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The diagnosis of lymphomas and leukemias requires hematopathologists to integrate microscopically visible cellular morphology with antibody-identified cell surface molecule expression. To merge these into one high-throughput, highly multiplexed, single-cell assay, we quantify cell morphological features by their underlying, antibody-measurable molecular components, which empowers mass cytometers to 'see' like pathologists. When applied to 71 diverse clinical samples, single-cell morphometric profiling reveals robust and distinct patterns of 'morphometric' markers for each major cell type. Individually, lamin B1 highlights acute leukemias, lamin A/C helps distinguish normal from neoplastic mature T cells, and VAMP-7 recapitulates light-cytometric side scatter. Combined with machine learning, morphometric markers form intuitive visualizations of normal and neoplastic cellular distribution and differentiation. When recalibrated for myelomonocytic blast enumeration, this approach is superior to flow cytometry and comparable to expert microscopy, bypassing years of specialized training. The contextualization of traditional surface markers on independent morphometric frameworks permits more sensitive and automated diagnosis of complex hematopoietic diseases. A scalable mass cytometry-based method for morphometrically classifying hematopoietic cells demonstrates diagnostic utility when applied to clinical samples., Author(s): Albert G. Tsai [sup.1] , David R. Glass [sup.1] [sup.2] , Marisa Juntilla [sup.1] , Felix J. Hartmann [sup.1] , Jean S. Oak [sup.1] , Sebastian Fernandez-Pol [sup.1] , [...]

The [beta]-selection checkpoint in [alpha][beta]T lymphocyte development occurs at the double negative (DN) 3 ([CD4.sup.-][CD8.sup.-][CD25.sup.+]c-[kit.sup.-]) stage, when further differentiation requires a signal from the newly rearranged TCR [beta] chain. Thymocytes with mutations in key signaling molecules in the phosphatidylinositol 3-kinase-Akt pathway manifest defects in survival, proliferation, and differentiation past the [beta]-selection checkpoint. However, little information is available regarding the role of Akt itself in thymocyte development. In this study, we explore the role of the two Akt isoforms most highly expressed in the thymus, Akt1 and Akt2, in early T cell development. Using several complementary approaches, we find that deletion of Akt1 results in only minor defects in thymocyte development. The [Akt1.sup.-/-][Akt2.sup.-/-] thymocytes manifest a severe developmental block at the DN3 stage and ultimately fail to repopulate the T cell compartment of an irradiated host. Further, we show that [Akt1.sup.-/-][Akt2.sup.-/-] DN3 cells have decreased glucose uptake and die in response to TCR stimulation in vitro. Study of thymocytes from the genetically altered mice suggests that the cause of the developmental defect is due to apoptosis, partially caused by decreased cellular growth and metabolism at the DN3 stage. Our results show that Akt protects thymocytes from cell death during the [beta]-selection checkpoint. apoptosis | metabolism | thymic development

Dyskeratosis congenita is a disease of impaired tissue maintenance downstream of telomere dysfunction. Characteristically, patients present with the clinical triad of nail dystrophy, oral leukoplakia, and skin pigmentation defects, but the disease involves degenerative changes in multiple organs. Mutations in telomere-binding proteins such as TINF2 (TRF1-interacting nuclear factor 2) or in telomerase, the enzyme that counteracts age related telomere shortening, are causative in dyskeratosis congenita. We present a patient who presented with severe hypoxemia at age 13. The patient had a history of myelodysplastic syndrome treated with bone marrow transplant at the age of 5. At age 18 she was hospitalized for an acute pneumonia progressing to respiratory failure, developed renal failure and ultimately, she and her family opted to withdraw support as she was not a candidate for a lung transplant. Sequencing of the patient's TINF2 locus revealed a heterozygous mutation (c.844C > T, Arg282Cys) which has previously been reported in a subset of dyskeratosis congenita patients. Tissue sections from multiple organs showed degenerative changes including disorganized bone remodeling, diffuse alveolar damage and small vessel proliferation in the lung, and hyperkeratosis with hyperpigmentation of the skin. Autopsy samples revealed a bimodal distribution of telomere length, with telomeres from donor hematopoietic tissues being an age-appropriate length and those from patient tissues showing pathogenic shortening, with the shortest telomeres in lung, liver, and kidney. We report for the first time a survey of degenerative changes and telomere lengths in multiple organs in a patient with dyskeratosis congenita., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The H2AX core histone variant is phosphorylated in chromatin around DNA double strand breaks (DSBs) and functions through unknown mechanisms to suppress antigen receptor locus translocations during V(D)J recombination. Formation of chromosomal coding joins and suppression of translocations involves the ataxia telangiectasia mutated and DNA-dependent protein kinase catalytic subunit serine/threonine kinases, each of which phosphorylates H2AX along cleaved antigen receptor loci. Using Abelson transformed pre-B cell lines, we find that H2AX is not required for coding join formation within chromosomal V(D)J recombination substrates. Yet we show that H2AX is phosphorylated along cleaved Igkappa DNA strands and prevents their separation in G1 phase cells and their progression into chromosome breaks and translocations after cellular proliferation. We also show that H2AX prevents chromosome breaks emanating from unrepaired RAG endonuclease-generated TCR-alpha/delta locus coding ends in primary thymocytes. Our data indicate that histone H2AX suppresses translocations during V(D)J recombination by creating chromatin modifications that stabilize disrupted antigen receptor locus DNA strands to prevent their irreversible dissociation. We propose that such H2AX-dependent mechanisms could function at additional chromosomal locations to facilitate the joining of DNA ends generated by other types of DSBs.