Fetal Red Blood Cells Research Articles

Fetal Growth Restriction and Elevated First Nucleated Red Blood Cell Counts in Term Brain-Damaged Infants Characterized by Fetal Heart Rate Abnormalities

Fetal Growth Restriction and Elevated First Nucleated Red Blood Cell Counts in Term Brain-Damaged Infants Characterized by Fetal Heart Rate Abnormalities

Fetal growth restriction and elevated first nucleated red blood cell counts in term brain-damaged infants characterized by fetal heart rate abnormalities

Fetal growth restriction and elevated first nucleated red blood cell counts in term brain-damaged infants characterized by fetal heart rate abnormalities

On the mechanism of tolerance to the Rh D antigen mediated by passive anti-D (Rh D prophylaxis)

Anti-D prophylaxis is the most successful clinical application of antibody-mediated immune suppression. Passive IgG anti-D is given to Rh D-negative women to prevent immunisation to foetal Rh D-positive red blood cells (RBC) and subsequent haemolytic disease of the newborn. Despite its widespread use and efficacy, the mechanism of action of this therapy is unproven. The known facts about the antigen, antibody response, dose of anti-D, RBC clearance and effects of the passive anti-D on subsequent primary and secondary immune responses are discussed in relation to recent information on ways by which immune responses may be suppressed. Most Rh D antigen sites on RBC are not bound by passive anti-D, and thus epitope masking (which may occur in experimental murine models using xenogeneic RBC) is not the reason why anti-D responses are prevented by administration of prophylactic anti-D. It is hypothesised that although clearance and destruction of the antigenic RBC may be a contributing factor in preventing immunisation, down-regulation of antigen-specific B cells through co-ligation of B cell receptors and inhibitory IgG Fc receptors must also occur.

Isolation of fetal nucleated red blood cells from maternal blood in normal and aneuploid pregnancies.

Fetal nucleated red blood cells (NRBC) have been widely reported in maternal blood during pregnancy. However, there is no consensus with regard to their presence in all pregnancies. Therefore, the usefulness of developing a fetal NRBC-based noninvasive method suitable for clinical prenatal diagnosis remains uncertain. Fluorescence in situ hybridization (FISH) method was used to evaluate the ability of one of our own monoclonal antibodies (mAb), 2B7.4, to isolate fetal NRBC from maternal blood by magnetic activated cell sorting (MACS). Our mAb was able to isolate from 25 to 822 NRBC from all of the 45 maternal blood samples included in this study. A correct diagnosis was achieved in 21 out of 24 pregnancies carrying trisomic fetuses (87.5%), with a fetal/maternal NRBC frequency of 8.4%. In contrast, a significantly lower percentage of fetal NRBC (0.2%) was observed in 22% of pregnancies carrying a chromosomally normal male fetus, that were correctly predicted. In conclusion, using 2B7.4 mAb we succeeded in isolating NRBC from the maternal blood samples, but most of the isolated cells were maternal in origin. Nevertheless, a higher number of fetal NRBC was found in the peripheral blood of pregnant women carrying aneuploid fetuses, which could allow development of a screening method for prenatal diagnosis of fetal aneuploidies.

568 The association of oligohydramnios with fetal nucleated red blood cell count

568 The association of oligohydramnios with fetal nucleated red blood cell count

Significantly higher number of fetal cells in the maternal circulation of women with pre-eclampsia.

Although the pathophysiology of pre-eclampsia is unknown, several studies have indicated that abnormal placentation early in pregnancy might play a key role. It has recently been suggested that this abnormal placentation may result in transfusion of fetal cells (feto-maternal transfusion) in women with pre-eclampsia. In the present study, fetal nucleated red blood cells were isolated from 20 women with pre-eclampsia and 20 controls using a very efficient magnetic activated cell sorting (MACS) protocol. The number of male cells was determined using two-color fluorescence in situ hybridization (FISH) for X and Y chromosomes. Significantly more XY cells could be detected in women with pre-eclampsia (0.61+/-1.2 XY cells/ml blood) compared to women with uncomplicated pregnancies (0.02+/-0.04 XY cells/ml blood) (Mann-Whitney U-test, p<0.001). These results suggest that fetal cell trafficking is enhanced in women with pre-eclampsia, and this finding may contribute to the understanding of the pathophysiology of the disease.

Fetal nucleated red blood cells from CVS washings: an aid to development of first trimester non‐invasive prenatal diagnosis

Fetal nucleated red blood cells (n-rbc) occur in the maternal circulation from 7 weeks of pregnancy. The enrichment of these cells from maternal blood will depend upon their stage of differentiation, which changes during development. We characterised the fetal n-rbc from chorionic villus sample (CVS) washings and used them to model first trimester non-invasive prenatal diagnosis. The ratio of epsilon- to gamma-globin-producing cells declined rapidly from 10 to 13 weeks, as did the ratio of nucleated to non-nucleated rbc. By 13 weeks the great majority of cells containing gamma- or epsilon-globin are anucleate. The fetal n-rbc were highly variable in size and density and sedimented over a wide density range with a high proportion (>80%) at a density overlapping with that of maternal rbc. We have devised an enrichment procedure using Orskoff lysis to differentially lyse the maternal cells followed by density centrifugation and separation using magnetic beads. This simple protocol allowed recovery of 70% (69+/-22%) of fetal cells when added at approximately 10 fetal cells/ml maternal blood. When 1 fetal cell/ml millilitre maternal blood was added (total volume 10 ml) the recovery was more variable but remained at approximately 70% (72+/-47%), with at least one fetal cell recovered in all cases.

FISH analysis of fetal nucleated red blood cells from CVS washings in cases of aneuploidy.

In chorionic villus sampling (CVS) the chromosome analysis is inconclusive in 1-2% of the samples. In many cases follow-up amniocentesis is performed. Fetal nucleated red blood cells (FNRBCs) are present in washings of chorionic villus samples. We wanted to establish whether analysis of these true fetal cells, using fluorescence in situ hybridization (FISH), could support the CVS karyotype. We analysed washings of first trimester chorionic villi from non-mosaic 45,X (n=6) and full trisomy 18 cases (n=7). FNRBCs were identified by immunostaining and FISH was performed with chromosome-specific probes for X, Y and 18. In all 13 samples FNRBCs were present (between 4 and 30 cells per sample). Five cases of monosomy X showed one X signal in 89-100% of the nuclei; in the other case 50% of the nuclei displayed one signal. In the trisomy 18 cases three spots were seen in 60-100% of the cells. The CVS aneuploidy was confirmed in FNRBCs in all samples, so FISH on FNRBCs can be used in cases of non-mosaic numerical chromosomal abnormalities. This test can confirm a CVS diagnosis of monosomy X or trisomy 18 and thus minimize the risk for false-positive diagnoses. An additional invasive test may be prevented.

Fetal nucleated red blood cells from CVS washings: an aid to development of first trimester non‐invasive prenatal diagnosis

Fetal nucleated red blood cells from CVS washings: an aid to development of first trimester non‐invasive prenatal diagnosis

Detection of fetal NRBCs in maternal blood of pregnant carriers of beta-thalassemia using anti-gamma and anti-epsilon monoclonal antibodies.

Fetal nucleated red blood cells (NRBCs) entering maternal circulation during pregnancy constitute a potential source of material for safe and reliable noninvasive prenatal diagnosis. The increased prevalence of beta-thalassemia mutations in countries like Greece may create a problem, making it difficult to distinguish between NRBCs of fetal or maternal origin. Use of Ab against embryonic hemoglobin epsilon may increase specificity for fetal NRBC detection. In the present study, Ab against embryonic hemoglobin epsilon was used in the first and second trimesters of pregnancy in order to determine if specificity for fetal NRBC detection could be increased.

Postpartum Chills Phenomenon: Is It a Feto-Maternal Transfusion Reaction?

Objective. To examine the theory that the postpartum shivering phenomenon is related to feto-maternal bleed during the third stage of labor.Methods. One hundred laboring low-risk women who had a normal vaginal delivery were observed for the presence of postpartum chills. The duration of the first and second stages of labor changes in body temperature, maternal and fetal blood types and the use of epidural anesthesia were recorded. Following the delivery maternal blood was examined for the presence of fetal red blood cells using the Kleihauer-Betke stain.Results. Complete data was available in 97 patients. Post-partum chills occurred in 31 of them (32%). Women with and without chills were similar in their maternal and gestational age, the use of epidural anesthesia, and length of second stage of labor. Women with chills delivered smaller babies but the difference did not reach significance. Maternal-fetal blood group incompatibility was significantly more common among shivering than non-shivering women (48% v...

Fetal nucleated red blood cell counts in peripheral blood of mothers bearing Down syndrome fetus.

Currently, prenatal detection of Down syndrome and other most common aneuploidies relies on invasive procedures such as amniocentesis and villocentesis, and on non-invasive screening tests such as second trimester maternal serum screening (Triple test), and first trimester screening (ULTRA-screen). However, it well known that invasive techniques carry a small risk of fetal loss, while both Triple test and ULTRA-screen are not diagnostic, may miss from 15 - 40 % of cases of Down syndrome, in addition to having a 5 - 8 % rate of false-positives. We now report clear evidence that the number of fetal nucleated red blood cells (FNRBCs) in the maternal circulation is remarkably higher in pregnant women carrying aneuploid fetuses, especially in cases of Down syndrome. These results are in agreement with the findings of other investigators using different methods, and suggest that the number of FNRBCs present in the maternal blood sample could be used as additional marker, in concert with existing screening tests, to improve non-invasive detection of Down syndrome, and other most common aneuploidies.

The application of magnetic cell sorter (MACS) to detect fetal cells in maternal peripheral blood.

The aim of the present study was to investigate the effectiveness of sorting fetal nucleated red blood cells (FNRBC) from maternal peripheral blood, particularly during early gestation periods, by a combination of specific gravity centrifugation and magnetic cell sorter (MACS). Without prior knowledge of the gender of the fetus, we determined gender by analyzing a Y-chromosome specific sequence by nested-PCR, using 10 ml of the peripheral blood of healthy primigravida women at different stages of gestation (first trimester: n = 17, second trimester: n = 13, and third trimester: n = 19). The results of this prenatal sex determination were compared to the sex of newborns. The specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV) of the present method during the first trimester were 100, 81.8, 100, and 75%, respectively; during the second trimester, 80, 50, 80, and 50%, respectively; and during the third trimester, 25, 63.6, 53.8, and 33.3%, respectively. The results show that this prenatal sex determination method has a highly accurate diagnostic rate during the first trimester, suggesting that it could be developed as a practical, non-invasive prenatal diagnostic technique for use during early gestation periods.

K + transport in red blood cells from human umbilical cord

The current study was designed to characterise K + transport in human fetal red blood cells, containing mainly haemoglobin F (HbF, and termed HbF cells), isolated from umbilical cords following normal parturition. Na +/K + pump activity was comparable to that in normal adult human red cells (which contain HbA, and are termed HbA cells). Passive (ouabain-resistant) K + transport was dominated by a bumetanide (10 μM)-resistant component, inhibited by [(dihydroxyindenyl)oxy]alkanoic acid (100 μM), calyculin A (100 nM) and Cl − removal, and stimulated by N-ethylmaleimide (1 mM) and staurosporine (2 μM) – all consistent with mediation via the K +-Cl − cotransporter (KCC). KCC activity in HbF cells was also O 2-dependent and stimulated by swelling and urea, and showed a biphasic response to changes in external pH. Peak activity of KCC in HbF cells was about 3-fold that in HbA cells. These characteristics are qualitatively similar to those observed in HbA cells, notwithstanding the different conditions experienced by HbF cells in vivo, and the presence of HbF rather than HbA. KCC in HbF cells has a higher total capacity, but when measured at the ambient PO 2 of fetal blood it would be similar in magnitude to that in fully oxygenated HbA cells, and about that required to balance K + accumulation via the Na +/K + pump. These findings are relevant to the mechanism by which O 2 regulates membrane transporters in red blood cells, and to the strategy of promoting HbF synthesis as a therapy for patients with sickle cell disease.

Derivated fetal haemoglobin as a marker for red cell age in the human fetus reflecting stimulated or impaired red blood cell production.

We have determined whether derivated fetal haemoglobin (dHbF, consisting of glycated and acetylated HbF) can be used as a cell age marker for fetal red blood cells (RBCs). Cord blood was obtained between 19 and 39 weeks of gestation from 28 alloimmunised anaemic fetuses (23 RhD+ and 5 Kell) and from 20 non-anaemic fetuses and newborns (controls). Density gradient centrifugation was applied to 36 samples (20 RhD+, 15 controls and 1 Kell) to obtain fractions of increasing cell age. Blood samples were used for measurements of mean cellular volume (MCV), mean cell haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), pyruvate kinase activity (PK) and derivated fetal haemoglobin (dHbF) by cation-exchange HPLC. Reticulocytes were counted only in the whole blood samples. In all density gradient separated RBC fractions, the values for MCV, MCH and PK activity decreased and those of MCHC and dHbF increased with increasing density (equivalent to increasing cell age). The mean density was lower for RBCs of the anaemic RHD group (1.072+/-0.007 g/ml) than for the non-anaemic controls (1.077+/-0.005 g/ml) (p<0.05) The RBC density of the Kell sensitised fetus did not differ from those of the controls. In the control group, the values of the cell age markers in whole blood changed significantly with the gestational age, showing an increase of mean age of the erythrocyte population. The best linear relationship was found for dHbF (y=6.28+0.17*weeks; r=0.84; p<0.001). In the anaemic RhD+ fetuses, the RBC age markers did not change with gestational age; the dHbF percentages were lower, and the MCV, MCH, PK values and the reticulocyte counts were higher than in the controls (0.05<p<0.001). The dHbF values of the Kell sensitised fetuses were above (p<0.01) and the reticulocyte counts were below normal (p<0.05) for gestational age. For the anaemic fetuses, a significant number of the dHbF values (86%) and of the reticulocyte counts (78%) differed from the values of the controls (p<0.01). The dHbF percentages in RhD+ fetuses showed the best correlation with the Hb deficit, which is a measure for anaemia (r=-0.81, p<0.0001). We conclude that the percentage derivated HbF may indicate whether the RBC production is normal for gestational age. It may in that sense reflect stimulated or impaired erythropoiesis in alloimmunised haemolytic anaemia.

Abnormal angiogenesis but intact hematopoietic potential in TGF-beta type I receptor-deficient mice

Deletion of the transforming growth factor beta1 (TGF-beta1) gene in mice has previously suggested that it regulates both hematopoiesis and angiogenesis. To define the function of TGF-beta more precisely, we inactivated the TGF-beta type I receptor (TbetaRI) gene by gene targeting. Mice lacking TbetaRI die at midgestation, exhibiting severe defects in vascular development of the yolk sac and placenta, and an absence of circulating red blood cells. However, despite obvious anemia in the TbetaRI(-/-) yolk sacs, clonogenic assays on yolk sac-derived hematopoietic precursors in vitro revealed that TbetaRI(-/-) mice exhibit normal hematopoietic potential compared with wild-type and heterozygous siblings. Endothelial cells derived from TbetaRI-deficient embryos show enhanced cell proliferation, improper migratory behavior and impaired fibronectin production in vitro, defects that are associated with the vascular defects seen in vivo. We thus demonstrate here that, while TbetaRI is crucial for the function of TGF-beta during vascular development and can not be compensated for by the activin receptor-like kinase-1 (ALK-1), functional hematopoiesis and development of hematopoietic progenitors is not dependent on TGF-beta signaling via TbetaRI.

ζ-, ε-, and γ-Globin mRNA in Blood Samples and CD71+ Cell Fractions from Fetuses and from Pregnant and Nonpregnant Women, with Special Attention to Identification of Fetal Erythroblasts

Information about the appearance of gamma-, epsilon-, and zeta-globin mRNAs in fetal erythroblasts during gestation and about the presence and amounts of these mRNAs in pregnant and nonpregnant women is important from the perspective of using these molecules as a marker of fetal erythroblasts. A specific marker is necessary for isolation and identification of fetal nucleated red blood cells from maternal blood samples for use in antenatal diagnosis of fetal genetic or chromosomal abnormalities. We used a very sensitive reverse transcription-PCR (RT-PCR) method, coamplification analysis of gamma- and epsilon-globin cDNA, and quantitative analysis of gamma-globin mRNA based on competitive RT-PCR to investigate these aspects. All adult whole-blood samples were negative for epsilon- and zeta-globin mRNA. Analyses of CD71(+) cell fractions showed that specimens from 19 of 20 nonpregnant and 10 of 14 pregnant women (at 9-13 weeks of gestation) were positive for gamma-globin mRNA (Fisher's exact test, P = 0.13), and those from 3 of 20 nonpregnant and 5 of 14 pregnant women were positive for zeta-globin mRNA (Fisher's exact test, P = 0.23). No epsilon-globin mRNA was detected in CD71(+) cell fractions from 1-mL blood samples from adults. CD71(+) cell fractions from eight fetal blood samples (at 17-20 weeks of gestation) were positive for all three globin mRNAs. We found no statistically significant difference between the amounts of gamma-globin mRNA in pregnant and nonpregnant women. This study indicates that epsilon-globin mRNA might function as a marker for fetal CD71(+) cells early in pregnancy. Although gamma-globin mRNA can be detected in CD71(+) cell fractions from most adults, these transcripts also may be of use because of a marked difference between adult and fetal values.

Antibodies to human fetal erythroid cells from a nonimmune phage antibody library.

The ability to isolate fetal nucleated red blood cells (NRBCs) from the maternal circulation makes possible prenatal genetic analysis without the need for diagnostic procedures that are invasive for the fetus. Such isolation requires antibodies specific to fetal NRBCs. To generate a panel of antibodies to antigens present on fetal NRBCs, a new type of nonimmune phage antibody library was generated in which multiple copies of antibody fragments are displayed on each phage. Antibody fragments specific for fetal NRBCs were isolated by extensive predepletion of the phage library on adult RBCs and white blood cells (WBCs) followed by positive selection and amplification on fetal liver erythroid cells. After two rounds of selection, 44% of the antibodies analyzed bound fetal NRBCs, with two-thirds of these showing no binding of WBCs. DNA fingerprint analysis revealed the presence of at least 16 unique antibodies. Antibody specificity was confirmed by flow cytometry, immunohistochemistry, and immunofluorescence of total fetal liver and adult RBCs and WBCs. Antibody profiling suggested the generation of antibodies to previously unknown fetal RBC antigens. We conclude that multivalent display of antibodies on phage leads to efficient selection of panels of specific antibodies to cell surface antigens. The antibodies generated to fetal RBC antigens may have clinical utility for isolating fetal NRBCs from maternal circulation for noninvasive prenatal genetic diagnosis. Some of the antibodies may also have possible therapeutic utility for erythroleukemia.

Postpartum chills phenomenon: is it a feto-maternal transfusion reaction?

To examine the theory that the postpartum shivering phenomenon is related to feto-maternal bleed during the third stage of labor. One hundred laboring low-risk women who had a normal vaginal delivery were observed for the presence of postpartum chills. The duration of the first and second stages of labor changes in body temperature, maternal and fetal blood types and the use of epidural anesthesia were recorded. Following the delivery maternal blood was examined for the presence of fetal red blood cells using the Kleihauer-Betke stain. Complete data was available in 97 patients. Post-partum chills occurred in 31 of them (32%). Women with and without chills were similar in their maternal and gestational age, the use of epidural anesthesia, and length of second stage of labor. Women with chills delivered smaller babies but the difference did not reach significance. Maternal-fetal blood group incompatibility was significantly more common among shivering than non-shivering women (48% vs. 20% respectively, p=0.006). Kleihauer-Betke test was positive in 11 women. The only two women in this group who experienced chills had maternal-fetal blood group incompatibility. Post-partum chills are a common phenomenon. It may be the clinical presentation of feto-maternal transfusion reaction. The small number of positive Kleihauer-Betke tests may reflect its low sensitivity in the detection of small feto-maternal bleeds.

Fetal hemoglobin-expressing nucleated red blood cell frequencies in pregnancies with intrauterine growth restriction

The objective of this study was to examine whether there is a difference in the frequency of fetal erythroblasts in maternal blood in pregnancies with intrauterine growth restriction (IUGR) as compared with normal pregnancies. Nucleated red blood cells (NRBC) were isolated from nine pregnant women with ultrasonically diagnosed IUGR (estimated fetal weight less than the 10th percentile for gestational age) and 11 women with appropriately grown fetuses. The frequency of fetal hemoglobin-expressing NRBC (FHE-NRBC) in maternal blood was evaluated by triple density centrifugation and anti-CD71+ magnetic cell sorting, followed by indirect immunocytochemistry for the detection of gamma-chain fetal hemoglobin. The number of FHE-NRBC in 10 ml maternal blood in the IUGR group was higher than in the control group (454.5 vs 56.7, p<0.05). This difference was even more pronounced when FHE-NRBC frequency was calculated relative to the total CD71+ population of cells. There were 118.9 FHE-NRBC per 10(5) CD71+ cells in IUGR pregnancies as compared with 11.5 cells in the control group (p<0.01). There was no difference in the total mean number of CD71+ mononuclear cells between the two groups. The observed increase in the frequency of fetal cells in the maternal circulation found in IUGR pregnancies may be a result of an increase in total NRBC in the fetal circulation or rather of abnormalities in placental structure. This phenomenon may assist in identifying pregnancies at risk for this complication early in the course of the pregnancy, even before actual growth restriction presents itself ultrasonically.