Comparative flow cytometry-based immunophenotyping analysis of peripheral blood leukocytes before and after fixation with paraformaldehyde.

Flow cytometry based immunophenotyping provides prime insight into cellular population composition and characteristics, and is widely used in basic and clinical research. Challenges in processing peripheral blood samples in a timely manner necessitate protocol adaptations and utilization of fixatives. Fixation, however, may introduce artifacts to the flow cytometry readout. We performed a comparative flow cytometry immunophenotyping analysis of 13 immune cell populations in the whole blood using a staining protocol with and without fixation step. Freshly procured human peripheral blood samples were stained with a panel of 33 fluorochrome-conjugated antibodies. Samples were processed using a protocol with or without a paraformaldehyde-based fixation step, and matching sample pairs were analyzed by flow cytometry. Our results show that paraformaldehyde-based fixation, in comparison to matched unfixed samples, did not significantly affect population distribution and frequency for: B cells, Plasmablasts, Dendritic cells, NK cells, Granulocytes, Neutrophils, Eosinophils, or Hematopoietic Stem/Progenitor Cells. However, fixation led to significant marker shifts in the subpopulation distribution in CD4, T regulatory, CD8, Monocytes, and Basophils. These results indicate the importance of pre-experimental assessment of fixation-introduced artifacts in the flow cytometry output when considering the feasibility of fresh processing. This is especially important for samples analyzed using comprehensive exploratory immunoprofiling panels.

Low expression of Abelson interactor-1 is linked to acquired drug resistance in Bcr-Abl-induced leukemia.

The basis for persistence of leukemic stem cells in the bone marrow microenvironment remains poorly understood. We present evidence that signaling cross-talk between α4 integrin and Abelson interactor-1 (Abi-1) is involved in the acquisition of an anchorage-dependent phenotype and drug resistance in Bcr-Abl-positive leukemia cells. Comparison of Abi-1 (ABI-1) and α4 integrin (ITGA4) gene expression in relapsing Bcr-Abl-positive CD34+progenitor cells demonstrated a reduction in Abi-1 and an increase in α4 integrin mRNA in the absence of Bcr-Abl mutations. This inverse correlation between Abi-1 and α4 integrin expression, as well as linkage to elevated phospho-Akt and phospho-Erk signaling, was confirmed in imatinib mesylate -resistant leukemic cells. These results indicate that the α4-Abi-1 signaling pathway may mediate acquisition of the drug-resistant phenotype of leukemic cells.

Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice.

Prostate cancer is one of the leading causes of cancer-related deaths in the United States and a leading diagnosed non-skin cancer in American men. Genetic mutations underlying prostate tumorigenesis include alterations of tumor suppressor genes. We tested the tumor suppressor hypothesis for ABI1/hSSH3BP1 by searching for gene mutations in primary prostate tumors from patients, and by analyzing the consequences of prostate-specific disruption of the mouse Abi1/Hssh3bp1 ortholog. We sequenced the ABI1/hSSH3BP1 gene and identified recurring mutations in 6 out of 35 prostate tumors. Moreover, complementation and anchorage-independent growth, proliferation, cellular adhesion and xenograft assays using the LNCaP cell line, which contains a loss-of-function Abi1 mutation, and a stably expressed wild-type or mutated ABI gene, were consistent with the tumor suppressor hypothesis. To test the hypothesis further, we disrupted the gene in the mouse prostate by breeding the Abi1 floxed strain with the probasin promoter-driven Cre recombinase strain. Histopathological evaluation of mice indicated development of prostatic intraepithelial neoplasia (PIN) in Abi1/Hssh3bp1 knockout mouse as early as the eighth month, but no progression beyond PIN was observed in mice as old as 12 months. Observed decreased levels of E-cadherin, ß-catenin and WAVE2 in mouse prostate suggest abnormal cellular adhesion as the mechanism underlying PIN development owing to Abi1 disruption. Analysis of syngeneic cell lines point to the possibility that upregulation of phospho-Akt underlies the enhanced cellular proliferation phenotype of cells lacking Abi1. This study provides proof-of-concept for the hypothesis that Abi1 downregulation has a role in the development of prostate cancer.

Hereditary spherocytosis: identification of several HS families with ankyrin and band 3 deficiency in a population of southwestern Poland.

Red blood cells of 17 patients out of seven families diagnosed with HS from the southwest of Poland were studied. In six families a deficiency of ankyrin was detected, and in one family a band 3 (anion-exchanger protein) deficiency was detected. Patients from six families with the ankyrin deficiency had a 19-51% decrease in ankyrin 2.1, while the family with the band 3 deficiency showed a 33% decrease in this protein content. All changes were statistically significant, as analysed by the Student t test (P<0.05). Analysis of haemolysis kinetics gives a reliable indication of altered osmotic properties of the spherocytic cells.