Stable phenotype of B-cell subsets following cryopreservation and thawing of normal human lymphocytes stored in a tissue biobank.

BACKGROUND: Cryopreservation is an acknowledged procedure to store vital cells for future biomarker analyses. Few studies, however, have analyzed the impact of the cryopreservation on phenotyping. METHODS: We have performed a controlled comparison of cryopreserved and fresh cellular aliquots prepared from individual healthy donors. We studied circulating B-cell subset membrane markers and global gene expression, respectively by multiparametric flow cytometry and microarray data. Extensive statistical analysis of the generated data tested the concept that "overall, there are no phenotypic differences between cryopreserved and fresh B-cell subsets." Subsequently, we performed an uncontrolled comparison of tonsil tissue samples. RESULTS: By multiparametric flow analysis, we documented no significant changes following cryopreservation of subset frequencies or membrane intensity for the differentiation markers CD19, CD20, CD22, CD27, CD38, CD45, and CD200. By gene expression profiling following cryopreservation, across all samples, only 16 out of 18708 genes were significantly up or down regulated, including FOSB, KLF4, RBP7, ANXA1 or CLC, DEFA3, respectively. Implementation of cryopreserved tissue in our research program allowed us to present a performance analysis, by comparing cryopreserved and fresh tonsil tissue. As expected, phenotypic differences were identified, but to an extent that did not affect the performance of the cryopreserved tissue to generate specific B-cell subset associated gene signatures and assign subset phenotypes to independent tissue samples. CONCLUSIONS: We have confirmed our working concept and illustrated the usefulness of vital cryopreserved cell suspensions for phenotypic studies of the normal B-cell hierarchy; however, storage procedures need to be delineated by tissue-specific comparative analysis.

Stable Phenotype Of B-Cell Subsets Following Cryopreservation and Thawing of Normal Human Lymphocytes Stored in a Tissue Biobank.

Background Cryopreservation is an acknowledged procedure to store vital cells for future biomarker analyses. Few studies, however, have analyzed the impact of the cryopreservation on phenotyping. Methods We have performed a controlled comparison of cryopreserved and fresh cellular aliquots prepared from individual healthy donors. We studied circulating B-cell subset membrane markers and global gene expression, respectively by multiparametric flow cytometry and microarray data. Extensive statistical analysis of the generated data tested the concept that "overall, there are phenotypic differences between cryopreserved and fresh B-cell subsets". Subsequently, we performed a consecutive uncontrolled comparison of tonsil tissue samples. Results By multiparametric flow analysis, we documented no significant changes following cryopreservation of subset frequencies or membrane intensity for the differentiation markers CD19, CD20, CD22, CD27, CD38, CD45, and CD200. By gene expression profiling following cryopreservation, across all samples, only 16 out of 18708 genes were significantly up or down regulated, including FOSB, KLF4, RBP7, ANXA1 or CLC, DEFA3, respectively. Implementation of cryopreserved tissue in our research program allowed us to present a performance analysis, by comparing cryopreserved and fresh tonsil tissue. As expected, phenotypic differences were identified, but to an extent that did not affect the performance of the cryopreserved tissue to generate specific B-cell subset associated gene signatures and assign subset phenotypes to independent tissue samples. Conclusions We have confirmed our working concept and illustrated the usefulness of vital cryopreserved cell suspensions for phenotypic studies of the normal B-cell hierarchy; however, storage procedures need to be delineated by tissue specific comparative analysis. © 2014 Clinical Cytometry Society.

Mastoiditis and Gradenigo's Syndrome with anaerobic bacteria.

BACKGROUND: Gradenigo's syndrome is a rare disease, which is characterized by the triad of the following conditions: suppurative otitis media, pain in the distribution of the first and the second division of trigeminal nerve, and abducens nerve palsy. The full triad may often not be present, but can develop if the condition is not treated correctly. CASE PRESENTATION: We report a case of a 3-year-old girl, who presented with fever and left-sided acute otitis media. She developed acute mastoiditis, which was initially treated by intravenous antibiotics, ventilation tube insertion and cortical mastoidectomy. After 6 days the clinical picture was complicated by development of left-sided abducens palsy. MRI-scanning showed osteomyelitis within the petro-mastoid complex, and a hyper intense signal of the adjacent meninges. Microbiological investigations showed Staphylococcus aureus and Fusobacterium necrophorum. She was treated successfully with intravenous broad-spectrum antibiotic therapy with anaerobic coverage. After 8 weeks of follow-up there was no sign of recurrent infection or abducens palsy. CONCLUSION: Gradenigo's syndrome is a rare, but life-threatening complication to middle ear infection. It is most commonly caused by aerobic microorganisms, but anaerobic microorganisms may also be found why anaerobic coverage should be considered when determining the antibiotic treatment.