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.     

A new method for high speed, sensitive detection of minimal residual disease

Investigations of rare cell types in peripheral blood samples, such as tumor, fetal, and endothelial cells, represent an emerging field with several potentially valuable medical applications. Peripheral blood is a particularly attractive body fluid for the detection of rare cells as its collection is minimally invasive and can be repeated throughout the course of the disease. Because the number of rare cells in mononuclear cells can be very low (1 in 10 million), a large number of cells must be quickly screened, which places demanding requirements on the screening technology. While enrichment technology has shown promise in managing metastatic disease, enrichment can cause distortions of cell morphology that limit pathological identification, and the enrichment targeting adds additional constraints that can affect sensitivity. Here, we describe a new approach for detecting rare leukemia cells that does not require prior enrichment. We have developed an immunocytochemical assay for identification of leukemia cells spiked in peripheral blood samples, and a high-speed scanning instrument with high numerical aperture and wide field of view to efficiently locate these cells in large sample sizes. A multiplex immunoassay with four biomarkers was used to uniquely identify the rare cells from leukocytes and labeling artifacts. The cytometer preserves the cell morphology and accurately locates labeled rare cells for subsequent high resolution imaging. The sensitivity and specificity of the approach show promise for detection of a low number of leukemia cells in blood (1 in 10 million nucleated cells). The method enables rapid location of rare circulating cells (25 M cells/min), no specific enrichment step, and excellent imaging of cellular morphology with multiple immunofluorescent markers. The cell imaging is comparable to other imaging approaches such as laser scan cytometry and image flow cytometry, but the cell analysis rate is many orders of magnitude faster making this approach practical for detection of rare cells.     

FISH analysis of all fetal nucleated cells in maternal whole blood: improved specificity by the use of two Y-chromosome probes.

Current cytogenetic approaches in noninvasive prenatal diagnosis focus on fetal nucleated red blood cells in maternal blood. This practice may be too restrictive because a vast proportion of other fetal cells is ignored. Recent studies have indicated that fetal cells can be directly detected, without prior enrichment, in maternal blood samples by fluorescence in situ hybridization (FISH) analysis for chromosomes X and Y (XY-FISH). In our blinded analysis of 40 maternal blood samples, we therefore examined all fetal cells without any enrichment. Initial examinations using conventional XY-FISH indicated a low specificity of 69.4%, which could be improved to 89.5% by the use of two different Y-chromosome-specific probes (YY-FISH) with only a slight concomitant decrease in sensitivity (52.4% vs 42.9%). On average, 12-20 male fetal cells/ml of maternal blood were identified by XY- and YY-FISH, respectively.     

A Single Cell Level Based Method for Copy Number Variation Analysis by Low Coverage Massively Parallel Sequencing

Copy number variations (CNVs), a common genomic mutation associated with various diseases, are important in research and clinical applications. Whole genome amplification (WGA) and massively parallel sequencing have been applied to single cell CNVs analysis, which provides new insight for the fields of biology and medicine. However, the WGA-induced bias significantly limits sensitivity and specificity for CNVs detection. Addressing these limitations, we developed a practical bioinformatic methodology for CNVs detection at the single cell level using low coverage massively parallel sequencing. This method consists of GC correction for WGA-induced bias removal, binary segmentation algorithm for locating CNVs breakpoints, and dynamic threshold determination for final signals filtering. Afterwards, we evaluated our method with seven test samples using low coverage sequencing (4∼9.5%). Four single-cell samples from peripheral blood, whose karyotypes were confirmed by whole genome sequencing analysis, were acquired. Three other test samples derived from blastocysts whose karyotypes were confirmed by SNP-array analysis were also recruited. The detection results for CNVs of larger than 1 Mb were highly consistent with confirmed results reaching 99.63% sensitivity and 97.71% specificity at base-pair level. Our study demonstrates the potential to overcome WGA-bias and to detect CNVs (>1 Mb) at the single cell level through low coverage massively parallel sequencing. It highlights the potential for CNVs research on single cells or limited DNA samples and may prove as a promising tool for research and clinical applications, such as pre-implantation genetic diagnosis/screening, fetal nucleated red blood cells research and cancer heterogeneity analysis.     

A novel method for depositing erythroid cells onto glass slides for fetal cell analysis.

BACKGROUND: We have developed a method for selecting erythroblasts from blood, the first step toward identifying fetal cells in maternal blood for diagnostic purposes. Because the selection method results in a large number of positive cells, we needed to develop new methods to deposit the cells onto slides and to modify in situ hybridization procedures to enable detection of fetal cells. METHODS: We utilized Nunc flaskettes to increase the slide surface area available for cell deposition. The ability of erythroid lineage cells to adhere to several surface modifications was examined. In situ hybridization methods were tested to find the best approach that is compatible with these cell preparations. RESULTS: The best glass slide coating for erythroid cells was found to be an antibody to glycophorin A, a red cell surface antigen. We were able to get excellent in situ hybridization signals in cells on flaskettes by modifying fixation and pretreatment parameters. CONCLUSIONS: The methods described here appear to be the best way of attaching a large number of erythroid lineage cells to slides and of detecting them by in situ hybridization.     

Measurement of erythrocytes on diagnostic slides by scanning electron microscopy

OBJECTIVE: To determine whether the measured sizes of erythrocytes in both paraffin-embedded sections and air-dried blood smears differ from values published in standard texts. STUDY DESIGN: Routinely prepared surgical pathology slides as well as an air-dried blood smear were viewed with a scanning electron microscope. Erythrocytes were measured using the instrument software. RESULTS: Erythrocyte size in the peripheral blood smear correlated well with textbook values, 7.2-7.9 microns. However, red blood cells within sectioned material from several laboratories showed a prominent decrease, ranging from 25% to 35%, as compared to textbook values, about 7 microns. CONCLUSION: Since cytologists and surgical pathologists often use the erythrocyte as a convenient marker on diagnostic slides, attention should be given to these observations in making sizing judgments.     

Unique monoclonal antibodies specifically bind surface structures on human fetal erythroid blood cells

Background

Continuing efforts in development of non-invasive prenatal genetic tests have focused on the isolation of fetal nucleated red blood cells (NRBCs) from maternal blood for decades. Because no fetal cell-specific antibody has been described so far, the present study focused on the development of monoclonal antibodies (mAbs) to antigens that are expressed exclusively on fetal NRBCs.Methods: Mice were immunized with fetal erythroid cell membranes and hybridomas screened for Abs using a multi-parameter fluorescence-activated cell sorting (FACS). Selected mAbs were evaluated by comparative FACS analysis involving Abs known to bind erythroid cell surface markers (CD71, CD36, CD34), antigen-i, galactose, or glycophorin-A (GPA). Specificity was further confirmed by extensive immunohistological and immunocytological analyses of NRBCs from umbilical cord blood and fetal and adult cells from liver, bone marrow, peripheral blood, and lymphoid tissues.Results: Screening of 690 hybridomas yielded three clones of which Abs from 4B8 and 4B9 clones demonstrated the desired specificity for a novel antigenic structure expressed on fetal erythroblast cell membranes. The antigenic structure identified is different from known surface markers (CD36, CD71, GPA, antigen-i, and galactose), and is not present on circulating adult erythroid cells, except for occasional detectability in adult bone marrow cells.Conclusions:The new mAbs specifically bind the same or highly overlapping epitopes of a surface antigen that is almost exclusively expressed on fetal erythroid cells. The high specificity of the mAbs should facilitate development of simple methods for reliable isolation of fetal NRBCs and their use in non-invasive prenatal diagnosis of fetal genetic status.     

Search for the optimal fetal cell antibody: results of immunophenotyping studies using flow cytometry

Fetal nucleated cells circulating in maternal peripheral blood are a noninvasive source of fetal DNA for prenatal genetic diagnoses. The successful isolation of fetal cells from maternal blood depends upon identification of differences between fetal and maternal cell surface antigen expression. To our best knowledge, a monoclonal antibody that binds only fetal blood cells has not yet been identified. We studied antigens recognized by six different monoclonal antibodies for their biologic expression on fetal blood cells as a function of gestational age, and compared their ability to bind fetal but not maternal cells. The results suggest a relationship between gestational age and nucleated cell surface antigen expression. The monoclonal antibodies FB3-2, H3-3, CD71 and 2-6B/6 are suitable reagents for first or early second trimester fetal cell isolation, although FB3-2 and H3-3 are more specific for fetal cells due to significantly lower expression of these antigens on maternal mononuclear cells. The observation that samples from fetuses with chromosome abnormalities or multiple structural anomalies express higher levels of these antigens indicates that these reagents will potentiate the detection of abnormal fetal cells in maternal blood samples. Received: 23 November 1996 / Accepted: 13 February 1997     

Clinical Problems: Quantitation of Transplacental Haemorrhage

On four occasions over a period of four years samples of adult blood to which known amounts of fetal blood had been added were distributed to 8-12 different laboratories taking part in clinical trials organized by an M.R.C. Working Party. Estimates were made of the proportion of fetal: adult red cells in the samples after preparing films by the acid-elution method. When the proportion of fetal: adult red cells was less than about 1:10,000, the highest and lowest estimates were separated by a factor of about 10. However, when the number of cells present was between about 1:100 and 1:1,000, most results were between half and twice the true number of cells present.It is pointed out that since fetal red cells are approximately 30% larger than adult red cells, and since only about 90% of fetal cells stain darkly in the acid-elution method, estimates of the proportion of darkly-staining cells in a film underestimate the volume of fetal red cells present by about one-third. A simple formula is proposed which corrects for this factor and which gives an estimate of the total volume of fetal red cells present, deduced from the ratio of fetal: adult red cells and assuming a maternal red cell volume at term of 1,800 ml.A method of screening blood films is suggested which, firstly, endeavours to standardize the density of adult red cells on films, and, secondly, takes into account the Poisson distribution. Thus limits are set for the number of fetal red cells which can be seen in scanning a given number of adult cells before the suspicion is aroused that a transplacental haemorrhage exceeding a certain amount is present.It is emphasized that the density of adult red cells on blood films varies very widely, and unless the cell density and the size of the low-power field are defined the practice of deducing the extent of transplacental haemorrhage from the number of fetal red cells seen per low-power field may lead to large errors.     

On the kinetics of erythroid cell differentiation in fetal mice. I. Microspectrophotometric determination of the hemoglobin content in erythroid cells during gestation

In a microspectrophotometric study, photographic emulsions and a computer are used for measuring the hemoglobin content of a large number (about 50,000) of erythroid cells in fetal mice. Histograms of the hemoglobin content in erythroid cells illustrate the kinetics of erythropoiesis in yolk sac derived nucleated cells in the fetal peripheral blood, in fetal liver, and in fetal spleen. After the occasional extrusion of their nucleus, yolk sac derived erythrocytes remain as “macrocytes” in fetal circulation two or three days longer than the nucleated yolk sac derived erythrocytes do. Erythrocytes in fetal liver have a constant hemoglobin content of 28 pg ² until day 17 of gestation. During further erythropoiesis in liver and then in the spleen, this amount is gradually adapted to the normal hemoglobin content in red blood cells of 16 pg.     

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.     

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。     

改良的PEP方法在无创性产前基因诊断中的应用

应用显微操作技术获取孕妇外周血中的单个有核红细胞,改良的PEP方法扩增单个有核红细胞的全基因组DNA;在此基础上,应用荧光标记聚合酶链反应扩增9个微卫星片段,进行基因型分析判定单个有核红细胞来源。综合性别和DMD基因内的数个STR位点连锁分析进行DMD基因诊断,应用PCR-STR连锁分析进行PKU基因诊断。结果显示,对10例DMD高危胎儿中的6例成功地进行了无创性产前基因诊断。同时对1例PKU也成功地进行了无创性产前基因诊断。改良的PEP方法扩增单个细胞的全基因组可以满足基因诊断的要求,是无创性产前基因诊断中一种极有价值的全基因组扩增的方法。 Abstract：We investigated the feasibility of using improved primer extension preamplificat ion method to diagnose DMD and PKU. The fetal nucleated red blood cells from the peripheral blood of pregnant women were detected and individually retrieved into glass capillary pipettes using a micromanipulator under microscopic observation. The whole genome of a single cell was amplified by improved primer extension preamplification (PEP).Genotypes were analyzed by amplifying the 9 STR fragments using fluorescence?PCR technique and NRBC's(nucleated red blood cell) origin w as determined.We diagnosed DMD prenatally using sex determination and linkage an alysis of several STR sites of dystrophin,and we diagnosed PKU prenatally using PCR?STR linkage analysis.6 of 10 potential DMD patients were diagnosed,includin g 1 male fetal patient,1 potential PKU patient was also diagnosed.The improved P EP method is a very valuable method of amplifying the whole genome of single cel ls,and the products of amplification are enough to the requirements of DNA in no n-invasive prenatal diagnosis.     

A practical application of computer pattern recognition research: the Abbott ADC-500 differential classifier.

The ADC-500 is a new blood cell differential classifier manufactured by Abbott Laboratories. It performs 500-cell leukocyte differentials on both normal and abnormal cells, evaluates red cell morphology and estimates platelet sufficiency at a rate of 40 to 50 samples per hour in stand-alone operation. The ADC-500 system consists of a spinner which prepares a uniform blood monolayer on a slide, a stainer which reproducibly stains the slide with Wright's stain, an encoder which attaches an instrument and human readable identification to the slide and an analyzer which accepts a stack of up to 50 slides, evaluates these slides and prints the results and the slide identification on report forms. The system's analysis rate, which represents a 5- to 10-fold increase over other commercially available differential counters, requires a number of specialized techniques for its realization. One key to this performance is the development of a high speed X-Y slide positioning stage which can move to a new cell and settle in 50 msec. Another is the high degree of parallelism used in the system structure and the pipelining of the data processing. A third is the development of uniform and repeatable sample preparation modules. Within the analyzer module, the autofocus, white cell acquisition and high resolution cell analysis systems are independent and operate in parallel. At the same time within the high resolution cell analysis system, one cell is acquired; the digitized image of a second processed; and a third is classified using pattern recognition techniques. All of these tasks, except focus, are under the control of a minicomputer system. Tests of the system reveal good accuracy and an improvement in precision due to the increase in the number of counted cells.     

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.     

Fetal cells in maternal blood: recovery by charge flow separation

Fetal blood cells can be recovered from the maternal circulation by charge flow separation (CFS), a method that obviates the risks associated with amniocentesis and chorionic villus sampling. By CFS, we processed blood samples from 13 women carrying male fetuses, 2 carrying fetuses with trisomy 21, and 1 who had delivered a stillborn infant with trisomy 18. On average more than 2000 fetal nucleated red blood cells were recovered per 20-ml sample of maternal blood. Recovery of fetal cells was confirmed by fluorescence in situ hybridization with probes for chromosomes Y, 18 and 21. After culturing of CFS-processed cells, amplification by the polymerase chain reaction revealed Y-chromosomal DNA in clones from four of six women bearing male fetuses, but not in clones from three women bearing female fetuses. Received: 8 January 1996 / Revised: 22 March 1996     

Detection and relocation of rare events. A comparative study using the laser scanning cytometer and the Metafer/RCDetect microscope scanning system

We compared instrumental analysis of enriched cord blood nucleated red blood cells (CB-NRBC) out of in vitro contamination preparations of dilutions of minute volumes of male cord blood into peripheral blood from nonpregnant women. This was done using the laser scanning cytometer (LSC) and the Metafer/RCDetect microscope scanning system, both allowing for relocation of positive cells defined on the basis of fluorescence parameters. Both instruments were efficient in performing scanning and relocation; a difference in the recovery of CB-NRBC was not significant and can be explained by the method of preparation used.     

Automated image detection and segmentation in blood smears.

A simple technique which automatically detects and then segments nucleated cells in Wright's giemsa-stained blood smears is presented. Our method differs from others in 1) the simplicity of our algorithms; 2) inclusion of touching (as well as nontouching) cells; and 3) use of these algorithms to segment as well as to detect nucleated cells employing conventionally prepared smears. Our method involves: 1) acquisition of spectral images; 2) preprocessing the acquired images; 3) detection of single and touching cells in the scene; 4) segmentation of the cells into nuclear and cytoplasmic regions; and 5) postprocessing of the segmented regions. The first two steps of this algorithm are employed to obtain high-quality images, to remove random noise, and to correct aberration and shading effects. Spectral information of the image is used in step 3 to segment the nucleated cells from the rest of the scene. Using the initial cell masks, nucleated cells which are just touching are detected and separated. Simple features are then extracted and conditions applied such that single nucleated cells are finally selected. In step 4, the intensity variations of the cells are then used to segment the nucleus from the cytoplasm. The success rate in segmenting the nucleated cells is between 81 and 93%. The major errors in segmentation of the nucleus and the cytoplasm in the recognized nucleated cells are 3.5% and 2.2%, respectively.     

Iodination-Coupled Tetrazonium and Ferric Ferricya-Nide Reduction Stains for Differentiating Red Blood Cells Containing Fetal or Adult Hemoglobin

Either the iodination-coupled tetrazonium reaction or the ferric ferricyanide reduction procedure can be used to differentiate red blood cells containing fetal hemoglobin (hemoglobin F) from those containing adult hemoglobin (hemoglobin A) in blood smears. Oxalated blood is diluted with 3 parts of physiological saline, and smears are made on slides. The air-dried slides are treated with absolute ethanol for 2 min, dried, and placed in phosphate-citrate buffer of pH 3.2-3.6 for 1 min at 37°C. They are then rinsed in distilled water, and dried for storage or stained at once by either the iodination-coupled tetrazonium or the ferric ferricyanide reduction procedure. Adult hemoglobin is extracted by the buffer, so that red blood cells containing fetal hemoglobin give a much darker stain than those containing adult hemoglobin. The hemoglobin S of patients with sickle-cell anemia behaves like adult hemoglobin.     

Prenatal asphyxia,hyperlacticaemia, hypoglycaemia,and erythroblastosis in growth retarded fetuses.

The umbilical venous oxygen and carbon dioxide tensions, pH, lactate and glucose concentrations, nucleated red cell (erythroblast) count, and haemoglobin concentration were measured in 38 cases of intrauterine growth retardation in which fetal blood sampling was performed by cordocentesis. The oxygen tension was below the normal mean for gestational age in 33 cases; in 14 it was below the lower limit of the 95% confidence interval for normal pregnancies. The severity of fetal hypoxia correlated significantly with fetal hypercapnia, acidosis, hyperlacticaemia, hypoglycaemia, and erythroblastosis. These findings indicate that "birth asphyxia" is not necessarily due to the process of birth.