Blood group D antigen content of nucleated red cell precursors.

The D antigen content of nucleated red cell precursors in human bone marrow was estimated using autoradiography and 125I-anti-D. D antigen first appeared in the pronormoblast, and the quantity of antigen progressively increased during red cell maturation. Maximal anti-D binding occurred on mature red blood cells. Pronormoblasts, basophilic normoblasts, polychromatophilic normoblasts, and orthochromatic normoblasts, respectively, had approximately 1/4, 1/2, 2/3, and 3/4 the quantity of antigen found on mature red cells. None of the other cell types were found in bone marrow labeled with anti-D.     

Globin synthesis during erythroid cell maturation in alpha thalassemia.

Globin chain synthesis was examined in erythroid cells of increasing maturity, fractionated from bone marrow of two patients with hemoglobin H disease and in one alpha thalassemia 1 heterozygote. In contrast to beta thalassemia where a gradient of alpha/beta chain ratios increasing with erythroid cell maturation is observed, in alpha thalassemia the alpha/beta chain ratio remains constant throughout maturation. This finding suggests that in alpha thalassemia there is no modification of the imbalance in globin chain synthesis either by increased alpha chain production or decreased beta chain synthesis in erythroid precursors. Furthermore, the constant alpha/beta ratio reflects a limited degree of beta chain destruction, indicating that the ability of the excess beta chains to associate into tetramers protects them from proteolytic digestion.     

Quantitatively different red cell/nucleated cell chimerism in patients with long-term, persistent hematopoietic mixed chimerism after bone marrow transplantation for thalassemia major or sickle cell disease

Background

Persistent mixed chimerism represents a state in which recipient and donor cells stably co-exist after hematopoietic stem cell transplantation. However, since in most of the studies reported in literature the engraftment state was observed in the nucleated cells, in this study we determined the donor origin of the mature erythrocytes of patients with persistent mixed chimerism after transplantation for hemoglobinopathies. Results were compared with the engraftment state observed in singly picked out burst-forming unit – erythroid colonies and in the nucleated cells collected from the peripheral blood and from the bone marrow.

Design and Methods

The donor origin of the erythrocytes was determined analyzing differences on the surface antigens of the erythrocyte suspension after incubation with anti-ABO and/or anti-C, -c, -D, -E and -e monoclonal antibodies by a flow cytometer. Analysis of short tandem repeats was used to determine the donor origin of nucleated cells and burst-forming unit – erythroid colonies singly picked out after 14 days of incubation.

Results

The proportions of donor-derived nucleated cells in four transplanted patients affected by hemoglobinopathies were 71%, 46%, 15% and 25% at day 1364, 1385, 1314 and 932, respectively. Similar results were obtained for the erythroid precursors, analyzing the donor/recipient origin of the burst-forming unit – erythroid colonies. In contrast, on the same days of observation, the proportions of donor-derived erythrocytes in the four patients with persistent mixed chimerism were 100%, 100%, 73% and 90%.

Conclusions

Our results showed that most of the erythrocytes present in four long-term transplanted patients affected by hemoglobinopathies and characterized by the presence of few donor engrafted nucleated cells were of donor origin. The indication that small proportions of donor engrafted cells might be sufficient for clinical control of the disease in patients affected by hemoglobinopathies is relevant, although the biological mechanisms underlying these observations need further investigation.     

SE-9000血液分析仪有核红细胞提示分析

目的:分析sysmex SE-9000血液分析仪有核红细胞(NRBC)提示的可信性.方法:收集仪器白细胞栏有NRBC提示的标本100份,无NRBC提示的标本300份,涂片镜检,并与仪器结果进行比较.结果:32份单项NRBC提示的标本,镜检只4份有NRBC,其阳性符合率为12.5%;68份有NRBC同时有未成熟粒细胞(Imm Gran)、原始细胞(Blasts)、异型/异常淋巴细胞(Aty/Abn Ly)、核左移(Left)中一项或多项异常提示的标本,镜检48份有NRBC,阳性符合率为70.5%; 226份无NRBC及其它异常提示的标本,镜检1份有NRBC,假阴性率为0.4%;74份无NRBC但有Imm Gran、Blasts、Aty/Abn Ly、Left中一项或多项异常提示的标本,镜检4份有NRBC,假阴性率5.4%.结论:SE-9000血液分析仪对单项NRBC提示的假阳性率高,对有NRBC提示并同时有白细胞异常提示的阳性符合率较好,可信度较高,假阴性率低,对NRBC的检查起到过筛作用,明确镜检对象,缩小镜检范围.     

单个胎儿有核红细胞的DNA分析

目的：获取孕妇外周血中的极少量胎儿有核红细胞（ＮＲＢＣ）,并进行单个ＮＲＢＣ的基因分析,探讨其在产前基因诊断中的价值。方法：将孕妇外周血进行不连续密度梯度离心,将分离后的细胞进行制片,光学显微镜下识别ＮＲＢＣ,然后用显微操作仪获取单个ＮＲＢＣ进行引物延伸预扩增（ＰＥＰ）及Ｙ染色体特异性ＤＹＺ１基因的聚合酶链反应（ＰＣＲ）,以确定其来源于胎儿。结果：单个ＮＲＢＣ的ＰＥＰ－ＰＣＲ法检测结果,６０％检出     

The molecular basis of alpha thalassemia in India. Its interaction with the sickle cell gene

The alpha globin genotype of a total of 282 Indians from Orissa state has been analyzed. The overall alpha thalassemia gene frequency is 0.29, most frequently caused by the -alpha 3.7 and -alpha 4.2 deletions. In one family a novel -alpha 3.5 deletion removing the alpha 1 globin gene with some of its flanking sequences has been found, suggesting further sequence homology of the alpha globin gene cluster 3' to the alpha 1 globin gene. Patients with sickle cell disease and alpha thalassemia had higher hemoglobin (Hb) levels, RBC counts, and Hb A2 levels, and lower reticulocyte counts, MCV, MCH, and Hb F levels than those with a normal alpha genotype. The frequency of splenomegaly was not influenced by the alpha globin genotype. A higher prevalence of alpha thalassemia was found in patients greater than or equal to 10 years of age than in the younger group, suggesting a possible advantageous effect of alpha thalassemia on the survival of patients with sickle cell disease.     

Pure red cell aplasia with inhibitor to erythroid precursors in serum

A patient with pure red cell aplasia (PRCA), who had the inhibitor to erythroid precursors in serum, is described. A 72-year-old female was referred to Nagoya National Hospital because of progressing anemia in April 1988. On admission, her hemoglobin was 4.8 g/dl, reticulocyte 0.8%, and bone marrow specimen contained only 1.2% erythroblast. On these bases, she was diagnosed as pure red cell aplasia. After small amount of blood was transfused, her hemoglobin and erythroblast in bone marrow (EBM) increased to 7.8 g/dl and 39.1%, respectively, and she was discharged. However, after a month, her hemoglobin dropped to 4.6 g/dl, reticulocyte to 0.1%, and EBM to 0%. Soon after corticosteroid therapy (prednisolone, 40 mg, daily) was started, a marked elevation of reticulocyte count was observed, and then her hemoglobin increased to 11.0 g/dl, and EBM to 31.6%. The reason for a transient spontaneous remission at the onset of her disease was occurred is unclear. The number of BFU-E in her bone marrow was within normal range, but it was suppressed significantly (65%) after the addition of her serum and the complement purified from rabbit plasma. This finding suggest the presence of inhibitor to erythroid precursors in her serum.     

Decreased globin messenger RNA in thalassemia detected by molecular hybridization

In previous studies of patients with β thalassemia, mRNA extracted from reticulocytes in peripheral blood when added to cell-free systems reproduces the deficient β-chain synthesis characteristic of intact cells. The present studies with specific probes for α and β mRNA were designed to decide whether the decreased β mRNA activity is due to the presence of abnormal or reduced β globin mRNA in these cells. Purified α and β complementary DNAs (cDNAs) have been synthesized with RNA-instructed DNA polymerase; α and β mRNAs isolated from heavy (β-producing) and light (α-producing) polyribosomes of rabbit reticulocytes were used as templates. Each of the cDNAs is more than 80% pure by the criterion of biological activity. The α cDNA labeled with [³²P]dCTP and the β cDNA labeled with [³H]dCTP have been added simultaneously to reaction mixtures containing various concentrations of mRNA from thalassemic and nonthalassemic subjects. The extent and rate of hybridization were determined, permitting a comparison of relative α and β mRNA content in the same annealing mixture. In six nonthalassemic patients, relatively equal amounts of hybridizable α and β mRNA appear to be present. In five of seven patients with β-thalassemia, significantly decreased amounts of β mRNA compared to α mRNA can be demonstrated. In two patients with Hemoglobin H disease, there is a decreased amount of α mRNA compared to β mRNA.     

Decreased red blood cell aggregation subsequent to improved glycaemic control in Type 2 diabetes mellitus.

AIMS: Reports of rheological changes following intensification of metabolic control are limited and not concordant. The present study was designed to test the hypothesis that intensification of management of Type 2 diabetes (T2DM) with diet, exercise and insulin improves haemorheological behaviour by reducing red blood cell (RBC) aggregation. METHODS: Blood was sampled from 55 subjects before and following 14 +/- 3 weeks of intensified management. RBC aggregation was measured in vitro for cells in plasma or in an aggregating 70 kD dextran solution. Plasma viscosity and whole blood viscosity were also measured. RESULTS: During treatment, fasting glucose fell 27%, HbA1c fell 21%, and serum triglycerides and total cholesterol fell 28% and 12%, respectively (P < 0.0001 for each). The extent and strength of RBC aggregation in plasma fell by 10-13% (P < 0.002). Similar decreases of RBC aggregation were seen for cells suspended in dextran (P < 0.002). Plasma viscosity decreased by 3% (P < 0.02) and high shear blood viscosity by 6-7% (P < 0.0001). Changes of RBC aggregation in plasma and in dextran were significantly correlated, supporting a cellular rather than a plasmatic origin for these changes. However, there were no significant correlations between RBC aggregation changes and changes of fasting glucose, HbA1c, serum triglycerides, serum cholesterol, or plasma fibrinogen. CONCLUSIONS: Intensified metabolic control results in a reduction of RBC aggregation that appears to be intrinsic to RBC. Since increased RBC aggregation can impair microcirculatory flow, it is possible that haemorheological factors may contribute to the reduction of microvascular complications resulting from improved metabolic control in T2DM.     

Beta thalassemia minor is a beneficial determinant of red blood cell storage lesion

Blood donor genetics and lifestyle affect the quality of red blood cell (RBC) storage. Heterozygotes for beta thalassemia (bThal⁺) constitute a non-negligible proportion of blood donors in the Mediterranean and other geographical areas. The unique hematological profile of bThal⁺ could affect the capacity of enduring storage stress, however, the storability of bThal⁺ RBC is largely unknown. In this study, RBC from 18 bThal⁺ donors were stored in the cold and profiled for primary (hemolysis) and secondary (phosphatidylserine exposure, potassium leakage, oxidative stress) quality measures, and metabolomics, versus sex- and age-matched controls. The bThal⁺ units exhibited better levels of storage hemolysis and susceptibility to lysis following osmotic, oxidative and mechanical insults. Moreover, bThal⁺ RBC had a lower percentage of surface removal signaling, reactive oxygen species and oxidative defects to membrane components at late stages of storage. Lower potassium accumulation and higher uratedependent antioxidant capacity were noted in the bThal⁺ supernatant. Full metabolomics analyses revealed alterations in purine and arginine pathways at baseline, along with activation of the pentose phosphate pathway and glycolysis upstream to pyruvate kinase in bThal⁺ RBC. Upon storage, substantial changes were observed in arginine, purine and vitamin B6 metabolism, as well as in the hexosamine pathway. A high degree of glutamate generation in bThal⁺ RBC was accompanied by low levels of purine oxidation products (IMP, hypoxanthine, allantoin). The bThal mutations impact the metabolism and the susceptibility to hemolysis of stored RBC, suggesting good post-transfusion recovery. However, hemoglobin increment and other clinical outcomes of bThal⁺ RBC transfusion deserve elucidation by future studies.     

Oxidative denaturation of red blood cells in thalassemia

We believe that on the basis of all available data, severe oxidative damage occurs in alpha- and beta-thalassemic RBCs, as depicted schematically in Fig 6. The differences in the severity and pattern of the oxidative damage may be related to the type and, perhaps, quantity of precipitated globin chains. The detrimental effect of the excess chains is multifold. In the process of globin-chain precipitation, free radicals are generated. The end product of the precipitated hemoglobin chains is heme, from which eventually iron and globin are liberated. Globin chains have been found to interact and disrupt the RBC membrane, damaging the cytoskeleton. The role of heme has not yet been studied in detail in thalassemic RBCs. However, there is some evidence that it participates in damaging RBCs in other types of hemoglobinopathies. Excess of iron is known to be a catalyst of peroxidation via the Fenton reaction, causing damage to the various RBC membrane components (lipids, proteins, etc). The denatured hemaglobin, in the form of hemichromes, aggregates with protein 3, forming Actual proof of excessive free radical production in thalassemia is still warranted. It will not be easy to document since the amount of superoxide dismutase in RBCs is above and beyond that required for neutralizing excess amount of superoxide. The more active radicals, particularly hydroxyl free radical, are difficult to measure because they are so active an interact immediately with any given substrate in their vicinity. In addition, we have to better understand the finding of excess membrane lipids in thalassemic RBCs and whether there are changes in the formation and propagation of lipid peroxidation in these cells compared with normal RBCs. Regarding the proteins, further understanding is required concerning the exact type and sites of oxidation that occurs in the beta-thalassemia 4.1 protein, and whether the damage found in alpha-thalassemia is due to oxidation of ankyrin itself or its entrapment within the complex of the precipitated hemichromes of beta chains. What is the role of the different globin chain oxidation and precipitation in generating such different cytoskeletal protein alterations? Another point that needs to be elucidated is the role of different kinds of antibodies that are attached to the newly exposed antigenic sites on the thalassemic RBC membranes.(ABSTRACT TRUNCATED AT 400 WORDS)