Metabolomics-based study on the significance of differential metabolite binding IgG isoforms in Hemolytic disease of newborn.

BACKGROUND: Hemolytic disease of the newborn (HDN) is a common condition that can have a severe impact on the health of newborns due to the hemolytic reactions it triggers. Although numerous studies have focused on understanding the pathogenesis of HDN, there are still many unanswered questions. METHODS: In this retrospective study, serum samples were collected from 15 healthy newborns and 8 infants diagnosed with hemolytic disease. The relationship between different metabolites and various IgG subtypes in Healthy, HDN and BLI groups was studied by biochemical technique and enzyme-linked immunosorbent assay (ELISA). Metabolomics analysis was conducted to identify the differential metabolites associated with HDN. Subsequently, Pearson's correlation analysis was used to determine the relation of these differential metabolites with IgG isoforms. The relationship between the metabolites and IgG subtypes was observed after treatment. RESULTS: The study results revealed that infants with hemolytic disease exhibited abnormal elevations in TBA, IgG1, IgG2a, IgG2b, IgG3, and IgG4 levels when compared to healthy newborns. Additionally, differences in metabolite contents were also observed. N, N-DIMETHYLARGININE showed negative correlations with TBA, IgG1, IgG2a, IgG2b, IgG3, and IgG4, while 2-HYDROXYBUTYRATE, AMINOISOBUTANOATE, Inosine, and ALLYL ISOTHIOCYANATE exhibited positive correlations with TBA, IgG1, IgG2a, IgG2b, IgG3, and IgG4. Through metabolomics-based research, we have discovered associations between differential metabolites and different IgG isoforms during the onset of HDN. CONCLUSION: These findings suggest that changes in metabolite and IgG isoform levels are linked to HDN. Understanding the involvement of IgG isoforms and metabolites can provide valuable guidance for the diagnosis and treatment of HDN.

The Kg-antigen, RhAG with a Lys164Gln mutation, gives rise to haemolytic disease of the newborn.

The Kg-antigen was first discovered in an investigation of a mother whose infant had haemolytic disease of the newborn (HDN). The antibody against the Kg-antigen is believed to be responsible for HDN. The Kg-antigen is provisionally registered under the number 700045, according to the Red Cell Immunogenetics and Blood Group Terminology. However, the molecular nature of the Kg-antigen has remained a mystery for over 30 years. In this study, a monoclonal antibody against the Kg-antigen and the recombinant protein were developed that allowed for the immunoprecipitation analysis. Immunoprecipitants from the propositus' red blood cell ghosts were subjected to mass spectrometry analysis, and DNA sequence analysis of the genes was also performed. A candidate for the Kg-antigen was molecularly isolated and confirmed to be a determinant of the Kg-antigen by cell transfection and flow cytometry analyses. The Kg-antigen and the genetic mutation were then screened for in a Japanese population. The molecular nature of the Kg-antigen was shown to be RhAG with a Lys164Gln mutation. Kg phenotyping further clarified that 0.22% of the Japanese population studied was positive for the Kg-antigen. These findings provide important information on the Kg-antigen, which has been clinically presumed to give rise to HDN.

Complications in pregnant women with sickle cell disease.

Pregnancy in women with sickle cell disease (SCD) is associated with increased maternal and fetal morbidity and mortality. Outcomes vary widely owing to methodological limitations of clinical studies, but overall, hypertensive disorders of pregnancy, venothromboembolism, poor fetal growth, and maternal and perinatal mortality are increased globally. Few therapeutic interventions have been explored other than prophylactic and selective transfusion therapy. Unfortunately, existing data are limited, and it remains unclear whether prophylactic use of chronic transfusions will improve pregnancy outcomes. Management of pregnant women with SCD is best accomplished with a multidisciplinary team that includes a sickle cell expert and an obstetrician familiar with high-risk pregnancies. Women with SCD should have individualized care plans that outline management of acute pain and guidelines for transfusion therapy. Neonates require close monitoring for neonatal abstinence syndrome and hemolytic disease of the newborn. Ideally all young women with SCD will have a "reproductive life plan" developed as a component of preconception counseling and health promotion. Research leading to improved pregnancy management focused on diminishing adverse maternal and neonatal outcomes is overdue. International collaborations should be considered to improve subject recruitment and foster timely completion of clinical trials. Additional therapeutic interventions outside of transfusion therapy should be explored.

Identification of red blood cell antibodies in maternal breast milk implicated in prolonged hemolytic disease of the fetus and newborn.

BACKGROUND: Alloantibodies against more than 50 non-ABO blood group antigens have been implicated in hemolytic disease of the fetus and newborn (HDFN) and are expected to wane within weeks after delivery. Persistent anemia leads to the hypothesis of continued exposure to red blood cell (RBC) alloantibodies via breast milk, which has been shown in a murine model and suggested in rare case reports. CASE REPORT: We report three cases of prolonged HDFN in two neonates with anti-D HDFN and one with anti-Jka HDFN. Patient 1 demonstrated 4+ anti-D serologic testing beyond 2 months; therefore, antibody testing was performed on maternal breast milk. METHODS: Maternal serum samples were tested for the presence of unexpected antibodies using standard Ortho gel card and 37 °C 60 minutes with anti-human globulin (AHG) tube saline methods. Antibody titrations were performed using the standard 37 °C 60 minutes to AHG tube saline method. Fresh breast milk samples were tested using the standard 37 °C 60 minutes to AHG tube saline method for both unexpected antibodies and titration study. Fresh breast milk from an O-positive, antibody-negative donor was used as control for any reactivity that may have been due to milk solids or proteins alone. RESULTS: Using a known methodology applied in a novel way to test breast milk for RBC alloantibodies, antibodies against fetal RBCs were identified in the maternal breast milk of three patients. CONCLUSION: Maternal RBC alloantibodies are present in breast milk and may be clinically significant in patients with prolonged recovery from HDFN.

Prevention of hemolytic disease of the fetus and newborn: what have we learned from animal models?

PURPOSE OF REVIEW: This review aims to highlight recent advances in our understanding of how anti-red blood cell (RBC) antibodies prevent erythrocyte immunization with an emphasis on new murine models. RECENT FINDINGS: New murine models with clinically relevant human erythrocyte antigens have been used to understand the alloimmunization process and its inhibition. The search to elucidate the mechanism of action of IgG-mediated inhibition of erythrocyte alloimmunization has provided new evidence in support of a potential role for epitope masking, immune deviation and/or antigen modulation in this process. In addition, recent evidence suggests that blends of monoclonal antibodies targeting nonoverlapping epitopes on the RBC surface can improve the efficacy of monoclonal antibodies approaching that of polyclonal IgG. SUMMARY: Animal models with defined alloantigens have helped to identify important mechanistic components that lead to alloimmunization and its inhibition by IgG. A better understanding of the underlying mechanisms leading to hemolytic disease of the fetus and newborn is required to develop the most effective prevention strategies for future patients.

Blood type biochemistry and human disease.

Associations between blood type and disease have been studied since the early 1900s when researchers determined that antibodies and antigens are inherited. In the 1950s, the chemical identification of the carbohydrate structure of surface antigens led to the understanding of biosynthetic pathways. The blood type is defined by oligosaccharide structures, which are specific to the antigens, thus, blood group antigens are secondary gene products, while the primary gene products are various glycosyltransferase enzymes that attach the sugar molecules to the oligosaccharide chain. Blood group antigens are found on red blood cells, platelets, leukocytes, plasma proteins, certain tissues, and various cell surface enzymes, and also exist in soluble form in body secretions such as breast milk, seminal fluid, saliva, sweat, gastric secretions, urine, and amniotic fluid. Recent advances in technology, biochemistry, and genetics have clarified the functional classifications of human blood group antigens, the structure of the A, B, H, and Lewis determinants and the enzymes that produce them, and the association of blood group antigens with disease risks. Further research to identify differences in the biochemical composition of blood group antigens, and the relationship to risks for disease, can be important for the identification of targets for the development of nutritional intervention strategies, or the identification of druggable targets. WIREs Syst Biol Med 2016, 8:517-535. doi: 10.1002/wsbm.1355 For further resources related to this article, please visit the WIREs website.

Transcutaneous bilirubin monitoring predicts unexplained late-onset hemolysis in a very low birthweight infant.

BACKGROUND: In term infants, transcutaneous bilirubin (TcB) monitoring can be used to predict hemolytic hyperbilirubinemia. However, it is not clear whether the technique can also be used to predict unexplained late-onset hemolysis in very low birthweight (VLBW) infants. CASE PRESENTATION: The case was an infant with a birthweight of 1154 g who developed unexplained late-onset hemolysis at 8 days of age. The hyperbilirubinemia rapidly worsened, and therefore both phototherapy and exchange transfusion were performed. TcB levels were measured using the JM-105 jaundice meter and found to have increased by >3 mg/dL since before the onset, demonstrating for the first time that the device clearly detects changes in hemolytic rate. CONCLUSIONS: Although TcB levels did not correspond directly with total serum bilirubin levels in VLBW infants, the two values exhibited parallel changes in this case. Therefore, serial TcB monitoring may be useful in the early prediction of unexplained late-onset hemolysis in VLBW infants.

Kell and Kx blood group systems.

The Kell and Kx blood group systems are expressed as covalently linked molecules on red blood cells (RBCs). The Kell blood group system is very polymorphic, with 35 antigens assigned to the system. The expression of Kell glycoprotein on RBCs is not critical to the erythrocyte function. However, the expression of KX is critical to normal morphology, and null mutations are associated with the McLeod neuroacanthocytosis syndrome. The immunogenicity of the K anigen is second only to the D anigen, and alloantibodies to Kell anigens can cause transfusion reactions and hemolytic disease of the fetus and newborn. Kell alloantibodies in pregnancy are known to suppress erythropoiesis, which can result in serious disease despite low amniotic bilirubin levels and low antibody titers. Late-onset anemia with reticulocytopenia is thought to be attributable to the continual suppression of erythropoiesis from residual alloantibody in the infant. Alloimmunization to XK protein is rare, and expressed polymorphisms have not been reported. Together these two blood group systems share an integral relationship in transfusion medicine, neurology, and musculoskeletal biology.

The effect of fetal anemia on fetal cardiac troponin T in pregnancies complicated by RhD alloimmunization.

OBJECTIVE: To study the effect of fetal anemia on fetal cardiac troponin T (cTnT) in pregnancies complicated by RhD alloimmunization. METHOD: Twenty pregnant women complicated by RhD alloimmunization who underwent intrauterine transfusion (IUT) for treatment of fetal anemia were studied. Immediately before IUT, fetal blood was obtained for hemoglobin and cTnT measurements. RESULTS: Complete measurements of hemoglobin and cTnT before IUT were obtained in 49 procedures, of which 20 were first-time. The regression analysis between hemoglobin z-score and cTnT values in 49 procedures showed significant negative correlation (r = -0.43, p = 0.002, Regression equation Log(cTnT) = -1.5057 + -0.07563 Hb z-score). Cardiac TnT values before first IUT were significantly associated with perinatal death. In the group with elevated cTnT (n = 7), fetal or neonatal death was more frequent (2 IUD and 2 NND) when compared to normal cTnT group (n = 13, 1 IUD) (57.1 vs. 7.7%, p = 0.031, Fisher's exact test). CONCLUSION: Fetal blood concentration of cTnT before IUT was negatively correlated to hemoglobin z-score, and levels of cTnT help to manage the pregnancies complicated by RhD alloimmunization.

The Duffy blood group system: a review.

Duffy was the first blood group mapped to an autosome (chromosome 1) using cytogenetic studies. Duffy antigens are located on a glycoprotein that can be found on erythrocytes and other cells throughout the body. Fy(a) and Fy(b) are products of their respective alleles (FY*A, FY*B). Fy(x) characterized by weak Fy(b) expression, is a result of an additional mutation in FY*B. The Fy(a-b-) phenotype, most commonly found in Blacks, occurs primarily as a result of GATA promoter region mutation upstream of the FY allele. This mutation prevents expression of Duffy glycoprotein on erythrocytes only, while permitting expression of nonerythroid cells. Other antigens include Fy3, Fy5, and Fy6. Antibodies to Duffy antigens are usually clinically significant and have been reported to cause hemolytic disease of the fetus and newborn. This review provides a general overview of the Duffy blood group system, including the role of the Duffy glycoprotein as a chemokine receptor (Duffy antigen receptor for chemokines) and in malarial infection.

The Gerbich blood group system: a review.

Antigens in the Gebrich blood group system are expressed on glycophorin C (GPC) and glycophorin D (GPD), which are both encoded by a single gene, GYPC. The GYPC gene is located on the long arm of chromosome 2, and Gebrich antigens are inherited as autosomal dominant traits. There are 11 antigens in the Gebrich blood group system, six of high prevalence (Ge2, Ge3, Ge4, GEPL [Ge10*], GEAT [Ge11*], GETI [Ge12*]) and five of low prevalence (Wb [Ge5], Ls(a) [Ge6], An(a) [Ge7], Dh(a) [Ge8], GEIS [Ge9]). GPC and GPD interact with protein 4.1R, contributing stability to RBC membrane. Reduced levels of GPC and GPD are associated with hereditary elliptocytosis, and Gebrich antigens act as receptors for the malarial parasite Plasmodium falciparum. Anti-Ge2 and anti-Ge3 have caused hemolytic transfusion reactions, and anti-Ge3 has produced hemolytic disease of the fetus and newborn (HDFN).

Lutheran.

The Lutheran blood group system consists of 19 antigens: four pairs of antithetical antigens--Lu(a)/Lu(b), Lu6/Lu9, Lu8/Lu14, and Au(a)/Au(b)--and 11 antigens of very high frequency. These antigens are located on four of the five immunoglobulin-like domains of both isoforms of the Lutheran glycoprotein. The LU gene is on chromosome 19 and comprises 15 exons. The two glycoprotein isoforms differ in the length of their cytoplasmic tails as a result of alternative splicing of intron 13. Lu(null) phenotype arises from homozygosity for inactivating mutations in the LU gene.The dominantly inherited Lu(mod) phenotype, In(Lu), results from heterozygosity for inactivating mutations in KLF1, the gene for the erythroid transcription binding factor EKLF. Clinically, antibodies of the Lutheran system are relatively benign. When hemolytic, they generally cause only mild, delayed hemolytic transfusion reactions or hemolytic disease of the fetus and newborn that can be treated by phototherapy. The Lutheran glycoproteins, which are members of the immunoglobulin superfamily of adhesion molecules and receptors, bind isoforms of laminin with alpha5 chains,components of the extracellular matrix abundant in vascular endothelia. The primary function of the Lutheran glycoproteins on RBCs could involve the transfer of maturing RBCs from the bone marrow to the peripheral circulation. They could also be involved in vascular occlusion and thrombotic events as complications of sickle cell disease and polycythemia vera, respectively.

Rh proteins: key structural and functional components of the red cell membrane.

Rh (Rhesus) proteins (D, CcEe) are expressed in red cells (RBC) in association with other membrane proteins (RhAG, LW, CD47 and GPB). By interacting with the spectrin-based skeleton through protein 4.2 and ankyrin, the Rh complex contributes to the maintenance of the mechanical properties of the erythrocyte membrane. The RH system is one of the most immunogenic and polymorphic human blood group system. Molecular basis of most Rh phenotypes, including the Rh(null) phenotype associated with hemolytic anemia, have been determined. The demonstration that the RHD-positive locus is composed of the RHD and RHCE genes, whereas the RHD gene is deleted in most RhD-negative individuals, allowed fetal RhD genotyping by non-invasive PCR assays for antenatal diagnosis of pregnancy at risk for Rh hemolytic disease of the newborn. In mammals, the Rh protein family includes two non-erythroid members, RhBG and RhCG, mainly expressed in liver and kidney, two organs specialized in ammonia genesis and excretion. Functional analyses in heterologous systems revealed that RhAG, RhBG and RhCG can mediate ammonium (NH(3) and/or NH(4)(+)) transport across the cell membrane and might represent mammalian specific ammonium transporters. Furthermore, recent studies performed in human and murine red blood cells (RBC) indicate that RhAG facilitates CH(3)NH(2)/NH(3) movement across the membrane and represents a potential example of gas channel. The crystallographic structure of the bacterial ammonia channel AmtB and functional studies showing that AmtB conducts NH(3) into reconstituted vesicles is fully consistent with these latter studies. In RBCs, RhAG may transport NH(3) to detoxifying organs like kidney and liver and with non-erythroid tissues orthologs may contribute to regulation of the acid-base balance.

Non-protein-bound iron and free radical damage in fetuses with rhesus haemolytic disease: influence of intrauterine transfusions.

OBJECTIVE: To determine iron-induced free radical damage in fetal rhesus haemolytic disease (RHD) before and after repeated intrauterine red blood cell transfusions and its relation to hydrops fetalis. DESIGN: Prospective, observational study. SETTING: Department of Obstetrics, Leiden University Medical Centre, the Netherlands. POPULATION: Fifty anaemic fetuses, including 13 hydropic ones, 9 preterm and 12 term neonates and 8 female non-pregnant adults. METHODS: Venous blood plasma samples were collected from 50 fetuses suffering from RHD preliminary to the first, and if appropriate, subsequent intrauterine red blood cell transfusions for determination of iron status including non-protein-bound iron (NPBI) and iron-binding primary antioxidant proteins, total plasma anti-oxidant capacity and its contributing secondary antioxidants (e.g. vitamin C, uric acid, sulphydryl groups and peroxidation products). Results were compared with values of healthy preterm and term neonates directly at birth and adult controls. Within the fetal haemolytic group, 13 hydropic fetuses were analysed as a separate group. MAIN OUTCOME MEASURES: Non-protein-bound iron, antioxidants, total antioxidant capacity and peroxidation products. Sub analysis of the outcome measures of the hydropic fetuses. RESULTS: RHD fetuses had at initial cordocentesis a significantly higher NPBI level and a significantly lower total plasma antioxidant capacity than control babies and adults. Their vitamin C tended to be more oxidised but lipid peroxidation had not increased, when compared with preterm babies. The repeated intrauterine red blood cell transfusions had a positive effect on the total antioxidant capacity of plasma and did not increase the concentration of NPBI. The hydropic fetuses, who had higher NPBI concentrations and lower plasma protein concentrations and total antioxidant capacity, did not show more peroxidation products in plasma than the non-hydropic fetuses. Fetuses without reversal of hydrops despite intrauterine transfusions showed decreasing levels of proteins with subsequent transfusions but peroxidation products remained constant. CONCLUSION: Repeated intrauterine red blood cell transfusions do not lead to free radical damage in the fetus in utero. Iron-induced free radical activity does not appear to play a causative role in the proceeding of hydrops fetalis in RHD.

Basis of hematopoietic defects in platelet-derived growth factor (PDGF)-B and PDGF beta-receptor null mice.

Platelet-derived growth factor (PDGF)-B and PDGF beta-receptor (PDGFR beta) deficiency in mice is embryonic lethal and results in cardiovascular, renal, placental, and hematologic disorders. The hematologic disorders are described, and a correlation with hepatic hypocellularity is demonstrated. To explore possible causes, the colony-forming activity of fetal liver cells in vitro was assessed, and hematopoietic chimeras were demonstrated by the transplantation of mutant fetal liver cells into lethally irradiated recipients. It was found that mutant colony formation is equivalent to that of wild-type controls. Hematopoietic chimeras reconstituted with PDGF-B(-/-), PDGFR beta(-/-), or wild-type fetal liver cells show complete engraftment (greater than 98%) with donor granulocytes, monocytes, B cells, and T cells and display none of the cardiovascular or hematologic abnormalities seen in mutants. In mouse embryos, PDGF-B is expressed by vascular endothelial cells and megakaryocytes. After birth, expression is seen in macrophages and neurons. This study demonstrates that hematopoietic PDGF-B or PDGFR beta expression is not required for hematopoiesis or integrity of the cardiovascular system. It is argued that metabolic stress arising from mutant defects in the placenta, heart, or blood vessels may lead to impaired liver growth and decreased production of blood cells. The chimera models in this study will serve as valuable tools to test the role of PDGF in inflammatory and immune responses. (Blood. 2001;97:1990-1998)

Amniotic fluid nitric oxide metabolites, cyclic guanosine 3',5' monophosphate and dimethylarginine in alloimmunized pregnancies.

OBJECTIVE: To determine the relationship between gestational age or the Liley index (the severity of fetal hemolysis) and the amniotic fluid total nitrite (NOx), cyclic guanosine 3',5 'monophosphate (cGMP) and dimethylarginine (DMA) concentrations. We hypothesized that the concentrations of these components change because of fetal growth or adaptation to fetal anemia. STUDY DESIGN: Amniotic fluids (n=64) were obtained between 23 and 37 weeks from fifty-three patients at risk for alloimmunization. Amniotic fluids from the pregnancies with a Liley index=1 were considered as controls (n=17). Creatinine (C, microMol) was determined with the Jaffé reagent, nitrite (NOx, microMol) with the Griess reagent, cGMP (nMol) by an enzyme immunoassay and DMA (microMol) after HPLC. Multiple regression analysis was used for separating the effects of growth and the estimated degree of anemia. RESULTS: The concentration of NOx, cGMP and DMA was not related to the Liley index or whether or not the fetuses needed blood transfusions. The concentrations of creatinine (C), NOx and cGMP increased during pregnancy (in weeks;W) (C=-69.2+6.28W; r2=0.532; P<0.0001, NOx=-17.6+1.29W; r2=0.106; P=0.01, cGMP=-20.9+1.05W; r2=0.414; P<0.0001). The DMA concentration (3.8+/-0.8(SD) and the NOx/creatinine ratio (181+/-110 mM/M) did not change with gestational age. The cGMP/creatinine ratios (microM/M) increased (cGMP/C=-41.8+4.31W; r2=0.134; P=0.007) whereas the DMA/creatinine ratio (mM/M) declined during pregnancy (DMA/C=73.1-1.34W; r2=0.278; P=0.0002). Consequently, the NOx/DMA and cGMP/DMA ratios increased (NOx/DMA=-6.96+0.43W; r2=0.105; P=0.02, cGMP/DMA=-5.9+0.29W; r2=0.391; P<0.0001). CONCLUSIONS: The concentrations in amniotic fluid of cGMP and NOX, but not of DMA increase during gestation. The cGMP/creatinine ratio increases also whereas that of DMA decreases. The changes in products of the NO-cGMP pathway are independent of mild to moderate fetal hemolysis and may result from fetal growth as well as from reduced inhibition of NO synthase by DMA. Gestational age related effects should be taken into account when analyzing nitric oxide metabolites in amniotic fluids.

Consumption of anti-D in the erythroblastotic fetus.

BACKGROUND: Maternal serum anti-D is actively transported across the placenta into the fetal blood where it adheres to D-positive erythrocytes. The anti-D coated red cells attach to Fc-receptors on fetal reticuloendothelial cells and are subsequently phagocytosed and hemolysed. It is not known if anti-D is consumed or recirculated in this process. MAIN QUESTION: Is anti-D in the fetus consumed in the hemolytic process in the erythroblastotic fetus and can the consumption be modulated by high-dose immunoglobulins (i.v.IG) given to the mother? METHODS: Fetal/maternal serum anti-D concentration ratios were calculated for simultaneously taken blood samples from three groups of Rh(D) immunized pregnant women; six women with fetuses who were of Rh(D) negative phenotype, 19 women with fetuses who were Rh(D) positive and received no treatment and, seven women who were treated with i.v.IG because they bore anemic, Rh(D) positive fetuses. RESULTS: Fetuses with a Rh(D) negative phenotype expressed an increase in fetal/maternal anti-D concentration ratios from 10 to 55% between 25 and 31 gestational weeks, while Rh(D) positive fetuses without i.v.IG treatment had stable values at the 10% level between 24 and 35 gestational weeks. During i.v.IG-treatment of the mothers, Rh(D) positive fetuses showed an increase in ratio from 10 to 30% between 26 and 34 gestational weeks. There was a statistically significant (p<0.001) difference between regression results of the three groups. CONCLUSIONS: Fetal anti-D is consumed in the hemolytic process and the consumption can be modulated by i.v.IG given to the mother.

Cytokines in fetal blood and amniotic fluid in Rh-immunized pregnancies.

OBJECTIVE: To determine fetal serum and amniotic fluid (AF) levels of interleukin (IL)-3, IL-6, granulocyte-macrophage colony-stimulating factor, stem cell factor, and erythropoietin, and to explore the relationship between cytokines and hemoglobin concentration, white blood cell count (WBC), and platelet count in fetuses affected by Rh immunization. METHODS: Thirty-four consecutive Rh-immunized patients in gestational weeks 19-33 were included. All patients were investigated by funipuncture and 13 by amniocentesis. The levels of IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, stem cell factor, and erythropoietin were estimated using commercially available immunoassays. RESULTS: There was a significant correlation between erythropoietin concentrations in fetal serum and AF (r = 0.54, P < .05), whereas none of the other cytokines showed a positive correlation between these two compartments. Fetal serum contained higher concentrations of IL-3, granulocyte-macrophage colony-stimulating factor, stem cell factor, and erythropoietin compared with AF. In contrast, the IL-6 level was significantly higher in AF compared with fetal serum (P = .002). Erythropoietin and IL-3 levels were both negatively correlated with fetal hemoglobin concentrations (r = -0.75, P = .02, and r = -0.67, P = .045). The fetal WBC correlated significantly with the fetal serum concentration of granulocyte-macrophage colony-stimulating factor (r = 0.38, P = .04). CONCLUSION: Human fetuses with anemia due to erythrocyte immunization exhibit an increased production of erythropoietin and IL-3. Other studied cytokines (such as stem cell factor, granulocyte-macrophage colony-stimulating factor, and IL-6) did not correlate with the degree of fetal anemia. Among the studied cytokines, only erythropoietin showed a positive correlation between fetal serum and AF.