Erythroblasts Research Articles

Machine learning of cell population data, complete blood count, and differential count parameters for early prediction of bacteremia among adult patients with suspected bacterial infections and blood culture sampling in emergency departments.

Bacteremia is a life-threatening complication of infectious diseases. Bacteremia can be predicted using machine learning (ML) models, but these models have not utilized cell population data (CPD). The derivation cohort from emergency department (ED) of China Medical University Hospital (CMUH) was used to develop the model and was prospectively validated in the same hospital. External validation was performed using cohorts from ED of Wei-Gong Memorial Hospital (WMH) and Tainan Municipal An-Nan Hospital (ANH). Adult patients who underwent complete blood count (CBC), differential count (DC), and blood culture tests were enrolled in the present study. The ML model was developed using CBC, DC, and CPD to predict bacteremia from positive blood cultures obtained within 4h before or after the acquisition of CBC/DC blood samples. This study included 20,636 patients from CMUH, 664 from WMH, and 1622 patients from ANH. Another 3143 patients were included in the prospective validation cohort of CMUH. The CatBoost model achieved an area under the receiver operating characteristic curve of 0.844 in the derivation cross-validation, 0.812 in the prospective validation, 0.844 in the WMH external validation, and 0.847 in the ANH external validation. The most valuable predictors of bacteremia in the CatBoost model were the mean conductivity of lymphocytes, nucleated red blood cell count, mean conductivity of monocytes, and neutrophil-to-lymphocyte ratio. ML model that incorporated CBC, DC, and CPD showed excellent performance in predicting bacteremia among adult patients with suspected bacterial infections and blood culture sampling in emergency departments.

Evaluation of the cytogenetic activity of the food dye Azorubine in a micronucleus test in mice

Introduction. Azorubine E122 (Carmoisine, Food Red 3) monoazo dye is used in the manufacture of desserts, caramels, sweets, marmalades, ice cream, alcoholic and non-alcoholic drinks, etc. The safety assessment of food additives includes the study of genotoxic potential. At the same time, for substances with a high or a small but long-term exposure (including food additives), in vivo tests are required.
 Materials and methods. The genotoxic activity of aqueous solutions of synthetic food azo dyes E122 Azorubin was studied by the micronuclear method on bone marrow cells in mice (males, hybrids F1 CBA × C57Bl6/j). The studied substances were injected into the stomach of mice at doses of 250–2000 mg/kg twice with an interval of 24 hours, with the preparation of bone marrow preparations 24 hours after the last administration. The frequency of micronucleated (MN) polychromatophilic erythrocytes (PCEs) was estimated on the base of the results of the analysis of 4000 PСЕ. The proportion of PСE among all red blood cells was determined by analyzing 500 cells per animal. 
 Results. There was no statistically significant increase in the frequency of PCE with MN over the current control with a double administration of Azorubine in all studied doses. After exposure at doses of 1000 and 2000 mg/kg, the incidence of MN PCEs slightly exceeded the upper limit of the 95% CI of the accumulated negative control and the effect was dose-dependent and statistically significant, which does not allow recognizing the answer as clearly negative.
 Limitations of the study are due to the methodology of the test: only cytogenetic disorders in a single tissue were analyzed under conditions of double enteral administration of the studied sample.
 Conclusion. An analysis of the frequency of MN PCEs in the bone marrow of mice after a double injection of Azorubine at doses of 250–2000 mg/kg made it possible to qualify the result of the experiment as uncertain.

Avian Erythrocytes and Agranulocytes - a Review

This study reports on the morphology of red blood cells and agranulocytes in various types of birds, such as rheas, emus, ostriches, chickens, and geese. Though all of them have the typical avian similarity in blood cells, yet there are some differences. Along with normal and mature erythrocytes, lymphocytes, and monocytes, the present study also focuses on rubricytes, reticulocytes, poikilocytes, polychromatophilic erythrocytes, reactive lymphocytes, pleomorphic monocyte nuclei, etc.

Spontaneous massive fetomaternal hemorrhage: two case reports and a literature review of placental pathology

BackgroundMassive fetomaternal hemorrhage (FMH) is a rare event during pregnancy that may cause severe fetal anemia or death.Case presentationThis paper reports two cases of fetomaternal hemorrhage with unexplained reasons. Both cases required emergency caesarean sections for non-reassuring fetal status and were treated with neonatal blood transfusion. Fetomaternal hemorrhage was confirmed via maternal Kleihauer-Betke test.ConclusionWe found parenchymal pallor, increased nucleated red blood cells (nRBCs), and syncytial knots (SKs) in the placentas, which are compatible with fetal anemia. Immunohistochemical staining indicated VEGF, CD34, and CD31 expression in the endothelial cells of the capillaries, characteristic of massive FMH placenta. This article also reviews the particular histopathological changes in FHM placenta according to the placental lesion classification system.

Human Endometrium Derived Induced Pluripotent Stem Cells Are Amenable to Directed Erythroid Differentiation

BACKGROUND:A protocol for using human endometrium derived induced pluripotent stem cells (iPSCs) to derive hematopoietic and erythroid lineages will be elaborated, through a two-phase culture system.METHODS:Discarded endometrial tissues were obtained from women receiving hysterectomy in their 4th to 5th decade due to benign uterine conditions. pCE-Sox2, Oct4, Klf4, L-Myc and Lin28 episomal vectors were used to electrotransfect the endometrial stromal cells. The first 8 days involves commitment to hematopoietic stem cells through embryoid body with robust expansion on murine bone marrow stromal cells. The second phase involves feeder free conditions with hydrocortisone, stem cell factor, interleukin-3, and recombinant EPO. After 22 days of feeder free culture, the expression profiles of CD235a+, CD34+, CD43+ and CD 71+ were analyzed by flow cytometry and Wright-Giemsa staining for differential counting. The oxygen carrying capacity of cultured RBCs was measured using a hemoxanalyser.RESULTS:As a result of inducing these cells via co-culture with murine stromal fibroblasts, all endometrium derived iPSCs were differentiated into erythroblasts with a stable yield of approximately 80% for polychromatic and orthochromatic normoblasts. The protocol for complete induction of erythroid lineage cells starting from human endometrial tissue via iPS cells has been optimized.CONCLUSION:Successful directed erythroid differentiation has occurred from human endometrium-derived iPS cells. A comprehensive process of actually deriving iPS cells using discarded surgical hysterectomy specimens to the erythroid fate has significance in that the scope of using human iPSC cell lines for tissue regeneration could be expanded in the future.

Fetal nucleated red blood cells in maternal peripheral blood for non-invasive prenatal diagnosis

Fetal nucleated red blood cells in maternal peripheral blood for non-invasive prenatal diagnosis

Diagnostic Value and Prognostic Significance of Nucleated Red Blood Cells (NRBCs) in Selected Medical Conditions.

Nucleated red blood cells (NRBCs) are premature erythrocyte precursors that reside in the bone marrow of humans of all ages as an element of erythropoiesis. They rarely present in healthy adults' circulatory systems but can be found circulating in fetuses and neonates. An NRBC count is a cost-effective laboratory test that is currently rarely used in everyday clinical practice; it is mostly used in the diagnosis of hematological diseases/disorders relating to erythropoiesis, anemia, or hemolysis. However, according to several studies, it may be used as a biomarker in the diagnosis and clinical outcome prognosis of preterm infants or severely ill adult patients. This would allow for a quick diagnosis of life-threatening conditions and the prediction of a possible change in a patient's condition, especially in relation to patients in the intensive care unit. In this review, we sought to summarize the possible use of NRBCs as a prognostic marker in various disease entities. Research into the evaluation of the NRBCs in the pediatric population most often concerns neonatal hypoxia, the occurrence and consequences of asphyxia, and overall neonatal mortality. Among adults, NRBCs can be used to predict changes in clinical condition and mortality in critically ill patients, including those with sepsis, trauma, ARDS, acute pancreatitis, or severe cardiovascular disease.

Red blood cells of Labeo rohita express Toll-like receptors, NOD- like receptors, interleukins, and interferon-I in response to Gram-negative bacterial infections and lipopolysaccharide stimulations.

Red blood cells (RBCs) are the most abundant cell types in the circulatory system of vertebrates. In fish, RBCs retain their nuclei throughout their lifetime and remain transcriptionally and translationally active. While their primary function is typically associated with gas exchange, recent reports indicate that nucleated red blood cells can play a significant role in regulating the body's innate immune response. The current article describes the innate immune role of red blood cells in rohu (Labeo rohita), a freshwater fish species that holds significant commercial importance in India and South-East Asian nations. From the whole blood and mucosal surface RBCs have been isolated through density gradient centrifugation with HiSep™LSM 1077 (density 1.007 ± 0.0010) and their purity has been confirmed by the Giemsa staining followed by microscopical observations. Toll-like receptors (TLR2, 3, 4, 5) and nucleotide oligomerization domain (NOD)-like receptors (NOD1 and NOD2) in RBCs of rohu fingerlings were observed to be significantly activated (P < 0.05) on infection with Aeromonas hydrophila and Edwardsiella tarda. This activation resulted in increased expression of interleukins (IL-8, IL-1β) and interferon (IFN)-I genes. The activation of TLR4, NOD1 and NOD2, as well as the expression of interleukins and IFN-I genes have been observed in both in vivo and in vitro stimulation of rohu RBCs with lipopolysaccharides. These findings highlight the importance of fish RBCs in enhancing innate immunity against various pathogenic invasions in rohu.

The impact of polycystic ovary syndrome on placental histopathology patterns in in-vitro fertilization singleton live births

IntroductionPregnant polycystic ovary syndrome (PCOS) patients are at increased risk for myriad obstetric complications, with the placenta thought to play a key role in their development. We aimed to evaluate placental histopathology patterns in placentas of women with PCOS who underwent in-vitro-fertilization (IVF). MethodsThis retrospective study utilized full gross and histopathologic assessment of placentas of all women who had IVF treatment and delivered at the Royal Victoria Hospital from 2009 to 2017, regardless of complications or mode of delivery. Pathologic findings included anatomic, inflammation, villous maturation, and vascular mal-perfusion features. Placentas of PCOS women were compared to those of ovulatory controls. Multivariate logistic regression was used to adjust results for confounding factors potentially associated with significant placental and perinatal characteristics. ResultsWomen with PCOS (n = 47) were more likely to develop gestational diabetes mellitus compared to ovulatory controls (n = 1121) (38.3% vs. 9.8%, p < 0.001). Placentas from PCOS women were more likely circumvallate placentas (aOR 8.3, 95%CI 1.9–37.3) and more likely to have a hypercoiled umbilical cord (aOR 6.8 95%CI 1.3–36.8) and villitis of unknown etiology (aOR 6.1, 95%CI 1.5–25.6). There was an increased likelihood of chorangiosis (aOR 2.7, 95% CI 1.3–5.8), evidence of fetal vascular malperfusion based on one criteria (aOR 2.7, 95%CI 1.1–7.4), or more than one criteria (aOR 6.4, 95%CI 1.6–25.9), more nucleated fetal red blood cells (aOR 5.2, 95%CI 1.1–24.5), and a higher likelihood of chorangiomas (aOR 9.4, 95%CI 1.6–55.1) in placentas from PCOS women than in controls. DiscussionIVF pregnancies’ placental histopathological characteristics are significantly impacted by an underlying diagnosis of PCOS, including important anatomic changes and vascular placental abnormalities.

Relationship Between the Coma Scores and Hemogram Parameters Measured by New-generation Devices.

To investigate the relationship between the coma scores-Glasgow coma scale (GCS), Sequential Organ Failure Assessment (SOFA), and Acute Physiological and Chronic Health Assessment (APACHE-II)-in intensive care unit (ICU) patients and the percentage of macrocytosis (%MAC), immature granulocyte (IG), cellular haemoglobin concentration (cHGB), nucleated red blood cell (NRBC), nucleated red cell/ white cell ratio (NR/W), hyperchromic ratio (%HPR), and platelet distribution width (PDW) values. A descriptive comparative study. Place and Duration of the Study:Medicine Faculty, University ofHarran,Turkiye, from December 2020 to May 2022. The hemogram parameters of the patient groups with a GCS of 3-8 (n=51) and a GCS of 9-15 (n=43) and a control group of 55 healthy volunteers were measured using the new-generation hemogram autoanalyzer AlinityHQ (Abbott, USA). These parameters were compared with the coma scores (GCS, SOFA, and APACHE-II) of the patients. There was a statistically significant difference in IG, %MAC and PDW values (p-values were 0.025, 0.011, and 0.004, respectively) and an inverse correlation with GCS scores (correlation coefficients were -0.247, -0.264, and -0.297, respectively) was observed. There was also a significant correlation between the SOFA scores and %HPR and cHGB (correlation coefficients were 0.234, -0.358, p-values were 0.025, 0.001, respectively), and the APACHE-II scores and NRBC and NR/W values (correlation coefficients were -0.270, -0.247, p values were 0.009, 0.017, respectively). While other haematological parameters other than PDW were not associated with coma scores, parameters measured using new-generation haematological devices (%MAC, IG, cHGB, NRBC, NR/W, and %HPR) were found to be associated with estimated coma scores. These parameters can therefore be used as simple, rapid prognostic biomarkers and assist researchers in the development of new scoring models. ICU, Hyper, Coma, Sofa, Apache.

Analytical errors in nucleated red blood cell enumeration.

Enumeration of nucleated red blood cells (NRBCs) in peripheral blood of dogs and cats is performed by manual counting during blood film evaluation. Automated methods have increased precision and accuracy; however, most analyzers cannot distinguish leukocytes and NRBCs. The Sysmex XN-V Series may distinguish NRBCs and leukocytes; however, analytical errors occur. We aimed to investigate cases with discrepant automated and manual NRBC counts, and to evaluate reasons for analytical errors. Data from samples with increased NRBCs were collected retrospectively and compared with manual counts performed on blood films using Spearman's correlation, Passing-Bablok agreement analysis, and Bland-Altman comparisons. Precision of the automated method and interobserver agreement of manual counts were evaluated. Cases with discrepant results were investigated. Agreement between the methods was good at ≤1NRBC ×109 /L in dogs and cats, and inadequate at ≥1NRBC ×109 /L. The automated method demonstrated a negative proportional difference to the manual method. Precision was good for the automated method (overall CV 7.1%) and interobserver agreement for the manual method was poor overall (mean CV 27.3%, range 0%-106.1%). Inaccuracies in NRBC enumeration by the automated method occurred with high hematocrits, the mergence of the cell fragments and leukocyte clouds, and the presence of earlier erythroid precursors. NRBC enumeration by the WNR channel on the Sysmex XN-1000 V is precise and has good agreement with manual counts in canine and feline blood samples at ≤1NRBC ×109 /L. Manual film review is indicated for samples with ≥1NRBC ×109 /L, earlier erythroid precursors, samples from greyhounds and dehydrated patients, and if gating errors are noted.

Nucleated Red Blood Cells Are Predictive of In-Hospital Mortality for Pediatric Patients.

We sought to establish whether nucleated red blood cells (NRBCs) are predictive of disposition, morbidity, and mortality for pediatric patients presenting to the emergency department (ED). A single-center retrospective cohort study examining all ED encounters from patients aged younger than 19 years between January 2016 and March 2020, during which a complete blood count was obtained. Univariate analysis and multivariable logistic regression were used to test the presence of NRBCs as an independent predictor of patient-related outcomes. The prevalence of NRBCs was 8.9% (4195/46,991 patient encounters). Patient with NRBCs were younger (median age 4.58 vs 8.23 years; P < 0.001). Those with NRBCs had higher rates of in-hospital mortality (30/2465 [1.22%] vs 65/21,741 [0.30%]; P < 0.001), sepsis (19% vs 12%; P < 0.001), shock (7% vs 4%; P < 0.001), and cardiopulmonary resuscitation (CPR) (0.62% vs 0.09%; P < 0.001). They were more likely to be admitted (59% vs 51%; P < 0.001), have longer median hospital length of stay {1.3 (interquartile range [IQR], 0.22-4.14) vs 0.8 days (IQR, 0.23-2.64); P < 0.001}, and median intensive care unit (ICU) length of stay (3.9 [IQR, 1.87-8.72] vs 2.6 days [IQR, 1.27-5.83]; P < 0.001). Multivariable regression revealed presence of NRBCs as an independent predictor for in-hospital mortality (adjusted odds ratio [aOR], 2.21; 95% confidence interval [CI], 1.38-3.53; P < 0.001), ICU admission (aOR, 1.30; 95% CI, 1.11-1.51; P < 0.001), CPR (aOR, 3.83; 95% CI, 2.33-6.30; P < 0.001), and 30-day return to the ED (aOR, 1.15; 95% CI, 1.15-1.26; P < 0.001). The presence of NRBCs is an independent predictor for mortality, including in-hospital mortality, ICU admission, CPR, and readmission within 30 days for children presenting to the ED.

Nucleated red blood cells: unexpected orchestrators of fetal immunity

Abstract Red blood cells (RBCs) are not typically considered active immune mediators. This dogma stems from the fact that RBC precursors discard organelles as they mature, thus losing the ability to alter gene expression in response to stimuli. Intriguingly, nucleated RBCs (nRBCs) circulate in human fetuses and neonates. Due to evolutionary pressure for successful reproduction, circulation of nRBCs during pregnancy is likely important. However, the role of nRBCs in uteroremains unknown. To define the role of human nRBCs, we queried single cell RNA-seq data and found that transcriptomics support nRBCs as putative immune mediators. Unexpectedly, we found that nRBCs express antigen processing &amp; presentation machinery. nRBCs constitutively express MHC II &amp; co-stimulatory molecules, hallmarks of specialized antigen-presenting cells. We further demonstrated that nRBCs internalize and cleave antigens. Currently, we aim to determine if nRBCs present antigens to T cells and to characterize the elicited response. In addition, we found that nRBCs express pattern recognition receptors capable of detecting pathogens and initiating an antimicrobial response. We demonstrated that nRBCs respond to viral infection through upregulation of antiviral genes, interferons/interferon-stimulated genes, MHC I, and pro-inflammatory cytokines/chemokines. These data suggest a putative functionality for nRBCs in mediating antiviral immunity in utero. We are currently investigating antiviral functions of nRBCs. Together, our findings shed light on an unexpected orchestrator of fetal immunity. Knowledge of the fetal immune system has potential to help us understand health and disease during pregnancy, as a neonate, and likely every stage of life after. Supported by grants from NIH/NIDDK 5T32DK007780, NIH/NIGMS K12GM081259, Burroughs Wellcome Fund 2022 Next Gen Pregnancy Initiative Award

Megakaryoblasts in pleural fluid in neonate with transient leukaemia of Down's syndrome.

Twin baby boy born at 35+2 weeks gestation with a birth weight of 2070 grams via emergency cesarean section secondary to abnormal CTG and foetal distress. Full blood count obtained at birth showed haemoglobin 159 g/L, total white cell count 99.54 × 109/L platelet count 779 × 109/L. Manual differential showed an absolute neutrophil count of 2.01 × 109/L absolute lymphocyte count of 45.27 × 109/L and blast count of 50.3 × 109/L (50%). Initial peripheral blood smear morphology review revealed a thrombocytosis, platelet anisocytosis and micromegakaryocytes. It also demonstrated neutropenia, myeloid left shift, large blasts with a high N/C ratio and nucleoli. Additionally, numerous nucleated red blood cells were seen with marked red cell dysplasia including binucleate forms and abnormal haemoglobinisation. There was no prenatal diagnosis of Down's syndrome (DS). The above blood results along with the baby's clinical signs raised suspicion of DS and subsequently transient leukaemia of DS. This baby was then transferred to the regional neonatal intensive care unit on day 2 of life. On admission, he was noted to have bilateral pleural effusion, synthetic liver dysfunction with elevated creatinine and ammonia levels as well as ascites. He was intubated, and ventilated and required inotropic support. Due to adverse features of elevated white cell count, hepatosplenomegaly, pleural effusion as well as ascites [1] and following the multidisciplinary team (MDT) discussion and parental consent, the patient was commenced on low dose cytarabine for 7 days. He tolerated that well, however, did experience mild cytopenias and pyrexic episodes, followed by recovery during the following two weeks with a reduction in pleural effusions, hepatosplenomegaly and reduction in oxygen requirement which enabled successful extubation and commencement of continuous positive airway pressure. Unfortunately, on day 19 of life he was noted to have slowly increasing oxygen requirements with re-accumulation of pleural effusions and increasing hepatosplenomegaly as well as increasing blast count in peripheral blood. He proceeded to require intubation and ventilation again as well as placement of a chest drain to remove pleural fluid to improve respiratory compliance. A review of pleural fluid morphology had shown frequent ‘blast-like’ cells; these were medium-sized with a high N/C ratio with finely condensed chromatin nucleoli and budded basophilic cytoplasm thought to most likely represent megakaryoblasts [2]. As a result of the deterioration, a decision by MDT was made to give another cycle of low-dose cytarabine on days 1 through 5. Following the second cycle of cytarabine, the patient again experienced mild cytopenias and pyrexic episodes, was treated with antibiotics and supportive management and transfusions of packed red cells. He had complete count recovery and no blast population identified morphologically on peripheral blood film on day 26 of the second cycle of cytarabine. His GATA1 mutation was subsequently found to be positive. He continued to improve and was discharged home on day 67 of life Figures 1-3. Karolina Chmielowiec—wrote the manuscript, Gary Beattie—manuscript review, obtained image, obtained and analysed flow cytometry data, Kathryn Clarke—manuscript review, obtained and analysed flow cytometry, Ellen McCloy—manuscript review, morphology review, Bethany Mitchell—manuscript review, morphology review, Christine Macartney—manuscript review, supervising consultant. All authors discussed the results and commented on the manuscript. None. The authors declare no conflict of interest. No funding was required for this submission. Not applicable. Not Applicable. Not Applicable All authors have contributed to the acquisition, analysis and interpretation of data as well as either drafting the paper or revising it critically. All authors approved the submitted and final versions. For original data, please contact [email protected]

Umbilical Cord Blood Bilirubin, Albumin, Reticulocyte Count, and Nucleated Red Blood Cells to Predict Subsequent Hyperbilirubinemia in Term Neonates: A Prospective Observational Study.

Hyperbilirubinaemia is one of the most important causes of re-admission in the early neonatal period. The socioeconomic factors are one of the most common reasons for early discharge in a developing country like India. This study aims to evaluate and analyze the statistical correlation of umbilical cord blood bilirubin, albumin, nucleated red blood cells (nRBC), and reticulocyte count as early predictors of neonatal hyperbilirubinemia. A prospective observational study was conducted from November 2015 to April 2017 in a tertiary care hospital in North Karnataka, India. Umbilical cord blood was collected at birth for analysis of bilirubin, albumin, reticulocyte count, and nRBC in term neonates. Total serum bilirubin (TSB) levels were estimated using the VITROS BuBc Slide method at 72 hours of life. Data were analyzed using SPSS version 23 (IBM Corp., Armonk, NY). A total of 200 term neonates were enrolled in the study, out of which 123 completed follow-ups. Of the 66 newborns who had cord bilirubin levels ≥1.75 mg/dl, 23 (34.8%) developed hyperbilirubinemia after 72 hours of life, whereas 10 of the 57 newborns (17.5%) whose cord bilirubin levels <1.75 mg/dl developed hyperbilirubinemia after 72 hours of life. Cord blood albumin of ≥3.75 g/dl was seen in 93 neonates, of which 18 (19.4%) developed hyperbilirubinemia after 72 hours of life and 15 (50%) with <3.75 g/dl developed hyperbilirubinemia after 72 hours of life. Cord reticulocyte count ≥4.95% was seen in 54 neonates; 20 (37.03%) developed hyperbilirubinemia, whereas in 69 neonates with <4.95%, 13 (18.84%) developed hyperbilirubinemia after 72 hours of life. Of the 62 neonates who had cord nRBC ≥3.5%, 28 (45.2%) neonates developed hyperbilirubinemia after 72 hours of life, whereas 5 of the 61 neonates (8.19%) with cord nRBC <3.5% developed hyperbilirubinemia after 72 hours of life. Cord blood bilirubin, albumin, reticulocyte counts, and nucleated RBC can be used as predictors of subsequent neonatal hyperbilirubinemia.

Placental lesions associated with stillbirth by gestational age, according to feature importance: Results from the stillbirth collaborative research network

IntroductionPrevious studies have identified lesions commonly found in placentas associated with stillbirth but have not distinguished across a range of gestational ages (GAs). The objective of this study was to identify lesions associated with stillbirths at different GAs by adapting methods from the chemical machine learning field to assign lesion importance based on correlation with GA. MethodsPlacentas from the Stillbirth Collaborative Research Network were examined according to standard protocols. GAs at stillbirth were categorized as: <28 weeks (extreme preterm stillbirth [PTSB]), 28–33′6 weeks (early PTSB), 34–36′6 weeks (late PTSB), ≥37 weeks (term stillbirth). We identified and ranked the most discriminating placental features, as well as those that were similar across GA ranges, using Kernel Principal Covariates Regression (KPCovR). ResultsThese analyses included 210 (47.2%) extreme PTSB, 85 (19.1%) early PTSB, 62 (13.9%) late PTSB, and 88 (19.8%) term stillbirths. When we compute the KPCovR, the first principal covariate indicates that there are four lesions (acute funisitis & nucleated fetal red blood cells found in extreme PTSB; multifocal reactive amniocytes & multifocal meconium found in term stillbirth) that distinguish GA ranges among all stillbirths. DiscussionThere are distinct placental lesions present across GA ranges in stillbirths; these lesions are identifiable using sophisticated feature selection. Further investigation may identify histologic changes across gestations that relate to fetal mortality.

DNA methylation age at birth and childhood: performance of epigenetic clocks and characteristics associated with epigenetic age acceleration in the Project Viva cohort

BackgroundEpigenetic age acceleration (EAA) and epigenetic gestational age acceleration (EGAA) are biomarkers of physiological development and may be affected by the perinatal environment. The aim of this study was to evaluate performance of epigenetic clocks and to identify biological and sociodemographic correlates of EGAA and EAA at birth and in childhood. In the Project Viva pre-birth cohort, DNA methylation was measured in nucleated cells in cord blood (leukocytes and nucleated red blood cells, N = 485) and leukocytes in early (N = 120, median age = 3.2 years) and mid-childhood (N = 460, median age = 7.7 years). We calculated epigenetic gestational age (EGA; Bohlin and Knight clocks) and epigenetic age (EA; Horvath and skin & blood clocks), and respective measures of EGAA and EAA. We evaluated the performance of clocks relative to chronological age using correlations and median absolute error. We tested for associations of maternal-child characteristics with EGAA and EAA using mutually adjusted linear models controlling for estimated cell type proportions. We also tested associations of Horvath EA at birth with childhood EAA.ResultsBohlin EGA was strongly correlated with chronological gestational age (Bohlin EGA r = 0.82, p < 0.001). Horvath and skin & blood EA were weakly correlated with gestational age, but moderately correlated with chronological age in childhood (r = 0.45–0.65). Maternal smoking during pregnancy was associated with higher skin & blood EAA at birth [B (95% CI) = 1.17 weeks (− 0.09, 2.42)] and in early childhood [0.34 years (0.03, 0.64)]. Female newborns and children had lower Bohlin EGAA [− 0.17 weeks (− 0.30, − 0.04)] and Horvath EAA at birth [B (95% CI) = − 2.88 weeks (− 4.41, − 1.35)] and in childhood [early childhood: − 0.3 years (− 0.60, 0.01); mid-childhood: − 0.48 years (− 0.77, − 0.18)] than males. When comparing self-reported Asian, Black, Hispanic, and more than one race or other racial/ethnic groups to White, we identified significant differences in EGAA and EAA at birth and in mid-childhood, but associations varied across clocks. Horvath EA at birth was positively associated with childhood Horvath and skin & blood EAA.ConclusionsMaternal smoking during pregnancy and child sex were associated with EGAA and EAA at multiple timepoints. Further research may provide insight into the relationship between perinatal factors, pediatric epigenetic aging, and health and development across the lifespan.

Modelling the erythroblastic island niche of dyserythropoietic anaemia type IV patients using induced pluripotent stem cells.

Introduction: Congenital dyserythropoietic anaemia (CDA) type IV has been associated with an amino acid substitution, Glu325Lys (E325K), in the transcription factor KLF1. These patients present with a range of symptoms, including the persistence of nucleated red blood cells (RBCs) in the peripheral blood which reflects the known role for KLF1 within the erythroid cell lineage. The final stages of RBCs maturation and enucleation take place within the erythroblastic island (EBI) niche in close association with EBI macrophages. It is not known whether the detrimental effects of the E325K mutation in KLF1 are restricted to the erythroid lineage or whether deficiencies in macrophages associated with their niche also contribute to the disease pathology. Methods: To address this question, we generated an in vitro model of the human EBI niche using induced pluripotent stem cells (iPSCs) derived from one CDA type IV patient as well as two iPSC lines genetically modified to express an KLF1-E325K-ERT2 protein that could be activated with 4OH-tamoxifen. The one patient iPSC line was compared to control lines from two healthy donors and the KLF1-E325K-ERT2 iPSC line to one inducible KLF1-ERT2 line generated from the same parental iPSCS. Results: The CDA patient-derived iPSCs and iPSCs expressing the activated KLF1-E325K-ERT2 protein showed significant deficiencies in the production of erythroid cells with associated disruption of some known KLF1 target genes. Macrophages could be generated from all iPSC lines but when the E325K-ERT2 fusion protein was activated, we noted the generation of a slightly less mature macrophage population marked by CD93. A subtle trend in their reduced ability to support RBC enucleation was also associated with macrophages carrying the E325K-ERT2 transgene. Discussion: Taken together these data support the notion that the clinically significant effects of the KLF1-E325K mutation are primarily associated with deficiencies in the erythroid lineage but it is possible that deficiencies in the niche might have the potential to exacerbate the condition. The strategy we describe provides a powerful approach to assess the effects of other mutations in KLF1 as well as other factors associated with the EBI niche.

Effect of RNA preservation methods on RNA quantity and quality of field-collected avian whole blood

The transcriptome comprises all RNA molecules in a sample, tissue, or organism. A limitation of comparative transcriptomic studies which compare gene expression between individuals often under some differing exposure or treatment, of wild avian populations continues to be sample preservation of high-quality RNA (i.e., ribonucleic acids that transfer, translate, and regulate the genetic code from DNA into proteins). Field sampling of wild bird blood provides challenges as RNA degradation progresses quickly, due in large part to the high nuclease content of avian blood and because cryopreservation is often not feasible at remote locations. The introduction of commercial buffers for preservation of RNA enables field-collected studies as these buffers deactivate nucleases which degrade target nucleic acids. We seek to compare the effectiveness of widely available RNA stabilizing buffers, RNAlater (Ambion) and DNA/RNA Shield (Zymo) at varying concentrations along with a dry ice-based flash freezing method to determine optimal preservation methods for field-collected avian blood samples. To determine optimal preservation methods, we assessed RNA quantity and quality metrics: RNA integrity numbers (RINe), rRNA ratios, and total RNA concentration. Nucleated red blood cells, a characteristic common across non-mammalian vertebrates, provide sufficient transcriptionally active material enabling the identification of potentially active gene regions from small and non-lethal samples (∼ 20 μl). A protocol was also optimized for total RNA extraction from avian blood samples with small starting volumes enabling sampling of birds with a minimum threshold of 5 g body mass. We found that RNA preservation buffers, RNAlater and DNA/RNA Shield at all concentrations provide sample protection from RNA degradation. We recommend that caution be exercised when using dry ice-based flash-freezing alone for sample preservation as these samples resulted in lower quality measures then samples in preservation buffer. Total RNA concentration was generally not affected by preservation treatment and may vary due to differences in initial sample volumes.

5599228 PITFALLS IN DIAGNOSING THROMBOTIC THROMBOCYTOPENIC PURPURA IN SICKLE CELL DISEASE

Background: Thrombotic microangiopathies are diagnosed by thrombocytopenia, microangiopathic haemolytic anaemia and evidence of end organ damage. In TTP this includes neurological symptoms, renal impairment and cardiac microthrombi. Confirmation of the diagnosis is severe deficiency (<10%) of the metalloproteinase ADAMTS13 and/or the presence of an inhibitor. The diagnosis of TTP in sickle cell disease (SCD) can be challenging as SCD is characterised by a chronic state of haemolysis often with very abnormal peripheral blood morphology so clinical features suggestive of TTP can be due to complications of SCD. More specifically, the coexistence of neurological impairment and thrombocytopenia seen in fat embolism syndrome (FES) complicating SCD can lead to an erroneous diagnosis of TTP. FES typically affects non-homozygous patients and those with a previously mild course of their illness manifesting with respiratory failure, neurological signs, thrombocytopenia and potential involvement of any system leading to high morbidity and mortality. Peripheral blood morphology typically shows very high number of nucleated red blood cells (nRBC). Aims: To identify published cases of TTP in patients with SCD and review the diagnostic approach. Methods: A PubMed literature search was performed using the terms “sickle cell” and “thrombotic thrombocytopenic purpura”/”TTP”. Results: Of 19 cases identified, 9 were individual case reports and a case series of 10. All patients had very mild course of their SCD. The median nadir platelet count was 38 x 109/L (7-70). 84% had neurological involvement either at presentation or shortly afterwards; primarily altered sensorium/drop in GCS. 79% patients also had severe type I respiratory failure either preceding or occurring simultaneously with the development of neurological signs and associated with extensive changes on chest imaging. Schistocytes were identified in all cases but in the vast majority at relatively low levels; at the same time, all showed large numbers of nRBCs. 18 of the 19 cases were published after 1998; in 15 there was no mention of ADAMTS13 testing at all. In the 3 cases with ADAMTS13 levels, all were normal; 2 samples were obtained before therapeutic plasma exchange (TPE) and 1 after. 18 patients received TPE and 1 an infusion of donor plasma. However, prior to TPE, all patients had already received red cell transfusion achieving Hb S levels of 30% or less. There were 2 (11%) deaths while the remainder of patients achieved complete recovery. One due to intracranial haemorrhage and the other, sepsis in the context of immunosuppression for prevention of TTP recurrence. The presence of early respiratory failure, the inconclusive morphological findings, the degree of thrombocytopenia, and the complete absence of confirmatory ADAMTS13 results bring the diagnosis of TTP to question in all cases. Given the clinical presentations, we suspect that some of these patients had FES. The positive outcomes may be explained by the fact that all patients received adequate red cell transfusion, but also the possible beneficial effect of TPE in acute complications of SCD with a hyper inflammatory component such as FES. Conclusion: The diagnosis of TTP in patients with SCD is challenging due to many overlapping features. It should not be made without confirmatory ADAMTS13 results and the possibility of alternative diagnoses should be explored.