Megakaryocytic dysfunction in myelodysplastic syndromes and idiopathic thrombocytopenic purpura is in part due to different forms of cell death.

Platelet production requires compartmentalized caspase activation within megakaryocytes. This eventually results in platelet release in conjunction with apoptosis of the remaining megakaryocyte. Recent studies have indicated that in low-risk myelodysplastic syndromes (MDS) and idiopathic thrombocytopenic purpura (ITP), premature cell death of megakaryocytes may contribute to thrombocytopenia. Different cell death patterns have been identified in megakaryocytes in these disorders. Growing evidence suggests that, besides apoptosis, necrosis and autophagic cell death, may also be programmed. Therefore, programmed cell death (PCD) can be classified in apoptosis, a caspase-dependent process, apoptosis-like, autophagic and necrosis-like PCD, which are predominantly caspase-independent processes. In MDS, megakaryocytes show features of necrosis-like PCD, whereas ITP megakaryocytes demonstrate predominantly characteristics of apoptosis-like PCD (para-apoptosis). Triggers for these death pathways are largely unknown. In MDS, the interaction of Fas/Fas-ligand might be of importance, whereas in ITP antiplatelet autoantibodies recognizing common antigens on megakaryocytes and platelets might be involved. These findings illustrate that cellular death pathways in megakaryocytes are recruited in both physiological and pathological settings, and that different forms of cell death can occur in the same cell depending on the stimulus and the cellular context. Elucidation of the underlying mechanisms might lead to novel therapeutic interventions.

Cost-effectiveness of pathogen inactivation for platelet transfusions in the Netherlands.

The objective of this study is to estimate cost-effectiveness of pathogen inactivation for platelet transfusions in the Netherlands. We used decision tree analysis to evaluate the cost-effectiveness of the addition of pathogen inactivation of pooled platelets to standard procedures for platelet transfusion safety (such as, donor recruitment and screening). Data on transfusions were derived from the University Medical Centre Groningen (the Netherlands) for 1997. Characteristics of platelet recipients (patient group, age, gender and survival) and data/assumptions on viral and bacterial risks were linked to direct and indirect costs/benefits of pathogen inactivation. Post-transfusion survival was simulated with a Markov model. Standard methods for cost-effectiveness were used. Cost-effectiveness was expressed in net costs per life-year gained (LYG) and estimated in baseline- and sensitivity analysis. Sensitivity was analysed with respect to various assumptions including sepsis risk, reduction of the discard rate and discounting. Stochastic analysis to derive 90% simulation intervals (SIs) was performed on sepsis risk. Net costs per LYG for pathogen inactivation were estimated 554,000 euro in the baseline-weighted average over the three patient groups (90% SI: 354,000-1092,500 euro). Sensitivity analysis revealed that cost-effectiveness was insensitive to viral risks and indirect costing, but highly sensitive to the assumed excess transfusions required and discounting of LYG. Given relatively high net costs per LYG that are internationally accepted for blood transfusion safety interventions, our estimated cost-effectiveness figures for pathogen inactivation may reflect acceptable cost-effectiveness in this specific area. Two main assumptions of our model were that the pathogen inactivation was 100% effective in preventing transmission of the pathogens considered and was not associated with major and/or costly adverse reactions. Validation of several crucial parameters is required, in particular the Dutch risk for acquiring and dying of transfusion-related sepsis.

Splenectomy for the treatment of thrombotic thrombocytopenic purpura.

Plasma exchange is the treatment of choice for patients with thrombotic thrombocytopenic purpura (TTP) and results in remission in >80% of the cases. Treatment of patients who are refractory to plasma therapy or have relapsing disease is difficult. Splenectomy has been a therapeutic option in these conditions but its value remains controversial. We report on a series of 33 patients with TTP who were splenectomised because they were plasma refractory (n = 9) or for relapsed disease (n = 24). Splenectomy generated prompt and unmaintained remissions in all except five patients, in whom remission was delayed (n = 4) or who died with progressive disease (n = 1). Four postoperative complications occurred: one pulmonary embolism and three surgical complications. Median follow-up after splenectomy was 109 months (range 28-230 months). The overall postsplenectomy relapse rate was 0.09 relapses/patient-year and the 10-year relapse-free survival (RFS) was 70% (95% CI 50-83%). In the patients with relapsing TTP, relapse rate fell from 0.74 relapses/patient-year before splenectomy to 0.10 after splenectomy (P < 0.00001). Two patients died from first postsplenectomy relapse. Although these results are based on retrospective data and that the relapse rate may spontaneously decrease with time, we conclude that splenectomy, when performed during stable disease, has an acceptable safety profile and should be considered in cases of plasma refractoriness or relapsing TTP to reach durable remissions and to reduce or prevent future relapses.

Intraoperative blood transfusion requirement is the main determinant of early surgical re-intervention after orthotopic liver transplantation.

Liver transplantation is the treatment of choice in selected patients with end-stage liver disease. Postoperative complications often require surgical re-intervention. This study is a retrospective single-centre study to assess the incidence and type of surgical re-intervention during the in-hospital period after liver transplantation and to identify predictors of this re-intervention. From 1994 to 2002, 231 consecutive adult liver transplantations were performed. Re-intervention was classified as biliary, vascular, bleeding, septicaemia, re-transplantation or as miscellaneous. One hundred and thirty-nine surgical re-interventions were performed in 79 of 231 patients (34%). Septicaemia (44%) and bleeding (27%) were the most frequent indications for re-intervention, followed by biliary (10%) re-intervention. Vascular re-intervention, re-transplantation, and re-intervention for miscellaneous reasons, were performed in 7% each. Of all analysed variables (gender, age, diagnosis, acute liver failure, Child-Pugh classification, Karnofsky score, previous abdominal surgery, creatinine clearance, prothrombin time, anti-thrombin, platelet count, surgical technique, cold ischaemia time, warm ischaemia time, functional anhepatic time, anatomic anhepatic time, revascularisation time, year of transplantation, aprotinin administration, transfused platelet concentrate, and red blood cell transfusion requirements), only the number of transfused red blood cell concentrates (RBCs) was identified as a predictor of surgical re-intervention. Median RBC transfusion requirement during liver transplantation was 2.9 l (range 0-18.8 l) in the re-intervention group compared with 1.5 l (range 0-13.4 l) in the non-re-intervention group (P<0.001). This study revealed intraoperative blood loss as the main determinant of early surgical re-intervention after liver transplantation and emphasises the need for further attempts to control blood loss during liver transplantation.

Pharmaco-economics of blood transfusion safety: review of the available evidence.

BACKGROUND AND OBJECTIVES: Pharmaco-economics provides a standardized methodology for valid comparisons of interventions in different fields of health care. The role of pharmaco-economics in the safety of blood and blood products has, however, been very limited to date. This review discusses the pharmaco-economic evaluations of strategies to enhance blood product safety that have been published in the scientific literature. MATERIALS AND METHODS: We reviewed pharmaco-economic methodology with special reference to cost-effectiveness analysis. We searched the literature for cost-effectiveness in blood product safety. RESULT: Net costs per quality adjusted life-year (QALY) gained varied from cost-saving for human immunodeficiency virus (HIV)- and hepatitis C virus (HCV) antibody screening and leucoreduction to several million US dollars per QALY gained for solvent-detergent treatment of plasma, nucleic acid amplification testing and HIV p24 antigen testing. CONCLUSIONS: To date the safety of blood transfusion has been largely determined by available technology, irrespective of pharmaco-economics. Net costs up to several million US dollars per QALY gained were found for interventions implemented.

In vitro megakaryocyte expansion in patients with delayed platelet engraftment after autologous stem cell transplantation.

Increasing the number of megakaryocytic cells in stem cell transplants by ex vivo expansion culture may provide an approach to accelerate platelet engraftment after high-dose chemotherapy. However, it is unknown if a relationship exists between the expansion potential of progenitor cells and the time to platelet engraftment in vivo. Therefore, we questioned if those patients who potentially would benefit most from expanded cell supplements are able to generate megakaryocytic cells efficiently in vitro. The in vitro megakaryocyte proliferation was analyzed from 19 leukapheresis samples from a group of multiple myeloma patients who all showed rapid neutrophil engraftment, but varied from 7 to 115 days post-transplant to achieve platelet levels >20x10(9)/l. CD34+ cells were isolated and analyzed for their potential to form megakaryocytic colonies (CFU-Mk) in colony assays and megakaryocytic (CD61+) cells in suspension cultures. The frequency and size of CFU-Mk and the expansion potential of CD61+ cells varied eightfold between individual patients. A similar range was found with CD34+ cells isolated from normal bone marrow (n=9). Rapid platelet engraftment occurred in patients receiving both high or low CFU-Mk doses and with high and low expansion of CD61+ cells. Four patients who experienced prolonged (>3 weeks) thrombocytopenia received low CFU-Mk doses, but the expansion potential was around median values or higher. Therefore, we conclude that the megakaryocyte proliferation is not impaired and that in vitro expansion could increase the number of megakaryocytic cells, although other factors could be more relevant in platelet engraftment in this group of patients.

Abnormal platelet adhesion on abdominal vessels in asymptomatic patients with paroxysmal nocturnal hemoglobinuria.

Platelet kinetic studies were performed in eight patients with paroxysmal nocturnal hemoglobinuria (PNH) but without symptoms or signs related to thrombosis. Indium-111 tropolonate labeled autologous platelets were used to determine mean platelet life and platelet production rate; total body images were also acquired. Three of seven patients with a thrombocytopenia showed a shortened mean platelet life, and the other five demonstrated a (nearly) normal platelet survival. Four of the seven patients with thrombocytopenia showed a decreased platelet production rate, but bone marrow cellularity was normo- to hypercellular. These observations suggest ineffective thrombopoiesis in 57% of the patients with PNH and thrombocytopenia. Total body imaging was performed during the platelet kinetic study in order to study organ uptake. Imaging of the abdominal vessels was demonstrated in all patients, suggesting enhanced adherence of platelets to the endothelium in patients with PNH. Since thrombosis of especially the abdominal vessels is a major cause of morbidity and mortality, the use of antithrombotic drugs might be considered despite the absence of abdominal complaints.

Mitochondrial disruption and limited apoptosis of erythroblasts are associated with high risk myelodysplasia. An ultrastructural analysis.

AIMS: To investigate the ultrastructural characteristics of erythroblasts in myelodysplasia (MDS) which might be of additional importance in understanding its pathogenesis. METHODS AND RESULTS: 22 patients were classified according to FAB (French-American-British classification), IPSS (international prognostic score system), cytogenetic risk factors and transfusion dependency. Using electron microscopy, in 77% of the cases, nuclear abnormalities consisting of disrupted membranes and cystic/dilated perinuclear spaces were noted. In a limited number of patients (n=7), a low percentage of apoptosis in the erythroid lineage (mean 3.1+/-1.6%; median 3%: range 1-6) (normal controls: <0.5%) could be noted, primarily in mature erythroblasts and significantly associated with spongiform nuclear features. In all patients extensive cytoplasmic vacuolization and myelin figures in erythroblasts were demonstrated. In 55% of the cases, enlarged and abnormal mitochondria were observed, significantly associated with iron-accumulation. A significant inverse relation existed between the absence of apoptosis and more advanced, or high risk disease and cytogenetic risk factors. Mitochondrial abnormalities were significantly correlated with high risk disease as well with an increase in transfusion dependency. CONCLUSIONS: These data indicate that in MDS apoptosis may play a role in early stages of disease. The overall prominent defects in mitochondria might be an additional defect that is involved in ineffective erythropoiesis.

Reduced transfusion requirements by recombinant factor VIIa in orthotopic liver transplantation: a pilot study.

BACKGROUND: Large transfusion requirements, i.e., excessive blood loss, during orthotopic liver transplantation (OLT) are correlated with increased morbidity and mortality. Recombinant factor VIIa (rF-VIIa) has been shown to improve hemostasis in a variety of conditions, but has never been studied in liver transplantation. METHODS: We performed a single-center, open-label, pilot study in adult patients undergoing OLT for cirrhosis Child-Pugh B or C, to assess efficacy and safety of rFVIIa. rFVIIa (80 microg/kg) was administered at the start of the operation, to be repeated according to predefined criteria. Packed red blood cells (RBC), fresh-frozen plasma, and platelet concentrates were administered according to predefined criteria. Perioperative transfusion requirements in study patients were compared with matched controls. RESULTS: Six patients were enrolled in the study. All received a single dose of rFVIIa. Transfusion requirements (given as median, with range in parentheses) were lower in the study group than in matched controls: 1.5 (0-5) vs. 7 (2-18) units of allogeneic RBC (P=0.006), 0 (0-2) vs. 3.5 (0-23) units of autologous RBC (P=0.043), total amount of RBC 3 (0-5) vs. 9 (4-40) units (P=0.002). Transfused fresh-frozen plasma was 1 (0-7) vs. 8 (2-35) units (P=0.011). Blood loss was 3.5 L (1.4-5.3) vs. 9.8 L (3.7-35.0) (P=0.004). One study patient developed a hepatic artery thrombosis at day 1 postoperatively. CONCLUSIONS: A single dose of 80 microg/kg rFVIIa significantly reduced transfusion requirements during OLT. Further study is needed to establish the optimally effective and safe dose of rFVIIa in orthotopic liver transplantation.

Effects of prednisone and splenectomy in patients with idiopathic thrombocytopenic purpura: only splenectomy induces a complete remission.

Idiopathic thrombocytopenic purpura (ITP) is a heterogeneous disease, whereby it is unclear if and in which way prednisone and splenectomy affect the platelet kinetics leading to a complete remission. To determine the effects of prednisone and splenectomy on the mean platelet life (MPL) and platelet production, platelet kinetic studies with Indium-111 tropolonate-labeled autologous platelets were performed in patients with ITP ( n=41). In 17 patients platelet kinetic studies were performed before and during prednisone treatment, and in 24 patients before and after splenectomy. MPL increased after prednisone therapy only in patients ( n=13) with a full recovery (FR, platelets >150 x 10(9)/l) and partial recovery (PR, 50 x 10(9)/l

6-Mercaptopurine, Still Valuable for the Palliative Treatment of Acute Myeloid Leukaemia.

Although 6-mercaptopurine (6-MP) is frequently used in the treatment of acute myeloid leukaemia (AML), its effect on disease progression has not been studied systematically. In a small retrospective analysis, we found that 6-MP could induce marked haematological improvement in a considerable number of AML patients who were not treated with intensive remission induction courses. Due to the inherent limitations of retrospective analyses, we then investigated prospectively in 51 consecutive patients over a 3-year period in a single centre, to what extent, oral 6-MP 250 mg twice a week could be beneficial to AML patients who were not-or no longer-eligible for intensive chemotherapy. Clinical response was scored according to changes in blood cell counts and dependency on blood transfusions. Thirteen patients (25%) were considered responders since they showed an increased platelet count from the first month after initiation of 6-MP onwards and they became independent of blood transfusions after 3 months. This effect lasted for 13 (range 7-30+) months. Median overall survival of this subgroup was 16.5 (6-33+) months. Ten patients (20%) had a shorter or incomplete response and a survival of 12 (6-30) months. Seven patients were lost to follow-up. Twenty-one (41%) failed to respond and survived for 4 (1.5-17) months. The response seemed not to be affected by previous chemotherapy, history of myelodysplasia, or karyotype abnormalities, but high leukocyte count initially was unfavourable. 6-MP thus can induce marked improvement of blood cell counts in a considerable proportion of AML patients who are not eligible for intensive chemotherapy, leading to good quality of life and a significant prolongation of survival.

The in vitro effects of cytokines on expansion and migration of megakaryocyte progenitors.

Increasing the number of megakaryocyte progenitors in stem cell transplants by ex vivo expansion culture may be an approach to accelerate platelet recovery in patients undergoing high-dose chemotherapy. We evaluated the effect of three different cytokine combinations on expansion, with special emphasis on the type of colony formation and migration of megakaryocytic cells. The number of clonogenic megakaryocyte progenitors (colony-forming units-megakaryocyte; CFU-Mk) with high- (> 20 cells/colony) and low-proliferative capacity (5-20 cells/colony) and the number of megakaryocytic (CD61+) cells were significantly increased by including interleukin 3 (IL-3) or IL-3 + IL-6 + IL-11 + Flt3-ligand to cultures containing megakaryocyte growth and development factor (MGDF) plus stem cell factor (SCF). No difference in the maturation of megakaryocytes from all three cytokine combinations to platelets were observed, as demonstrated by electron microscopy. In chemotaxis experiments, the migration towards stromal cell-derived factor 1 (SDF-1) was shown to be reduced for CD61+ cells and megakaryocyte progenitors cultured in other cytokines besides MGDF + SCF. The reduced migration was related to a lower expression of CXCR4, the receptor for SDF-1, on megakaryocytes from the proliferating cultures. These in vitro results demonstrate that expansion in IL-3 and other cytokines besides MGDF + SCF significantly impair the capacity of megakaryocytic cells to migrate.

Blood loss in orthotopic liver transplantation: a retrospective analysis of transfusion requirements and the effects of autotransfusion of cell saver blood in 164 consecutive patients.

Liver transplantation is associated with excessive blood loss. In order to identify factors influencing blood loss and to provide a basis for a pilot study to evaluate recombinant activated factor VII as a haemostatic agent, a retrospective study was performed in 164 consecutive patients with cholestatic or noncholestatic liver disease, who underwent orthotopic liver transplantation at a single centre between 1989 and 1996. Transfusion of allogeneic and autologous (cell saver) blood was used as a measurement of blood loss. Transfusion requirements were associated with age, gender, primary disease, Child-Pugh classification, serum levels of activated partial thromboplastin time, antithrombin III, urea and creatinine, platelet number, year of transplantation, length of cold ischaemia time and autologous blood transfusion. Of these variables, Child-Pugh classification (P = 0.001), urea (P = 0.0007), year of transplantation (P = 0.002), cold ischaemia time (P = 0.01) and autologous blood transfusion (P < 0.0001) were independent predictors of transfusion requirements by multivariate analysis. Thus, blood loss and transfusion requirements depend primarily on the severity of liver disease, quality of the donor liver, experience of the transplantation team and use of autologous (cell saver) blood transfusion. These findings emphasize the need for appropriate drug therapy and a critical reappraisal of current transfusion policy.

Autologous stem cell transplantation in multiple myeloma after VAD and EDAP courses: a high incidence of oligoclonal serum Igs post transplantation.

Thirty-seven patients with multiple myeloma (stage II and III, 65% increased beta2-microglobulin level) were prospectively treated with a median of 3.7 VAD courses (range 2-8) followed by cyclophosphamide (6 g/m2) in conjunction with G-CSF (5 microg/kg filgrastrim (n = 14), or 3.5 microg/kg lenograstrim (n = 22)), and peripheral stem cell (PSC) isolation. After regeneration this was followed by one EDAP course and high-dose melphalan (HDM, 200 mg/m2) in combination with re-infusion of PSC. Adequate stem cell mobilization was obtained with both G-CSF regimens. A median of 41x10(6) CD34+ cells/kg (range 4.5-161) was collected in a median of 1.6 leukapheresis procedures following filgrastrim (n = 14) and 24x10(6) CD34+ cells/kg (range 2. 3-80) in a median of 1.7 leukapheresis procedures following lenograstrim (n = 22) which indicated no significant difference (P = 0.24) between both G-CSF regimens. A rapid hematological recovery was obtained after HDM with reinfusion of a median of 9.3x10(6) CD34+ cells/kg. After the total courses the overall response was 84% with a complete remission rate of 30%. Currently the median overall survival is 44.0 months (95% CI 38.9-49.1) with a median follow-up of 33 months (range 3-51) and a median event-free survival of 29.0 months (95% CI 25.3-32.7) (n = 33). Post transplantation a high incidence of oligloclonal serum immunoglobulins (Igs) was observed. In 73% of the patients new oligoclonal or monoclonal serum bands were noticed 3 months post transplantation. IgG-lambda and IgG-kappa bands predominated. In 48% of the cases the oligoclonal Igs disappeared after a median follow-up of 22 months (range 8-36), whereas in 52% of the cases the oligoclonal Igs persisted with a median follow-up of 31 months (range 21-45), which did not correlate with a significant difference in overall, and event-free survival between both subgroups.

Excessive apoptosis of bone marrow erythroblasts in a patient with autoimmune haemolytic anaemia with reticulocytopenia.

We report a patient with autoimmune haemolytic anaemia (AIHA) with reticulocytopenia, who showed excessive apoptosis of erythroblasts. Ultrastructural analysis of bone marrow cells showed that 50% of erythroblasts had characteristic features of apoptosis, which was confirmed by staining with Annexin-V. In addition, in contrast to normal erythroblasts, Ig staining of the entire erythroblast population could be shown. These data show that apoptosis may contribute to the mechanism of reticulocytopenia in AIHA.

Platelet kinetic studies in patients with idiopathic thrombocytopenic purpura.

PURPOSE: To determine the value in diagnosis and treatment of mean platelet life, platelet production, and major sites of platelet destruction in patients with idiopathic thrombocytopenic purpura (ITP). PATIENTS AND METHODS: Sternal or posterior superior iliac spine bone marrow aspiration was performed in 141 patients. Platelet kinetic studies with Indium-111 tropolonate labeled autologous platelets were utilized to determine platelet production. RESULTS: Two subgroups of patients could be defined. The first group (n = 81, 58%) had normal or increased platelet production and increased peripheral platelet destruction. These patients fulfilled the conventional criteria for ITP, including reduced platelet survival time (mean +/- SD, 1.6 +/- 1.4 days). Forty-eight (59%) of these patients had increased splenic sequestration and 30 (88%) of the 34 patients who underwent splenectomy had a complete or partial remission. The second group (n = 60, 42%) had decreased platelet production, with significantly greater platelet survival times (3.6 +/- 2 days, P <0.0001). In this group, the proportion of patients with complete or partial response to splenectomy (62%) was somewhat lower (P = 0.09). These patients mainly had ineffective platelet production in the bone marrow. CONCLUSIONS: Platelet kinetic studies suggest that ITP is a heterogeneous disease that comprises two subgroups. Further studies are needed to validate these findings and to determine their effect on the choice and outcome of therapy.

Ischaemic colitis and lung infiltrates caused by extramedullary haematopoiesis in a patient with an acute erythroid leukaemia following polycythaemia vera.

A patient with 'spent' polycythaemia vera showed extensive extramedullary haematopoiesis (EMH) in non-haematopoietic tissue clinically resulting in an ischaemic colitis and respiratory symptoms due to lung infiltrates. On laboratory investigation, the EMH also included immature erythroblasts due to acute erythroid leukaemia. It is hypothesised that the abnormal homing of erythroid progenitors might be related to the abnormal expression of antigens, such as CD36.

CD34+/CD36- cells from myelodysplasia patients have a limited capacity to proliferate but can differentiate in response to Epo and MGF stimulation.

Myelodysplasia (MDS) is mostly characterized by a normal or increased number of normoblasts in the bone marrow and an impaired in vitro colony formation. In the present study we analyzed whether this might be due to a disconnection between proliferation and differentiation. CD34+/CD36- sorted bone marrow cells of 18 MDS patients were cultured in a clonogenic and suspension culture assay in the presence of erythropoietin (Epo) and mast cell growth factor (MGF). Burst-forming units erythroid (BFU-E, 75 +/- 88/10(4) CD34+ cells, X +/- s.d.) and colony-forming units E (CFU-E) were observed in eight of the 13 cases (62%) with refractory anemia with or without ring sideroblasts (RA and RARS) and one of the five cases with RA with excess of blasts or in transformation (RAEB and RAEB-T). Suspension cultures with CD34+/CD36- sorted cells with Epo plus MGF demonstrated an 8.9 +/- 6.5-fold expansion after 7 days in cases with >10 BFU-E/10(4) CD34+/CD36- cells while cases with <10 BFU-E/10(4) CD34+/CD36- cells demonstrated 1.0 +/- 0.8-fold expansion especially in cases with RAEB/RAEB-T. FACS and morphology analysis after 7 days of suspension culture demonstrated partial differentiation along the erythroid lineage in cases with RA/RARS (75%) and RAEB/RAEB-T (66%) reflected by the presence of erythroblasts and normoblasts with variable expression of CD34, CD36 and Glycophorin A. In cases with erythroid colony formation 69 +/- 24% of the cells were CD34-/CD36+ and in cases with <10 BFU-E/10(4) CD34+ cells 18 +/- 16% of cells were CD34-/CD36+. Iron staining showed the presence of ring sideroblasts in two cases with RARS indicating that the cells originate from the abnormal erythroid clone. Finally, it was shown that cases with an impaired proliferative response demonstrate an enhanced binding of Annexin-V on CD34+ cells during the first days of the cell suspension culture phase. These results suggest that a defect in the proliferative response is most pronouncedly expressed in MDS whereas a subpopulation of cells retain the capacity to differentiate between transition to a terminated stage.