anti-D Antibodies Research Articles

Determination of diethylstilbestrol based on biotin–streptavidin‐amplified time‐resolved fluoro‐immunoassay

A rapid and sensitive time-resolved fluoroimmunoassay (TR-FIA) based on the biotin-streptavidin amplification system was developed for the determination of diethylstilbestrol (DES). Europium-labelled streptavidin derivatives combined with europium and anhydride of diethylene triamine penta-acetic acid were used to label streptavidin; biotin was coupled with goat anti-rabbit IgG to form a biotin-goat anti-rabbit IgG bridge between streptavidin-europium and the anti-DES antibody in the immunoassay. The DES assay was carried out by measuring the fluorescence of Eu(3+) -SA at 615 nm. The presented method produced a wide linear range, 0.001-1000.0 ng/mL, and a detection limit up to 0.81 pg/mL for DES. The method was applied to determine DES in serum samples, with recoveries of 97.4-107.8% and RSD 1.32-4.04%. The assay results by the present method showed that biotin-streptavidin amplified TR-FIA for DES detection; it may offer high sensitivity and promising alternative special methods in biological samples.

The prevalence of irregular erythrocyte antibodies among antenatal women in Delhi.

Universal screening of all antenatal women, including D antigen-positive pregnant ones, is mandatory in most developed countries. However, no guidelines on this issue are available for developing countries such as India. Furthermore, there is limited information on immunisation rates in pregnant women (D antigen-positive and D antigen-negative) from India. We, therefore, studied the prevalence of alloantibodies among multigravida women in India. In this prospective study, carried out to detect the prevalence of alloantibodies among multigravida women in India, 3,577 multigravida women attending antenatal clinics were typed for ABO and D antigens and screened for alloantibodies by column agglutination technology. The medical history and detailed obstetric history of these women were reviewed and information recorded on any prior haemolytic disease of the foetus and newborn among siblings and/or blood transfusions. The overall prevalence of alloantibodies in this study was 1.25%. There was a statistically significant difference between alloimmunisation rates in the D antigen-negative and D antigen-positive groups (10.7% versus 0.12%, respectively). Anti-D antibody contributed to 78.4% of total alloimmunisations in our study. Anti-D was the most common culprit responsible for alloimmunisation. Other alloantibodies found included anti-C, anti-M, anti-S and anti-c. Large-scale population-based studies are required to assess the real magnitude of alloimmunisation in pregnant women in India.

Severe hemolytic disease of fetus and newborn caused by red blood cell antibodies undetected at first‐trimester screening (CME)

The objective was to determine clinical consequences of anti-D and non-D antibodies undetected at first-trimester screening for infant or fetus. This retrospective cohort study included all pregnant women with red blood cell (RBC) antibodies who were tested between 1993 and 2008. Data were obtained from the forms for tracking immunization at the transfusion department. Each form was analyzed for three data sets: the order of screening at which the antibodies were detected (initial or repeated screening), the order of pregnancy (first pregnancy or higher), and whether the antibodies caused severe hemolytic disease of fetus and newborn (HDFN). In D- women, anti-D was detected in 1.3% of cases. The anti-D was undetected in 72 (37%) cases on the first-trimester screening, of which eight cases were complicated by severe HDFN. In this group, three patients were primigravidae. An overall non-D incidence of 0.2% was observed. In 16 cases, non-D were undetected on the first-trimester screening (10 anti-c, two anti-E, two anti-C, one anti-S, and one case of anti-Rh17). Non-D antibodies undetected on initial screening caused 11 cases of severe HDFN (27% of all severe non-D HDFN). Ten of them were in multiparous women. Seven of 11 cases with severe HDFN that were missed were caused by anti-c. The third-trimester screening may detect RBC antibodies that were not present or detected on the first-trimester screening. Such screening may be especially relevant in D+ multiparous women due to the risk of HDFN.

Red blood cell antibody screening in pregnancy

Sir, Hemolytic disease of newborn (HDN) is defined as neonatal anemia and hyperbilirubinemia caused by an incompatibility between maternal and fetal red blood cells (RBCs).[1] In 98% cases it is caused due to ABO and Rh incompatibility and antibodies to other blood group antigens (Kell, c, E, C, Kidd, Duffy, M, and so on) are causative in remaining 2%.[2] More than 43 different RBC antigens have been reported to be associated with HDN.[3] Red cell antibody screening (RCAS) is a valuable tool in the detection of alloantibodies to other blood group systems (other than ABO and Rh) in the serum of patients during pregnancy or prior to transfusion. Red cell antibody identification (RCAI) should then be carried out on a larger panel of RBCs to precisely identify the antibody. In a prospective study carried out on 624 antenatal cases, RCAS was done using a 3-cell panel from Diamed, Switzerland. RCAI was carried out on cases that were positive for RCAS. RCAS was positive in 9 out of 624 cases—1.4% (excluding the 3 cases who had autoantibodies). After RCAI these were identified as anti-D antibody (6 cases, 66%), anti-D with anti-C antibody (2 cases, 22%), and anti-M antibody (1 case, 11%). The most common antibody identified remained anti-D. In 2 cases of Rh negative pregnancy, the RCAS was suggestive of anti-D. RCAI done, however, showed a combination of anti-D and anti-C. One case of anti-M was detected in a G2P1L1D1 lady. The first pregnancy was full-term normal delivery at home, however, the baby died after birth. The mother’s and baby’s blood group was O positive. RCAS done during second pregnancy was suggestive of anti-Duffy (Fya) or anti-M antibody. RCAI done showed anti-M antibody with dosage effect. The second pregnancy was postdated with Intrauterine growth retardation (IUGR,) and Lower segment caesarean section(LSCS) was done for fetal distress. The baby had hyperbilirubinemia and was Direct Coombs test (DCT) positive requiring phototherapy. Rh incompatibility continues to be a common cause for HDN. Patients with no prior history of sensitization can also develop anti-D as seen in 3 of our cases probably due to naturally occurring anti-Rh antibodies or antepartum hemorrhage. Despite the use anti-D immunization, 1%–2% of the cases are still sensitized. Anti-D immunization resulted in a favorable fetal outcome in the study. Anti-D complicated with anti-C presents with more severe HDN as seen in 1 patient who had a previous stillbirth and a hydrops baby despite receiving anti-D. Anti-C antibodies resulting in HDN requiring exchange transfusion have been reported.[4] Anti-M as a cause of HDN is rare as they are usually complete cold antibodies (IgM). However, it can be IgG type resulting in HDN.[3] HDN due to anti-M antibody can be mild to severe with stillbirth and cases requiring exchange transfusion have been reported.[5] Anti-M causing blood group discrepancy and crossmatch incompatibility has been reported in the Indian literature.[6] Antenatal detection of the non-anti-D causes of HDN requires RCAS. If RCAS is positive, the following steps are to be taken. RCAI should be done to identify the antibody. The spouse has to be screened for the presence of offending antigen and the pediatrician has to be alerted about delivery of a potentially sensitized infant. The blood bank should find a suitable antigen-negative donor for transfusion to baby and mother.

Diphtheria and Tetanus Antibody Persistence in Indian Pre-school Children and Response to a Booster Dose of DT Vaccine.

Despite effective vaccines, diphtheria (D) resurged recently in the former socialistic block, and tetanus (T) still occurs in less privileged countries. We studied the antibody persistence for D and T in Indian pre-school children who had received four doses of DTP vaccine and subsequently, the response to a booster dose. Anti-D and anti-T IgG antibodies prior to and one month after a DT vaccine were measured by ELISA in 223 healthy children of 4-6 years who had previously received four doses of the triple vaccine. Adverse reactions were monitored for one month. While 30% and 14% of subjects were susceptible to D and T, respectively, 98% and 100% of them attained seroprotection post-vaccination. Both responses were significant. Local, but not systemic reactions except fever were rather common. A high proportion of the Indian pre-school population is susceptible to D and T, despite of receiving four doses. The current policy of giving the fifth dose at this age is appropriate.

Early plasmapheresis followed by high‐dose γ‐globulin treatment saved a severely Rho‐incompatible pregnancy

An alloimmunized pregnancy induces anemia in the fetus and can ultimately lead to fetal hydrops and intrauterine fetal death. A woman with a severe Rho-incompatible pregnancy had experienced frequent pregnancies with fetomaternal transfusion without receiving RhIg and had high anti-D antibody titers present from early pregnancy. We succeeded in long-term inhibition of antibody production using plasmapheresis followed by high-dose γ-globulin treatment in early pregnancy.

Anti-nucleosome antibodies may predict lupus nephritis and severity of disease in systemic lupus erythematosus

Background/Objective Detection of anti-nucleosome antibodies in patients with systemic lupus erythematosus (SLE) has been well established and it is claimed that their presence is associated with disease activity. The objective of this study is to determine the diagnostic value of anti-nucleosome antibodies in the assessment of lupus nephritis and clinically active SLE. Methods The anti-nucleosome antibodies were evaluated in the serum of 200 Tunisian SLE patients at disease onset by a sensitive immunodot assay. Serum samples from each patient were also tested for ANA and anti-ds DNA antibody by IIF on Hep 2 cells and Crithidia luciliae respectively. During the follow-up, the patients were regularly monitored for clinical parameters. Global SLE activity was measured by the European Consensus Lupus Activity Measurement (ECLAM). Results The prevalence of anti-nucleosome and anti-dsDNA antibodies was 69% and 63.5% respectively. Anti-nucleosome antibodies were found to be 30.1% positive in SLE patients lacking anti-dsDNA antibody. 79.5% patients had active SLE at the first clinical examination. Anti-nucleosome antibodies were more sensitive than anti-dsDNA antibodies to detect active SLE (78% vs. 71.7%, P =0.19). 52.5% of SLE patients had renal involvement. Among these patients, the rate of anti-nucleosome positivity and anti-dsDNA were 77.1% and 67.6% respectively. The positivity of anti-nucleosome antibodies was significantly higher in patients with renal disease than the subjects without renal disease ( P = 0.009). Anti-nucleosome and anti-ds DNA antibodies were significantly correlated with disease activity ( P < 0.001 and P < 0.001 respectively). Conclusion Anti-nucleosome antibody reactivity may be a useful marker in the diagnosis and assessment of active SLE.

Is Transfusion of Rh Positive Red Blood Cells to ccDee Patients From a Cosmopolitan Population Safe?.

AbstractAbstract 1121▪ Introduction:D variant is a common phenotype in Africans. However, the allele multiplicity challenges the serological methods which often fail to detect D variant patients in such populations. Recently, two cases of strong anti-D allo-immunization occurred in our blood bank in ccDee patients who received transfusions with Rh positive red blood cells. The D phenotype of these patients was determined using two different monoclonal and one polyclonal anti-D antibodies, with normal agglutination (4+) observed. Considering these results, we decided to investigate the prevalence of D variant for the ccDee patients of our cosmopolitan population. The aim of this study was to determine whether ccDee blood recipients should systematically undergo molecular screening for D variant prior to transfusion in order to avoid anti-D allo-immunization. Materiel and methods:Fresh EDTA blood from 35 ccDee patients (22 Africans and 13 Europeans) was analyzed using both serological and molecular methods. Serological characterization was performed using two monoclonal and one human polyclonal anti-D antibodies. In addition, each sample was tested using an extended partial RhD typing set from Diamed (consisting of 12 monoclonal antisera). Molecular determination was performed using two polymerase chain reactions with sequence-specific priming (PCR-SSP) kits intended to diagnose either partial D or weak D phenotypes (BAGene, BAG, Lich, Germany). Result:Among the 35 patients screened, discordant results between serological and molecular methods were found in 12. While serological tests revealed a normal D phenotype for these patients with all anti-D antibodies tested, molecular tests diagnosed them as D variant (nine were DAU; two were weak D types 4.0 and 4.1; one was DAR). As expected, the vast majority (9/12) of these patients were of African descent. No discordance between serological and molecular screening was observed for the rest of the screened population (23 patients). Conclusion:Our study reveals that approximately one-third (12/35) of our ccDee patients present a D variant genotype despite normal serological results. It emphasizes the fact that transfusion of Rh positive red blood cells to ccDee patients (mainly of African origin) requires precautionary measures. Molecular D variant screening in ccDee patients should be performed in order to improve the handling of transfusions in ccDee patients. Disclosures:No relevant conflicts of interest to declare.

A Randomized Trial of the Safety and Benefit of Transfusion Vs. Standard Care In the Prevention of Sickle Cell-Related Complications In Adults: a Preliminary Report From the Phase II NHLBI Comprehensive Sickle Cell Centers (CSCC) Study of Neuropsychological Dysfunction and Neuroimaging Abnormalities In Neurologically Intact Adult Patients with Sickle Cell Disease

AbstractAbstract 3221 Background:Most adult sickle cell anemia patients have received transfusion therapy. However, prospective studies evaluating the efficacy of transfusions in preventing sickle cell-related complications are lacking. The Phase II Neuropsychological Adult Sickle Cell Anemia Study is a randomized trial of chronic transfusion vs. standard of care in patients with abnormal neurocognitive function in order to determine the safety and benefits of transfusion therapy on neurocognitive function. A secondary goal of the study is to evaluate the benefit of chronic transfusion on the frequency and severity of acute sickle cell events; this is a preliminary report of this specific aim. Methods:Eligibility required normal neurological exam, WAIS III PIQ score ≤ 90, hemoglobin ≤ 9 g/dL, hemoglobin SS electrophoresis, and age between 21 and 55 years. Patients were randomly assigned to receive either standard care or transfusions. The transfusion goal was to maintain a hemoglobin of 2 g/dL rise over baseline with matched red cells for D, C/c, E/e, and Kell antigens. The protocol required simple transfusions at approximately 4 week intervals. Chelation therapy was not part of the study design. Patients underwent serial clinical and laboratory evaluations with central analysis of all clinical and transfusion events and complications. Laboratory testing of subjects in the transfusion arm included quantitative hemoglobin S/A, hemoglobin concentration, ferritin levels, and red cell antibody screening; a full hematology/chemistry panel was performed for all subjects at baseline, the study mid-point, and at the end of the study. Results:There were 20 patients in the transfusion arm (TX arm) with a mean age of 29 years vs. 16 patients in the standard care arm (SC arm) with a mean age of 30.5 years. The baseline data in the TX arm was similar to the SC arm: hemoglobin 7.8 vs. 8.0 g/dL; hematocrit 22.6% vs. 23.1%; hemoglobin F 10.5% vs. 12.5%. Thirty-five percent of patients randomized to the TX arm had a history of acute chest syndrome (ACS) vs. 31% in the SC arm; 30% of patients in the TX arm were on hydroxyurea compared to 50% in the SC arm. The TX arm patients have received an average of 5.6 transfusions (2 units per transfusion) with only one subject requiring an acute transfusion (5%); in contrast, 4 SC arm patients (25%) were transfused for acute events for a total of 7 units (average 1.8 per patient). The transfusion therapy improved the average hematologic status of patients: hemoglobin S% decreased from 85% to 32% (p=0.0003); hemoglobin and hematocrit increased from 7.6 to 9.4 g/dL (p=0.0052) and 22% to 28%, (p=0.007), respectively. Bilirubin declined from 3.6 to 2.4 mg/dL (p=0.042). In contrast, only bilirubin showed a significant decrease in the SC arm. In the TX arm, serum ferritin rose an average of 1318 to 2368 (p=.001); there was no change in liver function. There were no clinical transfusion reactions in the 120 study transfusions (360 units); however, one patient on routine screening reported a transient anti-D antibody without clinical or laboratory changes. Clinical Results:Adverse events were higher in the SC arm. Total number of adverse events in the TX arm were 23 (1.2 per person) vs. 66 in (4.1 per person) in the SC arm. There were 5 hospitalizations in the TX arm and 21 in the SC care arm, with a median number of hospitalized days per hospitalization of 5.0 and 6.0 respectively. The total number of serious events was 6 in the TX arm (0.3 per person) vs. 23 in the SC arm (1.4 per person). The total number of vaso-occlusive events in the TX arm were 14 (0.7 per person) vs. 57 (3.6 per person) in the SC arm. Acute pulmonary events occurred in 25% (4 patients) of the SC arm vs. none in the TX arm. Conclusions:This is preliminary data from the first prospective randomized study of the safety and efficacy of transfusion therapy in adults with SCD. We demonstrate the safety of transfusion therapy. Compared to standard therapy, transfusions improve or stabilize critical laboratory markers, decrease serious sickle cell anemia-related adverse events, and decrease in hospitalizations. Increase in ferritin is an expected outcome in transfused patients since chelation was not a part of this transfusion protocol. On completion of the study, the potential benefits of transfusion therapy on sickle cell disease morbidity including neurocognitive function will be reported. Disclosures:Field:Novartis Pharm: Honoraria.

A retrospective study of positive antibody screens at delivery in Rh-negative parturients

Rhesus- (Rh-) negative women receiving anti-D antibodies antenatally often have a positive antibody screen at delivery. We investigated the incidence of positive antibody screens at delivery in this population and examined how the presence of positive antibody screens affected the time required to obtain type and screen or type and crossmatch results. Records of parturients who had type and screen or type and crossmatch done upon presentation for delivery from June to October 2007 were examined to determine estimated gestational age at admission, Rh-status, the presence of positive antibody screens, and the time interval from receipt of specimen in the blood bank to the availability of antibody screen results. Of the 480 specimens sent for type and screen or type and crossmatch, 20% of parturients were Rh-negative, with 57% of those demonstrating a positive antibody screen compared with 4% of the Rh-positive parturients (P < 0.01). In the Rh-negative group, 100% (95% CI 98-102) of positive antibody screens were anti-D antibodies. There was a longer median laboratory time for Rh-negative vs Rh-positive parturients (146 vs 65 min), for antibody positive vs antibody negative parturients (243 vs 65 min) (P < 0.001 for both), but not for Rh-positive/antibody positive vs Rh-negative/antibody positive patients (312 vs 218 min) (P = 0.09). The antibody screen was positive in 100% of Rh-negative parturients until 37 weeks gestation, after which there was a decline. Rh-negative parturients who receive anti-D antibodies antenatally have a higher incidence of positive antibody screens at delivery than Rh-positive parturients due to the presence of anti-D antibodies.

Index Of Suspicion In The Nursery

A 2.9-kg female infant is born at 35 weeks' gestation to a 33-year-old G4P3 woman via cesarean section due to chronic hypertension complicated by superimposed preeclampsia. The mother's blood type is A-negative; the antibody screen is positive for anti-D, anti-C, and anti-G antibodies; she has no prior history of sensitization; and her serologies are negative. The Apgar scores are 7 and 9 at 1 and 5 minutes, respectively. At birth, the infant appears pale and exhibits tachycardia, moderate hepatosplenomegaly, tachypnea, and mild respiratory distress requiring supplemental oxygen. Complete blood count and blood cultures are obtained, and ampicillin and gentamicin are started. The admission hematocrit is 21% (0.21), with a reticulocyte count of 64%. An umbilical venous catheter is placed for intravenous (IV) access. The infant receives a transfusion of packed red blood cells for significant anemia associated with tachycardia. Prophylactic phototherapy is started because of Rh sensitization. The cord blood bilirubin measures 11.4 mg/dL (195 mcmol/L). The infant's blood type is A-negative, and the antibody screen is positive for anti-D and anti-C antibodies, with a positive direct Coombs test. The red blood cell phenotype reveals the presence of “C” antigen. This is a very uncommon antigen in an Rh-negative infant, but the antigen had stimulated the anti-C antibodies that traversed the placenta and caused severe hemolysis of the newborn. At 7 hours of age, the infant's bilirubin is 16 mg/dL (273.6 mcmol/L). She is given two doses of intravenous immune globulin, and phototherapy is intensified. After 24 hours of intensive phototherapy, the infant develops a large reddish-purple discoloration on her abdomen that resembles a severe burn (Fig. 1). The well-demarcated, erythematous, purpuric rash is present over the areas closely exposed to the phototherapy lights and does not blanch with diascopy. No vesiculobullous eruptions, edema, or tenderness is noted. The unexposed …

Successful treatment with very low-dose etanercept in a patient with etanercept-induced liver dysfunction

Etanercept is a fusion protein that composes the soluble TNF receptor p75 and Fc region of immunoglobulin G1. Etanercept is usually administered subcutaneously at a dose of 10 or 25 mg twice a week in Japanese patients with rheumatoid arthritis (RA). There are a few reports of etanercept-induced liver dysfunction [1, 2]. A 37-year-old Japanese woman suffered from Raynaud’s phenomenon in 1996. She received a diagnosis of RA in 1998. Although she had high titre of antinuclear antibodies (1:10,240), she had no specific antibodies such as anti-ds DNA antibodies, anti-Scl-70 antibodies and antiSS-A antibodies. The serology for hepatitis B and C was negative. The medication programme for RA included sulfasalazine, bucillamine, methotrexate, mizoribine and actarit, which induced nausea, epigastric pain, persistent coughing, and a sensation of fatigue and numbness, respectively. Auranofin, penicillamine, aurothiomalate and tacrolimus each had no desirable effect. Mizoribine, bucillamine and actarit are used for RA only in Japan. Etanercept was initiated at a dose of 25 mg weekly to the female patient in January 2007. Her DAS28 score using CRP decreased from the level of high disease activity to the level of low disease activity 16 weeks after administration. However, her liver function tests such as ALT had deteriorated progressively since 12 weeks after etanerceptintroduction (Fig. 1). At the 20th week, etanercept was discontinued because of liver dysfunction, while the other medications maintained same. Because her liver function tests improved to a normal range at the 28th week, etanercept was resumed at a dose of 12.5 mg weekly at this point. Unfortunately, her liver function tests again showed abnormal 4 weeks after resumption, and etanercept was discontinued again. At the 36th week, etanercept was tried again at a dose of 12.5 mg every 10 days. Thereafter, her liver function tests remained within normal values, and her DAS28 score remained stable within good response by the EULAR criteria for 90 weeks (Fig. 1). Her hand X-ray did not deteriorate for 2 years (data not shown). She received etanercept at total doses of 37.5 mg monthly because of etanercept-induced and dose-dependent liver dysfunction. Her RA activity stabilised in good conditions with etanercept, which was used at even lower dose than the usual. Leak and Rincon-Aznar reported a similar patient with psoriatic arthritis, who experienced dose-related etanercept-induced liver toxicity [1]. It might

PREVENTION OF RhD ALLOIMMUNIZATION IN RhD NEGATIVE WOMEN

Despite the introduction of anti-D prophylaxis into clinical practice, RhD alloimmunization still presents a problem to date. The actual incidence of RhD alloimmunization in pregnant women remains unknown in most countries. Anti-D immunoglobulin is administered to RhD negative women at a fixed dose and in much greater amounts than is actually necessary. On the other hand, it is not possible to diagnose cases where greater doses are needed. To optimize the prevention of RhD alloimmunization in RhD negative women, it is important to diagnose conditions that lead to fetomaternal hemorrhage (FMH), precisely determine the volume and subsequently administer the appropriate dose of anti-D immunoglobulin. The possibility to accurately detect FMH and precisely determine its volume would enable more effective and less costly prevention of RhD alloimmunization. Anti-D immunoglobulin could be administered only in indicated cases and only in doses essentially necessary for prevention of RhD alloimmunization. The Cochrane and UpToDate databases of systematic reviews, as well as national guidelines, were reviewed. Due to the medical significance and indispensable economic costs associated with prevention of RhD alloimmunization, it would be appropriate to establish exact methodical guidelines. The text itself should be limited to a list of potentially sensitising events during which anti-D immunoglobulin should be administered to RhD negative women if anti-D antibodies are not already present. Following each potentially sensitising event, the minimal dose of anti-D immunoglobulin necessary for prevention of RhD alloimmunization should be determined. After 20 weeks of gestation, the volume of FMH should also be determined to specify the necessary dose of anti-D immunoglobulin.

Red blood cell membrane grafting of multi-functional hyperbranched polyglycerols

The covalent attachment of hydrophilic polymers or biopharmaceuticals to the surface of red blood cells (RBCs) has previously been shown as a relatively compatible and effective method for a range of applications. Here, the first example of cell-surface grafting with a hyperbranched and multi-functional macromolecule is described. A range (3 kDa–101 kDa) of dense, globular, and blood compatible hyperbranched polyglycerols (HPG) were synthesized and functionalized with cell-surface reactive, succinimidyl succinate groups (1–12 groups per polymer). Subsequently, HPG was grafted to the RBCs, which were analyzed using physical characterization techniques such as aqueous two-phase partitioning and particle electrophoresis. It was found that the extent of grafting was enhanced by increasing HPG molecular weight, the number of reactive groups per HPG, HPG concentration, and reaction time. Good in vitro cell viability – as measured by lipid peroxidation, hemoglobin oxidation, cell lysis, osmotic fragility, stability in fresh serum and aggregation behavior – was observed for grafting concentrations up to 4.8 m m. The multi-functional aspect of HPG is highlighted by the following observations: using fluorescein-labeled Anti-D (monoclonal) antibody and flow cytometry, the detection of cell-surface Rhesus (RhD) antigens were significantly reduced upon HPG grafting. Secondly, the potential for using HPG as a multi-functional, delivery agent was demonstrated by attaching fluorescent markers to the HPG via degradable linkages prior to grafting.

A case of pregnancy with Rhesus antibody and bicornuate uterus - a favourable outcome: a case report

IntroductionIn 1% of Rhesus negative women sensitisation occurs without any overt sensitising event during pregnancy. This accounts for late immunisation during a first pregnancy and is responsible for 18-27% of cases of alloimmunisation. The incidence of congenital uterine anomalies in a fertile population is 3.2% of which 5% are bicornuate uterus. Bicornuate uterus can lead to early miscarriages, preterm labor, fetal growth retardation and congenital malformations.Case presentationA 23-year-old lady in her first pregnancy developed Anti-D antibodies at 28 weeks of gestation without any known sensitising event. In view of increasing anti-D titres, at 36 weeks she was delivered. Incidentally during caesarean section she was found to have bicornuate uterus. The neonate was treated with phototherapy and blood transfusion following delivery.ConclusionRhesus antibodies when managed by close monitoring and timely delivery can lead to favourable outcome. Bicornuate uterus does not always lead to complications like miscarriage, growth retardation or preterm labour and does not need any special intervention.

The role of antenatal immunoprophylaxis in the prevention of maternal-foetal anti-Rh(D) alloimmunisation.

The first description of haemolytic disease of the newborn (HDN) can be traced back to 1609 and was made by a French midwife, Louise Bourgeois, who, from 1600, worked at the royal court of King Henry IV and Queen Marie de Medicis1–4. In the treatise that Bourgeois wrote in 1609 she described the birth of two twins3: the first had hydrops and died immediately, while the second, initially in a better condition, rapidly became jaundiced and, after having developed neurological symptoms (kernicterus), died 3 days after being born. Hydrops foetalis and kernicterus were correctly interpreted as two aspects of the same pathology only in 19325, when Diamond described foetal erythroblastosis secondary to severe haemolysis, although the cause was still unknown. A few years later, in 1938, Ruth Darrow correctly identified the (antibody-related) pathogenesis of HDN6, although erroneously attributing foetal haemoglobin the role of the culprit antigen, which was suggested to have induced a maternal antibody response after crossing the placenta. The true pathogenesis of the disease was definitively clarified in 1940 with the discovery of the Rhesus (Rh) blood group system by Landsteiner and Wiener7 and with the subsequent identification, in 1941, by Levine8, of the Rh(D) antigen. This antigen was, in fact, identified, in D-negative mothers, as being the cause of the immunisation occurring following transplacental passage of foetal D-positive red blood cells. The subsequent passage of maternal anti-D immunoglobulin G (IgG) across the placenta into the foetal circulation was recognised as the final event able to cause the spectrum of clinical events that characterise HDN. It did not take long before the risk of immunisation could be quantified1,3: i) 16% in the case of a Rh(D)-negative mother and a Rh(D)-positive, ABO-compatible foetus; ii) 2% in the case of a Rh(D)-negative mother and a Rh(D)-positive, ABO-incompatible foetus (about 20% of the cases); iii) overall risk of immunisation: 13.2%. Before 1945, about 50% of all foetuses with HDN died of kernicterus or hydrops foetalis. Subsequently, thanks to the progress in treatment, in industrialised countries the mortality decreased to 2–3%; this mortality rate was then very considerably further reduced (100-fold) with the introduction of anti-D immunoprophylaxis to prevent maternal-foetal anti-Rh(D) alloimmunisation 9. At the beginning of the 1960s, Stern demonstrated experimentally that the administration of anti-D IgG could prevent sensitisation to the Rh(D) antigen10; in the same period, other studies clarified the mechanism of Rh iso-immunisation in pregnancy and introduced the clinical practice of passive immunisation with anti-D IgG to protect Rh(D)-negative women from sensitisation against Rh(D)-positive red blood cells11–14. The successes obtained in studies of Rh(D)-negative male volunteers formed the experimental basis for clinical trials in pregnant Rh(D)-negative women15; these trials demonstrated that post-partum immunoprophylaxis decreased the incidence of post-pregnancy anti-Rh(D) immunisation from 12–13% to 1–2%15,16. Subsequently, in 1977, it was shown that 1.8% of Rh(D)-negative women, despite post-natal prophylaxis, continued to develop anti-D antibodies because of small transplacental haemorrhages during pregnancy17,18. One year later, a Canadian study by Bowman et al. showed, in 1,357 Rh(D)-negative primagravida, that the incidence of Rh(D) alloimmunisation could be reduced to 0.1% by prophylaxis with antenatal anti-D IgG, in addition to post-partum prophylaxis19. There is currently sufficient evidence demonstrating that antenatal anti-D prophylaxis also reduces the risk of Rh(D) immunisation in the next pregnancy to below the level of 0.4%. Forty years after Zipursky and Israels first proposed the use of anti-D IgG to reduce the incidence of Rh alloimmunisation in pregnancy14, immunoprophylaxis has drastically reduced the cases of Rh-induced HDN; nevertheless, this pathology continues to be relevant in 0.4 of 1,000 births (0.04%)20, for various reasons21: i) the possible occurrence of anti-D immunisation during the pregnancy (which occurs in about 1% of Rh(D)-negative women carrying a Rh(D)-positive foetus22); ii) the lack of efficacy of immunoprophylaxis because of the administration of an insufficient dose of anti-D IgG that is not congruent with the volume of the foetal-maternal haemorrhage; iii) immunoprophylaxis not administered; iv) possible errors in typing the pregnant or puerperal woman or the neonate; v) possible errors in transfusion therapy in women of child-bearing age.

Association of plasma soluble E-selectin and adiponectin with carotid plaque in patients with systemic lupus erythematosus

BackgroundSystemic lupus erythematosus (SLE) is associated with premature atherosclerosis but the mechanisms underlying this association are not understood. The role of endothelial dysfunction is hypothesized. MethodsIn predominantly non-Caucasian patients with SLE (N=119) and controls (N=71), carotid ultrasonography was performed and circulating endothelial cells (CECs), soluble endothelial protein C receptor and gene polymorphism at A6936G, soluble E-selectin (sE-selectin), and adiponectin were assessed. ResultsCarotid plaque was more prevalent among patients than controls (43% vs 17%, p=0.0002). Mean CCA IMT was greater in patients compared to controls (0.59±0.19mm vs 0.54±0.11mm, p=0.03). Among SLE patients, plaque was not associated with smoking, body-mass index, LDL, triglycerides, homocysteine, C-reactive protein, anti-ds DNA antibody, C3, C4, SLE activity, or medications. Age and levels of soluble E-selectin and adiponectin were significantly higher in the SLE patients with plaque compared to those without plaque in univariate and multivariate analyses. sE-selectin and adiponectin were found to serve as independent predictors of carotid plaque and that elevations were persistent over more than one visit. Unexpectedly, these biomarkers were present despite clinical quiescence. ConclusionPremature atherosclerosis is a consistent feature of SLE and extends across ethnicities. Higher levels of adiponectin may represent a physiological attempt to limit further endothelial damage already reflected by the elevation in sE-selectin and the observed increase in plaque represents overwhelming of this reparative process by atherogenic stimuli.

Administration of Anti-Platelet Antibodies Prevents the Anti-Platelet Immune Response and Bleeding Complications of Neonatal Immune Thrombocytopenia in a Murine Model.

Abstract 223 Background:The human platelet antigen (HPA) 1a is a potent immunogen located on the β3 integrin. Ten % of pregnant HPA1a negative women produce antibodies against the HPA1a antigen if the foetus is HPA1a positive. Fetal/neonatal immune thrombocytopenia (FNIT) can occur if the mother develops alloantibodies against fetal platelets, with intracranial haemorrhage as the most severe complication. The current opinion has been that immunization against the HPA1a antigen takes place during the first HPA1 non-compatible pregnancy. However, results from a large and recent screening study in Norway found that the majority (75%) of women were immunized around time of delivery, and not so often during pregnancy. This indicates that FNIT could be more similar to haemolytic disease of the newborn (HDN) than previously thought. To prevent HDN, antibody mediated immune suppression (AMIS) is induced by administration of anti-D antibodies in connection with RhD-negative pregnancies. The same principle could be used to prevent FNIT by administration of anti-HPA1a antibodies in HPA1a-negative pregnancies. We have previously established a murine model of FNIT using β3 integrin-deficient (β3−/−) mice. The first aim of the current project was to test whether administration of human anti-HPA1a IgG could suppress the anti-human platelet immune response in β3−/− mice after transfusion of human HPA1a positive platelets. For the second part of the project, we used a pure murine model to test whether administration of murine anti-β3 antibodies transfused after delivery could induce AMIS and prevent bleeding complications of FNIT in the subsequent pregnancies. Methods:Human/murine model: Human IgG from 5 donors with high levels of anti-HPA1a antibodies was purified by Protein G affinity chromatography. Purified IgG from one male donor without detectable anti-platelet specific antibodies was used as control IgG. Human platelets were isolated from an HPA1a positive donor. β3−/− mice were immunized by one tail vein transfusion with 2 × 106 human HPA1a positive platelets, with or without subsequent transfusion of 900ug human IgG (100% saturation). After 7 days, the mice were bled and sera collected. The anti-human platelet immune response was analyzed via flow cytometry, using FITC-conjugated goat anti-mouse IgG as detection antibody. Six mice were injected with anti-HPA1a containing IgG. Control IgG (n=6) or no IgG (n=4) were used as negative controls. Pure murine model: High-titer anti-β3 sera were produced by 4 weekly transfusions of 108 wild type (WT) platelets to β3−/− mice. Naïve β3−/− female mice were bred with naïve β3−/− male mice. Within 24 hours of delivery, the mother was transfused with 108 WT platelets with or without immediate transfusion of anti-β3 sera. The transfusions were repeated one week after delivery and the same females were bred again with WT male BALB/c mice. The anti-β3 immune response was analyzed via flow cytometry, using FITC-conjugated goat anti-mouse IgG. The FNIT phenotype was monitored and all live pups were bled from the carotid vein to determine platelet count. Results:Administration of purified anti-HPA1a IgG significantly suppressed the anti-human platelet immune response in β3−/− mice after transfusion of HPA1a positive platelets as compared with control IgG (p < 0.05). In the pure murine model of FNIT, the anti-β3 immune response was markedly suppressed during the subsequent pregnancy in the mice treated with anti-β3 sera. Two out of three mice receiving anti-β3 sera treatment delivered live pups with moderate thrombocytopenia without signs of haemorrhage (mean platelet count 217 ×106/mL). The third mouse receiving anti-β3 sera delivered dead pups. In contrast, all female mice (n = 3) without anti-β3 sera treatment miscarried. Conclusions:We have demonstrated in vivo that AMIS can be induced by administration of anti-platelet antibodies using a murine model of FNIT. Preliminary data indicates that bleeding complications of FNIT can be prevented with this prophylactic approach. Disclosures:Skogen:Prophylix Pharma a/s: Employment, Equity Ownership. Killie:Prophylix Pharma a/s: Equity Ownership. Husebekk:Prophylix Pharma a/s: Equity Ownership. Kjeldsen-Kragh:Prophylix Pharma a/s: Equity Ownership.

Mechanisms of anti-D action in the prevention of hemolytic disease of the fetus and newborn: what can we learn from rodent models?

Hemolytic disease of the fetus and newborn can be effectively prevented by administration of anti-D to the mother. In this setting, the IgG purified from the plasma of D-alloimmunized donors prevents the maternal immune response to D-positive red blood cells (RBC). Several monoclonal anti-D antibodies have recently been developed for potential use in the setting of hemolytic disease of the fetus and newborn; the functional assays used to assess the potential success of these antibodies have often assumed antigen clearance as the predominant mechanism of anti-D. Unfortunately, the in-vivo success of these monoclonal antibodies has thus far been limited. A similar inhibitory effect of IgG has been observed in animal models with a vast array of different antigens, referred to as antibody-mediated immune suppression (AMIS). Here, studies of AMIS are reviewed and the relevance of these findings for anti-D-mediated immunoprophylaxis is discussed. In animal models of AMIS, IgG-mediated antigen clearance was not sufficient for prevention of the antibody response to RBC. Furthermore, anti-RBC IgG inhibited B-cell priming to foreign RBC, but failed to prevent a T-cell response and immunological memory. The applicability of AMIS models for determining the true mechanism of anti-D, though uncertain, may nevertheless provide knowledge as to potential mechanisms of action of anti-RBC antibodies.

A Curious Case of Anti-D Antibody Titer

RhD alloimmunization remains a threat to 1% of the 10% of RhD-negative women in the United States who are giving birth to RhD-positive fetuses despite routine antenatal and postpartum administration of Rh(o)D immune globulin (RhIG). This report examines the clinical course of an RhD-negative woman who developed a high anti-D antibody titer during her pregnancy while carrying an RhD-positive female fetus yet had a negative antibody screen at the time she gave birth. Although she delivered a healthy newborn unaffected by hemolytic disease, subsequent pregnancies will be treated as though she is RhD alloimmunized. The discussion below includes possible causes for the abrupt rise in this woman's anti-D antibody titer, a review of the complex Rh system and cellular anamnestic response, and current fetal surveillance for hemolytic disease of the fetus and newborn.