Erythrocyte Survival Studies Research Articles

Measurement of blood volume with an enriched stable isotope of chromium (53Cr) and isotope dilution by combined gas chromatography-mass spectrometry

We used an established isotope dilution gas chromatography-mass spectrometry method to measure blood volumes with an enriched stable isotope of chromium. The results were compared with those of the conventional method, which involves radioactive 51Cr. The two methods were compared simultaneously in two male subjects of different sizes, and essentially identical volumes were obtained. The isotope dilution method is insensitive to contamination with natural (unenriched) chromium and can be used to measure all of the stable isotopes of chromium. The method has potential applications in simultaneous erythrocyte survival studies. The absence of radioactivity makes possible an accurate means of studying blood volume changes in children and during pregnancy.

Haemolytic effects of exercise

1. Exercise-induced haemolysis has been implicated in the sub-optimal iron status of endurance-trained athletes. Accordingly, erythrocyte survival studies using 51Cr were performed on male and female distance runners (n = 20) and sedentary control subjects (n = 10) in order to determine whether the rate of erythrocyte destruction was altered as a consequence of repetitive exercise training. 2. The chromium half-disappearance time of the male (25.4 +/- 3.6 days, mean +/- SD) but not the female (28.3 +/- 4.6 days) athletes was significantly lower than that of the male (33.1 +/- 4.5 days) and female (32.3 +/- 2.6 days) control subjects (P less than 0.01). The mean erythrocyte lifespan of the male and female distance runners (67.2 +/- 22.2 and 72.4 +/- 26.0 days, respectively) was significantly shorter than that of the non-exercising male and female subjects (113.4 +/- 31.0 and 114.1 +/- 29.0 days, respectively) (P less than 0.01). 3. There was no correlation between the mean erythrocyte lifespan and the haemoglobin concentration, serum ferritin levels, body mass, weekly training distance, number of years running or daily protein intake. The mean cell volume and reticulocyte count measured in the same athletes before and after completing a standard 42 km marathon race were within the normal range, whereas the plasma haemoglobin levels were elevated (77.0 +/- 50.5 mg/l) and the serum haptoglobin levels were decreased (0.89 +/- 0.4 g/l) at rest, with a further significant decrease after running (0.69 +/- 0.4 g/l) in the latter measurement (P less than 0.05). 4. It is concluded that the demonstrated increase in erythrocyte turnover may be sufficient to precipitate an iron deficiency in endurance athletes when dietary intake or absorption does not meet the accelerated erythropoietic demands.

Haemolytic effects of exercise.

1. Exercise-induced haemolysis has been implicated in the sub-optimal iron status of endurance-trained athletes. Accordingly, erythrocyte survival studies using 51Cr were performed on male and female distance runners (n = 20) and sedentary control subjects (n = 10) in order to determine whether the rate of erythrocyte destruction was altered as a consequence of repetitive exercise training. 2. The chromium half-disappearance time of the male (25.4 +/- 3.6 days, mean +/- SD) but not the female (28.3 +/- 4.6 days) athletes was significantly lower than that of the male (33.1 +/- 4.5 days) and female (32.3 +/- 2.6 days) control subjects (P less than 0.01). The mean erythrocyte lifespan of the male and female distance runners (67.2 +/- 22.2 and 72.4 +/- 26.0 days, respectively) was significantly shorter than that of the non-exercising male and female subjects (113.4 +/- 31.0 and 114.1 +/- 29.0 days, respectively) (P less than 0.01). 3. There was no correlation between the mean erythrocyte lifespan and the haemoglobin concentration, serum ferritin levels, body mass, weekly training distance, number of years running or daily protein intake. The mean cell volume and reticulocyte count measured in the same athletes before and after completing a standard 42 km marathon race were within the normal range, whereas the plasma haemoglobin levels were elevated (77.0 +/- 50.5 mg/l) and the serum haptoglobin levels were decreased (0.89 +/- 0.4 g/l) at rest, with a further significant decrease after running (0.69 +/- 0.4 g/l) in the latter measurement (P less than 0.05). 4. It is concluded that the demonstrated increase in erythrocyte turnover may be sufficient to precipitate an iron deficiency in endurance athletes when dietary intake or absorption does not meet the accelerated erythropoietic demands.

New possibilities in erythrocyte survival studies with the application of a Well-type germanium semiconductor detector

The application of a Well-type germanium semiconductor detector creates new possibilities in erythrocyte survival studies. Using 51Cr, the effective dose equivalent in a standard procedure can be reduced from approximately 780 to 2 microSv. The properties of the detector, namely, high efficiency and high energy resolution, also enable the application of two or even more radionuclides within one individual. This is demonstrated by some experiments utilizing a novel dual-label technique with 57Co tropolonate and 58Co tropolonate. Although this new dual-label technique needs further investigation, it underlines the potential possibilities of studying certain unanswered but clinically relevant questions in the field of transfusion medicine with the application of a Well-type germanium semiconductor detector.

Introduction of a Well-type germanium/lithium semiconductor detector in nuclear medicine

Semi-in-vivo diagnostic procedures in nuclear medicine are commonly carried out using conventional scintillation detectors. Modern semiconductor detectors have rarely been applied in this field until now. A particular example of this group is the Well-type germanium/lithium detector, a semiconductor detector which combines a high energy resolution with a high efficiency for gamma-radiation. The latter, combined with an excellent signal-to-background ratio, allows accurate detection of even very low activities. Patient doses can therefore be reduced considerably, as was demonstrated in erythrocyte survival studies, platelet survival studies and the Schilling test. The effective dose equivalent associated with erythrocyte survival studies, using the recommended 51Cr method, could be reduced from 780 to 2 μSv. The effective dose equivalent in platelet survival studies, using the recommended 111In method, could be reduced from 1400 to 14 μSv. For both procedures, the reliability was demonstrated by the results obtained with healthy controls. In the Schilling test, using 57Co, the effective dose equivalent could be reduced from 54 to 9 μSv. In addition, the high resolution of Ge-detectors enables selective and accurate determination of individual components in mixtures of isotopes. Therefore, with such detectors, low-level double-labeling procedures can be introduced in semi-in-vivo nuclear medicine, thus creating the possibility of studying certain unanswered but clinically relevant questions.

Erythrocyte survival studies in a rat myelogenous leukemia.

To determine the extent intrinsic erythrocyte defects and/or extrinsic factors were involved in anemia of rats bearing Shay chloroleukemia (SCL), survival of 3H-DFP labeled erythrocytes was studied in leukemic and nonleukemic hosts. Red blood cells labeled before induction of leukemia, were rapidly lost from the peripheral circulation of SCL rats in terminal stages of disease. However, labeled erythrocytes from terminal SCL animals displayed normal lifespans when transfused into nonleukemic controls. Thus the anemia of this leukemia probably resulted from extrinsic factors associated with the leukemic process. Hemorrhage appeared to be primarily responsible for the anemia of this disease.

Pathogenesis of anaemia in hamsters infected with Leishmania donovani.

The development of anaemia was studied in hamsters infected with Leishmania donovani. Haematocrit studies as well as erythrocyte survival studies using radioactive chromium (51Cr), gave evidence of a progressive anaemia during the course of infection. The erythrocytes were positive in direct antiglobulin tests using antisera to IgG and complement C3. Moreover, erythrophagocytosis as well as increased deposits of haemosiderin in the liver and the spleen, particularly in the areas of proliferating macrophages, were demonstrated. It would appear that the erythrocytes adsorb immune complexes on their surfaces and become liable to enhanced phagocytosis by the macrophages of the liver and the spleen.

Erythrocyte survival in sheep exposed to ozone.

Erythrocyte survival studies in the Dorset ewe using chromium 51 were performed. The purpose of the study was to determine if ozone exposure produces decreased cell survival which may be the result of premature erythrocyte aging. This strain of sheep has an erythrocyte glucose-6-phosphate dehydrogenase (G6PD) activity that is very low, being comparable to human A-variants with G6PD deficiency. Ozone exposure may produce hemolytic effects in G6PD deficients more readily than in erythrocytes with normal activity. A decrease in hematocrit was observed in the ozone exposed groups. With respect to red cell destruction, ozone does not appear to act immediately, but rather there appears to be a delayed effect. At 0.25 ppM ozone, the group reached the 50% remaining level an average of 1 day sooner than the control group. There was no significant difference between control and exposed groups at the 0.50 ppM and 0.70 ppM levels. Also, the results demonstrate a net decrease in hematocrit which is greater for 0.25 ppM ozone than any other exposure level. (RJC)

Erythrocyte agglomeration and survival studies in citrate-phosphate-dextrose (CPD) units.

We have previously reported increased tendency to agglomeration in CPD-stored erythrocytes. In the present study we investigated the deterioration pattern of fresh units, as detected by agglomeration, free hemoglobin levels, ATP levels, osmotic fragility, and certain enzymatic activities. When the negative test was converted to a positive one, the 24-h survival values of these units were determined by autotransfusion. A positive agglomeration occurred while viability was still above 70%. Fresh units had high ATP levels and negative agglomeration while old units had low ATP levels and positive agglomeration. However, in intermediate-storage units, no definite correlation between ATP and agglomeration was found. Agglomeration seems to reflect the degree of changes in stored erythrocytes. However, further studies are needed before this test may serve for prediction of post-transfusion viability.

Leukocyte labeling with technetium-99 m

Erythrocyte labeling with 51Cr or 99 m Tc is a well-established procedure in nuclear medicine. The labeled cells are useful in the measurement of red cell mass, in erythrocyte survival studies, and in the localization of erythrocyte pools. Attempts to label the leukocyte with the same radionuclides have not been as successful. This study reports on various laboratory parameters of labeling leukocytes with 99 m Tc. Microgram quantities of stannous chloride are used in the labeling process to reduce 99 m Tc-pertechnetate to a lower valence state. Other reducing agents such as ferrous ion and penicillamine are not effective. Pertechnetate itself is only loosely bound to the leukocyte. An electrophoretic technique is used to determine labeling yield. Differences in labeling efficiency between the cells from normal donors and the cells from chronic myelocytic leukemia (CML) donors have been detected. Leukocytes from normal donors generally label with greater efficiency than leukocytes from CML donors, but subject age is probably a contributing factor, with cells from young normal donors labeling more like cells from CML donors. The leukocyte and erythrocyte can be labeled under identical conditions in about the same yield. Labeled leukocytes retain good viability and phagocytic capability. Although autoradiographic studies are not completely conclusive because of the poor radiation characteristics of 99 m Tc, it appears possible that the -Sn- 99 m Tc label is on the surface of the cell and is not intracellular. The labeling process is being adapted to an aseptic process for future experimentation in animals and in human subjects.

Alteration of human erythrocyte membrane properties by complement fixation.

Erythrocyte survival studies of complement-coated radiolabeled erythrocytes have shown rapid removal of these cells from the peripheral blood with a return of these cells into the circulation within a few hours. We studied complement-coated human erythrocytes and measured surface charge and deformability, two parameters believed to be important in erythrocyte survival. Erythrocytes were coated with complement by two in vitro techniques: the addition of (a) low ionic strength sucrose, and (b) IgM cold agglutinins. Erythrocytes obtained from three patients with cold agglutinin disease were used as a source of in vivo complement-coated cells. No difference was found in surface charge as measured by electrophoretic mobility between erythrocytes from normal subjects and complement-coated erythrocytes from any of the three sources. When deformability was measured by filtration through 3-mum polycarbonate sieves, marked decreases in deformability were found in complement-coated erythrocytes. The filtration returned toward control levels by incubating the complement-coated erythrocytes in serum for 1 h and correlated with decreases in immune adherence. Using screen filtration pressure as a measure of deformability, a positive correlation between number of C3 molecules per erythrocyte and decreased deformability was found. C3b appeared responsible for the decreased deformability of the erythrocytes, since conversion of C3b to C3d resulted in a return of deformability toward normal. The data suggested that the sequestration of complement-coated human erythrocytes in the microvasculature can be explained in part by decreased deformability and changes in immune adherence.

The Use of I as a Membrane Protein Label for Erythrocyte Survival Studies

Abstract Radioactive iodide, incubated with erythrocytes in the presence of lactoperoxidase and hydrogen peroxide, is covalently bound to membrane proteins. Using this technique, rabbit erythrocytes were labeled with 125I, reinjected into the donors, and erythrocyte survivals determined. The values for the erythrocyte half-lives and the shape of the decay curves were comparable to those reported using DF32P. Following hemolysis of erythrocytes double labeled with 125I and 51Cr, radioactivity from both these isotopes appeared mainly in liver, lung, kidney, spleen, and urine. Enzymatic iodination provides a noneluting label of erythrocyte membrane proteins for the study of survival, sequestration and turnover of this red cell component.

Recommended methods for radioisotopic erythrocyte survival studies.

Recommended methods for radioisotopic erythrocyte survival studies.

The kinetics of urinary excretion of 51Cr from labeled erythrocytes in hemolytic anemia and the anemia of blood loss.

The urinary excretion of 51Cr was studied in conjunction with studies of erythrocyte survival, fecal blood loss, and erythrocyte volume in 54 patients with various hematologic disorders. It was confirmed that excretion of 51Cr is predominantly urinary, and the rate approximates a simple exponential function. In anemias unaccompanied by hemolysis or blood loss, the halftime for urinary excretion of erythrocyte-bound 51Cr (Tu½) ranged from 43 to 63 days. Tu½ was shortened in patients with hemolytic disease but prolonged in patients with severe blood loss; these deviations were proportional to the severity of hemolysis or blood loss, respectively. Determination of the rate of urinary 51Cr excretion may prove to be useful in differentiating various causes of anemia, especially because (1) it distinguishes severe or chronic blood loss from hemolysis, which is not possible with conventional erythrocyte survival studies; (2) the need for multiple venipunctures may be obviated; (3) fluctuations in blood volume or erythrocyte volume do not appear to affect the rate of urinary 51Cr excretion; (4) only a very small dose of radiochromium is needed.

61. Pyruvate kinase deficiency, a family study

A 3-year-old girl with pyruvate kinase deficiency has been investigated together with her family. Erythrocyte survival studies with 51Cr-labeled donor cells in the patient's circulation showed a half life of only 17 days. Direct Coomb's test was negative and cold agglutinin titres normal. Cholecystography showed gallstones and no gallbladder excretion. The pyruvate kinase activity in erythrocytes at 25° and pH 7.5 was only 9.5 and 6 μM substrate turnover/min 1011, respectively, on two occasion, whereas 2 SD of blood donors is 25.0–49.0. Corresponding activities for hexokinase, phosphofructokinase, phosphofructoaldolase, lactate dehydrogenase, glucose 6-phosphate dehydrogenase and glutathion reductase were either within or above normal range. In her crythrocytes adenosine triphosphate was low, adenosine diphosphate normal and 2,3-diphosphoglycerate high. Further studies seem to indicate that the pyruvate kinase in our patient has latered kinetic properties, an abnormal sequence of amino acids. The pyruvate kinase activity in crythrocytes from the patient's mother was low. Similar activities in crythrocytes from the patient's father and brother were at the lower limit of normal. One brother of the maternal grandmother had hemolytic anemia, thrombocytopenia and cholelithiasis, another brother had cholelithiasis and a sister had anemia. The result of further family studies will be reported.

Studies of erythrocyte kinetics using stable chromium and neutron activation analysis

The survival of erythrocytes is measured using50Cr labelling followed by neutron activation analysis. The measured half-life of50Cr tagged autologous erythrocytes in the rhesus monkey (17.9±2.7 d) compares favorably with the value of 17.1±1.4 d measured using the standard method of radioactive51Cr labelling. With the use of stable chromium as a tagging agent, erythrocyte kinetics can be monitored without the problems associated with the use of radioactive isotopes. The method presented is highly reproducible and eliminates the necessity of any chemical separation of chromium. The results of both50Cr and51Cr labelled erythrocyte survival studies are presented.

Hemolytic Anemia Caused By Penicillin

To the Editor:— Although washed normal human erythrocytes suspended in saline are rapidly hemolyzed when exposed to 25 mg/ml of cephalothin, no hemolysis was detected after exposure of whole blood mixed with anticoagulant acid citrate dextrose solution to 25 mg/ml of cephalothin for four hours. It seems likely that binding of cephalothin by plasma proteins inhibits its hemolytic action. The half-life of autologous 51 Crtagged cephalothin-treated erythrocytes was five days in a man with autoimmune hemolytic anemia. His hematocrit reading (16%) approximated that (19%) of the patient with antipenicillin antibodies at the time the survival studies were done. The proportions of erythrocytes, plasma proteins, and cephalothin were similar in the cephalothintreated erythrocyte survival studies of the two patients. It seems unlikely that the much shorter cephalothin-treated erythrocyte half-life of 135 minutes in the patient with antipenicillin antibodies was due to direct damage of the erythrocytes by cephalothin. However, the possibility

Effect of acetazolamide and magnesium therapy on erythrocyte survival in sickle-cell anaemia and sickle-cell-haemoglobin-C disease

Erythrocyte survival studies, with 51Cr were undertaken in 13 patients with sickle-cell disease, before and during treatment with acetazolamide and magnesium citrate. No significant improvement in erythrocyte life span was observed as a result of the treatment. Simultaneous observations on the number of circulating sickled cells, reticulocytes and haemoglobin level also showed no marked change during treatment. It is clear that acetazolamide and magnesium have no significant therapeutic effect on the haematological picture of these patients, but closely supervised use of magnesium might be valuable in diminishing the effects of thrombotic incidents.

A MAJOR PITFALL IN THE USE OF THE SAME RADIOISOTOPE FOR THE REPETITION OF ERYTHROCYTE AND OTHER SURVIVAL STUDIES.

A MAJOR PITFALL IN THE USE OF THE SAME RADIOISOTOPE FOR THE REPETITION OF ERYTHROCYTE AND OTHER SURVIVAL STUDIES.

Erythrocyte survival studies in experimental molybdenosis of sheep.

SummaryMedian survival times of the erythrocytes of sheep in experimental molybdenosis were determined using glycine-2-C14. The existence of 2 distinct populations of erythrocytes was observed in each sheep. Median survival times for the first erythrocyte populations in 2 sheep were 20 and 28 days, with 80 and 85 days for the second populations.