RESUMEN
The spun packed cell volume (PCV, hematocrit) is a key measurement on which are based hematology instrument calibration, reference range determination, and assignment of values to calibrators/controls. In 2001, the International Council for Standardization in Haematology (ICSH) recommended a Reference PCV method, which is fully traceable to the ICSH reference hemoglobin method. Because of its complexity, however, this method is impractical for occasional use in routine laboratories and is therefore intended primarily for use by manufacturers of capillary microhematocrit tubes, liquid calibrators, and multichannel analyzers. In response to the need for a simpler method--accessible to all routine laboratories--the ICSH offers this "Surrogate Reference" PCV procedure. It is traceable to the original ICSH Reference PCV method and is based on spun PCVs obtained using borosilicate capillary tubes with an already-known relationship to this reference procedure. This ICSH "Surrogate Reference" PCV method is substantially simpler, thus putting it within the reach of most routine hematology laboratories.
Asunto(s)
Hematócrito/normas , Recolección de Muestras de Sangre , Equipos y Suministros , Hematócrito/métodos , Humanos , Métodos , Estándares de ReferenciaRESUMEN
A convenient means of measuring apoptosis is described using an automated analyzer (MF-1; Sysmex), which normally provides rapid measurement of malarial parasites. By means of this MF-1 procedure, apoptotic cells exhibited characteristic changes of light scatter (size) and fluorescence (DNA content) relating to cell shrinkage and nuclear fragmentation of apoptosis.
Asunto(s)
Apoptosis/fisiología , Núcleo Celular/ultraestructura , Automatización/instrumentación , Automatización/métodos , Tamaño de la Célula , Supervivencia Celular , ADN/análisis , Citometría de Flujo/instrumentación , Citometría de Flujo/métodos , Células HL-60/citología , Humanos , Luz , Dispersión de RadiaciónRESUMEN
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.
Asunto(s)
Anemia Hemolítica/sangre , Anemia Ferropénica/sangre , Reticulocitos/fisiología , Análisis de Varianza , Femenino , Humanos , Masculino , Probabilidad , Recuento de Reticulocitos , Estadísticas no Paramétricas , TailandiaRESUMEN
The alternative application of an automated hematology analyzer, H*3 system, has been described for the detection of apoptosis. Apoptosis induction by the topoisomerase I inhibitor, camptothecin (CAM) on several cell lines is followed by typical morphological alterations. On the H*3 cytogram, measurement of CAM-treated cells revealed an increased population of cells with reduced size suggesting cell contraction during apoptosis. The decreased LUC/Lymph ratio also indicated the enhanced degree of apoptosis directly correlated with increasing CAM concentration and/or incubation period. Quantitative analysis shows a good correlation between the H*3 measurement and flow cytometry measurements of Annexin V-fluorescein isothiocyanate-labeled method. Thus, the H*3 measurement, under an appropriate adjustment, can be used as a rapid monitor for evaluating the degree of apoptotic changes in drug susceptibility testing of homogeneous cell samples.
Asunto(s)
Apoptosis , Citometría de Flujo/métodos , Apoptosis/efectos de los fármacos , Camptotecina/farmacología , Recuento de Células , Línea Celular , Inhibidores Enzimáticos/farmacología , Citometría de Flujo/instrumentación , Células HL-60 , Humanos , Inhibidores de Topoisomerasa I , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Fallo Renal Crónico/sangre , Lupus Eritematoso Sistémico/sangre , Reticulocitos , Anemia/sangre , Anemia/etiología , Estudios de Casos y Controles , Recuento de Eritrocitos , Índices de Eritrocitos , Citometría de Flujo , Humanos , Fallo Renal Crónico/complicaciones , Lupus Eritematoso Sistémico/complicaciones , Nefritis Lúpica/sangre , Nefritis Lúpica/complicacionesRESUMEN
OBJECTIVE: To evaluate the relationship between the status of serum vitamin A, E and hemoglobinopathies among Thai pregnant women. METHODS: This was a cross-sectional study in which serum vitamin A and E were assessed in 323 pregnant women with normal hemoglobin and 73 with hemoglobinopathies (47 with hemoglobin E and 26 with thalassemia) during the first trimester. RESULTS: There were no significant differences in the mean serum vitamin A, E concentrations and vitamin E/cholesterol ratio between pregnant women with normal hemoglobin and hemoglobinopathies, while confounding variables that might affect serum vitamin levels i.e. maternal age, gravida, BMI, gestational age, hematocrit, hemoglobin, mean corpuscular hemoglobin concentration and blood group were not different. CONCLUSION: The results of this study suggest that antenatal care in terms of micronutrients-vitamin A, E in Thai pregnant women with hemoglobinopathies should not be different from normal pregnant women.
Asunto(s)
Hemoglobinopatías/sangre , Complicaciones Hematológicas del Embarazo/sangre , Embarazo/sangre , Vitamina A/sangre , Vitamina E/sangre , Adulto , Estudios de Casos y Controles , Estudios Transversales , Femenino , Edad Gestacional , HumanosRESUMEN
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.
Asunto(s)
Anemia Ferropénica/diagnóstico , Hierro/metabolismo , Fallo Renal Crónico/complicaciones , Diálisis Peritoneal Ambulatoria Continua , Anemia Ferropénica/tratamiento farmacológico , Anemia Ferropénica/etiología , Eritropoyetina/uso terapéutico , Femenino , Humanos , Fallo Renal Crónico/terapia , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Proteínas RecombinantesRESUMEN
An attempt to overcome the uncertainties and errors of all processes in clinical laboratory has been done in systematic ways. To reach the stage of laboratory accreditation, quality and standard criteria in the laboratory must be developed. Two aspects of quality are considered as management and analysis. Input-output control is proposed to handle the process from the beginning of laboratory design until post analytical phase or the control at the outcome. The model of ten Ms including model, material, machine, man, mind, money, method, mechanism, measurement and menace and one O or outcome, is elaborated to cover pre-analytical, analytical and post-analytical phases. Laboratory accreditation is then an integral part of hospital accreditation with total quality management.
Asunto(s)
Acreditación , Laboratorios de Hospital/normas , Gestión de la Calidad Total/métodos , Humanos , Laboratorios de Hospital/organización & administración , Modelos Organizacionales , Estándares de Referencia , TailandiaRESUMEN
The International Council for Standardization in Haematology (ICSH), an international organization promoting international agreement on hematological testing, is now restructuring to strengthen its activities. In Asia, a diversity of testing methods exists and the resulting testing levels make it difficult to compare test results internationally among Asian countries. Fortunately, the ICSH is considering regionalizing its organization to 5 sub-societies to increase its activity, and we have been able to establish a new society, ICSH-Asia, under the ICSH umbrella.
Asunto(s)
Consejos de Planificación en Salud/organización & administración , Pruebas Hematológicas/normas , Hematología , Cooperación Internacional , Asia , Humanos , Estándares de ReferenciaRESUMEN
In Thailand, there are around 2,000 clinical laboratories in private and government hospitals, By the end of year 2004, all of these laboratories are required to use the same or comparable standard nationwide. Many laboratories are in the process of starting ISO/IEC Guide 25 for the fulfillment of laboratory accreditation. To run the standard system of hematology laboratories in Thailand, we have considered three main aspects: standard in process, method selection and academic interpretation. Because of the wide spectrum of blood diseases in Thailand: thalassemia, iron deficiency anemia and G6PD deficiency hemolytic disease, the analysis and interpretation of laboratory results using different technology are of great importance. National plan has thus set up in two direction, one for standard process and another for academic approach.
Asunto(s)
Pruebas Hematológicas/normas , Laboratorios de Hospital/normas , Garantía de la Calidad de Atención de Salud/organización & administración , Acreditación , Pruebas Hematológicas/instrumentación , Humanos , Cooperación Internacional , Laboratorios de Hospital/organización & administración , Personal de Laboratorio Clínico/educación , Personal de Laboratorio Clínico/provisión & distribución , Revisión por Expertos de la Atención de Salud , Estándares de Referencia , TailandiaRESUMEN
The results obtained with a WHO hemoglobin (Hb) colour scale were evaluated in a field study in Chibubur district in Java island by comparison with hemoglobin values obtained by an automated blood cell analyzer K-800 (Sysmex. Kobe, Japan). When the color scale test was performed following the instructions for use. Hb values observed were usually higher than the values obtained by the analyzer. Thirty microl blood was loaded on the filter paper and an 60 sec waiting period was used. The sensitivity of results obtained with the color scale was 23.3% (14/60), and specificity was 96.6% (58/60). We propose an additional testing method based on our results.
Asunto(s)
Anemia/diagnóstico , Hemoglobinometría/métodos , Niño , Color , Análisis Costo-Beneficio , Hemoglobinometría/economía , Hemoglobinometría/normas , Humanos , Indonesia , Valores de Referencia , Sensibilidad y Especificidad , Organización Mundial de la SaludRESUMEN
Hematology laboratory is generally required in the hospital. At the macroscale, hematology laboratories have served a large number of population. In Asia, more than 3,000 million people are potentially to use the hematology laboratory service, particularly the complete blood count. Since 1970s, automated technology has been introduced to Asia and as years passed by, technology diversity is increasing. However, there are considerable number of hematology laboratories that have no automated machine. They are still relied on manual technology which is still variable in spectrophotometer for hemoglobin determination, centrifuge for hematocrit and diluting pipet for cell counting. In particular, blood smear preparation and interpretation are very difficult to control for standardization from person to person and laboratory to laboratory. Different methodology and a large population in the huge geographical area in Asia, the agreement of standard criteria is greatly important. This report has shown strategy and action plan to reach the goal of hematology laboratory standardization in Asia.
Asunto(s)
Pruebas Hematológicas/normas , Cooperación Internacional , Laboratorios de Hospital/normas , Garantía de la Calidad de Atención de Salud , Asia , Humanos , Objetivos Organizacionales , Estándares de ReferenciaRESUMEN
Phospholipid asymmetry in the red blood cell (RBC) lipid bilayer is well maintained during the life of the cell, with phosphatidylserine (PS) virtually exclusively located in the inner monolayer. Loss of phospholipid asymmetry, and consequently exposure of PS, is thought to play an important role in red cell pathology. The anemia in the human thalassemias is caused by a combination of ineffective erythropoiesis (intramedullary hemolysis) and a decreased survival of adult RBCs in the peripheral blood. This premature destruction of the thalassemic RBC could in part be due to a loss of phospholipid asymmetry, because cells that expose PS are recognized and removed by macrophages. In addition, PS exposure can play a role in the hypercoagulable state reported to exist in severe beta-thalassemia intermedia. We describe PS exposure in RBCs of 56 comparably anemic patients with different genetic backgrounds of the alpha- or beta-thalassemia phenotype. The use of fluorescently labeled annexin V allowed us to determine loss of phospholipid asymmetry in individual cells. Our data indicate that in a number of thalassemic patients, subpopulations of red cells circulate that expose PS on their outer surface. The number of such cells can vary dramatically from patient to patient, from as low as that found in normal controls (less than 0.2%) up to 20%. Analysis by fluorescent microscopy of beta-thalassemic RBCs indicates that PS on the outer leaflet is distributed either over the entire membrane or localized in areas possibly related to regions rich in membrane-bound alpha-globin chains. We hypothesize that these membrane sites in which iron carrying globin chains accumulate and cause oxidative damage, could be important in the loss of membrane lipid organization. In conclusion, we report the presence of PS-exposing subpopulations of thalassemic RBC that are most likely physiologically important, because they could provide a surface for enhancing hemostasis as recently reported, and because such exposure may mediate the rapid removal of these RBCs from the circulation, thereby contributing to the anemia.
Asunto(s)
Membrana Eritrocítica/química , Eritrocitos/química , Fosfolípidos/química , Talasemia alfa/sangre , Talasemia beta/sangre , Anexina A5 , Membrana Eritrocítica/genética , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Colorantes Fluorescentes , Humanos , Microscopía Fluorescente , Fosfolípidos/genética , Talasemia alfa/genética , Talasemia beta/genéticaRESUMEN
The conventional approach to qualitative and quantitative analyses of hemoglobin (Hb) molecules for the diagnoses of hemoglobinopathies requires a combination of tests. We used an automated HPLC (VARIANT) system to study alpha-thalassemia and beta-thalassemia syndromes in Thailand. The beta-thalassemia short program is applicable to the diagnosis of alpha-thalassemia and beta-thalassemia disorders, including Hb H, EA Bart's disease, and EF Bart's disease, in adults, newborns, and fetuses. The system cannot quantify accurately certain Hb molecules, such as Hb H and Hb Bart's. The alpha-thalassemia short program was therefore developed and used to quantify Hb Bart's to detect alpha-thalassemia genotypes in cord blood. This automated HPLC system is an alternative approach to the diagnosis of complicated thalassemia syndromes in Thailand and Southeast Asia.
Asunto(s)
Hemoglobinopatías/diagnóstico , Diagnóstico Prenatal/métodos , Talasemia alfa/diagnóstico , Talasemia beta/diagnóstico , Adulto , Cromatografía Líquida de Alta Presión/métodos , Femenino , Sangre Fetal/química , Hemoglobina A2/análisis , Hemoglobinopatías/sangre , Hemoglobinas Anormales/análisis , Humanos , Recién Nacido , Fenotipo , Embarazo , Tailandia , Talasemia alfa/sangre , Talasemia beta/sangreRESUMEN
Hemoglobin Constant Spring (HbCS) is the most common nondeletional alpha-thalassemic mutation and is an important cause of HbH-like disease in Southeast Asia. HbCS variants have an almost normal mean cell volume (MCV) and the anemia is more severe when compared with other alpha-thalassemic variants. We explored the pathobiology of HbCS red blood cells (RBCs) because the underlying cause(s) of this MCV "normalizing" effect of HbCS and the more severe anemia are not fully explained. HbCS containing RBCs are distinctly overhydrated relative to deletional alpha-thalassemia variants, and the derangement of volume regulation and cell hydration occurs early in erythroid maturation and is fully expressed at the reticulocyte stage. Furthermore, the membrane rigidity and membrane mechanical stability of HbCS containing RBCs is increased when compared with HbH and alpha-thalassemia-1 trait RBCs. In seeking the cause(s) underlying these cellular alterations we analyzed membranes from HbCS and deletional alpha-thalassemic variants and found that in addition to oxidized beta-globin chains, oxidized alpha cs-globin chains are also associated with the membranes and their skeletons in HbCS containing RBCs. We propose that the membrane pathology of HbCS variants is caused by combination of the deleterious effects induced by membrane-bound oxidized alpha cs- and beta-globin chains. The membrane alterations induced by alpha cs chains are more akin to those induced by beta A-globin chains than those induced by the alpha A-globin chains that accumulate in the beta-thalassemias. Thus, each globin chain, alpha cs, alpha A, beta A, appears to produce its own form of membrane perturbation.
Asunto(s)
Membrana Eritrocítica/patología , Eritrocitos/patología , Hemoglobinas Anormales , Talasemia alfa/sangre , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Eritrocitos/ultraestructura , Humanos , TailandiaRESUMEN
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.
Asunto(s)
Transformación Celular Viral/fisiología , Virus del Dengue/fisiología , Endotelio/virología , División Celular/fisiología , Células Cultivadas , Endotelio/ultraestructura , Humanos , Factores de TiempoRESUMEN
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.
Asunto(s)
Eliptocitosis Hereditaria/sangre , Deformación Eritrocítica/fisiología , Malaria Falciparum/sangre , Humanos , Inmunidad Innata , Tailandia , Factores de TiempoRESUMEN
Thalassemia is an inherited hematological disorder which can generally be classified according to the affected globin imbalance (alpha- or beta-globin) into two main types, i.e. alpha-thalassemia and beta-thalassemia, respectively. There is a wide range of cellular abnormalities associated with thalassemic erythrocytes such as hypochromia, microcytosis, reduced cellular deformability and membrane oxidative damage. The red cell abnormalities lead to premature destruction with marrow erythroid hyperplasia and ineffective erythropoiesis. The abnormalities in thalassemic red blood cells have been found along the erythroid differentiation pathway other than the mature stage as previously shown in bone marrow erythroid precursors and in reticulocytes, the penultimate stage of erythroid differentiation. However, there is a lag in our understanding of the more primitive erythroid stages due to the difficult and hazardous marrow aspiration and heterogeneity of cells derived. We have utilized a novel method of Two-Phase Liquid Culture (TPLC) of beta-thalassemia/HbE erythroid precursors instead of conventional semisolid culture. This type of liquid culture can given higher cell yield with quite synchronous cell differentiation stages and easily be applied for other cellular analytical techniques. The peripheral blood mononuclear cells (PBMC) obtained from non-splenectomized and splenectomized beta-thalassemia/HbE patients were first cultured in medium supplemented with 5637 conditioned medium for a 6-day period (phase I) and then transferred to medium supplemented with recombinant human erythropoietin to allow the terminal differentiation of erythroid precursors (phase II). During the phase I or II, the cultured cells were periodically sampled to determine the cell number, cytocentrifuged on glass slides and stained with Wright stain for morphological assessment of their differentiation stages and analyzed flow cytometrically by staining with fluoresceinated anti-transferrin receptor (anti-CD71) and R-phycoerythrin-conjugated anti-glycophorin A. After assessment by flow cytometry, the remaining stained cells were cytocentrifuged on glass slides and photographed by a fluorescent microscope and a laser scanning confocal microscope. The results of morphological assessment, flow cytometric analysis and microscopic pictures will be presented.
Asunto(s)
Células Precursoras Eritroides/fisiología , Talasemia beta/sangre , Células Cultivadas , Eritroblastos/fisiología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , HumanosRESUMEN
Eighty-five patients who attended at Ramathibodi Hospital during November 1994 to June 1996 were investigated for thalassemia genotype, hemoglobin (Hb) typing and blood cell parameters. All patients were screened primarily for complete blood count using the Technicon H*3 automated hematology analyzer and Hb typing using the automated HPLC. Their genotypes were evaluated by in vitro gene amplification using primers for detection of common alpha-thalassemic genes found in the Thai population. We found 45 cases out of 85 were alpha-thalassemia trait with A2A typing, 10 were normal, 7 were alpha-thalassemia trait complicated by beta-thalassemia trait or HbE trait, 18 were HbH disease or HbH with Hb Constant Spring (HbH/CS), and 5 were AE Bart's disease. The alpha-thalassemia 1 trait had heterogeneity in red cell population as shown by increased red cell distribution width (RDW), the increased percent microcytic red cell (%Micro) and decreased mean cell volume (MCV). Red cell parameters in alpha-thalassemia 2 trait and HbCS trait were not significantly different from normal. The cases with coinheritance of alpha-thalassemia trait with beta-thalassemia trait or with HbE trait showed variation in their red cell parameters: one case showed less abnormal red cell parameters than those of uncomplicated alpha-thalassemia but the other two cases showed unimproved values. The homozygous alpha-thalassemia 2 showed similar red cell parameters to the alpha-thalassemia 1 trait. In conclusion, we can screen the alpha-thalassemia 1 trait and homozygous alpha-thalassemia 2 by using the simple red cell parameters such as the MCV and RDW; however, they must be confirmed for alpha-thalassemic genes. Unfortunately, red cell parameters of alpha-thalassemia 2 trait or HbCS trait were not different from those of normal subjects.
Asunto(s)
Eritrocitos/fisiología , Hemoglobina E/genética , Talasemia alfa/sangre , Talasemia beta/genética , Hemoglobinas/genética , Humanos , Tailandia , Talasemia alfa/genéticaRESUMEN
Presently genetic analyses for thalassemia types require relatively large amounts of heparinized blood (5 to 10 ml), and transport as well as degeneration of these sample is a problem in the developing world. We have developed a new method to simplify this procedure and obtain DNAs from small specimens. As experimental materials, thinly smeared blood on a glass slide or blood filtered with and adhered on polysthylene telephtalate (PST) fibers were used. These materials could be safely stored without interfering with DNA extraction for up to 3 months. The slide materials were digested with proteinase K, and DNA was extracted with Tris-EDTA-phenol:chloroform and precipitated with absolute ethanol. The PST specimens were washed with physiologic saline and treated in the same manner as described above. Products were easily amplified by PCR and digested with restriction endonucleases for beta thalassemia typing as well as for HLA-DQA1 gene typing. Results obtained by this method correlated well with previously reported incidences for thalassemia and HLA-DQA1 types in Thailand. This method can be used in the routine laboratory because it allows for stable and biosafe genetic analyses.