Evaluation of a 10color protocol as part of a 2tube screening panel for flow cytometric assessment of peripheral blood leukocytic subsets.

Peripheral blood (PB) immunophenotyping is commonly required for initial evaluation of various suspected disease entities. Several approaches have been proposed. The objective of this work is to explore the value of a 10color protocol developed in our laboratory for flow cytometric assessment of PB leukocytic subsets, as part of a 2tube screening panel. A combination of CD16/CD56/CD34/CD33/CD19/CD4/CD8/CD3/CD20/CD45 antibodies in 1 tube was applied routinely during flow cytometric analysis of PB samples for diagnostic purposes. The protocol was systematically complemented by a 2nd tube with anti-kappa, anti-lambda, CD5, CD19, and CD45 antibodies for adults and selected pediatric patients, and specifically oriented panels when necessary. 25 samples with no detectable neoplastic PB involvement and 31 samples with a hematolymphoid disorder were investigated retrospectively. The contribution of CD33 in the separation of leukocytic populations, as well as the benefits from the simultaneous assessment of CD20/CD19/CD45, CD16/CD56 and the detection of CD34+ cells were examined. The gating strategy with the use of CD33 provided additional information in certain cases. The protocol enabled recognition of differential expression of CD20 and CD45 in CD19+ cells with chronic lymphocytic leukemia phenotype, overall evaluation of NK and NK like T cells, estimation of CD16- granulocytes and CD56/CD16 expression in monocytes, as well as identification of minor cell subsets, such as CD34+ cells. The proposed 10color combination of antibodies analyzed in a standardized manner can offer significant information in the initial evaluation of PB samples, thus, guiding subsequent investigation if needed.

Bronchoalveolar lavage fluid and blood natural killer and natural killer T-like cells in cryptogenic organizing pneumonia.

BACKGROUND AND OBJECTIVE: Natural killer (NK) cells appear to be involved in the development of interstitial lung diseases (ILD). The purpose of this study was to investigate the involvement of NK and natural killer T (NKT)-like cells in two recognized cytotoxic ILD with systemic character, hypersensitivity pneumonitis (HP) and cryptogenic organizing pneumonia (COP), compared with idiopathic pulmonary fibrosis (IPF) and controls. METHODS: Bronchoalveolar lavage fluid (BALF) and peripheral blood (PBL) cells and lymphocyte subsets of 83 patients (26 with COP, 19 with HP and 38 with IPF) and 10 controls were prospectively studied by flow cytometry. RESULTS: The percentage of NK and NKT-like cells was lower in BALF than in PBL in all patient groups and controls. Patients with COP presented with statistically significantly higher NK and NKT-like cell counts in BALF compared with controls (P = 0.044 and P = 0.05 respectively) and IPF (P = 0.049 and P = 0.045 respectively). BALF NKT-like cell count correlated with PBL NKT-like cell count only in COP (r = 0.627, P = 0.002). In addition, a significant positive correlation between BALF NKT-like cell and PBL cytotoxic T CD8+ cell count was observed in COP (r = 0.562, P = 0.006) but not in HP, IPF or controls. CONCLUSIONS: Our study provides for the first time evidence for the implication of NKT-like cells in the pathogenesis of COP, as part of both localized and systemic cytotoxicity.

Ocrelizumab in Patients with Active Primary Progressive Multiple Sclerosis: Clinical Outcomes and Immune Markers of Treatment Response.

Ocrelizumab is a B-cell-depleting monoclonal antibody approved for the treatment of relapsing-remitting multiple sclerosis (RRMS) and active primary progressive MS (aPPMS). This prospective, uncontrolled, open-label, observational study aimed to assess the efficacy of ocrelizumab in patients with aPPMS and to dissect the clinical, radiological and laboratory attributes of treatment response. In total, 22 patients with aPPMS followed for 24 months were included. The primary efficacy outcome was the proportion of patients with optimal response at 24 months, defined as patients free of relapses, free of confirmed disability accumulation (CDA) and free of T1 Gd-enhancing lesions and new/enlarging T2 lesions on the brain and cervical MRI. In total, 14 (63.6%) patients and 13 patients (59.1%) were classified as responders at 12 and 24 months, respectively. Time exhibited a significant effect on mean absolute and normalized gray matter cerebellar volume (F = 4.342, p = 0.23 and F = 4.279, p = 0.024, respectively). Responders at 24 months exhibited reduced peripheral blood ((%) of CD19+ cells) plasmablasts compared to non-responders at the 6-month point estimate (7.69 ± 4.4 vs. 22.66 ± 7.19, respectively, p = 0.043). Response to ocrelizumab was linked to lower total and gray matter cerebellar volume loss over time. Reduced plasmablast depletion was linked for the first time to sub-optimal response to ocrelizumab in aPPMS.

Reduced expression of L-selectin in T-cells correlates with relative lymphocyte increase in patients with RRMS treated with natalizumab - functional implication towards PML risk.

Objectives: Natalizumab (NTZ), a treatment indicated for patients with highly active Relapsing - Remitting Multiple Sclerosis (RRMS), is known to induce increased relative frequency of lymphocytes. Progressive Multifocal Leukoencephalitis (PML) is a rare but serious adverse event related to NTZ. Moreover, reduced L-selectin (CD62L) expression in T-cells in cryopreserved samples of patients with RRMS under NTZ has been proposed as a biomarker of pre-PML state. We explore the association between L-selectin expression in T-cells and hematological parameters in freshly processed samples of patients with RRMS under NTZ.Methods: We studied L-selectin expression in patients with: RRMS under NTZ (n=34), fingolimod (FTY, n=14), interferon-beta (IFNß, n=22), glatiramer acetate (GA, N=17); in 9 patients with secondary progressive (SP) MS and in 6 healthy controls. Twenty-two patients under NTZ and 6 patients under FTY were followed for 18 months. One NTZ-treated patient developed PML during the study.Results: Patients under NTZ exhibited increased relative frequency of lymphocytes (40.02±1.45) compared to patients under first-line treatment (30.57±1.68, p<0.001) and to patients with SPMS (29±1.56, p=0.02), and a lower mean L-selectin expression in (69.39±1.73) compared to patients under first-line treatment (79.1±1.17, p=0.003). A negative correlation between the relative frequency of CD4+CD62L+ T-cells and the absolute lymphocyte counts (Pearson's r=0.367, p=0.033) was observed.Discussion: We hereby provide mechanistic insight in a possible pathway implicated in NTZ-related PML risk. These results further underline the need for thorough validation of L-selectin expression in T-cells as a potential pre-PML biomarker.

Long-term effect of continuous positive airway pressure therapy on inflammation markers of patients with obstructive sleep apnea syndrome.

STUDY OBJECTIVES: Several lines of evidence suggest immune system derangement in obstructive sleep apnea syndrome (OSAS) patients. However, no data exist on the long-term effect of continuous positive airway pressure (CPAP) treatment on systemic immunity. Hence, we sought to evaluate this effect on various immunological parameters in OSAS patients. DESIGN: Prospective case series. SETTING: Sleep unit of a general hospital. PATIENTS: Newly-diagnosed, nonsmoking, otherwise healthy OSAS male patients (n = 52) were evaluated on diagnosis and 6 months after CPAP treatment. According to compliance to CPAP use at 6-month follow-up, they were divided into 2 groups: group A (n = 32): good compliance (mean CPAP use > or = 4 h/night); and group B (n = 20): poor compliance (mean CPAP use < 4 h/night). INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: Blood samples were obtained at baseline and at the 6-month follow-up. Percentage and absolute count of lymphocyte subsets (by flow cytometry), serum TNF-alpha, IL-6, and uric acid levels were measured. No differences were recorded regarding the baseline anthropometric or sleep characteristics of the 2 groups. In group A, a significant decrease in the absolute count of total lymphocytes (P = 0.003), and of CD4+ cells (P = 0.001), and a decrease in TNF-alpha levels (P = 0.001) and uric acid levels (P < 0.001) was observed after CPAP application. On the contrary, no alterations occurred in any of the tested parameters in group B patients. CONCLUSIONS: The selective reduction of soluble and cellular immune response factors only in those OSAS patients who exhibited good compliance to CPAP therapy provides further evidence for an ongoing systemic immune process in OSAS.

CD5+ B lymphoproliferative disorder with subsequent development of plasma cell leukaemia: Diagnostic and aetiologic reasoning.

BACKGROUND: Plasma cell myeloma (PCM) has been sporadically reported to occur simultaneously or subsequently to mature B lymphoproliferative disorders (LPDs), predominantly chronic lymphocytic leukaemia (CLL). METHODS: We describe the clinical and laboratory findings of a 69-year-old male patient who developed plasma cell leukaemia (PCL) 8 years after an initial diagnosis of a low stage CD5+ B LPD and 3 years after treatment for LPD. RESULTS: The transition from a clinically indolent B LPD to an aggressive PCM was documented by bone marrow (BM) biopsy, while flow cytometric (FC) immunophenotyping conferred additional information by disclosing the co-existence of both disorders in BM and the presence of abnormal monotypic PCs in peripheral blood above PCL levels. Phenotypic findings suggested a discrete clonal origin of the two disorders. CONCLUSIONS: This report of PCL development in a patient with residual CD5+ B LPD, emphasizes the need for comprehensive diagnostic evaluation of such cases and scrutiny of their aetiological relationship, including FC immunophenotyping due to its high analytical sensitivity and multiparametric capacity compared to morphology or immunohistochemistry alone. © 2017 International Clinical Cytometry Society.

Utility of hematology analyzer and flow cytometry in timely and correct detection of circulating plasma cells: Report of three cases.

BACKGROUND: Circulating neoplastic plasma cells (PCs) are a common feature between classical plasma cell myeloma (PCM) and its aggressive variant, plasma cell leukemia (PCL), with their early suspicion and detection being of clinical importance for prognostic and diagnostic purposes. METHODS: Morphological and flow cytometric enumeration and characterization of peripheral blood (PB) neoplastic PCs were performed in 3 PCM patients after complete blood count (CBC) evaluation. RESULTS: Early suspicion of circulating PCs by initial CBC scatter plots and monocytosis led to morphological enumeration of PCs, with low level of concordance between morphologists. Flow cytometric PB analysis permitted accurate quantification and characterization of circulating clonal PCs. CONCLUSIONS: CBC findings can prompt early suspicion of circulating PCs in PCM patients leading to early and accurate flow cytometric analysis of clonal PCs in addition to morphological PB evaluation. The suggested combined approach proved to be most important in the PCL case with discordant morphological evaluations and marginal PC numbers according to current criteria. © 2016 International Clinical Cytometry Society.

Potential prognostic value of intracellular cytokine detection by flow cytometry in pulmonary sarcoidosis.

In pulmonary sarcoidosis, differential cytokine production in the lungs could be related to variable prognosis of patients at different stages of disease. Twenty patients with pulmonary sarcoidosis (10 at radiographic stage I and 10 at stages II-IV), as well as 10 age-matched healthy volunteers participated in the study. A 4-colour flow cytometric technique was used to measure interferon-γ (IFN-γ), interleukin (IL)-2, tumour necrosis factor-α (TNF-α), IL-4, and IL-13 production in phorbol myristate acetate (PMA)/ionomycin-stimulated CD4+ and CD8+ T cells from bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) of patients, and PB of control subjects. CD4+ T cells from patients showed higher expression of IFN-γ in BALF than in PB. Significant correlations were observed between the percentages of BALF CD4+ and CD8+ T cells expressing intracellular IFN-γ, IL-2, and TNF-α. Stage I patients had lower percentages of IFN-γ-producing CD4+ and CD8+ T cells, as well as TNF-α-producing CD8+ T cells, in BALF (but not in PB) than stage II-IV patients. A decreased TH1 and TC1 response was demonstrated in BALF of patients at stage I of disease, which could explain their anticipated better prognosis.