Morphology Antibody Chromosome Technique for Determining Phenotype and Genetic Status of the Same Cell

Sakari Knuutila1, Satu Mustjoki2

1 University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland, 2 Department of Medicine, Division of Hematology, Helsinki University Central Hospital, Helsinki, Finland
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 4.7
DOI:  10.1002/0471142905.hg0407s74
Online Posting Date:  July, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The morphology antibody chromosome (MAC) technique is a combination of methods that permits analysis of both phenotypic and genetic evaluation on a single interphase or mitotic cell as a basis for lineage analysis of neoplastic and normal cells. This unit describes MAC with sequential phenotypic analysis using antibody and an alkaline phosphatase anti‐alkaline phosphatase (APAAP) complex and genotypic analysis using in situ hybridization with either enzymatic or fluorescence detection. Alternate methods for phenotypic analysis are also described, which include the use of horseradish peroxidase–conjugated antibodies, fluorochrome‐conjugated antibodies, May‐Grunwald‐Giemsa cytochemical staining, and Sudan black B cytochemical staining. An additional protocol describes G‐ and C‐banding as alternatives to in situ hybridization (ISH) for genotyping MAC specimens. Support protocols describe methods for preparing specimens, cytospin preparations, in situ cultures, paraffin‐embedded or cryostat sections, and blood and bone marrow smears. Also described is a procedure for chromosome painting of previously GTG‐banded slides. An additional protocol is included for FISH analysis on sorted hematopoietic stem cells and its application in the detection of leukemic stem cells. For overcoming the drawbacks of scarcity and variability from case to case of malignant plasma cells in multiple myeloma FISH analyses, a protocol is included for the enrichment of plasma cells by immunomagnetic beads. Curr. Protoc. Hum. Genet. 74:4.7.1‐4.7.43. © 2012 by John Wiley & Sons, Inc.

Keywords: lineage specificity; immunophenotype; genomic alterations; chromosomes; fluorescence in situ hybridization; hematopoietic stem cells; multiple myeloma

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Sequential MAC Analysis Using APAAP Immunostaining and In Situ Hybridization
  • Alternate Protocol 1: Immunophenotyping Using HRPO‐Based Detection
  • Alternate Protocol 2: Immunophenotyping Using Fluorescence Detection
  • Alternate Protocol 3: Automated Capture of Immunofluorescence‐Positive Interphase Cells and Relocalization of the Cells After Fish Signal Detection
  • Alternate Protocol 4: Phenotyping Using May‐Grünwald‐Giemsa Cytochemical Stain
  • Alternate Protocol 5: Phenotyping Using Sudan Black B Cytochemical Stain
  • Alternate Protocol 6: Genetic Analysis by G‐ or C‐Banding of Chromosomes
  • Support Protocol 1: Preparation of Cytospin Slides
  • Support Protocol 2: Preparation of In Situ Cultures
  • Support Protocol 3: Preparation of Tissue Sections
  • Support Protocol 4: Preparation of Blood and Bone Marrow Smears
  • Basic Protocol 2: In Situ Hybridization on Previously GTG‐Banded Chromosomes
  • Basic Protocol 3: Fluorescence In Situ Hybridization on Sorted CD34+CD38– and CD34+CD38+ Hematopoietic Stem Cells
  • Basic Protocol 4: Fluorescence In Situ Hybridization on Sorted CD138+ Plasma Cells in Multiple Myeloma
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Sequential MAC Analysis Using APAAP Immunostaining and In Situ Hybridization

  Materials
  • Air‐dried slide containing cells for analysis (see Support Protocols protocol 81 to protocol 114)
  • Formaldehyde‐buffered acetone fixative (see recipe), 4°C
  • 0.05% (v/v) Triton X‐100 (BDH) in PBS (25 µl Triton X‐100 in 50 ml PBS)
  • PBS ( appendix 2D)
  • TBS/FBS (see recipe)
  • Mouse anti‐human primary antibody diluted 1:10 to 1:50 in TBS/FBS
  • Rabbit anti‐mouse Ig antiserum solution (see recipe)
  • APAAP mouse immunocomplex (Dakopatts) diluted 1:25 to 1:50 in TBS/FBS
  • APAAP substrate solution (see recipe; prepare immediately before use)
  • 5% Giemsa stain (see recipe)
  • 95% ethanol
  • 3:1 (v/v) methanol/acetic acid fixative
  • 0.01 N HCl
  • 0.1 to 1 mg/ml pepsin in 0.01 N HCl
  • Biotin‐ or digoxigenin‐labeled repeat‐sequence probe, single‐copy probe, or whole‐chromosome paint probe (unit 4.3)
  • Harris hematoxylin stain (Papanicolaou solution, Merck) diluted 1:1 in H 2O
  • 0.0025% (v/v) ammonia solution (10 µl 25% ammonia in 100 ml H 2O)
  • Mounting medium: Entellan (for enzymatic detection; Merck) or antifade mounting medium (for fluorescence detection; unit 4.3)
  • Coplin jars
  • Cytospin filters (Shandon/Lipshaw)
  • Moist chamber (Fig. )
  • 0.45‐µm filter membranes (Millipore)
  • Bright‐field microscope equipped with camera, stage micrometer, and 100× dry objective
  • Microscope equipped with camera, stage micrometer, fluorescence epi‐illumination, appropriate filter sets (see unit 4.4), and 100× objective (optional, for fluorescence detection)
  • Additional reagents and equipment for in situ hybridization with enzymatic detection (unit 4.3, protocol 1 and protocol 2) or fluorescence detection (unit 4.3, protocol 1) with signal amplification (unit 4.3, protocol 8) and DAPI or propidium iodide counterstaining (unit 4.3, protocol 1)

Alternate Protocol 1: Immunophenotyping Using HRPO‐Based Detection

  • 1% (v/v) H 2O 2 in methanol
  • Acetone/formaldehyde fixative: 0.007% to 0.03% (v/v) formaldehyde in acetone (10 to 40 µl 37% formaldehyde in 50 ml acetone), 4°C
  • PBS ( appendix 2D)
  • PBS/0.08% FBS: 0.08% (v/v) filter‐sterilized (1.2‐µm filter) FBS in PBS ( appendix 2D)
  • Horse serum diluted 1:50 in PBS/5% (v/v) FBS
  • PBS/0.2% FBS: 0.2% (v/v) filter‐sterilized FBS in PBS
  • Biotinylated anti‐mouse IgG diluted 1:250 in PBS/5% FBS
  • ABC kit (Vectastain) containing avidin DH and biotinylated horseradish peroxidase H (HRPO)
  • PBS/5% FBS: 5% (v/v) filter‐sterilized FBS in PBS
  • Immunoperoxidase substrate solution (see recipe)

Alternate Protocol 2: Immunophenotyping Using Fluorescence Detection

  • Acetone/formaldehyde fixative: 0.007% to 0.03% (v/v) formaldehyde in acetone (10 to 40 µl 37% formaldehyde in 50 ml acetone), 4°C
  • PBS/0.2% FBS: 0.2% (v/v) filter‐sterilized FBS in PBS ( appendix 2D)
  • Mouse anti‐human primary antibody diluted 1:10 to 1:30 in PBS/0.2% FBS
  • Fluorochrome‐conjugated goat anti‐mouse IgG secondary antibody (Cappel) diluted 1:200 in PBS/0.2% FBS
  • 0.5% (w/v) quinacrine dihydrochloride (unit 4.3)

Alternate Protocol 3: Automated Capture of Immunofluorescence‐Positive Interphase Cells and Relocalization of the Cells After Fish Signal Detection

  • Slide with cells for analysis
  • Vectashield mounting medium with DAPI
  • Immersion oil (Zeiss Immersol 518F)
  • 3:1 (v/v) methanol/acetic acid
  • 0.01 N HCl
  • 23 × 50–mm coverslips
  • Metafer scanning platform (MetaSystems; http://www.metasystems.de/); basic station comprised of:
    • Motorized Zeiss Axioplan 2 imaging microscope with motorized Märzhäuser scanning stage for 8 slides
    • CCD camera
    • Trackball
    • PC central unit (with keyboard and mouse)
    • Monitor
    • Printer
  • Metafer software with RCDetect (for detection and relocation of rare cells) and MetaCyte (for automatic multiparametric cell feature analysis) modules

Alternate Protocol 4: Phenotyping Using May‐Grünwald‐Giemsa Cytochemical Stain

  • Methanol
  • May‐Grünwald stain: May‐Grünwald eosin–methylene blue containing 50% (v/v) methanol (Merck)
  • Phosphate‐buffered water (PBW; see recipe)
  • Giemsa stain: 1.5 ml Giemsa (Merck) diluted in 60 ml PBW (see recipe; store stain ≤4 days at 4°C)

Alternate Protocol 5: Phenotyping Using Sudan Black B Cytochemical Stain

  • 37% (w/v) formaldehyde, 4°C
  • Sudan black B stain (see recipe), 20°C
  • 70% ethanol
  • 0.5% (w/v) quinacrine dihydrochloride (unit 4.3; optional)

Alternate Protocol 6: Genetic Analysis by G‐ or C‐Banding of Chromosomes

  • 60°C oven
  • Additional reagents and equipment for G‐ or C‐banding (unit 4.2)

Support Protocol 1: Preparation of Cytospin Slides

  Materials
  • Cells, e.g., heparinized blood, bone marrow specimen, suspended tumor cells, or cultured cells
  • Complete RPMI/20% FBS medium ( appendix 3G)
  • Mitogen: 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA, Sigma; for B lymphocytes), phytohemagglutinin (PHA, Sigma; for T lymphocytes), or other mitogen or growth factors (optional)
  • Colcemid (optional)
  • MAC hypotonic solution (see recipe), 20°C
  • 50‐ml tissue culture flask (Nunc)
  • 37°C, 5% CO 2 humidified incubator
  • 10‐ml disposable conical polypropylene centrifuge tubes
  • Cytospin 2 (Shandon/Lipshaw)
  • Ethanol‐cleaned glass slides
  • Cytospin filters (Shandon/Lipshaw)
  • Additional reagents and equipment for Ficoll‐Hypaque density‐gradient centrifugation of cells (unit 10.4) and counting cells using a hemacytometer ( appendix 3G)

Support Protocol 2: Preparation of In Situ Cultures

  Materials
  • Tumor cell suspension, or other cells for monolayer culture
  • Complete RPMI/20% FBS medium ( appendix 3G)
  • Colcemid
  • MAC hypotonic solution (see recipe)
  • 0.01 N HCl, 37°C
  • Pepsin: 0.1 to 1 mg/ml in 0.01 N HCl, 37°C
  • 15‐ml flaskette chamber slide (Nunc)
  • 37°C, 5% CO 2 humidified incubator
  • 72°C oven
  • Additional reagents and equipment for isolation and culture of cells (see protocol 8 and appendix 3G) and phenotyping and genetic study of cells for MAC analysis (see protocol 1 or 2 or Alternate Protocols protocol 21 to protocol 65)

Support Protocol 3: Preparation of Tissue Sections

  Material
  • 5‐µm paraffin‐embedded or cryostat tissue sections
  • Poly‐L‐lysine‐coated microscope slides (see recipe)
  • Xylene
  • Graded alcohol series: 70%, 95%, and 100% ethanol
  • 1% (v/v) H 2O 2 in methanol
  • 100% methanol
  • 1 M sodium thiocyanate, 72°C
  • 0.01 N to 0.1 N HCl, 37°C
  • Pepsin: 1 to 5 mg/ml in 0.01 N to 0.1 N HCl, 37°C
  • 60°C and 72°C ovens

Support Protocol 4: Preparation of Blood and Bone Marrow Smears

  Materials
  • Heparinized blood or bone marrow aspirate
  • 0.01 N HCl, 37°C
  • Pepsin: 0.1 to 1 mg/ml in 0.01 N HCl, 37°C
  • Ethanol‐cleaned microscope slides

Basic Protocol 2: In Situ Hybridization on Previously GTG‐Banded Chromosomes

  Materials
  • Unmounted or Entellan‐mounted slide with GTG‐banded metaphase cells (unit 4.2)
  • Xylene
  • Graded alcohol series: 70%, 80%, 90%, and 100% ethanol
  • Formaldehyde‐buffered acetone fixative (see recipe), 4°C
  • 2× SSC ( appendix 2D)/0.2% (v/v) Tween, 42°C
  • Whole‐chromosome paint probe (unit 4.3)
  • Coplin jars
  • Additional reagents and equipment for fluorescence in situ hybridization (FISH; unit 4.3)

Basic Protocol 3: Fluorescence In Situ Hybridization on Sorted CD34+CD38– and CD34+CD38+ Hematopoietic Stem Cells

  Materials
  • Fresh bone marrow samples
  • PBS/2 mM EDTA/0.5% BSA washing buffer (see recipe)
  • MidiMACS kit (Miltenyi Biotech) containing:
    • Fc receptor (FcR) blocking reagent (human IgG)
    • CD34 MicroBeads
    • MidiMACS columns
    • Magnetic holder
  • PBS ( appendix 2D)
  • CD34FITC antibody (BD, cat. no. 345801)
  • CD38PE antibody (BD, cat. no. 345806)
  • FACS buffer: PBS and 2 mM EDTA
  • Fresh methanol
  • Glacial acetic acid
  • Vysis BCR/ABL translocation probe (Abbott Molecular, cat. no. 31‐191032, dual color, dual fusion)
  • Hybridization buffer for probe (Abbott Molecular, cat. no. 30‐805826)
  • 0.01 M HCl: 10 ml 1 M HCl in 990 ml of distilled water
  • Pepsin (20 mg/ml)
  • 0.1× and 2× SSC ( appendix 2D)
  • 0.07 M NaOH
  • 70%, 96%, and absolute ethanol
  • Glue (e.g., Marabun Fixocum rubber cement)
  • 0.4× SSC/0.3% Igepal (or Tween, NP‐40) (20 ml 20× SSC and 3 ml 100% Igepal in 977 ml distilled water)
  • 2× SSC/0.1% Igepal (or Tween, NP‐40) (100 ml 20× SSC and 1 ml 100% Igepal in 899 ml distilled water)
  • VECTASHIELD mounting medium with DAPI (Vector Laboratories, cat. no. H‐1200)
  • Microscope and hemacytometer
  • Rotator, 4°C
  • Refrigerated centrifuge
  • 10‐ml tubes
  • Flow cytometer
  • FACS tubes
  • Cytospin slides (e.g., Thermo Shandon cytoslides, cat. no. 5991056)
  • 75°C heating block
  • 37°C incubator
  • 70°C water bath containing a Coplin jar for slide incubation
  • 22 × 22–mm (or smaller) glass coverslip
  • Hybridization box
  • Fluorescence microscope (60× or 100× magnification using appropriate filters detecting spectrum green and spectrum orange fluorochromes)
  • Additional reagents and equipment for Ficoll‐Hypaque density‐gradient centrifugation (unit 10.4)

Basic Protocol 4: Fluorescence In Situ Hybridization on Sorted CD138+ Plasma Cells in Multiple Myeloma

  Materials
  • Heparinized bone marrow aspirates
  • Additional reagents and equipment for density gradient centrifugation, immunomagnetic bead sorting, cytospin preparation, and FISH (see protocol 13)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Böyum, A. 1968. Isolation of leukocytes from human blood. Further observations. Scan. J. Clin. Lab. Invest. 21:31‐50.
   Cordell, J.L., Falini, B., Erber, W.N., Ghosh, A.K., Abdulaziz, Z., MacDonald, S., Pulford, K.A.F., Stein, H., and Mason, D.Y. 1984. Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti‐alkaline phosphatase (APAAP complexes). J. Histochem. Cytochem. 32:219‐229.
   El‐Rifai, W. and Knuutila, S. 1996. Fluorescent in situ hybridization on archival G‐banded slides. Cytogenet. Cell Genet. 73:322‐324.
   Funderud, S., Nustad, K., Lea, T., Vartdal, F., Gaudernack, G., Stenstad, P., and Ugelstad, J. 1987. Fractionation of lymphocytes by immunomagnetic beads. In Lymphocytes: A Practical Approach (G.G.B. Klaus, ed.) pp. 55‐65. IRL Press, Oxford.
   Kaplow, L.S. 1975. Substitute for benzidine in myeloperoxidase stains. Am. J. Clin. Pathol. 63:451.
   Kere, J., Knuutila, S., Ruutu, T., Leskinen, R., and de la Chapelle, A. 1988. Monocytic involvement by monosomy 7 preceded acute myelomonocytic leukemia in a patient with myelodysplastic syndrome. Leukemia 2:69‐73.
   Kibbelaar, R.E., van Kamp, H., Dreef, E.J., de Groot‐Swings, G., Kluin‐Nelemans, J.C., Beverstock, G.C., Fibbe, W.E., and Kluin, P.M. 1992. Combined immunophenotyping and DNA in situ hybridization to study lineage involvement in patients with myelodysplastic syndromes. Blood 79:1823‐1828.
   Knuutila, S. 1997. Lineage specificity in haematological neoplasms. Review article. Br. J. Haematol. 96:2‐11.
   Knuutila, S., Nylund, S.J., Wessman, M., and Larramendy, M.L. 1994a. Analysis of genotype and phenotype on the same interphase or mitotic cell. A manual of MAC (Morphology Antibody Chromosomes) methodology. Cancer Genet. Cytogenet. 72:1‐15.
   Knuutila, S., Teerenhovi, L., Larramendy, M.L., Elonen, E., Franssila, K., Nylund, S.J., Timonen, T., Heinonen, K., Mahlamäki, E., Winqvist, R., and Ruutu, T. 1994b. Cell lineage involvement of recurrent chromosomal abnormalities in hematologic neoplasms. Genes Chrom. Cancer 10:95‐102.
   Larramendy, M.L., Kovanen, P.E., and Knuutila, S. 1992. MAC (Morphology, Antibody, Chromosomes) method for study of cell proliferation in unfractionated human hematopoietic cell cultures. J. Histotechnol. 15:31‐38.
   Martín‐Subero, J.I., Chuboda, I., Harder, L., Gesk, S., Grote, W., Novo, F.J., Calasanz, M.J., and Siebert, R. 2002. Multicolor‐FICTION: Expanding the possibilities of combined morphologic, immunophenotypic, and genetic single cell analyses. Am. J. Pathol. 161:413‐420.
   Murdoch, A., Jenkinson, E.J., Johnson, G.D., and Owen, J.J.T. 1990. Alkaline phosphatase‐fast red, a new fluorescent label. Application in double labeling for cell surface antigen and cell cycle analysis. J. Immunol. Methods 132:45‐49.
   Mustjoki, S., Rohon, P., Rapakko, K., Jalkanen, S., Koskenvesa, P., Lundán, T., and Porkka, K. 2010. Low or undetectable numbers of Philadelphia chromosome positive leukemic stem cells (Ph+CD34+CD38neg) in chronic myeloid leukemia patients in complete cytogenetic remission after tyrosine kinase inhibitor therapy. Leukemia 24:219‐222.
   Perry, P.E. and Thomson, E.J. 1986. Immunogold labeling of metaphase cells. Cytogenet. Cell Genet. 41:121‐125.
   Price, C.M., Kanfer, E.J., Colman, S.M., Westwood, N., Barrett, A.J., and Greaves, M.F. 1992. Simultaneous genotypic and immunophenotypic analysis of interphase cells using dual‐color fluorescence: A demonstration of lineage involvement in polycythemia vera. Blood 80:1033‐1038.
   Schlegelberger, B., Weber‐Matthiesen, K., Eumann, S., Rathjen, M., and Grote, W. 1992. Detection of structural chromosome aberrations in immunophenotyped mitoses. Cytogenet. Cell Genet. 59:10‐11.
   Sikri, K.L., Foster, C.L., MacHugh, N., and Marshall, R.D. 1981. Localization of Tamm‐Horsfall glycoprotein in the human kidney using immuno‐fluorescence and immuno‐electron microscopical techniques. J. Anat. 132:597‐605.
   Stenman, S., Rosenqvist, M., and Ringertz, R. 1975. Preparation and spread of unfixed metaphase chromosomes for immunofluorescence staining of nuclear antigens. Exp. Cell Res. 90:87‐94.
   Strehl, S. and Ambros, P.F. 1993. Fluorescence in situ hybridization combined with immunohistochemistry for highly sensitive detection of chromosome 1 aberrations in neuroblastoma. Cytogenet. Cell Genet. 63:24‐28.
   Tarkkanen, M., Böhling, T., Heliö, H., Karaharju, E., Kaipainen, A., Szymanska, J., Elomaa, I., and Knuutila, S. 1993. A recurrent chondromyxoid fibroma with chromosome aberration ins(5;2) (q13;p21p25) and 2p deletion: A case report. Cancer Genet. Cytogenet. 65:141‐146.
   Teerenhovi, L., Knuutila, S., Ekblom, M., Rossi, L., Borgström, G.H., Tallman, J.K., Andersson, L., and de la Chapelle, A. 1984. A method for simultaneous study of the karyotype, morphology, and immunologic phenotype of mitotic cells in hematologic malignancies. Blood 64:1116‐1122.
   Thompson, F.H. 1991. Cytogenetic methodological approaches and findings in human solid tumors. In The ACT Cytogenetics Laboratory Manual, Second Edition (M.J. Barch, ed.) pp. 451‐488. Raven Press, New York.
   Tiainen, M., Popp, S., Parlier, V., Emmerich, P., Bellomo, M., Ruutu, T., Cremer, T., and Knuutila, S. 1992. Chromosomal in situ suppression hybridization of immunologically classified mitotic cells in hematologic malignancies. Genes Chrom. Cancer 4:135‐140.
   van den Berg, H., Vossen, J.M., Langlois van den Bergh, R., Bayer, J., and van Tol, M.J.D. 1991. Detection of Y chromosome by in situ hybridization in combination with membrane antigens by two‐color immunofluorescence. Lab. Invest. 64:623‐628.
   von Koskull, H. and Gahmberg, N. 1995. Fetal erythroblasts from maternal blood identified with 2,3‐ bisphosphoglycerate (BPG) and in situ hybridization (ISH) using Y‐specific probes. Prenatal Diagn. 15:149‐154.
   Waye, J.S., England, S.B., and Willard, H.F. 1987. Genomic organization of alpha satellite DNA on human chromosome 7: Evidence for two distinct alphoid domains on a single chromosome. Mol. Cell Biol. 7:349‐356.
   Weber‐Matthiesen, K., Winkemann, M., Müller‐Hermelink, A., Schlegelberger, B., and Grote, W. 1992. Simultaneous fluorescence immunophenotyping and interphase cytogenetics: A contribution to the characterization of tumor cells. J. Histochem. Cytochem. 40:171‐175.
   Weber‐Matthiesen, K., Deerberg, J., Müller‐Hermelink, A., Schlegelberger, B., and Grote, W. 1993a. Rapid immunophenotypic characterization of chromosomally aberrant cells by the FICTION method. Cytogenet. Cell Genet. 63:123‐125.
   Weber‐Matthiesen, K., Pressl, S., Schlegelberger, B., and Grote, W. 1993b. Combined immunophenotyping and interphase cytogenetics on cryostat sections by the new FICTION method. Leukemia 7:646‐649.
   Ziomek, C.A., Lepire, M.L., and Torres, I. 1990. A highly fluorescent simultaneous azo dye technique for demonstration of nonspecific alkaline phosphatase activity. J. Histochem. Cytochem. 38:437‐442.
Key References
   Knuutila et al., 1994a. See above.
  Compact review of simultaneous analysis of phenotype and genotype on a single cell.
   Knuutila, 1997. See above.
  Review of lineage specificity in hematologic neoplasms illustrating the power of MAC for study of tumor heterogeneity.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library