[Depressive symptoms are not associated with risks of rapid renal function decline or chronic kidney disease in middle-aged and elderly with normal kidney function: a longitudinal investigation].

OBJECTIVE: To explore the association between depressive symptoms and the risks of rapid decline in renal function and chronic kidney disease (CKD) in middle-aged and elderly with normal kidney function. METHODS: The residents aged 40- 75 years with eGFR≥60 mL·min-1·1.73 m-2 without proteinuria in Lanzhou region, who participated in the "REACTION" study carried out in 2011, were selected and followed up in 2014. A total of 4961 individuals with complete and qualified data from the two surveys were included in the subsequent analysis. Based on PHQ-9 questionnaire scores, the baseline population was divided into two groups with and without depressive symptoms. Cox proportional hazard analysis was used to compare the incidences of rapid renal function decline and CKD between the two groups and study the association of depressive symptoms with the risk of these renal conditions. RESULTS: PHQ-9 questionnaire scores were not found to correlate with baseline SCr, ALB, UACR or eGFR levels among the participarts (P>0.05). After a mean follow-up time of 3.4±0.6 years, 33.9% of the participants with depressive symptoms at baseline experienced a rapid decline in renal function and 3.6% progressed to CKD. During the follow-up, the incidence of rapid decline in renal function and the risk of developing CKD were not found to correlate with depressive symptoms in these participants (P>0.05) regardless of the type of the depressive syndromes. CONCLUSION: Depressive symptoms are not associated with the risks of rapid renal function decline or progression to CKD in middle-aged and elderly with normal kidney function.

[Triglyceride-glucose product is an independent risk factor for predicting chronic kidney disease in middle-aged and elderly population: a prospective cohort study].

OBJECTIVE: To explore the correlation of triglyceride-glucose product (TyG) index with chronic kidney disease (CKD) in elderly population in Lanzhou (Gansu Province, China). METHODS: From May to September, 2011, a total of 3868 middleaged and elderly individuals without CKD from 3 communities in Lanzhou were selected as the cohort study population and were followed for an average of 3.1 years (from June, 2014 to August, 2015). After excluding those with missing follow-up data, a total of 3439 individuals were included for analysis, who were divided according to the quartile of TyG index into Q1 group (TyG≤8.47), Q2 group (TyG 8.48-8.84), Q3 group (TyG 8.85-9.20) and Q4 group (TyG>9.20). The estimated glomerular filtration rate (eGFR) and urinary albumin/creatinine ratio (UACR) were used to evaluate the renal function of the participants. RESULTS: In this cohort, a high TyG index was found to correlate with a high risk of CKD (P < 0.05). Analysis of the follow-up data showed that the TyG index was significantly higher in patients who developed CKD during the follow-up than in those without CKD (P < 0.05). Logistic regression analysis showed that TyG index was an independent risk factor for abnormal eGFR and CKD (P < 0.05). CONCLUSION: A high TyG index is an independent risk factor for CKD in middle-aged and elderly population.

[Correlation of baseline serum 25-hydroxyvitamin D level with the risk of type 2 diabetes mellitus: a prospective cohort study].

OBJECTIVE: To investigate the correlation of baseline serum 25(OH) D level with the risk of type 2 diabetes mellitus (T2DM) and blood glucose control in diabetic patients among the middle-aged and elderly individuals in Chengguan District of Lanzhou, Gansu Province. OBJECTIVE: Residents aged 40 to 75 years in Lanzhou were selected from the "REACTION" study conducted in 2011 and had been followed up since 2014. A total of 5044 subjects with complete data from the two surveys were analyzed. Participants were divided into Q1, Q2, Q3, and Q4 subgroups based on quartiles of serum 25(OH)D level for comparison of the incidence of T2DM and blood glucose control. OBJECTIVE: Baseline 25(OH)D level was not found to correlate with FPG, 2h-PG or HbA1c levels among the residents (P>0.05). The participants were followed up for a mean of 3.4±0.6 years, and compared with those in Q1 group, the participants in Q2, Q3 and Q4 groups did not show significantly lowered risk of prediabetes or diabetes regardless of glucose tolerance status. Among the patients with T2DM, the compliance rate of glycemic control after the follow-up was significantly higher than that before the follow-up (63.4% vs 60.6%), and the levels of HbA1c, FPG, and 2h-PG decreased obviously after the follow-up. But compared with Q1 group, Q2, Q3 and Q4 groups showed no significant changes in glycemic control compliance rate or levels of HbA1c, FPG and 2h-PG after the follow-up (P>0.05). OBJECTIVE: There is no evidence that baseline 25(OH)D levels are associated with the risk of diabetes and blood glucose control in patients with T2DM.

Frequencies and geographic distributions of genetic mutations in transthyretin- and non-transthyretin-related familial amyloidosis.

Inherited forms of amyloidosis are rare; of these, transthyretin-related (ATTR) is the most common, but non-ATTR has been described as well. We studied a large case series of ATTR and a small series of non-ATTR to better determine the mutation frequencies and geographic distributions of these inherited forms of amyloidosis in the United States. We performed a retrospective cross-sectional study of 284 ATTR and non-ATTR patients seen at Mayo Clinic in Rochester, Minnesota, from 1 January 1970 through 29 January 2013. Mutations were identified by DNA sequencing, restriction fragment length polymorphism, or mass spectroscopy. The genetic testing method was unknown for several patients, but a small proportion were identified by family history or by classical clinical presentation associated with a specific mutation. The most common ATTR mutations were Thr60Ala (24%), Val30Met (15%), Val122Ile (10%), and Ser77Tyr (5%). Non-ATTR mutations included gelsolin (n = 3), apolipoprotein A-I (n = 6), apolipoprotein A-II (n = 1), fibrinogen A-α (n = 9), and lysozyme (n = 1). Although rare, ATTR and, to a lesser extent, non-ATTR are prevalent in the United States and should be considered for patients presenting in the appropriate clinical context.

Leptin receptor signaling inhibits ovarian follicle development and egg laying in chicken hens.

BACKGROUND: Nutrition intake during growth strongly influences ovarian follicle development and egg laying in chicken hens, yet the underlying endocrine regulatory mechanism is still poorly understood. The relevant research progress is hindered by difficulties in detection of leptin gene and its expression in the chicken. However, a functional leptin receptor (LEPR) is present in the chicken which has been implicated to play a regulatory role in ovarian follicle development and egg laying. The present study targeted LEPR by immunizing against its extracellular domain (ECD), and examined the resultant ovarian follicle development and egg-laying rate in chicken hens. METHODS: Hens that have been immunized four times with chicken LEPR ECD were assessed for their egg laying rate and feed intake, numbers of ovarian follicles, gene expression profiles, serum lipid parameters, as well as STAT3 signaling pathway. RESULTS: Administrations of cLEPR ECD antigen resulted in marked reductions in laying rate that over time eventually recovered to the levels exhibited by the Control hens. Together with the decrease in egg laying rate, cLEPR-immunized hens also exhibited significant reductions in feed intake, plasma concentrations of glucose, triglyceride, high-density lipoprotein, and low-density lipoprotein. Parallelled by reductions in feed intake, mRNA gene expression levels of AgRP, orexin, and NPY were down regulated, but of POMC, MC4R and lepR up-regulated in Immunized hen hypothalamus. cLEPR-immunization also promoted expressions of apoptotic genes such as caspase3 in theca and fas in granulosa layer, but severely depressed IGF-I expression in both theca and granulosa layers. CONCLUSIONS: Immunization against cLEPR ECD in egg-laying hens generated antibodies that mimic leptin bioactivity by enhancing leptin receptor transduction. This up-regulated apoptotic gene expression in ovarian follicles, negatively regulated the expression of genes that promote follicular development and hormone secretion, leading to follicle atresia and interruption of egg laying. The inhibition of progesterone secretion due to failure of follicle development also lowered feed intake. These results also demonstrate that immunization against cLEPR ECD may be utilized as a tool for studying bio-functions of cLEPR.

MeCP2 R168X male and female mutant mice exhibit Rett-like behavioral deficits.

Rett syndrome (RTT) is a regressive developmental disorder characterized by motor and breathing abnormalities, anxiety, cognitive dysfunction and seizures. Approximately 95% of RTT cases are caused by more than 200 different mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). While numerous transgenic mice have been created modeling common mutations in MeCP2, the behavioral phenotype of many of these male and, especially, female mutant mice has not been well characterized. Thorough phenotyping of additional RTT mouse models will provide valuable insight into the effects of Mecp2 mutations on behavior and aid in the selection of appropriate models, ages, sexes and outcome measures for preclinical trials. In this study, we characterize the phenotype of male and female mice containing the early truncating MeCP2 R168X nonsense point mutation, one of the most common in RTT individuals, and compare the phenotypes to Mecp2 null mutants. Mecp2(R168X) mutants mirror many clinical features of RTT. Mecp2(R168X/y) males exhibit impaired motor and cognitive function and reduced anxiety. The behavioral phenotype is less severe and with later onset in Mecp2(R168X/+) females. Seizures were noted in 3.7% of Mecp2(R168X) mutant females. The phenotype in Mecp2(R168X/y) mutant males is remarkably similar to our previous characterizations of Mecp2 null males, whereas Mecp2(R168X/+) females exhibit a number of phenotypic differences from females heterozygous for a null Mecp2 mutation. This study describes a number of highly robust behavioral paradigms that can be used in preclinical drug trials and underscores the importance of including Mecp2 mutant females in preclinical studies.

Biomechanical characterization of a low density silicone elastomer filled with hollow microspheres for maxillofacial prostheses.

An ideal material for maxillofacial prostheses has not been found. We created a novel material: silicone elastomer filled with hollow microspheres and characterized its biomechanical properties. Expancel hollow microspheres were mixed with MDX4-4210 silicone elastomer using Q7-9180 silicone fluid as diluent. The volume fractions of microspheres were 0, 5, 15, and 30% v/v (volume ratio to the total volume of MDX4-4210 and microspheres). The microspheres dispersed well in the matrix. The physical properties and biocompatibility of the composites were examined. Shock absorption was the greatest by the 5% v/v composite, and decreased with increasing concentrations of microspheres. The density, thermal conductivity, Shore A hardness, tear and tensile strength decreased with increasing concentrations of microspheres, while elongation at break increased. Importantly, the tear strength of all composites was markedly lower than that of pure silicone elastomer. Cell viability assays indicated that the composite was of good biocompatibility. The composite with a volume fraction of 5% exhibited the optimal properties for use as a maxillofacial prosthesis, though its tear strength was markedly lower than that of silicone elastomer. In conclusion, we developed a novel light and soft material with good flexibility and biocompatibility, which holds a promising prospect for clinical application as maxillofacial prosthesis.

Co-delivery of G-CSF and EPO released from fibrin gel for therapeutic neovascularization in rat hindlimb ischemia model.

OBJECTIVE: G-CSF and EPO have shown a notable capability in neovascularization. However, their use is limited because of untoward leucocytosis, erythrogenesis, and short half-life in the plasma. Herein, we examined whether G-CSF and EPO released from fibrin gel injected into ischemic tissues would synergistically promote neovascularization with limited systematic effects in a rat hindlimb ischemic model. METHODS AND RESULTS: In vivo study, group Gel received an intramuscular injection of fibrin gel; group Gel+G-CSF received fibrin gel containing human G-CSF; group Gel+EPO received fibrin gel containing human EPO; group Gel+G-CSF&EPO received fibrin gel containing G-CSF and EPO; group G-CSF&EPO received G-CSF and EPO. Through promoting the expression of SDF-1, local high concentration of EPO could traffic CXCR4+ cells mobilized by G-CSF to enhance neovascularization in ischemic muscle. The treatment with Gel+G-CSF&EPO was superior to the other treatments on blood flow reperfusion, capillary density, and α smooth muscle actin-positive vessel density. And this treatment induced a modest WBC count increase in peripheral blood. CONCLUSIONS: G-CSF and EPO released from fibrin gel had a combined effect on postischemia neovascularization. This treatment may be a novel therapeutic modality for ischemic peripheral artery disease.

The effect of surface properties on visible luminescence of nanosized colloidal ZnO membranes.

Luminescence properties of nanosized zinc oxide (ZnO) colloids depend greatly on their surface properties, which are in turn largely determined by the method of preparation. ZnO nanoparticles in the size range from 3 to 9 nm were prepared by addition of tetramethylammonium hydroxide ((CH3)4NOH) to an ethanolic zinc acetate solution. X-ray diffraction (XRD) indicates nanocrystalline ZnO membranes with polycrystalline hexagonal wurtzite structure. The ZnO membranes have a strong visible-emission intensity and the intensity depends upon hydrolysis time. The infrared spectra imply a variety of forms of zinc acetate complexes present on the surface of ZnO particles. The effect of the ZnO membrane surface properties on photoluminescence is discussed.

Fetal cell microchimerism in tissue from multiple sites in women with systemic sclerosis.

OBJECTIVE: The realization that fetal cells pass into the maternal circulation and can survive for many years has raised the question of whether fetal microchimerism can cause subsequent disease in the mother. Available data suggest that fetal-maternal transfusion may be related to some autoimmune diseases, notably systemic sclerosis (SSc). The goal of the current work was to identify and quantify tissue-specific fetal microchimerism in women with SSc. METHODS: We analyzed multiple tissue specimens obtained at autopsy from women with SSc as well as women who had died of causes unrelated to autoimmunity, using fluorescence in situ hybridization to detect the presence of male cells in women with sons. Tissues analyzed included adrenal gland, heart, intestine, kidney, liver, lung, lymph node, pancreas, parathyroid, skin, and spleen. RESULTS: Male cells were observed in tissue from at least 1 site in each woman with SSc and were found most frequently in spleen sections. After spleen, male cells were observed most frequently in lymph node, lung, adrenal gland, and skin tissue. The only tissue type in which male cells were not seen in any patient was pancreatic tissue. Male cells were not observed in tissue from women who had died of causes unrelated to autoimmunity. CONCLUSION: The results of this study suggest that fetal cells migrate from the peripheral circulation into multiple organs in women with SSc. All of the women studied had previously given birth to sons, so it is likely that these cells are of fetal origin. While the relevance of this finding to the pathogenesis of SSc remains to be elucidated, the presence of fetal cells in internal organs suggests that they could play a role in disease pathogenesis and that they may preferentially sequester in the spleen. The presence of these male cells may also be a result of disease, possibly through the migration of terminally differentiated and/or progenitor cells to areas of tissue damage.

Apoptosis in fetal nucleated erythrocytes circulating in maternal blood.

The purpose of this study was to determine if apoptosis occurs in fetal cells that have crossed into the maternal circulation, which would potentially explain the difference between the number of intact fetal cells and the amount of fetal DNA detectable in maternal plasma. We flow-sorted fetal nucleated erythrocytes (FNRBCs) using antibody to the gamma chain of fetal haemoglobin and confirmed them to be fetal in origin by FISH analysis using chromosome-specific probes. Fetal cells were then analysed microscopically for the presence of terminal UdTP nuclear end labelling (TUNEL) staining. Apoptotic change was observed in 42.7% of fetal NRBCs (106/246) and 3.5% of maternal cells (29/818). Results of this study indicate that a significant number of fetal cells in maternal blood are undergoing apoptosis at the time of sampling. Apoptosis may be one mechanism by which fetal cells are cleared by the maternal circulation.

The use of fluorescence in situ hybridization (FISH) on paraffin-embedded tissue sections for the study of microchimerism.

We describe here a simple and versatile method of fluorescence in situ hybridization (FISH) on paraffin-embedded tissue sections with specific application in the study of microchimerism, that is, the presence of intact foreign cells within an individual. This is accomplished through the use of X and Y chromosome-specific probes to identify the presence of male nuclei within a tissue section from a female, and vice versa. This technique requires only minor modification if at first the hybridization does not yield fluorescent signals of high quality. Analysis of a wide variety of tissue types is possible with this method, and multiple tissue types from one or more individuals can be processed in the same hybridization reaction. This robust FISH method has been used successfully in our laboratory to investigate fetal cell microchimerism in the following paraffin-embedded tissue types: skin, lung, thyroid, adrenal gland, lymph node, heart, spleen, liver, pancreas, kidney, and intestine.

Fluorescence in-situ hybridization analysis of chromosomal constitution in spermatozoa from a mosaic 47,XYY/46,XY male.

Sex-chromosome mosaicism in spermatozoa from a mosaic 47,XYY[20%]/46, XY[80%] male with fertility problems was assessed using triple-probe fluorescence in-situ hybridization (FISH) studies. Chromosome-specific probes for X, Y and 18 were used, and the possible outcomes were deduced. In normal haploid spermatozoa of the patient and a normal 46,XY male control, the X:Y ratio was close to 1:1. There was a significant difference in the total incidence of karyotypically abnormal spermatozoa between the patient and the 46, XY male control (2.31% versus 1.46%, P < 0.0001). The incidence of some types of disomic spermatozoa X+Y+18 (24,XY) and X+18+18 (24,X, +18), or diploid X+Y+18+18 (46,XY) spermatozoa was significantly increased in the patient's semen sample. There was, however, no significant difference in the incidence of disomic Y+Y+18 (24,YY) spermatozoa. Because the majority of the patient's spermatozoa was karyotypically normal, the aetiology of his fertility problems was unclear. These results add to the growing body of information regarding chromosome abnormalities in spermatozoa from men who are mosaic for sex chromosome abnormalities. In these men, FISH analysis of spermatozoa may be warranted to determine the relative percentages of abnormal cells, and to determine if in-vitro fertilization with preimplantation genetic diagnosis may increase the likelihood of a successful pregnancy.

Comparison of fetal cell recovery from maternal blood using a high density gradient for the initial separation step: 1.090 versus 1.119 g/ml.

The purpose of this study was to improve recovery of fetal nucleated erythrocytes (NRBCs) from maternal blood for non-invasive prenatal diagnosis. Peripheral blood samples were obtained from 27 women who had just undergone pregnancy termination at 6 to 23 weeks. Samples were split and mononuclear cells were isolated using Histopaque gradient at densities of 1.090 g/ml and 1.119 g/ml. CD45 depletion using magnetic activated cell-sorting, followed by flow-sorting with antibody to gamma-globin and fluorescence in situ hybridization (FISH) analysis, were used to evaluate the number of fetal NRBCs recovered. In samples separated with the 1.119 g/ml density gradient, the yield of true anti-gamma haemoglobin positive cells (median, 14. 9; range, 0-717.5) was significantly higher than that with the 1.090 g/ml density gradient (median, 4.9; range, 0-532.5). After FISH analysis, in the 14 samples in which the fetal karyotype differed from the mother, the median number of fetal NRBCs separated by the 1. 119 g/ml density gradient was 22.9 (2-717.5), which was significantly higher than that by the 1.090 g/ml gradient (median, 11.5; range, 0-532.5, p=0.022). Increased density of the gradient used for the initial enrichment of fetal cells results in improved fetal cell recovery in fresh post-termination blood samples, which may permit better non-invasive detection of fetal cells in maternal blood.

Fetal nucleated erythrocyte recovery: fluorescence activated cell sorting-based positive selection using anti-gamma globin versus magnetic activated cell sorting using anti-CD45 depletion and anti-gamma globin positive selection.

BACKGROUND: Fluorescence activated cell sorting (FACS)-based anti-gamma (gamma) positive selection and magnetic activated cell sorting (MACS)-based anti-CD45 depletion followed by anti-gamma positive staining have been two of the most frequently used methods to isolate fetal cells from maternal blood. To date, there has been no direct comparison of fetal cell recovery by these two methods. This study was designed to address this issue. METHODS: Fluorescence in situ hybridization (FISH) was performed on nucleated anti-gamma positive cells using X and Y probes. Twenty-four maternal blood samples were obtained immediately after elective termination of pregnancy to ensure a detectable number of fetal cells. RESULTS: The yield and purity of fetal nucleated erythrocytes (FNRBCs) was statistically higher in FACS sorted samples (P < 0.01). The specificity of staining for FNRBCs was statistically higher in MACS sorted samples (P < 0.01). CONCLUSIONS: The data from this study demonstrate that both techniques have benefits and limitations. FACS has the advantage of having higher yield, higher purity, higher FISH efficiency and ease in microscope analysis, and MACS has the advantage of having higher specificity and less cell loss during FISH.

Non-invasive exclusion of fetal aneuploidy in an at-risk couple with a balanced translocation.

A pregnant woman who was a carrier for a balanced chromosome translocation [46,XX, t(1;6) (p31;q14)] and who had had six miscarriages, declined invasive testing but agreed to non-invasive prenatal diagnosis by analysis of fetal cells in maternal blood. Monoclonal antibody (Mab) against the zeta (z) and gamma (gamma) chains of embryonic and fetal haemoglobin were used to identify fetal nucleated erythrocytes (FNRBC). There were no FNRBC detected at 7 weeks, one anti-z-positive FNRBC was detected at 11 weeks, and 12 anti-gamma-positive FNRBC were detected at 20 weeks. Fluorescent in-situ hybridization was performed using probes for chromosomes X, Y, 1 and 6 to identify fetal gender and the presence of an unbalanced chromosomal translocation. A tentative prenatal diagnosis was made of a female fetus disomic for chromosomes 1 and 6. A female infant with a 46,XX karyotype was born at term. This is the first attempt of exclusion of a chromosome translocation using fetal cells isolated from maternal blood. There is an advantage of using fetal cells isolated from maternal blood for non-invasive prenatal diagnosis in couples who have a history of multiple miscarriages due to a parental translocation, and who decline invasive testing in a pregnancy that continues to the second trimester.

Fetal cell recycling: diagnosis of gender and RhD genotype in the same fetal cell retrieved from maternal blood.

OBJECTIVE: Our aim was to develop a new technique, which we have termed fetal cell recycling, that combines the 2 powerful methods of fluorescence in situ hybridization and polymerase chain reaction to maximize the genetic information available from a small number of fetal nucleated erythrocytes obtained noninvasively from the blood of pregnant women. STUDY DESIGN: Blood samples were obtained from 4 Rh-negative women after elective termination of pregnancy at 7 to 17 weeks' gestation. Fetal nucleated erythrocytes were separated by flow sorting with antibody to the gamma chain of fetal hemoglobin. Fluorescence in situ hybridization with chromosome-specific probes was used to diagnose fetal gender. After fluorescence in situ hybridization analysis the fetal nucleated erythrocytes were recycled by a micromanipulation technique and deoxyribonucleic acid diagnosis was performed with polymerase chain reaction amplification of the RhD gene. RESULTS: Among the 4 case patients we detected a total of 101 fetal nucleated erythrocytes. All targeted cells were successfully retrieved with a micromanipulator. In each case we successfully performed both fluorescence in situ hybridization and polymerase chain reaction analysis. The predicted fetal gender and Rh status corresponded to the results obtained from fetal tissue. CONCLUSIONS: Fetal cell recycling combines the powers of highly sensitive molecular methods to maximize the genetic information available from a single fetal cell. This technique will permit noninvasive diagnosis of recessively inherited single-gene disorders.

Identification of fetal nucleated red cells in co-cultures from fetal and adult peripheral blood: differential effects of serum on fetal and adult erythropoiesis.

Seeking to optimize a novel method of isolating rare fetal erythroid cells in cultures from maternal blood, we have explored the effects of serum supplement on fetal and adult erythropoiesis. We used flow cytometry and sorting after labelling with antibodies to fetal haemoglobin (HbF) and adult haemoglobin (HbA). In adult blood-derived cultures, most nucleated red cells accumulated either only adult haemoglobin (F-A+) or a combination of fetal and adult haemoglobin (F+A+). Only a few were F+A-. Serum affected the proportions of adult cells expressing fetal haemoglobin (both F+A- and F+A+), which were minimized, but not eliminated altogether, with the use of charcoal-treated sera at low concentrations. In contrast, the expansion of fetal red cells, which made only fetal haemoglobin (F+A-) during at least one week of culture, was strongly increased with the use of charcoal treated sera, due to the removal of a charcoal-absorbable inhibitor. In co-cultures of fetal and adult erythroid cells, fetal cells could be enriched in the order of 200-fold by flow sorting with the F+A- criterion. However, since adult F+A- cells could not be suppressed completely, the purity of sorted fetal cells still depended on the relative numbers of fetal and maternal erythroid clonogenic cells in the blood sample. Thus, we demonstrate a method by which fetal nucleated red cells potentially present in maternal blood cultures can be identified and isolated from the vast majority of maternal erythroid cells, based on their correlated contents of fetal and adult haemoglobin.

Improvement of fetal cell recovery from maternal blood: suitable density gradient for FACS separation.

OBJECTIVE: To improve the recovery of fetal nucleated erythrocytes (NRBCs) from maternal blood for noninvasive prenatal genetic diagnosis. METHODS: Blood samples were obtained from 10 women at 8-22 weeks of gestation. Samples were split and mononuclear cells were isolated using 1.083 and 1.090 g/ml of Percoll solution. Flow sorting with antibody to fetal hemoglobin and fluorescence in situ hybridization (FISH) analysis were used to evaluate the number of fetal cells recovered. RESULTS: In samples separated with the 1.090 density gradient, the yield of true gamma-hemoglobin-positive cells (median 21.0, range 2.2-303.8) was 1.9 times higher than that in the 1.083 density (median 11.1, range 1.1-87.5), although it took 2. 1-fold longer time to flow sort the gamma-hemoglobin-positive cells. In 7 out of 10 cases, the number of gamma-hemoglobin-positive cells recovered from the 1.090 density gradient was 3 times or greater than that from 1.083 gradient. After FISH analysis, we detected a median of 13.3 (range 2.2-98.8) fetal NRBCs per 10-ml maternal blood in the 1.090 density gradient, whereas a median of 11.0 fetal NRBCs were detected in the 1.083 gradient (range 1.1-35.0). The number of fetal NRBCs in the 1.090 density was significantly higher than that in the 1.083. CONCLUSION: Increased Percoll density results in improved fetal cell recovery in fresh posttermination maternal samples. The increased yield of fetal cells using this gradient may permit better noninvasive detection of fetal chromosome as well as DNA abnormalities in maternal blood.

Fetal cell identifiers: results of microscope slide-based immunocytochemical studies as a function of gestational age and abnormality.

OBJECTIVE: We evaluated monoclonal antibodies to 3 cell surface and 3 intracellular antigens for their relative usefulness as markers to identify fetal cells in maternal blood. STUDY DESIGN: With indirect immunocytochemical labeling techniques, antigen expression was studied in 52 fetal blood samples as a function of gestational age, fetal karyotype, the presence of multiple anomalies detectable on ultrasonography, and anemia. RESULTS: A decline in the expression of these antigens as gestational age advanced was demonstrated. Samples from karyotypically abnormal fetuses, fetuses with multiple anomalies, and anemic fetuses showed an antigenic distribution that was immature for gestational age. In normal fetuses zeta globin and epsilon globin expression decreased after 12 to 14 weeks, potentially limiting the utility of these proteins as fetal cell markers in the isolation of fetal cells from maternal blood. CONCLUSIONS: The results of this study demonstrate a fetal developmental hematologic profile that varies with gestational age and also with pathologic condition. Antibodies to the gamma chain of fetal hemoglobin and the transferrin receptor (CD71) are the most useful fetal cell-identifying reagents.