Fetal Blood Cells Research Articles

Management and Treatment Outcomes of Hemolytic Disease of the Fetus and Newborn (HDFN)-A Retrospective Cohort Study.

Background: Hemolytic disease of the fetus and newborn (HDFN) is caused by maternal antibodies attacking fetal blood cell antigens. Despite routine antenatal anti-D prophylaxis, intrauterine transfusions (IUTs) are still needed in some HDFN cases. Methods: We conducted a retrospective cohort study on newborns with HDFN born in the 1st Department of Obstetrics and Gynecology of the Medical University of Warsaw. We analyzed 274 neonates with HDFN, identifying 46 who required IUT due to fetal anemia and 228 who did not. The laboratory results, management, and outcomes were compared between these groups. Results: Comparative analysis showed that newborns treated with IUT were more likely to have significant anemia, hyperbilirubinemia, and iron overload, indicated by a high ferritin concentration. These neonates more often required top-up transfusions, phototherapy, intravenous immunoglobulin infusions, and exchange transfusions. The length of stay was longer for newborns who received IUT. Conclusions: HDFN requiring IUT is associated with a greater number of complications in the neonatal period and more often requires additional treatment compared to HDFN not requiring IUT.

Evaluation of Four Humanized NOD-Derived Mouse Models for Dengue Virus-2 Infection.

Dengue is a significant public health problem with no specific viral treatment. One of the main challenges in studying dengue is the lack of adequate animal models recapitulating human immune responses. Most studies on humanized mice use NOD-scid IL2R gamma null (NSG) mice, which exhibit poor hematopoiesis for some cell populations. This study compares three humanized (hu) NOD-derived mouse models for dengue virus-2 (DENV-2) infection in the context of human cytokine expression. Three mouse strains (hu-NSG, hu-EXL, and hu-SGM3) received xenotransplants of human CD34+ fetal cord blood cells from a single donor, and one mouse strain received human peripheral blood mononuclear cells (hu-SGM3-PBMCs). All models exhibited infectious viruses in blood confirmed by plaque assay, but mice expressing human cytokines showed higher viremia compared to conventional NSG mice. The hu-SGM3-PBMCs model developed lethal infections, showing a significant increase in viremia and clinical signs. A detectable human cytokine response was observed in all the DENV-2-infected humanized mouse models. In conclusion, humanized NOD-derived mouse models expressing human cytokines offer a relevant platform for the study of dengue pathogenesis and antiviral therapies.

Development of a new staining protocol for the Kleihauer–Betke test to facilitate the reading of difficult cases

BackgroundThe Kleihauer–Betke (KB) test allows the detection of fetal red blood cells (containing fetal hemoglobin, HbF) in the maternal blood to identify and quantify potential fetal-maternal hemorrhages. In certain cases, detecting fetal red blood cells with conventional staining is difficult. False-positive results or overestimation of the quantity of fetal red blood cells may occur in cases of maternal hemoglobinopathy. In this study, we developed a new staining protocol to facilitate the reading of difficult smears and improve the precision of the quantification of fetal red blood cells; we also analyzed the performance of this new method. This study assessed blood samples with and without hemoglobin abnormalities, which present difficulties when interpreting the KB test.MethodsThe new staining formula is based on an improved elution technique and the use of a different stain instead of hematoxylin. To test this staining method, 16 samples from patients with abnormal hemoglobin electrophoresis and 14 samples from patients with normal hemoglobin electrophoresis were analyzed using the KB test with the classical staining method and the new staining method. In addition, a second series was prepared using the same samples spiked with fetal red blood cells from newborn blood, to compare the accuracy of the two methods in identifying fetal red blood cells.ResultsIn the 60 slides analyzed with both staining methods, we found that the new technique improved the accuracy from 78 to 85%; lowered the coefficient of variation between the operators, which decreased from 20.7% to 12.7%; increased the specificity in our population from 56 to 70%; and decreased the number of false-positive cases by 30%.ConclusionsWe successfully developed a new staining technique that facilitates the reading of difficult slides and improves the specificity of the detection of fetal red blood cells. This technique is recommended as a secondary method to use before sending the sample for additional exploration.

Classification of fetal and adult red blood cells based on hydrodynamic deformation and deep video recognition

Flow based deformation cytometry has shown potential for cell classification. We demonstrate the principle with an injection moulded microfluidic chip from which we capture videos of adult and fetal red blood cells, as they are being deformed in a microfluidic chip. Using a deep neural network - SlowFast - that takes the temporal behavior into account, we are able to discriminate between the cells with high accuracy. The accuracy was larger for adult blood cells than for fetal blood cells. However, no significant difference was observed between donors of the two types.

Single-cell multiomics of human fetal hematopoiesis define a developmental-specific population and a fetal signature.

Knowledge of human fetal blood development and how it differs from adult blood is highly relevant to our understanding of congenital blood and immune disorders and childhood leukemia, of which the latter can originate in utero. Blood formation occurs in waves that overlap in time and space, adding to heterogeneity, which necessitates single-cell approaches. Here, a combined single-cell immunophenotypic and transcriptional map of first trimester primitive blood development is presented. Using CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), the molecular profile of established immunophenotype-gated progenitors was analyzed in the fetal liver (FL). Classical markers for hematopoietic stem cells (HSCs), such as CD90 and CD49F, were largely preserved, whereas CD135 (FLT3) and CD123 (IL3R) had a ubiquitous expression pattern capturing heterogenous populations. Direct molecular comparison with an adult bone marrow data set revealed that the HSC state was less frequent in FL, whereas cells with a lymphomyeloid signature were more abundant. An erythromyeloid-primed multipotent progenitor cluster was identified, potentially representing a transient, fetal-specific population. Furthermore, differentially expressed genes between fetal and adult counterparts were specifically analyzed, and a fetal core signature was identified. The core gene set could separate subgroups of acute lymphoblastic leukemia by age, suggesting that a fetal program may be partially retained in specific subgroups of pediatric leukemia. Our detailed single-cell map presented herein emphasizes molecular and immunophenotypic differences between fetal and adult blood cells, which are of significance for future studies of pediatric leukemia and blood development in general.

16:00-16:10 Detection of microchimeric fetal cells in maternal peripheral blood during and after oocyte donation pregnancy

16:00-16:10 Detection of microchimeric fetal cells in maternal peripheral blood during and after oocyte donation pregnancy

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

LncRNA HCG27 Promotes Glucose Uptake Ability of HUVECs by MiR-378a-3p/MAPK1 Pathway.

Gestational diabetes mellitus (GDM) is the most common metabolic disorder during pregnancy. LncRNA HLA complex group 27 (HCG27) plays a crucial role in various metabolic diseases. However, the relationship between lncRNA HCG27 and GDM is not clear. This study aimed to verify a competing endogenous RNA (ceRNA) interaction regulation axis of miR-378a-3p/mitogen-activated protein kinase 1 (MAPK1) regulated by HCG27 in GDM. LncRNA HCG27 and miR-378a-3p were detected by RT-qPCR. The expression of MAPK1 in umbilical vein endothelial cells (HUVECs) was detected by RT-qPCR and that in the placenta by Western blotting. To explore the relationship among lncRNA HCG27, miR-378a-3p, MAPK1 and the glucose uptake ability of HUVECs, vector HCG27, si-HCG27, miR-378a-3p mimic and inhibitor were transfected to achieve overexpression and inhibition of HCG27 or miR-378a-3p. The interaction between miR-378a-3p and lncRNA HCG27 or MAPK1 was confirmed by the dual-luciferase reporter assay. Besides, glucose consumption by HUVECs was detected by the glucose assay kit. HCG27 expression was significantly decreased in both the placenta and primary umbilical vein endothelial cells, while the expression of miR-378a-3p was significantly increased in GDM tissues, and the expression of MAPK1 was decreased in GDM tissues. This ceRNA interaction regulation axis was proved to affect the glucose uptake function of HUVECs. The transfection of si-HCG27 could significantly reduce the expression of the MAPK1 protein. If the MAPK1 overexpression plasmid was transfected simultaneously with si-HCG27 transfection, the reduced glucose uptake in HUVECs resulting from the decrease in lncRNA HCG27 was reversed. MiR-378a-3p mimic can significantly reduce the mRNA expression of MAPK1 in HUVECs, whereas miR-378a-3p inhibitor can significantly increase the mRNA expression of MAPK1. The inhibition of miR-378a-3p could restore the decreased glucose uptake of HUVECs treated with si-HCG27. Besides, overexpression of lncRNA HCG27 could restore the glucose uptake ability of the palmitic acid-induced insulin resistance model of HUVECs to normal. LncRNA HCG27 promotes glucose uptake of HUVECs by miR-378a-3p/MAPK1 pathway, which may provide potential therapeutic targets for GDM. Besides, the fetal umbilical cord blood and umbilical vein endothelial cells collected from pregnant women with GDM after delivery could be used to detect the presence of adverse molecular markers of metabolic memory, so as to provide guidance for predicting the risk of cardiovascular diseases and health screening of offspring.

A novel enzyme-linked immunostaining technique based on silk membrane for the prenatal detection of fetomaternal haemorrhage.

Objective: Developing a simple, rapid, reliable, sensitive, and cost-effective method for prenatal detection of fetomaternal haemorrhage by combining multi-aperture silk membrane with enzyme-linked immunosorbent assay (ELISA), which does not require any complicated instruments and can be visually colored, so as to provide a new method for clinical detection of fetomaternal haemorrhage. Methods: As a carrier, a chemically treated silk membrane was used to immobilize anti-A/anti-B antibody reagent. PBS washed slowly after vertically dropping red blood cells. After adding biotin-labeled anti-A/anti-B antibody reagent, PBS is slowly washed, enzyme-labeled avidin is added, and TMB is used for color development after washing. Results: When there were both anti-A and anti-B fetal erythrocytes in pregnant women's peripheral blood, the final color was dark brown. When there are no anti-A and anti-B fetal red blood cells in pregnant women's peripheral blood, the final color development results do not change, which corresponds to the color of chemically treated silk membrane. Conclusion: The new enzyme-linked immunosorbent assay (ELISA) based on a silk membrane can distinguish fetal red blood cells from maternal red blood cells prenatally and can be used for prenatal detection of fetomaternal haemorrhage.

The layered development of mouse B and T Cells.

Traditional models of lymphopoiesis present B and T cell development as a linear process that initiates in the fetus and continues after birth in the bone marrow and thymus, respectively. However, this view of lymphocyte development is not in accord with reports, dating back several decades, indicating that the types of lymphocytes generated before and after birth differ. In this regard, selected γδ T cells, and those that utilize the Vγ3 receptor in particular, and innate-like B-1 B cells preferentially arise during fetal blood cell development. This review synthesizes data from multiple laboratories, with an emphasis on our own work using mouse models, demonstrating that innate and conventional B and T cells emerge in hematopoietic stem cell independent and dependent waves of development that are differentially regulated. This layering of lymphocyte development has implications for understanding the composition of the adult immune system and may provide insights into the origin of various lymphocytic leukemias.

Fetomaternal hemorrhage - case report

Maternal fetal leakage (FMH) is a well-known cause of fetal anemia. We define it as the transition of the fetal erythrocytes into the maternal circulation. Its severity is determined by measuring the level of fetal hemoglobin in the mother's blood using the Kleinhauer-Betke test or the cytometric method (FCM). The paper presents a case of a newborn with massive maternal-fetal leakage. The maternal blood Kleinhauer test result was greater than 5% of the fetal blood cells, and the initial neonatal hemoglobin level was 3.5 g / dl. Correct treatment of fetal anemia is presented. The additional studies performed allowed for the exclusion of other causes as a result of the extremely severe condition of the newborn after delivery. Early diagnosis of fetal-maternal leak and its treatment through execution.

The association between gestational diabetes mellitus and DNA damage in umbilical cord leukocytes and placental samples

Objective(s): To evaluate the relation between gestational diabetes mellitus (GDM) and maternal and/or fetal DNA integrity. Method: 59 pregnant women were classified into two groups on the basis of 75 g oral glucose tolerance test (OGTT) and glycemic profile (GP): Control group (OGTT and GP normal, n = 29) and GDM group (abnormal 75 g OGTT, n = 30). The umbilical cord blood and placental samples obtained from the maternal side were collected at the time of delivery. Alkaline comet assay was performed for the determination of DNA damage. The trial was approved with the protocol number 72867572.050.01.04-299082. Result(s): Body mass index (BMI), weight gain during pregnancy, glycemic means and fetal weight were increased in GDM group compared control group (p = .01, .0001, .04, and .01, respectively). In the GDM group, the number of large-for-gestational-age (LGA) infants was significantly higher compared to the nondiabetic group (p = .04). Tail DNA percentages in placental samples were higher in the GDM group compared to controls (p = .01); however, DNA integrity in umbilical cord leukocytes was similar between the groups (p = 0.1). In contrast to umbilical cord DNA damage, placental DNA damage showed positive correlation with maternal glycemia in the whole group and within each group. The positive association of placental DNA damage and GDM remained after adjusting for age, BMI, smoking, glycemia, gestational age at delivery, fetal weight at delivery, and delivery type (p = .01). Conclusion(s): Placental DNA damage is associated with GDM and placental cells seem to be more vulnerable to DNA damage compared to fetal blood cells.

Massive foeto-maternal transfusion: how long are foetal blood cells detectable in the maternal circulation?

Massive foeto-maternal transfusion: how long are foetal blood cells detectable in the maternal circulation?

Noninvasive Prenatal Testing in Immunohematology-Clinical, Technical and Ethical Considerations.

Hemolytic disease of the fetus and newborn (HDFN), as well as fetal and neonatal alloimmune thrombocytopenia (FNAIT), represent two important disease entities that are caused by maternal IgG antibodies directed against nonmaternally inherited antigens on the fetal blood cells. These antibodies are most frequently directed against the RhD antigen on red blood cells (RBCs) or the human platelet antigen 1a (HPA-1a) on platelets. For optimal management of pregnancies where HDFN or FNAIT is suspected, it is essential to determine the RhD or the HPA-1a type of the fetus. Noninvasive fetal RhD typing is also relevant for identifying which RhD-negative pregnant women should receive antenatal RhD prophylaxis. In this review, we will give an overview of the clinical indications and technical challenges related to the noninvasive analysis of fetal RBCs or platelet types. In addition, we will discuss the ethical implications associated with the routine administration of antenatal RhD to all pregnant RhD-negative women and likewise the ethical challenges related to making clinical decisions concerning the mother that have been based on samples collected from the (presumptive) father, which is a common practice when determining the risk of FNAIT.

Trophoblast antigens, fetal blood cell antigens, and the paradox of fetomaternal tolerance.

The paradox of fetomaternal tolerance has puzzled immunologists and reproductive biologists alike for almost 70 yr. Even the idea that the conceptus evokes a uniformly tolerogenic immune response in the mother is contradicted by the long-appreciated ability of pregnant women to mount robust antibody responses to paternal HLA molecules and RBC alloantigens such as Rh(D). Synthesizing these older observations with more recent work in mice, we discuss how the decision between tolerance or immunity to a given fetoplacental antigen appears to be a function of whether the antigen is trophoblast derived-and thus decorated with immunosuppressive glycans-or fetal blood cell derived.

Research Progress in Isolation and Enrichment of Fetal Cells from Maternal Blood

Prenatal diagnosis is an important means of early diagnosis of genetic diseases, which can effectively reduce the risk of birth defects. Free fetal cells, as a carrier of intact fetal genetic material, provide hope for the development of high-sensitivity and high-accuracy prenatal diagnosis technology. However, the number of fetal cells is small and it is difficult to apply clinically. In recent years, noninvasive prenatal diagnosis (NIPD) technology for fetal genetic material in maternal peripheral blood has developed rapidly, which makes it possible to diagnose genetic diseases by fetal cells in maternal peripheral blood. This article reviewed the current status of fetal cell separation and enrichment technology and its application in noninvasive prenatal diagnosis technology.

The Fetal-to-Adult Hematopoietic Stem Cell Transition and its Role in Childhood Hematopoietic Malignancies.

As with most organ systems that undergo continuous generation and maturation during the transition from fetal to adult life, the hematopoietic and immune systems also experience dynamic changes. Such changes lead to many unique features in blood cell function and immune responses in early childhood. The blood cells and immune cells in neonates are a mixture of fetal and adult origin due to the co-existence of both fetal and adult types of hematopoietic stem cells (HSCs) andprogenitor cells (HPCs). Fetal blood and immune cells gradually diminish during maturation of the infant and are almost completely replaced by adult types of cells by 3 to 4weeks after birth in mice. Such features in early childhood are associated with unique features of hematopoietic and immune diseases, such as leukemia, at these developmental stages. Therefore, understanding the cellular and molecular mechanisms by which hematopoietic and immune changes occur throughout ontogeny will provide useful information for the study and treatment of pediatric blood and immune diseases. In this review, we summarize the most recent studies on hematopoietic initiation during early embryonic development, the expansion of both fetal and adult types of HSCs and HPCs in the fetal liver and fetal bone marrow stages, and the shift from fetaltoadult hematopoiesis/immunity during neonatal/infant development. We also discuss the contributions of fetal types of HSCs/HPCs to childhood leukemias.

Isolation and characterization of fetal nucleated red blood cells from maternal blood as a target for single cell sequencing-based non-invasive genetic testing.

PurposeAlthough non‐invasive prenatal testing (NIPT) based on cell‐free DNA (cfDNA) in maternal plasma has been prevailing worldwide, low levels of fetal DNA fraction may lead to false‐negative results. Since fetal cells in maternal blood provide a pure source of fetal genomic DNA, we aimed to establish a workflow to isolate and sequence fetal nucleated red blood cells (fNRBCs) individually as a target for NIPT.MethodsUsing male‐bearing pregnancy cases, we isolated fNRBCs individually from maternal blood by FACS, and obtained their genomic sequence data through PCR screening with a Y‐chromosome marker and whole‐genome amplification (WGA)‐based whole‐genome sequencing.ResultsThe PCR and WGA efficiencies of fNRBC candidates were consistently lower than those of control cells. Sequencing data analyses revealed that although the majority of the fNRBC candidates were confirmed to be of fetal origin, many of the WGA‐based genomic libraries from fNRBCs were considered to have been amplified from a portion of genomic DNA.ConclusionsWe established a workflow to isolate and sequence fNRBCs individually. However, our results demonstrated that, to make cell‐based NIPT targeting fNRBCs feasible, cell isolation procedures need to be further refined such that the nuclei of fNRBCs are kept intact.

Novel Approaches to Develop Critical Reference Materials for Noninvasive Prenatal Testing: A Pilot Study.

Highly characterized reference materials are required to expand noninvasive prenatal testing (NIPT) for low incidence aneuploidies and microdeletions. The goal of this study was to develop reference materials for the development of next generation circulating cell-free DNA (ccfDNA) assays. This was a prospective study of pregnancies complicated by positive prenatal genetic screening. ccfDNA was isolated from maternal plasma and amplified. Lymphoblastoid cell lines were prepared from maternal peripheral blood mononuclear cells and fetal cord blood cells. Cells were Epstein-Barr virus immortalized and expanded. Amplified DNA and to a limited extent formulated lymphoblastoid-derived ccfDNA was tested in SNP-based and chromosome counting (CC) based massively parallel sequencing assays. Enrolled cases included fetuses with: T21 (2), T18 (1), T18-XXX (1), XYY (1), microdeletions (1), and euploid (2). Three lymphoblastoid cells lines were prepared. Genomic DNA was extracted from cell lines and fragmented to simulate ccfDNA. ccfDNA isolation yielded about 2000 usable genome equivalents of DNA for each case for amplification. Although the sonicated genomic DNA derived from lymphoblastoid cell lines did not yield results compatible with NIPT assays, when blinded, NIPT platforms correctly identified the amplified ccfDNA isolated from blood in the majority of cases. This study showed that maternal blood samples from pregnancies complicated by common chromosomal abnormalities can be used to generate materials for the development and evaluation of NIPT assays.

Effects of MMP-8 Production by LPS and TPCK Stimulation in Fetal Blood Cells

이 연구의 목적은 Matrix metalloproteinase-8 (MMP-8) 생성을 위한 태아 혈액 세포에서 Lipopolysaccharide (LPS) 자극의 효과와 Nuclear factor kappa B (NF-kB) 억제제인 Tosylphenylalanine chloromethyl ketone (TPCK)의 효과를 밝히는 것이다. 출생 시 제대혈은 선택적 제왕 절개 기간 동안 LPS (1μg/mL)를 사용하여 NF-kB 억제제인 TPCK (10 and 20 mM)의 유무에 관계없이 즉시 자극하였다. MMP-8의 농도는 1-6시간 후 상층액에서 특이 면역 분석으로 측정하였다. 그리고 NF-kB 활성은 웨스턴 블롯팅을 사용하여 측정하였다. 제대혈에 대한 LPS 자극은 시간이 경과함에 따라 MMP-8의 수준을 크게 높이는 반면, TPCK에 의한 NF-kB 억제제는 LPS 자극에 의한 MMP-8의 상승을 억제하였다. LPS 자극과 함께 제대혈에 의한 MMP-8 생성은 NF-kB 억제제인 TPCK에 의해 억제되었다. 우리의 실험 결과는 NF-kB가 태아 혈액 세포에서 LPS에 의해 유도된 MMP-8 생성에 대한 조절은 태아염증반응증후군의 진단 역할을 한다는 것을 시사한다.