Characterization of rapid flow cytometry chimerism purity panels which assess subset enrichment purity and nucleated red blood cell contamination utilizing Syto-16

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 < 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 < 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.

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.

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.

1. Intravenous injections in rabbits of F. Weber Co.'s Drawing Ink gave the following results: (a) With large doses, an outpouring of nucleated red blood cells, in many instances reaching high values (60,000 to 70,000 per c.mm.), accompanied by a slight initial rise of erythrocytes and hemoglobin, followed by a moderate anemia and in one animal, a severe anemia. (b) With smaller doses the presence of nucleated red cells in the peripheral blood was correspondingly less, the hemoglobin and erythrocytes increasing above the initial level. This increase was sustained for a considerable time, followed by a decrease to or below the initial level. (c) Pregnancy caused a marked suppression, and, shortly before delivery, a complete suppression of the normoblastosis. Nucleated red blood cells reappeared in the blood after the litter was cast. 2. The filtrate of Weber's Drawing Ink, obtained through a Berkefeld filter W, produced the same effects, somewhat enhanced, as the whole ink. 3. The bone marrow of the animals injected with Weber's ink and the filtrate, both showed a marked erythrocytic hyperplasia, with many open sinuses lined with nucleated red blood cells in all stages of maturation. 4. As the dialysed ink gave practically the same results, and the dialysate proved to be without any effect, the conclusion was drawn that a non-dialysable protective colloid was responsible for the marked stimulation of the hematopoietic organs. 5. The delivery of nucleated erythrocytes was interpreted as due to growth pressure induced by rapidly growing red blood cells as well as intrasinusoidal formation of erythrocytes. Pressure due to rapidly increasing phagocytic cells must also be considered. 6. It is fair to conclude that carbon particles as such stimulate endothelial cells mainly toward clasmatocyte formation without incapacitating the endothelial cells, while colloidal silver apparently had a toxic and incapacitating effect on this system of cells.

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.

Although nucleated red blood cells (nRBCs) are rarely found circulating in older children,1 they are commonly seen in the blood of newborns. They are primarily produced in the fetal bone...

Previous studies have shown that a stable presence of both donor and recipient haematopoietic derived cells after allogeneic haematopoietic stem cell transplantation (HSCT) occurs in approximately ten percent of the patients affected by β-Thalassemia. Once achieved this condition, defined as persistent mixed chimerism (PMC), the patients do not require additional red blood cells (RBCs) support and, regardless of the presence in some cases of an extremely low percentage of donor-derived nucleated cells, they are clinically cured by an incomplete, but functional graft. Most of the published papers have, however, investigated the impact of donor engraftment in the nucleated cells rather than in the mature erythrocytes. We have recently published a paper showing that in four long-term transplanted patients affected by hemoglobinopathies, characterized by the presence of few donor engrafted nucleated cells-both in the peripheral blood and in the bone marrow-the majority of the erythrocytes were of donor origin. Moreover we showed that the proportion of donor-derived erythroid precursors, determined by analyzing singularly picked-up burst-forming unit erythroid colonies, was equivalent to that observed in the mature nucleated cells rather than in the red blood cells. These results suggest that in patients characterized by the presence of PMC after HSCT a selective advantage of the donor erythroid precursors maturation might successfully contrast the problems bound to the recipient ineffective erythropoiesis. When genetically modified HSCT will be a possible option for treating Thalassemia Major, the co-existence of the repaired cells with those still expressing the genetic defect will be an expected scenario, not in an allogeneic, but in an autologous environment.

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.

Enhanced Oxalate Transport in Nucleated Red Blood Cells

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.

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.

We evaluated the nucleated red blood cell (NRBC) counts in all infants with very low birth weight admitted to our neonatal intensive care unit from 1983 to 1986. There were 374 infants with birth weights of 500 g to 1500 g admitted in the first 24 hours after birth, but 31 died before studies were obtained. Of the remainder, 282 were appropriate for gestational age (AGA) and 61 were small for gestational age (SGA). Over 80% of both AGA and SGA infants were inborn and were evaluated within three hours of delivery. Nucleated red blood cell counts were significantly increased in SGA infants compared with AGA infants. The percent of infants with NRBC counts higher than 4.0 and 10.0 x 10(9)/L were 48% and 26%, respectively, in SGA infants compared with 19% and 6%, respectively, in AGA infants. Similarly, the percent of SGA infants with more than 40 or 100 NRBCs per 100 white blood cells were 62% and 36%, respectively, compared with 25% and 6%, respectively, in AGA infants. Data for specific weeks of gestation are provided and the differences were present at each gestational age. Recent data in fetuses with growth retardation, when blood was obtained directly from the fetal umbilical vein, showed an association between elevated NRBC counts and chronic hypoxemia. When increased NRBC counts are seen soon after birth, the possibility of a chronic intrauterine insult should be considered.

To examine whether red blood cell transfusion in infants with anaemia of prematurity alters peripheral counts of red blood cell precursors, total white blood cells and white cell differential and platelets. In 18 consecutive stable premature infants with anaemia of prematurity, peripheral cell counts were prospectively recorded immediately before transfusion of 20 mL/kg packed red blood cells (given over 6 h), and at 48 h after completion of the transfusion. The median (interquartile range) haematocrit increased from 22.0% (21.3-24.0%) pre-transfusion to 37.0% (36.0-38.0%) post-transfusion (P < 0.001). Red-cell precursors decreased: median (interquartile range) reticulocytes from 3.7% (3.0-7.7%) to 3.7% (2.6-4.1%) (P = 0.03); and median (interquartile range) nucleated red blood cells from 0 G/L (0-0.2 G/L) to 0 G/L (0-0 G/L) (P = 0.03). The mean (SD) platelet count decreased from 420 G/L (154 G/L) to 313 G/L (101 G/L) (P = 0.001). The total white blood cell count and neutrophils did not change significantly; however, median (interquartile range) immature neutrophils decreased from 0.12 G/L (0.06-0.74 G/L) to 0.08 G/L (0.01-0.24 G/L) (P = 0.03). Lymphocytes, eosinophils, basophils and plasma cells remained unchanged. Monocytes increased (P = 0.01). Forty-eight hours after red blood cell transfusion to premature infants, there is an absolute decrease in red blood cell precursors, immature white blood cells and platelets, probably due to erythropoietin-suppression.

BackgroundGenome-wide DNA methylation (DNAm) studies have proven extremely useful to understand human hematopoiesis. Due to their active DNA content, nucleated red blood cells (nRBCs) contribute to epigenetic and transcriptomic studies derived from whole cord blood. Genomic studies of cord blood hematopoietic cells isolated by fluorescence-activated cell sorting (FACS) may be significantly altered by heterotopic interactions with nRBCs during conventional cell sorting.ResultsWe report that cord blood T cells, and to a lesser extent monocytes and B cells, physically engage with nRBCs during FACS. These heterotopic interactions resulted in significant cross-contamination of genome-wide epigenetic and transcriptomic data. Formal exclusion of erythroid lineage-specific markers yielded DNAm profiles (measured by the Illumina 450K array) of cord blood CD4 and CD8 T lymphocytes, B lymphocytes, natural killer (NK) cells, granulocytes, monocytes, and nRBCs that were more consistent with expected hematopoietic lineage relationships. Additionally, we identified eight highly differentially methylated CpG sites in nRBCs (false detection rate <5 %, |Δβ| >0.50) that can be used to detect nRBC contamination of purified hematopoietic cells or to assess the impact of nRBCs on whole cord blood DNAm profiles. Several of these erythroid markers are located in or near genes involved in erythropoiesis (ZFPM1, HDAC4) or immune function (MAP3K14, IFIT1B), reinforcing a possible immune regulatory role for nRBCs in early life.ConclusionsHeterotopic interactions between erythroid cells and white blood cells can result in contaminated cell populations if not properly excluded during cell sorting. Cord blood nRBCs have a distinct DNAm profile that can significantly skew epigenetic studies. Our findings have major implications for the design and interpretation of genome-wide epigenetic and transcriptomic studies using human cord blood.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0129-6) contains supplementary material, which is available to authorized users.

In order to search for a more reliable method of sorting fetal nucleated red blood cells (NRBCs) and DNA from maternal peripheral blood and to identify origin of NRBCs and DNA, NRBCs were isolated from peripheral blood of 88 pregnant women by density gradient centrifugation and fluorescence activated cell sorter (FACS) respectively. Nested polymerase chain reaction was used to detect normal male SRY gene from blood plasma DNA of 65 pregnant women. The results revealed that fetal NRBCs were found in 14 of 27 maternal samples by density gradient centrifugation. The number of cells was from 1 to 10. Using FACS, CD71+ cells were identified among all 61 samples. The frequency was (0.35±0.25)×10−2; The detectable rate of the SRY gene of blood plasma DNA from 46 women carrying male fetuses was 65.22% (30/46). Non-detectable rate for 19 women carrying female fetuses was 94.74% (18/19). It was concluded that the methods of sorting fetal NRBSs and DNA have already made great progress. The methods for fetal NRBCs and plasma DNA from maternal peripheral blood to diagnose genetic diseases seem to be the best methods of noninvasive prenatal diagnosis.