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Analysis of immunophenotype at second relapse of acute lymphoblastic leukemia in children 57Immunophenotyping was performed using cell suspension0.5-2.5 × 10 6 /ml. Cells were incubated with 1-3 monoclonal antibodies bound up with fluorochromesfor 15 minutes in the darkness. Afterwards, erythrocyteswere lysed with UtiLyse (DakoCytomation,Glostrup, Dannemark) or FACS Lysing Solution (BectonDickinson, Heidelberg, Germany), washed in PBS,and analyzed by flow cytometry.The following monoclonal antibodies and fluorochromeswere used: CD2-FITC/CD19-RPE, CD10-FITC/CD19-RPE, CD3-FITC/CD4-RPE/CD8-Cy5,CD5-FITC/CD20-RPE, TdT-FITC, CD7-FITC/CD13-RPE/CD33-RPE, CD34-PerCP-Cy5, CD38-FITC,HLA-DR (DakoCytomation or Becton Dickinson).Respective isotype controls required for the analysiswere used.In each case 5-10 thousands of cells were analyzedwith System II (Coulter) or CXP (Beckman Coulter)software. Subtypes of ALL were classifies as: prepre-B-ALL,common-pre-B-ALL, B-ALL and T-ALL.RESULTSBone marrow relapse occurred in 24/151 patients,including 13 girls (54.1%) and 11 boys (45.9%) withmedian age 10.5 years (range: 0.4-19.2 years). Thesepatients relapsed after 0.3-5.5 years (median 1.7 years)from the initial diagnosis. Very early relapse ocurred in11 patients, early relapse in 5 patients and late relapsein 8 patients. With respect to immunophenotype, 20patients had precursor-B-lineage ALL, 3 had T-ALLand 1 was undifferentiated, both at first diagnosis andat relapse.In 6/24 patients (5 girls, 1 boy) subsequent, secondbone marrow relapse of ALL occurred. Analysis ofimmunophenotype of lymphoblasts at first diagnosis,first and second relapse is presented in Table II.The basic immunophenotype was similar at secondrelapse in comparison to first diagnosis or first relapse.No immunological shift between T-lineage and B-lineage or between lymphoid and myeloid lineage wasobserved. However, changes in the expression of CD38antigen were observed. Although the analysis for presenceof CD38 was not done in all cases, its expressionwas relatively high at second relapse in 5/6 patients,and the antigen was usually present on almost all leukemiccells, while it was not often observed in earlierphases of disease. Expression of CD10 antigen disappearedin one patient (UPN 140), while it appeared inanother one (UPN 156) at second relapse. Expressionof CD34 disappeared in 2 patients.Table 2. Immunophenotype of lymphoblasts. Expression ofeach antigen is given as a percentage of cells expressingthis markerTabela 2. Immunofenotyp blastów. Ekspresja każdego antygenujest podana jako odsetek komórek wykazującychekspresję danego antygenuUPN ImmunophenotypUPN ImmunofenotypDiagnosisRozpoznanie52 Common-ALL CD19 82.6CD10 83.0CD34 86.0CD38 ND101 Common-ALL CD19 98.2CD10 99.5CD34 99.4CD38 ND115 Pro-B-ALL withCD2 coexpressionCD19 98.8CD10 4.28CD34 75.5CD38 NDCD2 35.5140 Common-ALL CD19 85.8CD10 82.1CD34 4.98CD38 ND156 Pro-B-ALL CD19 92.2CD10 0.3CD34 0.1CD38 ND163 Pro-B-ALL CD19 61.9CD10 0.05CD34 0.12CD38 58.3First relapsePierwsza wznowaCD19 92.4CD10 91.8CD34 91.9CD38 NDCD19 96.9CD10 73.1CD34 10.8CD38 97.0CD19 82.7CD10 0.4CD34 16.9CD38 NDCD2 66.1CD19 90.7CD10 85.6CD34 0.08CD38 6.60CD19 72.0CD10 0.4CD34 0.03CD38 81.0CD19 81.4CD10 0.10CD34 2.26CD38 87.0UPN – unique patent number (specyficzny numer pacjenta)ND – not done (nie oznaczono)DISCUSSIONSecond relapseDruga wznowaCD19 71.2CD10 71.2CD34 71.6CD38 NDCD19 95.0CD10 83.8CD34 4.33CD38 93.8CD19 94.0CD10 9.6CD34 0.0CD38 87.0CD2 79.4CD19 80.4CD10 3.1CD34 1.79CD38 51.5CD19 94.7CD10 94.0CD34 1.11CD38 92.8CD19 69.9CD10 0.30CD34 NDCD38 86.8In 24/151 (15.9%) patients, bone marrow relapsehas occurred. In six of them subsequent relapse tookplace. In immunophenotype analysis, a tendency toappearance of more immature antigens was observed.Increase of CD38 antigen and decrease of CD10 wasobserved in most of the patients. Various changes inimmunophenotype at relapse of ALL are relativelyfrequent phenomenon. Relapse of acute lymphoblasticleukemia frequently reveals more immature phenotypeof blasts and occurrence of unfavorable and complexcytogenetic abnormalities [3, 9, 10]. According to theoverall phenotype of the blast cells, phenotypicchanges usually reflect a trend towards more immatureantigenic profile in most cases [3, 6, 11, 12]. Both inour previous report [8] and current analysis, thechanges from diagnosis to relapse and then to the secondrelapse, affected mostly only one antigen. Thevariations were not associated with any clear maturationpattern. Thus, marker-shifts were found in most of