Naoyuki Maruo

Prognostic significance of loss of a chromosome 17p and p53 gene mutations in blast crisis of chronic myelogenous leukaemia

SUMMARY. In 31 cases of chronic myelogenous leukaemia (CML) we examined the prognostic significance of chromosomal loss of a 17p and p53 mutations at the onset of blast crisis (BC). p53 mutations were closely related to a shortened survival in CMLBC (P < 0.005 by the logrank test), whereas loss of a 17p by itself was not a poor prognostic indicator. The prognostic significance of loss of a 17p, however, emerged when combined with its predominance in the metaphases analysed. This predominance might easily and rapidly be screened by polymerase chain reaction‐based analysis in about half of the cases.

Shift in the megakaryocyte ploidy in MDS patients: microcytofluorometry with DAPI staining after destaining of Wright‐Giemsa stain

We applied DAPI (4′,6‐diamidino‐2‐phenylindole) staining to the determination of nuclear DNA content in single megakaryocytes in 12 normal subjects and 12 patients with myelodysplastic syndrome (MDS). After the megakaryocytes had been identified on Wright‐Giemsa stained smear and classified according to modified Feinendegens classification, they were photographed. Then Wright‐Giemsa stain was removed by immersion in 50% ethanol at 37°C for 1 h and 100% methanol at 37°C for 1 h. The specimens were then stained with DAPI solution (DAPI 0.01 mg/ml, pH 7.4 Tris‐EDTA‐2Na buffer solution and 0.01 M 2‐mercaptoethylamine hydrochloride mixed at the ratio of 0.5:98.5:1.0) for more than 30 min. The amount of nuclear DNA in the previously identified megakaryocytes was measured by microcytofluorometry.

Cytofluorometric analysis of cell proliferation and differentiation of the human erythroblasts.

SummaryThe method of cytofluorometric measurement of the contents of Hb and nuclear DNA on a single erythroid cell (Fukuda et al., 1975, 1977 a) was used for the quantitative analysis of the erythropoiesis in normal human bone marrow.The intracellular Hb in an erythroid cell was converted to fluorescent porphyrin after removing the Giemsa staining by irradiation with violet light in the presence of SH-donor (mercaptoethylamine hydrochloride, MEA) and its nuclear DNA was subsequently stained with pararosaniline Feulgen staining. With the two quantitative parameters, Hb content and DNA amount, the erythroid cells in normal human bone narrow were classified into 6 classes of different maturation stages (EI-EIV).The morphological characteristics of the most primitive erythroblast (EI cells) were described. The “proerythroblasts” which were identified on the bases of morphological criteria had in general aneuploid amounts of nuclear DNA with disproportional contents of Hb, thereby indicating that they are rather aberrations from the normal steps of cell maturation. The DNA amounts of “orthochromatic erythroblasts” (EV cells) showed continuous decrease from diploid range to almost zero suggesting that the removal of nuclear DNA from the erythroblast is not exclusively due to mechanical expulsion of a whole intact nucleus.

A microcytofluorometric method for quantitative and qualitative evaluation of CFU-E and BFU-E colonies

SummaryA new method has been developed for the precise identification of human bone marrow colony forming unit erythroid (CFU-E) and burst forming unit erythroid (BFU-E) colonies, and for determination of the hemoglobin contents using microcytofluorometry. The method relies on a photochemical reaction in which intracellular hemoglobin is converted into fluorescent porphyrin under violet light (λ=405 nm) in the presence of an SH-donor (mercaptoethylamine hydrochloride). The CFU-E and BFU-E colonies showed red fluorescence with two spectrum peaks at 600 and 650 nm when illuminated by violet light. These two peaks are consistent with those of porphyrin fluorescence. The porphyrin fluorescence was not inducible in colony forming unit granulocyte-macrophage (CFU-GM) colonies, while 20% of the CFU-GM colonies were false positive with respect to the conventional benzidine reaction. The photochemically inducible fluorescence began to appear in BFU-E colonies on the 4th day of culture, while the same colonies started to be positive for the benzidine reaction on the 9th day. Therefore, the photochemical reaction was more specific and sensitive than the benzidine reaction for the identification of CFU-E and BFU-E colonies. In addition, this method enabled us to measure the hemoglobin level in the cells forming the colonies because the intensity of the fluorescence was proportional to the amount of hemoglobin when the photochemical reaction was carried out for 50 min. As a result of qualitative and quantitative analysis of CFU-E colonies by this method, it was possible to detect the hemoglobin levels in the colonies from 1 of 4 cases of untreated acute nonlymphocytic leukemia and from 2 of 4 cases of myelodysplastic syndrome in which the hemoglobin levels were too low to be detected by the benzidine reaction. These cases, where the CFU-E colonies showed very low levels of hemoglobin, were associated with poor prognosis. Thus, our method is useful for identifying CFU-E colonies, determining their hemoglobin synthesis, and as a cue to predict the clinical course of the patients.