Ljiljana V. Vasovic

Visualization of macro-immune complexes in the antiphospholipid syndrome by multi-modal microscopy imaging

The antiphospholipid syndrome (APS) is an autoimmune thrombotic condition that is marked by autoantibodies against phospholipid-binding proteins. The mechanism(s) of thrombogenesis has (have) resisted elucidation since its description over thirty years ago. Nevertheless, a defining aspect of the disorder is positivity for clinical laboratory tests that confirm antibody binding to anionic phospholipids. It is remarkable that, to our knowledge, the binding of proteins from plasmas of APS patients to phospholipid has not been previously imaged. We therefore investigated this with high resolution microscopy-based imaging techniques that have not been previously used to address this question, namely atomic force microscopy and scanning electron microscopy. Atomic force microscopy imaging of APS plasmas incubated on an anionic planar phospholipid layer revealed the formation of distinct complex three-dimensional structures, which were morphologically dissimilar to structures formed from control plasmas from healthy patients. Likewise, scanning electron microscopy analysis of phospholipid vesicles incubated with APS plasmas in suspension showed formation of layered macro-immune complexes demonstrated by the significant agglomeration of a complex proteinaceous matrix from soluble plasma and aggregation of particles. In contrast, plasmas from healthy control samples bound to phospholipid vesicles in suspension generally displayed a more flattened, mat-like appearance by scanning electron microscopy. Scanning electron microscopy of plasma samples incubated on planar phospholipid layers and previously imaged by atomic force microscopy, corroborated the results obtained by mixing the plasmas with phospholipids in solution. Analysis of the incorporated proteins by silver stained SDS-polyacrylamide gel electrophoresis indicated considerable heterogeneity in the composition of the phospholipid vesicle-adsorbed proteins among APS patients. To our knowledge, these results provide the first images of plasma-derived APS immune complexes at high resolution, and show their consistent presence and heterogeneous compositions in APS patients. These findings demonstrate how high resolution microscopic techniques can contribute to advancing the understanding of an enigmatic disorder and may lay additional groundwork for furthering mechanistic understanding of APS.

Inverted erythrocyte membranes demonstrate β2GPI-antiphospholipid antibody interactions and membrane crosslinking

INTRODUCTION The antiphospholipid syndrome (APS) is an acquired autoimmune disorder predisposing patients to thrombosis or pregnancy complications. Since inverted erythrocyte membranes (iEMs) might provide a physiologically relevant source of anionic phospholipids, we studied the interactions of phospholipid-binding proteins and APS antibodies using iEMs. MATERIALS & METHODS iEMs were prepared from packed erythrocytes by hypotonic lysis. Phosphatidylserine (PS) exposure was confirmed by annexin A5 (A5) binding using fluorescence microscopy and flow cytometry. Binding of β2-glycoprotein I (β2GPI)-IgG immune complexes to iEMs was investigated with gel electrophoresis, western blot and flow cytometry. Functional involvement in coagulation was documented in the thrombin generation assay. RESULTS iEMs readily precipitated purified β2GPI as well as β2GPI from normal plasma and APS plasma. The plasma of APS patients provided higher levels of IgG binding to iEMs relative to healthy controls. Thrombin generation increased with increasing concentrations of iEMs, documenting that coagulation proteins bound to the exposed phospholipids. The LA effect was also distinguished in thrombin generation when comparing APS patients, as indicated by an increased lag time. Agglutination was observed after incubation with APS patient plasma and this was augmented by anti-human globulin. CONCLUSIONS In conclusion, iEMs can provide a more physiological approach than phospholipid vesicle-based tests for investigating APS and are more amenable to standardization than platelet membranes.

Intrauterine transfusions for severe fetal anemia and hydrops due to de novo εγδβ-thalassemia: INTRAUTERINE TRANSFUSIONS FOR εγδβ-THALASSEMIA

A 29-year-old G1P0 female at 24-6/7 weeks’ gestation was admitted for percutaneous umbilical cord blood sampling (PUBS) and intrauterine transfusion (IUT). Microarray testing from an amniocentesis sampling done for echogenic bowel, incidentally revealed a de novo heterozygous loss of 1.38 megabase within chromosomal band 11p15.4. This deletion encompassed the entire b-globin gene cluster and was diagnosed as ecdb-thalassemia (see figure—[left] first preintrauterine transfusion fetal cord sample smear showing erythroblasts, spectrum of maturing nucleated RBCs, polychromasia, anisocytosis, target cells, and spherocytes; Wright-Giemsa stain, 10003 magnification; [right] de novo heterozygous deletion of the entire b-globin gene cluster within the chromosomal band 11p15.4, schematic representation). The above prompted middle cerebral artery Doppler testing, which was remarkable for elevated peak systolic velocity of more than 1.5MoM consistent with severe fetal anemia. Ultrasonography showed hydrops with fetal ascites and pericardial effusion. Patient underwent a PUBS; hematocrit (Hct) was 21.9% and smear showed numerous target cells and spherocytes, likely from ABO incompatibility (mother O1, baby A1; see figure). A series of three IUT procedures led to complete resolution of hydrops and pericardial effusion and improvement of fetal anemia with a stable Hct of 43% upon delivery at 36 weeks’ gestation. The ecdb-thalassemias are extremely rare sporadic disorders caused by deletion of the b-globin gene cluster with or without deletion of the upstream b-locus control region. Approximately 30 heterozygous deletions are reported with the homozygous variant being incompatible with life. Severe fetal anemia results from pronounced chain imbalance with reduced c-globin (and thus reduced HbF production). Only four similar cases of IUTs have been reported in the literature but this is the first case of successful management of a de novo deletion leading to ecdb-thalassemia. Anemia typically resolves in early postnatal months with the adult phenotype being similar to b-thalassemia trait.

Hematopoietic Stem Cell Collections and Cellular Therapies

The advancement of hematopoietic progenitor cell (HPC) transplant has increased the chance of cure for a number of hematologic malignancies and other nonmalignant hematologic and congenital diseases. HPCs can be collected from a patient, stored, and transplanted back to the patient after chemotherapy/radiotherapy, as an autologous HPC transplant (HPCT). Infusion of HPCs from siblings or matched related donors or volunteer matched unrelated donors constitutes allogeneic HPCT. Finding an optimal donor for allogeneic HPCT by extensive human leukocyte antigen (HLA) matching between donor and recipient is the most important factor influencing transplant outcomes. The traditional source of HPCs is bone marrow; however, peripheral blood stem cells, mobilized to circulation and harvested by apheresis, have become the preferred graft source because of procurement of higher dosage and convenience of collection. If a full HLA match is not available, umbilical cord blood cells can be used, either as a single graft or in combination with other cellular therapy products (CTPs). A brief overview of donor matching and selection, choice of graft source, and CTP collection procedure are discussed. Laboratory graft characterization and processing, such as HPC CD34 + selection, CTP cryopreservation, storage, thawing, infusion, and impact of those parameters on transplant success and transplant-related outcomes, are reviewed. HPC infusion reactions and posttransplant monitoring including engraftment are outlined. New investigational products reaching clinical practice, such as expanded or genetically modified stem cells, immunotherapy, and regenerative CTP, are also discussed.

Transfusion Approaches in the Transplanted Patient

Blood transfusion support is essential in a transplant recipients recovery. A careful consideration needs to be given to balancing the benefits and risks of transfusions in the transplant population because of the inherent complexity of issues and management. This chapter provides a review of the unique management challenges posed by the hematopoietic progenitor cell transplant (HPCT) and solid organ transplant (SOT) recipients and how blood bank physicians must manage them. We review here the issues of ABO matching and major, minor, and bidirectional incompatibility and potential consequences of ABO-incompatible transplants in HPCT and SOT. The major focus is on safe and optimal transfusion support. The necessary standard modifications of blood components, such as irradiation and leukoreduction, are supplemented by the evolving and increasing use of pathogen-reduced blood products. Transfusion needs vary following transplant and engraftment because of dynamic changes in the hematopoietic compartment. Various strategies for the selection of blood components for transfusion in mismatched HPCT are compared because they changed over time based on new clinical paradigms. In addition, we have summarized strategies for platelet matching in the setting of refractory platelet response and special circumstances for granulocyte transfusions in patients who underwent a transplant. We also discuss special situations for transfusion approaches in pediatric transplants and complexities unique to patient populations that undergo long-term transfusion, such as patients with sickle cell and thalassemia. Our objective is to provide an informative guide for medical staff and clinician education in the transfusion management of patients who underwent a transplant based on clinical evidence that will result in the highest standards of care.

Validating and implementing the use of an infusion pump for the administration of thawed hematopoietic progenitor cells-a single-institution experience: VALIDATION OF INFUSION PUMP FOR PBSCs

Direct thaw and administration of previously cryopreserved peripheral blood stem cell products is a commonly used practice and should be performed rapidly to reduce cellular damage caused by dimethyl sulfoxide exposure. Cells are typically thawed at the bedside and infused by gravity through a high‐flow‐rate central venous catheter. An existing nontunneled catheter is occasionally used instead and often results in a slower infusion rate. To ensure expedient and consistent infusions, we validated and implemented the use of an infusion pump for thawed peripheral blood stem cells.

Comparison of time to engraftment between autologous patients receiving washed versus non-washed cryopreserved peripheral blood stem cell products

Abstract Washing cryopreserved peripheral blood stem cell (PBSC) products can decrease infusion-related adverse reactions but can also result in cell loss and reduced cell viability. To assess the risk and benefit of washing products, we compared the time to neutrophil and platelet engraftment between autologous patients that received washed products (n = 201) and non-washed products (n = 89). The effect of the other variables, including age, gender, diagnosis, transplant dose, method of stem cell mobilization, and growth factor support regimen post-transplant, was assessed. In multivariate analysis, direct thaw and infusion of non-washed products resulted in significantly faster neutrophil engraftment (p = .003) and platelet engraftment (p = .017) than washed products. The mean neutrophil and platelet engraftment times were 1.07 days faster and 2.27 days faster, respectively. In conclusion, direct thaw and infusion of cryopreserved PBSC without washing results in significantly shorter time to recovery of neutrophils and platelets after autologous transplantation.

Cryoprecipitate indications and patterns of use in the pediatric intensive care unit: inappropriate transfusions and lack of standardization

The dosage and indications for cryoprecipitate are not well studied for any patient population. Prior observational studies have suggested that 24% to 62% of cryoprecipitate transfusions are inappropriate, and there is limited information on patterns of cryoprecipitate use in children. The purpose of this retrospective study was to explore the indications and appropriateness of the use of cryoprecipitate in critically ill children.