Differences in levels of platelet-derived microparticles in platelet components prepared using the platelet rich plasma,buffy coat,and apheresis procedures

Background

There has been an increased interest in platelet-derived microparticles (PMPs) in transfusion medicine. Little is known about PMP status during the preparation of platelet concentrates for transfusion.

Microparticles from splenectomized β-thalassemia/HbE patients play roles on procoagulant activities with thrombotic potential

Thromboembolic events including cerebral thrombosis, deep vein thrombosis, and pulmonary embolism are major complications in β-thalassemia. Damaged red blood cells and chronic platelet activation in splenectomized β-thalassemia/HbE patients were associated with increased microparticles (MPs) releases into blood circulation. MPs are small membrane vesicles, which play important roles on coagulation. However, the role of MP in thalassemia is poorly understood. In this study, the effects of splenectomized-MPs on platelet activation and aggregation were investigated. The results showed that isolated MPs from fresh platelet-free plasma of patients and normal subjects directly induce platelet activation, platelet aggregation, and platelet-neutrophil aggregation in a dose-dependent manner. Interestingly, MPs obtained from splenectomized patients are more efficient in induction of platelet activation (P-selectin⁺) when compared to MPs from normal subjects (P < 0.05), tenfold lower than pathophysiological level, at 1:0.1 platelet MP ratio. Co-incubation of splenectomized-MPs with either normal-, non-splenectomized- or splenectomized-platelets at 1:10 platelet MP ratio increased platelet activation up to 5.1 ± 2.2, 5.6 ± 3.7, and 9.5 ± 3.0%, respectively, when normalized with individual baseline. These findings suggest that splenectomized patients were proned to be activated by MPs, and splenectomized-MPs could play an important role on chronic platelet activation and aggregation, leading to thrombus formation in β-thalassemia/HbE patients.     

Effects of donor age,donor sex,blood-component processing,and storage on cell-derived microparticle concentrations in routine blood-component preparation

Background

A number of factors cause increases in the number of cell-derived microparticles (MPs) in blood components. However, the overall effects of these factors on the concentration of MPs during routine blood-component preparation have not fully been elucidated.

Low cost CD4 enumeration using generic monoclonal antibody reagents and a two-color user-defined MultiSET protocol

BACKGROUND: The standard three-tube, three-color flow cytometric method utilizing the TriTEST reagents in conjunction with the MultiSET software commonly used in most laboratories in Thailand for CD4 enumeration is expensive and thus unavailable to most HIV-infected patients. A more affordable method, i.e., the PanLeucogating protocol using only two monoclonal antibody reagents, has been described but requires the use of the CellQUEST software that does not have automatic gating and reporting facilities. We describe a simple protocol that utilizes a two-color user-defined protocol with the automated MultiSET software for the acquisition, analysis, and reporting of CD4 results. METHODS: A two-color user-defined protocol was set up following instructions in the Becton Dickinson Biosciences MultiSET manual, adhering strictly to the information regarding the Gate and Attractor Hierarchy for analyzing various reagent combinations. This simple two-color user-defined MultiSET software was evaluated using generic monoclonal reagents in comparison with the standard TriTEST/MultiSET protocol. RESULTS: The two-color user-defined MultiSET software is easy to use. It requires only modification of the original MultiSET program and the results obtained are comparable with those derived from the standard TriTEST/MultiSET protocol. CONCLUSION: The use of this easy and reliable two-color user-defined MultiSET protocol represents an affordable alternative to CD4 testing in resource-poor settings.     

Low-cost CD4 enumeration in HIV-infected patients in Thailand

In Thailand, over one million people have been infected with HIV since the beginning of the epidemic. This has created a great burden on the country's limited health care budget. Monitoring CD4+ T-lymphocytes is important to determine the success of any antiretroviral therapy as well as HIV vaccine trials. However, the high cost of CD4 counts makes monitoring of every HIV-infected patient impossible in Thailand. Therefore, the development of affordable strategies is necessary in order to allow more HIV infected persons to access CD4 testing to control the disease. The current standard methods for enumeration of CD4+ T-lymphocytes are performed on whole blood by flow cytometric immunophenotyping using the 6-tube 2-color and 3-tube 3-color panels recommended by the Centers for Diseases Control (CDC). In this study, percentage CD4+ T-lymphocyte values (from 142 HIV-seropositive patients and 26 anti-HIV negative adult blood donors) generated by the use of just 2 reagents (CD45/CD4) in a 1-tube 2-color panel employing side scatter/CD45 morphospectral gating were compared to those obtained by state of the art methods. We also compared the use of generic monoclonal antibody reagents with commercial reagents and found the results to be comparable with an overall correlation coefficient (r) of more than 0.95 for both CD4+ and CD8+ T-lymphocytes. Bland-Altman analysis of the mean CD4 values plotted against the difference in values between the generic reagents and the commercial reagents showed no bias. The 1-tube 2-color method using generic monoclonal antibody reagents potentially permits more affordable but reliable CD4 testing and therefore could increase access for more HIV-infected patients in resource-poor countries.     

Flow rate calibration to determine cell-derived microparticles and homogeneity of blood components

Background

Cell-derived microparticles (MPs) are currently of great interest to screening transfusion donors and blood components. However, the current approach to counting MPs is not affordable for routine laboratory use due to its high cost.

Evaluation of a New Single-Parameter Volumetric Flow Cytometer (CyFlowgreen) for Enumeration of Absolute CD4+ T Lymphocytes in Human Immunodeficiency Virus Type 1-Infected Thai Patients

Use of the standard dual-platform flow cytometric method for determination of CD4⁺ T-lymphocyte counts, which needs both a flow cytometer (FCM) and hematological analyzer, would inevitably lead to increased variability. The development of new single-platform (SP) FCMs that provide direct CD4⁺ T-lymphocyte counts for improved assay precision and accuracy have recently attracted attention. This study evaluated one of those systems, CyFlow^green (Partec), a single-parameter SP volumetric FCM. The performance of CyFlow^green was compared with those of two reference standard SP microbead-based technologies of the three-color TruCOUNT tube with the FACScan FCM and a two-color FACSCount system (Becton Dickinson Biosciences). Absolute CD4⁺ and CD8⁺ T-lymphocyte counts in 200 human immunodeficiency virus type 1-seropositive blood specimens were determined. Statistical analysis for correlation and agreement were performed. A high correlation of absolute CD4 counts was shown when those obtained with CyFlow^green were compared with those obtained with the bead-based three-color TruCOUNT system (R² = 0.96; mean bias, −69.1 cells/μl; 95% confidence interval [CI], −225.7 to +87.5 cells/μl) and the FACSCount system (R² = 0.97; mean bias, −40.0 cells/μl; 95% CI, −165.1 to +85.1 cells/μl). The correlation of the CD4⁺ T-lymphocyte counts obtained by the two bead-based systems was high (R² = 0.98). Interestingly, CyFlow^green yielded CD4⁺ T-lymphocyte counts that were 21.8 and 7.2 cells/μl lower than those obtained with the TruCOUNT and the FACSCount systems, respectively, when CD4⁺ T-lymphocyte counts were <250 CD4⁺ T-lymphocyte counts/μl range or 17.3 and 5.8 cells/μl less, respectively, when CD4⁺ T-lymphocyte counts were <200 cells/μl. The single-parameter CyFlow^greenvolumetric technology performed well in comparison with the performance of the standard SP bead-based FCM system. However, a multicenter comparative study is needed before this FCM machine is implemented in resource-limited settings.     

Activated platelet-derived microparticles in thalassaemia

Thromboembolic complications have been documented in thalassaemia patients. The aggregability of abnormal red blood cells and the high level of membrane-derived microparticles (MPs) stemming from blood cells are thought to be responsible for the associated thrombotic risk. We investigated the number of MPs, their cellular origin and their procoagulant properties in beta-thalassaemia. Fresh whole blood was simultaneously stained for annexin V, cellular antigens and the known density beads. The procoagulant properties of these phosphatidylserine (PS)-bearing MPs were also measured by assessing the platelet factor-3-like activity in the blood. Flow cytometric results showed that splenectomised beta-thalassaemia/HbE patients had significantly higher levels of PS-bearing MPs than non-splenectomised beta-thalassaemia/HbE patients and normal individuals (P < 0.0001). There was a good correlation between PS-bearing MPs and PS-bearing platelets, reflecting the existence of chronic platelet activation in beta-thalassaemia/HbE patients (r(s) = 0.511, P < 0.001). The cellular origin of PS-bearing MPs showed mostly activated-platelet origin with adhesion (CD41a/CD62P/CD36). Moreover, the platelet procoagulant activity was higher in splenectomised beta-thalassaemia/HbE patients when compared with non-splenectomised (P < 0.05) and normal individuals (P < 0.01), and the amount correlated with PS-bearing MPs (rs = 0.560, P < 0.001). These findings suggest that MPs originate from activated platelets with a potential to aggravate thrombotic events when the numbers are excessive, as is commonly seen in splenectomised beta-thalassaemia/HbE patients.