Assay characteristics

The method is developed for accurate analysis of intracellular proteins in combination with extracellular antigens. The method enables detection and quantification of apoptosis-related proteins in a bulk of cells, but more importantly in very small cell populations as has been demonstrated in this paper for Bcl-2, Bcl-x_L, Mcl-1 and Bax expression in de novo AML cells and in minimal residual disease (MRD) cells in BM samples of AML patients at several intervals after start of therapy.

Protocol

BM sample preparation

Mononuclear cells from BM samples of de novo patients were isolated by ficoll gradient centrifugation, whereafter residual erythrocytes were lysed with ammonium chloride for 10 min at 4°C and washed.

In follow-up AML BM samples erythrocytes were lysed with ammonium chloride for 10 min at 4°C and washed. All nucleated cells were used for the pursuing staining procedure.

Extracellular staining

Cells (0.1–1 10⁶) are labelled for 15 min at room temperature with PE-, PerCP-, and APC-conjugated MoAbs (5 l/MoAb) defining a leukemia-associated phenotype (LAP), consisting of aberrant antigen combinations that are not or in very low frequences present in normal BM.^{1,2,3,4,5,6,7,8,9} In all cases, the FL1 channel is reserved for subsequent labelling of the intracellular proteins using FITC-conjugated antibodies.

Intracellular staining

After the surface labelling for the LAP, cells are washed with PBS+0.1% BSA and fixed in 100 l 1% paraformaldehyde during 5 min at RT. Hereafter, cells are permeabilised with 100 l 0.1% saponin for 15 min at RT and washed. After permeabilisation, an incubation for 30 min at 4°C with either 50 l FITC-conjugated mouse anti-human Bcl-2 (1:20) or polyclonal nonconjugated rabbit anti-human Bax (1:20), Bcl-x_L (1:20) or Mcl-1 (1:20) is performed. For Bcl-2, cells are washed and analysed immediately. Cells that are incubated with the nonconjugated antibodies against Bax, Bcl-x_L, and Mcl-1 are washed, and incubated for another 30 min at 4°C with FITC-conjugated anti-rabbit IgG (1:20). Following the second step incubation, cells are washed and analysed immediately on the flow cytometer.

Quantification

In order to quantitate the levels of fluorescence in LAP+cells, relative apoptosis-related protein expression is calculated using the following formula:

Time requirement

Total time: 2.5–5 h. The time indicated includes preparation of cells (0–2 h), bench work (2 h), measurement on flow cytometer (10–30 min) and analysis (10–30 min).

Materials

Hardware

FACS Calibur flow cytometer equipped with a Red Diode Laser (Becton Dickinson, Mountain View, CA, USA).

Software

Cellquest and Paint-A-gate are both from Becton and Dickinson.

Reagents and solutions

Saponin was from Sigma-Aldrich Chemie (Steinheim, Germany).

Antibodies

FITC-conjugated mouse anti-human Bcl-2 (clone 124) and FITC-conjugated anti-rabbit were from Dako Diagnostics BV (Glostrup, Denmark). The rabbit polyclonal antibodies Bax (P-19), Bcl-x_L (S-18) and Mcl-1 (S-19) were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

References

1. Campana D, Pui CH. Detection of minimal residual disease in acute leukemia: methodologic advances and clinical significance. Blood 1995; 85: 1416–1434. | PubMed | ISI | ChemPort |
2. San Miguel JF, Martinez A, Macedo A, Vidriales MB, Lopez-Berges C, Gonzalez M et al. Immunophenotyping investigation of minimal residual disease is a useful approach for predicting relapse in acute myeloid leukemia patients. Blood 1997; 90:2465–2470. | PubMed | ChemPort |
3. Terstappen LW, Safford M, Konemann S, Loken MR, Zurlutter K, Buchner T et al. Flow cytometric characterization of acute myeloid leukemia. Part II. henotypic heterogeneity at diagnosis. Leukemia 1992; 6: 70–80. | PubMed |
4. Venditti A, Buccisano F, Del Poeta G, Maurillo L, Tamburini A, Cox C, Battaglia A et al. Level of minimal residual disease after consolidation therapy predicts outcome in acute myeloid leukemia. Blood 2000; 96: 3948–3952. | PubMed | ISI | ChemPort |
5. San Miguel JF, Vidriales MB, Lopez-Berges C, Diaz-Mediavilla J, Gutierrez N, Canizo C et al. Early immunophenotypical evaluation of minimal residual disease in acute myeloid leukemia identifies different patient risk groups and may contribute to postinduction treatment stratification. Blood 2001; 98: 1746–1751. | Article | PubMed | ISI | ChemPort |
6. San Miguel JF, Ciudad J, Vidriales MB, Orfao A, Lucio P, Porwit-MacDonald A et al. Immunophenotypical detection of minimal residual disease in acute leukemia. Crit Rev Oncol Hematol 1999; 32: 175–185. | PubMed |
7. Macedo A, Orfao A, Ciudad J, Gonzalez M, Vidriales B, Lopez-Berges MC et al. Phenotypic analysis of CD34 subpopulations in normal human bone marrow and its application for the detection of minimal residual disease. Leukemia 1995; 9: 1896–1901. | PubMed | ISI | ChemPort |
8. Yin JA, Tobal K. Detection of minimal residual disease in acute myeloid leukemia: methodologies,clinical and biological significance. Br J Haematol 1999; 106: 578–590. | Article | PubMed | ChemPort |
9. Scolnik MP, Morilla R, de Bracco MM, Catovsky D, Matutes E. CD34 and CD117 are overexpressed in AML and may be valuable to detect minimal residual disease. Leukemia Res 2002; 26: 615–619.