Clasificación y métodos de diagnóstico de las membranopatías / Classification and diagnostic methods of membranopathies

Introducción: Las membranopatías son anemias hemolíticas hereditarias debidas a anomalías cualitativas o deficiencias cuantitativas de las proteínas del citoesqueleto del glóbulo rojo. Objetivo: Actualizar el diagnóstico de las membranopatías con la inclusión de las últimas recomendaciones del comité de grupos de expertos a nivel nacional e internacional. Métodos: Se realizó una revisión de la literatura en inglés y español, a través del sitio web PubMed y el motor de búsqueda Google académico, de artículos publicados en los últimos cinco años. Análisis y síntesis de la información: Las enfermedades de mayor interés clínico son: la esferocitosis, la eliptocitosis y la estomatocitosis hereditaria. Estas en general se heredan con carácter autosómico dominante pero existen formas que se transmiten con carácter recesivo, sin descartar posible mutación de novo. Para su diagnóstico se utilizan pruebas que incluyen el estudio de la morfología de los glóbulos rojos, la fragilidad osmótica, la lisis de glicerol acidificado, la criohemólisis hipertónica, la prueba de unión a la eosina-5'-maleimida por citometría de flujo, la electroforesis en gel de poliacrilamida con dodecilsulfato sódico y la ectacitometría. Conclusiones: Las membranopatías pueden sospecharse de manera preliminar teniendo en cuenta algunas alteraciones de la morfología eritrocitaria, aunque el diagnóstico se basa en estudios familiares y otros de carácter confirmatorio de la enfermedad, como los estudios moleculares. Los profesionales de la salud que atienden a pacientes jóvenes con anemia deben considerar la posibilidad de una anemia hemolítica por trastornos de la membrana eritrocitaria(AU)

ABSTRACT Introduction: Membranopathies are inherited hemolytic anemias due to qualitative abnormalities or quantitative deficiencies of red blood cell cytoskeletal proteins. Objective: to update the diagnosis of membranopathies with the inclusion of the latest recommendations from the committee of expert groups at the national and international level. Methods: A review of the literature in English and Spanish was carried out, through the PubMed website and the academic search engine Google, in articles published in the last five years. Analysis and synthesis of information: The diseases of greatest clinical interest are: spherocytocis, elliptocytosis and hereditary stomatocytosis. These are generally inherited with an autosomal dominant character but there are forms that are transmitted recessively, without ruling out a possible de novo mutation. For its diagnosis, tests are used that include the study of red blood cell morphology, osmotic fragility, acidified glycerol lysis, hypertonic cryohemolysis, eosin-5'-maleimide binding test by flow cytometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis and ectacytometry. Conclusions: Membranopathies can be preliminarily suspected taking into account some alterations in erythrocyte morphology, although the diagnosis is based on family studies and others confirming the disease, such as molecular studies. Healthcare professionals caring for young patients with anemia should consider the possibility of hemolytic anemia due to red cell membrane disorders(AU)

Análise qualitativa do teste de fragilidade osmótica para amostras processadas a fresco ou após 24 horas de incubação a 37ºC / Qualitative analysis of the osmotic fragility test for samples processed fresh or after 24 hours of incubation at 37ºC

Objetivo: Analisar qualitativamente o teste de fragilidade osmótica (F.O.) para amostras a fresco ou após incubação a 37°C. Métodos: Foram processadas 20 amostras de sangue periférico, coletadas em duplicata com 5mL em cada tubo com heparina, de pacientes com solicitação de F.O. como exame de rotina para processamento a fresco e após incubação por 24 horas em banho-maria a 37°C, em 13 tubos com concentrações variáveis de 0,1% a 0,9% de NaCl. Resultados: Foram analisadas 20 amostras de pacientes em sua maioria do gênero feminino 17/20 (85%), com idades entre 3 meses a 75 anos, para realização do teste de F.O. A análise qualitativa dos resultados mostrou que 9/20 (45%) amostras tiveram resultado concordante entre os testes de F.O. para amostras a fresco e após incubação a 37°C. Dos resultados discordantes, 8/11 (72,7%) resultados mostram fragilidade dos eritrócitos à hemólise nas amostras a fresco e curva normal (sem hemólise) após incubação da amostra a 37°C. Outros 3/11 (22,3%) resultados apresentaram curva normal (sem hemólise) no teste com amostra à fresco e resistência à hemólise no teste com a amostra após incubação a 37°C. Com o teste de Extato de Fisher não mostrou diferença estatística (p=0,5743) para as amostras processadas a fresco ou após incubação a 37°C. Conclusão: O teste de F.O. se mostrou mais eficiente quando a amostra testada foi analisada após incubação por 24 horas a 37°C em banho-maria, contudo não houve diferença estatística para resultados processados a fresco ou após incubação a 37°C.

Objective: Qualitatively analyze the osmotic fragility test (O.F.) for samples fresh or after incubation at 37°C. Methods: Twenty peripheral blood samples were processed, collected in duplicate with 5 ml in each tube with heparin, from patients with O.F. request. as a routine examination for fresh processing and after incubation for 24 hours in a water bath at 37°C, in 13 tubes with varying concentrations of 0.1% to 0.9% NaCl. Results: Twenty samples of patients were analyzed, mostly female, 17/20 (85%), aged between 3 months to 75 years, for the O.F. test. The qualitative analysis of the results showed that only 9/20 (45%) samples had a consistent result between the F.O. tests for fresh samples and after incubation at 37°C. From the discordant results, 8/11 (72.7%) results show fragility of erythrocytes to hemolysis in fresh samples and normal curve (without hemolysis) after sample incubation at 37o C. Other 3/11 (22.3%) results showed normal curve (without hemolysis) in the test with fresh sample and resistance to hemolysis in the test with the sample after incubation at 37o C. With the Fisher Extact test showing no statistical difference (p=0.5743) for samples processed fresh or after incubation at 37°C. Conclusion: The O.F. proved to be more efficient when the tested sample was analyzed after incubation for 24h at 37°C in a water bath, however, there was no statistical difference for results processed fresh or after incubation at 37°C.

Stability of refrigerated whole blood samples for osmotic fragility test

ABSTRACT Background: The osmotic fragility test (OFT), conventionally used for assisting the diagnosis of many erythrocyte disorders, is a manual and time-consuming analysis not daily performed in many medical laboratories. This study was aimed at defining the stability of whole blood samples used for assessing erythrocyte osmotic resistance. Methods: Twenty-one consecutive routine whole blood samples collected into 5.4 mg K2EDTA were tested immediately after collection (day 0) and at different time intervals afterward (day 1, 2, 3, 4, 7, 10 and 14) after storage at 4 °C. The OFT was performed with the Osmored Monotest (1.3% glycerol; Eurospital, Trieste, Italy). Results at the different time points were compared with those obtained at day 0 and with the reference change value (i.e., 33%). Results: The median value of both hyperosmolar and hyposmolar resistance increased from baseline, reaching statistical significance at day 7 for hyperosmolar resistance and at day 1 for hyposmolar resistance, respectively. The median relative increase of hemolysis percentage values become greater than the reference change value at day 3 for hyposmolar resistance, while this limit was never overcome for hyperosmolar resistance. A significant inverse association was found between the mean increase in hyperosmolar resistance and the baseline value of hyperosmolar resistance (r = −0.92), mean corpuscular volume (MCV; r = −0.46) or mean corpuscular hemoglobin (MCH; r = −0.44), as well as between the mean increase in hyposmolar resistance and the baseline value of hyposmolar resistance (r = −0.86), or patient age (r = −0.56). Conclusions: The sample stability seems critical for the OFT. Whole blood specimens should not be stored refrigerated at 4 °C for >2 days before testing.

Use of erythrocytes in cytotoxicity and toxicity assays of medicinal plant extracts: analysis of their application and bibliometric study / Utilización de eritrocitos en ensayos de citotoxicidad y toxidez de extractos de plantas medicinales: análisis de su aplicación y estudio bibliométrico

Plant species have been used for therapeutic purposes since ancient times and are still in use today since these products represent a source of raw material for the production of phytotherapeutic formulations. Screening and investigation of plants with pharmacological potential require the evaluation of characteristics related to their action, efficacy and safety in different steps. Among these steps, pre- clinical trials are used to evaluate the properties of the test product in in vitro experiments, such as cytotoxicity assays. Within this context, this study consists of a bibliometric analysis of some in vitro cytotoxicity and toxicity assays in erythrocytes used during bioprospecting of medicinal plants. The results demonstrated the wide application of erythrocytes to evaluate the biological effects of medicinal plant extracts. The methods were found to be valid and effective for the preliminary investigation of the in vitro cytotoxicity and toxicity of plant products.

El uso de especies vegetales para fines terapéuticos es una práctica histórica y todavía bastante actual, ya que estos productos pueden representar una fuente de materia prima para la producción de formulaciones fitoterápicas. En investigación de plantas con potencial farmacológico requiere la evaluación de su acción, eficacia y seguridad, a través de diferentes etapas. Entre estas, en los ensayos preclínicos se evalúan las propiedades del producto-prueba en experimentos in vitro, tales como ensayos de citotoxicidad, entre otros. En este aspecto, el presente estudio consiste en un análisis bibliométrico acerca de algunas pruebas de citotoxicidad y toxicidad in vitro en eritrocitos realizados en los ensayos de bioprospección de plantas medicinales. Los resultados evidencian la amplia utilización de eritrocitos para la evaluación de los efectos biológicos de extractos de plantas medicinales, apuntándolos como métodos válidos y eficaces para la investigación preliminar de la citotoxicidad y toxicidad in vitro de productos vegetales.

Two Korean Cases of Hereditary Spherocytosis Caused by Mutations in SLC4A1

Hereditary spherocytosis (HS) is caused by mutations in the SPTA1, SPTB, ANK1, SLC4A1, and EPB42 genes, all of which encode erythrocyte membrane proteins. Mutations in SLC4A1, which encodes band 3 protein, have rarely been reported as the causative factor among Korean patients with HS. Here, we report two Korean patients with HS carrying mutations in SLC4A1. Patient 1 was a 3-year-old girl with unremarkable past and family histories and was evaluated for anemia that was detected after a complete blood count. She was suspected of having HS considering the spherocytosis of her peripheral blood smear, increased osmotic fragility, hemolytic features in blood chemistry tests, and splenomegaly. Sequence analysis revealed that the patient harbored a single heterozygous missense mutation, c.2278C>T (p.Arg760Trp) in exon 17 of SLC4A1. Patient 2 was a 23-year-old man who had a prior history of intermittent jaundice. Although the patient did not have anemia, a genetic test for HS was performed due to evidence of hemolytic features in the blood chemistry test, splenomegaly, and a family history of HS. The test confirmed a single heterozygous missense mutation, c.2423G>T (p.Arg808Leu) in exon 18 of SLC4A1.

Cryohemolysis, erythrocyte osmotic fragility, and supplementary hematimetric indices in the diagnosis of hereditary spherocytosis

BACKGROUND: Hereditary spherocytosis (HS) is a chronic hemolytic anemia characterized by microspherocytes in the peripheral blood and increased erythrocyte osmotic fragility (EOF). This study evaluated the cryohemolysis test (CHT); initial hemolysis (IH); immediate and incubated hemolysis percentage in 5.5 g/L NaCl (H5.5); mean corpuscular hemoglobin concentration (MCHC); red blood cell distribution width (RDW); and Hb/MCHC, Hb/RDW, and MCHC/RDW ratios for the diagnosis of HS. METHODS: Data from 13 patients with HS were evaluated at the Instituto de Bioquímica Aplicada and compared with data from 14 unaffected individuals and 11 patients with anemia due to another etiology. Total blood and reticulocyte counts, CHT, and immediate and incubated EOF were performed in all subjects; sensitivity, specificity, efficiency, and Youden index (YI) were calculated. RESULTS: Eight patients with HS had MCHC ≥345 g/L, 10 had RDW ≥14.5%, 12 had IH >5.0 g/L, 11 had immediate H5.5 ≥5%, and 13 had incubated H5.5 ≥50% (the cut-off value to consider HS). The efficiency and YI were: immediate H5.5 (0.94–0.85), incubated H5.5 (0.89–0.82), IH (0.89–0.78), MCHC (0.87–0.62), CHT (0.84–0.54), and Hb/MCHC (0.71–0.56), respectively. The calculated ratios could distinguish subjects with HS from unaffected individuals (P 0.05). CONCLUSION: Although the CHT and supplementary hematimetric indexes were useful to differentiate individuals with SH from healthy controls, they cannot distinguish from anemias of other etiology. CHT and MCHC, in addition to EOF, are recommended for diagnosing HS patients because of their low cost and efficiency.

Alteraciones del sodio como causa de muerte en Patología Forense / Alterations of sodium as a cause of death in forensic pathology

ResumenLas alteraciones del sodio son un factor primordial o agravante que podrían condicionar o causar la muerte tanto en personas conocidas sanas como que en aquellas que presentan algún tipo de patología ya sea aguda o crónica. Dichas alteraciones en muchas ocasiones podrían pasar desapercibidas, dado su grandísima complejidad de detección, de tratamiento y en ocasiones de manejo clínico. Siendo estos factores que vuelven a dichas alteraciones un eje de la medicina clínica olvidada en muchas ocasiones por parte de los peritos encargados de realizar autopsias tanto clínicas como aquellas en las que priva algún interés de índole médico legal.

AbstractSodium alterations are a primary or aggravating factor that could cause death in both well-known and wellknown people, as well as those that present some type of pathology, either acute or chronic. These alterations in many occasions could go unnoticed, given their great complexity of detection, treatment and sometimes clinical management. These factors make these alterations an axis of clinical medicine often forgotten by experts in charge of performing clinical autopsies as well as those in which it deprives some interest of a legal medical nature

Establishment of Method for Detecting Red Blood Cell Osmotic Fragility by Flow Cytometry / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To establish a new method for detection of red blood cell osmotic fragility by using flow cytometry.</p><p><b>METHODS</b>The hypotension salt solution of different concentrations (0.70 ml normal saline+0.3 ml deionized water, 0.60 ml normal saline+0.40 ml deionized water and 0.55 ml normal saline+0.45 ml deionized water) were prepared with normal saline and deionized water, in which the red blood cells were suspended, and the residual red blood cells were detected by flow cytometer.</p><p><b>RESULTS</b>There was no significant difference in percentage of residual red blood cells between different time points detected by flow cytometer in 3 different hypotonic salt solutions. The percentage of residual red blood cells in B+C+D+E+F+G detected time region was different among 3 NaCl dilution groups. The percentage of residual red blood cells in normal control was lower than that in hemoglobinopathy group. The percentage of residual red blood cells in hereditary spherocytosis (HS) group was obviously lower than that in hemoglobinopathy and normal control groups. The comparison of 3 different dilution concentrations found that the second concentration (0.60 ml normal saline+0.40 ml deionized water) is more suitable to screen HS by FC500 flow cytometer.</p><p><b>CONCLUSION</b>The detection of red cell osmotic fragility by using flow cytometry is a simple, rapid, objective and economic way that can be an effective screening method for diagnose the HS.</p>

Coexistence of Gilbert Syndrome and Hereditary Spherocytosis in a Child Presenting with Extreme Jaundice / 대한소아소화기영양학회지

Gilbert syndrome is the most common inherited disorder of bilirubin glucuronidation. It is characterized by intermittent episodes of jaundice in the absence of hepatocellular disease or hemolysis. Hereditary spherocytosis is the most common inherited hemolytic anemia and is characterized by spherical, osmotically fragile erythrocytes that are selectively trapped by the spleen. The patients have variable degrees of anemia, jaundice, and splenomegaly. Hereditary spherocytosis usually leads to mild-to-moderate elevation of serum bilirubin levels. Severe hyperbilirubinemia compared with the degree of hemolysis should be lead to suspicion of additional clinical conditions such as Gilbert syndrome or thalassemia. We present the case of a 12-year-old boy with extreme jaundice and nausea. The diagnosis of hereditary spherocytosis was confirmed by osmotic fragility test results and that of Gilbert syndrome by genetic analysis findings.

Hematopoyesis extramedular en paciente con esferocitosis hereditaria asintomática / Extramedullary hematopoiesis in patients with asymptomatic hereditry spherocytosis

En diversas patologías con alteraciones en la producción de los elementos formes de la sangre pueden desarrollarse focos de hematopoyesis extramedular en diferentes sitios. Los más frecuentes son: bazo, hígado, ganglios linfáticos, y más raramente otros órganos como: glándulas adrenales, hillios renales, cartílagos, ligamentos, tejido adiposo, timo, pulmón, mediastino y duramadre de cráneo y columna. Generalmente el proceso es difuso pero pueden formarse grandes tumores de tejido hematopoyético. Las condiciones patológicas de la médula ósea más frecuentemente asociadas a hematopoyesis extramedular son: esferocitosis hereditaria, talasemia, síndromes mieloproliferativos con fibrosis medular, ocupación medular por patologías neoplásicas. presentamos un paciente de 45 años con esferocitosis hereditaria con masas de tejido hematopoyético extramedular paravertebral mediastinal que respondieron favorablemente a la esplenectomía.

Hereditary spherocytosis (HS) is a relatively common inherited hemolytic disorder in northern Europe and in the US. The reported prevalence of HS in Western countries is 1:5000. We describe a patient 45 years old, with hereditary spherocytosis with masses of mediastinal paravertebral extramedullary hematopoietic tissue, with a favorable response to splenectomy. The medical lieterature refers some cases of extramedullary hematopoiesis as a clinical expression of hereditary spherocytosis, mainly as thoracic masses with usually paravertebral localization. HS should be distinguished from other spherocytic hermolytic anemias. Diagnosis is usually made uring infancy or in young adults, but it can be at any moment of their life, until the seventh decade of life. Ocasionally, the diagnosis is first made in old age. The clinical expression of HS is highly variable, ranging from asymptomatic condition to a severe life-threatening hemolytic anemia. Laboratory features include spherocytosis, osmotic fragility, manifestations of hemolytic disease, elevated unconjugated bilirubin and reticulocytosis. The principal diagnostic test, RBC osmotic fragility, measures the surface/volume Ratio of the cells. The treatment of choice of HS in patients with inherited spherocytosis is splenectomy, which corrects hemolytic anemia. According to he literature, cases of failure following splenectomy have been reported.

Resistencia osmótica eritrocitaria en el diagnóstico de anemias hereditarias en Tucumán, Argentina / Red cell osmotic resistance in the hereditary anemias diagnosis in Tucumán, Argentina / Resistência osmótica eritrocitária no diagnóstico de anemias hereditárias em Tucumán, Argentina

Las anemias hereditarias más frecuentes en Tucumán (Argentina) son el rasgo beta talasémico (RBT), las hemoglobinopatías estructurales (HBP) y la esferocitosis hereditaria (EH). La resistencia osmótica eritrocitaria inmediata y 24 horas post-incubación constituye el método diagnóstico de la EH, y como tubo único (ROETU) es usada para cribado de RBT. El propósito del trabajo fue determinar el comportamiento de ROETU (4,0 y 5,5 g/L de NaCl) en el diagnóstico de anemias hereditarias. Se estudiaron 125 pacientes: 34 normales (GN), 59 con RBT (GRBT), 21 con HBP (GHBP) y 11 con EH (GEH), que fueron agrupados en niños (≤12 años), mujeres y hombres (>12 años). Se realizaron hemograma (Coulter AcT10 y Sysmex KX-21N), índices de Mentzer y de Shine&Lal, ROETU, hierro, transferrina y saturación de transferrina (Wiener Lab), reticulocitos (azul brillante de cresilo), prueba de falciformación y electroforesis de hemoglobina a pH alcalino y ácido. GRBT presentó anemia microcítica hipocrómica, y GEH y GHBP, anemia normocítica normocrómica. El hierro fue normal. GRBT y GHBP fueron resistentes en ROETU 4,0 g/L, aunque GRBT mostró mayor resistencia (p<0,05). GEH fue menos resistente que GN en ROETU 5,5 g/L (p<0,05). ROETU 4,0 y 5,5 g/L serían recomendables en el diagnóstico presuntivo de RBT y EH, respectivamente.

Beta thalassaemia trait (BTT), structural hemoglobinopathies (SHB) and hereditary spherocytosis (HS) are the most frequent hereditary anaemias in Tucumán (Argentina). Immediately and 24 hours post-incubation red cell osmotic resistance is the diagnosis method of HS, and as a single tube (RORST), it is used for clínicamenBTT screening. The purpose of this study was to determine the RORST (NaCl 4.0 and 5.5 g/L) behaviour in the diagnosis of hereditary anemia. The study encompassed 125 patients : 34 normal patients (NG), 59 with BTT (BTTG), 21 with SHB (SHBG) and 11 with HS (HSG), who were divided into children (≤12 years), women and men (> 12 years). Blood count (Coulter AcT10 and Sysmex KX-21N), Mentzer and Shine&Lal indexes, RORST, iron, transferrin and transferrin saturation (Wiener Lab), reticulocytes (brilliant cresyl blue), sickling and hemoglobin electrophoresis at alkaline and acid pH were performed. BTTG showed hypochromic microcytic anemia, and SHBG and HSG, normochromic normocytic anemia. Iron was normal. BTTG and SHBG were resistant in RORST 4.0 g/L, but BTTG showed more resistance (p<0.05). SHG was less resistant than NG at RORST 5.5 g/L (p<0.05). RORST at values of 4.0 and 5.5 g/L would be recommended for the presumptive diagnosis of BTT and SH, respectively.

As anemias hereditárias mais comuns em Tucumán (Argentina) são o traço beta talassemia minor (BTM), as hemoglobinopatias estruturais (HBP) e esferocitose hereditária (EH). A resistência osmótica dos eritrócitos imediata e 24 horas pós-incubação é o método de diagnóstico da EH, e como um único tubo (ROETU) é usado para a detecção de BTM. O objectivo deste estudo foi determinar o comportamento de ROETU (4,0 e 5,5 g/L de NaCl) para o diagnóstico de anemias hereditárias. Foram estudados 125 pacientes: 34 normais (GN), 59 com BTM (GBTM), 21 com HBP (GHBP) e 11 com EH (GEH), que foram reunidos em crianças (≤12 anos), mulheres e homens (>12 anos). Foi realizado hemograma (Coulter AcT10 e Sysmex KX-21N), índices de Mentzer e Shine&Lal, ROETU, ferro, transferrina e saturação de transferrina (Wiener Lab), reticulócitos (azul de cresil brilhante), teste de falcização e eletroforese de hemoglobina em pH alcalino e ácido. GBTM mostrou anemia microcítica hipocrômica, e GEH e GHBP, anemia normocítica normocrômica. O ferro foi normal. GRBT e GHBP foram resistentes em ROETU 4,0 g/L, mas GBTM mostrou maior resistência (p<0,05). GEH foi menos resistente que GN em ROETU 5,5 g/L (p<0,05). ROETU 4,0 e 5,5 g/L seria recomendado para o diagnóstico presuntivo da BTM e EH, respectivamente.

Effect of trehalose-loading on red blood cell membrane / 中国实验血液学杂志

This study was purposed to evaluate the effect of trehalose-loading on physiological and biochemistry properties of red blood cell (RBC) membrane. The samples were divided into the control group (RBC without trehalose loading) and the test group (RBC with trehalose loading). Osmotic fragility reaction was used to determine the osmotic fragility change of loaded RBC membrane in NaCl solution of different osmotic concentration. Flow cytometry and deformeter were used to assay the integrality and deformability of the RBC, respectively. The results showed that the NaCl solution osmotic concentrations were 160 mOsm and 121.4 mOsm, respectively when the haemolysis rate was 50% of the control group and the test group. Flow cytometry data demonstrated that incubation of RBC in a hypertonic trehalose solution resulted in a fraction of cells with different complexity that attached to little Annexin V-FITC, and that it could be removed by washing and resuspending the RBC in an iso-osmotic (300 mOsm PBS) medium. The deformability of the loaded RBC descend, the statistical difference was significant between control and test groups (P < 0.01). It is concluded that the membrane physiological and biochemistry stability and membrane integrality of RBC in a hyper osmotic pressure can be retained after trehalose loading.

A Case of Immune Hemolytic Anemia due to Autoantibodies Against C and e Antigens in a Patient with Paroxysmal Nocturnal Hemoglobinuria and Myelodysplastic Syndrome / 대한수혈학회지

Antiglobulin test-negative hemolytic anemia, thrombophilia, and marrow failure, such as aplastic anemia and myelodysplastic syndrome - refractory anemia (MDS-RA), are the primary clinical manifestations of paroxysmal nocturnal hemoglobinuria (PNH). Here, we report on a case of a 56-year-old male patient diagnosed with PNH, MDS-RA, and immune hemolytic anemia (IHA). The patient was transferred to the hospital with an impression of hemolytic anemia and pulmonary embolism. Positive results were observed on direct and indirect antiglobulin tests, and alloantibody, anti-C and anti-e, autoantibodies were identified. In addition, C and e antigens were found in Rh subgrouping. Therefore, due to the presence of autoantibodies against C and e antigens, we assumed that the cause of IHA was autoimmune reaction. Spherocytosis, increased osmotic fragility test, and positivity on direct and indirect antiglobulin tests were not considered characteristics of PNH. Therefore, without the presence of pulmonary embolism and MDS-RA, it is possible that autoimmune hemolytic anemia was considered the only reason for the hemolytic anemia, and that PNH could be overlooked. In patients with PH, use of washed RBCs during transfusion is not necessary. PNH screening test is recommended for patients who have experienced a thromboembolic event and intravascular hemolysis or MDS-RA. In order to obtain accurate information regarding the percentage of GPI-AP-deficient RBCs, flow cytometric analysis should be performed prior to transfusion.

Biomedical studies on lipid peroxidation and erythrocyte fragility during the process of aging

<p><b>OBJECTIVE</b>To investigate oxidative stress, hemoglobin percentage and erythrocyte osmotic fragility in various aging groups.</p><p><b>METHODS</b>A total of 200 healthy volunteers of both genders between age group 20-65 years were selected by random method. Determination of hemoglobin percentage was done employing modified cyanide method of Dacie and Lewis. The erythrocyte lysis was observed in hypotonic solution of buffered saline at varying concentrations and optical density was measured at 540 nm. The extent of lipid peroxidation in form of malondialdehyde was measured by thiobarbituric acid method.</p><p><b>RESULTS</b>The study found a significant decrease in hemoglobin percentage, increase in erythrocyte osmotic fragility and increased lipid peroxidation in form of malondialdehyde with increasing age.</p><p><b>CONCLUSIONS</b>Supplementation of antioxidants may prevent the oxidative injury in elderly group of subjects.</p>

Qualidade fisiológica de sementes de camomila [Chamomilla recutita (L. ) Rauschert] após envelhecimento acelerado e estresse salino / Physiological quality of chamomile [Chamomilla recutita (L. ) Rauschert] seeds after accelerated aging and salt stress

O objetivo do trabalho foi verificar a qualidade fisiológica de sementes da camomila submetidas a diferentes tempos de envelhecimento acelerado e ao estresse salino. Os experimentos foram realizados no Laboratório de Técnica de Sementes na PUCPR em São José dos Pinhais, Paraná. Os tempos de envelhecimento acelerado foram de 0, 24, 48, 72 e 96 h e o estresse salino foi induzido por soluções de cloreto de sódio com potenciais osmóticos de 0; -0,2; -0,4; -0,6; -0,8; -1,0 MPa, ambos com quatro repetições. Foram avaliados a germinação, o índice de velocidade de germinação (IVG), o tempo médio de germinação (TMG), a velocidade media de germinação (VMG) e a entropia. O delineamento experimental adotado foi o de blocos casualizados . Concluiu-se que as sementes de camomila tiveram sua qualidade fisiológica afetada após 24h de envelhecimento acelerado e sob estresse salino causado por solução de potencial osmótico de -0,6 MPa.

The aim of this study was to verify the physiological quality of chamomile seeds subjected to different accelerated aging periods and salt stress. The experiments were carried out in the Laboratory of Seed Technique, PUCPR, São José dos Pinhais, Paraná State, Brazil. The accelerated aging periods were 0, 24, 48, 72 and 96 h and the salt stress was induced by sodium chloride solutions with osmotic potentials of 0, -0.2, -0.4, -0.6, -0.8, -1.0 MPa, both with four replicates. The analyzed variables were: germination, germination velocity index (GVI), average germination time (AGT), average germination velocity (AGV) and entropy. Experimental design was in randomized blocks. Chamomile seeds had their physiological quality affected after 24 h of accelerated aging and under salt stress caused by the solution with osmotic potential of -0.6 MPa.

Erythrocytes as carrier for prednisolone: in vitro and in vivo evaluation

Resealed erythrocytes, as drug delivery system has tremendous potential to achieve site specificity and prolonged release of drug thereby enhancing therapeutic index and patient compliance. In the present investigation erythrocytes obtained from healthy volunteers were loaded with prednisolone using preswell dilution and dilution technique with two different cross-linking agents, glutaraldehyde and dimethylsulphoxide. Carrier erythrocytes, having acceptable loading parameters showed increased percentage drug content with the addition of cross-linking agents. In vitro drug release followed zero-order kinetics, haemoglobin content was found to be satisfactory and osmotic fragility study indicated that increased drug entrapment efficiency was found at 0.3%w/v concentration of sodium chloride [hypotonic solution]. In vivo tissue distribution studies were carried out for optimized formulation and order of distribution was found to be Liver>Lung>Kidney> Spleen. The developed drug delivery system is endowed with several exclusive advantages and hence holds potential for further research and clinical application

Flow-Assisted Differential Diagnosis of Hemolytic Anemia with Spherocytosis: A Case Report / 대한진단검사의학회지

In patients with hemolytic anemia associated with spherocytosis, differential diagnosis has to be made whether the hemolysis is immune-mediated or of non-immune origin. We report a case of hereditary spherocytosis in a 12-yr-old male child, in whom flow-assisted diagnosis was made. In this case, diagnosis was not determined because routine laboratory workups for hereditary spherocytosis yielded discrepant RESULTS: positive osmotic fragility test, positive direct antiglobulin test, and normal result in the red cell membrane protein sodium dodecyl succinimide polyacrylamide gel electrophoresis. However, all flow cytometry-based tests, such as osmotic fragility, direct antiglobulin, and eosin 5-maleimide binding test, yielded results compatible with hereditary spherocytosis. Additionally, in family study, the results of eosin 5-maleimide binding test suggested his disease being hereditary. In cases with diagnostic difficulties, flow cytometry may be used as an alternative tool, which can provide additional information in the differential diagnosis of hemolytic anemia with spherocytosis.

Policy for screening thalassaemia among the pregnant women by combined osmotic fragility and dichlorophenol-indolephenol tests

Thalassemia is very common in Thailand. To facilitate early diagnosis a reliable screening test is required. To see if a combination of osmotic fragility and dichlorophenol-indolphenol is better than electrophoresis for screening of thalassemia. Two tests i.e. osmotic fragility or dichlorophenol-indolephenol were done as a part of health policy to screen pregrant women in a community screening for thalassemia to see if these two tests are good in early detection of these cases. Two tests done together are easier to perform and can be done without cumbersome instruments and minimal manpower. Tests when interpreted with caution showed a very good screening test combination when compared with electrophoresis. As a part of health policy of Thailand combined method showed good results and were found successful for the policy. Osmotic fragility and dichlorophenol-indolephenol tests may be included in health policy for screening of thalassaemia among pregnant women

Prolonged Extreme Thrombocytosis in a Postsplenectomy Patient with Hereditary Spherocytosis

We report a case of prolonged extreme reactive thrombocytosis in a post-splenectomy patient with hereditary spherocytosis. A 29-year-old female patient presented with gall stones detected incidentally by abdominal ultrasonography. Her laboratory findings showed hemolytic anemia with spherocytosis on the peripheral blood smear and increased osmotic fragility. She was diagnosed with hereditary spherocytosis and underwent a laparoscopic cholecystectomy and splenectomy. After undergoing surgery, the hemolytic anemia was resolved but thrombocytosis was newly detected. Nineteen months after the splenectomy, the thrombocytosis was still persistent and extremely high. To our knowledge, this is the first report of a prolonged extreme reactive thrombocytosis after a splenectomy in Korea.

Low cost combination of DCIP and MCV was better than that of DCIP and OF in the screening for hemoglobin E.

OBJECTIVE: To evaluate hemoglobin E screening tests in a large scale of cases. MATERIAL AND METHOD: A cross-sectional descriptive study was conducted Whole blood obtained from subjects was evaluated for CBC, OF, DCIP, and hemoglobin typing. RESULTS: Five hundred twenty seven hemoglobin E and 280 reference subjects participated. DCIP's sensitivity, specificity, positive predictive value, and negative predictive value were 97.16%, 98.93%, 99.42%, and 95.19%, respectively. These values of OF were 69.12%, 80.00%, 86.67%, and 57.88%, respectively. In the combination of DCIP and OF gave rise to these values of 99.43%, 79.29%, 90.03%, and 96.67%, respectively. Finally the combination of DCIP and MCV < 80 fL resulted in these values to be 99.43%, 98.93%, 99.43%, and 98.93%, respectively. False positive and false negative rate were 1.07% and 0.57%, respectively. CONCLUSION: Combination of DCIP and MCVwas better than that of DCIP and OF in hemoglobin E screening.