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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.

ObjectiveTo compare the diagnostic performance of 10 mathematical formulae for identifying thalassemia trait in blood donors.MethodsCompete 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.ResultsIn 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 ConclusionsOur data indicate that the Shine and Lal formula has remarkable diagnostic performance in identifying donors with underlying thalassemia trait.

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.

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.

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.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.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.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.

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.

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 are asymptomatic and volunteers with these variants are eligible for blood donation.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.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.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.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.

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.

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.

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.

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.

In recent years, the use of point-of-care (POC) CD4 testing technologies has increased dramatically in Thailand and other Southeast Asian (SEA) countries. However, no data regarding external quality assessment of these POC CD4 testing technologies have been reported. The current study aimed to evaluate the performance of POC CD4 testing technologies, including BD FACSPresto, CyFlow, Guava, Muse, and PIMA, used in the participating laboratories in Thailand, Myanmar, Laos, Indonesia, and Nepal from 2014 to 2017. The results showed a trend toward an increasing number of participating laboratories using POC CD4 testing technologies between trial numbers 73 and 90. In the laboratories participating in the study, CyFlow technologies were the most used POC CD4 testing technologies. In addition, a trend of decreasing coefficients of variation (%CVs) was demonstrated across the trials, and the final trial showed an overall %CV of 14 %. In trial numbers 73 and 77, 57 % and 68 % of measurements showed standard deviation index (SDI) values ranging between − 1 and + 1. In trial numbers 80 and 90, the proportion of measurements with SDI values ranging between − 1 and + 1 had increased to 100 % and 72 %. These results suggest an improvement in POC CD4 testing in Thailand and other SEA countries.

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.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.This cross-sectional study analyzed 25 healthy volunteers and 69 β-thalassemia/HbE patients. The numbers of lymphocytes were determined using a UniCel DxH-800 and a standard flow cytometer using counting beads.In healthy volunteers, regression analysis of the lymphocyte counts using the two approaches showed an r2 0.85 and a p0.0001, and a Bland-Altman plot showed mean bias of +264 cells/μL. In β-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 β-thalassemia/HbE patients who had NRBCs100,001 cells/μL.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.

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 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.

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 ± 26,500 cells/μL; 5903‒166,156 cells/μL) vs. 30,058 ± 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.

The accuracy and reliability of CD4+ T-lymphocyte enumeration are crucial for HIV healthcare management. Thailand’s CD4 external quality assessment (EQA) program was implemented in 2003. This program aims to improve the quality of CD4 testing by providing the quality control material, remedial action, and technical training. In this article, the overall longitudinal performance of the participating laboratories from 2003 to 2015 is reported. The EQA blood samples were sent to the participating laboratories in Thailand and other Southeast Asian (SEA) countries. The participants were requested to enumerate CD3+ and CD4+ T-lymphocytes. The trimmed mean, standard deviation (SD), coefficient of variation (CV), and standard deviation index (SDI) of both fraction and number were calculated from the returned data. Our result indicated a continuously increasing number of participants. The response rate of 72 trials was 98 %. The outlier rates for the fraction and number of CD4+ T-lymphocytes were 3.3 % and 3.4 %, respectively. The average CV of the fraction and number of CD4+ T-lymphocytes was 7.1 % and 9.4 %, respectively. In addition, the analysis of our current EQA trial no. 072 revealed that more than 98 % of participating laboratories had SDI values between −2 and +2 for both fraction and number of CD3+ and CD4+ T-lymphocytes. In conclusion, the implementation of the CD4 EQA program in Thailand and other SEA countries has led to a reduction in the variability of the results of the CD4 count between laboratories and a continuous improvement in laboratory performance.

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 β-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

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.

CD4 testing is important for the clinical monitoring of patients with HIV/AIDS. Quality assurance is therefore an essential element to ensure that the test results are reliable, reproducible, traceable, and auditable. An external quality assessment scheme (EQAS) provides an assessment of the entire testing process, including pre- and post-analytical procedures. At present, there is an increased demand for EQAS owing to an increasing number of CD4 testing laboratories in many countries. However, participating in the existing international EQAS is extremely expensive, making its implementation in resource-limited settings difficult. An ideal alternative is to have an equally qualified local or regional CD4 EQAS, which is more practical and will have a more obvious impact on these countries. The present communication aims to establish the requirements and guidelines for implementing EQAS for CD4+ T-lymphocyte determination in individuals with HIV-1 infection in resource-limited regions, particularly in sub-Saharan African countries and in some countries in Southeast Asia. Guidelines including the recruitment of CD4 testing laboratories, pre-participation EQA workshops, and management of EQA blood samples along with the evaluation of the performance data are described. An example of participants’ registration form, details of EQAS blood panels, participants’ report form, and EQAS performance report form are also included in the guidelines. The guidelines presented herein will lay a foundation to help improve the reliability of CD4+ T-lymphocyte determination in resource-limited settings.

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

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 r 2 of 0.6 ( y = 0.69 x + 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 −31,142 platelets/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.

External quality assessment (EQA) of CD4 testing is important for clinically monitoring of patients with HIV/AIDS. EQA materials are limited to blood samples from normal blood donors with normal CD4+ T-lymphocyte levels. This study aimed to develop low-CD4+ T-lymphocyte blood samples for CD4 EQA. CD4+ T-lymphocyte-depleted blood samples were prepared using a magnetic bead separation technique. These CD4+ T-lymphocyte-depleted blood samples were mixed with undepleted whole blood samples to obtain low-CD4+ T-lymphocyte blood samples. The percentage and the number of CD4+ T-lymphocytes were determined using a flow cytometer. An evaluation study of this low-CD4+ T-lymphocyte blood sample was performed by sending to participating laboratories to investigate the potential use as CD4 EQA material. Our results showed that a magnetic separation technique could be used to prepare low-CD4+ T-lymphocyte blood samples. CD4+ T-lymphocytes in the low-CD4+ T-lymphocyte blood samples ranged from 15 % to 18 % and 160–300 cell/μl, respectively. In addition, CD4 testing of our low-CD4+ T-lymphocyte blood samples could be achieved following both the single- and the dual-platform flow cytometric approaches. A pilot EQA investigation revealed that the CV values of the number and percentage of CD4+ T-lymphocytes were 14 % and 10 %, respectively. Having the low-CD4+ T-lymphocyte blood samples in our CD4 EQA program should ensure reliability and credibility of CD4 testing in Thailand and other resource-poor countries.

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

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. Aim The aim of this study is to compare the PMP levels in platelet components prepared using the buffy coat (BC), platelet-rich plasma platelet concentrate (PRP-PC), and apheresis (AP) processes. Methods Platelet components were prepared using the PRP-PC and BC processes. Apheresis platelets were prepared using the Trima Accel and Amicus instruments. The samples were incubated with annexin A5-FITC, CD41-PE, and CD62P-APC. At day 1 after processing, the PMPs and activated platelets were determined using flow cytometry. Results Both the percentage and number of PMPs were higher in platelet components prepared using the Amicus instrument (2.6 ± 1.8, 32802 ± 19036 particles/μL) than in platelet components prepared using the Trima Accel instrument (0.5 ± 0.4, 7568 ± 5298 particles/μL), BC (1.2 ± 0.6, 12,920 ± 6426 particles/μL), and PRP-PC (0.9 ± 0.6, 10731 ± 5514 particles/μL). Both the percentage and number of activated platelets were higher in platelet components prepared using the Amicus instrument (33.2 ± 13.9, 427553 ± 196965 cells/μL) than in platelet components prepared using the Trima Accel instrument (16.2 ± 6.1, 211209 ± 87706 cells/μL), BC (12.9 ± 3.2, 140624 ± 41003 cells/μL), and PRP-PC (21.1 ± 6.3, 265210 ± 86257 cells/μL). Conclusions The study suggests high variability of PMPs and activated platelets in platelet components prepared using different processes. This result may be important in validating the instruments involved in platelet blood collection and processing.

SummaryIntroduction Heinz bodies are important to diagnosing and managing patients. However, microscopic examination of Heinz bodies has several disadvantages, demonstrating the need for a better method. We explored the potential use of flow cytometry to examine Heinz bodies. Methods Whole-blood samples were collected from patients deficient in G6PD and healthy volunteers. Acetylphenylhydrazine was used to induce formation of Heinz bodies in red blood cells (RBCs). Then, RBCs positive for Heinz bodies were examined using a FACSCanto II cytometer. Results RBCs treated with acetylphenylhydrazine formed Heinz bodies and emitted a broad spectrum of fluorescence that could be detected by flow cytometry. The maximum emission of fluorescence was observed at 45 min after the incubation with acetylphenylhydrazine. In addition, the fluorescence emitted was stable for at least 72 h. The flow cytometer could detect the RBCs positive for Heinz bodies even if they made up as little as 0.1% of the total RBC population. Furthermore, the percentage and number, respectively, of RBCs positive for Heinz bodies in G6PD-deficient patients and normal donors exhibited a mean ± standard deviation (SD) of 68.9 ± 27.5 vs. 50.9 ± 28.6 and 96 014 ±35 732 cells/μL vs. 74 688 ± 36 514 cells/μL. Conclusion Heinz bodies induced by acetylphenylhydrazine emit fluorescence, and this fluorescence could be examined using flow cytometry. Our study suggests the potential use of the developed method to investigate the formation of Heinz bodies in clinical samples.

Iron deficiency affects 500 million people, yet the molecular role of iron in gene expression remains poorly characterized. In addition, the alterations in global gene expression after iron chelation remain unclear and are important to assess for understanding the molecular pathology of iron deficiency and the biological effects of chelators. Considering this, we assessed the effect on whole genome gene expression of two iron chelators (desferrioxamine and 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone) that have markedly different permeability properties. Sixteen genes were significantly regulated by both ligands, whereas a further 50 genes were significantly regulated by either compound. Apart from iron-mediated regulation of expression via hypoxia inducible factor-1 alpha, it was noteworthy that the transcription factor p53 was also involved in iron-regulated gene expression. Examining 16 genes regulated by both chelators in normal and neoplastic cells, five genes (APP, GDF15, CITED2, EGR1, and PNRC1) were significantly differentially expressed between the cell types. In view of their functions in tumor suppression, proliferation, and apoptosis, these findings are important for understanding the selective antiproliferative effects of chelators against neoplastic cells. Most of the genes identified have not been described previously to be iron-regulated and are important for understanding the molecular and cellular effects of iron depletion.

Iron chelators have been reported to induce apoptosis and cell cycle arrest in cancer cells. Recent studies suggest broad and selective antitumor activity of the new iron chelator, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT; Whitnall et al., Proc Natl Acad Sci USA 2006;103:14901-14906). However, little is known concerning its effects on hematological malignancies. Using acute leukemia cells, the effect of Dp44mT on apoptosis, cell cycle, caspase-3 activation, and mitochondrial trans-membrane potential has been examined by flow cytometry. Dp44mT acted to induce a G(1)/S arrest in NB4 promyelocytic leukemia cells at low concentrations (0.5-2.5 microM), being far more effective than the clinically used chelator, desferrioxamine (DFO). Moreover, Dp44mT induced apoptosis of NB4 cells in a dose- and time-dependent manner with markedly less effect on nonproliferating cells. The apoptosis-inducing activity of Dp44mT was significantly more effective than DFO. Furthermore, this study also showed that Dp44mT had broad activity, inducing apoptosis in several types of acute leukemia and also multiple myeloma cell lines. Additional studies examining the cytotoxic mechanisms of Dp44mT showed that a reduction in the mitochondrial trans-membrane potential and caspase-3 activation could be involved in the mechanism of apoptosis. Our results suggest that Dp44mT possesses potential as an effective cytotoxic agent for the chemotherapeutic treatment of acute leukemia.

The advent of potent antiretroviral therapy in 1996 led to a revolution in the care of patients with human immunodeficiency virus (HIV) infection or AIDS. In countries such as Thailand, where antiretroviral drug therapy is now available due to the introduction of generic drugs, the provision of affordable and reliable CD4 testing for the initiation and monitoring of antiretroviral therapy has emerged as a vital issue. In Thailand, dual-platform (DP) flow cytometry immunophenotyping is the accepted standard method for the determination of the absolute counts of CD4+ T lymphocytes (6). The DP technique uses a flow cytometer (FCM), along with a hematology analyzer, which provides the absolute lymphocyte count, to provide the percentage of CD4+ T lymphocytes. The Centers for Disease Control and Prevention (CDC)-recommended three-tube, three-color monoclonal antibody panel with the lyse-and-wash or lyse-no-wash whole-blood method (6, 7) is commonly used in most regional hospitals. However, there are two main drawbacks of such a method: (i) many studies have highlighted problems associated with the DP-derived CD4+ T-lymphocyte count, especially in relation to the generation of an absolute lymphocyte count by the hematology analyzer (1, 3, 11, 16, 23); and (ii) the cost of DP FCM CD4 testing in Thailand remains relatively high ($12 to $20). Several single-platform (SP) flow cytometric technologies, including FCM volumetric counting and microfluorometry (8, 9, 13, 14, 19), as well as, most commonly, the bead-based flow cytometric method for measurement of the absolute CD4+ T-lymphocyte counts, have been developed and successfully evaluated (21, 25). Although this bead-based flow cytometric method eliminates the need for multiple technologies, it is still limited by the high cost of the currently available FCMs and fluorescent beads. Although alternative nonflow cytometric technologies are simple and less expensive, they have not been implemented widely due to their complexity, low-volume CD4 counting application (1 to 10 samples/day), and poor quality control (4, 5, 12, 22). The recent introduction of the inexpensive single-parameter CyFlowgreen FCM (Partec GmbH, Munster, Germany), which uses a single phycoerythrin (PE) conjugated-monoclonal antibody to CD4, has increased access to CD4+ count determinations for patients in areas that are not equipped with an FCM, particularly in district hospitals in the rural areas of Thailand. The purpose of this study was to evaluate this single-parameter SP CyFlowgreen technology as an alternative for determination of absolute CD4+ T-lymphocyte counts by comparing the values obtained with CyFlowgreen with those obtained by the standard SP bead-based systems, consisting of TruCOUNT tubes (Becton Dickinson Biosciences [BDB], San Jose, CA) with the FACScan (BDB) system and the FACSCount FCM (BDB). This study was done as part of the CDC Global AIDS Program, which supports the evaluation of alternative methodologies for CD4+ T-lymphocyte subset enumeration.

Background The current method of CD4 enumeration in Thailand, based on the three-tube, three-color method recommended by the Centers for Disease Control and Prevention, is expensive and thus unavailable to most patients who have the human immunodeficiency virus (HIV). Less expensive, simpler protocols (i.e., PanLeucogating and primary CD4 gating) have been described but require more published validation data to gain widespread acceptance. We describe a multicenter evaluation of the PanLeucogating method. Methods The PanLeucogating method using generic reagents was evaluated in comparison with the standard three-tube, three-color method using commercial reagents. Percentage of CD4+ T cells among lymphocytes and absolute CD4+ T-cell counts were determined in 611 HIV-infected individuals recruited from four sites. Linear regression and Bland-Altman tests were used for statistical analysis. Results The correlation of percentage of CD4+ T cells and absolute CD4+ T-cell counts obtained with the PanLeucogating strategy and the standard predicate method was high (r2 = 0.96 and 0.95, respectively, for the entire study population and r2 > 0.95 and 0.93, respectively, for each study group). Absolute CD4+ T-cell counts of the overall study pool and of the two subdivisions of absolute CD4+ T-cell counts (i.e., 0–250 cells/μl and >250 cells/μl) derived from the two methods demonstrated excellent agreement, with mean biases of +18 cells/μl, +11 cells/μl, and +24 cells/μl, respectively. Conclusions These observations demonstrate that CD4 enumeration by PanLeucogating is reliable and can be performed to an identical standard in a quality-assured network of collaborating laboratories as a new cost-effective approach to HIV monitoring. © 2005 Wiley-Liss, Inc.

Summary Background: Infection is one of the most common causes of death in β-thalassemia patients. This may be due in part to an underlying immunological abnormality. During the past decade, a subset of CD3+ T cells that express both CD4+CD8+ (DP) T-cells were discovered and have been described in several pathological conditions. However, phenotypic characterization of this unique T-lymphocyte subset in patients with β -thalassemia has not yet been investigated. Methods: Flow cytometry was used to determine the frequency of such CD4+CD8+(DP) cells in concert with frequencies of CD4+, CD8+, NKT cells and γδ-TCR T-lymphocytes in the peripheral blood of β-thalassemia/HbE patients. The frequencies of these lymphocyte subsets were compared with those in blood samples from healthy volunteers. Results: The results showed that the frequency of lymphocytes was significantly increased in splenectomized β-thalassemia/HbE patients but the frequencies of CD3+, CD4+ and CD8+ Tlymphocytes were not significantly different among the studied groups. However, analysis of unconventional T-lymphocytes revealed a significant increase in the frequency of CD4- CD8- in splenectomized β-thalassemia/HbE patients. The frequencies of CD4-CD8dim and CD4+CD8+ in β-thalassemia/HbE patients were similar to the controls. Further classification of the CD4+CD8+ cells revealed that β-thalassemia/HbE patient expressed significantly high levels of CD4brightCD8dim, with a marked increase found in non-splenectomized patients. Furthermore, significant increases in the frequency of γδ-TCR and NKT cells were also demonstrated in these splenectomized β-thalassemia/HbE patients. Conclusion: Our findings show the alteration of unconventional T-lymphocyte subsets in β- thalassemia/HbE patients, which may be responsible or may reflect the impaired immune response in β-thalssemia disease.

We have previously developed an affordable flow cytometric method for absolute cell count using glutaraldehyde-fixed chicken red blood cells. However, its use is limited to CD4+ T cells. In the current investigation, we studied the potential use of glutaraldehyde-fixed chicken RBCs to determine the number of residual white blood cells (rWBCs) in WBC-reduced blood component.Acridine orange (AO) was used to identify leucocytes in serial diluted blood samples ranging from 0.65 to 1,000 cells/microL. The absolute number of AO stained leucocytes were determined by using a known number of glutaraldehyde-fixed chicken RBCs on flow cytometer. The results were compared with the expected value and the absolute count determined by BD Leucocount (Becton Dickinson Bioscience). In addition, the stability of AO stained leucocytes and sample stability at various time points were measured. Reproducibility of the assay method was also addressed.There was a good correlation in the number of leucocytes between our new method and the expected numbers from serially diluted blood samples (r2 = 0.99, y = 1.04x + 0.50, p0.001). Furthermore, absolute leucocyte counts determined by the new method correlated well with those obtained from BD Leucocount (r2 = 0.99, y = 1.31 x - 6.37, p0.001). FL-1 intensity and the absolute number of AO stained leucocytes were stable for at least 24 hours after staining. Samples stored at 4 degrees C were stable for 48 hours and CV of the assay was at an acceptable level.This flow cytometric method for absolute leucocyte counts using AO and glutaraldehyde-fixed chicken RBCs is a simple, rapid, reliable and inexpensive method for routine monitoring of low levels of leucocytes in blood products.

Background Nitrite is a nitric oxide (NO) metabolite in tissues and blood, which can be converted to NO under hypoxia to facilitate tissue perfusion. Although nitrite is known to cause vasodilation following its reduction to NO, the effect of nitrite on platelet activity remains unclear. In this study, the effect of nitrite and nitrite+erythrocytes, with and without deoxygenation, on platelet activity was investigated. Methodology/Finding Platelet aggregation was studied in platelet-rich plasma (PRP) and PRP+erythrocytes by turbidimetric and impedance aggregometry, respectively. In PRP, DEANONOate inhibited platelet aggregation induced by ADP while nitrite had no effect on platelets. In PRP+erythrocytes, the inhibitory effect of DEANONOate on platelets decreased whereas nitrite at physiologic concentration (0.1 µM) inhibited platelet aggregation and ATP release. The effect of nitrite+erythrocytes on platelets was abrogated by C-PTIO (a membrane-impermeable NO scavenger), suggesting an NO-mediated action. Furthermore, deoxygenation enhanced the effect of nitrite as observed from a decrease of P-selectin expression and increase of the cGMP levels in platelets. The ADP-induced platelet aggregation in whole blood showed inverse correlations with the nitrite levels in whole blood and erythrocytes. Conclusion Nitrite alone at physiological levels has no effect on platelets in plasma. Nitrite in the presence of erythrocytes inhibits platelets through its reduction to NO, which is promoted by deoxygenation. Nitrite may have role in modulating platelet activity in the circulation, especially during hypoxia.

The frequency and absolute number of CD4+ T-lymphocytes continue to be one of the major clinical markers for management of HIV/AIDS. The present standard dual-platform (DP) three-color and two-color PanLeucogating flow cytometric (FCM) methods for most developing countries are either expensive if manufacturers' monoclonal antibody reagents are used or limited due to an insufficient supply of generic reagents. Clearly, more affordable FCM methods are needed.To develop a novel DP FCM method using biotin-streptavidin-fluorochrome labeling in combination with the two standard DP methods for 4 different white blood cells (WBC) using only one monoclonal antibody reagent.The percentage of CD4+ T-lymphocytes in 116 HIV-infected blood samples was determined using our new method. Results were compared with the two standard methods. Correlation and agreement of the pair method were determined using linear regression, Bland-Altman and percent similarity analysis.Our study showed that percentage of CD4+ T-lymphocyte values obtained from the new method correlated highly with the standard three-color and the two-color methods (r2 = 0.95 {n=52} and 0.97 {n=64}). The mean bias and percent similarity for the new method compared with the two standard methods were -0.53% (limit of agreement {LOA}:-5.22% to +4.16% with percent similarity of 99.28; and -0.22% with LOA of -3.42% to +2.98%, the percent similarity of 98.15, respectively.Our FCM method using biotin to label 4 different WBC samples followed by streptavidin staining is reliable for determination of CD4+ T-lymphocytes. Such an approach will significantly reduce the cost for monitoring HIV-infected patients in resource-limited settings.

Background: We have previously developed an alternative approach for undertaking absolute cell counting based upon flow-rate calibration using cell bead (FCB), in which cell bead (CB) can be used as a flow-rate calibration material for generating the absolute microparticle counts. Here, we extended our work of counting CD4+ T-lymphocytes in HIV-infected blood samples with the FCB method. Methods: CD4+ T-lymphocyte counts in EDTA blood samples from 30 healthy subjects and 80 HIV-1-infected patients were determined using TriTEST reagent. The absolute CD4+ T-lymphocytes were measured by FCB, and the results were compared with the absolute counting by commercial latex bead (CLB) or with flow rate-based calibration method (FR). Statistical correlation and agreement were analyzed using linear regression and Bland–Altman analysis. Results: There was no significant difference in the absolute number of CD4+ T-lymphocyte counts enumerated by FCB when compared with those two reference methods (CLB and FR). The absolute CD4+ T-lymphocyte counts obtained from FCB method was highly correlated with those obtained from CLB [r2= 0.99, y = 1.04x − 12.37, P < 0.001, and mean bias 11.96 cell/μl, limit of agreement (LOA) −57.82 – 81.74 cell/μl], FR method (r2 = 0.98; y = 0.97x − 3.13, P < 0.001, and mean bias −24.15 cell/μl, LOA −114.44 − 66.13 cell/μl). Conclusions: The use of FCB is comparable with the use of CLB and FR. This approach showed the effective in reducing cost for generating the absolute CD4+ T-lymphocyte counts. Such an approach should facilitate and ensure the success of the ongoing antiretroviral therapy program in resource-limited countries. © 2010 Clinical Cytometry Society

A CD4 count External Quality Assessment (EQA) program is important for the clinical monitoring of persons infected with HIV/AIDS. The purpose of the present study was to evaluate the CD4 EQA performance program of the flow cytometer laboratories that perform routine CD4 counts for these patients in Thailand. Stabilized whole blood samples were sent to participating laboratories to determine the percentage and absolute counts of CD4+ T-lymphocytes using their routine procedures. The data were analyzed and reports sent to the participants within one month. Most participating laboratories produced results that were within two standard deviations (SD) of the mean, while the average inter-laboratory coefficients of variation were less than 8% for CD4+ T-lymphocytes. This program was found to improve the reliability of CD4+ T-lymphocyte determinations. This test is becoming increasingly important as Thailand and other Southeast Asian countries scale up their national programs that provide access to antiretroviral therapy for persons living with HIV/AIDS.

CD4(+) T-lymphocyte count is an important marker in management of HIV-1-infected patients. The standard single-platform (SP) flow cytometric (FCM) CD4(+) testing that uses the known reference microbeads is expensive; more affordable alternatives are therefore needed. We evaluated the use of glutaraldehyde-fixed chicken red blood cells (CRBCs) as counting beads as an alternative for enumerating CD4(+) T-lymphocyte counts in 87 HIV-1-infected patients. Linear regression analyses revealed an excellent correlation of the SP FCM using CRBCs with the standard SP bead-based FCM method (percentages, r(2) > 0.99; absolute counts, r(2) > 0.98) over the entire range including the clinically relevant range. Mean percent bias for the CRBC method was +0.35% [limits of agreement (LOA): -1.86% to +2.57%]. For absolute CD4(+) T-lymphocytes, the mean biases was -47.76 cells per microliter (LOA: -191.34 to +98.81 cells/microL) with much lower bias for CD4 T-lymphocyte counts

Background: Various assays are used to enumerate peripheral blood absolute CD4+ T-lymphocytes. Flow cytometry is considered the gold standard for this purpose. However, the high cost of available flow cytometers and monoclonal antibody reagents make it difficult to implement such methods in the resource-poor settings. In this study, we evaluated a cheaper, recently developed single-platform microcapillary cytometer for CD4+ T-lymphocyte enumeration, the personal cell analyzer (PCA), from Guava® Technologies. Methods: CD4+ and CD8+ T-lymphocyte counts in whole blood samples from 250 HIV-1 infected Thais were determined, using a two-color reagent kit and the Guava PCA, and compared with the results obtained with two reference microbead-based methods from Becton Dickinson Biosciences: the three-color TruCOUNT™ tube method and the two-color FACSCount™ method. Statistical correlations and agreements were determined using linear correlation and Bland–Altman analysis. Results: Absolute CD4+ T-lymphocyte counts obtained using the Guava PCA method highly correlated with those obtained using TruCOUNT method (R2 = 0.95, mean bias +13.1 cells/μl, limit of agreement [LOA] −101.8 to +168.3 cells/μl). Absolute CD8+ T-lymphocyte counts obtained using the Guava PCA method also highly correlated with those obtained with the two reference methods (R2 = 0.92 and 0.88, respectively). Conclusion: This study shows that the enumeration of CD4+ T-lymphocytes using the Guava microcapillary cytometer PCA method performed well when compared with the two reference bead-based methods. However, like the two reference methods, this new method needs substantial technical expertise. © 2007 Clinical Cytometry Society.

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.

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. © 2006 International Society for Analytical Cytology

Vesicles are part of the red blood cells membrane which can be found in a small number in normal apoptotic process and increased in some diseases. In the present study, the authors measured the percentage of red blood cell vesicles in healthy subjects (n = 7), patients with alpha-thalassemia or Hemoglobin (Hb) H disease (n = 7), beta-thal/Hb E with nonsplenectomized (n = 5) and splenectomized (n = 7) before and after induction heated at 48.6 degrees C by using flow cytometry. It was found that the percentage of vesicles in every group were not statistically significantly different (p0.05) between pre and post incubation at 5 min. The percentage of vesicles of healthy subjects, beta-thal/Hb E nonsplenectomized patients and splenectomized patients were highest when induced by heating for 60 min. For patients with Hb H disease, the percentage of vesicles was maximum at 30 min when compared with healthy subjects, beta-thal/Hb E nonsplenectomized patients and splenectomized patients, respectively. In the present study, the authors report the significant increase of the percentage of vesicles in Hb H disease, beta-thal/Hb E nonsplenectomized and splenectomized after induction by heat when compared with healthy subjects. These findings may support the different pathology of the red blood cells found in alpha- and beta-thalassemia.

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.

Abstract 3189 Nitrite is now recognized as a circulating and storage form of nitric oxide (NO), whose biological effect is largely mediated by its chemical reduction to NO via the nitrite reductase activity of heme-containing proteins and other enzymatic reactions. NO activates soluble guanylyl cyclase to produce cGMP, leading to vasodilation that facilitates tissue oxygen supply, but which is also known to strongly inhibit platelet aggregation. Although the vasorelaxation effect of nitrite has recently been clarified, the effect of nitrite, especially at physiologic levels, on platelet function has not been well studied. We hypothesized that platelet inhibition by nitrite could occur under the conditions known to promote its reduction to NO, such as the presence of deoxygenated erythrocytes. We found that nitrite up to 100 μM had no effect on platelet aggregation in plasma; however, in the presence of erythrocytes inhibition of platelet aggregation by nitrite was observed, which was dependent on hematocrit. At 20% hematocrit, nitrite at physiologic concentration (0.1 mM) significantly inhibited platelet aggregation induced by either ADP or collagen. In contrast, DEANONOate, an NO donor, at low concentrations inhibited platelet aggregation in plasma, but its effect was abolished by erythrocytes at even 1% hematocrit. C-PTIO, a membrane impermeable NO scavenger, completely inhibited the effect of nitrite plus erythrocytes on platelets, suggesting that nitrite was converted to NO in or on the surface of the erythrocytes and then entered the platelets. Furthermore, deoxygenation increased the inhibitory effect of nitrite plus erythrocytes on platelets as demonstrated by a decrease of P-selectin expression (a marker of platelet degranulation) and a corresponding increase in cGMP levels in the platelets. We also found negative correlations between platelet aggregation induced by ADP and nitrite concentrations in whole blood and erythrocyte suspensions from various individuals. These results suggest an essential role of endogenous nitrite as a source of NO, produced by erythrocytes and perhaps other cells in the vasculature, in modulating platelet activity in the circulation. Although nitrite alone at physiological concentrations does not have a direct inhibitory effect on platelets, nitrite plus erythrocytes, which simulate the normal circulating blood, inhibit platelet aggregation and the effect is promoted by deoxygenation. The net effect is modulated by the tendency of oxyhemoglobin in erythrocytes to destroy NO and will likely depend on relative concentrations and the kinetics of the competing reactions, including the mechanisms by which NO activity may leave the erythrocyte. We suggest that these reactions may contribute to differences in the mechanisms of hemostasis in arterial and venous blood and might be the basis of pharmacological approaches to preventing thrombosis. Disclosures: Schechter: National Institutes of Health: I am a co-inventor of a patent for the use of nitrites in the treatment of cardiovascular diseases., Patents & Royalties.