Minimal residual disease (MRD) cells are thought to be responsible for the persistence and relapse of acute myeloid leukemia (AML). Flow cytometric MRD detection by the establishment of a leukemia-associated phenotype (LAP) at diagnosis can be used in 80% of AML patients, allowing detection and functional characterization of MRD in follow-up bone marrow. One of the mechanisms contributing to inefficient chemotherapy is apoptosis resistance. Measuring apoptosis parameters in MRD cells will help to unravel the importance of apoptosis resistance in AML. We therefore developed a four-color flow cytometry method that enables establishment of apoptosis-related protein expression such as Bcl-2, Bcl-xL, Mcl-1 and Bax at diagnosis and in MRD. Firstly, validation of this assay using Western blot analysis in five leukemia cell lines showed a significant correlation (R=0.70: P<0.0001). Secondly, the influence of the permeabilization procedure on LAP expression was investigated in 38 AML samples at diagnosis and in 42 MRD samples. Quantification of the frequency of LAP+ cells with and without permeabilization showed no significant differences (diagnosis: P= 0.57, follow-up: P= 0.43). The flow cytometric protocol thus enables analysis of apoptosis-related proteins at different stages of the disease, which will lead to a better understanding of the role of apoptosis resistance in the emergence of MRD in AML.
Complete remission rates as high as 70–80% have been reported in adult patients with acute myeloid leukemia (AML),1,2,3 but approximately 60–70% of the patients will eventually relapse because of the persistence of residual leukemia cells. These minimal residual disease (MRD) cells are undetectable by conventional morphological techniques. Detection of MRD at different stages of the disease, that is, after chemotherapy or in stem cell transplants, can provide useful information to tailor therapy, such as early intervention or purging of the stem cell graft. Several molecular and immunological methods have been developed to detect MRD cells, among which PCR and flow cytometry are the most suitable. Detection by flow cytometry (FC) is based on the idea that AML cells frequently show aberrant leukemia-associated phenotypes (LAPs) characterized by asynchronous antigen expression, crosslineage antigen expression and antigen overexpression.4,5,6,7,8,9,10,11,12 Although the use of FC has some drawbacks such as interpatient heterogeneity in antigen expression that requires a large panel of monoclonal antibodies (MoAbs) to cover all potential aberrancies, or the occurrence of phenotypic shifts,13,14,15 the FC method is very quick, sensitive and quantitative. Moreover, it is the only method that allows functional analysis on viable MRD cells thereby enabling to gain insight into the mechanisms by which the leukemia cells survive. These mechanisms include apoptosis resistance. A great deal of apoptosis research in AML has been focused on apoptosis-related proteins in de novo AML cells, and showed that AML patients with high Bcl-2 levels or high Bcl-2/Bax ratios at diagnosis have a poor clinical outcome.16,17,18,19,20 Analysis of apoptosis-related proteins in MRD cells that survived chemotherapy would offer insight into the role of apoptosis resistance in treatment failure in AML. For example, it might reveal characteristics present only in subpopulations at presentation of the disease. The aim of the present study was therefore to design a technique that enables detection and quantification of apoptosis-related proteins such as Bcl-2, Bcl-xL, Bax, Mcl-1 and p53 in MRD cells. With the conventional Western blot method, this would not be possible because of the limited number of MRD cells available. We have recently developed a technique that allows the detection of ABC transporter activity in MRD (van der Pol et al,21 Haematologica, 2003, 88, in press). The present approach was aimed to characterize the role of apoptosis-related proteins in MRD in AML. We have set up a four-color FC method and determined in five cell lines whether this method could reliably replace Western blot analysis and whether the permeabilization procedure necessary for the detection of intracellular apoptosis-related proteins would influence the profiles of cell surface antigen expression required for the identification of MRD cells.
Materials and methods
Human AML cell lines HL60, THP-1, KG1, the chronic myeloid leukemia blast crisis cell line K562 and the human histiocytic lymphoma cell line U937 were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). All cell lines, except KG-1 that was cultured in DMEM supplemented with 20% FCS, were cultured in RPMI supplemented with 10% FCS at 37°C in a humid atmosphere with 5% CO2. The cells were maintained at 0.1–1 × 106 cells/ml and were adjusted to a concentration of 0.3 × 106 cells/ml 24 h before experimental use.
Following informed consent, AML bone marrow samples were obtained at the time of diagnosis, at follow-up (after first, second and third cycle of chemotherapy, peripheral blood stem cell transplants, at regular time points after completion of treatment) and at relapse. FAB classification was as follows: M0 (n=5), M1 (n=5), M2 (n=8), M4 (n=3), M4eo (n=3), M5 (n=8), M6 (n=1), RAEB-t (n=2), unknown (n=3).
Unlabeled p53 (DO-7), unlabeled Bcl-2 (clone 124) mouse monoclonal antibody, FITC-conjugated mouse anti-human Bcl-2 (clone 124) and FITC-conjugated anti-rabbit were from Dako Diagnostics BV, The Netherlands. The rabbit polyclonal antibodies Bax (P-19), Bcl-xL (S-18) and Mcl-1 (S-19) were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
The MRD monoclonal antibody panel consisted of the following FITC-conjugated MoAbs: CD2 (T11, Coulter, Coultronics, Mergency, France), CD5 (DK23, Dako), CD7 (CLB-T-3A1/1,7, CLB, Amsterdam, The Netherlands), CD11c (KB90, Dako), CD13 (WM-47, Dako), CD15 (MMA, BD, Becton Dickinson, San Jose, CA, USA), CD19 (J4.119, Immunotech, Marseille, France), CD33 (CD33-4D3, Caltag, Caltag Laboratories, San Francisco, CA, USA), CD34 (8G12, BD), CD38 (T16, Immunotech), CD45 (J33, Immunotech), CD61 (RUUPL7F12, BD), CD65 (VIM2/GM-4102, AnderGrub, Kaumberg, Austria). PE-conjugated MoAbs were: CD2 (T11, Coulter), CD7 (8h8.1, Immunotech), CD11b (D12, BD), CD13 (My7, Coulter), CD15 (80H5, Immunotech), CD33 (My9, Coulter), CD34 (8G12, BD), CD38 (HB7, BD), CD45 (T29/33, Dako), CD56 (My31, BD), CD117 (104D2, BD), CD133 (CD133/1, Miltenyi Biotech, Bergisch Gladbach, Germany). PerCP-conjugated MoAbs were: CD4 (2D1, BD), CD19 (SJ25C1, BD), CD34 (8G12, BD), CD45 (2D1, BD), HLA-DR (L243, BD). APC-conjugated MoAbs were: CD2 (G11, Imgen, Antwerpen, Belgium), CD13 (22A5, Imgen), CD14 (MOP9, BD), CD19 (SJ25C1, BD), CD33 (P67.6, BD), CD34 (8G12, BD), CD38 (HB7, BD), CD45 (J33/2D1, Immunotech/BD), CD117 (104D2, BD), HLA-DR (T436, Caltag).
NaVO3, NaCl, Tris-HCl, EDTA, DTT, SDS, bromophenol blue, saponin and Triton X-100 were purchased from Sigma-Aldrich Chemie (Steinheim, Germany) and protease inhibitors (complete mini protease inhibitors) from Boehringer Mannheim, Mannheim, Germany. Molecular weight marker was the Bio-Rad rainbow marker (Bio-Rad Laboratories Ltd, Hertfordshire, UK).
Western blot analysis
Cell pellets of 1–5 × 106 cells were immediately frozen in liquid nitrogen and stored at –80°C until analysis. Frozen–thawed cell pellets were lysed in 50 μl/2 × 106 cells of lysing solution containing 0.06 mM NaVO3, 176 mM NaCl, 11.8 mM Tris-HCl pH 7.8, 5.9 mM EDTA, 1.2% Triton X-100 and protease inhibitors at 4°C for 30 min. To estimate the total protein level, a commercially available kit that employs the Bradford method22 was used. Sample buffer containing 0.19 M Tris-HCl pH 6.8, 30 mM DTT, 2% SDS, 0.1% bromophenol blue and 30% glycerol were added to the lysate in a ratio of lysate:sample buffer of 2:1. The samples were incubated at 100°C for 5 min. HL60 cell line was used as a positive control for Bcl-2, Bax, Bcl-xL and Mcl-1 expression and KG1 cell line was used as a positive control for p53 expression. After lysis, the cells were appropriately diluted in sample buffer so as to give a five-point standard curve for each blot by loading 20 μl containing 5, 10, 15, 20 and 25 μg (HL60) or 1, 3, 5, 7 and 10 μg (KG1) of total cellular protein. Each sample was subjected to SDS-polyacrylamide gel electrophoresis according to the method of Laemmli23 using the mini-protean II system supplied by Bio-Rad Laboratories Ltd (Hertfordshire, UK). Acrylamide gels (15%) were used for the analysis of all proteins. After electrophoresis, proteins were transferred to nitrocellulose membranes (Hybond-C; Amersham, Little Chalfont, UK) using the mini-protean II system (Bio-Rad Laboratories Ltd). After transfer, the blots were immersed for 1 h in a blocking buffer of 2% BSA in PBS containing 0.1% Tween (PBS-T). Primary antibody was diluted in blocking buffer and incubated with the blots for 1 h as follows: Bcl-2, Bax, Bcl-xL and Mcl-1 at 1:500 and p53 at 1:2000. The blots were washed thoroughly with PBS-T and incubated with secondary antibody (horseradish peroxidase (HRP) labeled anti-mouse or HRP labeled anti-rabbit, diluted 1:1000 in PBS-T) for 1 h. After a final wash in PBS-T, HRP was detected using Lumi-Light reagent (Boehringer Mannheim, Mannheim, Germany) as described in the manufacturer's instructions. Chemiluminescence was detected by autoradiography using X-ray film. The integrated optical density (IOD) of the resulting bands was determined by densitometry using a Bio-Rad densitometer. The identity of the target protein was determined on the basis of a molecular weight marker. A standard curve of the positive reference cell line was produced for each blot by plotting the IOD of each standard band against the amount (μg) of total protein cell lysate loaded. For each cell line, the IOD was compared with the standard curve of the positive control cell lines. The amount of specific protein in the cell line was then presented as a ratio relative to the control cell line. This way of quantification, instead of using a house-keeping protein, was chosen to avoid that differences in size and volume, as in primary AML samples, contribute to a quantification error.
Establishment of LAPs and MRD detection
The phenotypical analysis of de novo AML was performed on whole bone marrow or mobilized peripheral blood samples upon staining with FITC-, PE-, PerCP- and APC-conjugated MoAbs. A detailed description of the establishment of a LAP and MRD detection has previously been published.21,24
Apoptosis-related protein detection in cell lines, de novo AML and in MRD by flow cytometry
For a detailed description of the procedure and quantification of apoptosis-related protein expression, see the appendix, ‘Method in focus’.
The Spearman ρ correlation test was used to show the correlation between the FC and Western blot analysis. The Wilcoxon signed ranks test was performed to compare MRD percentages with and without the permeabilization procedure. All statistical analyses were performed using the SPSS software program. A P-value <0.05 was considered as significant.
Results and discussion
Comparison of apoptosis-related protein detection by flow cytometry and Western blot analysis
To prove that the flow cytometric method described here can be reliably used in AML samples at diagnosis and in MRD in AML, flow cytometric detection of apoptosis-related proteins was first compared with Western blot analysis, which we considered to be the golden standard. The methods showed to correlate well in a linear fashion for Bcl-2, Bcl-xL, Mcl-1, Bax and p53 (R=0.7; P<0.0001). We conclude that Bcl-2, Bcl-xL, Mcl-1, Bax and p53 can be reliably detected by flow cytometry, which is in accordance with a recent study in AML only investigating Bcl-2.25
Morphological and phenotypical preservation after permeabilization procedure
If simultaneous analysis of apoptosis-related proteins and MRD by FC is to be performed accurately, the membrane permeabilization step required for intracellular protein detection should not modify the cell surface labeling. Although the FSC/SSC characteristics undergo changes by the permeabilization procedure, it is possible to exclude debris characterized by a low FSC/SSC (Figures 1a and b). After the permeabilization procedure, the blast population, characterized by a low CD45 expression and a low SSC (Lacombe et al,26 see Figures 1c and d), is comparable to the fresh sample as shown in Figure 1g. Furthermore, Figures 1c and d illustrate the usefulness of the CD45 staining in combination with SSC to exclude neutrophils, monocytes and lymphocytes from MRD phenotyping. This gating is of particular importance since it has been described that neutrophils differ considerably of normal and leukemia progenitor cells in Bcl-2 and Bcl-xL mRNA content.27
Since the identification of MRD relies on the detection of LAPs,21,24 the effect of the permeabilization procedure on the phenotypical analysis of diagnosis and follow-up bone marrow AML material was studied. Figures 1e and f illustrate the approach using an AML with blasts expressing CD117 with coexpression of CD7 (LAP: CD117+ CD7+) at diagnosis. A small influence of the permeabilization procedure on the expression of the surface antigens CD117 and CD7 can be seen, although the percentages within the selected gates did not change (Figure 1e–g). To determine the preservation of all surface antigens involved in the study during the permeabilization procedure, we tested 38 diagnosis (17 cases with a CD34+ LAP, 13 cases with a CD117+ LAP, three cases with a CD133+ LAP and five cases with a phenotype including both CD34 and CD117 as a progenitor marker) and 42 MRD samples. Results are presented as LAP+ cells as a percentage of the progenitor cell population (CD34+ or CD117+ or CD133+). As can be seen in Figure 2, percentages of LAP+ cells at diagnosis and in follow-up samples with and without the permeabilization procedure correlated well, showing a close to y=x relation. In this cohort, none of the antigens that were used (as described in Figure 2) were affected by the permeabilization procedure. From these results we conclude that the permeabilization procedure can be applied to the phenotypical analysis of both de novo and MRD cells without loss of cells of interest.
Detection of intracellular apoptosis-related proteins in de novo AML and MRD
An example of an AML at diagnosis (Figure 3, left column) and a sample from the same patient after the first cycle of chemotherapy (Figure 3, middle and right columns) is demonstrated. At diagnosis, the AML presented with blasts (R2, Figure 3b) expressing CD34 and with coexpression of CD2 (CD34+ CD2+ cells; see R5, Figure 3c) MRD cells, present in the BM sample after first cycle of chemotherapy, show up in R5 in Figure 3d. Gating on the LAP+ MRD cells now offers the possibility to analyze the protein expression at different time points of the disease. Accurate gating on leukemia CD34+ cells is of extreme importance since the presence of normal progenitor cells (CD34+ CD2−, R6 in Figure 3d) might easily affect the results. Such would be especially important with differences between normal and leukemia progenitors as described, for example, for Bcl-2 and Bcl-xL mRNA levels.27 Indeed, the MRD cells in R5 (Figure 3d) have a different Bcl-2 expression than the CD34+ CD2− normal cells in R6 (11.5 vs 8.5, respectively, not shown).
This method can not only be extended to other cell populations that can be phenotypically discriminated from each other, for instance blast cells (CD45 low), lymphocytes (CD45 high cells) or normal progenitor cells (CD34+ CD2− cells) but also to other apoptosis-related proteins. As an example, Bax expression of the above-described populations is shown (right column, Figure 3).
In addition, the apoptosis-related proteins investigated in this study can be extended to other known members of the Bcl-2 family (A1, Bcl-w, Bak, Bad, Bcl-xS) or members of the recently emerging important family of inhibitors of apoptosis proteins, IAPs: XIAP, ILP2, c-IAP1, c-IAP2, ML-IAP, NIAP, Survivin, Apollon.28,29,30,31,32 Moreover, the availability of many directly labeled monoclonal antibodies and polyclonal antibodies makes this method very flexible so that it can be used in several studies using cells in suspension, especially in studies aimed at small populations that cannot be measured with conventional methods such as Western blot analysis.
In conclusion, the multiparametric flow cytometric protocol presented in this paper permits a very accurate analysis of apoptosis-related proteins in a complex mixture of cell populations such as that found in follow-up AML bone marrow containing MRD cells and can give important data on the role of apoptosis-resistance in AML. An ongoing study using this approach will offer great help to unravel the mechanisms contributing to the emergence of MRD and chemotherapy failure in AML, and will eventually help to direct future therapies.
Lowenberg B, Downing JR, Burnett A . Acute myeloid leukemia. N Engl J Med 1999; 341: 1051–1062.
Burnett AK, Goldstone AH, Stevens RM, Hann IM, Rees JK, Gray RG et al. Randomised comparison of addition of autologous bone-marrow transplantation to intensive chemotherapy for acute myeloid leukemia in first remission: results of MRC AML 10 trial. UK Medical Research Council Adult and Children's Leukemia Working Parties. Lancet 1998; 351: 700–708.
Harousseau JL, Cahn JY, Pignon B, Witz F, Milpied N, Delain M et al. Comparison of autologous bone marrow transplantation and intensive chemotherapy as postremission therapy in adult acute myeloid leukemia. The Groupe Ouest Est Leucemies Aigues Myeloblastiques (GOELAM). Blood 1997; 90: 2978–2986.
Campana D, Pui CH . Detection of minimal residual disease in acute leukemia: methodologic advances and clinical significance. Blood 1995; 85: 1416–1434.
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.
Terstappen LW, Safford M, Konemann S, Loken MR, Zurlutter K, Buchner T et al. Flow cytometric characterization of acute myeloid leukemia. Part II. Phenotypic heterogeneity at diagnosis. Leukemia 1992; 6: 70–80.
Venditti A, Buccisano F, Del Poeta G, Maurillo L, Tamburini A, Cox C et al. Level of minimal residual disease after consolidation therapy predicts outcome in acute myeloid leukemia. Blood 2000; 96: 3948–3952.
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.
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.
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.
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.
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.
Baer MR, Stewart CC, Dodge RK, Leget G, Sule N, Mrozek K et al. High frequency of immunophenotype changes in acute myeloid leukemia at relapse: implications for residual disease detection (Cancer and Leukemia Group B Study 8361). Blood 2001; 97: 3574–3580.
Macedo A, San Miguel JF, Vidriales MB, Lopez-Berges MC, Garcia-Marcos MA, Gonzalez M et al. Phenotypic changes in acute myeloid leukemia: implications in the detection of minimal residual disease. J Clin Pathol 1996; 49: 15–18.
Oelschlagel U, Nowak R, Mohr B, Thiede C, Ehninger G, Schaub A et al. Specificity of immunophenotyping in acute promyelocytic leukemia. Cytometry 2000; 42: 396–397.
Stoetzer OJ, Nussler V, Darsow M, Gullis E, Pelka-Fleischer R, Scheel U et al. Association of bcl-2, bax, bcl-xL and interleukin-1 beta-converting enzyme expression with initial response to chemotherapy in acute myeloid leukemia. Leukemia 1996; 10 (Suppl 3): S18–S22.
Campos L, Rouault JP, Sabido O, Oriol P, Roubi N, Vasselon C et al. High expression of bcl-2 protein in acute myeloid leukemia cells is associated with poor response to chemotherapy. Blood 1993; 81: 3091–3096.
Lauria F, Raspadori D, Rondelli D, Ventura MA, Fiacchini M, Visani G et al. High bcl-2 expression in acute myeloid leukemia cells correlates with CD34 positivity and complete remission rate. Leukemia 1997; 11: 2075–2078.
Maung ZT, MacLean FR, Reid MM, Pearson AD, Proctor SJ, Hamilton PJ et al. The relationship between bcl-2 expression and response to chemotherapy in acute leukemia. Br J Haematol 1994; 88: 105–109.
Karakas T, Maurer U, Weidmann E, Miething CC, Hoelzer D, Bergmann L . High expression of bcl-2 mRNA as a determinant of poor prognosis in acute myeloid leukemia. Ann Oncol 1998; 9: 159–165.
van der Pol MA, Pater JM, Feller N, Westra AH, van Stijn A, Ossenkoppele GJ et al. Functional characterization of minimal residual disease for P-glycoprotein and multidrug resistance protein activity in acute myeloid leukemia. Leukemia 2001; 15: 1554–1563.
Bradford MM . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248–254.
Laemmli UK . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680–685.
Feller N, Schuurhuis GJ, van der Pol MA, Westra AH, Weijers GWD, van Stijn A et al. High percentage of CD34 positive cells in autologous AML peripheral blood stem cell products reflects inadequate in vivo purging and low chemotherapeutic toxicity in a subgroup of patients with poor clinical outcome. Leukemia 2003 17: 68–75.
Dragowska WH, Lopes de Menezes DE, Sartor J, Mayer LD . Quantitative fluorescence cytometric analysis of Bcl-2 levels in tumor cells exhibiting a wide range of inherent Bcl-2 protein expression: correlation with Western blot analysis. Cytometry 2000; 40: 346–352.
Lacombe F, Durrieu F, Briais A, Dumain P, Belloc F, Bascans E et al. Flow cytometry CD45 gating for immunophenotyping of acute myeloid leukemia. Leukemia 1997; 11: 1878–1886.
Andreeff M, Jiang S, Zhang X, Konopleva M, Estrov Z, Snell VE et al. Expression of Bcl-2-related genes in normal and AML progenitors: changes induced by chemotherapy and retinoic acid. Leukemia 1999; 13: 1881–1892.
Vaughan AT, Betti CJ, Villalobos MJ . Surviving apoptosis. Apoptosis 2002; 7: 173–177.
Milella M, Kornblau SM, Estrov Z, Carter BZ, Lapillonne H, Harris D et al. Therapeutic targeting of the MEK/MAPK signal transduction module in acute myeloid leukemia. J Clin Invest 2001; 108: 851–859.
Jia L, Srinivasula SM, Liu FT, Newland AC, Fernandes-Alnemri T, Alnemri ES et al. Apaf-1 protein deficiency confers resistance to cytochrome c-dependent apoptosis in human leukemic cells. Blood 2001; 98: 414–421.
Shinozawa I, Inokuchi K, Wakabayashi I, Dan K . Disturbed expression of the anti-apoptosis gene, survivin, and EPR-1 in hematological malignancies. Leukemia Res 2000; 24: 965–970.
Tamm I, Kornblau SM, Segall H, Krajewski S, Welsh K, Kitada S et al. Expression and prognostic significance of IAP-family genes in human cancers and myeloid leukemias. Clin Cancer Res 2000; 6: 1796–1803.
The Dutch Cancer Foundation financially supported this work.
About this article
Cite this article
van Stijn, A., Kok, A., van der Pol, M. et al. A flow cytometric method to detect apoptosis-related protein expression in minimal residual disease in acute myeloid leukemia. Leukemia 17, 780–786 (2003). https://doi.org/10.1038/sj.leu.2402885
- minimal residual disease
- apoptosis resistance
- acute myeloid leukemia
Enhancement in alpha-tocopherol succinate-induced apoptosis by all-trans-retinoic acid in primary leukemic cells: role of antioxidant defense, Bax and c-myc
Molecular and Cellular Biochemistry (2008)
Phosphoinositide 3-kinase/Akt signaling pathway and its therapeutical implications for human acute myeloid leukemia
Gene expression profiling of minimal residual disease in acute myeloid leukaemia by novel multiplex-PCR-based method