Colony-forming units optimization and human papillomavirus detection in umbilical cord blood.

INTRODUCTION: Analysis of several parameters is required for adequate quality control in umbilical cord blood units (UCBU) when used for therapeutic purposes. OBJECTIVE: To optimize colony-forming units (CFU) from clonogenic cultures and to detect the human papillomavirus (HPV) genome in UCBU. METHODS: One hundred and forty-one umbilical cord blood (UCB), segment or CFU samples from UCBU clonogenic cultures were included. DNA extraction, quantification and endogenous GAPDH gene PCR amplification were carried out. Subsequently, HPV L1 gene was detected using the MY09/MY11 and GP5/GP6+ oligonucleotides. PCR products were analyzed with electrophoresis in agarose gel. CFU-extracted purified DNA was analyzed by electrophoresis in agarose gel, as well as some DNAs, using the sequence-specific priming technique. RESULTS: CFU-extracted DNA concentration was higher in comparison with that of UCB (p = 0.0041) and that of the segment (p < 0.0001), as well as that of UCB in comparison with that of the segment (p < 0.0001). All samples were positive for GAPDH amplification and negative for MY09/MY/11 and GP5/GP6+. CONCLUSIONS: Cryopreserved UCBUs were HPV-negative. Obtaining CFU DNA from clonogenic cultures with high concentrations and purity is feasible.

INTRODUCCIÓN: Se requiere analizar diversos parámetros para el control de calidad adecuado de las unidades de sangre de cordón umbilical (USCU) cuando se utilizan con fines terapéuticos. OBJETIVO: Optimizar las unidades formadoras de colonias (UFC) de cultivos clonogénicos y detectar el genoma del virus del papiloma humano (VPH) en USCU. MÉTODOS: Se incluyeron 141 muestras de sangre de cordón umbilical (SCU), de segmento y de UFC de cultivos clonogénicos de USCU. Se realizó extracción de ADN, cuantificación y amplificación por PCR del gen endógeno GAPDH. Se detectó el gen L1 del VPH con los oligonucleótidos MY09/MY11 y GP5/GP6+; los productos de PCR se migraron en electroforesis de agarosa. El ADN purificado de las UFC se analizó mediante electroforesis de agarosa y algunos ADN, con la técnica sequence specific priming. RESULTADOS: La concentración de ADN extraído de UFC fue superior comparada con la de SCU (p = 0.0041) y la de segmento (p < 0.0001); así como la de SCU comparada con la de segmento (p < 0.0001). Todas las muestras fueron positivas para la amplificación de GAPDH y negativas para MY09/MY11 y GP5/GP6+. CONCLUSIONES: Las USCU criopreservadas fueron VPH netativas; además, es factible obtener ADN en altas concentraciones y con alta pureza a partir de UFC de los cultivos clonogénicos.

Optimización de unidades formadoras de colonias y detección del virus de papiloma humano en sangre de cordón umbilical / Colony-forming units optimization and human papillomavirus detection in umbilical cord blood

Resumen Introducción: Se requiere analizar diversos parámetros para el control de calidad adecuado de las unidades de sangre de cordón umbilical (USCU) cuando se utilizan con fines terapéuticos. Objetivo: Optimizar las unidades formadoras de colonias (UFC) de cultivos clonogénicos y detectar el genoma del virus del papiloma humano (VPH) en USCU. Métodos: Se incluyeron 141 muestras de sangre de cordón umbilical (SCU), de segmento y de UFC de cultivos clonogénicos de USCU. Se realizó extracción de ADN, cuantificación y amplificación por PCR del gen endógeno GAPDH. Se detectó el gen L1 del VPH con los oligonucleótidos MY09/MY11 y GP5/GP6+; los productos de PCR se migraron en electroforesis de agarosa. El ADN purificado de las UFC se analizó mediante electroforesis de agarosa y algunos ADN, con la técnica sequence specific priming. Resultados: La concentración de ADN extraído de UFC fue superior comparada con la de SCU (p = 0.0041) y la de segmento (p < 0.0001); así como la de SCU comparada con la de segmento (p < 0.0001). Todas las muestras fueron positivas para la amplificación de GAPDH y negativas para MY09/MY11 y GP5/GP6+. Conclusiones: Las USCU criopreservadas fueron VPH netativas; además, es factible obtener ADN en altas concentraciones y con alta pureza a partir de UFC de los cultivos clonogénicos.

Abstract Introduction: Analysis of several markers is required for adequate quality control in umbilical cord blood units (UCBU) when are used for therapeutic purposes. Objective: To optimize colony-forming units (CFU) from clonogenic cultures and to detect the human papillomavirus (HPV) genome in UCBU. Methods: One hundred and forty-one umbilical cord blood (UCB), segment or CFU samples from UCBU clonogenic cultures were included. DNA extraction, quantification and endogenous GAPDH gene PCR amplification were carried out. Subsequently, HPV L1 gene was detected using the MY09/MY11 and GP5/GP6+ oligonucleotides. PCR products were analyzed with electrophoresis in agarose gel. CFU-extracted purified DNA was analyzed by electrophoresis in agarose gel, as well as some DNAs, using the SSP technique. Results: CFU-extracted DNA concentration was higher in comparison with that of UCB (p = 0.0041) and that of the segment (p < 0.0001), as well as that of UCB in comparison with that of the segment (p < 0.0001). All samples were positive for GAPDH amplification and negative for MY09/MY11 and GP5/GP6+. Conclusions: Cryopreserved UCBUs were HPV-negative. Obtaining CFU DNA from clonogenic cultures with high concentrations and purity is feasible.

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.