Serum Protein Research Articles

B-307 Comparative studies on serum quantitative immunoglobulin assay and serum protein electrophoresis for the diagnosis of multiple myeloma

Abstract Background Various clinical laboratory tests are valuable in enhancing the diagnosis of monoclonal gammopathy. The nephelometric-based serum quantitative immunoglobulin test provides great utility to understand the magnitude of antibody-mediated responses and is useful in interpreting Serum protein electrophoresis (SPEs). In clinical practice, several (SPEs) are ordered without a preliminary order of serum quantitative immunoglobulins but pathologists can and do order the test for comparison. However, there is seldom any data in the literature describing the correlative relationship between the two tests. It is therefore imperative to create an evidenced-based system of test-ordering for the laboratory investigation of the presence of monoclonal protein. The goal of the project is to compare the results of quantitative serum immunoglobulin assay and SPE in order to determine their concordance. Methods In this preliminary phase of the project, retrospective results of quantitative serum immunoglobulin assay and SPEs that were performed simultaneously were retrieved from the electronic medical records. The span of data extended to the past 1 year. If there is an additional immunofixation (IFE) order associated with the SPE, the IFE report was also retrieved for further comparison. Based on the etiology of multiple myeloma, results of calcium, red blood cell count and creatinine were also pooled and analyzed. Results of free light chain assay and associated kappa-lambda ratios were also obtained. All the data obtained were entered into and filtered in excel spreadsheet program and subsequently analyzed with GraphPad prism. Results 1000 patient results were analyzed, of which 404 were males and 596 were females. 200 of the patients had either SPE or quantitative immunoglobulin performed which had the following age demographic; 23 were between 18-39 years, 52 were 40-59 years and 125 were 60-89 years. This data suggests geriatric population are more likely to be seen for multiple myeloma suspicion. There were 60 concurrent orders. Of this, there was 65.5% agreement between the serum quantitative immunoglobulin assay and SPE or IFE while 34.5% showed a mismatch. 26% of the orders for quantitative immunoglobulins assay had no follow-up SPE. A high Kappa/lambda ratio was concordant with a positive SPE 83% of the time. But only 66% of normal kappa/lambda ratio matched a normal SPE. Conclusions Here, we have shown that in addition to the kappa/lambda ratio, the quantitative immunoglobulins test has a considerable agreement with SPE and may be an important preliminary test. The test could be useful to rule out monoclonal gammopathy, confirm polyclonal pattern or embolden a normal SPE interpretation. The present research will have a direct impact on the diagnosis and monitoring of monoclonal gammopathy and enhance appropriate test utilization. The research will improve the development of a better ordering policy for laboratory operations with respect to the work up of multiple myeloma. As more data is unraveled, better understanding will be gained into the relationship between quantitative immunoglobulin and SPE for better patient care.

B-241 Next-generation monitoring: M-inSightclinical application in multiple myeloma

Abstract Background The improvement of multiple myeloma (MM) therapy effectiveness has increased the need for precise, sensitive, and dynamic monitoring of minimal residual disease (MRD). Current MRD assessment methods that are based on bone marrow aspirates, pose limitations due to the invasiveness of the procedure and potential heterogeneous sampling. Recognizing the need for non-invasive, frequent testing, a targeted mass spectrometry, M-InSight®, (MS)-based MRD blood-test was developed. This innovative assay identifies and tracks clonotypic peptides from the patient's monoclonal immunoglobulin, offering a blood-based alternative with equivalent sensitivity to existing bone marrow reliant techniques.Here we employed M-InSight® to sequence and select clonotypic peptides, enabling highly specific, ultra-sensitive quantification of the M-protein. This study examines the correlation between M-protein quantification using M-InSight® and conventional serum protein electrophoresis (SPEP) values routinely obtained in clinical settings. Given the established role of M-protein as a biomarker for MM diagnosis and longitudinal monitoring, ensuring concordance between emerging methodologies and existing standards is vital. Methods Serum samples were obtained from the clinical laboratory at the University of Miami and characterized using SPEP (capillary electrophoresis, Sebia), immunotyping (Sebia), and free light chain quantitation (The binding site). Patients with M-protein concentrations ≥0.2 g/dL by SPEP were included. Longitudinal samples were collected. Subsequently, all samples were analyzed using M-InSight®. The initial sample for each patient underwent de novo sequencing of the M-protein by mass spectrometry and was also used to calibrate the concentration with the known value from SPEP. An in-house bioinformatics algorithm aligned, sequenced, and selected patient-specific clonotypic peptides. These peptides were targeted for the M-inSight® follow up assay allowing the quantification of M-protein. Results A variety of M-protein isotypes were successfully sequenced (IgGK, IgGL, IgAK, IgAL, IgMK, IgML, free K, free L, IgDL, biclonal) at concentrations ranging from 0.2 to 2.9 g/dL. M-protein was successfully sequenced and clonotypic peptides were identified in 57 out of 59 patients using the initial sample. A total of 109 serum samples collected during maintenance or post treatment were analyzed, 50 of them were follow-up samples and were used to correlate M-protein values from both techniques. M-protein quantification by M-Insight showed high concordance with the one obtained by SPEP, with a correlation value of 0.88 (intercept set to 0, slope 0.91. Pearson = 0.886, p-value <0.001). Out of 57 patients whose M-protein was sequenced, 55 were positive by SPEP and were successfully quantified by M-InSight®. Furthermore, M-InSight® detected and quantified M-protein on samples of the 2 remaining patients that showed M-protein values below SPEP detection limits, highlighting the higher sensitivity of M-InSight®. Conclusions M-inSight® assay successfully identified clonotypic peptides in 57 out of 59 patients which were used to quantify M-protein concentration and showed a strong concordance with SPEP and affirming the utility of M-InSight® as a reliable method for routine monitoring of MM. The ability to seamlessly integrate M-InSight® into routine monitoring protocols offers clinicians a valuable tool for MRD monitoring, tracking disease progression and response to therapy with enhanced sensitivity and specificity.

B-342 Teclistamab interference in serum protein electrophoresis and serum immunofixation electrophoresis in patients with recurrent or relapsed multiple myeloma

Abstract Background Serum protein electrophoresis (SPEP) and serum immunofixation electrophoresis (IFE) analyses are mainstays in the initial diagnosis, risk stratification, and evaluating treatment response in patients with multiple myeloma (MM) in accordance with the International Myeloma Working Group criteria. Published reports have demonstrated that therapeutic monoclonal antibodies (mAbs) used to treat MM (e.g. anti-CD38 IgG-kappa) are detectable by SPEP and IFE, which may interfere with accurate evaluation of complete response. Recently, Teclistamab (Tecvayli®; Janssen Biotech, Horsham Township, PA), a humanized bispecific IgG-lambda mAb targeting B-cell maturation antigen (BCMA) and CD3 on T cells was approved for treating patients with penta-refractory MM. In contrast with other IgG-kappa therapeutic mAbs, the degree to which Teclistamab may interfere with detection and quantification of monoclonal proteins is unknown. Therefore, we sought to assess the interference of Teclistamab therapy on routine SPEP and IFE analysis. Methods Patients treated with Teclistamab were identified by retrospective review of electronic medical records. Teclistamab (90 mg/mL) was spiked into a remnant patient sample with IgG lambda MM at a final concentration of 10 μg/mL, which corresponds to ½Cmax values in patients infused with Teclistamab. SPEP and IFE were performed using the Helena SPIFE Touch instrument. Computer-assisted densitometry was used to quantify monoclonal proteins in the gamma region of SPEP gels. Results In several patients with refractory MM treated with Teclistamab, IFE analysis revealed IgG lambda or lambda light chain monoclonal proteins, which were inconsistent with previously-identified monoclonal proteins (e.g. IgG kappa). In one patient diagnosed with IgG lambda MM, spiking 10 μg/mL Teclistamab increased M-spike abundance by approximately 0.15 g/dL when quantified by SPEP. Conclusions In patients with relapsed or refractory MM, treatment with Teclistamab may confound accurate clinical assessment of complete response by SPEP and IFE methods. Increased awareness of this preanalytical interference is necessary to guide clinical decision making.

B-102 The Impact of Protein / Protein Interactions on Serum Free Light Chain Assay Calibration

Abstract Background Since 2001, Serum Free Light Chain measurements have become established as routine clinical laboratory tests. Katzmann (2002) published a reference interval based on 282 normal samples establishing a ratio reference range 0.26-1.65. Whilst kappa and lambda measurements are of little diagnostic value, kappa/lambda ratio gives a strong indication of monoclonal gammopathy. In recent years, it has been suggested that the ratio may have changed. Here we present investigations identifying protein/protein interactions that may account for the discrepancies between QC release and real-world data; identifying a remediation step which will allow verification of the published ratio. Methods The manufacturers QC release data was reviewed for kappa, lambda and kappa/lambda ratio for all lots from 2018-2023, and compared to published data. To mimic field observation, an accelerated stability study was performed on standard calibrators and protein/protein interactions identified using western blot. Subsequently, calibrators were depleted of alpha-1 antitrypsin (A1AT) and other serum proteins using specific affinity chromatography; accelerated stability was repeated. A calibrator of pure polyclonal free light chains (FLC) in human serum albumin was produced in the presence/absence of A1AT and additional interfering serum proteins. Results At release, the average kappa reference range was 7.2-29.5 mg/L (62 kits, 9 different US blood donor sera collections and 3852 data points) and 7.7-24 mg/L for lambda (78 kits, 9 different US blood donor sera and 4664 data points), with a kappa/lambda ratio of 0.45-1.69. There was minimal change over 5yrs to these measurements. Analysis of FLC calibrators during an accelerated stability identified interactions between kappa FLC and ubiquitous serum proteins, including A1AT. The standard calibrator activity declined by 14.9±0.1% (mean±SEM) over a 4-month accelerated period @22oC, equivalent to 16 months real time. Calibrators depleted of A1AT and specific additional serum proteins showed significantly less decline in activity 1.4±1.4%, p<0.001. When blood donor sera were analysed using the standard calibrators following 4 months at 22oC, the results showed an increase in the reported kappa concentrations compared to those generated from frozen calibrators (19.0[15.4-22.5] to 21.3[17.0-26.0] mg/L, 12.5±3.8%, p<0.001). This observation was mirrored in kappa/lambda ratio (1.3[1.1-1.5] to 1.4[1.2-1.7] mg/L, 12.5±3.8%, p<0.001) and when clinical patient samples were assessed (30.1[14.0-47.1] to 34.3[15.5-56.9] mg/L, 13.8±1.4%, p<0.001). The depleted calibrator did not report an increase in sample results. FLC+HSA calibrators, when spiked with either physiological concentrations of A1AT or other serum proteins, showed a 16.7% and 15.3% decrease in activity respectively, compared to FLC+HSA calibrators without additional proteins. Conclusions At release, US blood donor sera have different absolute kappa mg/L compared to the published reference interval, but these changes do not invalidate the published kappa/lambda ratio, in contrast to recent reports and have not changed since 2018. An unpredictable kappa/protein interaction was observed, particularly with A1AT in a time dependent fashion, which correlated to a reduction in calibrator activity. Removal of A1AT and other specific serum proteins remediated the issue, and the kappa/lambda ratio was verified irrespective of the age of the calibrator.

B-337 Mass Spectrometry to evaluate treatment response in patients with Multiple Myeloma. A comparative study versus Immunofixation

Abstract Background The International Myeloma Working Group (IMWG) defines treatment response categories based on monoclonal protein (MP) changes by serum protein electrophoresis (SPE) and immunofixation (sIFE). Patients that after treatment remain positive by sIFE while SPE is negative, are categorized as very good partial response (VGPR) and, if sIFE becomes negative, patients would be considered in complete remission (CR). However, the interpretation of sIFE is qualitative and subjective. In this study we evaluate the discrepancies between sIFE and Mass Spectrometry (MS) to evaluate tumor burden and the accuracy of treatment response and it´s clinical impact in terms of progression free survival (PFS). Methods Patient samples were automatically prepared on the EXENT-iP®500 liquid handler, and sample spots were analysed using the EXENT-iX®500 MALDI-TOF mass spectrometer. Light chain spectral peaks were identified by EXENT-iQ® software and then quantified indirectly in combination with total immunoglobulins concentrations by immunoturbidimetry in the Optilite® platform (The Binding Site, part of Thermo Fisher Scientific). MPs were also identified and quantified by SPE and sIFE. Results From the 430 samples from the GEM12, GEM14 and GEM-CESAR clinical trials analyzed at pre-determined time points to evaluate treatment response (post-Induction, post-ASCT, post-cons, 1st and 2nd year after maintenance), MS identified the M protein in 45% of cases (193/430) and sIFE was positive in 32% (137/430). Combining the results of both methods, we found that MS and sIFE were concordant in 76% of cases (326/430), 26.28% IFE+/ MS+ and 49.53% IFE-/ MS-. Considering sIFE as a reference, the negative predictive value (NPV) of MS was 0.8987, with a sensitivity and a specificity of 0.8248 and 0.7270, respectively (p<0.0001). Nevertheless, the positive predictive value (PPV) was 0.5855 which could be explained by the discordances observed between IFE and MS techniques (5.58% of patients IFE+/MS- and 18.6% of patients IFE-/MS+). Indeed, when the 137 patients in VGPR (IFE+ patients) were analyzed according to MS status we found that patients IFE+/ MS+ had a trend to an inferior mPFS in comparison to IFE+/MS- (5.96 years vs not reached). These discordances were even more noticeable when comparing the 293 patients in CR (IFE- patients) with MS status, since IFE-/ MS+ patients had a mPFS of 4.65 years vs mPFS not reached for IFE-/ MS- patients (p=0.0001). Conclusions Our data confirms that IFE sensitivity limitations can affect the evaluation of treatment response in patients with MM, being more noticeable when the presence of MP band in sIFE is not clear because is very fuzzy or oligoclonal bands appear. Consequently, heterogeneity in interpretation practices across labs is a major cause of misattributed response categories, especially on those patients achieving treatment responses >VGPR. In an era of highly effective MM treatments that induce quick and deep responses, we believe IMWG response criteria should be updated with an MS response category.

B-089 Pseudo-clonal Increased IgG4 with Polyclonal Distribution Demonstrated by MALDI-TOF-MS in a Pediatric Patient with Tricho-Hepato-Enteric Syndrome (THES)

Abstract Background THES is a rare autosomal recessive syndromic enteropathy caused by pathogenic variants in two genes, the Tetratricopeptide Repeat Domain 37 (TTC37) and Ski2 Like RNA Helicase (SKIV2L), that encode components of the human SKI complex involved in RNA degradation. This disease is characterized by several clinical features with variable penetrance including intractable congenital diarrhea, wooly hair abnormalities, intrauterine growth restriction, facial dysmorphism, immune dysfunction, liver abnormalities, and skin hyperpigmentation. Hypogammaglobulinemia with low IgG and normal to low IgM levels have been described in these cases which may require intravenous immunoglobulin (IVIG) replacement therapy. Some cases have a reduced population of switched memory B cells, T cells with reduced IFN-γ secretion, and hyporesponsive NK cells. Our patient is a 4-year-old female reported to have a homozygous TTC37 mutation with clinical manifestations of TPN dependence, liver fibrosis with portal hypertension, splenomegaly, developmental delay, thrombocytopenia and anemia requiring transfusion, and a history of hypogammaglobinemia requiring IVIG replacement. Patient would go on to develop elevated immunoglobin levels leading to additional testing described below. Methods Serum total protein analysis performed using the using Siemens Atellica CH Total Protein II test (Weichselbaum method using biuret reagent). Serum immunoglobin (IgA, IgG and IgG), IgG subclass, and kappa/lambda free light chain level analysis performed by turbidimetric photometry assay using a Binding Site Optilite analyzer. Serum protein electrophoresis and immunotyping performed using CAPILLARYS 2 capillary zone electrophoresis and immunosubtraction with antisera. Serum protein immunofixation performed using Sebia Hydrasys 2 with Hydragel 9 IF Maxi kit. Confirmation and final determination of serum immunoproteins performed at our reference laboratory using MASS-FIX (MALDI-TOF MS) matrix-assisted laser desorption/ionization-time of flight mass spectrometry after patient serum applied to 5 separate immunoaffinity resins specific to immunoglobulin G, A, M, K, and L for purification. Results Initial testing showed a broadly migrating beta-gamma region IgG kappa monotypic restriction measuring 1.7 g/dL, with predominantly kappa subtraction and trivial lambda subtraction by immunosubtraction; serum free kappa/lambda light chain ratio was increased at 22.94. Serum immunoglobins and free light chains measured (mg/dL): IgA 738 (15 - 160), IgM 48 (50 - 240), IgG 2,805 (405 - 1,160), free kappa 20.42 (0.33 - 1.94), and free lambda 0.89 (0.57 - 2.63). IgG subclasses measured (mg/dL): IgG1 533 (320 - 950), IgG2 174 (50 - 340), IgG3 16.9 (10.0 - 120.0), IgG4 1,221.1 (2.0 - 110.0). Additional serum protein values included (g/dL): total protein 8.10 (6.00 - 8.30), albumin 2.97 (3.43 - 4.84), alpha-1 0.32 (0.21 - 0.44), alpha-2 0.52 (0.54 - 0.97), beta globulin 3.42 (0.65 - 1.03), and gamma globulin 0.87 (0.70 - 2.30). The polyclonal nature of the IgG4 spike was later demonstrated by the MASS-FIX mass spectrograph. Conclusions MASS-FIX is a useful addition to the toolset used in the diagnosis of possible monoclonal gammopathies and protein disorders, especially where broad monotypic restrictions are observed or in patients with abnormal presentations for monoclonal gammopathies. Using current standard techniques, the above case could have been incorrectly classified as a monoclonal process resulting in unnecessary cost and incorrect treatment.

B-311 Relation of Free Immunoglobulin Ligh Chain Concentration to Overflow Proteinuria

Abstract Background Free immunoglobulin light chains are small proteins that undergo substantial glomerular filtration. When high serum concentrations of free light chains occur in multiple myeloma, the high filtered load of light chains exceeds the uptake capacity of proximal tubules, and overflow proteinuria ensues. Massive excretion of light chains can lead to cast formation and renal injury. The present study sought to determine the relation of serum light chain concentration to overflow proteinuria. Methods Results for patients with plasma cell disorders were reviewed as approved by an institutional review board. A Binding Site Optilite analyzer measured serum free light chain concentrations. Urine and serum protein electrophoresis were performed on Helena agarose gels. A total of 170 paired serum and 24-hour urine specimens were evaluated for 36 patients with increased kappa light chains and 26 patients with increased lambda light chains. Results Overflow proteinuria with either immunoglobulin light chain presented with light chain as the major urinary protein and limited increases of urinary albumin. Light chain excretion exceeded albumin excretion when light chain excretion surpassed 100 mg/d. Similar results were obtained for cases with increased kappa and lambda free light chains. There was a general relation of increased serum kappa and lambda light chains and urinary excretion of the corresponding light chain. Limited urinary excretion of light chains (<120 mg/d) was seen when the serum light chain concentration was < 250 mg/L, representing an approximate threshold for substantial overflow proteinuria. Overflow proteinuria exceeding 2,000 g/d was seen only when serum free light chain concentrations exceeded 800 mg/L. However, a few patients with high serum light chain values (>1,000/L) had low urinary light chain excretion that was discordant from the relation of serum light chain concentration to light chain excretion. Serum light chain values >4,000 mg/L (N = 10) were all associated with overflow proteinuria of > 1,500 mg/d and, in 7 cases, >10,000 mg/d. In a few cases, there was evidence that serum light chain assays overestimated light chain concentrations. In one case, the serum free light chain value was twice the total protein and about 12-fold higher than the serum M-spike, suggestive of substantial overestimation. Conclusions Defining the relation between serum free light chain concentrations and the magnitude of overflow proteinuria helps identify patients at low and high risk of massive overflow proteinuria that can cause renal injury. Limited overflow proteinuria (<120 mg/d) occurred when serum light chain concentration was < 250 mg/L, and substantial overflow proteinuria occurred when serum light chains exceeded 4,000 mg/L Serum free light chain concentrations above 250 mg/L were incompletely predictive of urinary free light chain excretion; a few discordant cases with high measured serum light chain concentrations (>1,000 mg/L) and low excretion of light chains were observed. Further investigation is indicated for cases with possible overestimation of free light chains or discordance between serum light chain values and urinary light chain excretion. Unexpected results in some cases could relate to polymerization of free light chains as previously reported.

B-165 The EXENT System reliably distinguishes daratumumab from endogenous monoclonal immunoglobulins in multiple myeloma patients

Abstract Background The use of therapeutic monoclonal antibodies (t-mAbs) in the treatment of multiple myeloma (MM) has created challenges in the clinical laboratory. Co-migration of t-mAbs (predominantly IgGK) and the endogenous M-protein during serum protein electrophoresis can result in inability to distinguish between a patient’s M protein and the t-mAb. This can hinder accurate assessment of therapeutic responses or may lead to extensive workup with immunofixation electrophoresis and gel-shift assays for accurate confirmation of a complete response, or to exclude the emergence of a new clone. There is a need for an analytically specific routine method to identify t-mAb interference. MALDI-TOF mass spectrometry is an especially promising tool, as due to its ability to identify M proteins’ molecular mass, it has the potential to recognize any t-mAbs with known amino acid composition. We have used the EXENT® Immunoglobulin Isotypes (GAM) Assay for the EXENT Analyzer (The Binding Site, part of Thermo Fisher Scientific) to assess the reproducibility of daratumumab (anti-CD38, IgGK) detection in an analytical study, and assessed the EXENT System’s ability to detect daratumumab in a cohort of MM patients. Methods The expected mass to charge ratio (m/z) of the +2-charge state of daratumumab’s kappa light chain was calculated based on published information. Daratumumab was spiked into normal human serum at 1.0 g/L, and this material was tested in 5 replicates a day over 5 days. In addition, 109 samples from 34 MM patients receiving daratumumab, carfilzomib, lenalidomide and dexamethasone (Dara-KRd) treatment were also tested. Samples were obtained after 14 or 28 days following daratumumab administration in various treatment cycles, ranging from cycle 4 to end-of-treatment. Results The expected m/z for the +2-charge state of daratumumab’s kappa light chain was calculated as 11,691.01 to 11,693.1. In the reproducibility experiment, the median m/z was 11691.3, within the expected range. The difference between the lowest and highest m/z was 1.3 across the 25 replicates. Daratumumab was detected in 103 out of 109 samples (97.3%): 51/57 (89.5%) in IgGK samples, and in all IgGL (23) and IgA (29) samples. In two IgGK patients, where daratumumab was not detected in 2 out of 4 samples and in 1 out of 3 samples, respectively, the patients’ own M proteins were in the proximity (24.8 and 27.1 m/z) of daratumumab’s m/z. In the other three cases where daratumumab was not modeled by the algorithm, the daratumumab peak overlapped with the endogenous M-protein's adduct peak (estimated m/z 11,688) in one sample; it was masked by the shoulder of a large (8.42 g/L) IgGK M-protein in another sample; and no obvious reason was identified in the third sample. There was no difference in the detection rate between 14 days and 28 days post administration. Conclusions The EXENT Immunoglobulin Isotypes (GAM) Assay has successfully identified the presence of daratumumab in approximately 90% of IgGK patient samples in addition to all other (non-IgGK) MM samples. These results demonstrate the potential of the EXENT Immunoglobulin Isotypes (GAM) Assay for recognizing known t-mAb interference.

B-075 Evaluation of patients with monoclonal gammopathy of undetermined significance using Optilite Freelite assays

Abstract Background Monoclonal gammopathy of undetermined significance (MGUS) is a pre-malignant disorder with a progression risk to multiple myeloma of 1% per year. Patients with MGUS may produce monoclonal intact immunoglobulins (Ig) and/or free light chains (FLC). The International Myeloma Working Group (IMWG) recommendations for the use of FLC measurements in MGUS are to establish the diagnosis of light chain only MGUS (LC-MGUS) and further to risk-stratify newly diagnosed MGUS. Once identified, MGUS is longitudinally monitored at intervals dependent on the risk of transformation. The IMWG recommends serum protein electrophoresis (SPEP) for monitoring of known MGUS with intact Ig, however others have published benefits of additionally monitoring with FLC. In this study, we evaluated the diagnostic sensitivity and specificity of the Optilite® Freelite® Kappa Free Kit and the Optilite Freelite Lambda Free Kit (The Binding Site, part of Thermo Fisher Scientific) in patients with MGUS, and the monitoring performance of Freelite results measured in serial samples from patients with clinically stable and progressive MGUS. Methods Serum samples from 234 patients with clinically diagnosed MGUS and 140 disease controls, taken at single time points, and serial samples (≥3) from 49 patients with MGUS were tested with the Freelite assays. Intact M-protein presence and isotype was confirmed in MGUS samples with immunofixation electrophoresis (IFE). In patients clinically diagnosed with LC-MGUS, the presence of detectable M protein on IFE was not required. The disease control cohort (non-MGUS) had disorders commonly associated with elevated polyclonal immunoglobulins (e.g autoimmune, liver, and renal diseases), confirmed by total IgG and/or IgA and/or IgM results and the absence of M-protein by IFE. Patients represented a diverse population from both European and US sources. Results The overall diagnostic sensitivity of the Optilite Freelite assays was 59.4%, calculated as the frequency of abnormal free light chain ratio (rFLC) in intact immunoglobulin MGUS, and abnormal rFLC with concurrent elevated involved FLC level in LC-MGUS. The diagnostic sensitivity was: 58.0% in non-IgM MGUS, 41.7% in IgM MGUS, 66.7% in biclonal MGUS and 100% in LC-MGUS. In intact (IgG/IgA/IgM) kappa MGUS, the diagnostic sensitivity was 67.5%, versus 41.5% in intact (IgG/IgA/IgM) lambda MGUS. The diagnostic specificity was 86.4%. In the 49 patients with serial samples, concentrations of involved FLC were stable in 93.3% of patients with clinically stable MGUS, whereas in patients with clinically progressive MGUS an increasing trend was seen in 50% of cases. Conclusions These results demonstrate that the Optilite Freelite Kappa and Lambda assays produce clinically relevant results that are useful in the evaluation of MGUS.

B-319 Kappa and Lambda Free Light Chains Quantification in Serum Samples with 3 or 4 Monoclonal Components Detected by Serum Immunofixation

Abstract Background Serum free light chain (FLC) quantification is a diagnostic criterion for monoclonal gammopathy. Oligoclonal expansion is defined when two or more small bands are detected in the gamma region of serum protein electrophoresis (SPEP) or immunofixation (SIFE) within the context of infectious diseases, autoimmune diseases, or some lymphoproliferative processes. This study aims to investigate the frequency of FLC disturbances in samples with oligoclonal pattern of 3 or 4 monoclonal bands observable in SIFE. Methods We have analyzed 7553 consecutive samples during the period of January to December 2023 in which both determination of FLC and SIFE were ordered in a large central laboratory at São Paulo, Brazil. FLC quantification and SIFE analysis were performed with FreeLite assay and Optilite system (The Binding Site, UK) and SPIFE 3000 (Helena Laboratories, USA), respectively. Results In our analysis, 119 samples (50 males, 69 females, median age 69 years (range 37-90)), with 3 or 4 monoclonal bands were identified, constituting 1.57% of the overall study population. These samples were then categorized into two groups. The first group had normal FLC Ratio (range 0.26 to 1.65) and the second group had altered FLC Ratio (outside 0.26-1.65 range). Seventy-seven samples (65%) had altered FLC ratio, with kappa elevation being the most common alteration (51 samples). Within these samples, median FLC ratio was 2.99 (range 1.72-719.05). Lambda elevation was present in 26 samples, with median FLC ratio 0.04 (range 0.005-0.16). Conclusions Presence of 3 or more oligoclonal bands in SIFE analysis may be indicative of various pathological processes (benign and malignant) and also be observed in post-transplantation. In this study, we have observed that most of these samples present altered FLC ratio, which indicate monoclonal disturbances in the production of free light chains. Those disturbances may be associated with a lymphoproliferative disease (e.g. multiple myeloma) or may be transient. Clinicians should be aware of the need for follow-up of these cases.

B-101 Assessing the Performance of Cerebrospinal Fluid Kappa Immunoglobulin Free Light Chain as a Diagnostic Marker for Multiple Sclerosis Using Automated Chart Review

Abstract Background Multiple sclerosis (MS) is an immune-mediated central nervous system (CNS) demyelinating disease which is diagnosed based on CNS lesions' spatial and temporal dissemination. The 2017 revised McDonald Criteria include cerebrospinal fluid (CSF) specific oligoclonal bands (OCBs) to indicate dissemination in time, but their assessment is labor-intensive and requires a paired serum sample. Kappa immunoglobulin free light chain in CSF (KCSF) has emerged as a more sensitive but less specific MS biomarker, measured from a single CSF sample. Our institution's MS testing panel uses KCSF initially to rule out MS, with reflex to OCB for positive confirmation if KCSF is borderline positive (≥0.06 mg/dL). However, manual correlation of laboratory and clinical data hinders the direct comparison of the diagnostic performance between these markers. Herein we report a retrospective assessment of KCSF and OCB diagnostic performance using automated chart review. Methods Between January 1st, 2021 and December 31st, 2022, there were 1,237 patients with simultaneously measured OCB and KCSF. CSF-specific OCBs were measured by separating CSF and serum proteins using an agarose isoelectric focusing gel with a pH gradient from 3-10 (Sebia). After transfer to nitrocellulose paper, antisera were applied to visualize IgG specific bands and quantify CSF-specific OCBs. KCSF was quantitated using nephelometry by applying free light chain antisera (Binding Site) to CSF samples using a Siemens BNII analyzer. An automated chart review process utilizing keyword matching was implemented to identify patients with MS. An initial filter phase required presence of the term “multiple sclerosis”, while a second required the presence of any of the following terms: “primary progressive,” “secondary progressive,” “relapsing remitting,” “progressive relapsing,” or “tumefactive” to identify patients with diagnosed MS. The automated method’s performance was then compared to a manual chart review of 200 randomly selected patients. Subsequently, the automated process was used to determine the MS diagnosis status of all 1,237 patients. Results Of the 200 manually reviewed patients, 44 MS diagnoses were found. The automated process correctly identified 41 of these with a resulting sensitivity and specificity of 93.18% and 95.51%, respectively. Applied to the entire cohort, the automated review identified 275 MS diagnoses. For the entire cohort, the area under the curve (AUC) for the receiver operating characteristic (ROC) curves for OCB and KCSF were 0.824 (95% confidence interval: 0.794, 0.854) and 0.808 (0.777, 0.839) respectively. Our institution’s diagnostic cutoff of ≥2 for OCB yielded a sensitivity and specificity of 74.2% (68.3%, 79.1%) and 84.9% (73.8%, 89.5%), respectively. The best combination for sensitivity and specificity for KCSF with the Youden index yielded a cutoff ≥0.0654 mg/dL (sensitivity of 79.6% [73.1%, 84.0%] and specificity of 72.4% [62.8%, 76.8%]), which is similar to previous publications. This supports the use of automated chart review and suggests the KCSF cutoff has been stable over time. Conclusions KCSF exhibits comparable MS diagnostic performance to OCB yet only requires one CSF sample and can be measured rapidly. When validated, mining unstructured data allows for test performance characterization in large patient cohorts without the need for manual chart review.

Haptoglobin buffers lipopolysaccharides to delay activation of NFκB.

It has remained yet unclear which soluble factors regulate the anti-inflammatory macrophage phenotype observed in both homeostasis and tumourigenesis. We show here that haptoglobin, a major serum protein with elusive immunoregulatory properties, binds and buffers bacterial lipopolysaccharides to attenuate activation of NFκB in macrophages. Haptoglobin binds different lipopolysaccharides with low micromolar affinities. Given its abundance, haptoglobin constitutes a buffer for serum-borne lipopolysaccharides, shielding them to safeguard against aberrant inflammatory reactions by reducing the amount of free lipopolysaccharides available for binding to TLR4. Concordantly, NFκB activation by haptoglobin-associated lipopolysaccharides was markedly delayed relative to stimulation with pure lipopolysaccharide. Our findings warrant evaluation of therapeutic benefits of haptoglobin for inflammatory conditions and re-evaluation of purification strategies. Finally, they allow to elucidate mechanisms of enhanced immunosuppression by oncofetal haptoglobin.

B-137 Sepsis risk prediction using machine learning (ML) and routine blood markers up to 1 week before emergency admission

Abstract Background Sepsis is a major global health concern, responsible for approximately 50 million cases and 11 million deaths annually, representing 20% of global mortality. It is the leading cause of hospital readmissions and deaths, with associated costs exceeding $38 billion per year. Alarmingly, 80% of sepsis cases occur outside the hospital, and 70% of patients visit an outpatient facility within a week of admission. Early diagnosis and appropriate treatment could prevent 80% of sepsis-related deaths. Our study demonstrates that machine learning models hold significant promise in identifying sepsis risks using routine blood tests up to 1 week before admission. Methods For our study, we utilized a dataset of over 25,000 patient records, including both sepsis and non-sepsis cases, sourced from MIMIC-IV (version 2.2), a comprehensive electronic health record dataset from Beth Israel Deaconess Medical Center, Boston, MA. We employed a gradient boosted model with 300 trees, a maximal depth of 30 layers, and gain ratio as the criterion for attribute selection. The model's performance was evaluated using 10-fold cross-validation, demonstrating optimal performance. Input parameters included age, gender, and results of routine blood markers such as complete blood counts, differential counts, comprehensive metabolic panels, and lipid panels recorded up to 1 week before sepsis diagnosis. Results Our model achieved remarkable predictive accuracy for sepsis risk, with an area under the receiver operating characteristic curve (AUC) of 0.99 and accuracy of 0.99. Notably, the model achieved 1.0 sensitivity, 0.99 specificity, 0.99 positive predictive value, 1.0 negative predictive value, 0.99 F measure, and 0.99 Youden index. Calcium, protein, liver enzymes, hematocrit, white blood cells, and cholesterol were identified as key contributors to sepsis risk prediction. Importantly, prediction accuracy remained consistent up to 1 week prior to diagnosis. Conclusions In conclusion, dysregulated intracellular calcium handling during sepsis is associated with an intensified inflammatory response, cellular death, and subsequent organ dysfunction. Low serum protein levels, particularly albumin, are linked to poor outcomes in sepsis. Recent studies have identified liver dysfunction as an early event in sepsis, underscoring its significance in the disease process. Elevated bilirubin levels are associated with an increased risk of mortality. Low hematocrit (HCT) levels are linked to higher 30-day mortality and serve as an independent prognostic biomarker in sepsis. Monocytes play a significant role in sepsis pathophysiology, undergoing various changes including preserved phagocytic activity, increased reactive oxygen species and nitric oxide generation, and decreased production of inflammatory cytokines such as IL-6 and TNF-alpha. Septic patients often exhibit lymphopenia, affecting CD4 and CD8 T cells, B cells, and natural killer (NK) cells. Hypocholesterolemia is common in sepsis and serves as an important early prognostic factor, with low plasma concentrations associated with poor outcomes. Since every hour of delayed treatment increases the risk of death by 4%-9%, changes in routine blood markers detected by machine learning models can provide early indications of impending sepsis up to 1 week before diagnosis.

Key biomarkers in type 2 diabetes patients: A systematic review.

Type 2 diabetes mellitus (T2DM) is not just a local health issue but a significant global health burden, affecting patient outcomes and clinical management worldwide. Despite the wealth of studies reporting T2DM biomarkers, there is an urgent need for a comparative review. This review aims to provide a comprehensive analysis based on the reported T2DM biomarkers and how these are linked with other conditions, such as inflammation and wound healing. A comparative review was conducted on 24 001 study participants, including 10 024 T2DM patients and 13 977 controls (CTL; age 30-90 years). Four main profiles were extracted and analysed from the clinical reports over the past 11 years: haematological (1084 cases vs. 1458 CTL), protein (6753 cases vs. 9613 CTL), cytokine (975 cases vs. 1350 CTL) and lipid (1212 cases vs. 1556 CTL). This review provides a detailed analysis of the haematological profile in T2DM patients, highlighting fundamental changes such as increased white blood cells and platelet counts, accompanied by decreases in red blood cell counts and iron absorption. In the serum protein profile, a reduction in albumin and anti-inflammatory cytokines was noted along with an increase in globulin levels and pro-inflammatory cytokines. Furthermore, changes in lipid profiles were discussed, specifically the decreases in high-density lipoprotein (HDL) and the increases in low-density lipoprotein (LDL) and triglycerides. Understanding the changes in these four biomarker profiles is essential for developing innovative strategies to create diagnostic and prognostic tools for diabetes management.

A-149 Comparison of Mass Spectrometry to Sickle Solubility for Confirmation of Low Percentages of Hemoglobin S

Abstract Background Hemoglobin S (HbS) causes sickling disorders. In the laboratory, confirmation of HbS in patient samples with varying percentages is essential. Assessment is routinely performed using capillary electrophoresis (CE) and/or high-performance liquid chromatography. Sickle solubility (SS) exploits decreased solubility of HbS under reducing conditions and is a confirmatory test. SS is unreliable with low HbS, elevated HbF, severe anemia, erythrocytosis, hyperglobulinemia, extreme leukocytosis, hyperlipidemia, or increased serum proteins. Infant samples commonly have low percentages of HbS, high HbF, and limited sample volume. Intact globin chain Mass Spectrometry (MS) is used to identify abnormal hemoglobin variants by detecting the mass change (normal α/β/γ mass =15127/15867/15996 amu), respectively). MS has potential advantages in sensitivity and efficiency over SS and requires lower input sample volume. Herein we compare the utility of MS versus SS for confirmation of HbS. Methods Twenty-three EDTA-anticoagulated blood samples containing low percentages of HbS were identified. SS was performed on 30μL washed packed cells (requires 200μL whole blood) per standard methods. Intact Quadrupole Time-of-Flight MS (Accurate-Mass Q-TOF LC/MS 6520, Agilent Technologies, California) was performed using 10μL whole blood sample in acetonitrile solution reagent and results were compared. HbS and HbF levels were assayed by capillary electrophoresis (Sebia, France) per standard methods. Results Select results are summarized in Table 1. SS detected HbS percentages to 4.8% when HbF was low but not 6% HbS when Hb F is high. MS showed no HbF interference and identified HbS peaks (at 15837 amu) down to 3.1%. HbS percentages <3.1 were undetected by both methods. Conclusions Overall, MS was superior to SS for confirming HbS, particularly in samples with low variant percentage and/or elevated HbF. MS is also optimal when sample volume is minimal, such as for infants. Percentages of HbS <3% require methods such as IEF or DNA sequencing for confirmation.

Falsely Abnormal Serum Protein Electrophoresis after Administration of Intravenous Immunoglobulins (IVIG): A Retrospective Cohort Study

Intravenous immunoglobulin (IVIG) therapy, used in several neurologic, hematologic, immunologic and dermatologic conditions, is known to interfere with the results of some serum laboratory tests. We analyzed the potential interference of IVIG on serum protein electrophoresis (SPEP) by reviewing more than a decade of SPEP studies performed by the clinical immunology laboratory of the Johns Hopkins Hospital. Of the total 100,350 SPEP performed between January 1, 2013 and December 31, 2023, 395 contained the keyword IVIG in the pathologist report, contributed by 348 patients confirmed to have received IVIG by chart review. Of the 348 patients, 20 (6%) had a M-spike on SPEP suggestive of monoclonal gammopathy, while 328 (94%) did not have it. Of the 20 patients, 14 received IVIG within 30 days from the SPEP collection date, while 6 received beyond 30 days. Serum immunofixation electrophoresis (SIFE) and clinical follow up showed no evidence of monoclonal gammopathy in 5 of the 14 patients. Overall, this 11-year retrospective cohort study showed that 5 of 348 (1.4%) patients treated with IVIG and tested by SPEP had a false M-spike, that is a spike not confirmed to be caused by a monoclonal gammopathy by subsequent studies. Although small, the false positive rate of 1.4% suggests that integrating knowledge of recent IVIG administration into the pathologist report would reduce SPEP misdiagnosis.

Advancing Albumin Isolation from Human Serum with Graphene Oxide and Derivatives: A Novel Approach for Clinical Applications.

This study introduces a novel, environmentally friendly albumin isolation method using graphene oxide (GO). GO selectively extracts albumin from serum samples, leveraging the unique interactions between GO's oxygen-containing functional groups and serum proteins. This method achieves high purification efficiency without the need for hazardous chemicals. Comprehensive characterization of GO and reduced graphene oxide (rGO) through techniques such as X-ray diffraction (XRD) analysis, Raman spectroscopy, scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FTIR) confirmed the structural and functional group transformations crucial for protein binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analyses demonstrated over 95% purity of isolated albumin, with minimal contamination from other serum proteins. The developed method, optimized for pH and incubation conditions, showcases a green, cost-effective, and simple alternative for albumin purification, promising broad applicability in biomedical research and clinical applications.

Changes in the serum proteome profile of patients with neuroborreliosis, foresters, and patients treated according to ILADS method.

The aim of study was to evaluate the changes in proteomic profile of human serum induced by the development of tick-borne neuroborreliosis (NB), before/after therapy, patients treated with prolonged multidrug therapy according to ILADS (International Lyme and Associated Diseases Society), and foresters frequently exposed to tick bites. A proteomics approach was used to analyze the expression of proteins in serum of patients and sex/age-matched healthy donors. The analysis was performed using SDS-PAGE/LC-MS/MS (Q-Exactive OrbiTrap mass spectrometer). Obtained results indicated changes in the serum proteome of patients with NB putting attention to the proteins involved mainly in calcium transport/metabolism and signaling molecules that differ patients before and after classic therapy. Moreover, ILADS treated patients have different protein distribution than patients from other groups, what is the consequence of prolonged antibiotic therapy. In the case of foresters, the most important result is the increased β-secretase level. Obtained results may contribute to a better understanding of the mechanism of the development of tick-borne diseases, as well as will allow create new opportunities for its rapid and more effective therapy. However, further studies, on larger patients groups, are needed to apply them in clinical practice.

Serum apolipoprotein H determines ferroptosis resistance by modulating cellular lipid composition

Ferroptosis is a regulated cell death process dependent on iron, triggered by the accumulation of lipid peroxidation. The environmental context significantly impacts cellular sensitivities to ferroptosis. Serum, constituting the extracellular fluid composition in vivo, provides crucial environmental biomolecules. In this study, we investigated the influence of sera on ferroptosis induction, pinpointing the serum protein apolipoprotein H (APOH) as a pivotal inhibitor of ferroptosis. Moreover, we elucidated that APOH suppresses ferroptosis by activating the phosphoinositide 3-kinase (PI3K)-AKT-sterol regulatory element-binding proteins (SREBPs) pathway, thereby elevating stearoyl-CoA desaturase (SCD) levels and augmenting cellular monounsaturated fatty acid-containing phospholipids (MUFA-PLs). Furthermore, ApoHinfer, the peptide derivative of the active region of APOH, mimics its ferroptosis inhibitory activity. Our findings underscore the critical role of serum protein APOH in the inhibition of ferroptosis and indicates potential therapeutic applications in treating cancer and diseases associated with ferroptosis.

Localization of albumin with correlative super resolution light- and electron microscopy in the kidney

The functioning of vertebrate life relies on renal filtration of surplus fluid and elimination of low-molecular-weight waste products, while keeping serum proteins in the blood. In disease, however, there is leak of serum proteins and tracing them to identify the leaking position within tissue with a nanometer resolution poses a significant challenge. Correlative microscopy integrates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Using chemical tagging of albumin with synthetic fluorophores we achieve protein-specific labeling that preserve their post-embedding fluorescence after high-pressure freezing and freeze-substitution of murine kidney tissue. Using advanced registration techniques for super-resolution correlative light and electron microscopy, we can localize the labeled albumin with a high precision in the x-y plane of electron micrographs and cartograph its distribution. Thereby we can quantify the albumin concentration and measure a linear reduction gradient across the kidney filtration barrier. Our study shows the feasibility of combining different microscopy contrasts for tracing fluorescently labeled protein markers with super resolution in various tissue samples and opens new perspectives for correlative imaging in volume electron microscopy.