Predictors of red blood cell transfusion after cardiac surgery: a prospective cohort study

Abstract OBJECTIVE To identify predictors of red blood cell transfusion (RBCT) after cardiac surgery. METHOD A prospective cohort study performed with 323 adults after cardiac surgery, from April to December of 2013. A data collection instrument was constructed by the researchers containing factors associated with excessive bleeding after cardiac surgery, as found in the literature, for investigation in the immediate postoperative period. The relationship between risk factors and the outcome was assessed by univariate analysis and logistic regression. RESULTS The factors associated with RBCT in the immediate postoperative period included lower height and weight, decreased platelet count, lower hemoglobin level, higher prevalence of platelet count <150x10 3/mm3, lower volume of protamine, longer duration of anesthesia, higher prevalence of intraoperative RBCT, lower body temperature, higher heart rate and higher positive end-expiratory pressure. The independent predictor was weight <66.5Kg. CONCLUSION Factors associated with RBCT in the immediate postoperative period of cardiac surgery were found. The independent predictor was weight.

Correlation of discocyte frequency and ATP concentration in preserved blood: A morphological indicator of red blood cell viability

Red blood cells (RBC) are viable if kept in an adequate preservative solution, although gradual changes in morphology and metabolism may occur. There is a gradual decrease in adenosine-5'-triphosphate (ATP) concentration, pH, glucose consumption, and enzyme activity during preservation. The normal discocyte shapes are initially replaced by echinocytes and stomatocytes and, at final stages, by spherocytes, the last step before splenic sequestration. Post-transfusional survival has been correlated with the ATP concentration. RBC preserved in ADSOL, a solution containing adenine, dextrose, sodium chloride, and mannitol, are viable for transfusion for up to 6 weeks. Erythrocytes from 10 blood units taken from healthy adult donors were preserved for 12 weeks in ADSOL at 4oC. We now report a significant correlation (r2 = 0.98) between the percentage of discocytes (89 to 7%) and ATP (100 to 10%) concentration in ADSOL-preserved RBC. The results suggest that the percent of discocyte shapes used as an indicator of ATP concentration may be a useful indicator for quality control of RBC viability in centers which have limited assay facilities.

Relationship between plasma and red blood cell concentrations of quinine in Brazilian children with uncomplicated Plasmodium falciparummalaria on oral therapy

We determined the relationship between plasma and red blood cell concentrations of quinine in children with uncomplicated falciparum malaria from an endemic area of Amazonian region. Quinine was determined by high performance liquid chromatography with ultraviolet detection. In the steady state the ratio between plasma and red blood cell quinine concentration was 1.89 ± 1.25 ranging from 1.05 to 2.34. This result demonstrated that quinine do not concentrate in red blood cell of Brazilian children and characterize the absence of interracial difference in this relationship.

Detection and quantification of Duffy antigen on bovine red blood cell membranes using a polyclonal antibody

Babesiosis is one of the most important diseases affecting livestock agriculture worldwide. Animals from the subspecies Bos taurus indicus are more resistant to babesiosis than those from Bos taurus taurus. The genera Babesia and Plasmodium are Apicomplexa hemoparasites and share features such as invasion of red blood cells (RBC). The glycoprotein Duffy is the only human erythrocyte receptor for Pasmodium vivax and a mutation which abolishes expression of this glycoprotein on erythrocyte surfaces is responsible for making the majority of people originating from the indigenous populations of West Africa resistant to P. vivax. The current work detected and quantified the Duffy antigen on Bos taurus indicus and Bos taurus taurus erythrocyte surfaces using a polyclonal antibody in order to investigate if differences in susceptibility to Babesia are due to different levels of Duffy antigen expression on the RBCs of these animals, as is known to be the case in human beings for interactions of Plasmodium vivax-Duffy antigen. ELISA tests showed that the antibody that was raised against Duffy antigens detected the presence of Duffy antigen in both subspecies and that the amount of this antigen on those erythrocyte membranes was similar. These results indicate that the greater resistance of B. taurus indicus to babesiosis cannot be explained by the absence or lower expression of Duffy antigen on RBC surfaces.

Changes in red blood cell osmotic fragility induced by total plasma and plasma fractions obtained from rats bearing progressive and regressive variants of the Walker 256 tumor

Two variants (A and B) of the widely employed Walker 256 rat tumor cells are known. When inoculated sc, the A variant produces solid, invasive, highly metastasizing tumors that cause severe systemic effects and death. We have obtained a regressive variant (AR) whose sc growth is slower, resulting in 70-80% regression followed by development of immunity against A and AR variants. Simultaneously with the beginning of tumor regression, a temporary anemia developed (~8 days duration), accompanied by marked splenomegaly (~300%) and changes in red blood cell osmotic fragility, with mean corpuscular fragility increasing from 4.1 to 6.5 g/l NaCl. The possibility was raised that plasma factors associated with the immune response induced these changes. In the present study, we identify and compare the osmotic fragility increasing activity of plasma fractions obtained from A and AR tumor bearers at different stages of tumor development. The results showed that by day 4 compounds precipitating in 60% (NH4)2SO4 and able to increase red blood cell osmotic fragility appeared in the plasma of A and AR tumor bearers. Later, these compounds disappeared from the plasma of A tumor bearers but slightly increased in the plasma of AR tumor bearers. Furthermore, by day 10, compounds precipitating between 60 and 80% (NH4)2SO4 and with similar effects appeared only in plasma of AR tumor bearers. The salt solubility, production kinetics and hemolytic activity of these compounds resemble those of the immunoglobulins. This, together with their preferential increase in rats bearing the AR variant, suggest their association with an immune response against this tumor.

Melatonin and N-acetyl-serotonin cross the red blood cell membrane and evoke calcium mobilization in malarial parasites

The duration of the intraerythrocytic cycle of Plasmodium is a key factor in the pathogenicity of this parasite. The simultaneous attack of the host red blood cells by the parasites depends on the synchronicity of their development. Unraveling the signals at the basis of this synchronicity represents a challenging biological question and may be very important to develop alternative strategies for therapeutic approaches. Recently, we reported that the synchrony of Plasmodium is modulated by melatonin, a host hormone that is synthesized only during the dark phases. Here we report that N-acetyl-serotonin, a melatonin precursor, also releases Ca2+ from isolated P. chabaudi parasites at micro- and nanomolar concentrations and that the release is blocked by 250 mM luzindole, an antagonist of melatonin receptors, and 20 mM U73122, a phospholipase C inhibitor. On the basis of confocal microscopy, we also report the ability of 0.1 µM melatonin and 0.1 µM N-acetyl-serotonin to cross the red blood cell membrane and to mobilize intracellular calcium in parasites previously loaded with the fluorescent calcium indicator Fluo-3 AM. The present data represent a step forward into the understanding of the signal transduction process in the host-parasite relationship by supporting the idea that the host hormone melatonin and N-acetyl-serotonin generate IP3 and therefore mobilize intracellular Ca2+ in Plasmodium inside red blood cells.

Risk factors for alloimmunization by patients with sickle cell disease

Blood transfusion in patients with sickle cell disease (SCD) is limited by the development of alloantibodies to erythrocytes. In the present study, the frequency and risk factors for alloimmunization were determined. Transfusion records and medical charts of 828 SCD patients who had been transfused and followed at the Belo Horizonte Blood Center, Belo Horizonte, MG, Brazil, were retrospectively reviewed. Alloimmunization frequency was 9.9% (95% CI: 7.9 to 11.9%) and 125 alloantibodies were detected, 79% of which belonged to the Rhesus and Kell systems. Female patients developed alloimmunization more frequently (P = 0.03). The median age of the alloimmunized group was 23.3 years, compared to 14.6 years for the non-alloimmunized group (P < 0.0001). Multivariate analyses were applied to the data for 608 hemoglobin (Hb) SS or SC patients whose number of transfusions was recorded accurately. Number of transfusions (P = 0.00006), older age (P = 0.056) and Hb SC (P = 0.02) showed independent statistical associations with alloimmunization. Hb SC patients older than 14 years faced a 2.8-fold higher (95% CI: 1.3 to 6.0) risk of alloimmunization than Hb SS patients. Female Hb SC patients had the highest risk of developing alloantibodies. In patients younger than 14 years, only the number of transfusions was significant. We conclude that an increased risk of alloimmunization was associated with older patients with Hb SC, specially females, even after adjustments were made for the number of transfusions received, the most significant variable.

Acute effects of resistance exercise and intermittent intense aerobic exercise on blood cell count and oxidative stress in trained middle-aged women

The aim of this study was to compare the effect of an intermittent intense aerobic exercise session and a resistance exercise session on blood cell counts and oxidative stress parameters in middle-aged women. Thirty-four women were selected and divided into three groups: RE group (performing 60 min of resistance exercises, N = 12), spinning group (performing 60 min of spinning, N = 12), and control group (not exercising regularly, N = 10). In both exercise groups, lymphocytes and monocytes decreased after 1-h recuperation (post-exercise) compared to immediately after exercise (P < 0.05). Immediately after exercise, in both exercised groups, a significant increase in TBARS (from 16.5 ± 2 to 25 ± 2 for the spinning group and from 18.6 ± 1 to 28.2 ± 3 nmol MDA/mL serum for the RE group) and protein carbonyl (from 1.0 ± 0.3 to 1.6 ± 0.2 for the spinning group and from 0.9 ± 0.2 to 1.5 ± 0.2 nmol/mg protein for the RE group) was observed (P < 0.05). A decrease in antioxidant activities (non-protein sulfhydryl, superoxide dismutase, catalase) was also demonstrated with a negative correlation between damage markers and antioxidant body defenses (P < 0.05). These results indicate that an acute bout of intermittent or anaerobic exercise induces immune suppression and increases the production of reactive oxygen species, causing oxidative stress in middle-aged and trained women. Furthermore, we demonstrated that trained women show improved antioxidant capacity and lower oxidative damage than sedentary ones, demonstrating the benefits of chronic regular physical activity.

Removal of Trypanosoma cruzi by white cell-reduction filters: an electronmicroscopic study

White cell (WBC)-reduction filters have been shown to be effective in removing infectious agents from infected blood products. In this study, the mechanisms of Trypanosoma cruzi (T. cruzi) retention by WBC-reduction filters were assessed. Human packed red blood cell (PRBC) and platelet concentrate (PC) samples were contaminated with T. cruzi organisms (Y strain; 3.4 x 10(6)/ml), and then filtered using WBC-reduction experimental filters that provided about 3 log10 WBC removal. Transmission electron microscopy sections showed that T. cruzi parasites were removed from contaminated PRBC and PC samples primarily by mechanical mechanism without interacting with filter fibbers or blood cells. In addition, we found that T. cruzi parasites were also removed by a direct fibber adhesion. These data indicate that T. cruzi parasites are removed from infected blood not only by mechanical mechanism but also by biological mechanism probably mediated by parasite surface proteins.

Hemolytic disease of the newborn due to anti-U

Anti-U is a rare red blood cell alloantibody that has been found exclusively in blacks. It can cause hemolytic disease of the newborn and hemolytic transfusion reactions. We describe the case of a female newborn presenting a strongly positive direct antiglobulin test due to an IgG antibody in cord blood. Anti-U was recovered from cord blood using acid eluate technique. Her mother presented positive screening of antibodies with anti-U identified at delivery. It was of IgG1 and IgG3 subclasses and showed a titer of 32. Monocyte monolayer assay showed moderate interaction of Fc receptors with maternal serum with a positive result (3.1%). The newborn was treated only with 48 hours of phototherapy for mild hemolytic disease. She recovered well and was discharged on the 4th day of life. We conclude that whenever an antibody against a high frequency erythrocyte antigen is identified in brown and black pregnant women, anti-U must be investigated.

Kinetics of antigen specific and non-specific polyclonal B-cell responses during lethal Plasmodium yoelii malaria

In order to study the kinetics and composition of the polyclonal B-cell activation associated to malaria infection, antigen-specific and non-specific B-cell responses were evaluated in the spleens of mice infected with Plasmodium yoelii 17 XL or injected with lysed erythrocytes or plasma from P. yoelii infected mice or with P. falciparum culture supernatants. Spleen/body weigth ratio, numbers of nucleated spleen cells and Immunoglobulin-containing and Immunoglobulin-secreting cells increased progressively during the course of infection,in parallel to the parasitemia. A different pattern of kinetics was observed when anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell plaque forming cells response were studied: maximum values were observed at early stages of infection, whereas the number of total Immunoglobulin-containing and Immunoglobulin-secreting cells were not yet altered. Conversely, at the end of infection, when these latter values reached their maximum, the anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell specific responses were normal or even infranormal. In mice injected with Plasmodium-derived material, a higher increase in antigen-specific PFC was observed, as compared to the increase of Immunoglobulin-containing and Immunoglobulin-secreting cell numbers. This suggested a "preferential" (antigen-plus mitogen-induced) stimulation of antigen-specific cells rather than a generalized non-specific (mitogen-induced) triggering of B-lymphocytes. On the basis of these and previous results, it is suggested that polyclonal B-cell activation that takes place during the course of infection appears as a result of successive waves of antigen-specific B-cell activation.

Apoptosis of non-parasitised red blood cells in Plasmodium yoelii malaria

Recently, while studying erythrocytic apoptosis during Plasmodium yoelii infection, we observed an increase in the levels of non-parasitised red blood cell (nRBC) apoptosis, which could be related to malarial anaemia. Therefore, in the present study, we attempted to investigate whether nRBC apoptosis is associated with the peripheral RBC count, parasite load or immune response. To this end, BALB/c mice were infected with P. yoelii 17XL and nRBC apoptosis, number of peripheral RBCs, parasitaemia and plasmatic levels of cytokines, nitric oxide and anti-RBC antibodies were evaluated at the early and late stages of anaemia. The apoptosis of nRBCs increased at the late stage and was associated with parasitaemia, but not with the intensity of the immune response. The increased percentage of nRBC apoptosis that was observed when anaemia was accentuated was not related to a reduction in peripheral RBCs. We conclude that nRBC apoptosis in P. yoelii malaria appears to be induced in response to a high parasite load. Further studies on malaria models in which acute anaemia develops during low parasitaemia are needed to identify the potential pathogenic role of nRBC apoptosis.

Antibody recognition of Plasmodium falciparum infected red blood cells by symptomatic and asymptomatic individuals in the Brazilian Amazon

In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms.

Detection and analysis of apoptosis in peripheral blood cells from breast cancer patients

Apoptosis is a well-known specific process of cell death that normally occurs in physiological situations such as tissue or organ development and involution. During tumor growth there is a balance between proliferation and cell death which involves apoptotic mechanisms. In the present study genomic DNAs from 120 breast tumor biopsies were analyzed by agarose gel electrophoresis and none of them presented the fragmentation pattern characteristic of the apoptosis process. However, 33% of the 105 breast cancer patients clearly showed the apoptotic pattern when DNA from blood cells was analyzed. None of the DNAs from healthy volunteer blood cells showed any trace of apoptosis. Since the breast cancer patients were not receiving chemo- or hormone therapy, the possible relationship between blood cortisol levels and the apoptotic pattern found in patient blood cells was investigated. Using a chemoluminescence immunodetection assay, similar cortisol levels were observed in breast cancer patient sera presenting or not apoptotic blood cells and in healthy volunteer sera. Analysis of the clinical data obtained from 60 of these patients showed that patients bearing tumors of smaller size (under 20 mm) were more susceptible to the apoptotic effect in blood cells. According to the Elston grade, it was observed that 7 of 12 patients with grade III tumors (58%) presented apoptotic peripheral blood cells, in contrast to 10 of 48 patients with grade I and grade II tumors. These observations may reflect the immunosuppression characteristic of some breast cancer patients, which may contribute to tumor growth. Therefore, further studies are necessary to elucidate the factor(s) involved in such massive blood cell death.

The calcium-dependent protease of Loxosceles gaucho venom acts preferentially upon red cell band 3 transmembrane protein

Eighty micrograms red blood cell (RBC) ghosts from patients who had previously exhibited the cutaneous form of loxoscelism (presenting localized dermonecrosis) and the viscerocutaneous form of loxoscelism (presenting dermonecrosis, hemoglobinuria, hematuria, and jaundice) and from controls were incubated with 2.5 µg crude Loxosceles gaucho venom in 5 mM phosphate buffer, pH 7.4, at 37ºC. Among all membrane proteins, quantitative proteolysis of the important integral transmembrane protein 3 increased with venom dose and with incubation time from 30 to 120 min, as demonstrated by gel densitometry. Similar quantitative data were obtained for RBC ghosts from patients and from control subjects, a fact that argues against the possibility of genetic factors favoring the hemolytic viscerocutaneous form. These data suggest that the clinical forms may be different types of the same disease, with the viscerocutaneous form being the result of large amounts of intravascularly injected venom and the superficial form being the result of in situ venom action. Since protein 3 is a housekeeping integral membrane protein, whose genetic deficiency leads to hemolytic anemia, it is reasonable to relate it to the hemolysis which occurs in the viscerocutaneous form of loxoscelism. The venom protease responsible for the process was not inhibited after 120-min incubation by 0.2 mM paramethylsulfonyl fluoride or by 0.2 mM N-ethylmaleimide but was inhibited by 25 mM ethylenediaminetetraacetic acid (a calcium-chelating agent) in 5 mM phosphate buffer at pH 7.4, which suggests that the enzyme is a calcium-dependent metalloprotease.