The role of erythrocyte aggregation in the abnormal hemorheology of multiple myeloma patients.

OBJECTIVES: The aim of this study is to clarify whether increased aggregation of red blood cells (RBCs) of multiple myeloma (MM) patients is caused by changes in plasma chemical composition or is associated with alterations in RBC properties and in addition, to suggest an approach to revert the enhanced aggregation in MM toward normal. MATERIALS AND METHODS: 40 blood samples of MM patients and suspensions of control RBCs in MM plasma were examined. In addition, RBC aggregation in MM blood was studied in the presence of dextrans with mean molecular weights of 9.6 and 40 kDa (D9.6 and D40). A method based on electrical and dielectric properties of cellular suspensions was used to study RBC aggregation. In this method, a lower aggregation index demonstrates a higher aggregability. RESULTS: The mean values of aggregation index for whole blood of healthy individuals, control cells in MM plasma and MM blood sample are 19.0, 7.2 and 3.2%, respectively. The kinetics of RBC aggregation slow down with the decrease in the fraction of MM plasma. No correlation was found between RBC aggregation and the immunoglobulin plasma level. Addition of D9.6 to MM blood reverts the enhanced aggregation toward normal. DISCUSSION: The findings that RBC aggregation changes in the following order: MM blood > normal RBCs in MM plasma > control blood sample, suggest that surface-active plasma molecules play a role in enhanced aggregation in MM. The surface concentration of these molecules and hence RBC aggregability is reduced in the presence of dextrans due to their competitive adsorption onto RBC membrane. Because the end-to-end distance of D40 is quite comparable with the Debye length, the effect of this particular dextran on RBC aggregation is negligible.

Possible oxidative stress involvement in congenital dyserythropoietic anemia type 1.

INTRODUCTION: Congenital dyserythropoietic anemia type 1 (CDA1) patients may suffer from iron overload, associated with oxidative damage. The aim of this study was to evaluate possible involvement of oxidative stress in the pathogenesis of CDA1. STUDY DESIGN: : Blood samples from 10 children diagnosed as CDA1 patients from five Bedouin families, were studied. In this study, activities of superoxide dismutase and catalase were evaluated as well as methemoglobin, plasma total thiols, plasma total antioxidant capacity and glycerol lysis time. RESULTS: Normal values were found for superoxide dismutase, methemoglobin, trolox equivalent antioxidant capacity and total plasma thiols in CDA1 patients. However average catalase levels were significantly reduced (P<0.001) and glycerol lysis test was significantly prolonged (P<0.001). Ferritin levels, which were slightly increased in all patients, positively correlated with catalase values (r = 0.74, P = 0.022). CONCLUSION: Oxidative stress has not been proven in CDA1 pediatric patients. Some indications of oxidative damage exist, but it may not be directly related to the mechanism of anemia.

Quantitative determinations of microcytic-hypochromic red blood cell population and glycerol permeability in iron-deficiency anemia and beta thalassemia minor.

The Hl/H2 Technicon automated cell analyzer measures, in addition to the usual red blood cell (RBC) parameters, subpopulations of microcytic (M) and hypochromic (H) red blood cells. The M/H ratio may be useful in the differential diagnosis of iron-deficiency anemia (IDA) and beta thalassemia minor (Thal). Thirty-three iron-deficient patients and 26 thalassemia patients were studied. The M/H ratio was found to be higher in thalassemia patients than in IDA patients. Using a cut-off point of 1.9 M/H ratio, the calculated discriminant efficiency was 88%. When glycerol lysis values were determined at 70 s as a cut-off point, the discriminant efficiency was slightly higher, at 91%. Thus, the combination of the M/H ratio and the glycerol lysis time (GLT) improves the discriminant efficiency and provides a good diagnostic tool to differentiate between the two microcytic-hypochromic anemias. The study suggests that the M/H ratio together with the GLT could serve as a useful screening tool, prior to the application of other more sophisticated methods.

Effect of vitamin K1 on glucose-6-phosphate dehydrogenase deficient neonatal erythrocytes in vitro.

AIM: To determine whether vitamin K1, which is routinely administered to neonates, could act as an exogenous oxidising agent and be partly responsible for haemolysis in glucose-6-phosphat-dehydrogenase (G-6-PD). METHODS: G-6-PD deficient (n = 7) and control (n = 10) umbilical cord blood red blood cells were incubated in vitro with a vitamin K1 preparation (Konakion). Two concentrations of Vitamin K1 were used, both higher than that of expected serum concentrations, following routine injection of 1 mg vitamin K1. Concentrations of reduced glutathione (GSH) and methaemoglobin, indicators of oxidative red blood cell damage, were determined before and after incubation, and the mean percentage change from baseline calculated. RESULTS: Values (mean (SD)) for GSH, at baseline, and after incubation with vitamin K1 at concentrations of 44 and 444 microM, respectively, and percentage change from baseline (mean (SD)) were 1.97 + 0.31 mumol/g haemoglobin, 1.89 +/- 0.44 mumol/g (-4.3 +/- 13.1%), and 1.69 +/- 0.41 mumol/g (-14.5 +/- 9.3%) for the G-6-PD deficient red blood cells, and 2.27 +/- 0.31 mumol/g haemoglobin, 2.09 +/- 0.56 mumol/g (-7.2 +/- 23.2%), and 2.12 +/- 0.38 mumol/g (-6.0 + 14.1%) for the control cells. For methaemoglobin (percentage of total haemoglobin), the corresponding values were 2.01 +/- 0.53%, 1.93 +/- 0.37% (-0.6 +/- 17.4%) and 2.06 +/- 0.43% (5.7 +/- 14.2%) for the G-6-PD deficient red blood cells, and 1.56 +/- 0.74%, 1.70 +/- 0.78% (12.7 +/- 21.9%), and 1.78 +/- 0.71% (20.6 +/- 26.8%) for the control red blood cells. None of the corresponding percentage changes from baseline was significantly different when G-6-PD deficient and control red blood cells were compared. CONCLUSIONS: These findings suggest that G-6-PD deficient red blood cells are not at increased risk of oxidative damage from vitamin K1.

Red blood cell permeability to thiol compounds following oxidative stress.

The permeability of red blood cells (RBCs) to thiol containing compounds, reduced glutathione (GSH) and N-acetyl cysteine (NAC), has been studied in control adult and neonatal cells and after oxidative stress. NAC penetrates the cell membrane easily while GSH hardly permeates. We measured their capacity to enhance intracellular non-protein thiols (NPSH), after inducing damage to the membrane by formation of defects. Diamide, phenazine methosulfate (PMS) and t-butyl hydroperoxide (BHP) were chosen as exogenous oxidants, each inducing damage by a different mechanism. Our data indicate that although neonatal cells are more sensitive to oxidative stress, only membrane damage induced by diamide, renders adult and neonatal cells permeable to GSH. NAC treatment enhances thiol levels in cells exposed to oxidizing agents, as well as in control cells.

Effect of radiation on red cell membrane and intracellular oxidative defense systems.

Ionizing radiation is currently used for prevention of transfusion associated graft versus host disease (TAGVHD). As radiation damage is associated with the production of activated oxygen species, the aim of this study was to observe the immediate effect of ionizing radiation on red cell membrane and intracellular oxidative defense systems. Neonatal and iron deficiency (IDA) cells, known for their increased sensitivity to oxidative stress, were chosen and compared with normal cells. Irradiation was performed in doses of 1500 cGy, 3000 cGy and 5000 cGy. GSH and methemoglobin levels and the activity of different antioxidant enzymes, measured under optimal in vitro conditions, were preserved in all cells after irradiation. Only radiation at the highest does of 5000 cGy, caused significant potassium leakage in neonatal cells and insignificant increase in IDA cells. Thus, cells with increased sensitivity to oxidative stress are more susceptible to damage by ionizing radiation than normal cells.

Prolonged storage of red cells: the effect of pH, adenine and phosphate.

Prolonged storage of red blood cells (RBCs) at 4 degrees C results in decreased intracellular ATP levels with diminished posttransfusion survival. Meryman described a preservative medium, exceptional in its capacity to increase these intracellular levels during the first weeks of storage and later in maintaining adequate levels, for extended storage periods. We modified this medium, investigated its constituents, and found that its ATP-preserving effect was unrelated to its tonicity or to the presence of mannitol. Throughout storage, RBC potassium leakage and lactate production were moderate. No evidence of osmotic swelling was detected. In spite of high ATP levels, the cells became echinocytes, thus discounting a direct correlation between shape and metabolic status. The most striking finding in this study was that the prestorage pH of the blood unit (pH 7.0), has a crucial contribution in elevating nucleotide levels in a medium containing high levels of phosphate (18-40 mM) and adenine. We suggest that a combined effect of optimal pH, adenine, glucose and phosphate in the medium contributes to the ability of the RBCs to synthesize the necessary purine nucleotides by the 'salvage pathway'.

Iron deficiency anemia: recovery from in vitro oxidative stress.

Red blood cells in iron deficiency anemia (IDA) have a decreased activity of essential antioxidant enzymes. The present study examined the effect of in vitro exposure to oxidative agents in IDA cells and their recovery capacity. Red cells of 26 IDA patients and 10 healthy subjects were examined. Cells of IDA patients had higher levels of reduced glutathione (GSH), and normal methemoglobin and malonyldialdehyde (MDA) levels. Exposure to butyl hydroperoxide revealed a dose-dependent sensitivity in IDA cells, with extensive GSH depletion and increased MDA levels. These changes were partially reversible by incubation with dithiothreitol. Exposure to phenazine methosulfate, to produce intracellular superoxide ions, resulted in moderate GSH depletion and methemoglobin production. IDA cells were more sensitive than control cells to high concentrations of this substance. This effect was further augmented by preincubation with a superoxide dismutase inhibitor. Our data demonstrate that IDA cells are more susceptible to oxidation but have good capacity for recovery.

Effect of vitamin K on neonatal erythrocytes.

This study investigated the possible oxidative effect of vitamin K3 (menadione) and Vitamin K1 (Konakion) on neonatal erythrocytes by controlled in vitro exposure. Menadione caused only mild morphological changes and did not decrease ATP levels. However, it oxidized intracellular hemoglobin to methemoglobin in neonatal cells more than in adult cells. Reduced glutathione contents were higher in neonatal cells, but less available for antioxidant protection. Konakion did not increase methemoglobin levels in newborn infants after a prophylactic injection. In vitro exposure to Konakion did not affect reduced glutathione and ATP levels, nor did it oxidize hemoglobin. However, extensive morphological changes were observed, attributed to the effect of its solvent. Therefore, it seems that menadione, which is no longer administered to newborns, causes oxidative stress in neonatal cells whereas Konakion, the current vitamin K1, does not, either in in vitro studies or by therapeutic administration.

Predisposing factors in bladder calculi. Review of 100 cases.

One hundred patients, aged twenty to ninety-two years, underwent 111 procedures for removal of bladder calculi. Most patients (88) had some type of bladder outlet obstruction. Two types of stones were identified: those that had apparently formed in the upper tract and been trapped in the bladder (17 cases) and those that appeared to have formed in the bladder in the presence of various types of outlet obstruction. Stone analysis revealed uric acid stones in 50 percent, calcium oxalate stones in 19 percent, and stones of mixed composition in 31 percent. Five patients had metabolic abnormalities predisposing to stone formation; in 2 cases, these abnormalities were discovered during the evaluation for stone disease. Treatment depended on stone characteristics, associated pathology, and the general health of the patient. A review of the literature with regard to the morbidity and mortality of combining treatment of vesical calculi and bladder outlet obstruction secondary to prostatic obstruction is included.

Oxidative stress in newborn erythrocytes.

Phenylhydrazine (PHZ) exposure is used to study in vitro red cell aging mechanisms dependent on Hb oxidation. The effect of PHZ on normal neonatal red blood cells was studied in unseparated blood and after separation into light and heavy cells. PHZ caused more extensive morphologic changes in neonatal than in adult red blood cells. PHZ exposure of neonatal cells caused less reduced glutathione depletion than in adult cells. Although we found the same total amount of oxidized Hb in both cells, a well-defined oxidation product of Hb was demonstrated by Mössbauer spectra only in neonatal cells. This oxidation product was not methemoglobin but a trivalent, high-spin iron compound. All neonatal cell populations were more sensitive to PHZ than were adult ones, as demonstrated by the presence of Heinz bodies at low PHZ concentration, which did not affect adult cells. These studies demonstrate greater sensitivity of neonatal cells to PHZ in all density-separated populations.

Prolonged storage of red cells with ammonium chloride and mannitol.

Recently, a new preservation medium consisting of ammonium chloride added to adenine, glucose, mannitol, citrate, and potassium phosphate, was described. Unexpectedly, the predominant effect on red cell storage was an initial elevation of ATP levels, followed by remarkable maintenance of these levels at 12 to 18 weeks with acceptable 24-hour survival. The aim of the study reported here was to investigate the reasons for the advantageous effects of the different constituents. With the new preservation medium, ATP content was maintained at acceptable levels for at least 8 weeks with low spontaneous hemolysis. However, similarly high ATP levels were also maintained in the absence of ammonium chloride. Comparable results were obtained on substituting sodium for potassium salts in the new medium. When the main cation in the preservation medium was replaced by sodium, the addition of ammonium or rubidium chloride did not provide an advantage. Therefore, ammonium chloride was not essential for this medium, whereas mannitol does seem to be essential in a solution whose content of nonpermeant solutes is hypotonic.

Storage-induced changes in human newborn red cells.

Fetal red cells are well suited for intrauterine life; however, little is known about their response to postnatal environments. The purpose of this work was to investigate the metabolic and membrane changes affecting newborn red cells during their exposure to storage in citrate-phosphate-dextrose (CPD) and citrate-phosphate-dextrose-adenine (CPDA-1). The findings suggest that newborn red cells are affected more by storage than are adult cells. These accelerated storage changes in the red cells of newborns may be related indirectly to the rapid adenosine triphosphate (ATP) decline. As is the case with adult red cells, fetal cells withstand storage in CPDA-1 better than in CPD. The storage lesion in these cells was partly reversible, as in adult cells, by incubation with adenosine. It was therefore concluded that newborn red cells obtained from placentas and stored for several weeks in CPD or CPDA-1 media or other media that improve the metabolic profile of these cells may be acceptable for transfusion.

Liquid stored erythrocytes: agglomeration, morphology, sialic acid, and toluidine blue uptake studies.

Increased tendency to reversible agglomeration in erythrocytes in outdated units has been previously demonstrated. This phenomenon occurred while post transfusion viability was still acceptable. In the present study erythrocytes of units with early agglomeration were investigated and compared with those with late agglomeration. There was no morphological characterization of these erythrocytes and their sialic acid content and the toluidine blue uptake were not lower than in late agglomeration. Thus, it seems that agglomeration reflects membranal changes, but is not directly related to the sialic acid content of the membrane or to its toluidine blue uptake.

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.

Changes in agglomeration of human red blood cells in liquid storage in CPD media.

A procedure which may distinguish between old and new CPD blood units in liquid state is described. It is based on the observation of increased tendency to reversible agglomeration in old erythrocytes in liquid preservation. Erythrocytes clump together when they are mixed with low ionic strength solutions in pH range of 5.2 to 6.5. We found that liquid-stored erythrocytes show an augmented tendency to agglomerate in 0.24M sucrose, pH 7.2. The tendency increases with storage so that in the fourth week, more than 70% of the units show agglomeration under these conditions. The addition of minute amounts of sodium chloride may prevent agglomeration. As the cells age, higher salt concentration is required to prevent agglomeration. A short incubation of washed cells with adenosine may reverse the tendency of outdated erythrocytes to agglomerate, concomitantly with reestablishment of initial ATP level. However, depletion of the ATP of fresh cells with fluoride does not induce agglomeration. A 20 hour incubation of units at 37 C with CPD revealed an increased sensitivity of older units, with low ATP and positive agglomeration. This test may help in distinguishing between outdated and younger units in the blood bank.

Late spontaneous rupture of ileal conduit.

A case of late spontaneous rupture of an ileal conduit, obstructed by a fibrous band, is presented. Resection of the proximal segment of the conduit bearing the perforation resulted in complete recovery.