Thawing methods do not affect cell viability of CD45+ and CD34+ cells, but long-term cryopreservation of umbilical cord blood units generally decreases cell viability.

INTRODUCTION: Umbilical cord blood units (UCBUs) are collected and cryopreserved in biobanks for a future transplant. Hematopoietic stem cells and hematopoietic progenitor cells (HSC/HPC) present in UCB can be damaged due to factors such as the cryopreservation process, the thawing process, and prolonged storage time. METHODS: UCBUs (n = 27) were obtained from the Biobank of the National Center of Blood Transfusion (NCBT) from Mexico. They contained three attached segments of UCBU, including 1.0-2.3 × 106 CD34+ cells prior to cryopreservation and were stored during the period from 2003 to 2015. Each UCB segment was thawed with three different methods and CD34 cells, CD45 cells, and 7-AAD were identified by flow cytometry. Furthermore, we carried out CFU assays, and trypan blue staining. RESULTS: Viable CD45+ (vCD45+) cells, vCD34+ cells, CFU, and percentage of E-Clone were not statistically significant among three different thawing methods. The number of vCD45+ and vCD34+ cells diminished in the three thawing methods compared with the same cells prior to their cryopreservation (p < 0.0001). vCD45+ and vCD34+ cells diminished in the ≥10-year cryopreservation group (p < 0.001). In addition, percentage of recovery of vCD45+ and vCD34+ cells diminished in this same group (p = 0.013 and p < 0.0001, respectively). CONCLUSION: The thawing methods did not affect either cell viability (vCD45+ and vCD34+ cells) or pluripotency (CFU, percentage of E-Clone) in attached segments of UCBUs. The ≥10-year cryopreservation time in attached segments of UCBUs alters the number of vCD45+ and vCD34+ cells; however, it does not affect their pluripotency.

[Enfermedad de Chagas en México].

Chagas disease, which is caused by Trypanosoma cruzi, is considered to be the most serious parasitic disease in America. It is transmitted mainly by triatominae ("kissing bugs"). Mazzoti reported the first two human cases in Mexico. The form of transmission is by parasites entering the organism in feces of the insect, by blood transfusion, from mother to child, by organ transplant and laboratory accidents. In Mexico, 1.1 million people are estimated to be infected; the incidence in 2012 was 0.70 per 1,00,000 population. In 2017, the highest incidence rates were registered in Yucatán, Oaxaca and Hidalgo. The infection causes cardiomyopathies and mega-organs of the digestive tract. Diagnosis in the acute phase is by parasitological approach and, in the chronic phase, by laboratory screening studies. In Mexico's blood banks, screening for Chagas disease is mandatory; from 2007 to 2016, seroprevalence has decreased from 0.40 to 0.32 due to the improvement of donor selection processes and the ad hoc questionnaire. The targets of the parasite are neurons and smooth and myocardial muscle cells. The association of neuronal and smooth muscle destruction defines the presentation of chagas mega-syndromes. Initial manifestations of the disease can go unnoticed; 5% show apparent signs and symptoms and 30% will progress to the chronic asymptomatic phase. Currently available treatments have effect in the acute phase. For the control of Chagas disease, the Specific Action Program for the Prevention and Control of Chagas Disease (PAE Chagas 2013-2018) is available to initiate activities aimed at eliminating transfusion and congenital transmission and controlling vector transmission. The success of medical care depends on oportune detection, early etiological treatment and coverage broadening. On the other hand, monitoring and screening of pregnant women living in risk areas and blood and organ donors universal screening will enable the elimination congenital and transfusion transmission.

La enfermedad de Chagas, causada por el Trypanosoma cruzi, está considerada como la parasitosis más grave en América. Se transmite principalmente por triatominos (chinches). El doctor Mazzoti reportó los dos primeros casos humanos en México. La forma de transmisión es por la entrada al organismo de los parásitos en heces del insecto, por transfusión sanguínea, de madre a hijo, por trasplante de órganos y por accidentes de laboratorio. En México se estima que 1.1 millones de personas están infectadas; la incidencia en 2012 fue de 0.70 por 100 000 habitantes. En 2017, las mayores tasas de incidencia se registraron en Yucatán, Oaxaca e Hidalgo. La infección ocasiona miocardiopatías y megaórganos del tracto digestivo. El diagnóstico en fase aguda es por abordaje parasitológico y en fase crónica, por estudios de tamizaje por laboratorio. En los bancos de sangre de México, el estudio de la enfermedad de Chagas es de observancia obligatoria; de 2007 a 2016, la seroprevalencia ha disminuido de 0.40 a 0.32 debido a la mejora de los procesos de selección al donante y al cuestionario ad hoc. Los blancos del parásito son las células neuronales y las de los músculos liso y miocárdico. La asociación de la destrucción neuronal y del músculo liso define la presentación de los síndromes megachagásicos. Las manifestaciones iniciales de la enfermedad pueden pasar desapercibidas; 5 % de los pacientes presenta signos y síntomas aparentes y 30 % evolucionará a la fase crónica asintomática. Los tratamientos actuales tienen efecto en la fase aguda. Para el control de la enfermedad de Chagas se dispone del Programa de Acción Específico para la Vigilancia Prevención y Control de la Enfermedad de Chagas (PAE Chagas 2013-2018), encaminado a eliminar la transmisión transfusional y congénita y a controlar la transmisión vectorial. De la detección oportuna, el tratamiento etiológico temprano y la ampliación de cobertura depende el éxito de la atención médica. Por su parte, la vigilancia y tamizaje de las mujeres embarazadas que viven en zonas de riesgo y el tamizaje universal de donadores de sangre y órganos harán posible la eliminación de la transmisión connatal y transfusional.

HLA frequency in candidates to transplant without compatible cord blood at the National Center of Blood Transfusion (Mexico).

INTRODUCTION: Umbilical Cord Blood Units (UCBU) for transplantation, are a therapeutic possibility for patients with a wide range of oncohaematological diseases and other immunologic disorders. The search of compatible donors for bone marrow transplantation is increasingly difficult for patients of mixed ethnicity. The aim of this work was determine the HLA frequency of candidates for transplantation without compatible UCBU at the National Center of from Blood Transfusion (NCBT) - Mexico. MATERIAL AND METHODS: A retrospective analysis of candidates to transplant without compatible UCBU was performed in the archives from 2003 to 2016 at the NCBT. HLA class I and II genotyping of candidates was performed by medium resolution methods: Sequence Specific Primer and/or Specific Sequence Oligonucleotide. HLA frequencies were obtained by including individuals without any particular bias to a phenotype and strict statistical and genetic analysis of populations were done. The database in www.allelefrequencies.net was used in order to identify the ethnic origin of the most frequent alleles. RESULTS: Three hundred and sixty-four candidates without compatible UCBU for transplantation were identified. The most frequent haplotype HLA I and II were: HLA-A*02/02, 02/24, 24/24, 02/68, 01/24 and 24/68; HLA-B*39/39, 35/51, 44/44, 44/40, 35/40 and 35/35; HLA-DRB1*04/04, 13/07, 04/13, 13/13 and 03/11. The ethnic origins of the analyzed data were represented in most cases by Amerindians, Caucasics, Orientals, Asians, Arabs and Africans. CONCLUSION: This work shows the existence of a broad genetic diversity of candidates for transplantation with UCBU, making it difficult to find compatible units considering donors only from the capital.

Bactericidal effect of γ-radiation with 137Cesium in platelet concentrates.

INTRODUCTION: γ-radiation is a method that was originally designed for inactivation of T lymphocytes in blood and blood components in order to prevent transfusion associated-graft versus host disease (TA-GVHD). Klebsiella pneumoniae, Escherichia coli, Enterococcus faecium and Staphylococcus epidermidis strains are important pathogens in blood banks since they have been related to post-transfusional sepsis. This study was conducted to demonstrate that γ-radiation is effective in reducing the viability of bacteria in platelet concentrates (PC). MATERIAL AND METHODS: Klebsiella pneumoniae, E. coli, E. faecium and S. epidermidis strains were adjusted at 101 to 106 CFU/ml and used in artificial contamination assays in PC. Contaminated platelet concentrates were subjected to γ-radiation with doses of 2500 cGy in a 137Cesium irradiator. The average of surviving bacteria at different bacterial concentrations, logarithmic reduction values (LRV) and bacterial death after γ-radiation percentage was calculated. RESULTS: Escherichia coli and K. pneumoniae were eliminated in 101 to 103 CFU; in contrast with 104 to 106 CFU, the LRV were 2.4, 2.6 and 2.6 for E. coli and 3.3, 2.7 and 3.0 for K. pneumoniae strains at 104, 105 and 106 CFU respectively. For Gram-positive strains, 101 CFU in PC, the inactivation post γ-radiation was not completed. Logarithmic reduction values post γ-radiation were 0.8 to 1.2 for E. faecium and S. epidermidis strains respectively. CONCLUSION: γ-radiation cannot be an alternative for the inactivation of pathogens in PC, because of the bacterial concentration and pathogen nature - being resistant to γ-radiation, the Gram-positive bacteria.

Enfermedad de Chagas en México / Chagas disease in Mexico

Resumen México es un país endémico para la enfermedad de Chagas, donde dos terceras partes del territorio pueden ser consideradas en riesgo de transmisión vectorial, es decir que 1'100,000 individuos podrían estar infectados con Trypanosoma cruzi y 29'500,000 en riesgo de contraer la infección. En la morbimortalidad del padecimiento son importantes las características de la vivienda, condiciones biológicas, ambientales y factores socioculturales. El tamizaje en bancos de sangre, a la fecha, es de observancia obligatoria con una cobertura mayor al 92%. El diagnóstico no se establece frecuentemente debido al desconocimiento de la enfermedad por parte del personal de salud y de la población. La fase aguda generalmente pasa desapercibida y en la crónica, la patología se presenta principalmente en el corazón, con evolución lenta. La patogénesis de la miocardiopatía crónica es muy compleja y se presentan lesiones con mayor frecuencia en el sistema nervioso autónomo y miocardio, lo que genera trastornos en la conductibilidad y contractilidad del órgano. Se describen los principales mecanismos patogénicos implicados en el desarrollo de la enfermedad.

Abstract Mexico is a country endemic for Chagas disease in which two thirds of the territory can be considered at risk of vector-borne infection. This means that 1.1 million people could be infected with Trypanosoma cruzi and 29.5 million at risk of infection. Dwelling characteristics of poverty in these rural areas linked with biological conditions, lifestyle, environmental and sociocultural factors are important in the morbidity and mortality of the disease. Nowadays, the screening for the parasite is mandatory and at least 92% of blood banks are covered. The inadequate knowledge of the disease by the health personnel and the population limits the possibility of the diagnosis. The acute phase of the disease courses undetected. The main affected organ in Chagas disease is the heart, with a slow evolution; the pathogenesis of chronic cardiomyopathy is complex and lesions occur mainly in the autonomic nervous system and myocardium leading to disturbances in the conductivity and contractility of the organ. The main pathogenic mechanisms involved in the development of the disease are described.

A Large Proportion of the Mexican Population Remained Susceptible to A(H1N1)pdm09 Infection One Year after the Emergence of 2009 Influenza Pandemic.

BACKGROUND: The 2009 H1N1 influenza pandemic initially affected Mexico from April 2009 to July 2010. By August 2010, a fourth of the population had received the monovalent vaccine against the pandemic virus (A(H1N1)pdm09). To assess the proportion of the Mexican population who remained potentially susceptible to infection throughout the summer of 2010, we estimated the population seroprevalence to A(H1N1)pdm09 in a serosurvey of blood donors. METHODS: We evaluated baseline cross-reactivity to the pandemic strain and set the threshold for seropositivity using pre-pandemic (2005-2008) stored serum samples and sera from confirmed A(H1N1)pdm09 infected individuals. Between June and September 2010, a convenience sample serosurvey of adult blood donors, children, and adolescents was conducted in six states of Mexico. Sera were tested by the microneutralization (MN) and hemagglutination inhibition (HI) assays, and regarded seropositive if antibody titers were equal or exceeded 1:40 for MN and 1:20 for HI. Age-standardized seroprevalence were calculated using the 2010 National Census population. RESULTS: Sera from 1,484 individuals were analyzed; 1,363 (92%) were blood donors, and 121 (8%) children or adolescents aged ≤19 years. Mean age (standard deviation) was 31.4 (11.5) years, and 276 (19%) were women. A total of 516 (35%) participants declared history of influenza vaccination after April 2009. The age-standardized seroprevalence to A(H1N1)pdm09 was 48% by the MN and 41% by the HI assays, respectively. The youngest quintile, aged 1 to 22 years, had the highest the seroprevalence; 61% (95% confidence interval [CI]: 56, 66%) for MN, and 56% (95% CI: 51, 62%) for HI. CONCLUSIONS: Despite high transmission of A(H1N1)pdm09 observed immediately after its emergence and extensive vaccination, over a half of the Mexican population remained potentially susceptible to A(H1N1)pdm09 infection. Subsequent influenza seasons with high transmission of A(H1N1)pdm09, as 2011-2012 and 2013-2014, are compatible with these findings.

Molecular characterization of microbial contaminants isolated from Umbilical Cord Blood Units for transplant

ABSTRACT Disposal of Umbilical Cord Blood Units due to microbial contamination is a major problem in Cord Blood Banks worldwide as it reduces the number of units available for transplantation. Additionally, economic losses are generated as result of resources and infrastructure used to obtain such units. Umbilical Cord Blood Units that showed initial microbial contamination were subject to strains isolation, identification, and characterization by sequencing the 16S rRNA gene and Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR). Moreover, tests of antimicrobial resistance/sensitivity and phenotypic activities that may play an important role in microbial infection were performed. Microbial contamination was detected in 120 Umbilical Cord Blood Units (2.31%) in the period from 2003 to 2013. The most frequently isolated strains were Enterococcus faecium, followed by Staphylococcus epidermidis, Escherichia coli, Enterococcus faecalis, Staphylococcus haemoliticus, Klebsiella pneumoniae, Enterococcus durans, Lactobacillus helveticus, Enterococcus hiriae and Roseomonas genomospecies 5. The ERIC-PCR assays revealed a wide genetic diversity in some strains although belonging to the same genus and specie, indicating different sources of contamination. Broad-spectrum penicillins, third generation cephalosporins, aminoglycosides, and fluoroquinolones showed lower inhibitory activity on the tested strains. All strains were proteolytic, 67.69% were amylase-positive, 27.6% hemolysis-positive, and 34.71% nuclease-positive. The most common sources of contamination were: vaginal flora, digestive tract, and skin flora, highlighting the need for staff training in good manufacturing practices in collection SCU since all contaminants identified are part of the microbial flora of the donors. Implications and consequences in the therapeutic use of Umbilical Cord Blood Units for transplantation contaminated by multiresistant bacteria in immunocompromised patients are discussed.

Molecular characterization of microbial contaminants isolated from Umbilical Cord Blood Units for transplant.

Disposal of Umbilical Cord Blood Units due to microbial contamination is a major problem in Cord Blood Banks worldwide as it reduces the number of units available for transplantation. Additionally, economic losses are generated as result of resources and infrastructure used to obtain such units. Umbilical Cord Blood Units that showed initial microbial contamination were subject to strains isolation, identification, and characterization by sequencing the 16S rRNA gene and Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR). Moreover, tests of antimicrobial resistance/sensitivity and phenotypic activities that may play an important role in microbial infection were performed. Microbial contamination was detected in 120 Umbilical Cord Blood Units (2.31%) in the period from 2003 to 2013. The most frequently isolated strains were Enterococcus faecium, followed by Staphylococcus epidermidis, Escherichia coli, Enterococcus faecalis, Staphylococcus haemoliticus, Klebsiella pneumoniae, Enterococcus durans, Lactobacillus helveticus, Enterococcus hiriae and Roseomonas genomospecies 5. The ERIC-PCR assays revealed a wide genetic diversity in some strains although belonging to the same genus and specie, indicating different sources of contamination. Broad-spectrum penicillins, third generation cephalosporins, aminoglycosides, and fluoroquinolones showed lower inhibitory activity on the tested strains. All strains were proteolytic, 67.69% were amylase-positive, 27.6% hemolysis-positive, and 34.71% nuclease-positive. The most common sources of contamination were: vaginal flora, digestive tract, and skin flora, highlighting the need for staff training in good manufacturing practices in collection SCU since all contaminants identified are part of the microbial flora of the donors. Implications and consequences in the therapeutic use of Umbilical Cord Blood Units for transplantation contaminated by multiresistant bacteria in immunocompromised patients are discussed.

Propagation capacity of bacterial contaminants in platelet concentrates using a luciferase reporter system.

INTRODUCTION: Currently the use of molecular tools and techniques of Genetic Engineering in the study of microbial behavior in blood components has replaced the employment of classical methods of microbiology. This work focuses on the use of a novel lux reporter system for monitoring the contaminating propagation capacity of bacteria present in platelet concentrates under standard storage conditions in the blood bank. METHODS: A miniTn5 promotor probe carrying the lux operon from Photorhabdus luminiscens (pUTminiTn5luxCDABEKm2) was used to construct four bacterial bioluminescent mutants: Escherichia coli, Salmonella typhi, Proteus mirabilis and Pseudomonas aeruginosa. Luminescent mutants were used for contamination tests with 20 CFU in platelet concentrates bags and were stored under standard storage conditions in the blood bank (100 rpm at 22 °C). The measurements of luminous activity and optical density were used to monitor bacterial proliferation during 7 days (168 h). RESULTS: During the exponential growth phase (log) of bacterial strains, a lineal correlation between luminous activity vs biomass was observed (R(2) = 0.985, 0.976, 0.981) for E. coli::Tn5luxCDABEKm2, P. mirabilis::Tn5luxCDABEKm2 and P. auriginosa::Tn5luxCDABEKm2, respectively. The above indicates that metabolic activity (production of ATP) is directly related to biomass in this phase of microbial growth. While conducting experiments, the inability to propagate S. typhi::Tn5luxCDABEKm2 was detected. We can speculate that platelet concentrates contain specific components that prevent the propagation of S. typhi. CONCLUSION: The use of luxCDABE system for the quantification of luminous activity is a rapid and sensitive alternative to study the propagation and auto-sterilization of bacterial contaminants in platelet concentrates.

[Blood Safety in the XXI century. Transfusion transmitted infectious diseases. International and Mexican view]. / Enfermedades infecciosas transmitidas por transfusión. Panorama internacional y en México.

Currently worldwide, the transfusion of blood components cannot be done without residual risks, as compared to those countries with a high human development index, mostly in Europe, that have blood donation systems based on 100% repeat volunteer donors and use molecular biology techniques in screening for infectious diseases. In Latin America and the Caribbean countries, prevention of transfusion-transmissible diseases requires special and different strategies due to several factors: the high prevalence of replacement donors, their specific geographical location, climate, genetic, and sociocultural status of the population make them vulnerable to endemic diseases such as dengue, malaria, and Chagas disease. Thus it is necessary to create local approaches to increase blood safety and achieve the goals set by the Pan American Health Organization.

Prevalencia del virus humano herpes 7 en donadores de sangre mexicanos / Prevalence of human herpes viruz 7 in Mexican blood donors

El herpes virus humano 7 (HHV-7) fue aislado recientemente de las células CD4 de individuos sanos; actualmente se realizan estudios de suposible asociación con enfermedades de humanos. Se estudiaron 200 muestras de sangre de candidatos a donadores asintomáticos del banco de sangre del Hospital General de México, utilizando la prueba de inmunofluorescencia indirecta (IFA) en células SupT1 infectadas con el HHV-7 en la Universidad de Colonia, Alemania. El 83.5 por ciento de las muestras fueron de varones y el 16.5 por ciento de mujeres; provenían de 12 diferentes estados de la república mexicana con predominio del Distrito Federal (60.5 por ciento) y del Estado de México (28 por ciento) con edad promedio de 29.2 años. El HHV-7 fue detectado en el 98.5 por ciento de los casos a diferentes titulaciones; el 84.5 por ciento presentó títulos elevados (ò 1:80). El 1 por ciento resultó positivo a hepatitis B, el 2 por ciento a sífilis, y el 0.5 por ciento a brucella. Todos fueron negativos a hepatitis C así como al VIH. La elevada prevalencia de HHV-7 deberá aclararse en investigaciones futuras que permitan determinar la posible asociación de títulos altos con infección activa, así como el significado de ésta en relación a enfermedad

Utilidad de la impronta de ganglio linfático en el diagnóstico del linfoma / Lymph node imprints in the diagnosis of lymphoma

Para conocer la utilidad de la impronta de ganglio linfático, se revisó el archivo de citomorfología del laboratorio de Estudios Espaciales del Servicio de Hematología en el período de febrero de 1983 a diciembre de 1986 y se correlacionó con los reportes de histopatología de la Unidad de Patolog del Hospital General, S.S. Se revisaron 44 improntas de pacientes con diagnóstico en 31 casos (72%) y no habiendo correlación diagnóstico en 13 casos (28%). Los resultados de este estudio justifican la introducción de la impronta de ganglio dentro de los exámenes de procedimientos diagnóstico en pacientes con probable linfoma. Su uso sicesivo y la adquisición de mayor experiencia del observador hará que este porcentaje se mejore sin que reemplace el diagnóstico histopatológico .

Estudio clinopatológico de los linfomas no Hodgkin de testículo / Clinical and pathological study of the testicle linphomas no Hodgkin type up to data papers

Para conocer la frecuencia de los linfomas no Hodgkin (LNH) con afección testicular y sus características clínicas y anatomopatológicas, se revisaron los archivos de Hematología y Patología de enero de 1975 a diciembre de 1984. En el servicio de Hematología se encontraron 479 casos de LNH, de los cuales 263 (54.9%) fueron del sexo masculino y de éstos, seis (2.3%) presentaron afección testicular. Un caso se consideró enfermedad primaria, tres enfermedad sistémica y dos complicación tardía. La edad promedio fue de 50 años. De acuerdo a la clasificación internacional (CI), cinco casos correspondieron a linfomas de linfocitos poco diferenciados con patrón difuso. La sobrevida mayor correspondió al caso primario, con 60 meses. El promedio de sobrevida del grupo fue de cinco meses. En la Unidad de Patología se encontraron 148 casos de autopsia con LNH, de éstos 92 (62.1%) fueron del sexo masculino, de los cuales 10 (10.8%) mostraron participación testicular. En dos de los 10 casos se conocía la participación testicular y en seis fue hallazgo de autopsia, en un caso se localizó en epidídimo y en otro en condones espermáticos. La edad promedio fue de 49 años. Cinco casos presentaron participación extraganglionar además de la testicular: tres en piel, un nasal y cuatro en sistema nervioso central. De acuerdo a la CI, uno correspondió a grado de malignidad bajo, seis a grado de malignidad intermedio y tres a grado de malignidad alto. De acuerdo a la clasificación de Rappaport tres fueron linfomas histiocíticos, tres de linfocitos poco diferenciados, uno de linfocitos bien diferenciados, un plasmocitoma, un sarcoma inmuno blásico y un caso de micosis fungoides .

Correlación clínico-pronostica en el linforma no-hodgkin: valoración de la nueva clasificación propuesta en un estudio retrospectivo / Clinico prognostic correlation in non-Hodgkin lymphoma: value of the new classification proposed in a retrospective study

Se revisaron en forma retrospectiva los datos clínicos e histológicos de 282 pacientes con linfoma No-Hodgkin, estudiados en el Hospital General de México de la S.S.A. Se establecieron los diagnósticos y correlaciones pronósticas de acuerdo a la nueva formulación internacional. Los resultados muestran que la nueva clasificación ofrece buena precisión pronóstica y adecuada reproducibilidad al patólogo. Los linfomas más frecuentes fueron los de grado intermedio de malignidad (60.9%), seguidos de los de alto grado (30.1%) y los menos frecuentes fueron los de bajo grado (8.8%). La mayor parte de los pacientes estaban en estadio clínico IV, sin diferencias significativas entre los tres grupos. El porcentaje de defunciones correlacionó adecuadamente en los tres grupos (bajo grado 24%, grado intermedio 32.5% y alto grado 47%). Los linfomas foliculares constituyeron un bajo porcentaje, en contraste con lo informado por autores extranjeros. Se mencionan algunos aspectos comparativos con las clasificaciones de Rappaport y de Lukes y Collins