Kasama Sukapirom

Activated platelet-derived microparticles in thalassaemia.

Thromboembolic complications have been documented in thalassaemia patients. The aggregability of abnormal red blood cells and the high level of membrane‐derived microparticles (MPs) stemming from blood cells are thought to be responsible for the associated thrombotic risk. We investigated the number of MPs, their cellular origin and their procoagulant properties in β‐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 β‐thalassaemia/HbE patients had significantly higher levels of PS‐bearing MPs than non‐splenectomised β‐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 β‐thalassaemia/HbE patients (rs = 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 β‐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 β‐thalassaemia/HbE patients.

Performance evaluation of the Alere PIMA CD4 test for monitoring HIV-infected individuals in resource-constrained settings.

Background: Enumeration of CD4+ T-lymphocytes is important in the management of HIV. However, standard laboratory systems based on flow cytometry are expensive, complicated, and thus unavailable to most resource-limited settings where a low-cost and fully automated point-of-care CD4 testing system is required. In attempts to address this issue, a study was conducted to validate the Alere PIMA point-of-care CD4 test. Method: Duplicate values of the absolute number of CD4+ T-lymphocytes in 203 HIV-infected blood samples obtained using the PIMA system were compared with the two predicate single-platform FACSCount and the dual-platform FACSCan (Becton Dickinson Biosciences). Results: The overall absolute CD4+ T-lymphocyte count obtained using the PIMA system correlated highly with the FACSCount (r2 = 0.957; mean bias, -54.2 cells/μL; limit of agreement, -190.9 to +82.5 cells/μL) and the FACSCan (r2 = 0.957; mean bias -44.0 cells/μL; limit of agreement, -179.7 to +91.6 cells/μL). Good correlation and low biases were also observed for samples with CD4+ T-lymphocyte count ranges of 0 to 200 and 0 to 350 cells/μL. Additionally, there was no significant difference in absolute CD4+ T-lymphocyte counts noted between the duplicate samples using the PIMA system. Conclusions: This new point-of-care product is a simple and reliable system and should contribute significantly to the simplification of performing CD4 testing and thus increase access for patients in resource-limited settings. The inability to obtain values for the frequency (%) of CD4+ T-lymphocyte count is one limitation of the PIMA system, the addition of which would be of value for clinical staging or monitoring in HIV-infected pediatric patients.

Comparison of 5 Flow Cytometric Immunophenotyping Systems for Absolute CD4+ T-Lymphocyte Counts in HIV-1-Infected Patients Living in Resource-Limited Settings

Enumeration of CD4+ T lymphocytes is important in management of HIV-infected patients. However, CD4 testing by current gold standard bead-based flow cytometer (FCM) system is expensive for developing countries. This study compared 2 affordable volumetric FCMs with the 3 predicate FCM systems. CD4+ T-lymphocyte counts on blood samples from 150 HIV-1-infected Thai patients were determined in parallel by 5 FCM systems: the 2 single-platform volumetric FCM systems, Guava and CyFlowgreen; the 2 standard single-platform bead-based systems (2-color FACSCount and the TriTEST/TruCOUNT tube using a FACSCalibur FCM); and the dual-platform TriTEST system. Correlation and agreement were analyzed using linear regression and Bland-Altman analysis. Results from these 2 volumetric systems gave similar results and excellent correlation: R2 > 0.93; mean biases ranged from +6.3 to +24.1 cells per microliter more for the Guava. In contrast, the CyFlowgreen showed the lowest values with R2 > 0.97; mean biases ranged from −9.8 to −27.6 cells per microliter. This indicates that the absolute CD4+ T-lymphocyte counts determined by CyFlowgreen are <FACSCount<DP TriTEST<TriTEST/TruCOUNT<Guava. Although the use of these 2 volumetric FCMs could make CD4+ T-lymphocyte enumeration more affordable in resource-poor settings, variations among these systems should be considered if these are to be interchanged.

New BD FACSCount™ CD4 reagent system for simultaneous enumeration of percent and absolute CD4 T‐lymphocytes in HIV‐1‐infected pediatric patients

Absolute CD4+ T‐lymphocyte counts are used in the initiation and monitoring of antiretroviral therapy in HIV‐infected patients. Becton Dickinsons (BD) FACSCount™ system was introduced 12 years ago as a dedicated instrument for enumeration of absolute CD4+ T‐lymphocytes. However, this system does not provide percent CD4+ T‐lymphocyte that is the required monitoring parameter in pediatric patients. We evaluated a new BD FACSCount CD4 software and reagents for simultaneous percent and absolute CD4+ T‐lymphocytes in HIV‐infected blood.

Platelet activation and platelet–leukocyte interaction in β-thalassemia/hemoglobin E patients with marked nucleated erythrocytosis

Patients with thalassemia, an inherited hemolytic anemia, have increased risk of hypercoagulable complications. A whole blood flow cytometric (FCM) method has been used for studies of platelet activation and platelet–leukocyte aggregation in these patients. However, this FCM method presents technical difficulties because of the high proportion of immature red blood cells (RBCs) in these patients. A protocol for the simultaneous measurement of platelet activation and their aggregation with leukocyte populations in whole blood using four-color FCM which excluded immature RBC was devised, and evaluated for the evaluation of platelet function in patients with β-thalassemia/hemoglobin E (HbE). Whole blood from these patients and from healthy volunteers was stained for platelet activation and platelet–leukocyte aggregates using anti-CD42a, anti-CD62P, anti-CD45 and glycophorin A (GPA) conjugated with different fluorochromes. Our FCM method is simple, effective and based on the assumption that GPA is present on all immature RBCs, but is not expressed on CD45+ leukocytes. Results from the studies showed that blood samples from these patients contained a high frequency of circulating activated platelets (CD42a+/CD62P+) when compared to samples from healthy individuals. The percentage of platelet–neutrophil, platelet–monocyte—but not platelet–lymphocyte—aggregates were also elevated in both thalassemia genotypes with marked increase in patients who had undergone splenectomy. These findings suggest that platelets adhere to neutrophils and monocytes are activated which support the clinical observation that splenectomized thalassemia patients have an increased risk of arterial or venous thrombotic manifestations.

Clinical evaluation of a simple image cytometer for CD4 enumeration on HIV‐infected patients

Affordable, easy‐to‐use, and reliable CD4+ T lymphocyte enumeration systems are needed in resource‐constrained settings to monitor HIV.

Pinostrobin from Boesenbergia pandurata Is an Inhibitor of Ca2+-Signal-Mediated Cell-Cycle Regulation in the Yeast Saccharomyces cerevisiae

Upon searching plant extracts for inhibitors of the Ca2+ signaling pathway using the zds1Δ-yeast proliferation based assay, a crude rhizome extract of Boesenbergia pandurata was found to be strongly positive, and from this extract pinostrobin, alpinetin, and pinocembrin chalcone were isolated as active components. Further biochemical experiments confirmed that pinostrobin possesses inhibitory activity on the Ca2+ signals involved in the control of G2/M phase cell cycle progression in Saccharomyces cerevisiae.

The Use of Glutaraldehyde-Fixed Chicken Red Blood Cells as Counting Beads for Performing Affordable Single-Platform CD4+ T-Lymphocyte Count in HIV-1-Infected Patients

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, r2 > 0.99; absolute counts, r2 > 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/μL) with much lower bias for CD4+ T-lymphocyte counts <200 cells per microliter (LOA: −31.92 to +22.95 cells/μL). The use of CRBCs is comparable with the use of commercial microbeads. This has resulted in major cost savings to resource-limited countries where the health care system is under increasing pressure to operate cost effectively. This can greatly facilitate and ensure the success of the ongoing antiretroviral therapy program in these countries.

Flow cytometric quantitation of opsonophagocytosis and intracellular killing of Candida albicans using a whole blood microassay

Flow cytometric assays for quantifying polymorphonuclear leucocyte (PMN) function involve peripheral blood fractionation methods. However, most are of limited use for conditions like pediatrics and thalassemia, as they may require large blood volumes or complicated by red blood cell contamination. In whole blood assay, 500‐μl aliquots of normal or thalassemic whole blood were incubated with bis‐carboxyethyl‐carboxyfluorescein pentaacetoxymethylester (BCECF‐AM)‐labeled Candida albicans cells and phycoerythrin‐conjugated anti‐CD13 monoclonal antibody for PMNs. Phagocytosis was quantitated by gating on CD13 label PMNs and determining the frequency of phagocytosed yeast using flow cytometry. The killing activity was quantitated by estimating the number of viable fluorescence retaining yeast cells liberated following lysis of PMNs. In normal control or thalassemia samples, nonphagocytosed yeast and PMNs with or without phagocytosed yeast were readily distinguished by two‐color dot plot, permitting phagocytosis estimates. Viable and nonviable yeast killed by whole blood PMNs were distinguished by side scatter and fluorescence, permitting killing estimates. The patterns seen using whole blood was similar to that seen with fractionated PMNs. Data from thalassemia patients showed similar opsonophagocytosis but slightly decreased intracellular killing of yeast cells when compared with normal subjects. This assay provides an alternative method to assess PMN function in small blood samples, which could be especially useful in conditions like thalassemia and pediatric patients.

Characterisation and immuno-stimulating activity of polysaccharides from Thai medicinal plants

Water-soluble polysaccharides were isolated from the tubers of Butea superba Roxb. and Pueraria candollei Wall. Ex Benth. var. mirifica (Shaw et Suvat.) C. Niyomdham, the leaves of Centella asiatica (L.) Urb, Ocimum basilicum L., Psidium guajava and Andrographis paniculata (Burn. f.) Nees, the stems of Cymbopogon citratus (Stapf ExG), and the fruits of Psidium guajava and Scaphium scaphigerum. The immunological impacts of the polysaccharides on T-lymphocyte proliferation in vitro was investigated by flow cytometric (immunofluorescence) analysis using staphylococcal enterotoxin B (SEB) as a positive control. It was found that the polysaccharides enhanced T-lymphocyte proliferation, ranging from 4.5 to 27.0% at a concentration of 100 µg mL−1, while the activity of SEB was 13.3%. The medicinal plants showing the highest immuno-stimulating activity were the tubers of Butea superba Roxb. The water-extracted tubers contained 60.0% (w/w) carbohydrates with 6.6% (w/w) uronic acid. The major constituent monosaccharides of the tubers were 28.2 mol% galactose, 10.5 mol% arabinose and 36.4 mol% glucose.