Donor-Derived Cell-Free DNA to Diagnose Graft Rejection Post-Transplant: Past, Present and Future

Donor-derived cell-free DNA (dd-cfDNA) is a non-invasive biomarker that is more sensitive and specific towards diagnosing any graft injury or rejection. Due to its applicability over all transplanted organs irrespective of age, sex, race, ethnicity, and the non-requirement of a donor sample, it emerges as a new gold standard for graft health and rejection monitoring. Published research articles describing the role and efficiency of dd-cfDNA were identified and scrutinized to acquire a brief understanding of the history, evolution, emergence, role, efficiency, and applicability of dd-cfDNA in the field of transplantation. The dd-cfDNA can be quantified using quantitative PCR, next-generation sequencing, and droplet digital PCR, and there is a commendatory outcome in terms of diagnosing graft injury and monitoring graft health. The increased levels of dd-cfDNA can diagnose the rejection prior to any other presently used biochemistry or immunological assay methods. Biopsies are performed when these tests show any signs of injury and/or rejection. Therefore, by the time these tests predict and show any unusual or improper activity of the graft, the graft is already damaged by almost 50%. This review elucidates the evolution, physiology, techniques, limitations, and prospects of dd-cfDNA as a biomarker for post-transplant graft damage and rejection.

Radioligand Assay-Based Detection of Antibodies against SARS-CoV-2 in Hospital Workers Treating Patients with Severe COVID-19 in Japan

This study aimed to clarify whether infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is prevalent among the staff of a hospital providing treatment to patients with severe coronavirus disease 2019 (COVID-19) using radioligand assay (RLA). One thousand samples from the staff of a general hospital providing treatment to patients with severe COVID-19 were assayed for SARS-CoV-2 nucleocapsid protein (N) IgG using RLA. Nine patients with COVID-19 who had been treated in inpatient settings and had already recovered were used as control subjects, and 186 blood donor samples obtained more than 10 years ago were used as negative controls. Four of the 1000 samples showed apparently positive results, and approximately 10 or more samples showed slightly high counts. Interestingly, a few among the blood donor samples also showed slightly high values. To validate the results, antibody examinations using ELISA and neutralizing antibody tests were performed on 21 samples, and chemiluminescence immunoassay (CLIA) was performed on 201 samples, both resulting in a very high correlation. One blood donor sample showed slightly positive results in both RLA and CLIA, suggesting a cross-reaction. This study showed that five months after the pandemic began in Japan, the staff of a general hospital with a tertiary emergency medical facility had an extremely low seroprevalence of the antibodies against SARS-CoV-2. Further investigation will be needed to determine whether the slightly high results were due to cross-reactions or a low titer of anti-SARS-CoV-2 antibodies. The quantitative RLA was considered sensitive enough to detect low titers of antibodies.

Complement activating ABO IgM/IgG act synergistically to cause erythrophagocytosis: implications among minor ABO incompatible platelet transfusions

Background Typically minor ABO incompatible platelet products are transfused without any incident, yet serious hemolytic transfusion reactions occur. To mitigate these events, ABO ‘low titer’ products are used for minor ABO incompatible transfusions. We sought to understand the role of IgG and complement activation by anti-A on extravascular hemolysis. Samples evaluated: i) Group O plasma from a blood donor whose apheresis platelet product resulted in an extravascular transfusion reaction, ii) Group O plasma from 12 healthy donors with matching titers that activated complement (N = 6) or not (N = 6), and iii) Group O sera from 10 patients with anti-A hemolysin activity. Monocytes from healthy donors were co-incubated with anti-A-sensitized fluorescently-labeled Group A1+ RBCs with and without fresh Group A serum, as a source of complement C3, and phagocytosis was analyzed by flow cytometry. The plasma and sera had variable direct agglutinating (IgM) and indirect (IgG) titers. Results None of 12 selected samples showed monocyte-dependent erythrophagocytosis with or without complement activation. The donor sample causing a hemolytic transfusion reaction and 2 of the 10 patient sera with hemolysin activity showed significant erythrophagocytosis (>10%) only when complement C3 was activated. The single donor plasma and two sera demonstrating significant erythrophagocytosis had high IgM (≥128) and IgG titers (>1024). The donor plasma anti-A was IgG1, while the patient sera were an IgG3 and an IgG1 plus IgG2. Conclusion High anti-A IgM/IgG titers act synergistically to cause significant monocyte erythrophagocytosis by activating complement C3, thus engaging both Fcγ- and CR1-receptors.

The characteristics of blood donors in England and Wales

This chapter explores the characteristics of blood donors in England and Wales, considering a study made in the summer and autumn of 1967 with the assistance of the Ministry of Health and the National Blood Transfusion Service. The study examines regional trends and statistics relating to donor populations and donor reporting rates for the general public, institutions — comprising factories, offices, and universities — and the Defence Services. The general conclusion which emerges is that the donor sample broadly resembles the population in respect of age, sex, and marital status when account is taken of the possible effects of the age-incapacity and reproductive factors. Moreover, for most age groups, the general public donor is more representative of the national population than the institutional donor or the total of all donors. The institutional and Defence Services donor tends on the whole to be younger.

KIR Ligand Incompatibility in HLA-Identical Sibling Nonmyeloablative Hematopoietic Stem Cell Transplantation for Hematologic Malignancies.

Natural killer (NK) cell alloreactivity based on inhibitory killer immunoglobulin-like receptor (KIR)-ligand incompatibility (i.e., missing KIR ligand) in the graft-vs-host (GVH) direction has been described to favorably influence engraftment, GVHD, and graft-vs-tumor (GVT) effect following haploidentical or matched unrelated HSCT in patients with hematologic malignancies. The degree and effect of KIR-ligand incompatibility has recently been explored following HLA-identical sibling HSCT (Hsu et al., Blood2005;105:4878). Based on a murine model, we developed a clinical trial with a goal of deliberately inducing a GVHD-free mixed chimeric platform following nonmyeloablative conditioning (consisting of equine ATG, cyclophosphamide, thymic irradiation, and a brief course of cyclosporine) and HLA-matched sibling HSCT. Donor leukocyte infusions (DLI) were given as early as five weeks post-HSCT to patients without GVHD in an attempt to achieve a conversion to full donor chimerism (FDC) with the goal of fully capturing GVT effect with little or no GVHD. In this study we hypothesized that KIR-ligand incompatibility in the GVH direction and KIR-ligand compatibility in the host-vs-graft (HVG) direction would reduce the rate of graft failure, decrease the incidence of GVHD, and improve overall survival following HLA-matched sibling HSCT. Fourteen transplant recipients (bone marrow, n=9; peripheral blood stem cell, n=5) with refractory hematologic malignancies (NHL, n=7; HD, n=3; MM, n=2; CLL, n=1; AML, n=1) were analyzed. KIR typing was accomplished using PCR amplified DNA from both donor and recipient patient samples. Typing of the amplified DNA was performed using the Lifecodes KIR-SSO typing kits (Tepnel Lifecodes Corporation). By using the SSO (sequence-specific oligonucleotides) technology in conjunction with the Luminex Instrument, KIR loci were identified for each patient and donor sample. KIR-ligand (HLA) incompatibility in the GVH and HVG directions were assessed based on HLA and KIR genotyping (KIR2DL1, KIR2DL2, KIR2DL3 and KIR3DL1) of 14 donors and 12 of the 14 recipients. Six of the 14 patients eventually lost their grafts despite DLI. Seven patients spontaneously achieved FDC and one converted to FDC following DLI. The missing KIR ligand analysis showed 12 patients (86%, n=12/14) with KIR-ligand incompatibility in the GVH direction (1 missing ligand, n=9; 2 missing ligands, n=3) and 10 patients (83%, n=10/12) with KIR-ligand incompatibility in the HVG direction (1 missing ligand, n=9; 2 missing ligands n=1). The presence or the degree of KIR-ligand incompatibility in GVH or HVG direction was not found to be predictive of spontaneous FDC or graft rejection. There was no significant relationship between the number of missing KIR ligands in either direction and the development of acute or chronic GVHD. This study shows a higher rate of KIR-ligand incompatibility in the GVH (86%) and HVG (83%) directions in the setting of HLA-matched sibling HSCT than previously reported. Although the small number of patients does not allow for statistically meaningful conclusions regarding clinical outcome, the observation of a high incidence of KIR-ligand incompatibility in this population justifies the study of larger patient cohorts to determine the influence of NK cell alloreactivity on transplant outcomes.