Computational protocol: Hematopoietic Stem Cells in Neonates: Any Differences between Very Preterm and Term Neonates?

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Protocol publication

[…] The enumeration of HSPC was conducted in whole UCB using BD Trucount tubes (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s instruction. 100 µl whole UCB were added by reverse pipetting to BD Trucount tubes and stained with anti- CD45− FITC, anti- CD34− PE, anti- CD38− PE- Cy7, anti- CD10− PE- CF594, 7-AAD (all BD Biosciences) and anti- CD133– APC (Miltenyi Biotechnology, Bergisch, Gladbach, Germany). After immunofluorescence staining, erythrocytes were lysed with ammonium chloride lysis solution and analyzed within one hour by flow cytometry (LSRII, BD Biosciences) and analysed using FlowJo software (TreeStar Inc., Ashland, OR, USA). For enumeration of HSPC in UCB, CD34+ cells were gated according to the modified ISHAGE criteria (CD45dim/7-AAD-/CD34+ cells). CD10 was added to exclude B- lymphoid progenitors. The number of cells/µl was calculated as [(# gated cells/# acquired beads) * (# of beads per test/test volume). [...] Statistical analysis was performed with IBM SPSS Statistics 21. Kolmogorov- Smirnov test was performed to prove normal distribution. Student’s T- Test was used to analyze normal distributed data. In case of non- normal distribution, data were further analyzed using Mann- Whitney- U test. Bonferroni correction for multiple testing was used. Differences of quantitative variables were compared by Mann- Whitney U test and qualitative variables with Fisher’s exact test. Correlation between variables was determined using Spearman’s rank correlation coefficient. Multiple linear backward regression was performed to evaluate the effects of obstetric, perinatal and neonatal factors on the UCB HSC count. A p<0.05 was considered as statistically significant. […]

Pipeline specifications

Software tools FlowJo, SPSS
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens
Diseases Sjogren-Larsson Syndrome