Non-Perturbative Identification and Subtyping of Amyloidosis in Human Kidney Tissue with Raman Spectroscopy and Machine Learning.

Amyloids are proteins with characteristic beta-sheet secondary structures that display fibrillary ultrastructural configurations. They can result in pathologic lesions when deposited in human organs. Various types of amyloid protein can be routinely identified in human tissue specimens by special stains, immunolabeling, and electron microscopy, and, for certain forms of amyloidosis, mass spectrometry is required. In this study, we applied Raman spectroscopy to identify immunoglobulin light chain and amyloid A amyloidosis in human renal tissue biopsies and compared the results with a normal kidney biopsy as a control case. Raman spectra of amyloid fibrils within unstained, frozen, human kidney tissue demonstrated changes in conformation of protein secondary structures. By using t-distributed stochastic neighbor embedding (t-SNE) and density-based spatial clustering of applications with noise (DBSCAN), Raman spectroscopic data were accurately classified with respect to each amyloid type and deposition site. To the best of our knowledge, this is the first time Raman spectroscopy has been used for amyloid characterization of ex vivo human kidney tissue samples. Our approach, using Raman spectroscopy with machine learning algorithms, shows the potential for the identification of amyloid in pathologic lesions.

Non-crystalline light chain proximal tubulopathy, a morphologically protean entity.

BACKGROUND: Light chain proximal tubulopathy (LCPT) is a rare form of paraprotein-related disease, occurring in two main histopathological forms: crystalline and non-crystalline. The clinicopathological features, treatment strategies and outcomes, especially of the non-crystalline form, are not well described. METHODS: We conducted a single-centre retrospective case series of 12 LCPT patients, 5 crystalline and 7 non-crystalline, between 2005 and 2021. RESULTS: The median age was 69.5 years (range 47-80). Ten patients presented with CKD and significant proteinuria (median estimated glomerular filtration rate of 43.5 ml/min/1.73 m2; urine protein:creatinine ratio 328 mg/mmol). Only six patients had known haematological disease at the time of renal biopsy. Multiple myeloma (MM) was diagnosed in seven patients cases and monoclonal gammopathy of renal significance (MGRS) in five patients. A clone was detected in all cases combining serum/urine electrophoresis and free light chain (LC) assays. Crystalline and non-crystalline variants had similar clinical presentations. For the non-crystalline variant, a diagnosis was reached based on a combination of CKD without another cause, haematological workup, LC restriction on immunofluorescence and abnormalities on electron microscopy (EM). Nine of 12 patients received clone-directed treatment. Patients who achieved haematological response (including all non-crystalline LCPT) had improved renal outcomes over a median follow-up of 79 months. CONCLUSIONS: The non-crystalline variant may go unrecognised because of its subtle histopathological features and requires EM to distinguish it from 'excessive LC resorption without tubular injury'. Clone-directed treatment with good haematological response improves renal outcomes in both variants but limited data exist in MGRS. Multicentre prospective studies are needed to better define the clinicopathological characteristics associated with poor outcomes and optimize treatment strategies in patients with MGRS.

Clinic-pathological characteristics of rare tubulointerstitial diseases. / å°‘è§è‚¾å°ç®¡é—´è´¨æ€§ç–¾ç— çš„ä¸´åºŠåŠç— ç†ç‰¹å¾åˆ†æž.

OBJECTIVES: Tubulointerstitial diseases is one of the common causes of renal dysfunction. Some rare pathological types are easy to be misdiagnosed and missedly diagnosed because of their low prevalence and relatively insufficient understanding, which affects the treatment and prognosis of patients. This study aims to explore clinical manifestations and pathological characteristics of several rare tubulointerstitial diseases, and therefore to improve their diagnosis and treatment. METHODS: A total of 9 363 patients diagnosed by renal biopsy in the Department of Nephrology, Second Xiangya Hospital, Central South University from November 2011 to September 2021 were selected. Six cases of light chain cast nephropathy (LCCN), 2 cases of light chain proximal tubulopathy (LCPT), 1 case of LCCN with LCPT, 4 cases of genetic tubulointerstitial disease, and 6 cases of non-genetic related tubulointerstitial lesion were screened out, and their clinical manifestations and renal biopsy pathological results were collected, compared, and analyzed. RESULTS: Patients with LCCN presented with mild to moderate anemia, microscopic hematuria, and mild to moderate proteinuria. Compared with patients with LCPT, proteinuria and anemia were more prominent in patients with LCCN. Five patients with LCCN and 2 patients with LCPT had elevated serum free kappa light chain. Five patients with LCCN presented clinically with acute kidney injury (AKI). Two patients with LCPT and 1 patient with LCCN and LCPT showed CKD combined with AKI, and 1 LCPT patient presented with typical Fanconi syndrome (FS). Five patients with LCCN, 2 patients with LCPT, and 1 patient with LCCN and LCPT were diagnosed with multiple myeloma. Five patients with LCCN had kappa light chain restriction in tubules on immunofluorescence and a "fractured" protein casts with pale periodic acid-Schiff (PAS) staining on light microscopy. Immunohistochemical staining of 2 LCPT patients showed strongly positive kappa light chain staining in the proximal tubular epithelial cells. And monoclonal light chain crystals in crystalline LCPT and abnormal lysosomes and different morphological inclusion bodies in noncrystalline LCPT were observed under the electron microscope. Six patients with LCCN were mainly treated by chemotherapy. Renal function was deteriorated in 1 patient, was stable in 4 patients, and was improved in 1 patient. Two patients with LCPT improved their renal function after chemotherapy. Four patients with genetic tubulointerstitial disease were clinically presented as CKD, mostly mild proteinuria, with or without microscopic hematuria, and also presented with hyperuricemia, urine glucose under normal blood glucose, anemia, polycystic kidneys. Only 1 case had a clear family history, and the diagnosis was mainly based on renal pathological characteristics and genetic testing. Compared with patients with non-genetic related tubulointerstitial lesion, patients with genetic tubulointerstitial disease had an earlier age of onset, higher blood uric acid, lower Hb and estiated glomemlar fitration (eGFR), and less edema and hypertension. Renal pathology of genetic tubulointerstitial disease presented tubular atrophy and interstitial fibrosis, abnormal tubular dilation, glomerular capsuledilation, and glomerular capillary loop shrinkage. Glomerular dysplasia and varying degrees of glomerular sclerosis were observed. Genetic tubulointerstitial disease patients were mainly treated with enteral dialysis, hypouricemic and hypoglycemic treatment. Two genetic tubulointerstitial disease patients had significantly deteriorated renal function, and 2 patients had stable renal function. CONCLUSIONS: Patients with AKI or FS, who present serum immunofixation electrophoresis and/or serum free kappa light chain abnormalities, should be alert to LCCN or LCPT. Renal biopsy is a critical detection for diagnosis of LCCN and LCPT. Chemotherapy and stem cell transplantation could delay progression of renal function in patients with LCCN and LCPT. If the non-atrophic area of the renal interstitium presents glomerular capsule dilatation, glomerular capillary loop shrinkage, and abnormal tubular dilatation under the light microscopy, genetic tubulointerstitial disease might be considered, which should be traced to family history and can be diagnosed by genetic testing.

A method to obtain purified free light chain monomers and dimers from urine samples of patients with multiple myeloma.

Antibody light chains are synthesized in excess by plasma cells, and this excess can be secreted into biological fluids as dimers or monomers in various proportions. Structural differences between monomers or dimers of free light chains (FLC) can affect their biological functions and possibly their pathogenicity. They also may exhibit differential immune reactivity, perhaps explaining discrepant quantifications when measured by different immunoreagents. Having purified FLC monomers and dimers available can be useful for studying their properties. Here we propose a simple preparatory procedure to purify FLC monomers and dimers from urine samples of patients with plasma cell disorders. Two representative urine samples containing lambda or kappa FLC were loaded into a nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel strips containing separate monomers and dimers were excised, electroeluted, and the FLC recovered. The FLC were recovered from SDS-PAGE gel in sufficient amounts to be quantified by UV and two automated nephelometric assays immunochemical. The procedure was found to be simple, reproducible, and with a high yield, thus offering the opportunity to compare different assays. Not all urine samples are suitable for this procedure, but this approach allows for the purification of FLC monomers and dimers from many selected urine samples which maintain their oligomeric organization.

Salivary free light chains and salivary immunoglobulins as potential non-invasive biomarkers in primary Sjögren's syndrome.

BACKGROUND: B cells contribute significantly to the pathogenesis of primary Sjögren's syndrome (pSS). Free light chains (FLCs) are generated during the production of immunoglobulins (Igs) and are surrogates of B cell activity. We hypothesized that salivary FLCs and salivary Igs could represent salivary gland inflammation and therefore, serve as biomarkers in pSS. METHODS: Patients >18 years old fulfilling the American College of Rheumatology / European League Against Rheumatism (EULAR) 2016 criteria for pSS and age-matched healthy and disease controls (sicca non-pSS, rheumatoid arthritis, systemic lupus erythematosus) were recruited for this cross-sectional study. FLCs in saliva and serum were measured by immunoturbidimetry. Serum and salivary Igs were measured by nephelometry and enzyme-linked immunosorbent assay, respectively. Area under the receiver operator characteristic curve was determined. The factors influencing the serum and salivary FLCs in pSS were determined using backward linear regression. RESULTS: A total of 78 patients with pSS, 76 healthy controls and 62 disease controls were recruited. Median EULAR SS disease activity index (interquartile range) was 1 (3.75). Serum FLCκ and FLCλ, salivary FLCλ, serum and salivary IgG, salivary IgM was significantly higher in the pSS group compared to the controls. Areas under the curve for salivary FLCλ, serum FLCκ, serum and salivary IgG were 0.75, 0.72, 0.78 and 0.77, respectively. Regression analysis showed that salivary FLCκ, salivary FLCλ and salivary IgG were associated with positive salivary gland histopathology. Use of immunosuppressants and glucocorticoids was associated with lower values of salivary parameters. CONCLUSION: Salivary FLCλ and salivary IgG were significantly different between pSS and control groups and could be potential non-invasive biomarkers in pSS. These findings should be confirmed in larger longitudinal studies.

AL amyloidosis: untangling new therapies.

Systemic light chain (AL) amyloidosis is a protein misfolding disorder characterized by the deposition of abnormal immunoglobulin light chains in fibrillary aggregates, resulting in end-organ damage. Several unique challenges face treating physicians, including delayed diagnosis, advanced vital organ involvement, and morbidity with treatment. Aggressive supportive care and risk-adapted application of plasma cell-directed therapies are the cornerstones of management. The therapeutic revolution in multiple myeloma will likely further expand the arsenal against plasma cells. Careful investigation of these agents will be critical to establish their role in this fragile population. The promise of fibril-directed therapies to restore organ function remains despite early disappointments. In this review, we discuss new therapies to tackle AL amyloidosis using a case-based approach.

Kappa free light chain index as a diagnostic biomarker in multiple sclerosis: A real-world investigation.

Kappa free light chain (KFLC) index, a measure for intrathecal production of free kappa chains, has been increasingly recognized for its diagnostic potential in multiple sclerosis (MS) as a quantitative alternative to IgG oligoclonal bands (OCBs). Our objective was to investigate the sensitivity, specificity, and overall diagnostic accuracy of KFLC index in MS. KFLC index was prospectively determined as part of the diagnostic workup in patients with suspected MS (n = 327) between May 2013 and February 2020. Patients with clinically isolated syndrome (CIS), radiologically isolated syndrome (RIS), and MS had markedly higher KFLC index (44.6, IQR 16-128) compared with subjects with other neuro-inflammatory disorders (ONID) and symptomatic controls (SC) (2.19, IQR 1.68-2.98, p < 0.001). KFLC index had a sensitivity of 0.93 (95% CI 0.88-0.95) and specificity of 0.87 (95% CI 0.8-0.92) to discriminate CIS/RIS/MS from ONID and SC (AUC 0.94, 95% CI 0.91-0.97, p < 0.001). KFLC index and intrathecal fraction (IF) KFLC had similar accuracies to detect MS. Treatment with disease-modifying therapy (DMT) did not influence the level of KFLC index and it was not affected by demographic factors or associated with degenerative or inflammatory biomarkers in cerebrospinal fluid (CSF). KFLC index in MS diagnostics has methodological advantages compared to OCB and is independent to subjective interpretation. Moreover, it is an attractive diagnostic tool since the diagnostic specificity and sensitivity of KFLC index are similar with that of OCBs and KFLCIF and better than for IgG index. We show that KFLC index was influenced neither by DMT nor by demographic factors or other inflammatory or degenerative processes in MS as determined by biomarkers in CSF.

Light chain deposition disease presenting as an atrial mass: a case report and review of literature.

Light chain deposition disease (LCDD) also known as nonamyloidotic immunoglobulin deposition disease is a rare systemic disorder due to the abnormal deposition of immunoglobulin in multiple organs caused by the clonal proliferation of B lymphocytes and plasma cells. Renal involvement is the most common with cardiac manifestations being the most common extra renal presentation of the disease. Renal involvement is not always associated with LCDD. Isolated cardiac involvement can manifest in a wide variety of ways: heart failure, cardiomyopathy, arrhythmias, angina, myocardial infarction, etc. We hereby present an unusual case of 59-year-old female who presented to clinic for routine follow up. A murmur on physical exam was evaluated with echocardiogram which led to the discovery of an incidental right atrial mass. Cardiac magnetic resonance imaging was completed 6 months later for follow up which showed increasing size of the mass. The mass was excised and found to be consistent with LCDD. To the best of our knowledge, this is the first reported case of LCDD manifesting as an atrial mass. Through this case report and review of literature we would like to generate awareness among our fellow pathologists and clinicians to maintain a high level of suspicion for LCDD as it can manifest in many unusual ways, with or without kidney involvement.

MALDI mass spectrometry imaging unravels organ and amyloid-type specific peptide signatures in pulmonary and gastrointestinal amyloidosis.

PURPOSE: Amyloidosis is a disease group caused by pathological aggregation and deposition of peptides in diverse tissue sites. Recently, matrix-assisted laser desorption/ionization mass spectrometry imaging coupled with ion mobility separation (MALDI-IMS MSI) was introduced as a novel tool to identify and classify amyloidosis using single sections from formalin-fixed and paraffin-embedded cardiac biopsies. Here, we tested the hypothesis that MALDI-IMS MSI can be applied to lung and gastrointestinal specimens. EXPERIMENTAL DESIGN: Forty six lung and 65 gastrointestinal biopsy and resection specimens with different types of amyloid were subjected to MALDI-IMS MSI. Ninety three specimens included tissue areas without amyloid as internal negative controls. Nine cases without amyloid served as additional negative controls. RESULTS: Utilizing a peptide filter method and 21 known amyloid specific tryptic peptides we confirmed the applicability of a universal peptide signature with a sensitivity of 100% and a specificity of 100% for the detection of amyloid deposits in the lung and gastrointestinal tract. Additionally, the frequencies of individual m/z-values of the 21 tryptic marker peptides showed organ- and tissue-type specific differences. CONCLUSIONS AND CLINICAL RELEVANCE: MALDI-IMS MSI adds a valuable analytical approach to diagnose and classify amyloid and the detection frequency of individual tryptic peptides is organ-/tissue-type specific.

Monoclonal free light chain detection and quantification: Performances and limits of available laboratory assays.

The detection and quantification of immunoglobulin free light chains in serum and urine is recommended for the diagnosis and monitoring of monoclonal gammopathies according to the guidelines of the International Myeloma Working Group (IMWG). Several tests are currently available in the clinical laboratory to detect and quantify free light chains but although quality, efficiency, and effectiveness have been improved, the results are still variable and poorly harmonized and standardized. The present review article wants to analyze these aspects, with a keen eye on techniques, such as mass spectrometry, that could replace in the practical clinical laboratory the current methods including Bence-Jones protein assay and free light chain immunoassays.

MASS-FIX for the detection of monoclonal proteins and light chain N-glycosylation in routine clinical practice: a cross-sectional study of 6315 patients.

Immunoenrichment-based matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), termed MASS-FIX, offers several advantages over immunofixation for the detection and isotyping of serum monoclonal protein, including superior sensitivity and specificity, the ability to differentiate therapeutic monoclonal antibodies, and the rapid identification of light chain (LC) N-glycosylation. We identified 6315 patients with MASS-FIX performed at our institution since 2018. Of these, 4118 patients (65%) with a wide array of plasma cell disorders (PCD), including rare monoclonal gammopathies of clinical significance, had a positive MASS-FIX. Two-hundred twenty-one (5%) of the MASS-FIX positive patients had evidence of LC N-glycosylation, which was more commonly identified in IgM heavy chain isotype, kappa LC isotype, and in diagnoses of immunoglobulin light chain (AL) amyloidosis and cold agglutinin disease (CAD) compared to other PCD. This cross-sectional study describes the largest cohort of patients to undergo MASS-FIX in routine clinical practice. Our findings demonstrate the widespread utility of this assay, and confirm that LC N-glycosylation should prompt suspicion for AL amyloidosis and CAD in the appropriate clinical context.

Mass spectrometry for the evaluation of monoclonal proteins in multiple myeloma and related disorders: an International Myeloma Working Group Mass Spectrometry Committee Report.

Plasma cell disorders (PCDs) are identified in the clinical lab by detecting the monoclonal immunoglobulin (M-protein) which they produce. Traditionally, serum protein electrophoresis methods have been utilized to detect and isotype M-proteins. Increasing demands to detect low-level disease and new therapeutic monoclonal immunoglobulin treatments have stretched the electrophoretic methods to their analytical limits. Newer techniques based on mass spectrometry (MS) are emerging which have improved clinical and analytical performance. MS is gaining traction into clinical laboratories, and has replaced immunofixation electrophoresis (IFE) in routine practice at one institution. The International Myeloma Working Group (IMWG) Mass Spectrometry Committee reviewed the literature in order to summarize current data and to make recommendations regarding the role of mass spectrometric methods in diagnosing and monitoring patients with myeloma and related disorders. Current literature demonstrates that immune-enrichment of immunoglobulins coupled to intact light chain MALDI-TOF MS has clinical characteristics equivalent in performance to IFE with added benefits of detecting additional risk factors for PCDs, differentiating M-protein from therapeutic antibodies, and is a suitable replacement for IFE for diagnosing and monitoring multiple myeloma and related PCDs. In this paper we discuss the IMWG recommendations for the use of MS in PCDs.

Prognostic impact of rapid reduction of involved free light chains in multiple myeloma patients under first-line treatment with Bendamustine, Prednisone, and Bortezomib (BPV).

INTRODUCTION: Light chain involvement is observed in almost every patient (pt) with newly diagnosed multiple myeloma (MM). Owing to a relatively short half-life, rapid reduction in the involved free light chain (iFLC) is of potential prognostic value. METHODS: This retrospective analysis included 92 pts with newly diagnosed MM treated with bendamustine, prednisone, and bortezomib (BPV). RESULTS: After a median number of two (range 1-5) BPV cycles, the majority of pts (n = 86; 93%) responded with either sCR (n = 21), CR (n = 1), nCR (n = 25), VGPR (n = 20), or PR (n = 19). PFS and OS at 48 months were 39% and 67%, respectively. At baseline, 79 out of 92 pts (86%) had iFLC levels above the upper standard level and an abnormal ratio of involved to uninvolved free light chain ≥ 8. In a subgroup analysis of these pts, we evaluated the prognostic importance of an early reduction of the iFLC during the first two BPV cycles. A reduction ≥ 50% of the iFLC on day 8 of the first cycle was observed in 31 of 69 pts. These pts had a significantly better median PFS of 49 months as compared to 20 months in 38 pts with a lower iFLC reduction (p = 0.002). In contrast, OS did not differ significantly with a 48 months survival of 77% vs 69% (p > 0.05). CONCLUSION: These results indicate that a rapid decrease in the iFLC on day 8 is an early prognostic marker for newly diagnosed MM pts undergoing BPV treatment.

Improved CE(SDS)-CZE-MS method utilizing an 8-port nanoliter valve.

Capillary sieving electrophoresis utilizing SDS (CE(SDS)) is one of the most applied methods for the analysis of antibody (mAb) size heterogeneity in the biopharmaceutical industry. Inadequate peak identification of observed protein fragments is still a major issue. In a recent publication, we introduced an electrophoretic 2D system, enabling online mass spectrometric detection of generic CE(SDS) separated peaks and identification of several mAb fragments. However, an improvement regarding system stability and handling of the approach was desired. Here, we introduce a novel 8-port valve in conjunction with an optimized decomplexation strategy. The valve contains four sample loops with increased distances between the separation dimensions. Thus, successively coinjection of solvent and cationic surfactant without any additional detector in the second dimension is enabled, simplifying the decomplexation strategy. Removal efficiency was optimized by testing different volumes of solvents as presample and cationic surfactant as postsample zone. 2D measurements of the light and heavy chain of the reduced NIST mAb with the 8-port valve and the optimized decomplexation strategy demonstrates the increased robustness of the system. The presented novel set-up is a step toward routine application of CE(SDS)-CZE-MS for impurity characterization of proteins in the biopharmaceutical field.

Masson Trichrome and Sulfated Alcian Blue Stains Distinguish Light Chain Deposition Disease From Amyloidosis in the Lung.

Light chain deposition disease, characterized by nonamyloidogenic deposits of immunoglobulin light chains, is rare in the lung and possibly underdiagnosed due to low clinical suspicion and lack of readily accessible tests. We encountered a case of pulmonary light chain deposition disease (PLCDD) in which light chain deposits appeared crimson red with a Masson trichrome (MT) stain and salmon pink with a sulfated Alcian blue (SAB) stain. This prompted us to characterize a series of PLCDD cases and assess the utility of MT and SAB stains to distinguish them from amyloidosis. From the pathology archives of 2 institutions spanning 10 years, we identified 11 cases of PLCDD, including 7 diagnosed as such and 4 determined retrospectively. The deposits in all cases of PLCDD stained crimson red with MT and salmon pink with SAB, while the cases of pulmonary amyloid (n=10) stained blue-gray and blue-green, respectively. The immunoglobulin light chain nature of the deposits was confirmed in 10 of 11 cases by either immunofluorescence microscopy (n=5) or mass spectrometry (n=5). Transmission electron microscopy revealed osmiophilic, electron-dense deposits in all cases analyzed (n=3). An extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue type was diagnosed in 10 cases and 1 represented a plasma cell neoplasm. Our study highlights the importance of considering PLCDD in the differential diagnosis of amyloid-like deposits in the lung and the value of performing MT and SAB stains to distinguish between PLCDD and amyloidosis.

The impact of COVID-19 in the management of AL amyloidosis and Immunoglobulin Deposition Disease: A single-center experience.

INTRODUCTION: Patients with AL amyloidosis and immunoglobulin deposition diseases (IDD) are vulnerable during the COVID-19 pandemic due to the immune compromise from the plasma cell disorder and therapy-related immune defects. We describe a local experience in providing care for patients with AL amyloidosis and IDD. METHOD: Patient treatment and disease status since the beginning of the pandemic on March 11, 2020, as declared by WHO, were collected and analyzed. RESULTS: Ninety-six patients with AL amyloidosis and IDD were included. Four patients with IDD and 22 patients with systemic AL amyloidosis were receiving treatment during the pandemic. Since the pandemic, patients' treatments were discontinued if they achieved VGPR or better postinduction. Seven patients discontinued all treatment after achieving VGPR, and others required treatment modifications. 28 patients have been tested for COVID-19, and all tests have been negative. Three patients died since the pandemic, two from organ complications of systemic AL amyloidosis and one from an unrelated cause. CONCLUSION: The management of AL amyloidosis and IDD must be individualized on the clinical characteristics, centers' access to care under the pandemic restrictions, and the epidemiological aspects of the outbreak.

Persistent challenges with treating multiple myeloma early.

Over the past decade, 2 strategies have advanced the treatment of patients with multiple myeloma and its precursor diseases. First, the definition has changed to include patients without end organ damage, who previously would not have been treated. Second, there is widespread enthusiasm for treating high-risk, smoldering multiple myeloma. In this commentary, we explore the evidence supporting these therapeutic expansions. Although early treatment adds cost and therapeutic burden, it remains unknown whether survival and health-related quality of life are improved by early treatment. Herein, we consider the implications of diagnostic expansion in multiple myeloma.

Salivary free light chains as a new biomarker to measure psychological stress: the impact of a university exam period on salivary immunoglobulins, cortisol, DHEA and symptoms of infection.

INTRODUCTION: Measurement of immunoglobulin free light chains (FLCs) in saliva can serve as a non-invasive biomarker in health and behavioural research. FLCs have been explored in relation to physiological stress but FLC responses to psychological stress and their relationship with infections remain unknown. This study aimed to investigate the impact of exam period stress on salivary FLCs alongside other established biomarkers of stress and whether FLCs relate to symptoms of infection. METHODS: 58 healthy adults studying at university completed saliva samples and questionnaires in a period without exams (baseline), and again prior to the start of an exam period. Saliva samples were assessed for FLCs, IgA, cortisol and dehydroepiandrosterone (DHEA). Measures of life events stress, perceived stress, anxiety and depression were completed. Students also reported incidence and severity of symptoms of infection and rated general well-being at baseline, prior to, during and after the exam period. Exercise, sleep and alcohol consumption were also assessed at both timepoints. RESULTS: FLCs secretion rates were significantly lower at the exam period compared to baseline (p < .01), with reductions of 26% and 25% for κ FLC and λ FLC, respectively. In agreement, salivary IgA secretion rate was lower at exams (non-significant trend, p = .07). Cortisol concentration significantly increased at exams (p < .05) while DHEA did not change, leading to an increase in the cortisol:DHEA ratio (p = .06). Depression (p < .05) and anxiety increased from baseline to exams and life stress reported in the build up to the exam period was higher compared with baseline (p < .001). Well-being significantly decreased from baseline to exams (p < .01). The proportion of participants reporting infection symptoms (70%) was unchanged between baseline and prior to exams. No significant relationships were found between FLCs or other saliva parameters and infection symptoms, well-being or stress/psychological measures. Changes in saliva parameters between timepoints were independent of health behaviours. CONCLUSIONS: Salivary FLCs are responsive to life events stress and corroborate with IgA. This preliminary study highlights the potential utility of FLCs as a new salivary biomarker in stress research.