L-plastin associated syndrome of immune deficiency and hematologic cytopenia.

BACKGROUND: LCP1 encodes L-plastin, an actin-bundling protein primarily expressed in hematopoietic cells. In mouse and fish models, LCP1 deficiency has been shown to result in hematologic and immune defects. OBJECTIVE: This study aimed to determine the nature of a human inborn error of immunity resulting from a novel genetic variant of LCP1. METHODS: We performed genetic, protein, and cellular analysis of PBMCs from a kindred with apparent autosomal dominant immune deficiency. We identified a candidate causal mutation in LCP1, which we evaluated by engineering the orthologous mutation in mice and Jurkat cells. RESULTS: A splice-site variant in LCP1 segregated with lymphopenia, neutropenia, and thrombocytopenia. The splicing defect resulted in at least 2 aberrant transcripts, producing an in-frame deletion of 24 nucleotides, and a frameshift deletion of exon 8. Cellular analysis of the kindred revealed a proportionate reduction of T and B cells and a mild expansion of transitional B cells. Similarly, mice carrying the orthologous genetic variant exhibited the same in-frame aberrant transcript, reduced expression Lcp1 and gene dose-dependent leukopenia, mild thrombocytopenia, and lymphopenia, with a significant reduction of T-cell populations. Functional analysis revealed that LCP1c740-1G>A confers a defect in platelet development and function with aberrant spreading on collagen. Immunologic analysis revealed defective actin organization in T cells, reduced migration of PBMCs from patients, splenocytes from mutant mice, and a mutant Jurkat cell line in response to CXCL12; impaired germinal center B-cell expansion after immunization; and reduced cytokinesis during T cell proliferation. CONCLUSIONS: We describe a unique human hematopoietic defect affecting neutrophils, lymphocytes, and platelets arising from partial LCP1 deficiency.

An Optimized Method of Collecting Murine Peripheral Blood and Dilution Correction for Accurate Blood Cell Enumeration.

Accurate measurement of whole blood counts from mice is an essential quantitative tool across the fields of vascular cell biology. In particular, the measurement of platelet counts can be challenging as the process relies upon good phlebotomy technique, the inclusion of a sufficient amount of the appropriate anticoagulant, and very often dilution of the sample to meet the sample volume requirements of an automated analyzer. To minimize sample dilution, blood collection tubes pre-coated with the anticoagulant can be used; however, these are expensive and prone to blood clotting issues. Here, we describe a simple dilution correction method that accurately calculates blood-to-anticoagulant dilutions to generate appropriate volumes for automated blood cell analysis while minimizing blood clotting. We also discuss some simple steps that can be incorporated into blood collection methods to avoid artefacts during blood collection. Blood count data analysis involving volume correction and clot exclusion can significantly reduce variable blood cell count values among healthy untreated littermates. It also detects subtle changes in blood cell counts, mainly of platelets and RBCs in experimental settings, which can be masked in the absence of careful and precise volume correction. Blood count analysis with volume correction precisely determines mouse whole blood cell counts for investigators. The decreased variability in cell count values reduces the number of experimental animals required for meaningful analysis. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: An optimized method of collecting murine peripheral blood and dilution correction for accurate blood cell enumeration.