Effect of swab pooling on the Accula point-of-care RT-PCR for SARS-CoV-2 detection.

Introduction: Swab pooling may allow for more efficient use of point-of-care assays for SARS-CoV-2 detection in settings where widespread testing is warranted, but the effects of pooling on assay performance are not well described. Methods: We tested the Thermo-Fisher Accula rapid point-of-care RT-PCR platform with contrived pooled nasal swab specimens. Results: We observed a higher limit of detection of 3,750 copies/swab in pooled specimens compared to 2,250 copies/swab in individual specimens. Assay performance appeared worse in a specimen with visible nasal mucous and debris, although performance was improved when using a standard laboratory mechanical pipette compared to the transfer pipette included in the assay kit. Conclusion: Clinicians and public health officials overseeing mass testing efforts must understand limitations and benefits of swab or sample pooling, including reduced assay performance from pooled specimens. We conclude that the Accula RT-PCR platform remains an attractive candidate assay for pooling strategies owing to the superior analytical sensitivity compared to most home use and point-of-care tests despite the inhibitory effects of pooled specimens we characterized.

Can red blood cell function assays assess response to red cell-modifying therapies?

BACKGROUND: Red blood cell (RBC)-modifying therapies have provided new opportunities for patients with sickle cell disease, although the absence of validated biomarkers of RBC function is a barrier to FDA approval and clinical adoption. Flow Adhesion (FA) and Mechanical Fragility (MF) biomarkers objectively stratify individuals with SCD into pro-adhesive vs pro-hemolytic phenotypes respectively, which may potentially help predict therapeutic responses. OBJECTIVE: A Phase 3 clinical trial to determine the effectiveness of vepoloxamer, an RBC-modifying therapy in sickle cell disease (SCD), failed to meet its primary clinical outcome. The aim of this study was to determine whether standardized flow adhesion and mechanical fragility bioassays could differentiate cellular level "responders" from "non-responders" to vepoloxamer treatment. METHODS: Standardized biomarkers of RBC function (adhesion and mechanical fragility) were utilized in this study to assess the effect of veploxamer on blood samples collected from SCD subjects and to determine whether our assays could differentiate cellular-level "responders" from "non-responders" to vepoloxamer treatment. A Wilcoxon signed-rank test was used to test for differences in adhesion in response to varying vepoloxamer treatments and a Wilcoxon Mann-Whitney test was used to assess differences in mechanical fragility, pre- and post-vepoloxamer treatment. A p-value<0.05 was considered significant. RESULTS: In this study, we report that in vitro treatment with vepoloxamer reduced adhesion by >75%in 54%of patient samples and induced changes in the membranes of sickle erythrocytes (SSRBCs) making sickle cells behave more like normal erythrocytes (AARBCs) in terms of their resistance to hemolysis. CONCLUSION: This study demonstrates that the standardized flow adhesion and mechanical fragility biomarkers described here may be useful tools to predict clinical responders to RBC-modifying therapies.

Erythropoietic properties of human induced pluripotent stem cells-derived red blood cells in immunodeficient mice.

Transfusion of red blood cells (RBCs) is a life-saving intervention for anemic patients. Human induced pluripotent stem cells (iPSC) have the capability to expand and differentiate into RBCs (iPSC-RBCs). Here we developed a murine model to investigate the in vivo properties of human iPSC-RBCs. iPSC lines were produced from human peripheral blood mononuclear cells by transient expression of plasmids containing OCT4, SOX2, MYC, KLF4, and BCL-XL genes. Human iPSC-RBCs were generated in culture supplemented with human platelet lysate, and were CD34- CD235a+ CD233+ CD49dlow CD71low ; about 13% of iPSC-RBCs were enucleated before transfusion. Systemic administration of clodronate liposomes (CL) and cobra venom factor (CVF) to NOD scid gamma (NSG) mice markedly promoted the circulatory survival of human iPSC-RBCs following transfusion. While iPSC-RBCs progressively decreased with time, 90% of circulating iPSC-RBCs were enucleated 1 day after transfusion (CD235a+ CD233+ CD49d- CD71- ). Surprisingly, human iPSC-RBCs reappeared in the peripheral circulation at 3 weeks after transfusion at levels more than 8-fold higher than at 1 h after transfusion. Moreover, a substantial portion of the transfused nucleated iPSC-RBCs preferentially homed to the bone marrow, and were detectable at 24 days after transfusion. These results suggest that nucleated human iPSC-derived cells that homed to the bone marrow of NSG mice retained the capability to complete differentiation into enucleated erythrocytes and egress the bone marrow into peripheral blood. The results offer a new model using human peripheral blood-derived iPSC and CL/CVF-treated NSG mice to investigate the development and circulation of human erythroid cells in vivo.

BMI1 enables extensive expansion of functional erythroblasts from human peripheral blood mononuclear cells.

Transfusion of red blood cells (RBCs) from ABO-matched but genetically unrelated donors is commonly used for treating anemia and acute blood loss. Increasing demand and insufficient supply for donor RBCs, especially those of universal blood types or free of known and unknown pathogens, has called for ex vivo generation of functional RBCs by large-scale cell culture. However, generating physiological numbers of transfusable cultured RBCs (cRBCs) ex vivo remains challenging, due to our inability to either extensively expand primary RBC precursors (erythroblasts) or achieve efficient enucleation once erythroblasts have been expanded and induced to differentiation and maturation. Here, we report that ectopic expression of the human BMI1 gene confers extensive expansion of human erythroblasts, which can be derived readily from adult peripheral blood mononuclear cells of either healthy donors or sickle cell patients. These extensively expanded erythroblasts (E3s) are able to proliferate exponentially (>1 trillion-fold in 2 months) in a defined culture medium. Expanded E3 cells are karyotypically normal and capable of terminal maturation with approximately 50% enucleation. Additionally, E3-derived cRBCs can circulate in a mouse model following transfusion similar to primary human RBCs. Therefore, we provide a facile approach of generating physiological numbers of human functional erythroblasts ex vivo.

Refractory thrombotic thrombocytopenic purpura related to checkpoint inhibitor immunotherapy.

BACKGROUND: Checkpoint inhibitors enhance T-lymphocyte-mediated antitumor responses, resulting in increased survival for patients with neoplastic disease. However, a subset of patients receiving checkpoint inhibitor therapy may experience adverse complications that include the development of autoimmune conditions, such as thrombotic thrombocytopenic purpura (TTP). Given the potential etiologic differences of checkpoint inhibitor-related autoimmunity, TTP that develops in the presence of checkpoint inhibitors may be refractory to current treatment methods and therefore may require additional treatment and prognostic consideration. CASE REPORT: Herein, we describe the unique clinical course of a patient who was treated with the combined checkpoint inhibitors nivolumab and ipilimumab for Stage IV malignant melanoma, who subsequently developed TTP. Unlike many patients with TTP, this patient failed to develop a sustained response to therapeutic plasma exchange. Additional use of steroids, anti-CD20, and plasma cell-targeting therapy (bortezomib) also failed to substantially reverse thrombocytopenia in a sustainable fashion. During this time, her melanoma progressed, and she ultimately succumbed. CONCLUSION: This case illustrates not only that TTP may be a potential complication of checkpoint inhibitor therapy, but also that TTP developing in this setting may result in an unpredictable response to commonly employed TTP treatment modalities. Ultimately, checkpoint inhibitor-related TTP may require distinct management approaches and prognostic considerations.

Cross-sectional analysis of adhesion in individuals with sickle cell disease using a standardized whole blood adhesion bioassay to VCAM-1.

Sickle cell disease (SCD) is characterized by frequent and unpredictable vaso-occlusive episodes (VOEs) that lead to severe pain, organ damage, and early death. Lack of reliable biomarkers that objectively define VOEs remains a critical barrier to improving the care for SCD patients. VOEs result from a complex interplay of cell-cell interactions that promote micro-vascular occlusion. Earlier studies demonstrated that sickle erythrocytes are more adherent than non-sickle erythrocytes and established a direct link between adhesion and frequency of VOEs. We developed a standardized, flow-based adhesion bioassay to assess the adhesive properties of SCD blood samples. The current study provides a cross-sectional analysis of steady state adhesion in SCD patients presenting at monthly out-patient hematology visits. Steady state adhesion varied from patient-to-patient. Adhesion positively correlated with reticulocyte percent and WBC count although there was no significant relationship between adhesion and platelets or hemoglobin in this study. Additionally, steady state adhesion indices were significantly lower in SCD subjects receiving hydroxyurea therapy when compared to the untreated group. The well-plate based microfluidic flow adhesion bioassay described in this report may provide a platform to identify SCD subjects with severe disease phenotypes, predict impending VOEs, and monitor response to current and developing therapies.

VLA-4 blockade by natalizumab inhibits sickle reticulocyte and leucocyte adhesion during simulated blood flow.

Very Late Antigen-4 (VLA-4, α4ß1-integrin, ITGA4) orchestrates cell-cell and cell-endothelium adhesion. Given the proposed role of VLA-4 in sickle cell disease (SCD) pathophysiology, we evaluated the ability of the VLA-4 blocking antibody natalizumab to inhibit SCD blood cell adhesion. Natalizumab recognized surface VLA-4 on leucocytes and reticulocytes in whole blood from SCD subjects. SCD reticulocytes were positive for VLA-4, while VLA-4 staining of non-SCD reticulocytes was undetectable. Titrations with natalizumab revealed the presence of saturable levels of VLA-4 on both SCD reticulocytes and leucocytes similar to healthy subject leucocytes. Under physiological flow conditions, the adhesion of SCD whole blood cells and isolated SCD leucocytes to immobilized vascular cell adhesion molecule 1 (VCAM-1) was blocked by natalizumab in a dose-dependent manner, which correlated with cell surface receptor binding. Natalizumab also inhibited >50% of whole blood cell binding to TNF-α activated human umbilical vein endothelial cell monolayers under physiological flow at clinically relevant concentrations (10 to 100 µg/ml). This indicates that VLA-4 is the dominant receptor that drives SCD reticulocyte and mononuclear cell adhesion to VCAM-1 and that the VLA-4 adhesion to VCAM-1 is a significant contributor to SCD blood cell adhesion to endothelium. Thus, VLA-4 blockade may be beneficial in sickle cell disease.

Extensive plexiform neurofibroma in a premature neonate.

We describe a premature neonate with an extensive plexiform neurofibroma. Prenatal ultrasound at 32 weeks of gestation was normal. Postnatal examination was significant for a palpable left neck mass. Magnetic resonance imaging (MRI) of the head demonstrated a mass involving the left cavernous sinus with spreading to the left orbital region. MRI of the neck was positive for extensive adenopathy, left more than right, with extension into the deep face region and infratemporal fossa on the left side. MRI of the chest, abdomen, and pelvis demonstrated a mass extending from the superior mediastinum to the left pelvic retroperitoneal region, including the mesenteric vasculature and spinal canal at multiple levels with compression of the spinal cord. Biopsy of the left neck mass confirmed for plexiform neurofibroma. A careful search of the literature revealed no previous report of such an extensive neurofibroma in a premature neonate. Surgical decompression in this premature neonate was not possible because of the extensive nature of the disease; it is known that neurofibroma is non-respondent to chemotherapy and radiotherapy. Therefore, alternative treatment is needed to improve the outcome.

Increased erythrocyte adhesion to VCAM-1 during pulsatile flow: Application of a microfluidic flow adhesion bioassay.

Sickle cell disease (SCD) is characterized by microvascular occlusion mediated by adhesive interactions of sickle erythrocytes (SSRBCs) to the endothelium. Most in vitro flow adhesion assays measure SSRBC adhesion during continuous flow, although in vivo SSRBC adhesive interactions occur during pulsatile flow. Using a well-plate microfluidic flow adhesion system, we demonstrate that isolated SSRBCs adhere to vascular cell adhesion molecule (VCAM-1) at greater levels during pulsatile versus continuous flow. A significant increase in adhesive interactions was observed between all pulse frequencies 1 Hz to 2 Hz (60-120 beats/min) when compared to non-pulsatile flow. Adhesion of isolated SSRBCs and whole blood during pulsatile flow was unaffected by protein kinase A (PKA) inhibition, and exposure of SSRBCs to pulsatile flow did not affect the intrinsic adhesive properties of SSRBCs. The cell type responsible for increased adhesion of whole blood varied from patient to patient. We conclude that low flow periods of the pulse cycle allow more adhesive interactions between sickle erythrocytes and VCAM-1, and sickle erythrocyte adhesion in the context of whole blood may better reflect physiologic cellular interactions. The microfluidic flow adhesion bioassay used in this study may have applications for clinical assessment of sickle erythrocyte adhesion during pulsatile flow.