Rh Immunoglobulin Research Articles

A rapid gel agglutination test for the determination of fetomaternal haemorrhage

Determination of fetomaternal haemorrhage (FMH) remains an area of difficulty. In most cases, prophylactic Rh immunoglobulin is usually administered to affected women without testing for foetal red blood cells (RBC). Here, we describe a new particle gel immunoassay (PaGIA) for the determination FMH (FMH-PaGIA). Superparamagnetic particles were coated with monoclonal anti-D and mixed with ethylenediaminetetraacetic acid-anticoagulated blood samples from D-negative pregnant women. The particles were isolated using a magnetic particle concentrator and then placed into the reaction chamber of a gel card. Agglutinated particles on top or dispersed through the gel matrix indicated the presence of D-positive cells. After the test was adapted to detect >or=0.3% D-positive RBC, randomly selected postpartum samples from 208 women were analysed in parallel with the Kleihauer-Betke test (KBT). In addition, all discrepancies were further analysed by flow cytometry. A total of 203 of the 208 postpartum samples were negative in both tests. One sample reacted positive with both assays. Two samples were strongly positive in the new FMH-PaGIA, but negative in the KBT. A serological re-examination revealed that both women were D positive. The KBT gave a false-positive result in two cases because of hereditary persistence of haemoglobin F. The new test is specific, easy to perform and can be done at any time in all laboratories.

Update on HDFN: new information on long-standing controversies

Abstract Hemolytic disease of the fetus and newborn (HDFN) results from maternal IgG antibodies that cross the placenta to the fetal circulation during gestation and cause RBC destruction and complications before birth (HDF), or anemia and hyperbilirubinemia after birth (HDN), or both. In its most severe form, HDF produces hydrops fetalis, which is characterized by total body edema, hepatosplenomegaly, and heart failure and can lead to intrauterine death. Before discovery of Rh immunoglobulin (RhIG), HDFN from anti-D was a significant cause of perinatal mortality or long-term disability. Routine administration of RhIG to D– women during pregnancy and shortly after the birth of D+ infants effectively reduced the incidence of HDFN caused by anti-D. Maternal alloimmunization to other RBC antigens in the Rh, Kell, and other blood group systems can not be routinely prevented and these antibodies can also cause HDFN. Advances in prenatal care, noninvasive monitoring, and intrauterine transfusion have improved the outlook for affected pregnancies to the extent that even hydrops fetalis can be reversed and effectively treated in many cases. This review will provide an update on the current issues in prevention and treatment of HDFN, emphasizing recent insights into long-standing controversies regarding maternal weak D phenotypes and D alloimmunization, noninvasive fetal diagnosis and monitoring of affected pregnancies, and recent treatment guidelines. Immunohematology 2006;22:188–195.

Disease Versus Disease: How One Disease May Ameliorate Another

Systemic disease, either genetic or acquired, may prevent or decrease the severity of another disease. These observations have led to important therapeutic advances. The best-known examples are Edward Jenner's use in 1798 of cowpox to prevent smallpox and J.B. Haldane's 1942 observation that erythrocyte disorders such as thalassemia and sickle cell disease modify the severity of malaria. Patients with and carriers of cystic fibrosis may have genetic resistance to tuberculosis and/or secretory diarrhea. The beneficial effects of undernutrition have led to therapeutic diets for seizures, celiac disease, type 2 diabetes, and inflammatory bowel disease. Finasteride for prostatic hypertrophy was developed after the observation that patients with male pseudohermaphrodism resulting from 5-alpha-reductase mutations do not develop prostatic hypertrophy. Rh immunoglobulin for Rh hemolytic disease prevention followed the observation that ABO incompatibility prevented Rh sensitization. The natural immunosuppression of measles may cause remission of nephrosis, and that of leprosy prevents psoriasis. Patients with one form of agammaglobulinemia (X-linked) never get Epstein-Barr virus infection, and patients with another form (common variable) are seemingly cured by HIV infection. HIV/AIDS is prevented or modified by co-receptor mutations (notably the CCRDelta32 chemokine mutation), HIV-2, or GB virus C infection. Additional exploration of these genetic, infectious, and metabolic influences on disease severity may provide new therapeutic approaches to HIV and other diseases.

Anti‐D alloimmunization after D‐mismatched allogeneic hematopoietic stem cell transplantation in patients with hematologic diseases

The de novo development of anti-D after D-mismatched allogeneic hematopoietic stem cell transplantation (AHSCT) is a possibility that must be considered. The transfusion of D- blood components after AHSCT has been recommended but anti-D alloimmunization in this setting has been studied little. Thus, the aim of this study was to analyze anti-D formation after D-mismatched AHSCT. Thirty patients with a hematologic disease who underwent D-mismatched AHSCT were retrospectively studied. Support therapy included red blood cells (RBCs) and platelet (PLT) concentrates (PCs) from whole-blood donations and PLTs from apheresis. After AHSCT, patients received D+ PCs without administering Rh immunoglobulin (RhIG). An antibody screening to detect anti-D was performed by low-ionic-strength saline-indirect antiglobulin test with the tube test. Fifteen D+ patients received stem cells (SCs) of D- donors and 15 D- patients received SCs of D+ donors. After AHSCT, patients received a median of 11.5 (range, 0-32) D- RBC units. D+ patients received 682 (83%) of 825 PLT units from D+ donors, and D- patients received 573 (85%) of 678 PLT units from D+ donors. None of the 30 patients developed anti-D after a median follow-up of 32 weeks (range, 4-310 weeks). Anti-D alloimmunization after performing a D-mismatched AHSCT is infrequent in patients with hematologic diseases although patients receive D-mismatched PLT transfusions without RhIG administration.

Long-term cost-efficacy of rituximab in immune thrombocytopenic purpura

Sir, Prior to 1981, the only effective treatments for increasing platelet count in immune thrombocytopenic purpura (ITP) were corticosteroids and splenectomy. More recently, intravenous immunoglobulin (IVIg) and Rh immunoglobulin (IV RhIg) have demonstrated efficacy. Other treatments, including danazol, azathioprine, cyclophosphamide, vinca alkaloids and cyclosporin A, are supported by small and mostly uncontrolled clinical trials. When conventional therapy fails, ITP may be a challenging clinical situation, as long-term corticosteroids have severe side-effects, and IVIg loses its efficacy over the long term. Monoclonal antiCD20 antibodies (rituximab) appear to be effective in ITP, but controlled clinical trials are lacking.1 We present a case of refractory ITP with a sustained response to rituximab, and discuss the cost/benefit ratio of other …

Rh(D) Immunoglobulin – Where Does it Come From?

Australian Rh(D) Immunoglobulin is produced by CSL Bioplasma using plasma containing anti‐D collected by the Australian Red Cross Blood Service (ARCBS) from Australian voluntary blood donors. The ARCBS Rh Project was established, initially in New South Wales in 1967, with an aim of providing an adequate supply of Australian derived Rh(D) Immunoglobulin. In 1999, the NHMRC ‘Guidelines on the prophylactic use of Rh(D) Immunoglobulin (Anti‐D) in obstetrics’ stated that universal prophylaxis with Rh(D) Immunoglobulin to RhD negative women with no preformed anti‐D antibodies at 28 and 34 weeks gestation is generally regarded as best practice. With the availablity of Rh(D) Immunoglobulin at that time, the NHMRC recommended a phased implementation commencing with all Rh(D) negative primigravidae with no preformed anti‐D antibodies (Phase 1). Phase 1 commenced in late 2002. The NHMRC recommemnded that full antenatal prophylaxis (phase 2) would be implemented when domestic supplies of Rh(D) Immunoglobulin increased sufficiently to cover the increased demand. The ARCBS plays an integral part in the implementation of universal antenatal prophylaxis by being the supplier of plasma containing anti‐D for production and as the distributor of the final Rh(D) Immunoglobulin product. In 1999, the ARCBS supplied an average of 15 × 106 IU of anti‐D per month to CSL Bioplasma. Implementation of phase 1 occurred following the introduction of two new products; the Australian derived Rh(D) Immunoglobulin 250 IU dose for first trimester indications and the temporary importation of WinRho SDFTM for postnatal use. Implementation of phase 2 and 3 requires the ARCBS to increase the amount of anti‐D collected to an average of 30 × 106 IU and 45 × 106 IU per month, respectively. Phase 3 of the program is full antenatal prophylaxis supported entirely by domestic product. In 2002, additional funding was allocated to the ARCBS Rh Project to implement strategies to ensure that the input targets for phase 2 and 3 are achieved. In mid 2004, following months of primary immunisation and boosting of both new and existing donors, input levels began to consistently meet or exceed 30 × 106 IU, and planning for phase 2, implementation began. This presentation will give an overview of the strategies used by the ARCBS to increase the volume of anti‐D collected from these donors.

Karl landsteiner and his major contributions to haematology

Karl landsteiner and his major contributions to haematology

The Accessibility of Abortion Services In the United States, 2001

Abortion services are provided in hospitals, doctors' offices and various types of clinics, but about two-thirds of procedures are performed in specialized abortion clinics. While this system appears to work well for most women, some women seeking abortion face obstacles related to distance, cost, harassment and special medical conditions. Nine percent of nonhospital abortion patients must travel more than 100 miles and 18 percent travel 50 to 100 miles for services. The average woman having a first-trimester nonhospital abortion paid $251 in 1989. Fees were higher in facilities with small abortion caseloads. An abortion at 10 weeks' gestation in a hospital cost an average of $1,757. Charges for abortions at 16 weeks averaged $509 in abortion clinics, compared with $1,539 in hospitals for curettage and $2,246 for instillation procedures. Some women face other barriers: Only 43 percent of all abortion facilities offer services past 12 weeks, and 27-37 percent of nonhospital facilities say they do not treat patients who test positive for the human immunodeficiency virus (HIV), the virus that causes AIDS. Women who need special services such as an administration of Rh immunoglobulin, general anesthesia or HIV testing usually pay extra for these services. In addition, 85 percent of nonhospital facilities that serve 400 or more abortion patients a year reported some form of antiabortion harassment in 1988, most commonly picketing; there was virtually no change in this proportion between 1985 and 1988.Aspects of abortion services that affect their accessibility to U.S. women are described. Following an overview of the types of providers (both hospital and non-hospital), consideration is given to barriers to abortion, including distance, cost, harassment by antiabortion demonstrators, length-of-gestation constraints, and the reluctance of many facilities to provide services to those who test positive for HIV. The author concludes that abortion services will become increasingly difficult and expensive to obtain.

Determination of RhD Zygosity: Comparison of a Double Amplification Refractory Mutation System Approach and a Multiplex Real-Time Quantitative PCR Approach

Rh isoimmunization and hemolytic disease of the newborn still occur despite the availability of Rh immunoglobulin. For the prenatal investigation of sensitized RhD-negative pregnant women, determination of the zygosity of the RhD-positive father has important implications. The currently available molecular methods for RhD zygosity assessment, in general, are technically demanding and labor-intensive. Therefore, at present, rhesus genotype assessment is most commonly inferred from results of serological tests. The recent elucidation of the genetic structure of the prevalent RHD deletion in Caucasians, as well as the development of real-time PCR, allowed us to explore two new approaches for the molecular determination of RhD zygosity. Two methods for RhD zygosity determination were developed. The first was based on the double Amplification Refractory Mutation System (double ARMS). The second was based on multiplex real-time quantitative PCR. For the double ARMS assay, allele-specific primers were designed to directly amplify the most prevalent RHD deletion found in RhD-negative individuals in the Caucasian population. The multiplex real-time quantitative PCR assay, on the other hand, involved coamplification and quantification of RHD-specific sequences in relation to a reference gene, albumin, in a single PCR reaction. A ratio, DeltaCt, based on the threshold cycle, was then determined and reflects the RHD gene dosage. The allele-specific primers of the double ARMS assay reliably amplified the RHD-deleted allele and therefore accurately distinguished homozygous from heterozygous RhD-positive samples. The results were in complete concordance with serological testing. For the multiplex real-time quantitative PCR assay, the DeltaCt values clearly segregated into two distinct populations according to the RHD gene dosage, with mean values of 1.70 (SD, 0.17) and 2.62 (SD, 0.29) for the homozygous and heterozygous samples, respectively (P: <0.001, t-test). The results were in complete concordance with the results of serological testing as well as with the double ARMS assay. Double ARMS and real-time quantitative PCR are alternative robust assays for the determination of RhD zygosity.

Evaluation of the Safety of Rh Immunoglobulin by Monitoring Viral Markers among Rh–Negative Female Blood Donors

Background and Objectives: Although immunoglobulin (Ig) preparations including RhIg have been noted for their record of safety, recent reports of hepatitis C virus (HCV) transmission by some Ig preparations have raised concern. This analysis examined the safety of RhIg manufactured in the US by comparing the prevalence and incidence of viral markers in Rh–negative and Rh–positive female blood donors. Materials and Methods: Demographic and viral marker data were analyzed for allogeneic donations collected from female donors of childbearing age (17–49 years of age) from April 1992 to May 1996. Prevalence and incidence rates were calculated for HCV, human immunodeficiency virus (HIV), and hepatitis B surface antigen (HBsAg). Results: Of the 624,939 female donors included in the study, 96,355 (15.4%) were Rh–negative and 528,584 (84.6%) Rh–positive. There were no significant differences in the prevalence of HCV and HIV between Rh–negative and Rh–positive female donors. HBsAg prevalence was significantly higher among non–white compared to white donors. Following implementation of the more sensitive EIA 2.0 screening test for HCV in April 1992, prevalence and incidence rates declined over time at similar rates for Rh–negative and Rh–positive female donors. Conclusions: Rh–negative female donors had similar prevalence and incidence rates for most viral markers compared to Rh–positive female donors. This analysis supports the historical safety of RhIg.

Evaluation of the Safety of Rh Immunoglobulin by Monitoring Viral Markers among Rh-Negative Female Blood Donors

Evaluation of the Safety of Rh Immunoglobulin by Monitoring Viral Markers among Rh-Negative Female Blood Donors

Noninvasive Testing for Fetal Anemia

Despite the introduction of anti-D immune globulin (Rh immunoglobulin) for the prevention of anti-D red-cell alloimmunization, hemolytic disease of the newborn continues to occur because of the lac...

Quantitating fetomaternal hemorrhages of D+ red cells using an FITC-conjugated IgG monoclonal anti-D by flow cytometry: a case report

Several methods for quantitating fetomaternal hemorrhages (FMHs) have been described; these include the Kleihauer-Betke and red cell rosetting tests, and flow cytometry that uses an indirect antiglobulin technique, employing either FITC-conjugated IgG/unlabeled anti-D or streptavidin conjugates with biotinylated anti-D to enumerate D+ red cells in maternal blood. We have used a recently described directly conjugated FITC anti-D for direct flow cytometric (direct FC) quantitation of FMH in a patient who presented with a large fetal bleed (approx. 80mL) as determined using the Kleihauer method. We compared the efficacy of the direct FC technique to the roset- ting and Kleihauer tests in estimating the quantity of Rh immunoglobulin (RhIg) to be administered to the mother to suppress Rh alloimmunization. Both the Kleihauer and the direct FC gave precise estimates of 80mL for the size of bleed, whereas the rosetting test failed to be as precise. The former tests predicted that a 10,000 iu dose (2,000 μ g) of RhIg would be adequate; the lack of alloanti-D in a maternal follow-up sample obtained 9 months after delivery supported this prediction and underlined the reliability of the direct FC method as an alternative to the Kleihauer method for quantitating large FMHs. Immunohematology 1997;13:12–14.

Testing the Mother's Blood to Determine Fetal Rh Type

Rh-negative pregnant women can become sensitized to Rh antigens on the surface of fetal red blood cells that cross into the maternal circulation. Despite the use of prophylactic Rh immunoglobulin, an estimated 10.6 of 10,000 live births …

The Accessibility of Abortion Services in the United States

Abortion services are provided in hospitals, doctors' offices and various types of clinics, but about two-thirds of procedures are performed in specialized abortion clinics. While this system appears to work well for most women, some women seeking abortion face obstacles related to distance, cost, harassment and special medical conditions. Nine percent of nonhospital abortion patients must travel more than 100 miles and 18 percent travel 50 to 100 miles for services. The average woman having a first-trimester nonhospital abortion paid $251 in 1989. Fees were higher in facilities with small abortion caseloads. An abortion at 10 weeks' gestation in a hospital cost an average of $1,757. Charges for abortions at 16 weeks averaged $509 in abortion clinics, compared with $1,539 in hospitals for curettage and $2,246 for instillation procedures. Some women face other barriers: Only 43 percent of all abortion facilities offer services past 12 weeks, and 27-37 percent of nonhospital facilities say they do not treat patients who test positive for the human immunodeficiency virus (HIV), the virus that causes AIDS. Women who need special services such as an administration of Rh immunoglobulin, general anesthesia or HIV testing usually pay extra for these services. In addition, 85 percent of nonhospital facilities that serve 400 or more abortion patients a year reported some form of antiabortion harassment in 1988, most commonly picketing; there was virtually no change in this proportion between 1985 and 1988.

Rh immunization by the partial D antigen of category DVa

Reported here is the first example of a partial D antigen stimulating the production of anti-D: stimulation was of fetal origin. During her second pregnancy, anti-D developed in the serum of a D-negative mother who had received Rh immunoglobulin after the birth of her first D-positive child. Her second baby had moderate neonatal jaundice and was successfully treated by phototherapy. Subsequently the red cells of the father and of the first child were shown to carry a partial D antigen of category DVa type. Six available batches of Rh immunoglobulin reacted with DVa cells.

Direct quantitation of IgG subclasses 1, 2, and 3 bound to red cells by Rh1 (D) antibodies.

The authors developed quantitative radioimmunoassays to allow direct measurement of total human IgG and individual IgG subclasses among antibodies bound to cell surfaces. The assays use four mouse monoclonal radioiodinated antibodies, one that reacts equally well with all four human IgG subclasses and three that are specific for human IgG subclasses 1, 2, or 3. The assays were used to analyze IgG subclass composition in 21 high-titer anti-D samples from Rh-negative volunteers immunized for Rh immunoglobulin production. Anti-D activity was restricted primarily to the IgG1 and IgG3 subclasses. Eleven of 21 sera demonstrated red cell antibodies with a marked predominance of IgG1 (87 +/- 3.6% of total IgG antibody, +/- SEM) and low levels of IgG3 (1.4 +/- 0.73%). In the remaining 10 sera, IgG3 made up a greater proportion of total IgG antibody (32 +/- 3.8%), although IgG1 was still predominant (61 +/- 4.1%). This observed dichotomy in the IgG subclass profiles of different anti-D sera may be a consideration in the selection of anti-D sera for the production of the immunoglobulin used in the prophylaxis of Rh-incompatible pregnancies.

Maternal erythrocytes in the fetal circulation. The immunocytochemical identification of minor populations of erythrocytes.

Maternal erythrocytes (Rh+) have been identified in the fetal circulation of Rh- infants by the use of a simple immunocytochemical staining technic. Placental or capillary blood from an Rh- infant of an Rh+ mother was incubated in sequence with Rh immunoglobulin, rabbit antihuman IgG, and finally goat antirabbit IgG-alkaline phosphatase conjugate. Maternal cells were found in 14% (11 of 80) of placental vein samples and 10% (2 of 20) of capillary samples. The volume of maternal blood present in the fetal circulation was calculated to be 0.06-2.3 mL with the use of placental vein samples and 0.6-1.25 mL with the use of capillary blood.

Rh immunoglobulin utilization after ectopic pregnancy

To assess the use of Rh immunoglobulin (RhlG) after ectopic pregnancy, we reviewed the charts of 305 patients treated from 1975 through 1978 at a large metropolitan hospital. We compared these patients with 389 who had had spontaneous abortions and been treated at the same hospital in 1975. The rate of ascertainment of Rh type was significantly higher for the group with ectopic pregnancy than for the group with spontaneous abortion (98.4% versus 95.1%; p < 0.05). Nevertheless, presumably fertile RhlG candidates after ectopic pregnancy were 3.3 times more likely not to receive RhlG than candidates after spontaneous abortion (64.3% versus 19.4%; p < 0.01). Patients with ectopic pregnancy are an important part of the “RhlG utilization gap”; the mechanism for providing prophylaxis for patients needs to be improved.

Antifertility effects from immunization of female baboons with C-terminal peptides of HCG beta subunit

Anecdotal evidence has suggested an association of intravenous drug abuse with alloimmunization; however, published data are limited to case reports.The purpose of this study was to determine whether women with a history of intravenous drug abuse have an increased risk of alloimmunization.A retrospective cohort study was performed with the use of data from a single-center blood bank and perinatal database from 2008–2014. Blood bank data were used to identify women with alloimmunization, which was defined as a positive antibody screen in pregnancy not due to naturally occurring antibodies, agglutinins, autoantibodies, or Rh immunoglobulin administration. Intravenous drug abuse was ascertained from a comprehensive database that has captured all drug abuse in pregnancy since 2008. For women who contributed >1 pregnancy to the database, only the most recent pregnancy was included. The rates of alloimmunization among women with a history of intravenous drug abuse and general obstetric populations were calculated and compared. The distribution of alloantibody types, proportion of Rh-group alloantibodies, and patient Rh status were assessed for intravenous and non–intravenous drug abuse–associated alloimmunization. Characteristics and outcomes between intravenous and non–intravenous drug abuse–associated alloimmunization were assessed for women with clinically significant alloantibodies.Alloimmunization was more common in women with a history of intravenous drug abuse (11/305 women; 3.6%) compared to women without a history of intravenous drug abuse (288/16,022 women; 1.8%; relative risk, 2.00; 95% confidence interval, 1.11–3.62). Needle-sharing was present in 7 and suspected in 4 women with an intravenous drug abuse history. Among women with a history of intravenous drug abuse, none had a history of transfusion or traditional risk factor for alloimmunization. The distribution of alloantibodies was different between intravenous drug abuse– and non–intravenous drug abuse–associated alloimmunization. Rh group alloantibodies and Rh-negative status were more common in women with a history of intravenous drug abuse. Among Rh-negative women with a history of intravenous drug abuse, 50% of RhD alloimmunization cases occurred in nulliparous women. The rate of multiple alloantibodies was not different between intravenous drug abuse– and non–intravenous drug abuse–associated alloimmunization.Maternal history of intravenous drug abuse is associated with an increased risk of alloimmunization. Approximately 1 in 30 intravenous drug abuse women may be diagnosed with an alloantibody in pregnancy. Given the current US opioid epidemic, increased vigilance in screening is required. Needle-sharing represents a possible mechanism for intravenous drug abuse–associated alloimmunization; however, limited obstetric care, failure to obtain Rh immunoglobulin, or failure to identify early pregnancy loss cannot be excluded.