Distribution of Red Blood Cell Alloantibodies Among Transfusion-Dependent ß-Thalassemia Patients in Different Population of Iran: Effect of Ethnicity.

The best approach for prevention of alloimmunization in ß-thalassemia (ß-thal) patients is perfect matching of all red blood cell (RBC) antigens associated with clinically significant antibodies, but this is expensive and may limit the blood supply. Knowing the most common alloantibodies in transfusion-dependent ß-thal patients make it possible to establish more cost-effective matching strategies for high-risk antigens. With this in mind, we intended to determine the most common alloantibodies in different parts of Iran. A total of 480 alloimmunized ß-thal major (ß-TM) patients who were referred to the Tehran Adult Thalassemia Clinic in Tehran, Iran from all provinces between 2015 and 2017, were included in this study. Antibody screening was performed on the fresh serum of all patients. Subsequently, the specification of antibodies was identified using a panel of recognized blood group antigens. Anti-K was the most common alloantibody detected in ß-TM patients in all regions of Iran. The prevalence of this antibody reached to 37.7% in the western area, but in southeastern region, anti-E was predominant. Interestingly, the rare alloantibody anti-Kpa was detected with a high prevalence in the western region. The antibodies against E and D antigens were also encountered with high prevalence in most regions of the country. The present study demonstrated the distribution of alloantibodies in alloimmunized transfusion-dependent ß-thal patients from diverse ethnic and racial backgrounds of the Iranian population. The results of this study can be used as a basis to establish cost-effective RBC phenotyping and matching strategies for high-risk antigens in donors and chronic transfusion recipients in different regions of Iran.

Simple method for screening of alpha-thalassaemia 1 carriers.

Alpha-thalassaemia 1 genetic disorder occurs when there is a deletion of two linked alpha-globin genes. The interaction between these abnormal genes leads to the most severe type of thalassaemia disease, haemoglobin (Hb) Bart's hydrops fetalis. The identification of alpha-thalassaemia 1 carriers and genetic counselling are essential for the prevention and control of severe thalassaemia diseases. In this study, we have developed a rapid screening method for identifying alpha-thalassaemia 1. A sandwich-type immunochromatographic (IC) strip test was developed, using the generated monoclonal anti-Hb Bart's antibody, to trace the Hb Bart's in haemolysates. When assayed by our IC strip test, all alpha-thalassaemia 1, HbH disease, HbH-Constant Spring (H-CS) disease, HbH-CS and heterozygous HbE (CSEA) Bart's disease, and homozygous alpha-thalassaemia 2 showed positive results. No false negative results were observed in these blood samples. In alpha-thalassaemia 2 heterozygotes, 83% of them showed positive reactivity. Among HbE (both homozygotes and heterozygotes), beta-thalassaemia (heterozygotes, homozygotes and beta-thalassaemia/HbE) and normal subjects, the IC strip test revealed negative reactivity of 100, 85 and 97%, respectively. These results indicate that this novel immunodiagnostic kit, in combination with red blood cell indices, is suitable for screening and ruling out mass populations for the presence of alpha-thalassaemia 1.

Hydrops fetalis caused by homozygous alpha-thalassemia and Kidd antigen alloimmunization in a Chinese woman.

We report a case with hydrops fetalis resulting from homozygous alpha-thalassemia and alloimmune hemolytic anemia due to anti-JK3. This is one of the few cases with immune- and nonimmune-induced hydrops fetalis.

Production of a mouse hybridoma secreting monoclonal antibody highly specific to hemoglobin Bart's (gamma4).

Hemoglobin (Hb) Bart's (gamma4) was isolated and purified from Hb Bart's hydrops fetalis syndrome blood by CM-Sephadex C-50 chromatography. The isolated Hb Bart's was analyzed for its purity by high performance liquid chromatography. Balb/c mice were immunized intraperitoneally with Hb Bart's. The immunized mouse splenic cells were hybridized with mouse myeloma, X63-Ag8.653, by polyethylene glycol. There were 12 hybridoma clones, out of several thousand culture wells, secreting antibody against purified Hb Bart's. However, when those 12 monoclonal antibodies (mAb) were tested with Hb Bart's (gamma4), HbF (alpha2gamma12), HbH, HbE, and HbA2, there was only 1 hybridoma clone secreting mAb highly reactive to Hb Bart's with very low reactivity to HbF. A rabbit polyclonal antibody with relative high reactivity to Hb Bart's compared to HbF with the ratio of 2.4:1 was also produced by affinity column chromatography for the purpose of developing an enzyme-linked immunosorbent assay (ELISA) base for qualitative and quantitative determination of Hb Bart's in adult hemolysates. Preliminary results in quantitative determination of Hb Bart's in Hb solution of 3 alpha thalassemia families having at least 1 child with HbH disease and 6 normal subjects indicated that it was possible to quantify Hb Bart's by our developed ELISA with appropriate sensitivity and specificity.

Chicken egg yolk antibodies specific for the gamma chain of human hemoglobin for diagnosis of thalassemia.

Immunoglobulin Y (IgY) technology was used to generate anti-hemoglobin Bart's (Hb Bart's) IgY antibodies (Abs) for development into an enzyme-linked immunosorbent assay (ELISA) test for thalassemia diagnosis. Hb Bart's purified from the hemolysate of a patient with Hb Bart's hydrops fetalis (homozygous alpha-thalassemia) was used to immunize a chicken via the pectoralis muscle. After water dilution and sodium sulfate precipitation, 40 to 70 mg of IgY could be extracted from an egg. IgY, first detected in sera 2 weeks after immunization, reached the highest titer at week 4, and the titer remained stable for at least 2 weeks before declining. The pattern of Ab response in the yolk was the same as in the serum but was somewhat delayed. The IgY Abs produced reacted with gamma globin, Hb Bart's, Hb F, normal cord hemolysate (Hbs F plus A), and Hb Bart's hydrops fetalis (Hbs Bart's plus Portland) and to a lesser degree with beta globin, Hb A, Hb A2 and adult hemolysate (Hbs A plus A2), but the Abs did not react with alpha globin. Immunoaffinity purification with Hb A coupled to Sepharose was used to isolate an unbound IgY that reacted with Hb F, Hb Bart's, and gamma globin, and this IgY was used to develop an ELISA test for thalassemia diagnosis. The results of direct ELISA analyses of 336 hemolysate samples from individuals with various known thalassemia genotypes and phenotypes and from healthy individuals confirmed the specificity of the polyclonal Abs for Hbs containing Hb F and Hb Bart's. This specificity, which was due to the Abs' strong reactivity in cases of pathologic thalassemic diseases and weak reactivity in cases of nonpathologic thalassemic diseases, depended on the levels of Hb Bart's and Hb F.

Simple non-invasive prenatal detection of Hb Bart's disease by analysis of fetal erythrocytes in maternal blood.

OBJECTIVE: To investigate a simple non-invasive technique for early detection of Hemoglobin (Hb) Bart's disease. METHOD: Maternal blood smears from 8 known Hb Bart's pregnancies and 40 at-risk pregnancies were investigated. Maternal peripheral blood smears were stained with fluorescence-labeled monoclonal antibodies against alpha- and embryonic zeta-globin chains. RESULTS: Fetal nonnucleated red blood cells, stained with anti-zeta but not with anti-alpha globin antibodies were found in 15 out of 16 affected pregnancies but were not detected in 23 out of 24 unaffected pregnancies. CONCLUSION: Results showed that non-invasive immunofluorescence staining of maternal blood is a feasible approach for screening Hb Bart's disease before ultrasound manifestation in affected pregnancies.

Detection of fetal NRBCs in maternal blood of pregnant carriers of beta-thalassemia using anti-gamma and anti-epsilon monoclonal antibodies.

Fetal nucleated red blood cells (NRBCs) entering maternal circulation during pregnancy constitute a potential source of material for safe and reliable noninvasive prenatal diagnosis. The increased prevalence of beta-thalassemia mutations in countries like Greece may create a problem, making it difficult to distinguish between NRBCs of fetal or maternal origin. Use of Ab against embryonic hemoglobin epsilon may increase specificity for fetal NRBC detection. In the present study, Ab against embryonic hemoglobin epsilon was used in the first and second trimesters of pregnancy in order to determine if specificity for fetal NRBC detection could be increased.

Alloimmunization and erythrocyte autoimmunization in transfusion-dependent thalassemia patients of predominantly asian descent.

The development of hemolytic alloantibodies and erythrocyte autoantibodies complicates transfusion therapy in thalassemia patients. The frequency, causes, and prevention of this phenomena among 64 transfused thalassemia patients (75% Asian) were evaluated. The effect of red blood cell (RBC) phenotypic differences between donors (mostly white) and Asian recipients on the frequency of alloimmunization was determined. Additional transfusion and patient immune factors were examined. 14 (22%) of 64 patients (75% Asian) became alloimmunized. A mismatched RBC phenotype between the white population, comprising the majority of the donor pool, and that of the Asian recipients, was found for K, c, S, and Fyb antigens, which accounts for 38% of the alloantibodies among Asian patients. Patients who had a splenectomy had a higher rate of alloimmunization than patients who did not have a splenectomy (36% vs 12.8%; P =.06). Erythrocyte autoantibodies, as determined by a positive Coombs test, developed in 25% or 16 of the 64 patients, thereby causing severe hemolytic anemia in 3 of 16 patients. Of these 16, 11 antibodies were typed immunoglobulin G [IgG], and 5 were typed IgM. Autoimmunization was associated with alloimmunization and with the absence of spleen (44% and 56%, respectively). Transfused RBCs had abnormal deformability profiles, more prominent in the patients without a spleen, which possibly stimulated antibody production. Transfusion of phenotypically matched blood for the Rh and Kell (leukodepleted in 92%) systems compared to blood phenotypically matched for the standard ABO-D system (leukodepleted in 60%) proved to be effective in preventing alloimmunization (2.8% vs 33%; P =.0005). Alloimmunization and autoimmunization are common, serious complications in Asian thalassemia patients, who are affected by donor-recipient RBC antigen mismatch and immunological factors.

[Effects of abnormal Hb on red cell membranes].

Unstable hemoglobin disorders are due to substitutions or deletions of amino acids which alter the normal tertiary structure of hemoglobin and/or decrease heme-binding to globin. These changes result in enhanced oxidation to methemoglobin, rapid conversion of methemoglobin to hemichrome and sometimes heme loss, which leads to denaturation and precipitation as Heinz bodies. This process is associated with marked oxidative membrane damage, such as crosslinking of membrane proteins, membrane lipid peroxidation, hemin-induced destabilization of cytoskeletal protein interactions, and increased permeability to potassium ions. The damaged erythrocytes are sequestered in the spleen, where Heinz bodies are "pitted" or the entire cell is phagocytized by macrophages. The precise mechanisms leading to hemolysis are not fully understood. However, one hypothesis involves hemichrome binding to the cytoplasmic domain of band 3, leading to clustering of band 3 in the membrane and immunologic recognition of the redistributed band 3 by autologous senescent antibodies. This theory is based on immunologic findings rather than deformability changes, and it is consistent with many features of unstable hemoglobins.

In vivo antagonism of a T cell response by an endogenously expressed ligand.

3.L2 T cell receptor transgenic T cells are activated by the 64-76 peptide of the mouse hemoglobin d beta chain [Hb(64-76)], and their response is antagonized by the position 72 alanine substitution of this peptide (A72). To test the effect of this altered peptide ligand (APL) on 3.L2 T cell function in vivo, a transgene expressing A72 in major histocompatibility complex II positive cells (A72tg) has been introduced into mice. We demonstrate that 3.L2 T cells, when transferred to A72tg+ mice show a dramatically reduced proliferative response to Hb(64-76). Identical decreased responses were observed using T cells that developed in either A72tg+ or A72tg- hosts. This affect was not attributable to diminished precursor frequency, anergy, or competition for binding to I-Ek molecules. These results unequivocally demonstrate in vivo antagonism by an endogenous APL and characterize a class of self-peptides that, although inefficient in causing deletion in the thymus, effectively modulate T cell responses in the periphery.

Utilization of monoclonal-antibody-based assay (HemoCard in screening for and differentiating between genotypes of sickle cell disease and other hemoglobinopathies.

Sickle cell disease covers a group of conditions in which pathology may be attributed to the presence of sickle hemoglobin (HbS). The identification of HbS and other variants including those in combination with HbS is commonly achieved by cellulose acetate electrophoresis at alkaline pH. Because many hemoglobin variants with similar charges have similar electrophoretic migration patterns, they are difficult to differentiate by electrophoresis. The HemoCard assays address this concern through the use of monoclonal antibodies capable of specifically recognizing the unique amino acid substitution in the variant hemoglobin. The panel of HemoCard monoclonal antibodies confirms the absence and presence of HbA, HbC, HbE, HbS, and other sickling hemoglobin variants. The combination of alkaline cellulose acetate electrophoresis and HemoCard assays allows the technologist to reach a final conformation of both common and much less common sickle cell disease genotypes, combinations of HbS with other hemoglobins that ordinarily do not produce sickle cell disease, and other clinically important hemoglobinopathies including HbE/beta-thalassemia and hemoglobin C disease.

Epitope mapping of human factor IX inhibitor antibodies.

We have determined the location of epitopes on the factor IX for three haemophilia B inhibitor antibodies (HB-1, HB-3, HB-7) and a monoclonal anti-factor IX inhibitory antibody (designated 65-10). The main binding region of HB-1, HB-3 and HB-7 was 155YVNSTEAETI164 (residues 155-164), 167NITQSTQSFN176 and 156VNSTEAETI164, respectively. The binding region of 65-10 was 168ITQSTQSFNDFTRVV182, which included the cleavage site (180R-V181) for activation by factor XIa. By neutralization experiments using two peptides, 156VNSTEAETI164 and 167NITQSTQSFN176, the degree of neutralization of anti-factor IX IgG purified by protein A was determined. Neutralization of three antibodies, HB-1, HB-3 and HB-7, in the presence of 10 mM of the peptides 156VNSTEAETI164 was 30.1%, 0% and 10.8%, respectively, and in the presence of 4 mM of 167NITQSTQSFN176 it was 0%, 13.5% and 17.3%, respectively. On the other hand, when plasmas of patients instead of purified IgG were used for neutralization, 10 mM of 156VNSTEAETI164 and 4 mM of 167NITQSTQSFN176 failed to neutralize the inhibitor in the plasmas.

Peroxidase/monoclonal antibody conjugates for the study of hemoglobinopathies.

Monoclonal antibodies (mAbs) to normal human hemoglobins (Hbs) A and F and to variant Hbs C and G-Philadelphia were conjugated to horseradish peroxidase (HRP) and used in qualitative or quantitative enzyme-linked immunosorbent assays (ELISAs). Conjugates with output molar HRP/IgG ratios close to 2.0 had higher avidity for the cognate antigens than those with ratios above or below 2.0. The analytical sensitivities of the conjugates ranged from 0.2 to 4 ng of hemolysate containing the target hemoglobin, and it was not related to the input or the output HRP/IgG ratios. The overall imprecision for the qualitative ELISA was below 8%, and the accuracy for the identification of Hbs C and G-Philadelphia was 100% as compared with established methods. Quantitative determinations of HbA based upon direct dose-response curves showed an analytical sensitivity of 1% and an imprecision < or = 11%. The most significant application of the HbA assay was in the differential diagnosis of hemoglobinopathies associated with partial or total suppression of HbA synthesis. Competitive dose-response curves for the HRP/anti-gamma conjugate allowed the quantification of HbF in the clinically significant range of 0.5-10%, with an imprecision < or = 12%. It is concluded that the incorporation of HRP/mAb conjugates into the ELISA technique offers a simpler, more rapid, yet specific alternative for the measurement of hemoglobins.

Determination of hemoglobin Bart's in alpha thalassemia traits by two-site immunoradiometric assay. I. Purification of hemoglobin Bart's and preparation of specific anti-Hb Bart's.

This research report describes methods for the preparation of hemolysate and the isolation and purification of hemoglobins Bart's, A, A2, E, F and H. Procedures for the preparation of anti-Hb Bart's by injecting purified Hb Bart's into rabbits is indicated in the time schedule. The rabbit antisera were evaluated by antigen-antibody reaction in agar gel. Although the antiserum reacted with Hb Bart's but not with Hb A, A2,E and H, it also cross-reacted with Hb F. After the rabbit antisera were absorbed with Hb F, the antisera were highly specific because it only reacted with Hb Bart's. The purified specific anti-Hb Bart's was labelled with radioactive 125I by chloramine-T method. After passing through Sephadex G-100 column, the 125I labelled specific anti-Hb Bart's was obtained in the first peak. This radioactive labelled anti-Hb Bart's was ready to use in the two-site immunoradiometric assay.

Enzyme immunoassay for the identification of hemoglobin variants.

We have prepared monospecific antibodies to Hbs D-Los Angeles, J-Baltimore, O-Arab and J-Paris-I and developed an enzyme immunoassay (ELISA) for their identification in hemolysates. Hbs in adult or cord blood hemolysates were coated to the wells of microtiter plates and reacted with the appropriate antisera followed by the detection system which contains anti-rabbit IgG/peroxidase conjugate and the substrate tetramethylbenzidine. Sixty-nine samples were tentatively considered to contain the above hemoglobin variants by isoelectrofocusing and the identity of 83% of them was confirmed by ELISA. Some of the non-reacting hemolysates were shown by amino acid sequence analysis to contain Hbs Korle-Bu, D-Ibadan, G-Copenhagen and the new variant Chandigarh. This ELISA offers specificity and simplicity for the confirmatory identification of hemoglobin variants.

Enhanced reaction with Vicia graminea lectin and exposed terminal N-acetyl-D-glucosaminyl residues on a sample of human red cells with Hb M-Hyde Park.

A sample of polyagglutinable red cells was obtained from a healthy individual (group O, N) possessing a hemoglobin (Hb) variant called Hb M-Hyde Park. The sialic acid content of the individual's red cells is 90 percent of normal, and his cells are agglutinated by monoclonal but not lectin anti-Tn, a panel of lectins specific for N-acetylgalactosamine (or galactose), and N-acetylglucosamine. Enhanced agglutination reactions were obtained with Vicia graminea, Ulex europaeus, and human anti-I and -i. Using various enzyme treatments and different methods of labeling cell surface components, two defective cell membrane sites have been identified: one associated with the O-linked oligosaccharides on sialoglycoproteins and the other associated with exposed N-acetylglucosaminyl residues located on membrane components of apparent molecular weights 88,000 to 130,000 and 46,000 to 73,000 (probably the Band 3 and Band 4.5 regions, respectively).