Figure 2 - uploaded by Urs Wilgen
Content may be subject to copyright.
Selected examples of findings on protein electrophoresis and combined light chain immunofixation (CLIF). Panels A and C are examples of serum protein electrophoresis (PE) with panels B and D the superimposable CLIF results. 1: An example of a false positive on both PE and CLIF due to faint oligoclonal banding in the γ -region that was fully characterised with routine immunofixation electrophoresis (IFE) and isoelectric focussing. 2: A fibrinogen band visible in the β - γ region on PE, but not detected with CLIF. 3: A trace monoclonal IgG λ -band detected by CLIF and subsequently confirmed on IFE. 4: An IgA κ band detected in the β -region with CLIF, but missed with PE.
Source publication
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 electrophores...
Contexts in source publication
Context 1
... agreement between the adjudicated reference diag- nosis and CLIF ( κ Cohen 0.85) was significantly improved over that of PE ( κ Cohen 0.73). Both PE and CLIF were equally prone to identifying faint bands as potentially monoclo- nal (p = 0.27) and in the vast majority (38 PE; 42 CLIF) of these cases multiple small oligoclonal bands could be demonstrated with the definitive methods of IFE and IEF ( Figure 2 , example 1). Haemolysis (two cases) and fibrinogen (one case, Figure 2 , example 2) resulted in small bands detected on PE, but not with CLIF. ...
Context 2
... PE and CLIF were equally prone to identifying faint bands as potentially monoclo- nal (p = 0.27) and in the vast majority (38 PE; 42 CLIF) of these cases multiple small oligoclonal bands could be demonstrated with the definitive methods of IFE and IEF ( Figure 2 , example 1). Haemolysis (two cases) and fibrinogen (one case, Figure 2 , example 2) resulted in small bands detected on PE, but not with CLIF. In six CLIF cases we concluded that an application artefact was mis- interpreted as a possible band. ...
Similar publications
Approximately 10% of all haematologic cancers are related to Multiple Myeloma (MM). Whole-body 18F-FDG PETCT is an extremely useful imaging tool for the assessment of patients with MM. The software developed in this research performs a pixel thresholding based segmentation and a semi-automatic placement of regions of interest at key anatomical site...
Simple Summary
Multiple Myeloma (MM) can be a diagnostic challenge as it often presents with unspecific symptoms in patients in general practice. Serum-free light chain (sFLC) ratio is suggested to replace urine protein electrophoresis (UPE) in the diagnostic work-up of myeloma. We aimed to investigate the performance of the sFLC ratio in general p...
Citations
... A screening uIFE test with a mixture of anti-κ and λ light chain 24 , or a mixture of albumin, α1-microglobulin, anti κ and λ antisera 25 is performed to detect BJP prior to setting up traditional urine BJP-IFE with pentavalent anti-IgG, -IgA, -IgM and anti-κ and -λ chains antiserum. This procedure displays the advantage that free light κ and λ chains may be tested in a single lane, identifying negative samples, which can be rapidly ruled out. ...
Objective:
Bence Jones proteinuria (BJP) refers to monoclonal free immunoglobulin light chains detected in urine, deriving from the clonal expansion of plasma cells in the bone marrow in patients with plasma cell dyscrasias, associated with monoclonal gammopathies of uncertain origin. This review summarizes routinely diagnostic procedures to assess BJP highlighting critical steps of pre-analytical, analytical, and post-analytical phases.
Qualitative and quantitative methods:
The best option for BJP detection is the first morning void urine sample and immunofixation electrophoresis detection technique (IFE) the recommended method, with the employment of specific polyvalent antisera. Other qualitative tests for a quick evaluation of BJP are currently available. Densitometric analysis performed on the 24-hour urine is the recommended method to quantify BJP. To overcome the 24-hour collection, it is possible to use morning urine sample and correlate the assessed value of BJP to creatininuria. In addition to the traditional ones, we here reviewed screening methods currently used to avoid false negatives and reduce the time around test (TAT), together with immunochemical quantification methods for increased sensitivity, after checking BJP by IFE. Mass spectrometry emerges as a new challenge in the determination of BJP.
Conclusions:
The employment of different based-assays methods may be useful for diagnostic purposes to improve the accuracy of BJP monitoring in monoclonal gammopathies.
... Given that the serum κ FLC /λ FLC ratio increases due to impaired renal FLC clearance [18,19], we studied if FLCs play a role in AKI by comparing the κ FLC and λ FLC levels to the maximum serum creatinine levels measured during the hospital stay in patients with acute HFRS (1 st day of hospitalization). Indeed, both κ FLC and λ FLC levels positively correlated with the maximum creatinine values (Fig 2A and 2B), suggesting that FLC levels could serve as prognostic indicator in estimating the severity of AKI in PUUV-HFRS. ...
In humans, orthohantaviruses can cause hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). An earlier study reported that acute Andes virus HPS caused a massive and transient elevation in the number of circulating plasmablasts with specificity towards both viral and host antigens suggestive of polyclonal B cell activation. Immunoglobulins (Igs), produced by different B cell populations, comprise heavy and light chains; however, a certain amount of free light chains (FLCs) is constantly present in serum. Upregulation of FLCs, especially clonal species, associates with renal pathogenesis by fibril or deposit formations affecting the glomeruli, induction of epithelial cell disorders, or cast formation in the tubular network. We report that acute orthohantavirus infection increases the level of Ig FLCs in serum of both HFRS and HPS patients, and that the increase correlates with the severity of acute kidney injury in HFRS. The fact that the kappa to lambda FLC ratio in the sera of HFRS and HPS patients remained within the normal range suggests polyclonal B cell activation rather than proliferation of a single B cell clone. HFRS patients demonstrated increased urinary excretion of FLCs, and we found plasma cell infiltration in archival patient kidney biopsies that we speculate to contribute to the observed FLC excreta. Analysis of hospitalized HFRS patients’ peripheral blood mononuclear cells showed elevated plasmablast levels, a fraction of which stained positive for Puumala virus antigen. Furthermore, B cells isolated from healthy donors were susceptible to Puumala virus in vitro , and the virus infection induced increased production of Igs and FLCs. The findings propose that hantaviruses directly activate B cells, and that the ensuing intense production of polyclonal Igs and FLCs may contribute to acute hantavirus infection-associated pathological findings.
... [5] It is common practice for laboratories to perform immunofixation only if a paraprotein is present on SPEP, despite studies indicating the superiority sensitivity of immunofixation in detecting low level monoclonal bands missed by conventional electrophoresis techniques. [8,9] Although modern high resolution electrophoresis can be as sensitive as immunofixation, [10] it is not in widespread use and has not been tested in POEMS syndrome cases which classically manifest small but significantly relevant monoclonal gammopathies. The data from our clinical cohort demonstrates a critical low level monoclonal band would not have been detected in 23% of cases by SPEP methodology only. ...
Background
Prompt diagnosis and early treatment prevents disability in Polyneuropathy Organomegaly Endocrinopathy Monoclonal-protein and Skin Changes (POEMS) syndrome. Delay in diagnosis is common with 55% of patients initially incorrectly diagnosed with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). Patients are often treated with intravenous immunoglobulin which is both expensive and ineffective in the treatment of POEMS. Testing patients with acquired demyelinating neuropathy with serum vascular endothelial growth factor (VEGF) more accurately identifies POEMS syndrome than the current standard of care. Incorporating VEGF testing into screening could prevent misdiagnosis and reduce costs.
Methods
We used observed treatment information for patients in the University College London Hospital’s POEMS syndrome database (n=100) and from the National Immunoglobulin Database to estimate costs associated with incorrect CIDP diagnoses across our cohort. We conducted a model-based cost-effectiveness analysis to compare the current diagnostic algorithm with an alternative which includes VEGF testing for all patients with an acquired demyelinating neuropathy.
Results
Treatment associated with an incorrect CIDP diagnosis led to total wasted healthcare expenditures of between £808 550 and £1 111 756 across our cohort, with an average cost-per-POEMS-patient misdiagnosed of £14 701 to £20 214. Introducing mandatory VEGF testing for patients with acquired demyelinating neuropathy would lead to annual cost-savings of £107 398 for the National Health Service and could prevent misdiagnosis in 16 cases per annum.
Conclusions
Misdiagnosis in POEMS syndrome results in diagnostic delay, disease progression and significant healthcare costs. Introducing mandatory VEGF testing for patients with acquired demyelinating neuropathy is a cost-effective strategy allowing for early POEMS diagnosis and potentially enabling prompt disease-directed therapy.
... Non è descritto in letteratura un metodo unico per concentrare le urine al fine della determinazione della PBJ. Alcuni Autori indicano di concentrare le urine a partire da un fattore 10 (10x) fino a un fattore 200 (200x) (7,(37)(38)(39)(40)(41), mentre altri suggeriscono di valutare preventivamente la concentrazione delle proteine totali urinarie per poi usare un fattore di concentrazione variabile al variare della proteinuria (16,37,39). ...
... In letteratura sono stati descritti metodi di immunofissazione rapida da utilizzarsi come esame di primo livello che impiegano miscele di antisieri anti-κ e λ totali (talora in aggiunta anche antisieri anti IgG, IgA, IgM, anti-albumina e anti-alfa1 microglobulina) cimentati su un'unica corsia (40,50,51). Questi metodi consentirebbero di evidenziare i campioni negativi sottoponendo alla conferma in immunofissazione classica i campioni dubbi o positivi. ...
INTRODUZIONE La ricerca, la caratterizzazione e la quantificazione della proteina di Bence Jones (PBJ) è fondamentale per l'inquadramento e il monitoraggio del paziente affetto da discrasia plasmacellulare (DP) (1-7). Le DP sono un gruppo eterogeneo di patologie caratterizzate dalla proliferazione di un singolo clone di cellule B che ABSTRACT Update of the Italian Society of Clinical Biochemistry (SIBioC) Consensus document on the detection and quantification of the Bence Jones protein. Bence Jones protein (BJP) refers to urine monoclonal free immunoglobulin light chains produced by the clonal expansion of a plasma cell in the bone marrow. BJP is strongly associated with systemic amyloidosis AL, light chain deposition disease, and multiple myeloma; less frequently, BJP may be recognized either in patients with monoclonal gammopathies of uncertain significance (MGUS) and with other plasma cell dyscrasias or in patients with malignant non-Hodgkin's lymphomas and chronic lymphocytic leukemia. This paper contains updated recommendations for the detection and the measurement of BJP in clinical practice from the Working Group "Proteins" of the Italian Society of Clinical Biochemistry (SIBioC), with specific indications for improving all the steps of the preanalytical, analytical, and postanalytical phases. The first morning void is the urine sample recommended for BJP detection, while 24-hours urine collection is preferred for BJP quantification. Native urine cannot be used for samples with low or very low content in urine total protein; in these cases, samples should be concentrated by using specific disposables, such as ultrafiltration membranes retaining proteins with molecular weight around 10 kDa. The required degree of concentration may vary according to sensitivity of the electrophoretic method utilized and the protein content of the sample. The detection of BJP may be performed directly by the recommended method agarose gel immunofixation (IFE) with specific polyvalent immunoglobulin antisera IgG-IgA-IgM, total and light chains; alternatively, an electrophoretic screening may be acceptable to rule out negative test results. However, positive test results should be confirmed by IFE. Tests based on immunometric methods can be used neither as screening test, nor for the BJP quantification; however, it could be useful for monitoring purposes, provided that the renal function of the patient is preserved. BJP measurement should be performed by the densitometric scanning of the electrophoretic peak corresponding to BJP, and results should be expressed as ratio of the BJP peak percentage to the urine total protein. Test results should be always integrated by standardized interpretative comments included in the laboratory reports.
... Basile et al. [7] described a method using pentavalent immunofixation electrophoresis (Penta-IFE) composed of a mix of IgG, IgA, IgM, light chains κ and λ antisera. Jenner et al. [8] prepared an in-house mix of Sebia κ and λ light chains antisera in a 1:1 ratio. In our opinion, BJTS offers some advantages: it can be used with unconcentrated urine specimens, it is ready for use which eliminates risks of error and simplifies procedures; it allows to identify both free light chains and intact immunoglobulins, thus avoiding any heavy chains diseases being missed; lastly, it may help evaluate electrophoretic performance due to the presence of albumin also in physiological urine samples. ...
We describe the advantages of using a new BJTS antiserum to screen for Bence Jones protein (BJP) and as a marker of proteinuria. With a single lane in urinary immunofixation electrophoresis, BJTS antiserum can detect BJP, as well as urinary albumin and α1-microglobulin, which are important markers of glomerular and tubular kidney damage respectively. Our study indicates that BJTS antiserum can improve laboratory cost-efficiency and time-effectiveness; it can also provide physicians with important information not only concerning BJP, but also regarding potential kidney damage.
... The test could be improved by using a mixture of antisera to κ and λ free and bound light chains that would reduce the background staining of the gel when using pentavalent antiserum and thus lead to a decrease in false positives [4]. ...
... Replacing these tests with high-throughput and less labour-intensive tests implies cost reduction as well as simpler result management (since immunochemical tests do not require interpretative comments). For example, the systematic introduction of combined κ and λ light chain immunofixation as an initial screening test for laboratory investigation of all urines resulted in a significant turn-around-time reduction [4]. ...
... Reference ranges have been defined and validated [5][6][7] and are as follows: N latex assay kappa FLC 7-22 mg/L, lambda FLC 8-27 mg/L, kappa:lambda ratio 0.31-1.56 and Freelite assay kappa FLC 3-19 mg/L, lambda FLC 6-26 mg/L, kappa:lambda ratio 0.26-1.65 with renal specific reference range 0.37-3.10. Screening immunofixation electrophoresis (IFE) was performed on all pre-haemodialysis serum samples using Sebia reagents and equipment (Sebia, Cedex, France); a combined IFE antisera was used [11]. ...
Background:
Quantification of serum free light chains (FLC) is important in the diagnosis of plasma cell diseases where an abnormal kappa:lambda ratio infers a population of monoclonal plasma cells. The Freelite™ and N Latex assays have been validated in populations without kidney disease but there is a paucity of data relating to the use of these assays in end stage kidney disease (ESKD). The aim of the study was to compare FLC assay performance in ESKD patients on haemodialysis.
Methods:
Cross-sectional multi-centre study comparing the performance of the two assays on 112 haemodialysis patients without known paraproteinaemia. We quantified FLC pre- and post-dialysis using both the N Latex and the Freelite assays.
Results:
FLC levels were elevated by both assays. Lambda FLC levels were considerably higher by the N Latex assay. Using the proposed renal reference range for Freelite (0.37-3.1) all but one patient had normal kappa:lambda FLC ratios. In contrast, there were no abnormal FLC ratios pre-dialysis using the N Latex assay. This was due to lambda FLC reading significantly higher by the N Latex assay. Kappa and lambda FLC levels decreased with dialysis but remained elevated above the normal range. The excess of lambda FLC by N Latex persisted post-dialysis but was somewhat attenuated. Dialysis adequacy and dialysis modality predicted clearance of kappa and lambda FLC by both assays.
Conclusions:
The N Latex assay reported significantly higher pre-dialysis lambda FLC concentrations compared with the Freelite assays. Clinicians should be aware of the need for a separate renal reference range for interpreting FLC ratio using the Freelite assay but not for the N Latex assay in ESKD patients.
... Screening immunofixation electrophoresis (sIFE) is a modified immunofixation (IFE) procedure that employs a single application of combined antisera directed against heavy and/or light chains to detect a paraprotein. The technical aspects and diagnostic performance of sIFE is dealt with in detail elsewhere [1,2]. The clinical rationale behind detecting small paraproteins and the analytical characteristics of the available procedures will be briefly discussed to justify the recommendation that sIFE should be the initial laboratory procedure to investigate paraproteins in complex serum and urine matrices. ...
... Of the available diagnostic techniques standard agarose or capillary PE has the worst ability to detect paraproteins with a detection limit of 0.5-1 g/L that depends on background staining of residual polyclonal immunoglobulin. This is further degraded when these bands migrate in non-γ regions [2,3]. All newly detected bands with a localised migration on PE needs to be further investigated with IFE to confirm monoclonality. ...
... Screening IFE (sIFE) is a modified IFE that employs a single application of combined antisera (GAMκλ or κλ) to detect a paraprotein. This leverages the improved analytical sensitivity and specificity of immune typing techniques and approaches the efficiency of standard electrophoretic techniques [1,2]. The principle is graphically illustrated in Figure 1 where samples were applied in an identical sequence on a standard PE and a sIFE gel. ...
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.
POEMS syndrome (Polyneuropathy, Organomegaly, Endocrinopathy, Monoclonal gammopathy, Skin disorder) is a rare disease characterised by an inflammatory polyneuropathy an a monoclonal plasma cell dyscrasia. POEMS syndrome causes some of the most significant disability and mortality of any inflammatory neuropathy. The pathophysiology is unknown but recognised to be cytokine mediated, notably driven by vascular endothelial growth factor, however little is known about the other mediators at play. This thesis collates clinical data from the largest POEMS cohorti in Europe in order to study the characteristic disease features, optimise therapies and identify factors that influence outcome. Utilising our POEMS sample biobank, we carry out highly sensitive immunoassays to study the cytokines released in POEMS syndrome, and whether they correlate with disease activity. We go on to study the proteome of POEMS syndrome through mass spectrometry, to uncover the biological pathway involved and identify a number of novel, potentially pathogenic molecules. Fluid biomarkers of neuropathy in POEMS syndrome and related neuropathies are additionally explored. The development and optimisation of a homebrew immunoassay for peripherin, a peripheral nerve specific biomarker is detailed. The potential clinical utility of this biomarker is compared against that of serum neurofilament light. Finally, we attempt to model the neuropathogenesis of POEMS neuropathy in vitro using a novel human induced pleuripotent stem cell derived neuronal culture system.
