A Monoclonal Antibody with Potential for Aiding Non-invasive Prenatal Diagnosis : Utility in Screening of Pregnant Women at Risk of Preeclampsia
Servicio de Análisis Clínicos, Hospital San Agustín, Avilés, Spain (AF); Servicio de Bioquímica (BP,FVA) and Servicio de Ginecología (AE), Hospital Universitario Central de Asturias, Oviedo, Spain (AE); Division of Laboratory Medicine, Department of Pathology and Immunology, Washington University, St. Louis, Missouri (JHL); and Servicio de Bioquímica, Departamento de Bioquímica y Biología Molecular, Universidad de Oviedo, Spain (FVA)
Correspondence to: Francisco V. Alvarez, Servicio de Bioquímica, Hospital Universitario Central de Asturias, Celestino Villamil s/n, 33006, Oviedo, Asturias, Spain. E-mail: falvarez{at}arrakis.es
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
(J Histochem Cytochem 53:345350, 2005)
Key Words: erythroblasts fetal cells preeclampsia pregnancy-induced hypertension magnetic cell sorting fluorescence in situ hybridization
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Many researchers have selected nucleated red blood cells (NRBC) as the target cells for the development of a non-invasive method of prenatal diagnosis in maternal blood. Erythroblasts have several advantages over other types of fetal cells, such as their relative abundance in the early fetal circulation, the expression of antigens that allows their enrichment and identification, and their short life span, which precludes the isolation of fetal cells from previous pregnancies (Pearson 1967; Sitar et al.1997
; Bianchi and Lo 2001
).
Because of the scarcity of fetal cells in maternal circulation, approaches are needed to enrich fetal erythroblasts from maternal peripheral blood (Steele et al. 1996). Magnetic cell sorting has been the most widely employed method. This approach is based on the binding of an immunomagnetically labeled monoclonal antibody (mAb) to a cell-surface antigen present on fetal NRBC. The most widely used mAbs for fetal NRBC isolation are those directed against the transferrin receptor (CD71) (Gänshirt et al. 1992
).
Preeclampsia is a multisystem disorder characterized by hypertension and proteinuria that occurs late in the second or, more frequently, in the third trimester of pregnancy. The disease adversely affects 3-5% of pregnancies and is one of the most important causes of maternal and fetal mortality and morbidity in developed countries. It is associated with substantial risks, such as intrauterine growth restriction with attendant complications for the fetus, prematurity, and death. The mother becomes at risk of seizures (eclampsia), renal failure, pulmonary edema, stroke, and death (Roberts and Redman 1993; Ness and Roberts 1996
). The pathogenesis of preeclampsia is still not completely understood, but placental dysfunction is thought to be the primary cause of the disease (Redman 1991
; Dekker and Sibai 1998
; Redman and Sargent 2001
; Solomon and Seely 2004
). Although the symptoms of preeclampsia become apparent only in the second half of pregnancy, the underlying pathological causes in the placenta occur much earlier. No reliable test exists to identify women at risk of developing the disorder early enough in their pregnancies to permit preventive treatment. In the last few years, however, several studies described the elevation in fetal cell traffic and the increased release of fetal DNA into maternal circulation of pregnant women affected by preeclampsia (Holzgreve et al. 1998
; Holzgreve and Hahn 1999
; Lo et al. 1999
; Al-Mufti et al. 2000
; Jansen et al. 2001
).
We have developed several anti-CD71 mAbs against fetal erythroblasts (Alvarez et al. 1999) and have optimized a protocol for their isolation from peripheral maternal blood (Prieto et al. 2001
). In a previous study done with pregnant women at risk of fetal aneuploidy, we found that one of our mAbs, 2F6.3, isolated a higher number of erythroblasts than a commercially available anti-CD71 antibody (data not shown). Therefore, we selected this mAb for further investigations.
In this study, we compare the efficacy of our 2F6.3 mAb versus a commercial anti-CD71 antibody in the isolation of erythroblasts from blood samples of pregnant women with pregnancy-induced hypertension (PIH), who thus are at risk of developing preeclampsia, and from a control group of pregnant women without suggested clinical features of any disease, at similar gestational ages.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patients
Pregnant women attending the Department of Obstetrics and Gynecology at the Hospital Universitario Central de Asturias (Oviedo, Spain) were recruited for the study with informed consent approved by Human Studies Committee of our hospital. Maternal blood samples (30 ml) were collected from 30 pregnant women suffering PIH and from 13 pregnant women, matched for gestational age, without suggested clinical features of developing preeclampsia (control group). Blood was collected into Vaccutainer tubes (Becton Dickinson, Plymouth, UK) containing EDTA as anticoagulant.
PIH was defined as a sustained rise in diastolic blood pressure to 90 mm Hg or higher above previous values. The pregnant women in the control group were not hypertensive.
The mean gestational ages were 35 weeks (range: 2140 weeks) and 34 weeks (range: 3236) for the PIH and control groups, respectively.
NRBC Enrichment from Maternal Blood
We processed blood samples within 1 hr from being drawn. We carried out Double Histopaque (Sigma, St Louis, MO) density gradient centrifugation as described elsewhere (Prieto et al. 2001). Both plasm/1.077 and 1.077/1.107 interfaces were collected separately and split into two aliquots. We processed the aliquots as paired samples using two different mAbs against the transferrin receptor: 2F6.3, and a commercial anti-CD71 antibody (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany). We incubated cell suspensions with 2F6.3 mAb or the commercial magnetically labeled anti-CD71 antibody for 15 min at 4C. We carried out a second incubation with magnetic microbeads conjugated to rat anti-mouse IgG2 antibody (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) for the fraction processed with 2F6.3 antibody, because this mAb is not magnetically labeled. Labeled cells in both fractions were immediately separated on miniMACS columns (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Aliquots of the positively selected cells were cytospun onto slides (Cytospin 3, Shandon, Astmoor, UK).
Identification and Detection of NRBC
Slides were air-dried overnight before being fixed in methanol and stained with diaminobenzidine (DAB, Sigma, St Louis, MO) to detect the presence of hemoglobin and with hematoxylin to counterstain the nuclei. NRBC were identified according to their morphology and DAB staining. The total number of NRBC on the entire cytospun area was enumerated.
Florescence In Situ Hybridization Analysis
We treated the slides with HCl in 70% ethanol for 5 min to remove hematoxylin. We then incubated the slides with RNase A (Sigma, St Louis, MO) in a humidified chamber for 30 min at 37C. After two washings (2 x SSC and then PBS), the slides were treated, for 10 min at 37C, with a 0.0075% solution of pepsin in 0.01 N HCl and then washed three times with PBS for 5 min. The slides were fixed in a 4% formaldehyde/PBS solution for 5 min. After two PBS washings, slides were dehydrated in series of 70%, 85%, and 100% ethanol.
We performed dual-color fluorescence in situ hybridization (FISH) analysis using Spectrum CEP Y green and Spectrum CEP X orange directly labeled probes (Vysis Inc., Downers Grove, IL). We sealed the slides with rubber cement and placed them in a prewarmed humidified incubator (Hybrite, Vysis). They were incubated for 2 min at 90C for denaturation, and then hybridization was carried out overnight at 37C. The next day, rubber cement was removed, and the slides were washed three times in 50% formamide/2 x SSC for 10 min at 46C, following by two washings at 46C (first 10 min in 2 x SSC and then 5 min in 2 x SCC/0.1% Tween 20). Cells were counterstained with 4,6-diamidino-2-phenyl-indol (DAPI II; Vysis, Weisbaden-Delkenheim, Germany) and analyzed with a fluorescence microscope (Leica DMR, Heerbrugg, Switzerland).
Data Analysis
The data were analyzed by SPSS 12.0 Statistical Software Package for Windows (SPSS Inc., Chicago, IL). Because data distribution was not Gaussian, comparison between the number of NRBC isolated using 2F6.3 and the Miltenyi anti-CD71 mAb was carried out using nonparametric tests (Wilcoxon Signed Ranks Test and U-Mann Whitney Test). A p value of <0.05 was considered statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Twelve of the 30 pregnant women with PIH developed preeclampsia. We compared the number of NRBC obtained from the women who developed preeclampsia with the number from the control group. 2F6.3 antibody isolated a higher median number of NRBC in preeclampsia samples (500 versus 262, respectively), but the difference was not statistically significant. The median of the ratio of total cells/NRBC was significantly higher in preeclampsia group, however: 1 NRBC in 3537 total cells, versus 1 NRBC in 8502 total cells in normotensive samples (p<0.05; U-Mann Whitney Test).
We used FISH to analyze maternal samples from pregnant women carrying a male fetus to verify fetal NRBC recovery. We processed blood samples from 10 women with PIH and from 6 normotensive pregnant women with both antibodies. In pregnant women at risk of developing preeclampsia, we observed fetal erythroblasts (NRBC with one X and one Y signal) in 60% of the samples processed with 2F6.3 mAb but in only 30% of the same samples processed with the Miltenyi antibody. In the six normotensive pregnancies bearing male fetuses, we found fetal cells in half of the samples tested with both antibodies. The number of fetal NRBC was significantly higher in the samples processed with 2F6.3 mAb (p<0,05; Wilcoxon Signed Ranks Test). In the PIH group, the median percentage of fetal erythroblasts was 0.8% (range: 0.012.5%) and 0.0% (range: 0.07.7%) for 2F6.3 and the Miltenyi anti-CD71 antibody, respectively. In the control group, the medians were 1.8% (range: 0.027.3) and 0.0% (0.014.3), respectively. There were no statistically significant differences between the percentage of nuclei with one X and one Y signal in hypertensive and normal pregnancies. Table 3 presents FISH analysis with 2F6.3 and the commercial anti-CD71 antibodies in both groups.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, one of our antibodies, 2F6.3, demonstrated a higher efficacy in isolating NRBC from peripheral blood of pregnant women at risk of developing preeclampsia than a commercial anti-CD71 antibody; 2F6.3 mAb isolated a significantly higher number of NRBC with less maternal contamination. When we compared these antibodies in normotensive pregnant women, we observed the same result. In a previous study performed with samples from pregnant women at risk of carrying an aneuploidy fetus, 2F6.3 mAb also isolated more NRBC than other antibodies (unpublished data). These findings suggest that this antibody could be a useful tool for non-invasive prenatal diagnosis.
The causes of preeclampsia remain unclear, but the underlying changes leading to the disorder occur early in pregnancy, before manifestation of the symptoms. Gänshirt et al. (1994) described the elevation of the number of erythroblasts in pregnancies affected by preeclampsia. Studies performed on samples collected during the second trimester indicate that this disorder occurs early in those pregnancies in which preeclampsia subsequently develop (Holzgreve et al. 2001b
; Zhong et al. 2001
; Cotter et al. 2002
; Zhong et al. 2002
). Our data showed a higher number of NRBC in the blood of patients with PIH than in control samples, but the increase was not statistically significant (p>0.05). Not all hypertension in pregnancy is caused by preeclampsia. Gestational hypertension without proteinuria or other systemic manifestations is frequently confused with preeclampsia but usually has a benign course (Sibai 1996
). In this study, only 12 of the 30 pregnant women included in the group at risk for preeclampsia actually developed the disorder. We did not find statistically significant differences in the number of NRBC isolated from preeclampsia and from normotensive samples, but we observed a tendency for an enhanced number of NRBC in preeclampsia cases. A feature observed in all the reports, as well as in our study, is that the number of erythroblasts was not increased in all cases of preeclampsia, and a degree of overlap was seen between preeclampsia and control women groups (Hahn and Holzgreve 2002
).
We used FISH probes for X and Y chromosomes to quantify and compare the recovery of fetal NRBC in samples from PIH and normotensive pregnant women carrying male singleton fetuses. Using FISH, Holzgreve et al. (1998) showed a significant proportion of fetal erythroblasts in preeclampsia (up to 50%), but our findings suggest that NRBC isolated in the maternal circulation of the PIH group are predominantly maternal in origin.
The number of fetal NRBC isolated was relatively low with both antibodies but was significantly higher in samples processed with 2F6.3 mAb. The proportion of fetal erythroblasts obtained by our group was comparable to that described by other researchers (Al-Mufti et al. 2000). Several authors have described difficulties that occur during FISH analysis of the sorted erythroblasts. Micromanipulation and PCR analysis of a single fetal cell has been described as a more efficient method than FISH to analyze fetal erythroblasts (von Eggeling et al, 1997
; Troeger et al. 1999
; Zhong et al. 2002
). FISH requires careful handling of cells at all stages of the treatment because of the risk of loss. The scarcity of fetal erythroblasts in the maternal circulation, even after enrichment, requires high hybridization efficiency for DNA probes. In this study, hybridization efficiency median was 90% for both mAbs. The efficiency of 2F6.3 antibody in isolating a higher number of NRBC with less maternal contamination, however, could explain why FISH analysis correctly identified fetal sex in 60% of pregnant women carrying a male fetus when this antibody was used for the enrichment, whereas the commercial anti-CD71 mAb correctly identified only 30% of the male fetuses.
Although increased trafficking of fetal nucleated cells in women suffering preeclampsia has been described, in this study FISH analysis did not show differences between the number of fetal NRBC isolated in the PIH and control groups. In this study, only 12 of the 30 pregnant women with PIH actually developed preeclampsia. Additional research will also be required to investigate whether abnormal fetal cell traffic of NRBC may be detectable even before the development of the clinical signs of preeclampsia. The presence of fetal erythroblasts in maternal blood, in combination with other biochemical markers, would be useful in screening for preeclampsia. The 2F6.3 antibody has demonstrated to be an efficient tool for NRBC recovery from peripheral maternal blood.
![]() |
Acknowledgments |
---|
We thank all the women who participated in this study and all the physicians who collaborated by sending us maternal blood samples.
![]() |
Footnotes |
---|
Received for publication May 18, 2004; accepted December 16, 2004
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Al-Mufti R, Hambley H, Albaiges G, Lees C, Nicolaides KH (2000) Increased fetal erythroblasts in women who subsequently develop pre-eclampsia. Hum Reprod 15:16241628
Alvarez FV, Olander J, Crimmins D, Prieto B, Paz A, Alonso R, Porter S, et al. (1999) Development, characterization and use of monoclonal antibodies made to antigens expressed on the surface of fetal nucleated red blood cells. Clin Chem 45:16141620
Bianchi DW (1999) Fetal cells in the maternal circulation: feasibility for prenatal diagnosis. Br J Haematol 105:574583[CrossRef][Medline]
Bianchi DW, Lo YMD (2001) Fetomaternal cellular and plasma DNA trafficking: the yin and the yang. Ann NY Acad Sci 945:119131
Cotter AM, Martin CM, O'Leary JJ, Daly SF (2002) Increased maternal cell trafficking in early pregnancy is associated with an increased risk of pre-eclampsia. Hypertens Pregnancy 21:65[CrossRef][Medline]
Dekker GA, Sibai BM (1998) Etiology and pathogenesis of preeclampsia: current concepts. Am J Obstet Gynecol 179:13591375[Medline]
Gänshirt D, Burschyk M, Garritsen HSP, Helmer L, Miny P, Horst J, Schneider HP, et al. (1992) Magnetic cell sorting and the transferrin receptor as a potential means of prenatal diagnosis from maternal blood. Am J Obstet Gynecol 166:13501355[Medline]
Gänshirt D, Borjesson-Stoll R, Burschyk M, Garrisen HS, Neusser M, Smeets FW, Velasco M, et al. (1994) Successful prenatal diagnosis from maternal blood with magnetic-activated cell sorting. Ann NY Acad Sci 731:103114[Medline]
Gänshirt D, Garritsen HSP, Holzgreve W (1995) Fetal cells in maternal blood. Curr Opin Obstet Gynaecol 7:103108[Medline]
Hahn S, Holzgreve W (2002) Fetal cells and cell-free fetal DNA in maternal blood: new insights into pre-eclampsia. Hum Reprod Update 8:501508
Holzgreve W, Ghezzi F, Di Naro E, Maymon E, Gänshirt D, Hahn S (1998) Disturbed fetomaternal cell traffic in preeclampsia. Obstet Gynecol 91:669672
Holzgreve W, Hahn S (1999) Novel molecular biological approaches for the diagnosis of pre-eclampsia. Clin Chem 45:451452
Holzgreve W, Hahn S (2001a) Prenatal diagnosis using fetal cells and free DNA in maternal blood. Clin Perinatol 28:353365[Medline]
Holzgreve W, Li JC, Steinborn A, Kulz T, Sohn C, Hodel M, Hahn S (2001b) Elevation in erythroblast count in maternal blood before the onset of pre-eclampsia. Am J Obstet Gynecol 184:165168[CrossRef][Medline]
Jansen MW, Korver-Hakkennes K, van Leenen D, Visser W, in't Veld PA, de Groot CJ, Wladimiroff JW (2001) Significantly higher number of fetal cells in the maternal circulation of women with pre-eclampsia. Prenat Diagn 21:10221026[CrossRef][Medline]
Leung TN, Zhang J, Lau TK, Chan LYS, Lo YMD (2001) Increased maternal plasma fetal DNA concentrations in women who eventually develop preeclampsia. Clin Chem 47:137139
Lo YMD (1994) Non-invasive prenatal diagnosis using fetal cells in maternal blood. J Clin Pathol 47:10601065[Medline]
Lo YMD, Lo ESF, Watson N, Noakes L, Sargent IL, Thilaganathan B, Wainscoat JS (1996) Two-way cell traffic between mother and fetus: biologic and clinical implications. Blood 88:43904395
Lo YMD, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, Wainscoat JS (1997) Presence of fetal DNA in maternal plasma and serum. Lancet 350:485487[CrossRef][Medline]
Lo YMD, Leung TN, Tein MSC, Sargent IL, Zhang J, Lau TK, Haines CJ, et al. (1999) Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clin Chem 45:184188
Lo YMD (2000) Fetal DNA in maternal plasma: biology and diagnostic applications. Clin Chem 46:19031906
Ness RB, Roberts JM (1996) Heterogeneous causes constituting the single syndrome of pre-eclampsia: a hypothesis and its implications. Am J Obstet Gynecol 175:13651370[Medline]
Pearson HA (1967) Life-span of the fetal red blood cell. J Pediatr 70:166171[Medline]
Prieto B, Alonso R, Paz A, Cándenas M, Venta R, Ladenson JH, Alvarez FV (2001) Optimization of nucleated red blood cell (NRBC) recovery from maternal blood collected using both layers of a double density gradient. Prenat Diagn 21:187193[CrossRef][Medline]
Redman CW (1991). Current topic: pre-eclampsia and the placenta. Placenta 12:301308[Medline]
Redman CW, Sargent IL (2001) The pathogenesis of preeclampsia. Gynecol Obstet Fertil 29:518522[Medline]
Roberts J, Redman C (1993) Pre-eclampsia: more than pregnancy-induced hypertension. Lancet 341:14471451[CrossRef][Medline]
Sibai BM (1996) Treatment of hypertension in pregnant women. New Engl J Med 335:257264
Sitar G, Manenti L, Farina A (1997) Characterization of the biophysical properties of human erythroblasts as a preliminary step to the isolation of fetal erythroblasts form maternal blood for noninvasive prenatal genetic investigation. Haematologica 82:510[Medline]
Solomon CG, Seely EW (2004) Preeclampsia: searching for the cause. N Engl J Med 350:641642
Steele CD, Wapner RJ, Smith JB, Haynes MK, Jackson LG (1996) Prenatal diagnosis using fetal cells isolating from maternal peripheral blood: a review. Clin Obstet Gynecol 39:801813[CrossRef][Medline]
Troeger C, Zhong XY, Burgemeister R, Minderer S, Tercanli S, Holzgreve W, et al. (1999) Approximately half of the erythroblasts in maternal blood are of fetal origin. Mol Hum Reprod 5:11621165
von Eggeling F, Michel S, Günther M, Schimmel B, Claussen U (1997) Determination of the origin of single nucleated cells in maternal circulation by means of random PCR and a set of length polymorphism. Hum Genet 99:266270[CrossRef][Medline]
Zhong XY, Holzgreve W, Hahn S (2001) Circulatory fetal and maternal DNA in pregnancies at risk and those affected by pre-eclampsia. Ann NY Acad Sci 945:138140
Zhong XY, Holzgreve W, Hahn S (2002) The levels of circulatory fetal DNA in maternal plasma are elevated prior to onset of pre-eclampsia. Hypertens Preg 21:7783[Medline]