Reticulocyte analysis in iron deficiency anemia and hemolytic anemia.

Reticulocyte analysis was studied in 28 anemic patients, 15 with iron deficiency anemia (IDA), and 13 with hemolytic anemia including 9 glucose 6 phosphate dehydrogenase deficiency (G6PD def.), and 4 with G6PD def. combined with HbE trait or alpha thalassemia trait (alpha thal trait). The reticulocyte analysis among these patients showed the increased number of reticulocyte percentage with low degree of maturation in both IDA and G6PD def. patients. The significantly decreased reticulocyte hemoglobin content (CHr) was found in IDA (CHr = 21.74 +/- 4.61 pg in IDA vs 28.41 +/- 1.34 pg in normal; p-value = < 0.0001), whereas, increased CHr was found in G6PD def. patients. In addition, the G6PD def. patients also showed a significant increase in mean corpuscular reticulocyte volume (MCVr) when compared to normal (MCVr = 132.0 +/- 8.39 fl. in G6PD def. vs 110.39 +/- 5.09 in normal; p-value = < 0.0001). However, a significant decrease in MCVr was found in IDA patients (MCVr = 95.89 +/- 8.57 fl.; p-value = < 0.0001 vs normal). From this study, we can suggest that the reticulocyte hemoglobin content (CHr) and mean corpuscular reticulocyte volume (MCVr) are the important defects in patients with iron deficiency anemia.

Reticulocyte analysis in systemic lupus erythematosus and chronic renal failure using flow cytometry.

The number and maturation of circulating reticulocytes were measured in patients with systemic lupus erythematosus (SLE) and chronic renal failure (CRF) using an automated hematological analyzer (Technicon H*3 RTX) for their erythropoietic activities. Both SLE and CRF patients had increased reticulocyte numbers with a low degree of maturation. The SLE patients had no changes in mean reticulocyte corpuscular volume (MCVr) as compared to normal subjects (110.20 +/- 15.43 fl. in SLE and 110.39 +/- 5.09 fl. in normal), whereas CRF patients had significantly increased mean corpuscular reticulocyte volume (MCVr = 120.99 +/- 8.09 fl., p-value = 0.0019 as compared with normal). Three cases of SLE with nephrotic syndrome (NS) had high degree of MCVr (113.4, 125.0 and 133.1 fl., respectively). The renal involvement in SLE patients and CRF patients may associate with increased reticulocyte corpuscular volume.

Thalassemic serum inhibits endothelial cell mitosis in vitro.

Human umbilical vein endothelial cells were cultured in vitro using Iscove's Modified Dulbecco's Medium (IMDM) supplemented with either pooled normal human serum, or pooled thalassemic serum, or autologous umbilical cord serum, or fetal bovine serum. The mitotic activity was determined under the inverted phase contrast microscope and the number of mitotic cells was counted. Our results showed that the mitotic cells decreased in endothelial cell culture with thalassemic serum as compared with normal human serum, autologous umbilical cord serum or fetal bovine serum. The percentage of mitotic cells decreased on day 3 in the presence of beta-thalassemia/HbE serum from both splenectomized and non-splenectomized patients as compared with normal or autologous umbilical cord serum. In the presence of alpha-thalassemic serum, a similar outcome was also observed. From this study we can conclude that the thalassemic sera might contain factors which affect the endothelial cell growth and proliferation by inhibiting mitosis in vitro.

Possible evidence of endothelial cell activation and disturbance in thalassemia: an in vitro study.

Activation of vascular endothelium is considered as an important facet of inflammation, thrombosis, and vasculitis. Activated endothelial cells express a number of immunologically relevant surface markers which are not detected in dormant condition. These surface markers on endothelial cell may involve in adhesion reaction and migration of blood cell components. We demonstrated increased level of the soluble adhesion molecules in circulating blood of both alpha- and beta-thalassemic patients. These adhesion molecules are theoretically known to be released from endothelial cells. The adhesion molecules included soluble Intercellular Adhesion Molecule-1 (sICAM-1), soluble E-Selectin (ELAM-1), soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1), and von Willebrand Factor (vWF). The levels of these adhesion molecules were measured in serum from 32 thalassemic patients and 10 control healthy subjects. As compared to normal, increased sICAM-1 was found in beta-thal/HbE patients with non-splenectomy; BE-NS (p = 0.002), increased ELAM-1 in beta-thal/HbE patients with splenectomy; BE-S (p = 0.01) and HbH with Hb Constant Spring; HbH/CS (p = 0.001), and increased sVCAM-1 in BE-NS; (p = < 0.0001) and BE-S (p = 0.002). Significant increase in von Willebrand Factor (vWF), a marker for endothelial cell, was shown in BE-S (p = 0.04) as compared to normal. Adhesion molecules were also markedly demonstrated in the supernatant of in vitro culture of human vascular endothelial cell in the presence of 30% thalassemic serum, and these adhesion molecules were also detected on the surface of the cells by using the technic of laser scanning confocal microscope and direct immunofluorescence.

Malaria protection in hereditary ovalocytosis: relation to red cell deformability, red cell parameters and degree of ovalocytosis.

In the culture of red cells with Plasmodium falciparum, erythrocytes from both Thai patients and subjects (patient's parents) with hereditary ovalocytosis have a protective effect against malarial infection. High percentage of ovalocyte (75-100%) was found in patients whereas their parents had lower percentage (25-50%). Invasion index (II) and multiplication ratio (MR) of P. falciparum in these abnormal red cells from the patients were significantly decreased as compared to those in normal red cells (patients: II = 1.52 +/- 0.91, MR = 8.83 +/- 6.73; normal subjects: II = 4.45 +/- 1.51, MR = 25.23 +/- 6.25). This suggests that the red cells from these patients had significant degree of malaria protection. The significant protection was also shown in red cells from the parent group (II = 1.86 +/- 0.81, MR = 15.69 +/- 3.50). Although the parents had lower ovalocyte percentage, degree of protection against malaria parasite was as effective as those found in patients with high ovalocytic red cells. This has been confirmed by statistical analysis showing nonsignificant difference in II value between the two groups. In contrast, red cells of both groups had poor deformability (deformability index, DI) as compared to the normal group. No statistically different DI values were demonstrated between the two. This indicates that poorly deformable red cells, not their ovalocytic shape, make a significant contribution to limitation of malaria parasite invasion. The MR values in patients were less than those found in the parent group but statistical analysis showed no significant difference. Reduced MR values were found with increased numbers of microcytic, hyperchromic and hypochromic red cells in patients.

Dengue viruses induce cell proliferation and morphological changes of endothelial cells.

Replication of dengue viruses (type 1, 2, 3 and 4) in vitro in endothelial cells from human umbilical cord vein was demonstrated by virus titers and immunofluorescent antibody studies. Both showed highest peak at Day 6 after inoculation and declined to origin at Day 14. Some of the cultured endothelial cells detached from the culture well. Most of these floating cells were rarely viable as shown by failure in trypan blue exclusion whereas the adhering cells are mostly viable. More frequent and higher intensity of immunofluorescent positive cells were found in the detached cells as compared to adhering cells. The virus titers in the supernatant and in the adhering cell population were comparable, although floating cells were maximally 26.2% of the total cultured endothelial cells. Many floating cells and occasional adhering cells had numerous blebs on their surface. Endothelial cell proliferation was markedly increased after virus inoculation as compared with the control. Increased number of mitotic cells was also observed in the dengue virus-endothelial cell culture. Comparing among the four types, dengue type 4 induced highest peaks of cell proliferation and cell mitosis at Day 10 after inoculation. Dengue type 2 had the highest virus titers both in adhering cells and in supernatant at Day 6 as compared with other types.

Surface and total tissue factor activity of endothelial cells.

With a technic that was developed by us, we found that normal human umbilical vein endothelial cells (HUVEC) in culture characteristically had very little tissue factor (TF) activity either on the surface or in the cells which had been disrupted. In the presence of endotoxin (E. coli O26:B6), a trigger for thrombosis in septicemic patients, we could not detect an increased TF activity of HUVEC on its surface. However, an increase in TF (total TF) was detected after disruption of the cells. The increase in total TF was dose-dependent. Endotoxin at the concentration of 10 micrograms/ml caused around 5 fold increase in total TF activity compared to that of HUVEC in the absence of endotoxin.

Alterations and pathology of thalassemic red cells: comparison between alpha- and beta-thalassemia.

Two main types of thalassemia have been categorized according to defective production of the globin gene ie alpha-thalassemia and beta-thalassemia. We report different red cell abnormalities between these two types. The study included 139 thalassemic patients including 91 patients with hemoglobin (Hb) H disease (52 cases with the classical genotype and 39 cases with Hb Constant Spring) and 48 were beta-thalassemia/Hb E disease. The deformability index of thalassemic red cells measured by laser diffractometer was significantly lower than that of normal red cells. Increased susceptibility of the thalassemic red cells to monocyte phagocytosis was markedly noted. Few sialic acid molecules were scattered on red cell surface of thalassemic red cells. Reticulocytes with delayed maturation stage were also observed in thalassemia indicating enhanced release from the bone marrow. The alpha-thalassemic red cells had relatively better deformability, increased susceptibility to phagocytosis, reduced sialic acid content and greater degree irregular distribution of sialic acid on red cell surface as compared to beta-thalassemic red cells. The alpha-type with hemoglobin Constant Spring (Hb CS) had increased percentage of reticulocyte and young reticulocyte (high fluorescent intensity) as compared to beta-thalassemic red cells. The different abnormalities between alpha- and beta-thalassemic red cells may lead to different mechanism of red cell destruction and different severity of the disease.

Dengue virus and endothelial cell: a related phenomenon to thrombocytopenia and granulocytopenia in dengue hemorrhagic fever.

Adhesion to endothelial cells by blood cells was assessed by measuring the cell number of each blood cell component in the supernatant after exposing blood cells to dengue-infected endothelial cells for 0, 10, 20 and 30 minutes. White blood cells, neutrophils, lymphocytes, platelets, and large lymphocytes or large unstained cells (LUC) preferentially bound to dengue-infected endothelial cells as compared to the control endothelial cells. P values were 0.0096 for total leukocytes and platelets, 0.006 for lymphocytes, and 0.001 for neutrophils and LUC. Monocytes basophils and eosinophils had no interaction with dengue-infected endothelial cells. The increased binding of neutrophil and platelet to endothelial cell may explain neutropenia and thrombocytopenia in DHF patients.

Thalassemic serum impairs endothelial cell growth in vitro.

Endothelial cells cultured for 3 days in the presence of hemoglobin H pooled sera had significantly decreased cell proliferation compared to those in normal serum. Inhibition was demonstrated at a concentration of 20% pooled serum in the cultured medium. Further decrease was shown in the presence of 30% pooled hemoglobin H sera. Sera from two genotypes of Hb H disease (alpha-thal 1/alpha-thal 2 and alpha-thal 1/Hb Constant Spring) had the same degree of inhibitory effect. Pooled sera from beta-thal/Hb E patients (both splenectomized or nonsplenectomized cases) had no such inhibitory effect. However, at day 4 and 5, the growth pattern relatively declined. Bilirubin at a concentration greater than 4.0 mg% in the medium 199 also caused significant decrease in cell proliferation. Since the diluted Hb H serum had bilirubin less than 4.0 mg%, the inhibitory effect of the pooled HbH serum is thus not due to effect of bilirubin. The difference between HbH and beta-thal/HbE sera in terms of inhibition of endothelial cell proliferation is the new finding that needs further investigation to explain vascularization and hemostasis in the patients of these two genotypes.

Red blood cell and platelet induced differentiation of endothelial cells into capillary-like structure.

We cultivated endothelial cells of human umbilical vein origin in the presence of red blood cells and platelet-rich plasma, and observed the phenomena which occurred in the petri dish under phase contrast microscopy. Many small particles were observed after an overnight incubation. We washed the dish two times, then added thrombin to the dish. The network of thread-like strands appeared within 10 to 20 minutes of the addition, and at the same time the small particles adhered on the surface of the strands, swelled and fused gradually to cover the surface of the strands completely. Within 30 to 60 minutes the network of the strands changed into a capillary-like structure. These phenomena were not observed if we omitted red blood cells or platelet-rich plasma. Studies by transmission electron microscopy revealed that the inner surface of the lumen of the structure was covered with cells. The cells isolated from the lumen by trypsin grew to confluence in the conventional culture medium, and showed vWF antigen on their surface. These observations indicated that the method described is useful for in vitro study of angiogenesis.

Interaction between endothelial cells and thalassemic red cells in vitro.

Erythrocytes from 45 patients with thalassemia and/or hemoglobinopathies were studied for their cytoadherence property to the vascular endothelial cells in vitro. In plasma free medium, erythrocytes from patients with beta-thal/Hb E both splenectomized and nonsplenectomized, HbH diseases (alpha-thal 1/alpha-thal 2 and alpha-thal 1/Hb Constant Spring genotypes) and homozygous Hb E subjects bind to endothelial cells at a greater number as compared to the binding cell number of normal erythrocytes (p-value < 0.05 in all types). Addition of autologous platelet-rich plasma or whole blood to the culture system causes further increase in the number of adhering beta-thalassemia red cells. Platelet-rich plasma had more enhancement effect than the whole blood. However, no such enhancement of both platelet-rich plasma and whole blood was demonstrated in the culture of normal or alpha-thalassemia erythrocytes. Increased binding between red cells and endothelial cells may contribute to the greater risk of vascular occlusion in thalassemic patients.

An unusual adhesion between red-cells and platelets in falciparum malaria.

A Thai female patient infected with P. falciparum had 80 per cent P. falciparum infected red cells at ring stage in the peripheral blood smear. The complications included anemia, thrombocytopenia, acute renal failure and pulmonary edema. A marked decrease in platelets number, low hemoglobin, low hematocrit and decreased red blood cell count were detected. More than 70 per cent of total platelets detected in the blood smear were binding to parasitized red blood cells. The number of binding platelets declined with decreasing per cent parasitized red cells. It was also noted that some platelets (10-20%) adhered to nonparasitized red cells. An increased number of large lymphocytes was shown by increased numbers of large unstained cells (LUC) by H* 1 automated analyzer. The peripheral blood smear showed abnormal binding of platelets to the infected red cells more frequently than to non infected red cells and free platelets on the day of high parasitemia. This abnormal phenomena was related to the number of platelets in the circulation. When the parasitized red cells were not detected in the blood smear, the number of platelets in the circulation had returned to normal.

Cytometric analysis of paroxysmal nocturnal hemoglobinuria erythrocytes.

Erythrocytes from six cases of paroxysmal nocturnal hemoglobinuria (PNH) were analysed by the H*1 hematology analyser using laser inspection for each individual red cell. The computer program categorized the red cells into 9 groups based on the data of red cell volume and intracellular hemoglobin concentration. The only 46.3 +/- 10.9 per cent (Mean +/- SD) of PNH red cells were normochromic normocytic. Hypochromia (hemoglobin concentration less than 28 g/dL) with normal red cell volume (between 60 to 120 fL) were found in 22.8 +/- 19.7 per cent and with large cell volume (greater than 120 fL) = 14.9 +/- 4.5 per cent of total red cells. Large cells with normal hemoglobin concentration were found in 12.9 +/- 13.2 per cent. The red cells had increased heterogeneity in red cell volume measured as red cell distribution width (RDW), Mean +/- SD = 23.6 +/- 2.3 per cent and increased heterogeneity in their hemoglobin concentration distribution with (HDW) (3.4 +/- 0.5 g/dL). The high RDW was resulted from increased number of varying size of macrocytes and the high HDW was caused by the increased number of hypochromic red cells. Heterogeneity in PNH red cell population described in this study has been postulated to relate with the clonal abnormality of the PNH red cells.