Microparticle Counts Research Articles

Comparison of Clot-Based vs Chromogenic Factor Xa Procoagulant Phospholipid Activity Assays

We compared 2 commercial plasma procoagulant phospholipid activity (PPA) assays, chromogenic, using bound annexin V to capture phosphatidylserine-containing microparticles, and clot-based. In both, anionic phospholipids accelerated activation of prothrombin by factor Xa. PPA levels were lower in the chromogenic vs the clot-based assay, with poor correlation between methods: normal samples, mean ± SD, 27 ± 17 vs 590 ± 414 nmol/L (n = 24; r(2) = 0.29) and patient samples, mean ± SD, 45 ± 44 vs 401 ± 330 nmol/L (n = 51; r(2) = 0.26). Recovery of phosphatidylserine added to normal, heparinized, and warfarin plasma samples averaged 109% ± 39% using the chromogenic assay but was higher and more varied (mean ± SD, 176% ± 59%) in the clot-based assay. Lupus anticoagulants caused low recovery in both assays. Removal of microparticles by 0.22-μm filtration reduced PPA by 91% in the clot-based and 65% in the chromogenic assay. The clot-based assay showed higher correlation (r(2) = 0.82 vs 0.23) with flow cytometric platelet microparticle counts. The 2 assays measure different aspects of PPA in plasma, with the chromogenic assay primarily measuring smaller microparticles.

Red cell microparticle enumeration: validation of a flow cytometric approach

There is growing interest in the clinical application of red blood cell (RBC) microparticle (MP) enumeration as they have been postulated to be effectors of coagulation and inflammation following transfusion and in sickle cell disease. No uniform approach in MP enumeration exists and a key limitation is the lack of an internal validation process. We present and validate a flow cytometric approach where an internal standard is utilized. Glycophorin A(+) Annexin V(+) events were enumerated using MPs isolated from RBC units or plasma samples obtained from volunteers. A mixture of absolute counting (7·6 μm) and calibration beads (0·5, 0·9 and 3 μm) at a fixed ratio was added to each sample. RBC MPs were initially selected based upon a fluorescence threshold, and the 0·5- and 0·9-μm beads defined the upper and lower light scatter distribution of MPs. The ratio of 7·6:3-μm bead events was used as an internal standard to validate the precision of MP enumeration across samples (coefficient of variation = 2·5-7·2%) and remained constant in both platelet-rich plasma (PRP) and platelet-free plasma (PFP). RBC MP counts increased in both PRP and PFP obtained from whole blood stimulated with ionophore and increasing calcium concentrations, with PRP showing higher MP counts than PFP at every concentration studied. This method is a useful strategy to detect RBC MP counts across bio-samples provided that the flow cytometer can reliably discriminate the size of the calibration beads.

Number of Microparticles Generated During Acute Myocardial Infarction and Stable Angina Correlates with Platelet Activation

Elevated levels of circulating microparticles (MPs) have been reported in patients with acute myocardial infarction (AMI) and coronary artery disease. Platelet activation and inflammation have been recognized during AMI and stable angina (SA). We hypothesize that the origin and count of MPs in AMI and SA patients are related to markers of inflammation and platelet activation. Platelet, monocytes and endothelial MPs and surface P-selectin were determined in 12 AMI patients, 10 SA patients and 9 controls by flow cytometry. Plasma P-selectin, CD40 ligand (sCD40L) and interleukin 6 (IL-6) levels were evaluated by ELISA methods. The total MP count was compared in control subjects, AMI, and SA patients: 12,765 (8465) vs. 38,750 (11,931) vs. 29,715 (12,072) counts/μl (p = 0.01), respectively. Patients with AMI displayed higher levels of total and platelet origin- tissue factor-positive (CD42/CD142) MPs than patients with SA: 72.8 (6.2) vs. 56.2 (6.4) %, p = 0.001. Levels of soluble P-selectin were significantly elevated in patients with AMI as compared to SA patients: 146 (6.5) vs. 107 (2.7) ng/mL, p = 0.005; significant correlation between total MP count and relative number of CD34, CD51, CD42-positive MPs, and the P-selectin expression was observed in patients with AMI. Platelet activation in AMI is associated with increased generation of MPs not only from platelets, but also monocytes and endothelial cells. It suggests that interactions between platelets, monocytes and endothelial cells play an important role in the pathogenesis of myocardial ischemia.

Hormone replacement therapy leads to increased plasma levels of platelet derived microparticles in postmenopausal women

Whereas prevention of cardiovascular diseases by hormonal replacement therapy is still part of an ongoing debate, well-defined data are available relating hormonal replacement therapy to an elevated risk of venous thrombosis and embolism. Although it seems that venous thrombosis in patients treated with hormonal replacement therapy is linked to changes in plasmatic coagulation, less is known about the role of platelet-derived microparticles, as well as endothelial cell-derived microparticles. In this prospective case-control study, levels of microparticles were investigated in postmenopausal women receiving hormone replacement therapy (n = 15) and compared to age-matched controls (n = 15). Total count of microparticles and the subgroup of microparticles derived from endothelial cells did not differ in the investigated groups. In contrast, median levels of microparticles derived from platelet/megacaryocyte were higher in women taking hormonal replacement therapy (5,244 × 10(6)/l) than in controls (2,803 × 10(6)/l; p = 0.040). Furthermore, hormonal replacement therapy led to a higher plasma level of microparticles derived from activated platelets, exposing P-selectin (136 × 10(6)/l vs. 58 × 10(6)/l; p = 0.011), or exposing CD63 (171 × 10(6) vs. 91 × 10(6)/l; p = 0.011) compared to the control group. Higher concentrations of microparticles derived from (activated) platelets/megacaryocytes were present in postmenopausal women taking hormonal replacement therapy. This finding indicates a procoagulant state in these women and might play a role in the development of venous side effects. In contrast, levels of endothelial cell-derived microparticles did not differ.

P-selectin glycoprotein ligand-1 positive microparticles in allogeneic stem cell transplantation of hematologic malignancies

P-selectin and its receptor P-selectin glycoprotein ligand-1 (PSGL-1) mediate adhesion between leukocytes, tumor cells (including leukemias and lymphomas), and platelets, and play an important role in hematopoiesis, T cell activation, and cancer growth and metastasis. As microparticles (MPs) are released from activated or apoptotic cells, there should be significant numbers of circulating PSGL-1-bearing MPs in the blood of patients undergoing allogeneic stem cell transplantation (alloSCT). We enumerated PSGL-1-expressing MPs in plasma samples from 30 consecutive patients with hematologic disorders at different time points during the course of alloSCT by flow cytometry and analyzed their relation to cell counts, patient characteristics, and clinical outcome. Median follow-up time of surviving patients was 1,772 days (range 1272-1981 days). Nineteen patients (63.3%) died, 10 due to progression of disease (33.3%). The PSGL-1 MPs significantly declined during conditioning therapy but increased again after transfusion of donor cells and even more at the time of engraftment. Numbers >250/μL after graft transfusion were associated with a shorter time to engraftment for patients receiving fresh peripheral stem cell grafts (median, 15 vs. 21days; p = 0.049). Furthermore, low PSGL-1 MP values at those two time points were associated with a higher risk of progress/relapse in univariate analysis (p = 0.008-0.014; hazard ratio [HR] = 15.0-42.0) with cumulative incidences at 5 years of 81.8% versus 28.6% and 85.7% versus 20.0%, respectively. In conclusion, PSGL-1 microparticles show a characteristic course during alloSCT and their possible association with relapse/progress requires further evaluation of the PSGL-1/P-selectin interaction in leukemias and lymphomas.

A new microparticle size calibration standard for use in measuring smaller microparticles using a new flow cytometer

Microparticle size measurements are often calibrated on flow cytometers using polystyrene microspheres that forward scatter more light vs. particle diameter than cellular microparticles. We compared theoretical with measured forward angle light scattering on the LSRII, FC500 and Apogee A40 using polystyrene and silica microspheres vs. synthetic lipid vesicles and platelets, then compared plasma microparticle counts using different calibration strategies. Polystyrene and silica microspheres with higher refractive indices forward scattered more light with a wavelength of 488 nm for a given size microparticle than did lipid vesicles or platelets. The LSRII and FC500 did not count many, and were unable to separate by size, polystyrene microspheres <0.5 μm in diameter. On the Apogee A40, polystyrene microspheres could be separated by size down to 0.2 μm, and a polystyrene microsphere 0.4 μm in diameter produced the same forward scatter relative intensity as a 1-μm lipid or cellular microparticle. Using the new calibrator, the Apogee A40 found 80 000-4 000 000 μL(-1) total microparticles, 11 000-350 000 μL(-1) annexin V positive microparticles and 6000-350 000 μL(-1) platelet microparticles <1 μm in plasma samples. The Apogee A40 was able to resolve size differences in polystyrene microspheres down to 0.2 μm and microparticles down to 0.4 μm. On the Apogee A40 we propose using a 0.4-μm polystyrene microsphere as equivalent to a 1-μm cellular microparticle for size calibration. Using this calibrator, the Apogee A40 detected higher numbers of total, platelet and annexin V positive microparticles than were found using a Megamix gate.

Relation Between Endothelial Microparticles and Endothelial Function Following Acute Exercise in Men with CAD

Purpose: Microparticles (MP) are small membranous vesicles shed from formed elements such as circulating platelets and vascular endothelial cells. They express surface antigens characteristic of their cell-of-origin and contain DNA, RNA, miRNA and intracellular proteins. Depending on the dynamic morphological state of the parent cell, phenotypically varied MP may be released into the circulation. MP serve not only as markers, but also potential mediators of inflammation, coagulation and endothelial dysfunction. This study examined the relation between total MP, endothelial MP (EMP), platelet MP (PMP) and endothelial function (a validated surrogate measure of early atherosclerosis) in response to acute exercise in men with coronary artery disease (CAD). Methods: Eight men with CAD (65.7 ± 4.5 yr, VO2max 18.6 ± 4.1 ml/kg/min, BMI 29.7±3.3 kg/m-2) underwent 20 min of treadmill walking at 66 % VO2max. Endothelial dependent dilation of the brachial artery was assessed before and 1 h following the acute bout of exercise. Vessel diameter was determined using high resolution vascular ultrasonography (SonoSite, MicroMaxx) from flow mediated dilation (FMD) following 5 min of forearm occlusion. Endothelial independent vasodilation (EID) was measured following the administration of glyceryl trinitrate (0.4 mg). MP counts were enumerated by flow cytometry from platelet poor plasma (PPP) stored at –80°C. MP were characterized based on size (0.5 – 1.0 μm sizing gate) and their ability to bind annexin V. MP had also to bind to CD62E or CD41 to be of endothelial or platelet origin respectively. Results: Compared to baseline, FMD was significantly increased at 60 min following the acute bout of exercise. There was no change in EID. There was significant relation between the percent change in total MP (annexin V+ events) and the percent changes in flow mediated dilation 60 min after the exercise bout and between the percent change in EMP (annexin V+/CD62E+ events) and the percent changes in flow mediated dilation 60 min after the exercise bout. Conclusion: Changes in endothelial function in response to acute exercise may be mediated by alterations in circulating levels of MP.

Cantilever-based micro-particle filter with simultaneous single particle detection

Currently, separation of whole blood samples on lab-on-a-chip systems is achieved via filters followed by analysis of the filtered matter such as counting of blood cells. Here, a micro-chip based on cantilever technology is developed, which enables simultaneous filtration and counting of micro-particles from a liquid. A hole-array is integrated into a micro-cantilever, which is inserted into a microfluidic channel perpendicular to the flow. A metal pad at the apex of the cantilever enables an optical read-out of the deflection of the cantilever. When a micro-particle is too large to pass a hole in the cantilever, clogging of the holes increases the flow resistance of the cantilever. This causes a bending of the device, which can be detected by the optical read-out system. By arranging an array of such cantilevers with different hole sizes, separation by size can be achieved. In this paper a proof of concept of the device is demonstrated by filtering and counting 20 µm polystyrene beads dispersed in an aqueous solution.

A microfluidic multichannel resistive pulse sensor using frequency division multiplexing for high throughput counting of micro particles

In this work we demonstrate an on-chip multiplexed multichannel resistive pulse sensor (Coulter counter) for high throughput counting of microscale particles. The design, fabrication and testing of this device are presented. The high throughput counting is a result of using multiple parallel microfluidic channels to analyze the sample. Detection is achieved by using frequency division multiplexing; each microchannel is modulated with its own known and unique frequency, a combined measurement is made across a single pair of electrodes, and the measured signal is demodulated to determine the signal across each individual channel. Testing results using 30 µm polystyrene particles demonstrate that the throughput of the multiplexed device gets improved 300% over a single-channel device; this is achieved by simultaneously detecting particles through the device's four parallel channels. In addition, the ac modulation method used in this paper reduces the polarization effect on the microelectrodes, and thereby allows for measurement of the particle sizes with significantly reduced error. The multiplexed detection principle can be extended to a larger number of channels to further improve the throughput, without increasing the external detection electronics.

Decline in platelet microparticles contributes to reduced hemostatic potential of stored plasma

IntroductionIn an effort to administer life-saving transfusions quickly, some trauma centers maintain thawed plasma (TP). According to AABB, TP is approved for transfusion for up to five days when stored at 1 - 6°C. However, the alterations in microparticles (MP) contained in the plasma, which are an integral component of plasma's hemostatic capacity, are not well characterized. We report on MP changes in TP between its initial thaw (FFP-0) and five days (FFP-5) of storage. Materials and MethodsFFP units (n=30) were thawed at 37°C and kept refrigerated for five days. Phenotypes of residual cells, which include platelets, erythrocytes, leukocytes, monocytes, endothelial cells, and MP counterparts of each cell type, were analyzed by flow cytometry. Functional assays were used for MP procoagulant activity, plasma thrombin generation, and clotting properties (thromboelastography). ResultsIn FFP-0 the majority (94%) of residual cells were platelets, along with significant levels of platelet MPs (4408×103/L). FFP-5 showed a decline in MP count by 50% (p<0.0001), and procoagulant activity by 29% (p<0.0001). FFP-5 exhibited only 54% (p<0.0001) of the potential for thrombin generation as FFP-0, while thromboelastography indicated a slower clotting response (p<0.0001) and a longer delay in reaching maximum clot (p<0.01). Removal of MP by filtration resulted in reduced thrombin generation, while the MP replacement restored it. ConclusionsDecline in MP with storage contributes to FFP-5's reduced ability to provide the hemostatic potential exhibited by FFP-0, suggesting the presence of platelet MPs in freshly TP may be beneficial and protective in the initial treatment of hemorrhage.

Flow cytometric diagnostic assessment of cell-derived microparticles is severely confounded by immune complexes in rheumatoid arthritis

Background and objectivesMicroparticles/microvesicles are readily detectable in various biological fluids, and have recently been shown to be highly specific and sensitive biomarkers, for example, in tumours. Numerous autoimmune diseases including...

Surveillance of megakaryocytic function by measurement of CD61‐exposing microparticles in allogeneic hematopoietic stem cell recipients

Increasing evidence suggests that circulating microparticles (MP) exposing CD61 originate predominantly from megakaryocytes. Dramatic changes in megakaryocytic homeostasis are regularly observed following allogeneic hematopoietic stem cell transplantation (HSCT) and associated with transplantation-associated complications. We studied MP plasma levels prospectively in healthy subjects (n = 10) and allogeneic HSCT recipients (n = 19) twice weekly from the start of conditioning therapy up to day 30. A total of 224 measurement points were evaluated. MP were isolated, double-stained with annexin V and anti-CD61, and analyzed by flow cytometry. In uncomplicated HSCT, we found a correlation between platelet and CD61-exposing MP count, which resulted in a constant ratio of MP per platelet. The ratio was increased in patients with active hematological malignancies before transplantation and normalized during conditioning therapy. After take, the MP ratio increased, whereas infections and microangiopathic hemolytic anemia did not affect the ratio. In patients with GvHD, a decreased MP ratio was observed depending on the grade of GvHD, possibly indicating megakaryocytic damage. The MP ratio was able to discriminate between toxic, septic, and GvHD-induced hyperbilirubinemia. We first describe CD61+ MP levels during allogeneic HSCT and postulate that the MP ratio might be a useful biomarker for the surveillance of megakaryocytes during HSCT.

Decreases in CD31 and CD47 Levels on the Cell Surface during Etoposide-Induced Jurkat Cell Apoptosis

Engulfment of apoptotic cells is regulated by 'eat me' and 'don't eat me' signals on the cell surface. Alterations to the 'eat me' signals have been well described; however, very little is known about the 'don't eat me' signals on the cell surface during apoptosis. In the present study, apoptosis of Jurkat cells was induced by treatment with topoisomerase II inhibitor etoposide, and then the CD31 and CD47 levels on the apoptotic cell surface and in microparticles were estimated by flow cytometry and immunoblotting methods in the presence of caspase, metalloproteinase, and Rho-associated coiled-coil containing protein kinase 1 (ROCK1) inhibitors. The CD31 and CD47 levels on the cell surface of apoptotic Jurkat cells had decreased after treatment with etoposide. These decreases in CD31 and CD47 levels on the apoptotic cell surface were almost completely suppressed by the caspase 3 inhibitor, Ac-DEVD-CHO, and partially suppressed by caspase 8 (Ac-IETD-CHO) and caspase 9 (Ac-LEHE-CHO) inhibitors but not by the metalloproteinase inhibitors GM6001 and TAPI-0. Microparticle counts in culture supernatants were higher during etoposide-induced apoptosis. The ROCK1 inhibitor, Y27632, suppressed blebbing formation and microparticle release. Moreover, flow cytometry and immunoblotting revealed CD31 and CD47 in the microparticles. These results indicate that CD31 and CD47 were released by the apoptotic Jurkat cells into the culture supernatant in microparticles, but not in soluble forms, resulting in decreased levels on the apoptotic cell surface.

Microparticle and cell counting with digital microfluidic compact disc using standard CD drive

Lab-on-chip medical diagnostics in a global health setting would greatly benefit from highly portable, cost effective and readily available devices. Digital compact disc (CD) and the corresponding detection device-CD drives-for personal computers are extremely affordable and distributable worldwide, therefore they can be immediately used in global health applications if empowered with molecular and cellular biosensing functions. Here we present a novel digital microfluidic CD device derived from conventional music or data CD and demonstrate its preliminary application of counting polystyrene microparticles and living cells in minute-volume fluidic samples. No other detection instruments except for a standard CD drive in a personal computer is used for reading and decoding the quantitative liquid sample information from the digital microfluidic CD. The results presented herein are the first step towards creating a truly portable, low-cost and ubiquitously accessible device-health diagnostic compact disc (HDCD)-for biosensing and health diagnostics, especially in remote or impoverished settings with limited medical infrastructure and healthcare workers.

Pre-analytical and analytical issues in the analysis of blood microparticles

Results of plasma microparticles (MPs) measurements reported in the literature vary widely. This is clearly not only related to the lack of well-standardised MP assays, but also to variations in pre-analytical conditions. In this review we will discuss the pre-analytical variables related to plasma and MP preparation which may affect MP analysis. Additionally we will address several analytical issues in commonly used MP assays and briefly discuss some novel approaches for the detection and characterisation of MPs. Ideally MP measurements should be performed in plasma, freshly prepared directly after blood withdrawal. As platelet contamination seems to be one of the major pre-analytical problems in processing plasma for MP measurement, the use of platelet-free plasma may be preferred. When frozen-thawed plasma is used, especially PMP and annexinV-positive MP counts should be interpreted with caution. When flow cytometry is chosen as a method for quantification of MPs, some analytical conditions should be standardised, e.g. settings of the flow cytometer, quality of the antibodies, and use of counting beads. Fluorescence-nanoparticle tracking analysis and atomic force microscopy can accurately count nanosized MPs, but unfortunately the operational procedures of both methods are still time consuming and they give no information on the functional properties of MPs. The MP-TF activity assay provides information on MPs carrying active TF, regardless of their parental origin. Ultimately, standardisation of pre-analytical procedures and the introduction of reliable and rapid methods for the measurement of MPs are urgently needed to facilitate their use as biomarker in the pathophysiology of diseases.

The microbubble or the microparticle?

decompression sickness (DCS) has long been attributed to physical forces exerted by inert gas bubbles that may form in tissues, resulting in vascular occlusion and tissue disruption. Bubble formation occurs when a decrease in ambient pressure exceeds the rate at which soluble inert gas (e.g.,

Endothelial microparticle formation in moderate concentrations of homocysteine and methionine in vitro.

Microparticles (MPs) are small membrane vesicles released by stimulated or apoptotic cells, including the endothelium. Hyperhomocysteinemia (HHcy) is a blood disorder characterized by an increase in the plasma concentrations of total homocysteine (Hcy). The plasma Hcy level is determined by environmental factors (dietary habits, i.e. the intake of folic acid, FA) and genetic factors (N 5,N 10-methylenetetrahydro-folate reductase, MTHFR, polymorphism 677C>T). To evaluate whether moderate Hcy concentrations induce endothelial MP formation, the role of FA supplementation and the influence of MTHFR polymorphism were analysed. Human umbilical vein endothelial cells (HUVEC) were treated in vitro with 50 μM of Hcy and methionine (Met). The MP number and apoptotic phenotype were analyzed using flow cytometry. Increasing doses of FA (5, 15 and 50 μM) were used to reduce the HHcy effect. The MTHFR 677C>T polymorphism was determined. HUVEC stimulated by Hcy produced significantly more MPs than HUVEC under the control conditions: 3,551 ± 620 vs 2,270 ± 657 kMP (p = 0.02). Supplementation with FA at concentrations of 5, 15 and 50 μM reduced the MP count in the cell culture supernatant to 345 ± 332, 873 ± 329, and 688 ± 453 kMP, respectively (p = 0.03). MTHFR 677C>T heterozygosity was associated with a significant increase in MP formation after stimulation with Hcy compared to the control conditions: 3,617 ± 152 vs 1,518 ± 343 kMP (p = 0.02). Furthermore, the MTHFR genotype altered MP formation after Met loading. On average, 24% of the entire MP population was apoptotic (annexin V-positive). Endothelial function impairment due to HHcy is related to MP shedding, which may involve platelets and other blood and vascular cells. MP shedding is a physiological response to moderate HHcy.

Overcoming Limitations of Microparticle Measurement by Flow Cytometry

Circulating microparticles are submicron vesicles released from cell membranes in response to activation or apoptosis. Acknowledgment of their role both as markers and pathogenic effectors in thrombosis, inflammation, and the spread of cancer has increased the interest of their measurement in clinical practice. However, assessment of their clinical use is impeded by technological issues. Among the different methodologies available, flow cytometry is the most commonly used technique. This review addresses flow cytometry limitations in microparticle measurement that may be subdivided into three domains: sizing, probing, and counting. This article also covers the various standardization strategies and technological improvements that have been proposed to overcome these limitations. New tools using size-calibrated beads and recent progress in instrumentation have opened new avenues to improve detection of microparticle populations of smaller sizes. Significant optimization in microparticle detection is also expected from the use of new fluorescent dyes and the establishment of practical recommendations. Finally, absolute counting of microparticles will also benefit from adapted bead-based strategies or, alternatively, from the generalized availability of volumetric systems. Overall, recent technological improvements maintain flow cytometry as a highly competitive analytical method to measure microparticles. Challenging these evolutions in pathological situations is a mandatory step to validate their real impact in clinical practice.

Preventing Platelet-Derived Microparticle Formation—and Possible Side Effects—With Prestorage Leukofiltration of Whole Blood

Platelet-derived microparticles (PDMPs) probably function in hemostasis, thrombosis, inflammation, and transfusion-related immunomodulation. To compare PDMP levels of leukocyte-filtered and unfiltered whole blood during storage. Ten whole blood donations were collected and processed. Half of each collection was filtered, half remained unfiltered, and both halves were measured for red cell, white cell, and platelet (PLT) content before storage. Samples were drawn on days 0, 1, 2, 3, 5, 7, 14, 21, 28, and 35 and analyzed by flow cytometry. Leukocyte filtration lowered prestorage PDMP and PLT counts by an average of 72% and 99%, respectively. Prestorage PDMP counts were 123 +/- 51/microL in unfiltered whole blood supernatant versus 34 +/- 18/microL after filtration. Prestorage PLT counts were 190 +/- 49/microL in unfiltered whole blood supernatant versus 2 +/- 4/microL after filtration. Moreover, PDMP and PLT counts in filtered whole blood remained low throughout storage, typically below 100/microL. In contrast, unfiltered whole blood PDMP- and PLT-gated events increased approximately 2 log during storage, with the peak number of PLT-gated events tending to coincide with the peak number of PDMP-gated events (4 donors) or to come after the peak number of PDMP-gated events (6 donors). Leukocyte filtration of whole blood lowers prestorage PDMP and PLT counts. Platelet-derived microparticle and PLT counts remain low throughout 35 days of storage. In contrast, PDMP- and PLT-gated events increase significantly in unfiltered whole blood. The nature of PLT-gated events in stored blood warrants further investigation.

Enumeration of the absolute CD4 T‐lymphocyte count by cell‐bead assay

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. 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. 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 [r(2)= 0.99, y = 1.04x - 12.37, P < 0.001, and mean bias 11.96 cell/microl, limit of agreement (LOA) -57.82 - 81.74 cell/microl], FR method (r(2) = 0.98; y = 0.97x - 3.13, P < 0.001, and mean bias -24.15 cell/microl, LOA -114.44 - 66.13 cell/microl). 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.