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1. Journal Club Presentation Dr. Ajit Kumar Singh MD(Laboratory Medicine )2 nd Year PGT Chittaranjan National Cancer Institute Moderator Dr. Subhranshu Mandal MD Microbiology Associate Professor Department of laboratory medicine Chittaranjan National Cancer Institute

2. Received April 19, 2022; accepted July 01, 2022. https://doi.org/10.1093/jalm/jfac068 © American Association for Clinical Chemistry 2022

3. About the authors Department of Pathology, Medical College of Georgia at Augusta University, Augusta, GA 30912, USA; Department of Laboratory Medicine , University of California at San Francisco, 1001 Potrero Ave., SFGH 5, San Francisco CA 94110, USA; Department of Population Health Sciences, Medical College of Georgia at Augusta University, Augusta, GA 30912, USA Gurmukh Singh Brooke M. Whitaker Roni J. Bollag Alan H. B. Wu Hongyan Xu

4. Critical Appraisal Of Title Contextual Clear Directional toward research PICO Population mentioned Intervention is well explained Comparator group are specified Outcome is clearly stated Reflect Aim and objective

5. Critical appraisal of abstract Structured abstract Indicative abstract Comprehensive in its contents Appropriate in its length Information abstracted from full article

6. Reason of choosing this article Relevant and novel nature SFLC assay is sensitive marker and has been included in the diagnostic criteria of MM and related disorders by IMWG(2014) Non – secretory multiple myeloma(NSMM)

7. Introduction In the process of immunoglobulin production by plasma cells and lymphocytes, light chains are generally produced in more than heavy chains The excess light chains circulate in blood and filtered by the glomerulus and are detectable in urine. Lambda light chains also occur as dimers (larger size) , longer half-life of lambda light chains as compared to kappa Monoclonal immunoglobulins are produced by neoplastic lymphocytes and plasma cells, plasma cell disorders

8. Introduction Oligoclonal immunoglobulin pattern detected in cerebrospinal fluid of patients with multiple sclerosis. Monoclonal immunoglobulins are also noted in AL amyloidosis (light chain amyloid), light and heavy chains deposition diseases , POEMS syndrome and TEMPI syndrome Some non-neoplastic auto-immune disorders associated with monoclonal immunoglobulins

9. Introduction Classical monoclonal gammopathies consist of monoclonal gammopathy of undetermined significance (MGUS), smoldering/asymptomatic myeloma (SMM), and multiple myeloma (MM). Variable amount of shift in the results for serum free light chains (SFLC) has been observed for the freelite assay, especially towards higher kappa/lambda ratio this has led to a risk of misdiagnosis Different SFLC assays are also variably affected by renal failure

10. Methodology and Study Design This study compares the Diazyme Laboratories' SFLC assay with the industry-standard Freelite assay DSFLCA is based on a latex-enhanced immunoturbidimetric assay Freelite assay based on Optilite analyzer platform utilizing latex particles coated with affinity-purified polyclonal antibodies to detect kappa and lambda free light chains

11. Methodology and Study Design This study was a combined effort of 3 medical school-affiliated medical centers and Diazyme Laboratories, a division of General Atomics Residual serum specimens from clinically indicated diagnostic and follow-up testing for monoclonal gammopathies were used for the study No specimens were collected solely for the purpose of the study Residual serum specimens were stored at−20 °C. Specimens were shipped to Diazyme Laboratories on dry ice for serum free light chain quantification by the Diazyme method

12. Inclusion and Exclusion Criteria Inclusion criteria included: Confirmed diagnosis of MM At least 3 serum specimens collected at different phases of illness and treatment Availability of clinical information to assess response level Exclusion criteria included: If fewer than 3 serum specimens were available and Patients had MGUS and SMM but a diagnosis of MM was not established.

13. Aim and objective Aim: Understanding the Significance of SFLC Assays Comparative Analysis of SFLC Assays Clinical Implications and Future Considerations Objective: The study compares the Diazyme Laboratories' SFLC assay with the industry-standard Freelite assay for monitoring monoclonal gammopathies, focusing on the role of assay in MM management

14. Clinical Practice Guidelines in Oncology on Multiple Myeloma The clinical status of patients was classified by criteria defined by the IMWG and Version 1.2020/National Comprehensive Cancer Network (NCCN) In this study, a total of 541 specimens were collected from 169 patient at - 3 sites, Augusta University-Medical College of Georgia (AU), San Francisco General Hospital (SFGH), and University of California San Francisco Health(UCSF).

15. Clinical Practice Guidelines in Oncology on Multiple Myeloma .

16. Result The response criteria established by clinicians are often based on multiple factors besides FLC results. FLC results evaluated alone might lead to errant response The clinical disease response categories were condensed into2 clinical status categories: “Progression” and “ Noprogression .” Patient with “Progression” consist of those with monitoring events defined as “ Progressive Disease”. Patient with “No Progression” consist of those with monitoring events defined as “Good Response, Moderate Response, and Stable Disease Response” categories.

17. Comparison of responses based on DSFLCA and Freelite assay

18. Discussion Clinical Implications : The implications of SFLC assays in diagnosing and monitoring plasma cell disorders, particularly in the context of MM, emphasizing their role in tracking disease progression and treatment response. Future Recommendations : Future advancements in SFLC quantification, highlighting the potential impact of assay drift on diagnosis and treatment decisions and the need for accurate measurements in managing MM. Operational Efficiency : Comparability of the Diazyme assay with the Freelite method, providing insights into the potential for front-end automation and improved operational efficiency, shaping the future landscape of SFLC assays in clinical practice.

19. Conclusion The results of the Diazyme Human Kappa and Lambda FLC assay showed good concordance with IMWG/NCCN clinical assessment and the results of the industry standard, Binding Site Freelite assay.

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21. What are free light chains (FLC)? Free light chains are produced by plasma cells - these are the cells responsible for the production of antibodies, also called immunoglobulins. An antibody is made up of heavy chains and light chains. The heavy and light chains are bound together to form an intact immunoglobulin. These two proteins are produced independently and assembled within the plasma cells. The light chains are produced in excess and therefore they can be found also as free proteins in the serum. This is why they are called serum free light chains. Free light chains are an excellent biomarker to evaluate the uncontrolled growth of plasma cells, typical of blood cancers such as MM, as they can often be found at concentrations proportional to the tumour burden

22. What are Freelite assays? Freelite assays are based on polyclonal antisera, targeting kappa and lambda free light chains respectively. Freelite assays are highly sensitive and specific. The analytical sensitivity and specificity of Freelite assays is achieved by the use of latex particles coated with affinity purified polyclonal antibodies. Freelite assays can be used in serum, EDTA and lithium heparin plasma, urine, and cerebrospinal fluid (CSF) using the Binding Site Optilite ® automated Analyser .

23. The normal reference ranges for serum free light chains ( sFLC ) are generally as follows: Kappa Free Light Chains: 3.3−19.4 mg/L1 Lambda Free Light Chains: 5.71−26.3 mg/L 1Kappa/Lambda Ratio: 0.26−1.65

24. The Freelite assay is a laboratory procedure that involves the following steps: Sample Collection: A blood sample is taken from the patient, typically in an outpatient setting Quantitative Analysis: The sample is sent to a laboratory where it undergoes quantitative analysis to measure the levels of kappa and lambda serum free light chains Calibration Curve: Initially, a series of calibrators with known antigen concentrations are assayed to produce a calibration curve. This curve plots measured light scatter against antigen concentration Sample Assay: Samples with unknown antigen concentrations are then assayed, and the results are interpreted using the calibration curve

25. in serum, plasma, and urine, the assay can aid in the diagnosis and monitoring of monoclonal gammopathies, such as: MM including light chain multiple myeloma, oligo-secretory Multiple Myeloma and non-secretory Multiple Myeloma, AL amyloidosis Lymphocytic neoplasms Waldenström's macroglobulinaemia (WM) Light chain deposition disease (LCDD)

26. Introduction The heavy chains are alpha, gamma, mu, delta, and epsilon for IgA, IgG, IgM, IgD , and IgE , respectively. Light chains may be kappa or lambda. One immunoglobulin has only one type of heavy and one type of light chain Through a combination of DNA rearrangement, permutation, and combination of heavy and light chains and somatic hypermutation during maturation of antibody producing cells, it is possible to generate over10 16 different types of immunoglobulins

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