Kleihauer抗酸染色法标记母血中的胎儿有核红细胞

以18例孕7~25周的孕妇外周血为材料, 经Percoll不连续密度梯度离心初步富集胎儿有核红细胞。然后用Kleihauer抗酸染色法进行标记, 结果阳性胎儿有核红细胞的胞浆呈深红色, 而母亲的有核红细胞胞浆无色。显微操作法获取单个胎儿有核红细胞, 经全基因组扩增后, 产物进行性别鉴定及STR连锁分析检测, 验证有核红细胞的来源, 并完成9例杜氏肌营养不良(Duchenne muscular dystrophy,DMD)的无创性产前基因诊断。应用Kleihauer抗酸染色法标记胎儿有核红细胞, 它是一种快速、简单、直接的化学染色方法, 更易于推广到临床应用。     

Fetal nucleated cells in maternal peripheral blood: frequency and relationship to gestational age

To determine the frequency of fetal nucleated cells in maternal peripheral blood during different stages of pregnancy, 50 primigravidas were investigated by determining the frequency of cells with the Y chromosome using fluorescence in situ hybridization (FISH) of Y-specific repetitive sequences of the DYZ1 family. Polymerase chain reaction (PCR) amplifying the same part of the DYZ1 used as the probe in FISH and a single-copy Y-specific fragment was also carried out for genomic DNA from the same samples. Cells with the hybridization signal were detected by FISH at and after 15 weeks of pregnancy in all pregnant women who gave birth to boys. The ratio of cells with the signal to those without the signal ranged from 1 in 144,000 to 1 in 4,000 with a tendency to increase as the pregnancy advanced. The frequency of fetal cells estimated by the PCR experiments was significantly and positively correlated with that found by FISH. The present study suggests that fetal nucleated cells increase in maternal peripheral blood with advancing gestation, from less than 1 in 100,000 nucleated cells in the first trimester to around 1 in 10,000 at term. These frequencies were much lower than those reported by cytological methods.     

母体外周血中胎儿有核红细胞的分离和富集方法的研究进展

母体外周血中分离的胎儿有核红细胞（fNRBCs）包含胎儿完整的遗传信息,可用于无创产前诊断。fNRBCs的分离和富集方法主要分为三类：物理分选法、抗原-抗体结合分离法和增殖法。不同的方法获得的fNRBCs的数量和纯度不同,多种方法联合使用可以提高富集产物中fNRBCs的纯度和数量。本文就母体外周血中fNRBCs的分离和富集方法进行综述。     

Detection and relocation of cord blood nucleated red blood cells by laser scanning cytometry

BACKGROUND: Fetal nucleated red blood cells (NRBC) present in the peripheral blood of pregnant women at low frequency are a potential target for noninvasive prenatal diagnostics. METHODS: CD71-enriched cells from male cord blood (CB) were stained for the gamma chain of HbF (Hb-gamma) and cytocentrifuged. Fluorescence in situ hybridization (FISH) was done for the Y chromosome. Following staining of the nucleus with TO-PRO-3, laser scanning cytometry was performed. Artificial mixtures of small volumes of male CB and blood drawn from nonpregnant females were analyzed. RESULTS: In CB, 59% of events double positive for Hb-gamma and TO-PRO-3 were identified as CB-NRBC. In contamination studies, male fetal CB-NRBC were identified perfectly on the basis of morphologic characteristics and FISH reactivity following relocation and visual assessment. Mean recovery was 8.7%. CONCLUSIONS: Laser scanning cytometry of preenriched fetal NRBC may offer a promising way for noninvasive prenatal diagnostics. This is because it provides a virtual enrichment step and the position on the slides of cells visually confirmed to correspond to fetal NRBC is known. Further experimental procedures on well-defined and located target cells may be feasible.     

The effect of neuraminidase on the relative surface charge-associated properties of rat red blood cells of different ages

Approx. 70% of the sialic acid on the rat erythrocyte surface is susceptible to cleavage by neuraminidase (Vibrio cholerae). Neuraminidase treatment results in a reduction in the partition coefficient (K) of the red cells in a charged dextran-poly(ethylene glycol) aqueous phase system and in the electrophoretic mobility of the cells. Countercurrent distribution of rat neuraminidase-treated erythrocytes, containing 59Fe-labeled mature red cells of distinct age, indicates that (a) the electrophoretic mobilities of red cells in different cavities along the extraction train increase with increasing K, as is the case with untreated erythrocytes, and (b) the cell age-related differences in surface charge-associated properties are neither eliminated nor altered by the enzyme action.     

Partial oxidative-stress perturbs membrane permeability and fluidity of fish nucleated red blood cells

We investigated the influence of partial oxidative stress on permeability and fluidity of nucleated fish red blood cells for simulating nucleated somatic cells. Peroxide value indicating lipid hydroperoxide level in nucleated red blood cells of common carp (Cyprinus carpio) increased with increasing body size. We detected that oxidation of nucleated red blood cells led to the degraded PUFA compositions and accelerated the permeability of calcein and ATP in the nucleated red blood cells restrictedly oxidized with 1 mM AAPH treatment for 30 min at 21 degrees C in the dark. Using fluorescence probes, PC3P, we found that oxidative stress reduced the membrane fluidity of nucleated red blood cells. It was also observed that AAPH had no significant influence on the osmotic fragility and electrophoretic profiles of red blood cell proteins. These results suggest that partial oxidative-stress, even if failure to fragment the membrane, may affect membrane permeability of fish nucleated red blood cells for an important energy molecule, ATP.     

Arrestin from nucleated red blood cells binds to bovine rhodopsin in a light-dependent manner

Using a panel of monoclonal antibodies, it has previously been demonstrated that the cytosol of nucleated red cells (trout and turkey) contains a protein similar to arrestin, a soluble protein found so far only in the photosensitive cells and which, by binding to photoexcited rhodopsin, inhibits the phototransduction process. The role of this arrestin-like protein in non-photosensitive cells is questionable. In this report we present evidence that partially purified red blood cell arrestin (RBC arrestin) behaves functionally like bovine retinal arrestin: it binds to phosphorylated bovine rhodopsin only when this receptor has been photoactivated. Thus RBC arrestin and bovine retinal arrestin are closely related both structurally and functionally. By analogy with the function of retinal arrestin, it is proposed that RBC arrestin is involved in desensitization of membrane transport proteins and/or adrenergic receptors.     

Sodium-23 NMR relaxation times in nucleated red blood cells and suspensions of nuclei.

The relaxation behavior of intracellular 23Na in suspensions of chicken erythrocytes and of their nuclei was investigated. The transverse magnetization was found to decay biexponentially. The average relaxation rates for the nucleated chicken erythrocytes are considerably shorter than the average relaxation rates obtained for dog and human nonnucleated red blood cells. Of particular significance is the twofold decrease in the short component of T2. Calculations based on the measured 23Na NMR relaxation rates in suspensions of nuclei indicate that most of the difference between the relaxation rates in the mammalian as compared to the chicken erythrocytes, can be accounted for by the contribution of the nuclei in the latter.     

Rosette-forming T cells in human peripheral blood at different ages

Rosette-forming T cells (RFC) were studied in 51 human volunteers, ranging in age from 16 to 69 yr. Subjects were divided in six groups, according to age (Table 1). The percentage of RFC was constant in Groups 2, 3, and 4 (20–49 yr) and slightly but significantly higher in Group 1. On the contrary, it dropped significantly in Groups 5 and 6. This drop in per cent of RFC is coincident with a drop of thymic function; it should be taken in account whenever RFC are studied as a clinical test.     

Fetal cells in the maternal blood

Summary In an attempt to stimulate fetal cells in the maternal blood to mitotic division, peripheral blood lymphocytes were cultured from ten primiparous women and six multiparous women. In the case of the ten primiparous women, PWM was used to stimulate lymphocytes in 3- and 7-day cultures made at the 16th, 20th, 24th, and 28th week of gestation. Altogether, 10565 mitoses were analyzed after quinacrine staining of cells from five mothers who each subsequently gave birth to a male infant, and not a single XY mitosis was found.In the case of the multiparous women, lymphocyte cultures, with PHA or LPS as mitogen and MLC, were initiated between the 13th and 20th week of pregnancy. Four of the mothers were pregnant with a male child, and two with a female child. From cultures of each of the four mothers expecting a boy, a total of 9721 mitoses were analyzed after quinacrine staining, and not a single XY mitosis was found. However, one XY cell was found in the culture from one of the two women who delivered a girl. The XY mitosis probably originated from a pregnancy 8 months earlier which terminated in a male infant.In an attempt to culture and obtain good chromosome preparations from small numbers of cells, it was shown that a good mitotic response and good chromosome preparations could be obtained from as few as 6000 lymphocytes.     

Fetal gender determination and BclI polymorphism using nucleated erythrocytes in maternal blood.

This study demonstrated determination of fetal gender from nucleated red blood cells (NRBCs) in maternal blood and attempted to apply prenatal diagnosis of hemophilia A using BclI DNA polymorphism. Venous blood was drawn from 20 pregnant women, and NRBCs were recovered by magnetic activated cell sorting and anti-GPA (glycophorin A) immunostaining. After microdissector isolation of the NRBCs, primer extension preamplification (PEP) and nested PCR of the amelogenin gene were performed to determine fetal gender. We also performed PEP and nested PCR of BclI polymorphism to verify the validity of prenatal diagnosis of hemophilia A. DNA amplification was achieved in 107 cells (51.9%) and fetal gender determined with 65.0% accuracy. Unfortunately, we could not verify the validity within the scope of this study. However, in a larger number of cases that are informative in BclI polymorphism, we will be able to identify patients affected by hemophilia A using fetal NRBCs in maternal blood.     

Genomic DNA fragmentation in red blood cells of the bullfrog during metamorphosis

Red blood cells (RBC) of the bullfrog ( Rana catesbeiana ) contain larval-type hemoglobin (Hb) during the larval period. At the beginning of metamorphosis, RBC containing adult-type Hb appear and two types of RBC coexist in the systemic circulation. During the metamorphic climax, RBC with larval-type Hb disappear from the circulation and, simultaneously, RBC containing adult-type Hb begin to circulate. These two types of RBC were separated by Percoll density gradient centrifugation to examine the molecular size of the genomic DNA of each population. DNA fragmentation was detected only in new RBC with adult-type Hb that appeared in the systemic circulation and remained throughout post-metamorphic life. Semiquantification of DNA on agarose gel showed that the degree of DNA fragmentation was highest at the metamorphic climax. As the existence of DNA fragments suggested endonucleolytic cleavage, nuclease activity was examined by an activity gel system and in vitro circular plasmid DNA digestion assays. The latter revealed that both types of RBC possess endonucleolytic activity throughout the pre- and post-metamorphic periods. Assays of endogenous endonucleolytic activities under different divalent ionic conditions suggested that mobilization of intracellular Ca²⁺-Mg²⁺ induces genomic DNA fragmentation in adult-type RBC.