Congenital Hemolytic Anemias: Is There a Role for the Immune System?

Congenital hemolytic anemias (CHAs) are a heterogeneous group of rare hereditary conditions including defects of erythrocyte membrane proteins, red cell enzymes, and disorders due to defective erythropoiesis. They are characterized by variable degree of anemia, chronic extravascular hemolysis, reduced erythrocyte life span, splenomegaly, jaundice, biliary lithiasis, and iron overload. Although few data are reported on the role of the immune system in CHAs, several immune-mediated mechanisms may be involved in the pathogenesis of these rare diseases. We reported in ~60% of patients with hereditary spherocytosis (HS), the presence of naturally-occurring autoantibodies (NAbs) directed against different membrane proteins (α- and ß-spectrin, band 3, and dematin). Positive HS subjects showed a more hemolytic pattern and NAbs were more evident in aged erythrocytes. The latter is in line with the function of NAbs in the opsonization of damaged/senescent erythrocytes and their consequent removal in the spleen. Splenectomy, usually performed to reduce erythrocyte catheresis and improve Hb levels, has different efficacy in various CHAs. Median Hb increase is 3 g/dL in HS, 1.6-1.8 g/dL in pyruvate kinase deficiency (PKD), and 1 g/dL in congenital dyserythropoietic anemias (CDA) type II. Consistently with clinical severity, splenectomy is performed in 20% of HS, 45% of CDAII, and in 60% of PKD patients. Importantly, sepsis and thrombotic events have been registered, particularly in PKD with a frequency of ~7% for both. Furthermore, we analyzed the role of pro-inflammatory cytokines and found that interleukin 10 and interferon γ, and to a lesser extent interleukin 6, were increased in all CHAs compared with controls. Moreover, CDAII and enzymatic defects showed increased tumor necrosis factor-α and reduced interleukin 17. Finally, we reported that iron overload occurred in 31% of patients with membrane defects, in ~60% of CDAII cases, and in up to 82% of PKD patients (defined by MRI liver iron concentration >4 mg Fe/gdw). Hepcidin was slightly increased in CHAs compared with controls and positively correlated with ferritin and with the inflammatory cytokines interleukin 6 and interferon γ. Overall the results suggest the existence of a vicious circle between chronic hemolysis, inflammatory response, bone marrow dyserythropoiesis, and iron overload.

Do red cell alloantibodies continue to challenge breast fed babies?

BACKGROUND: Newborns have limited specific immune capability at birth, owing to delayed and constrained development of adaptive immunity. To supplement this period the mother passively transfers antibodies to the child either transplacentally or through breast milk. When maternal alloimmunisation occurs through foreign or fetal red cell surface antigens, stimulating the production of immunoglobulin G (IgG) antibodies, these IgG antibodies can cross the placenta and cause haemolytic disease of the fetus and the newborn. OBJECTIVE: We present two case reports of a neonate and an infant in whom IgG red cell alloantibodies were transferred through maternal breast milk. METHODS: Maternal serum, baby's serum and expressed breast milk samples were tested for the presence of red cell alloantibodies using gel card. Antibody screening, antibody identifications and titres alongside monospecific direct antiglobulin test, IgG subtypes were performed using the standard methods. RESULTS: In the first case, a 6-month-old child was incidentally found to have positive antibody screen. Anti-KELL1 was identified, which was also present in maternal serum and breast milk. The second neonate was evaluated for haemolysis and was found to have anti-D. Anti-D was also detected in the maternal serum and breast milk. Both babies did not have any sensitising events. The first baby was asymptomatic, but the second baby had ongoing haemolysis until 1 month. CONCLUSION: We report that maternal anti-KELL1 and anti-D antibodies were present in breast milk and were capable of being transferred to a feeding child. Our case report also raises interesting and unanswered immunologic fundamentals that should be considered in neonates with unexplained anaemia or delayed and persistent haemolysis.

Retrospective analysis of HDFN due to ABO incompatibility in a single institution over 6 years.

OBJECTIVES: To study the rate of ABO haemolytic anaemia of fetus and newborn (HDFN) in one institution over 6 years. BACKGROUND: ABO major incompatibility between mothers and their newborns occurs in about 10% of births. So, mothers with an O blood group may form IgG-class antibodies against A and B antigens, which could pass across the placenta and lead to a variable degree of HDFN in the newborn. METHODS: At our institution, we have reviewed data regarding ABO-based HDFN in the last 6 years. RESULTS: We found that, in 28 089 deliveries, an ABO major incompatibility between mothers and newborns occurs in 11% of cases, with 72% of O/A and 28% of O/B incompatibility. In turn, 23% of these newborns had an eluate-confirmed positive direct antiglobulin test [DAT; 74% (511) were due to anti-A and 26% (179) to anti-B], with 1·0% requiring invasive treatments (exchange transfusion or intravenous immunoglobulin). Overall, 2·5% of the total newborns had a positive DAT for an anti-A or anti-B antibody, and 0·11% required invasive treatment in addition to phototherapy for their HDFN. CONCLUSIONS: Serological ABO HDFN is a relatively frequent event when an O-A/O-B incompatibility between mothers and their newborn occurs and, in most cases, translates into a self-limiting disease, with a small number of newborns requiring invasive treatments. The DAT test, although not predictive of disease severity, appears to be a useful tool to monitor babies born from O-A/O-B-incompatible pregnancies and to identify those who may require treatment.

Increased red cell turnover in a line of CD22-deficient mice is caused by Gpi1c: a model for hereditary haemolytic anaemia.

CD22, an inhibitory co-receptor of the BCR, has been identified as a potential candidate gene for the development of autoimmune haemolytic anaemia in mice. In this study, we have examined Cd22(tm1Msn) CD22-deficient mice and identified an increase in RBC turnover and stress erythropoiesis, which might be consistent with haemolysis. We then, however, eliminated CD22 deficiency as the cause of accelerated RBC turnover and established that enhanced RBC turnover occurs independently of B cells and anti-RBC autoanti-bodies. Accelerated RBC turnover in this particular strain of CD22-deficient mice is red cell intrinsic and appears to be the consequence of a defective allele of glucose phosphate isomerase, Gpi1(c). This form of Gpi1 was originally derived from wild mice and results in a substantial reduction in enzyme activity. We have identified the polymorphism that causes impaired catalytic activity in the Gpi1(c) allele, and biochemically confirmed an approximate 75% reduction of GPI1 activity in Cd22(-/-) RBCs. The Cd22(-/-).Gpi1(c) congenic mouse provides a novel animal model of GPI1-deficiency, which is one of the most common causes of chronic non-spherocytic haemolytic anaemia in humans.

The Lbw2 locus promotes autoimmune hemolytic anemia.

The lupus-prone New Zealand Black (NZB) strain uniquely develops a genetically imposed severe spontaneous autoimmune hemolytic anemia (AIHA) that is very similar to the corresponding human disease. Previous studies have mapped anti-erythrocyte Ab (AEA)-promoting NZB loci to several chromosomal locations, including chromosome 4; however, none of these have been analyzed with interval congenics. In this study, we used NZB.NZW-Lbw2 congenic (designated Lbw2 congenic) mice containing an introgressed fragment of New Zealand White (NZW) on chromosome 4 encompassing Lbw2, a locus previously linked to survival, glomerulonephritis, and splenomegaly, to investigate its role in AIHA. Lbw2 congenic mice exhibited marked reductions in AEAs and splenomegaly but not in anti-nuclear Abs. Furthermore, Lbw2 congenics had greater numbers of marginal zone B cells and reduced expansion of peritoneal cells, particularly the B-1a cell subset at early ages, but no reduction in B cell response to LPS. Analysis of a panel of subinterval congenic mice showed that the full effect of Lbw2 on AEA production was dependent on three subloci, with splenomegaly mapping to two of the subloci and expansions of peritoneal cell populations, including B-1a cells to one. These results directly demonstrated the presence of AEA-specific promoting genes on NZB chromosome 4, documented a marked influence of background genes on autoimmune phenotypes related to Lbw2, and further refined the locations of the underlying genetic variants. Delineation of the Lbw2 genes should yield new insights into both the pathogenesis of AIHA and the nature of epistatic interactions of lupus-modifying genetic variants.

HO-1 polymorphism as a genetic determinant behind the malaria resistance afforded by haemolytic disorders.

Malaria affects thousands of people around the world representing a critical issue regarding health policies in tropical countries. Similarly, also haemolytic diseases such as sickle cell disease and thalassemias are a concern in different parts of the globe. It is well established that haemolytic diseases, such as sickle cell disease (SCD) and thalassemias, represent a resistance factor to malaria, which explains the high frequencies of such genetic variants in malaria endemic areas. In this context, it has been shown that the rate limiting enzyme heme oxygenase I (HO-1), responsible for the catabolism of the free heme in the body, is an important resistance factor in malaria and is also important in the physiopathology of haemolytic diseases. Here, we suggest that allelic variants of HO-1, which display significant differences in terms of protein expression, have been selected in endemic malaria areas since the HO-1 enzyme can enhance the protection against malaria conferred by haemolytic diseases This protection apply mainly in what concerns protection against severe malaria forms. Therefore, HO-1 genotyping would be fundamental to determine resistance of a given individual to lethal forms of malaria as well as to common clinical complications typical to haemolytic diseases and would be helpful in the establishment of public health politics.

Mapping of Char10, a novel malaria susceptibility locus on mouse chromosome 9.

Resistance to blood-stage malaria in AcB55 and AcB61 is caused by a loss of function mutation in pyruvate kinase (Pklr(I90N)). Likewise, pyruvate kinase (PK) deficiency in humans is protective against Plasmodium replication in vitro. We identified a third AcB strain, AcB62 that also carries the Pklr(I90N) mutation. However, AcB62 mice were susceptible to P.chabaudi infection and showed high levels of parasite replication (54-62% peak parasitemia). AcB62 mice showed the hallmarks of PK deficiency-associated anemia similar to AcB55/61 with reticulocytosis, splenic red pulp expansion, tissue iron overload, and increased expression of iron metabolism proteins. This suggests that malaria susceptibility in AcB62 is not because of absence of PK deficiency-associated pathophysiology. To map novel genetic factors affecting malaria susceptibility in AcB62, we generated an informative F2 population using AcB62 (Pklr(I90N)) and CBA-Pk(slc) (Pklr(G338D)) as progenitors and identified a novel locus on chromosome 9 (Char10; LOD=7.24) that controls peak parasitemia. A weaker linkage to the Pklr region of chromosome 3 (LOD=3.7) was also detected, a finding that may reflect the segregation of the two defective Pklr alleles. AcB62 alleles at both loci are associated with higher peak parasitemia. These results identify Char10 as a novel locus modulating severity of malaria in the context of PK deficiency.

Multifocal osteomyelitis caused by Candida dubliniensis.

Candida dubliniensis is an emerging fungal pathogen, especially in immunodeficient patients. We report what is to the best of our knowledge the first case of multifocal osteomyelitis following disseminated infection in a patient after haematopoietic stem cell transplantation. PFGE for typing of C. dubliniensis was developed and the necessity of long-term antifungal therapy is discussed.

Generation and phenotyping of mCd59a and mCd59b double-knockout mice.

CD59 is a membrane protein inhibitor of the membrane attack complex (MAC) of complement. Humans express only one, whereas mice express two CD59 genes. We previously reported the targeted deletion of the mCd59b gene in which absence of mCd59b together with an unintended down regulation of mCd59a caused hemolytic anemia with spontaneous platelet activation. To confirm the complement role in the hemolytic anemia caused by abrogation of mCd59 function, we have developed a mCd59a and mCd59b double knock out mice and analyzed its phenotype in complement sufficient and deficient (C3(-/-)). We report here that total abrogation of mCd59 function in mCd59ab(-/-) mice results in complement-mediated hemolytic anemia that is rescued by the deficiency of C3 in compound mCd59ab(-/-)/C3(-/-) mice.

Pyruvate kinase deficiency: correlation between enzyme activity, extent of hemolytic anemia and protection against malaria in independent mouse mutants.

AcB55, AcB61 and CBA/N-Pk(slc) mice carry loss of function mutations in the erythrocyte specific pyruvate kinase gene (Pklr). In AcB55 and AcB61 (Pklr(I90N)) PK deficiency is protective against blood-stage malaria. The mechanistic basis of protection against malaria is unknown and was studied in these two mutant alleles in vivo. The Pklr(G338D) mutation of the CBA/N-Pk(slc) mutant is shown to be more deleterious than the Pklr(I90N) allele with respect to enzymatic activity and severity of hemolytic anemia, with a more dramatic reduction in the half-life of erythrocytes (increased turnover) in the CBA/N-Pk(slc) mice. The CBA/N-Pk(slc) mice are also shown to be highly resistant to infection with Plasmodium chabaudi AS when compared to CBA/J and CBA/N controls. Resistance to malaria, measured as lower levels of blood-stage replication of P. chabaudi, rapid elimination of infected erythrocytes and increased survival to infection, was greater in the Pklr(G338D) mutant, CBA/N-Pk(slc), than in the Pklr(I90N) mutant strains, AcB55/AcB61. These results strongly suggest a correlation between severity of PK-deficiency and extent of protection against malaria. Additionally, the protective effect is independent of the genetic background on which the Pklr mutations occurred.

[A quantitative determination of IgG anti-D subclasses by Elisa in hemolytic disease of the newborn]. / Etude quantitative des sous-classes d'IgG anti-D par Elisa au cours de la maladie hémolytique néonatale.

The quantification of IgG anti-D subclasses is one of the most important parameters considered in the assessment of the severity of hemolytic disease of the newborn. Traditionally IgG subclassing is performed using qualitative haemagglutination methods, difficult to interpret. A quantitative enzyme-linked immunosorbent assay (Elisa) was implemented for measuring IgG anti-D subclasses in 20 sera collected from 14 RhD-immunized pregnant women. All 4 IgG subclasses were detected in the 20 sera tested. The mean proportion of IgG1 was 52.8%. The mean proportion of IgG3 was 30.7%. The mean proportions of IgG2 and IgG4 were 14.5 and 1.9% respectively. A good correlation between the sum of IgG subclasses and the severity of HDN was found. Severe HDN occurred when both IgG1 and IgG3 were present. IgG1 anti-D was the predominant subclass in 4 of the 8 severe cases.

[Autoimmune hemolytic anemia with complement-positive direct antiglobulin test]. / Anemia hemolítica autoinmune con prueba de antiglobulina positiva a complemento.

Autoimmune hemolytic anemia (AIHI) is an infrequent disease in the pediatric age group. Its diagnosis is given by the direct antiglobulin test (DAT) or Coombs' test, which determines which type of globulin (IgG or complement) is the cause of the hemolysis. The type of globulin involved determines the etiology of AIHI, which is usually confirmed by positive results of other laboratory investigations such as cold agglutinin determination or the Donath-Landsteiner test. We present three cases of AIHI. DAT was positive to complement with diverse etiology: warm antibody with IgG-negative DAT, cold agglutinins associated with infectious mononucleosis, and Doth-Landsteiner antibodies. In all patients, empirical treatment with corticosteroids was initiated. The treatment was withdrawn or continued, depending on the final etiology of AIHI.

[Immune cytopenias in newborns]. / Cytopénies immunes néonatales.

Rhesus D haemolytic disease of the newborn (RH HDN) and neonatal PlA1 alloimmune thrombocytopenia (NAT) are the main immune cytopenias affecting fetal red blood cells or platelets through maternal antibodies. During RH HDN, fetal anaemia and neonatal hyperbilirubinaemia may progress, if untreated, towards fetal death and neonatal kernicterus. Likewise, during NAT, intracranial haemorrhage may occur antenally, at delivery or postnatally. Fetal and neonatal transfusion therapy, pre-term delivery, and intensive phototherapy avoid or greatly reduce the incidence of these complications. However, the best treatment of RH HDN is to prevent primary anti-D immunisation in Rh negative pregnant women through passive immunotherapy with Rh immune globulin.

Haemolytic disease of the newborn due to anti-Ce.

A baby was readmitted to the hospital 3 days after delivery when she developed jaundice. At admission, the direct antiglobulin test was also found to be positive. The baby required emergency exchange transfusion. A strongly reacting IgG anti-Ce (Rh7) was found in the serum of the mother. Severe haemolytic disease of the newborn due to anti-Ce is very rare. The mother's serum had been screened and found negative for red cell antibodies at 16 weeks gestation but the test was not repeated later in pregnancy when this antibody might have been detected.

Neonatal lupus erythematosus with microvascular hemolysis.

A female, term newborn born to a mother with a history of idiopathic thrombocytopenic purpura and antinuclear antibodies, single-stranded A antibody, and IgM anticardiolipin antibodies presented with immune thrombocytopenia, disseminated intravascular coagulation (DIC), microangiopathic hemolytic anemia, and a characteristic lupus rash in the periorbital areas. She responded to combined treatment with dexamethasone and intravenous immunoglobulin (IVIG). At age 9 months, she was readmitted with severe thrombocytopenia, DIC, and microangiopathic hemolytic anemia. She again responded to IVIG. This suggests that microangiopathic hemolysis can be a presenting symptom in neonatal lupus erythematosus and that reoccurrence of the microangiopathic hemolysis may occur even after the disappearance of lupus antibodies.

Survival of donor cells 25 years after intrauterine transfusion.

Persistence of donor leukocytes in the circulation of recipients of intrauterine transfusion (IUT) has been observed up to 5 years after birth. The aim of this study was to determine whether transfusions with nonirradiated, nonleukocyte-depleted donor blood during the fetal period resulted in long-term immunomodulation of the recipient. Twenty-four surviving IUT recipients between 1966 and 1976 were tested for autoimmune disease and autoantibodies at follow-up. Ten had sex-mismatched donors and were therefore informative for chimerism studies using fluorescence in situ hybridization (FISH). Seven female recipients could be tested for chimerism using a Y- chromosome-specific polymerase chain reaction (PCR) because they received at least 1 IUT from a male donor. Nine recipients could be studied for cytotoxic T-lymphocyte precursor (CTLp) and helper T-lymphocyte precursor (HTLp) frequencies because the original donors were available for testing. All surviving IUT recipients were in good health at the time of the examination, and routine laboratory testing revealed no abnormalities. None of the IUT recipients were chimeric as determined by FISH analysis, but Y-chromosome-specific sequences were detected by PCR in 6 of the 7 women. However, the CTLp and HTLp frequencies of the IUT recipients against the donors were comparable to those of the controls. The current study provides evidence that IUT can result in the persistence of donor cells in the recipient for a period longer than 20 years but that it is not associated with immunotolerance or with signs of chronic antigenic stimulation. (Blood. 2000;95:2709-2714)