Prevalence of thrombophilia-associated genetic risk factors in blood donors of a regional hospital in southern Brazil

ABSTRACT Introduction: Thromboembolic events occur due to an imbalance in the hemostasis and some factors associated with this condition can be inherited. In order to evaluate the frequency of genotypes considered to be common hereditary risk factors for thrombophilia associated with venous thrombosis (g.1691G>A and g.20210G>A) and hyperhomocysteinemia (g.677C>T and g.1298A>C), samples from voluntary healthy blood donors at the Hospital de Clínicas de Porto Alegre were tested. Methods: We examined 325 blood samples from blood donors collected from October 2017 to July 2018. Blood was collected on filter paper and the DNA was extracted for single nucleotide polymorphisms (SNPs) analysis using the qualitative real time polymerase chain reaction. Results: The calculated frequencies of each genetic variant in heterozygosity were 4% for the FV gene (g.1691G> A), 4% for the F2 gene (g.20210G> A) and 42% and 39% for methylenetetrahydrofolate reductase (MTHFR), g.677C>T and g.1298A>C, respectively. Only the genetic variants of MTHFR were found in homozygosity, with frequencies of 14% and 6% (g.677C>T and g.1298A>C), respectively. Discussion: Altogether, these results describe the frequencies of genetic variants associated with venous thrombosis and hyperhomocysteinemia in the analyzed group and are important to enhance our current knowledge about the genetic profiles of Brazilian blood donors.

Standardization of an organic DNA extraction method from dried blood spots and its downstream molecular applications in neonatal screening and diagnostic confirmation of lysosomal disorders

Introduction: Dried blood spot (DBS) samples have been used for diagnostic purposes since their introduction in the neonatal screening of phenylketonuria almost 50 years ago. The range of its application has been extended to modern approaches, such as next-generation sequencing (NGS) for molecular genetic testing. This study aimed to evaluate the use of a standardized organic method for DNA extraction from DBS samples in the diagnostic setting.Methods: The clinical applicability of the method was tested using 3 samples collected from a newborn screening project for lysosomal storage diseases, allowing the determination of the genotype of the individuals. DNA was extracted from 3 3-mm diameter DBS punches. Quality, purity, and concentration were determined, and method performance was assessed by standard polymerase chain reaction, restriction length polymorphism, Sanger sequencing, and targeted NGS.Results: Results were compared with the ones obtained from DNA samples extracted following the internally validated in-house extraction protocol that used 6 3-mm punches of DBS and samples extracted from whole blood.Conclusion: This organic method proved to be effective in obtaining high-quality DNA from DBS, being compatible with several downstream molecular applications, in addition to having a lower cost per sample

Quantification of peripheral blood CD34+ cells prior to stem cell harvesting by leukapheresis: a single center experience

ABSTRACT Background: Due to laboratory logistic issues, our center has traditionally scheduled peripheral blood stem cell harvests based on timing from the start of mobilization. This has proved to be useful in some cases, but also resulted in many fruitless harvests due to poor mobilization. In order to improve the efficiency of collections and compare the effectiveness of peripheral blood CD34+ cells as a predictor with data from other reports, this study analyzed the implementation of this routine. Methods: Peripheral blood and leukapheresis samples were quantified by flow cytometry and the association between these parameters was assessed. Results: Sixty-six consecutive leukapheresis samples were collected from 34 patients after the collection of peripheral blood samples for CD34+ quantification. A moderate positive correlation was observed between peripheral blood CD34+ cell count and total CD34+ cell count/kg (r = 0.596; p-value < 0.001). A multivariable regression model also confirmed this association and allowed the estimation that for every increase in five CD34+ cells/µL in the peripheral blood, a mean increase of 0.38 × 106 CD34+ cells/kg could be predicted. Demographic characteristics, baseline comorbidities and mobilization regimen did not influence final CD34+ cell count in this sample. Conclusions: As observed in other centers, quantification of peripheral blood CD34+ progenitor cells is a strong predictor of effectiveness to guide stem cell harvesting. Due to the results of this study, a modification in the peripheral blood stem cell harvesting logistics was implemented at our center in order to incorporate this routine.