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.

Immature Platelet Fraction in Septic Patients: Clinical Relevance of Immature Platelet Fraction is Limited to the Sensitive and Accurate Discrimination of Septic Patients From Non-Septic Patients, Not to the Discrimination of Sepsis Severity

BACKGROUND: The immature platelet fraction (IPF) reflects the degree of reticulated platelets. We evaluated performances of IPF as a biomarker for the discrimination of septic patients from non-septic patients and sepsis severity. METHODS: Total 312 patients admitted between March and July 2013 were enrolled and samples were obtained at admission. Lactate (LA), procalcitonin (PCT), C-reactive protein (CRP), immature granulocyte fraction (IG), immature reticulocyte fraction (IRF), and IPF were analyzed as sepsis biomarkers and their performances were compared. RESULTS: The performance of IPF (area under the curve [AUC]=0.868) in the discrimination of septic patients from non-septic patients was comparable to PCT/CRP/LA/IG (AUC=0.923/0.940/0.781/0.812, P=0.233/0.106/0.186/0.353, respectively), and was significantly better than the IRF (AUC=0.658, P=0.007). Sensitivity (89.8%, 95% confidence interval [CI] 84.9-99.8%) and accuracy (83.2%, 95% CI 78.8-90.0%) of IPF were the best among all biomarkers. The performance of IPF in discriminating septic patients from non-septic patients with local infection showed similar results. However, the IPF could not efficiently discriminate sepsis severity (AUC=0.599), similar to other biomarkers (AUC=0.519-0.752). CONCLUSIONS: The IPF possessed high sensitivity/accuracy in discriminating septic patients from non-septic patients, regardless of local infection status. However, the IPF did not efficiently discriminate sepsis severity. The clinical relevance of IPF as a sepsis biomarker is, therefore, limited to sensitive and accurate discrimination of septic patients from non-septic patients, not discrimination of sepsis severity.

Reticulocyte assessment as an indirect evaluation and elaboration of erythroid marrow output in the groups of pregnant women in Maiduguri, Nigeria

Nutritional anaemia is one of Nigeria's major public health problem among pregnant women. In this locality, no large scale study has been done to assess bone marrow activity in groups of pregnant women using Reticulocyte assessment. This result will serve as indirect evaluation and elaboration of erythroid marrow output in the pregnant women. To assess bone marrow activity in groups of pregnant women using Reticulocyte assessment as an indirect evaluation. 150 women covering the three trimesters of pregnancy were recruited into the study, prospectively. 101 multiparous and 49 primiparous pregnant women, consisting of 33 in the first trimester, 74 in the second trimester and 43 in the third trimester were investigated. Red blood cell count, Reticulocyte count and assessment were conducted by standard methods. The patients in the third trimester had statistically significant [P<0.05] lower reticulocyte values than those in the first and second trimesters. The primiparous pregnant women had statistically significant [P<0.05] higher reticulocyte values than the multiparous pregnant women. Absolute reticulocyte count results were 50.0 +/- 15.2, 60.0 +/- 18.4, 34.0 +/- 12.8 and 48.0 +/- 12.0 for first, second and third trimesters and combined group, respectively. Reticulocyte index was 1.5 +/- 0.6, 1.9 +/- 0.8, 0.8 +/- 0.6 for first, second and third trimesters, respectively and 1.5 +/- 0.5 for combined group. Reticulocyte production index was 0.9 +/- 0.2, 1.1 +/- 0.4, 0.7 +/- 0.5 and 0.7 +/- 0.4 for first, second and third trimesters and combined group, respectively. In this study, bone marrow activity as assessed by reticulocyte studies is on the lower side of the normal range, more so in the third trimester of pregnancy. Severe anaemia during pregnancy therefore remains endemic despite intervention measures such as the distribution of iron and folate tablets. One of the problems yet uninvestigated is the bone marrow turnover as affected by some other nutritional differences as well as malaria, heavy loads of helminths, and other inflammatory or infectious diseases. A successful strategy to combat anaemia, therefore, should address all the casual factors after their elucidation

Recombinant human erythropoietin in anemia of prematurity.

OBJECTIVE: To evaluate safety and efficacy of recombinant human erythropoietin (r-HuEPO)in reducing the need for red cell transfusions in anemia of prematurity. METHODS: forty -two preterm infants (gestational age <32 weeks) were randomly assigned to a "treatment" group (r-HuEPO 400 units/kg every alternate day * 10 doses) or "no treatment" (control) group. All infants on enteral feeds received oral iron 3 mg/kg/day, graded up to 6 mg/kg/day. RESULTS: Higher reticulocyte counts in week 2 and 3 and higher hemoglobin levels in week 4 were noted after treatment with r-HuEPO. Despite stumulated erythropoiesis, the frequency of transfusions could not be reduced with r-HuEPO therapy.Overall, Phlebotomy losses, frequency and volume of redcell transfusions were significantly more in neonates with birthweight <1000 grams compared with those with birthweight >1000 grams (p<0.05). Associated side effects of r-HuEPO such as neutropenia,sepsis, hypertension or increased risk of late death did not occur. CONCLUSION:r-HuEPO therapy was safe without any side effects.Inability of r-HuEPO therapy to minimize red cell transfusions for anemia of prematurity may be explained by a relatively strict red-cell transfusion policy and the desired degree of treatment effect.