To assess the clinical value of determination of the interferon (IFN)-producing capacity of patients, IFN production induced by Sendai virus (HVJ) in vitro was measured in cell cultures of whole blood from patients with various diseases. IFN production in patients with lung cancer, myelodysplastic syndromes, noninsulin-dependent diabetes mellitus, pulmonary tuberculosis, and asymptomatic HIV-1 infection was lower than that in healthy persons. Furthermore, periodic measurements of IFN production revealed decreasing IFN producing capacities in patients with lung cancer with progression of the tumor stage. However, increased IFN-producing capacities were observed in patients with tuberculosis after standard therapy. Further experiments showed that the main type of IFN induced in whole blood cultures was IFN-alpha, and decreased IFN production in patients did not result from a decreased number of leukocytes but rather from an impairment of cellular IFN production. The evaluation of IFN production in whole blood cell cultures may be a feasible method of assessing the impaired immune status.
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 Feinendegen's classification, they were photographed. Then Wright-Giemsa stain was removed by immersion in 50% ethanol at 37 degrees C for 1 h and 100% methanol at 37 degrees 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. The maximum population of megakaryocytes ploidy was in 16N in normal subjects, 8N in 10/12 MDS patients, and 4N in the remaining two patients. These findings suggest impairment of the development of the megakaryocytes nucleus in the MDS patients.
A technique for cytofluorometric measurement of contents of Hb and nuclear DNA on a single erythroid cell is described.Smears of mouse bone marrow were fixed with methanol and treated with 0.2 M mercaptoethylamine hydrochloride (MEM) dissolved in 1.5 M perchloric acid, irradiated by UV light under a fluorescence microscope. This is a modification of method of Granick et al. (1965) to convert hemoglobin into fluorescent porphyrin. The smears were then stained with Feulgen nuclear reactions.The non-specific fluorescence in the background was eliminated by pre-, or post-irradiation method with the wavelength 543 nm (Fujita and Fukuda, 1974). The amounts of Feulgen nuclear DNA and intracellular porphyrin converted from heme or Hb were determined by cytofluorometric measurement on a single erythroid cell. With the two quantitative parameters. erythroid cells in bone marrow were classified into seven classes of different maturation stages. "Proerythroblasts" which were identified on the bases of morphological criteria had in general aneuploid amounts of nuclear DNA with disproportional contents of Hb showing rather aberrations from normal steps of cell maturation.The DNA amounts of "orthochromaticerythroblasts" showed continuous decrease from diploid range to almost zero suggesting that the removal of nuclear DNA is not exclusively due to mechanical expulsion of a whole intact nucleus.
The microcytofluorometrical method was applied to determine the relative hemoglobin (Hb) content in the bone marrow colony-forming unit-erythroid (CFU-E) colonies from 6 patients with myelodysplastic syndromes (MDS) and 10 healthy subjects. This method relies on a photochemical reaction, by which intracellular Hb is converted into fluorescent porphyrin using a 0.2 M mercaptoethylamine solution (an SH donor) and violet light (λ = 405 nm). The relative Hb content was determined as a function of the intensity of emitted porphyrin fluorescence. The number of colonies identified by porphyrin fluorescence was smaller in MDS patients than in normal subjects. The relative Hb content was also lower in MDS patients than in normal subjects. In addition, the coefficient of variation of the relative Hb content in the CFU-E colonies was larger in MDS patients than in normal subjects. These findings suggest that colonies with low relative Hb content undergo impaired erythropoiesis and that the CFU-E colonies undergoing the impaired erythropoiesis are mixed with CFU-E colonies showing normal erythropoiesis in the bone marrow of MDS patients.
A 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 (lambda = 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.(ABSTRACT TRUNCATED AT 250 WORDS)
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 CML-BC (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.
The megakaryocytic ploidy was microfluorometrically measured in 12 normal controls and 15 myelodysplastic syndrome (MDS) patients using DAPI (4',6-diamidino-2-phenylindole) staining after destaining of the Wright-Giemsa (WG) stain. MDS patients had slightly more immature megakaryocytes when compared with normal controls. The megakaryocytic ploidy distribution had a peak at 16N in normal controls, at 8N in the 11 of the 15 MDS patients, and at 4N in the remaining 4 patients, which is suggestive of impaired polyploidization in MDS patients. In MDS, micromegakaryocytes were shown not to be immature but much more impaired in polyploidization than non-micromegakaryocytes. However, there was no difference in the megakaryocytic ploidy pattern among the type of the modification of Feinendegen' classification in each case for both the normal controls and the MDS patients, suggesting that the megakaryocytic ploidy is probably determined at the maturation level of the megakaryoblasts or the precursor cells. The study of megakaryocytic ploidy before and after therapy in the case of refractory anemia with excess of blasts might suggest that the remission of MDS patients might be qualitatively different from that seen in acute leukemia patients. Furthermore, the DNA histogram of the megakaryocytes from one of the two MDS patients obtained by the new method, which is able to determine the amount of DNA in the immunologically identified megakaryocytes microfluorometrically, using the monoclonal anti-glycoproteins IIb/IIIa antibody on bone marrow smears, showed a shift towards small ploidy compared with those defined on the basis of WG staining. This finding indicates that the micromegakaryocytes or the megakaryoblasts which could not be identified morphologically can be identified immunologically.(ABSTRACT TRUNCATED AT 250 WORDS)
A microcytofluorometrical DNA measurement was basically studied and was applied to single megakaryocytes previously identified on a Wright-Giemsa stained smear. The smear was first photographed and the location of each megakaryocyte was recorded on a cell map. The smear was then bleached with 50% acid ethanol and absolute methanol, and re-stained with 4',6-diamidino-2-phenylindole (DAPI) reagent (pH 7.4) at 4 degrees C. Nuclear blue fluorescence was observed and the intensity of this fluorescence was proportional to the amount of DNA with the coefficient of variation (CV) of 3.6% when stained for 30 min. After 30 min DAPI staining, the DNA measurement was microcytofluorometrically performed in single megakaryocytes which had been morphologically classified into 4 groups on the basis of cytoplasmic maturation, Bessis' classification, assessed on Wright-Giemsa-stained bone-marrow smears from normal human beings. The histograms of the cells did not show any difference in DNA ploidy distribution among the classes: that is, the DNA histograms disclosed ploidy distribution from 4 N to 64 N with the largest population of 16 N. These findings suggest that nuclear DNA synthesis is completed before platelet production starts. This method is useful for comparing the morphological features and DNA content of single megakaryocytes.
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