Gonadal function of young patients with beta-thalassemia following bone marrow transplantation.

Bone marrow transplantation (BMT) can induce short- and long-term impairment of gonadal function. Patients with beta-thalassemia represent a special group, as their primary diagnosis and its treatment modalities are responsible for gonadal dysfunction. To address the effect of BMT on puberty and gonadal function, we investigated 25 patients (12 males) with thalassemia who received allogenic BMT during childhood or adolescence and at the post-transplant evaluation were at an age that the pubertal process should have started. Pubertal stage by Tanner of breast and pubic hair, as well as testicular volume were assessed pre-BMT once and post-BMT at least twice. Menstrual history was recorded. FSH, LH, testosterone and estradiol levels were also determined. The impact of BMT appears to be different in the two sexes. Males seem to have higher tolerance, as all males who were pubertal at the time of BMT had normal testosterone, and all but one normal gonadotropin levels. From those who were prepubertal at BMT, 62% proceeded to normal pubertal development. Post-menarcheal females seem to be an extremely sensitive group to the deleterious effect of the transplantation process, as 100% of the post-menarcheal females exhibited amenorrhea and elevated gonadotropin levels. These findings are important for pre- and post-BMT counseling.

The oral manifestations of chronic graft-versus-host disease (cGVHD) in paediatric allogeneic bone marrow transplant recipients.

The oral manifestations of chronic graft-versus-host disease (cGVHD) in eight allogeneic bone marrow transplant (BMT) paediatric recipients were studied clinically, and lip biopsies were performed in seven of them. A prominent lichenoid reaction was observed in four patients, two with accompanying ulceration. Superficial mucoceles were present in three children. Clinically obvious xerostomia was seen in seven patients. Lip biopsies were positive and correlated with the clinical manifestations. Both clinical and histological findings confirmed the diagnosis of cGVHD. In three additional children, with systemic manifestations indicating cGVHD, the oral mucosa was clinically and histologically normal, and the systemic manifestations were, thus, attributed to drug reactions. The above findings indicate the high value of oral examination in diagnosing or confirming paediatric cGVHD. Superficial mucoceles, reported for the first time in paediatric recipients, seem to be important in the early diagnosis of cGVHD.

Combined umbilical cord blood and bone marrow transplantation in the treatment of beta-thalassemia major.

The authors report on three children with beta-thalassemia major, class II, III, and III according to the Pesaro classification, with a body weight of 16, 62, and 50 kg, respectively, who received grafts using both umbilical cord blood (UCB) and bone marrow (BM) stem cells from their HLA-matched siblings. The number of UCB nucleated cells collected was 2 x 10(7)/kg, 1.2 x 10(7)/kg, and 2.5 x 10(7)/kg, respectively, and was considered insufficient to secure engraftment. The authors increased the number of hematopoetic progenitors by harvesting BM from the same donors. All 3 patients showed prompt engraftment with neutrophil recovery on days 17, 18, and 17 post-transplant, respectively, and platelet recovery on days 19, 25, and 22 post-transplant, respectively. One patient had remarkably increased HbF of values 31, 19, and 12% at 3, 6, and 12 months post-transplant, respectively, which were accompanied by an increase in the G gamma/A gamma ratio, suggesting UCB-derived hematopoetic reconstitution. All patients are alive and transfusion independent 23, 18, and 16 months post-transplant, respectively. For patients with homozygous beta-thalassemia who are at high risk of graft failure, either because of major prior alloimmunization or an insufficient amount of UCB stem cells, combined transplantation with UCB and BM could offer a quick and safe alternative therapy.

A functional hierarchy among the CD34+ hematopoietic cells based on in vitro proliferative and differentiative potential of AC133+CD34(bright) and AC133(dim/)-CD34+ human cord blood cells.

The 5-transmembrane receptor AC133 is expressed on a subpopulation of human hematopoietic cells that includes the CD34(bright) cells. We evaluated the developmental potential of AC133+CD34(bright) and AC133(dim/-)CD34+ cells isolated from 5 cord blood (CB) samples by studying the in vitro proliferative and differentiative potential of each population in both progenitor and mature cell expansion cultures. Seven-day culture of AC133+CD34(bright) cells with a cytokine combination favoring primitive progenitor cells causes a significant increase in CD34+, CFU-C and noncycling stem/progenitor cells HPP-Q (High Proliferative Potential-Quiescent), whereas culture of AC133(dim/-)CD34+ cells shows a limited increase in committed progenitor cells only. HPP-Q cells were not found in freshly isolated AC133(dim/-)CD34+ nor in expanded CD34+ cells derived from AC133(dim/-)CD34+ cells. No statistically significant difference was observed between the 1-week expanded AC133+ and the initial AC133+CD34(bright) cells regarding their clonogenic efficiency (CE), while expanded CD34+ cells derived from AC133(dim/-)CD34+ cells exhibited a decreased CE. Subexpansion of the reselected AC133+ derived from AC133+CD34(bright) cells reveals a further increase of stem/progenitor cells and the 14-day expanded AC133+ cells reveal an unchanged CE. Subexpansion of reselected 7-day CD34+ cells derived from AC133(dim/-)CD34+ cells was not possible. Culture of AC133+CD34(bright) cells in cytokines that favor megakaryopoiesis or erythropoiesis resulted in a significant expansion of CD41+ and CD71+ cells, respectively; AC133(dim/-)CD34+, in comparison, showed a limited potential to megakaryocytic differentiation and a decreased production of erythroid cells. Our data indicate that early high proliferating stem/progenitor cells and early committed progenitors are present in AC133+CD34(bright) cells, but not in AC133(dim/-)CD34+ cells; the latter represent late committed progenitors with limited proliferative potential.

Clinical value of bone marrow cultures in childhood pure red cell aplasia.

PURPOSE: We assessed the value of marrow cultures for defining the pathophysiology, diagnosis, and therapeutic response to immunosuppressive therapy in childhood pure red cell aplasia (PRCA). PATIENTS AND METHODS: Patients were evaluated either at diagnosis (n = 23) or at the time of treatment failure (n = 2). Twelve patients had transient erythroblastopenia of childhood (TEC), 4 had Diamont-Blackfan anemia (DBA), and 9 had acquired sustained PRCA (A-Su-PRCA). Bone marrow mononuclear cells were cultured with combination of human recombinant (rhu) erythropoietin (EPO), granulocyte monocyte colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), Interleukin 3 (IL-3), either with or without stem cell factor (SCF), and burst forming unit of erythroid (BFU-E) growth was assessed. RESULTS: The combination of growth factors without SCF failed to induce any erythropoiesis (BFU-E < 10/10(5) mononuclear cells) in 10 patients (2 with TEC, 2 with DBA, and 6 with A-Su-PRCA), although the growth of erythroid colonies was substantially lower in the remaining patients than in controls (45.5 +/- 15.4 versus 91.7 +/- 12.7, p < 0.05). Addition of SCF restored erythropoiesis in all but 6 patients (5 with A-Su-PRCA and 1 with DBA). Five of 6 nonresponders did not respond to any immunomodulating therapy; of the 5, 3 had or developed some evidence of myelodysplasia. CONCLUSION: Our data indicate that in vitro colony studies might prove to be a useful diagnostic tool, because erythropoiesis' poor response to growth factors, including SCF, may suggest the diagnosis of myelodysplasia. Moreover, it may have predictive value; in cases of PRCA, regardless of etiology, poor growth of erythropoietic colonies may predict refractoriness to immunomodulating therapy.

[Clinical evaluation of various preventive methods for non-hemolytic transfusion reactions]. / Evaluation clinique de différentes méthodes préventives des réactions transfusionnelles non hémolytiques.

A study has been carried out on the incidence of non-hemolytic transfusion reaction on a group of patients suffering from thalassemia. Of this group, the rate of reactions per patient, based on the relationship between the number of patients with non-hemolytic transfusion reactions and the total number had risen to 31.8%. 83.7% of the patients with non-hemolytic transfusion reactions did not give a positive reaction to lymphocytotoxicity. 654 patients having, or not having shown a non-hemolytic transfusion reaction received washed red cell concentrates prepared extemporaneously. This process allowed the rate of reaction per patient to drop to 3.9%. The transfusion of deleucocytated red cell concentrates by filtration, carried out on a group of 188 patients, made the rate of reaction per patient drop to 2.8%. As regards to rate reaction per patient, there is no significant difference statistically between these two groups, however, it must be pointed out that the administration of filtered red cell concentrates, by deleucocytation, notably improves the incidence of a feverish reaction, while the administration of washed red cell concentrates has an important impact on allergic reactions. Interestingly, in solution, the washed red cell concentrates have the added advantage of having only very small quantities of free iron or vasoactive proteic derivatives. The new four bag system, now allows us to collect, separate and wash in a closed circuit. Compared to the traditional method this system has the advantage of assuring greater efficiency and security. In conclusion, for the first time, the administration of washed red cell concentrates on patients who receive regular transfusions, may represent a good procedure, combined or not with deleucocytation by filtration, as to prevent the occurrence of non-hemolytic reactions.

Red cell alloantibodies in patients with thalassemia.

We present the results of tests carried out to detect alloimmunization against red cells in 1,200 patients (607 males and 593 females), transfused and followed up during the period 1981-1987 in our hospital. Of these patients, 1,135 were thalassemic and 65 had sickle cell/beta-thalassemia. In 162 patients who received blood matched for the AB0, rhesus and Kell systems from their first transfusion, the immunization rate was very low (3.7%). In a pilot group consisting of 83 patients with the same clinical characteristics, who received blood matched only for the AB0 and Rh-D antigens, there was a significant difference in the frequency of alloantibodies (15.7%, p less than 0.001). Of 1,038 patients who received blood only matched for AB0 and Rh-D 244 (23.5%) with one or more red cell alloantibodies were identified. Of these 1,038 patients, 973 were exclusively thalassemic. In 220 (22.6%) of them, alloantibodies were found. The sickle cell beta-thalassemia patients presented alloantibodies with a higher frequency (36.9%, 24/65). Only one antibody was found in 114 patients (51.8%) and two or more in 106 patients (48.2%). The alloimmunization significantly concerned the rhesus (34.0%) and Kell (29.8%) systems. Anti-Kell was most often identified (28.5%). Alloimmunization appears considerably lower in patients in whom blood transfusion is started before the age of 3 than in those in whom it is started after that age (20.9 vs. 47.5%, p less than 0.0001).