PMC3222265_01
Flow cytometric analysis of bone marrow. Flow cytometric diagnosis of AML M4 was established. (A) CD45/Side light scatter (SSC) dot plot picture with two gates set around myeloblast cells and monoblast cells. (B) Fluorescence (Fl) 1/Fl2 dot plot presentation of myeloblast cells (right dot plots - CD34+/ CD33+/-/CD13+/ CD11b-/CD64-/HLADR-) and monoblast cells (left dot plots - CD34-/CD33+/ CD13+/CD11b+/CD64+/H LADR+).
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F1
AML -M4 (Figure 1). Cytogenetic analysis was performed of 18 metaphase plates of bone marrow which showed: 46, XX,(7)-(q32;q36),t(8;21)(q22;q22),del(7)(q32;q36). A complex aberrant karyotype was found and not conforming to the sex, which led to a cytogenetic analysis of peripheral blood lymphocyte culture stimulated with phytochemaglutinin, due to which the karyotype 47, XXY was found, corresponding to Klinefelter's syndrome.
The karyotype corresponds to Klinefelter's syndrome. No structural chromosomes were detected: complete cytogenetic response regarding t(8;21)(q22;q22). Routine cytogenetic analysis was performed on metaphase chromosomes from bone marrow samples using a direct method and after short-term 24 hours or 48 hours culturing. Metaphases were analyzed using GTG differentially stained chromosomes at a discriminatory level of 300-400 bands per haploid count. Karyotypic findings were interpreted and described according to the International System for Human Cytogenetic Nomenclature (ISCN, 2009).
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F2
Hematological remission with complete cytogenetic response (CCyR) of the disease was achieved after the third induction course containing mitoxantron, eteposide and cytarabine, documented by means of flow cytometric and cytogenetic analysis in October 2006 (Figure 2).
Detection of residual AML1-ETO transcripts by Nested Primers (NP) Reverse Transcription Polymerase Chain Reaction (RT-PCR).(A) Control amplification of beta-Actin cDNA as a marker for RNA integrity and efficiency of copy DNA synthesis. (B) First round PCR for AML1-ETO transcripts - absence of product of amplification. (C) Second round PCR for AML1-ETO transcripts - weak positive reaction corresponding to relatively low number of AML1-ETO transcripts. PCR assay for AML1-ETO fusion trasnscripts.
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F3
At the end of the second year flow cytometric, cytogenetic and molecular analysis (Figure 3) was carried out once again of bone marrow, which con- firmed the achieved remission.