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Context.--Spurious platelet counts from automated methods have been reported in patients with abnormal red blood cells. However, there is no specific study regarding performance of platelet counts by automated methods in patients with thalassemia. Objective.--To investigate the performance of automated platelet counts, including impedance (PLT-I) and optical fluorescent (PLT-O and PLT-F) methods, and compare them with the international reference method (IRM) for platelet counting in patients with thalassemia. Design.--Two hundred forty-nine thalassemia specimens from various subtypes were examined. PLT-I, PLTO, and PLT-F from a Sysmex XN analyzer were evaluated and compared against the IRM. Demographic data, platelet counts, and red blood cell parameters are shown. Comparability between evaluated methods and IRM, as well as test characteristics, is presented. Factors involving inaccurate PLT-I were analyzed. Results.--Specimens with platelet counts ranging from 31 X [10.sup.3]/[micro]L to 932 X 103/iL were included. Most patients were patients with thalassemia major. Correlation between PLT-I and IRM was lower than that of the other methods in overall patients. PLT-O and PLT-F were correlated to IRM when classifying patients according to clinically significant platelet ranges. All automated methods had acceptable sensitivities; however, specificity of PLT-I was low for diagnosis of thrombocytopenia. High RDW-CV (red blood cell distribution width-coefficient of variation) was an independent factor of inaccurate PLT-I measurement. Conclusions.--Among the evaluated methods, PLT-I was the method least correlated to IRM, with PLT-O and PLT-F comparable to IRM in patients with thalassemia. Optical platelet counts and careful blood smear examination are recommended alternative platelet counting methods, depending on the clinical setting. (Arch Pathol Lab Med. 2017; 141:830-836; doi: 10.5858/arpa.2016-0222-OA), Platelet count is the component of complete blood count that is used to diagnose quantitative defect in platelets. Accuracy of platelet count is essential for determining bleeding risk in patients. [...]

For development of a long-lasting protective malaria vaccine, it is crucial to understand whether Plasmodium-induced memory B cells (MBCs) or plasma cells develop and stably contribute to protective immunity, or on the contrary the parasite suppresses antibody responses by inducing MBC dysfunction. The expansion of T-bethi atypical MBCs is described in chronic Plasmodium falciparum-exposed individuals. However, it remains unclear whether accumulation of T-bethi atypical MBCs is indicative of a protective role or rather an impaired function of the immune system in malaria. Here, the phenotypic and functional features of T-bethi atypical MBCs were studied in P. vivax patients living in an area of low malaria transmission. During P. vivax infection, the patients produced a twofold higher frequency of T-bethi atypical MBCs compared to malaria non-exposed individuals. This distinct atypical MBC subset had a switched IgG phenotype with overexpression of activation markers and FcRL5, and decreased Syk phosphorylation upon BCR stimulation. Post-infection, expansion of T-bethi IgG+ atypical MBCs was maintained for at least 3 months. Further studies of the contribution of T-bethi atypical MBC function to humoral immunity showed that synergizing IFN-γ with TLR7/8 and IL-21 signals was required for their differentiation into plasma cells and antibody secretion. [ABSTRACT FROM AUTHOR]

A novel in vitro culture system using variable concentrations of biotin/streptavidin to label red blood cells (RBCs) that allows for the simultaneous comparison of growth rates in Plasmodium falciparum malaria parasite in four heterogeneous target RBC populations is described. Donor RBCs containing both P. falciparum‐infected RBCs and non‐infected RBCs at 0.5% parasitemia were first labeled with 7‐hydroxy‐9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one) succinimidyl ester (DDAO‐SE) followed by co‐culture with a mixture of equal numbers of four differentially biotin/streptavidin labeled RBC populations. After two to three schizogonic growth cycles, co‐cultures were harvested and stained with streptavidin‐phycoerythrin (SA‐PE) followed by staining of parasite‐infected RBCs with nucleic acid fluorochrome SYBR Green I. To demonstrate the application of this method, some target RBC populations that had sialic acid residues removed using neuraminidase treatment were mixed with RBC populations without enzymatic treatment and incubated with donor parasitized RBCs strain W2 (sialic acid‐dependent) or 3D7 (sialic acid‐independent). Significant less susceptibility to malaria parasite invasion was obtained with enzyme‐treated RBC populations when compared with non‐treated RBCs in blood samples from the same individual when using malaria parasite strain W2, whereas no difference in percent parasitemias was noted following infection with malaria parasite strain 3D7. This novel malaria culture method is cheap and provides increased sensitivity for direct comparison of parasite growth over time of any of the four RBC populations under identical conditions and eliminates the experimental bias due to contaminated donor RBCs. The application of biotin‐labeled RBCs will therefore provide a better understanding of invasion phenotype‐specific host–parasite interactions and the extent of complex malaria invasion mechanism. © 2019 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Summary: Dengue virus (DENV) infection is considered one of the most important mosquito‐borne diseases. It causes a spectrum of illness that could be due to qualitative and/or quantitative difference(s) of the natural killer (NK) cell responses during acute DENV infection. This view prompted us to perform a detailed phenotypic comparative characterization of NK cell subsets from DENV‐infected patients with dengue fever (DF), patients with dengue haemorrhagic fever (DHF) and healthy controls. The activation/differentiation molecules, CD69 and CD57 and a variety of tissue homing molecules were analysed on the CD56hi CD16− and CD56lo CD16+ NK cells. Although there was no increase in the frequency of the total NK cells during DENV infection compared with the healthy individuals, there was a significant increase in the frequency of the CD56hi CD16− subset and the frequency of CD69 expression by both NK cell subsets during the febrile phase of infection. We also found an increase in the frequencies of cells expressing CD69 and CD57 in the CD56lo CD16+ subset compared with those in the CD56hi CD16− subset. Moreover, although the CD56lo CD16+ subset contained a high frequency of cells expressing skin‐homing markers, the CD56hi CD16− subset contained a high frequency of cells expressing bone marrow and lymph node trafficking markers. Interestingly, no differences of these NK cell subsets were noted in samples from patients with DF versus those with DHF. These findings suggest that activation and differentiation and the patterns of tissue homing molecules of the two major NK cell subsets are different and that these might play a critical role in the immune response against acute DENV infection. [ABSTRACT FROM AUTHOR]

Innate immune responses are reasoned to play an important role during both acute and chronic SIV infection and play a deterministic role during the acute stages on the rate of infection and disease progression. NK cells are an integral part of the innate immune system but their role in influencing the course of SIV infection has been a subject of debate. As a means to delineate the effect of NK cells on SIV infection, use was made of a Janus kinase 3 (JAK3) inhibitor that has previously been shown to be effective in the depletion of NK cells in vivo in nonhuman primates (NHP). Extensive safety and in vitro/in vivo PK studies were conducted and an optimal dose that depletes NK cells and NK cell function in vivo identified. Six chronically SIV infected rhesus macaques, 3 with undetectable/low plasma viral loads and 3 with high plasma viral loads were administered a daily oral dose of 10 mg/kg for 35 days. Data obtained showed that, at the dose tested, the major cell lineage affected both in the blood and the GI tissues were the NK cells. Such depletion appeared to be associated with a transient increase in plasma and GI tissue viral loads. Whereas the number of NK cells returned to baseline values in the blood, the GI tissues remained depleted of NK cells for a prolonged period of time. Recent findings show that the JAK3 inhibitor utilized in the studies reported herein has a broader activity than previously reported with dose dependent effects on both JAK2 and JAK1 suggests that it is likely that multiple pathways are affected with the administration of this drug that needs to be taken into account. The findings reported herein are the first studies on the use of a JAK3 inhibitor in lentivirus infected NHP. [ABSTRACT FROM AUTHOR]

Comprehensive studies of the frequencies and absolute numbers of the various cell lineages that synthesize IL-17 in the blood and corresponding gastrointestinal (GI) tissues, their correlation with CD4+ Tregs, CD8+ Tregs, total and IFN-α synthesizing plasmacytoid dendritic cells (pDC) relative to plasma viral load in SIV infection has been lacking. The unique availability of SIV infected rhesus macaques (RM) classified as Elite Controllers (EC), and those with Low, Intermediate and High Viral Loads (HVL) provided a unique opportunity to address this issue. Results of these studies showed that EC demonstrated a remarkable ability to reverse changes that are induced acutely by SIV in the various cell lineages. Highlights of the differences between EC and HVL RM within Gastro-intestinal tissues (GIT) was the maintenance and/or increases in the levels of IL-17 synthesizing CD4, CD8, and NK cells and pDCs associated with slight decreases in the levels of CD4+ Tregs and IFN-α synthesizing pDCs in EC as compared with decreases in the levels of IL-17 synthesizing CD4, CD8 and NK cells associated with increases in pDCs and IFN-α synthesizing pDCs in HVL monkeys. A previously underappreciated role for CD8+ Tregs was also noted with a moderate increase in ECs but further increases of CD8+ Tregs with increasing VL in viremic monkeys. Positive correlations between plasma VL and decreases in the levels of Th17, Tc17, NK-17, CD4+ Tregs and increases in the levels of CD8+ Tregs, total and IFN-α synthesizing pDCs were also noted. This study also identified 2 additional IL-17+ subsets in GIT as CD3−/CD8+/NKG2a− and CD3+/CD8+/NKG2a+ subsets. Studies also suggest a limited role for IFN-α synthesizing pDCs in chronic immune activation despite persistent up-regulation of ISGs. Finally, elevated persistent innate immune responses appear associated with poor prognosis. These findings provide an initial foundation for markers important to follow for vaccine design. [ABSTRACT FROM AUTHOR]

HIV viral load is more reliable tool for monitoring treatment throughout the course of HIV/AIDS, but the test may be expensive in resource-limited settings. Therefore, enumeration of CD4 T-lymphocyte count remains important in these settings. This study evaluated the performance of BDFACSPresto, a near-patient CD4 counter planned to be used in primary healthcare clinics in Thailand. Results of percent, absolute CD4 count and hemoglobin (Hb) on the FACSPresto were compared with the TriTEST/TruCOUNT/BDFACSCalibur method and a Sysmex hematology analyzer. Phase I of the study was performed in an ISO15189 laboratory. Both percentage and absolute values showed Passing–Bablok slopes within 0.98–1.06 and 0.97–1.13, mean Bland–Altman biases of +1.2% and +20.5 cells/µL, respectively. In phase II, venous and some capillary blood samples were analyzed in four primary healthcare clinics. The results showed good correlation between capillary and venous blood. For venous blood samples, regression lines showed slopes of 1.01–1.05 and 1.01–1.07 for all percentage and absolute values. The overall mean biases were +0.9% and +17.0 cells/µL. For Hb, Passing–Bablok regression result gave slope within 1.01–1.07 and mean bias of −0.06 g/dL. Thus, CD4 enumeration in blood by the FACSPresto is reliable and can be performed to an identical standard at primary healthcare clinics. [ABSTRACT FROM AUTHOR]

Objective: To explore cooperation between activated naïve (aNAV) B cells and CD4 + T cells in the pathogenesis of SLE through autoantibody production, T-cell differentiation and inflammatory cytokine secretion., Methods: Peripheral blood mononuclear cell samples were obtained from 31 patients with SLE and used to characterise phenotype of aNAV B cells (n=14) and measured the phosphorylation of B-cell receptor (BCR) signalling molecules (n=5). Upregulation of T-cell costimulatory molecules after BCR and toll-like receptor (TLR)-7/TLR-8 stimulation was detected in cells from four subjects. To explore the role of these cells in SLE pathogenesis via T cell-dependent mechanisms, four subjects were analysed to detect the promotion of CD4 + T-cell activation and antibody-secreting cell (ASC) differentiation after CD4 + T-cell-B-cell cocultures. The aNAV B cells from four patients were used to assess cytokine secretion., Results: The aNAV B cells of patients with SLE had increased expression of surface CD40, HLA-DR and interleukin-21 receptor (IL-21R) and FCRL5 molecules. With BCR stimulation, these cells greatly increased PLCγ2 phosphorylation. Integrated BCR and TLR-7/TLR-8 signals induced overexpression of CD40, CD86, IL-21R and HLA-DR on lupus aNAV B cells. In T-cell-B-cell cocultures, lupus aNAV B cells (with upregulated costimulatory molecules) promoted CD4 + T-cell proliferation and polarisation toward effector Th 2 and Th 17 cells. Importantly, in this coculture system, CD4 + T-cell signals enhanced aNAV B-cell differentiation into auto-ASCs and produced anti-DNA antibodies. The interaction between CD4 + T cell and aNAV B cell increased production of inflammatory cytokines (IL-6, IL-8 and IL-23)., Conclusion: Cooperation between aNAV B cells and CD4 + T cells contributed to SLE pathogenesis by promoting both differentiation of pathogenic T cells (Th 2 and Th 17 ) and autoantibody secretion., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)