Fetal Blood Cells Research Articles

Dynamic Changes in Fetal Microchimerism in Maternal Peripheral Blood Mononuclear Cells, CD4+ and CD8+ cells in Normal Pregnancy

Objective Cell trafficking during pregnancy results in persistence of small populations of fetal cells in the mother, known as fetal microchimerism (FMc). Changes in cell-free fetal DNA during gestation have been well described, however, less is known about dynamic changes in fetal immune cells in maternal blood. We have investigated FMc in maternal peripheral blood mononuclear cells (PBMC) longitudinally across gestation. Study design Thirty-five women with normal pregnancies were studied. FMc was identified in PBMC, CD4+ and CD8+ subsets employing quantitative PCR assays targeting fetal-specific genetic polymorphisms. FMc quantities were reported as fetal genome equivalents (gEq) per 1,000,000 gEq mother’s cells. Poisson regression modeled the rate of FMc detection. Main outcome measure FMc in PBMC. Results The probability of detecting one fetal cell equivalent increased 6.2-fold each trimester [Incidence Rate Ratio (IRR) 95% CI: 1.73, 21.91; p = 0.005]. Although FMc in PBMC was not detected for the majority of time points, 7 of 35 women had detectable FMc during pregnancy at one or more time points, with the majority of positive samples being from the third trimester. There was a suggestion of greater HLA-sharing in families where women had FMc in PBMC. FMc was detected in 9% of CD4+ (2/23) and 18% of CD8+ (3/25) subsets. Conclusions FMc in PBMC increased as gestation progressed and was found within CD4+ and CD8+ subsets in some women in the latter half of gestation. A number of factors could influence cellular FMc levels including sub-clinical fetal-maternal interface changes and events related to parturition. Whether FMc during pregnancy predicts persistent FMc and/or correlates with fetal-maternal HLA relationships also merits further study.

Identification of candidate surface antigens for non-invasive prenatal diagnosis by comparative global gene expression on human fetal mesenchymal stem cells

Transplacental passage of circulating first-trimester fetal mesenchymal stem cells (fMSC) raises the prospect of harvesting fetal cells in maternal blood. Despite high sensitivity in model systems, negative selection and culture strategies yield fMSC only rarely in post-termination maternal blood. The different adhesion molecule profile of fMSC to competitor maternal cell types suggests that improved positive selection strategies may facilitate non-invasive prenatal diagnosis. We aimed to identify surface antigens specific to fMSC and not maternal peripheral blood lymphocytes (PBL), using genome-wide analysis of actively expressed transcripts. Maternal PBL and fMSC cultured from first-trimester blood, liver and bone marrow were assessed for global gene expression by Affymetrix U133Plus2.0 arrays. Data were analysed using Affymetrix GCOS01.2. Transcripts present in all fMSC (n = 9) but absent in all PBL samples (n = 3) were selected for further analysis of cell-surface membrane molecules by RT-PCR and immunocytochemistry. Of 1544 genes expressed in fMSC and not maternal PBL, filtering for cell-surface molecules yielded 159 genes. Of these, 29 had a mean expression ratio of >300 (P < 0.001), which represented 18 unique genes, and their positive expression in all fMSC samples was confirmed by RT-PCR. Candidates for non-invasive prenatal diagnosis were chosen for further analysis by immunocytochemistry. Surface expression of OSMR and COL1 proteins on all fMSC, but no maternal PBL was confirmed. Identification of novel surface antigens on circulating human fMSC and not maternal PBL facilitates positive selection strategies for isolating fMSC for non-invasive prenatal diagnosis.

Diagnosis ofin uteroParvovirus B19 infection and maternal immune response – the relevance of linear epitopes and advanced serologic testing

Parvovirus B19 (B19V) infection in utero causing fetal anemia and non-immune hydrops fetalis (NIHF) is a potentially life-threatening event for the fetus. Postexpositional non-invasive diagnosis is based on maternal IgG/IgM response and detection of viral genome in maternal blood. Serologic testing directs prenatal follow-up. Fetal infection is confirmed by polymerase chain reaction or in situ hybridization in fetal blood and/or amniotic fluid cells. The performance of serologic tests is significant in order to direct pre- and perinatal care at rational use of resources. Timing of diagnostic procedures and knowledge of the time course of infection in pregnant, asymptomatic women are critical. IgM negative testing in the presence of prolonged viremia may complicate individual risk analysis in pregnancy. Recently, advanced IgG avidity assays and epitope-type specific assays (IgG ETS EIA) have been re-evaluated. Epidemiology, clinical relevance and management of B19V infection in pregnancy. A review of the current literature (November 1984 - May 2009) and evaluation of current information on performance and predictive value of molecular and VP1/VP2 antigen-based IgG tests directed at the diagnosis of materno-fetal B19V infection and detection of past immunity. New aspects of B19V-associated fetal disease other than anemia/NIHF are also covered. An overview of immunology and clinical relevance of B19V infection in pregnancy, of the potential value of advanced serologic testing and fields of future research. In the absence of a commercially available vaccine, serologic tests remain important tools in individual risk analysis of pregnant women exposed to B19V. Sequential application of IgG avidity and IgG ETS EIAs may improve risk stratification and timing of invasive testing in B19V-exposed pregnancies, in particular with IgM-negativity and/or persistent DNAemia. Prospective evaluation of these test systems correlated to fetal outcome in order to reduce fetal morbidity and mortality as well as the overall burden of disease of B19V with regard to fetal malformation may be subject to future research.

The role of antenatal immunoprophylaxis in the prevention of maternal-foetal anti-Rh(D) alloimmunisation.

The first description of haemolytic disease of the newborn (HDN) can be traced back to 1609 and was made by a French midwife, Louise Bourgeois, who, from 1600, worked at the royal court of King Henry IV and Queen Marie de Medicis1–4. In the treatise that Bourgeois wrote in 1609 she described the birth of two twins3: the first had hydrops and died immediately, while the second, initially in a better condition, rapidly became jaundiced and, after having developed neurological symptoms (kernicterus), died 3 days after being born. Hydrops foetalis and kernicterus were correctly interpreted as two aspects of the same pathology only in 19325, when Diamond described foetal erythroblastosis secondary to severe haemolysis, although the cause was still unknown. A few years later, in 1938, Ruth Darrow correctly identified the (antibody-related) pathogenesis of HDN6, although erroneously attributing foetal haemoglobin the role of the culprit antigen, which was suggested to have induced a maternal antibody response after crossing the placenta. The true pathogenesis of the disease was definitively clarified in 1940 with the discovery of the Rhesus (Rh) blood group system by Landsteiner and Wiener7 and with the subsequent identification, in 1941, by Levine8, of the Rh(D) antigen. This antigen was, in fact, identified, in D-negative mothers, as being the cause of the immunisation occurring following transplacental passage of foetal D-positive red blood cells. The subsequent passage of maternal anti-D immunoglobulin G (IgG) across the placenta into the foetal circulation was recognised as the final event able to cause the spectrum of clinical events that characterise HDN. It did not take long before the risk of immunisation could be quantified1,3: i) 16% in the case of a Rh(D)-negative mother and a Rh(D)-positive, ABO-compatible foetus; ii) 2% in the case of a Rh(D)-negative mother and a Rh(D)-positive, ABO-incompatible foetus (about 20% of the cases); iii) overall risk of immunisation: 13.2%. Before 1945, about 50% of all foetuses with HDN died of kernicterus or hydrops foetalis. Subsequently, thanks to the progress in treatment, in industrialised countries the mortality decreased to 2–3%; this mortality rate was then very considerably further reduced (100-fold) with the introduction of anti-D immunoprophylaxis to prevent maternal-foetal anti-Rh(D) alloimmunisation 9. At the beginning of the 1960s, Stern demonstrated experimentally that the administration of anti-D IgG could prevent sensitisation to the Rh(D) antigen10; in the same period, other studies clarified the mechanism of Rh iso-immunisation in pregnancy and introduced the clinical practice of passive immunisation with anti-D IgG to protect Rh(D)-negative women from sensitisation against Rh(D)-positive red blood cells11–14. The successes obtained in studies of Rh(D)-negative male volunteers formed the experimental basis for clinical trials in pregnant Rh(D)-negative women15; these trials demonstrated that post-partum immunoprophylaxis decreased the incidence of post-pregnancy anti-Rh(D) immunisation from 12–13% to 1–2%15,16. Subsequently, in 1977, it was shown that 1.8% of Rh(D)-negative women, despite post-natal prophylaxis, continued to develop anti-D antibodies because of small transplacental haemorrhages during pregnancy17,18. One year later, a Canadian study by Bowman et al. showed, in 1,357 Rh(D)-negative primagravida, that the incidence of Rh(D) alloimmunisation could be reduced to 0.1% by prophylaxis with antenatal anti-D IgG, in addition to post-partum prophylaxis19. There is currently sufficient evidence demonstrating that antenatal anti-D prophylaxis also reduces the risk of Rh(D) immunisation in the next pregnancy to below the level of 0.4%. Forty years after Zipursky and Israels first proposed the use of anti-D IgG to reduce the incidence of Rh alloimmunisation in pregnancy14, immunoprophylaxis has drastically reduced the cases of Rh-induced HDN; nevertheless, this pathology continues to be relevant in 0.4 of 1,000 births (0.04%)20, for various reasons21: i) the possible occurrence of anti-D immunisation during the pregnancy (which occurs in about 1% of Rh(D)-negative women carrying a Rh(D)-positive foetus22); ii) the lack of efficacy of immunoprophylaxis because of the administration of an insufficient dose of anti-D IgG that is not congruent with the volume of the foetal-maternal haemorrhage; iii) immunoprophylaxis not administered; iv) possible errors in typing the pregnant or puerperal woman or the neonate; v) possible errors in transfusion therapy in women of child-bearing age.

Method for isolation of floating fetal RBCs from maternal circulation based on their inherent magnetic property

Isolation of floating fetal cells from maternal circulation has immense potential in diagnosing of various genetic alterations in the developing fetus. Currently, non-invasive fetal cell isolation methods include fluorescence/magnetic activated cell sorting that use antibodies specific to fetal cells. Apart from being complex and expensive the biggest challenge associated with these cell sorting methods is low concentration of fetal cells in maternal peripheral blood. In order to make the complete process much simpler and effective, we propose a novel method for isolation of floating fetal cells that uses a continuous flow of maternal blood for effectively harvesting a higher fetal cell volume compared to any other existing method. The isolation mechanism is based on the difference in the magnetic susceptibility of fetal hemoglobin (HbF- α 2 γ 2) and maternal hemoglobin (HbA- α 2 β 2). HbF has high oxygen saturation capacity (diamagnetic) compared to HbA (paramagnetic), and this difference in saturation is further enhanced by presence of 2,3-bisphosphoglycerate (2,3-BPG). When placed in magnetic field, these cells get separated based on the difference ( p ≤ 0.001) in their magnetophoretic mobility. This separation method may also be used for detection of fetomaternal hemorrhages and also treatment of Rh incompatibility.

Amniotic fluid embolism.

The disastrous entry of amniotic fluid into the maternal circulation leads to dramatic sequelae of clinical events, characteristically referred to as Amniotic fluid embolism (AFE). The underlying mechanism for AFE is still poorly understood. Unfortunately, this situation has very grave maternal and fetal consequences. AFE can occur during labor, caesarean section, dilatation and evacuation or in the immediate postpartum period. The pathophysiology is believed to be immune mediated which affects the respiratory, cardiovascular, neurological and hematological systems. Undetected and untreated it culminates into fulminant pulmonary edema, intractable convulsions, disseminated intravascular coagulation (DIC), malignant arrhythmias and cardiac arrest. Definite diagnosis can be confirmed by identification of lanugo, fetal hair and fetal squamous cells (squames) in blood aspirated from the right ventricle. Usually the diagnosis is made clinically and by exclusion of other causes. The cornerstone of management is a multidisciplinary approach with supportive treatment of failing organs systems. Despite improved modalities for diagnosing AFE, and better intensive care support facilities, the mortality is still high.

Rhesus Alloimmunisation: Forgotten but not Gone

The prophylactic use of rhesus immunoglobulin has led to a significant reduction in the incidence of haemolytic disease of the newborn secondary to rhesus alloimmunisation, which now only occurs in 1 in 1000 live births. Despite prophylaxis, antibodies to the D antigen are still the commonest cause of rhesus isoimmunisation. Although now a rare pregnancy disorder rhesus isoimmunisation continues to be a challenging obstetric complication and while the face of this disease has undergone many changes over recent years, the principles of management remain unchanged. Pregnancies at risk of the disease are identified by past obstetric history and the antenatal detection of red cell antibodies with serial measurement of antibody levels. In cases of heterozygous paternity the real risk of fetal disease can be determined using new DNA techniques to determine the fetal blood cell type with tissue obtained from amniocentesis, and now by molecular analysis of circulating fetal DNA in the maternal plasma and serum. Sensitised pregnancies are monitored for the development of fetal anaemia. Since its introduction into clinical practise by Liley over 40 years ago, spectral analysis of amniotic fluid bilirubin as an indirect measure of fetal haemolysis has been the primary method of screening for fetal anaemia. Noninvasive evaluation is now possible with the use of doppler ultrasound to detect changes to fetal blood flow secondary to fetal anaemia, most commonly using the middle cerebral artery, and ultrasound evaluation of fetal haemopoetic organs and for the diagnosis of hydrops. Ultrasound guided intrauterine transfusion of red blood cells remains the treatment for the severely affected fetus. Perinatal survival rates in the non hydropic fetus are reported at over 90% and long term studies have revealed normal neurological outcomes for more than 90% cases.

Utilization of Anti-RhD in the Emergency Department After Blunt Trauma

In the United States, trauma occurs in 6% to 7% of pregnancies. Its severity may range from critical injuries where the mother's life is at risk, to apparently minor injuries, which might not be associated with any worrisome symptoms. One of the risks associated with a traumatic event is fetomaternal hemorrhage--the transfer of fetal blood cells into the maternal circulation. If the maternal blood type is rhesus negative and the fetus is rhesus positive, even a small amount of blood can cause the mother to develop antibodies against the fetal Rho D antigen, thus becoming sensitized. In subsequent pregnancies, this can lead to hemolytic disease of the fetus or newborn, which, if severe, is associated with total body edema, hepatosplenomegaly and heart failure, and intrauterine death. Although there are no published studies specific to the US population, poor awareness of the risk of sensitization following trauma and underutilization of anti-RhD in the emergency department has been reported in countries such as Canada and the United Kingdom. This article reminds caregivers of the risk of rhesus sensitization following blunt trauma, in order that they can administer anti-RhD appropriately and hemolytic disease of the fetus or newborn can be prevented.

The effect of pregnancy on production of maternal endogenous hematopoietic stem cells

Fetal microchimerism refers to the presence of fetal cells in maternal blood and tissues during pregnancy. This microchimerism may result from trafficking of fetal and maternal blood across the placenta during pregnancy. Physiological changes in the maternal blood cellular milieu are also recognized during pregnancy and in the early postpartum period. Earlier studies showed that maternal blood contains CD34 + hematopoietic stem cells (HSCs) that bear paternal genetic markers or male phenotype, suggesting that these cells circulated to the mother from male fetuses during pregnancy. Other studies showed that these maternal HSCs have significantly lower expansion potential than their fetal counterparts. We have recently shown increased percentages of CD34 + HSCs in peripheral blood of pregnant and parous women. Herein, we hypothesize that pregnancy stimulates the production of endogenous CD34 + HSCs of maternal origin, a phenomenon which potentially could favor postpartum regenerative capacity.

In Vitro Effects of Artesunate on Rat Embryonic and Fetal Blood Cells

In Vitro Effects of Artesunate on Rat Embryonic and Fetal Blood Cells

Detection of Fetal Sex in the Peripheral Blood of Pregnant Women

Objective: To choose the best method to examine fetal sex in maternal blood as early as possible and evaluate the quantitative change of fetal-free DNA in maternal plasma. Method: One hundred and fifty pregnant women were studied at 5–9 completed weeks of gestation. Fetal cells were isolated using lymphocyte separation liquid and 3% gelatin. Furthermore, fluorescence in situ hybridization was used to examine the terminal of the Y chromosome. Nested polymerase chain reaction (PCR) and real-time fluorescence quantitative PCR (FQ-PCR) were used to amplify the SRY gene of the plasma DNA extracted from the same 150 samples of maternal blood. Sequential analysis was performed using FQ-PCR during the whole pregnancy on 32 pregnant women carrying male fetuses. Results: Using fluorescence in situ hybridization, we can find male fetal cells in maternal blood as early as the 49th day. We can also find free fetal DNA in maternal plasma as early as the 49th and the 42nd day of pregnancy using nested PCR and FQ-PCR. The amount of fetal DNA was increasing with the gestation week. The standard value of every gestation week was obtained by FQ-PCR. Conclusion: FQ-PCR was the best method to detect fetal sex in early pregnancy. There is a principle of quantitative change of free fetal DNA in maternal plasma.

Identification and quantification of fetal red blood cells in maternal blood by a dual‐color flow cytometric method: evaluation of the Fetal Cell Count kit

As an alternative to the cumbersome Kleihauer-Betke test (KBT), flow cytometry represents a powerful method for the identification and quantification of fetal red blood cells (RBCs) in maternal circulation. The aim of this study was to evaluate the Fetal Cell Count kit (IQ Products), an innovative flow cytometric method, based on the combination of antibodies directed, respectively, against fetal hemoglobin (HbF) and carbonic anhydrase (CA), a marker expressed after birth, to discriminate fetal RBCs from adult F cells containing HbF. The investigation was performed by two French laboratories that compared the data obtained by flow cytometry and KBT in 455 pregnant or just-delivered women as well as in 124 artificial mixtures containing from 0.01 to 5.00 percent cord cells. The FL1/FL2 histogram allowed distinction between fetal RBCs (HbF+, CA-), F cells (HbF+, CA+), and adult RBCs (HbF-, CA+). The limits of detection and quantification were determined at 0.03 and 0.10 percent or 0.02 and 0.05 percent when analyzing 100,000 or 200,000 events, respectively. Linearity was demonstrated between 0.01 and 5.00 percent fetal cells in the mixtures (r = 0.95, p < 0.01). A good correlation between fluorescence-activated cell sorting (FACS) and KBT results was obtained with artificial mixtures (r = 0.94, p < 0.01). From the 405 Kleihauer-negative samples, none were identified as positive by FACS. Among the 50 Kleihauer-positive samples, 6 were shown not to contain fetal cells but F cells by FACS. With this new dual-color flow cytometric method, accurate evaluation of fetomaternal hemorrhage was achieved even in the face of HbF of maternal origin.

Two‐way trafficking of Annexin V positive cells between mother and fetus: determination of apoptosis at delivery

The aim of this study was to quantitate apoptosis in maternal circulation and umbilical cord blood (UCB) at delivery. The proportion of fetal cells in maternal blood as well as that of maternal cells in UCB was also determined. Three milliliters of peripheral blood was collected from nine women during labor. Five women delivered males and four delivered females. Immediately after delivery, 3 mL UCB was collected. Ten microliters was used to quantitate apoptosis by the ethidium bromide assay (EthBr) and from the remaining blood, Annexin V positive cells were isolated by MACS. The Median apoptosis rate in maternal samples was 25% (19-34) and in UCB 20% (16-28). Annexin V positive cells were present in all samples analyzed. As shown by Fluorescence in situ hybridization (FISH) in maternal samples, cells with an XY hybridization pattern were identified in cases with male newborns in a median concentration of 1.7% (1.6-2.1). On the corresponding UCB, a median of 1.2% (0.8-1.6) XX cells were detected. The study demonstrates the existence of a bidirectional transfer of fetal and maternal cells under apoptosis across the placenta and provides useful information regarding use of UCB for transplantation.

Spot Counting to Locate Fetal Cells in Maternal Blood and Tissue: A Comparison of Manual and Automated Microscopy

Fetal cell detection in maternal tissue requires an accurate, efficient, and reproducible microscopy method. Our objective was to compare manual scoring to a commercially available automated scanning system for the detection of chromosome signals by fluorescence in situ hybridization (FISH). X and Y chromosome FISH signals were detected on slides of calibrated mixtures of blood, paraffin-embedded liver sections, and post-termination blood. For manual scoring (400x magnification), the number of cells located and duration of scoring were recorded. For automated scanning using the Metasystems Metafer3/Metafer4 Scanning System (200x magnification), duration of scanning, number of gallery images generated, duration of manual review of gallery images, and number of confirmed fetal cells were recorded. From all slides the number of target fetal cells located by manual and automated microscopy was highly correlated (r = 0.90). However, automated scanning required on average 4-fold more time than manual scoring (P < 0.0001), with an average automated scanning time of 9.7 h per slide compared with 2.4 h per slide when scored manually. In general, the accuracy of automated and manual microscopy is comparable, although manual scoring is more efficient because of the level of magnification necessary for automated scanning of cells, and a large number of gallery images generated by automated scanning that must then be reviewed manually. This suggests that when rapid analysis is required (i.e., clinical situations), manual microscopy is preferable. In contrast, automated scanning may have advantages over manual microscopy when time constraints are less imposed (i.e., research situations).

Fetal cord blood mononuclear cells collected at term from HIV-1 infected women harbor transcriptionally active integrated HIV proviral DNA

Strategies to limit perinatal transmission of human immunodeficiency virus (HIV) include antepartum/intrapartum antiretroviral therapy and pre-labor cesarean section with resulting reduction in the vertical transmission rate from 25% to 2%. In the current study we examined fetal cord blood cells for the presence of integrated provirus which is generally not detectable by current methods used to screen neonatal blood. Placental tissue and cord blood were obtained from 21 HIV infected women who received prenatal care and delivered at Grady Memorial Hospital in Atlanta, Georgia. Prenatal care was provided in accordance with current guidelines for care of HIV+ parturients. All specimens were obtained under Emory University IRB approved protocols. RNA and DNA were prepared from isolated placental or cord blood mononuclear cells (CBMCs) by standard methods. Integrated provirus or HIV cDNA standard concentrations were then determined by a two step modified absolute concentration Nested Real-Time qPCR assay. Realtime PCR measurements of multiply spliced (MS) or unspliced (US) HIV mRNA transcripts were conducted to determine active transcription. Quantitative measurements of Y chromosomal DNA by real-time PCR were performed for select maternal/fetal blood pairs using DYS14 primer pairs to confirm the absence of maternal contamination of fetal cord blood. Placental cells isolated from these tissues all contained detectable levels of integrated HIV proviral DNA. Five (24%) of the corresponding samples of fetal CBMCs also had detectable levels of HIV proviral DNA, possibly suggesting a higher rate of intrauterine transmission than has been reported. Furthermore, all HIV+ CBMCs had detectable levels of MS and US proviral mRNA transcripts indicating that integrated HIV proviral DNA in all infected CBMCs was transcriptionally active. Findings suggest that even with use of antiretroviral therapy in the ante-and intrapartum periods some fetuses harbor integrated HIV proviral DNA that may retain the potential to become transcriptionally active.

Comparative developmental toxicity of 2,3,7,8-tetrachlorodibenzo- p-dioxin in the hamster, rat and guinea pig

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) is a persistent environmental contaminant capable of causing a wide variety of adverse health effects including teratogenesis and altered development. The objective of this study was to compare the developmental toxicity of TCDD in the hamster, rat and guinea pig, which in mature animals exhibit a relatively low, medium and high sensitivity to TCDD, respectively. A single oral dose of TCDD was administered to pregnant rats (0, 1.5, 3.0, 6.0 or 18.0 μg/kg) on gestation day 10, pregnant hamsters (0, 1.5, 3.0, 6.0 or 18.0 μg/kg) on gestation day 9 and pregnant guinea pigs (0, 0.15 or 1.5 μg/kg) on gestation day 14 with fetal analysis on gestation day 20, 15 and 56, respectively. The developmental toxicity of TCDD in the three species included increased fetal mortality, alterations to fetal body weight, body length, organ weight and significant changes to the fetal white blood cell differential counts. Additionally, teratogenic responses were observed in the hamster and rat consisting of cleft palate, kidney congestion, hydronephrosis and intestinal hemorrhaging. Furthermore, the results from this study demonstrate that despite the up to 5000-fold interspecies variability to the acute lethal potency of TCDD observed in mature guinea pigs, rats and hamsters, the developing fetus is uniquely vulnerable to gestational TCDD exposure and displays approximately a 10-fold variability in fetal lethal potency in these species. Together, these results will assist efforts to reduce the uncertainty in the risk assessment for TCDD in sensitive populations, such as the developing embryo and fetus.

Direct quantification of fetal cells in maternal blood by real‐time PCR

Fetal cells in maternal blood still present an enticing alternative for the development of a safe and efficacious non-invasive method for prenatal diagnosis. However, most enrichment methods are very tedious and have failed to realise this long sought after goal. We developed a simple, robust TaqMan real-time PCR assay to directly quantify male fetal cells in maternal blood using the multi-copy DYS14, without the need for any additional enrichment procedure. The sensitivity of the DYS14 assay was evaluated by using female genomic DNA spiked with male DNA or male cells. The specificity of the DYS14 assay was evaluated by examining 40 adult blood samples and 44 maternal blood samples from pregnant women with known fetal gender. Direct quantification of fetal cells in maternal blood was performed in 14 of the maternal blood samples by the DYS14 assay. The results were compared to those by SRY assay. The sensitivity of DYS14 PCR assay was found to be higher than that of SRY assay for the detection of fetal cells. The number of fetal cells in maternal blood did not exceed 2/mL blood. The presence of cell-free fetal DNA in maternal blood could lead to erroneous quantification of fetal cell DNA. The number of fetal cells detected in blood cell pellets, which had been unwashed to remove the cell-free fetal DNA, was indeed significantly higher than those in extensively washed samples. We quantified the fetal cells in maternal blood without fetal cell enrichment procedures by TaqMan real-time PCR for a multi-copy DYS14 locus. Our assay is sensitive and also suitable for automation, and may be a useful tool for the determination of fetal-maternal cell trafficking, such as microchimerism.

Identification and partial characterization of soluble and membrane-bound KDN(deaminoneuraminic acid)-glycoproteins in human ovarian teratocarcinoma PA-1, and enhanced expression of free and bound KDN in cells cultured in mannose-rich media

KDN (Deaminoneuraminic acid, or deaminated neuraminic acid) is a minor but biosynthetically independent member of the sialic acid. Human occurrence of KDN has already been established, although its level is so little that it is often undetectable by conventional sialic acid analysis. Elevated expression of KDN in fetal cord blood cells and some malignant tumor cells have been reported. However, in mammalian cells and tissues KDN mostly occurs as the free sugar and little occurred conjugated to glycolipids and/or glycoproteins. A positive correlation between the ratio of free KDN/free Neu5Ac in ovarian adenocarcinomas and the stage of malignancy has been noted for diagnostic use. We hypothesized that elevated expression of KDN in mammalian systems may be closely related to elevated activities of enzymes involved in the formation of sialoglycoconjugates and/or aberrant supply of the precursor sugar, mannose, used in the biosynthesis of KDN. In this study we used human ovarian teratocarcinoma cells PA-1 to further analyze KDN expression in human cells. Major findings reported in this paper are, (i) a 30 kDa KDN-glycoprotein immunostainable with monoclonal antibody, mAb.kdn3G, (specific for the KDNalpha2 --> 3Galbeta1--> epitope) and sensitive to KDNase was identified in the membrane fraction of the cell: (ii) a 49 kDa KDN-glycoprotein that is not reactive with mAb.kdn3G but is sensitive to KDNase was identified in the soluble fraction: and (iii) PA-1 cells showed unique response to mannose added to the growth medium in that the levels of both free and bound forms of KDN are elevated. This is the first report on the identification of mammalian KDN-glycoproteins by chemical and biochemical methods.

Quantification of all fetal nucleated cells in maternal blood in different cases of aneuploidies

We quantified all fetal nucleated cells (FNCs) per unit volume of maternal blood in different aneuploid pregnancies using molecular cytogenetic techniques. Seven cases of male trisomy 18, two triploidies (69,XXX), two 47,XXX, one 47,XXY, one 47,XYY, one male trisomy 13, and one case of 47,XY,r(22),+r(22) were analyzed. Whole blood samples were obtained from 15 women between 17 and 29 gestational weeks and harvested without using fetal cell enrichment procedures. Fluorescence in situ hybridization and primed in situ labeling were performed to identify the FNCs. All slides were manually scanned to quantify those cells. We have identified 4-20 FNCs/ml of maternal blood in the cases of trisomy 18; 10 and 25 FNCs/ml in the two cases of triploidy; 16 and 14 FNCs/ml, respectively, in the two X trisomies; 19 FNCs/ml in the 47,XXY; 26 FNCs/ml in the 47,XYY; nine FNCs/ml in the trisomy 13; and 10 FNCs/ml in the case of r(22). To detect all FNCs in all aneuploid pregnancies, we have used a very simple method that minimizes the manipulation steps to avoid losing fetal cells. The number of FNCs identified in aneuploid pregnancies was 2-5 times higher than in normal pregnancies. This higher number of FNCs will favor the design of a non-invasive pre-natal test.

Sharpening the Tools: A summary of a National Institutes of Health workshop on new technologies for detection of fetal cells in maternal blood for early prenatal diagnosis

In 2003 the National Institute of Child Health and Human Development (NICHD) sponsored a workshop entitled “Sharpening the Tools”, which was designed to explore the then current state of prenatal diagnosis and screening using fetal cells in maternal blood. The goals of the workshop were to: review the then current state of the field and assess present capabilities, identify future research needs and challenges in this area, identify promising new and innovative approaches for future exploration, and provide a written summary of the conference for public distribution. The workshop featured brief presentations by experts from a wide range of scientific fields and by innovative bioengineering and technology leaders from academic centers and private industry. The workshop was divided into presentations on target cells, target approaches for separation, genetic and protein analysis, and ‘out of the box’ (bioengineering) approaches. The passage of time since the workshop has allowed an objective assessment of where the research has progressed. A 2006 update on the field is included at the end of the summary.