Convalescent plasma treatment for SARS-CoV-2 infected high-risk patients: a matched pair analysis to the LEOSS cohort.

Establishing the optimal treatment for COVID-19 patients remains challenging. Specifically, immunocompromised and pre-diseased patients are at high risk for severe disease course and face limited therapeutic options. Convalescent plasma (CP) has been considered as therapeutic approach, but reliable data are lacking, especially for high-risk patients. We performed a retrospective analysis of 55 hospitalized COVID-19 patients from University Hospital Duesseldorf (UKD) at high risk for disease progression, in a substantial proportion due to immunosuppression from cancer, solid organ transplantation, autoimmune disease, dialysis. A matched-pairs analysis (1:4) was performed with 220 patients from the Lean European Open Survey on SARS-CoV-2-infected Patients (LEOSS) who were treated or not treated with CP. Both cohorts had high mortality (UKD 41.8%, LEOSS 34.1%). A matched-pairs analysis showed no significant effect on mortality. CP administration before the formation of pulmonary infiltrates showed the lowest mortality in both cohorts (10%), whereas mortality in the complicated phase was 27.8%. CP administration during the critical phase revealed the highest mortality: UKD 60.9%, LEOSS 48.3%. In our cohort of COVID-19 patients with severe comorbidities CP did not significantly reduce mortality in a retrospective matched-pairs analysis. However, our data supports the concept that a reduction in mortality is achievable by early CP administration.

Comparison of Volumetric and Bead-Based Counting of CD34 Cells by Single-Platform Flow Cytometry.

BACKGROUND: Over 2,000 people a year in the United Kingdom need a bone marrow or blood stem cell transplant. It is important to accurately quantify the hematopoietic stem cells to predict whether the transplant will be successful in replenishing the immune system. However, they are present at low frequency, which complicates accurate quantification. The current gold standard method is single-platform flow cytometry using internal reference counting beads to determine the concentration of CD34 cells. However, volumetric flow cytometers have the ability to measure the acquisition volume, which removes the need for reference beads for calculation of cell concentrations. METHOD: In this study, we compared both methods for calculating CD34 cell concentrations in volumetric cytometers, using either the volume reading or the number of reference beads for calculation. In addition, the uncertainty of measurement for each method was estimated. RESULTS: The results show that both methods have similar uncertainties of measurement. Regression analysis showed low to no statistical difference in CD34 cell concentrations obtained with each method. CONCLUSIONS: Overall, this study suggests that the volumetric method is a valid approach but that the adoption of this technology may be hindered without some form of external calibration of volume readings to increase confidence in the measurement. © 2019 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

Inflammatory response after acute ischemic stroke.

BACKGROUND AND PURPOSE: This study aimed to characterize the time course of inflammatory parameters after acute ischemic stroke. METHODS: We serially determined high sensitivity C-reactive protein (CRP), fibrinogen, and leukocyte counts at 10 time points between days 1 and 90 after ischemic stroke and in control subjects. RESULTS: CRP did not significantly change, whereas fibrinogen increased after stroke. At all time points, CRP and fibrinogen were higher than in healthy control subjects, but not risk factor control subjects. The leukocyte count declined after stroke and was significantly elevated as compared to both control groups only on day 1 but not later. NIHSS levels were positively correlated with CRP and fibrinogen at all time points. Larger infarcts were associated with a higher CRP and leukocyte counts on day 90. Treatment with aspirin was associated with lower values for all three inflammatory parameters in the subacute phase after ischemia. CONCLUSIONS: The course after stroke was different between the parameters of inflammation. Only the leukocytes followed the paradigm of an acute phase response.

Human alloantibody anti-Mart interferes with Mac-1-dependent leukocyte adhesion.

The CD11b/CD18 integrin plays a crucial role in cell-cell adhesion processes. Recently, we described a case of severe neonatal alloimmune neutropenia (NAIN) caused by an alloantibody against a variant of the CD11b subunit (Mart alloantigen). Allele-specific transfected cells allowed us to demonstrate that an H61R point mutation is directly responsible for the formation of Mart epitopes. No difference in the adhesion capability between H61 and R61 homozygous neutrophils was observed. Functional analysis showed that anti-Mart inhibited Mac-1-dependent adhesion of neutrophils and monocytic U937 cells to fibrinogen, intercellular adhesion molecule-1 (ICAM-1), receptor for advanced glycation end product (RAGE), and glycoprotein Ibalpha but not to junctional adhesion molecule-C or urokinase plasminogen activator receptor (uPAR). Accordingly, anti-Mart blocked neutrophil and U937 cell adhesion to endothelial cells and platelet-leukocyte aggregate formation in whole blood under high shear. Other sera of anti-Mart from mothers of infants without NAIN did not show inhibitory properties. We conclude that anti-Mart antibodies with different functional properties exist. This is supported by our findings that anti-Mart antibodies have different abilities to inhibit cell-cell adhesion, to enhance the respiratory burst of neutrophils, and to recognize different epitopes at the N-terminal region of CD11b. In conclusion, some anti-Mart alloantibodies interfere with Mac-1-dependent cellular functions of neutrophils, cause NAIN, and may be used as tools for studying Mac-1-dependent functions.

Platelet function under aspirin, clopidogrel, and both after ischemic stroke: a case-crossover study.

BACKGROUND AND PURPOSE: Combined antiplatelet agents may offer additive protection over single drugs after stroke. We investigated whether platelet activation is reduced under combined aspirin and clopidogrel compared with each drug alone. METHODS: In a case-crossover study, 31 patients with previous atherothrombotic or lacunar stroke who were treated with aspirin (100 to 300 mg/d) received clopidogrel (75 mg/d) and both aspirin and clopidogrel for 4 weeks. Platelet function in whole blood was studied after each treatment period and in healthy control subjects to assess activation-dependent antigens CD62p and CD63 by flow cytometry and collagen/epinephrine (CEPI-CT) and collagen/ADP (CADP-CT) closure times with the platelet function analyzer PFA-100, which investigates platelet-related function under shear stress. RESULTS: CD62p expression and CD63 expression were not different under the 3 treatment regimens. CD63 but not CD62p expression was lower in control subjects than in stroke patients regardless of the antiplatelet treatment (P<0.05). CEPI-CT was prolonged under aspirin and aspirin plus clopidogrel compared with clopidogrel monotherapy (P<0.0001). CADP-CT was longer under combination therapy than under aspirin (P=0.0009) or clopidogrel (P=0.0074) or in control subjects (P=0.0010), mainly because of strong prolongation in a patient subgroup (28%). CONCLUSIONS: CD63 expression reflecting the release of platelet lysosomes is consistently increased after stroke and incompletely suppressed by treatment with aspirin, clopidogrel, or both. The strong prolongation of CADP-CT under combined aspirin and clopidogrel in a patient subgroup may indicate a lower risk of thrombosis but also a higher risk of hemorrhage. The predictive value of platelet activation parameters requires investigation in prospective studies.

Course of platelet activation markers after ischemic stroke.

BACKGROUND AND PURPOSE: The aim of this study was to evaluate the time course of platelet activation after ischemic stroke and to investigate whether platelet activation and inflammation are correlated with each other. METHODS: We serially determined expression of p-selectin (CD62p) and lysosome-associated membrane protein (CD63) by platelets using flow cytometry at 10 time points between days 1 and 90 in patients after ischemic stroke (n=50), in healthy subjects (n=30), and in risk factor control subjects (n=20). Furthermore, we correlated leukocyte count, C-reactive protein, and fibrinogen levels with platelet activation markers. RESULTS: CD62p and CD63 expression was higher on day 1 after stroke than in both control groups (P<0.005 for both). CD62p expression rapidly declined, whereas CD63 expression remained significantly elevated until day 90. Stroke severity and different medication for secondary stroke prevention did not influence CD62p or CD63 expression. Platelet activation markers and inflammatory parameters were not correlated with each other at any time point after stroke. CONCLUSIONS: The initial increase in both CD62p and CD63 expression by platelets is followed by a differential regulation of both parameters after stroke. The rapid decrease in CD62p expression may be caused by shedding from the cell surface. Its persistent elevation makes CD63 a good candidate for studies on predictors for stroke recurrence. Our findings suggest that the expression of CD62p and CD63 by platelets is regulated independently from inflammatory indexes.

The junctional adhesion molecule 3 (JAM-3) on human platelets is a counterreceptor for the leukocyte integrin Mac-1.

The recently described junctional adhesion molecules (JAMs) in man and mice are involved in homotypic and heterotypic intercellular interactions. Here, a third member of this family, human JAM-3, was identified and described as a novel counterreceptor on platelets for the leukocyte beta2-integrin Mac-1 (alphaMbeta2, CD11b/CD18). With the help of two monoclonal antibodies, Gi11 and Gi13, against a 43-kD surface glycoprotein on human platelets, a full-length cDNA encoding JAM-3 was identified. JAM-3 is a type I transmembrane glycoprotein containing two Ig-like domains. Although JAM-3 did not undergo homophilic interactions, myelo-monocytic cells adhered to immobilized JAM-3 or to JAM-3-transfected cells. This heterophilic interaction was specifically attributed to a direct interaction of JAM-3 with the beta2-integrin Mac-1 and to a lower extent with p150.95 (alphaXbeta2, CD11c/CD18) but not with LFA-1 (alphaLbeta2, CD11a/CD18) or with beta1-integrins. These results were corroborated by analysis of K562 erythroleukemic cells transfected with different heterodimeric beta2-integrins and by using purified proteins. Moreover, purified JAM-3 or antibodies against JAM-3 blocked the platelet-neutrophil interaction, indicating that platelet JAM-3 serves as a counterreceptor for Mac-1 mediating leukocyte-platelet interactions. JAM-3 thereby provides a novel molecular target for antagonizing interactions between vascular cells that promote inflammatory vascular pathologies such as in atherothrombosis.