Evaluation of RET-He values as an early indicator of iron deficiency anemia in pregnant women

Abstract Introduction During pregnancy, women are at an increased risk of developing iron-deficiency anemia. Objective The objective of this study was to assess the diagnostic performance of the reticulocyte hemoglobin equivalent (RET-He) in the early detection of iron-deficiency anemia in a group of pregnant women and to establish a reference range for this parameter in a group of control individuals. Method: A total of 60 patients and 130 control subjects were included in the study. Blood samples collected from the subjects were submitted to a complete blood count and a serum ferritin test and the data were analyzed by comparing the groups and ROC curves. Results The reference range found for the RET-He was between 29.75pg and 38.24pg, with a median of 35pg. The receiver operating characteristic (ROC) curve analysis for the ferritin parameter showed an area under the curve of 0.732 for the RET-He, 0.586 for hemoglobin, 0.551 for the mean corpuscular hemoglobin concentration and 0.482 for the mean corpuscular volume. Conclusion Early diagnosis of iron deficiency anemia in pregnancy is essential to prevent damage to both maternal and fetal health. The RET-He presents an excellent potential as an auxiliary tool for the diagnosis of iron deficiency in pregnant women.

Morbilidad y mortalidad de 599 pacientes con drepanocitosis en el Instituto de Hematología e Inmunología / Morbidity and mortality of 599 sickle cell disease patients in the Institute of Hematology and Immunology

Introducción: La drepanocitosis es la anemia hemolítica congénita más común del mundo. Entre el 5 y 15 por ciento de la población mundial es portadora de la hemoglobina S y en Cuba, la frecuencia es de 3,08 por ciento, lo que representa un problema de salud pública. Objetivo: Caracterizar el cuadro clínico, el perfil hematológico y la probabilidad de supervivencia de los pacientes con drepanocitosis en el Instituto de Hematología e Inmunología. Método: Se realizó estudio descriptivo, longitudinal y retrospectivo, que incluyó todos los enfermos seguidos, al menos dos años, en la institución, entre enero de 1973 y diciembre del 2009. Resultados: Se incluyeron 599 pacientes (285 masculinos), 439 SS/Sβ0tal y 160 SC/Sβ+tal. El seguimiento medio fue de 17,6±9,5 años. Predominaron los pacientes entre 20 y 59 años. Los eventos clínicos más frecuentes fueron las crisis vasoclusivas dolorosas, las infecciones, el síndrome torácico agudo y las complicaciones hepáticas. Los valores de reticulocitos, plaquetas, leucocitos y hemoglobina fetal fueron significativamente mayores en los pacientes SS/Sβ0tal; no así la hemoglobina total que fue mayor en los SC/Sβ+tal. La probabilidad de supervivencia global de los pacientes a los 45 años fue de 69 por ciento. Los accidentes vasculares encefálicos (17,5 por ciento), las complicaciones hepáticas (17,5 por ciento) y las cardíacas (14,28 por ciento) fueron las principales causas de muerte. Conclusiones: La distribución demográfica y por hemoglobinopatías, el cuadro clínico, y el perfil hematológico fueron similares a los encontrados en pacientes de otras regiones geográficas, excepto la frecuencia de complicaciones hepáticas que fue mayor. La probabilidad de supervivencia fue similar con los mejores centros de atención en el mundo(AU)

Introduction: Sickle cell disease is the most common congenital hemolytic anemia in the world. Between 5 to 15 percent of the world population is a carrier of hemoglobin S and in Cuba, the frequency is 3.08 percent, which represents a public health problem. Objective: To characterize the clinical picture, the hematological profile, and the probability of survival of patients with sickle cell disease at the Institute of Hematology and Immunology. Method: A descriptive, longitudinal and retrospective study was carried out, which included all patients followed up for at least two years at the institution between January 1973 and December 2009. Results: 599 patients (285 male), 439 SS/Sβ0tal and 160 SC/Sβ+tal, were included. The mean follow-up was 17.6±9.5 years. Patients between 20 and 59 years old predominated. The most frequent clinical events were painful vasocclusive crises, infections, acute chest syndrome, and liver complications. The reticulocytes, platelets, leukocytes and fetal hemoglobin values ​​were significantly higher in the SS/Sβ0tal patients, but not the total hemoglobin, which was higher in the SC/Sβ+tal. The overall survival probability of patients at 45 years was 70 percent. Stroke (17.5 percent), liver complications (17.5 percent), and cardiac complications (14.28 percent) were the main causes of death. Conclusions: The demographic distribution and by hemoglobinopathies, the clinical events, and the hematological profile were similar to those found in patients from other geographic regions, except the frequency of liver complications, which was higher. The probability of survival was comparable with the best care centers in the world(AU)

Characteristics of bone marrow compensatory erythropoiesis in hereditary spherocytosis / 中华血液学杂志

Objective: To reveal the compensatory features of bone marrow (BM) erythropoiesis in hereditary spherocytosis (HS) and to explore the effect of diferent hemoglobin levels on this compensation. Methods: Clinical and laboratory data of patients with HS were collected, and the peripheral blood absolute reticulocytes counts value was taken as the surrogate parameter to evaluate the ability of erythropoiesis compensation. BM erythropoiesis compensation in HS with diferent degrees of anemia were evaluated. Results: ①Three hundred and two patients were enrolled, including 115 with compensated hemolytic disease, 74 with mild anemia, 90 with moderate anemia, and 23 with severe anemia. ②Hemoglobin (HGB) was negatively correlated with serum erythropoietin in the decompensated hemolytic anemia group (EPO; rs=-0.585, P<0.001) . ③The median absolute reticulocyte count (ARC) of HS patients was 0.34 (0.27, 0.44) ×10(12)/L, up to 4.25 times that of normal people. The maximum ARC was 0.81×10(12)/L, about 10 times that of normal people. The median ARC of patients with compensated hemolytic disease was 0.29 (0.22, 0.38) ×10(12)/L, up to 3.63 times that of normal people. The median ARC of patients with hemolytic anemia was 0.38 (0.30, 0.46) ×10(12)/L, which was significantly higher than the patients with compensated hemolytic disease, up to 4.75 times that of normal people (z=4.999, P=0.003) . ④ ARC was negatively correlated with HGB in the compensated hemolytic disease group (rs=-0.177, P=0.002) and positively correlated with HGB in the decompensated hemolytic anemia group (rs=0.191, P=0.009) . There was no significant difference in the ARC among patients with mild, moderate, and severe anemia (χ(2)=4.588, P=0.101) . ⑤The median immature reticulocyte production index of the mild, moderate, and severe anemia groups was 13.1% (9.1%, 18.4%) , 17.0% (13.4%, 20.8%) , and 17.8% (14.6%, 21.8%) , respectively; the mild anemia group had lower index values than the moderate and severe anemia groups (P(adj) values were both<0.05) , but there was no significant difference between the latter groups (P(adj)=1.000) . The median immature reticulocyte count of patients in the mild, moderate, and severe groups was 5.09 (2.60, 7.74) ×10(10)/L, 6.24 (4.34, 8.83) ×10(10)/L, and 7.00 (3.07, 8.22) ×10(10)/L, respectively; there was no significant difference among the groups (χ(2)=3.081, P=0.214) . Conclusion: HGB can be maintained at a normal level through bone marrow erythropoiesis, while red blood cells are reduced in HS. However, once anemia develops, the bone marrow exerts its maximum erythropoiesis capacity and does not increase, regardless of anemia aggravation or serum EPO increase.

Use of the reticulocyte channel warmed to 41 °C of the XN-9000 analyzer in samples with the presence of cold agglutinins

ABSTRACT Objectives The purpose of this study was to compare data obtained from the reticulocyte channel (RET channel) heated to 41 °C with those obtained from impedance channel (I-Channel) at room temperature in the samples with the mean corpuscular hemoglobin concentration (MCHC) < 370 g/L and in samples with the MCHC > 370 g/L, in the presence of cold agglutinins. Methods In this study, 60 blood samples (group 1) with the MCHC < 370 g/L (without cold agglutinins) and 78 blood samples (group 2) with the MCHC > 370 g/L (with cold agglutinins) were used to compare the two analytical channels of the XN-9000 analyzer in different preanalytical conditions. The parameters evaluated in both groups were the following: red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean cell volume (MCV), RBC-most frequent volume (R-MFV), mean hemoglobin concentration (MCH) and mean cellular hemoglobin concentration (MCHC). Results The results of this study showed an excellent correlation with both channels of the XN-9000 analyzer in samples with and without cold agglutinins, except for the MCHC. The bias between the values obtained in the I-channel and those obtained in the RET channel of both groups was insignificant and remained within the limits of acceptability, as reported by Ricos et al. for all considered parameters, except for MCHC. Conclusions The presence of cold agglutinins in blood samples can be detected by a spurious lowering of the RBC count and by a spurious increase in the MCHC. The RET channel represents a great opportunity to correct the RBC count in a rapid manner without preheating. However, neither methodology can completely solve the residual presence of cold agglutinins in all samples, despite the MCHC values being < 370 g/L.

Utilidad clínica del equivalente de hemoglobina reticulocitaria en niños en hemodiálisis / Clinical usefulness of the reticulocyte hemoglobin equivalent in children on hemodialysis

Introducción. La anemia por deficiencia de hierro en hemodiálisis es frecuente. Para su detección se utiliza la ferritina sérica y la saturación de transferrina (ST). El equivalente de hemoglobina reticulocitaria (RET-He) es un análisis que no se modifica en estados inflamatorios y refleja directamente el hierro disponible en médula ósea.Objetivo. Explorar la capacidad diagnóstica de deficiencia absoluta de hierro del RET-He y evaluar su correlación con marcadores tradicionales de ferropenia.Población y métodos. Estudio retrospectivo que comparó RET-He con ferritina y ST en niños en hemodiálisis atendidos en el Hospital Garrahan entre julio de 2016 y julio de 2019. Resultados. En 164 observaciones realizadas en 40 niños, se encontró correlación positiva débil entre hemoglobina y RET-He (r 0,35, p < 0,001), positiva significativa entre ST y RET-He (r 0,52, p < 0,001), negativa baja entre hemoglobina y ferritina (r -0,19, p 0,02) y ausencia de correlación entre hemoglobina y ST (r 0,05, p 0,5). El 50 % presentaba anemia; la ferropénica fue por marcadores tradicionales en el 13 % y por RET-He en el 44 %. El RET-He mostró sensibilidad del 90,9 % (IC 95: 57,1-99,5 %), especificidad del 74,5 % (IC 95: 66,7-81 %), valor predictivo negativo del 99,1 % (IC 95: 94,5-99,9 %) y positivo del 20,4 % (IC 95: 10,7-34,7 %) para detectar anemia ferropénica con valor de corte de 29 picogramos.Conclusiones. Pese a su capacidad limitada, el RET-He como biomarcador de deficiencia de hierro aumenta la detección de anemia ferropénica en niños en hemodiálisis

Introduction. Iron-deficiency anemia is common in hemodialysis patients. Serum ferritin and transferrin saturation (TS) are used for its detection. The reticulocyte hemoglobin equivalent (RET-He) is a marker that is not altered by inflammatory conditions and directly reflects iron availability in the bone marrow.Objective. To explore the diagnostic capability of RET-He to detect absolute iron deficiency and assess its correlation with traditional markers of iron deficiency.Population and methods. Retrospective study comparing RET-He with ferritin and TS in children on hemodialysis seen at Hospital Garrahan between July 2016 and July 2019.Results. In 164 observations carried out in 40 children, a weak positive correlation was found between hemoglobin and RET-He (r = 0.35, p < 0.001), a significant positive correlation between TS and RET-He (r = 0.52, p < 0.001), a low negative correlation between hemoglobin and ferritin (r = -0.19, p = 0.02), and a lack of correlation between hemoglobin and TS (r = 0.05, p = 0.5). Anemia was observed in 50 %; iron-deficiency anemia was detected by traditional markers in 13 % and by RET-He in 44 %. RET-He showed a sensitivity of 90.9 % (95 % CI: 57.1-99.5 %), a specificity of 74.5 % (95 % CI: 66.7-81 %), a negative predictive value of 99.1 % (95 % CI: 94.5-99.9 %), and a positive predictive value of 20.4 % (95 % CI: 10.7-34.7 %) to detect iron-deficiency anemia with a cut-off value of 29 pg.Conclusions. Despite its limited capability, the use of RET-He as a biomarker of iron deficiency increases the detection of iron-deficiency anemia in children on hemodialysis.

Avaliação do conteúdo de hemoglobina do reticulócito em pacientes anêmicos sob tratamento dialítico / Evaluation of the reticulocyte hemoglobin content in anemic patients in dialytic treatment

A anemia é uma complicação importante na doença renal crônica (DRC), culminando com o aumento da morbidade e mortalidade, tornando-se fundamental a busca de marcadores hematológicos que permitam seu diagnóstico precoce. Este artigo teve como objetivo revisar na literatura estudos que investigaram a associação entre o conteúdo de hemoglobina contida nos reticulócitos com a anemia em pacientes com DRC em tratamento dialítico e gerar evidências de sua importância na prática clínica. Foi realizada a busca nas bases de dados eletrônicas: Medline e Web of Science. O período de busca definido foi de janeiro de 1997 a dezembro de 2017. O conteúdo de hemoglobina dos reticulócitos avalia a hemoglobina contida nos reticulócitos, sendo que estes, após liberação da medula óssea, permanecem no sangue periférico de um a quatro dias antes de completar sua maturação, conferindo-lhe maior especificidade em refletir a disponibilidade de ferro aos precursores hematopoiéticos, além de fornecer uma avaliação precoce da resposta eritropoética frente ao tratamento de ferro IV. Os artigos selecionados nessa revisão demonstraram que o conteúdo de hemoglobina do reticulócito, além de refletir precocemente a disponibilidade de ferro aos precursores hematopoiéticos, o mesmo não sofre interferência de citocinas inflamatórias, tornando-o um marcador eficaz no monitoramento da cinética do ferro em pacientes em diálise.

Anemia is an important complication in chronic kidney disease (CKD), culminating with the increase in morbidity and mortality, making it fundamental to search for hematological markers that allow its early diagnosis. This review aimed to review in the literature studies that investigated the association between hemoglobin content in reticulocytes and anemia in patients with CKD in dialysis and to generate evidence of its importance in clinical practice. The hemoglobin content of the reticulocytes evaluates the hemoglobin contained in the reticulocytes, which, after release of the bone marrow, remain in the peripheral blood 1 to 4 days before their maturation completes, giving it greater specificity in reflecting the availability of iron to the reticulocytes. Hematopoietic precursors, in addition to providing an early assessment of the erythropoietic response to IV iron treatment. The articles selected in this review have demonstrated that the reticulocyte hemoglobin content in addition to early reflection of the availability of iron to the hematopoietic precursors does not suffer interference from inflammatory cytokines, making it an effective marker in the monitoring of iron kinetics in dialysis patients.

Effects of ketoprofen in spermatogenesis of mice exposed to continuos hypobaric hypoxia / Efecto del ketoprofeno en la espermatogénesis de ratones expuestos a hipoxia hipobárica continua

Hypoxia hypobaric (HH) can cause alterations at testicular level, with temperature increase, intrascrotal alteration and deterioration of spermatogenesis. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as ketoprofen have anti-angiogenic properties, and can decrease testicular abnormalities. The objective of the study was to evaluate the effect of ketoprofen on spermatogenesis of mice exposed to continuous hypobaric hypoxia. 78 Mus musculus CF-1 male mice 3 to 4 months old were used and subjected to HH in chamber at 4200 m. They were divided into 13 groups (G) of 6 animals: 10 with HH cycles (1, 2, 3, 4 and 8, lasting 8.3 days each cycle, two groups each) and 3 in normoxia (Nx). Intraperitoneal ketoprofen 25 mg/kg was administered every 4 days. Euthanasia of these animals was performed at the end of each cycle and in the case the Nx groups at the end of cycles 1, 4 and 8. Percentage of microhematocrit and reticulocytes were measured in blood smears and a morphometric and histopathological analysis of the height of the epithelium, the tubular diameter and the diameter of the tubular lumen was made. It was shown that hematocrit increases continuously up to 8 cycles, while reticulocytes increase up to 3 cycles. Continuous HH decreases the tubular diameter in a sustained manner and proportional to HH cycles, and the height increased only in the groups subjected to 8 cycles. The groups treated with ketoprofen saw a decrease in angiogenesis, presenting some degree of protection at the testicular level.

La hipoxia hipobárica (HH) puede provocar alteraciones a nivel testicular, con aumento de la temperatura, alteración intraescrotal y deterioro de la espermatogénesis. Los antiinflamatorios no esteroidales (AINEs) como el ketoprofeno tienen propiedades antiangiogénicas, pudiendo disminuir las alteraciones testiculares. El objetivo de estudio fue evaluar el efecto del ketoprofeno en la espermatogénesis de ratones expuestos a hipoxia hipobárica continua. Se utilizaron 78 ratones macho Mus musculus CF-1 de 3 a 4 meses de edad y se sometieron a HH en cámara a 4200 m. Se dividieron en 13 grupos (G) de 6 animales: 10 con ciclos de HH (1, 2, 3, 4 y 8, con duración de 8,3 días cada ciclo, dos grupos cada uno) y 3 en normoxia (Nx). Se administró ketoprofeno intraperitoneal 25 mg/kg cada 4 días. La eutanasia de estos animales se realizó al final de cada ciclo y en el caso los grupos Nx al final de los ciclos 1, 4 y 8. Se midió porcentaje de microhematocrito y reticulocitos en frotis de sangre y se hizo un análisis morfométrico e histopatológico de la altura del epitelio, el diámetro tubular y el diámetro de la luz tubular. Se evidenció que el hematocrito aumenta de manera continua hasta los 8 ciclos, en cambio los reticulocitos aumentan hasta los 3 ciclos. La HH continua disminuye el diámetro tubular de forma sostenida y proporcional a los ciclos de HH, y la altura aumentó sólo en los grupos sometidos a 8 ciclos. Los grupos tratados con ketoprofeno se vio una disminución de la angiogénesis, presentando algún grado de protección a nivel testicular.

Adherence to iron prophylactic therapy during pregnancy in an urban regional hospital in South Africa

Background: Iron and folic acid supplementation plays a major role in the prevention and control of iron-deficiency anaemia in pregnancy. Therefore, this study assesses adherence to prophylactic iron supplementation during the antenatal period in South Africa. Methods: An observational study was conducted in a regional hospital from January to December 2016. HIV-uninfected(n= 100) and HIV-infected (n= 100)] women were enrolled and subdivided into three groups: (a)≤34 weeks (n= 33), (b)34­36 weeks (n= 34) and (c)≥37 weeks (n= 33) gestational age respectively. A structured questionnaire was used for data collection. Data were coded and statistically analysed using SPSS software. Pill count and self-reported data from women (n= 24) at≤34 weeks and 34­36 weeks reflected < 50% adherence and 46% non-adherence, being higher in the HIV-infected women (75%). Nausea was the commonest side effect across all trimesters (79. 2%). Adherence (27.8%) and non-adherence (72.1%) to iron, folic acid and calcium supplementation were found in 88% of women. Conclusion: This study found that adherence to micronutrient supplementation is low in pregnancy, albeit higher in HIV-infected women receiving antenatal care at a regional hospital in Durban, South Africa

Etiologyical Analysis of 133 Patients with Elderly Macrocytic Anemia and Diagnostic Significance of Laboratory Tests / 中国实验血液学杂志

OBJECTIVE@#To study the etiology of macrocytic anemia in elderly patients and to evaluate the diagnostic significance of laborotory tests.@*METHODS@#133 elderly macrocytic anemia patients, whose age>60 years old, hemoglobin100 fL, and bone marrow cell test was performed, and these patients were grouped according to diseases, and the bilirubin, lactate dehydrogenase, folic acid, vit B12 and serum ferritin were tested, then the results of tests were compared and analyzed.@*RESULTS@#The majority of the cases were diagnosed as megaloblastic anemia (MA), myelodysplasia syndrome (MDS), acute leukemia/multiple myeloma (AL/MM) and hemolytic anemia (HA). Usually HA was a simple anemia, while others were accompanied by decrease of other 1 or 2 series. HA patients were often with significant high level of well volume (MCV), red cell distribution width(RDW), reticulocytes (RC) and indirect bilirubin (IBIL) (P<0.01). However, MA patients were often with high level of LDH. Serum ferritin (SF) level was significantly higher in both MDS and AL/MM groups (P<0.01).@*CONCLUSION@#Common causes of macrocytic anemia in elderly patients are MA, MDS, AL/MM and HA. The combination detection of MCV, RDW, RC, LDH, IBIL and SF contributes to enhancing the accuracy of diagnosis.

The role of Lactobacillus reuteri DSM 17938 for the absorption of iron preparations in children with iron deficiency anemia / 소아과

PURPOSE: To determine whether Lactobacillus reuteri DSM 17938 plays a role in absorption of iron preparations given to children with iron deficiency anemia (IDA). METHODS: We performed a quasi-experimental study involving pre- and postintervention tests using a control group in North Sulawesi province, Indonesia, between July and September 2017. We conducted a single-blind controlled trial that included primary school children who were diagnosed with IDA based on reticulocyte hemoglobin equivalent (Ret-He) levels <27.8 pg/L. RESULTS: A total of 66 children were randomized into 2 groups. Thirty-four children received iron preparations with the addition of L. reuteri DSM 17938 (group 1), whereas the other 32 received iron preparations alone (group 2). The baseline Ret-He levels before intervention were similar in both groups. After 14 days of intervention, mean Ret-He level in group 1 changed from 24.43±1.64 to 28.21±1.72 pg/L (P=0.000). Mean Ret-He level in group 2 changed from 24.31±1.42 to 27.03±2.14 pg/L (P=0.000). Statistical analysis showed a significant increase in Ret-He levels in both groups; Ret-He levels were significantly higher in the experimental group than in the control group (P<0.05). CONCLUSION: Children with IDA receiving iron preparations with L. reuteri DSM 17938 for 14 days show higher Ret-He levels than those receiving iron preparations alone.

Hemoglobina de reticulocito y su importancia en el diagnóstico temprano de anemia ferropénica / Reticulocyte hemoglobin and its importance in early diagnosis of iron deficiency anemia

Resumen Introducción: El contenido de hemoglobina de reticulocitos (CHr), es un parámetro en la biometría hematológica automatizada que proporciona información sobre el contenido de hierro, por ello se ha utilizado como un marcador de la biodisponibilidad del hierro en la eritropoyesis, permite su detección en una etapa temprana de la anemia ferropénica y otras patologías como inflamación crónica, enfermedad renal crónica; además realizar monitoreo de terapias con eritropoyetina y hierro. Objetivo: Exponer la aplicabilidad de la CHr como un parámetro en el diagnóstico precoz de la anemia por deficiencia de hierro, así como su medición e interpretación. Materiales y métodos: Se realizó la revisión de artículos científicos en inglés y español en las bases de datos PubMed, ScienceDirect, LILACS y Medline, usando descriptores validados en Medical Subject Headings (MeSH), considerando periodo de publicabilidad del 80% inferior a 5 años. Resultados: Se describe la importancia, aplicabilidad, determinación e interpretación de este parámetro como biomarcador específico hemático temprano en el diagnóstico de deficiencia de hierro antes de presentarse cambios morfológicos eritroides. Conclusiones: La CHr es un parámetro de gran utilidad en el diagnóstico temprano de anemia ferropénica y otras patologías como deficiencia funcional de hierro, estados de inflamación crónica y enfermedad renal crónica.

Abstract Introduction: The reticulocyte hemoglobin content (CHr) is a parameter in automated hematological biometrics, which can provide information on the iron content. So it has been used as a marker of the bioavailability of iron in the erythropoiesis, it allows its detection at an early stage of iron deficiency anemia and other pathologies such as chronic inflammation, chronic kidney disease; in addition to monitoring therapy with erythropoietin and iron. Objective: To expose the applicability of CHr as a parameter in the early diagnosis of iron deficiency anemia, as well as its measurement and interpretation. Materials and methods: The review of scientific articles in English and Spanish was carried out in the PubMed, ScienceDirect, LILACS and Medline databases, using descriptors validated in Medical Subject Headings (MeSH), considering the publication period of 80% less than 5 years. Results: The importance, applicability, determination and interpretation of this parameter is described as an early specific biomarker in the blood in the diagnosis of iron deficiency before presenting morphological changes occurring during terminal erythroid differentiation. Conclusions: CHr is a very useful parameter in the early diagnosis of iron deficiency anemia and other pathologies such as functional deficiency, chronic inflammation states and chronic renal disease.

Citometría de flujo en reticulocitos de sangre periférica como indicador de inestabilidad cromosómica en pacientes con gliomas de alto grado / Flow cytometry in peripheral blood reticulocytes as a marker of chromosome instability in highgrade glioma patients

Resumen Introducción. La cuantificación de la inestabilidad cromosómica es un parámetro importante para evaluar la genotoxicidad y la radiosensibilidad. Las técnicas convencionales requieren cultivos celulares o laboriosos análisis microscópicos de cromosomas o núcleos. La citometría de flujo en reticulocitos ha surgido como una alternativa para los estudios in vivo, ya que reduce los tiempos de análisis e incrementa hasta en 20 veces el número de células analizables. Objetivos. Estandarizar los parámetros de citometría de flujo requeridos para seleccionar y cuantificar reticulocitos micronucleados (RET-MN) a partir de muestras de sangre periférica, y cuantificar la frecuencia de esta subpoblación anormal como medida de inestabilidad citogenética en sendas poblaciones de voluntarios sanos (n=25) y pacientes (n=25) recién diagnosticados con gliomas de alto grado antes de iniciar el tratamiento. Materiales y métodos. Las células sanguíneas se marcaron con anti-CD71-PE para reticulocitos, anti- CD61-FITC para la exclusión de plaquetas y yoduro de propidio para detectar el ADN en reticulocitos. La fracción celular MN-RETCD71+ se seleccionó y se cuantificó con un citómetro de flujo automático. Resultados. Se describió detalladamente la estandarización de los parámetros citométricos, con énfasis en la selección y la cuantificación de la subpoblación celular MN-RETCD71+. Se establecieron los niveles basales de MN-RETCD71+ en la población de control y en los pacientes se encontró un incremento de 5,2 veces antes de iniciar el tratamiento (p<0,05). Conclusión. Los resultados evidenciaron la utilidad de la citometría de flujo acoplada a la marcación de las células RETCD71+ como método eficiente para cuantificar la inestabilidad cromosómica in vivo. Se sugieren posibles razones del incremento de micronúcleos en células RETCD71+ de pacientes con gliomas.

Abstract Introduction: The quantification of chromosomal instability is an important parameter to assess genotoxicity and radiosensitivity. Most conventional techniques require cell cultures or laborious microscopic analyses of chromosomes or nuclei. However, a flow cytometry that selects the reticulocytes has been developed as an alternative for in vivo studies, which expedites the analytical procedures and increases up to 20 times the number of target cells to be analyzed. Objectives: To standardize the flow cytometry parameters for selecting and quantifying the micronucleated reticulocytesCD71+ (MN-RET) from freshly drawn peripheral blood and to quantify the frequency of this abnormal cell subpopulation as a measure of cytogenetic instability in populations of healthy volunteers (n =25), and patients (n=25), recently diagnosed with high-grade gliomas before the onset of treatment. Materials and methods: Blood cells were methanol-fixed and labeled with anti-CD-71-PE for reticulocytes, antiCD-61-FITC for platelet exclusion, and propidium iodide for DNA detection in reticulocytes. The MN-RETCD71+ cell fraction was selected and quantified with an automatic flow cytometer. Results: The standardization of cytometry parameters was described in detail, emphasizing the selection and quantification of the MN-RETCD71+ cellular fraction. The micronuclei basal level was established in healthy controls. In patients, a 5.2-fold increase before the onset of treatment was observed (p <0.05). Conclusion: The data showed the usefulness of flow cytometry coupled with anti-CD-71-PE and anti- CD-61-FITC labeling in circulating reticulocytes as an efficient and high resolution method to quantify chromosome instability in vivo. Finally, possible reasons for the higher average of micronuclei in RETCD71+ cells from untreated high-grade glioma patients were discussed.

In vitro kinetics of reticulocyte subtypes: maturation after red blood cell storage in additive solution-1 (AS-1)

ABSTRACT Background: Reticulocytes are immature red blood cells containing RNA remnants. Their population kinetics has been documented under various in vivo and in vitro conditions, including after storage of red blood cells in blood banks. The purpose of this study was to describe the influence of blood bank storage on the kinetics of reticulocyte disappearance by in vitro culturing. Method: Samples of reticulocyte-enriched fractions (Percoll density-gradient) were obtained over different storage times from six red blood cell units stored in additive solution-1 (AS-1). Reticulocyte fractions were then cultured in enriched media at 37 ºC and analyzed by flow cytometry with thiazole orange taking into account hemolysis. Results: Density-gradient enriched reticulocyte fractions were obtain from standard red blood cell units with <1% of reticulocytes. An exponential drop of reticulocytes was observed in cultures. The time taken for reticulocyte disappearance in cultures was shorter with increased blood bank storage time (144 ± 46 h at 0.5 weeks of storage and 15 ± 14 h in the sixth week). High fluorescence reticulocytes disappeared completely in 42.5 ± 8.5 h, medium fluorescence reticulocytes in 73.4 ± 20.8 h and low fluorescence reticulocytes in 269.9 ± 98.8 h in red blood cell units stored for half a week. These times significantly decreased in red blood cell units stored for more time. Conclusion: In vitro reticulocyte disappearance was significantly faster after prolonged storage of red blood cell units at 4 ºC. The in vitro half-life at 0.5 weeks of storage was not significantly different from the values reported for fresh venous blood, but after the sixth week of storage, the half-lives were shorter. The possible explanation is that blood bank storage does not cause irreversible damage to the human reticulocyte maturational machinery.

Necessity of Reticulocyte Calibration for More Accurate and Precise Results

No abstract available.

Hemopoese em desnutrição proteica: caracterização do estroma medular e avaliação da participação do cálcio / Hematopoiesis in protein malnutrition: characterization of bone marrow stroma and evaluation of calcium participation

A desnutrição proteica continua sendo um dos principais problemas nutricionais do mundo. Trabalhos de nosso laboratório e de outros autores evidenciam que entre as alterações presentes na desnutrição proteica, está a alteração do tecido hemopoético, com modificações em componentes da matriz extracelular, alterações no ciclo celular da célula tronco/progenitora hemopoética, redução da produção de precursores hemopoéticos, tanto na série eritrocitária como na série leucocitária, levando a anemia e leucopenia. Os mecanismos de participação do Ca2+ nas células da medula óssea são pouco conhecidos, porém, sabe-se que ele atua no processo de hemopoese. Têm sido descrito que elevações da concentração de Ca2+ citoplasmático induzem a proliferação e diferenciação de células mielóides. A ação dessa via em indivíduos desnutridos também é pouco conhecida. Este estudo tem como objetivo avaliar o estabelecimento da celularidade medular in vitro, bem como investigar mecanismos moleculares envolvidos na proliferação e diferenciação dessa celularidade, além de avaliar a ação do cálcio na presença da interleucina-3 em células-tronco hemopoéticas murinas e sua modulação para avaliar alterações na via das MAPKs. Camundongos C57BL/6, machos e adultos foram submetidos à desnutrição proteica e, após a perda de aproximadamente 20% de seu peso corporal, as células da medula óssea foram colhidas. Essas células foram imunofenotipadas, além de reagirem com anticorpos específicos para caracterização da célula-tronco hemopoética e proteínas da via de sinalização de cálcio intracelular. Observamos que a celularidade do estroma medular em cultura de longa duração de animais desnutridos é alterada, principalmente em células de origem mesenquimal, que aparecem em maior número em desnutridos ao longo dos dias de cultura. Além disso, as ondas de cálcio intracelular estavam diminuídas em animais desnutridos, bem como as proteínas p-PKC, p-PLCy, CAMKII, p-AKT e p-STAT5 não respondem ao estímulo de IL-3, levando a uma deficiência da expressão das MAPK: ERK 1/2, JNK e p38. A desnutrição proteica pode causar alterações na celularidade estromal da medula óssea e na diferenciação das células tronco hemopoéticas pela via das MAPKs estimulada por IL-3

Protein malnutrition remains one of the world's major nutritional problems. Studies from our laboratory and others shown that alterations in protein malnutrition include hemopoietic tissue alterations, changes in extracellular matrix components, changes in the hemopoietic stem/progenitor cell tissue, reduction in the production of hemopoietic precursors, in the erythroid series as in the mieloyd series, leading to anemia and leukopenia. Mechanisms of Ca2+ participation in bone marrow cells are poorly understood, but no hemopoiesis has been developed. Elevations of cytoplasmic Ca2+ concentration in proliferation and differentiation of myeloid cells were included. Such an action through malnourished animals is also a little known. This study aims to evaluate the establishment of cellularity in vitro as well as investigate the molecular involvement in cell proliferation and differentiation, as well as to evaluate the action of calcium in the presence of IL-3 in hemopoietic stem cells and its modulation by analytical evaluations in the MAPKs pathway. C57BL/6, male adult mices were subjected to protein restriction and, after loss of approximately 20% of their body weight, bone marrow cells were harvested. These were immunophenotyped in addition to specific activation terms for the hemopoietic stem cell and intracellular signaling pathway proteins. We observed that the bone marrow cells in long-term culture of malnourished animals is altered, mainly in cells of mesenchymal origin, which appears in greater numbers in undernourished throughout the days of culture. In addition, as intracellular calcium waves decreased in malnourished animals, as well as the p-PKC, p-PLC, CAMKII, p-AKT and p-STAT5 proteins did not respond to IL-3, sugesting expression of the expression of MAPK: ERK 1/2, JNK and p38. Protein malnutrition may have changes in bone marrow capacity and differentiation of hemopoietic stem cells through IL-3-stimulated MAPKs

The research of hemoglobin mass on athletic biological passport for blood doping detection / 中国应用生理学杂志

OBJECTIVE@#To research the changes of blood parameters on different time points after micro-rHuEPO injection on young men and hope to provide evidences for Athletic Biological Passport (ABP) using in the detection of blood doping.@*METHODS@#Fourteen health young men were injected with micro-rHuEPO for 7 weeks, twice per one week.The subjects were treated with ferralia 105 mg every day during the injections.The control group was administrated with the same volume of saline solution injection and placebo 105 mg.The blood parameters (red blood cell, hemoglobin, reticulocyte, total hemoglobin, plasma volume, etc) were tested 11 times (7 days before the first injection, 3, 10, 17, 24, 31, 38 and 45 days after the first injection, 1, 2 and 3 weeks post the seventh week of last injection).By analyzed these total and concentration parameters, hope to know the effective of two kinds of parameters on ABP.@*RESULTS@#The levels of RBC and[Hb] were increased sharply after two weeks of injection and reached the peak in 5-6 weeks (9-10%, <0.01).It sustained till 3 weeks after the last injection.Total hemoglobin was increased significantly during the whole procedure of injections and reached the peak in week 5 (10%, <0.01) till 1 week after the last injection.Red blood cell volume was increased with the EPO injection and reached the peak in week 5 (<0.01), but blood volume wasn't increased significantly.Plasma volume was decreased with EPO injection and it was hemoconcentration.@*CONCLUSIONS@#It can enhance the total and concentration parameters through 7-week micro-rHuEPO injections and the total haemoglobin is more sensitive.So tHb can be used to detect blood doping in ABP.At the end of the last injection, the total blood parameters, such as the total hemoglobin, returned to normal, while the concentration index remained highly, possibly as a result of blood concentration.

Niveles de eritropoyetina y reticulocitos en residentes de bajas alturas migrantes a medianas alturas / Erythropoietin and reticulocyte levels in lowland residents migrating to middle altitudes

RESUMEN Introducción: La eritropoyetina es una alfa globulina glicosilada con producción renal en más del 90% en la vida adulta. Es la principal hormona en el mantenimiento constante de la masa eritrocitaria, aunque existen modificaciones en sus niveles asociados con el tabaquismo, anemias, EPOC y la migración de bajas a medianas o altas alturas. Esto último desencadena un proceso hipóxico que puede llegar a producir mal agudo de montaña. Objetivo: Describir el comportamiento de la eritropoyetina, el recuento de reticulocitos y su influencia, en procesos de adaptación a la altura. Metodología: Estudio descriptivo de corte transversal que incluyó 11 participantes provenientes de bajas alturas a quienes se les determinó la concentración sérica de eritropoyetina y el recuento de reticulocitos en un periodo de 28 días. Resultados: Ocho de los participantes presentaron un ascenso progresivo en los niveles séricos de eritropoyetina, uno mantuvo una curva plana y dos presentaron comportamiento atípico respecto a lo reportado en la literatura. Conclusión: La eritropoyetina es un factor fundamental que marca el comienzo de la eritropoyesis, cuya finalidad es mejorar el aporte de oxígeno en procesos de adaptación a la altura. Además, la hipoxia es un factor determinante en el inicio y desarrollo del mal agudo de montaña. El recuento de reticulocitos depende del estímulo proliferativo y anti-apoptótico de la eritropoyetina, así como de las concentraciones séricas de vitamina B12, hierro y ácido fólico.

ABSTRACT Introduction: EPO is a glycosylated alpha globulin produced in more than 90% by kidneys through adult life, being a key hormone that regulated the erythrocytic mass. However, there are some modifications in the levels of this hormone that may be related to smoking, anemia, EPOC and migration to from low to higher altitudes, inducing hypoxic processes. Depending on the individual, it may produced the disease named as acute mountain sickness. Objective: To describe erythropoietin level modifications, reticulocytes count and its influence, on the adaptive process to altitude. Methodology: This is a transversal descriptive study including 11 participants from low altitudes places, whose EPO serum concentration and reticulocytes count was determined during 28 days. Results: Eight participants presented a progressive increase in EPO serum levels, one participant exhibited a constant level and two more showed atypical results according to previous literature. Conclusion: EPO is a key factor for determining the erythropoiesis beginning, as its objective is to improve the oxygen provision during altitude adaptation processes by increasing its concentration in blood due to hypoxic stimulus. Besides, hypoxia is a determinant factor in the beginning and development of acute mountain sickness. The reticulocytes count depends also on the EPO proliferative and anti-apoptotic stimulus, and on the serum concentrations of B12 vitamin, iron and folic acid.

2 Cases of Beta-thalassemia Minor in Korea / 임상소아혈액종양

In Korea, recent epidemiologic studies show that the incidence of β-thalassemia is increasing as the influx of South-East Asian population increases and molecular technologies develop. However, many patients are still misdiagnosed as iron deficiency anemia (IDA). All patients with microcytic anemia need to perform evaluation including reticulocyte index, Mentzer index, and iron studies. Considering the increasing incidence of β-thalassemia, hemoglobin beta globulin (HBB) gene sequencing should be performed if suspicious. In our cases, patients whose parents were both Koreans were confirmed to have β-thalassemia with a substitution in c1, ATG>GTG, and deletion of the HBB gene. In Korea, initiation condon ATG>AGG (20.9%) is most common mutation, followed by codon 17 (A>T) (17.6%), codon 121 (G>T) (12.1%), and so on. We report two cases of β-thalassemia diagnosed by genetic testing for microcytic anemia.