Cytofluorometric Measurement of Contents of Nuclear Dna and Intracellular Porphyrin Converted From Heme or Hemoglobin on a Single Erythroid Cell

Abstract

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.

Cytofluorometric Quantitation of the Hemoglobin and Dna Contents of a Single Erythroid Cell

“…This is probably due to the contamination of Hb in the serum after partial hemolysis during preparation of the smear. The application of post-irradiation (4) with violet excitation light on photochemically treated specimens is thought to be effective in eliminating increased fluorescence in the background, because porphyrin fluorescence is known to be relatively stable for violet light (5) . The authors are now testing the effects of violet irradiatiation on subsequent Feulgen staining.…”

“…Another problem in performing the quantitative convention of Hb to porphyrin is the disturbance due to the chemical lability of heme derivatives against oxidation and heating, especially in acidic circumstances (2,5). For example, the overheating of specimens, due to drying with excessively hot air should be avoided.…”

“…One of the photochemically treated smears of human bone marrow (40 mintreated) was subsequently stained with Feulgen reaction, according to the method described previously (3,4,5,6) . Hydrolysis was carried out in 1 N HCl for 5 min at 60+0.5°C.…”

“…For observation and cytofluorometry of the porphyrin and Feulgen fluorescences, a digital microfluorometer (Olympus-MMSP-FR-II) equipped with an incident illumination system was used as described previously (3,4,5,6). Excitation beams for the nuclear Feulgen DNA and porphyrin were obtained by using the green (Amax=543 nm, QA1'2=1.8 nm, transmission, T=92%) and violet (Amax =405 nm, dA112=1.8 nm, T=92%) interference filters respectively.…”

“…A method for the cytofluorometric determination of the amounts of Feulgen nuclear DNA and Hb in a single erythroid cell was reported upon previously (5). The intracellular Hb in an erythroid cell was photochemically converted to fluorescent porphyrin and its nuclear DNA was subsequently stained with pararosaniline Feulgen reaction.…”