Clopidogrel, which is one of the most prescribed antiplatelet medications in the world, is given to stroke survivors for the prevention of secondary cardiovascular events. Clopidogrel exerts its antiplatelet activity via antagonism of the P2Y12 receptor (P2RY12). Although not widely known or considered during the initial clinical trials for clopidogrel, P2RY12 is also expressed on microglia, which are the brain’s immune cells, where the receptor facilitates chemotactic migration toward sites of cellular damage. If microglial P2RY12 is blocked, microglia lose the ability to migrate to damaged sites and carry out essential repair processes. We aimed to investigate whether administering clopidogrel to mice post-stroke was associated with (i) impaired motor skills and cognitive recovery; (ii) physiological changes, such as survival rate and body weight; (iii) changes in the neurovascular unit, including blood vessels, microglia, and neurons; and (iv) changes in immune cells. Photothrombotic stroke (or sham surgery) was induced in adult male mice. From 24 h post-stroke, mice were treated daily for 14 days with either clopidogrel or a control. Cognitive performance (memory and learning) was assessed using a mouse touchscreen platform (paired associated learning task), while motor impairment was assessed using the cylinder task for paw asymmetry. On day 15, the mice were euthanized and their brains were collected for immunohistochemistry analysis. Clopidogrel administration significantly impaired learning and memory recovery, reduced mouse survival rates, and reduced body weight post-stroke. Furthermore, clopidogrel significantly increased vascular leakage, significantly increased the number and appearance of microglia, and significantly reduced the number of T cells within the peri-infarct region post-stroke. These data suggest that clopidogrel hampers cognitive performance post-stroke. This effect is potentially mediated by an increase in vascular permeability post-stroke, providing a pathway for clopidogrel to access the central nervous system, and thus, interfere in repair and recovery processes.

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Background: As part of routine quality monitoring of frozen hematopoietic progenitor cell (HPC) products we perform Trypan Blue (TB) viability upon thawing. The sixth edition of FACT standards requires an assay for viable CD34 be performed on HPC products intended for hematopoietic reconstitution (D8.1.3.2). The eighth edition of AABB standards also requires a test for viability (5.17A.4.a). TB dye exclusion is a simple and rapid test for differentiating total living cells from nonviable cells, but dye uptake assessment can be subjective and CD34 specific measurement is not possible. Measuring cell viability by flow cytometry (flow) using the flurochrome 7-amino actinomycin (7-AAD) is less vulnerable to subjectivity and allows for cell-type specific assessment, but is more expensive, time-intensive, and requires specialized laboratory equipment and advanced technical expertise. Therefore, we evaluated TB compared to CD45 7-AAD viability, and CD45 7-AAD compared to CD34 7-AAD viability in order to establish that TB is an acceptable alternative to CD34 viability for frozen-thawed HPC collected by apheresis HPC(A). Methods: TB viability was performed with TB solution (.4%) with HPC(A) sample re-suspended in alpha Minimum Essential Media. Samples submitted to the flow lab were aliquoted from HPC(A) products suspended in dimethyl sulfoxide, then washed twice in buffer and stained within one hour of receipt by the flow lab. Analysis used the International Society of Hematotherapy and Graft Engineering (ISHAGE) protocol. The mean viability, mean difference, 95% confidence interval (CI) were calculated, and statistical significance was determined using the paired t-test. Results: For frozen-thawed HPC(A) samples (n = 39), mean CD45 7-AAD viability was 82.4% (CI 79.9 to 84.9%), and mean TB viability was 80.0% (CI 78.0 to 82.0%). The average difference is 2.4% which is not statistically significant (P = .12). Mean CD45 7-AAD viability was 82.3% (CI 79.6 to 84.9%) and mean CD34 7-AAD viability was 96.9% (CI 95.8 to 98.0%) (n = 33) where CD34 7-AAD viability was consistently greater than CD45 7-AAD viability by 14.6% (mean, CI 12.1 to 17.1%). Conclusion: For frozen-thawed HPC(A), overall viability measured with TB is not significantly different from viability measured by CD45 7-AAD. Furthermore, CD34 7-AAD viability is always greater than CD45 7-AAD viability. Therefore, we established that our process using TB viability for quality assessment of frozen-thawed HPC(A) products is an acceptable alternative to CD34 viability of each product and in compliance with D8.1.3.2 and 5.17A.4.a.

Background:The initial HCT evaluation can be overwhelming for a patient and caregiver. The appointment involves visits with the multi-disciplinary team who communicate an extensive amount of information. The patient and caregiver are given valuable written material of the HCT process that reinforces the verbal discussion. Even with written and verbal communication tools, patients reported gaps in knowledge about the HCT process. Patients also report feeling overwhelmed with the amount of verbal and written material presented. In addition, education teaching and method dispersed to patients and their caregivers vary among HCT Nurse Coordinators. Objectives: The aims of the project were to: 1) Increase the patient's and caregiver's understanding of HCT through a visual and oral presentation tool; 2) Increase patient's and caregiver's retention and knowledge level of HCT process; and 3) Ensure standardization of the patient education material taught by the HCT Nurse Coordinator. Method: A power point presentation was created for use during the first HCT evaluation visit. The HCT Nurse Coordinator accessed the presentation and referred to the images and photographs during the discussion of HCT. Included in the presentation, but not limited to, are images of stem cells, central lines, the hemapheresis department and equipment, and the injection teaching process. Pictures of our outpatient and new 23-bed inpatient HCT units were also included. Findings: Visualizations help to increase patient knowledge about line placement, hemapheresis, and the HCT process. Also, providing photographs of critical HCT processes for retention of material decreases anxiety and allows patients to be better prepared for HCT. By utilizing the power point presentation during the initial evaluation visit, the HCT Nurse Coordinators can consistently provide the same information taught to each patient. Conclusion: The addition of a visual presentation tool during the initial HCT evaluation visit can reinforce verbal and written resources for patients and caregivers. By utilizing the visual presentation tool, HCT Nurse Transplant Coordinators can provide consistent information regarding the HCT process and increases the patient's knowledge level.