Urine Immunofixation Electrophoresis for Diagnosis of Monoclonal Gammopathy: Evaluation of Methods for Urine Concentration.

BACKGROUND: Examination of urine by immunofixation electrophoresis (UIFE) is one of the tests recommended for screening and monitoring of monoclonal gammopathies, especially multiple myeloma. Unlike the serum free light chain measurement, a positive result on urine immunofixation is diagnostic for monoclonal immunoglobulin light chains. Urine is usually concentrated, generally by membrane filtration, prior to electrophoresis. METHODS: Alternative methods to membrane filtration for urine concentration were examined. Residual urine specimens submitted for urine protein electrophoresis were concentrated by precipitation of the proteins by ammonium sulfate salt precipitation, precipitation with ethanol and acetonitrile, and by desiccation. The concentrated specimens were subjected to immunofixation electrophoresis using antisera to free light chains (FLC). The results were compared with those from conventional immunofixation electrophoresis using specimens concentrated by membrane filtration. RESULTS: Ammonium sulfate, ethanol, and acetonitrile precipitation results were less than satisfactory. Concentration by desiccation provided results comparable, if not better than, those by membrane filtration and conventional UIFE. The cost of desiccation is minimal compared to more than $5.00/specimen cost of concentration by membrane filtration. The differences in the results with conventional UIFE and the method described here are likely due to (a) variability in the reactivity of different antisera to free monoclonal light chains, and (b) obscuration of monoclonal free light chains by co-migration with intact immunoglobulin monoclonal proteins. CONCLUSIONS: Concentrating urine by desiccation for immunofixation electrophoresis is technically simple, inexpensive, and provides results comparable to concentrating by membrane filtration. Using FLC provides a more sensitive assay than using conventional antisera.

Long-term alliance for pathology equipment and technology acquisition: a paradigm shift for improved quality and efficiency.

OBJECTIVE: Personnel costs are the largest single budget item in the clinical laboratory, other major expenses being equipment, analyzers, blood and blood components, and cost of day-to-day consumables. This report describes our experience with developing a long-term relationship with a single major vendor as a paradigm shift from the traditional multiple vendors, multiple contracts, and recurrent extended negotiations. Our objective was to develop a long-term approach for replacement of effete equipment and upgrades to operations in a pathology and laboratory medicine department in collaboration with vendors providing equipment and services. METHODS: Major vendors were invited to visit the department to analyze the workload and workflow and suggest integrated solutions to meet the goals of the department. Multiple iterations of the proposals were evaluated, and a recommendation made to the medical center leadership. The vendor, the medical center, and the department jointly developed a 15-year partnership plan to improve the operations of pathology services. The agreement encompasses a range of management and performance criteria for both sides. The salient items discussed were laboratory staffing, turnaround time, workload change, test insourcing, reference laboratory costs, and scholarly productivity and teaching. RESULTS: The agreement reduced laboratory staffing by 21%, eliminated stat tests by reducing the turnaround time for routine tests to less than 45 minutes for 90% of tests, with an increase of 9.1% in the number of tests, Cost avoidance in salary and reference laboratory costs was $3,424,136/year against an expected target of $2 million in total savings, despite not including cost avoidance from promoting appropriate use of laboratory testing for inpatients and increase in revenue from increase in ambulatory testing. Vizient score in laboratory utilization improved from the 94th to 76th percentile. Scholarly output increased by more than 100%. CONCLUSION: This model of a long-term alliance with a chosen vendor led to improvements in quality and efficiency.

Quantification of Free Immunoglobulin Light Chains in Urine.

BACKGROUND: The serum-free immunoglobulin light chain assay has been recommended as a screening test for monoclonal gammopathy. We evaluated the usefulness of urine free immunoglobulin light concentration for selection of specimens for immunofixation electrophoresis. METHODS: Using kits from The Binding Site for Freelite ®, we validated examination of urine for measuring free κ and λ light chains. The results of urine free light chain concentrations were evaluated to ascertain if the results could be used to reduce the number of specimens requiring urine protein immunofixation electrophoresis. RESULTS: In the 515 specimens examined, there was no evidence of monoclonal gammopathy or history of monoclonal gammopathy in 331. Monoclonal κ or λ light chains were detectable in 42 and 30 specimens, respectively. There was history of κ or λ chain associated monoclonal gammopathy in 62 and 50 patients, respectively. In the 38 monoclonal κ positive urine specimens, with light chain data, κ/λ ratio was >5.83 in all specimens. In 27 specimens positive for monoclonal λ light chains, with light chain data, the urine λ/κ ratio was > 0.17 in 24 of 27 specimens and > 0.041 in all specimens. In patients without monoclonal gammopathy all specimens had a κ/λ ratio of >5.83 or λ/κ ratio >0.17. CONCLUSIONS: The Freelite ® assay from The Binding Site is suitable for quantification of free light chains in urine. In patients with known history of monoclonal gammopathy, urine immunofixation electrophoresis may be omitted in specimens with κ/λ ratio of <5.83 for κ associated lesions and λ/κ ratio of <0.041 for λ associated lesions. However, the results do not support using this test for first-time urine testing for monoclonal light chains as it is not predictive of positive result, nor does it exclude a monoclonal light chain in urine.

Urine Protein Immunofixation Electrophoresis: Free Light Chain Urine Immunofixation Electrophoresis Is More Sensitive than Conventional Assays for Detecting Monoclonal Light Chains and Could Serve as a Marker of Minimal Residual Disease.

BACKGROUND: Immunoglobulin monoclonal light chains (MLCs) in serum and urine are markers for monoclonal gammopathy and could serve as markers of minimal residual disease (MRD) in multiple myeloma (MM). Excretion of MLCs in urine is known to result in renal damage and shorter survival in patients with LC-predominant MM. METHODS: Retrospective review of urine immunofixation in 1738 specimens at 3 medical centers was conducted to assess the utility of urinalysis for diagnosis and monitoring of monoclonal gammopathy. We tested 228 stored urine specimens via the modified urine immunofixation method, using antisera to assay free LCs (FLCs). RESULTS: Our review of urine immunofixation results and medical records validated the theory that the only meaningful value-added finding was detection of monoclonal free light chains. Examination of 228 urine specimens using our novel method revealed 18.4% additional positive results. The rate of incremental findings for lambda LCs was nearly 3-fold higher than for kappa LCs. CONCLUSIONS: The new method of urine immunofixation is significantly more sensitive and more efficient than the conventional method for detecting MLCs in urine. The new assay appears to be sensitive enough to prove that MLCs serve as a marker of MRD in MM.

Pathology Job Search and Interview: Perspectives on the United States Experience.

CONTEXT.­: Pathology training is focused on the attainment of clinical, diagnostic, and administrative skills. Preparation for employment search and the interview process are often neglected. Given that a near majority of pathology trainees in the United States are graduates of foreign medical schools, training in the job search and interview process according to local customs, norms, and expectations has greater salience for individuals new to the United States. OBJECTIVE.­: To offer perspectives on 2 components of the job search process: (1) finding a suitable job opening in academic and private practice settings and (2) preparing for an interview. We have provided a set of common interview questions and suggested preparatory methodology. The differences in the process and expectations in academic settings and private practice operations are highlighted. Engaging in the job search process early and networking are emphasized. We have also suggested approaches for pathology teachers and mentors in guiding trainees in a job search and preparation for an interview. DATA SOURCES.­: The information and opinions expressed in this communication are based on the personal experiences of 4 senior pathologists in academic and private practice settings. CONCLUSIONS.­: Start networking early. Leverage contacts with teachers, attending pathologists, senior residents, and people at national meetings to locate appropriate job opportunities. Seek assistance from attending pathologists in preparing a curriculum vitae and cover letter. Prepare for the questions that may come up in an interview. A dress rehearsal for an interview is strongly recommended.

Serum Free Light Chain Quantification Testing: Comparison of Two Methods for Disease Monitoring.

BACKGROUND: Levels of free immunoglobulin light chains in serum and urine are a sensitive measure of dysregulated immunoglobulin synthesis. The development of an assay for free light chains in serum was a major advance in laboratory testing for monoclonal gammopathies. The original assay by The Binding Site, called Freelite®, has been in common use in laboratory monitoring of monoclonal gammopathies. Two clinical entities, myeloma-defining condition and light chain-predominant multiple myeloma, rely on quantitative measurements of serum free light chains. METHODS: Using polyclonal antisera specific to free light chains, Diazyme Laboratories developed a latex immunoturbidimetric assay for quantification of human kappa and lambda serum free light chains. We evaluated the Diazyme assay by comparing the results of kappa and lambda free light chain quantification, and kappa/lambda ratio with the results on the same specimens by the Freelite method. We also compared the correlation of the 2 methods to evaluate response to treatment and to changes in clinical status of patients with multiple myeloma. RESULTS: The results of Freelite and Diazyme methods are comparable. There was no statistically significant difference in the performance of the 2 assays for quantification of light chains, kappa/lambda ratio, or correlation of clinical parameters from patients with multiple myeloma at various stages of monitoring the disease in 2 geographically diverse laboratory and clinical environments. CONCLUSIONS: The Diazyme method is comparable to Freelite and provides an opportunity to add the test to front-end automation and improvement in efficiency of the assay.

Light Chain-Predominant Multiple Myeloma Subgroup: Impaired Renal Function Correlates with Decreased Survival.

OBJECTIVE: Patients with light chain-predominant multiple myeloma have been shown to exhibit shorter survival. Retrospective comparison of clinical and laboratory data was undertaken to ascertain the likely cause(s) of this observation. METHODS: Records of patients with multiple myeloma seen at 1 institution revealed 316 patients with conventional and 71 patients with light chain-predominant multiple myelomas with secretion of intact immunoglobulins. Laboratory and clinical findings in the 2 groups were compared. RESULTS: Patients with light chain-predominant multiple myeloma had a significantly higher death rate, a higher rate of chronic dialysis, a lower estimated glomerular filtration rate and serum albumin, a significantly higher urine protein concentration, and a significantly higher prevalence of hypertension and blood transfusion requirements. Other clinical and laboratory parameters surveyed were not significantly different between the 2 groups. CONCLUSION: The shorter survival of patients with light chain-predominant multiple myeloma is clearly associated with renal damage caused by excess free immunoglobulin light chains. Renal damage may be ameliorated by early aggressive treatment with chemotherapy, plasmapheresis, and dialysis; a multi-institutional prospective controlled trial would be needed to test this hypothesis.

Accurate Quantification of Monoclonal Immunoglobulins Migrating in the Beta Region on Protein Electrophoresis.

BACKGROUND: The concentration of monoclonal immunoglobulins (Igs) in neoplastic monoclonal gammopathic manifestations is generally measured by densitometric scanning of the monoclonal peaks on gel or by measuring absorbance at 210 nm in capillary electrophoresis (CE). For monoclonal Igs migrating in the beta region, measurement is complicated by the major beta-region proteins, namely, transferrin and C3. METHODS: C3 interference in densitometry was eliminated by heat treatment of serum, and monoclonal Igs were quantified by densitometry of the residual band. The immunochemical measurement of transferrin was converted to its equivalent densitometric quantity. For monoclonal Ig migrating with transferrin, the contribution of the latter was removed by subtracting the converted transferrin concentration from the combined densitometric quantification of the band. With CE, monoclonal Ig was measured by using immunosubtraction (ISUB) to guide demarcation. RESULTS: The results obtained using the C3 depletion and transferrin subtraction method were lower and yet comparable to the results derived from using CE measurement guided by ISUB. As we expected, the results from both methods were lower than those derived from a perpendicular drop measurement of the peak or via nephelometric assay of the involved isotype. DISCUSSION: Accurate measurement of monoclonal Igs is important for the diagnosis and monitoring of monoclonal gammopathic manifestations. Determination of serum free light chain concentration per gram of monoclonal Ig is an essential measure for the diagnosis of light chain-predominant multiple myeloma. The method described herein improves accuracy of measurements for monoclonal Igs migrating in the beta region, without the need for special reagents or equipment.

Multiple myeloma: Detection of free monoclonal light chains by modified immunofixation electrophoresis with antisera against free light chains.

INTRODUCTION: Neoplastic monoclonal gammopathies are frequently associated with production of excess free monoclonal light chains. A sensitive method for detecting free monoclonal light chains in serum could provide a marker for residual/minimal residual disease and as an adjunct to serum protein electrophoresis to serve as a screening method for monoclonal gammopathies. METHODS: Conventional serum immunofixation electrophoresis was modified by applying undiluted serum samples, and staining for serum free light chains with antisera specific to free light chains. Washing/blotting of gels was enhanced to remove residual proteins that did not bind to the antibodies including intact monoclonal immunoglobulins. Results from this modified immunofixation electrophoresis were compared with results from commercially available MASS-FIX/MALDI assay. RESULTS: Monoclonal free kappa light chains could be detected to a concentration of about 1.78 mg/L and monoclonal free lambda light chains to a concentration of about 1.15 mg/L without the need for special equipment. Comparison of these thresholds with parallel results from a commercially available MASS-FIX/MALDI assay revealed the modified electrophoretic method to be more sensitive in the context of free monoclonal light chains. CONCLUSIONS: Modified serum immunofixation electrophoresis has been shown to detect low levels of monoclonal free light chains at a sensitivity suitable for the method to be used in detecting minimal residual disease, and potentially in a screening assay for monoclonal gammopathies. The disparity in the results with commercially available MASS-FIX/MALDI assay is postulated to be due to limited repertoire of reactivity of nanobodies of camelid origin.

Light Chain Multiple Myeloma: High Serum Free Light Chain Concentrations Portend Renal Damage and Poorer Survival.

BACKGROUND: Monoclonal immunoglobulins provide an indication of the tumor burden in patients with plasma cell neoplasms. Higher concentrations of serum free light chains in light chain predominant multiple myeloma have been shown to correlate with a poorer outcome. We examined the correlations of serum free light chain concentrations in light chain myelomas with survival, estimated glomerular filtration rate (eGFR) and other clinical and pathological parameters. METHODS: Records of patients with light chain multiple myelomas were reviewed. Highest concentration of serum free light chains for each patient were plotted to ascertain an inflection/change point. Survival, eGFR, and other clinical and pathological parameters were compared between the low and high light chain concentration groups. RESULTS: Plotting serum free light chain concentrations revealed an inflection point at a concentration of 455 mg/L apportioning patients in to 2 subgroups: 39 patients with low light chain concentrations and 26 patients with high concentrations. The high concentration group had more unfavorable pathology in bone marrow examination in terms of higher neoplastic plasma cell burden and high-risk cytogenetics. The survival rate and eGFR in the high concentration group were significantly worse than in the low concentration group. CONCLUSIONS: As noted for light chain predominant multiple myeloma, high serum free light chain concentration in light chain multiple myelomas are associated with higher renal disease burden and shorter survival. Monitoring of serum free light chain concentrations and customizing treatments to address this parameter are warranted.

A rapid near-patient detection system for SARS-CoV-2 using saliva.

The highly infectious nature of SARS-CoV-2 necessitates the use of widespread testing to control the spread of the virus. Presently, the standard molecular testing method (reverse transcriptase-polymerase chain reaction, RT-PCR) is restricted to the laboratory, time-consuming, and costly. This increases the turnaround time for getting test results. This study sought to develop a rapid, near-patient saliva-based test for COVID-19 (Saliva-Dry LAMP) with similar accuracy to that of standard RT-PCR tests. A lyophilized dual-target reverse transcription-loop-mediated isothermal amplification (RT-LAMP) test with fluorometric detection by the naked eye was developed. The assay relies on dry reagents that are room temperature stable. A device containing a centrifuge, heat block, and blue LED light system was manufactured to reduce the cost of performing the assay. This test has a limit of detection of 1 copy/µL and achieved a positive percent agreement of 100% [95% CI 88.43% to 100.0%] and a negative percent agreement of 96.7% [95% CI 82.78-99.92%] relative to a reference standard test. Saliva-Dry LAMP can be completed in 105 min. Precision, cross-reactivity, and interfering substances analysis met international regulatory standards. The combination of ease of sample collection, dry reagents, visual detection, low capital equipment cost, and excellent analytical sensitivity make Saliva-Dry LAMP particularly useful for resource-limited settings.

Immune subtraction for improved resolution in serum protein immunofixation electrophoresis and antibody isotype determination in a patient with autoantibody.

Heavy chain isotypes of low level monoclonal immunoglobulins are sometimes obscured in serum immunofixation electrophoresis (SIFE) by a heavy background of polyclonal immunoglobulins. However, accurate determination of the heavy chain isotype is essential for a complete diagnosis, as isotype determination of autoantibodies may have relevance in determining therapeutic procedures. Immune subtraction (IS) was employed in a patient with neuropathy and GD1a autoantibody. IS allowed identification of the cognate heavy chain related to a lambda light chain restriction noted on initial SIFE as well as isotype determination of the autoantibody. Antisera specific to individual heavy and light chains were used for depletion of specific immunoglobulin types. Depletion of kappa light chain associated immunoglobulins allowed unequivocal determination of the isotype of lambda light chain-associated low level monoclonal band to be IgG Lambda. Selective depletion of kappa, lambda, gamma and mu heavy chain immunoglobulins was employed to determine IgG Kappa isotype of the auto-antibody.

Engaging Pathology Residents in Clinical Chemistry: The Essential Ingredient Is a Committed Teacher.

BACKGROUND: Pathology residents are thought to show a lack of interest in clinical chemistry, therefore potentially graduating from training programs unprepared to function as laboratory directors and clinical consultants. METHODS: A structured program of tutorials based primarily on Henry's textbook, supplemented by recent review articles; a question bank of about 600 questions to emphasize key concepts; requirement for performing and presenting quality improvement projects; participation in on-site CAP inspections; review of reference laboratory test requests; and involving residents in scholarly activity have resulted in sustained, transferable, and significant improvements in engagement, knowledge, competence, and examination scores. RESULTS: The primary parameter for measuring change in resident competence and engagement were improvements in resident in-service examination (RISE) scores, publications in peer-reviewed journals, and receipt of awards. The revised program produced significant improvement in RISE scores in clinical chemistry, over and above the improvements in the general residency program. The residents were authors on 12 publications in peer-reviewed PubMed listed journals in the 5-year period since revision in the clinical chemistry curriculum compared to no publications in clinical chemistry in the 5-year period before the new curriculum. Over the past 2 years, 6 of the 11 publications by graduating residents were in clinical chemistry, and 6 of 7 awards for research were garnered by residents engaged in clinical chemistry investigations. All of the residents passed their clinical pathology boards on first attempt since the change compared to 2 failures in the prior 5-year period. CONCLUSIONS: The structured program described here is important as a template that could be adopted by any pathology training program. The question bank developed by this program is a valuable and transferable aid. However, success of such a program is dependent on the commitment of a knowledgeable, dedicated, and passionate teacher.

Analysis of Multiple Bands on Serum Protein Immunofixation Electrophoresis: Challenge in Interpretation of Clonality in a Patient with Light Chain-Predominant Multiple Myeloma.

Sera from patients with multiple myeloma usually display a single monoclonal immunoglobulin band on serum protein immunofixation electrophoresis. Multiple bands may be seen if the myeloma is bi- or triclonal or if the monoclonal immunoglobulin has rheumatoid factor activity. We describe a patient with light chain-predominant IgA lambda myeloma; the patient's serum displayed 2 spatially distinct bands reacting for alpha heavy and lambda light chains. The methods used to establish monoclonality are addressed.

Can EEG Predict Response to Atomoxetine in attention deficit hyperactivity disorder at 1 Week?

Objective: The objective of the study is to predict the early changes in electroencephalography (EEG) at 1 week and its correlation to clinical response at 6 weeks after treatment with atomoxetine in children with ADHD. Method: In 50 children (6-14 years) with ADHD (Diagnostic and Statistical Manual of Mental Disorders [5th ed.; DSM-5]), Vanderbilt ADHD Parent Rating Scale (VADPRS) and Vanderbilt ADHD Teachers Rating Scale (VADTRS) were applied at baseline, 1, 4, and 6 weeks. EEG was recorded using International 10-20 System of electrode placement at baseline and at 1 week after atomoxetine treatment. EEG changes at 1 week after atomoxetine therapy was correlated to clinical response at 6 weeks. Results: Patients were classified as responders or nonresponders based on the VADPRS/VADTRS findings. After 1 week of treatment, responders' theta cordance values were decreased, whereas nonresponders' values didn't decrease significantly. Conclusion: Patients with decreased theta cordance values, especially in the left temporoparietal region, at 1 week were likely to respond to atomoxetine while those without any such change were likely to be nonresponders.

Skene's Gland Adenocarcinoma: Borrowing From Prostate Cancer Experience for the Evaluation and Management of a Rare Malignancy.

OBJECTIVE: To determine if adenocarcinoma of the Skene's glands in women, which has a histological and immunohistochemical appearance similar to prostate cancer, can be evaluated and managed with the same tools we use for prostate cancer. METHODS: Serum prostate-specific antigen kinetics, 3D multiparametric (MP) magnetic resonance imaging (MRI), fluciclovine F-18 positron emission tomography (PET), and androgen deprivation therapy (ADT) were employed in a case of Skene's gland adenocarcinoma. RESULTS: The 3D MP MRI clarified the anatomy of the primary lesion and fluciclovine F-18 PET significantly improved our ability to stage the tumor prompting pelvic lymph node dissection that may have otherwise not been performed. ADT resulted in a significant impact on prostate-specific antigen kinetics despite the patient having a testosterone level in the normal range for a postmenopausal woman. CONCLUSION: Despite the rarity of Skene's gland adenocarcinoma, we can employ many of the tools at our disposal for the evaluation and management of prostate cancer to benefit the women found to have this malignancy.

Light Chain Predominant Intact Immunoglobulin Monoclonal Gammopathy Disorders: Shorter Survival in Light Chain Predominant Multiple Myelomas.

BACKGROUND: A proportion of intact immunoglobulin (Ig)-producing multiple myelomas (MMs) was observed to secrete much higher amounts of free light chains (LCs) than usual. OBJECTIVES: To determine the change point between usual and LC-predominant intact Ig-secreting MMs and other monoclonal gammopathic manifestations and the biological significance of the observation. METHODS: We conducted retrospective examination of laboratory findings in 386 MM, 27 smoldering MM, and 179 monoclonal gammopathy of undetermined significance (MGUS) cases that secreted intact Igs. We recorded the highest levels of involved serum free LC, highest ratio of involved to uninvolved LC, highest concentration of involved LC per g of monoclonal Ig, and highest value for ratio of involved to uninvolved LCs divided by the monoclonal Ig concentration. Each data set was sorted into kappa- and lambda LC-associated lesions. Length of time, in months, between diagnosis and last contact with the patients having myeloma was recorded. RESULTS: Change point analysis of data revealed a subgroup of cases with distinctly higher levels of free LCs. In myelomas, including plasma cell leukemias, 16.4% of myelomas with kappa LCs and 22.3% of myelomas with lambda LCs, the LC secretion was distinctly higher than in the remaining cases, by a combination of 4 parameters, listed herein. Corresponding figures for smoldering myeloma (SMM) and monoclonal gammopathy of undetermined significance (MGUS) were 12.5, 27.3, 3.8, and 6.8, respectively. Ten of the 13 (77%) cases of plasma cell leukemia) and all cases of IgD myeloma (n = 4) showed excess secretion of serum free LCs. Among IgG and IgA myelomas, including plasma cell leukemias, the LC-predominant lesions had shorter survival, by an average of 22.5 months. CONCLUSIONS: In total, 18.4% of MMs, including plasma cell leukemias, secrete distinctly higher amounts of serum free LCs than other intact Ig-secreting myelomas and confer significantly lower survival. Quantification of monoclonal serum free LCs may be useful in this subgroup in monitoring progress and potentially in ascertaining minimal residual disease. The findings also stress the need for separate criteria for kappa and lambda LC associated monoclonal gammopathic manifestations. The significantly shorter survival of patients with LC-predominant myelomas warrants consideration in prospective trials of treatments.