流式细胞术产前非侵入性定量检测胎-母出血的研究

目的　建立产前非侵入性定量检测RhD阴性孕妇胎 母出血的流式细胞术实验方法。方法　RhD阴性红细胞与RhD阳性红细胞按一定比例混合 ,用抗 D作为一抗结合D+ 红细胞 ,以羊抗人IgGF(ab′) 2 FITC作为二抗 ,连接抗 D ,应用流式细胞术检测D+ 红细胞占D-红细胞的比例 ,建立抗 D及抗人IgGF(ab′) 2 FITC的量效关系 ,确定两者最佳使用剂量。在此实验条件下 ,进行流式细胞术检测值与实际值的相关性分析 ,判定可以检测出的D-红细胞中混有D+ 红细胞的准确出血量范围 ,应用建立的流式细胞术方法测定 9例RhD阴性孕妇外周血中的胎 母出血量。结果　抗 D的有效使用剂量为 1∶4 0 0稀释 ,10 0 μl/5× 10 6细胞 ;羊抗人IgGF(ab′) 2 FITC的有效使用剂量为 2 5 μg/5× 10 6细胞 ;流式细胞术测定比例与已知实际比例的相关系数r =0 998;准确检测的胎 母出血量为 (0 .6～ 30 )ml。 9例RhD阴性孕妇外周血中除 2例未检出D+ 细胞而无法判断外 ,其余 7例均检测出胎 母出血 ,胎 母出血量为 (1.2～ 6 .4 8)ml。结论　针对孕期发生胎 母出血反应的RhD阴性孕妇 ,可以应用流式细胞术方法在产前准确无损伤地定量检测胎 母出血量 ,指导临床预防、治疗Rh新生儿溶血病     

Analysis of factors affecting quantification of fetomaternal hemorrhage by flow cytometry

BACKGROUND: An analysis was carried out to determine the sources and extent of errors encountered in the quantitation of the volume of fetomaternal hemorrhage (FMH) by flow cytometry. Different assay conditions were compared, to define the simplest, most accurate protocol. STUDY DESIGN AND METHODS: D-, D+, and artificial FMH (mixtures of D+ and D- RBCs) were stained either by a direct method (using FITC-conjugated IgG3 D MoAb [BRAD-3]), with or without dual labeling with PE-conjugated anti-GPA, or by indirect methods (using polyclonal anti-D followed by FITC- or biotin-conjugated anti-IgG reagents). Cells were selected for flow cytometric analysis on the basis of either forward or side scatter (log FSC/log SSC) characteristics or of GPA+ labeling or were unselected. The numbers of events labeled with anti-D were determined from histograms. For some samples, 10 replicates of 500,000 events each were analyzed. RESULTS: Background fluorescent events in 10 directly labeled gated D- samples ranged from 0.007 to 0.023 percent, equivalent to 0.15- to 0.51-mL FMH. Both the use of a gate on log FSC/SSC or the selection of GPA+ events only resulted in a reduction in FMH of 0.3 mL or less. The intra-assay variation in FMH, or sampling error, was found to be approximately 10 percent at low artificial FMH (<10 mL) but greater (< or =50% with a CV of 15%) with D- samples. Direct staining was quicker and produced a lower background than indirect staining. CONCLUSION: The inherent sampling error that is due to the random distribution of rare events throughout the blood sample contributed greatly to the variation in the volume of FMH calculated by flow cytometry. The FMH should not be underestimated. For a routine assay, a simplified protocol and calculation will be sufficiently accurate to determine the dose of prophylactic anti-D that should be given to the patient.     

Detection of fetal red cells in fetomaternal hemorrhage using a fetal hemoglobin monoclonal antibody by flow cytometry

BACKGROUND: The laboratory determination of the level of fetal cells in maternal circulation remains an important support in the obstetrical management of women with suspected uterine trauma and in the proper dose administration of anti-D for prevention of Rh hemolytic disease of the newborn. Limitations in the sensitivity and precision of the widely used manual Kleihauer-Betke test have prompted an increased utilization of flow cytometric methods for fetal cell detection in maternal blood samples. STUDY DESIGN AND METHODS: Murine monoclonal antibodies directed against fetal hemoglobin (HbF) were developed, conjugated to fluorescein isothiocyanate, and used in a multiparametric flow cytometric assay developed for the quantitation of fetal red cells. A rapid intracellular staining method using brief glutaraldehyde fixation and Triton X-100 permeabilization prior to monoclonal antibody incubation was developed, along with optimization of the flow cytometric analysis protocol for the analysis of 50,000 cells. The performance of the assay was assessed for linearity and precision and correlated with the Kleihauer-Betke acid elution method. RESULTS: The anti-HbF flow cytometric method showed good correlation with the Kleihauer-Betke method (r2 = 0.86) and superior precision with a CV < 15 percent for blood samples with > 0.1 percent fetal cells. Analysis of 150 blood samples from nonpregnant adults, including individuals with elevated HbF due to hemoglobinopathies and hereditary persistence of HbF, gave a mean value of 0.02 percent fetal cells, and all results were less than 0.1 percent. CONCLUSIONS: The anti-HbF flow cytometric method for detection of fetal cells offers a simple, reliable, and more precise alternative to the Kleihauer-Betke manual technique for the assessment of fetomaternal hemorrhage. The method has additional potential applications for the study of HbF levels or frequency of adult red cells with low levels of HbF (F cells) in individuals with hemoglobinopathies.     

Massive fetomaternal hemorrhage: clearance of fetal red blood cells after intravenous anti-D prophylaxis monitored by flow cytometry

BACKGROUND: The clearance of D+ red blood cells (RBCs) from the circulation in D- individuals mediated by passively administered anti-D occurs by opsonization with the antibody and subsequent removal in the spleen. Few data exist on the kinetics of clearance of large volumes of D+ RBCs from the maternal circulation by anti-D in clinical cases of massive fetomaternal hemorrhage (FMH). CASE REPORT: A 33-year-old D- woman delivered a D+ female infant by emergency cesarean section for suspected fetal anemia. A massive FMH, initially estimated to be approximately 142 mL of RBCs, was found. In addition to the standard dose of intramuscular (IM) anti-D (300 microg) given immediately after delivery, 2700 microg of anti-D was administered intravenously (IV). The clearance of D+ fetal cells from the maternal circulation was monitored by flow cytometry in samples obtained on a daily basis using anti-D. The mother had no detectable anti-D 6 months after delivery. RESULTS: No clearance of fetal cells was apparent after the insufficient dose of IM anti-D. The IV administration of anti-D caused accelerated clearance of D+ fetal RBCs with a t1/2 of 24.5 hours. D+ reticulocytes comprised 4.2 percent of all D+ cells in the maternal circulation at delivery suggesting acute fetal blood loss. CONCLUSIONS: The approach used in this report allowed a detailed analysis of the kinetics related to the clearance of fetal D+ RBCs. Simultaneous measurements of fetal reticulocytes and fetal RBCs in maternal blood may establish the timing of an FMH.     

Use of a directly conjugated monoclonal anti-D (BRAD-3) for quantification of fetomaternal hemorrhage by flow cytometry

BACKGROUND: Determination of the volume of fetal D-positive cells in the circulation of D-negative women after delivery is carried out to determine whether additional prophylactic anti-D should be given to the mother. Although the Kleihauer-Betke test is still widely used to calculate the fetomaternal hemorrhage, increasing use is being made of flow cytometry. STUDY DESIGN AND METHODS: A conjugated monoclonal anti- D was prepared by labeling purified BRAD-3 (IgG3) with fluorescein isothiocyanate (FITC-BRAD-3). This reagent was used to label D-positive red cells by a one-step procedure: 5 microL of washed cells were incubated with 50 microL of FITC-BRAD-3 (50 micrograms/mL) at 37 degrees C for 30 minutes; then the cells were washed and 500,000 events were analyzed by flow cytometry. RESULTS: The FITC-BRAD-3 reagent effectively labeled D-positive cells. The percentage of D-positive cells in mixtures containing more than 0.04 percent D-positive cells in D-negative cells was accurately determined by using this reagent and flow cytometry. Although the Kleihauer-Betke test was more accurate than this one-step flow cytometric method at quantifying fetomaternal hemorrhage of < 1 mL, the flow cytometric method was more accurate in the 1- to 7-mL fetomaternal hemorrhage range of 1 to 7 mL (whole-blood equivalents). Analysis of 175 clinical samples for fetomaternal hemorrhage gave consistent quantification results with the three methods used: the Kleihauer-Betke test, flow cytometry with FITC-BRAD- 3, and flow cytometry with polyclonal anti-D followed by FITC-anti-IgG. CONCLUSION: Labeling of samples with FITC-BRAD-3 was simple and rapid. By flow cytometric analysis, good separation of D-positive from D- negative cells was obtained, and fetomaternal hemorrhage of > 1 mL was quantified accurately.     

A combined flow cytometry-based method for fetomaternal hemorrhage and maternal D

BACKGROUND: The fetomaternal bleed assay by flow cytometry is a semiquantitative test used to determine the volume of fetomaternal hemorrhage (FMH). We have enhanced this method by combining the fetomaternal bleed assay with a D antibody in the same tube to determine the maternal D as well as the need for Rh immune globulin (RhIG) administration. STUDY DESIGN AND METHODS: The performance of the FMB/D assay was compared against the Kleihauer‐Betke (KB) acid elution method for the quantitation of fetal bleed and tube method for the maternal D determination. Peripheral blood cells from the pregnant women were fixed and permeabilized, incubated with monoclonal antibodies directed against fetal hemoglobin (Hb F) and D, and analyzed by flow cytometry. The relative concentration of Hb F‐containing cells to adult cells was used to determine the volume of the FMH. In addition, the cells were identified as D+ or D?. RESULTS: D typing of nonpregnant adult donors matched in all cases with the tube method with a mean value of 0.01% Hb F‐positive cells (n = 25). D typing of pregnant or postpartum samples matched in all cases (n = 100), and the RhIG dose calculated by the percentage of Hb F for each method matched 97 percent (n = 36; p = 0.324). The one discrepant sample was consistent with the acknowledged overestimation when using the KB method. CONCLUSION: The highly precise anti‐Hb F flow cytometric method was combined with maternal D determination by anti‐D. This method correlates well with standard assays eliminating the need for additional testing without sacrificing clinical information.     

Semiautomated data analysis of flow cytometric estimation of fetomaternal hemorrhage in D- women

BACKGROUND: Accurate and reliable measurement of the volume of fetal D+ cells in D- women is required for adequate anti-D prophylaxis. A semiautomated flow cytometry assay based on a standardized calibration curve that was created with simulated fetomatemal hemorrhage (FMH) mixtures was developed. STUDY DESIGN AND METHODS: A calibration range of 0.083- to 2-percent D+ cells in the D-RBC mixtures (2-44 mL calculated FMH) was analyzed by use of a flow cytometer (XL-MCL, Coulter Electronics Ltd). Linear regression analysis of the calibration curve data with computer software (Excel, Microsoft) allowed semiautomated determination of the FMH volume. To optimize the assay, fresh versus frozen and thawed RBCs, RBCs from adults who are heterozygous for D or cord RBCs, and indirect- or direct-labeling techniques were evaluated by use of MoAbs. RESULTS: Fresh RBCs from adults heterozygous for D were chosen for routine use, although equivalent calibration curves were obtained with all cells tested (n = 12 calibration assays; r2 = 0.999; mean SD, 14%). A monoclonal anti-D reagent (Therad 10, Diagnostics Scotland) worked well in both indirect-(anti-IgG F(ab)-FITC) and direct-(anti-D-FITC) labeling methods compared to the use of BRAD-3 FITC. In routine practice, the FMH volumes obtained were mainly lower than those obtained in the Kleihauer Betke test when there was less than 4 mL of FMH. CONCLUSION: Semiautomated data acquisition and calibration curve analysis represents a further step toward standardization of flow cytometry for accurate FMH quantification and facilitates evaluation and control of day-to-day variations between laboratories, flow cytometers, and operators.     

Detection and quantitation of fetomaternal hemorrhage

Failure to administer additional doses of Rh immune globulin (RhIG) to patients with excessive fetomaternal hemorrhage (FMH) is one of the causes of continued Rh isoimmunization. We compared the fetal cell ratio (FCR) with the other laboratory methods currently used at our hospital to quantitate FMH, ie, fetal cell preparation (FCP), RhIG cross-match with maternal serum, and indirect Coombs' testing 24 to 48 hours after administration of RhIG. The incidence of excessive FMH as detected by the various methods was 3.6% (cross-match), 9.6% (Coombs'), 18% (FCP), and 66% (FCR). The methods currently used do not accurately quantify FMH. The FCR is the most sensitive test but it has a high false-positive rate and thus does not appear to be clinically useful. We suggest that repeating the indirect Coombs' test may provide a practical alternative for determining the need for additional doses of RhIG.     

Subcellular localization-dependent changes in EGFP fluorescence lifetime measured by time-resolved flow cytometry

Intracellular protein transport and localization to subcellular regions are processes necessary for normal protein function. Fluorescent proteins can be fused to proteins of interest to track movement and determine localization within a cell. Currently, fluorescence microscopy combined with image processing is most often used to study protein movement and subcellular localization. In this contribution we evaluate a high-throughput time-resolved flow cytometry approach to correlate intracellular localization of human LC3 protein with the fluorescence lifetime of enhanced green fluorescent protein (EGFP). Subcellular LC3 localization to autophagosomes is a marker of the cellular process called autophagy. In breast cancer cells expressing native EGFP and EGFP-LC3 fusion proteins, we measured the fluorescence intensity and lifetime of (i) diffuse EGFP (ii) punctate EGFP-LC3 and (iii) diffuse EGFP-ΔLC3 after amino acid starvation to induce autophagy-dependent LC3 localization. We verify EGFP-LC3 localization with low-throughput confocal microscopy and compare to fluorescence intensity measured by standard flow cytometry. Our results demonstrate that time-resolved flow cytometry can be correlated to subcellular localization of EGFP fusion proteins by measuring changes in fluorescence lifetime.OCIS codes: (260.2510) Fluorescence, (170.6920) Time-resolved imaging, (140.3518) Lasers, frequency modulated, (170.1530) Cell analysis     

Quantification of glycophorin A and glycophorin B on normal human RBCs by flow cytometry

BACKGROUND: The quantification of antigens and proteins on RBCs has been achieved by different approaches. Flow cytometry allows the results of the earliest studies to be to reappraised because it offers the possibility of measuring the immunofluorescence intensity of single cells and integrating the individual data of a large number of cells within a very short time. STUDY DESIGN AND METHODS: Flow cytometry was used in this work to analyze the binding of four MoAbs to glycophorin A (GPA) and glycophorin B (GPB). RBCs in their native state (nonfixed) were utilized. To avoid the agglutination problem, cells were disaggregated before measurements, dates were taken on 20,000 events on the single-cell region, and the fluorescence intensity of the principal peak present in the fluorescence histograms was used for the analysis. The quantification of sites per RBC was estimated by applying the Langmuir adhesion model. RESULTS: The numbers of GPA and GPB sites obtained for samples from healthy donors were similar to those found in the literature (1.86-4.9) x 10(5) and (0.48-1.61) x 10(5) for GPA and (0.21-1.14) x 10(5) and (0.47-0.88) x 10(5) for GPB. Differences between antibodies were found that depend on the binding site of each one. CONCLUSION: A simple method to quantify antigen sites on RBCs was developed. It could be applied whenever one antibody is assumed to bind exactly one antigen.     

Doppler sonography for predicting fetal anemia caused by massive fetomaternal hemorrhage

Fetomaternal hemorrhage (FMH) can cause severe anemia in the fetus. Untreated, this may cause hydrops or even fetal death. However, correct diagnosis of FMH followed by blood transfusion can prevent these life-threatening consequences. We describe two cases in which fetal anemia was suspected because of maternal reporting of decreased or absent fetal movements, the detection of a sinusoidal heart rate pattern and increased blood flow velocities of the middle cerebral artery and umbilical vein. Together with the Betke-Kleihauer test showing fetal cells in the maternal circulation, this led to the correct diagnosis of severe fetal anemia caused by FMH. A Cesarean section was performed within a few hours. Both neonates were severely anemic and received immediate blood transfusions. They are currently alive and well.     

Thematic workshop on fluorescence compensation settings in multicolor flow cytometry

In his program of thematic one-day workshops, the French Association of Cytometry had organized a workshop dedicated to the fluorescence compensation settings in multicolor flow cytometry. This special day was in honor of our past President Jean Luc D'Hautcourt who has been involved in the quality of the use of flow cytometry in its clinical and research purposes. Review on fluorescence phenomena, compensation rules, settings, and few observed confounding situations were presented.     

流式细胞术计数血小板

目的　建立流式细胞仪计数血小板的实验方法。方法　用PE标记抗人CD61抗体标记全血中血小板 ,同时加入 5 μl已知浓度的FITC 微球溶液。流式细胞仪检测血小板 (FL2 )和微球 (FL1)的数量 ,换算出患者血小板浓度。结果　流式细胞仪法与血细胞计数仪法计数结果无显著性差异 ,低血小板组两法相关程度低于正常血小板组 ,提示流式细胞仪计数血小板能排除多种干扰因素 ,最低检出血小板量为 0 15 0× 10 9/L。该方法CV =5 4 % (Plt=2 2 0×10 9/L)和 11 1% (Plt=1 5 7× 10 9/L)。结论　流式细胞仪法适合对低血小板标本血小板的精确计数。     

Bacteria detection by flow cytometry

Since bacterial infection of the recipient has become the most frequent infection risk in transfusion medicine, suitable methods for bacteria detection in blood components are of great interest. Platelet concentrates are currently the focus of attention, as they are stored under temperature conditions, which enable the multiplication of most bacteria species contaminating blood donations. Rapid methods for bacteria detection allow testing immediately before transfusion in a bed-side like manner. This approach would overcome the sampling error observed in early sampling combined with culturing of bacteria and would, at least, prevent the transfusion of highly contaminated blood components leading to acute septic shock or even death of the patient. Flow cytometry has been demonstrated to be a rapid and feasible approach for detection of bacteria in platelet concentrates. The general aim of the current study was to develop protocols for the application of this technique under routine conditions. The effect of improved test reagents on practicability and sensitivity of the method is evaluated. Furthermore, the implementation of fluorescent absolute count beads as an internal standard is demonstrated. A simplified pre-incubation procedure has been undertaken to diminish the detection limit in a pragmatic manner. Additionally, the application of bacteria detection by flow cytometry as a culture method is shown, i.e., transfer of samples from platelet concentrates into a satellite bag, incubation of the latter at 37 degrees C, and measuring the contaminating bacteria in a flow cytometer.     

Analysis of platelets by flow cytometry

Transfusion medicine has become a multi-disciplinary field with many recent technical developments and the flow cytometer has had a significant impact in transfusion medicine, especially at the level of platelet immunobiology. Many routine tests in platelet immunology are now performed by flow cytometry laboratories, including assessment of platelet-associated allo- and auto-antibodies and complement components. Platelet analysis by flow cytometry has been applied to detection of platelet antigens, platelet surface-bound proteins, platelet activation, measurement of reticulated platelets, intracellular calcium studies, and the measurement of platelet microparticles in vivo and in vitro. This review will focus on the use of the flow cytometer in these applications in investigations of platelet immunology.     

实时荧光PCR法和流式细胞术检测HLA-B27的结果分析

目的比较实时荧光PCR法和流式细胞术两种方法检测人类白细胞抗原B27(HLA-B27)的临床应用价值。方法分别采用实时荧光PCR法和流式细胞术两种方法检测225例疑似强直性脊柱炎患者血中HLA-B27,比较和分析两种方法的检测结果。结果 95.11%样本检测结果相同,差异不具有统计学意义(P0.05),将存在差异的标本进行基因测序后,结果与实时荧光PCR的检测结果一致。结论两种方法检测HLA-B27均具有较高的灵敏度和特异度,实时荧光PCR法在结果的准确性上更优于流式细胞术。