Biomedical subjects
Publications and source records attributed to Y Brossard.
Umbilical cord blood T cells are less functional. Different explanations have been proposed. In this study we analyze the Vbeta T cell cord blood repertoire. All the Vbeta families are expressed. We found only the overexpression of three Vbeta: Vbeta 5-1, Vbeta 5-2, Vbeta 21-2.
Parents of a sample of 76 same sexed pairs of twins aged 3 to 9 months completed a mailed similarity questionnaire. It included the Bonnelykke et al.'s questionnaire and a four anthropological variable scale. To improve each of these two methods, three other combined methods were carried out and results were compared with the biological zygosity diagnosis. The Bonnelykke et al.'s classification combined with anthropological scale (method 4) gave only 1.2% misclassified in the whole sample. It is concluded that zygosity diagnosis using this type of procedure to distinguish MZ and DZ pairs would be important not only for epidemiological study but also for pediatricians and parents.
We have compared the sensitivity and specificity of four PCR methods of RHD gene detection using different sets of primers located in the regions of highest divergence between the RHD and RHCE genes, notably exon 10 (method I), exon 7 (method II), exon 4 (method III) and intron 4 (method IV). Methods I-III were the most sensitive and gave a detectable signal with D-pos/D-neg mixtures containing only 0.001% D-positive cells. Moreover, method II could detect the equivalent DNA amount present in only three nucleated cells in the assay without hybridization of PCR products, whereas the sensitivity of the other methods was 10-50 times less. Investigation of D variants indicated that false-negative results were obtained with method II (D(IVb) variant), method III (D(VI) and DFR variants) and method IV (D(VI) variants), but not method I. Weak D (D(u)) was correctly detected as D-positive by all methods, but most cases of Rh(null) appeared as false-positives, as they carry normal RH genes that are not phenotypically expressed. Some false-positive results were obtained with method I in a few Caucasian DNA samples serotyped as RhD-neg but carrying a C- or E-allele, whereas a high incidence of false-positives was found among non-Caucasian Rh-negative samples by all methods. In the Caucasian population, however, we found a full correlation between the predicted genotype and observed phenotype at birth of 92 infants. Although we routinely use the four methods for RHD genotyping, a PCR strategy based on at least two methods is recommended.
We measured the changes in cutaneous bilirubin (Br) and serum Br photoisomers in two groups of 5 jaundiced newborn infants treated by intensive phototherapy (IP), one with blue light and the other with green light. Cutaneous Br was measured with a transcutaneous jaundice meter and photoisomers were measured by HPLC. Cutaneous Br decreased in the two groups as soon as IP began, and the skin was completely bleached within 3 h with blue light only. Rebound occurred which was more marked after blue light IP. The main serum photoproduct was the 4Z-15E isomer, which reached a steady-state level within 1 h in both groups, whereas the lumirubin and 4E-15Z Br concentrations were slightly higher after green light IP. These data indicate that blue light is more suitable for IP, although this is not clearly explained by the production of more lumirubin.
OBJECTIVE: To examine twin similarities on biparietal diameter (BPD) measurements by zygosity (monozygotic [MZ] and dizygotic [DZ]) and chorionicity (monochorionic [MC] and dichorionic [DC]) and their evolution during pregnancy. METHOD: A sample of 54 pairs of twins (43 DZ sets, 11 MZ sets [7 MC and 4 DC]) was constructed using retrospective data. Despite the small sample size, our data were complete, and, for the first time we measured different fetal parameters on digital ultrasound images outside routine examination. The intraexaminer and interexaminer reliability of BPD measurement was significant (r = .95, P < .001). RESULTS: In this study, developmental results indicate significant linear regression coefficient (R) through the whole period of gestation (r = .96, P < .001), though product moment correlations comparing the periods of gestation two by two are weaker. The distribution of BPD values was slightly wider at the 28th week and markedly wider at the 32nd week than that at the 18th and 23rd weeks. The intraclass correlations of DZ and MZ (MC and DC) twins were examined at the 18th, 23rd, 28th and 32nd weeks of amenorhea. The intraclass correlations of DZ twins were significant through the whole period of gestation (r = .45, P = .001; r = .27, P = .04; r = .36, P = .008; and r = .42, P = .002, respectively), whereas the intraclass correlations of MZ twins were significant only at the 18th, 23rd and 28th weeks (r = .73, P = .002; r = .69, P = .005; r = .49, P = .047, respectively). We found significant within-variance differences not only between DZ and DC-MZ but also between DC-MZ and MC-MZ in late gestation. CONCLUSION: Our analysis of twin BPD development demonstrated that zygosity and chorionicity type are both important determinants of twin fetal development.
OBJECTIVE: Our purpose was to evaluate the potential of midtrimester fetal blood sampling to detect in utero human immunodeficiency virus type 1 infection. STUDY DESIGN: Ultrasonographically guided fetal blood sampling was performed before pregnancy termination in 28 women infected with human immunodeficiency virus type 1. Mean gestational age was 22 weeks (range 15 to 29 weeks). Samples were tested for human immunodeficiency virus with undissociated p24 antigen and deoxyribonucleic acid polymerase chain reaction or viral isolation by cell culture. After terminations fetal thymuses were also tested for human immunodeficiency virus infection by polymerase chain reaction. Polymerase chain reaction was also performed on maternal cells to confirm that the primer pairs used were able to detect human immunodeficiency virus type 1 strains present. RESULTS: All fetal blood samples had negative results by p24 antigen and polymerase chain reaction or virus culture and all fetal thymuses were negative by polymerase chain reaction. CONCLUSION: Early in utero human immunodeficiency virus infection appears infrequent, supporting the hypothesis that mother-to-child transmission predominantly occurs late in pregnancy. Therefore midtrimester prenatal diagnosis is not currently of use to women in making reproductive decisions.
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PURPOSE: To determine the frequency of maternal-fetal hemorrhage at or above 1 microliter of maternal whole blood. METHODS: Seventy-three mothers whose red blood cells bore an Rh antigen (Rh D, Rh c, Rh E) that was absent on red blood cells of their newborns were identified and a new cytological method, the Kleihauerimmunogold-silver-staining technique, was applied on the blood of their neonates to detect and quantify maternal red blood cells. Stringent precautions were taken to avoid contaminations of neonatal blood samples by adult red blood cells. RESULTS: Maternal red blood cells were present in 3 newborns, a frequency of 4% (95% Cl: 1-11%), and the estimated volumes of hemorrhage were 0.8, 1.5, and 101 microliters of maternal whole blood. No obstetric factor was clearly associated in this limited study with the occurrence of maternal-fetal hemorrhage. CONCLUSIONS: Mother-to-fetus microtransfusion greater than 1 microliter is infrequent at or near delivery, and it may be observed after an uncomplicated pregnancy and vaginal delivery.
BACKGROUND: The platelet count increases transiently after treatment with polyclonal anti-D in about 50 percent of D+ patients with autoimmune thrombocytopenic purpura (AITP). The effect is usually attributed to macrophage Fc-receptor blockade by antibody-coated red cells. As polyclonal anti-D is in limited supply, prospective testing was performed on a monoclonal anti-D (MoAb D) in such patients. STUDY DESIGN AND METHODS: Seven D+ patients with chronic AITP received MoAb D intravenously at doses of 47 to 95 microg per kg of body weight. Response was assessed by studying platelet count increment. Hemolysis and red cell-bound MoAb D were measured before and after MoAb D administration. RESULTS: MoAb D red cell binding was demonstrated in all patients at a ratio higher than that observed in AITP patients successfully treated with polyclonal anti-D. However, little or no platelet count increment was observed in six patients, while a transient response was observed in only one (platelet count 97 x 10(9)/L before MoAb D infusion and 163 x 10(9)/L 4 days later). Furthermore, because five patients showed signs of hemolysis and two became anemic, higher doses of MoAb D should be used only with caution in patients with AITP. CONCLUSION: The MoAb D used in this study cannot be proposed as an alternative treatment for patients with AITP.
OBJECTIVE: To assess trends in HIV infection among sexually transmitted disease (STD) patients. DESIGN: Repeated unlinked anonymous survey, 1991-1993. SETTING: STD clinics in Paris, France. SUBJECTS: Patients (n = 4354) with a new suspected STD. METHODS: HIV antibody testing, using blood from syphilis samples. RESULTS: HIV prevalences were stable over time in all transmission groups. One-third of homo-/bisexual men were HIV-positive. Prevalence was 2.5 times higher among heterosexual patients from Africa or the Caribbean than among those from other countries. Among patients under 25 years of age prevalence significantly decreased from 4.3% in 1991 to 0.8% in 1993 (P = 0.01). Among homo-/bisexual men, despite a 50% reduction in the incidence of STD, the absolute number of those newly HIV-infected remained stable; median age increased from 28 years in 1991 to 32 years in 1993 (P = 0.02). Among heterosexuals, trends in HIV incidence were difficult to assess: recently infected patients were more likely to be identified in 1993 than in 1991, since the proportion of patients who reported a recent HIV-negative test increased over time. CONCLUSION: Prevalence studies contribute to define specific subgroups which should be targeted for prevention (HIV-positive or older homosexuals, heterosexuals from Africa and the Caribbean). Despite a decrease in both overall STD incidence and HIV prevalence among patients aged under 25 years, overall HIV incidence has not decreased, at least among homo-/bisexual men in whom recent HIV infections occurred at a high rate overall, and increased in those aged 35 years or more. Sentinel site-based HIV seroprevalence studies are best interpreted in the light of results obtained from different populations and through routine surveillance of STD.
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OBJECTIVE: To estimate the prevalence of in utero transmission of HIV-1 through the second trimester. MATERIAL AND METHODS: One hundred consecutive, unselected, intact fetuses, beyond 15 weeks gestational age (mean, 22.4 weeks) were studied. These were obtained following spontaneous intrauterine deaths (n = 4), miscarriages (n = 4), and elective mid-trimester terminations (n = 92), eight of which were fetuses with malformations from HIV-1-positive pregnancies. Coded DNA extracts from the fetal thymuses were tested blindly by polymerase chain reaction in three laboratories using a total of six different primer pairs. RESULTS: Two thymuses tested positive [95% confidence interval (Cl), 0.2-7]. Results from the three laboratories were consistent in all 100 cases. The two fetuses with HIV in the thymus both tested positive in other organs, demonstrating systemic HIV infection. The first fetus, whose mother had advanced AIDS, had died in utero and had diffuse toxoplasmosis. The second died following extremely premature delivery in a pregnancy complicated by repeated bleeding. HIV infection was observed in none of the 92 fetuses that resulted from elective mid-trimester terminations (95% Cl, 0-4). CONCLUSION: The frequency of early in utero HIV infection appears to be low, compared with transmission rates in infants born to HIV-1-infected mothers, suggesting that transmission occurs mostly later in pregnancy and/or at delivery. Specific risk factors may have implications in the occurrence of early as opposed to late transmission.
Umbilical cord blood is considered an alternate source of hematopoietic stem cells in bone marrow transplantation. However, its use might be hampered by contamination of neonatal blood with maternal cells, which could contribute unacceptably to graft-vs.-host disease (GVHD) after transplant. In a previous study (Socié et al., Blood 83:340, 1994), we used polymerase chain reaction (PCR) amplification of minisatellite sequences (sensitivity 1-0.1%) to address the question of this contamination. In a single case among 47 analyzed, we were able to detect a maternal-specific allele in the cord blood sample. We have now studied the same cord samples using a highly sensitive, allele-specific PCR amplification method. A maternal allele could be discriminated from neonate alleles in 10 cases and maternal cells were detected in all 10 cord blood samples. These cells amounted to 10(-4) to 10(-5) of cord blood nucleated cells. In three cases, cord blood separated cell subpopulations could be analyzed and were found to contain maternal cells at about the same level. The presence of maternal cells at such a low level in cord blood samples probably would have no effect on GVHD in a clinical setting of transplantation but raises interesting questions in terms of materno-fetal immune tolerance and transmission of viruses (in particular human immunodeficiency virus) from infected mother to child.
Since our first report in 1989, 26 transplants by means of umbilical cord blood have been reported. Furthermore, systematic studies of the feasibility of using banked placental blood for bone marrow reconstitution of unrelated recipients on a large scale are in progress worldwide. However, already by 1989, it was pointed out that the use of cord blood might be hampered by contamination of neonatal blood with maternal cells contributing unacceptably to graft-versus-host disease (GVHD). In the present study, we used the polymerase chain reaction (PCR) amplification of 2 minisatellite sequences (33.6 and MS 51) to address this question. The sensitivity of PCR amplification of minisatellite sequences is known to be of 1% to 0.1%. This sensitivity has been confirmed in the present study, in which a dilution analysis was performed for each experiment in which cell separation was performed. The inclusion of the dilution experiment in these analyses allowed us to estimate the relative amount of contaminating maternal cells, if any. Among 47 cases (31 whole blood analyses, 10 gradient separations, and 6 subpopulation separations), the coamplification of the 2 minisatellites sequences allowed the discrimination of maternal and neonate alleles in 42 cases (89%). In 1 case, we were able to detect a child-specific allele in a mother's whole blood sample, thus validating our approach to search for maternal cells in cord blood. In a single other case, we were able to detect a maternal-specific allele in the cord blood sample. This maternal specific allele was detected in the whole blood, polymorphonuclear cell, and lymphocyte fractions. Comparison of the signal intensity obtained with these 3 cord blood samples to the result of the dilution experiment performed in the same analysis led to an estimate of 1 to 5% maternal cells in the polymorphonuclear cell fraction and 0.1% to 1% maternal cells in the whole blood and lymphocyte cell fractions. In conclusion, our study indicates that maternal cells are very rarely present in the cord blood collected at birth because we detected them in only 1 of 47 cases. More importantly, when detected, they were present at very low level in the lymphocyte cell fraction (0.1% to 1%). However, although small, this amount of cells may result in GVHD in a susceptible recipient. Because the method we used allows the detection of maternal cells within cord blood from 10(4) nucleated cells, it would thus be of interest in a cord blood banking perspective.
Two complementary surveys were carried out in the 89 hospital units of South-Eastern France which deal with pregnant women. Firstly, in November 1991, medical chiefs of these units were interviewed face-to-face about their current HIV screening policy. Secondly, between Jan 27 and March 22, 1992, all women at the end of their pregnancy attending these units were included in an anonymous unlinked seroprevalence survey, irrespective of pregnancy outcome (n = 11,056). The goal of the research was to compare HIV prenatal screening policies and seroprevalences by pregnancy outcomes in order to contribute to the public debate initiated on that issue by the French health authorities. The seroprevalence survey showed a global prevalence rate of 0.43% (CI 95% = 0.32-0.54) with the prevalence among women who had an elective abortion (0.56%) being more than twice that among women who delivered (0.22%). However, routine HIV screening was more frequent toward women coming for regular prenatal care than for women seeking abortion. A systematic procedure for obtaining women's consent for HIV testing only existed in a minority of units. Only 23 out of the 62 units offering both antenatal and termination services to women had the same screening policy for women attending the different services. The research confirmed that a mandatory requirement would not improve HIV screening policy during prenatal care. However, less emphasis on women who have opted for termination of pregnancy, an absence of appropriate counselling and information procedures, and pressures on HIV-infected women to terminate current pregnancies and discourage future ones strongly suggest that HIV prenatal screening in French hospitals remains mainly focussed on fetal concerns, without sufficient attention towards the needs of women at risk of HIV infection.
After anti-RhD, anti-Rhc is the most important red cell alloantibody which can cause haemolytic disease of the newborn (HDN) when the mother is Rhc-negative and the fetus Rhc-positive. We report here the development of polymerase chain reaction (PCR) assays which detect the presence of the Rhc alleles in amniotic cells by the use of allele-specific primers (ASP). It is expected that such determination will help in the management of pregnancies at risk of Rhc haemolytic disease. In the course of this study we have similarly performed PCR-ASP experiments to detect fetal RHE alleles since, in rare cases, anti-RhE can also cause HDN.
We had a case where risk of rhesus D feto-maternal immunisation occurred following failure to diagnose feto-maternal haemorrhage (HFM); and it was shown up by rhesus negative mother with a rhesus positive fetus being diagnosed as having has a massive HFM only three days after delivery. Giving the mother the standard dose of Anti-D immunoglobulin without a previous test to find out how serious the HFM was showed that we do not test for this normally. So it seems to us necessary when considering prophylaxis of rhesus D immunisation to go back to first principles and carry out Kleihauer's test particularly when neonatal anaemia is found in the child.
BACKGROUND: The effectiveness of phototherapy in lowering serum bilirubin levels in neonates varies inversely with the rate and degree of hemolysis. Combining this therapy with albumin perfusion could enhance its effectiveness. This study examines total, unconjugated and unbound fractions of bilirubin in infants treated by phototherapy alone or by phototherapy plus albumin. PATIENTS AND METHODS: The files of 211 neonates treated from January 1990 to March 1991 for severe neonatal jaundice were analysed. Jaundice was due to ABO incompatibility in 113 cases and hereditary hemolytic anemia in 6 cases; its cause was unknown in 92 patients. Other causes of jaundice such as Rh incompatibility, premature delivery before 34 gestational weeks and neonatal infections were excluded from the study. All 211 neonates were given phototherapy from admission with similar light energy. 114 babies (group I) received only phototherapy, while the 97 others (group II) were also given human albumin, (1.5 g/kg), during the first 2 hours of phototherapy. RESULTS: The decrease in serum unconjugated and unbound bilirubin after 4 hours of phototherapy was 34% in group I and 45% in group II (p < 0.0005). There was no difference between both groups after 24 hours of phototherapy. Other factors such as the initial concentration of serum unconjugated and unbound bilirubin, age at the onset of therapy, and ethnic skin color also influenced the decrease in bilirubin after 4 and 24 hours of phototherapy, in addition to albumin perfusion. CONCLUSIONS: Albumin perfusion plus phototherapy appears to induce a rapid and early decrease in unconjugated, unbound bilirubin, the fraction that is potentially neurotoxic, while phototherapy alone acts over a longer period.