Assessment of inter-operator variability in peripheral monocyte subset gating strategy using flow cytometry in patients with suspected acute stroke.

BACKGROUND: Innovative tools to reliably identify patients with acute stroke are needed. Peripheral monocyte subsets, that is, classical-Mon1, intermediate-Mon2, and non-classical-Mon3, with their activation marker expression analyzed using flow-cytometry (FCM) could be interesting cell biomarker candidates. AIM: To assess the inter-operator variability in a new peripheral monocyte subset gating strategy using FCM in patients with suspected acute stroke. METHODS: In BOOST-study ("Biomarkers-algOrithm-for-strOke-diagnoSis-and Treatment-resistance-prediction," NCT04726839), patients ≥18 years with symptoms suggesting acute stroke within the last 24 h were included. Blood was collected upon admission to emergency unit. FCM analysis was performed using the FACS-CANTO-II® flow-cytometer and Flow-Jo™-software. Analyzed markers were CD45/CD91/CD14/CD16 (monocyte backbone) and CD62L/CD11b/HLA-DR/CD86/CCR2/ICAM-1/CX3CR1/TF (activation markers). Inter-operator agreement (starting from raw-data files) was quantified by the measure distribution and, for each patient, the coefficient of variation (CV). RESULTS: Three operators analyzed 20 patient blood samples. Median inter-operator CVs were below the pre-specified tolerance limits (10% [for Mon1 counts], 20% [Mon2, Mon3 counts], 15% [activation marker median-fluorescence-intensities]). We observed a slight, but systematic, inter-operator effect. Overall, absolute inter-operator differences in fractions of monocyte subsets were <0.03. CONCLUSION: Our gating strategy allowed monocyte subset gating with an acceptable inter-operator variability. Although low, the inter-operator effect should be considered in monocyte data analysis of BOOST-patients.

Is platelet function testing at the acute phase under P2Y12 inhibitors helpful in predicting bleeding in real-life patients with acute coronary syndrome? The AVALANCHE study.

BACKGROUND: In patients with acute coronary syndrome (ACS), current international guidelines recommend newer potent and predictable P2Y12 inhibitors as first-line treatment despite a greater bleeding risk compared with clopidogrel. AIM: To determine if platelet function testing can predict bleeding in real-life patients with ACS treated with newer P2Y12 inhibitors. METHODS: In this retrospective study, all consecutive adults admitted to the Lariboisière University Hospital for ACS, whatever the P2Y12 inhibitor prescribed, who had platelet function testing (vasodilator-stimulated phosphoprotein phosphorylation [VASP] index and aggregation tests) during the initial hospital stay were included. Follow-up was performed to record bleeding events according to the Bleeding Academic Research Consortium (BARC) classification. RESULTS: A total of 364 patients were included, treated with ticagrelor (n=123), prasugrel (n=105) or clopidogrel (n=136); 42.3% after an ST-segment elevation myocardial infarction, 27.1% after a non-ST-segment elevation myocardial infarction and 30.6% with unstable angina. Mean age was 64±11 years. Median VASP index was significantly lower with the newer P2Y12 inhibitors (14% under ticagrelor, 14% under prasugrel) than with clopidogrel (42%). Despite these differences in the degree of platelet inhibition, the occurrence of bleeding (BARC 2-5) during follow-up was 7.7% overall, and was similar for all P2Y12 inhibitors (ticagrelor 8.9%; prasugrel 6.6%; clopidogrel 7.4%). For each P2Y12 inhibitor, it was impossible to determine a VASP index threshold under which bleeding was significantly greater during follow-up. Similarly, ADP-induced aggregation was more profoundly inhibited by ticagrelor and prasugrel than by clopidogrel, but this did not allow a threshold to be set for increased haemorrhagic risk. CONCLUSIONS: Despite the substantial occurrence of bleeding in patients with ACS during follow-up, neither the VASP index nor platelet aggregation test results measured at the acute phase were helpful in predicting bleeding risk. Whether platelet function testing could be helpful later in the course of treatment remains to be evaluated.

Comparison of Circulating Immune Cells Profiles and Kinetics Between Coronavirus Disease 2019 and Bacterial Sepsis.

OBJECTIVES: Although clinical presentation of coronavirus disease 2019 has been extensively described, immune response to severe acute respiratory syndrome coronavirus 2 remains yet not fully understood. Similarities with bacterial sepsis were observed; however, few studies specifically addressed differences of immune response between both conditions. Here, we report a longitudinal analysis of the immune response in coronavirus disease 2019 patients, its correlation with outcome, and comparison between severe coronavirus disease 2019 patients and septic patients. DESIGN: Longitudinal, retrospective observational study. SETTING: Tertiary-care hospital during the first 2020 coronavirus disease 2019 outbreak in France. PATIENTS: All successive patients with confirmed severe acute respiratory syndrome coronavirus 2 infection admitted to the emergency department, medical ward, and ICU with at least one available immunophenotyping performed during hospital stay. MEASUREMENTS AND MAIN RESULTS: Between March and April 2020, 247 patients with coronavirus disease 2019 were included and compared with a historical cohort of 108 severe septic patients. Nonsevere coronavirus disease 2019 patients (n = 153) presented normal or slightly altered immune profiles. Severe coronavirus disease 2019 (n = 94) immune profile differed from sepsis. Coronavirus disease 2019 exhibited profound and prolonged lymphopenia (mostly on CD3, CD4, CD8, and NK cells), neutrophilia, and human leukocyte antigen D receptor expression on CD14+ monocytes down-regulation. Surprisingly, coronavirus disease 2019 patients presented a unique profile of B cells expansion, basophilia, and eosinophilia. Lymphopenia, human leukocyte antigen D receptor expression on CD14+ monocytes down-regulation, and neutrophilia were associated with a worsened outcome, whereas basophilia and eosinophilia were associated with survival. Circulating immune cell kinetics differed between severe coronavirus disease 2019 and sepsis, lack of correction of immune alterations in coronavirus disease 2019 patients during the first 2 weeks of ICU admission was associated with death and nosocomial infections. CONCLUSIONS: Circulating immune cells profile differs between mild and severe coronavirus disease 2019 patients. Severe coronavirus disease 2019 is associated with a unique immune profile as compared with sepsis. Several immune features are associated with outcome. Thus, immune monitoring of coronavirus disease 2019 might be of help for patient management.

Multicentric experience with interferon gamma therapy in sepsis induced immunosuppression. A case series.

BACKGROUND: The sepsis-induced immunodepression contributes to impaired clinical outcomes of various stress conditions. This syndrome is well documented and characterized by attenuated function of innate and adaptive immune cells. Several pharmacological interventions aimed to restore the immune response are emerging of which interferon-gamma (IFNγ) is one. It is of paramount relevance to obtain clinical information on optimal timing of the IFNγ-treatment, -tolerance, -effectiveness and outcome before performing a RCT. We describe the effects of IFNγ in a cohort of 18 adult and 2 pediatric sepsis patients. METHODS: In this open-label prospective multi-center case-series, IFNγ treatment was initiated in patients selected on clinical and immunological criteria early (< 4 days) or late (> 7 days) following the onset of sepsis. The data collected in 18 adults and 2 liver transplanted pediatric patients were: clinical scores, monocyte expression of HLA-DR (flow cytometry), lymphocyte immune-phenotyping (flow cytometry), IL-6 and IL-10 plasma levels (ELISA), bacterial cultures, disease severity, and mortality. RESULTS: In 15 out of 18 patients IFNγ treatment was associated with an increase of median HLA-DR expression from 2666 [IQ 1547; 4991] to 12,451 [IQ 4166; 19,707], while the absolute number of lymphocyte subpopulations were not affected, except for the decrease number of NK cells 94.5 [23; 136] to 32.5 [13; 90.8] (0.0625)]. Plasma levels of IL-6 464 [201-770] to 108 (89-140) ng/mL (p = 0.04) and IL-10 from IL-10 from 29 [12-59] to 9 [1-15] pg/mL decreased significantly. Three patients who received IFNγ early after ICU admission (<4 days) died. The other patients had a rapid clinical improvement assessed by the SOFA score and bacterial cultures that were repeatedly positive became negative. The 2 pediatric cases improved rapidly, but 1 died for hemorrhagic complication. CONCLUSION: Guided by clinical and immunological monitoring, adjunctive immunotherapy with IFNγ appears well-tolerated in our cases and improves immune host defense in sepsis induced immuno suppression. Randomized clinical studies to assess its potential clinical benefit are warranted.

Perioperative management of a patient with Glanzmann thrombasthenia undergoing a coronary artery bypass graft surgery: a case report.

: We report herein the successful perioperative management of a 57-year-old man with a type I Glanzmann thrombasthenia undergoing coronary artery bypass graft surgery and right carotid endarterectomy. The patient suffered from several lesions in the three major coronary arteries and in the right carotid necessitating surgery. Prophylactic human leukocyte antigen (HLA)-matched platelets transfusions were continuous administrated before, and through the immediate perioperative period. Posttransfusion platelet recovery was monitored using flow cytometry to determine the percentage of circulating platelet expressing CD61 (ß3). No bleeding complications occurred during and following the procedure. The patient did not develop HLA antibodies or αIIbß3 antibodies. Thrombophilia screening revealed a heterozygous G20210A prothrombin gene mutation. The patient also suffered from an atrial fibrillation, necessitating anticoagulation therapy. During the hospital stay, a treatment with vitamin K antagonists for stroke prevention was initiated. The patient was discharged 8 days following surgery, and no further complications occurred during the 6 months follow-up.

A case of daptomycin-induced immune thrombocytopenia.

We report a case of severe daptomycin-induced immune thrombocytopenia in a patient treated for methicillin-resistant Staphylococcus epidermidis and ampicillin-resistant Enterococcus faecalis bacteremia acquired in an intensive care unit. Serum antibodies bound to platelets in the presence of daptomycin on flow cytometry. There was no evidence of other causes of thrombocytopenia. The patient died of brain herniation complicating extensive cerebral hemorrhage. To our knowledge, this is the first described case of daptomycin-induced thrombocytopenia.

Normal levels of peripheral CD19(+) CD5(+) CLL-like cells: toward a defined threshold for CLL follow-up -- a GEIL-GOELAMS study.

BACKGROUND: The development of flow cytometry as a useful tool for the detection of minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) is potentially hampered by the fact that a normal subset of B-cells with a similar immunophenotype is present in the peripheral blood. This subset of CLL-like cells is not well defined in terms of frequency. METHODS: Here, we performed a multicenter study with a panel of four-color antibody combinations possibly useful for the detection of MRD in CLL, to establish the levels of normal CLL-like cells in 49 healthy controls. ROC curves established the upper level of such cells at 4 × 10(-4) . The two best combinations were further applied to 419 samples from 117 treated CLL patients. RESULTS: The combinations CD19/CD5/CD43/CD79b and CD19/CD5/CD81/CD22 appeared very robust and well correlated to enumerate normal CLL-like cells in a lysis no-wash approach. In follow-up samples from CLL patients, they disclosed only 9.8% of the samples within the normal range. In more than 90% of the cases, it was thus possible to report confidently on the absence or presence of MRD in these patients. CONCLUSIONS: This manuscript reports on the frequency of CD19(+) CD5(+) B-cells in normal peripheral blood and confirms the combinations recommended by the European research initiative on CLL as being performing to assess remaining CLL cells above a threshold of 4 × 10(-4) white blood cells.