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.

Radioimmunoassay for abnormal hemoglobins.

A sensitive and specific radioimmunoassay has been developed for the identification or quantification of the human hemoglobin variants S, C, D-Los Angeles, E, G Philadelphia, Russ, O Arab, Beograd, J Paris I, G San Jose, Q Iran, Korle Bu, and F Malta I. In the immunoassay, monospecific antibody preparations are used which recognize the single amino acid substitution in the variant polypeptide chail and do not cross-react with normal hemoglobins or hemoglobin variants containing a different amino acid exchange at the same position.

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.

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.

Immunochemical properties of abnormal hemoglobins C-Harlem (beta 6 Glu replaced by Val, beta 73 Asp replaced by Asn), S (beta 6 Glu replaced by Val), Korle Bu (beta 73 Asp replaced by Asn), Vancouver (beta 73 Asp replaced by Tyr), and Mobile (beta 73 Asp replaced by Val).

Antibodies were made to three mutant hemoglobins, each containing different single amino acid substitutions at beta 73:Hb Korle Bu (Asp replaced by Asn), Hb Mobile (Asp leads to Val), Hb Vancouver (Asp replaced by Tyr); and to one mutant hemoglobin, Hb C-Harlem, containing two substitutions in the beta chain (beta 6 Glu replaced by Val, as in Hb S, and beta 73 Asp replaced by Asn, as in Hb Korle Bu). The antiserum to Hb C-Harlem contained two antibody populations, each specific for one mutant amino acid residue. The antiserum to Hb Vancouver was completely specific for this mutant and did not cross-react with Hb Mobile and Hb Korle Bu; however, antiserum to Hb Korle Bu partially cross-reacted with Hb Mobile and to a smaller degree with Hb Vancouver. Antiserum to Hb Mobile exhibited even less cross-reactivity with Hb Korle Bu and C-Harlem and none with Hb Vancouver. These and previous studies indicate the involvement of at least three independent areas in the beta chain as antigenic determinant sites. It appears that the three mutants at beta 73 elicit the formation of antibodies which have a gradation in their specificity due to the nature of the amino acid sidechain.

Toward a system for detecting somatic-cell mutations. V. Preparation of fluorescent antibodies to hemoglobin hasharon, a human alpha-chain variant.

Antibodies against the abnormal human hemoglobin, Hb Hasharon (alpha 47 Asp leads to His), were raised in horse and purified by absorption against Sepharose 4B to which normal hemoglobins or Hb Hasharon were bound. The purified, non-precipitating antibodies were tested for specificity against normal hemoglobins and Hb Hasharon by immunodiffusion in the presence of anti-horse IgG, and by exposing mixtures of normal and Hb Hasharon-containing red cells to the antibodies after conjugation of the latter with fluorescein isothiocyanate. The ease with which antibodies specific for different variant hemoglobins have been prepared, and their potential for identifying individual erythrocytes that contain these hemoglobins by virtue of somatic mutation, underscore their value as aids to detection and analysis of mutational events in human subjects.

A hemolytic plaque assay for the detection of red blood cells carrying abnormal or mutant hemoglobin.

A simple hemolytic plaque assay for the detection of red blood cells carrying abnormal or mutant hemoglobin is described. The assay is based on the use, as indicator cells, of sheep red blood cells coated with monospecific antibody against the hemoglobin variant, another complement fixing multispecific anti-hemoglobin antibody as developing serum and an antibody against red cell ghosts which can lyse only the red cells of the species being studied and not the indicator sheep red cells. The test red cells are mixed with the indicator sheep red cells, the developing antiserum and the anti-ghost antibody. The mixture is transferred into thin chambers prepared with 2 microscope slides and incubated at room temperature (25 degrees C). The indicator sheep red cells around each test red cell carrying abnormal or mutant hemoglobin are lysed resulting in the formation of a plaque. Instead of anti-hemoglobin-coated sheep red blood cells, protein A-coated sheep red blood cells may be used as indicator cells provided the developing antiserum is monospecific for the hemoglobin variant. When sheep red blood cells coated with an antibody specific for DBA/2J mouse hemoglobin were used in the assay, only red cells from DBA/2J mice (carrying d hemoglobin) formed plaques while C57BL/6J red cells (carrying s hemoglobin) did not.

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.

Umbilical vein blood flow measurement in nonimmune hydrops fetalis.

Twenty-five measurements of fetal umbilical vein blood flow were performed in 22 cases of nonimmune hydrops fetalis using real-time and pulsed Doppler duplex ultrasound. The umbilical vein diameter, blood velocity, and blood flow in fetuses with hemoglobin Bart's hydrops fetalis were usually higher than those in fetuses with hydrops fetalis from other causes. Umbilical vein blood flow measurement appears to be an effective technique for differentiating hemoglobin Bart's from non-Bart's hydrops in this series. This hemodynamic characteristic of umbilical vein blood flow may be helpful in determining the etiology of nonimmune hydrops fetalis.

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.

[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.

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.

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.

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.

Hemoglobins Lepore and anti-Lepore.

The structure, properties, genetics, and clinical and biochemical expression of hemoglobins Lepore (deltabeta) and anti-Lepore (betadelta) are described. In addition to the three Lepore variants (Lepore Hollandia, Lepore Baltimore and Lepore Washington) at least four anti-Lepore variants (Miyada, P Nilotic (P Congo), Coventry and Lincoln Park) are known at the present time. All known hemoglobins Lepore and anti-Lepore are products of non-homologous crossing-over between the delta and the beta genes. Although the Hb Lepore condition is expressed phenotypically and clinically as beta thalassemia, the presence of about 10% of Hb Lepore distinguishes the condition hematologically from beta thalassemia. Data on the hematological and biochemical expression of this hemoglobinopathy are presented. In contrast to the anemia in the Lepore condition, there is no phenotypic evidence of thalassemia in persons with hemoglobin anti-Lepore, because no beta chain deficiency accompanies the latter condition. Although no adequate explanation has been advanced concerning the factors which maintain a low synthesis of the Lepore and anti-Lepore chains, it has been suggested that multiple rare codons may introduce rate-limiting steps or that the deltabeta and betadelta mRNAs may be unstable. Data on the geographical distribution and structural identification of Hb Lepore are presented.

Generation of a monoclonal antibody specific for Hb G-Philadelphia [alpha 2(68)(E17)Asn----Lys beta 2] and development of an immunoassay.

A murine hybridoma was generated which secreted a monoclonal antibody (Mab) that specifically recognized the alpha 2(68)(E17)Asn----Lys beta 2 substitution of Hb G-Philadelphia. Hybridomas were produced by fusion of RBF/DnJ immune splenic lymphocytes with FOX-NY murine myeloma cells and selected in adenine-aminopterin-thymidine (AAT) medium. Culture fluids were screened by ELISA for antibody reacting with Hb G-Philadelphia but not Hb A. One such culture was cloned by limiting dilution, expanded and injected into pristane-primed, cyclophosphamide-suppressed BALB/c mice for ascites production. An enzyme-linked immunoassay was developed by conjugating hemoglobin in hemolysates or purified hemoglobins to the plastic surface of wells of a microtiter plate. The ascites fluid containing the Hb G-Philadelphia Mab was added to the wells followed by goat anti-mouse IgG conjugated with horseradish peroxidase. After the addition of substrate (tetramethylbenzidine), a deep blue color developed, signifying a positive reaction. We analyzed 58 hemolysates (17 adult, 41 cord) containing a G-variant along with 28 control hemolysates (12 cords comprising FA, FAC, FAS, FSS, FCC phenotypes; 16 adults consisting of AA, AS, SS, SC, S-beta thal, AD-Los Angeles phenotypes). Of the 58 hemolysates containing a G-variant, 53 were positive by ELISA and confirmed by radioimmunoassay (RIA). Four of the five hemolysates negative for Hb G-Philadelphia were shown to be Hb G-Montgomery by RIA. None of the control hemolysates were positive. The assay could be completed in 1 hr and represents a technological advance in hemoglobin identification.

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.