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Objective: To report our experience in preventing RhD maternal isoimmunization by using anti-D gamma globulin among Rh-negative women., Material and Methods: Between 1982 and 1995, immunologic and hematologic data were collected from all Rh-negative women seen at Mexico's National Perinatology Institute. Women at risk of Rh isoimmunization were given a prophylactic dose of 150 micrograms of anti-D gamma globulin., Results: A total of 4,857 Rh-negative women were seen during the study period (4.85% of the total population of women seen at the Institute), 629 (13.0%) of whom developed RhD isoimmunization; 542 (86.2%) of these women were already isoimmunized when first seen at our Institute. Twenty-two women (3.5%) developed isoimmunization even after receiving a proper dose of anti-D gamma globulin. Prophylaxis was given to 2,605 women (53.6%); 2,039 received a single dose, and 475 two doses. Prophylaxis failed in 22 cases; four were women with multiple pregnancy and 18 developed obstetric pathologic conditions., Conclusions: The use of anti-D gamma globulin resulted in a reduction of maternal Rh isoimmunization to less than one case per 1,000 women. Failures to prevent isoimmunization were associated to additional obstetric conditions and to lack of adherence to prevention guidelines.

Unlabelled: The aim of the study is to analyze the outcome of pregnancies complicated by Rh-isoimmunization for the period 1996-2001 and to outline the aspects of optimization of the obstetrical conduct., Material and Methods: The current study includes 39 pregnant women with Rh-isoimmunisation to whom amniocentesis and cordocentesis was performed. All cases were analyzed using medical history, serology (indirect Coombs, PAP test), ultrasound examination, amniocentesis, cordocentesis, NST., Results: From 39 pregnancies, complicated by severe Rh-isoimmunization 36 resulted in a live delivery, 2 resulted in intrauterine death of the fetus and 1 in early neonatal death. The titre of the antibodies is of prognostical value only in the first isoimmunised pregnancy. In this case there is a reliable correlation between the condition of the newborn and the zone from the Liley curve, antenatally found. With history of former immune pregnancies with unfavourable perinatal outcome most precise information about the condition of the foetus gives the cordocentesis., Conclusion: In all of the discussed cases the Rh-isoimmunization is a result of no andi-D immunoglobulin profilaxis post partum or following abortion. That is why the efforts should be directed towards conduction of proper profilaxis to all Rh-negative pregnant women.

Anti-D prophylaxis is currently applied in France after birth of an RhD positive infant, after interruption of pregnancy and after some antenatal immunizing events (amniocentesis...). However this program does not cover all the prenatal exposures to fetal RhD antigen, and maternal Rh immunization continues to occur. DNA RhD genotyping of the fetus is now reliably performed on amniotic fluid, and pre diagnostic studies on fetal DNA extracted from maternal plasma are promising. The widespread use of fetal RhD genotyping on maternal blood would allow the antenatal administration of Rh immunoglobuline in all Rh negative patients bearing an Rh positive fetus, insofar as it would preclude exposing the other Rh negative patients to the above plasma derived and rather expensive blood product.

In order to find out whether or not the incidence rate of RhD-immunization has increased in pregnant women who were not given antenatal prophylaxis a survey was carried out comprising 620 women. It was evident that the number of new immunizations had decreased, both in absolute numbers and in relation to the number of births during the years 1991-1998. Hence prophylactic anti-D seems not to be indicated, neither for this group of women nor for women who have had a spontaneous abortion.

In spite of the progress made since 1970 in specific prevention by anti-rhesus immunoglobulins, and improved management of at-risk pregnancies, allo-immunization due to the erythrocytic Rh 1 antigen (formerly known as Rhesus D or Rh D) remains widespread. In fact, anti-Rh 1 antibodies currently constitute over one-third of the immune antibodies detected after pregnancy. The prevention of allo-immunization against the Rh 1 antigen is therefore still problematical, and concerns approximately one pregnant woman in seven. The etiology and pathology of fetal hemolytic disease have been recalled, and the treatment approach during pregnancy and delivery has been carefully examined. Tests for quantifying the risk of fetomaternal hemorrhage have also been described. This approach aims at improving the methods of preventing allo-immunization (e.g., during pregnancy and delivery) and the efficacy of treatment. It is also stated that if the necessary preventive action is not taken in cases of allo-immunization due to to the Rh 1 antigen, this should be considered a grave medical fault.

Serology for the presence of anti-D after RhD-incompatible platelet transfusions was performed in 24 RhD-negative patients with haematological disease and 59 RhD-negative patients with non-haematological disease. None of the patients were given prophylaxis with anti-D to prevent RhD immunization. Eight out of 59 (13.5%) non-haematology patients developed detectable anti-D, whereas 0 out of 24 (0%) of the haematology patients formed anti-D (P = 0.06). The risk of alloimmunization after RhD-incompatible platelet transfusions using platelet concentrates prepared by modern technical methods appears to be small in patients with haematological disease, but is significant in non-immunocompromised patients.

Replacement of the hyperimmune anti-Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti-RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high-affinity anti-RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin-treated red blood cells enabled the isolation of two high-affinity Fab-expressing phage clones. LD-6-3 and LD-6-33, specific for RhD. These showed a novel reaction pattern by recognizing the D variants D(III), D(IVa), D(IVb), D(Va), D(VI) types I and II. D(VII), Rh33 and DFR. Full-length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growth-adapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti-RhD antibody adapted to pilot-scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody-dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti-RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.

Objective: To determine and evaluate policies and procedures related to weak D phenotype testing and terminology and the administration of Rh immune globulin in selected clinical situations. Design, Setting, and Participants.-Institutions participating in the College of American Pathologists 1999 J-A Comprehensive Transfusion Medicine Survey program were asked to respond to a series of supplementary questions related to weak D phenotype testing and Rh immune globulin administration. More than 3500 institutions and transfusion services participated., Results: Most supplementary questions elicited more than 3000 responses. Despite no clinical or regulatory mandate, 58. 2% of transfusion services routinely perform an antiglobulin test for the weak D phenotype in patients who test negative with anti-D reagents. Significant differences were found concerning the transfusion of blood components to patients with the weak D phenotype and the administration of Rh immune globulin to these individuals. At least one patient with the weak D phenotype with anti-D alloantibody formation was observed during a 12-month period by 31.8% of transfusion services., Conclusions: Significant variability concerning policies and procedures related to weak D typing and terminology was found in this survey. Transfusion of blood components to patients with the weak D phenotype and the administration of Rh immune globulin also demonstrated variations. Anti-D alloantibody formation by patients with the weak D phenotype may not be as rare as previously thought. Additional study related to the clinical significance of these results is warranted.

Background: Passively acquired blood group alloantibodies are detected regularly after infusions of IV Rh immune globulin (RhIG) for the treatment of immune thrombocytopenic purpura (ITP) in D+ patients., Study Design and Methods: Blood samples from 16 D+ patients with ITP were tested after treatment with IV RhIG for the presence of passively acquired alloantibodies. Similar studies were conducted for three D- patients after injections of IM RhIG for Rh immunoprophyl-axis. Four production lots of IV RhIG and 2 lots of IM RhIG were tested for the presence of alloantibodies., Results: All 16 D+ patients with ITP developed a positive DAT, as well as positive antibody detection test results, after infusions of IV RhIG. All postinfusion plasma samples contained anti-D, as well as one or more additional antibodies, usually anti-C, -E, -G, -V, or -Fy(a). Eluates from patients' RBCs with positive DAT results contained multiple passively acquired alloantibodies. Multiple alloantibodies were detected in samples of different production lots of IV RhIG or IM RhIG. No acute transfusion reactions were observed in five D+ patients with ITP who had been treated with IV RhIG and had been given serologically incompatible D+ RBCs. After injections of IM RhIG, the only passively acquired alloantibody detected was anti-D., Conclusion: Plasma samples from D+ patients with ITP treated with IV RhIG regularly contained anti-D and multiple other passively acquired Rh, Duffy, or Kidd system alloantibodies. Postinfusion RBC samples all had positive DAT results with eluates containing anti-D and multiple other Rh, Duffy, or Kidd system antibodies. The consistent detection of multiple passively acquired alloantibodies after IV RhIG, in contrast to the detection of anti-D only after IM RhIG, reflects the immediate effect of the entire (bolus) dose of RhIG by the IV route, the dose for treating ITP that is approximately 10 times the dose for Rh immunoprophylaxis, and the expected serologic incompatibility with recipients' D+ RBCs.

Monoclonal anti-D has proved impossible to make in rodent systems. Human monoclonal anti-D has been produced using EBV transformed peripheral B cells, coupled with fusions to myeloma cell lines. More recently molecular biology techniques have been used to produce monoclonal anti-D. The range of monoclonal anti-D produced is considered. The selection of monoclonal anti-D for use as blood grouping reagents for typing donors and recipients is reviewed--all types of D positive should be typed as positive when donors are considered. However, DVI patients should be typed as D negative. Considerations for the development of monoclonal anti-D for prophylactic use are reviewed.

Background: A woman may develop Rh-negative antibodies during her first pregnancy when her fetus is Rh-positive. Antibodies develop most frequently after the 28th week of gestation., Objectives: The objective of this review was to asses the effects of giving antenatal anti-D immunoglobulin at 28 weeks or more of pregnancy on the incidence of RhD alloimmunisation when given to Rhesus negative mothers without anti-D antibodies., Search Strategy: We searched the Cochrane Pregnancy and Childbirth Group trials register, Cochrane Controlled Trials Register, and bibliographies. Date of last search: December 1998., Selection Criteria: Randomised trials in Rhesus negative women without anti-D antibodies given anti-D after 28 weeks of pregnancy, compared with no treatment or placebo., Data Collection and Analysis: Data were extracted by one reviewer and double entered., Main Results: Two eligible trials, which involved over 4500 women, compared anti-D prophylaxis with no treatment. Although the data suggested, when women receive anti-D at 28 and 34 weeks gestation, a reduced incidence of immunisation during pregnancy (0R O.44, 95% CI 0.18-1.12), after the birth of a Rhesus positive infant (OR 0.44, 95% CI 0.18-1.12), and within 12 months after birth of a Rhesus positive infant (OR 0.44, 95% CI 0.19-1.01), none of these differences were statistically significant. In the trial, which used the larger dose of anti-D (100ug; 500IU), there was a clear reduction in the incidence of immunisation at 2-12 months following birth in women who had received Anti-D at 28 and 34 weeks (OR 0.22 95% CI 0.05-0.88). No data were available for the risk of RhD alloimmunisation in a subsequent pregnancy. No differences were observed in the incidence of neonatal jaundice., Reviewer's Conclusions: The risk of RhD alloimmunisation during or immediately after a first pregnancy is about 1.5%. Administration of 100ug (500IU) anti-D at 28 weeks and 34 weeks gestation to women in their first pregnancy can reduce this risk to about 0.2% without, to date, any adverse effects. Although such a policy is unlikely to confer benefit or improve outcome in the present pregnancy, fewer women will have Rhesus D antibodies in their next pregnancy. Adoption of such a policy will need to consider the costs of prophylaxis against the costs of care for women who become sensitised and their affected infants, and local adequacy of supply of anti-D gammaglobulin.

Background: The development of Rh immunisation and its prophylactic use since the 1970s has meant that severe Rhesus D (RhD) alloimmunisation is now rarely seen., Objectives: The objective of this systematic review was to assess the effects of giving anti-D to Rhesus negative women, with no anti-D antibodies, who had given birth to a Rhesus positive infant., Search Strategy: We searched the Cochrane Pregnancy and Childbirth Group trials register, the Cochrane Controlled Trials Register, MEDLINE (from 1966 to January 1999) and reference lists of relevant articles. Date of last search of Cochrane Controlled Trials Register: January 1999., Selection Criteria: Randomised trials in Rhesus negative women without antibodies who were given anti-D immunoglobulin postpartum compared with no treatment or placebo., Data Collection and Analysis: Assessments of inclusion criteria, trial quality and data extraction were done by each author independently. Initial analyses included all trials. Other analyses assessed the effect of trial quality, ABO compatibility and dose., Main Results: Six eligible trials compared postpartum anti-D prophylaxis with no treatment or placebo. The trials involved over 10,000 women, but trial quality varied. Anti-D lowered the incidence of RhD alloimmunisation six months after birth (relative risk 0.04, 95% confidence interval 0.02 to 0.06), and in a subsequent pregnancy (relative risk 0.12, 95% confidence interval 0. 07 to 0.23). These benefits were seen regardless of the ABO status of the mother and baby and when anti-D was given within 72 hours of birth. Higher doses (up to 200 micro grams) were more effective than lower doses (up to 50 micro grams) in preventing RhD alloimmunisation in a subsequent pregnancy., Reviewer's Conclusions: Anti-D, given within 72 hours after childbirth, reduces the risk of RhD alloimmunisation in Rhesus negative women who have given birth to a Rhesus positive infant. However the evidence on the optimal dose is limited.

Purpose: Prenatal and postnatal prophylaxis of the Rh (D) haemolytic disease of the newborn have clearly reduced the number of cases but still there are alloimmunizations., Patients and Methods: All cases detected in our Hospital in the last 24 years have been reviewed and possible causes analyzed., Results: From a total of 10,332 deliveries in Rh (D) negative women we have detected 114 anti-D in 86 women. In 74 women anti-D was the only antibody and in 12 there were more antibodies. Data were managed in 3-year periods and we see a progressive decrease in the incidence of alloimmunization with a minimum of 0.03 per 1000 pregnancies in the period 89-91 and a posterior progression to an incidence of 0.12 in the last 3-year period 95-97. The causes were: pregnancies before 1970 in 31, incorrect prophylaxis in 12, despite a correct prophylaxis in 6, previous pregnancies without complete information about the prophylaxis in 13, previous transfusion in 6, previous pregnancies or transfusion in 8 and indetermined in 10., Conclusion: It is desirable to reduce at minimum the number of Rh (D) alloimmunizations by strictly following the prophylaxis protocols.

Anti-RhD immunoglobulin was administered to RhD-negative women based on estimates of fetal bleed size obtained using a direct immunofluorescence flow cytometric technique employing a FITC-conjugated monoclonal human anti-D (BRAD 3). The effectiveness of the dose administered was assessed by (i) measuring the fraction of RhD-positive fetal cells in the maternal circulation at d0, and between d2 and d10 post RhD Ig administration, (ii) quantifying the amount of anti-D detectable in maternal plasma following RhD Ig injection in the perinatal period and (iii) assessing maternal serum for the presence of immune anti-D in follow-up samples taken 3 months to 3 years after delivery. Fifty-four women were assessed, 29 having fetal bleeds in excess of 4 mL. Follow-up samples were received from 20/29 mothers after RhD Ig administration; 43-99% and 69-99% of fetal cells had been cleared by d2/3 and d5/6, respectively, in 14/20 mothers, whereas less than 50% had been cleared in the remaining mothers. Long-term follow-up samples were obtained from eight of the 29 mothers (four with bleeds >/=20 mL, two with bleeds >95 mL): none had detectable anti-D in the serum 4 months to 3 years after delivery despite the persistence of up to 36% fetal RhD-positive cells in the maternal circulation six days after delivery.

Background: The D immunoprophylaxis program has successfully reduced the incidence of Rh hemolytic disease of the newborn (HDN), but it has also reduced the availability of plasma-derived polyclonal anti-D, which constitutes the current therapeutic product. Human monoclonal anti-D from hybridoma cell lines may be an acceptable alternative, and clinical efficacy of each anti-D is being evaluated in several centers., Study Design and Methods: This study represents the largest assessment (outside of the International Workshops) of human D monoclonal antibodies for potential therapeutic use. The in vitro biologic activity and immunologic and serologic reactivity of a coded panel of 20 D antibodies (THERAD) was investigated. The bioassays used were lymphocyte (K-cell) antibody-dependent cell-mediated cytotoxicity (ADCC), monocyte ADCC, and monocyte chemiluminescence, which together reflect the processes involved in antibody-coated red cell destruction in vivo. From this panel, six antibodies (THERADs 14, 19, 22, 23, 27, and 28, comprising 3 IgG1 and 3 IgG3 D monoclonal antibodies) were further selected to investigate the effects of blending in the three bioassays., Results: Several THERAD blends displayed greater activity than their component parts, in the range of 6 to 124 percent. There was no evidence to suggest functional blocking effects with this restricted panel of antibodies., Conclusion: The THERAD blends containing both IgG1 and IgG3 anti-D appeared to be the most functionally active, as did blends containing antibodies to two distinct D epitopes. This in vitro evidence has important implications for the future formulation of an effective monoclonal preparation for the prevention of Rh HDN.

The reduction in the incidence of Rh D alloimmunization is a prototype for the effectiveness of preventive medicine. Some controversies remain, however, such as the use of anti-D immune globulin in patients with either threatened abortion or antenatal hemorrhage. Similarly, it may not be cost-effective either to screen all Rh D-negative patients with an indirect Coombs test at 24-28 weeks of gestation or to screen all postpartum patients for excessive fetomaternal hemorrhage.

Alloimmunization to the D blood group antigen following the transfusion of D-positive red blood cells to a D-negative recipient may be prevented in most persons by a prompt and adequate dose of Rho (D) immune globulin (RhIG). Until recently, the only RhIG approved by the US Food and Drug Administration (FDA) for this indication required intramuscular injection, an inconvenient and painful route for the relatively large volume that may be required. We describe the successful prevention of Rh alloimmunization following the unintentional transfusion of D-positive red blood cells to a D-negative infant by the intravenous infusion of WinRho SD, a new RhIG that is FDA-approved for prevention of post-transfusion Rh alloimmunization by intravenous administration. We believe that this more convenient and less painful approach should be the treatment of choice for preventing Rh alloimmunization following the transfusion of D-positive red cells to a D-negative recipient.

Background: The ability to determine fetal RhD Status noninvasively is useful in the treatment of RhD-sensitized pregnant women whose partners are heterozygous for the RhD gene. The recent demonstration of fetal DNA in maternal plasma raises the possibility that fetal RhD genotyping may be possible with the use of maternal plasma., Methods: We studied 57 RhD-negative pregnant women and their singleton fetuses. DNA extracted from maternal plasma was analyzed for the RhD gene with a fluorescence-based polymerase-chain-reaction (PCR) test sensitive enough to detect the RhD gene in a single cell. Fetal RhD status was determined directly by serologic analysis of cord blood or PCR analysis of amniotic fluid., Results: Among the 57 RhD-negative women, 12 were in their first trimester of pregnancy, 30 were in their second trimester, and 15 were in their third trimester. Thirty-nine fetuses were RhD-positive, and 18 were RhD-negative. In the samples obtained from women in their second or third trimester of pregnancy, the results of RhD PCR analysis of maternal plasma DNA were completely concordant with the results of serologic analysis. Among the maternal plasma samples collected in the first trimester, 2 contained no RhD DNA, but the fetuses were RhD-positive; the results in the other 10 samples were concordant (7 were RhD-positive, and 3 RhD-negative)., Conclusions: Noninvasive fetal RhD genotyping can be performed rapidly and reliably with the use of maternal plasma beginning in the second trimester of pregnancy.

Background: Standard post-delivery administration of anti-D, together with further anti-D for events known to result in fetomaternal haemorrhage (FMH) during pregnancy, has reduced the incidence of alloimmunisation in RhD women to 0.83-1.5% in the UK. Residual alloimmunisation occurs mainly for two reasons: i) failure to administer sufficient anti-D at the correct time after known at-risk events, either during pregnancy or at delivery; and ii) alloimmunisation during pregnancy as a result of 'silent' FMH. The RhD antigen is well developed by 6 weeks' gestation and the fetoplacental blood volume increases during pregnancy. Studies show that 3% of pregnant women have FMH in the first trimester, 12% in the second, and 45% in the third. Analysis of alloimmunisation in primigravidae clearly shows that on average, 90% are detectable after 28 weeks' gestation. Additional anti-D prophylaxis during the course of pregnancy, starting at 28 weeks, can reduce alloimmunisation to a minimum by protecting against occult FMH. The identification of intrapartum alloimmunisation as being the 'true' cause of alloimmunisation is best assessed by studying first pregnancies, and rigorous analysis of antenatal anti-D efficacy should preferably include observation in second pregnancies to avoid underestimation of alloimmunisation. Ideally there should be no exclusions, i.e. treating both arms of the study on an 'intention to treat' basis, otherwise there will be an overestimate of efficacy under routine practice conditions. Initial safety concerns about effects of antenatal anti-D on the fetus have not been confirmed in practice., Options: Although there is only one randomised controlled clinical trial (with small numbers) demonstrating a further reduction in alloimmunisation following antenatal administration of anti-D meriting a grade A recommendation (see appendix II), the total body of evidence of efficiency is compelling. Whilst two doses of 300 microg are effective, this is no more so than the single dose in practice, and as it requires considerably more anti-D immunoglobulin, it is probably not cost effective. If a single dose is to be given, it is too late at 34 weeks, and 28 weeks is to be recommended. If divided doses are to be given at 28 and 34 weeks, 50 microg is insufficient, and 100 microg is recommended. Two dose regimes can be recommended, as follows: i) single dose of 300 microg at 28 weeks--the results of the single 300 microg dose in first pregnancies is limited to the Canadian study with observed reduction from 1.6% (45/2768) in concurrent nonrandomised controls to 0.18% (2/1086) in the treatment group. ii) two doses of 100 microg at 28 and 34 weeks--the two controlled studies give similar results in first pregnancies. A reduction in alloimmunisation is seen from 1.11% (4/360) in controls to <0.28% (0/362) in the treatment group in the French study , and from 0.95% (19/2000) in controls to 0.32% (4/1238) in the treatment group in the English study. Whilst anti-D is in limited supply, it is more cost effective to restrict antenatal prophylaxis to first pregnancies. It is also probable that a single dose of 250 microg (as used in Europe) will be as effective in practice as the 300 microg dose, given the limitations of the anti-D quantification assay, and the vial overfill introduced by manufacturers, but this has not been formally proven in clinical trials. The number of RhD deaths is now very low, even with standard postpartum prophylaxis, but there is a systematic underreporting in the UK, due to early fetal deaths being recorded as 'abortion' rather than as haemolytic disease of the newborn (HDN). There have been no systematic studies on the reduction in mortality observed with antepartum anti-D. Nevertheless, it is self-evident that if immunisation is largely prevented, then so will fetal morbidity and mortality.

Prognosis in cases of erythrocyte immunisation has improved continuously over the past decades. Morbidity and mortality have been reduced by improvements in management, including screening programmes, non-invasive ultrasound evaluation and invasive procedures. The article provides an outline of the latest developments in the management of erythrocyte immunisation, and several controversial issues are discussed, such as antibody screening, strategies for the reduction of antibody titres, and the organisation of care.