Spatial analysis of photoplethysmography in cutaneous squamous cell carcinoma.

The primary treatment of the common malignancy squamous cell carcinoma is surgical removal. In this process, sufficient tissue removal is balanced against unnecessary mutilation. We recently presented a remote photoplethysmography algorithm, which revealed significant differences between processed video recordings of cancer biopsy areas and surrounding tissue. The aim of this study was to investigate whether spatial analyses of photoplethysmography data correlate with post-excision pathological analyses and thus have potential to assist in tumour delineation. Based on high speed video recordings of 11 patients with squamous cell carcinoma, we examined different parameters derived from temporal remote photoplethysmography variations. Signal characteristics values in sites matching histological sections were compared with pathological measures. Values were ranked and statistically tested with a Kendall correlation analysis. A moderate, negative correlation was found between signal oscillations and the width and transversal area of squamous cell carcinoma in the frequencies below 1 Hz and specifically from 0.02 to 0.15 Hz. We have presented a correlation between frequency content and prevalence of cancer based on regular video recordings of squamous cell carcinoma. We believe this is supported by published findings on malignant melanoma. Our findings indicate that photoplethysmography can be used to distinguish SCC from healthy skin.

Photoplethysmography for demarcation of cutaneous squamous cell carcinoma.

A video processing algorithm designed to identify cancer suspicious skin areas is presented here. It is based on video recordings of squamous cell carcinoma in the skin. Squamous cell carcinoma is a common malignancy, normally treated by surgical removal. The surgeon should always balance sufficient tissue removal against unnecessary mutilation, and therefore methods for distinction of cancer boundaries are wanted. Squamous cell carcinoma has angiogenesis and increased blood supply. Remote photoplethysmography is an evolving technique for analysis of signal variations in video recordings in order to extract vital signs such as pulsation. We hypothesize that the remote photoplethysmography signal inside the area of a squamous cell carcinoma is significantly different from the surrounding healthy skin. Based on high speed video recordings of 13 patients with squamous cell carcinoma, we have examined temporal signal differences in cancer areas versus healthy skin areas. A significant difference in temporal signal changes between cancer areas and healthy areas was found. Our video processing algorithm showed promising results encouraging further investigation to clarify how detailed distinctions can be made.

[Enlarged vestibular aqueduct in a ten-year-old girl].

Enlarged vestibular aqueduct (EVA) is a malformation in the inner ear and occurs in approximately 1-12% of the hearing-impaired individuals. A ten-year-old girl was seen at the emergency room (ER) because of sudden hearing loss on the right ear. The patient was known with sudden deafness on the left ear. In the ER she appeared completely deaf. A head CT-scan showed EVA, and she was treated with cochlear implant. This case report illustrates the importance of performing MR- or CT-scan, especially when diagnosing children with sudden hearing loss after a head injury.

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.

Validation and implementation of a method for microarray gene expression profiling of minor B-cell subpopulations in man.

BACKGROUND: This report describes a method for the generation of global gene expression profiles from low frequent B-cell subsets by using fluorescence-activated cell sorting and RNA amplification. However, some of the differentiating compartments involve a low number of cells and therefore it is important to optimize and validate each step in the procedure. METHODS: Normal lymphoid tissues from blood, tonsils, thymus and bone marrow were immunophenotyped by the 8-colour Euroflow panel using multiparametric flow cytometry. Subsets of B-cells containing cell numbers ranging from 800 to 33,000 and with frequencies varying between 0.1 and 10 percent were sorted, subjected to mRNA purification, amplified by the NuGEN protocol and finally analysed by the Affymetrix platform. RESULTS: Following a step by step strategy, each step in the workflow was validated and the sorting/storage conditions optimized as described in this report. First, an analysis of four cancer cell lines on Affymetrix arrays, using either 100 ng RNA labelled with the Ambion standard protocol or 1 ng RNA amplified and labelled by the NuGEN protocol, revealed a significant correlation of gene expressions (r ≥ 0.9 for all). Comparison of qPCR data in samples with or without amplification for 8 genes showed that a relative difference between six cell lines was preserved (r ≥ 0.9). Second, a comparison of cells sorted into PrepProtect, RNAlater or directly into lysis/binding buffer showed a higher yield of purified mRNA following storage in lysis/binding buffer (p < 0.001). Third, the identity of the B-cell subsets validated by the cluster of differentiation (CD) membrane profile was highly concordant with the transcriptional gene expression (p-values <0.001). Finally, in normal bone marrow and tonsil samples, eight evaluated genes were expressed in accordance with the biology of lymphopoiesis (p-values < 0.001), which enabled the generation of a gene-specific B-cell atlas. CONCLUSION: A description of the implementation and validation of commercially available kits in the laboratory has been examined. This included steps for cell sorting, cell lysis/stabilization, RNA isolation, RNA concentration and amplification for microarray analysis. The workflow described in this report will enable the generation of microarray data from minor sorted B-cell subsets.