Anti-CD38 monoclonal antibody interference with blood compatibility testing: Differentiating isatuximab and daratumumab via functional epitope mapping.

BACKGROUND: There are two FDA-approved anti-CD38 monoclonal antibodies for treatment of multiple myeloma: isatuximab and daratumumab. Owing to expression of CD38 on reagent red blood cells (RBCs), these antibodies interfere with indirect antiglobulin tests (IATs). We sought to understand differences in such interference by performing binding experiments. STUDY DESIGN AND METHODS: In vitro experiments to compare the binding to RBCs of isatuximab and daratumumab alone or in the presence of a mouse anti-human CD38 antibody (HB-7 or AT13/5) or a nicotinamide adenine dinucleotide-analog CD38 inhibitor were performed and quantified by flow cytometry, imaging, mass spectrometry, surface plasmon resonance, and LigandTracer technologies. Serologic testing was performed on plasma samples spiked with isatuximab or daratumumab. RESULTS: CD38 expressed on RBCs can be directly bound by daratumumab, whereas isatuximab requires a co-factor, such as HB-7, AT13/5, or a CD38 inhibitor, suggesting that the isatuximab epitope on RBCs is masked in vitro. Daratumumab samples more frequently showed interference and had stronger reactions than isatuximab samples. Dithiothreitol treatment was equally effective in mitigating the interference caused by either drug. DISCUSSION: Both isatuximab and daratumumab interfere with IATs but at different magnitudes, reflecting distinct binding to CD38 on RBCs. From the binding studies, we conclude that the isatuximab epitope on RBCs is masked in vitro and binding requires a certain CD38 conformation or co-factor. This circumstance may explain why interference is seen only in a subset of patients receiving isatuximab when compared with interference seen in most patients on daratumumab therapy.

Impact of new myeloma agents on the transfusion laboratory.

Monoclonal antibody (mAb) therapy targeting CD38 and CD47 antigens expressed on cancer cells has transformed therapy options for patients with multiple myeloma as well as other haematological and non-haematological malignancies. While the on target effects of these new drugs highlight the promise of precision cancer therapeutics, the unintended, off target binding of drugs to red blood cells (RBCs) and platelets has required transfusion service laboratories (TSL) and immunohaematology reference laboratories (IRL) to innovate and rapidly set up processes and testing protocols to overcome the significant interference in routine pre-transfusion tests caused by these agents. Binding of anti-CD38 and anti-CD47 drugs to reagent RBCs leads to false positive pan-agglutination during the antihuman globulin phase of testing, making it difficult to rule out underlying alloantibodies, and leading to delays in setting up compatible units for RBC transfusion. Anti-CD47 agents can also interfere with ABO/Rh typing studies. Several methods to successfully mitigate interference have been described, such as treatment of reagent RBCs with reducing agents or enzymes, allogeneic RBC adsorption studies and drug specific neutralisation assays; all methods have limitations. TSLs should select an approach that best fits their workflow and expertise and takes into consideration their level of access to specialised outside testing, local blood supplier capabilities, and the type of patient population served. For platelet refractory patients, samples should be tested by platelet antibody assays that are known to be unaffected by drug therapy. RBC transfusion support for multiple myeloma patients receiving anti-CD38 or anti-CD47 drugs can be optimised by establishing good communication between the clinical teams and TSLs, building electronic notification processes, and ensuring timely completion of baseline pre-transfusion testing and RBC phenotype/genotype prior to starting therapy. Staff education, standardisation of laboratory mitigation measures, and implementation of testing algorithms that consider mAb-induced interference when working up a pan-agglutinin help to significantly decrease delays that would otherwise result if standard methods were employed to complete antibody identification studies.

Sepsis from an apheresis platelet contaminated with Acinetobacter calcoaceticus/baumannii complex bacteria and Staphylococcus saprophyticus after pathogen reduction.

BACKGROUND: Strategies to reduce platelet (PLT) bacterial contamination include donor screening, skin disinfection, sample diversion, bacterial culture, pathogen reduction (PR), and day-of-transfusion tests. We report bacterial sepsis following a pathogen-reduced PLT transfusion. CASE REPORT: An adult male with relapsed acute lymphoblastic leukemia was successfully treated for central catheter-associated Staphylococcus aureus bacteremia. A peripherally inserted central catheter (PICC) was placed. Chills, rigors, and flushing developed immediately after PICC-infused pathogen-reduced PLTs, progressing to septic shock requiring intensive care management. METHODS: PICC and peripheral blood (PB), transfused bag saline flushes (TBFs), environmental samples, and the pathogen-reduced untransfused co-component (CC) were cultured. Plasma metagenomic and bacterial isolate whole-genome sequencing; PLT mitochondrial DNA (mtDNA) testing of untransfused CC and TBF; CC testing for amotosalen (S-59)/S-59 photoproducts; isolate PR studies (INTERCEPT); and TBF polymerase chain reaction for recipient Y-chromosome DNA were performed. RESULTS: PB and PICC cultures grew Acinetobacter calcoaceticus/baumannii complex (ACBC). TBF was gram-positive; mass spectrometry identified ACBC and Staphylococcus saprophyticus (SS). CC Gram stain and cultures were negative. Environmental cultures, some done after decontamination, were ACBC/SS negative. Posttransfusion patient plasma and TBF ACBC sequences were genetically identical. No Y-chromosome signal was detected in TBF. S-59 photoproducts and evidence of mtDNA amplification inhibition were found in the CC. Spiking PR studies showed >5.9-log inactivation for both isolates. Donor skin cultures for Acinetobacter were negative. CONCLUSION: CC sterility, PR studies, residual S-59 photoproducts, and mtDNA amplification inhibition suggest successful PR. Unidentified environmental sources and inherent or acquired bag defects may have contributed to postmanufacturing pathogen-reduced PLT contamination.

Investigating Transfusion-related Sepsis Using Culture-Independent Metagenomic Sequencing.

BACKGROUND: Transfusion-related sepsis remains an important hospital infection control challenge. Investigation of septic transfusion events is often restricted by the limitations of bacterial culture in terms of time requirements and low yield in the setting of prior antibiotic administration. METHODS: In 3 gram-negative septic transfusion cases, we performed metagenomic next-generation sequencing (mNGS) of direct clinical blood specimens in addition to standard culture-based approaches utilized for infection control investigations. Pathogen detection leveraged IDSeq, a new open-access microbial bioinformatics portal. Phylogenetic analysis was performed to assess microbial genetic relatedness and understand transmission events. RESULTS: mNGS of direct clinical blood specimens afforded precision detection of pathogens responsible for each case of transfusion-related sepsis and enabled discovery of a novel Acinetobacter species in a platelet product that had become contaminated despite photochemical pathogen reduction. In each case, longitudinal assessment of pathogen burden elucidated the temporal sequence of events associated with each transfusion-transmitted infection. We found that informative data could be obtained from culture-independent mNGS of residual platelet products and leftover blood specimens that were either unsuitable or unavailable for culture or that failed to grow due to prior antibiotic administration. We additionally developed methods to enhance accuracy for detecting transfusion-associated pathogens that share taxonomic similarity to contaminants commonly found in mNGS library preparations. CONCLUSIONS: Culture-independent mNGS of blood products afforded rapid and precise assessment of pathogen identity, abundance, and genetic relatedness. Together, these challenging cases demonstrated the potential for metagenomics to advance existing methods for investigating transfusion-transmitted infections.

Sepsis Attributed to Bacterial Contamination of Platelets Associated with a Potential Common Source - Multiple States, 2018.

During May-October 2018, four patients from three states experienced sepsis after transfusion of apheresis platelets contaminated with Acinetobacter calcoaceticus-baumannii complex (ACBC) and Staphylococcus saprophyticus; one patient died. ACBC isolates from patients' blood, transfused platelet residuals, and two environmental samples were closely related by whole genome sequencing. S. saprophyticus isolates from two patients' blood, three transfused platelet residuals, and one hospital environmental sample formed two whole genome sequencing clusters. This whole genome sequencing analysis indicated a potential common source of bacterial contamination; investigation into the contamination source continues. All platelet donations were collected using apheresis cell separator machines and collection sets from the same manufacturer; two of three collection sets were from the same lot. One implicated platelet unit had been treated with pathogen-inactivation technology, and two had tested negative with a rapid bacterial detection device after negative primary culture. Because platelets are usually stored at room temperature, bacteria in contaminated platelet units can proliferate to clinically relevant levels by the time of transfusion. Clinicians should monitor for sepsis after platelet transfusions even after implementation of bacterial contamination mitigation strategies. Recognizing adverse transfusion reactions and reporting to the platelet supplier and hemovigilance systems is crucial for public health practitioners to detect and prevent sepsis associated with contaminated platelets.

Development and Implementation of Real-Time Web-Based Dashboards in a Multisite Transfusion Service.

BACKGROUND: In hospital transfusion services, visualization of blood product inventory in the form of web-based dashboards has the potential to improve the workflow and efficiency of blood product inventory management. While off-the-shelf "business intelligence" solutions by external vendors may offer the ability to display and analyze blood bank inventory data, laboratories may lack resources to readily access this technology. Using in-house talent, our transfusion service developed real-time, web-based dashboards to replace manual processes for managing both blood product inventory and cooler tracking at two large academic hospital blood banks. METHODS: Dashboards were developed using Hypertext Markup Language, Cascading Style Sheets, and Hypertext Preprocessor scripting/programming languages. Data are extracted in real time from Sunquest (v7.3) Laboratory Information Systems Database (InterSystems Cache) and are refreshed every 2 min. Data are hosted internally by our institution's web servers and are accessed on a webpage via Microsoft Group Policy shortcuts. RESULTS: Dashboards were designed and implemented to provide a fully customizable, dynamic, and secure method of displaying blood product inventory and blood product cooler status. Transfusion service staff utilized dashboard data to maintain adequate blood product supply, modify blood product replacement orders to prevent excess inventory, and transfer short-dated blood products between our facilities to minimize wastage. CONCLUSIONS: Dashboard technology can be readily implemented at hospital transfusion services with minimal capital expenditure. The implementation of real-time web-based dashboards for blood product inventory and cooler management at our centers facilitated on-demand blood product monitoring and replaced a tedious, manual process with a user-friendly and intuitive electronic tool.

Prolonged ceftriaxone-induced immune thrombocytopenia due to impaired drug clearance: a case report.

BACKGROUND: Antibody-mediated drug-induced thrombocytopenia (DIT) typically requires the presence of the sensitizing drug in the plasma. Therefore, platelet (PLT) counts often start to recover 1 to 2 days after discontinuation of the offending medication. We present a case of ceftriaxone-induced DIT that resulted in severe, prolonged thrombocytopenia. CASE REPORT: A 65-year-old woman with liver and renal insufficiency was transferred to our hospital for liver transplant evaluation. Two days after a 5-day course of ceftriaxone, her PLT count declined from a stable baseline of approximately 70 × 10(9) /L to a value of 3 × 10(9) /L, with coincident onset of mucocutaneous purpura. Her PLT count remained in the 1 × 10(9) to 6 × 10(9) /L range until her death 13 days later, despite intravenous immune globulin, steroids, and PLT transfusions. The persistently low PLT count impeded central catheter placement for hemodialysis and possible therapeutic plasmapheresis. A strong ceftriaxone-dependent, PLT-reactive antibody was identified in a sample drawn 7 days after ceftriaxone was last administered, and ceftriaxone remained detectable in her serum for at least 8 days after the last dose. CONCLUSION: A ceftriaxone-dependent, PLT-reactive antibody was responsible for the persistent thrombocytopenia in this patient. Although DIT is generally expected to improve within a few days of drug discontinuation, impaired drug clearance can significantly alter the outcome. This case highlights the importance of altered drug metabolism and clearance in critically ill patients, especially those with combined hepatic and renal dysfunction. DIT should be strongly suspected in patients with acute thrombocytopenia, and all treatment options to reduce serum drug levels should be seriously considered.

Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection.

Retroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) long terminal repeats (LTRs) may have significantly reduced genotoxicity relative to the conventional retroviral vectors used in recent, otherwise successful clinical trials. Large-scale production of such vectors is problematic, however, as the introduction of SIN vectors into packaging cells cannot be accomplished with the traditional method of viral transduction. We have derived a set of packaging cell lines for HIV-based lentiviral vectors and developed a novel concatemeric array transfection technique for the introduction of SIN vector genomes devoid of enhancer and promoter sequences in the LTR. We used this method to derive a producer cell clone for a SIN lentiviral vector expressing green fluorescent protein, which when grown in a bioreactor generated more than 20 L of supernatant with titers above 10(7) transducing units (TU) per milliliter. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells that produced similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human interleukin 2 receptor common gamma chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 x 10(7) TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1).

Correction of murine hemophilia A by hematopoietic stem cell gene therapy.

A serious complication of current protein replacement therapy for hemophilia A patients with coagulation factor VIII (FVIII) deficiency is the frequent development of anti-FVIII inhibitor antibodies that preclude therapeutic benefit from further treatment. Induction of tolerance by persistent high-level FVIII synthesis following transplantation with hematopoietic stem cells expressing a retrovirally delivered FVIII transgene offers the possibility of permanently correcting the disease. Here, we transplanted bone marrow cells transduced with an optimized MSCV-based FVIII oncoretroviral vector into immunocompetent hemophilia A mice that had been conditioned with a potentially lethal dose of irradiation (800 cGy), a sublethal dose of irradiation (550 cGy), or a nonmyeloablative preparative regimen involving busulfan. Therapeutic levels of FVIII (42, 18, and 11% of normal, respectively) were detected in the plasma of the transplant recipients for the duration of the study (over 6 months). Moreover, subsequent challenge with recombinant FVIII elicited at most a minor anti-FVIII inhibitor antibody response in any of the experimental animals, in contrast to the vigorous neutralizing humoral reaction to FVIII that was stimulated in naive hemophilia A mice. These findings represent an encouraging advance toward potential clinical application and long-term amelioration or cure of this progressively debilitating, life-threatening bleeding disorder.

Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions.

The genes for the alpha and beta chains of a highly reactive anti-MART-1 T-cell receptor were isolated from T-lymphocytes that mediated in vivo regression of tumor in a patient with metastatic melanoma. These genes were cloned and inserted into MSCV-based retroviral vectors. After transduction, greater than 50% gene transfer efficiency was demonstrated in primary T-lymphocytes stimulated by an anti-CD3 antibody. The specificity and biologic activity of TCR gene-transduced T-cells was determined by cytokine production after coculture of T-cells with stimulator cells pulsed with MART-1 peptide. The production of interferon-gamma and granulocyte macrophage-colony stimulating factor (GM-CSF) was comparable to highly active MART-1 specific peripheral blood lymphocytes (PBL) in the amount of cytokine produced and transduced cells recognized peptide pulsed cells at dilutions similar to cytotoxic T lymphocyte (CTL) clones. Human leukocyte antigen (HLA) class I restricted recognition was demonstrated by mobilization of degranulation marker CD107a, by cell lysis, by cytokine production, and by proliferation in the presence of HLA-A2-positive but not HLA-A2-negative melanoma cell lines. Similar data was obtained when tumor-infiltrating lymphocytes (TIL) were transduced with the TCR genes, converting previously nonreactive cells to tumor reactive cells. TCR-transduced T-cells are thus attractive candidates for evaluation in cell transfer therapies of patients with cancer.

Sustained phenotypic correction of hemophilia a mice following oncoretroviral-mediated expression of a bioengineered human factor VIII gene in long-term hematopoietic repopulating cells.

Hematopoietic stem cells (HSCs) are an attractive target cell population for hemophilia A gene therapy because of their capacity to regenerate the hematolymphoid system permanently following transplantation. Here we transplanted bone marrow (BM) cells transduced with a splicing-optimized MSCV oncoretroviral vector expressing a secretion-improved human factor VIII gene into immunocompromised hemophilic mice that had received a reduced dose conditioning regimen. An enhanced green fluorescent protein (EGFP) reporter gene linked to an encephalomyocarditis virus internal ribosome entry site was incorporated into the vector to allow preselection of transduced cells and facile evaluation of engraftment. Sustained expression of EGFP was demonstrated in the peripheral blood, and therapeutic levels of factor VIII were detected in the plasma of the majority of the recipients for the duration of the observation period (up to 22 weeks). Coordinate expression of factor VIII and EGFP (up to 19 weeks) was transferred to secondary BM transplant recipients, indicating that long-term repopulating HSCs had been successfully gene modified. Notably, the hemophilic phenotype of all treated mice was corrected, thus demonstrating the potential of HSC-directed oncoretroviral-mediated factor VIII gene transfer as a curative therapeutic strategy for hemophilia A.

Development of improved factor VIII molecules and new gene transfer approaches for hemophilia A.

Hemophilia A, the most common inherited bleeding disorder, is caused by deficiency or functional defects in coagulation factor VIII (fVIII). Conventional treatment for this disease involves intravenous infusions of plasma-derived or recombinant fVIII products. Although replacement therapy effectively stops the bleeding episodes, it has a risk of transmission of viral blood-borne diseases and development of neutralizing antibodies that inactivate the administered fVIII protein. Hemophilia A is an attractive candidate for application of gene therapy approaches because the therapeutic window is wide and even modest elevation of fVIII levels will correct the hemophilic phenotype. Ongoing preclinical investigations utilize animal models of hemophilia A, including genetically fVIII-deficient mice and naturally fVIII-deficient dogs, to optimize vectors, transgenes and target cell populations for Phase I clinical trials. In this review, we outline the progress in understanding the mechanisms of fVIII turnover, which provides a basis for development of improved fVIII molecules with prolonged half-life in the circulation. We discuss the possibility of incorporating these improved fVIII molecules as transgenes into self-inactivating lentiviral vectors carrying chromatin insulator sequences, representing a new generation of gene delivery vehicle, to target hematopoietic stem cells and endothelial cells. The use of hematopoietic stem cells as the target cell population may prevent inhibitor formation to transduced fVIII by induction of immune tolerance. Alternatively, endothelial cells may support optimal synthesis of fVIII and myeloablative conditioning of patients with radiation or chemotherapy may not be required for efficient engraftment of the engineered cells. Collectively, these proposed advances represent promising prophylactic strategies toward long-term correction of the coagulation defect in this progressively debilitating, life-threatening disease.