Flow cytometry "Ogata score" for the diagnosis of myelodysplastic syndromes in a real-life setting. A Latin American experience.

INTRODUCTION: Flow cytometry (FC) is a helpful tool for the diagnosis of myelodysplastic syndrome (MDS). Different FC score systems have been developed. The "Ogata score" is a simple diagnostic score that has been validated having a sensitivity of 69% and a specificity of 92% in low-risk MDS. We aimed to study the feasibility and the utility of the "Ogata score" for the diagnosis of MDS among Latin America (LA) Laboratories. METHODS: This is a case and control study conducted in LA institutions members of Grupo Latinoamericano de Mielodisplasia (GLAM). A total of 146 MDS patients and 57 control patients were included. "Ogata score" was calculated. RESULTS: The sensitivity of "Ogata score" was 75.6% (95% CI, 66.8-81.3), specificity was 91.2% (95% CI, 79.7-96.7), PPV was 95.6% (95% CI, 88.5-98.3), and NPV was 65.4% (95% CI, 49.1-71.9). In low/intermediate-1 IPSS patients group, the sensitivity was 70.1% (95% CI, 60.2-78.2), specificity was 91.2% (CI-95%, 79.7-96.7), PPV was 94.2% (95% CI, 86.4-97.8), and NPV was 62.1% (95% CI, 53.0-78.7). In the group of patients "without MDS specific markers" (patients without ring sideroblasts, blast excess, or chromosomal abnormalities), the sensitivity was 66.7% (CI-95%, 55.8-76.0), specificity was 91.2% (95% CI, 79.7-96.7), PPV was 92.3% (95% CI, 82.2-97.1), and NPV was 63.5% (95% CI, 51.9-73.5). CONCLUSIONS: The diagnostic power found in this study was similar to the reported by Della-Porta et al. Also in LA, the analysis was made in modern equipment with acquisition of at least 100 000 events which permits a good reproducibility of the results.

Esferocitosis hereditaria: experiencia clínica y diagnóstica en Argentina / Hereditary spherocytosis: clinical and diagnostic experience in Argentina / Esferocitose hereditária: experiência clínica e diagnóstica na Argentina

Los objetivos del presente estudio fueron: a) Analizar las características demográficas y clínicas de nuestra población al diagnóstico; b) Evaluar si las pruebas más recientes presentan ventajas sobre las tradicionales; c) Confirmar la frecuencia de las distintas deficiencias de proteínas de membrana; d) Establecer la relación entre severidad y resultado de las pruebas o tipo de deficiencia. Se analizaron 359 individuos estudiados desde 2007, cuando se incorporaron criohemólisis hipertónica (CH), citometría de flujo con eosina-5'- maleimida (5'EMA-CF), FOE por citometría de flujo (FOE-CF) y electroforesis de proteínas de membrana (SDS-PAGE) al estudio de laboratorio clásico, fragilidad osmótica eritrocitaria (FOE) y autohemólisis (AH). Criterios diagnósticos para Esferocitosis Hereditaria (ESH): esferocitos en frotis y dos pruebas positivas. Se identificaron 174 pacientes con ESH y 22 portadores sanos. El 74,9% eran menores de 12 años. La transmisión fue dominante en el 83,1% de los casos. Tuvieron manifestaciones neonatales 89,1%. Las pruebas con mayor sensibilidad fueron CH (92,0%), FOE diferida (91,1%) y 5'EMA-CF (88,5%). En los 125 pacientes en quienes se realizaron CH, 5'EMA-CF y FOE-CF se observó que todos tenían al menos una prueba positiva; 122 (97,6%) tuvieron dos o tres positivas. Las deficiencias más frecuentes fueron ankirina y espectrina. No hubo diferencia en el resultado de las pruebas entre los subgrupos de severidad. Se concluye que las deficiencias más frecuentes en Argentina son ankirina y espectrina, coincidiendo con otras poblaciones latinoamericanas. El uso simultáneo de CH, 5'EMA-CF y FOE-CF permite diagnosticar más del 97% de los casos. La incidencia de manifestaciones neonatales es elevada.

The aims of this study were (a) to assess demographic and clinical aspects of our population at diagnosis; (b) to evaluate diagnostic accuracy of hypertonic cryohemolysis (HC), eosin-5'-maleimide flow cytometry (EMA-FC) and flow cytometric osmotic fragility (OF-FC) in relation to standard screening tests osmotic fragility (OF) and autohemolysis (AH); (c) to confirm the previously reported prevalence of membrane proteins defects; and (d) to assess the relationship between severity of anemia and results of confirmatory tests. Since 2007, the following tests were available in our laboratory: OF, AH, HC, EMA-FC, OF-FC and SDS-PAGE of membrane proteins. Diagnostic criteria for hereditary spherocytosis were spherocytes in blood smear plus ≥2 positive tests. Data from 359 individuals were analyzed: 174 HS patients and 22 silent carriers were detected; 74.9% of patients were less than 12 years old; 83.1% of them showed a dominant inheritance pattern; antecedent of neonatal jaundice/anemia was registered in 89.1%. Tests with higher sensitivity were: HC (92.0%), incubated OF (91.1%), and EMA-FC (88.5%). HC, EMA-FC and OF-FC were simultaneously performed on 125 patients: each of them had at least 1 positive test; 122 (97.6%) had 2 or 3 positive tests. Ankyrin and spectrin were the most frequently found protein deficiencies. Comparison of test results in relation to severity of anemia showed no difference between groups. It can be concluded that compared toother Latin American countries, ankyrin and spectrin were the most frequent protein deficiencies. Simultaneous performing of HC, EMA-FC and OF-FC enabled diagnosing HS in more than 97% of patients. A high incidence of neonatal jaundice/anemia was observed.

Os objetivos do presente estudo foram: a) analisar as características demográficas e clínicas de nossa população ao diagnóstico; b) Avaliar se as provas mais recentes apresentam vantagens sobre as tradicionais; c) Confirmar a frequência das diversas deficiências de proteínas de membrana; d) Establecer a relação entre severidade e resultado das provas ou tipo de deficiência. Foram analisados 359 indivíduos estudados desde 2007, quando se incorporaram crio-hemólise hipertônica (CH), citometria de fluxo com eosina-5'-maleimida (5'EMA-CF), FOE por citometria de fluxo (FOE-CF) e eletroforese de proteínas de membrana (SDS-PAGE) ao estudo de laboratório clássico - fragilidade osmótica eritrocitária (FOE) e auto-hemólise (AH). Critérios diagnósticos para ESH: esferócitos em esfregaço e duas provas positivas. Foram identificados 174 pacientes com ESH e 22 portadores sadios. 74,9% eram menores de 12 anos. A transmissão foi dominante em 83,1%. Tiveram manifestações neonatais 89,1%. As provas com maior sensibilidade foram CH (92,0%), FOE diferida (91,1%) e 5'EMA-CF (88,5%). Nos 125 pacientes aos quais lhes realizaram CH, 5'EMA-CF e FOE-CF se observou que todos tinham no mínimo uma prova positiva; 122 (97,6%) tiveram duas ou três positivas. As deficiências mais frequentes foram anquirina e espectrina. Não houve diferença no resultado das provas entre os subgrupos de severidade. Conclui-se que as deficiências mais frequentes na Argentina são anquirina e espectrina, as quais coincidem com outras populações latinoamericanas. O uso simultâneo de CH, 5'EMA-CF e FOE-CF permite diagnosticar mais de 97% dos casos. A incidência de manifestações neonatais é elevada.

Red blood cell aquaporin-1 expression is decreased in hereditary spherocytosis.

Aquaporin-1 (AQP1) is the membrane water channel responsible for changes in erythrocyte volume in response to the tonicity of the medium. As the aberrant distribution of proteins in hereditary spherocytosis (HS) generates deficiencies of proteins other than those codified by the mutated gene, we postulated that AQP1 expression might be impaired in spherocytes. AQP1 expression was evaluated through flow cytometry in 5 normal controls, 1 autoimmune hemolytic anemia, 10 HS (2 mild, 3 moderate, 2 severe, and 3 splenectomized), and 3 silent carriers. The effect of AQP1 inhibitors was evaluated through water flow-based tests: osmotic fragility and hypertonic cryohemolysis. Serum osmolality was measured in 20 normal controls and 13 HS. The effect of erythropoietin (Epo) on AQP1 expression was determined in cultures of erythroleukemia UT-7 cells, dependent on Epo to survive. Independent of erythrocyte size, HS patients showed a lower content of AQP1 in erythrocyte membranes which correlated with the severity of the disease. Accordingly, red blood cells from HS subjects were less sensitive to cryohemolysis than normal erythrocytes after inhibition of the AQP1 water channel. A lower serum osmolality in HS with respect to normal controls suggests alterations during reticulocyte remodeling. The decreased AQP1 expression could contribute to explain variable degrees of anemia in hereditary spherocytosis. The finding of AQP1 expression induced by Epo in a model of erythroid cells may be interpreted as a mechanism to restore the balance of red cell water fluxes.

Use of capillary blood to diagnose hereditary spherocytosis.

We studied 31 children with hemolytic anemia, or with positive family history for hereditary spherocytosis (HS), to assess the reliability of capillary blood samples for the diagnosis. HS was diagnosed in 20 patients. Cryohemolysis (CH) was positive in 94% and eosin-5'-maleimide flow cytometry in 90% of them, whereas flow cytometric osmotic fragility was positive in 94%. Capillary blood sampling showed to be useful for the diagnosis. Simultaneous use of these three tests allows confirming diagnosis in 100% of patients. The use of very small blood volumes (300 µl) allows an earlier diagnosis in neonates and small infants.

A prospective study to assess the predictive value for hereditary spherocytosis using five laboratory tests (cryohemolysis test, eosin-5'-maleimide flow cytometry, osmotic fragility test, autohemolysis test, and SDS-PAGE) on 50 hereditary spherocytosis families in Argentina.

This prospective study was carried out to assess the usefulness of five laboratory tests in the diagnosis of hereditary spherocytosis (HS), based on the correlation of erythrocyte membrane protein defects with clinical and laboratory features, and also to determine the membrane protein deficiencies detected in Argentina. Of 116 patients and their family members tested, 62 of them were diagnosed to have HS. The specificity of cryohemolysis (CH) test was 95.2%, and its cut-off value to distinguish HS from normal was 2.8%. For flow cytometry, cut-off points of 17% for mean channel fluorescence (MCF) decrease and 14% coefficient of variation (CV) increase showed 95.9% and 92.2% specificity, respectively. Both tests showed the highest percentages of positive results for diagnosis. Either CH or flow cytometry was positive in 93.5% of patients. In eight patients, flow cytometry was positive only through CV increase. Protein defects were detected in 72.3% of patients; ankyrin and spectrin were the most frequently found deficiencies. The CV of the fluorescence showed significantly higher increases in moderate and severe anemia than in mild anemia (p = 0.003). Severity of anemia showed no other correlation with tests results, type of deficient protein, inheritance pattern, or neonatal jaundice. CH and flow cytometry are easy methods with the highest diagnostic accuracy. Simultaneous reading of mean channel fluorescence (MCF) decrease and CV increase improve diagnostic usefulness of flow cytometry. This test seems to be a reliable predictor of severity. The type of detected protein deficiency has no predictive value for outcome. Predominant ankyrin and spectrin deficiencies agree with reports from other Latin American countries.

Severe pandemic 2009 H1N1 influenza disease due to pathogenic immune complexes.

Pandemic influenza viruses often cause severe disease in middle-aged adults without preexisting comorbidities. The mechanism of illness associated with severe disease in this age group is not well understood. Here we find preexisting serum antibodies that cross-react with, but do not protect against, 2009 H1N1 influenza virus in middle-aged adults. Nonprotective antibody is associated with immune complex-mediated disease after infection. We detected high titers of serum antibody of low avidity for H1-2009 antigen, and low-avidity pulmonary immune complexes against the same protein, in severely ill individuals. Moreover, C4d deposition--a marker of complement activation mediated by immune complexes--was present in lung sections of fatal cases. Archived lung sections from middle-aged adults with confirmed fatal influenza 1957 H2N2 infection revealed a similar mechanism of illness. These observations provide a previously unknown biological mechanism for the unusual age distribution of severe cases during influenza pandemics.

Inmunodepresión secundaria a traumatismo encefalocraneano / Immunodepression induced by traumatic brain injury

Introducción: Los pacientes que sobreviven a la injuria inicial por trauma severo presentan con elevada frecuencia complicaciones infecciosas, sépticas y disfunción multiorgánica. El traumatismo de cráneo (TEC) parece ser un factor de riesgo independiente en relación con la aparición de esas complicaciones. Los mecanismos causales estarían relacionados a una parálisis de la inmunidad celular inducida por el TEC. Objetivos: Analizar el grado de alteración de la competencia inmunológica en pacientes con TEC severo, determinado por los niveles plasmáticos de las citokinas IL-10, IL-6 y TNF-O y el nivel de expresión de HLA-DR de los monocitos sanguíneos CD14+. Pacientes y métodos: Se incorporaron 15 pacientes ingresados con TEC severo (GCS 8). Ninguno de los pacientes había recibido corticoides ni catecolaminas. Trece voluntarios normales se utilizaron como controles...(AU)

Inmunodepresión secundaria a traumatismo encefalocraneano / Immunodepression induced by traumatic brain injury

Introducción: Los pacientes que sobreviven a la injuria inicial por trauma severo presentan con elevada frecuencia complicaciones infecciosas, sépticas y disfunción multiorgánica. El traumatismo de cráneo (TEC) parece ser un factor de riesgo independiente en relación con la aparición de esas complicaciones. Los mecanismos causales estarían relacionados a una parálisis de la inmunidad celular inducida por el TEC. Objetivos: Analizar el grado de alteración de la competencia inmunológica en pacientes con TEC severo, determinado por los niveles plasmáticos de las citokinas IL-10, IL-6 y TNF-Ó y el nivel de expresión de HLA-DR de los monocitos sanguíneos CD14+. Pacientes y métodos: Se incorporaron 15 pacientes ingresados con TEC severo (GCS ¾ 8). Ninguno de los pacientes había recibido corticoides ni catecolaminas. Trece voluntarios normales se utilizaron como controles...

Liberación precoz de la citokina antiinflamatoria IL10 en el trauma severo de craneo / Early release of the antiinflammatory cytokine IL-10 in traumatic brain injury

Los pacientes que sobreviven a la injuria inicial de un trauma severo presentan con elevada frecuencia complicaciones infecciosas, sépticas y disfunción multiorgánica. Los mecanismos causales estarían relacionados a una alteración de la inmunidad celular inducida por el trauma y mediada en parte por la a expresión de citokinas antinflamatorias. Se evaluaron los niveles plasmáticos de la citokina antinflamatoria IL-10 y de la citokina proinflamatoria TNF-a en 15 pacientes ingresados con traumatismo encefalocraneano (TEC) severo predominante. Ninguno de los pacientes había recibido corticoides ni catecolaminas; 13 voluntarios normales se utilizaron como controles. Al ingreso los niveles plasmáticos de IL-10 fueron significativamente mayores en los pacientes que en los controles: 41.8 (17.3-265.4) pg/mL vs. 2.2 (1.4-2.7) pg/mL, p<0.001 (test de Mann-Withney). Los niveles de IL-10 no fueron diferentes entre la primera (menos de 6 horas post-trauma) y la segunda muestra (4 horas después) (test de Wilcoxon). Los niveles plasmáticos de TNF-a fueron semejantes en los pacientes respecto a los controles. Estos resultados muestran que los pacientes con TEC severo desarrollan precozmente una respuesta con elevación significativa de los niveles plasmáticos de IL-10 y que podría explicar, por lo menos en parte, la situación de inmunodepresión inducida por el TEC.

Liberación precoz de la citokina antiinflamatoria IL10 en el trauma severo de craneo / Early release of the antiinflammatory cytokine IL-10 in traumatic brain injury

Los pacientes que sobreviven a la injuria inicial de un trauma severo presentan con elevada frecuencia complicaciones infecciosas, sépticas y disfunción multiorgánica. Los mecanismos causales estarían relacionados a una alteración de la inmunidad celular inducida por el trauma y mediada en parte por la a expresión de citokinas antinflamatorias. Se evaluaron los niveles plasmáticos de la citokina antinflamatoria IL-10 y de la citokina proinflamatoria TNF-a en 15 pacientes ingresados con traumatismo encefalocraneano (TEC) severo predominante. Ninguno de los pacientes había recibido corticoides ni catecolaminas; 13 voluntarios normales se utilizaron como controles. Al ingreso los niveles plasmáticos de IL-10 fueron significativamente mayores en los pacientes que en los controles: 41.8 (17.3-265.4) pg/mL vs. 2.2 (1.4-2.7) pg/mL, p<0.001 (test de Mann-Withney). Los niveles de IL-10 no fueron diferentes entre la primera (menos de 6 horas post-trauma) y la segunda muestra (4 horas después) (test de Wilcoxon). Los niveles plasmáticos de TNF-a fueron semejantes en los pacientes respecto a los controles. Estos resultados muestran que los pacientes con TEC severo desarrollan precozmente una respuesta con elevación significativa de los niveles plasmáticos de IL-10 y que podría explicar, por lo menos en parte, la situación de inmunodepresión inducida por el TEC. (AU)