EXPERIMENTAL BONE MARROW REACTIONS

The ontogeny of the glucose-6-phosphatase enzyme in human embryonic and fetal red blood cells.

Apoptosis and erythrocyte senescence share the common feature of exposure of phosphatidylserine (PS) in the outer leaflet of the cells. Western analysis showed that mature red cells contain Fas, FasL, Fas-associated death domain (FADD), caspase 8, and caspase 3. Circulating, aged cells showed colocalization of Fas with the raft marker proteins Galpha(s) and CD59; the existence of Fas-associated FasL, FADD and caspase 8; and caspase 8 and caspase 3 activity. Aged red cells had significantly lower aminophospholipid translocase activity and higher levels of PS externalization in comparison with young cells. In support of our contention that caspases play a functional role in the mature red cell, the oxidatively stressed red cell recapitulated apoptotic events, including translocation of Fas into rafts, formation of a Fas-associated complex, and activation of caspases 8 and 3. These events were independent of calpain but dependent on reactive oxygen species (ROS) as evident from the effects of the ROS scavenger N-acetylcysteine. Caspase activation was associated with loss of aminophospholipid translocase activity and with PS externalization. ROS was not generated by treatment of cells with t-butyl hydroperoxide at 10 degrees C, and Fas did not translocate into rafts. Concomitantly, neither formation of a Fas-associated signaling complex nor caspase activation could be observed, supporting the view that translocation of Fas into rafts was the trigger for the chain of events leading to caspase 3 activation. Our data demonstrate for the first time the novel involvement of Fas/caspase 8/caspase 3-dependent signaling in an enucleated cell leading to PS externalization, a central feature of erythrophagocytosis and erythrocyte biology.

The composite response of the erythron to exogenous erythropoietin has been studied in normal, splenectomized, and polycythemic mice. After stimulation the normal animal doubled its marrow nucleated red cells by the 3rd day with little further change by the 5th. Nucleated red cells within the spleen began to increase sharply on the 2nd day and, by the 5th, exceeded those in the marrow. The total nucleated erythroid response represented a fourfold increase. Reticulocytes lagged behind the expansion of the nucleated red cell mass, but by the 5th day the original ratio was re-established. Hemoglobin synthesis was increased, but the ratio of hemoglobin synthesized in nucleated red cells and reticulocytes was basically unchanged. Early displacement of marrow reticulocytes into circulation and the production of a larger red cell also occurred. No evidence of a change in the number of erythroid mitoses was found; only a slight decrease in the average cell cycle time was demonstrated. Thus, whereas erythropoietin stimulation induced several changes in erythropoiesis, the increased number of cells entering into the maturing pool appeared to be of greatest quantitative significance.Splenectomy reduced the proliferative response of the erythron over 5 days stimulation to three-fourths that found in the normal animal. This difference, also reflected in a proportionately lower reticulocyte response and increment in circulating red cell mass, suggests that erythropoiesis within the mouse marrow is spatially or otherwise restricted and that the spleen provided a supplemental area of erythroid expansion.

IntroductionIn critically ill patients, the appearance of nucleated red blood cells (NRBCs) in blood is associated with a variety of severe diseases. Generally, when NRBCs are detected in the patients' blood, the prognosis is poor.MethodsIn a prospective study, the detection of NRBCs was used for a daily monitoring of 383 medical intensive care patients.ResultsThe incidence of NRBCs in medical intensive care patients was 17.5% (67/383). The mortality of NRBC-positive patients was 50.7% (34/67); this was significantly higher (p < 0.001) than the mortality of NRBC-negative patients (9.8%, 31/316). Mortality increased with increasing NRBC concentration. Seventy-eight point six percent of the patients with NRBCs of more than 200/μl died. The detection of NRBCs is highly predictive of death, the odds ratio after adjustment for other laboratory and clinical prognostic indicators being 1.987 (p < 0.01) for each increase in the NRBC category (0/μl, 1 to 100/μl, 101 to 200/μl, and more than 200/μl). Each step-up in the NRBC category increased the mortality risk as much as an increase in APACHE II (Acute Physiology and Chronic Health Evaluation II) score of approximately 4 points. The mortality of patients who were NRBC-positive on the day of relocation from the intensive care unit to a peripheral ward was 27.6% (8/27). This was significantly higher than the mortality of patients who were NRBC-negative on the relocation day (8.6%, 28/325; p < 0.01). On average, NRBCs were detected for the first time 14 days (median, 3 days) before death.ConclusionThe routine analysis of NRBCs in blood is of high prognostic power with regard to mortality of critically ill patients. Therefore, this parameter may serve as a daily indicator of patients at high mortality risk. Furthermore, NRBC-positive intensive care patients should not be relocated to a normal ward but should obtain ongoing intensive care treatment.

Observations on the Phosphatase Content of Blood and Bone Marrow Cells in Normal and Pathologic Hemopoiesis

Presence of peripheral blood nucleated red blood cell (NRBC) is associated with pathological conditions and leads to the overestimation of white blood cell count in automated haematology analysers (HA). The authors evaluated NRBC enumeration by a new HA Sysmex XN (XN) to demonstrate the precision and comparability to manual count (MC) at the various NRBC values. Specimens with initially NRBC positive were included. For precision assessment, 8 levels of NRBCs were repeatedly analysed. For comparison study, 234 specimens were analysed by both XN and MC. For precision study, the percentage of coefficient of variation ranged from 14% to 45.6% and 1.2% to 4.4% for MC and XN, respectively. For comparison study between XN and MC, NRBCs ranged from 0% to 612.5%. Regression analysis demonstrated an r(2) of 0.98. The mean bias of 14.1% with 95% limits of agreement between -48.76% and 76.95% was found. The NRBC counts from XN appeared to be more in accordance with MC when the NRBCs were lower than 200% with the concordance rate of 94.2%. The automated NRBC enumeration by XN was precise and could replace the traditional MC, especially for the specimens with NRBCs lower than 200%.

INTRODUCTION: Nucleated red blood cells and immature granulocytes are not normally detected in the blood of healthy adults. We aimed to investigate the effect of nucleated red blood cells and immature granulocytes on mortality in order to identify critically ill patients who were admitted to the emergency department, at high risk of death, and who was not traumatic. MATERIAL AND METHODS: This study was performed retrospectively in the emergency department of a tertiary education and research hospital between January 2021 and June 2021. All patients who died out of trauma and patients who were discharged from the emergency department on the same day were included. Nucleated red blood cells and immature granulocytes parameters were compared between the two groups. The primary outcome was all-cause death in the emergency department. RESULTS: Of the 188 patients included in the study, 129 (68.6%) were male. Nucleated red blood cells (1.88 ± 6.9/μL; 0.02 ± 0.08), % immature granulocytes (2.91 ± 3.04/μL; 0.58 ± 1.63) and immature red blood cells in deceased patients’ granulocyte count (0.38 ± 0.46/μL; 0.04 ± 0.04) was significantly more significant than the control group (p &lt; 0.001). When the area under the curve was examined, the highest value was found in nucleated red blood cells (Area under the curve = 0.920, p &lt; 0.001). In multivariate regression analysis, high nucleated red blood cells, immature granulocyte count, and white blood cell levels were associated with all-cause mortality in the emergency department. CONCLUSIONS: High nucleated red blood cells and immature granulocyte levels may be associated with increased mortality during admission to the emergency department.

In a cross-sectional study of 13 chromosomally abnormal fetuses, umbilical venous blood was obtained by cordocentesis at 17-32 weeks' gestation. Fetal blood transferrin receptor (CD71) expression (mean = 79.8 per cent, range = 60-98 per cent) and nucleated red cell count (mean = 10.4 x 10(9) per 1, range = 1.0-25.0 x 10(9) per 1) were significantly higher than the appropriate normal mean for gestation (z = 3.92, P < 0.0001 and z = 3.69, P < 0.001, respectively). These haematological changes in chromosomally abnormal fetuses would facilitate their prenatal diagnosis by analysis of fetal nucleated red blood cells isolated from the maternal circulation on the basis of CD71 expression.

Erythrocyte superoxide dismutase activities were measured in 45 subjects, 15 each of beta 0-thalassaemia/haemoglobin (Hb) E disease, Hb H disease, and normal. The erythrocyte superoxide dismutase activities were significantly higher in the patients with beta 0-thalassaemia/Hb E and Hb H diseases than in the normal subjects. The increase of erythrocyte superoxide dismutase activities is most likely due to abnormalities specific to thalassaemic red cells rather than an increased number of younger red cells for reticulocytes and nucleated red blood cells did not affect the enzyme activity. Patients with beta 0-thalassaemia/Hb E disease with lower haemoglobin concentration had significantly higher superoxide dismutase activities. In all 45 subjects haemoglobin concentrations and superoxide dismutase activities were inversely correlated (r = -0.60 (p less than 0.001)). This indicates that the amounts of superoxide generated in the red cells may, at least partly, determine severity of red cell damage and thus severity of disease; the increased superoxide dismutase activity in thalassaemia is a response to superoxide generated in greater amounts because of accumulation of excessive globin chains and iron in the red cells. The superoxide dismutase activities in Hb H disease, an alpha-thalassaemic disease, were found to be strikingly increased, higher than in beta 0-thalassaemic disease or other conditions.

In a retrospective case control study we aimed to evaluate whether infants and children with nucleated red blood cells (NRBCs) in their peripheral blood smears after cardiopulmonary bypass (CPB) had longer bypass times than controls without NRBCs. On review of a 3-year period, 58 children with NRBCs after CPB (and without NRBCs prior to CPB) were identified (cases). A random sample of 100 children without NRBCs after CPB over the same period served as controls. The median age (range) of the children with NRBCs and without NRBCs was 0.6 years (2 days to 20 years) and 1.4 years (2 days to 16 years), respectively (p = 0.03). The children with NRBCs had a significantly longer bypass time than the controls (mean, standard deviation (SD): 114 min, 50 vs 79 min, 46 min; p < 0.0001). For the patients with postoperative polychromasia alone, the mean CPB time (111 min, SD 46 min) was also significantly longer than the respective time in the controls (p < 0.001). Markers of organ dysfunction (renal failure, use of inotropic support, time of endotracheal intubation, stay in intensive care unit and stay in hospital) were significantly more frequent/longer in the NRBC group. Post-CPB release of NRBCs is associated with longer CPB time. This alteration may be part of the CPB-related systemic inflammatory response syndrome.

In Brief Objective To assess the frequencies of nucleated red blood cells (RBCs) in maternal blood during different stages of gestation and postpartum. Methods Peripheral venous blood samples were collected longitudinally from 38 pregnant women from the first trimester to 3 months postpartum. Nucleated RBCs were isolated by using a triple-density gradient with Histopaque (Sigma Diagnostics, St. Louis, MO) and identified by Kleihauser-Betke acid stain. Results The number of nucleated RBCs steadily increased from 3.9 (standard error 0.6) per 107 nucleated cells in early gestation (6–10 weeks) to 112.0 (standard error 7.5) per 107 nucleated cells near term and decreased rapidly after delivery. The number of nucleated RBCs was not related to the gender of the fetus or the ABO blood type compatibility between the mother and fetus. Conclusion The number of nucleated RBCs in the maternal blood increase progressively throughout pregnancy, with some variation from subject to subject. The number of nucleated red blood cells in maternal blood increases as gestation advances, regardless of maternal-fetal blood type compatibility or fetal gender.

The aim of this study was to evaluate the influence of pre-eclampsia on the cord and maternal nucleated red blood cell (NRBC) count. Immediately after delivery, 1 mL of maternal venous blood and 1 mL of cord blood from 50 pre-eclamptic and 150 healthy pregnant women were collected separately in tubes containing 1.5 mg ethylene diamine tetra-acetic acid. Blood smears were prepared and stained using the Giemsa method. The number of NRBC per 100 leukocytes in maternal and cord blood was counted and compared between the two groups using SPSS software package for Windows. Any correlation of the NRBC count in maternal and umbilical cord blood was also evaluated. P-values < 0.05 were considered significant. The mean (+/-SD) NRBC per 100 white blood cell (WBC) level in cord blood of newborns in the pre-eclamptic group (18.2 +/- 31.8, range 0-142) was significantly greater than in the control group (6.2 +/- 8.1, range 0-36). Low birth weight and intrauterine growth restriction showed a statistically significant relationship with abnormal NRBC count in pre-eclamptic patients. A significant correlation was found between the maternal and cord blood NRBC count in the pre-eclamptic group. Fetal response to utero-placental insufficiency in pre-eclampsia leads to elevated NRBC in the cord blood, particularly in the presence of low birth weight and intrauterine growth restriction. The positive correlation between maternal and cord blood NRBC counts in pre-eclamptic patients indicates that maybe the hypoperfused placenta plays a role in the correlated alteration of the maternal and fetal NRBC count.

BackgroundThe Sysmex XN haematology instrument performs automatic reflex testing, depending on sample results. A nucleated red blood cell (NRBC) count is provided on all samples. The instrument has a smaller...

The detection of nucleated red blood cells (NRBCs) in blood of patients suffering from a variety of severe diseases is known to be highly associated with increased mortality. Blood analyzers to routinely measure NRBC concentrations are now available. However, the diagnostic and prognostic significance of this parameter for intensive care patients has not been evaluated. Using a Sysmex XE-2100 analyzer, NRBC concentrations were determined in blood samples from 421 patients treated in intensive care units (general and accident surgery, cardiothoracic surgery, and internal medicine) of a university hospital. NRBCs were found at least once in 19.2% of all patients. The mortality of NRBC-positive patients (n=81) was 42.0% (n=34); this was significantly higher (p<0.001) than the mortality of NRBC-negative patients (5.9%, n=340). The NRBC concentration was 115+/-4x10(6)/l (median 40x10(6)/l; range 20-2930x10(6)/l) at initial detection of NRBCs in the blood. Mortality increased with increasing NRBC concentration and increasing frequency of occurrence. With regard to in-hospital mortality, NRBCs in blood showed sensitivity and specificity of 63.0% and 87.2%, respectively. The detection of NRBCs is highly predictive of death, the odds ratio after adjustment for other laboratory prognostic indicators being 1.01 (p<0.01) for each increase in the NRBC concentration of +1x10(6)/l. NRBCs were detected for the first time, on average, 13 days (median 8 days) before death. The routine analysis of NRBCs in blood is of high prognostic power with regard to in-hospital mortality of critically ill patients. Therefore, this parameter may serve as an early indicator for patients at increased mortality risk.

A Novel Alpha Specrtin Mutation Causing Severe Innefective Erythropoiesis