Charge flow separation: quantification of nucleated red blood cells in maternal blood during pregnancy

We set out to ascertain the numbers of fetal cells that enter the maternal blood stream during pregnancy. Samples of 15-16 ml of whole blood were collected from 225 women--mostly 10-18 weeks pregnant--and then processed by charge flow separation, a novel method based on free flow electrophoresis in a buffer counterflow gradient. After their recovery in four different separation instruments, nucleated red blood cells (NRBC) were enumerated histologically. In some cases fetal NRBC were identified and enumerated by fluorescence in situ hybridization with probes for the X and Y chromosomes and fetal haemoglobin mRNA. Recoveries were consistent among the four separation instruments: the median numbers of NRBC obtained were 4190, 1590, 2805 and 3860. Our data show that approximately 30 per cent of those cells were fetal. Thus, recent reports on the separation of fetal NRBC by other methods, give underestimates of their frequency in maternal blood.     

Strategies for rare-event detection: an approach for automated fetal cell detection in maternal blood

This article explores the feasibility of the use of automated microscopy and image analysis to detect the presence of rare fetal nucleated red blood cells (NRBCs) circulating in maternal blood. The rationales for enrichment and for automated image analysis for "rare-event" detection are reviewed. We also describe the application of automated image analysis to 42 maternal blood samples, using a protocol consisting of one-step enrichment followed by immunocytochemical staining for fetal hemoglobin (HbF) and FISH for X- and Y-chromosomal sequences. Automated image analysis consisted of multimode microscopy and subsequent visual evaluation of image memories containing the selected objects. The FISH results were compared with the results of conventional karyotyping of the chorionic villi. By use of manual screening, 43% of the slides were found to be positive (>=1 NRBC), with a mean number of 11 NRBCs (range 1-40). By automated microscopy, 52% were positive, with on average 17 NRBCs (range 1-111). There was a good correlation between both manual and automated screening, but the NRBC yield from automated image analysis was found to be superior to that from manual screening (P=.0443), particularly when the NRBC count was >15. Seven (64%) of 11 XY fetuses were correctly diagnosed by FISH analysis of automatically detected cells, and all discrepancies were restricted to the lower cell-count range. We believe that automated microscopy and image analysis reduce the screening workload, are more sensitive than manual evaluation, and can be used to detect rare HbF-containing NRBCs in maternal blood.     

The destructive capabilities of the synovium in the haemophilic joint

OBJECTIVE: To determine the fetal Rhc genotype by using the polymerase chain reaction (PCR) amplification procedure and maternal blood at the different steps of the fetal cell enrichment process. METHODS: Maternal peripheral venous blood samples were obtained from 11 pregnant women homozygous for the C antigen before amniocentesis. Three were not alloimmunized and eight were alloimmunized. The fathers were known to be heterozygous or homozygous for the c antigen by serologic testing. The mononuclear cell layer was isolated from maternal blood and flow sorted using monoclonal antibodies to CD36 or CD71 and glycophorin A. This was followed by PCR of the blood, mononuclear cells, and the sorted cells with allele-specific primers to RhCc genes. Gel electrophoresis was performed to predict fetal Rhc genotype. The fetal RhCc genotype was confirmed by serologic and DNA testing. RESULTS: All infants were positive for the Rhc gene. The positive fetal Rhc genotype was determined correctly in three of the 11 maternal blood samples without enrichment, in six of the nine mononuclear cell samples, and in seven of the eight sorted cell samples. The fetal genotype from one sorted sample was predicted to be homozygous C. One infant was determined by serology on cord blood to be negative for the c antigen, but repeated infant DNA amplification was consistent with the c genotype. CONCLUSION: Noninvasive fetal Rhc genotyping can be determined by PCR amplification of the rare fetal cells in maternal blood. These data reaffirm that enrichment of maternal blood for fetal cells is necessary to improve the sensitivity of the test.     

Prenatal diagnosis of trisomy 13 on fetal cells obtained from maternal blood after minor enrichment

In a pilot study to establish fetal nucleated red blood cell (NRBC) detection in maternal blood, trisomy 13 was diagnosed by FISH analysis at 11 weeks' gestation. The NRBCs were detected after a single-step ficoll density gradient enrichment. In blood samples taken both before and after CVS, 52 and 80 NRBCs, respectively, were found to be positive for fetal haemoglobin. In 47 per cent of these cells, FISH analysis for X and Y chromosomes confirmed the fetal sex. Moreover, 48 per cent of these NRBCs showed three fluorescent signals for a chromosome 13 probe, which confirmed the diagnosis of trisomy 13, previously detected at CVS karyotyping. This is the first report of non-invasive prenatal diagnosis of trisomy 13, i.e., pre-CVS, in the first trimester. The high number of fetal NRBCs detected indicates a connection with aneuploidy, probably due to early impairment of the feto-maternal barrier.     

Oramucosal delivery of LHRH: pharmacokinetic studies of controlled and enhanced transmucosal permeation

A method base on the two-compartment model was developed to measure quantitative cerebral blood flow (CBF) and partition coefficient (lambda) of IMP from dynamic SPECT and single arterial blood sampling. In this method, the linear differential equation of two-compartment model, Yokoi proposed, was employed and quantitative CBF and lambda values were measured with the standard input function calibrated by single arterial sampling. The input function was derived from the standard input function scaled by a factor determined by the single arterial blood sample. This new technique was applied to 5 normal volunteers (Ages ranged from 25 to 29 yr., average 26 yr.). The optimal time to calibrate the standard input function in the individual study and optimal the period of the upper limit time to which input function is integrated from IMP administration for analysis of the equation were determined to minimize the difference between integration of the calibrated standard input function and of the individual input function. Minimization of the difference yields an optimal calibration time (4 to 10 min after IMP administration) and the period of the upper limit time (8 to 60 min after acquisition start). Comparison of CBF and lambda values obtained by the graphical method using the calibrated standard data and individual input function were performed. It should be noted that CBF values were in good agreement between the two methods, respectively (r = 0.92, P<0.01; r = 0.72, p = 0.01). This method is easy to estimate CBF and lambda by only single arterial blood sampling and IMP dynamic SPECT, and useful for routine studies.     

Primary angiosarcoma of the breast

In order to study the effects of propylthiouracil (PTU) on the fetal thyroid and serum concentration, 9 women undergoing therapeutic abortion at the second trimester of pregnancy were given a single dose of 50, 100 and 150 mg PTU respectively. The concentrations of PTU in the maternal and fetal serum and those in the fetal thyroid tissues were determined with a high-performance liquid chromatographic method, the lowest limit of which was 25 micrograms/L. After an oral bolus of PTU, the PTU concentrations in the maternal serum reached a peak level after one hour and there was a wide individual variation of the peak value. The study showed PTU could be transferred through the placenta from the maternal blood to the fetal blood and highly accumulated in the fetal thyroid tissues. The fetal serum PTU concentrations correlated with those of their mothers at the same time (r = 0.7084, P < 0.02) and the concentrations in the fetal thyroid tissues could be evaluated by the maternal serum peak level after an oral bolus. PTU didn't inhibit the fetal thyroid peroxidase activity even when the maternal serum PTU concentration reached 4 mg/L, which is the lowest effective therapeutic concentration of PTU in adults.     

A highly sensitive polymerase chain reaction method reveals the ubiquitous presence of maternal cells in human umbilical cord blood

Umbilical cord blood is considered an alternate source of hematopoietic stem cells in bone marrow transplantation. However, its use might be hampered by contamination of neonatal blood with maternal cells, which could contribute unacceptably to graft-vs.-host disease (GVHD) after transplant. In a previous study (Socié et al., Blood 83:340, 1994), we used polymerase chain reaction (PCR) amplification of minisatellite sequences (sensitivity 1-0.1%) to address the question of this contamination. In a single case among 47 analyzed, we were able to detect a maternal-specific allele in the cord blood sample. We have now studied the same cord samples using a highly sensitive, allele-specific PCR amplification method. A maternal allele could be discriminated from neonate alleles in 10 cases and maternal cells were detected in all 10 cord blood samples. These cells amounted to 10(-4) to 10(-5) of cord blood nucleated cells. In three cases, cord blood separated cell subpopulations could be analyzed and were found to contain maternal cells at about the same level. The presence of maternal cells at such a low level in cord blood samples probably would have no effect on GVHD in a clinical setting of transplantation but raises interesting questions in terms of materno-fetal immune tolerance and transmission of viruses (in particular human immunodeficiency virus) from infected mother to child.     

Lack of association between urinary creatinine and ethanol concentrations and urine/blood ratio of ethanol in two successive voids from drinking drivers

Primer extension preamplification (PEP), which can amplify sequence-independently a limited quantity of DNA as a whole, allows multiple DNA analyses by polymerase chain reaction (PCR). This technique may be applicable to forensic cases, especially in cases where only small amounts of DNA are available. To define the accuracy and sensitivity of PEP, the DNA typing results of nine loci (TH01, MCT118, HLA-DQ alpha, amelogenin, LDLR, GYPA, HBGG, D7S8, GC) by PCR with PEP (PEP-PCR) were compared with those by PCR without PEP. Both results were identical to each other for each sample tested. Furthermore, amplification of an initial genomic DNA by PEP was found to range from 15 to 600 times of the initial quantity and the efficiency of PEP may depend on the sequences flanking the loci tested. These results indicate that PEP can increase typing potential of PCR from forensic samples.     

Potential role of transforming growth factor-beta 1 in terminating the immune response in patients with Guillain-Barré syndrome

Currently, amniocentesis, chorionic villus sampling (CVS) and fetal blood sampling are used to obtain fetal cells for genetic diagnosis. These invasive procedures pose a small but not negligible risk for the fetus. Efforts have been directed towards the enrichment of fetal cells, such as erythroblasts, from maternal blood and progress has been made in the diagnosis of some chromosomal disorders and in sex determinations. We now report the detection of point mutations in single gene disorders using this method of prenatal diagnosis by enriching fetal cells from maternal blood by magnetic cell sorting followed by isolation of pure fetal cells by microdissection. In two pregnancies at risk for sickle cell anaemia and beta-thalassaemia, we successfully identified the fetal genotypes. Thus, prenatal diagnosis of single gene disorders by recovering fetal cells from maternal circulation appears to be a feasible approach.     

Application of enzyme immunoassay for postchemotherapy evaluation of human strongyloidiasis

Primer extension preamplification (PEP) increases the scope and capacity of single cell genetic diagnosis by generating sufficient template to perform multiple subsequent DNA analyses using the polymerase chain reaction. We report the simultaneous analysis of single cells at five commonly deleted dystrophin exons and at the ZFX/ZFY loci. Ninety three percent of PEP reactions with single amniocytes, chorionic villus cells and blastomeres were successful, and a blinded analysis of single lymphoblasts from affected males resulted in 93% diagnostic accuracy, demonstrating its applicability in preimplantation prevention of Duchenne muscular dystrophy. Transfer of unaffected male embryos and improved diagnostic reliability are achieved with the ability to perform replicate multilocus analyses from the same blastomere.     

Serum homocysteine level and protein intake are related to risk of microalbuminuria: the Hoorn Study

OBJECTIVE: Currently, prenatal diagnosis of chromosome abnormalities requires invasive techniques such as amniocentesis and chorionic villus sampling that carry small but finite risks of fetal loss. A noninvasive approach is to isolate fetal cells from maternal blood by flow sorting followed by genetic interphase analysis with fluorescence in situ hybridization. Because the ratio of fetal to maternal cells is relatively low after flow sorting and to detect 90% to 95% of fetal aneuploidies associated with serious birth defects, a 5-color fluorescent in situ hybridization strategy is necessary for simultaneous detection of chromosomes X, Y, 13, 18, and 21 in all flow-sorted nuclei recovered from a specimen. STUDY DESIGN: Fetal nucleated red blood cells were isolated from maternal blood in 40 cases (10.4 to 27.0 weeks' gestation) by flow cytometry on the basis of positive selection of CD71+ (transferrin receptor), CD45-, and LDS751 staining. Each case was evaluated for 5-color fluorescent in situ hybridization efficiency by determining the percentage of flow-sorted nuclei containing 8 hybridization signals for chromosomes X, Y, 13, 18, and 21. RESULTS: A total of 42,312 flow-sorted nuclei from maternal blood samples were analyzed. In 5 of 16 (31%) cases with a male fetus, 0.16% of nuclei scored were identified as fetal by the presence of 1 signal each for chromosomes X and Y. Fetal trisomy 21 nuclei were accurately detected in 2 cases with a female fetus, each of which was subsequently confirmed. CONCLUSIONS: Five-color interphase fluorescent in situ hybridization analysis can be used to effectively analyze rare fetal aneuploid nuclei in enriched flow-sorted cells isolated from maternal blood.     

Rapid trisomy diagnosis (21, 18, and 13) using fluorescent PCR and short tandem repeats: applications for prenatal diagnosis and preimplantation genetic diagnosis

PURPOSE AND METHODS: Prenatal diagnosis of fetal trisomies is usually performed by cytogenetic analysis from amniotic fluid. This requires lengthy laboratory procedures and high costs and is unsuitable for large-scale screening of pregnant women. An alternative method, which is rapid and inexpensive and may potentially be suitable for diagnosing trisomies even from single fetal cells, is the fluorescent polymerase chain reaction (F-PCR) using polymorphic small tandem repeats (STRs). RESULTS: In this paper we present data demonstrating that fluorescent PCR amplification of STRs can be used for rapid diagnosis of trisomy 21, trisomy 18, and trisomy 13 and can be successfully applied to both prenatal diagnosis and diagnosis of single cells. This study also reports significant numbers of prenatal diagnoses using quantitative fluorescent PCR. CONCLUSIONS: We believe that further studies of greater numbers of samples will determine the absolute reliability of this technique. These results also provide a model for trisomy diagnosis from single cells using multiple STR markers for either preimplantation genetic diagnosis or, potentially, diagnosis from fetal cells isolated from maternal blood.     

Nucleated red blood cells in cord blood of singleton term neonates

OBJECTIVE: This study aims to establish normal values for nucleated red blood cells in term singletons and factors associated with their elevation. STUDY DESIGN: Cord blood was prospectively collected from term singleton gestations from Feb. 1 to July 31, 1995. Umbilical vein white blood cells and nucleated red blood cells were counted and umbilical arterial pH was determined. Medical records provided maternal and neonatal information. RESULTS: Cord blood from 1112 cases was obtained and evaluated for nucleated red blood cells per 100 white blood cells. Nine outliers were censored (nucleated red blood cells per 100 white blood cells = 126 to 830); five cases were excluded because of missing data. The mean value of nucleated red blood cells per 100 white blood cells was 8.55, the SD was 10.27, and the range was 0 to 89. The value did not very by maternal tobacco or drug use, anemia, fetal presentation, or mode of delivery. Both maternal diabetes and meconium were associated with elevated values, p < 0.01. Apgar scores and cord pHs showed trends toward inverse proportionality to the number of nucleated red blood cells per 100 white blood cells. CONCLUSION: The mean number of nucleated red blood cells per 100 white blood cells was 8.55, with a wide range and SD. Elevated values may be associated with markers of intrauterine hypoxia such as meconium, lower Apgar scores, and lower pH values.     

A study on thallium-201 SPECT in brain metastases of lung cancer: with special reference to tumor size and tumor to normal brain thallium uptake ratio

The activity of prolyl endopeptidase (PEP), a serine proteinase, has been found to be significantly lower in the blood of patients with major depression than in normal volunteers. The present study investigates plasma PEP activity in 25 major depressed, 10 manic, and 14 schizophrenic subjects versus 30 normal volunteers. It also examines the effects of antidepressants, valproate, and neuroleptic drugs on plasma PEP activity. PEP activity was significantly lower in major depressed subjects than in normal volunteers and in patients with mania and schizophrenia. In depressed subjects, plasma PEP activity was significantly increased during treatment with antidepressant drugs, such as fluoxetine. Plasma PEP activity was significantly increased in manic and schizophrenic subjects compared with normal volunteers. In manic subjects, short-term treatment with valproate had a significant suppressive effect on PEP activity. No significant effects of neuroleptics on PEP activity could be found in the schizophrenic patients. The results support the hypothesis that lower PEP activity could play a role in the pathophysiology of major depression, while increased PEP activity may be related to psychotic conditions, such as mania and schizophrenia.     

Systolic time intervals of the left ventricle in neuromuscular diseases

In 64 patients having neuromuscular disease and in 11 healthy persons, preejection period (PEP), ejection time index (LVETI) and PEP/LVET ratio of the left ventricle were studied by indirect method. PEP was significantly longer, ejection time index was significantly shorter and PEP/LVET ratio was significantly higher in patients with progressive muscular dystrophy than healthy persons. In 75% of patients having this disease LVETI was shortened and only in 30% of them Ecg abnormalities could be detected. These findings are postulated to be due to diffusely decreased contractility of the left ventricular heart muscle in progressive muscular dystrophy.     

Renal cancer: preoperative evaluation with dual-phase three-dimensional MR angiography

Using a highly sensitive allele-specific PCR amplification method, we have previously shown that maternal cells could be detected in all 10 cord bloods tested. This raised the question of whether maternal cells are released into cord blood during the process of delivery or whether they are already present during pregnancy. We have now used the same PCR method to detect the presence of maternal cells in nine fetal blood samples collected at different gestational ages. Maternal cells were detected in eight samples obtained between 24 and 35 weeks of gestation. They were estimated to amount between 10(-4) and 10(-5) of nucleated fetal blood cells. In two cases mononuclear and polymorphonuclear cell fractions were separated by Ficoll gradient centrifugation and maternal cells were detected as comparable levels in both fractions. Maternal cells could not be detected in the one fetal blood sample obtained at 20 weeks of gestation, suggesting that maternal cells could appear at detectable levels in fetal blood during the third trimester of pregnancy. These results are discussed in terms of materno-fetal immune tolerance and of transmission of viruses (and more specifically of the human immunodeficiency virus) from mother to child.     

Biapenem pharmacokinetics in healthy volunteers and in patients with impaired renal function

Biapenem (CAS 120410-24-4) is a new broad spectrum antibiotic agent from the group of carbapenem antibiotics. Results of a pharmacokinetic study in eight volunteers, 17 patients with variant degrees of renal impairment and in addition 13 haemodialysis patients, both on (n = 8) and off dialysis (n = 5), are reported. A single dose of 500 mg biapenem was administered i.v. over 30 min; blood and urine samples were collected up to 24 h post infusion in volunteers and up to 48 h in patients. Concentrations were determined by microbiological assay using the cup plate method. The tolerance was good. The renal function in patients was determined using single shot 51chromium-EDTA clearance. The calculation of pharmacokinetic parameters was performed non-compartmentally as well as based on an open two-compartment model. Although the compound is eliminated extrarenally in considerable amounts (approximately 46% in volunteers), an important prolonged elimination in renal dysfunction was found. This was mainly due to decreased renal elimination but also partly due to decreased extrarenal clearance. A dose reduction factor (DRF) is calculated derived from the ratio of the areas under the serum concentration curve (AUC), in normal and impaired renal function. Dosage suggestions are made. The compound is eliminated considerably by haemodialysis. It is therefore recommended that biapenem is given after haemodialysis or in double the dose on haemodialysis days.     

A prospective evaluation of children under the age of 5 years living in the same household as adults with recently diagnosed pulmonary tuberculosis

Lung injury is a well-documented adverse effect of cardiopulmonary bypass. The mechanism of injury is not fully understood, but pulmonary hypoxia may be a factor. Post-operative pulmonary epithelial permeability (PEP) in ventilated versus non-ventilated lungs was measured within 2 h of return to the intensive care unit using a 99Tcm-diethylenetriamine pentaacetate aerosol technique. A portable scintillation detector system was required. Sodium iodide detectors have been used previously with this technique but are cumbersome. This study used mini caesium iodide detectors (Oakfield Instruments, Oxon, UK), which can be attached directly to the patient and are more suited to the intensive care setting. The clearance half-time from lung to blood (T1/2LB) was measured in 31 patients (62 lungs). The mean (+/- S.E.M.) clearance half-times were 42.3 +/- 2.7 and 45.7 +/- 3.8 min for non-ventilated and ventilated lungs respectively, with a mean difference of 3.4 +/- 3.1 min (P > 0.05). We conclude that, using this technique, no significant difference in PEP is observed between ventilated and non-ventilated lungs in patients undergoing cardiopulmonary bypass.     

Prenatal diagnosis of triploidy and trisomy 21 through fetal erythroblasts isolated from maternal blood

BACKGROUND: A long-sought goal of medical genetics has been the development of prenatal diagnostic procedures that do not endanger the conceptus. The safety of noninvasive methods for prenatal diagnosis would be especially attractive because they could be extended to all pregnant women, regardless of their ages or histories. Noninvasive prenatal diagnosis for the entire population might be possible recovering fetal cells from maternal blood. For this purpose, we have studied fetal erythroblasts. MATERIALS AND METHODS: To evaluate the potential of the method for clinical use, we studied maternal blood samples from 11 women referred to us for prenatal diagnosis between 15 and 20 weeks of gestation. For simple and effective enrichment of fetal nucleated erythrocytes from peripheral maternal blood, we combined a triple density gradient and magnetic-activated cell sorting (MACS) of anti-CD71 transferrin receptor antibody labeled cells. The isolated cells were analysed by using dual-colour interphase fluorescent in situ hybridization (FISH) with X-, Y-, 18- and 21-specific DNA probes. RESULTS: Chromosomal abnormalities detected on enriched fetal cells include trisomy 21 and triploidy. CONCLUSIONS: Based on the current results it is suggested that the technique described here is a simple, fast, efficient and reliable method for non invasive prenatal diagnosis.     

Noninvasive quantification of iodine-123-iomazenil SPECT

The feasibility of a noninvasive method for quantification of [123I]iomazenil binding using a standardized arterial input function and a single venous blood sample was assessed in normal volunteers. METHODS: Serial SPECT images and blood data from six healthy male volunteers after intravenous injection of [123I]iomazenil were used. The standardized input function was derived by averaging the six subjects' arterial curves. Individual input functions were estimated by calibrating the standardized input function with one-point venous blood radioactivity concentration. Ligand transport (K1) and receptor binding were computed from the estimated input functions and two separate SPECT scans using a table look-up procedure based on a three-compartment, two-parameter model. Reference values for K1 and receptor binding were determined from the serial SPECT data and individual arterial curves using a three-compartment, three-parameter model and curve fitting. RESULTS: Analyses of the error caused by the calibration in relation to the time postinjection revealed that the optimal calibration time was 30 min postinjection. Receptor binding obtained by this simplified method correlated well with the reference values (r = 0.941) and was estimated within an error of 10% in the cerebral cortical regions. Although the estimated K1 showed relatively poor correlation (r = 0.699) with the reference value, it was an excellent relative measure in each subject. CONCLUSION: Our method provided an absolute measure of the benzodiazepine receptor binding and a relative measure of ligand transport from two SPECT scans and a venous blood sample. This method would be useful for quantitative assessment of benzodiazepine receptors in clinical settings.