Thrombopoietin-independent generation of platelet-like particles from megakaryoblastic cells.

The use of megakaryoblastic leukemia MEG-01 cells can help reveal the mechanisms of thrombopoiesis. However, conventional in vitro activation of platelet release from MEG-01 cells requires thrombopoietin, which is costly. Here, we aim to develop a more straightforward and affordable method. Synchronization of the MEG-01 cells was initially performed using serum-free culture, followed by spontaneous cell differentiation in the presence of serum. Different stages of megakaryoblast differentiation were classified based on cell morphology, DNA content, and cell cycle. The MEG-01 cells released platelet-like particles at a level comparable to that of the thrombopoietin-activated MEG-01 cells. The platelet-like particles were distinguishable from PLP-derived extracellular vesicles and could express P-selectin following ADP activation. Importantly, the platelet-like particles induced fibrin clotting in vitro using platelet-poor plasma. Therefore, this thrombopoietin-independent cell synchronization method is an effective and straightforward method for studying megakaryopoiesis and thrombopoiesis.

Diagnostic Performance of 10 Mathematical Formulae for Identifying Blood Donors with Thalassemia Trait.

OBJECTIVE: To compare the diagnostic performance of 10 mathematical formulae for identifying thalassemia trait in blood donors. METHODS: Compete blood counts were conducted on peripheral blood specimens using the UniCel DxH 800 hematology analyzer. Receiver operating characteristic curves were used to evaluate the diagnostic performance of each mathematical formula. RESULTS: In the 66 donors with thalassemia and 288 subjects with no thalassemia analyzed, donors with thalassemia trait had lower values for mean corpuscular volume and mean corpuscular hemoglobin than subjects without thalassemia donors (77 fL vs 86 fL [P < .001]; 25 pg vs 28 pg [P < .001]). The formula developed by Shine and Lal in 1977 showed the highest area under the curve value, namely, 0.9. At the cutoff value of <1812, this formula had maximum specificity of 82.35% and sensitivity of 89.58%. CONCLUSIONS: Our data indicate that the Shine and Lal formula has remarkable diagnostic performance in identifying donors with underlying thalassemia trait.

Blood Donors with Thalassemic Trait, Glucose-6-Phosphate Dehydrogenase Deficiency Trait, and Sickle Cell Trait and Their Blood Products: Current Status and Future Perspective.

The use of blood products for different medical purposes has increased in recent years. To meet increasing demand, some blood centers allow volunteer donors with thalassemic trait, glucose-6-phosphate dehydrogenase deficiency (G6PD) trait, and sickle cell trait (SCT) to donate blood if their hemoglobin values fall within acceptable ranges and show no signs of hemolysis. Currently, there are no standard guidelines or policies regarding the use or management of blood products obtained from these donors. However, in recent years, there has been advanced research on eligible donors who have these underlying conditions. In this review, we summarize the current knowledge from in vitro and in vivo studies regarding donor characteristics, changes in physical and biochemical parameters in blood products during processing and storage, and posttransfusion efficacy of blood products. In addition, we discuss some unresolved issues concerning blood products from thalassemic trait, G6PD-deficiency trait, and SCT donors.

Urinary red blood cell-derived microparticles and phosphatidylserine-exposing red blood cells in glomerular and non-glomerular hematuria patients.

BACKGROUND: Distinguishing glomerular hematuria (GH) from non-glomerular hematuria (NGH) is important for treating the cause of hematuria. We aimed to determine red blood cell-derived microparticles (RMPs) and phosphatidylserine (PS)-exposing red blood cells (RBCs) and evaluate their use for diagnosing GH and NGH patients. METHODS: All patients received a physical assessment and urological examination. Dysmorphic RBCs (dRBCs) and acanthocytes were examined using a light microscope. The urinary RMPs and PS-exposing RBCs were determined using flow cytometry. RESULTS: The ratio of RMPs to RBCs was higher in GH patients (n = 29) than in NGH patients (n = 29) (1.06 vs. 0.18). The value of the sum of the PS-exposing RBCs plus RMPs divided by the number of RBCs was higher in GH patients than in NGH patients (48.3% vs. 19.4%). The percentage of RBCs was higher in GH patients than in NGH patients (54.5% vs. 21.8%). Similarly, both the percentages of acanthocytes and of non-acanthocytes were higher in GH patients than in NGH patients (29% vs. 7.7% and 25.4% vs. 14.2%, respectively). The ROC-AUC of the number of PS-exposing RBCs plus RMPs divided by the number of RBCs was 0.9 (95% CI, 0.82-0.97), and the RMPs:RBCs ratio was 0.88 (95% CI, 0.79-0.98). The ROC-AUCs of the dRBCs and acanthocytes were 0.85 (95% CI, 0.78-0.95) and 0.88 (95% CI, 0.8-0.97), respectively. CONCLUSIONS: Patients with GH have higher numbers of urinary RMPs and PS-exposing RBCs. These parameters have the potential to be predictive tools for classifying GH in the future.

Accuracy of lymphocyte counts from UniCel DxH 800 in ß-thalassemia/HbE patients having various numbers of nucleated red blood cells.

BACKGROUND: The UniCel® DxH-800 is an automated cell counter widely used in laboratories. However, the effects of increased nucleated red blood cells (NRBCs) on the lymphocyte counts obtained using the UniCel DxH-800 have not been fully elucidated. OBJECTIVE: The study's objective was to compare lymphocyte counts obtained using the DxH-800 and those obtained using flow cytometry in various ranges of NRBCs. METHODS: This cross-sectional study analyzed 25 healthy volunteers and 69 ß-thalassemia/HbE patients. The numbers of lymphocytes were determined using a UniCel DxH-800 and a standard flow cytometer using counting beads. RESULTS: In healthy volunteers, regression analysis of the lymphocyte counts using the two approaches showed an r2 0.85 and a p < 0.0001, and a Bland-Altman plot showed mean bias of +264 cells/µL. In ß-thalassemia/HbE patients, regression analysis of the lymphocyte counts obtained using an automated cell counter and a flow cytometer showed an r2 of 0.06, a p = 0.028, and a Bland-Altman plot showed the mean bias of +1,509 cells/µL. In addition, a high degree of discrepancy in the lymphocyte counts was observed in ß-thalassemia/HbE patients who had NRBCs > 100,001 cells/µL. CONCLUSIONS: The present study demonstrated that the UniCel DxH-800 performed well in enumerating lymphocytes in specimens that contained various numbers of NRBCs. However, a high number of NRBCs may interfere with lymphocyte counts obtained using the counter.

Storage Duration and Red Blood Cell-Derived Microparticles in Packed Red Blood Cells Obtained from Donors with Thalassemia.

OBJECTIVE: To address the effects of storage duration on red blood cell (RBC)-derived microparticles (RMPs) in packed RBCs from donors who have thalassemia. MATERIALS AND METHODS: Packed RBCs were prepared according to laboratory routine. The quantity of RMPs was determined using FACSCalibur and counting beads. RESULTS: Across durations of storage, the packed RBCs from donors with thalassemia (n = 28) and healthy volunteers (n = 104) showed average RMPs to be 47,426 (10,139‒127,785) particles/µL vs 49,021 (13,033‒126,749) particles/µL, respectively (P = .63). The peak RMP levels in donors with thalassemia and healthy volunteers, respectively, were shown in products from storage days 34 and 38. Both groups showed a trend toward a positive association between RMP concentration and the duration of storage in packed RBC bags stored under blood bank conditions. CONCLUSION: Our results suggest that storage-induced RMP release has similar effects in stored packed RBCs obtained from both donors with thalassemia and healthy volunteers.

Cell-Derived Microparticles in Blood Products from Blood Donors Deficient in Glucose-6-Phosphate Dehydrogenase.

OBJECTIVE: To quantitate the microparticles (MPs) in whole blood and blood products obtained from blood donors who are deficient in glucose-6-phosphate dehydrogenase (G6PD). METHODS: The current study analyzed whole blood and blood components prepared from 49 blood donors with G6PD deficiencies and 98 with G6PD-normal results. Packed red blood cells (PRBCs), platelet concentrate (PC), and plasma were prepared according to transfusion laboratory procedures. MP concentrations were determined using a flow cytometer. RESULTS: Blood components prepared from donors with G6PD deficiency were characterized by higher red blood cell-derived MP (RMP) concentration in PRBCs (25,526 vs 18,738 particles/µL) but lower concentrations of platelet-derived MPs (PMPs; in whole blood and PC), leukocyte-derived MPs (LMP; in whole blood and plasma) and total MP (in PC), compared with those from donors with G6PD-normal test results. CONCLUSIONS: These results suggest that differences in G6PD status may account for variation in RMP levels during processing.

Cell-Derived Microparticles in Blood Products from Thalassemic Blood Donors.

OBJECTIVE: To determine the number of cell-derived microparticles (MPs) in blood products obtained from donors who have thalassemia. METHODS: Packed red blood cells (PRBCs), plasma, and platelet concentrate (PC) were prepared according to routine procedures. We used flow cytometry to quantitate the concentration of MPs. RESULTS: The results of a comparison of MP levels in unprocessed whole blood showed that the concentration of all MPs in the donors without thalassemia trait (n = 255) was higher than in donors with thalassemia trait (n = 70). After processing, increased concentrations of MPs were documented in both groups. Among the blood components, PRBC showed higher platelet-derived MP concentrations in donors with thalassemia than in donors without thalassemia. However, PC showed higher concentrations of total MPs in donors without thalassemia than in donors with that condition. CONCLUSIONS: Our results suggest little influence of thalassemia-trait status on changes in MP concentrations in blood components.

Effects of Cell-Derived Microparticles on Immune Cells and Potential Implications in Clinical Medicine.

In the past few years, interest has increased in cell-derived microparticles (MPs), which are defined by their size of from 0.1 to 1 µm, and can be derived from various cell types, including endothelial cells, leukocytes, red blood cells (RBCs), and platelets. These MPs carry negatively charged phosphatidylserine (PS) on their surfaces and proteins packaged from numerous cellular components. MPs that have been shed by the body can play important roles in the pathophysiology of diseases and can affect various biological systems. Among these systems, the immune components have been shown to be modulated by MPs. Therefore, understanding the roles of MPs in the immune system is crucial to developing alternative therapeutic treatments for diseases. This review describes the effects of MPs on various immune cells and provides plausible potential applications of the immune-modulating properties of MPs in clinical medicine.

Quantitation of phosphatidylserine-exposing platelets and platelet-derived microparticles in platelet products: A new strategy to improve efficacy of platelet transfusion.

Platelet transfusion is an effective therapy to prevent or treat bleeding. Considering the different clinical purposes of transfusion, it is necessary to assess the quality of platelet products prepared in transfusion laboratories. So far, there is no solution to the problem of how best to do this. Here, we summarize the quantitation of phosphatidylserine (PS)-exposing platelets and platelet-derived microparticles (PMPs) in platelet products using previously reported data. Because PS promotes the assembly and enhances the activity of coagulation factors, classifying platelet products according to their concentrations of PS-exposing platelets and PMPs will improve the therapeutic treatment of transfusion recipients.

Novel WT1 Target Genes: IL-2, IL-2RB, and IL-2RG Discovered during WT1 Silencing Using Lentiviral-Based RNAi in Myeloid Leukemia Cells.

Wilms' tumor 1 (WT1) is a transcription factor which plays a major role in cell proliferation, differentiation, survival, and apoptosis. WT1 was first identified as a tumor suppressor gene in Wilms' tumor. However, overexpression of WT1 has been detected in several types of malignancy including some types of leukemia. To investigate the molecular mechanism underlying WT1-mediated leukemogenesis, lentiviral-based siRNA was employed as a tool to suppress WT1 expression in the myeloid leukemia cell line, K562. Successfully, both WT1 RNA and protein levels were downregulated in the leukemia cells. The silencing of WT1 resulted in significant growth inhibition in WT1-siRNA-treated cells for 40 ± 7.0%, 44 ± 9.5%, and 88 ± 9.1% at 48, 72, and 96 hours posttransduction as compared with the control cells, respectively. By using apoptosis detection assays (caspase-3/7 activity and Annexin V-FITC/PI assays), WT1 silencing induced a higher degree of early and late apoptosis in siRNA-treated K562 as compared with the control cells. Interestingly, the expression of survival signaling genes, IL-2, IL-2RB, and IL-2RG, was also suppressed after WT1-siRNA treatment. In addition, the WT1 silencing also inhibited the S phase of the cell cycle and induced cell death. Our results indicated that WT1 silencing by siRNA can suppress cellular proliferation, induce apoptosis, and reduce S phase fraction of K562 cells. Moreover, transcriptional modulation of IL-2, IL-2RB, and IL2-2RG expression by WT1 was likely involved in this phenotypic change. Overall, this study confirmed the oncogenic role of WT1 in myeloid leukemia and discovered the new target genes of WT1 which are likely involved in WT1-mediated leukemogenesis.

Quantitation of Cell-Derived Microparticles in Blood Products and Its Potential Applications in Transfusion Laboratories.

Cell-derived microparticles (MPs) are small fragments released from various cells when they are activated or undergo apoptosis. In the field of transfusion medicine, a number of studies have documented increased levels of MPs in blood products, which have been associated with multiple factors, including donor variability, blood component processing, and storage. In addition, transfusions that contain high levels of MPs are linked to posttransfusion complications. Considering the clinical importance of MP levels, transfusion laboratories should routinely screen blood products for them. However, this practice is not yet applied routinely, perhaps in part because of a lack of understanding of how to apply MP data to transfusion medicine. We describe the methods used to quantitate MPs in blood components and discuss the application of these quantitative data in routine transfusion laboratories in order to manage quality, improve the outcomes of transfusions, and minimize their complications.

Affordable, Reliable Dual-Platform Approach to Quantitating Phosphatidylserine-Exposing Platelets in Platelet Components.

OBJECTIVE: To compare the number of phosphatidylserine (PS)-exposing platelets obtained using the dual-platform approach and bead-based flow cytometry. METHODS: Platelets were enumerated using the ADVIA 2010i instrument (Siemens AG). The numbers and percentages of PS-exposing platelets in 175 platelet products were determined using a FACSCalibur flow cytometer (Becton, Dickinson and Company) and counting beads. RESULTS: Our results showed good correlation (r2 = 0.96; P <.001) between the PS-exposing platelets obtained using counting beads and the dual-platform approach. The results of Bland-Altman analysis showed a bias of +46,449 cells per µL and a limit of agreement (LOA) from -197,863 to 290,762 cells per µL. Also, 8 measurements (5.0%) revealed a number of PS-exposing platelets outside the LOA ranges. Further, 21 measurements (12.0%) revealed greater than 2-fold changes in the number of PS-exposing platelets. CONCLUSIONS: The results suggest that the dual-platform approach is affordable and reliable for quantitating PS-exposing platelets as part of monitoring the quality of platelet products.

Quantitation of phosphatidylserine-exposing platelets in platelet concentrate prepared in routine blood transfusion laboratory.

BACKGROUND: Phosphatidylserine (PS) plays important roles in platelets' pro-coagulant function. However, little is known about assessing this molecule in platelet concentrates (PCs) prepared for routine blood transfusion service. AIM: To quantitate the number of PS-exposing platelets in PCs prepared in a routine transfusion laboratory. METHODS: PC products were prepared according to routine laboratory procedure. The numbers of PS-exposing platelets in the PCs and in unprocessed whole blood were determined using flow cytometry. RESULTS: A cross-sectional study of 253 PCs found that they had significantly increased numbers of PS-exposing platelets compared to unprocessed whole blood (47,439 ±â€¯26,500 cells/µL; 5903‒166,156 cells/µL) vs. 30,058 ±â€¯12,958 cells/µL; 8,154-86,606 cells/µL). A heterogeneity study demonstrated that 6% and 2% of the measured PCs and of unprocessed donor whole blood, respectively, showed an increase in the number of PS-exposing platelets that was greater than 2 fold. CONCLUSIONS: The study suggested that the number PS-exposing platelets in PC prepared in a routine transfusion laboratory differs. However, assessment of the number of PS-exposing platelets in platelet products could be a valid measure to use in managing the quality of platelet processing in routine laboratories.

Replication and cytokine profiles of different subgenotypes of enterovirus 71 isolated from Thai patients in peripheral blood mononuclear cells.

Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are common causative agents of mild and self-limiting symptoms of childhood hand, foot, and mouth disease (HFMD). However, some EV71-infected HFMD patients can develop severe neurological and/or fatal cardiopulmonary complications. In Thailand, HFMD associated with the EV71 subgenotypes C4a and B5 were reported to be associated with diverse outcomes. However, variations in enterovirus subgenotypes and virulence factors have not been fully elucidated; this study elucidated these variations in peripheral blood mononuclear cells (PBMCs) exposed to different subgenotypes of isolated enteroviruses for 24 and 48 h. Following infection, viral titers were determined by plaque assay. Infected cells and intracellular cytokines were quantified using flow cytometry, and multiplex assay was used to examine cytokine release. All isolated subgenotypes showed replication capability in PBMCs; specifically, the replication titer of EV71 C4a tended to be higher than titers of EV71 B5 and CA16. Additionally, the infectivity of EV71 B5 was higher in monocytes than in lymphocytes. Compared with EV71 B5, EV71 C4a and CA16 had greater ability to induce intra- and extracellular cytokine responses. These findings provide new insights into variations in cellular immune responses to different EV71 subgenotypes isolated from Thai patients, which should be considered for the development of vaccines and therapeutic agents.

Thalassemia trait and G6PD deficiency in Thai blood donors.

BACKGROUND: Thalassemia trait and G6PD deficiency are asymptomatic and volunteers with these variants are eligible for blood donation. AIMS: This study aimed to investigate prevalence and hematologic profiles of blood donors with thalassemia trait and G6PD deficiency and the influence of these abnormalities have on donor retention and blood component preparation. METHODS: Prospectively recruited blood donors were investigated for thalassemia and G6PD deficiency. Characteristic data, hematologic profiles, proportions of prepared blood components, donor return rate within 12 months and adverse reactions in patients receiving red cell transfusions were compared among thalassemia trait, G6PD deficiency, and normal donors. RESULTS: In Thai blood donors, thalassemia trait prevalence was 21.1% and G6PD deficiency prevalence based on G6PD activity was 7.7%. Blood donors with thalassemia trait had significantly lower hemoglobin, MCV, and MCH than blood donors without thalassemia trait (Hb 13.55 ± 1.00 vs. 13.96 ± 1.25 g/dL, MCV 76.70 ± 6.69 vs. 87.01 ± 5.10 fL, and MCH 25.06 ± 2.17 vs. 28.67 ± 1.91 pg, all respectively and all p < 0.01). However, the hematologic profiles of blood donors with G6PD deficiency were not significantly different from the hematologic profiles of blood donors with normal G6PD activity. No significant difference was observed among thalassemia trait, G6PD deficiency, and normal donors relative to donor retention and blood component preparation. CONCLUSION: The high prevalence of thalassemia trait and G6PD deficiency in Thai blood donors observed in this study does not adversely affect donor retention and blood component preparation.

Comparison of Phosphatidylserine-Exposing Red Blood Cells, Fragmented Red Blood Cells and Red Blood Cell-Derived Microparticles in ß-Thalassemia/HbE Patients.

OBJECTIVE: To determine the number and intensity of phosphatidylserine (PS) expression of the red blood cells (RBCs), fragmented RBCs, and RBC-derived microparticles (RMPs) in patients with ß-thalassemia/hemoglobin (Hb)E. METHODS: We used flow cytometry to determine the number and levels of PS expression. RESULTS: The number of PS-exposing RBCs was statistically significantly higher (P <.001) than that of PS-exposing fragmented RBCs or RMPs. In contrast, the intensity of PS expression was significantly higher (P <.001) in RMPs than in RBCs or fragmented RBCs. Our study showed a trend of association between RBC distribution width (RDW) and both the number of fragmented RBCs and RMPs and their intensity of PS expression. CONCLUSION: In ß-thalassemia/HbE, PS-exposing RBCs, fragmented RBCs, and RMPs all differed in their numbers and their intensity of PS expression. The effects of these differences among PS-exposing populations on the pathophysiology of the disease require further investigation.

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. AIM: To evaluate the effects of donor age, donor sex, blood-component preparation, and storage on MP concentrations. METHODS: Flow cytometry was used to quantitate the number of whole blood-derived MPs. RESULTS: The total MP concentration was similar in male and female donors (26,044 ± 1254 particles/µL vs. 27,696 ± 1584 particles/µL). The total MP concentration did not differ significantly among the different age groups: 18-30 years (28,730 ± 1600 particles/µL), 31-40 years (24,972 ± 5947 particles/µL), and 41-58 years (25,195 ± 1727 particles/µL). However, the total number of MPs in fresh plasma (152,110 ± 46,716 particles/µL) was significantly higher (p < 0.05) than that in unprocessed whole blood (26,752 ± 985 particles/µL), fresh packed red blood cells (PRBCs) (28,574 ± 1028 particles/µL), and platelet concentrate (PC) (33,072 ± 1858 particles/µL). Furthermore, the total numbers of MPs in stored PRBCs and fresh-frozen plasma (FFP) were significantly higher (p < 0.05) than those in fresh PRBCs and fresh plasma, respectively. CONCLUSIONS: The study suggests that donor factors, blood-component processing and storage contribute to the MP concentration in routine blood-product preparation. The findings can improve quality control and management of blood-product manufacturing in routine transfusion laboratories.

Corrected Lymphocyte Percentages Reduce the Differences in Absolute CD4+ T Lymphocyte Counts between Dual-Platform and Single-Platform Flow Cytometric Approaches.

OBJECTIVE: To determine whether a corrected lymphocyte percentage could reduce bias in the absolute cluster of differentiation (CD)4+ T lymphocyte counts obtained via dual-platform (DP) vs standard single-platform (SP) flow cytometry. METHODS: The correction factor (CF) for the lymphocyte percentages was calculated at 6 laboratories. The absolute CD4+ T lymphocyte counts in 300 blood specimens infected with human immunodeficiency virus (HIV) were determined using the DP and SP methods. RESULTS: Applying the CFs revealed that 4 sites showed a decrease in the mean bias of absolute CD4+ T lymphocyte counts determined via DP vs standard SP (-109 vs -84 cells/µL, -80 vs -58 cells/µL, -52 vs -45 cells/µL, and -32 vs 1 cells/µL). However, 2 participating laboratories revealed an increase in the difference of the mean bias (-42 vs -49 cells/µL and -20 vs -69 cells/µL). CONCLUSIONS: Use of the corrected lymphocyte percentage shows potential for decreasing the difference in CD4 counts between DP and the standard SP method.

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. AIM: The current study aimed to investigate the potential use of flow-rate calibration for counting MPs in whole blood, packed red blood cells (PRBCs), and platelet concentrates (PCs). METHODS: The accuracy of flow-rate calibration was investigated by comparing the platelet counts of an automated counter and a flow-rate calibrator. The concentration of MPs and their origins in whole blood (n=100), PRBCs (n=100), and PCs (n=92) were determined using a FACSCalibur. The MPs' fold-changes were calculated to assess the homogeneity of the blood components. RESULTS: Comparing the platelet counts conducted by automated counting and flow-rate calibration showed an r2 of 0.6 (y=0.69x+97,620). The CVs of the within-run and between-run variations of flow-rate calibration were 8.2% and 12.1%, respectively. The Bland-Altman plot showed a mean bias of -31,142platelets/µl. MP enumeration revealed both the difference in MP levels and their origins in whole blood, PRBCs, and PCs. Screening the blood components demonstrated high heterogeneity of the MP levels in PCs when compared to whole blood and PRBCs. CONCLUSIONS: The results of the present study suggest the accuracy and precision of flow-rate calibration for enumerating MPs. This flow-rate approach is affordable for assessing the homogeneity of MPs in blood components in routine laboratory practice.