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Background: For appropriate management of hemolytic disease of the fetus and newborn (HDFN), it is important to detect irregular red cell antibody in the antenatal period. Though it is a simple one-step method, it is not part of routine antenatal screening in many developing countries. To reiterate the importance of antenatal antibody screening, we have assessed the frequency and clinical significance of irregular red cell antibodies in our patient population., Materials and Methods: A prospective study was carried out from October 2013 to May 2015 at a tertiary care center from south India. All antenatal samples received by the laboratory for red cell antibody screening were screened using a commercial three-cell screening panel. Antibody identification along with further Immunohematological techniques as required were performed for cases with positive screening results. Neonates of the alloimmunized cases were followed up to determine the clinical significance of the antibody., Results: A total of 2336 antenatal mothers were screened for red cell antibodies. The overall rate of alloimmunization in the study group was 2.27%. Alloimmunization rate among RhD-negative pregnancies was 6.9%. Other than anti-D (49%), we identified anti-D + anti-C (5%), anti-G (5%), anti-c (5%), anti-E (2%), anti-e (2%), anti-H (Bombay phenotype) (7%), anti-M (2%), anti-Lea (2%), anti-Leb (12%), and autoantibodies (9%) in the maternal serum. Anti-D, anti-D + anti-C, anti-G, anti-c, and anti-H were found to be clinically significant in this study., Conclusion: This study showed that 1 in 125 RhD-positive pregnancies can develop red cell alloantibodies. Hence, implementing routine antenatal antibody screening irrespective of RhD status is essential.

Fetal nucleated red blood cells (nRBCs) are rare in maternal circulation, but their presence constitutes a potential source of non-invasive prenatal genetic diagnosis. This study was undertaken to establish a non-invasive prenatal genetic diagnosis method using isolated fetal nRBCs. A multi-step method including triple density gradient and magnetic activated cell sorting (MACS) using CD45 and CD71, cytospin centrifugation, K-B staining, and glycophorin A-immuno fluorescence in situ hybridization (GPA-immuno FISH) was performed. The study population included 65 patients from 8 to 41 weeks of gestation, and fetal nRBC was separated from all cases. The number of fetal nRBCs retrieved was 12.8 +/- 2.7 in 8 to 11 gestational weeks, 15.2 +/- 6.5 in 12 to 18 gestational weeks, 16.4 +/- 6.5 in 19 to 23 gestational weeks, 10.6 +/- 3.2 in 24 to 28 gestational weeks, and 5.5 +/- 1.9 in 35 to 41 gestational weeks: the mean number of nRBCs collected from 20 ml of maternal peripheral blood was 13.7 +/- 6.2. The highest value of yield was 45.6% from 12 to 18 weeks gestation. The fetal sex determination confirmed by amniocentesis or chorionic villus sampling showed 100% sensitivity and 91.7% specificity for males; 91.7% sensitivity and 100% specificity for females. We showed that fetal cells can be reliably enriched from maternal blood and that they can be used for detecting specific chromosomes by FISH with a specificity superior to current non-invasive methods.

We have developed a method that allows the prenatal DNA diagnosis of ornithine transcarbamylase (OTC) deficiency by using a single fetal nucleated erythrocyte (NRBC) isolated from maternal blood. OTC gene analysis of a male patient (TF) with early onset OTC deficiency was performed by single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing. To investigate the possible prenatal diagnosis of OTC deficiency, maternal blood was obtained at 13 weeks of gestation of a subsequent pregnancy, from the mother of patient TF. NRBCs in the maternal blood were separated by using the density gradient method and then collected with a micromanipulator. The entire genome of a single NRBC was amplified by primer extension preamplification (PEP). The human leukocyte antigen (HLA)-DQ alpha genotype and sex were determined from small aliquots of the PEP product. The HLA-DQ alpha genotype of each of the parents of the male patient was also determined. Once a single NRBC had been identified as being of fetal origin, the OTC gene was analyzed by using the restriction fragment length polymorphism (RFLP) method. DNA analysis revealed a point mutation in exon 9 of the OTC gene in the OTC-deficient patient (TF). All NRBCs retrieved from maternal blood were successfully identified as being of fetal origin by HLA-DQ alpha genotyping and sex determination. RFLP analysis demonstrated that the fetal OTC gene was normal. This is the first study to successfully diagnose OTC deficiency prenatally, by using a single fetal NRBC from the maternal circulation. Such prenatal DNA diagnosis is non-invasive and can be applied to other genetic diseases, including autosomal and X-linked diseases.

Fetal nucleated red cells which pass into the maternal circulation during pregnancy are a potential cell source for non-invasive prenatal genetic diagnosis. To sort these rare cells with a high degree of specificity, we focussed our attention on the erythropoietin receptor, a strictly erythroid-specific antigen. We first labelled these receptors with biotin-(sialyl)-erythropoietin, then isolated the erythroid cells by magnetic beads conjugated with streptavidin in a MiniMACS (magnetic cell separator). The effectiveness of this strategy for the enrichment of fetal cells was evaluated by assessing its accuracy for gender prediction in 18 male-bearing pregnancies. Polymerase chain reaction (PCR) results on maternal blood samples sorted for Epo-r and CD71 antigens displayed similar sensitivity (55% Epo-r, 61% CD71) in detecting Y-specific sequences while immunocytochemical studies on four maternal blood samples, sorted after increasing the binding time of the ligand to Epo-r (8 h), showed a substantial improvement in fetal cell recovery and purity. We conclude that sorting by Epo-r/biotin-(sialyl)-erythropoietin provides effective enrichment of fetal nucleated red cells allowing the possibility of direct prenatal cytogenetic analysis by multiprobe fluorescent in-situ hybridization (FISH).

Nucleated erythrocytes found in the venous blood of pregnant women have been seen as the most promising type of cell for prenatal diagnosis of fetal abnormalities because of their short life span and their scarcity in the venous blood of healthy nonpregnant women. However, this review demonstrates that a great proportion of the nucleated erythrocytes found in venous blood during pregnancy are of maternal origin and that pregnancy per se induces nucleated erythrocytes of maternal origin to appear in the peripheral blood. More efficient and specific enrichment techniques are therefore needed to obtain purified nucleated erythrocytes of fetal origin in such quantities that reliable prenatal diagnoses can be performed.

Using a micropipette aspiration technique, erythrocyte membrane shear elastic modulus was determined for 23 patients with Duchenne muscular dystrophy, for 14 female carriers, and for three control groups (age matched boys and adult males and females). On average the elastic modulus was slightly greater than normal for both affected males and female carriers. However, there was overlap in the values from all groups. The decreased deformability of dystrophic membranes demonstrated by the test is not great enough to be used for carrier detection nor to make the possibility of prenatal diagnosis likely.

The absence of peroxisomes in patients with the cerebrohepatorenal syndrome of Zellweger leads to several biochemical abnormalities, including deficient synthesis of plasmalogens as well as accumulation of very long-chain fatty acids and intermediates in bile acid biosynthesis. Accumulation of very long-chain fatty acids in serum and fibroblasts has hitherto been used most extensively for diagnosis. Due to the relatively small amounts of the very long-chain fatty acids also in the Zellweger patients, and the risk for interfering impurities, such analyses are difficult. Direct assay of plasmalogens is also relatively difficult and time-consuming. In this report, we describe a relatively simple method for diagnosis, based on gas-liquid chromatography of a lipid extract of erythrocytes after methyl transesterification. The alpha, beta-unsaturated ether in the plasmalogen is converted to the dimethylacetal of the corresponding aldehyde, and the relative amount of plasmalogen is thus reflected by the ratio between 18:0 dimethylacetal and methyl stearate as well as by the ratio between 16:0 dimethylacetal and methyl palmitate. The ratio 18:0 dimethylacetal/methyl stearate was found to be 0.28 +/- 0.03 (mean +/- SD) after analyses of erythrocytes from healthy or non-Zellweger infants, but less than 0.02 in erythrocytes from three infants with the Zellweger syndrome. Preliminary work with amniotic fluid suggests that this analysis may be suitable also for prenatal diagnosis of the Zellweger syndrome.

Prenatal diagnosis of inborn errors of metabolism has been possible only if the enzyme affected is expressed in amniotic fluid cells grown in culture. Arginase is essentially undetectable in normal human fibroblasts, amniotic fluid, and amniotic fluid cells but is present in high amounts in red blood cells. It is absent in the red blood cells of patients with liver arginase deficiency. The properties of the enzyme in the red cells of healthy children and adults were compared to those of the enzyme obtained from cord blood red cells of 13--20-week fetuses obtained at hysterotomy. The activities, heavy metal requirements, heat stability, pH optimum, kinetic properties, and reaction with anti-arginase antibody were examined. Both enzyme species were either identical or substantially similar by all criteria. The adult and fetal enzymes are, therefore, probably determined by the same structural gene. Fetal red cells obtained during amniocentesis and amnioscopy should then be a suitable tissue to use to make the prenatal diagnosis of arginase deficiency.

Seventy-two fetal blood samples obtained by umbilical vein puncture under ultrasound guidance for prenatal diagnosis and monitoring purposes were tested for the expression of 38 blood group antigens. The gestational age at sampling ranged from 18 to 34 wk with 76% of fetuses between 20 and 25 wk. Compared to adult red blood cells, the following antigens showed either abnormally decreased frequency, or significantly reduced expression: A, B, A1, H, Lua, Lub, Lea, Leb, P1, P, and I. Selected anti-I and anti-i cold agglutinins, active at room temperature, were used at appropriate dilution, in a rapid slide or spin test to check for possible mixture of the sample with maternal blood and were shown to detect contaminations up to 5%. The test has proved particularly helpful for immediate and in process assessment of foetal origin of the sampled blood.

The purpose of the present review is to try to highlight recent advances in the application of microfluidic technology on non-invasive prenatal diagnosis (NIPD). The immunoaffinity based microfluidic technology is the most common approach for NIPD, followed by size-based microfluidic methods. Immunoaffinity microfluidic methods can enrich and isolate circulating fetal extravillous trophoblasts (fEVTs) or fetal nucleated red blood cells (fnRBCs) for NIPD by using specific antibodies, but size-based microfluidic systems are only applied to isolate fEVTs. Most studies based on the immunoaffinity microfluidic system gave good results. Enough fetal cells were obtained for chromosomal and/or genetic analysis in all blood samples. However, the results from studies using size-based microfluidic systems for NIPD are less than ideal. In conclusion, recent advances in microfluidic devices make the immunoaffinity based microfluidic system potentially a powerful tool for cell-based NIPD. However, more clinical validation is needed. [ABSTRACT FROM AUTHOR]

Noninvasive prenatal diagnosis (NIPD) aims to detect fetal-related genetic disorders before birth by detecting markers in the peripheral blood of pregnant women, holding the potential in reducing the risk of fetal birth defects. Fetal-nucleated red blood cells (fNRBCs) can be used as biomarkers for NIPD, given their remarkable nature of carrying the entire genetic information of the fetus. Here, we review recent advances in NIPD technologies based on the isolation and analysis of fNRBCs. Conventional cell separation methods rely primarily on physical properties and surface antigens of fNRBCs, such as density gradient centrifugation, fluorescence-activated cell sorting, and magnetic-activated cell sorting. Due to the limitations of sensitivity and purity in Conventional methods, separation techniques based on micro-/nanomaterials have been developed as novel methods for isolating and enriching fNRBCs. We also discuss emerging methods based on microfluidic chips and nanostructured substrates for static and dynamic isolation of fNRBCs. Additionally, we introduce the identification techniques of fNRBCs and address the potential clinical diagnostic values of fNRBCs. Finally, we highlight the challenges and the future directions of fNRBCs as treatment guidelines in NIPD. [ABSTRACT FROM AUTHOR]

The current gold standard for the definitive diagnosis of fetal aneuploidy uses either chorionic villus sampling (CVS) or amniocentesis, both of which are which are invasive procedures carrying a procedure-related risk of miscarriage of up to 0.1–0.2%. Non-invasive prenatal diagnosis using fetal nucleated red blood cells (FNRBCs) isolated from maternal peripheral venous blood would remove this risk of miscarriage since these cells can be isolated from the mother's blood. We aimed to detect whole-chromosome aneuploidies from single nucleated fetal red blood cells using whole-genome amplification followed by massively parallel sequencing performed on a semiconductor sequencing platform. Twenty-six single cells were picked from the placental villi of twelve patients thought to have a normal fetal genotype and who were undergoing elective first-trimester surgical termination of pregnancy. Following karyotyping, it was subsequently found that two of these cases were also abnormal (one trisomy 15 and one mosaic genotype). One single cell from chorionic villus samples for two patients carrying a fetus with trisomy 21 and two single cells from women carrying fetuses with T18 were also picked. Pooled libraries were sequenced on the Ion Proton and data were analysed using Ion Reporter software. We correctly classified fetal genotype in all 24 normal cells, as well as the 2 T21 cells, the 2 T18 cells, and the two T15 cells. The two cells picked from the fetus with a mosaic result by CVS were classified as unaffected, suggesting that this was a case of confined placental mosaicism. Fetal sex was correctly assigned in all cases. We demonstrated that semiconductor sequencing using commercially available software for data analysis can be achieved for the non-invasive prenatal diagnosis of whole-chromosome aneuploidy with 100% accuracy. [ABSTRACT FROM AUTHOR]

Background/Aim. Pregnancy-induced red blood cell (RBC) alloimmunization is important not only because of the possible negative effects on subsequent pregnancy outcomes in case the fetus carries the antigen but also because of the optimal trans-fusion management in cases of obstetric hemorrhage. Timely detection of RBC antibodies is part of testing, prevention, and treatment strategy, aimed at achieving better outcomes for alloimmunized mothers with an affected fetus. The aim of the study was to determine the frequency and specificity of alloantibodies among pregnant women from the South Bačka District, Serbia, with special attention to the incidence of anti-D alloantibodies. Methods. A retrospective study was conducted in the Blood Transfusion Institute of Vojvodina and covered the period from January 1, 2010, to December 31, 2019. Screening and antibody identification were performed by an indirect antiglobulin test in gel-microcards (ID-Card Liss/Coombs) with two test RBC (ID-DiaCell I-II screening cells, Bio-Rad, Cressier, Switzerland) on an automated system (IH-500, Bio-Rad). Results. Among 25,694 tested pregnant women, 1.38% were actively immunized, while 1.12% of women acquired antibodies in the current pregnancy. Among 3,622 (14.09%) RhD-negative women, 1.77% produced anti-D antibodies during the ongoing pregnancy. Distribution of anti-body specificity was: anti-D 23.34%, anti-M 11.85%, anti-E 9.41%, anti-K 9.41%, anti-C 5.92%, anti-Fyb 5.92%, anti-c 3.13%, anti-S 3.13%, anti-Lea 3.13%, anti-Leb 3.13%, anti-Cw 1.75%, anti-Jka 1.40%, anti-P 1.05%, anti-Lub 0.70%, anti-Fya 0.35%, autoantibody of undetermined specificity 0.70%, and irregular antibodies of undetermined specificity 15.68%. Conclusion. Immunoglobulin prophylaxis has led to a significant reduction in the frequency of D-alloimmunization among pregnant women in the South Bačka District over the last ten years. However, the incidence of anti-D antibodies is still significantly higher than in published data for developed countries. We also identified the other, less commonly present, clinically significant antibodies. There is a need to introduce uniform recommendations for immunohematological testing in pregnancy on the territory of the Republic of Serbia in accordance with modern requirements.

Background: Hemoglobinopathies refer to a range of diseases, in which the hemoglobin (Hb) molecule’s synthesis or structure is aberrant. It is passed down through the generations (inherited). Hb C illness, Hb S-C disease, sickle cell anemia, and thalassemia are all part of this group. Hemoglobinopathies are the most frequent genetically inherited red blood cell condition on the planet. Hb variant screening and precise identification have become more crucial in the antenatal diagnosis and prevention of Hb diseases. Aims and Objectives: For Hb analysis, cation exchange high-performance liquid chromatography (HPLC) has emerged as the method of choice. This research was brought up to diagnose numerous hemoglobinopathies and also to measure distinct Hb fractions. Materials and Methods: Four hundred and seventy participants were examined for hemoglobinopathies in the investigation. Different fractions of Hb were compared to patients with normal Hb after diagnosis. Results: Normal HPLC, SCA (Hb SS), sickle cell trait (SCT), and -thalassemia trait were discovered in 110 (23.4%), 130 (27.6%), 210 (44.6%), and 20 (4.2%) individuals, respectively, out of 470 patients. Rare hemoglobinopathies such as tenacious fetal Hb (PFH), Hb E, Hb D-Punjab, and Hb Q India accounted for 15 (3.1%) of the sufferers. Patients with SCA had a mean percent of Hb S, Hb F, Hb A, and Hb A2 of 81.6±74.6, 22.9±13.9, 3.1±6.1, and 2.5±1.02, respectively. Hb F was 22.9±13.9% on average, which was much higher than normal and SCT. A high HbA2 level (>3.5%) indicated the existence of the β-thalassemia trait. Conclusion: In the included section of the population, SCD is the most common kind of hemoglobinopathy, both homozygous and heterozygous. Hb F concentrations are observed to be elevated in SCA patients, and it is thought to be a key element in protecting patients from problems. Raised Hb A2, which indicates the presence of the β-thalassemia trait, was also discovered in this area, and it is suspected when the value is >3.7%. Cation exchange HPLC is ideal for routine hemoglobinopathies research due to its high resolution, short test time, and precise quantification. [ABSTRACT FROM AUTHOR]

Hemolytic disease of the fetus and newborn is a consequence of maternal immune response to fetal red blood cells antigens inherited from the father, that the mother does not possess, stimulated by antepartum or intrapartum fetomaternal hemorrhage. As a result, fetal erythrocytes are destroyed, leading to various degrees of anemia and hyperbilirubinemia, with high perinatal morbidity and mortality rates. This review's purpose is to reveal the importance of the alloantibodies beyond RhD and ABO system, the updated algorithms used in the diagnosis and management of the disease and the importance of RhD universal immune prophylaxis practice. For this purpose, the database from PubMed and UpToDate was searched for literature reviews, case studies, society guidelines and retrospective studies in English regarding hemolytic disease of the fetus and newborn from 2013 to March 2022. Anti D prophylaxis protocols, early diagnosis and proper intrauterine and postpartum treatment help significantly reduce the disease's burden. [ABSTRACT FROM AUTHOR]

Objective: To investigate factors associated with fetal fraction and to develop a new predictive method for low fetal fraction before noninvasive prenatal testing. Methods: The study was a retrospective cohort analysis based on the results of noninvasive prenatal testing, complete blood count, thyroxin test, and Down's syndrome screening during the first or second trimester in 14,043 pregnant women. Random forests algorithm was applied to predict the low fetal fraction status (fetal fraction < 4%) through individual information and laboratory records. The performance of the model was evaluated and compared to predictions using maternal weight. Results: Of 14,043 cases, maternal weight, red blood cell, hemoglobin, and free T3 were significantly negatively correlated with fetal fraction while gestation age, free T4, pregnancy-associated plasma protein-A, alpha-fetoprotein, unconjugated estriol, and β-human chorionic gonadotropin were significantly positively correlated with fetal fraction. Compared to predictions using maternal weight as an isolated parameter, the model had a higher area under the curve of receiver operating characteristic and overall accuracy. Conclusions: The comprehensive predictive method based on combined multiple factors was more effective than a single-factor model in low fetal fraction status prediction. This method can provide more pretest quality control for noninvasive prenatal testing. [ABSTRACT FROM AUTHOR]

Objectives: To develop a prenatal ultrasound staging system for placenta accreta spectrum (PAS) disorders in women with placenta previa and to evaluate its association with surgical outcome, placental invasion and the clinical staging system for PAS disorders proposed by the International Federation of Gynecology and Obstetrics (FIGO).Methods: This was a secondary retrospective analysis of prospectively collected data from women with placenta previa. We classified women according to the following staging system for PAS disorders, based upon the presence of ultrasound signs of PAS in women with placenta previa: PAS0, placenta previa with no ultrasound signs of invasion or with placental lacunae but no evidence of abnormal uterus-bladder interface; PAS1, presence of at least two of placental lacunae, loss of the clear zone or bladder wall interruption; PAS2, PAS1 plus uterovescical hypervascularity; PAS3, PAS1 or PAS2 plus evidence of increased vascularity in the inferior part of the lower uterine segment potentially extending into the parametrial region. We explored whether this ultrasound staging system correlates with surgical outcome (estimated blood loss (EBL, mL), units of packed red blood cells (PRBC), fresh frozen plasma (FFP) and platelets (PLT) transfused, operation time (min), surgical complications defined as the occurrence of any damage to the bladder, ureters or bowel, length of hospital stay (days) and admission to intensive care unit (ICU)) and depth of placental invasion. The correlation between the present ultrasound staging system and the clinical grading system proposed by FIGO was assessed. Prenatal and surgical management were not based on the proposed prenatal ultrasound staging system. Linear and multiple regression models were used.Results: Two-hundred and fifty-nine women were included in the analysis. Mean EBL was 516 ± 151 mL in women with PAS0, 609 ± 146 mL in those with PAS1, 950 ± 190 mL in those with PAS2 and 1323 ± 533 mL in those with PAS3, and increased significantly with increasing severity of PAS ultrasound stage. Mean units of PRBC transfused were 0.05 ± 0.21 in PAS0, 0.10 ± 0.45 in PAS1, 1.19 ± 1.11 in PAS2 and 4.48 ± 2.06 in PAS3, and increased significantly with PAS stage. Similarly, there was a progressive increase in the mean units of FFP transfused from PAS1 to PAS3 (0.0 ± 0.0 in PAS1, 0.25 ± 1.0 in PAS2 and 3.63 ± 2.67 in PAS3). Women presenting with PAS3 on ultrasound had significantly more units of PLT transfused (2.37 ± 2.40) compared with those with PAS0 (0.03 ± 0.18), PAS1 (0.0 ± 0.0) or PAS2 (0.0 ± 0.0). Mean operation time was longer in women with PAS3 (184 ± 32 min) compared with those with PAS1 (153 ± 38 min) or PAS2 (161 ± 28 min). Similarly, women with PAS3 had longer hospital stay (7.4 ± 2.1 days) compared with those with PAS0 (3.4 ± 0.6 days), PAS1 (6.4 ± 1.3 days) or PAS2 (5.9 ± 0.8 days). On linear regression analysis, after adjusting for all potential confounders, higher PAS stage was associated independently with a significant increase in EBL (314 (95% CI, 230-399) mL per one-stage increase; P < 0.001), units of PRBC transfused (1.74 (95% CI, 1.33-2.15) per one-stage increase; P < 0.001), units of FFP transfused (1.19 (95% CI, 0.61-1.77) per one-stage increase; P < 0.001), units of PLT transfused (1.03 (95% CI, 0.59-1.47) per one-stage increase; P < 0.001), operation time (38.8 (95% CI, 31.6-46.1) min per one-stage increase; P < 0.001) and length of hospital stay (0.83 (95% CI, 0.46-1.27) days per one-stage increase; P < 0.001). On logistic regression analysis, increased severity of PAS was associated independently with surgical complications (odds ratio, 3.14 (95% CI, 1.36-7.25); P = 0.007), while only PAS3 was associated with admission to the ICU (P < 0.001). All women with PAS0 on ultrasound were classified as having Grade-1 PAS disorder according to the FIGO grading system. Conversely, of the women presenting with PAS1 on ultrasound, 64.1% (95% CI, 48.4-77.3%) were classified as having Grade-3, while 35.9% (95% CI, 22.7-51.6%) were classified as having Grade-4 PAS disorder, according to the FIGO grading system. All women with PAS2 were categorized as having Grade-5 and all those with PAS3 as having Grade-6 PAS disorder according to the FIGO system.Conclusion: Ultrasound staging of PAS disorders is feasible and correlates with surgical outcome, depth of invasion and the FIGO clinical grading system. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

Background: Noninvasive prenatal testing (NIPT) based on cell-free DNA in maternal circulation has been accepted worldwide by the clinical community since 2011 but limitations, such as maternal malignancy and fetoplacental mosaicism, preclude its full replacement of invasive prenatal diagnosis. We present a novel silicon-based nanostructured microfluidics platform named as "Cell Reveal™" to demonstrate the feasibility of capturing circulating fetal nucleated red blood cells (fnRBC) and extravillous cytotrophoblasts (EVT) for cellbased noninvasive prenatal diagnosis (cbNIPD). Methods: The "Cell Reveal™" system is a silicon-based, nanostructured microfluidics using immunoaffinity to capture the trophoblasts and the nucleated RBC (nRBC) with specific antibodies. The automated computer analysis software was used to identify the targeted cells through additional immunostaining of the corresponding antigens. The identified cells were retrieved for whole genome amplification for subsequent investigations by micromanipulation in one microchip, and left in situ for subsequent fluorescence in situ hybridization (FISH) in another microchip. When validation, bloods from pregnant women (n = 24) at gestational age 11-13+6 weeks were enrolled. When verification, bloods from pregnant women (n = 5) receiving chorionic villus sampling or amniocentesis at gestation age 11+4-21 weeks with an aneuploid or euploid fetus were enrolled, followed by genetic analyses using FISH, short tandem repeat (STR) analyses, array comparative genomic hybridization, and next generation sequencing, in which the laboratory is blind to the fetal genetic complement. Results: The numbers of captured targeted cells were 1-44 nRBC/2 ml and 1-32 EVT/2 ml in the validation group. The genetic investigations performed in the verification group confirmed the captured cells to be fetal origin. In every 8 ml of the maternal blood being blindly tested, both fnRBC and EVT were always captured. The numbers of captured fetal cells were 14-22 fnRBC/4 ml and 1-44 EVT/4 ml of maternal blood. Conclusions: This report is one of the first few to verify the capture of fnRBC in addition to EVT. The scalability of our automated system made us one step closer toward the goal of in vitro diagnostics. [ABSTRACT FROM AUTHOR]

Objective To evaluate the repercussions of recent changes to the cut-offs used in the first screening step of the pre-marital screening programme for thalassaemia prevention in Iran. Methods The profiles of 984 subjects referred to a genetic laboratory, and the tests of 242 parents of children with thalassaemia major were assessed for red blood cell (RBC) indices, haemoglobin (Hb) A2 levels and results of Hb electrophoresis. Results Of 407 suspected thalassaemia minor (STM) cases, 18 proved positive for thalassaemia minor on molecular analysis (18/407, confidence interval 2.6–6.9%). If the revised screening cut-offs had been used to determine who would undergo molecular analysis, two of these cases would not have been identified. Only 4.4% of suspected cases with lower than normal RBC indices (mean corpuscular volume <80 fl and mean corpuscular Hb <27 pg) and HbA2 (<3.5%) were diagnosed with thalassaemia minor. Conclusion The thalassaemia major prevention programme is performed in two separate steps. One step involves the screening of subjects and identification of β-thalassaemia minor, suspected cases for thalassaemia minor (STM), and normal subject groups. The other step concerns the identification of thalassaemia minor in the STM group. Changing the cut-offs at the first screening step does not result in significant improvement from an economic view, and is associated with significant risk at the second screening step. [ABSTRACT FROM AUTHOR]

At present, prevention of thalassaemia and sickle cell disease is the only realistic approach to control the birth of new patients in countries having high numbers of carriers. This is fully justified because avoiding the birth of an ever increasing number of patients may allow a more effective use of the available resources in improving the management of the patients surviving today and alleviate the already overloaded public health system from the inevitable tremendous and ever increasing cost. Moreover, prenatal diagnosis may help couples at risk to have non-thalassaemic children. Greece is one of the countries where the mean frequency of carriers is approximately 7.5 per cent (population 11 million) and has set up a nationwide programme for carrier identification in the early seventies; this is provided through a dozen of specific Units attached to the major Blood Transfusion Services of the country, on a voluntary basis and free of charge. Spread of information through mass media, the schools, and other groups has greatly contributed in creating the necessary sensitization; obstetricians and antenatal Clinics are also instrumental to this effect. Prenatal diagnosis is offered centrally (Athens) and covers satisfactorily the estimated needs (500- 600 annually); the total number has already exceeded 35,000. According to information obtained from the major paediatric hospitals all over the country, the number of thalassaemia major or SCD admitted for treatment over the last ten years has been around 15 yearly (instead of an estimate of 120-130). [ABSTRACT FROM AUTHOR]

Background: Our previous study has shown that prenatal exposure to X-ray irradiation causes cerebral hypo-perfusion during the postnatal development of central nervous system (CNS). However, the source of the hypo-perfusion and its impact on the CNS development remains unclear. The present study developed an automatic analysis method to determine the mean red blood cell (RBC) speed through single microvessels imaged with two-photon microscopy in the cerebral cortex of rats prenatally exposed to X-ray irradiation (1.5 Gy). Methodology/Principal Findings: We obtained a mean RBC speed (0.960.6 mm/sec) that ranged from 0.2 to 4.4 mm/sec from 121 vessels in the radiation-exposed rats, which was about 40% lower than that of normal rats that were not exposed. These results were then compared with the conventional method for monitoring microvascular perfusion using the arteriovenous transit time (AVTT) determined by tracking fluorescent markers. A significant increase in the AVTT was observed in the exposed rats (1.960.6 sec) as compared to the age-matched non-exposed rats (1.260.3 sec). The results indicate that parenchyma capillary blood velocity in the exposed rats was approximately 37% lower than in non-exposed rats. Conclusions/Significance: The algorithm presented is simple and robust relative to monitoring individual RBC speeds, which is superior in terms of noise tolerance and computation time. The demonstrative results show that the method developed in this study for determining the mean RBC speed in the spatial frequency domain was consistent with the conventional transit time method. [ABSTRACT FROM AUTHOR]

One of the ongoing challenges for the accurate diagnosis and treatment of children with fetal alcohol spectrum disorders (FASD) is the difficulty of confirming whether a mother drank during her pregnancy. Commonly used screening questionnaires often are unreliable, and current established biomarkers of alcohol consumption are not sensitive enough for use with many pregnant women. These limitations underscore the critical need to develop novel biomarkers with greater sensitivity for detecting moderate levels of drinking during pregnancy for longer periods of time after the last drinking episode. In addition, developing reliable biomarkers of fetal alcohol effects that can identify children at risk for adverse neurobehavioral outcomes could lead to behavioral interventions earlier in development. The use of animal models of FASD in biomarker development could accelerate progress in this challenging field of research. [ABSTRACT FROM AUTHOR]

Invasive procedures for prenatal diagnosis are associated with increased risk of abortion; thus, development of noninvasive procedures would be beneficial. Based on the observation that embryonic nucleated red blood cell (NRBC) crosses the placenta and enters the circulation of pregnant women, the ability to identify such cell would allow development of such procedures. Identification of NRBCs in blood samples would be possible provided that specific antibodies are available. Here we have isolated recombinant antibodies using phage display. From the panel of antibody fragments specifically recognising ϵ-Hb, one was chosen for further characterization, DAb1. DAb1 binds to ϵ-Hb both in Western blots and immunocytochemistry. Several ϵ-Hb positive cells were detected in a blood sample taken as postchorionic villus sampling (CVS). To evaluate the sensitivity of themethod, K562 cells (which express ϵ-Hb) were spiked in a blood sample followed by staining in solution and FACS analysis. [ABSTRACT FROM AUTHOR]

Methylmercury (MeHg) is one of the most hazardous substances that affects the fetus through fish consumption. The objective of this study was to evaluate the changes in the level of exposure to MeHg by assessing the mercury (Hg) concentrations of the segmental hair at parturition and 3 months after parturition, and to study their correlations with the total Hg concentrations of maternal and cord red blood cells (RBCs) and neonatal hair as biomarkers of fetal exposure to MeHg at parturition. In total, 40 paired samples of maternal hair from the scalp, maternal and cord RBCs, and 21 samples of neonatal hair from the scalp were collected at parturition. In addition, 19 samples of maternal hair from the scalp were collected at 3 months after parturition. The maternal hair samples were cut into 1cm segments from the scalp end toward the tip. The geometric mean of the Hg concentrations in cord RBCs was approximately 1.6 times higher than that in the maternal RBCs, and a strong correlation coefficient (r=0.91) was found between them. The increase or decrease in the Hg concentrations of the segmental hair during gestation differed largely among individuals. The correlation coefficients between the Hg concentrations of the segmental hair and cord RBCs were the strongest (r=0.90) in the hair segment 1cm from the scalp and decreased gradually with the distance from the scalp. The correlation coefficients between the Hg concentrations of the segmental hair collected at 3 months after parturition and maternal RBCs were over 0.9 in the hair segments 5 and 6cm from the scalp, suggesting that the time required for the incorporation of Hg from the blood into a growing hair was very short. The geometric mean of Hg concentrations in the neonatal hair at parturition was similar to that in the maternal hair 1cm from the scalp at parturition, and they exhibited a strong correlation (r=0.95). The findings of this study indicate that maternal hair close to the scalp at parturition and neonatal hair are useful biomarkers of fetal exposure to MeHg at parturition. In addition, the segmental maternal hair throughout gestation is essential to obtain important information on MeHg exposure during the different sensitive windows or bolus MeHg exposure during gestation. [Copyright &y& Elsevier]

The isolation and analysis of fetal cells from maternal blood would allow non-invasive prenatal genetic screening and diagnosis. Over the past decade, progress has been made towards this goal using various enrichment strategies and analysis by fluorescence in-situ hybridization with chromosome-specific probes and PCR. One method that is currently being explored involves culturing fetal cells. Developing conditions which allow the number of fetal-derived cells to expand in culture and the number of maternally derived cells to be suppressed in culture may lead to a new selection process for obtaining fetal cells. Culturing of fetal cells from maternal blood could make possible conventional metaphase analysis of fetal cells for diagnosis of chromosomal abnormalities. [ABSTRACT FROM AUTHOR]

Background: Antenatal screening for maternal antibodies can detect the likelihood of the baby being affected by hemolytic disease of the newborn. At present, in most centers, Rh D incompatibility is the only cause for hemolytic disease of the newborn for which screening is routinely done. Routine testing for maternal antibodies using a screening panel that detects other immune antibodies in addition to anti D was introduced in our centre in January, 2007. Since then, approximately 700 pregnant women are screened per month. Aims: To detect relative frequencies of maternal sensitization to D and to other blood group antigens with a view towards aiding to develop protocols for better antenatal care and management of pregnancies in sensitized women. Materials and Methods: Blood grouping and Rh typing is done for all pregnant women at the time of their first antenatal checkup in Christian Medical College, Vellore. Simultaneously, screening for antibodies is done using indirect Coombs method employing a commercially derived screening panel of red blood cells (Surgiscreen; Ortho-Clinical Diagnostics). The antibody titre is determined at the time of detection. Results: To date, 26 cases of maternal sensitization to blood group antigens have been detected. Fifteen (57.7%) of the sensitised patients were Rh negative. Eleven (42.3%) patients were detected to have anti Rh D. The presumptive characterization of antibodies in the remaining patients are as follows: anti-C/anti-S - four patients; anti-C/anti-S - three patients; anti-M/anti-Lub - two patients; anti-C/ anti-Fyb - two patients; anti-E/anti-Fyb - two patients; anti-Leb/anti- N - one patient; anti-JKb/anti-Leb/anti-P - one patient. The antibody titre was significantly high (1:2048; anti-M/anti-Lub) in one patient at presentation. The remainder had titres below 1:32. Conclusion: Anti-Rh D in our study was responsible for only 42.3% of maternal immune antibodies to blood group antigens. Possibly the high degree of awareness regarding Rh hemolytic disease of the newborn and widespread prophylactic usage of anti-D may be responsible for reduced prevalence of sensitization to D. Notably, 4 out of 15 (26.7%) Rh negative women in our study showed sensitization to antigens other than D. It is hoped that antenatal identification of other blood group related antibodies and follow-up titres of the same through pregnancy can aid the clinician to anticipate a baby that may suffer from hemolytic disease that is not Rh related, and to take necessary steps in management including procuring appropriate blood for an exchange transfusion if required. [ABSTRACT FROM AUTHOR]