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.

C3 Glomerulonephritis Associated with Monoclonal Gammopathy of Renal Significance.

INTRODUCTION: Monoclonal gammopathy of renal significance (MGRS) is described as a hematologic condition characterized by nephrotoxicmonoclonal proteins produced by a non-malignant B-cell or plasma cell clone. Nevertheless, MGRS can cause serious renal lesions, leading to high morbidity. In C3 glomerulonephritis, a monoclonal protein can cause renal damage indirectly. Acting as an autoantibody, the protein cannot be detected in the kidney biopsy, promoting the dysregulation of the alternative pathway of the complement system. MATERIAL AND METHODS: This non-systematic review was based on a comprehensive search in databases and scientific journals, such as PubMed, Nature Reviews Nephrology and Kidney International, including the terms 'C3 Glomerulonephritis' and 'Monoclonal gammopathy of renal significance'. We review the pathophysiology, presentation, diagnosis, differential diagnosis and treatment of C3 glomerulonephritis associated with MGRS. DISCUSSION: With the increasing understanding of the complex interaction between monoclonal gammopathy and renal damage, such as C3 glomerulonephritis, it becomes clear that an early recognition is crucial, as Ig-directed therapy might improve outcomes. In this context, and in order to maximize the chance of a correct diagnosis, renal biopsy is mandatory to determine the exact nature of the lesion, and the severity of renal disease. Conclusion: It is important to make an early diagnosis of MGRS-associated C3 glomerulonephritis in order to prevent not only the progression to a hematological malignancy, but also end-stage renal disease.

Introdução: A gamopatia monoclonal de significado renal (MGRS) é descrita como uma doença hematológica caracterizada pela existência de proteínas monoclonais nefrotóxicas produzidas por um clone não maligno de células B ou plasmócitos. A MGRS pode causar lesões renais graves, levando a elevada morbilidade. Na glomerulonefrite C3, a proteína monoclonal pode causar indiretamente lesão renal. A proteína atua como auto-anticorpo, não sendo detetada na biópsia renal, promovendo a desregulação da via alternativa do complemento. Material e Métodos: Esta revisão não sistemática foi baseada numa pesquisa abrangente com recurso a base de dados e revistascientíficas, como a PubMed, Nature Reviews Nephrology e Kidney International, utilizando os termos 'Glomerulonefrite C3' e 'Gamopatia monoclonal de significado renal'. Apresentamos uma revisão da fisiopatologia, apresentação clínica, diagnóstico, diagnóstico diferencial e tratamento de glomerulonefrite C3 associado a MGRS Discussão: Com a crescente compreensão da complexa interação entre a gamopatia monoclonal e a lesão renal, como é exemplo a glomerulonefrite C3, torna-se claro que um reconhecimento precoce é crucial, dado que a terapia dirigida à Ig pode melhorar o resultado. Neste contexto, para maximizar a probabilidade de um diagnóstico correto, uma biópsia renal é necessária para determinar a natureza exata da lesão e a severidade da doença renal. Conclusão: É importante realizar um diagnóstico precoce de glomerulonefrite G3 associada a MGRS de modo a prevenir não apenas a progressão para uma neoplasia hematológica, mas também para doença renal terminal.

Epidemiological and immunochemical parameters of monoclonal plasma cell dyscrasias of 2121 cases in Algeria.

BACKGROUND: Plasma cell dyscrasias (PCD) are a heterogeneous group of diseases characterized by the expansion of monoclonal bone marrow plasma cells that produce a monoclonal immunoglobulin (M-component). PURPOSE: This is a retrospective study that describes the epidemiological, immunochemical features and etiology of monoclonal gammopathies diagnosed between 1998 and 2016 in the Teaching Hospital Beni-Messous of Algiers. PATIENTS AND METHODS: 2121 cases of monoclonal gammopathies (MG) were collected during this period. Serum/urine protein electrophoresis, serum/urine immunofixation and serum free light chain measurements were used to demonstrate M protein. RESULTS: The middle age of the patients at the time of the diagnosis were 62.96 ± 13.19 years with extremes ranging from 07 to 99 years. The study included 1013 (47, 76 %) men and 1108 (52, 23 %) women with a sex ratio 0,91. Isotypes repartition was: IgG (60.91 %), IgA (17.91 %), light chain (10.46 %), IgM (6.6 %), IgD (1.03 %) and IgE (0.09 %) of cases. The most frequent diagnosis was: Multiple Myeloma (55.20 %), followed by monoclonal gammopathy of undetermined significance (34.13 %). CONCLUSION: In our study, two particularities were noted. There is no male predominance in Algerian PCD patients. Moreover, we observed a higher frequency of light chain multiple myeloma and lower frequency of IgM isotype compared to western studies.

Concordance of Capillary Electrophoresis and Conventional Gel Electrophoresis in Two Different Groups of Patients with.

BACKGROUND: Serum and urinary protein electrophoresis play an important role in the identification of monoclonal gammopathy. Recently, capillary electrophoresis (CE) has been adapted in many clinical laboratories because of several advantages such as short turnaround time, automation, and high reproducibility. However, there have been unsolved concerns for the concordance between conventional gel and automated capillary electrophoresis methods for protein separation in clinical specimens. In this study, we investigated the diagnostic performance of both methods for detecting monoclonal (M) protein. METHODS: From February 2012 to August 2015, a total of 3,013 CE tests were performed in our hospital. Among these cases, we reconfirmed results of CE (Capillary 2, Sebia, Lysse, France) with those of conventional agarose gel electrophoresis (GE) (Hydragel 4IF, Sebia, Lisses, France) in 28 specimens from 24 patients with newly diagnosed monoclonal gammopathy (group 1). In addition, 22 cases from 15 patients with previously diagnosed monoclonal gammopathy presenting indeterminate or suspicious results on CE (group 2) were also reconfirmed with GE. RESULTS: We compared the results between the two electrophoresis methods in two different groups of patients with newly diagnosed discrete monoclonal peaks vs. pre-existing monoclonal gammopathy with obscure results in follow-up courses. In group 1, agreement rate was 100% (28/28) and there was no discrepant result between these two electrophoresis methods. In contrast, group 2 showed 86.4% (19/22) agreement rate and 0.67 Cohen's kappa value (95% confidence interval, 0.51 - 1.02). CONCLUSIONS: According to our results, both electrophoresis methods can be used with the same level of assurance at the time of initial diagnosis for monoclonal gammopathy. However, in patients with previously diagnosed monoclonal gammopathy in follow-up course after appropriate treatments, discordant results can be observed due to the reduced amount of M proteins. Therefore, we suggest that some ambiguous cases with very small amounts of M components require a combination of both CE and GE methods for accurate interpretation to confirm the presence of M proteins.

Laboratory testing in monoclonal gammopathy of renal significance (MGRS).

Recently, monoclonal gammopathy of renal significance (MGRS) reclassified all monoclonal (M) gammopathies that are associated with the development of a kidney disease but do not meet the definition of symptomatic multiple myeloma (MM) or malignant lymphoma. The purpose was to distinguish the M gammopathy as the nephrotoxic agent independent from the clonal mass. The diagnosis of MGRS obviously depends on the detection of the M-protein. More importantly, the success of treatment is correlated with the reduction of the M-protein. Therefore, familiarity with the M-protein tests is a must. Protein electrophoresis performed in serum or urine is inexpensive and rapid due to automation. However, poor sensitivity especially with the urine is an issue particularly with the low-level M gammopathy often encountered with MGRS. Immunofixation adds to the sensitivity and specificity but also the cost. Serum free light chain (sFLC) assays have significantly increased the sensitivity of M-protein detection and is relatively inexpensive. It is important to recognize that there is more than one assay on the market and their results are not interchangeable. In addition, in certain diseases, immunofixation is more sensitive than sFLC. Finally, novel techniques with promising results are adding to the ability to identify M-proteins. Using the time of flight method, the use of mass spectrometry of serum samples has been shown to dramatically increase the sensitivity of M-protein detection. In another technique, oligomeric LCs are identified on urinary exosomes amplifying the specificity for the nephrotoxic M-protein.

A Patient with Abnormal Kidney Function and a Monoclonal Light Chain in the Urine.

Monoclonal gammopathy is increasingly recognized as a cause of kidney injury. These renal conditions behave differently than ones without monoclonal gammopathy and require specific treatment. To avoid misdiagnosis, testing for paraprotein should be performed in addition to vasculitis and autoimmune diseases serologies in adults with unexplained AKI or proteinuria. Because the prevalence of monoclonal gammopathy is much more common than glomerular diseases, the nephrotoxicity of the monoclonal protein must be confirmed before cytotoxic therapy is initiated. This can only be done by a kidney biopsy. After a monoclonal gammopathy of renal significant is verified, the evaluation should then focus on the identification of the pathologic clone, because therapy is clone specific. We present this patient to illustrate the clinical presentation of a patient with renal dysfunction and a monoclonal gammopathy. This patient is also used to discuss the diagnostic process in detail when monoclonal gammopathy-associated renal disease is suspected.

Strengths and weaknesses of methods for identifying monoclonal free light chains of Ig: examples from two cases with renal disease.

BACKGROUND: Serum free light chain (FLC) analysis with ratio and urine immunofixation electrophoresis (IFE) are both available for routine use in helping to detect plasma cell dyscrasia and related diseases. CASES: Case reports showing one serum positive for serum FLC but that showed a hook effect and overestimated the amount of monoclonal FLC while urine IFE was negative for Bence Jones protein, and a second serum that showed elevated FLC κ and λ but a normal κ/λ ratio, while urine IFE was positive for Bence Jones protein. CONCLUSIONS: These two techniques complement one another. Neither of the techniques is truly quantitative, and both exhibit methodological defects.

Screening immunofixation should replace protein electrophoresis as the initial investigation of monoclonal gammopathy: Point.

The reliable detection of paraprotein in serum and urine is the primary purpose of electrophoretic procedures in clinical laboratories. Screening immunofixation electrophoresis (sIFE) employs a single application of antisera directed against heavy and light chains that facilitates the detection of paraproteins that migrate in the non-γ region or that are below the detection limit of protein electrophoresis. These paraproteins that are missed by routine electrophoresis occur in up to 27.3% of newly investigated and 13.6% of monitored patients. Small paraproteins missed by conventional electrophoretic techniques are clinically important in the diagnosis and monitoring of malignant plasma and B-cell disorders. The superior diagnostic performance of sIFE makes it suitable as the initial laboratory procedure to investigate paraproteins in complex serum and urine matrices.

A Rare Case of Acquired Fanconi's Syndrome With Monoclonal Gammopathy in an Infant.

BACKGROUND: Monoclonal gammopathies associated with acquired Fanconi's syndrome (AFS) have been reported in the adult population. AFS is characterized by renal dysfunction resulting in proteinuria, aminoaciduria, hypophosphatemia, glucosuria, and hyperchloremic metabolic acidosis. In this case report, we document the clinical and laboratory findings of a preterm infant with features of both AFS and monoclonal gammopathy in the urine. METHODS: Clinical suspicion of AFS prompted the following laboratory studies to be performed: urine protein electrophoresis (UPEP), urine immunofixation, and urine amino acid analysis with high performance liquid chromatography (HPLC). RESULTS: Urine amino acid analysis revealed aminoaciduria. On UPEP, nonselective glomerular proteinuria was seen with a faint band in the gamma region. Urine immunofixation confirmed the presence of a monoclonal IgG lambda component with free monoclonal lambda light chains. CONCLUSION: To the best of our knowledge, this is the first case of pediatric AFS reported with a monoclonal gammopathy and monoclonal free light chains.

Should routine laboratories stop doing screening serum protein electrophoresis and replace it with screening immune-fixation electrophoresis? No quick fixes: Counterpoint.

Monoclonal gammopathies are characterised by the production of a monoclonal immunoglobulin or free light chains by an abnormal plasma cell or B-cell clone and may indicate malignancy or a precursor (MGUS). There is currently no consensus on the initial test or combination of tests to be performed in suspected monoclonal gammopathies but serum protein electrophoresis and urine protein electrophoresis are commonly requested as initial investigations. If abnormal, immunofixation electrophoresis is then performed to confirm the presence of paraprotein and to determine its heavy and light chain type. Recently, some groups have developed simplified "screening" IFE methods for use in parallel to SPEP for the detection monoclonal gammopathies. We argue here that screening IFE may be of benefit in clinical laboratories using SPEP with poor resolution in the ß-region, assisting in the detection of mainly IgA paraprotein, but may be of less benefit in laboratories utilising higher resolution gels. Further it may increase the detection of trace bands of questionable clinical significance, representing transient phenomena in infectious and auto-immune conditions or very low risk MGUS. The increased detection of these bands using screening IFE would require further patient follow up, possibly causing unnecessary patient anxiety and additional follow up healthcare costs.

The Cutoff Level for Urine Protein in Urine Immunofixation Electrophoresis.

BACKGROUND: Immunofixation electrophoresis (IFE) maintains its importance in diagnosing monoclonal gammopathies. In particular, urine IFE detects free light chains (FLC) in urine samples even at low concentrations and offers higher sensitivity compared to serum electrophoresis and serum IFE. The aim of the present study was to determine the place and significance of quantitative urinary protein measurement before IFE in interpreting the results of subsequent IFE and to determine the most appropriate protein concentrations for the appearance of bands. METHODS: The records of a total of 600 patients, who underwent screening for Bence Jones proteinuria using IFE on 24-hour urine, were retrospectively reviewed. Urine IFE was performed using Helena SAS-I and SAS-I devices. The total protein concentration in the urine was quantitatively determined by the Pyrogallol red method, and the urine albumin level was determined using the immunoturbidimetric method. These analyses were measured on an Olympus/Beckmann AU5800. RESULTS: The evaluation of IFE results revealed that 311 patients had normal results, 108 patients had monoclonal bands, five patients had biclonal bands, 28 had polyclonal bands, and 148 patients had various degrees of proteinuria. ROC curves were created in order to determine the most appropriate urinary protein and albumin levels to observe bands in IFE. Accordingly, urine baseline protein level (mg/dL) showed the highest AUC value (cutoff value: 19.4 mg/dL, sensitivity: 92%, specificity: 98.2%, AUC: 0.972). CONCLUSIONS: The present study showed that quantitative protein measurement before IFE eliminated the disadvantages associated with the IFE method and its interpretation.

Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to detect monoclonal immunoglobulin light chains in serum and urine.

RATIONALE: Use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to monitor serum and urine samples for endogenous monoclonal immunoglobulins. MALDI-TOFMS is faster, fully automatable, and provides superior specificity compared to protein gel electrophoresis (PEL). METHODS: Samples were enriched for immunoglobulins in 5 min using Melon Gel™ followed by reduction with dithiothreitol for 15 min to separate immunoglobulin light chains and heavy chains. Samples were then desalted using C4 ZipTips, mixed with sinapinic acid matrix, and analyzed on a Bruker Biflex III MALDI-TOF mass spectrometer. RESULTS: Monoclonal immunoglobulin light chains were identified in serum and urine samples from patients with a known monoclonal gammopathy using MALDI-TOFMS with minimal sample preparation. CONCLUSIONS: MALDI-TOFMS can identify a monoclonal immunoglobulin in serum and urine samples. The molecular mass of the monoclonal immunoglobulin light chain is obtained providing unprecedented specificity compared to PEL. In addition, the methodology can be automated, making it a practical alternative to PEL.

[Urinary investigations in the diagnosis and monitoring of monoclonal gammopathies in daily practice]. / Place des explorations urinaires dans le diagnostic et le suivi des gammapathies monoclonales en pratique quotidienne.

The management of monoclonal gammopathies remains a public health issue with an incidence greater than 3% of the population over 50 years. Laboratory investigations, including urinary investigations play a key role in the diagnosis and monitoring of the patients. Urinary investigations are not recommended when screening monoclonal gammopathies. However, the initial laboratory evaluation of the monoclonal gammopathies systematically relies on renal function and proteinuria assessment. Urinary proteins electrophoresis combined with urinary proteins immunofixation are also recommended in the initial evaluation, with the exception of the Waldenström's disease. In some cases, serum investigations remain negative whereas urinary investigations confirm the presence of a monoclonal component. National and international recommendations have also been published about the monitoring of monoclonal gammopathies. The biological monitoring of monoclonal gammopathy of undetermined significance is mostly done by serum tests. Urinary investigations are commonly included in the response criteria in case of multiple myeloma or AL amyloidosis. Laboratory investigations like serum free light chain assay tend to decrease the need of urinary investigations in the monoclonal gammopathies. However, these urinary investigations currently maintain a leading role in the diagnosis and monitoring of monoclonal gammopathies.

Combined light chain immunofixation to detect monoclonal gammopathy: a comparison to standard electrophoresis in serum and urine.

BACKGROUND: The purpose of this study was to evaluate a combined κ and λ light chain immunofixation (CLIF) as a screening tool to detect monoclonal immunoglobulins in serum and urine. A secondary aim was to investigate the impact on workflow and reagent utilisation of a systematic implementation of CLIF in addition to routine protein electrophoresis (PE) on all samples. METHODS: Light chain antisera (κ and λ) were mixed in a 1:1 ratio and loaded in the same sequence as the PE to create a superimposable image. RESULTS: The CLIF procedure agreed significantly better with standard immunofixation procedures in the serum and urine. In 33 (22%) new patients and in 114 (15%) follow-up patients CLIF detected a band missed by PE in serum. In 34 (4.5%) of previously categorised cases the monoclonal band was below the detection limit of CLIF in serum, but still detectable by conventional immunofixation electrophoresis. In one case (0.7%) a band in a urine specimen was missed by CLIF compared to 70 (49%) missed by PE. After the systematic introduction of CLIF turn-around-times (TATs) and utilisation of laboratory consumables decreased significantly (p<0.001). CONCLUSIONS: A systematic implementation of CLIF led to the detection of monoclonal bands missed by PE with an improvement in TATs and a decrease in cost.

[Crystalline keratopathy due to kappa chains in a monoclonal gammopathy]. / Queratopatía cristalina por cadenas kappa en gammapatía monoclonal.

CASE REPORT: The following case shows corneal crystal formation in a patient in whom thee systemic work-up led to the diagnosis of a monoclonal gammopathy with increased monoclonal immunoglobulin G (IgG). We present the corneal signs and subsequent haematological investigations undertaken to establish this important association. DISCUSSION: Systemic work-up of a patient with corneal deposits showed a monoclonal gammopathy with increased monoclonal immunoglobulin (IgG-type kappa). Corneal crystals, a rare, but significant, clinical finding, may be the initial presentation in a patient with monoclonal gammopathy.