Kohlhagen, Mindy

ABSTRACT Background Monoclonal gammopathy of thrombotic significance (MGTS) is a recently described chronic prothrombotic condition characterized by monoclonal anti-PF4 antibodies that are detected above the polyclonal antibody background in patient sera (i.e. present as monoclonal gammopathy of undetermined significance, MGUS). Due to conflicting data in the published literature on antibody clonality in heparin-induced thrombocytopenia (HIT), we evaluated clonality and abundance of anti-PF4 antibodies in HIT, including investigating whether an MGUS, if present in HIT, represents the causative anti-PF4 antibody. Methods Blood samples from 15 patients with HIT were subject to Platelet Factor 4-dependent antigen-based and functional tests. The unmanipulated serum antibody repertoire and isolated anti-PF4 antibodies were subjected to mass spectrometric evaluation. Results Two of the 15 HIT patients had an IgG MGUS. Notably, anti-PF4 antibodies were not synonymous with the MGUS antibody in either of the two patients. Eight of the 15 patients demonstrated monoclonal anti-PF4 antibodies, however, none of the anti-PF4 antibodies were detectable as an MGUS upon evaluation of the entire serum antibody repertoire, reflecting their low abundance. In the seven patients with multiple anti-PF4 antibodies, non-monoclonality was confirmed by analysis of deglycosylated antibody heavy chains. Conclusions Anti-PF4 HIT antibodies are monoclonal in approximately 50% of HIT patients, however, antibody abundance is low such that they are not detectable over the polyclonal IgG background (i.e. are MGUS-negative), differentiating HIT from MGTS. This observation helps explain the transient nature of HIT relative to the persistent prothrombotic state seen in MGTS.

ABSTRACT Background Platelet factor 4‐polyanion enzyme‐linked immunosorbent assays (ELISAs) are considered highly sensitive for diagnosing heparin‐induced thrombocytopenia (HIT), such that current practice guidelines recommend use of ELISA‐negative results to exclude HIT. Once HIT is ruled out, alternative, non‐heparin‐based anticoagulant treatments are ceased, and heparin reintroduction frequently occurs. Methods Antigen‐based and PF4‐dependent functional testing were used to study PF4/polyvinylsulfonate ELISA‐negative platelet‐activating antibodies in HIT‐suspected patients. Results Three patients with clinical presentations consistent with HIT tested negative in an ELISA using PF4‐polyvinylsulfonate (PF4/PVS), an antigenic target very commonly used for HIT antibody screening. All three patients demonstrated PF4‐dependent platelet activation in functional testing that was sensitive to blockade of platelet FcγRIIa receptors and inhibited by high concentrations of heparin, consistent with pathogenic HIT antibodies. Functional testing‐based screening of 500 ELISA‐negative patients identified three additional patients whose sera activated platelets in a PF4‐ and FcγRIIa‐dependent manner and had clinical histories consistent with HIT. Five of the six ELISA‐negative HIT patients were re‐exposed to heparin, which precipitated a decrease in platelet counts in all re‐exposed patients, and one patient developed a new thrombus. Conclusions Recognition of ELISA‐negative HIT is critical to avoid harm due to the cessation of alternative anticoagulation therapy and re‐exposure of these patients to heparin.

Abstract Introduction In 2018, our institution implemented a MALDI-TOF mass spectrometry (MS) method (Mass-Fix) to detect myeloma associated M-proteins in blood. Currently, the lab has performed more than 500K tests by Mass-Fix. While Mass-Fix has been found to be more sensitive and specific than IFE, an ESI-Q-TOF (High Resolution Mass-Fix) method has demonstrated the potential to detect M-proteins at significantly lower levels. Blood minimal residual disease (MRD) detection in lieu of bone marrow (BM) is desirable since blood is easier and less expensive to obtain and analyze. This feature can allow for more frequent MRD monitoring for myeloma patients. Methods 66 residual blood samples from myeloma patients with varying M-protein varying isotypes were diluted into healthy blood to estimate the limit of detection of HR-Mass-Fix method. 179 Rochester Mayo Clinic patients who had a clinically ordered BM MRD assessment by next generation flow (NGF) cytometry (2 x 10-6), a blood sample available within 7 days of the BM biopsy, and a pre-treatment measured M-protein mass by Mass-Fix were utilized to assess the concordance of the BM and blood MRD status. Results The average limit of detection (LOD) of the HR-Mass-Fix was found to be significantly lower than Mass-Fix (0.0003 g/dL and 0.01 g/dL, respectively). The treated patient cohort utilized for comparison consisted of 145 myeloma, 21 AL amyloid, 4 LPL lymphoma and 9 other PCDs. Comparing the Mass-Fix and HR-Mass-Fix results, 100% of blood positive MRD results by Mass-Fix were positive by HR-Mass-Fix. HR-Mass-Fix detected residual M-proteins (mass within +/- 1 Da of pre-treatment) in 29 samples (40%) that were negative by Mass-Fix. Thus, HR-Mass-Fix increased the overall blood MRD positivity rate from 57% by Mass-Fix to 78%. The BM NGF MRD positivity was lower at 54%. The overall agreement of blood and BM by Mass-Fix was 71% (kappa 0.4) which decreased to 0.63 (kappa 0.3) with HR-Mass-Fix. Most of the disagreements were HR-Mass-Fix Blood (+)/BM NGF (-). Blood M-protein MRD appeared to be affected by M-protein half-lives. To determine this, the M-protein isotype distribution of Blood/BM MRD status was compared the M-protein isotype distribution of the overall cohort. The half-lives are as follows: IgG 23 days, IgA 6 days, IgM 5 days and FLC 0.25 days. When both blood and BM were positive, the isotype distribution was not significantly different than the overall cohort. For blood(+)/BM(-) patients, IgG (with the longest half-life) were significantly overrepresented compared to the overall cohort (74% vs 51%) . For blood(-)/BM(+) cases, there were no IgG M-proteins, and the majority were FLC patients. These results suggest that patients with shorter half-life M-proteins (non-IgG) are more likely to be Blood MRD(-) than patients with longer half-life M-proteins. M-protein rate changes over time will likely be needed to resolve the M-protein half- life affect. Conclusions HR-Mass-Fix can significantly increase the detection of M-proteins in comparison to our current Mass-Fix method. Ig recycling was found to be a likely reason for the disagreement between BM and blood. Therefore, a negative blood MRD status by HR-Mass-Fix is most likely to be the most significant in predicting patient outcomes.

ABSTRACT BACKGROUND Platelet factor 4-polyanion enzyme-linked immunosorbent assays (ELISAs) are considered highly sensitive for diagnosing heparin-induced thrombocytopenia (HIT), such that current practice guidelines recommend use of ELISA-negative results to exclude HIT. Once HIT is ruled out, alternative, non-heparin-based anticoagulant treatments are ceased, and heparin reintroduction frequently occurs. METHODS Antigen-based and PF4-dependent functional testing were used to study PF4/polyvinyl sulfonate ELISA-negative platelet-activating antibodies in HIT-suspected patients and mice immunized with PF4/heparin. RESULTS Three patients with clinical presentations consistent with HIT tested negative in an ELISA using PF4-polyvinylsulfonate (PF4/PVS), an antigenic target very commonly used for HIT antibody detection. All three patients demonstrated PF4-dependent platelet activation in functional testing that was sensitive to blockade of platelet FcγRIIa receptors and inhibited by high concentrations of heparin, consistent with pathogenic HIT antibodies. Functional testing-based screening of 500 ELISA-negative patients identified three patients whose sera activated platelets in a PF4- and FcγRIIa-dependent manner, and had clinical histories consistent with HIT. Five of the six ELISA-negative HIT patients were re-exposed to heparin, which precipitated a decrease in platelet counts in all re-exposed patients, and one patient developed a new thrombus. To advance the study of ELISA-negative HIT antibodies, mice were immunized with PF4/heparin, and functional and antigenic assays were simultaneously used to successfully identify an ELISA-negative, PF4-dependent platelet-activating murine monoclonal antibody that recapitulated the serological characteristics of ELISA-negative HIT patients. CONCLUSIONS Recognition of ELISA-negative HIT is critical to avoid harm due to the cessation of alternative anticoagulation therapy and re-exposure of these patients to heparin.

Abstract Background Monoclonal antibody therapies (t-mabs) are known to interfere in serum immunofixation, as they show up as a discrete band. Until recently, most of the t-mabs were IgG kappa monoclonal immunoglobulins, and laboratories were familiar with the clue of a cathodal migrating IgG kappa in a patient sample, suggesting this could be daratumumab or another t-mab in the report. However, with the advent of novel antibody-based medications, identification via traditional laboratory methods has grown more complex. Teclistamab-cqyv is an IgG4 lambda T-cell engager bispecific antibody targeting B-cell maturation antigen (BCMA) and CD3 on T cells, used in relapsed and refractory multiple myeloma. The goal of this study was to determine if teclistamab could be detected on serum immunofixation and mass-fix. Methods Residual waste serum samples with no monoclonal protein via mass-fix (a MALDI-TOF based assay used in place of serum immunofixation) and variable polyclonal background were used to spike teclistamab (n=3). Briefly, one sample had total IgG < 500 mg/dL measured by nephelometry on a Siemens BNII (431 mg/dL, hypo gamma), a second sample had IgG within reference intervals (779 mg/dL, normal gamma) and the third sample had IgG > 1600 mg/dL (1950 mg/dL, polyclonal hypergamma). Each sample was divided into 3 aliquots and spiked with teclistamab (Tecvayli, Johnson &Johnson) obtained from the pharmacy, at final concentrations of 10, 20, and 40 mg/dL in the serum samples. The nine artificial samples were subjected to immunofixation electrophoresis (Sebia) and a new monoclonal protein study using mass-fix, a laboratory developed test. Results In the serum immunofixation experiments, a single discrete IgG lambda band was visible in the hypogamma and normal gamma samples (Table 1) when teclistamab was present at 20 mg/dL or 40 mg/dL. No bands were observed for the 10 mg/dL spike in hypos or normals. No teclistamab was detected at any concentration in the hypergamma sample. When mass-fix was performed, monoclonal protein spikes consistent with the expected mass of the two unique lambda light chains associated with teclistamab (22624Da and 22636Da) were identified in the hypo and normal gamma samples at the 3 concentrations tested: 10, 20, and 40 mg/dL. No mass-fix spikes were seen above the polyclonal spectra in the hypergamma samples. Conclusion The polyclonal background of immunoglobulins in a serum specimen plays a significant role in the identification of any monoclonal protein. Teclistamab is a new IgG lambda t-mab with potential to show up as an interference in both immunofixation and mass-fix, depending on the amount of polyclonal IgG present. Without positive identification, teclistamab presence could complicate interpretation of a patient’s response to treatment. This proof-of-concept study shows that mass-fix can detect smaller concentrations of teclistamab than immunofixation, therefore would be more prone to identifying this t-mab than labs employing the gel-based immunofixation method.

Abstract Background The diagnosis of monoclonal gammopathies relies on the detection of monoclonal immunoglobulins (M-proteins) in serum or urine. Mass-Fix utilizes matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to detect and quantify M-proteins. This method employs immunoaffinity enrichment using antibodies against heavy chains (HC) and light chains (LC), followed by MS analysis to determine M-protein isotypes. Mass-Fix has also revealed novel M-protein characteristics, including LC glycosylation and fragmentation, facilitated by measuring the mass of LC. LC glycosylation is as an independent risk factor for disease progression of monoclonal gammopathy of undetermined significance (MGUS), while light chain fragments have been postulated as the origin of amyloid light chain (AL) plaques. Since Mass-Fix enriches intact immunoglobulins before dissociating LCs, it potentially enhances fragment detection. However, the precise nature and clinical significance of these fragments remain to be fully elucidated. Methods Mass-Fix serum results from Mayo Clinic patients tested from September 10, 2018, to October 1, 2024, were reviewed for the presence of M-protein and LC fragments. The frequency and the isotype of fragment LC fragments was determined in the M-protein positive cohort (n=15410). Patients diagnosed with either MGUS (n=6996), AL amyloidosis (n=315), or both (n=32) who had positive M-protein results on Mass-Fix, either prior to or within one month after the diagnosis, were included for a retrospective clinical association study of LC fragments and disease. To validate that these fragments were not artifacts of MS analysis, PAGE gel analysis and Western blots were performed using clinical residual serum from 9 patients: 2 IgG/kappa and 3 IgG/lambda patients with detectable LC fragments and 2 IgG/kappa and 2 IgG/lambda patients without fragments. Samples were purified using antibodies against gamma heavy chains and reduced with TCEP prior to gel electrophoresis and Western blot. Results Of the 15410 patients with monoclonal proteins detected by Mass-Fix, 2896 (18.7%) had detectable LC fragments; kappa fragments in 1533 and lambda fragments in 1363. Most often, the fragments were observed in pairs, with combined molecular weight typically equaled that of the full-length monoclonal LC present in the same sample. LC fragments were observed more frequently among patients diagnosed with AL amyloidosis (118/347=34%) compared to those without the diagnosis (685/6996=10%), with an odds ratio of 4.75 (95% CI, 3.75-6.01). PAGE with Coomassie blue staining revealed bands with sizes representing LC fragments in the five M-protein samples with detectable LC fragments on Mass-Fix, while no such bands observed in those without LC fragments. Western blot using anti-kappa or anti-lambda antibodies confirmed the identity of the bands with higher molecular weight as a fragment of LC in each of the IgG/kappa and IgG/lambda samples. Conclusion Mass-Fix is able to discriminate LC fragments from intact LC based on their sizes. The detection of LC fragments with Western blot without ionization suggests a biological origin rather than analytical artifacts from MS. The clinical association study suggests that LC fragments may have utility in increasing the suspicion of AL amyloidosis in M-protein positive patients with LC fragments, potentially facilitating earlier intervention and improving patient outcomes.

Background The detection, isotyping and quantitation of over-expressed immunoglobulin(s) (Igs) (M-protein) is key for diagnosing and monitoring patients with monoclonal gammopathies. Traditionally, our lab has used a combination of agarose gel protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE) for these purposes. In 2018, a mass spectrometric method (Mass-Fix) was implemented to replace IFE for M-protein isotyping. This work documents the validation of a quantitative version (Q-Mass-Fix) to replace both SPEP and IFE. Methods 24,645 samples submitted for M-protein testing between 7/2018 and 9/2023 with quantitative IgG, IgA and IgM measurements, Mass-Fix analysis and SPEP results were utilized for this study. A separate cohort of 100,000 M-protein negative samples were utilized to mathematically model the spectral shapes IgG, IgA and IgM polyclonal background. To detect M-proteins, software was developed that fits the polyclonal Ig portion of the spectra and M-proteins are detected as peaks above the polyclonal background. M-proteins isotypes are determined by matching similar peaks in spectra in 5 sperate immuno enrichments (IgG, IgA, IgM, kappa and lambda). M-protein quantitation was obtained by multiplying total M-protein isotype quantity by the fraction area of the M-protein in the spectra. M-protein dilutional studies were used to examine the linearity and level of detection of Q-Mass-Fix. Results SPEP and Q-Mass-Fix M-protein quantitation had the best agreement between the range of 10 to 40 g/L. Below 10 g/L, Q-Mass Fix had on average, lower M-protein values. Above 40 g/dL, Q-Mass-Fix values were on average, higher than SPEP. The automated isotyping assignment was 98.8 percent consistent with the isotype assigned by visual inspection. Dilutional studies revealed that Q-Mass-Fix quantitation was linear over a range of 70 to 0.04 g/L (slope 1.0, r2 0.99) with an analytical limit of quantitation of 0.1 g/L and a limit of detection of 0.04 g/L. Conclusion Q-Mass-Fix represents a significant improvement in comparison to our current SPEP method. Q-Mass-Fix is more capable of removing the interference of polyclonal Igs below 10 g/L and is not subjective to the dye saturation effect at high M-protein levels. The increased analytical measuring range and improved limit of detection of detection could be beneficial in tracking patient's response. Q-Mass Fix is more automated than SPEP and the workflow in our laboratory.