Alloimmune Disease Research Articles

The Alloantigenic Sites of α3α4α5(IV) Collagen: PATHOGENIC X-LINKED ALPORT ALLOANTIBODIES TARGET TWO ACCESSIBLE CONFORMATIONAL EPITOPES IN THE α5NC1 DOMAIN

The Alloantigenic Sites of α3α4α5(IV) Collagen: PATHOGENIC X-LINKED ALPORT ALLOANTIBODIES TARGET TWO ACCESSIBLE CONFORMATIONAL EPITOPES IN THE α5NC1 DOMAIN

Transfusion‐Related Risks of Intradermal Allogeneic Lymphocyte Immunotherapy: Single Cases in a Large Cohort and Review of the Literature

Lymphocyte immunotherapy (LIT) is applied in infertility treatment. Moreover, it has been suggested for prevention of rhesus D-hemolytic disease and as a vaccine for reduction of human immunodeficiency virus-1 susceptibility. Although transfusion-related problems have been rarely reported they were a matter of debate. Here we discuss extensive single-center experience with intradermal LIT for implantation failure and recurrent miscarriages. Retrospective 2- to 3-year follow-up of in vitro fertilization couples treated during 1996-2002 (feedback 2,848/3,041 = 93%), registering 930 deliveries. Prospective survey for acute reactions for 2000-2003 (feedback 2,687/3,246 = 83%). Review of the literature. Infections of the patient and transplant rejection later in life are minor residual risks. Post-transfusion purpura was suspected once but not verified. Anaphylaxis or malignancy were not promoted. Fetal/newborn alloimmune disease (severe hemolytic disease, thrombocytopenia, neutropenia) were not observed. Based on microbiological, immunological, and hematological testing the risks of intradermal LIT are low.

Neonatal hemochromatosis: It's OK to say “NO” to antioxidant-chelator therapy

Neonatal hemochromatosis (NH) is a rare medical condition presenting as severe liver disease in the newborn period. The natural history of patients with NH is grim; when not stillborn, most infants present in the first days or weeks of life with clinical features of liver failure. Within the first few weeks of life, most succumb to the complications of end-stage liver disease if not successfully rescued by liver transplantation. Given its rarity and severity, NH has been difficult to study and frustrating to treat. Rarely, spontaneous recovery without disease-specific medical intervention or liver transplantation is observed in patients with NH. In this issue of Liver Transplantation, Rodrigues and co-workers describe the presentation, management and outcomes of the largest series of patients with NH reported to date: 19 infants evaluated over a 12-year time span at the King’s College Hospital, London. Very few centers in the world have reported on more than a few patients with NH. 1-3

Noninvasive Prenatal Diagnosis of Fetal Rhesus D

Rhesus (Rh) D blood group incompatibility between the pregnant woman and her fetus is a significant problem due to the possibility of maternal alloimmunization and consequent hemolytic disease of the newborn. The RhD-negative blood group is found in 15% of whites, 3-5% of black Africans, and is rare in Asians. Advances in both our understanding of the RHD locus and its variants, as well as technical improvements in the extraction and amplification of cell-free fetal DNA in maternal plasma, have led to incorporation of noninvasive diagnosis of RHD genotype into routine prenatal care in the United Kingdom, France, and the Netherlands. In this commentary we examine the experience to date with large-scale clinical trials performed in the European Union, describe approaches to reduce false-positive and false-negative results, and review ongoing research to standardize assays and reduce costs using automated assays. False-negative cases are mainly due to either a lack of fetal DNA in the maternal sample due to early gestation or insensitive methods. False-positive cases are due to genotypic variants observed in individuals of African ancestry. Noninvasive prenatal diagnosis of fetal Rhesus D genotype is sensitive and accurate and has been widely validated in Europe. The United States should begin to undertake clinical trials to bring this technology to patient care as soon as possible.

NEONATAL HEMOCHROMATOSIS (NH) DUE TO GESTATIONAL ALLOIMMUNITY: DETECTION OF MATERNAL ALLOANTIBODY AND PREVENTION OF RECURRENCE

Background: NH is a severe fetal liver disease. We hypothesized that NH is an alloimmune gestational disease and showed that treatment with high-dose IVIG during gestation prevents lethal recurrence (Lancet 2004;364:1690-8). The aims of this study were: 1. Identify the fetal target of alloimmunity in order to develop a serologic test for alloantibody; and 2. Extend the trial of gestational treatment in order to further determine its effectiveness. Methods and Results: Subjects were women whose last pregnancy ended in a bona fide case of NH; controls were unaffected women and spouses of subjects. IgG isolated by protein-A sepharose binding was tested for immunoreactivity with fetal liver proteins by western blot and 2-D PAGE immunoblot. IgG from NH subjects consistently reacted at high dilution with a specific protein in fetal (but not adult) human, mouse and calf liver, whereas control IgG at low titer at most weakly detects this protein, which is tentatively identified as a fetal isoform of hemeoxygenase-1 (HO-1). This protein isolated from fetal calf liver is being used to develop a sandwich immunoassay for quantitative, high throughput determination of alloantibody. Subjects were offered gestational treatment (IVIG 1g/kg weekly from 18 wks gestation) in subsequent pregnancies. To date, 29 subjects have enrolled. All 25 babies born to date survived with medical care alone (p < 0.00001 versus previous gestations); 5 women are currently receiving treatment; and 1 subject suffered miscarriage at 18 weeks before treatment began. We became aware after the fact that a woman beginning off-protocol treatment at 24-25 weeks had a severely affected baby who died. She subsequently tested strongly positive for alloantibody. Conclusions: NH is often the result of maternal alloimmunity against fetal liver and recurrence can be prevented by high-dose IVIG during gestation, beginning at 18 weeks or earlier. We speculate that depletion of fetal hepatic HO-1 activity results in loss of cellular antioxidant and sensitivity to iron-induced oxidant injury.

19 Maternal Alloimmunization and Hemolytic Disease: Italian and Foreign Immigrants Newborns

The aim of this work is to present 40 newborns affected by alloimmunization: 26 Italians (0.18%) and 14 foreign immigrants (0.56%). 23 of them had anti D antibodies, 10 anti D+C, 1 anti D+C+E, 4 anti c, 1 anti jka, 1 anti Kell. 12 italians newborns had anti D antibodies, 7 anti D+C, 1 anti D+C+E, 4 anti c, 1 anti Kell, 1 anti jka. 11 foreign immigrants newborns had anti D antibodies, 3 anti D+C. Exchange tranfusion (ET), immunoglobulin infusion, phototerapy, and red cell transfusion (T) were performed depending on the severity of the immunization. Italian women direct Coombs' titer was 1/64. A titer >1/8000 was found only in foreign immigrants women. 9 patients required fetal transfusions (FT). Mean gestational age was 36.2 weeks, mean neonatal weight was 2795 grams. Mean hemoglobin at birth was 14.2 g/dl. Mean hematocrit was 41.6% 15 newborns received 1 ET. Greatest bilirubin value was 39 mg/dl. After discharge blood samples were collected every 3 weeks for hemoglobin, hematocrit, reticulocyte count and direct Coombs test. All the patients presented regular neurologic development and growth. 15 newborns required at least 1 T. According to our experience,a newborn requires FT if Coombs' titer of yhe mother at birth is >1/256, ET if it is >1/128, T if it is >1/128. It was always performed one of them if the titer was >1/2048. Because a increasing number of foreign immigrants, the incidence of Rh isoimmunization is raising. Foreign immigrants newborns developed the majority of alloimmunization because of the lack of maternal prophylaxis, with highest titers and most severe illnesses. It would be important to create an international screening and prophylaxis program in order to obtain again an reduction of Rh isoimmunization and to avoid fetal death and severe handicap.

Neonatal Hemochromatosis: Is It an Alloimmune Disease?

Neonatal hemochromatosis (NH) has been defined clinically as severe neonatal liver disease in association with extrahepatic siderosis in a distribution similar to that seen in HFE-associated hereditary hemochromatosis (1). Though it is generally considered a rare disease, it is one of the most commonly recognized causes of liver failure in the neonate and a frequent indication for liver transplantation in the first 3 months of life (2–4). Its etiology and pathogenesis are as yet unknown. Indeed, it has been considered to be a syndrome in which a number of primary etiologies, such as infection, genetic-metabolic disease and toxic insult, lead to a common phenotype. We have hypothesized that much, if not all, NH is a consequence of gestational alloimmune disease. This review presents the clinical evidence leading to this hypothesis, the data we have collected in its support, and the direction our investigations are taking to ultimately prove or disprove it.

Unusual antibody titer in a severe case of rhesus incompatibility

Unusual antibody titer in a severe case of rhesus incompatibility

Does antenatal maternal administration of phenobarbital prevent exchange transfusion in neonates with alloimmune hemolytic disease?

Does antenatal maternal administration of phenobarbital prevent exchange transfusion in neonates with alloimmune hemolytic disease?

Transfusion medicine illustrated. Maternofetal transfusion detected by gel-based typing.

At our center, ABO and Rh typing of all newborns is performed on umbilical cord blood samples with tube-based testing; gel testing is an ancillary strategy used to resolve weak agglutination and other problems. We present two infants with unusual results. The RBCs of Infant 1 were agglutinated, by use of tube testing, with reagent anti-B, but not with anti-A. With gel testing, a mixed-field reaction (two bands) was seen with reagent anti-B (left figure, Case A). The mother typed as group B. The RBCs of Infant 2 were strongly agglutinated with reagent anti-B, but only weakly with anti-A, by use of tube testing. With gel typing, a weak mixed-field reaction was seen with reagent anti-A (middle figure, Case B). The mother typed as group A. A Kleihauer-Betke (acid elution) smear of each specimen revealed the presence of adult RBCs (pale ghosts) in addition to fetal RBCs (bright pink) (right figure, Case 1, Kleihauer-Betke test). In Case 1, the proportion of maternal cells was 32 percent; in Case 2, 20 percent. Each infant's sample had been drawn by use of direct venipuncture into the umbilical cord. We concluded that the RBCs of fetus 1 were group O, with maternal B cells in the sample; the RBCs of fetus 2 were group B, with maternal A cells, both due to maternofetal transfusion. Gel agglutination is known to be more discriminatory than tube testing in the detection of dual populations of RBCs. Trafficking of RBCs occurs bidirectionally between fetal and maternal circulations. The implications of fetal-to-maternal transfusion include maternal alloimmunization and hemolytic disease of the newborn; the consequences of maternal-to-fetal transfusion are less well understood.

Acute Flare-up of Conjunctival Graft-versus-host Disease with Eosinophil Infiltration in a Patient with Chronic Graft-versus-host Disease

Chronic graft-versus-host disease (cGvHD) is a disabling alloimmune disease. Acute flare-ups of alloimmune reactions can occur even at the chronic clinical stage necessitating modulation of immunosuppression therapy. We studied conjunctival lesions of a patient with an acute flare-up in cGvHD. Along with severe GvHD lesions, biopsies showed eosinophils with pathological signs of activation. Since eosinophil recruitment and activation is observed in flare-ups of patients with autoimmune or inflammatory bowel diseases, we suggest that activated eosinophils in target organs may be a marker of evolutive lesions in alloimmune reactions as in other kinds of inflammatory diseases.

Antibody-induced anemia in fetal sheep: model for hemolytic disease of the fetus and newborn.

Our purpose was to produce a condition analogous to alloimmune hemolytic disease of the fetus and newborn by infusing antierythrocyte antibodies in fetal sheep. Antierythrocyte antibodies were infused intravascularly into late-gestation ovine fetuses over a 10-day period. Fetal blood was sampled daily for complete blood cell counts, blood gases, iron, erythropoietin (EPO), and electrolyte concentrations. Red cell mass (RCM) and blood volume were determined every other day using indicator dilution techniques. Results were compared with eight similarly aged control animals. Statistical analysis included Student t test, three-factor analysis of variance, and least squares regression. The hematocrit in seven fetal sheep receiving antibody infusion declined significantly by 10.3 +/- 1.7%, whereas it increased in control animals 2.3 +/- 0.6% (P <.001). RCM was reduced by 18.9 +/- 3.2% over the 10-day protocol while increasing 34.1 +/- 4.2% in control animals, representing more than a 50% difference in RCM (P <.001). Fetal EPO was significantly increased with lower hematocrit and lower PO(2) (P <.001). As fetal hematocrit declined below 25%, lactate and reticulocytes also increased (P <.001). Plasma iron concentration was not significantly altered (P =.47). The chronically catheterized fetal sheep is a viable model for studying immunologically induced fetal anemia as hematocrit can be titrated and the fall in RCM and hematocrit are associated with fetal hypoxia and elevated EPO as occurs in the anemic human fetus. Furthermore, there appears to be a threshold degree of anemia required to elicit responses as the fetal EPO, PO(2), and lactate appeared unresponsive until hematocrit fell below 25%.

Treatment of Hemolytic Disease of the Newborn Caused by Anti-Kell Antibody With Recombinant Erythropoietin

Recent data suggest that antibody-mediated suppression of erythroid progenitors may contribute to the anti-Kell-induced alloimmune hemolytic disease of the newborn (HDN). A 32-week-old girl who was positive for Kell was born to a mother who was negative for Kell but known to have anti-Kell antibodies. After birth, the baby had HDN and hyperbilirubinemia develop (peak bilirubin 21 mg/dL at day 9 of life). which was treated with phototherapy. Although the hyperbilirubinemia resolved, she became progressively anemic (hematocrit 22%) with an inappropriately low reticulocyte response (1.1%) and erythropoietin (EPO) level (20 mU/mL). To avoid the need for a blood transfusion, she was treated with recombinant erythropoietin (rEPO) and oral iron supplements. One week after starting EPO, the reticulocyte count increased to 9.1%. Erythropoietin therapy was continued for a total of 9 weeks, with resolution of her anemia at the end of therapy (hematocrit 35%). Thus, we were able to successfully treat the anemia with rEPO with avoidance of blood transfusion. This patient demonstrates that the antibody-mediated erythroid suppression in Kell alloimmune anemia can be overcome by rEPO. Recombinant erythropoietin should therefore be considered in the management of infants with severe or hypoproliferative anti-Kell-associated anemia.

Benefits and risks of fetal red-cell transfusion after 32 weeks gestation

Objective: To compare the outcome after intrauterine transfusion (IUT) between fetuses treated before and those treated after 32 weeks gestation. Setting: National referral center for intrauterine treatment of red-cell alloimmunization in The Netherlands. Study Design: Retrospective evaluation of an 11 year period, during which 209 fetuses were treated for alloimmune hemolytic disease with 609 red-cell IUTs. We compared fetal and neonatal outcome in three groups: fetuses only treated before 32 weeks gestation (group A, n=46), those treated both before and after 32 weeks (group B, n=117), and those where IUT was started at or after 32 weeks (group C, n=46). Results: Survival rate was 48% in group A, 100% in group B, and 91% in group C. Moreover, fetuses in group A were hydropic significantly more often. Short-term perinatal loss rate after IUT was 3.4% in the 409 procedures performed before 32 weeks and 1.0% in the 200 procedures performed after 32 weeks gestation. Conclusion: Perinatal losses were much more common in fetuses only treated before 32 weeks gestation. Two procedure-related perinatal losses in 200 IUT after 32 weeks remain a matter of concern because of the good prospects of alternative extrauterine treatment.

The outcome of pregnancy in Kell alloimmunisation.

To assess management and outcome of pregnancies with anti-Kell in the West Midlands in the UK over 13 years. A retrospective review of casenotes. A regional referral clinic for red cell alloimmune disease and fetal medicine unit at a university hospital. Sixty-five pregnancies were identified in 52 Kell-sensitised women with Kell positive partners from the records of the Birmingham Blood Transfusion Centre. Information from the casenotes was entered on a database and comparisons were made using the SPSS for Windows statistics package. Mode of sensitisation, degree of fetal or neonatal anaemia, need for transfusion, gestation at delivery, birthweight and pregnancy outcome. Alloimmunisation was transfusion-related in 29 pregnancies and pregnancy-induced in 33. The cause could not be identified in three cases. There were 22 proven Kell positive fetuses, of which 18 were affected, in which alloimmunisation was pregnancy-related in 12 cases and transfusion-related in five. Antibody titres and amniotic fluid OD450 were not helpful in management. Severe or very severe disease occurred in 50% of the affected pregnancies (9/18). There was no difference in pregnancy outcome between transfusion or pregnancy induced sensitisation. Anti-Kell alloimmunisation is an uncommon cause of serious anaemia in a significant proportion of affected pregnancies. There appears to be no difference between that caused by pregnancy or transfusion. Estimation of fetal haemoglobin concentration by cordocentesis is recommended, as antibody titres and amniocentesis are not helpful.

Interaction of CTLA-4 (CD152) with CD80 or CD86 inhibits human T-cell activation.

Occupancy of CTLA-4 (cytotoxic T-lymphocyte antigen-4 or CD152) negatively regulates the activation of mouse T lymphocytes, as indicated by the fate of CTLA-4-deficient mice, by the impact of anti-CTLA-4 monoclonal antibodies (mAbs) on mouse T-cell activation in vitro and by the impact of CTLA-4 blockade on the course of experimental tumoral, autoimmune, alloimmune or infectious disease in this animal. The function of human CTLA-4, however, remains less clear. The expression and function of human CTLA-4 were further explored. CTLA-4 was expressed under mitogenic conditions only, its expression being, at least partially, dependent on the secretion of interleukin-2. Memory T cells expressed CTLA-4 with faster kinetics than naive T cells. The functional role of human CTLA-4 was assessed utilizing a panel of four anti-CTLA-4 mAbs that blocked the interaction between CTLA-4 and its ligands. These mAbs, in immobilized form, profoundly inhibited the activation of T cells by immobilized anti-CD3 mAb in the absence of anti-CD28 mAb, but co-stimulated T-cell activation in the presence of anti-CD28 mAb. Finally, and importantly, blockade of the interaction of CTLA-4 with its ligands using soluble anti-CTLA-4 mAbs, in intact form or as Fab fragments, enhanced T-cell activation in several polyclonal or alloantigen-specific CD80- or CD80/CD86-dependent assays, as measured by cytokine production, cellular proliferation or cytotoxic responses. It is concluded that interaction of CTLA-4 with its functional ligands, CD80 or CD86, can down-regulate human T-cell responses, probably by intracellular signalling events and independent of CD28 occupancy.

Frequencies of platelet‐specific alloantigen systems 1–5 in three distinct ethnic groups in Brazil

The human platelet antigen (HPA) systems are related to immune platelet disorders as well as to the development of occlusive vascular disease. Several distinct biallelic HPA systems are known, and a heterogeneous distribution of HPA alleles has been described among distinct ethnic groups. In this study we genotyped 320 carefully selected individuals from three distinct ethnic groups in Brazil (Caucasians, Blacks and Amazonian Indians) for the HPA-1, -2, -3, -4 and -5 systems. A similar prevalence for all HPA alleles was found in Brazilians of Caucasian and Black descent. These data contrast with those reported for similar ethnic groups in other countries. Among the Amazonian Indians, no b allele of the HPA-1, -4 and -5 systems was identified. The data presented here could be useful in the diagnosis of alloimmune platelet disease, in genetic counselling and in the development of screening programmes for HPA-related diseases.

Alloimmune haemolytic disease towards the year 2000

Alloimmune haemolytic disease towards the year 2000

Developmental biology of erythropoiesis

A newborn infant represents the culmination of developmental events from conception through organogenesis. Red cells are critically important for survival and growth of the embryo. During development, erythropoiesis occurs in two distinct forms. The first ‘primitive’ form consists of nucleated erythroblasts that differentiate within the blood vessels of the extraembryonic yolk sac. The second ‘definitive’ form consists of anucleate erythrocytes that differentiate within the liver and third trimester bone marrow of the fetus. While adult bone marrow and cord blood now serve as sources of stem cells for the treatment by transplantation of genetic and malignant diseases, the developmental origin of hematopoietic stem cells has not been determined. During the third trimester the fetus grows rapidly and the production of red cells is approximately 3–5 times that of adult steady state levels. Birth brings dramatic changes in oxygenation and erythropoietin production that result in a tenfold drop in red cell production and in a transient ‘physiologic’ anemia. Other causes of fetal and infant anemias have their origins in development processes. These include globin gene switching in α and β thalassemia, the expression of red cell antigens in alloimmune hemolytic disease, and the poorly understood defects in the regulation of erythropoiesis in Diamond Blackfan anemia. Even in the adult, vestiges of fetal erythropoiesis are evident during transient states of accelerated erythroid expansion. A better understanding of the development of erythropoiesis will bring improvements in the treatment of anemia, not only in the newborn, but also in the fetus and the adult.

The disappearance of fetal and donor red blood cells in alloimmunised pregnancies: a reappraisal.

To determine the proportional reduction per day in the number of fetal and donor red blood cells from the fetal circulation after intrauterine intravascular transfusions. A retrospective study of 302 transfusions in 101 fetuses. The Department of Obstetrics and Gynaecology of the University Medical Centre Leiden, The Netherlands. We measured the haematocrit in fetal samples both before and after repeated intravascular intrauterine transfusion in fetuses with alloimmune disease. The percentage of fetal erythrocytes was determined in Kleihauer-Betke stained smears. The decline of fetal, donor and mixed red blood cells was calculated by dividing the proportional decrease of the haematocrit values of the number of days between transfusions, also after correction for changes in fetoplacental volumes. Results (given as mean [SD]) are derived from the proportional changes of haematocrit per day. The interval between the first and second transfusion (15.5 days [SD 5.2]) was shorter than between subsequent transfusions (means ranging from 21.4 to 21.9 days; P < or = 0.0001). The decline per day of mixed, and of donor red blood cells, calculated without corrections for volume changes did not differ from those corrected for volume changes resulting from the transfusion and from fetal growth (correction factor 1.1 [SD 0.4]). Since the coefficient of variance is smaller for the uncorrected decline values, this type of calculation is preferable for clinical purposes. The disappearance of fetal erythrocytes after the first transfusion (6.1%/day [SD 2.9]) was faster than that of mixed fetal and donor red blood cells (3.2%/day [SD 1.2]; P < 0.0001) and of donor cells alone (1.4%/day [SD 1.6]; P < 0.0001). The decline of the mixed red blood cell population became the same as that of the donor cells (2.2%/day [SD 0.8]) after the second transfusion. This decline of donor cells was higher than after the first transfusion (1.4%/day [SD 1.6]; P < 0.05). After the first transfusion the fetal erythrocytes disappeared faster after transplacental puncture of the umbilical cord (6.6%/day [SD 2.8]) than after transamniotic punctures (5.4%/day [SD 2.7]; P = 0.05). The mixed red blood cell also decreased faster (3.5%/day [SD 1.3] versus 2.8%/day [SD 0.9]; P < 0.01). The fast disappearance of fetal erythrocytes, especially after transplacental punctures, shows that the interval between the first and second transfusion needs to be shorter than that for intervals between subsequent transfusions. The number of donor erythrocytes declines by approximately 2% per day.