Section 1C: Assessment of the functional activity and IgG Fc receptor utilisation of 64 IgG Rh monoclonal antibodies. Coordinator's report.

Sixty-four IgG Rh monoclonal antibodies (Mabs) submitted to the Fourth International Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens were characterised and tested in quantitative functional assays at five laboratories. The biological assays measured the ability of anti-D to mediate phagocytosis or extracellular lysis of RBC by IgG Fc receptor (Fc gamma R)-bearing effector cells. Interactions of RBC pre-sensitised with anti-D (EA-IgG) with monocytes in chemiluminescence (CL) assays were found proportional to the amount of IgG anti-D on the RBC. Using antibodies to inhibit Fc gamma RI, Fc gamma RII or Fc gamma RIII, the only receptor utilised in the monocyte CL and ADCC assays for interactions with EA-IgG1 was found to be Fc gamma RI. In these assays, enhanced interactions were promoted by EA-IgG3 and additional Fc gamma receptors may have contributed. IgG2 anti-D was not reactive in these assays and EA-IgG4 promoted weak reactions through Fc gamma RI. A macrophage ADCC assay showed that haemolysis of EA-IgG3 was greater than that of EA-IgG1, mediated mainly through Fc gamma RIII. In ADCC assays using lymphocytes (NK cells) as effector cells and papainised RBC target cells, only a minority of IgG1 anti-D Mabs were shown to be able to mediate haemolysis in the presence of monomeric IgG (AB serum or IVIg). These interactions were mediated solely through Fc gamma RIII. Haemolysis via Fc gamma RIII may depend on the presence of certain sugars on the oligosaccharide moiety of IgG. Most Mabs (IgG1, IgG2, IgG3 and IgG4) elicited intermediate, low or no haemolysis in these assays. Blocking studies indicated that low activity IgG1 and IgG4 anti-D utilised only Fc gamma RI. Other IgG1 and IgG3 Mabs appeared to promote haemolysis through Fc gamma RI and Fc gamma RIII while IgG2 was inhibited by Mabs to both Fc gamma RII and Fc gamma RIII, suggesting a variety of Fc gamma R are utilised for anti-D of low haemolytic activity. Excellent agreement between the results of the lymphocyte ADCC assays and antibody quantitation was observed between the participating laboratories.

Seroprevalence of human herpes virus 8 antibody in populations at high or low risk of transfusion, graft, or sexual transmission of viruses.

BACKGROUND: The routes of transmission of human herpes virus 8 (HHV-8) remain unclear. In particular, HHV-8 transmission by blood components and organ transplantation is still debated and raises public health issues. The objective of this study was to determine the prevalence of anti-HHV-8 in selected populations of persons or patients with or without risk factors for the transmission of viral infections, in order to determine the routes of HHV-8 transmission. STUDY DESIGN AND METHODS: A total of 1431 persons or patients at low or high risk of sexually, blood-, or graft-transmitted viral infections were tested by means of a standardized immunofluorescence serologic assay detecting anti-HHV-8. RESULTS: The persons or patients could be classified into three distinct groups according to anti-HHV-8 prevalence: a low prevalence group (0.0% to 5.0%), including healthy blood donors, healthy pregnant women, multiply transfused patients with thalassemia major, and IV drug users; an intermediate prevalence group (5.0% to 20.0%), including organ donors, kidney transplant recipients, and multiply transfused patients with sickle cell disease; a high prevalence group (>20.0%), including HIV-negative persons at high risk of sexually-transmitted viral infections, and HIV-infected homosexual men and heterosexuals. CONCLUSION: The sexual route appears to be the main route of HHV-8 transmission; bloodborne transmission of HHV-8, if it exists, is rare. In contrast, organ transplantation recipients might be exposed to HHV-8 transmission by the transplanted organ, which raises the issue of systematic screening of organ donors.

Early changes in cutaneous bilirubin and serum bilirubin isomers during intensive phototherapy of jaundiced neonates with blue and green light.

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.

A highly sensitive polymerase chain reaction method reveals the ubiquitous presence of maternal cells in human umbilical cord blood.

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.

HIV prenatal screening in south-eastern France: differences in seroprevalence and screening policies by pregnancy outcome.

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.

[Massive fetomaternal hemorrhage and prevention of fetomaternal Rhesus incompatibility. The failure of our present system of prevention]. / Hémorragie foeto-maternelle massive et prévention de l'incompatibilité Rhésus foeto-maternelle. Une faille de l'organisation actuelle de la prévention.

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.

Hematopoietic progenitors cells in cord blood.

Human umbilical cord blood was evaluated as an alternative to bone marrow as a source of stem cells for hematopoietic transplantation. In order to define an optimal collection procedure, we have studied the parameters that influence the collection, handling and storage of cord blood. We have attempted to correlate the quality of the samples with obstetrical and neonatal parameters. Using culture techniques we have studied the long term viability of the cells. Cell separation was also investigated. Our results suggest that most of the sample collected could be suitable for transplantation, in term of progenitors. However the observed variability between samples suggest that an efficient control of the quality of the samples is important.

Clinical applications of stem cell transfusion from cord blood and rationale for cord blood banking.

Umbilical cord blood collected and cryopreserved at birth contains enough hematopoietic progenitor stem cells for engraftment. HLA identical sibling cord blood transplant has been performed for the first time, in a child with Fanconi anemia. Three years latter, this child is alive with a complete donor type bone marrow. Since this first attempt, several other patients with other diseases have been transplanted successfully. Cord blood banking is a safe and easy procedure. Due to the high proliferative capacity of neonatal hematopoietic progenitors and to the relative immunological functional immaturity of neonatal lymphocytes cord blood cells could be used for matched unrelated or partially mismatched transplants.

[The perinatal transmission of the hepatitis B virus in the Paris area]. / Transmission périnatale du virus de l'hépatite B dans la région de Paris.

HBsAg was detected in 152 pregnant women among 6,605 (2.3%) screened in the prenatal clinics of four hospitals representative of the Paris metropolitan area. In 98% of cases, HBsAg positivity indicated chronic HBV carrier status. Among patients born out of continental France (47% of screened women, 79% of positive women) relative risk of chronic infection was 6 in Asians, 5.5 in Africans, and 4 in French women born in non-continental France. No significant difference in medical history was seen between HBsAg-positive and HBsAg-negative patients, in any of the birthplace groups. In women born out of continental France, number of children and crowding in the home were correlated with HBsAg-positivity; these correlations were not found in French women born in continental France. In non-African, non-Asian women, screening on the basis of medical, social and familial criteria (simulated in this study) would not be effective. Routine screening for HBsAg in pregnancy is advocated. The cost of the prevention of each case of perinatally acquired chronic HBV infection by routine screening followed by prophylactic treatment of a risk neonates was estimated at 180,000 French Francs (35,000 dollars). This approach is the only means of preventing the long-term life-threatening complications of chronic HBV infection in the 600 neonates born each year in France to HBsAg-positive mothers.

HIV proteins absent from placentas of 75 HIV-1-positive women studied by immunohistochemistry.

Recent epidemiological and virological data suggest that the incidence of maternofetal transmission of HIV-1 infection is between 20 and 30%. The available evidence points to a possible role of peri- and postnatal contamination, but the isolation of HIV from fetuses shows that transplacental transmission also occurs. We attempted to detect, by means of an immunohistochemical method, HIV proteins in frozen placentas from 75 HIV-1-positive women (30 at term, 45 induced abortions). In addition, in situ hybridization using HIV-specific probes was performed in three cases. Neither HIV proteins nor nucleic acid sequences were detected, but CD4+ mononuclear cells were present in the chorion and villi, regardless of the clinical and biological status of the mother (particularly in the nine cases in which the infants were infected). There are several possible mechanisms involving the placenta in the maternofetal transmission of HIV, including active transport of the HIV-immunoglobulin G complex via Fc receptors on trophoblastic cells, passive transplacental passage of HIV during a viraemic episode, the passage of infected maternal cells, and infection of the placenta itself. The methods we used could not rule out the presence of HIV DNA provirus within the genome of placental cells. In any event, immunohistochemical detection of HIV proteins in the placenta is not a technique suitable for the prenatal diagnosis of HIV infection or for identifying newborns likely to develop HIV infection.

Collection of placental blood with a view to hemopoietic reconstitution.

Collection of placental blood in a sterile and closed system is a simple, safe and efficient procedure. One hundred and fifty eight units of fetal blood were collected by applying the same requirements of quality and safety as those defined for blood products in blood banks. No adverse effects were seen in mothers or their newborns.

Hematopoietic stem cell potential from umbilical cord blood.

We studied the conditions of collection and isolation of hematopoietic cells from cord blood in order to optimise the sampling. A statistically significant correlation was found between the total stem cell content of the samples and the time of delivery suggesting that the quantity of hematopoietic stem cells available is higher when cord blood collection is performed earlier during pregnancy. Attempt to isolate the white cells resulted in a dramatic loss of stem cells. Factors affecting cell recovery and purification must be investigated in order to optimize cord blood cell banking.

Paris cooperative study on HIV sero-positivity in pregnancy: preliminary results.

PIP: Researchers tested the sera of 10,277 pregnant women who visited 9 prenatal clinics in Paris, France or its environs between February 17, 1987 - August, 17, 1987. The women also completed a questionnaire about risk factors. Laboratory personnel tested for HIV using and ELISA technique, and referred positive sera for further screening to a virology laboratory in Tours. Researchers already knew that 43 cases were HIV positive, 26 of whom had an induced abortion and 17 chose to continue the pregnancy. 30 patients tested positive who did not know they carried HIV: 8 in the abortion group; 19 in the pregnancy group; 1 in the miscarriage group; and 1 in the ectopic pregnancy group. The overall HIV prevalence rate was 7.1/1000. The researchers further defined the prevalence rate by taking only into account the discovered HIV positive patients, since the others were already known and included in HIV statistics. In the induced abortion group, the prevalence rate was 6.42% and in the pregnancy group 2.18%. The leading mode of transmission for the 73 HIV positive women was intravenous (IV) drug use, followed by being from an endemic country. By comparing HIV testing results and the questionnaires of the discovered HIV positive women, researchers would have selected 93% of these women. On the other hand, 35.8% of all the patients responded positively to at least 1 item. This shows that it is difficult to develop an adequate questionnaire and select an appropriate sample size. The researchers hope to improve the specificity of the questionnaire, in order to reduce the number of patients to be screened for HIV.^ieng