Platelet transfusions from D+ donors to D- patients: a 10-year follow-up study of 1014 patients.

BACKGROUND: Current guidelines recommend that platelets (PLTs) from D- donors should be given to D- patients. However, such evidence comes from studies with a limited number of included patients that reported an incidence of anti-D alloimmunization to be up to 19%. We thus decided to extend these findings by examining anti-D alloimmunization at our institution, where PLT transfusions from D+ donors are transfused to D- patients because of logistic constraints. STUDY DESIGN AND METHODS: From April 1999 to December 2009, we retrospectively reviewed the clinical and transfusion records of all D- patients who received PLT transfusions from D+ donors at our hospital. PLT concentrates (PCs) were obtained from apheresis and from whole blood donations. RhIG was not administered after the transfusion of PCs from D+ donors. The antibody screen test to detect anti-D was performed by low-ionic-strength solution indirect antiglobulin test using the gel test. RESULTS: Our series comprises 1014 D- patients who received 5128 PLT transfusions from D+ donors (89% were pooled PCs). We had 315 (31.1%) patients who had a blood sample to analyze the presence of anti-D 4 or more weeks after the first D+ PLT transfusion with a median follow-up of 29 weeks (range, 4-718 weeks). Anti-D developed in 12 (3.8%) of these 315 patients. CONCLUSIONS: The frequency of anti-D alloimmunization of D- patients after receiving pooled PCs from D+ donors is low. The transfusion of D-incompatible pooled PCs without immunoprophylaxis to D- men or D- women without childbearing potential seems a reasonable and safe alternative.

G-CSF increases the expression of VCAM-1 on stromal cells promoting the adhesion of CD34+ hematopoietic cells: studies under flow conditions.

OBJECTIVE AND METHODS: The knowledge of the mechanisms underlying the adhesive processes that lead to homing and/or mobilization of hematopoietic progenitor cells, and the influence of blood rheology, is still limited. We analyzed the impact of flow conditions on the adhesion of CD34+ peripheral blood progenitor cell (PBPC) to the adhesive proteins fibronectin, laminin, and collagen, and to stromal cells. RESULTS: Under static conditions, all the adhesive substrata assayed promoted adhesion of CD34+ PBPC, being higher on the stromal cells. Under flow conditions, adhesion of CD34+ PBPC was remarkable on stromal cells while insignificant onto the purified proteins. Exposure of stromal cell monolayers to granulocyte colony-stimulating factor (G-CSF) further enhanced PBPC adhesion. This effect correlated with the activation of p38 MAPK and with an increase in the expression of VCAM-1 on stromal cells exposed to G-CSF. In inhibitory assays, both an antibody to the G-CSFR and a specific inhibitor of the p38 MAPK blocked the effects induced by the cytokine. CONCLUSION: Our results provide direct evidence that in stromal cells G-CSF activates the signaling protein p38 MAPK, inducing expression of the adhesion receptor VCAM-1. This mechanism seems to promote adhesion of CD34+ cells on stromal cells and could play a potential role in homing events.

Platelet membrane fragments enhance the procoagulant effect of recombinant factor VIIa in studies with circulating human blood under conditions of experimental thrombocytopenia.

The mechanism of action of recombinant factor VIIa (rFVIIa), which is being considered as an alternative treatment for the control of bleeding episodes in patients with thrombocytopenia, has not been fully characterized. This study was undertaken to explore the effects of rFVIIa and platelet microvesicles on hemostasis in an experimental model of thrombocytopenia. Damaged arterial segments were exposed to thrombocytopenic blood (shear rate 600 s(-1)) either with or without the addition of rFVIIa and/or platelet microvesicles. The presence of fibrin and platelets on the subendothelium were morphometrically quantified and immunolocalization techniques and electron microscopy were used for a more detailed analysis. Both rFVIIa and platelet microvesicles consistently improved fibrin formation on the damaged vascular subendothelium, and microvesicles were shown to be localized at different levels of the fibrin lattice. Further, under conditions of moderate thrombocytopenia, addition of platelet microvesicles potentiated the procoagulant action of rFVIIa. This effect may be due to the phospholipid surface provided by the platelet microvesicles. These studies support the concept that, under conditions of thrombocytopenia, both rFVIIa and platelet microvesicles enhance fibrin formation at sites of vascular damage.

Erythropoietin triggers a signaling pathway in endothelial cells and increases the thrombogenicity of their extracellular matrices in vitro.

We demonstrate that exposure of cultured human endothelial cells to rHuEPO resulted in a dose-dependent increase in the tyrosine kinase activity, with phosphorylation of JAK-2 followed by rapid phosphorylation of STAT-5. Simultaneously, rHuEPO induced long-lasting phosphorylation of MAPK p42/44. Activation of this signaling pathways was directly associated with an increase in the thrombogenic properties of the extracellular matrix generated by these cells, when they were exposed to flowing blood. The enhancement in the reactivity of the resulting extracellular matrix towards platelets was associated with a higher expression of tissue factor. All these effects were blocked by an antibody to the EPO receptor and by specific inhibitors of tyrosine phosphorylation. The observed action of rHuEPO on endothelial cells seemed to be specifically triggered by the subsequent events that follow receptor binding, and occurred even at pharmacological concentrations of the cytokine. Our results indicate that rHuEPO has a direct action on the endothelium, increasing the reactivity of the underlying extracellular matrix towards platelets, effect that may be attributed to an increase in the expression of TF.

Granulocyte colony-stimulating factor increases expression of adhesion receptors on endothelial cells through activation of p38 MAPK.

BACKGROUND AND OBJECTIVES: Granulocyte colony-stimulating factor (G-CSF) is specific for the granulocytic cell line, although receptors for this cytokine have been found in other cell types including endothelial cells. These observations prompted us to investigate the potential effect of G-CSF on the endothelium. DESIGN AN METHODS: Endothelial cell monolayers were exposed to G-CSF to evaluate: i) signal transduction mechanisms, ii) expression of adhesion receptors at the cell surface, and iii) leukocyte adhesion on EC monolayers. RESULTS: Exposure of human umbilical vein endothelial cells (EC) in culture to G-CSF resulted in the activation of the signal transduction pathways JAK/STAT (JAK-1, STAT-1 and STAT-3) and RAS/MAPK (MAPK p42/44 and p38 MAPK). We also observed significantly increased expression of the adhesion receptors, E-selectin (ELAM-1), vascular endothelial cell adhesion molecule-1 and intracelleular adhesion molecule-1 at the cell surface in response to G-CSF, increases that were followed by an augmented adhesion of leukocytes on the previously exposed EC monolayers. These effects were blocked by the presence of SB203580, a p38 MAPK inhibitor, by U0126, a MAPK p42/44 inhibitor, and by inhibiting the G-CSF receptor with a specific antibody. INTERPRETATION AND CONCLUSIONS: Our results demonstrate that G-CSF increases the expression of adhesion receptors on EC, promoting leukocyte adhesion. This effect seems to be triggered by the signaling events that follow receptor binding. Results from experiments using specific inhibitors suggest that activation of p38 MAPK is required to promote expression of adhesion receptors in endothelial cells and the recruitment of leukocytes in response to G-CSF.

Leukoreduced buffy coat-derived platelet concentrates photochemically treated with amotosalen HCl and ultraviolet A light stored up to 7 days: assessment of hemostatic function under flow conditions.

BACKGROUND: Amotosalen plus ultraviolet A light photochemical treatment (PCT) inactivates high titers of bacteria, and other pathogens, in platelet concentrates (PCs) potentially allowing the storage of platelets (PLTs) for up to 7 days. Adhesion and aggregation of PLTs to injured vascular surfaces are critical aspects of PLT hemostatic function. STUDY DESIGN AND METHODS: Two ABO-identical leukoreduced buffy coat-derived PCs in additive solution were mixed and divided: one-half underwent PCT (PCT-PCs) and the other was kept as a control (C-PCs); both were stored under standard conditions. The total number of paired PCs studied was nine. Samples were taken on Day 1 (before PCT) and after 5 and 7 days of storage. The adhesion and aggregation capacities were evaluated under flow conditions in a ex vivo perfusion model. RESULTS: Compared to control, PCT resulted in a decrease in PLT count of 6.5 percent (p = 0.004) and 10.2 percent (p = 0.008) after 5 and 7 days' storage, respectively (n = 9). PLT interaction with subendothelium was mainly in form of adhesion. The surface covered by PCT PLTs on Day 1 was 26.0 +/- 4.2 percent (mean +/- SEM). On Day 5, PCT-PCs showed a covered surface of 20.9 +/- 2.2 percent, and the C-PCs, 20.6 +/- 1.6 percent. After 7 days, PCT-PCs produced a nonsignificant higher PLT deposition compared to control (27.1 +/- 2.9% vs. 21.2 +/- 2.8%, p = 0.06). CONCLUSION: PCT of PCs and storage up to 7 days was associated with a 10.2 percent decrease in PLT count due to processing losses compared to C-PC. PLT adhesive and aggregating capacities under flow conditions of PCT-PCs were similar to C-PCs and remained well preserved for up to 7 days of storage.

Anti-D alloimmunization after D-mismatched allogeneic hematopoietic stem cell transplantation in patients with hematologic diseases.

BACKGROUND: The de novo development of anti-D after D-mismatched allogeneic hematopoietic stem cell transplantation (AHSCT) is a possibility that must be considered. The transfusion of D- blood components after AHSCT has been recommended but anti-D alloimmunization in this setting has been studied little. Thus, the aim of this study was to analyze anti-D formation after D-mismatched AHSCT. STUDY DESIGN AND METHODS: Thirty patients with a hematologic disease who underwent D-mismatched AHSCT were retrospectively studied. Support therapy included red blood cells (RBCs) and platelet (PLT) concentrates (PCs) from whole-blood donations and PLTs from apheresis. After AHSCT, patients received D+ PCs without administering Rh immunoglobulin (RhIG). An antibody screening to detect anti-D was performed by low-ionic-strength saline-indirect antiglobulin test with the tube test. RESULTS: Fifteen D+ patients received stem cells (SCs) of D- donors and 15 D- patients received SCs of D+ donors. After AHSCT, patients received a median of 11.5 (range, 0-32) D- RBC units. D+ patients received 682 (83%) of 825 PLT units from D+ donors, and D- patients received 573 (85%) of 678 PLT units from D+ donors. None of the 30 patients developed anti-D after a median follow-up of 32 weeks (range, 4-310 weeks). CONCLUSION: Anti-D alloimmunization after performing a D-mismatched AHSCT is infrequent in patients with hematologic diseases although patients receive D-mismatched PLT transfusions without RhIG administration.

Cofactors associated with liver disease mortality in an HBsAg-positive Mediterranean cohort: 20 years of follow-up.

The risk of developing liver cancer in hepatitis B virus (HBV) carriers differs across geographical areas, suggesting that exposure to other risk factors may contribute to HBV-linked cancer risk. Our study estimates the mortality due to liver disease and the role of other risk factors in a Spanish HBV cohort. 2,352 hepatitis B surface antigen (HBsAg)-positive and 15,504 HBsAg-negative subjects were identified among blood donors during 1972-1985 and were followed until December 2000 through the Mortality Registry. Clinical examination and an epidemiological questionnaire were performed on 1,000 HBsAg-positive survivors during 1994-1996. In subjects deceased from liver disease, medical records were revised and relatives were interviewed. A nested case-control analysis was conducted comparing both groups. In HBsAg-positive men, an excess mortality from liver cancer [standardized mortality ratio (SMR): 14.1; 7.7-23.6], cirrhosis (SMR: 10.5; 7.0-15.1), haematological neoplasms (SMR: 3.2; 1.2-6.9) and AIDS was detected (SMR: 5.5; 2.2-11.4). In women, an excess was found for cirrhosis (SMR: 7.2; 1.4-21.1). Progression factors to liver disease were alcohol intake [odds ratio (OR): 6.3; 3.1-12.8], diabetes (OR: 3.6; 1.3-9.6), HBV replication (OR: 50.0; 14.9-167.3) and hepatitis C virus (HCV) infection (OR: 27.4; 7.1-107.7). In conclusion, in Spain after 20 years of follow-up, chronic HBV exposure appears as a major risk factor for liver cancer among men and for cirrhosis in both sexes. The risk of death from liver disease among HBV carriers with the presence of HBV replication, HCV, alcohol consumption and diabetes was significantly increased and suggests synergism among these exposures and HBV. Mortality from haematological neoplasms was detected and could be associated to HIV coinfection. These results support screening and adequate follow-up among HBsAg-positive subjects at high risk to develop liver disease, particularly when these risk cofactors are present.

Effect of holding buffy coats 4 or 18 hours before preparing pooled filtered PLT concentrates in plasma.

BACKGROUND: Filtered PLT concentrates (PCs) were prepared in plasma pooling three (for children) or six buffy coats (BCs; for adults) after holding them a maximum of 4 hours (blood bags collected in the afternoon) or 18 hours (blood bags collected in the morning). STUDY DESIGN AND METHODS: With flow cytometry, PCs prepared after holding BCs 4 or 18 hours were compared. BCs removed from whole-blood donations in quadruple bag packs ("top-top") were held 4 or 18 hours before pooling them with a sterile connecting device. After the BCs were centrifuged, the supernatant was transferred through a BC filter (Autostop, Pall Medical) to a CLX bag. Samples for analysis were collected from the whole-blood bag, BCs, and PCs immediately after preparation and after 1, 3, 5, and 7 days of storage on a flat-bed agitator at 22 +/- 2 degrees C. The main PLT membrane glycoproteins (GPs, IIb-IIIa, IV, and Ibalpha), some of their ligands (fibrinogen, fibronectin, and VWF), activation-dependent antigens (CD62P and CD63), and procoagulant activity markers (annexin V and bound coagulation FV-Va) have been studied. RESULTS: In the 12 PCs (six pools of 3 units each group) studied, a minor increase in activation markers during preparation was observed. During the storage, a significant increase in the expression of GPIIb-IIIa, CD62P, CD63, annexin V, and FVa was measured. After 5 days of storage, only the percentage of PLTs with bound fibrinogen was significantly greater in PCs prepared after holding BCs for 4 hours. CONCLUSION: In PCs prepared after holding BCs 4 or 18 hours before pooling and filtering, only a minor significant difference in the percentage of PLTs with bound fibrinogen was found after 5 days of storage. This difference is probably of little, if any, transfusional significance.

Possible hemostatic effect of synthetic liposomes in experimental studies under flow conditions.

BACKGROUND AND OBJECTIVES: The possibility of developing synthetic platelet substitutes is a subject of current interest. We explored the possible hemostatic effect of synthetic phospholipid incorporated in multilamellar vesicles (MLVs) or intermediate unilamellar vesicles (IUVs) using a well-characterized experimental system with circulating human thrombocytopenic blood (10 min, 250 s(-1)). DESIGN AND METHODS: The ability of the liposomes containing different combinations of dipalmitoylphosphatidylcholine (DPPC), phosphatidylethanolamine (PE) and dipalmitoylphosphatidylserine (DPPS) to promote fibrin formation (%F) on the damaged subendothelium was morphometrically evaluated. Generation of thrombin in the system was monitored through prothrombin fragment F1+2 determination. RESULTS: IUV liposomes containing DPPC, 1DPPS:9DPPC, 1DPPS:3DPPC, 1PE:1DPPC increased fibrin deposition on the subendothelium (53.87 +/- 11.0%; 39.76 +/- 6.75%; 40.69 +/- 10.54% and 32.22 +/- 7.35%, respectively vs. thrombocytopenic blood 11.5 +/- 1.2%; p<0.05), while 9PE:1DPPS IUV liposome failed to promote a procoagulant effect. MLV liposomes containing DPPC alone, 1DPPS:3DPPC and 1PE:1DPPC showed a positive effect on fibrin deposition (85.50 +/- 5.95%, 59.86 +/- 11.55% and 43.73 +/- 7.84% respectively; p<0.05). However, no effect was observed in those experiments performed with liposomes containing 3DPPS:1DPPC. After perfusion experiments, the coagulation system became activated, but differences were not statistically significant vs. control experiments, except for MLV liposomes containing DPPC alone (p<0.05). INTERPRETATION AND CONCLUSIONS: These results confirm that, at an experimental level, liposomes containing phospholipids could potentially be used to improve hemostasis in patients with quantitative or qualitative platelet disorders.

Tiempo de tromboplastina parcial activado, que no activada / Activated partial thromboplastin time

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¿Plasmaféresis? Probablemente quieredecir un recambio plasmático / Plasmapheresis? It probably means plasma exchange

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Soporte hepático bioartificial en la insuficiencia hepática aguda grave. Primer caso tratado en España / Bioartificial liver support for acute liver failure. First case treated in Spain

FUNDAMENTO: En el último decenio se ha asistido a un aumento creciente en la investigación dirigida a desarrollar sistemas de soporte hepático artificial. Por primera vez se han diseñado sistemas híbridos que incorporan biorreactores que contienen hepatocitos a través de los que circula la sangre o plasma de los enfermos con insuficiencia hepatocelular. Se pretende que estos hepatocitos puedan sustituir, al menos en parte, la función del hígado enfermo y de esta manera mejorar el pronóstico de los pacientes con hepatopatías graves agudas o crónicas. OBSERVACIÓN CLÍNICA: En el presente artículo describimos el primer caso tratado en España en el contexto de un estudio aleatorizado, controlado, multicéntrico e internacional dirigido a investigar la eficacia y seguridad de un sistema de soporte hepático bioartificial basado en hepatocitos porcinos criopreservados en pacientes con insuficiencia hepática aguda grave o disfunción primaria del injerto. RESULTADOS: En esta primera experiencia se completaron dos sesiones de tratamiento antes de que la paciente, afectada de un cuadro de insuficiencia hepática aguda grave, pudiera ser sometida a un trasplante hepático urgente, que se completó con éxito. Tras más de dos años de seguimiento la paciente sigue una vida normal y no ha presentado ningún efecto adverso relacionado con el procedimiento de soporte hepático bioartificial al que se sometió. CONCLUSIÓN: Se empieza a disponer de sistemas de soporte hepático bioartificial aplicables en la práctica clínica, si bien deberá esperarse a que se demuestren su seguridad y eficacia antes de aconsejar su uso generalizado (AU)