Clinical Chemistry AACC Online Job Center
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 45: 1614-1620, 1999;
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Alvarez, F. V.
Right arrow Articles by Ladenson, J. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Alvarez, F. V.
Right arrow Articles by Ladenson, J. H.
Related Collections
Right arrow Molecular Diagnostics and Genetics
Right arrow Oak Ridge Conference
(Clinical Chemistry. 1999;45:1614-1620.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Development, Characterization, and Use of Monoclonal Antibodies Made to Antigens Expressed on the Surface of Fetal Nucleated Red Blood Cells

Francisco V. Alvarez1,2,a, Jitka Olander3, Dan Crimmins3, Belén Prieto1, Ana Paz1, Rebeca Alonso1, Sharon Porter3, Jay Hess4, Robert D. Crist5, Yvonne Landt3 and Jack H. Ladenson3

1 Servicio de Análisis Clínicos, Hospital San Agustín, Aviles 33400 Asturias, Spain.

2 Department of Biochemistry, University of Aviedo, 33006 Asturias, Spain.

3 Washington University School of Medicine, Department of Pathology, Division of Laboratory Medicine, 660 S. Euclid Ave., St. Louis, MO 63110.

4 Washington University School of Medicine, Department of Pathology, 660 S. Euclid, St. Louis, MO 63110.

5 Comprehensive Health, Overland Park, Kansas City, KS.
a Address correspondence to this author at: Servicio de Análisis Clínicos, Hospital San Agustín, c/Camino de Heros, 4 Avilés, 33400 Asturias, Spain. Fax 34 985 123025; e-mail falvarez{at}arrakis.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
For prenatal genetic testing (1)(2), an attractive strategy is to isolate fetal cells from peripheral maternal blood and genetically test them for inherited disease. Fetal nucleated red blood cells (NRBCs)1 are good candidates for the purpose of prenatal diagnosis for several reasons. They are mononuclear, abundant in first trimester fetal blood, and rare in adult peripheral blood (3)(4). In addition, nucleated erythrocytes express several antigens, such as the transferrin receptor expressed on rapidly dividing cells, that might be useful targets for enriching samples of maternal blood. These cells also produce unique fetal hemoglobin chains such as {zeta}, {epsilon}, and {gamma} (5), providing useful markers of fetal cells. Finally, erythrocytes are unlikely to persist from one pregnancy to the next (6). However, controversy exists regarding the origin of the NRBCs in maternal blood: some authors state that most nucleated erythrocytes in first trimester maternal blood are of maternal origin (7)(8).

The rarity of fetal cells in maternal circulation necessitates several enrichment techniques. A single, double, or triple density gradient is commonly used before separation strategies such as fluorescence-activated cell sorting (FACS) (9)(10)(11)(12), magnetic activated cell sorting (13)(14)(15), immunomagnetic beads (16)(17), antibody-conjugated columns (18), and charge flow separation (19)(20).

The most common strategy to enrich NRBCs uses a monoclonal antibody (mAb) that binds a cell surface antigen present on fetal NRBCs. The most widely used mAbs for fetal NRBC isolation are those directed against the transferrin receptor (CD71) (21)(22), the thrombospondin receptor (CD36) (23), and glycophorin A, a red cell sialoglycoprotein (21)(24).

To improve the purity and yield of fetal NRBCs isolated from maternal blood and to investigate the biology of materno-fetal transfer of cells, we produced and characterized murine mAbs to human fetal liver NRBCs and used some of these antibodies to isolate NRBCs from maternal blood.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
preparation of fetal NRBCs
Fetal liver tissue samples were obtained from medically approved terminations at 15–20 weeks of gestation. NRBCs were isolated essentially as described by Bhat et al. (25) except that Histopaque 1107 was used at the bottom of the double density gradient.

A negative procedure was used to remove leukocytes. Cells (2 x 109) in 2 mL of phosphate-buffered saline containing 5 mmol/L EDTA, 1 g/L human serum albumin, and 0.5 g/L NaN3 (PBS/HSA buffer) were incubated with 50 µL of microbeads coated with antibodies to CD45, CD14, CD15, or CD16 (Myltenyi Biotec) for 30 min at 4 °C. The cell-microbead mixture was washed and passed over a Myltenyi (Myltenyi Biotec) type CS+ magnetic separation column (2 x 108 bound cell capacity) according to the instructions of the manufacturer. The purity of the resulting NRBC preparation was assessed by visually examining cytospins (Shandon cytospin 3) stained with the nuclear stain giemsa and with 3,3'-dimethoxybenzidine, which stains hemoglobin.

development of hybridomas
Mice were immunized by intraperitoneal injections of 3 x 101 to 6 x 101 fetal NRBCs every 2 weeks for 6 weeks. All mice received an additional injection of NRBCs 72 h before fusion. Cell fusion was performed by the method of Waldman (26) with the P3 x 63-Ag8.653 myeloma fusion partner by the Washington University Hybridoma Center (St. Louis, MO). The hybridomas were screened for binding to cell surface antigens on fetal NRBCs by FACS analysis. Fetal NRBCs (40 µL) resuspended in PBS/HSA (1 x 101 cells) were incubated in 96-well plates with 10 µL of hybridoma supernatant for 30 min at 4 °C with agitation. The positive and negative controls were antibodies to CD71 and CD45, respectively (Becton Dickinson). After two washes, the cells were incubated as above with 40 µL of a 1:50 dilution of goat anti-mouse IgG Fab conjugated to fluorescein isothiocyanate (Sigma Diagnostics). The cells were washed again and resuspended in 500 µL of PBS for analysis in a FACScan (Becton Dickinson).

Adsorption of positive hybridomas to adult red blood cells (RBCs), subtypes A and B, eliminated mAbs to mature RBC antigens. We incubated 1 x 108 adult blood cells with 200 µL of hybridoma supernatant for 30 min at 4 °C with gentle agitation. After centrifugation, 150 µL of supernatant was incubated with 1 x 101 fetal NRBCs and screened as described above. The same supernatants without adsorption were analyzed concurrently. Only the hybridomas with unchanged positive fluorescence were considered specific for fetal NRBCs.

Anti-fetal NRBC mAbs were produced in ascites fluid and purified by protein A-agarose chromatography (Genex).

immunohistochemistry
The human T-cell lymphoblastoid cell line CEM, which expresses 5- to 100-fold more CD71 than normal resting lymphocytes (27), was purchased from American Type Culture Collection. White blood cells (WBCs) were isolated on Histopaque gradients (28) and were activated by incubation with 2 mg/L Concanavalin A (Sigma Diagnostics) for 48 h at 37 °C and 5% CO2. Cytospins of cells were fixed in methanol for 5 min at room temperature. The Vectastain anti-mouse IgG ABC-AP kit (Vector Laboratories) was used according to manufacturer's instructions. Binding was visualized with Vector Red alkaline phosphatase substrate and a hematoxylin counter stain (Sigma Diagnostics). The slides were viewed on an Olympus B 40x microscope.

mAb COMPETITION STUDIES
mAbs were radioiodinated using Iodogen (Pierce Chemical) according to manufacturer's recommendations, with a mean specific activity of 4 x 107 cpm/µg (280 Ci/mmol). For competition studies, 1 x 105 freshly purified fetal NRBCs were incubated with ~3 x 104 cpm 125I-labeled mAb (2 x 10-3 to 4 x 10-3 µg) and 10 µg of unlabeled competing mAb in a total volume of 100 µL PBS containing 10 g/L bovine serum albumin (BSA) for 2 h at 4 °C. The cells were pelleted at 2000g for 10 min at 4 °C and washed twice by resuspending in 200 µL of PBS/BSA/azide (PBS containing 10 g/L BSA and 0.5 g/L NaN3). The final pellet was counted in a gamma counter (Multiprias; Packard Instruments).

immunopurification of fetal nrbc antigens
Fetal NRBCs at 1 x 1010 to 4 x 1010 cells/L were lysed in 0.01 mol/L Tris, pH 8.0, 0.14 mol/L NaCl, 2.5 g/L NaN3, 0.1 mmol/L Pefabloc® (Boehringer Mannheim), 5 nmol/L iodoacetamide, 0.2 kIU/L aprotinin, and 5 mL/L Triton X-100 on ice for 1 h with occasional mixing.

Nuclei were removed by centrifugation at 300g for 10 min at 4 °C, and the resulting supernatant was made 2 g/L sodium deoxycholate. Unsolubilized membranes were removed by centrifugation at 48 000g for 1 h at 4 °C. Antigen was immunopurified from the supernatant as described by Springer (29) except that 0.1-mL columns of immunoabsorbant were used. The mean yield was 50 µg protein per 1 x 109 NRBCs, estimated by the Bradford (30) protein assay (Bio-Rad). Purified antigen was stored at -70 °C until use.

antigen identification
Antigen was digested with a CNBr mixture consisting of 239 µL of 880 mL/L formic acid (Aldrich), 46 µL of 0.35 g/L affinity-purified antigen, and 15 µL of 5 mol/L CNBr in acetonitrile (Aldrich) and reacted overnight. Samples were electrophoresed on 4–20% sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) minigels (31) with subsequent electrotransfer to PSQ-grade polyvinylidene difluoride membranes (Sigma), using a three-buffer semidry blotting system (32)(ESA); the stained bands were Edman sequenced (31). The unstained membrane was analyzed by Western blotting, using Amersham Life Sciences AuroProbe BL plus gold-labeled goat anti-mouse as the secondary mAb, and bands were detected using the silver enhancement kit.

enrichment of NRBCs FROM MATERNAL BLOOD
After informed consent was obtained under a protocol approved by San Agustín Hospital, 25-mL blood samples were collected from 252 pregnant women into Vacutainer Tubes containing EDTA anticoagulant. The majority of the women were average risk patients participating in routine prenatal screening recruited from clinical sites throughout the Principado de Asturias. In all cases, gestational age was confirmed by sonogram. Because amniocentesis was performed in only a few cases, we analyzed most of the samples without knowledge of fetal gender.

Mononuclear cells from peripheral maternal blood were isolated on a double histopaque gradient. The washed cells were resuspended in PBS/BSA/azide to a final concentration of 1 x 101 cells per 100 µL and incubated with 3 µg of one of our mAbs for 20 min at 4 °C. The cells were then incubated with magnetic microbeads conjugated to rat anti-mouse IgG antibody (20 µL per 107 cells; Miltenyi Biotech) for 20 min at 4 °C and separated on the Miltenyi miniMACS magnetic column type MS (1 x 107 bound cell capacity) as described by the manufacturer. The NRBCs were enumerated on cytospins.

pcr
Sorted cells were pelleted at 10 000g and snap frozen. For PCR, the cells were resuspended in 35 µL of water and boiled for 10 min. The following primer sequences were used:

Primers XES14 and XES15 flanked a single copy of Y-specific sequence consisting of 600 bp; primers SRY4 and SRY5 flanked a 80-bp sequence nested within the 600-bp sequence (33).

PCR mixture containing 1.7 mmol/L MgCl2, 0.2 mmol/L each dNTP, 200 ng of each primer (Integrated DNA Technologies), and 1 U of Taq polymerase (Boehringer Mannheim) in 50 µL of the PCR buffer provided by the manufacturer. The first of two amplifications was with primers Xes14 and Xes15; the reaction was denatured at 94 °C for 10 min and then underwent 34 cycles of denaturation at 94 °C for 1 min, annealing at 58 °C for 45 s, and elongation at 72 °C for 1, followed by a final 10 min at 72 °C. The second amplification was performed with 10 µL of the reaction product in fresh reagents and using the primers SRY4 and SRY5; a 2-min denaturation at 94 °C was followed by 34 cycles of denaturation at 94 °C for 20 s, annealing at 58 °C for 45 s, and elongation at 72 °C for 1 min, and then by a final 10 min at 72 °C.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NRBCs purified from fetal liver and used as immunogen are depicted in Fig. 1 , which shows that the majority of the cells are erythroblasts and NRBCs. From the group of antibodies specific for NRBCs in FACS analysis, four of the most effective were selected for further characterization. Table 1 summarizes the staining data for these four mAbs plus commercial antibodies to CD71. The mAbs stained NRBCs, CEM cells, and some mature RBCs, but not WBCs. This demonstrated specificity of the mAbs for NRBCs and implicated CD71 as the antigen recognized because the CEM cells overexpress this protein (27). Activated lymphocytes are also reported to express the transferrin receptor (27), and the in vitro-activated WBCs did stain albeit weakly. The staining of some mature RBCs may simply indicate that these are reticulocytes, which still display some transferrin receptor (34). Staining was not observed with a-CD71 (Becton Dickinson), the mAb to CD71 frequently used for sorting NRBCs, even at concentrations up to 10 mg/L, whereas our mAbs stained effectively at concentrations of 0.1–4 mg/L. This same a-CD71 mAb does bind to NRBCs because it was used as a positive control in our FACS screen of the hybridomas. Competitive binding experiments (data not shown) indicated that two of our mAbs (4B2.1 and 2F6.3) react with a similar epitope, whereas the other two (2E11.3 and 2B7.4) recognize a different one. Thus, our mAbs recognize at least two epitopes. The data also indicate that the Becton Dickinson a-CD71 mAb either binds to a third epitope or that it binds with much lower avidity.



View larger version (111K):
[in this window]
[in a new window]
  		Figure 1. Fetal NRBCs purified from fetal livers by negative selection (see Materials and Methods) and stained with Wright-Giemsa and 3,3'-dimethoxybenzidine.


View this table:
[in this window]
[in a new window]
  		Table 1. Staining by the mAbs of methanol-fixed cytospin preparations of five different cell types.

To confirm the identity of the antigen as transferrin receptor, we used our mAb immobilized on agarose as affinity columns to immunopurify antigen from fetal liver NRBCs. Two Coomassie-stained bands of approximately equal intensity were seen that migrate in the 85–110 kDa range under reducing conditions on 4–20% SDS-PAGE (Fig. 2 , lane 2). A Western blot of a duplicate lane indicated that only the ~110-kDa band reacted with a commercial anti-human transferrin receptor mAb H68.4 (Zymed) specific to residues 3–28 of the cytoplasmic tail (Fig. 2 , lane 3). Immunoblots with our mAbs yielded only a very weakly stained ~110-kDa band even after reaction with gold-labeled secondary mAb and three 45-min rounds of silver enhancement (data not shown).



View larger version (59K):
[in this window]
[in a new window]
  		Figure 2. Reduced SDS-PAGE on 4–20% gel of NRBC membrane antigen immunopurified using mAb 2F6.3.

Lane 1, molecular standards; lane 2, Coomassie blue-stained sample (~6 µg); lane 3, Western blot of duplicate lane immunostained with H68.4, a transferrin receptor mAb (Zymed). Arrow, 80-bp Y-specific amplified fragment.

In a separate experiment, the immunopurified proteins were transferred to polyvinylidene difluoride membranes and then Edman sequenced. No sequence was found for the ~110-kDa species, but the ~85-kDa band gave an 11-residue N-terminal sequence identical to human transferrin. The inability to sequence the 110-kDa antigen suggested a blocked NH2 terminus, thus requiring an internal amino acid sequence (35) for positive identification. Five major bands were discernible after CNBr digestion that corresponded to molecular masses of ~36, 29, 22.5, 17, and 14 kDa (not shown). Sequencing of 9–12 residues of these fragments allowed identification of the parent protein as the transferrin receptor, which is known to exist as a transmembrane, glycosylated, disulfide-linked homodimer (36). Under our experimental conditions, transferrin copurified with its receptor.

Data on the enrichment of NRBCs from maternal blood using our four mAbs are presented in Fig. 3 . Blood was obtained from 252 pregnant women at 15–18 weeks of gestation and was processed between 2 and 24 h after collection. The absolute number of NRBCs isolated from 25 mL of maternal blood ranged from 0 to 419 with three of the mAbs and from 3 to 336 with mAb 2B7.4. This mAb isolated 3–10 NRBCs in 10% of the samples, 11–50 NRBCs in 60% of samples, and 51–336 NRBCs in 30% of the maternal blood samples. We also found that the median and mean numbers of isolated NRBC were higher in samples processed between 2 and 6 h after maternal blood collection and that maternal WBC counts were higher in older samples (data not shown). In four samples in which the sex of the fetus was known, the enriched NRBC sample was split into three aliquots: one for microscopic examination and two for Y-chromosome PCR. Fig. 4 shows the PCR results for these four samples of enriched NRBCs. The PCR results confirmed the sex of the fetus established by amniocentesis.



View larger version (30K):
[in this window]
[in a new window]
  		Figure 3. Percentage of samples within the given range of NRBCs isolated per 25 mL of maternal blood for each mAb tested.

The total number of samples tested with each mAb was 78 with 2B7.4, 68 with 2E11.3, 57 with 2F6.3, and 48 with 4B2.1. mAb 2B7.4 isolated three or more NRBCs in all samples. The NRBC/leukocyte ratios were as follows: 1:4 to >1:300 for 2B7.4; 1:22 to >1:300 for 2E11.3; 1:5 to >1:300 for 2F6.3; and 1:7 to >1:300 for 4B2.1. The yield of NRBCs using mAb 2B7.4 was significantly better than the other three antibodies (P <=0.0002, Wilcoxon rank-sum test).



View larger version (89K):
[in this window]
[in a new window]
  		Figure 4. PCR amplification of single-copy Y-specific sequence from NRBC-isolated maternal blood.

Lanes 1 and 8, molecular markers; lane 2, male positive control (5 male WBCs + 1000 female WBCs); lane 3, female positive control (1000 female WBCs); lanes 4 and 5, male fetus; lanes 6 and 7, female fetus. The arrow at 80 bp indicates a male fetus.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Purification of fetal NRBCs for prenatal diagnosis presents a difficult challenge. The number of these cells in peripheral maternal blood has been reported to range from 1:104 to 1:08 WBCs (3)(19)(21)(37)(38). Because in adult women there are 6 x 101 to 9 x 101 WBCs per milliliter of blood, this translates into a reported range of 1–1800 NRBCs per 20 mL of maternal blood. However, the reported ranges of recovered NRBCs are much lower than this range (39)(40)(41) with the exception of Wachtel et al. (19).

The inconsistent presence of NRBCs in all pregnancies (7)(37)(39)(42) and the low yield reported suggest several questions that remain to be answered before the clinical utility of isolating fetal NRBCs for prenatal genetic diagnosis can be established. These questions involve whether fetal cells are present in all pregnancies and the extent to which the number of fetal cells are dependent on fetal status, gestational time, or maternal complications. To address these questions, we decided to develop reagents that might give better yields of fetal cells.

Our strategy was to generate high avidity, specific mAbs against fetal NRBCs by immunizing mice with fetal NRBCs purified from fetal livers. Our screening protocol selected mAbs that had high affinity for NRBCs but not for mature RBCs. The prominent surface antigen was identified as transferrin receptor (CD71), and competitive binding studies showed that mAbs 2E11.3 and 2B7.4 bind a similar epitope, whereas 4B2.1 and 2F6.3 bind to a different epitope on the transferrin receptor.

The weak reactivity of mAbs 4B2.1 and 2E11.3 in Western blots suggests that the mAbs recognize conformationally dependent epitopes on the receptor that are destabilized under denaturing conditions (43). Because 2E11.3 binds a different epitope than 4B2.1 and 2F6.3, both epitopes are conformationally sensitive.

The NRBC enrichment procedure, consisting of a double density gradient followed by magnetic activated cell sorting with four of our mAbs, was applied to 252 maternal blood samples. The mean number of NRBCs obtained varied with the mAb (Fig. 3Up ). More NRBCs were isolated when mAb 2B7.4 was used than the other three mAbs were used; 2E11.3 isolated the least. The mean number of NRBCs obtained using our enrichment procedure (0–419 per 25 mL of blood) is similar to that (0–220 per 30 mL of blood) reported by Oosterwijk et al. (44). However, we isolated one or more NRBCs in 96% of the samples, whereas Oosterwijk et al. detected NRBCs in only 66% of their cases. Our NRBC/leukocyte (purity) ratio was improved compared with these results (44) and those reported elsewhere (20)(21)(39)(41). An important factor in this ratio appeared to be the time between blood collection and gradient separation, which was also observed by Oosterwijk et al. (45). The lowest WBC contamination was observed when the cells were processed within 6 h of collection. Because our samples came from throughout Asturias, many of them could not be processed that quickly; however, all were processed within 24 h. mAb 2B7.4 not only isolated the highest number of NRBCs (>10 cells in 90% of the samples), but it isolated NRBCs in all 78 consecutive maternal blood samples studied, a success rate not described in the literature previously. Thus, 2B7.4 isolates sufficient NRBCs for fluorescence in situ hybridization and/or PCR in 90% of the cases and in the remaining 10% should provide sufficient cells for PCR analysis. It should be emphasized that the high NRBC yields with our mAbs were obtained in blood samples from a healthy population of pregnant women, not from a high-risk population in which higher yields of NRBCs have been reported (38).

To determine whether any of the NRBCs isolated from maternal blood were of fetal origin, PCR for the Y and X chromosomes was performed on some of the samples. In two cases in which a male pregnancy was known, the PCR correctly identified the Y chromosome, indicating a fetal source for the NRBCs (Fig. 4Up ). Subsequent PCR analysis detected the Y chromosome in 40% of 50 samples, and follow-up information will provide the gender at delivery (F.V. Alvarez, personal communication).

Further optimization of the enrichment procedure may be possible. For example, selective lysis of mature RBCs after the density gradient spin to remove reticulocytes, which may still express some transferrin receptor, may improve yields with mAbs 4B2.1 and 2F6.3. The simultaneous use of mAbs to different epitopes on the transferrin receptor may also increase yield. Work is in progress to identify the isolated NRBCs as fetal or maternal in origin, using fluorescence in situ hybridization for the Y chromosome and immunostaining for {gamma} or {epsilon} fetal hemoglobin.

In conclusion, our mAbs, especially 2B7.4, successfully enriched NRBCs from maternal blood samples with low maternal WBC contamination. Thus, the mAbs we have developed against the transferrin receptor on fetal NRBCs possess high specificity for this type of cell and allow the separation of NRBCs from maternal blood, which may be useful in assessing fetal genetic disorders.


   Acknowledgments
 
This project was supported by the Fondo de Investigacion Sanitaria (98/1530), Fundacion para el Fomento en Asturias de la Investigacion Cientifica Aplicada y la Tecnologia (PB-SAL 98-02), and Fundacion Inocente Inocente. F.V. Alvarez was supported in part by a fellowship from the Fondo de Investigaciones Sanitarias (95/5205). We thank Parveen Chand for expert technical assistance on flow cytometry, Curt Parvin for statistical assistance, and Ann Barczewski for technical assistance. We thank all of the women who participated in this study, and Drs. Elena Vega, Ana Escudero, Paloma Diaz Velazquez, Maria Fernandez-Marmiesse, and all of the physicians who collaborated in sending us maternal blood samples. The antibodies described in this paper are under option for exclusive license at this time through the Washington University Center of Technology Management.


   Footnotes
 
1 Nonstandard abbreviations: NRBC, nucleated red blood cell; FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline; HSA, human serum albumin; RBC, red blood cell; WBC, white blood cell; BSA, bovine serum albumin; and SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ashwood ER. Clinical chemistry of pregnancy. In: Burtis CA, Ashwood ER, eds. Tietz textbook of clinical chemistry, 2nd ed. Philadelphia, PA: WB Saunders, 1994:2107pp..
  2. Tabor A, Philip J, Madsen M, Bang J, Obel EB, Norgaard-Pedersen B. Randomized controlled trial of genetic amniocentesis in 4606 low-risk women. Lancet 1986;1:1287-1293. [ISI][Medline] [Order article via Infotrieve]
  3. Steele CD, Wapner RJ, Smith JB, Haynes MK, Jackson LG. Prenatal diagnosis using fetal cells isolated from maternal peripheral blood: a review. Clin Obstet Gynecol 1996;39:801-813. [ISI][Medline] [Order article via Infotrieve]
  4. Lamvu G, Kuller JA. Prenatal diagnosis using fetal cells from maternal circulation. Obstet Gynecol Survey 1997;52:433-437. [Medline] [Order article via Infotrieve]
  5. Mesker WE, Ouwerkerk-van Velzen MCM, Oosterwijk JC, Bernini LF, Golbus MS, Kanhai HHH, et al. Two-color immunocytochemical staining of gamma and epsilon type haemoglobin in fetal red cells. Prenat Diagn 1998;18:1131-1137. [ISI][Medline] [Order article via Infotrieve]
  6. Pearson HA. Life-span of the fetal red blood cell. J Pediatr 1967;70:166-171. [ISI][Medline] [Order article via Infotrieve]
  7. Slunga-Tallberg A, Wessman M, Ylinen K, von Koskull H, Knuutila S. Nucleated erythrocytes in enriched and unenriched peripheral venous blood samples from pregnant and nonpregnant women. Fetal Diagn Ther 1994;9:291-295. [ISI][Medline] [Order article via Infotrieve]
  8. Slunga-Tallberg A, El-Rifai W, Keinanen M, Ylinen K, Kurki T, Klinger K, et al. Maternal origin of nucleated erythrocytes in peripheral venous blood of pregnant women. Hum Genet 1995;96:53-57. [ISI][Medline] [Order article via Infotrieve]
  9. Elias S, Price J, Dockter M, Wachtel S, Tharapel A, Simpson JL, Klinger KW. First trimester prenatal diagnosis of trisomy 21 in fetal cells from maternal blood. Lancet 1992;340:1033.[ISI][Medline] [Order article via Infotrieve]
  10. Herzenberg LA, Bianchi DW, Schroder J, Cann HM, Iverson GM. Fetal cells in the blood of pregnant women: detection and enrichment by fluorescence-activated cell sorting. Proc Natl Acad Sci U S A 1979;76:1453-1455. [Abstract/Free Full Text]
  11. Iverson GM, Bianchi DW, Cann HM, Herzenberg LA. Detection and isolation of fetal cells from maternal blood using the fluorescence-activated cell sorter (FACS). Prenat Diagn 1981;1:61-73. [ISI][Medline] [Order article via Infotrieve]
  12. Bianchi DW, Flint AF, Pizzimenti MF, Knoll JHM, Latt SA. Isolation of fetal DNA from nucleated erythrocytes in maternal blood. Proc Natl Acad Sci U S A 1990;87:3279-3283. [Abstract/Free Full Text]
  13. Gènshirt-Ahlert D, Borjesson-Stoll R, Burschyk M. Detection of fetal trisomies 21 and 18 from maternal blood using triple gradient and magnetic cell sorting. Am J Reprod Immunol 1993;30:194-201.
  14. Gènshirt-Ahlert D, Burschyk M, Garritsen HSP, Helmer L, Miny P, Horst J, et al. Magnetic cell sorting and the transferrin receptor as potential means of prenatal diagnosis from maternal blood. Am J Obstet Gynecol 1992;166:1350-1355. [ISI][Medline] [Order article via Infotrieve]
  15. Busch J, Huber P, Pfluger E, Miltenyi ST, Holtz J, Radbruch A. Enrichment of fetal cells from maternal blood by high gradient magnetic cell sorting (double MACS) for PCR-based genetic analysis. Prenat Diagn 1994;14:1129-1140. [ISI][Medline] [Order article via Infotrieve]
  16. Mueller UW, Hawes CS, Wright AE, Petropoulos A, Deboni E, Firgaira TA, et al. Isolation of fetal trophoblast cells from peripheral blood of pregnant women. Lancet 1990;336:197-200. [ISI][Medline] [Order article via Infotrieve]
  17. Camaschella C, Alfarano A, Gottardi E. Prenatal diagnosis of fetal hemoglobin Lepore-Boston disease on maternal peripheral blood. Blood 1990;75:2102-2106. [Abstract/Free Full Text]
  18. Hall JM, Adams S, Williams S, Rehse MA, Layton TJ, Molesh DA. Purification of fetal cells from maternal blood using an avidin-biotin immunoaffinity column. Ann N Y Acad Sci 1994;731:115-127. [ISI][Medline] [Order article via Infotrieve]
  19. Wachtel SS, Sammons D, Twitty G, Utermohlen J, Tooley E, Phillips O, et al. Charge flow separation: quantification of nucleated red blood cells in maternal blood during pregnancy. Prenat Diagn 1998;18:455-463. [ISI][Medline] [Order article via Infotrieve]
  20. Wachtel SS, Sammons D, Manley M, Wachtel G, Twitty G, Utermohlen J, et al. Fetal cells in maternal blood: recovery by charge flow separation. Hum Genet 1996;98:162-166. [ISI][Medline] [Order article via Infotrieve]
  21. Price JO, Elias S, Wachtel S, Klinger K, Dockter M, Tharapel A, et al. Prenatal diagnosis with fetal cells isolated from maternal blood by multiparameter flow cytometry. Am J Obstet Gynecol 1991;165:1731-1737. [ISI][Medline] [Order article via Infotrieve]
  22. Bianchi DW, Mahr A, Zickwolf GK, Houseal TW, Flint AF, Klinger KW. Detection of fetal cells with 47, XY, +21 karyotype in maternal peripheral blood. Hum Genet 1992;90:368-370. [ISI][Medline] [Order article via Infotrieve]
  23. Bianchi DW, Zickwolf GK, Yih MC, Flint AF, Geifman OH, Erikson MS, et al. Erythroid-specific antibodies enhance detection of fetal nucleated erythrocytes in maternal blood. Prenat Diagn 1993;3:293-300.
  24. Wachtel S, Elias S, Price J, Wachtel G, Phillips O, Shulman L, et al. Fetal cells in the maternal circulation: isolation by multiparameter flow cytometry and confirmation by polymerase chain reaction. Human Reprod 1991;6:1466-1469. [Abstract/Free Full Text]
  25. Bhat NM, Bieber MM, Teng NNH. One step separation of human fetal lymphocytes from nucleated red blood cells. J Immunol Methods 1990;131:147-149. [ISI][Medline] [Order article via Infotrieve]
  26. Waldman MHC. Production of murine monoclonal antibodies. Methods Hematol 1986;13:1-20.
  27. Larrick JW, Cresswell P. Modulation of cell surface iron transferrin receptors by cellular density and state of activation. J Supramol Struct 1979;11:579-586. [ISI][Medline] [Order article via Infotrieve]
  28. English D, Anderson BR. Single step separation of red blood cells, granulocytes and mononuclear leukocytes on discontinuous density gradient of ficoll-hypaque. J Immunol Methods 1974;5:249-252. [ISI][Medline] [Order article via Infotrieve]
  29. Springer TA. Isolation of proteins using antibodies. In: Coligan JE, Kruisbeck AM, Margulies DH, Shevach EM, Strober W, eds. Current protocols in immunology. New York: John Wiley & Sons, 1992:821pp..
  30. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal Biochem 1976;72:248-254. [ISI][Medline] [Order article via Infotrieve]
  31. Crimmins DL, McCourt DW, Thomas RS, Scott MG, Macke K, Schwartz BD. In situ chemical cleavage of proteins immobilized to glass-fiber and polyvinylidenedifluoride membranes. Cleavage at tryptophan residues with 2-(2'-nitrophenylsulfenyl)-3-methyl-3'-brominodolenine to obtain internal amino acid sequence. Anal Biochem 1990;187:27-38. [ISI][Medline] [Order article via Infotrieve]
  32. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods 1984;10:203-209. [ISI][Medline] [Order article via Infotrieve]
  33. Sinclair AH, Berta P, Palmer MS, Hawkins JR, Griffiths BL, Smith MJ, et al. A gene from human sex determining region encoding a protein. Nature 1990;346:240-244. [Medline] [Order article via Infotrieve]
  34. Loken MR, Shah VO, Dattilio KL, Civin CI. Flow cytometric analysis of human bone marrow. I. Normal erythroid development. Blood 1987;69:255-263. [Abstract/Free Full Text]
  35. Scott MG, Crimmins DL, McCourt DW, Tarrand JJ, Eyerman ML, Nahm MH. A simple in situ cyanogen bromide cleavage method to obtain internal amino acid sequence of proteins electroblotted to PVDF membranes. Biochem Biophys Res Comm 1988;155:1353-1359. [ISI][Medline] [Order article via Infotrieve]
  36. Enns CA, Rutledge EA, Williams AM. The transferrin receptor. Biomembranes 1996;4:255-287.
  37. Hamada H, Arinami T, Kubo T, Hamaguchi H, Iwasaki H. Fetal nucleated cells in maternal peripheral blood: frequency and relationship to gestational age. Hum Genet 1993;91:427-432. [ISI][Medline] [Order article via Infotrieve]
  38. Bianchi DW, Williams JM, Sullivan LM, Hanson FW, Klinger KW, Shuber AP. PCR quantitation of fetal cells in maternal blood in normal and aneuploid pregnancies. Am J Hum Genet 1997;61:822-829. [ISI][Medline] [Order article via Infotrieve]
  39. Reading JP, Huffman JL, Wu JC, Reading JP, Huffman JL, Wu JC, et al. Nucleated erythrocytes in maternal blood: quantity and quality of fetal cells in enriched populations. Mol Hum Reprod 1995;10:2510-2515.
  40. Little M-T, Langlois S, Wilson RD, Lansdrop PM. Frequency of fetal cells in sorted sub-populations of nucleated erythroid and CD34+ hematopoetic progenitor cells from maternal peripheral blood. Blood 1997;89:2347-2358. [Abstract/Free Full Text]
  41. Bianchi DW, Shuber AP, De Maria M, Fougner AC, Klinger KW. Fetal cells in maternal blood: determination of purity and yield by quantitative polymerase chain reaction. Am J Obstet Gynecol 1994;171:922-926. [ISI][Medline] [Order article via Infotrieve]
  42. Nakagome Y, Seke S, Nagafuchi S, Nakahori Y, Sato K. Absence of fetal cells in maternal circulation at a level of 1 in 25,000. Am J Med Genet 1991;40:506-508. [ISI][Medline] [Order article via Infotrieve]
  43. Harlow E, Lane D, eds. Immunoblotting. In: Antibodies: a laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratories, 1988:474..
  44. Oosterwijk JC, Mesker WE, Ouwerkerk-van Velzen MCM, Knepflé CFHM, Wiesmeijer KC, Beverstock GC, et al. Fetal cell detection in maternal blood: a study in 236 samples using erythroblast morphology, DAB and HbF staining, and FISH analysis. Cytometry 1998;32:178-185. [ISI][Medline] [Order article via Infotrieve]
  45. Oosterwijk JC, Mesker WE, Ouwerkerk-van Velzen MCM, Knepflé CFHM, Wiesmeijer KC, van den Burg MJM, et al. Development of a preparation and staining method for fetal erythroblasts in maternal blood: simultaneous immunocytochemical staining and FISH analysis. Cytometry 1998;32:170-177. [ISI][Medline] [Order article via Infotrieve]



The following articles in journals at HighWire Press have cited this article:


Home page
 J. Histochem. Cytochem.Home page
A. Fernandez, B. Prieto, A. Escudero, J. H. Ladenson, and F. V. Alvarez
A Monoclonal Antibody with Potential for Aiding Non-invasive Prenatal Diagnosis: Utility in Screening of Pregnant Women at Risk of Preeclampsia
J. Histochem. Cytochem., March 1, 2005; 53(3): 345 - 350.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
G. Coata, F. Tilesi, M. Fizzotti, V. Lauro, L. Pennacchi, A. Tabilio, and G. C. Di Renzo
Prenatal Diagnosis of Genetic Abnormalities Using Fetal CD34+ Stem Cells in Maternal Circulation and Evidence They Do Not Affect Diagnosis in Later Pregnancies
Stem Cells, November 1, 2001; 19(6): 534 - 542.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Alvarez, F. V.
Right arrow Articles by Ladenson, J. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Alvarez, F. V.
Right arrow Articles by Ladenson, J. H.
Related Collections
Right arrow Molecular Diagnostics and Genetics
Right arrow Oak Ridge Conference

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS