anti-D Ig Research Articles

Terminating Routine Cord Blood RhD Typing of the Newborns to Guide Postnatal Anti-D Immunoglobulin Prophylaxis Based on the Results of Fetal RHD Genotyping

Introduction: Targeted routine antenatal prophylaxis with anti-D immunoglobulin (Ig) only to RhD-negative pregnant women who carry RhD-positive fetuses (determined by fetal RHD genotyping) has reduced D-alloimmunization significantly when administered in addition to postnatal prophylaxis. Achieving high analysis sensitivity and few false-negative fetal RHD results will make RhD typing of the newborn redundant. Postnatal prophylaxis can then be given based on the result of fetal RHD genotyping. Terminating routine RhD typing of the newborns in cord blood will streamline maternity care. Accordingly, we compared the results of fetal RHD genotyping with RhD typing of the newborns. Methods: Fetal RHD genotyping was performed, and antenatal anti-D Ig was administered at gestational week 24 and 28, respectively. Data for 2017–2020 are reported. Results: Ten laboratories reported 18,536 fetal RHD genotypings, and 16,378 RhD typing results of newborns. We found 46 false-positive (0.28%) and seven false-negative (0.04%) results. Sensitivity of the assays was 99.93%, while specificity was 99.24%. Conclusion: Few false-negative results support the good analysis quality of fetal RHD genotyping. Routine cord blood RhD typing will therefore be discontinued nationwide and postnatal anti-D Ig will now be given based on the result of fetal RHD genotyping.

Emergency Clinicians interpretation and application of Anti-D guidelines

Objective: The objective of this study was to audit the use of anti-D immunoglobulin (anti-D) against the current Australian guidelines in one large inner-city referral hospital over three years and critique the practice identified. Background: Pregnant patients who have a D-negative (RhD negative) blood type are at risk of D alloimmunisation if a potentially sensitising event occurs during pregnancy or birth. The administration of anti-D Ig can prevent complications related to alloimmunisation. Potentially sensitising events commonly present to the emergency department requiring the administration of anti-D Ig in line with current guidelines. Study Design and Methods: This is a retrospective cohort study of all patients who received anti-D Ig in a large inner-city emergency department (ED) over three years (July 2014 – June 2017). Indications for administration were scrutinised against current guidelines by experienced clinicians. Results: A total of 228 patients received anti-D Ig, with the majority being less than twelve weeks in gestation (169, 74.1%). Anti-D Ig was administered without support from the guidelines in 81 (35.5%) patients, with a lack of documented sensitising event in 77 (95%) of these cases. Discussion and Conclusion: There were inconsistencies amongst clinicians who prescribe anti-D Ig in the ED, and a lack of the application of current guidelines. This may stem from a lack of empirical evidence about the need for anti-D Ig in the most common group presenting to EDs, those under twelve weeks in gestation. Current guidelines also fail to take into consideration future need, which could be incorporated in future, ED specific anti-D Ig guidelines. Implications for research, policy, and practice: This audit identified overuse of anti-D Ig in the ED. This may stem from the absence of evidence for its use in pregnant patients under 12 weeks in gestation. To reduce unsupported use, further data on alloimmunisation rates following potentially sensitising events in early pregnancy would be helpful. Additional guidelines specific to patients under 12 weeks in gestation, and presenting to the emergency department may reduce some unsupported usage. What is already known about the topic? Pregnant patients who have an D-negative blood type are at risk of D alloimmunisation when a sensitising event occurs. It is common for patients to present to the emergency department with complications in early pregnancy Previous work has shown that there is variation in anti-D Ig administration in the emergency department. What this paper adds: There is significant use of anti-D Ig in the emergency department that is outside of current guidelines. The current guidelines may not serve the needs of the majority of presentations in the emergency department. Further data on alloimmunisation rates following potentially sensitising events in patients less then twelve weeks of gestation would be useful.

Immune Thrombocytopenia Purpura

Key Points Two Types: Acute and Chronic Diagnosis: Established by excluding known causes of thrombocytopenia Treatment: Depends on the platelet count and the presence and severity of bleeding. When therapy is indicated, the primary treatment options for the newly diagnosed patient are: Corticosteroids Intravenous immunoglobulin Intravenous anti-D Ig

Anti-D monoclonal antibodies from 23 human and rodent cell lines display diverse IgG Fc-glycosylation profiles that determine their clinical efficacy

Anti-D immunoglobulin (Anti-D Ig) prophylaxis prevents haemolytic disease of the fetus and newborn. Monoclonal IgG anti-Ds (mAb-Ds) would enable unlimited supplies but have differed in efficacy in FcγRIIIa-mediated ADCC assays and clinical trials. Structural variations of the oligosaccharide chains of mAb-Ds are hypothesised to be responsible. Quantitative data on 12 Fc-glycosylation features of 23 mAb-Ds (12 clones, 5 produced from multiple cell lines) and one blood donor-derived anti-D Ig were obtained by HPLC and mass spectrometry using 3 methods. Glycosylation of mAb-Ds from human B-lymphoblastoid cell lines (B) was similar to anti-D Ig although fucosylation varied, affecting ADCC activity. In vivo, two B mAb-Ds with 77–81% fucosylation cleared red cells and prevented D-immunisation but less effectively than anti-D Ig. High fucosylation (>89%) of mouse-human heterohybridoma (HH) and Chinese hamster ovary (CHO) mAb-Ds blocked ADCC and clearance. Rat YB2/0 mAb-Ds with <50% fucosylation mediated more efficient ADCC and clearance than anti-D Ig. Galactosylation of B mAb-Ds was 57–83% but 15–58% for rodent mAb-Ds. HH mAb-Ds had non-human sugars. These data reveal high galactosylation like anti-D Ig (>60%) together with lower fucosylation (<60%) as safe features of mAb-Ds for mediating rapid red cell clearance at low doses, to enable effective, inexpensive prophylaxis.

Can we safely stop testing for Rh status and immunizing Rh-negative women having early abortions? A comparison of Rh alloimmunization in Canada and the Netherlands

ObjectiveThe objective of this study was to compare Rh alloimmunization rates in two countries (Canada and the Netherlands) with completely different policies regarding abortion-related use of anti-D immunoglobulin to ultimately determine any benefit in use. In the Netherlands, the policy is to offer anti-D immunoglobulin to Rh-negative women having spontaneous abortions over 10 weeks 0 days gestation and induced abortions over 7 weeks 0 days. In Canada, it is recommended to offer all Rh-negative women having induced or spontaneous abortions anti-D immunoglobulin. MethodsWe used public databases to obtain the population data, the number of births, the abortion rates (the percentage of women having induced abortions in one year) and the Rh-negativity rates (percentage of Rh negative women) in Canada and the Netherlands. Both countries do routine prenatal blood screening and we obtained the rates of clinically significant antibodies from public databases. ResultsIn nearly 2 million blood samples from pregnant women in both Canada and the Netherlands, the prevalence of clinically significant antibodies was statistically lower in the Netherlands: 4.21 (95% CI: 4.12 to 4.30) and 4.03 (95% CI: 3.93 to 4.12) per 1000, respectively. Canada and the Netherlands had small differences in rates of abortion (1.9 per 100 vs 1.2 per 100) and of Rh negativity (13.0% vs 14.5%). ConclusionDespite different anti-D Ig treatment policies, we found a similar prevalence of clinically significant perinatal antibodies among women in Canada and the Netherlands. ImplicationsOur findings suggest that The Dutch policy of not treating Rh-negative women having spontaneous abortions under 10 weeks’ or induced abortions under 7 weeks’ gestation can be safely adopted by other countries.

HLA-DQB1*0201 phenotype and severe primary RhD immunization

Purpose of Investigation: Although post-delivery administration of anti-D immunoglobulin (anti-D Ig) and antenatal prophylaxis the residual prevalence of RH alloimmunization is due to an incomplete application of the guidelines or to clinical conditions that foster mechanisms of immunization. Materials and Methods: The authors report a case of devastating RhD alloimmunization in a secundipara, group 0 Rh negative, without potentially immunizing events. Results: Anti-D antibodies were screened at 6/2 and at 13/4 weeks and were found negative. Fetal echocardiography at 25/0 weeks showed dilated right cardiac sections and a mild pericardial effusion. Titer of anti-D antibodies was (1:1024) with negative Kleihauer-Betke. At 27/0 weeks she had an increase of anti-D antibody titer (1:8192), peak systolic velocity in the middle cerebral artery suggested severe fetal anemia. A positive HLA-DQB1 allele *0201 was found, and she gave birth to an infant with severe immune-mediated hemolysis (HB: 3.8 g/dl) who was discharged 62 days after birth due to perinatal complications. Conclusion: HLA-DQB1*0201 phenotyping can improve the surveillance and treatment of non-sensitized D negative patients.

Maternal hepatitis C (HCV) infection and Anti-D immunoglobulin therapy: study testing antibodies, RNA and Genotype of HCV in Baghdad

Introduction: Hepatitis C virus (HCV) infection is a serious health problem. It is a major contributor to end-stage liver disease. Worldwide, 1–8% of all pregnant women were infected. Women with viral hepatitis may be at an increased risk of pregnancy complications. There are several obstetrics intervention acts as risk factors, which are specific to women pertaining the HCV infection; anti-D immunoglobulin (Ig) therapy may be one of them. Our objectives were to estimate the prevalence of HCV antibodies (anti-HCV), RNA, and genotype distribution among women with anti-D Ig therapy.Materials and methods: A cross sectional study was conducted. A sample of 154 Rhesus negative (Rh − ve) pregnant women regardless of the anti-D Ig therapy was collected. Anti-HCV were tested using third generation enzyme immunoassay (EIA-3) and immunoblot assay (Lia Tek-111), subsequently. In addition, 89 serum samples were subjected to molecular analysis using RT-PCR and DNA enzyme immunoassay (DEIA) method for the detection of HCV-RNA and genotypes.Results: Anti-HCV, and HCV-RNA seroprevalence were significantly higher (17.1, 35.5%) among women with anti-D Ig than their counter group (6.4, 13.16%), p = .038, .018, respectively. Significant direct positive dose response correlation (r = 0.78, p = .005) had been seen between number of anti-D Ig therapy and anti-HCV seropositive rate. Anti-D Ig therapy act as a risk factor (odds ratio (OR) = 3.01, 95%CI: 1.01–8.9) especially from the third dose onward. Women with anti-D Ig therapy were at higher risk (3.6 times more) of positive HCV-RNA (OR =3.6, 95%CI =1.19–10.837). Genotype HCV-1b showed higher prevalent (52.9%) among the recipients of anti-D Ig therapy while genotype HCV-3a (6.6%) was the lowest.Conclusions: Our study showed that Anti-D immunoglobulin therapy acts as a risk factor for acquiring HCV infection. Screening for HCV should be recommended for all recipients of anti-D Ig. Not only HCV antibodies but HCV-RNA detection being recommended for the diagnosis of HCV infection.A brief rational: Pregnant women with HCV infection are at risk of adverse obstetric outcome. Anti-D Ig therapy may be a risk factor for HCV infection. Hence, we conducted a cross sectional study with the objectives to estimate the prevalence of HCV antibodies (anti-HCV), RNA, and genotype distribution among women with anti-D Ig therapy. We found that anti-HCV and HCV-RNA seroprevalence were significantly higher in women with anti-D Ig. In addition, women with anti-D Ig therapy were 3.6 times more at risk of positive HCV-RNA with genotype HCV-1b showed higher prevalence. Therefore, anti-D Ig therapy is a risk factor for acquiring HCV infection and we recommend screening for HCV for all recipients of anti-D Ig. In addition, the diagnosis of HCV infection, should be made with HCV antibodies and HCV-RNA detection.

Comparison of the Therapeutic Effect of Anti-D IG and IVIG in Children with Acute Immune Thrombocytopenic Purpura Attending a Children’s Medical Center: A Randomized, Double-Blind, Controlled Clinical Trial

Background: This study aimed at comparing the therapeutic effect of anti-D immunoglobulin (anti-D IG) and Intravenous Immunoglobulin (IVIG) in children with acute immune thrombocytopenic purpura (ITP). Methods: This Randomized double-blind, controlled clinical trial was conducted on 98 children attending a Children’s medical center during year 2008. The selected participants were allocated to IVIG and anti-D IG groups using the balanced-block randomization method. The platelet count, hemoglobin level, and side effects of the medications were evaluated on days 1, 3, 7, 14, and 21 after drug injection. Results: After drug injection, the platelet count increased in both groups (P 0.05). The hemoglobin level also decreased after injection in both groups (P 0.05). Regarding the ability of the drugs to increase the platelet count, 56% of the patients in the anti-D IG group and 52% of the children in the IVIG groups had platelet counts of more than 20 000 during the 24 hours after drug administration with no difference between the 2 groups (P = 0.836). The incidence of drug adverse effects, including fever and chills (4.1% in the anti-D group versus 10.4% in the IVIG group), severe hemolysis (4.5% in the anti D group versus 0% in the IVIG group) and headache (6.25% in the anti-D group versus 4.1% in the IVIG group), had no significant difference between the two groups (P > 0.05). Conclusions: Intravenous Immunoglobulin and anti-D have similar effectiveness in the treatment of children with acute ITP. Considering the fewer side effects of anti-D, it may be a suitable replacement for IVIG in ITP patients.

Midwives' experience of offering anti-D immunoglobulin to women: The importance of choice

Background:Informed decision-making around anti-D immunoglobulin (Ig) is important but complex, requiring consideration of individual factors to reach the most appropriate choices for individual women.Aims:This research aimed to develop understanding of midwives' practices when offering anti-D Ig to RhD-negative women.Methods:A descriptive, qualitative study was used. Two focus groups were held with 11 midwife participants from two Scottish maternity units. Data were analysed using thematic analysis within a framework approach.Findings:When offering anti-D Ig, midwives were limited in their ability to fully engage with women in a process of individual informed decision-making, due partly to their own knowledge and understanding and partly to organisational culture and support.Conclusions:When interventions are recommended and offered routinely, it is challenging to extend the principles of woman-centred individualised care to facilitate meaningful decision-making. More effort is required to understand the difficulties faced by midwives.

Identification of genetic polymorphisms that predict responder/non-responder profiles to the RhD antigen.

Regular plasma donors who produce high titre anti-D immunoglobulin (Ig) are overseen by the Australian Red Cross Blood Service RhD Program. New donors to the program are immunised with small amounts of RhD-positive RBCs, whilst donors who have developed anti-D due to previous RhD-incompatible blood transfusion or pregnancy are boosted with RhD-positive RBCs to maintain a high level of serum anti-D Ig. A significant proportion of primarily immunised individuals do not respond to RhD immunisation and are therefore unnecessarily exposed to the risks involved in RBC sensitisation. We genotyped 184 anti-D donors for ∼9000 immunological and inflammatory genetic polymorphisms on an Affymetrix GeneChip, and validated the results with a High-Resolution Melt analysis assay. We built and validated a predictive logistic regression model using High Responder and Non-Responder anti-D donors that incorporated highly-associated polymorphisms and gender. High Responder and Non-Responder profiles in anti-D donors were significantly associated with a shortlist of 13 genetic polymorphisms and sex of the donor. The derivation of a logistic regression model showed an accuracy rate of 92.6% that was subsequently validated as 60.0% with an independent set of donor samples. This study has developed a logistic regression model and a genotyping assay that can predict the responder profiles of anti-D donors and could potentially be applied to new donors and transfusion-dependent patients in a clinical setting. Additionally, target polymorphisms identified in immunological genes could help to elucidate the immunomodulatory pathways regulating the immune response to the RhD antigen, and to other RBC antigens.

Routine administration of Anti-D: the ethical case for offering pregnant women fetal RHDgenotyping and a review of policy and practice

BackgroundSince its introduction in the 1960s Anti-D immunoglobulin (Anti-D Ig) has been highly successful in reducing the incidence of haemolytic disease of the fetus and newborn (HDFN) and achieving improvements to maternal and fetal health. It has protected women from other invasive interventions during pregnancy and prevented deaths and damage amongst newborns and is a technology which has been adopted worldwide. Currently about one third of pregnant women with the blood group Rhesus D (RhD) negative in the UK (approximately 40,000 women per year in England and Wales), receive antenatal Anti-D Ig in pregnancy when they do not require it because they are carrying a RhD negative fetus. Since 1997, a test using cell free fetal DNA (cffDNA) in maternal blood has been developed to identify the genotype of the fetus and can be used to predict the fetal RhD blood group.DiscussionThis paper considers whether it is ethically acceptable to continue administering antenatal Anti-D Ig to all RhD negative women when fetal RHD genotyping using maternal blood could identify those women who do not need this product.SummaryThe antenatal administration of Anti-D Ig to a third of RhD negative pregnant women who carry a RhD negative fetus and therefore do not need it raises important ethical issues. If fetal RHD genotyping using maternal blood was offered to all RhD negative pregnant women it would assist them to make an informed choice about whether or not to have antenatal Anti-D Ig.

BCSH guideline for the use of anti-D immunoglobulin for the prevention of haemolytic disease of the fetus and newborn.

BCSH guideline for the use of anti-D immunoglobulin for the prevention of haemolytic disease of the fetus and newborn.

Delays and omissions in administering anti-D Ig are putting newborns at risk

Delays and omissions in administering anti-D Ig are putting newborns at risk

Anti-D administration after spontaneous miscarriage for preventing Rhesus alloimmunisation.

During pregnancy, a Rhesus-negative (Rh-negative) woman may develop antibodies if her fetus is Rh-positive, which can cause fetal morbidity or mortality in following pregnancies, if untreated. To assess the effects of administering anti-D immunoglobulin (Ig) after spontaneous miscarriage in a Rh-negative woman, with no anti-D antibodies. We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (31 December 2012). Randomised controlled trials (RCT) in Rh-negative women without antibodies who were given anti-D Ig following spontaneous miscarriage compared with no treatment or placebo treatment following spontaneous miscarriage as control. Two review authors independently assessed trials for inclusion and trial quality. Two review authors extracted data and checked it for accuracy. We included one RCT, involving 48 women who had a miscarriage between eight to 24 weeks of gestation. Of the 19 women in the treatment group, 14 had therapeutic dilatation & curettage (D&C) and five had spontaneous miscarriage; of the 29 women in the control group, 25 had therapeutic D&C and four had spontaneous miscarriage. The treatment group received 300 µg anti-D Ig intramuscular injection and were compared with a control group who received 1 cc homogenous gamma globulin placebo.This review's primary outcomes (development of a positive Kleihauer Betke test (a test that detects fetal cells in the maternal blood; and development of RhD alloimmunisation in a subsequent pregnancy) were not reported in the included study.Similarly, none of the review's secondary outcomes were reported in the included study: the need for increased surveillance for suspected fetal blood sampling and fetal transfusions in subsequent pregnancies, neonatal morbidity such as neonatal anaemia, jaundice, bilirubin encephalopathy, erythroblastosis, prematurity, hypoglycaemia (low blood sugar) in subsequent pregnancies, maternal adverse events of anti-D administration including anaphylactic reaction and blood-borne infections.The included study did report subsequent Rh-positive pregnancies in three women in the treatment group and six women in the control group. However, due to the small sample size, the study failed to show any difference in maternal sensitisation or development of Rh alloimmunisation in the subsequent pregnancies. There are insufficient data available to evaluate the practice of anti-D administration in an unsensitised Rh-negative mother after spontaneous miscarriage. Thus, until high-quality evidence becomes available, the practice of anti-D Immunoglobulin prophylaxis after spontaneous miscarriage for preventing Rh alloimmunisation cannot be generalised and should be based on the standard practice guidelines of each country.

Comparison of anti-D immunoglobulin, methylprednisolone, or intravenous immunoglobulin therapy in newly diagnosed pediatric immune thrombocytopenic purpura

This study aimed to evaluate the efficacy, cost, and effects of anti-D immunoglobulin (anti-D Ig), methylprednisolone, or intravenous immunoglobulin (IVIG) therapy on the development of chronic disease in children who are Rh-positive with diagnosed immune thrombocytopenic purpura (ITP). Children with newly diagnosed ITP and platelet count <20,000/mm(3) were prospectively randomized to treatment with anti-D Ig (50 μg/kg), methylprednisolone (2 mg/kg/day), or IVIG (0.4 g/kg/day, 5 days). Sixty children with a mean age of 6.7 years were divided into three equal groups. No difference was observed between platelet counts before treatment and on day 3 of treatment. However, platelet counts at day 7 were lower in the methylprednisolone group than in the IVIG group (P = 0.03). In the anti-D Ig group, hemoglobin and hematocrit levels were significantly lower at the end of treatment (P < 0.05). Chronic ITP developed in 30% of the anti-D Ig group, 35% of the methylprednisolone group, and 25% of the IVIG group, but no significant difference was noted among the groups. The cost analysis revealed that the mean cost of IVIG was 7.4 times higher than anti-D Ig and 10.9 times higher than methylprednisolone. In the treatment of ITP in childhood, one 50 μg/kg dose of anti-D Ig has similar effects to IVIG and methylprednisolone. Among patients who were treated with anti-D Ig, serious anemia was not observed, and the cost of treatment was less than that of IVIG treatment.

A novel approach to the evaluation of bleeding-related episodes in patients with chronic immune thrombocytopenia

Objective:In clinical studies of patients with severe thrombocytopenia, rescue treatments are used to prevent or stop bleeding. Estimating risk reductions of bleeding for clinical study medications can be challenging. This study evaluated a new and possibly more accurate way of assessing the effects of a treatment intervention on bleeding-related outcomes. We developed a composite endpoint, termed bleeding-related episodes (BRE).Research design and methods:BREs were assessed in a post-hoc analysis of patients with chronic immune thrombocytopenia (ITP) who participated in two romiplostim, phase 3, placebo-controlled studies. Patients received romiplostim or placebo once weekly for 24 weeks. A BRE was defined as an actual bleeding event and/or the use of rescue medication. In total, 125 patients (41 placebo, 84 romiplostim) with platelet counts <30 K were enrolled.Clinical trial registration:NCT00102323/NCT00102336.Results:The rate of all BREs across all studies was reduced by 56% in patients receiving romiplostim compared with placebo. The rate of BREs using immunoglobulin (IVIg or anti-D Ig) was reduced by 89% in patients receiving romiplostim compared with placebo. BREs were more frequent in both groups at platelet counts <50 × 109/L. Results were similar between splenectomized and nonsplenectomized patients. We believe that prior to the development of this tool, bleeding events were underdiagnosed. The BRE tool allowed the identification of multiple interventions within bleeding episodes, which may have required separate interventions and were therefore considered to be additional BREs.Conclusions:In this study, the composite endpoint of a bleeding event and the use of rescue medication within close proximity of the bleeding event appears to be feasible and informative. The BRE tool allows for more precise understanding of the effect of rescue therapies in ITP and has broader applications to future clinical trials where assessment of bleeding risk can be complicated or masked by rescue interventions.Limitations:This was a post hoc analysis. The assignment of platelet counts to a BRE was based on the platelet count on the first day of a BRE, which may not reflect the platelet count during the entire episode, and the assignment of platelet counts was based on the estimation required for events that occurred between weekly measurements.

Routine antenatal anti‐D prophylaxis – is the protection adequate?

Prophylactic anti-D given during pregnancy can be detected in current indirect antiglobulin tests (IAT). Using this to measure the persistence of prophylactic anti-D, this study set out to determine whether there is an association between anti-D detectable at delivery and the RhD status of the foetus and/or the duration of the pregnancy post the standard 28 week dose of routine antenatal anti-D prophylaxis (RAADP). The study also investigated the detection rates of anti-D at delivery when given in a two dose regime or a one dose regime. All IAT screening was undertaken using fully automated Diamed gel technology. The results from 407 women were included in the two dose regime study, and 157 in the one dose regime study. 160/407 (39%) women receiving one dose of prophylactic anti-D had no detectable anti-D at delivery. 123/157 (78%) women on the one dose regime had no detectable anti-D at delivery. No association was found between detectable anti-D at delivery and the RhD status of the infant in either study arm. A strong association was found between detectable anti-D Ig at delivery and the duration of the pregnancy post the 28 week dose in each study arm. Our data show that neither the two dose nor the one dose regime appear to provide adequate cover at delivery for a large percentage of pregnant women. This appears to be associated with the duration of the pregnancy past the 28 week dose but not associated with the RhD status of the foetus.

Innate immune genes synergize to predict increased risk of chronic disease in hepatitis C virus infection

Hepatitis C is a common infection with significant morbidity and mortality, and only a minority of patients successfully clear the infection. Identification of factors that influence disease progression in HCV infection is difficult owing to the lack of well-defined patient cohorts. However, recent evidence supports a role for the innate immune system in virus clearance. In this study, we investigated innate immune genes for their contribution to disease progression in a unique cohort of well-controlled HCV-infected patients. The Irish cohort of HCV patients is uniquely homogenous; patients were infected with a single genotype of HCV from contaminated anti-D Ig. We genotyped 543 infected patients, including 247 patients who spontaneously resolved infection, for natural killer (NK) cell-associated killer cell Ig-like receptors (KIR) genes and the recently reported IL28B (IFNλ3) SNP. The NK cell gene KIR2DS3 was significantly increased in patients with chronic infection [odds ratio (OR) 1.90, 95% confidence interval (CI) 1.25-2.90, P < 0.002]. The IL28B "T" allele was also significantly increased in chronically infected patients (OR 7.38, 95% CI 4.93-11.07, P < 10(-8)). The presence of both markers synergized to significantly increase the risk of chronic infection over either factor alone (OR 20.11, 95% CI 9.05-44.68, P < 10(-7)). In functional experiments, we found that IL28A significantly inhibited IFN-γ production by NK cells. Thus, we demonstrate a functional link between NK cells and type 3 IFN. Our findings may contribute to the development of a prognostic test for HCV and identify therapeutic strategies for the clinical management of HCV-infected patients.

Quantification of Feto-Maternal Hemorrhage: Selection of Techniques for a Resource-Poor Setting

Background: Though RhD sensitization in RhD-negative mothers is now almost eradicated in the developed world, it continues to be a major health problem in developing nations like India. Inadequate immunoprophylaxis is the main reason. Adequate dose calculation of anti-D Ig is possible through estimation of correct feto-maternal hemorrhage (FMH) volume. In this regard, different methods have been used. Methods: We evaluated three quantitative techniques of estimating FMH: the Kleihauer-Betke test (KBT) and two flow cytometry (FC) techniques, i.e., the indirect immunofluorescence technique (IIFT) and direct immunofluorescence technique (DIFT). Stock solutions of both RhD-positive and D-negative cells were made, and 7 serial dilutions of RhD-positive cord cells in D-negative adult cells were prepared. Result: Both KBT and FC approximated the expected concentration of fetal RhD-positive cells in all mixtures tested. In both methods, an underestimation of fetal RhD-positive cells was observed when their expected concentration was ≧0.75%. Conclusion: Though FC is the most sensitive of all techniques, very few laboratories in developing nations can afford such a costly device, so it will be prudent for them to use KBT as the gold standard due to its rapidity, simplicity and affordability.

Detection of fetal RhD gene from maternal blood

Hemolytic disease of the newborn (HDN) is a clinic phenomenon which occurs during pregnancy due to the Rhesus (Rh) D alloimmunization between a Rh (-) pregnant woman, who has become sensitive to RhD antigens, and her Rh (+) fetus. As a result of the attack of maternal RhD antibodies on fetal RhD antigens, fetal anemia, HDN and fetal death may occur. % 40 of Rh (-) pregnant women carry Rh (-) fetus. However, all Rh (-) pregnant women are offered anti-D Immunoglobulin (Anti-D Ig) at 28 weeks' gestation in case of fetomaternal haemorrhage, so the pregnant women carrying Rh (-) fetus are exposed to blood products unnecessarily. Although the RhD of fetus can be detected, methods used for prenatal diagnosis recently are invasive tests and they can result in abortion in a certain percentage. The discovery of circulating cell-free fetal nucleic acids in maternal plasma has opened up new possibilities for non invasive prenatal diagnosis. The aim of this study was to detect prenatal RhD by analysing the presence of the RhD gene of fetal DNA in maternal blood. Total free DNA was isolated from the blood of 19 Rh (-) pregnant women, who had RhD alloimmunization with their husbands, in the 11-14 th week of their pregnancy. The existence of a gene in isolated DNA was investigated with TaqMan prob and "Real-time PCR" method by using primers belonging to exon 7 of RhD gene. Using a quantitative real-time PCR assay, the presence of RhD gene sequences was evaluated in the serum of patients at the onset of pregnancy. We have analyzed 19 Rh (-) pregnant women. Twelve of them were Rh (-) and the rest of them were 7 Rh (+). After birth the baby's blood groups were concordant with our results. The results obtained by RhD primer were analysed. The possibility of detection of fetal RhD gene in maternal blood contributed to noninvasive prenatal diagnosis.