Immunoglobulin Interference Research Articles

Abstract 1497: A unique strategy to arm iPSC-derived CAR-T cells to perform antibody-directed cellular cytotoxicity to facilitate multi-antigen targeting

Abstract Although chimeric antigen receptor (CAR)-T cells have been transformational for several liquid tumors, their effectiveness in treating a variety of solid tumors has been limited in part because the heterogeneity of tumor-associated antigens found in many bulky tumors represents a major obstacle to the development and the delivery of effective CAR-T cell therapies in cancer. Here we demonstrate that iPSC-derived CAR-T (CAR-iT) cells are an ideal off-the-shelf approach that can be engineered to perform antibody-dependent cell-mediated cytotoxicity (ADCC) to facilitate multi-antigen targeting with the introduction of various monoclonal antibodies (mAbs). Under this strategy, while the CAR is equipped to predominantly target antigen 1, targeting additional tumor associated antigens through the use of various mAbs can be leveraged through ADCC. To this end, CAR-iT cells were genetically edited at the iPSC stage to uniformly express a novel high-affinity variant (158V), non-cleavable CD16A (hnCD16) Fc receptor to recognize and bind to the Fc fragment of mAbs and facilitate ADCC. When combined with therapeutic mAbs targeting HER2, EGFR, or PDL1, the cytolytic efficacy of hnCD16+ CAR-iT cells was significantly enhanced compared to parental CAR-iT cells in several tumor models, including ovarian, HER2+ breast, and triple-negative breast cancers, underscoring the flexibility of a combined CAR and ADCC – mediated targeting approach. Importantly, although both CD16A and CAR signaling share common CD3ζ pathways, simultaneous activation did not appreciably attenuate the activation and effector function of CAR-iT cells. Moreover, hnCD16+ CAR-iT cells demonstrated enhanced control in an established triple negative breast cancer solid tumor xenograft model. The control of antigenically heterogenous tumors by hnCD16+ CAR-iT cells was also significantly improved when compared to parental CAR-iT cells. To confirm selectivity and avoidance of immunoglobulin interference with functional output, various in vitro cellular cytotoxicity assays were conducted with IgG1 to confirm that hnCD16 requires crosslinking to elicit ADCC while maintaining full potentiation of CAR function. Additional in vitro and in vivo studies are ongoing and will be presented. Collectively, the data demonstrates that CAR-iT cells can be further modified with hnCD16 Fc receptor to elicit ADCC and the combination of CAR and hnCD16 is a unique approach to tackling tumor heterogeneity often found in bulky tumors. Citation Format: Martin Hosking, Soheila Shirinbak, Joy Grant, Wen-I Yeh, Yijia Pan, Mochtar Pribadi, Amit Mehta, Amanda Yzaguirre, Philip Chu, Shohreh Sikaroodi, Angela Gentile, Bruce Walcheck, Jianming Wu, Anhco Nguyen, Bahram Valamehr. A unique strategy to arm iPSC-derived CAR-T cells to perform antibody-directed cellular cytotoxicity to facilitate multi-antigen targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1497.

Pseudohypercreatininemia as an Initial Presentation of Waldenstrom’s Macroglobulinemia

Chronic kidney disease (CKD) is a major public health problem. Estimated glomerular filtration rates based on serum creatinine values are used to diagnose this condition, and accuracy of laboratory testing is a crucial step in diagnosing and managing these patients efficiently in a timely manner. Spuriously elevated creatinine values have been reported due to the interference of other substances, including paraproteins. Paraproteins can interfere with the determination of various clinically important analytes in an unpredictable manner. While mild to moderate degrees of interference affecting the measurement of serum creatinine values have been previously reported, we present a case of a patient with markedly elevated serum creatinine who was referred for emergent assessment for possible consideration of renal replacement therapy. A thorough history and repeat laboratory investigations at several laboratories ultimately revealed the initial serum creatinine level to be spuriously high, i.e. “pseudohypercreatinimea” due to immunoglobulin interference. Further investigations ultimately led to the diagnosis of Waldenstrom’s macroglobulinemia and the patient was appropriately treated. However, this case sheds light on the importance of being aware of a spuriously high creatinine value and its possible causes, as it can have significant diagnostic implications. World J Nephrol Urol. 2016;5(4):83-84 doi: https://doi.org/10.14740/wjnu282w

The pH of chemistry assays plays an important role in monoclonal immunoglobulin interferences

ObjectivesImmunoglobulin paraproteins can interfere with multiple chemistry assays. We want to investigate the mechanisms of immunoglobulin interference. Design and methodsSerum samples containing paraproteins from the index patient and eight additional patients were used to investigate the interference with the creatinine and total protein assays on the Beckman Coulter AU5400/2700 analyzer, and to determine the effects of pH and ionic strength on the precipitation of different immunoglobulins in these patient samples. ResultsThe paraprotein interference with the creatinine and total protein assays was caused by the precipitation of IgM paraprotein in the index patient's samples under alkaline assay conditions. At extremely high pH (12–13) and extremely low pH (1–2) and low ionic strength, paraprotein formed large aggregates in samples from the index patient but not from other patients. ConclusionsThe pH and ionic strength are the key factors that contribute to protein aggregation and precipitation which interfere with the creatinine and total protein measurements on AU5400/2700. The different amino acid sequence of each monoclonal paraprotein will determine the pH and ionic strength at which the paraprotein will precipitate.

Monoclonal and polyclonal immunoglobulin interference in a conjugated bilirubin assay.

Monoclonal proteins can interfere with the conjugated bilirubin assay on the Beckman Coulter AU5400 and AU2700 instruments and produce spurious results. Protein depletion eliminates the interference, suggesting that monoclonal proteins cause the problem. To determine the interference rate with the Beckman Coulter AU5400 and AU2700 conjugated bilirubin assay and to identify the interfering substance. Beckman Coulter bilirubin results from 33,720 samples analyzed during 6 months were evaluated for interference. On a subset of samples, protein G columns were used to specifically remove immunoglobulin G (IgG) to determine the cause of the interference. Another 117 samples containing a (known) monoclonal protein were selected to determine the interference rate. From 26 different patients, 103 samples had conjugated bilirubin results greater than the total bilirubin for a false-positive rate of 0.3%. Removal of IgG from a subset of those samples with IgG monoclonal protein and increased polyclonal IgG eliminated the conjugated bilirubin interference. In separate, selected samples containing monoclonal proteins, the false-positive interference rate was 7.7% (9 of 117) and was greatest when the monoclonal protein concentration was greater than 4 g/dL. Another 11 of the 117 samples (9.4%) with monoclonal proteins exhibited assay interference by producing false-negative results. The overall interference rate, therefore, was 17.1%. The false-positive interference rate for the Beckman Coulter conjugated bilirubin assay was 0.3% for routinely analyzed serum/plasma samples. In addition to monoclonal proteins, we found that polyclonal immunoglobulins can also interfere with the conjugated bilirubin assay. The overall interference rate was 17.1% for samples with a monoclonal protein.

Two Suspected Cases of Immunoglobulin-Mediated Interference Causing Falsely Low Vancomycin Concentrations with the Beckman PETINIA Method

To describe 2 recent cases of suspected immunoglobulin-mediated interference with the Beckman Coulter particle-enhanced turbidimetric inhibition immunoassay (PETINIA) used to measure vancomycin serum or plasma concentrations and to review the existing literature. A 64-year-old woman with a history of multiple immune-related comorbidities received vancomycin for treatment of a prosthetic joint infection growing coagulase-negative Staphylococcus spp. A 33-year-old man with a history of Felty syndrome received vancomycin for the treatment of methicillin-resistant Staphylococcus aureus pneumonia. Both patients had multiple vancomycin trough concentrations determined using the Beckman Coulter PETINIA method and had measured concentrations reported as less than 4 mg/L despite appropriate vancomycin dosing for their size, age, and organ function. The patients' serum was then tested by alternative methods, which reported vancomycin concentrations consistent with those expected with the patients' dosing regimens. Immunoglobulins are well known for interfering with chemistry assays. It is suspected that high levels of immunoglobulins in these 2 patients interfered with the accurate measurement of serum vancomycin concentrations. An objective causality assessment supported the interactions as being definite and probable, respectively. Two other cases of suspected immunoglobulin interference with vancomycin measurement have been reported in the literature when vancomycin concentrations were initially measured using the Beckman Coulter PETINIA method. In vitro studies support the hypothesis that immunoglobulin interference with vancomycin measurement by this method occurs and is a graded phenomenon. These cases suggest a high likelihood of immunoglobulin-mediated interference with the Beckman Coulter PETINIA method, which results in the reporting of falsely low vancomycin serum concentrations. When vancomycin concentrations do not correlate with what would be expected clinically, clinicians should have a high index of suspicion for potential assay interference.

Immunoglobulin interference in serum follicle-stimulating hormone assays: autoimmune and heterophilic antibody interference

Interference in immunoassay caused by endogenous immunoglobulin is a cause of incorrect laboratory results that can drastically affect patient management. Two cases of immunoglobulin interference in serum follicle-stimulating hormone (FSH) assays are presented. These cases illustrate two common mechanisms for false-positive interference in two-site (sandwich) immunoassays. The first case describes a circulating autoimmune FSH immunoglobulin complex ('macro'-FSH), which has not been previously described for FSH, and the second a cross-linking antibody directed against the assay reagents. Immunoglobulin interference was detected and characterized using a combination of method comparison, immunosubtraction and size exclusion chromatography.

The blood coagulation mechanism in multiple myeloma.

Many cancers are associated with hypercoagulability, including multiple myeloma. At least four possible reasons for hypercoagulability have been described in myeloma patients: interference of immunoglobulins on fibrin structure, procoagulant autoantibody production, effects of inflammatory cytokines on endothelium, and acquired activated protein C (APC) resistance. Moreover, injury to endothelium, either by tumor cells or by chemotherapy, may predispose to thrombosis by causing upregulation of adhesion molecules, allowing adhesion of blood cellular elements (platelets, lymphocyte, neutrophils, and tumor cells, which secrete thrombogenic as well as angiogenic substances). In most cases, the pathogenesis of a thrombotic complication in myeloma patients remains unexplained. Administration of chemotherapy may play a larger role in the thrombotic process than a specific abnormality does because thrombotic complications become more prominent after the start of treatment. The recently reported evidence of a non-factor V Leiden APC resistance has increased our understanding of the pathophysiology of this hypercoagulable state.

Interference of immunoglobulin G (IgG) antibodies in IgA antibody determinations of Chlamydia pneumoniae by microimmunofluorescence test

In the microimmunofluorescence test for measuring immunoglobulin A (IgA) antibodies against Chlamydia pneumoniae, removal of interfering IgG antibodies made IgA antibody reactivity patterns in 952 serum samples easier to interpret, prozone effects disappeared, and titers increased, especially in the sera with high IgG titers. IgA rheumatoid factors did not interfere in the assay.

Interference of Immunoglobulins in the Radioimmunoassay of Human β-Endorphin

The interference of immunoglobulins in the radioimmunoassay (RIA) of human beta-endorphin was investigated. Human IgM showed no cross-reactivity. Human IgA showed a weak cross-reaction, but the dilution curve of IgA did not show parallelism with the standard curve of beta-endorphin, thus indicating its antigenic difference. The dilution curves of human IgG showed 0.18% displacement with respect to the human beta-endorphin standard curve, with good parallelism. Moreover, five patients with multiple myeloma of the IgG type showed falsely elevated beta-endorphin levels. We investigated the possibility that certain IgGs may be responsible for the displacement of [125I]beta-endorphin in the beta-endorphin kit. The apparent beta-endorphin level of plasma from multiple myeloma patients was markedly decreased after affinity chromatography of the serum on protein A-Sepharose. In another 3 patients with multiple myeloma, we examined IgG interference by measuring the beta-endorphin levels in their lyophilized IgG diluted with saline. The results demonstrated high values of 20.2, 25.5 and 21.2 pmol/l respectively, also showing good parallelism. These immunological parallels to human beta-endorphin verify that a part of the amino acid sequence of human IgG is similar to that of human beta-endorphin. Consequently, in the measurement of beta-endorphin with polyclonal antibody, the results may sometimes be spuriously high due to cross-reaction with IgG, e.g., in patients with IgG myeloma. To avoid IgG interference, a specific monoclonal antibody to synthetic beta-endorphin should be used rather than polyclonal antibodies.

Interference of immunoglobulins in two glucagon radioimmunoassays.

Radioimmunoassays of glucagon in plasma may be complicated by interaction with other substances of high molecular mass. Precipitates of such substances with ammonium sulfate showed, after isoelectric focusing, two fractions having glucagon immunoreactivity. One fraction (pl approximately 10) evidently is associated with the Fc portion (but not the Fab portion) of purified polyclonal immunoglobulin G (IgG). Equal amounts of purified monoclonal IgG of various subclasses, especially IgG 1, gave different "glucagon" readings, suggesting that some IgG may interfere more strongly than others. The other fraction (pI 5-6) appeared less consistently, and on gel chromatography appeared to be slightly larger than IgG. Together these fractions add about 50-100 ng/L to the immunoreactive glucagon values in plasma. Therefore methods in which glucagon is extracted before assay should be used for determining the concentration of glucagon present physiologically.

Immunoglobulin interference with phosphorus and chloride determinations with the Coulter Chemistry.

Journal Article Immunoglobulin interference with phosphorus and chloride determinations with the Coulter Chemistry. Get access R A Landowne R A Landowne Search for other works by this author on: Oxford Academic Google Scholar Clinical Chemistry, Volume 25, Issue 6, 1 June 1979, Pages 1189–1190, https://doi.org/10.1093/clinchem/25.6.1189a Published: 01 June 1979