Ahnond Bunyaratvej

Platelet Counting by the RBC/Platelet Ratio Method: A Reference Method

The International Council for Standardization in Haematology (ICSH) and the International Society of Laboratory Hematology (ISLH) recommend the counting of specifically labeled platelets relative to the RBCs with a fluorescence flow cytometer, together with an accurate RBC count determined with a semiautomated, single-channel aperture-impedance counter as a reference method for the enumeration of platelets. Fresh EDTA-anticoagulated venous blood specimens are measured within 4 hours of the draw. The specimen is prediluted (1:20) and the platelets labeled with two monoclonal antibodies specific to a cluster of differentiation common to all platelets. A final 1:1,000 dilution is made and at least 50,000 events with a minimum of 1,000 platelet events are counted with a flow cytometer to determine the RBC/platelet ratio. The platelet count is then calculated from this ratio and the RBC concentration of the original blood specimen.

Indices of Iron status in continuous ambulatory peritoneal dialysis patients

Data for iron-status indices in continuous ambulatory peritoneal dialysis patients are limited. The reliability of commonly used indices for the diagnosis of iron-deficiency anemia in peritoneal dialysis patients is still unknown. To study diagnostic values of iron-status indices, including serum ferritin, transferrin saturation, reticulocyte hemoglobin content, and bone marrow-stainable iron, 21 stable anemic peritoneal dialysis patients who have been treated with erythropoietin and oral iron supplementation for more than 3 months were enrolled in this study. The mean age was 51.4 +/- 2.9 years; dialysis duration, 28.7 +/- 5.1 months; initial hemoglobin, 8.4 +/- 0.2 g/dL; erythropoietin dosage, 71 +/- 2 micro/kg/wk; serum albumin, 3.5 +/- 0.1 g/dL; intact parathyroid hormone (PTH), 233 +/- 44 ng/mL; serum ferritin, 643 +/- 135 ng/mL; transferrin saturation, 33.93% +/- 3.9%; and reticulocyte hemoglobin content, 31.6 +/- 4 pg. Bone marrow aspiration was performed in all patients to determine marrow iron content and exclude hematological disorders. All patients were treated with 1, 000 mg of intravenous ferric saccharate infusion in two divided doses more than 1 week apart. Patients who responded to the iron infusion within 3 months by increasing serum hemoglobin of greater than 1 gm/dL more than baseline were defined as being functional iron deficient before the intravenous iron infusion. Serum ferritin, transferrin saturation, and reticulocyte hemoglobin content were followed serially after iron infusion. Fifteen patients (71.4%) responded to the iron administration, indicating iron deficiency. Nine of 13 (69%) patients with the presence of bone marrow-stainable iron still responded to intravenous iron supplementation, suggesting functional iron deficiency. Absence of bone marrow-stainable iron was not a sensitive marker for the diagnosis of iron deficiency, 25% sensitivity. No single value of iron-status indices that can definitely exclude iron-deficiency anemia in peritoneal dialysis patients was found. Therefore, failure to increase hemoglobin concentration after intravenous iron administration should be shown before excluding iron-deficiency anemia as a cause of poor erythropoietic response to erythropoietin therapy.

Exploring stemness gene expression and vasculogenic mimicry capacity in well- and poorly-differentiated hepatocellular carcinoma cell lines

Vasculogenic mimicry (VM) is the phenomenon where cancer cells mimic endothelial cells by forming blood vessels. A stem cell-like phenotype has been proposed to be involved in this tumor plasticity. VM seems to correlate with metastasis rate, but there have been no reports on the effects of pro-metastatic and pro-angiogenic factors or hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) on VM formation of hepatocellular carcinoma (HCC) cells. Here, we determine VM capacity and expression of stemness genes (Oct4, Sox2, Nanog and CD133) in well- and poorly-differentiated HCC cell lines. The poorly-differentiated cell line SK-Hep-1 with mesenchymal features (high invasiveness and expressing Vimentin, with no E-cadherin) could form VM in vitro, while the well-differentiated cell line HepG2 did not form VM. There was no correlation between expression of stemness genes and intrinsic VM capacity. However, HGF but not VEGF, could induce VM formation in HepG2, concomitant with epithelial-mesenchymal transition (EMT), de-differentiation and increased expression of stemness genes. Our results show that the role of stemness genes in VM capacity of HCC cells is likely to depend on differentiation status.

Defective spectrin dimer self‐association in thalassemic red cells

The relative proportions of spectrin tetramer and dimer forms extrated from red cell membranes in a low ionic strength buffer at 4°C were determined for 15 normal subjects, 27 subjects with α‐thalassemia (7 α‐thalassemia trait, 9 Hb H disease (α‐thal 1/α‐thal 2) and 11 Hb H with Hb Constant Spring (CS), 23 subjects with β‐thalassemia (6 β‐thalassemia trait, 5 homozygous β‐thalassemia, 11 β°‐thalassemia with Hb E and 1 β +‐thalassemia with Hb E), 6 subjects with Hb E (2 homozygous and 4 carriers) and 1 subject with combined α‐thal 1/Hb CS and Hb E (AE Barts disease). In all subjects (except carriers of Hb E and 1 splenectomized case of β°‐thal/Hb E) spectrin dimer forms were elevated when compared to levels in normal controls, but there were no significant differences between carrier and disease forms. Conversion of spectrin dimers to tetramers at 30° C was reduced in the thalassemic subjects with disease but was within normal range for thalassemic carriers.

Erythrocyte Volume and Haemoglobin Concentration in Haemoglobin H Disease: Discrimination between the Two Genotypes

Erythrocyte volume and haemoglobin concentration in individual red cells from 62 patients with Hb H disease: 37 with H genotype (alpha-thalassaemia 1/alpha-thalassaemia 2) and 25 H/CS genotype (alpha-thalassaemia 1/Hb Constant Spring) were measured using the H*1 haematology analyser. All 25 cases with H/CS genotype, the more severe genotype, had microcytes (red cells with a volume smaller than 60 fl) less than 35% and hypochromic red cells (red cells with haemoglobin concentration less than 28 g/dl) more than 35%. A discriminant function, the ratio between the percentage of hypochromic red cells and the percentage of microcytes (Hypo/Micro), was proposed. Most of the H/CS patients (76%) had Hypo/Micro greater than 2.5 whereas those of H patients (82%) were below 2.5. Red cell volume histograms were also characteristically different between the two genotypes: the H/CS had a peak between 60 and 90 fl while the H genotype showed a peak at or very close to 60 fl, indicating a greater degree of microcytosis. Increased hypochromia with a slight decrease in cell size of H/CS red cells suggests that the poor degree of haemoglobinization has no linkage or very little role in disturbing the synthesis of membrane proteins and their assembly to the plasma membrane.

Inhibitory effect of β°-thalassaemia/haemoglobin E erythrocytes on Plasmodium falciparum growth in vitro

Abstract The growth of Plasmodium falciparum in erythrocytes from individuals with β∘-thalassaemia or haemoglobin (Hb) E, or both, was assessed in vitro . A significant inhibitory effect on the growth of the parasite was found only with erythrocytes from individuals doubly heterozygous for β∘-thalassaemia and HbE. The inhibitory effect was particularly marked with erythrocytes from splenectomized β∘-thalassaemia/HbE patients. The protective effect was related to HbF, Hb levels and shape abnormalities of the variant erythrocytes.

In vitro transdifferentiation of corneal epithelial-like cells from human skin-derived precursor cells.

The damage of human corneal cells encounter with the problem of availability of corneal cells for replacement. Limitation of the source of corneal cells has been realized. An attempt of development of corneal epithelial-like cells from the human skin-derived precursor (hSKPs) has been made in this study. Combination of three essential growth factors: epidermal growth factor (EGF), keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) could demonstrate successfully induction of hSKPs to differentiation into corneal cells.The induced cells expressed the appearance of markers of corneal epithelial cells as shown by the presence of keratin 3 (K3) by antibody label and Western blot assay. The K3 gene expression of induced hSKPs cells as shown by reverse transcription-polymerase chain reaction (RT-PCR) technology was also demonstrated. The presence of these markers at both gene and protein levels could lead to our conclusion that the directional transdifferentiation of hSKPs cells into corneal epithelial cells was successfully done under this cell induction protocol. The finding shows a newly available stem cell source can be obtained from easily available skin. Cells from autologous human skin might be used for corneal disorder treatment in future clinical application.

Number and Maturation of Reticulocytes in Various Genotypes of Thalassaemia as Assessed by Flow Cytometry

Ineffective erythropoiesis is a prominent defect leading to anaemic status in thalassaemic patients. Reticulocyte enumeration in the peripheral blood is a non-aggressive method of measuring bone marrow erythropoietic activity. We used an automated reticulocyte counter (Sysmex R-3000) to determine the number and maturation level of circulating reticulocytes among various types of thalassaemia: non-splenectomized beta-thalassaemia/haemoglobin E (beta E) and splenectomized cases (beta E-S), classical haemoglobin H disease (H), haemoglobin H disease with haemoglobin Constant Spring (H/CS), homozygous haemoglobin Constant Spring (CS/CS), homozygous haemoglobin E (EE), heterozygous thalassaemics and other rare combinations. Haemoglobin H disease has a higher absolute count than beta-thalassaemia (beta E), indicating relatively better compensatory erythropoiesis in haemoglobin H disease. Those with CS genes (H/CS and CS/CS) have poorer reticulocyte maturation than any other type of thalassaemia with rather high absolute numbers, especially in H/CS. This indicates a severer degree of ineffective erythropoiesis in beta-thalassaemia (beta E), which reflects an insufficient rise in reticulocyte number in comparison with alpha-thalassaemia (H). The presence of haemoglobin Constant Spring is associated with abnormally low reticulocyte maturation due to enhanced erythrocyte production or direct effect of Constant Spring globin itself, both still unexplained with the current information. The splenectomized beta E has increased reticulocyte number and cells with high DNA content, probably nucleated red cells, designated as the upper particle count parameter. However, there is the same degree of reticulocyte maturation in non-splenectomized and splenectomized beta E patients, suggesting a role for splenic pooling of reticulocytes.

Haemoglobin-E trait and the clinical course of malaria in Thai soldiers

1. BENNETT JM, CATOVSKY D, DANIEL MT, et al. Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 1982: 189-199. 2. ECONOMOPOULOS T, TATHAKIS N, FOUDOULAKIS A, et al. Myelodysplastic syndromes: Analysis of 13 1 cases according to the FAB classification. Eur J Haematol 1987: 38: 338-344. 3. CAZZOLA M, BAROSI G, GOBBI PG, et al. Natural history of idiopathic refractory sideroblastic anaemia. Blood 1988: 7 1 :

Different Patterns of Intraerythrocytic Inclusion Body Distribution in the Two Types of Haemoglobin H Disease

Electron microscopic examination of intraerythrocytic inclusion bodies induced by methylene blue was carried out in 7 non-splenectomized patients with haemoglobin (Hb) H disease. In classical Hb H disease with the genotype of alpha-thalassaemia 1/alpha-thalassaemia 2 the inclusion bodies were mostly membrane-associated. In contrast, in Hb H disease of alpha-thalassaemia 1/Hb Constant Spring genotype the majority of inclusion bodies were in a floating stage. Possible mechanisms for the unexpected difference are discussed.