Multiply Transfused Patients Research Articles

Bone Marrow Iron Concentration as a Marker of Iron Accumulation and Marrow Expansion in Patients with Beta Thalassemia Major.

Liver iron concentration (LIC) is a known and accurate marker of iron accumulation and is widely utilized to monitor iron chelation therapy in multiply transfused patients with Beta thalassemia major. Iron concentration in the bone marrow has not been studied and reported before. We utilized atomic absorption spectrophotometry to measure the iron content of marrow biopsy (BIC) in 102 thalassemia patients in various phase of treatment, 74 of them during the course of the disease and 28 patients after successful allogeneic marrow transplant. We observed BIC values below 0.5 mg/g dry weight in 7 healthy donors used as controls. Mean and median BIC were 4.67 and 2.70 (range 0.05 – 59.9) mg/g dw, in 101 valuable patients. Bone Marrow iron concentration (BIC) was calculated at the same time of LIC for each patient and a ratio LIC/BIC was generated. LIC/BIC ratio below 3, ratio between 3 and 10, and above 10 identified three categories of patients each with significant linear correlation between LIC and BIC (0.74; 0.76; 0.81 respectively). Twenty eight patients were in the first category, 48 in the second, and 25 in the third. Mean BIC was 9.47, 3.7, 1.2 mg/g dw and mean LIC was 12.7, 19.6, 22.3 mg/g dw respectively for CAT1, CAT2 and CAT3. Patients were also classified in class of risk for transplant, based on hepatomegaly, presence of liver fibrosis and history of regular or irregular iron chelation. BIC was higher in class 3 patients and in the irregularly chelated patients. Patients in class 1 were prevalently in CAT1 or 2, patients in class 3 were prevalently in CAT2 or 3. BIC value in each of the 3 categories correlated significantly with the whole body iron (WBI) amount, and WBI was significantly different in the three categories being lower in CAT1, that have higher BIC ( 3927 mg; 5894 mg, 7030 mg in CAT1, 2 and 3; p 0.01). Iron chelation quality (regular vs irregular) correlated with BIC value and with BIC category, majority of regularly chelated patients were in the Category 1 vs Category 2 or 3 (p 0.01). Patients before transplant were prevalently in CAT1 and 2, while those post transplant were mostly in CAT3, twelve patients that were studied both before and after transplant, changed from cat 1 to 2 or from cat 2 to 3. Their BIC changed significantly decreasing after BMT (median BIC was 7.8 before transplant and 2.25 after, p=0.002) To investigate the role of genetic hemochromatosis mutations, H63D region and Hamp region in 63 patients were also studied. Mean BIC was 3.08 in 16 patients with H63D mutation compared to 5.59 in those without mutation, and 11/16 had BIC below the median, p=0.03. Significantly more patients with H63D mutation were in CAT3 (p 0.02). Apparently H63D mutation favours accumulation of iron in the body, that was higher in CAT3, but participate also to lowering utilization of introduced iron. In conclusion, patients in category 1 had lower LIC and higher BIC, comprehended 50% of the patients in class 1 of risk, and 45% of the regularly chelated patients. On the contrary, majority of patients post transplant independently of having or not performed an iron removal program were in CAT3. We can draw conclusion that BIC related categories, as described here, give information on the balance between accumulation and utilization of iron. Patients belonging to BIC-Cat 1 correspond to patients that have been treated adequately and with lower iron body burden, better chelation history. They have higher BIC value and lower LIC value. They also are in pre-transplant phase, with active beta-thalassemia, when ineffective erythropoiesis and marrow expansion are more prominent. Further studies will be necessary to confirm the association of marrow iron content with the marrow functionality and expansion.

Management of hepatitis virus infections

Management of hepatitis virus infections

Prevalence of HLA antibodies in transfused patients with and without red blood cell antibodies

BACKGROUND: Multiply transfused patients are at increased risk of developing red cell (RBC) antibodies, as well as antibodies to HLA. Although pretransfusion testing screens for RBC antibodies, no such testing is routinely performed for HLA antibodies. Determining which patients are more likely to make HLA antibodies may be important for patients undergoing elective surgery where platelets (PLTs) may be required. It is hypothesized that patients with RBC alloantibodies may be more likely to have HLA antibodies than previously transfused patients without RBC antibodies. STUDY DESIGN AND METHODS: Blood was collected from 53 adult male surgical patients with RBC alloantibodies and a control group of 69 similar male patients with a history of previous transfusions but no evidence of RBC alloimmunization. The samples were tested for the presence of immunoglobulin G Class I HLA antibodies by enzyme-linked immunosorbent assay. RESULTS: Of the 53 samples from patients with RBC alloantibodies, 12 (22.6%) also had HLA antibodies, whereas only 7 (10.1%) of the 69 patients in the control group had HLA antibodies (p < 0.03). CONCLUSIONS: There is a significant difference between the rates of HLA alloimmunization in male patients with RBC antibodies versus multiply transfused patients without RBC antibodies. Screening for HLA antibodies may be warranted in patients with RBC alloantibodies who might require PLT transfusion support for elective surgery.

Effect of intravenous deferoxamine in multiply transfused patients

Purpose : Multiple transfusions in patients with chronic anemia can result in excessive iron deposition in tissues and organs. Effective iron chelation therapy in chronically transfused patients can only be achieved when iron chelators remove sufficient amounts of iron equivalent to those accumulated in the body from transfusions, thus leading to maintain body iron load at a non-toxic level. This study was retrospectively carried out to investigate the effect of intravenous iron chelation therapy with deferoxamine in patients who have received multiple transfusions. Methods : From March 2005 to January 2007, 15 patients who have received multiple transfusions were included in this study. Transfusion dependent patients were defined as those receiving >1 packed red blood cell (RBC) units/month for at least 6 months. They received intravenous deferoxamine for 7 days (10-30 mg/kg/day, 24 hour continuous infusions). Before and after deferoxamine infusions and 3 months later, we compared serum iron, TIBC, and ferritin in transfusion dependent patients and transfusion independent patients. Results : There were 6 males and 9 females and their age range was 5.6-21.3 (median 8.3) years. Transfusion dependent patients were 7 and 8 were transfusion independent states after stem cell transplantation or chemotherapy. There was no significant change in ferritin level after deferoxamine treatment for the transfusion dependent patients but significant falling of ferritin level was observed for the transfusion independent patients 3 months later compared with baseline ferritin level (P=0.046). Some adverse events were observed but symptoms were mild and tolerable. Conclusion : Seven days of intravenous deferoxamine was safe and effective in transfusion independent patients. In transfusion dependent patients, chelation therapy should be maintained, in order to minimize or prevent iron accumulation and storage in the tissues.

A comparison of two automated methods for the detection and identification of red blood cell alloantibodies.

The aim of this study was to compare the routine use of two automated systems (OrthoAutoVue Innova, microcolumn, and Immucor Galileo, solid phase) for the screening and identification of irregular red blood cell alloantibodies in samples, analysed in our Transfusion Service during 6 months of normal activity. The study focused particularly on an evaluation of the repeatability of the screening tests, the identification of antibody specificities and the identification of antibodies in samples showing discordant results. Overall 2,229 samples from potential blood donors (A), multiply transfused patients with blood disorders (DH), potential transfusion recipients (TS), and external cases (E) were studied. The protocols were carried out according to the manufacturers recommendations. The screening tests detected 78 samples that were positive with both systems, while 18 were positive only with Immucor and 11 only with Ortho (thus, overall, Immucor detected 96 positive samples and Ortho 89 positive samples). The use of the respective identification panels enabled us to identify the antibodies in 65 samples with Immucor and in 61 samples with the Ortho system; 74 antibodies were identified with Immucor (55 with a single specificity and 19 with mixed specificities) and 68 antibodies with Ortho (51 and 17, respectively). In the remaining cases (31 samples for Immucor and 28 for Ortho), the antibody specificity was not identified. The two systems were found to be essentially similar. The Immucor system revealed a greater number of antibodies, mainly because of its greater sensitivity at detecting anti-D antibodies. Both systems showed a repeatability of over 85%, demonstrating that automation of immunohaematological tests is advantageous. The specificity of the antibody was identified in 68% of the samples. Furthermore, using the two systems led to the identification of ten new antibodies (6 anti-D, 2 anti-E, 1 anti Le(a), and 1 anti-Vel), which would not have been detected had only one of the two methods been used.

Microchimerism: a lasting legacy of transfusion?

Microchimerism: a lasting legacy of transfusion?

Quantification of siderophages in bronchoalveolar fluid in transfusional and primary pulmonary hemosiderosis

Transfusional iron overload may occur in the lungs. We hypothesized that quantitating siderophages in the bronchoalveolar fluid (BALF) of heavily transfused patients may prove to be a useful tool in determining lung iron overload in transfusion-dependent patients. The study included six patients (7-20 years) with thalassemia major (TM) who had received multiple blood transfusions, one with hereditary spherocytosis (four blood transfusions) and one with sickle cell disease (never transfused); they were compared to three children with idiopathic pulmonary hemosiderosis (IPH) (2.5-7.0 years) as positive controls. Fiberoptic bronchoscopy with bronchoalveolar lavage was performed in seven patients under general anesthesia for elective surgery and the rest were bronchoscoped electively under sedation. Spirometry was also performed in eight patients. There was no significant difference between children with TM and IPH in siderophages as percentage of total count (95% CI -31.0 to 1.5, P = 0.068). There were positive relationships between both mean serum ferritin values during the preceding year and the total number of units of transfused blood, and percent siderophage count among multiply transfused patients (P = 0.010, P = 0.052, respectively); similar findings were noted for the Golde score (P = 0.001, P = 0.031, respectively). None of the patients showed lung function impairment. In conclusion, in this small study, we found that the BALF of multiply transfused patients with benign hematological disorders contain similar numbers of siderophages to that of patients with IPH; this is strongly suggestive of secondary pulmonary hemosiderosis. The correlation between the patients' serum ferritin, and the BALF siderophages suggests that the later may serve as a marker of pulmonary iron overload in patients requiring blood transfusion and appear to be more sensitive than standard pulmonary function tests.

Prevalence of HLA antibodies in transfused patients with and without red cell antibodies

Multiply transfused patients are at increased risk of developing red cell (RBC) antibodies, as well as antibodies to HLA. Although pretransfusion testing screens for RBC antibodies, no such testing is routinely performed for HLA antibodies. Determining which patients are more likely to make HLA antibodies may be important for patients undergoing elective surgery where platelets (PLTs) may be required. It is hypothesized that patients with RBC alloantibodies may be more likely to have HLA antibodies than previously transfused patients without RBC antibodies. Blood was collected from 53 adult male surgical patients with RBC alloantibodies and a control group of 69 similar male patients with a history of previous transfusions but no evidence of RBC alloimmunization. The samples were tested for the presence of immunoglobulin G Class I HLA antibodies by enzyme-linked immunosorbent assay. Of the 53 samples from patients with RBC alloantibodies, 12 (22.6%) also had HLA antibodies, whereas only 7 (10.1%) of the 69 patients in the control group had HLA antibodies (p < 0.03). There is a significant difference between the rates of HLA alloimmunization in male patients with RBC antibodies versus multiply transfused patients without RBC antibodies. Screening for HLA antibodies may be warranted in patients with RBC alloantibodies who might require PLT transfusion support for elective surgery.

Fludarabine, cyclophosphamide and horse antithymocyte globulin conditioning regimen for allogeneic peripheral blood stem cell transplantation performed in non-HEPA filter rooms for multiply transfused patients with severe aplastic anemia

Multiply transfused patients of severe aplastic anemia are at increased risk of graft rejection. Five such patients underwent peripheral blood stem cell transplantation from HLA-identical siblings with a fludarabine-based protocol. The conditioning consisted of fludarabine 30 mg/m(2)/day x 6 days, cyclophosphamide 60 mg/kg/day x 2 days and horse antithymocyte globulin (ATG) x 4 days. Two different ATG preparations were used: ATGAM (dose 30 mg/kg/day x 4 days) or Thymogam (dose 40 mg/kg/day x 4 days). Engraftment: median time to absolute neutrophil count (ANC) >0.5 x 10(9)/l was 11 days (range: 8-17) and median time to platelet count >20 x 10(9)/l was 11 days (range: 9-17). At a median follow-up of 171 days (range: 47-389), there has been no graft rejection and all patients are in complete remission. Acute GVHD (grade 1) occurred in one patient only. Chronic GVHD developed in two patients (extensive in one and limited in another). The transplants were performed in non-HEPA filter rooms. In only one patient, systemic antifungal therapy (voriconazole) was used. The use of Thymogam brand of ATG for conditioning is being reported for the first time. Our experience suggests that this fludarabine-based protocol allows rapid sustained engraftment in high-risk patients without significant immediate toxicity.

Transfusion of Leukoreduced RBC Results in Immunization to Minor Histocompatibility Antigens by Crosspriming into Recipient MHC Class I Pathways.

Background: HLA matched related bone marrow transplantation (BMT) following myeloablative treatment with busulfan, cyclophosphamide and ATG is an effective therapy for children with severe sickle cell disease (SCD). However, a five percent risk for transplant related mortality precludes its use in less severely affected children, and a much higher risk in adults prevents its use in older patients. Low dose total body irradiation based conditioning regimens may provide a safer approach to BMT for patients with SCD. However, although these non-myeloablative regimens reliably allow for sustained engraftment in patients with malignant diseases, there is a high incidence of graft rejection in patients with SCD. One distinct variable in the sickle cell population is chronic transfusion with multiple units of red blood cells (RBC), which may lead to immunization to antigens present on donor bone marrow. Because BMTs in this setting are HLA matched, any immunization responsible for increased rejection is likely against minor histocompatibility antigens (mHAs). It has been assumed that contaminating leukocytes in RBC units are the main source of immunization to mHAs. However, a recent analysis of patients undergoing HLA matched BMT for aplastic anemia performed by the International bone marrow transplant registry, indicates that rejection of multiply transfused patients remains high despite the use of leukoreduced blood products. We hypothesized that RBC are alone sufficient to immunize to mHAs through consumption of donor RBC by recipient antigen presenting cells (APC) and crosspresentation of donor peptides in recipient MHC I molecules.Methods: Chicken Ovalbumin (OVA) is a well studied antigen. We converted OVA into a model RBC associated antigen using a maleimide-sulfhydryl based crosslinking strategy to couple OVA to RBC (OVA-RBC). To monitor activation of CD8+ T cells specific for OVA, we utilized mice transgenic for a T cell receptor that recognizes an OVA peptide 257–264 (SIINFEKL) presented by MHC class I H-2Kb (OT-I mice). Leukoreduced (OVA-RBC) from B10.BR (H-2k) donor mice were transfused into C57BL/6 (H-2b) mice that had been adoptively transferred with OT-I T cells. Activation, and expansion of OT-I cells was assessed by flow cytometry using specific tetramer reagents.Results: Although their lifespan is somewhat decreased, OVA-RBC continue to circulate for 10–14 days with stable persistence of OVA on the RBC. In response to transfusion of OVA-RBC, OT-I T cells expanded 49 fold compared to untransfused mice (p value = .0002). This was antigen specific, as no expansion was seen with RBC crosslinked to a third party antigen (hen egg lysozyme). Moreover, the observed T cell expansion required that the antigen be associated with the RBC, as no expansion was observed after injection with soluble OVA. This experimental design precludes direct presentation of OVA by donor cells, as donor cells do not express the MHC (H-2Kb) to which OT-I T cells are restricted.Discussion: Based upon the above data, we conclude that mHAs on transfused RBC crossprime into the MHC class I pathway of recipient APC. Since human hematopoietic progenitor cells express many of the known mHAs, this observation provides a mechanism by which chronic transfusion of even stringently leukoreduced RBC may result in sufficient mHA immunization to increase the frequency of BMT rejection.

Allosensitization in Patients Receiving Multiple Blood Transfusions

Extended antigen (C, E, K) matching decreased the incidence of alloantibody (alloAB) and autoantibody (autoAB) formation, in addition to eliminating transfusion reactions in the multiply transfused sickle cell disease patients. AlloAB formation possibly transforms the immune system into a hyperactive state leading to further and earlier alloAB and autoAB formation. However, additional CEK matching results in marked overuse of Rh-negative packed red blood cell (pRBC) units, 30 minutes' extra time of a skilled technologist, and 153 dollars extra CEK reagent cost per unit to find CEK-matched pRBCs for every transfusion for these multiply transfused patients.

Fludarabine-based conditioning for allogeneic stem cell transplantation for multiply transfused patients with Fanconi's anemia

A fludarabine-based protocol (fludarabine (25 mg/m(2)/day x 6 days), cyclophosphamide (10 mg/kg/day x 2 days) and ATG (ATGAM 10 mg/kg/day x 4 days)) was used in four multiply transfused Fanconi's anemia (FA) patients aged 5-15 years to reduce rejection during allogeneic bone marrow transplantation (BMT). Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and mini methotrexate. The graft source was G-CSF-stimulated bone marrow or peripheral blood stem cells (PBSC) in two patients each. All patients engrafted with median time to ANC>500/mm(3) being 14 days (range: 12-17) and unsupported platelet count >20 ,000/mm(3) being 13 days (range: 11-18). One patient had secondary graft rejection on day 56 and expired on day 69 due to fungal pneumonia. One patient who developed acute myeloid leukemia on day 56 underwent successful induction with cytosine and daunorubicin followed by peripheral blood stem cell (PBSC) rescue on day 70 and is presently in remission with complete donor chimerism and grade I GVHD. At a median follow-up of 13 months (range: 4-21), three patients (75%) are well with complete donor chimerism. Addition of fludarabine to the conditioning regimen for BMT in FA can provide additional immunosuppression for engraftment without increasing toxicity.

Editorial

The transfusion of red blood cells (RBCs) is now considered a well-settled and essential therapy. However, some difficulties and constraints still occur, such as long-term blood product shortage, blood donor population aging, known and yet unknown transfusion-transmitted infectious agents, growing cost of the transfusion supply chain management, and the inescapable blood group polymorphism barrier. Red blood cells can be now cultured in vitro from human hematopoietic, human embryonic, or human-induced pluripotent stem cells (hiPSCs). The highly promising hiPSC technology represents a potentially unlimited source of RBCs and opens the door to the revolutionary development of a new generation of allogeneic transfusion products. Assuming that in vitro large-scale cultured RBC production efficiently operates in the near future, we draw here some futuristic but realistic scenarios regarding potential applications for alloimmunized patients and those with a rare blood group. We retrospectively studied a cohort of 16,486 consecutive alloimmunized patients (10-year period), showing 1 to 7 alloantibodies with 361 different antibody combinations. We showed that only 3 hiPSC clones would be sufficient to match more than 99% of the 16,486 patients in need of RBC transfusions. The study of the French National Registry of People with a Rare Blood Phenotype/Genotype (10-year period) shows that 15 hiPSC clones would cover 100% of the needs in patients of white ancestry. In addition, one single hiPSC clone would meet 73% of the needs in alloimmunized patients with sickle cell disease for whom rare cryopreserved RBC units were required. As a result, we consider that a very limited number of RBC clones would be able to not only provide for the need for most alloimmunized patients and those with a rare blood group but also efficiently allow for a policy for alloimmunization prevention in multiply transfused patients.

Age-specific prevalence, transmission and phylogeny of TT virus in the Czech Republic

BackgroundTT virus is prevalent worldwide, but its prevalence and genotype distribution in Central and East-Europe has not been determined. The high prevalence of TTV in multiply-transfused patients points to the importance of a parenteral mode of transmission, but since more than half of the general population is infected other possible routes of transmission must be considered.MethodsIn our study, we investigated the epidemiology, transmission and phylogeny of TTV in the Czech Republic. The following groups were selected: a control group of 196 blood donors, 20 patients with hemophilia, 49 intravenous drug users, 100 sex workers, 50 penitentiary prisoners, 208 healthy children aged 1 to 14 years, 54 cord blood samples, 52 patients with non-A-E hepatitis, 74 patients with hepatitis C, and 51 blood donors with increased ALT levels. Primers specific for the non-coding region were used. The genotype distribution was studied in 70 TTV-positive samples.ResultsThe prevalence rate of TTV among the Czech population was 52.6%. We have shown that TTV is not transmitted prenatally. Children were infected after birth with two peaks: one at the age of two years and the other after the beginning of primary school. Adults have shown a further increase in the TTV prevalence with age. The highest TTV prevalence was found in the group of patients who had received multiple blood transfusions. The TTV prevalence rate in subjects at an increased risk of sexual transmission was not significantly higher than in the general population. Genotypes G2 and G1 were most prevalent among the Czech population, followed by G8 and G3. The subjects positive for markers of HBV and/or HCV infection tested significantly more often TTV DNA positive, which is suggestive of a common route of transmission of these three infections.ConclusionsThis study on TTV prevalence, mode of transmission and age-specific prevalence is the most extensive study performed in Central and Eastern Europe. It showed insights into the epidemiology of TTV infection, but failed to associate TTV infection with clinical manifestations.

Radiotherapy-Based Conditioning Is Effective Immunosuppression for Patients Undergoing Transplantation with T-Cell Depleted Stem Cell Grafts for Severe Aplasia.

The combination of cyclophosphamide and ALG is highly effective in inducing engraftment in patients with aplastic anaemia undergoing allogeneic stem cell transplantation. However, the incidence of graft vs. host disease remains substantial. We studied the outcome of individuals with aplastic anaemia conditioned with a radiation containing regimen followed by the infusion of stem cell grafts that had been depleted of lymphocytes with CAMPATH-1H. The conditioning regime consisted of fractionated (f) total body irradiation 8 Gy followed by f total nodal irradiation 6 Gy. Additionally, patients received cyclophosphamide 60 mg/kg on 2 consecutive days. Cytokine mobilised peripheral blood grafts from HLA identical siblings were T-cell depleted with CAMPATH-1H “in the bag”. Cyclosporin was commenced on day -1 and continued until day +90. Seventeen heavily transfused patients with aplastic anaemia with a median age of 18 years (range 14–56) were studied. Median time from diagnosis to transplantation was 172 days (range 34–443). The median CD34+ cell number infused was 3.47 x106/kg (range 1.03–18.4). All patients engrafted. Recovery was fast and patients reached 0.5 x 109/L polymorphs by median day 11 (range 9–17) days. Toxicity from the conditioning included grade 4 haematological toxicity in all. Other major toxicity was gastrointestinal mucosal damage, which exceed grade 2 in only 2 instances. 1 patient developed thrombotic thrombocytopenic purpura which responded to substitution of cyclosporin with tacrolimus and plasmapheresis. Another, who had normal blood counts died of infection on day 241. Chimerism studies at 6 months post transplantation confirmed donor origin of haematopoiesis in all 7 patients tested. None of the patients developed acute or chronic graft vs. host disease. There was no delayed graft failure and 94% of patients survive disease free, at a median of 1303.5 days (range 216–2615) from graft infusion. In this cohort of multiply transfused patients, the radiation containing schedules described in this study led to universal engraftment with limited toxicity despite T-cell depletion. No patient developed graft vs. host disease or late graft failure. Lower doses of radiation containing conditioning should be explored further.

Hematopoietic stem cell transplantation for multiply transfused patients with sickle cell disease and thalassemia after low-dose total body irradiation, fludarabine, and rabbit anti-thymocyte globulin

Patients with sickle cell disease (N = 3) and thalassemia (N = 1) with high-risk features received hematopoietic stem cell transplantations (HCT) to induce stable (full or partial) donor engraftment. Patients were 9-30 years of age. Fludarabine, rabbit anti-thymocyte globulin (ATG), and 200 cGy total body irradiation were administered pre-transplant. Patients received bone marrow (N = 3) or peripheral blood stem cells (N = 1) from HLA-identical siblings, followed by mycophenolate mofetil and cyclosporine for post-grafting immunosuppression. Significant lymphopenia, but only moderate neutropenia and thrombocytopenia developed post transplant. No grade IV nonhematological toxicities or acute graft-versus-host disease (GVHD) were observed. At 3 months after transplantation, three of four patients had evidence of donor myeloid chimerism (range, 15-100%). However, after post transplant immunosuppression was discontinued, graft rejection occurred in all but one patient. This patient is now doing well 27 months post transplant with full donor engraftment. One patient died after a second transplant, and another patient experienced a stroke as her graft was being rejected. These results suggest that stable donor engraftment after nonmyeloablative HCT is difficult to achieve among immunocompetent patients with hemoglobinopathies and that new approaches will need to be developed before wider application of this transplantation method for hemoglobinopathies.

Radiotherapy-based conditioning is effective immunosuppression for patients undergoing transplantation with T-cell depleted stem cell grafts for severe aplasia

We studied the outcome of individuals with aplastic anemia conditioned with a radiation-containing regimen followed by an infusion of stem cell grafts that had been depleted of lymphocytes with CAMPATH-1H (antiCD52; humanised). The conditioning regime consisted of fractionated (f) TBI 8 Gy followed by f total nodal irradiation (TNI) 6 Gy. In addition, patients received CY 60 mg/kg on 2 consecutive days. Cytokine-mobilized peripheral blood grafts from HLA-identical siblings were T-cell depleted with CAMPATH-1H 'in the bag'. CsA was commenced on day -1 and continued until day +90. Seventeen heavily transfused patients with aplastic anemia, median age 18 years (range 14-56 years), were studied. The median time from diagnosis to transplantation was 172 days (range 34-443 days). The median CD34(+) cell number infused was 3.47 x 10(6)/kg (range 1.03-18.4 x 10(6)/kg). All patients engrafted. Recovery was fast and patients reached 0.5 x 10(9)/L polymorphs by median day 11 (range 9-17 days). Toxicity from the conditioning included grade 4 hematologic toxicity in all patients. Another major toxicity was gastrointestinal mucosal damage, which exceeded grade 2 in two instances. One patient developed thrombotic thrombocytopenic purpura, which responded to substitution of CsA with tacrolimus and plasmapheresis. Another patient, who had normal blood counts, died of infection on day 241. Chimerism studies at 6 months post-transplantation confirmed the donor origin of hematopoiesis in all seven patients tested. None of the patients developed acute or chronic GvHD. There was no delayed graft failure and 94% of patients had survived disease free at a median of 1303.5 days (range 216-2615 days) from graft infusion. In this cohort of multiply transfused patients, the radiation-containing schedules described in this study led to universal engraftment with limited toxicity despite T-cell depletion. No patient developed GvHD or late graft failure. Lower doses of radiation-containing conditioning should be explored further.

Fludarabine based conditioning regimen for allogeneic stem cell transplantation for multiply transfused patients with Fanconi’s anemia

A fludarabine-based protocol (fludarabine (25 mg/m/day × 6 days), cyclophosphamide (10 mg/kg/day × 2 days) and ATG (ATGAM 10 mg/kg/day × 4 days)) was used in four multiply transfused Fanconi's anemia (FA) patients aged 5-15 years to reduce rejection during allogeneic bone marrow transplantation (BMT). Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and mini methotrexate. The graft source was G-CSF-stimulated bone marrow or peripheral blood stem cells (PBSC) in two patients each. All patients engrafted with median time to ANC>500/mm being 14 days (range: 12-17) and unsupported platelet count >20 ,000/mm being 13 days (range: 11-18). One patient had secondary graft rejection on day 56 and expired on day 69 due to fungal pneumonia. One patient who developed acute myeloid leukemia on day 56 underwent successful induction with cytosine and daunorubicin followed by peripheral blood stem cell (PBSC) rescue on day 70 and is presently in remission with complete donor chimerism and grade I GVHD. At a median follow-up of 13 months (range: 4-21), three patients (75%) are well with complete donor chimerism. Addition of fludarabine to the conditioning regimen for BMT in FA can provide additional immunosuppression for engraftment without increasing toxicity.

HTLV-I and HTLV-II infections in volunteer blood donors and high-risk groups in Lebanon.

A serosurvey for Human T-cell Lymphotropic virus type 1 (HTLV-I)/HTLV-II was conducted in 1,900 blood donors, 120 pregnant women and 436 high-risk group patients in Beirut, Lebanon. One of the 1,900 blood donors was anti-HTLV-I/II-seroreactive on screening by enzyme immunoassay (EIA) but was indeterminate by Western blot and negative by polymerase chain reaction. None of the other 556 subjects studied was seroreactive by EIA. The credibility of the zero prevalence of HTLV-I/II infection among the Lebanese blood donors is supported by the absence of seroreactivity of antibodies in the multiply transfused patients. It seems therefore that the prevalence of HTLV-I/II appears to be less than 1 in 2,456 in the Lebanese population and hence, HTLV-I/II infection does not appear to require routine screening in Lebanon.

TT virus genotype changes frequently in multiply transfused patients with hemophilia but rarely in patients with chronic hepatitis C and in healthy subjects.

TT virus (TTV), a novel DNA virus, was originally thought to be transmitted by transfusion. However, nonparenteral transmission is recently suspected to be a major mode of transmission. To investigate the possibility of reinfection with TTV in multiply transfused patients and to evaluate the significance of transfusion transmission of TTV in patients with hemophilia, serial changes in TTV genotype were investigated in three groups. Serial changes in TTV genotype were investigated in 16 multiply transfused patients with hemophilia, 16 age-matched patients with chronic hepatitis C, and 16 age-matched healthy subjects. Mixed infection with multiple TTV genotypes was common in all groups. However, changes in TTV genotype were frequent in patients with hemophilia (15/16; 93.8%) but rare in patients with chronic hepatitis C and in healthy subjects (each group: 1/16; 6.3%). Changes in TTV genotype were frequently observed in multiply transfused patients with hemophilia but not in patients with chronic hepatitis or in healthy subjects without risk of transfusion transmission. This difference may suggest that exposure to TTV or even reinfection occurs frequently in patients with hemophilia, which could be evidence of transfusion transmission of TTV in this population.