Nucleated Erythrocytes Research Articles

More than hemoglobin - the unexpected diversity of globins in vertebrate red blood cells.

In many multicellular organisms, oxygen is transported by respiratory proteins, which are globins in vertebrates, between respiratory organs and tissues. In jawed vertebrates, eight globins are known which are expressed in a highly tissue-specific manner. Until now, hemoglobin (Hb) had been agreed to be the only globin expressed in vertebrate erythrocytes. Here, we investigate for the first time the mRNA expression of globin genes in nucleated and anucleated erythrocytes of model vertebrate species by quantitative real-time reverse transcription PCR (qRT-PCR). Surprisingly, we found transcripts of the whole gnathostome globin superfamily in RBCs. The mRNA expression levels varied among species, with Hb being by far the dominant globin. Only in stickleback, a globin previously thought to be neuron-specific, neuroglobin, had higher mRNA expression. We furthermore show that in birds transcripts of globin E, which was earlier reported to be transcribed only in the eye, are also present in RBCs. Even in anucleated RBCs of mammals, we found transcripts of myoglobin, neuroglobin, and cytoglobin. Our findings add new aspects to the current knowledge on the expression of globins in vertebrate tissues. However, whether or not the mRNA expression of these globin genes has any functional significance in RBCs has to be investigated in future studies.

Analysis of blood cell autophagy distribution in hematologic diseases by transmission electron microscope

目的研究血细胞自噬在血液病中的分布特征及其可能病理机制。方法回顾性分析3 277例血液病患者电镜标本，利用透射电子显微镜观察血细胞自噬现象，分析血细胞自噬在血液病中的分布特点，结合临床检测和诊断结果分析其病理机制。结果检出15例患者伴有成熟粒细胞或有核红细胞自噬现象，其中骨髓增生异常综合征6例，急性白血病2例，再生障碍性贫血、纯红细胞再生障碍性贫血、地中海贫血、缺铁性贫血、淋巴瘤、多发性骨髓瘤和真性红细胞增多症各1例。15例患者中粒细胞自噬11例，有核红细胞自噬4例。9例伴细胞凋亡，6例伴细胞溶解，5例伴细胞巨幼样变。结论血液病成熟粒细胞和有核红细胞自噬多见于骨髓增生异常综合征，细胞凋亡、巨幼样变和溶解等病态造血可能诱发细胞自噬。

Erythropoietin Receptors and IgG Autoantibody Expression on Nucleated Erythrocytes in Some Cases of Immuno-Related Pancytopenia.

In recent years, we have detected autoantibodies in bone marrow (BM) hemopoietic cells in some patients with idiopathic cytopenia of undetermined significance (ICUS), termed immunorelated pancytopenia (IRP). However, we know little about the targets of these autoantibodies. Twenty-six newly diagnosed IRP patients with IgG autoantibody on nucleated erythrocytes and 20 healthy donors as controls were enrolled in this study. The serum erythropoietin (EPO) level was examined by ELISA. Expression of EPO receptor (EPOR) and IgG autoantibody on the membrane of nucleated erythrocytes were detected by flow cytometry before and after autoantibody stripping. The serum EPO level of the untreated patients was 199.9 ± 106.4 mIU/mL, which was significantly higher than that of normal controls (13.2 ± 8.41 mIU/mL, p < 0.01). EPOR expression on nucleated erythrocytes in the patients was 1.38 ± 0.73%, lower than that of normal controls (2.33 ± 1.73%), but there was no significant difference; EPOR in these patients was inversely correlated with IgG autoantibody on erythrocytes (r = -0.479, p = 0.013). The regression equation was Y = 0.116-0.479X; EPOR expression on the membrane increased significiantly (5.63 ± 4.99%, p < 0.01) after stripping the autoantibodies. After immunosuppressive treatment, median hemoglobin increased from 72 g/L to 98 g/L, and median reticulocytes increased from 1.46% to 3.56%. IgG autoantibodies might block or competitively inhibit EPOR on nucleated erythrocytes in some cases of ICUS.

A Study of Naturally Acquired Canine Babesiosis Caused by Single and Mixed Babesia Species in Zambia: Clinicopathological Findings and Case Management.

A retrospective and prospective analysis of clinical records of dogs diagnosed with Babesia infections was carried out for the years 2000 to 2013 from practices in Lusaka, Zambia. Records of 363 dogs with confirmed Babesia infections were analysed using demographic factors including sex, breed, age, and clinical signs in relation to haematological findings and Babesia species. The clinical and laboratory findings observed are described as well as Babesia species identification. The study included 18 breeds and the highest proportion were mongrels (32.2%), males representing 64.5% of the population. The most common presenting problems were anorexia (65.3%) and lethargy/weakness (65.3%). The most common clinical signs were fever (87.3%), pallor (52.3%), lymphadenopathy (47.4%), and presence of ticks (44.9%). Anaemia (96.4%) and nucleated erythrocytes (42.2%) were the most common laboratory findings. A mixed infection of Babesia rossi and Babesia gibsoni was present in 59.7% of dogs, whilst 8% and 32.2% had B. rossi and B. gibsoni as a single infection, respectively. Case management mainly involved therapy with tetracyclines and imidocarb and was usually accompanied by clinical improvement. This study highlights, for the first time, the presence of B. gibsoni in natural dog populations in Zambia, where previously only B. rossi was reported.

Evaluation of Cord Blood - Haematological Scoring System as an Early Predictive Screening Method for the Detection of Early Onset Neonatal Sepsis.

Sepsis is one of the major causes of neonatal morbidity and mortality. Early recognition and diagnosis of neonatal sepsis are difficult because of the variable and non-specific clinical presentation of this condition. Hence, there is a need for early predictive screening method for neonatal sepsis. To evaluate the role of Cord blood Haematological Scoring System as an early predictive screening method for detection of early onset neonatal sepsis and also to identify the neonates who are at risk of developing neonatal sepsis using cord blood. The present prospective cross-sectional study was conducted by taking 153 cord blood samples of full term newborns immediately after delivery in the hospital. Pre-term, still birth and emergency deliveries were excluded from the study. The cord blood was collected and analysed for various Haematological parameters like Total leucocyte count, Absolute Neutrophil count, Immature to mature Neutrophil ratio, immature to mature ratio, Neutrophil morphology, nucleated erythrocytes, platelet count, micro erythrocyte sedimentation rate. Blood cultures were performed as gold standard for diagnosing neonatal sepsis. Chi-square test, Risk ratio, risks in exposed and risk in unexposed were performed. Of 153 newborns for analysis, 59 (38.56%) developed sepsis. The haematological scoring system found that an abnormal immature to total neutrophil ratio, Neutropenia, micro erythrocyte sedimentation rate followed by an abnormal immature to mature neutrophil ratio were the most sensitive indicators in identifying infants with sepsis. The study also found that higher the score, the greater the certainty of sepsis being present. The haematological scoring system using cord blood can be considered as an early predictive screening method for detection of early onset neonatal sepsis. Identifying the risk of developing sepsis early can prevent morbidity and mortality of the neonates.

Triggering of Suicidal Erythrocyte Death Following Boswellic Acid Exposure

Background/Aims: The antinflammatory natural product boswellic acid is effective against cancer at least in part by inducing tumor cell apoptosis. Similar to apoptosis of nucleated cells erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i), energy depletion, ceramide formation and p38 kinase activation. The present study tested, whether and how boswellic acid induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca²⁺]_i from Fluo3-fluorescence, ceramide abundance utilizing specific antibodies, reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofuorescein diacetate (DCFDA) fluorescence, and cytosolic ATP concentration utilizing a luciferin-luciferase assay kit. Results: A 24 hours exposure of human erythrocytes to boswellic acid (5 µg/ml) significantly increased the percentage of annexin-V-binding cells (to 9.3 ±0.9 %) and significantly decreased forward scatter. Boswellic acid did not significantly modify [Ca²⁺]_i, cytosolic ATP, ROS, or ceramide abundance. The effect of boswellic acid on annexin-V-binding was significantly blunted, but not abolished by p38 kinase inhibitors skepinone (2 µM) and SB203580 (2 µM). Conclusions: Boswellic acid stimulates cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part dependent on p38 protein kinase activity.

In Vitro Differentiation of Embryonic Stem Cells into Hematopoietic Lineage: Towards Erythroid Progenitor's Production.

Embryonic stem cells (ESCs) differentiation via embryoid body (EB) formation is an established method that generates the three germ layers. However, EB differentiation poses several problems including formation of heterogeneous cell populations. Herein, we described a differentiation protocol on enhancing mesoderm derivation from murine ESCs (mESCs) using conditioned medium (CM) from HepG2 cells. We used this technique to direct hematopoiesis by generating "embryoid-like" colonies (ELCs) from murine (m) ESCs without following standard formation of EBs. Our CM-mESCs group yielded an almost fivefold increase in ELC formation (p ≤ 0.05) and higher expression of mesoderm genes;-Brachyury-T, Goosecoid, and Flk-1 compared with control mESCs group. Hematopoietic colony formation from CM-mESCs was also enhanced by twofold at days 7 and 14 with earlier colony commitment compared to control mESCs (p ≤ 0.05). This early clonogenic capacity was confirmed morphologically by the presence of nucleated erythrocytes and macrophages as early as day 7 in culture using standard 14-day colony-forming assay. Early expression of hematopoietic primitive (ζ-globin) and definitive (β-globin) erythroid genes and proteins was also observed by day 7 in the CM-treated culture. These data indicate that hematopoietic cells more quickly differentiate from CM-treated, compared with those using standard EB approaches, and provide an efficient bioprocess platform for erythroid-specific differentiation of ESCs.

Morphology and water permeability of red blood cells from green sea turtle (Chelonia mydas).

The morphology and diffusional water permeability (P d) of red blood cells (RBCs) from green sea turtle (GST) (Chelonia mydas) are presented for the first time. The RBCs had an ellipsoidal shape with full-axis lengths (diameters): D = 14.4μm; d = 10.2μm; h = 2.8μm. The values of P d (cms(-1)) were 5.1 × 10(-3) at 15°C, 5.7 × 10(-3) at 20°C, 6.3 × 10(-3) at 25°C, 6.8 × 10(-3) at 30°C, and 7.9 × 10(-3) at 37°C (i.e., significantly higher than in human RBCs in which it was measured to be 4.2 × 10(-3) at 25°C, 5.0 × 10(-3) at 30°C, and 6.2 × 10(-3) at 37°C). There was a lack of inhibition of P d of GST RBCs by p-chloromercuribenzoate (PCMB), a well-known inhibitor of the RBC water channel proteins (WCPs). The activation energy of water diffusion (E a,d) in GST RBCs was 15.0 ± 1.6kJmol(-1) which is lower than the E a,d for human RBCs (~25kJmol(-1)). These results indicate that in the membrane of GST RBCs, there were no WCPs that were inhibited by the mercurial reagent, while the lipid bilayer of this membrane is unusually permeable to water. This is likely to be a phylogenetically old trait, like that found in amphibians and even the later birds, all of which have nucleated erythrocytes; and it is also likely to be a result of the animal's adaptation to a herbivorous diet (algae and seagrasses).

Cellular characterization of thrombocytes in Xenopus laevis with specific monoclonal antibodies

Platelets are produced from megakaryocytes (MKs) in the bone marrow. In contrast, most nonmammalian vertebrates have nucleated and spindle-shaped thrombocytes instead of platelets in their circulatory systems, and the presence of MKs as thrombocyte progenitors has not been verified. In developing a new animal model in adult African clawed frog (Xenopus laevis), we needed to distinguish nucleated thrombocytes and their progenitors from other blood cells, because the cellular morphology of activated thrombocytes resembles lymphocytes and other cells. We initially generated two monoclonal antibodies, T5 and T12, to X. laevis thrombocytes. Whereas T5 recognized both thrombocytes and leukocytes, T12 specifically reacted to spindle-shaped thrombocytes. The T12(+) thrombocytes displayed much higher DNA ploidy than nucleated erythrocytes, and they expressed CD41 and Fli-1. In the presence of CaCl2, adenosine diphosphate, thrombin, or various collagens, T12(+) thrombocytes exhibited aggregation. These thrombocytes were located predominantly in the hepatic sinusoids and the splenic red pulp, suggesting that both organs are the sites of thrombopoiesis. Notably, circulating thrombocytes exhibited lower DNA ploidy than hepatic thrombocytes. Intraperitoneal administration of T12 produced immune thrombocytopenia in frogs, which reached a nadir 4 days postinjection, followed by recovery, suggesting that humoral regulation maintained the number of circulating thrombocytes. Although differences between MKs and thrombocytes in X. laevis remain to be defined, our results provide further insight into MK development and thrombopoiesis in vertebrates.

Coupling of membrane and metabolic functions in nucleated erythrocytes of Scorpaena porcus L. Under hypoxia in vivo and in vitro

The in vivo and in vitro effect of hypoxia (0.57–8.17 mg O2l−1) on functional characteristics of nucleated erythrocytes of the benthic scorpionfish Scorpaena porcus L. was studied. The cellular systems in this species were established to be characterized by a balanced suppression of membrane and metabolic functions in conditions of external oxygen deficit (in vivo experiments). It manifests itself in the capacity of cells to maintain the intracellular ATP concentration and transmembrane Na+ and K+ gradients within the norm at low activity of Na+, K+-ATPase and hexokinase. This phenomenon appears to be underlain by the reduction in the number of active plasma membrane ion channels diminishing energetic costs of cationic gradient maintenance (metabolic arrest phenomenon). The same is also evidenced by the rise in FDA and R123 fluorescence intensity in erythrocyte suspensions under hypoxia (in vitro experiments). The mechanisms underlying this phenomenon are discussed.

The asymmetric membrane structure of erythrocytes from Crucian carp studied by atomic force microscopy

Cell membranes play a key role in cellular activities. Fish erythrocytes, prototype of the nucleated erythrocytes of lower vertebrates, are predominantly oval, biconvex discs with elliptical nucleus and much larger in size than human erythrocytes. In attempts to disclose the correlation of membrane structure between fish and human erythrocytes, we used in situ atomic force microscope (AFM) combined with single-molecule force spectroscopy to study the membrane structure of Crucian carp erythrocytes under quasi-native conditions. Our results revealed the asymmetric distribution of proteins in Crucian carp erythrocyte membrane: the outer leaflet of membrane is rather smooth without any proteins, whereas the inner leaflet of membrane is very rough with dense proteins. The asymmetry of fish erythrocyte membrane structure fits well with the semi-mosaic model of human erythrocyte membrane structure. This similarity of membrane structure between human and fish erythrocytes extends the semi-mosaic model of erythrocytes membrane structure to a wider range of species.

Characterisation of the green turtle’s leukocyte subpopulations by flow cytometry and evaluation of their phagocytic activity

Phagocytosis is a fundamental aspect of innate immunity that is conserved across many species making it a potentially useful health-assessment tool for wildlife. In non-mammalian vertebrates, heterophils, monocytes, macrophages, melanomacrophages, and thrombocytes all have phagocytic properties. Recently, B lymphocytes from fish, amphibians, and aquatic turtles have also showed phagocytic capacity. Phagocytes can be studied by flow cytometry; however, the use of this tool is complicated in reptiles partly because nucleated erythrocytes complicate the procedure. We separated green turtle leukocytes by density gradient centrifugation and identified subpopulations by flow cytometry and confocal microscopy. Additionally, we assessed their ability to phagocytize Fluorspheres and Ovoalbumin-Alexa. We found that heterophils and lymphocytes but not monocytes could be easily identified by flow cytometry. While heterophils from adults and juvenile turtles were equally able to phagocytize fluorspheres, adults had significantly more phagocytic ability for OVA-Alexa. Lymphocytes had a mild phagocytic activity with fluorospheres (27-38%; 39-45%) and OVA-Alexa (35-46%; 14-22%) in juvenile and adult green turtles, respectively. Confocal microscopy confirmed phagocytosis of fluorospheres in both heterophils and lymphocytes. This provides the first evidence that green turtle lymphocytes have phagocytic activity and that this assay could potentially be useful to measure one aspect of innate immunity in this species.

Embryonic hematopoiesis

Blood cells are continually produced from a pool of progenitors that derive from hematopoietic stem cells (HSCs). In vertebrates, the hematopoietic system develops from two distinct waves or generation of precursors. The first wave occurs in the yolk sac, in mammals or equivalent embryonic structure, and produces nucleated primitive erythrocytes that provide the embryo with the first oxygen transporter and are, therefore, essential for the viability of the embryo. The yolk sac also produces myeloid cells that migrate to the central nervous system and to the skin to form the microglia and skin specific macrophages, the Langerhans cells. The second wave occurs in the dorsal aorta and produces multipotential hematopoietic progenitors. These cells are generated once in the lifetime from mesoderm derivatives closely related to endothelial cells, during a short period of embryonic development. Newly generated cells do not reconstitute the hematopoietic compartment of conventional recipients; therefore, they are designated as immature or pre-HSCs. They undergo maturation into adult HSCs in the aorta or in the fetal liver accompanied by the expression of MHC class I, CD45, CD150, Sca-1 and the absence of CD48. Differentiation of HSCs first occurs in the fetal liver, giving rise to mature blood cells. HSCs also expand in the fetal liver, and in a short time period (four days in the mouse embryo), they increase over 40-fold. HSCs and progenitor cells exit the fetal liver and colonize the spleen, where differentiation to the myeloid lineage and particular lymphoid subsets is favored.

P-gp expression in brown trout erythrocytes: evidence of a detoxification mechanism in fish erythrocytes

Blood is a site of physiological transport for a great variety of molecules, including xenobiotics. Blood cells in aquatic vertebrates, such as fish, are directly exposed to aquatic pollution. P-gp are ubiquitous “membrane detoxification proteins” implicated in the cellular efflux of various xenobiotics, such as polycyclic aromatic hydrocarbons (PAHs), which may be pollutants. The existence of this P-gp detoxification system inducible by benzo [a] pyrene (BaP), a highly cytotoxic PAH, was investigated in the nucleated erythrocytes of brown trout. Western blot analysis showed the expression of a 140-kDa P-gp in trout erythrocytes. Primary cultures of erythrocytes exposed to increasing concentrations of BaP showed no evidence of cell toxicity. Yet, in the same BaP-treated erythrocytes, P-gp expression increased significantly in a dose-dependent manner. Brown trout P-gp erythrocytes act as membrane defence mechanism against the pollutant, a property that can be exploited for future biomarker development to monitor water quality.

Noninvasive Prenatal Test Raises Concern Over Screening Strategies and the Role of Ultrasound

Noninvasive Prenatal Test Raises Concern Over Screening Strategies and the Role of Ultrasound

Differentiation of nucleated erythrocytes using gata1 reporter medaka fish

Differentiation of nucleated erythrocytes using gata1 reporter medaka fish

Low RPS14 expression in MDS without 5q ‐ aberration confers higher apoptosis rate of nucleated erythrocytes and predicts prolonged survival and possible response to lenalidomide in lower risk non‐5q‐ patients

Haploinsufficiency of the ribosomal protein S14 (RPS14) has been identified as a causal factor in myelodysplastic syndrome (MDS) with isolated del (5q). This study was carried out to investigate RPS14 expression in MDS without 5q deletion and the role of lower expressed RPS14 plays in pathogenesis and the prognosis and lenalidomide-response predicting of non-5q-MDS. The expression level of RPS14 transcription was detected in 156 MDS patients without 5q-. The apoptosis of bone marrow (BM) nucleated erythrocytes was also analyzed. Furthermore, patient prognosis with lower or normal RPS14 expression was analyzed, and the role of RPS14 expression in lenalidomide-response prediction was evaluated. The reduced RPS14 expression occurred in 83 of 156 (53.2%) non-5q-patients. Patients with RPS14 lower expression presented higher platelet counts in the peripheral blood compared with RPS14 normal patients (P = 0.012). The lower RPS14 expression status was inversely correlated with increased apoptosis ratio in nucleated erythrocytes from BM (r = -0.54, P = 0.013). Patients with lower RPS14 expression have a higher 2-yr survival probability than normal RPS14 cases in the international prognosis scoring system (IPSS) lower risk group (90.8% vs. 71.7%; P = 0.018). A multivariate analysis showed RPS14 expression status was an independent predictor for survival in lower risk MDS patients without 5q deletion. Twelve patients were treated with lenalidomide. Five of seven patients achieved an erythroid response in the lower RPS14 expression group (5/7, 71.4%), compared with zero responses in the five normal RPS14 patients (P = 0.018). Lower RPS14 expression in MDS patients without 5q deletion is associated with increased apoptosis of nucleated erythrocytes in lower risk MDS. Additionally, lower RPS14 predicts prolonged survival and possible response to lenalidomide in lower risk MDS patients.

Effect of Bushen Yiqi Huoxue recipe on placental vasculature in pregnant rats with fetal growth restriction induced by passive smoking

Interactions of vascular endothelial growth factor (VEGF) with receptors VEGFR1/Flt1 and VEGFR2/Flk1, and those of angiopoietins (Ang-1, Ang-2) with receptor Tie2 play important roles in placental angiogenesis. This study investigated vascular morphology and expression of these angiogenic factors in rat placenta on the day 15, 18, 21 of gestation (D15, D18 and D21). The rats were randomly assigned into 3 groups: normal group, model group [fetal growth restriction (FGR) model], and Bushen Yiqi Huoxue (BYHR) recipe treatment group (BYHR group, the pregnant rats with FGR were treated with BYHR recipe). Morphological analysis indicated that during initial villous formation, fetal nucleated erythrocytes (FNEs) appeared in maternal blood sinus (MBS). Subsequently, FNEs were surrounded by endothelial cells to form fetal capillary (FC) and then by trophoblast cells to form villi. As pregnancy proceeded, FC density increased progressively with increasing endothelial identification staining (EIS) in normal and BYHR groups. Whereas, villous formation was suppressed, normal increase in FC density was impaired and EIS was weakened in model group. Quantitative PCR analysis showed that VEGF and Flk1 mRNA increased over gestation in all groups, indicating that VEGF might play a pivotal role in FC growth during late gestation. VEGF mRNA was increased on D15, while decreased on D21 in model group as compared with normal group and BYHR group. Immunohistochemically, Ang-2 protein was highly expressed in FNEs, gradually disappeared as villi matured, and decreased over gestation in all groups, indicating that Ang-2 might play a pivotal role in villous formation, which was further supported by decreased Ang-2 mRNA and protein expression in model group on D15. Ang-1 mRNA, Tie2 mRNA and Ang-1/Ang-2 ratio increased from D15 to D18 in all groups as placenta matured. Ang-1 mRNA, Tie2 mRNA and Ang-1/Ang-2 ratio were decreased on D18 in model group as compared with normal and BYHR groups, indicating delayed maturity of FGR placenta. Alterations in angiogenic factors may result in altered placental vasculature and cause placental insufficiency. BYHR recipe could balance the angiogenic factors to promote the formation and maturation of FGR placental vasculature.

A single cell gene detection using micro-tweezers and the microchamber polymerase chain reaction for the fetal DNA analysis

Here, we described a method to analyze a single nucleated fetal erythrocyte by a microchamber array and micro-tweezers. For non-invasive prenatal diagnosis, an extremely small number of fetal nucleated red blood cells need to be properly obtained from the maternal circulation. Fluorescence-activated cell sorting, magnetic-activated cell sorting or charge flow separation is usually used for isolation of fetal cells from maternal blood. However, these methods have low recovery rates, and are plagued by difficulties in accurately isolating fetal cells from maternal cells. Therefore, in this study we used micro-tweezers which were fabricated using micromachining techniques for picking up single fetal cells. The micro-tweezers have two thin probes whose distance can be regulated by applying voltage, and is able to sandwich single cell. This is a manual procedure, and not a high-throughput technique. However, it can obtain single cells accurately, thereby avoiding false-positive results that can occur by including other cells and/or genomic DNA derived from maternal cells. Microchamber PCR was performed to analyze for single fetal cells placed onto a microchamber array by micro-tweezers. Using this method, we successfully amplified the Rhesus blood group D antigen (RhD) gene from a single fetal nucleated red blood cell.

The PRAME and WT1 Transcripts Constitute a Good Molecular Marker Combination for Monitoring Minimal Residual Disease in Myelodysplastic Syndromes

Abstract 3853 BackgroundWith the incorporation of more aggressive therapies such as hematopoietic stem cell transplantation (HSCT), more myelodysplastic syndromes (MDS) patients fall into a status of minimal residual disease (MRD). The sensitive molecular markers are required to monitor MRD and predict relapse. The Wilms' tumor gene (WT1) is now a widely accepted molecular marker of MDS. However, WT1 itself could not fully meet the demands because a subset of MDS patients does not overexpress WT1, and the increase of WT1 expression compared with the normal control of MDS patients was mainly within 2-log. We wondered if the preferentially expressed antigen of melanoma (PRAME) gene could supplement WT1. MethodsThe PRAME and WT1 transcript levels were simultaneously measured in 312 bone marrow samples collected from newly diagnosed MDS patients and 27 samples from non-malignant cytopenia patients. Bone marrow samples from 14 MDS patients after their disease progression were also detected. To evaluate the value of combined detection of WT1 and PRAME transcripts, one hundred and eleven BM samples collected from 17 MDS patients during their treatment were tested them simultaneously (chemotherapy alone: 1 patient; HSCT:16 patients). Bone marrow samples from six MDS patients and five normal controls were sorted into the blasts (CD34+), nucleated erythrocytes (CD71+), immature myeloid cells (CD33+CD34-), and lymphocyte (CD45+high, low SSC) fractions by flow cytometry and measured the PRAME and WT1 transcript levels, respectively. We had previously established that the upper limits of the PRAME and WT1 transcript levels tested in normal bone marrow samples were 0.28% and 0.50%, respectively. ResultsNone of the 27 non-malignant cytopenia patients overexpressed PRAME (median 0.085%, range 0.01%-0.28%) and WT1 (median 0.095%, range 0.0089%-0.36%). Both WT1 and PRAME were commonly overexpressed in MDS. Both the overexpression frequency and the >1-log increase expression frequency of PRAME were similar to those of WT1 (74.4 % vs 77.6%; 51.6% vs 49.0%; p>0.05), and 88.1% of the patients overexpressed at least one marker. Moreover, the frequencies of PRAME expression with higher degrees of increase were significantly higher compared with those of WT1 expression (>2-log increase: 30.8% vs 3.8%; >3-log increase: 9.0% vs 0%; all p<0.001). PRAME had a higher log increase than WT1 in 53.3% of the patients with overexpressed WT1. For samples collected from newly diagnosed MDS patients, both the WT1 and PRAME transcript levels were significantly correlated with the percentage of blasts in bone marrow (r=0.35 and 0.22, all p<0.001). After disease progression, both WT1 and PRAME expressions prominently increased in six patients, only WT1 markedly increased in six patients, only PRAME markedly increased in one patient, and neither WT1 nor PRAME increased in one patient. Both PRAME and WT1 transcript levels generally fluctuated within the normal range after HSCT in all 10 patients in continuous complete remission (PRAME: 58/64 samples, WT1: 58/64 samples). Six out of seven patients were predicted relapse by the combined detection: sustained overexpression or significant increase over the normal range of both WT1 and PRAME in three patients, earlier by PRAME than WT1 or by PRAME alone in three patients. The expression patterns of WT1 differed from those of PRAME in the sorted cell fractions: WT1 is overexpressed in blasts and is significantly decreased in relative mature cells. Whereas, PRAME is mainly overexpressed not only by stem cells and blasts but also by the relative mature cells. ConclusionThe combined detection of WT1 and PRAME transcripts in newly diagnosed MDS patients could find more suitable and sensitive molecular marker for them compared to detecting WT1 alone. The PRAME and WT1 transcripts constitute a good molecular marker combination for monitoring minimal residual disease in MDS. Disclosures:No relevant conflicts of interest to declare.