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.