Identification of hemoglobins within individual rhesus monkey (Macaca mulatta) erythrocytes.

Rabbit and goat antibodies to monkey adult and fetal hemoglobin were prepared and purified to apparent monospecificity. After conjugation with fluorescein isothiocyanate, the antibodies were employed to identify the hemoglobin types within individual cells in peripheral erythrocyte smears. The percentage of neonatal monkey erythrocytes containing fetal hemoglobin was found to decrease with time. The existence of adult or fetal hemoglobin in the erythrocytes appeared to be mutually exclusive.     

Utilization of a differential hemoglobin elution procedure as a rapid assay for identification of embryonic and adult chicken red blood cells.

1. A hemoglobin elution-staining procedure has been developed for distinguishing embryonic chick red blood cells from adult chicken red blood cells. 2. Adult hemoglobin is eluted from red blood cells with 1.9 M potassium phosphate buffer, pH 7.2; whereas, embryonic hemoglobin is retained within the cells and gives positive staining with erythrosin B. 3. The hemoglobin elution-staining pattern during development can be correlated with two embryonic hemoglobins as detected by polyacrylamide gel electrophoresis. 4. The series of red blood cells staining with erythrosin B correspond to the primary erythrocyte series suggesting that hemoglobin expression during development is correlated with different cell populations.     

Ultra-rare-event detection performance of a custom scanning cytometer on a model preparation of fetal nRBCs

BACKGROUND: The performance of a fully automated scanning cytometer incorporating previously reported high-precision autofocus and accurate image segmentation was evaluated for the detection of "ultra-rare" cells using a model of fetal nucleated red blood cells (fnRBCs) in the maternal circulation. These distinctive scanning cytometry techniques were expected to markedly improve sensitivity and specificity. METHODS: Normal adult blood and fetal red blood cells were stained with fluorescein isothiocyanate-conjugated anti-fetal hemoglobin and 4,6-diamidino-2-phenylindole, a nuclear dye. Adult cells were spiked with fetal cells to create ratios of about 1 fnRBC in 10(7) nucleated cells and deposited in monolayers on slides using centrifugal cytology. Rare-event performance parameters were reviewed, formalized, and applied to test the new instrument using this cell model. RESULTS: Fifteen slides were analyzed to establish performance by comparison with manual detection, and four sets of four slides each were then scanned to explore the limit of detection. Results were an average sensitivity of 91%, an average specificity error of 12.3 false-positives per million cells, and repeatability of 100% at a cell analysis rate of 862 Hz. With addition of a quick interactive step subsequent to scanning, the false-positive rate dropped to a total of only one artifact over the 15 experiments. The instrument succeeded at locating 1 fnRBC in 20 million adult cells, the lowest limit of detection tested. CONCLUSION: This consistently high performance, coupled with the capability of scanning arbitrarily large numbers of cells, validates the considerable potential of precise high-speed autofocus and accurate real-time image segmentation for ultra-rare event detection.     

Simplified flow cytometric method for fetal hemoglobin containing red blood cells

Assay of fetal hemoglobin (HbF) and/or HbF containing red blood cells (F+ cells) is essential for monitoring sickle cell and thalassemic patients, especially during treatment with HbF stimulators. Some previous flow cytometric methods contain several washing steps. This simplified method contains no washing step and takes less than an hour to perform. The %F+ cells in five mixtures of fetal red blood cells with adult red blood cells were nonsignificantly different in the original and simplified procedure. The %F+ cells of 12 patients compared in these two procedures were also not significantly different. The intra- and interassay %CVs do not exceed 3% and 7% respectively. EDTA, citrate, or heparin is suitable as anticoagulant and the samples can be stored at 4 degrees C for up to 2 weeks. The %F+ cells and %HbF [by high-performance liquid chromatography (HPLC)] of 83 samples were highly significantly correlated regardless of diagnosis. In conclusion, this new simplified flow cytometric method for F+ cells is simple, convenient, rapid, reproducible, and could be applied for monitoring sickle cell and thalassemic patients as an alternative to HPLC, where this is unavailable. It can also be applied as a fetal cell assay in fetomaternal hemorrhage.     

Prooxidative effects of TEMPO on human erythrocytes

The prooxidative effects of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) were observed in human erythrocytes. Incubation of red blood cells with the membrane-permeable TEMPO leads to a decrease in the concentration of intracellular reduced glutathione, accompanied by the reduction of TEMPO. Extracellular ferricyanide inhibited the loss of glutathione and reduction of TEMPO. TEMPO induced glutathione release from the cells and oxidation of hemoglobin to methemoglobin; ferricyanide prevented these effects. These results indicate that TEMPO may act as an oxidant to erythrocytes, whilst extracellular ferricyanide protects against its effects.     

The demonstration of ferrihemochrome intermediates in heinz body formation following the reduction of oxyhemoglobin A by acetylphenylhydrazone.

Interaction of acetylphenylhydrazine with oxyhemoglobin A in a hemolysate or in intact red cells resulted in the formation of ferrihemochromes as shown by a characteristic optical spectrum. The same optical spectrum was observed in a suspension of red cell ghosts containing numerous Heinz bodies. Electron paramagnetic resonance of actylphenylhydrazine-incubated red cells disclosed the presence of previously identified reversible ferrihemochromes, which can be reduced to functional hemoglobin, and irreversible ferrihemochromes, which cannot be reduced to functional hemoglobin. (Ferrihemochromes are defined as low spin forms of ferric hemoglobin having heme ligands endogenous to the protein structure). In contrast, only irreversible ferrihemochromes could be observed in ghosts containing Heinz bodies. In addition both optical and magnetic features of sulfhemoglobin were observed in an acetylphenylhydrazine-treated red cell hemolysate. Similar optical features are produced by the interaction of aromatic nitrogen-containg reductants with purified oxyhemoglobin in the presence of (NH4)2S. This reaction is not effected by the presence of catalase, suggesting that H2O2 is not an intermediate of the reaction. It is concluded that the mechanism of action of acetylphenylhydrazine with oxyhemoglobin is two-fold, ultimate reduction to high spin ferric hemoglobin followed by ferrihemochrome formation. Thus it appears that the pathway of denaturation of hemolytic anemias and thalassemia or induced by chemical reagents, entails a common route involving the formation of ferric hemoglobin by a reductive mechanism, followed by reversible ferrihemochromes, irreversible ferrihemochromes, and ultimately, precipitation.     

A Two Step Stain for Normal and Leukemic Monocytes Using Two Different Dyes Applied in Sequence

A staining procedure for monocytes in specimens of blood and bone marrow was developed. The technique was a two step procedure in which unfixed cells were exposed first to a methanolic solution of C.I. basic blue 54. Next, an aqueous alkaline buffered solution of C.I. basic blue 141 was added to the first staining solution. After staining for 10 min in the solution with two stains, slides or coverslips were washed for 5 sec in pH 5.6 phosphate buffer and drained dry. The cytoplasm of monocytes stained intensely deep purple and frequently nuclei were stained red. Similar staining was not found in other types of normal or abnormal blood and bone marrow cells.     

Modifcation of the French Azure C Tissue Stain

A staining procedure for monocytes in specimens of blood and bone marrow was developed. The technique was a two step procedure in which unfixed cells were exposed first to a methanolic solution of C.I. basic blue 54. Next, an aqueous alkaline buffered solution of C.I. basic blue 141 was added to the first staining solution. After staining for 10 min in the solution with two stains, slides or coverslips were washed for 5 sec in pH 5.6 phosphate buffer and drained dry. The cytoplasm of monocytes stained intensely deep purple and frequently nuclei were stained red. Similar staining was not found in other types of normal or abnormal blood and bone marrow cells.     

Rehydration of air-dried smears. An alternative method for cytologic analysis of exfoliative cells

OBJECTIVE: To compare the cytologic characteristics of gastric mucosal cell smears prepared by air drying and rehydration prior to alcohol fixing with cells wet fixed in alcohol. STUDY DESIGN: Gastric mucosal cells were obtained from 55 consecutive patients undergoing gastroscopy. Paired smears were made, one immediately fixed in 95% ethanol for 20 minutes (wet fixed [WF]) and the other air dried for at least 20 minutes prior to rehydration with normal saline for 30 seconds and fixation in 95% ethanol for 20 minutes (air dried/rehydrated/fixed [ARF]). Both slides were stained by the Papanicolaou method. Coded slides were examined blind and graded 1 (superior), 2 (satisfactory) or 3 (poor) with respect to staining of chromatin, nuclear membrane, nucleoli, cytoplasm/cell border and group morphology. Histology confirmed a benign disease process or normal mucosa. RESULTS: Comparing grade 1 versus grades 2 and 3, ARF slides were significantly better than WF slides for all cytologic features (P < .05). Comparing grade 1 and 2 versus grade 3, there was no significant difference between ARF and WF slides (P > .05) (chi 2 analysis). CONCLUSION: The cytologic features of ARF smears of gastric cells were equal or superior to those of WF smears. This method of preparing smears is simpler and avoids some of the problems of ethanol fixation of wet smears.     

The Interplay between Molten Globules and Heme Disassociation Defines Human Hemoglobin Disassembly

Hemoglobin functions as a tetrameric oxygen transport protein, with each subunit containing a heme cofactor. Its denaturation, either in vivo or in vitro, involves autoxidation to methemoglobin, followed by cofactor loss and globin unfolding. We have proposed a global disassembly scheme for human methemoglobin, linking hemin (ferric protoporphyrin IX) disassociation and apoprotein unfolding pathways. The model is based on the evaluation of circular dichroism and visible absorbance measurements of guanidine-hydrochloride-induced disassembly of methemoglobin and previous measurements of apohemoglobin unfolding. The populations of holointermediates and equilibrium disassembly parameters were estimated quantitatively for adult and fetal hemoglobins. The key stages are characterized by hexacoordinated hemichrome intermediates, which are important for preventing hemin disassociation from partially unfolded, molten globular species during early disassembly and late-stage assembly events. Both unfolding experiments and independent small angle x-ray scattering measurements demonstrate that heme disassociation leads to the loss of tetrameric structural integrity. Our model predicts that after autoxidation, dimeric and monomeric hemichrome intermediates occur along the disassembly pathway inside red cells, where the hemoglobin concentration is very high. This prediction suggests why misassembled hemoglobins often get trapped as hemichromes that accumulate into insoluble Heinz bodies in the red cells of patients with unstable hemoglobinopathies. These Heinz bodies become deposited on the cell membranes and can lead to hemolysis. Alternatively, when acellular hemoglobin is diluted into blood plasma after red cell lysis, the disassembly pathway appears to be dominated by early hemin disassociation events, which leads to the generation of higher fractions of unfolded apo subunits and free hemin, which are known to damage the integrity of blood vessel walls. Thus, our model provides explanations of the pathophysiology of hemoglobinopathies and other disease states associated with unstable globins and red cell lysis and also insights into the factors governing hemoglobin assembly during erythropoiesis.     

ERYTHROPOIESIS DURING AMPHIBIAN METAMORPHOSIS : III. Immunochemical Detection of Tadpole and Frog Hemoglobins (Rana catesbeiana) in Single Erythrocytes

Rabbit antibodies specific for the major tadpole and frog hemoglobin components of R. catesbeiana were used for the detection of the two hemoglobins inside single cells. The antisera, after fractionation by ammonium sulfate precipitation and diethylaminoethyl (DEAE)-cellulose chromatography, were conjugated with fluorescein isothiocyanate for the antifrog hemoglobin serum and tetramethylrhodamine isothiocyanate for the antitadpole hemoglobin serum. The conjugated fractions, refractionated by stepwise elution from a DEAE-cellulose column, were used for the fluorescent staining of blood smears, liver tissue imprints, and smears of liver cell suspensions. Both simultaneous and sequential staining with the two fluorescent preparations indicated that larval and adult hemoglobins were not present within the same erythrocyte during metamorphosis. In other experiments, erythroid cells from animals in metamorphosis were spread on agar containing specific antiserum. Precipitates were formed around the cells which contain the particular hemoglobin. The percentages of cells containing either tadpole or frog hemoglobin were estimated within the experimental error of the method. The data showed that the two hemoglobins are in different cells. It is concluded that the hemoglobin change observed during the metamorphosis of R. catesbeiana is due to the appearance of a new population of erythroid cells containing exclusively frog hemoglobin.     

Reactions of Hemoglobiniferous Cells to Aced and Basic Dyes under Varying Conditions of H-Ion Activity

1. An attempt has been made to apply Loeb's concept of the amphoteric nature of proteins for the discrimination of suspected hemoglobiniferous substances from known hemoglobiniferous substances according to their reactions to acid and basic dyes with reference to the isoelectric point of hemoglobin (pH 6.8).

2. The substances in the cytoplasm of known hemoglobiniferous cells (red blood corpuscles, normoblasts and erythroblasts) of the lymph nodes, spleen and bone marrow of the albino rat, when suspended in buffered dye-sucrose solutions, retain eosin on the acid side of pH 7.0, but the substances of the Russell bodies, suspected of being hemoglobiniferous, do not retain eosin at all; and the cytoplasm of the plasma cells, also alleged to be slightly hemoglobiniferous, only retains eosin on the acid side of pH 6.4.

3. The only basic dye used which did not precipitate in buffer solutions was methylene blue. This did not react with hemoglobin in accordance with Loeb's concept, because it did not penetrate mature red blood corpuscles and in those immature erythrocytes which it did penetrate it was precipitated by the reticulum.

4. Therefore, from the results obtained with the acid dye, it is tentatively concluded that the substance in the Russell bodies and in the cytoplasm of the plasma cells are not hemoglobiniferous because they do not react as do the substances in known hemoglobiniferous cells with reference to the isoelectric point of hemoglobin.

5. More investigation, however, must be carried out on both fresh and fixed material before a final unequivocal answer can be made to this problem.     

The mechanism of the ferric ferricyanide reduction reaction

Synopsis The ferric ferricyanide reaction has been generally considered to depend on the reduction of ferricyanide to ferrocyanide and, in the presence of the ferric ion, the consequent production of Prussian Blue. In testing the ability of ferricyanide to prevent the azo-coupling reactions of enterochromaffin cells and of the noradrenaline islets of the adrenal medulla, ferricyanide proved to be an effective oxidant in alkaline solution, but failed to quinonize the two catechols below pH 6. Similar results are obtained in oxidative blocking of cutaneous sulphydryl sites against the ferric ferricyanide reaction and against staining by the mercaptide acid azo-coupling reaction of Lillie & Glenner (1965).Ferric ferricyanide solutions are used at pH 2–2.5 and the ferric ion is an effective quinonizing and thiol-oxidizing agent at this pH level; the ferricyanide ion is not. The ferric ferricyanide reaction depends on the reduction of ferric to ferrous ions in the presence of ferricyanide, with the production of Turnbull's Blue.     

Maternal-Fetal Oxygen Transfer in Lower Vertebrates

There exists a difference in oxygen affinity between fetal andmaternal bloods in almost all vertebrates examined and thisdifference in affinities probably facilitates oxygen transferto the fetus. It is likely that the high oxygen affinity offetal blood represents a biochemical pre-adaptation from anancestral oviparous embryo for oxygen uptake in a relativelyhypoxic environment. In most cases, the maternal-fetal differencein blood oxygen affinities is due to the characteristics ofthe fetal red cell and not due to any changes in the adult redcell during pregnancy. These characteristics are based on thepresence of a unique fetal hemoglobin with an intrinsicallyhigh affinity for oxygen or on the absence of high red cellconcentrations of organic phosphates—allosteric modulatorsof hemoglobin function. However, in several species of snake,representing different families, it appears that pregnancy isassociated with apronounced decrease in the oxygen affinityof the adult red cell. This suggests that the blood of the pregnantfemale is better able to unload oxygen to the fetus than couldthe blood of thenonpregnant adult. The maternal-fetal differencein blood oxygen affinities in these species isprobably due tothe characteristics of the fetal red cell as well as to thechange in the affinity of the adult cell during pregnancy. Nonetheless,although the magnitude of the pregnancy-associated change inoxygen affinity of the adult cell in these snakes suggests thatit is physiologically significant, the actual significance remainsto be determined.     

Diferric transferrin reduction by K562 cells. A critical study

This paper critically examines the redox activity of K562 cells (chronic myelogenous leukemia cells) and normal peripheral blood lymphocytes (PBL). Ferricyanide reduction, diferric transferrin reduction, and ferric ion reduction were measured spectrophotometrically by following the time-dependent changes of absorbance difference characteristic for ferricyanide disappearance and for the formation of ferrous ion:chelator complexes. Bathophenanthroline disulfonate (BPS) and ferrozine (FZ) were used to detect the appearance of ferrous ions in the reaction mixtures when diferric transferrin or ferric reduction was studied. Special attention was devoted to the analysis of time-dependent absorbance changes in the presence and absence of cells under different assay conditions. It was observed and concluded that: (i) FZ was far less sensitive and more sluggish than BPS for detecting ferrous ions at concentrations commonly used for BPS; (ii) FZ, at concentrations of at least 10-times the commonly used BPS concentrations, seemed to verify the results obtained with BPS; (iii) ferricyanide reduction, diferric transferrin reduction and ferric ion reduction by both K562 cells and peripheral blood lymphocytes did not differ significantly; and (iv) earlier values published for the redox activities of different cells might be overestimated, partly because of the observation published in 1988 that diferric transferrin might have loosely bound extra iron which is easily reduced. It is suggested that the specific diferric transferrin reduction by cells might be considered as a consequence of (i) changing the steady-state equilibrium in the diferric transferrin-containing solution by addition of ferrous ion chelators which effectively raised the redox potential of the iron bound in holotransferrin, and (ii) changing the steady-state equilibrium by addition of cells which would introduce, via their large and mostly negatively charged plasma membrane surface, a new phase which would favor release and reduction of the iron in diferric transferrin by a ferric ion oxidoreductase. The reduction of ferricyanide is also much slower than activities reported for other cells which may indicate reduced plasma membrane redox activity in these cells.     

Hypoxia-stimulated reduction of doxyl stearic acids in human red blood cells. Role of hemoglobin.

Nitroxide free radicals are under active investigation for their potential use as metabolically responsive contrast agents in electron paramagnetic resonance and nuclear magnetic resonance imaging. The metabolism in human red blood cells of lipid-soluble nitroxides, doxyl stearic acids (DSA), has been investigated. We observed that under normoxia DSA were stable in red blood cells for at least 2 h, but hypoxia stimulated spin label reduction. Complete signal recovery after air or ferricyanide oxidation suggested the formation of hydroxylamine during hypoxia. DSA reduction was found to be dependent upon the position of the nitroxide ring in the fatty acid chain with the reduction rate higher when the -NO degree of the doxyl ring was closer to the fatty acid carboxylic end. The reduction kinetics of DSA with the doxyl ring nearest to the carboxylic end (5DSA) was bifasic. A rapid reduction of about half of the 5DSA was observed in the first hour and, thereafter, a slow reduction process become predominant. The slope of the slow reduction abruptly decreased below 5 microM, thus suggesting a concentration-dependent membrane-cytoplasm translocation of 5DSA. The reducing activity of the red blood cell (RBC) was completely recovered in the cell lysate. Under hypoxia, purified hemoglobin and myoglobin reduced 5DSA and a complete recovery of the signal was obtained after air reoxidation. Globin did not reduce 5DSA, while methemoglobin showed only a small reduction of 5DSA, thus suggesting that ferrous-heme was involved in the hypoxic reduction of DSA. both DSA localization and the characteristics of intracellular reductant (hemoglobin) are responsible for the high stability of DSA in the RBC.     

Chloroquine-sensitive transplasmalemma electron transport in Tetrahymena pyriformis: a hypothesis for control of parasite protozoa through transmembrane redox

Plasma membrane electron transport was studied in a protozoan cell, Tetrahymena pyriformis, by assaying transmembrane ferricyanide reduction and the reduction of iron compounds. The rates of ferricyanide reduction varied between 0.5 and 2.5 mumol/g dry wt. per min, with a pH optimum at 7.0-7.5. Other active non-permeable electron acceptors, with redox potentials from +360 to -125 mV, were cytochrome c, hexaammine ruthenium chloride, ferric-EDTA, ammonium ferric citrate, and indigo di-, tri- and tetrasulfonates. It was found that Tetrahymena cells can reduce external electron acceptors with redox potentials at pH 7.0 down to -125 mV. Ferricyanide stimulates ciliary action. Transmembrane ferricyanide reduction by Tetrahymena was not inhibited by such mitochondrial inhibitors as antimycin A, 2-n-heptyl-4-hydroxyquinoline N-oxide, or potassium cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Tetrahymena appears to involve a plasma membrane electron transport chain similar to those of other animal cells. As in other cells, the transmembrane electron transport is associated with proton release which may be involved in internal pH control. The transmembrane redox system differs from that of mammalian cells in a 20-fold greater sensitivity to chloroquine and quinacrine. The Tetrahymena ferricyanide reduction is also inhibited by chlorpromazine and suramin. Sensitivity to these drugs indicates that the transplasma membrane electron transport and associated proton pumping may be a target for drugs used against malaria, Trypanosomes and other protozoa.     

Oxygen equilibrium characteristics of adult and fetal hemoglobin of Japanese monkey (Macaca fuscata).

Adult hemoglobin and fetal hemoglobin were obtained from Japanese monkey (Macaca fuscata) and their oxygen equilibrium characteristics were studied. (1) The oxygen affinity of fetal hemoglobin was higher than that of adult hemoglobin both in the presence and absence of 2,3-diphosphoglycerate. The presence of diphosphoglycerate lowers the oxygen affinity of adult hemoglobin much greater than does that of HbF and the diphosphoglycerate levels of red cells of adult and newborn monkeys are about the same. (2) The intensity of the Bohr effect, as expressed by -deltalogP50/deltapH, at pH 7.4 was in the order of fetal hemoglobin-diphosphoglycerate greater than adult hemoglobin-diphosphoglycerate greater than fetal hemoglobin greater than adult hemoglobin.     

Intracellular flavonoids as electron donors for extracellular ferricyanide reduction in human erythrocytes.

Reduction of extracellular ferricyanide [Fe(CN)(6)](-3) to ferrocyanide by intact cells reflects the activity of a trans-plasma membrane oxidoreductase that, in human red blood cells, utilizes ascorbic acid as an electron donor. We herein report that the flavonoids quercetin and myricetin, while inhibiting dehydroascorbic acid uptake-and thus the erythrocyte ascorbic acid content-effectively stimulate the extracellular reduction of ferricyanide. Other flavonoids such as rutin, acacetin, apigenin, and genistein do not show the same effect. The notion that quercetin or myricetin may serve as an intracellular donor for a trans-plasma membrane oxidoreductase is supported by the following lines of evidence: (i) they afford direct reduction of ferricyanide; (ii) extracellular reduction of ferricyanide was not mediated by direct effects of the flavonoids released by the cells and was abolished by the sulphydryl reagent parachloromercuribenzenesulfonic acid (pCMBS); (iii) the intracellular concentrations of quercetin or myricetin well correlate with increases in ferricyanide reduction; (iv) the intracellular concentration of the flavonoids dramatically declines after ferricyanide exposure. Taken together, the results presented in this study demonstrate that myricetin and quercetin, which accumulate in large amounts in red blood cells, act as intracellular substrates of a pCMBS-sensitive trans-plasma membrane oxidoreductase. This may represent a novel mechanism whereby these flavonoids exert beneficial effects under oxidative stress conditions.     

Adenylate kinase isoenzyme patterns in tissues of people of different phenotype

Three distinct isoenzyme sets have been demonstrated for adenylate kinase (AK) from human tissues by the technique of starch gel electrophoresis. In adult and fetal muscle, liver, kidney, brain, spleen, lung, and leukocytes the same set was found, though individual bands had different intensities. In adult red cells and in fetal red cells separate and distinctive isoenzyme sets were observed. However, in all tissues, adult and fetal, the phenotype of an individual was recognizable by the presence or absence of a characteristic cathodal band. The apparent anomaly of the same phenotype being expressed in different isoenzyme sets was resolved by the demonstration that in adult and fetal red cells AK complexed with hemoglobin. The implications of this complex are briefly discussed.Supported by grants from the Scottish Hospital Endowments Research Trust (HERT 309) and the Muscular Dystrophy Group of Great Britain.