduction
