直接胎儿穿刺取血法及并发症的研究

目的　探讨 B超引导下直接胎儿穿刺取血的适应证、胎儿血判定方法、穿刺方法与并发症的关系。　方法　对 90例宫内妊娠 16～ 36周、有医疗穿刺指征的孕妇 ,在 B超引导下进行直接胎儿血穿刺。穿刺前后分别进行胎心监护 ,穿刺后 2 h及 2 4h B超了解胎盘情况。　结果　 90例穿刺病例中无胎儿丢失及胎盘早剥等严重母儿并发症。穿刺过程中胎心变化发生率为 35 .6 % (32 / 90 ) ,均在 6 0 s内恢复 ;穿刺后 4h内出现胎心变化发生率为 2 .2 % (2 / 90 ) ;胎心变化与取血量及穿刺部位正相关 r分别为 0 .2 7及 0 .36。穿刺后 2 h及 2 4h出现胎盘变化 1例 ,发生率为 1.1%。 90例无因穿刺并发症需紧急终止妊娠者 ;胎儿娩出后脐带未见特殊异常。　结论　在高分辨度彩超定位下 ,直接胎儿穿刺取血是安全可靠的。由胎儿血直接提供的有关胎儿染色体、宫内感染、代谢状况、内分泌状况及血液病学等相关因素的资料 ,是进行准确的胎儿宫内诊断不可替代的重要依据     

Fetal blood sampling

Fetal blood sampling is now performed in many centres through different approaches (fetoscopy, placentacentesis, cardiac puncture, umbilical cord needling, intrahepatic vein puncture) for prenatal diagnosis of congenital defects, management of intrauterine growth retardation and fetal therapy. One hundred and thirty-nine fetal blood samples have been performed during a 10 month period at Queen Charlotte's Maternity Hospital, London, using an individualized approach. One failure to obtain fetal blood occurred and there were four fetal losses, three of which followed an intrauterine transfusion in very severely affected fetuses. Two of these losses were associated with peculiar circumstances (see above). The procedure-related risk is nowadays more difficult to evaluate than in the past, when most fetal blood samplings were carried out in the second trimester for prenatal diagnosis. Most case studies, and ours as well, are not homogeneous and high-risk patients such as those with Rhesus disease or intrauterine growth retardation are also included. It seems, however, that transabdominal needling of the cord, at either placental or fetal insertion, is a low-risk procedure although a larger number of cases should be collected to draw definite conclusions about sampling from the intrahepatic vein. Fetoscopy also has a low risk in experienced hands, but the training period is certainly longer and the application in the second half of the pregnancy has been limited to a few cases. It is likely to be used only very little in the future. A flexible approach to fetal blood sampling allows the best choice of technique and utilizes the advantages of each technique.     

Quantitative FISH analysis and in vitro suspension cultures of erythroid cells from maternal peripheral blood for the isolation of fetal cells

Several techniques for the enrichment of nucleated fetal red blood cells present in maternal blood have been reported. Here we describe the use of a quantitative fluorescence in situ hybridization (FISH) method and in vitro suspension cultures of erythroid cells from newborn cord blood and maternal peripheral blood. Together with a rapid high performance liquid chromatography (HPLC) method, that allows us to determine as few as 100 cells containing haemoglobin F (HbF), we have scrutinized the reported enrichment methods for fetal nucleated cells in peripheral maternal blood. One hundred FISH analyses on maternal peripheral blood were performed. The method comprises a cell lysis method for depletion of red cells with minimal losses of nucleated cells, uniform numbers of cells (750 000 cells each) on microscopic slides, and inclusion of internal controls to monitor the efficacy of hybridization. Twenty-six cultures of pure erythroid progenitor cells from maternal peripheral blood were analysed for the expansion of fetal cells. To generate these in vitro cultures, nucleated cells from 10-20 ml of peripheral blood from 26 pregnant women were grown in media containing growth factors and hormones to yield over 10(7) of immature erythroid cells within two weeks. Of those, 13 cultures were from pregnancies with confirmed male fetuses. A total of approximately 8x10(8) maternal cells were added into tissue culture medium for these 13 cultures, resulting in about 2x10(8) nearly pure erythroid cells after two weeks. Whereas fetal cells, alone or added into cultures of peripheral blood, grow rapidly and can be detected quantitatively, we could not find any fetal cells in cultures from maternal blood. Likewise, in 7.5x10(7) peripheral blood cells probed by FISH analysis (half of which were from pregnancies with male fetuses) no single Y chromosome was detected. In summary, suspension cultures of erythroid cells can be established routinely and easily. With the quantitative FISH technique used, 750 000 cells per slide can be screened reliably for cells with Y chromosomes. However, the stringent quality-criteria and most elaborate methods indicate that fetal cells in maternal peripheral blood can not be found using the current technology.     

An audit of outcome in intravascular transfusions using the intrahepatic portion of the fetal umbilical vein compared to cordocentesis

INTRODUCTION: Maternal red cell alloimmunization is a potential cause of perinatal morbidity and mortality. The outcome of severe disease has been transformed by the use of in-utero and particularly, fetal intravascular transfusion. In the majority of instances this is performed by cordocentesis. However, this cohort study represents the experience in a large tertiary referral centre in performing fetal intravascular transfusions via the intrahepatic vein (IHV). METHODS: Over an 8-year period, 1997-2004, 221 in-utero transfusions (IUT) were performed for rhesus disease in 66 pregnancies. 86% had severe fetal anaemia caused by anti-D, 10.6% by anti-Kell and 3.4% by anti-c. The median maternal age of the cohort was 31 years (range 19-43). The median gestation at initial IUT was 25 weeks (interquartile range (IQR) 23-29 weeks). RESULTS: A median number of three IUT were performed in each fetus (IQR 2-5) with a median haemoglobin at first fetal blood sampling of 7.3 g% (IQR 4.6-8.8 g%) (73% < or =5 SD and 27% < or =2 SD). Of the total intravascular transfusions, 170 were performed via the IHV (71.7%), 33 via cordocentesis (13.9%) and 1 by intracardiac puncture (0.5%). There were 'transient' bradycardias complicating 4.1% of all transfusions and amniorrhexis following 1.4%. 92% of babies were live born at a median gestation of 34 weeks (range 21-38) with a birth weight centile of 50 (range 3-90). There was no significant difference in intravascular transfusion complication rate when the procedure was performed via the IHV (7.6%) as compared to cord root puncture (3.0%) (Fisher's exact test, p < 0.47). CONCLUSION: IUT performed by fetal IHV puncture is safe and carries no excess morbidity when performed for severe rhesus disease.     

Fetal blood sampling from the intrahepatic vein: analysis of safety and clinical experience with 214 procedures

Transabdominal fetal blood sampling under ultrasonic guidance was performed at the intrahepatic vein on 214 occasions in 177 fetuses. In 72 cases, an intravascular transfusion was also attempted at the same site. In 91.1% of the samplings, more than 1 mL of pure fetal blood was obtained, and in 89.9% of transfusions, fetal hematocrit or platelet concentration was raised to a satisfactory level. Fetal bradycardia and intraperitoneal bleeding occurred in 2.3% of the cases. Among fetuses at low risk, there was only one intrauterine death, which occurred 3 weeks after the procedure, and one spontaneous abortion in a patient with twin pregnancy. In fetuses with Rh/Kell alloimmunization or perinatal alloimmune thrombocytopenia, the survival rate was 86%. Four liver enzymes were assayed in the blood of 13 fetuses that underwent transfusions at the intrahepatic vein and 13 controls in whom the site of sampling was the umbilical vein at the placental cord insertion. No differences were found between the groups at the subsequent transfusion 2-5 weeks later. The intrahepatic vein is an alternate site of sampling/transfusion when access is difficult or failure occurs at the placental cord insertion. This approach minimizes the risks of fetal blood loss, fetomaternal hemorrhage, arterial vasospasm, and cord tamponade.     

Fetal blood sampling and pregnancy loss in relation to indication

OBJECTIVE--To assess the relation between the indication for fetal blood sampling and pregnancy loss following the procedure. DESIGN--Retrospective study. SETTING--The tertiary referral Fetal Medicine Units at Guy's and University College Hospitals, London. SUBJECTS--Women undergoing diagnostic fetal blood sampling in four groups: (1) 94 having prenatal diagnosis with normal ultrasound findings; (2) 94 with a structural fetal abnormality; (3) 30 having fetal assessment and (4) 35 with non-immune hydrops. INTERVENTIONS--Freehand ultrasound guided fetal blood sampling from umbilical cord, intrahepatic vein or fetal heart. MAIN OUTCOME MEASURES--Pregnancy losses were divided into those within 2 weeks and those 2 weeks after the procedure, obstetric accidents and neonatal deaths. RESULTS--The 253 patients had fetal blood sampled on 268 occasions. Fifty-one pregnancies were terminated. Overall, 51 of the remaining 202 desired continuing pregnancies were lost, of which 19 (9%) were lost within 2 weeks of the procedure. After exclusion of the pregnancies that were terminated, the procedure-related losses within 2 weeks of sampling were 1 in 76 (1%), 5 in 76 (7%), 4 in 29 (14%) and 9 in 36 (25%) in groups 1, 2, 3 and 4 respectively. CONCLUSIONS--The risk of fetal blood sampling is increased in abnormal pregnancies, reflecting the underlying pathology and this must be taken into account when counselling patients before the procedure.     

Management of anti-Rhesus-D antibodies in pregnancy: a review from 1994 to 1998

OBJECTIVE: To review our management of anti-Rhesus-D antibodies in pregnancy over a 5-year period in order to assess possible changes in the management or prognosis which may have developed with time. METHOD: Retrospective analysis of prospectively collected data from 31 pregnancies with maternal anti-D levels >4 IU/ml and in which the fetus was Rhesus positive. RESULTS: There were a total of 30 amniocenteses, 8 cordocenteses, and 54 fetal blood transfusions performed. When undertaken as the first procedure, the mean gestational age at amniocentesis was 30 weeks as compared with 25 weeks for fetal blood sampling/transfusion (p < 0.05). The median anti-D level at the first procedure was 24 IU/ml for amniocentesis and 64 IU/ml for fetal blood sampling. Of the 54 blood transfusions, 43 were intravascular, 4 were intraperitoneal, and 7 transfusions were both intravascular and intraperitoneal. CONCLUSIONS: Intravascular as opposed to intraperitoneal transfusions were found to be the main method of transfusion in the later years in this study, a finding which was expected with improved sonographic equipment. Apart from this, management and prognosis of anti-D red cell isoimmunisation in pregnancy have remained relatively stable since the 1980s. Amniocentesis was useful in the management of such pregnancies, especially as an initial procedure in the cases with a lower initial anti-D level. In this series 90% of the fetuses requiring blood transfusion, but were without hydrops, survived, whereas this was about 70%, if they had become hydropic (this latter figure was reduced by 2 hydropic deaths before 20 weeks' gestation in the same very severely affected woman).     

Elevation of prostaglandin levels in pregnancies complicated by premature rupture of the membranes.

Premature labour and infection (both maternal and neonatal) are common outcomes of pregnancies complicated by premature rupture of the membranes (PROM). Both conditions are associated with elevation of prostaglandin levels. It therefore seems likely that prostaglandins may be increased in patients with pregnancies complicated by PROM. The aim of this study was to test this hypothesis by comparing prostaglandin levels in pregnancies complicated by PROM to those from control pregnancies with intact membranes. Paired fetal and maternal samples were obtained from nine patients with pregnancies complicated by PROM. The median onset of PROM was 28 weeks (range 22-31 weeks). Fetal blood was obtained by cordocentesis, ultrasound guided puncture of the umbilical cord and this was performed at a median of 4 days (range 1-28) following rupture of the membranes. Delivery occurred at a median gestational age of 33 weeks (range 28-37). Paired samples were also obtained from 12 "control" pregnancies with intact membranes and no evidence of infection or premature labour. The indications for cordocentesis in this group were rapid fetal karyotyping following the ultrasonic detection of small fetal structural abnormalities or fetal blood grouping following the detection of high maternal haemolytic antibody titre. Sampling was performed at a median of 27 weeks of gestation (range 22-32). Prostaglandin levels were assessed by determination of the level of the metabolic compound bicyclo-PGEM by radioimmunoassay.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Accuracy of the haemoglobin alkaline denaturation test for detecting maternal blood contamination of fetal blood samples for prenatal karyotyping.

The haemoglobin alkaline denaturation test was routinely performed in 183 fetal blood samples obtained by cordocentesis for prenatal karyotyping by adding 0.1 ml of the blood into a glass test tube containing 5 ml of water and 0.3 ml of 10 per cent KOH as the alkali reagent. The mixture was agitated gently and read at 2 minutes, at which time it was interpreted as a pure fetal blood sample or contaminated with maternal blood according to the change in colour. In order to determine the accuracy of this test to detect maternal blood contamination, the results were compared with the number of fetal and maternal cells detected by standard cytogenetic techniques in those blood samples obtained from male fetuses (n=97). Among these samples, the haemoglobin alkaline denaturation test gave an adult haemoglobin reaction in two cases (2.1 per cent); both samples showed different degrees of maternal 46,XX cells in the metaphases examined (29 of 30 cells in one case and 2 of 31 cells in the other). Conversely, of the 95 samples which gave a fetal haemoglobin reaction, the cytogenetic analysis did not reveal any maternal cells in the metaphases analysed (median 30 cells, range 20-65). We concluded that the haemoglobin alkaline denaturation test is an accurate method for excluding clinically significant maternal blood contamination of fetal blood samples obtained for prenatal karyotyping. This simple, inexpensive technique provides immediate information and, therefore, can be safely incorporated as a bedside test for analysis during fetal blood sampling procedures.     

Fetomaternal transfusion and pregnancy outcome after cordocentesis

OBJECTIVE: To study the extent of fetomaternal transfusion and the outcome of pregnancy after cordocentesis. MATERIAL AND METHODS: 268 women underwent percutaneous fetal umbilical cord blood sampling for fetal karyotyping between 15 and 26 gestations of weeks. Complete follow-up was available in 221 (82.5%) of the cases. Cordocentesis was performed under continuous real-time ultrasound guidance. The duration of the procedure and the post-procedural bleeding time was counted in seconds. Fetomaternal transfusion was calculated by using the measurements of the maternal serum levels of alpha-fetoprotein before and after the procedure. The data were analyzed by Student's t and multiple regression tests. RESULTS: The maximum and mean amounts of fetomaternal transfusion were 1.067 and 0.061 ml, respectively. Twenty percent or more alpha-fetoprotein elevation was in 35.4% of the cases. Positive correlation was found between bleeding time after cordocentesis and fetomaternal transfusion (r = 0.174, p < 0.0129) as well as between the duration of the procedure (r = 0.165, p < 0.0171) and the amount of fetomaternal transfusion. Comparing the cordocentesis at the placental insertion site and at the free cord loop, a smaller amount of fetomaternal transfusion was observed (p < 0.0123) in the latter. Transplacental passage was associated with a higher amount of fetomaternal transfusion (p < 0.0067). No association was found between the extent of fetomaternal transfusion and the outcome of pregnancy. The fetal loss related to the cordocentesis was 0.50%. CONCLUSIONS: The extent of fetomaternal transfusion was influenced by the subsequent four parameters: procedural time, bleeding time, puncture site and transplacental penetration. The lack of the association between the degree of fetomaternal transfusion and the outcome of pregnancy, along with the low (0.50%) post-procedural fetal loss rate, suggest that cordocentesis is clinically a safe procedure.     

经母血采集胎儿细胞行产前诊断的最佳时间探讨

目的：探讨利用母血循环中胎儿细胞进行产前诊断的最佳采血时间。方法：对４１例孕龄为６～１４周的妇女连续取血，采用套式聚合酶链反应技术检测人类Ｙ染色体特异的锌指蛋白基因（ＺＦＹ）。结果：１９例妊娠男性胎儿妇女外周血ＺＦＹ随着孕龄的增加，其胎儿单拷贝基因的检出率增高，其中孕６周时检出率为１／１９（５．３％），孕１１周时为１３／１９（６８．４％），而到孕１４周时，则达到１８／１９（９５．０％）；对２２例妊娠女性胎儿妇女外周血进行ＺＦＹ检测时，无一例假阳性结果，这一检测方法在妊娠早期进行胎儿性别鉴定的总准确率达到９７．８％（４０／４１）。结论：利用母血循环中胎儿细胞进行产前诊断的最佳采血时间应在妊娠１４周，同时提示胎儿细胞最早进入母血循环中的时间在不同个体间存在明显的差异。     

Fetal blood sampling via the umbilical cord using a needle guided by ultrasound. Report of 66 cases

Pure fetal blood has been aspirated in utero from the umbilical vein near the placental insertion of the cord using a twenty gauge needle under ultrasound guidance. Sixty-six samples were taken on 63 pregnancies between 17 and 32 weeks of gestation. One to two millilitres of blood can be obtained easily without amniotic fluid dilution or contamination by maternal blood, as confirmed by the measurements of the mean corpuscular volume, the histogram distribution of the red blood cells and the hematocrit. In all cases the Kleihauer test and isoelectrofocusing of the hemoglobins were performed. Coagulation factors were also studied in 60 cases. In 17 cases a medical abortion was voluntarily induced after the procedure, and the follow-up was normal during the observation period after sampling. In the other cases, pregnancies have continued normally and twelve healthy babies have already been born.     

Initial fetal platelet counts predict the response to intravenous gammaglobulin therapy in fetuses that are affected by PLA1 incompatibility

OBJECTIVE: Fetal alloimmune thrombocytopenia is the result of maternal fetal platelet antigen incompatibility; intracranial hemorrhage is its most serious complication. Our previous studies have demonstrated an inability to accurately predict fetal platelet counts in this disorder. The goal of the present investigation was to identify factors that would predict the response of the fetal platelet count to therapy so that use of fetal blood sampling could be minimized. STUDY DESIGN: Patients who were eligible for the study were all those who (1) had alloimmune thrombocytopenia secondary to Pl(A1) (HPA-1a, Zw(A)) platelet antigen incompatibility, (2) were treated with maternally administered intravenous immunoglobulin at 1 g/kg of body weight per week, with or without low dose steroids, and (3) had percutaneous fetal blood sampling before the initiation of therapy (first fetal blood sampling) and again 3 to 7 weeks afterwards (second fetal blood sampling). RESULTS: In this retrospective review, 74 patients who were affected by alloimmune thrombocytopenia had a median platelet count of 21,000 per microliter at the first fetal blood sampling and 47,000 per microliter at the second fetal blood sampling, with a median increase in platelet count of 24,000 per microliter. Response to treatment was defined as either (1) an improvement in platelet count (the second fetal blood sampling greater than the first fetal blood sampling, and second fetal blood sampling > 20,000 per microliter) or (2) a minimal decline in platelet count (the first fetal blood sampling > or = 40,000 per microliter and the difference between the first and second fetal blood sampling < or = 10,000 per microliter). The first fetal blood sampling had prognostic value for the second fetal blood sampling (P = .0001), although the previous sibling birth platelet count and history of sibling intracranial hemorrhage did not predict the platelet count at the first or second fetal blood sampling or the change in platelet count between the samplings. When the patients were segregated to first fetal blood sampling of > 20,000 per microliter versus < or = 20,000 per microliter, the response rates for the 2 groups were 89% (33/37 patients) versus 51% (19/37 patients; P = .001). CONCLUSION: In fetal alloimmune thrombocytopenia secondary to Pl(A1) platelet antigen incompatibility, fetuses with platelet counts > 20,000 per microliter at the initiation of therapy were predicted to maintain their platelet count at the second fetal blood sampling at > 20,000 per microliter. The characteristics of the previous sibling, as previously reported, did not predict the initial fetal blood sampling, the second fetal blood sampling, or the response to treatment.     

Rise in maternal serum alpha-fetoprotein concentration after chorionic villus sampling and the possibility of isoimmunization

Maternal serum alpha-fetoprotein concentrations were determined by radioimmunoassay in 72 patients immediately before and after chorionic villus sampling for prenatal diagnosis. Fifty percent showed a rise of greater than or equal to 5 ng/ml. Assuming such rises represent fetal blood crossing the intervillous space, in 14% of the cases greater than or equal to 60 microliters of fetal blood was transferred at the time of chorionic villus sampling. A positive correlation was found between the magnitude of rise in maternal serum alpha-fetoprotein levels and the amount of villi removed (r = 0.39, p less than 0.001). When cases were examined by number of passes, a greater rise in maternal serum alpha-fetoprotein levels was noted with multiple passes than with single passes for a given sample size. The transfer of greater than or equal to 60 microliters of fetal blood suggests that maternal sensitization to fetal antigens may occur after chorionic villus sampling. During biopsy, as small a sample of villi as necessary for diagnosis should be taken with as few catheter passes as possible.     

Prenatal diagnosis of X-linked mental retardation with fragile (X) using fetoscopy and fetal blood sampling

Pure fetal blood, (uncontaminated with maternal blood), was obtained from two male fetuses at risk for X-linked mental retardation with fragile(X) at Xq27-28 by direct vision fetoscopy and fetal blood sampling. Both were shown to have this fragile site on the X chromosome while nine other fetal blood samples from pregnancies at risk for other X-linked diseases, or haemoglobinopathies did not show fragile sites at Xq27-28, and a blood sample from an abortus showed only 1 fragile site in 95 mitoses. Both pregnancies were terminated, cultures established from fetal tissues, and the diagnosis confirmed in each case. The problems of demonstrating the fragile site in tissues other than fetal blood in these pregnancies (such as amniotic fluid cells or fibroblasts from fetal tissues) are discussed.     

Recent advances in non-invasive prenatal DNA diagnosis through analysis of maternal blood

Prenatal diagnosis of aneuploidy and single-gene disorders is usually performed by collecting fetal samples through amniocentesis or chorionic villus sampling. However, these invasive procedures are associated with some degree of risk to the fetus and/or mother. Therefore, in recent years, considerable effort has been made to develop non-invasive prenatal diagnostic procedures. One potential non-invasive approach involves analysis of cell-free fetal DNA in maternal plasma or serum. Another approach utilizes fetal cells within the maternal circulation as a source of fetal DNA. At the present time, fetal gender and fetal RhD blood type within RhD-negative pregnant women can be reliably determined through analysis of maternal plasma. Furthermore, genetic alterations can be diagnosed in the maternal plasma when the mother does not have the alterations. However, the diagnosis of maternally inherited genetic disease and aneuploidy is limited using this approach. Non-invasive prenatal diagnosis through examination of intact fetal cells circulating within maternal blood can be used to diagnose a full range of genetic disorders. Since only a limited number of fetal cells circulate within maternal blood, procedures to enrich the cells and enable single cell analysis with high sensitivity are required. Recently, separation methods, including a lectin-based method and autoimage analyzing, have been developed, which have improved the sensitivity of genetic analysis. This progress has supported the possibility of non-invasive prenatal diagnosis of genetic disorders. In the present article, we discuss recent advances in the field of non-invasive prenatal diagnosis.     

Arginine vasopressin in amniotic fluid, arterial and venous cord plasma and maternal venous plasma

High concentrations of arginine vasopressin (AVP) in arterial umbilical cord blood at the time of delivery have been attributed to either a generalized increase in the activity of the fetal endocrine system at the onset of labor or to fetal asphyxia. We measured AVP in amniotic fluid, arterial and venous cord blood and in maternal venous blood from 13 patients at 38-40 weeks of gestation at the time of elective cesarean section with a nonasphyxic fetus (group I), in amniotic fluid from 19 patients at 15-17 weeks of gestation (group II) and in venous blood from 13 nonpregnant control subjects (group III). Our results showed a high concentration of AVP in the amniotic fluid both in the middle and at the end of normal pregnancy and at the same level as in arterial cord blood, whereas AVP in the venous cord blood was significantly lower and at the same level as in the maternal venous blood and in the control group. It is concluded that the fetus produces AVP and this is at least not solely caused by fetal asphyxia or related to parturition.     

Detection of fetal cells with common chromosomal aneuploidies in maternal blood.

This study was designed to address the feasibility of detection of fetal cells with chromosome abnormalities in maternal blood. Peripheral venous blood samples were collected from 150 pregnant women 1 day to 8 weeks before invasive examinations (amniocentesis, chorionic villus sampling, or fetal blood sampling). Fetal nucleated red blood cells were isolated by using a triple-density gradient followed by magnetic-activated cell sorting to select CD71 cells. Fluorescence in situ hybridization (FISH) with probes specific for chromosomes X, Y, 13, 18, and 21 was used to detect fetal cells from aneuploid pregnancies. The hybridization efficiency was greater than 98% for each probe in normal controls, and approximately 20% of all cells failed to hybridize after magnetic-activated cell sorting. Of the 10 aneuploid pregnancies identified with invasive procedures, trisomic fetal cells were identified in eight. The frequency of fetal cells in the sorted specimens ranged from 0 to 223 per 10,000 maternal nucleated cells. Although the aneuploid fetal cells could be successfully detected in eight of 10 patients in the current study, the existence of some limiting factors, such as scarcity of fetal cells and poor hybridization efficiency of FISH, raises questions about its clinical suitability for routine use.     

How useful is the in vitro expansion of fetal CD34+ progenitor cells from maternal blood samples for diagnostic purposes?

Fetal cells present in the maternal circulation are a potential source of fetal DNA that can be used for the development of a prenatal diagnostic test. Since their numbers are very low, amplification of fetal cells has been discussed for a long time. So far, most studies have focused on culturing fetal erythroid cells. In this study, we evaluated whether limiting numbers of fetal haemopoietic progenitor cells present in an excess of maternal cells were able to overgrow the maternal component. Therefore, we used a model system in which limiting numbers of male CD34+ umbilical cord blood cells were diluted in 400 000 female CD34+ peripheral blood cells. The number of XY positive cells derived from umbilical cord blood was determined using two-colour in situ hybridization with X and Y chromosomal probes. We demonstrated a 1500-fold relative expansion of male umbilical cord blood cells over the peripheral blood component after three weeks of liquid culture, which also corresponded to the extent of expansion of CD34+ cells derived from 20-week fetal blood. However, application of the same culture protocol to maternal blood samples obtained at 7-16 weeks of gestation showed no preferential growth of fetal haemopoietic progenitor cells. This study, therefore, suggests that fetal primitive haemopoietic progenitor cells do either not circulate in maternal blood before 16 weeks of gestation, or require different combinations/concentrations of cytokines for their in vitro expansion.