XE-2100血细胞分析仪在检测妊娠高血压疾病孕妇外周血中NRBCs的意义

目的:比较妊娠高血压疾病(HDP)组孕妇和正常妊娠孕妇外周血中的有核红细胞(NRBCs)的数量并鉴定其来源,探讨XE-2100血细胞分析仪用来检测HDP孕妇外周血NRBCs的应用价值。方法:利用XE-2100血细胞分析仪对孕周为30-38周、年龄为23-41岁的10例正常孕妇组、10例先兆子痫轻度组、11例先兆子痫重度组和10例子痫组孕妇外周血有核红细胞进行检测;对检测出NRBCs的14名HDP孕妇进行HbF酸洗脱试验,鉴定其NRBCs的来源。结果:HDP孕妇外周血中的NRBCs大多为胎儿源性(70·97±0·17%);在先兆子痫轻度组、先兆子痫重度组和子痫组孕妇的外周血NRBCs数量较正常孕妇有显著的升高(所有P值<0·05);XE-2100用于检测HDP病人外周血中的NRBCs有临床应用价值。     

孕妇外周血胎儿细胞中巨细胞病毒基因的检测及意义

目的利用孕妇外周血中胎儿细胞进行无创性产前诊断巨细胞病毒宫内感染的新方法。方法(1)采用显微操作技术从273例孕妇外血中分离单个胎儿有核红细胞。(2)应用多重引物原位合成技术(primed in situ labeling，PRINS)检测76例孕妇外周血HCMV-DNA阳性标本的单个胎儿细胞的SRY基因和HCMV-DNA基因。(3)应用引物延伸预扩增法(primed extension preamplification，PEP)及聚合酶链反应(PCR)检测273例孕妇外周血中单个胎儿细胞的SRY基因和HCMV-DNA基因。结果(1)应用显微操作技术分选胎儿细胞的分选率为100％。(2)PRINS技术检测SRY基因的敏感性为97．56％(40／41)，特异性为100％(35／35)。检测HCMV-DNA基因的敏感性为92．68％(38／41)，特异性为100％(35／35)。(3)PEP及PCR方法检测SRY基因的敏感性为97．39％(149／153)，特异性为99．17％(119／120)，检测HCMV-DNA基因的敏感性为95．12％(39／41)，特异性为100％(232／232)。结论应用PRINS技术、PEP方法及PCR技术对孕妇外周血中单个胎儿细胞进行非创伤性产前诊断巨细胞病毒宫内感染是一种敏感性高特异性强的新方法，可能具有广阔的临床应用前景。     

单抗CD71用于荧光激活细胞分选法富集分选母血中的胎儿细胞

目的:研究转铁蛋白受体(CD71)抗体对单个核细胞的标记情况,探讨该单克隆抗体识别胎儿细胞的有效性。方法:选用CD71抗体对92例正常孕妇外周血、15例未孕妇女外周血和15例胎儿脐血单个核细胞进行标记,并用于荧光激活细胞分选法富集分选母血中的胎儿有核红细胞。结果:孕10～35周的孕妇外周血中CD71阳性细胞的比例为(5.59±4.83)×10-2,与未孕妇女外周血及胎儿脐血中CD71阳性细胞的比例比较,均差异有极显著性(P<0.001)。结论:孕妇外周血中存在胎儿细胞,单抗CD71可以作为一种有效识别抗体用于母血中胎儿细胞的分选,从而为进一步的无创性产前诊断工作打下基础。     

应用密度梯度离心结合免疫磁珠法富集孕妇外周血中胎儿NRBC的实验研究

目的研究利用不连续密度梯度离心结合免疫磁珠方法,分选孕妇外周血中的胎儿有核红细胞(NRBC)。方法采集21名孕周在12-28周的孕妇外周血10ml,经不连续密度梯度离心后,再用免疫磁珠法分选和富集胎儿NRBC,同时对富集前后的孕妇外周血进行瑞氏染色和胎儿血红蛋白(fetal hemoglobin,HbF)特异性抗体标记、识别。结果未经分选、富集,21名孕妇外周血有核细胞层涂片经瑞氏染色检查均未发现NRBC;梯度离心富集后21名孕妇中有11例外周血有核细胞涂片找到1～8个NRBC;再经免疫磁珠分离后有12例涂片发现1～7个NRBC,同时进行HbF特异性抗体标记,证实其NRBC阳性涂片中均存在胎源性NRBC。结论本研究初步建立了分选、富集胎儿NRBC的实验方法,通过进一步的方法学改进,有望用于产前基因筛查的研究。     

应用密度梯度离心结合免疫磁珠法富集孕妇外周血中胎儿NRBC的实验研究

目的 研究利用不连续密度梯度离心结合免疫磁珠方法,分选孕妇外周血中的胎儿有核红细胞(NRBC).方法 采集21名孕周在12-28周的孕妇外周血10ml,经不连续密度梯度离心后,再用免疫磁珠法分选和富集胎儿NRBC,同时对富集前后的孕妇外周血进行瑞氏染色和胎儿血红蛋白(fetal hemoglobin,HbF)特异性抗体标记、识别.结果 未经分选、富集,21名孕妇外周血有核细胞层涂片经瑞氏染色检查均未发现NRBC;梯度离心富集后21名孕妇中有11例外周血有核细胞涂片找到1～8个NRBC;再经免疫磁珠分离后有12例涂片发现1～7个NRBC,同时进行HbF特异性抗体标记,证实其NRBC阳性涂片中均存在胎源性NRBC.结论 本研究初步建立了分选、富集胎儿NRBC的实验方法,通过进一步的方法学改进,有望用于产前基因筛查的研究.     

引物延伸预扩增后STR分型在鉴定母血中胎儿有核红细胞的应用

目的 探讨一种鉴定母血中胎儿有核红细胞（NRBC）的方法，为无创性产前基因诊断创造必要条件。方法 联苯胺染色识别孕妇外周血中NRBC，经显微操作获取，并以引物延伸预扩增（PEP）对单个NRBC进行全基凶组扩增后，用短串联重复序列（STR）分析其基因型，与父母基因型比对，确定该细胞的来源。结果 28例轻型β地中海贫血孕妇外周血样本中每例发现NRBC4～13个／5ml，经鉴定每例有胎儿NRBC2～8个／5ml，约43．6％的NRBC来源于胎儿。结论 PEP后STR基因型分析能有效鉴定孕妇外周血中NRBC的来源，使应用单个胎儿NRBC进行产前基因诊断成为可能。     

利用孕妇外周血中胎儿有核红细胞行无创产前性别诊断的初步研究

目的建立一种利用孕妇外周血中分选出的胎儿有核红细胞（FNRBC）进行无创伤性产前诊断的有效方法。方法收集5例妊娠7～9周的人流妇女的绒毛及外周血20ml,采用抗-CD45和抗ε链双标细胞,流式细胞仪分选CD45dim的细胞经显微挑选出ε-链免疫荧光抗体阳性的胎儿有核红细胞,再采用单细胞多重置换扩增技术（MDA）进行全基因组扩增,以扩增产物为模板,常规PCR检测Alphoid基因片段,确定胎儿性别,与绒毛PCR结果比较。结果抗-ε链、抗-CD45双标记的5例标本流式分选CD45dim细胞后,所有标本均观察到血红蛋白ε链阳性细胞。怀有2例男性和1例女性孕妇的外周血分选出的阳性细胞经MDA和PCRAlphoid基因片段扩增,显示1例全基因组扩增失败;1例诊断为男胎,1例为女性,与绒毛PCR性别检测结果完全符合。结论ε-抗体、CD45双标的CD45dim细胞经流式分选后结合显微操作技术从母血中挑选出ε-血红蛋白阳性细胞,经全基因组扩增可以有效获取足够量的研究模板,保证后续分析的准确性,可应用于性连锁遗传疾病的无创性产前诊断。     

PCR技术用于胎儿性别鉴定的方法学探索

目的 建立一种实用可靠的方法应用于胎儿早期性别鉴定,为伴性遗传病及早采取终止妊娠提供科学依据.方法 对40个孕妇在不同的妊娠时期4,8,16 w抽取外周血,分离血浆和有核红细胞,提取DNA,聚合酶链反应(PCR)扩增性别决定性基因(SRY和DYZ).并对相应孕妇出生的婴儿进行性别采访,比较不同条件PCR检测胎儿性别的准确度.结果 妊娠4 w外周血浆准确度为0.825,有核红细胞准确度为0.850;8 w血浆准确度为0.875,有核红细胞为0.850;16 w时血浆的准确度为0.975,有核红细胞也为0.975.结论 母亲外周血浆可用于胎儿性别鉴定,但妊娠期不能少于16 w.     

应用新型抗胎儿血红蛋白抗体富集母体循环中胎儿有核红细胞

目的 利用多肽合成技术制备抗胎儿血红蛋白(fetal hem-oglobin,HBF)γ链的抗体,探讨其用于检测孕妇外周血中胎儿有核红细胞(nucleated red blood cell,NRBC)进行无创性产前基因诊断的可行性.方法 针对胎儿血红蛋白的特异性抗原表位,选定第69～78位HbF-γ特异的11个氨基酸残基的肽段为免疫原,将人工合成的胎儿血红蛋白γ链的多肽与载体蛋白(KLH)偶联,佐剂乳化后免疫羊,制备羊抗人胎儿血红蛋白的抗血清,经蛋白G纯化,HbF特异性抗体标记、识别、显微操作法富集32例孕周为22～39周的孕妇外周血中的胎儿有核红细胞,引物延伸预扩增后,利用9个短串联重复序列(short tandem repeat,STR)的复合扩增方法对富集的阳性细胞进行扩增,用于孕妇外周血中富集的HbF阳性细胞胎儿来源的遗传学鉴定.结果 经HbF多克隆抗体标记,32名孕妇外周血中均发现与HbF呈阳性反应的胎儿NRBC,并具有鲜明的形态学特征,光学显微镜下可见NRBC细胞质呈棕黄色,核浆比例较低,苏木素复染后胞核呈蓝色,明显区别于其他细胞,每份样本出现NRBC 0.6～1.8个/ml,共计183个,平均为1.3个/ml,经STR多态性基因位点鉴定,准确率为90.6%.结论 利用多肽合成技术制备的抗胎儿血红蛋白γ链的抗体能有效识别母血中的胎儿有核红细胞,可应用于无创性产前基因诊断,具备良好的应用前景.     

母亲血液中的胎儿细胞:磁性细胞分离和免疫表型定型及FISH确证

在妊娠期间胎儿细胞进入母体循环系统,这可为产前诊断提供一种安全、可靠的非侵入性测验手段。不过,合适的细胞类型的选择和富集的方法是需要优先考虑的重要问题。多数研究者认为选择胎儿的有核红细胞(NRBCs)较为恰当,因为它们属于单个核细胞,胎儿血中的数量充盈,相对地分化较好而且生命期限较短。在40例妊娠妇女怀孕16到18周期间收集外周血样品20ml。富集NRBCs,作者采用了阴性磁性细胞分离(MACs),     

母血中胎儿有核红细胞的分离及无创性产前基因诊断研究

目的 从母血循环中分离、富集、鉴定和提取胎儿有核红细胞，建立遗传病无创性产前基因诊断方法。方法 取２５名妊娠８～３６周的孕妇静脉血，加载在细胞分离液Ｈｉｓｔｏｐａｑｕｅ１．０７７上分离单个核细胞（ＭＮＣ）；用包被ＣＤ７１抗体的ＤｙｎａｂｅａｄｓＭ－４５０ＣＤ２１免疫磁珠或包被ＣＤ４５抗体的ＤｙｎａｂｅａｄｓＭ－４５０ＣＤ４５免疫磁珠富集ＭＮＣ中的胎儿有核红细胞：ａｎｔｉ－ξ－ｂｉｏｔｉｎ或ａｎｔｉ     

密度梯度离心结合Keihuaer抗酸染色法富集孕妇外周血中FNRBC的研究

目的 探索建立一种简便、快速、较低成本的用于孕妇外周血胎儿有核红细胞(FNRBC)富集的实验方法.方法 采集18名孕周在8～16周的孕妇外周血10 ml,经Percoll不连续密度梯度离心初步分离后,运用Keihuaer抗酸染色法对玻片上的细胞进行染色标记,显微镜下观察、辨别FNRBC的特殊染色及形态,对阳性涂片者进一步行胎儿血红蛋白(HBF)特异性抗体标记以证实其胎源性.结果 经Keihuaer抗酸染色处理,阳性FNRBC的胞浆染色呈深红色,核蓝色,而母亲的有核红细胞胞浆不着色.18名孕妇外周血中有14名可检出FNRBC,平均1～6个阳性FNRBC/病例,阳性检出率为77.8%.14例FNRBC阳性玻片经HBF抗体标记亦证实相同结果,而4例Keihuaer抗酸染色阴性病例亦表现HBF标记阴性,两者检出符合率100.0%.结论 密度梯度离心结合Keihuaer抗酸染色法用于富集FNRBC 具有较高的特异性,实验操作方法简便、快速,有望用于无创性产前基因诊断的推广应用.     

Development, characterization, and use of monoclonal antibodies made to antigens expressed on the surface of fetal nucleated red blood cells.

BACKGROUND: Current methods for obtaining fetal cells for prenatal diagnosis are invasive and carry a small (0.5-1.0%) but definite risk of miscarriage. An attractive alternative would be isolation of fetal cells from peripheral maternal blood using antibodies with high specificity and avidity. METHODS: To generate antibodies, we purified nucleated red blood cells (NRBCs) from fetal livers and used them as the immunogen to generate monoclonal antibodies (mAbs) directed against surface antigens. RESULTS: The four antibodies recognized at least two conformationally sensitive epitopes of the transferrin receptor. Isolation of NRBCs from 252 maternal blood samples using these antibodies in magnetic activated cell sorting after an initial density gradient centrifugation yielded 0-419 NRBCs per 25 mL of maternal blood. One antibody, 2B7.4, not only isolated the highest number of NRBCs (>10 in 90% of the samples) but also isolated these NRBCs in 78 consecutive maternal samples. CONCLUSION: Antibody 2B7.4 shows promise for the isolation of NRBCs from maternal blood and should allow studies concerning the source of these cells, fetal vs maternal, and the factors controlling their prevalence.     

孕妇外周血胎儿有核红细胞富集的孕周选择

目的 利用抗转铁蛋白受体（CD71）抗体和抗白细胞共同抗原（CD45）抗体双标法，检测不同孕周孕妇外周血中有核红细胞的比例，以期选择最佳孕周进行胎儿有核红细胞富集。方法 采用抗-CD71抗体和抗-CD45抗体双标法，应用流式细胞术（flow cytometry，FCM）检测81例不同孕周孕妇外周血中有核红细胞的比例，并与未孕组和脐血组进行对照。结果 孕7～34周孕妇外周血中CD71^＋／CD45^dim细胞的比例为（0．107土0．063）×10^-2，与未孕组和脐血组相比差异均有统计学意义；CD71^＋／CD45^dim细胞在外周血中的比例在孕17～18周达峰值，为（0．144土0．096）×10^-2。结论 不同孕周孕妇外周血有核红细胞比例不同，孕17～18周比例最高。利用母血循环中的胎儿有核红细胞进行产前诊断，其最佳时间可选择在妊娠17～18周。     

Cesarean section due to fetal distress increases the number of stem cells in umbilical cord blood

BACKGROUND: Umbilical cord blood (UCB) can be used as hematopoietic stem cell source for transplantation. The success of a transplantation is highly correlated with the number of total nucleated cells (TNCs) and CD34+ cells in the UCB. Certain obstetric factors increase the yield of stem cells in the UCB. It is necessary to evaluate optimal conditions in labor to decrease the rate of sample rejection due to low cell count. No data exist regarding the difference between primary and secondary Cesarean sections in terms of efficacy of stem cell harvesting. STUDY DESIGN AND METHODS: Seventy-nine consecutive UCB units from women who had a Cesarean section between 1997 and 2003 were included. The number of TNCs, CD34+ cells, colony-forming units (CFUs), white blood cells (WBCs), nucleated red blood cells (NRBCs), and the total collection volume were compared between cases with primary and secondary Cesarean section. RESULTS: UCB obtained after a Cesarean section due to fetal distress has significantly higher numbers of TNCs, CD34+ cells, NRBCs, and WBCs compared to elective Cesarean section. Of the cases with secondary Cesarean section due to fetal distress, 67 percent resulted in UCB units with sufficient TNC numbers (> or =80 x 10(7) TNCs) compared to 42 percent of the cases with primary Cesarean section. CONCLUSION: Fetal distress increases the number of hematopoietic stem cells mobilized into UCB. Particular effort should be made to collect UCB from newborns who experienced fetal distress.     

Poor prognosis indicated by nucleated red blood cells in peripheral blood is not associated with organ failure of the liver or kidney.

BACKGROUND: The appearance of nucleated red blood cells (NRBCs) in peripheral blood is associated with a variety of severe diseases. When NRBCs are detected in blood, this is generally associated with increased mortality. METHODS: In a prospective study, NRBCs and other laboratory parameters were measured daily in the peripheral blood of surgical intensive care patients. The appearance of NRBCs was analyzed in relation to laboratory indicators of organ injury. RESULTS: A total of 284 surgical intensive care patients were included in this study. The mortality of NRBC-positive patients was 44.0% (40/91). This was significantly higher (p<0.001) than the mortality of NRBC-negative patients (4.2%, 8/193). Mortality increased with the NRBC concentration and the length of the NRBC-positive period. Multiple logistic regression analysis of several other clinical and laboratory risk indicators revealed a significant association between NRBCs and increased mortality, with an odds ratio of 1.95 (95% CI 1.35-2.82; p<0.001) for each increment in NRBC category (0, 1-40, 41-80, 81-240 and >240 NRBC/microL). After the initial detection of NRBCs in blood, there were no significant increases in creatinine concentrations or alanine aminotransferase activity. However, the appearance of NRBCs coincided with increasing C-reactive protein and thrombocyte concentrations. CONCLUSIONS: The detection of NRBCs in blood of surgical intensive care patients is of prognostic power with regard to patient mortality. This prognostic significance of NRBCs was independent of some clinical and other laboratory risk parameters. The appearance of NRBCs in blood was not associated with kidney failure or lesion of the liver.     

Successful diagnosis of fetal gender using conventional PCR analysis of maternal serum

BACKGROUND: Fetal DNA has been found in maternal plasma and serum. Diagnosis of fetal gender using maternal plasma and serum has been attempted in an effort to develop a new noninvasive method of prenatal diagnosis. METHODS: Peripheral blood samples were obtained from 61 pregnant women at 10-17 weeks of gestation before amniocentesis. DNA was extracted from 800 microL of each plasma or serum sample. To detect the Y-chromosome-specific sequences DYS14 and DYZ3 in the maternal plasma and serum, 40 cycles of PCR were carried out for each DNA extract. The PCR products were analyzed by 2.5% agarose gel electrophoresis and ethidium bromide staining, and the results were compared with the results of the cytogenetic analyses of amniocentesis. RESULTS: Cytogenetic analysis of amniocentesis revealed that 31 pregnant women had a male fetus and the remaining 30 pregnant women had a female fetus. Both DYS14 and DYZ3 were detected in 27 of the 31 plasma samples obtained from pregnant women carrying a male fetus and in all of 31 serum samples obtained from the same women. Neither DYS14 nor DYZ3 was detected in either the plasma or serum samples obtained from any of the 30 pregnant women carrying a female fetus. CONCLUSION: PCR analysis of maternal serum can be used to diagnose fetal gender.