Free Light Chain Concentrations Research Articles

Free light chains and heavy/light chains in monitoring POEMS patients.

POEMS syndrome is defined by Polyneuropathy, Organomegaly, Endocrinopathy, Monoclonal gammopathy and Skin changes. The vascular endothelial growth factor (VEGF) appears to play a key role in the pathogenesis of the syndrome, and its concentrations are deemed to correlate to disease activity. The aim of the present study was to verify whether other biochemical markers including serum free light chains (FLC) and heavy/light chains (HLC) would be of value in monitoring POEMS patients. Fifty-three serum samples were collected from seven POEMS patients at diagnosis and during a follow-up period (range 14-56 months). VEGF was measured using an ELISA method, while FLC and HLC concentrations were measured using Binding Site reagents on a BNII (Siemens) nephelometer. At diagnosis all patients presented high VEGF concentrations, while the κ/λFLC ratio (FLCr) was within the reference range. Four patients had abnormal HLC, HLCκ/HLCλ (HLCr) and FLC values. The relationship between the trend of VEGF and both HLC and FLC during the follow-up was analysed by means of Cohen's κ coefficient. VEGF and HLC values displayed a significant κ-Cohen (0.537, p=0.002) in all chemotherapy-responder patients while in non-responders it did not. Conversely, in both responders and non-responders, VEGF and FLC values did not attain a significance on κ-Cohen analysis. In three out of four responders HLCr values increased, thus reflecting an improved clinical condition. The findings made in the present study indicate that HLC, either as intact immunoglobulin or as HLCr, may provide useful information, particularly in identifying responders and confirm that the role of FLC is unreliable in monitoring patients with POEMS syndrome.

Accurate Risk Stratification Identifies Patients with AL Amyloidosis Benefiting Most from Upfront Bortezomib Combinations: A Study of Treatment Outcomes in 984 Patients

Background. In the last decade major advances have been made in the management of AL amyloidosis, such as the use of free light chains (FLC) and cardiac biomarkers in diagnosis, risk assessment, and evaluation of response, and the introduction of novel agents in the therapeutic armamentarium. However, available studies on the natural history of this disease are based on data collected before this era, and large comparative studies on novel agents are still lacking. We report the outcome of the 984 consecutive patients diagnosed between 2004 and 2014 at the Pavia Amyloidosis Research and Treatment Center (ARCT).Methods. Baseline evaluation and therapeutic strategy were protocolized. Patients were prospectively followed for response and survival. Response was assessed by intent to treat.Results. Overall, the heart was involved in 76% (Mayo stage III 39%), the kidney in 66% (stage III 15%), the liver in 14%, the soft tissues in 17%, and the peripheral nervous system (PNS) in 12%. Isolated involvement of the heart or kidney was observed in 17% and 14% of cases, respectively, while the liver or PNS were the only organs involved in <1% of patients. Most common upfront treatments were melphalan-dexamethasone [MDex, 354 patients, overall hematologic response (HR) 51%, very good partial or complete response (VGPR/CR) 37%], cyclophosphamide-bortezomib-dexamethasone (CyBorD, 295 patients, HR 53%, VGPR/PR 39%), and bortezomib-MDex [BMDex, 128 patients, HR 64% (P=0.010 compared to MDex; P=0.033 compared to CyBorD), VGPR/PR 52% (P=0.002 compared to MDex; P=0.0011 compared to CyBorD)]. Amongst responders, exposure to bortezomib upfront was associated with a longer time to second-line treatment (median 43 months vs. not reached, P=0.004). After a 36-month median follow-up of living patients, overall median survival was 40 months. Mayo stage predicted survival, with 86%, 57%, and 27% of stage I, II, and III patients surviving 3 years, respectively (P<0.001). Stage III patients could be divided into 2 groups based on whether NT-proBNP was below (stage IIIa) or above (stage IIIb) 8500 ng/L (3 year survival 38% vs. 15%, P<0.001). Fifty-nine percent of stage IIIb patients died in the first 6 months, and 76% within 1 year from diagnosis. Mayo stage I could also be divided into 2 groups, with stage Ia patients having both normal alkaline phosphatase and dFLC <100 mg/L (3 year survival 97% vs. 76%, P<0.001). The only 3 deaths in stage Ia patients were not disease-related, while liver and heart failure were responsible of most deaths in stage Ib. We propose a novel 5-stage system for prognostication of survival (Figure). In the overall population, the outcome was not different in patients treated upfront with MDex, CyBorD, and BMDex (48%, 55%, and 55% at 3 years, respectively, P=0.720). However, exposure to bortezomib upfront was associated with longer survival in stage Ib patients (95% vs. 73% surviving 3 years, P=0.044), while there was no significant advantage of BMDex or CyBorD over MDex in the other stages. A high FLC burden (dFLC >180 mg/L) was associated with poorer survival in stage II patients treated with MDex (38% vs. 68% at 3 years, P<0.001) and CyBorD (50% vs. 70% at 3 years, P=0.033), but not in subjects treated with BMDex (67% vs. 70% at 3 years, P=0.401).Conclusion. The outcome of patients with AL amyloidosis is extremely heterogeneous, but can be accurately predicted based on biomarkers of organ damage and FLC concentration. The extensive use of upfront bortezomib prolongs the duration of response, but does not translate in a general improvement of survival. This might be due to the fact that most patients refractory to or relapsed after MDex were rescued with bortezomib. However, accurate stratification reveals subgroups of patients in whom bortezomib combinations are most effective. In stage Ib patients bortezomib triplets grant the best outcome, while the combination of bortezomib, melphalan, and dexamethasone should be preferred in subjects with moderate cardiac dysfunction and elevated FLC burden. [Display omitted] DisclosuresOff Label Use: Bortezomib in AL amyloidosis. Merlini:Millennium Takeda: Honoraria; Janssen Cilag: Honoraria.

Free Light Chain Ratio As a Simple Prognostic Marker in a Single Institution Experience

Multiple myeloma (MM) is a plasma cell dyscrasia characterized by bone marrow plasma cell infiltration and the secretion of monoclonal immunoglobulin (Ig) in the serum or urine. Median survival in MM patients is approximately 5 years and significant morbidity may be experienced. The course is progressive and although, always incurable, the prognosis is highly variable. The two more widely used staging systems in MM are by Durie and Salmon and the International Staging System (ISS). Others have been studied, including serum free light chain (sFLC) concentrations and ratio (sFLCr).Methods: We measured sFLC in 27 newly diagnosed MM (21 Intact Ig MM, 5 light chain MM, 1 non secretory MM) at our center (Hospital del Salvador, Santiago de Chile) from October 2013 until June 2015. The sFLCr was calculated with the involved monoclonal light chain as the numerator. The median sFLCr was 17. Patients were divided into a "low group" (<17 sFLCr) and a "high group" (>17 sFLCr). We also analyzed these patients using the cut off (sFLCr of 50) previously reported by Garcia de Veas Silva et al. [Hematology reports 2015; 7 (s1) p23] The median follow-up was 16 months.Results: During the period of study there were 8 deaths (29,7%). Seven (87,5%) of these deaths presented with an ISS score of 3 (table 1). Mortality rates were lower in the group of patients with "low" sFLCr (15,3%, 2 deaths in a group of 13 patients), as compared to patients with a "high" sFLCr (42,9%, 6 deaths in a group of 14 patients) (table 2).Using the cutoff established by Garcia de Veas Silva et al, the mortality rate for patients with sFLCr >50 was 66,7% vs. 11,1% in for patients in the <50 sFLCr group (table 3).Discussion: Although a short follow up period was available for analysis, we believe these results are promising. sFLCr can be used as an easy prognostic indicator in newly diagnosed, symptomatic MM, especially when high risk patients (>50 sFLCr) are identified. The introduction of biomarkers in the evaluation of MM patients will enable better risk assessment and rational follow up.Table 1International Staging System. Stratification of our study population.ISSN PatientsN DeathsMortality (%)120021218,3313753,9Table 2Mortality rate in our study population using the median sFLCr as a cut off value.Mortality MMN PatientsN DeathsMortality (%)All27829,6sFLCr>1714642,9sFLCr<1713215,4Table 3Mortality rate in our population using the published cut off sFLCr value.Mortality MMN PatientsN DeathsMortality (%)All27829,6sFLCr>509666,7sFLCr<5018211,1 DisclosuresDelgado:The Binding Site: Employment.

Serum Versus Urine Free Light Chains for Assessment of Monoclonal Gammopathies

IntroductionMonoclonal gammopathies comprise a spectrum of disorders including Monoclonal Gammopathy of Undetermined Significant (MGUS), Smoldering Multiple Myeloma (SMM), and Active Multiple Myeloma (MM) characterized by production of monoclonal immunoglobulin heavy and/or light chains. Prior to availability of the FREELITE™ (Binding Site Ltd; Birmingham, UK) assay for measurement of immunoglobulin free light chains (FLC), laboratory monitoring of these disorders used predominantly SPEP, quantitation of immunoglobulin heavy chains (quantitative immunoglobulins), and 24 urine collection for total protein and UPEP to extrapolate production of immunoglobulin light chains. The FREELITE™ assay has up to 3-log increased sensitivity (1.5-3.0 mg/L) for detection of free light chains over standard electrophoresis (500-2,000 mg/L) and immunofixation (150-500 mg/L), and since its introduction, has been an integral tool in diagnosis and monitoring of monoclonal gammopathies. This assay detects more plasma cell disorders than SPEP, UPEP and IFE combined due to its higher sensitivity and ability to derive the ratio of affected to unaffected light chain. Measurement of urine FLC using FREELITE™ has not been integrated into standard practice due to presumed variability in FLC concentration due to changes in glomerular filtration, variability in tubular reabsorption of light chains, and lack of data regarding this use. In our practice, we routinely use random urine samples instead of 24 hour urine collections which are cumbersome and suffer from poor patient compliance.Methods:The study was approved by the Stratton VA Medical Center Institutional Review Board. As it has been our practice to obtain both random urine along with serum for FLC, we retrospectively reviewed patients diagnosed with monoclonal gammopathies and compared random urine free light chains measured by FREELITE™ to serum FLC and serum quantitative immunoglobulins. Data was analyzed for correlation using Pearson product moment correlation. P values of >0.05 were considered significant.Results:We identified 23 individuals, all male (consistent with VA population). Mean (±SD) age was 68±10 years at diagnosis, creatinine 1.3±0.5 mg/dl, and 9±6 pairs of data points per patient. Five (5) had MGUS, 5 SM, and 13 MM (2 light chain only). Results are illustrated in the Table. Normalization of urine results using concurrent serum and urine creatinine did not change the statistical significance of any of the results.TableCorrelation (p<0.05) between affected serum immunoglobulin, urine FLC, and serum FLCSerum ImmunoglobulinUrine FLCSerum FLC#(%) of PatientsYESYESno2(10)YESnoYES6(29)YESYESYES5(24)Nonono7(33)YESYES11(48)DiscussionWhile serum FLC is adequate in the majority of patients for monitoring monoclonal gammopathies, urine FLC correlates as well as serum FLC in about ½ of the patients. In addition, in a small number of individuals, urine FLC correlates with serum total serum immunoglobulin better than serum FLC. We feel that random urine FLC is useful for monitoring monoclonal gammopathies, and in a minority of instances, provides more accurate assessment of disease activity than serum. DisclosuresNo relevant conflicts of interest to declare.

Diagnostic and Prognostic Value of the Cerebrospinal Fluid Concentration of Immunoglobulin Free Light Chains in Clinically Isolated Syndrome with Conversion to Multiple Sclerosis.

Background and objectiveIn this study, we evaluated the diagnostic and prognostic significance of cerebrospinal fluid free light chains (CSF FLC) at the time of clinically isolated syndrome (CIS).MethodsWe compared FLC-parameters at the moment of CIS in patients with conversion to multiple sclerosis (MS) after 2 years (CIS-MS), patients who remained stable both clinically and radiologically after 2 years (CIS-nonMS), patients with non-inflammatory neurologic diseases (NIND) as a comparison group and patients with other inflammatory neurologic diseases (IND) with intrathecal oligoclonal bands (OCB) synthesis. ROC-analysis was conducted to define FLC-assay characteristics and cut-off values. We also compared FLC-concentrations in CIS patients to determine their OCB-status. A correlation analysis was performed between FLC-concentrations and the expanded disability scale score (EDSS), annualized relapse rate (ARR) and MRI-activity (i.e., number of new and gadolinium-enhancing (Gd+) lesions) in patients.ResultsThe levels of kappa-FLC (k-FLCCSF) and lambda-FLC (λ-FLCCSF) as well as kappa- and lambda-quotients (Q-k and Q-λ) were elevated in CIS-MS compared to the CIS-nonMS and NIND groups. These levels did not differ significantly when compared with the IND group. We identified several patients with high k-FLCCSF and λ-FLCCSF in OCB-negative CIS and IND groups. The level of k-FLCCSF production was significantly higher in OCB-positive patients in the CIS-MS group compared to the CIS-nonMS group. The concentrations of k-FLCCSF and Q-k in the CIS-MS group showed significant correlation with the level of EDSS after 2 years (k-FLC: r = 0.4477,p = 0.0016; Q-k: r = 0.4621, p = 0.0016). λ-FLCCSF and Q-λ inversely correlated with the number of Gd+ lesions (CSF λ-FLC: r = -0.3698, p = 0.0223; Q-λ: r = -0.4527, p = 0.0056).ConclusionThe concentration of CSF FLC predicts conversion to MS within 2 years following CIS. OCB-positive patients with an early conversion have a higher concentration of CSF-FLC. We have also shown a prognostic significance of k-FLCCSF for future EDSS-progression.

Validation of serum free light chain reference ranges in primary care.

The demand for measurement of serum immunoglobulin free kappa (κ) and lambda (λ) light chains has increased. The κ:λ ratio is used to assist in diagnosis/monitoring of plasma cell disorders. The binding site reference range for serum-free light chain κ:λ ratios of 0.26-1.65 was derived from healthy volunteers. Subsequently, a reference range of 0.37-3.1 for patients with chronic kidney disease has been proposed. Elevated free light chain concentrations and borderline raised free light chain ratios also may be found in polyclonal gammopathies and with other non-renal illnesses. This assessment was conducted to validate the established free light chain reference ranges in individuals from primary care. A total of 130 samples were identified from routine blood samples collected in primary care for routine biochemistry testing and estimated glomerular filtration rate calculation. The median and range of κ:λ ratios found in each estimated glomerular filtration rate group used for chronic kidney disease classification were higher than previously described. This was the case for individuals with normal or essentially normal renal function with estimated glomerular filtration rates>90, (0.58-1.76) and estimated glomerular filtration rate of 60-90 mL/min/1.73 m(2), (0.71-1.93). Individuals with estimated glomerular filtration rate 15-30, (0.72-4.50) and estimated glomerular filtration rate <15 ml/min/1.73 m(2) (0.71-4.95) also had higher values when compared to the current renal reference range of 0.37-3.10. Elevation of free light chain-κ:λ ratios may occur in the absence of a reduced renal function shown by a normal estimated glomerular filtration rate and in the presence of reduced renal function by estimated glomerular filtration rate when comparing results with the established reference ranges. Explanations include choice of analytical systems or the presence of other concurrent non-plasma cell illness.

Abstract 19078: Early Predictors of Survival to Heart Transplantation in Patients With Amyloid Light-chain Cardiomyopathy

Background: Amyloid cardiomyopathy progresses more rapidly and has different manifestations than more common cardiomyopathies. Nevertheless, survival of selected cardiac amyloid patients after orthotopic heart transplantation (OHT) followed by autologous stem cell transplant is similar to other restrictive cardiomyopathies. The importance of clinical presentation and pre-transplantation characteristics on outcome in patients with AL amyloid cardiomyopathy evaluated for heart transplantation is still not well defined. Methods: The impact of echocardiographic, hemodynamic, and clinical parameters on outcome in 46 patients evaluated for OHT and enrolled in the International Consortium for Cardiac Amyloid Transplant (iCCAT) database was studied. Cox proportional hazards models of time to death were used. End point was death after transplant evaluation censored at transplant. Results: The median age at the time of transplant evaluated was 57.6 (+/- 9.6) years, and the mean time from presentation to listing was 27 (+/- 26) days. 19 (41%) patients underwent OHT after an average wait time of 72 days. Univariate risk factors associated with death after initial evaluation were cardiac output (p=0.012), right ventricular stroke work index (p=0.033), left ventricular end diastolic dimension (p=0.024), left ventricular outflow tract velocity time integral (p=0.019), mean arterial pressures (p=0.005), NT pro-BNP (p=0.007), presence of pleural effusion (p=0.005), as well as elevated kappa and lambda serum free light chain concentrations (p=0.004). Whereas left ventricular ejection fraction did not correlate with death after evaluation, increased right ventricular wall thickness remained a strong mortality predictor in a multivariate model with light chain difference and cardiac output (HR 1.6 for every 1 mm increase in thickness, p=0.017). Conclusions: Pleural involvement, high light chain burden, and right ventricular infiltration predicted death while awaiting a donor organ and may be markers of more systemic disease. Ultimately, this could affect transplant candidate selection and allocation strategies.

Elevated Concentrations of Serum Immunoglobulin Free Light Chains in Systemic Lupus Erythematosus Patients in Relation to Disease Activity, Inflammatory Status, B Cell Activity and Epstein-Barr Virus Antibodies.

ObjectiveIn this study, we examined the concentration of serum immunoglobulin free light chains (FLCs) in systemic lupus erythematosus (SLE) patients and investigated its association with various disease parameters in order to evaluate the role of FLCs as a potential biomarker in SLE. Furthermore, FLCs’ association with Epstein-Barr virus (EBV) antibodies was examined.MethodsUsing a nephelometric assay, κFLC and λFLC concentrations were quantified in sera from 45 SLE patients and 40 healthy controls. SLE patients with renal insufficiency were excluded in order to preclude high concentrations of serum FLCs due to decreased clearance.ResultsSerum FLC concentrations were significantly elevated in SLE patients compared to healthy controls (p<0.0001) also after adjusting for Ig levels (p<0.0001). The concentration of serum FLCs correlated with a global disease activity (SLE disease activity index (SLEDAI)) score of the SLE patients (r = 0.399, p = 0.007). Furthermore, concentrations of FLCs correlated with titers of dsDNA antibodies (r = 0.383, p = 0.009), and FLC levels and SLEDAI scores correlated in the anti-dsDNA-positive SLE patients, but not in anti-dsDNA-negative SLE patients. Total immunoglobulin (IgG and IgA) concentrations correlated with FLC concentrations and elevated FLC levels were additionally shown to associate with the inflammatory marker C-reactive protein and also with complement consumption determined by low C4 in SLE patients. Collectively, results indicated that elevated serum FLCs reflects increased B cell activity in relation to inflammation. SLE patients had an increased seropositivity of EBV-directed antibodies that did not associate with elevated FLC concentrations. An explanation for this could be that serum FLC concentrations reflect the current EBV activity (reactivation) whereas EBV-directed antibodies reflect the extent of previous infection/reactivations.ConclusionSLE patients have elevated concentrations of serum FLCs that correlate with global disease activity scores and especially serologic markers for active disease. These findings are suggestive of circulating FLCs having potential as a new supplementary serologic biomarker in SLE.

DIAGNOSTIC AND PROGNOSTIC SIGNIFICANCE OF INTRATHECAL SYNTHESIS OF IMMUNOGLOBULIN FREE LIGHT CHAINS IN MULTIPLE SCLEROSIS

Increased intrathecal synthesis and oligoclonal banding of immunoglobulins (Ig) in cerebrospinal fluid (CSF) are major immunological findings in multiple sclerosis. Free light chains are fragments of Ig molecules that are produced in parallel to oligoclonal Ig, and their production reflects total Ig synthesis within central nervous system. Assessment of intrathecal kappaand lambda free light chain production within CNS compartment will help to improve diagnosis and prediction of outcomes in multiple sclerosis. Concentrations of kappa and lambda free light chains and clonality of immunoglobulin pattern synthesis were studied in paired CSF and serum samples of 151 patients, including 92 patients with multiple sclerosis, and 33 patients with clinically isolated syndrome which later was transformed into the definite multiple sclerosis. A control group consisted of 26 patients with other inflammatory diseases of central nervous system. Several Ig parameters have been determined in the patients, i.e., Ig clonality in CSF; concentrations of free light chains (both kappaand lambda-) in CSF; as well as their indexes and ratios. It was established that synthesis of kappa free light chains were significantly elevated in patients with multiple sclerosis. Moreover, the amounts of free light Ig chains in patients with positive oligoclonality test were significantly higher than in cases without intrathecal oligoclonal synthesis. With respect to diagnostic significance, the kappa quotient proved the best available option for diagnosis of multiple sclerosis. Its combination with Ig oligoclonality assays caused a decrease in false-negative diagnostics by 42%. Diagnostic benefit of the kappa quotient could be also confirmed by the data of ROC analysis. Also concentration of lambda free light chains in cerebrospinal fluid showed a negative correlation with conversion terms of clinically isolated syndrome to evident multiple sclerosis. Therefore, the indexes of free Ig light chains enables more precise diagnostics, like as more efficient severity evaluation of multiple sclerosis.

Quantitation of free light chains in the cerebrospinal fluid reliably predicts their intrathecal synthesis.

The results of free light chains quantitation in the cerebrospinal fluid were recently compared with the presence of cerebrospinal fluid-restricted oligoclonal IgG, but not oligoclonal free kappa light chains and oligoclonal free lambda light chains. We therefore aimed to compare the performance of the quantitative tests with the qualitative one for the same molecule. Seventy-five paired cerebrospinal fluid and serum samples were analysed for oligoclonal IgG, oligoclonal free kappa light chains and oligoclonal free lambda light chains. Cerebrospinal fluid and serum free kappa and lambda light chains were quantified using Freelite™ kits on SPA Plus analyzer. ROC curves were analysed for the prediction of intrathecal synthesis and compared for cerebrospinal fluid concentration, cerebrospinal fluid/serum quotient (QfLC) and index (QfLC/QAlbumin). The presence of cerebrospinal fluid-restricted oligoclonal free kappa light chains and oligoclonal free lambda light chains bands was used as reference. No statistically significant differences were observed among cerebrospinal fluid concentration, QfLC and index for the prediction of free light chain intrathecal synthesis. Each parameter was able to predict the occurrence of cerebrospinal fluid-restricted oligoclonal free light chain bands (AUCs 0.932-0.999). However, we noted elevated cerebrospinal fluid free light chain concentrations in the absence of cerebrospinal fluid-restricted oligoclonal free light chain bands in two patients with very high serum free light chain values. Quantitation of cerebrospinal fluid free light chains reliably predicts their intrathecal synthesis. Yet, cerebrospinal fluid/serum quotient may still be preferred to correct for high serum free light chain concentrations. An appropriate formula should be sought to correct for blood-cerebrospinal fluid barrier status.

Plasmapheresis in cast nephropathy: yes or no?

Renal failure is a frequent complication of multiple myeloma and portends a poor prognosis. Plasmapheresis has been suggested as an adjunct to chemotherapy to halt or reverse renal injury associated with multiple myeloma. The purpose of this article is to review the rationale for using plasmapheresis for this indication and then provide a discussion of the evidence regarding its use. The outcome of patients with multiple myeloma has improved considerably in recent years, mostly owing to the introduction of new highly effective chemotherapeutic agents. However, patients with renal failure who do not recover independent renal function continue to have very poor prognosis. Recent evidence now indicates that an early and sustained reduction in circulating free light chains (FLCs) is associated with improved renal recovery in patients with myeloma kidney. Extracorporeal removal of FLCs with plasmapheresis, or other techniques, can achieve rapid and sustained reduction in serum FLC concentration in patients with acute myeloma kidney. Unfortunately, there is currently no convincing evidence in the literature that the addition of mechanical removal of FLC to standard chemotherapy translates into clinical benefits for patients. Plasmapheresis is theoretically attractive as a means of rapidly lowering serum FLC burden in the hope of reducing nephrotoxicity in patients with multiple myeloma. However, the role of plasmapheresis in improving renal prognosis and patient survival remains to be demonstrated.

Comparison of Free Light Chain Assay with Protein Electrophoresis for Screening and Monitoring PTLD

Background: Post-transplant lymphoproliferative disorder (PTLD) is primarily diagnosed histologically using tissue biopsy. Free light chain (FLC) assay and serum protein electrophoresis (SPE) have both been studied as tools to screen and monitor PTLD. However, limited data are available to compare these two assays in a well characterized patient population. It is also not clear what reference ranges should be adopted for the FLC assay in a post-transplant population.Method: Blood samples from 169 patients receiving a variety of solid organ transplants were analyzed for FLCs and screened for gammopathies by SPE/IFE.Results: Compared with non-PTLD patients, PTLD patients had higher mean, median and upper 95 percentile range of both κ and λ FLCs (p ranging from 0.0002 to 0.024). The mean, median and 95 percentile range of κ:λ ratio were similar between the two groups. PTLD patients were more likely to have polyclonal or monoclonal FLC elevations (p = 0.04). They also showed a higher frequency of gammopathy abnormalities (p = 0.0052). Nonetheless, neither FLC assay nor SPE demonstrated a clear association with the timing of PTLD diagnosis. FLC concentrations in non-PTLD recipients were higher than those in the general healthy population (95 percentile range: κ, 0.60-8.33 mg/dL vs. 0.33-1.94 mg/dL; λ, 0.77-7.08 mg/dL vs. 0.571-2.63 mg/dL) but the κ:λ ratio was similar to that of the healthy group (0.26-1.65).Conclusions: Our results suggested that elevated FLC concentrations and gammopathy abnormalities were both associated with PTLD. Therefore, FLC assay and SPE should be used conjunctively for screening PTLD among solid organ transplant recipients. For this application, the data showed that a higher upper limit of κ and λ FLC levels and normal κ:λ ratio should be used as diagnostic reference ranges. Additionally, neither method was clearly associated with the timing of PTLD diagnosis, indicating that they may be unsuitable for monitoring PTLD in the post-transplant population.Table 1Longitudinal measurements of serum/plasma free light chains and SPE/IFE in eight PTLD cases.Type of transplant and type of PTLDSamplesDays from PTLD diagnosisaκ FLC, mg/dLλ FLC, mg/dLκ /λSPE/IFELiver transplant, B-cell PTLD1.1 1.2– 6162.33b1.824.966.010.47 0.303no band –cLiver transplant, B-cell PTLD2.1 2.2 2.3 2.4 2.5-145* -126 84 141 4280.338 0.79 0.335 0.476 2.530.62 1.02 1.06 1.15 1.980.545 0.775 0.316 0.414 1.28no band no band 1 IgG ©µ, 1 IgG λ 1 IgG ©µ, 1 IgG λ no bandLiver transplant, polymorphic hyperplasia3.1 3.2-77 2084.197.185.93.380.71 2.122 IgG ©µ, 2 λ FLC–Liver transplant, B-cell PTLD4.1 4.2 4.361 272 5374.643.257.111.16.6510.960.418 0.489 0.648no band no band no bandLiver transplant, B-cell PTLD5.1 5.2 5.3 5.4 5.59 12 393 429 476305.59570.721 1.01 1.2474.251921.67 1.72 2.564.114.980.432 0.587 0.4841 IgG ©µ, 1 λ FLC 2 IgM ©µ no band no band no bandLiver transplant, B-cell PTLD6.1 6.2 6.3 6.462 153 174 1883.165.583.972.142.923.913.813.371.08 1.43 1.04 0.635– 1 IgG ©µ 1 IgG ©µ 1 IgG ©µKidney transplant, PTLD7.1 7.215 301.4 1.681.58 2.060.89 0.82no band no bandLung transplant, Non-Hodgkin lymphoma8.1-604.284.580.94no banda Positive values indicate time points before PTLD diagnosis, while negative values indicate time points after PTLD diagnosis.b Numbers in bold format indicates values above ULN.c SPE/IFE results not available due to insufficient sample volume. DisclosuresKuhn:The Binding Site, Inc: Employment.

Quantitative Assay of Immunoglobulin Free Light Chains (FLC): Evaluation of Monoclonal Versus Polyclonal Antibody and Immunoturbidimetric Versus Immunonephelometric Detection Technology

Introduction. It has been rarely reported that a laboratory test introduced so rapid and radical changes in diagnostic algorithm as is the case with the quantitative assay of free light chains of immunoglobulins (FLC) in serum and its role in the diagnostic algorithm of monoclonal gammopathies. Since the first description of immunoassay in year 2001 until today, new evidence has continuously been reported in the literature that confirm the clinical usefulness of this test in diagnosis, monitoring and prognosis of monoclonal plasma-proliferative diseases, especially diseases of light chains such as primary amyloidosis, light chain deposition disease (LCDD) or light chain multiple myeloma (LCMM) and nonsecretory multiple myeloma (NSMM). Recently, a commercial test has become available on the market that uses polyclonal antibodies to specific epitopes of free light chains which are hidden in the intact immunoglobulin molecules. In 2011, a commercial immunoassay was launched on the market that uses monoclonal instead of polyclonal antibodies, reducing the variability between different series of reagents and controlling excess antigen in the sample.Aim. The aim of this study was to evaluate monoclonal versus polyclonal antibody and immunoturbidimetric versus immunonephelometric detection technology. Does different detection tehnology – besides different used antibody – contribute to greater variability in results?Method. In this study we compared results of 40 samples measured with polyclonal antibody (The Binding Site Ltd., Birmingham, UK) and monoclonal antibody (N Latex FLC, Siemens Healthcare Diagnostics, Marburg, Germany). In other 40 samples we compared results achieved with different antibodies and different analytical platforms (Siemens Nephelometer with Roche Cobas). Results were statistically analyzed using MedCalc software.Results. Results are shown in Table 1. Comparing all results, it is evident that there is at least proportional error when comparing different antibodies and different analytical systems. Although it is known that immunoturbidimetry is less sensitive than immunonephelometric method, greater discrepancies in results were not found. When we categorized patients as positive and negative according to manufacturer’s reference interval for kf/lf ratio, agreement between groups with different antibody and same detection technology was 63% (weighted kappa 0.30). Agreement between groups with different antibodies and different detection technology was 86% (weighted kappa 0.22). Although we have not measured the same samples when testing antibody and analytical platform, the selected analytical platform has, according to our results, no additional impact on the variability of results.Abstract 5680. Table 1Comparison of FLC results using different antibody and different analytical platformsMethodFLC kappa polyclonal Ab Nephelometer SiemensFLC kappa monoclonal Ab Nephelometer SiemensFLC lambda polyclonal Ab Nephelometer SiemensFLC lambda monoclonal Ab Nephelometer SiemensResults (min-max)6.59-5210.006.34-1600.001.67-3010.001.00-1600.00Passing-Bablok fitintercept (95% Cl)8.2442.9255 to 14.92491.0945-1.5910 to 5.5631slope (95% Cl)0.5950.4564 to 0.78521.87981.5336 to 2.1045Correlation rs (p<0.0001)0.9110.887MethodFLC kappa polyclonal Ab Cobas RocheFLC kappa monoclonal Ab Nephelometer SiemensFLC lambda polyclonal Ab Cobas RocheFLC lambda monoclonal Ab Nephelometer SiemensResults (min-max)2.40-453.201.10-342.002.30-452.1010.30-301.20Passing-Bablok fitintercept (95% Cl)2.622-3.8207 to 9.30230.1585-3.9731 to 4.5892slope (95% Cl)1.51.2972 to 1.99330.74160.6174 to 0.8708Correlation rs (p<0.0001)0.8730.929Conclusion. Physicians and especially clinical biochemists must be aware of the technical shortcomings of this test, such as the variability between different series (lots) reagents, non-linearity, unreliable detection of excess antigen and overestimation of FLC concentrations due to nonspecific interference or polymerization.Although initial results are not discouraging, it will be necessary to collect much more evidence, especially bearing in mind that use of monoclonal antibodies along with advantages has certain disadvantages. In the future, it will probably be necessary to incorporate into the guidelines a recommendation to report the method used, like for other tumor markers. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Serum free light chains in patients with HIV infection: their association with markers of disease severity and antiretroviral use

AimSerum free light chain measurements are used to follow-up and manage patients with monoclonal gammopathies, and abnormal ratios are associated with risk of progression in certain diseases. B cell dysfunction...

Comparison of the free and total light chain assays in serum and urine samples with immunofixation electrophoresis for detecting monoclonal proteins in patients with monoclonal gammopathy

ABSTRACT Monoclonal protein (M-protein) is produced by a malignant clone of plasma cells. Detected in serum and/or urine, this typically indicates multiple myeloma (MM) or other monoclonal gammopathy (MG). In a majority of MM cases, with the production of intact monoclonal immunoglobulin (Ig), malignant plasmocytes and/or B lymphocytes often produce excessive amounts of free light chains (FLCs). Excessive synthesis of FLCs lowers the ability of renal proximal tubules to re-absorb FLCs, which results in abnormally high levels of FLCs in the urine (Bence Jones protein, BJP). In laboratory practice, there are tests available for the quantitative measurement of only FLCs κ and λ or for total light chains (TLCs). These tests measure both free forms and bound in the (Ig) molecules forms as light chains that are evident in the serum and in urine. The purpose of this study was to evaluate the FLCs and TLCs approaches in screening serum and urine samples of patients with MM, doing so in comparison to the results of immunofixation (IFE) assessment. A second purpose was to assess the suitability of the collected material for obtaining the most reliable results. The results of serum FLCs (sFLCs) assays suggest that this approach is of the highest reliability and diagnostic usefulness in the detection of MG with excess production of FLCs, in comparison to other available tests. In our work, when κ band light chains were detected in serum IFE (sIFE), 91% patients had their FLCs concentrations beyond the reference range, whereas 89% patients had increased λ FLCs when λ band light chains were detected in sIFE. We also found abnormal sFLC κ/λ ratios in 86.4% and 88.9% of all subject patients who had κ or λ band light chains detected in their sIFE, respectively.

THU0508 Sparing Methotrexate Results in a Less Stringent Control of Plasma Cell and Immunoglobulin Free Light Chain Production in Seropositive Patients with Rheumatoid Arthritis

BackgroundClinical trials revealed that tumor necrosis factor alpha (TNF-α) blockade is more effective if administered with concomitant methotrexate (MTX).ObjectivesBased on an observation made when studying B cell subsets in patients...

Diagnosis of Cardiac Amyloidosis by Skin Lesions

In June 2011, a 74-year-old man was admitted for recurrent heart failure. He had hypertension, diabetes mellitus, renal failure due to nephrosclerosis and diabetic nephropathy, prostate cancer, tuberculosis, and gouty arthritis, and had received a parathyroidectomy. In 2009, he had received a mechanical prosthesis to treat aortic stenosis. Cardiac auscultation was rhythmic, with no murmurs, and with normal prosthetic sounds. The patient presented petechiae, purpura, papules, and spontaneous ecchymosis at the eyelids, groin, neck, and axillas (Figure 1), with onset after initiation of acenocoumarol in 2009. The electrocardiogram demonstrated sinus rhythm with a long P-R interval, right bundle branch block with left anterior hemiblock, and low voltage, consistent with infiltrative cardiomyopathy (Figure 2). The echocardiogram demonstrated left ventricular hypertrophy with increased echogenicity (Figures 3A and B), ejection fraction of 45%, and a restrictive pattern on pulsed Doppler (Figure 3C: increased E/A ratio) and tissue Doppler (Figure 3D: decreased E’ and A’ peaks). Examination of skin biopsy specimens with Congo red stain showed amyloidosis secondary to monoclonal gammopathy, with positive immunohistochemistry for lambda light chains. He was treated with melphalan and dexamethasone, which were discontinued because of a favorable hematological response, with normalization of serum free light chain concentrations. Thereafter, he was hospitalized several times for heart failure and ultimately died of this cause in 2013. The purpura, petechiae, and ecchymosis were due to intracutaneous bleeding, resulting from a fragile vascular status secondary to amyloid infiltration. This is sometimes the first and only sign of systemic amyloidosis. Although these features are not specific to this disease, they may be an aid to its diagnosis and prompt treatment in the appropriate clinical context. Figure 3. Figure 1. I

Serum free light chains and the adaptive immune response in usual and alpha-1-antitrypsin deficiency-related chronic obstructive pulmonary disease

BackgroundChronic obstructive pulmonary disease (COPD) is a chronic inflammatory condition with a complex pathophysiology. In COPD, the immune response involves the adaptive immune system, although the antigenic stimulus driving this response is unknown. During the process of antibody synthesis an excess of free light chains (FLCs) are produced and secreted into the circulation. High polyclonal FLC concentrations have been found in several autoimmune and inflammatory conditions such as systemic lupus erythematosus and rheumatoid arthritis. Thus, FLCs are being investigated as a biomarker of immune activation, disease activity, and severity in these conditions. Our aim was to establish whether serum FLCs could be a marker of phenotypic variation or predict poor outcome in patients with usual and alpha-1-antitrypsin deficiency (A1ATD)-related COPD. MethodsWe measured FLC concentrations in 294 COPD patients with A1ATD-related and 85 patients with usual COPD using the Freelite serum FLC assay (The Binding Site Ltd, Birmingham, UK). We then compared combined (κ and λ) FLC (cFLC) concentrations in different subgroups defined by the presence of chronic bronchitis, colonisation of the lower respiratory tract, and subsequent mortality. In addition, we measured the estimated glomerular filtration rate (eGFR) of patients included in the study. FindingsOur preliminary data showed that in A1ATD-related COPD and usual COPD, cFLC concentrations were significantly higher in patients with chronic bronchitis than in those without bronchitis (p=0·008 vs p=0·047). Patients with A1ATD-related COPD who were chronically colonised with pathogenic organisms in their stable state also had significantly higher cFLC concentrations than did those who were not colonised (p=0·036). A multiple regression analysis showed that eGFR and the presence of chronic bronchitis significantly predicted cFLC concentrations in patients with A1ATD: renal function was the strongest predictor (β=−0·326, p<0·0001) followed by the presence of chronic bronchitis (β=0·147, p=0·01). A similar trend was seen in the usual COPD group although this did not meet statistical significance (eGFR β=−0·220, p=0·055; chronic bronchitis β=0·224, p=0·061). The 33 A1ATD patients who subsequently died also had significantly higher cFLC concentrations (p=0·005) than did those still alive. InterpretationThe higher cFLC concentrations in patients with A1ATD-related COPD who have chronic bronchitis and colonised lower respiratory tracts could mean that there is a greater adaptive immune response in these individuals. The drive behind this response might come directly from microorganisms or through self-perpetuating autoimmune events. Within the specialty of COPD clinical management, there is a move towards a more individualised approach to treating different clinical phenotypes. Measurement of serum FLCs might have a potential role as a biomarker to help stratify patients. The higher cFLC concentrations in patients with renal impairment is unsurprising in view of their renal clearance, but raised polyclonal FLCs have also been shown to be a predictor of mortality in the general population. Our mortality results suggest that FLCs could have a role in risk stratification for patients with COPD, but further investigation is needed. FundingGrifols Therapeutics.

Isotype Matched Paired Immunoglobulin Suppression Identifies High Risk Multiple Myeloma Patients and Is Independent Of Bone Marrow Infiltration Suggesting Significant Variability In The HLC Matched Pair Suppressive Activity Of Individual Myeloma Clones

IntroductionMultiple myeloma (MM) is characterised by the production of monoclonal immunoglobulins (MIg), both intact and a free light chain (FLC) component. The production of MIg can be associated with suppression of the non-involved isotypes which can easily be measured by conventional techniques while identification of a possible suppression of the HLC matched pair isotype was not possible before the introduction of the HLC assay. Here we assess the frequency and biologic relevance of the suppression of HLC pair matched isotypes and non-involved isotypes and correlate these findings and those of MIg measurements with bone marrow plasma cell (BMPC) infiltration. Patients and Methods113 previously untreated MM patients (72 IgG (46κ, 26λ) and 41 IgA (23κ, 18λ)) with a median age of 65 years (range 32-86 years) were enrolled and followed for up to 13 years (median: 4 range 0.25-13 years). Serum FLC (FLCκ/FLCλ) and HLC concentrations (IgGκ/IgGλ and IgAκ/IgAλ) measurements were made using polyclonal antisera assays (FreeliteTM, Binding Site, Birmingham, UK. and, HevyliteTM, Binding Site, Birmingham, UK) and results compared to standard serum assessments (SPE, IFE, β2M, Albumin, LDH and CRP) and bone marrow plasma cell (BMPC) infiltration. Severe non-involved isotype or HLC pair suppression was defined as >50% lower of than the published lower normal ranges for the relevant isotypes. Correlation and survival analysis was performed using the SPSS v 21 programme. ResultsMedian overall survival was 46 months (range 3-158). IgA MIg production correlated weakly to BMPC infiltration at presentation (Pearsons 0.327; p=0.026) but to no other marker. Uninvolved IgA HLC did not correlate to BMPC infiltration but had a weak correlation to LDH (Pearsons 0.436; p=0.002). By contrast, IgG MIg production showed significant correlation to ß2M (Pearsons 0.53; p<0.001), with uninvolved IgG HLC having no significant correlations. There was a greater degree of monoclonal production in IgG patients than in IgA patients (p=0.049). Severe IgG and IgM suppression was noted in 13/41 (31%) of IgA patients with 78% of patients having either isotype suppressed. In IgA MM patients IgM suppression was more commonly identified than IgG (32/41 v 13/41 patients respectively) and IgG suppression was always concomitant with IgM suppression. Severe IgA and IgM suppression was noted in 36/72 (50%) of IgG patients with 69% of patients having either isotype suppressed. In contrast to IgA patients there was no difference between the incidence of IgA or IgM suppression. Severe isotype matched pair suppression was more frequently noted in IgG compared to IgA patients (66% vs. 60%, p<0.01). Severe HLC pair suppression correlated with a decreased OS (46.6 vs. 84.4 months, p<0.044). By contrast neither non-involved isotype nor monoclonal Ig production was correlated with outcome. Discussion and ConclusionThe correlation between IgG MIg and BMPC infiltration indicates a greater uniformity in the inter-individual MIg production rate in patients with IgG compared to those with IgA MIg, which were only weakly correlated with BMPC infiltration. Part of this finding may also be due to greater variations in the catabolic rate of individual IgG MIgs. The absence of a correlation between the degree of HLC pair suppression and BMPC infiltration suggests significant variation in the ability of individual clones in suppressing the isotope matched pair. This uncoupling between the proportion of myeloma cells and isotype suppression was previously unknown. Myeloma clones with greater HLC matched isotype pair suppression reflect a more aggressive phenotype which correlates with short survival. Suppression of the non-involved isotype, in contradistinction, showed no prognostic relevance. Disclosures:Young:The Binding Site Group Ltd: Employment. Harding:The Binding Site Group Ltd: Employment.

High-Cut Off Dialysis In Multiple Myeloma Patients With Dialysis Dependent Acute Renal Failure Shows Durable Renal Recovery: A Long-Term Follow Up Analysis

Acute kidney injury (AKI) either in primary diagnosis or at relapse is one of the frequent complications in multiple myeloma (MM). Myeloma cast nephropathy is induced by mechanical occlusion of the distal tubule by casts consisting of free light chains (FLC) and Tamm Horsfall protein. Impairment of renal function affects prognosis, with particularly poor outcomes in patients requiring hemodialysis with a historically reported median survival of 3-4 months. Introduction of novel therapeutic agents in MM therapy has improved MM response as well as renal outcome mainly due to a short time to initial response and consecutively reduction of the toxic FLC component. Extracorporeal light chain elimination in addition to chemotherapeutic treatment has been discussed controversially so far. We and others introduced the high-cut off (HCO) dialysis which allows effective elimination of FLC in an extended standard dialysis protocol. Recently, we demonstrated the efficacy of combined systemic and extracorporeal therapy regarding time to FLC reduction and renal recovery in a first case series of MM patients with dialysis-dependent AKI (Heyne et al., Ann Haematol 2012). Here, we report long-term renal outcome and overall survival data.19 MM patients with dialysis dependent AKI were treated between November 2006 and October 2009 with HCO dialysis in parallel to systemic chemotherapy. The applied chemotherapy was chosen by the treating haematologist. All data was calculated by intent-to-treat (ITT) analysis. Progression-free survival (PFS) was analysed from the first day of HCO dialysis to progression or death, whichever occurred first. Overall survival was calculated from the first day of HCO dialysis to death. For statistical analysis GraphPad Prism (GraphPad Software Inc., La Jolla/CA, USA) and R (R Foundation for Statistical Computing, Vienna) biostatistical software was used.The ITT cohort consisted of 19 patients. Median age was 69 (range 50-83) years. 10 patients had newly diagnosed, 9 relapsed or refractory MM. All patients had ISS stage III disease. Median serum FLC concentration at baseline was 8,580 (1 590-66 100) mg/L. Median serum creatinine was 6.8 (4.4-11.6) mg/dL, median eGFR 7.0 (3.3-10.9) mL/min/1.73m². Four (21%) early deaths occurred in the first three months of treatment, 2 patients with newly diagnosed and 2 patients with relapsed disease. Chemotherapy consisted mainly of bortezomib based regimens. As we reported previously, sustained renal recovery was achieved in 73.7% or 14/19 patients with a median time to independence of hemodialysis of 15 (4-64) days. In the long-term follow up analysis with a median follow-up of 62 months, 11/14 dialysis independent patients remained free of dialysis during further disease course. 3 patients again requiring dialysis all showed terminally refractory disease. Median PFS of the IIT population was 7.8 months for primary diagnosed and 4.8 months for relapsed and refractory patients, median OS of the whole population was 9.5 (range 2.8-not reached) months for primary diagnosed patients and 4.8 (range 1.0-19.8) months for the relapsed and refractory group.Combination of systemic treatment and HCO dialysis in patients with myeloma cast nephropathy and dialysis dependent renal failure results in durable renal remissions in the majority of patients. Close monitoring of patients and early treatment intervention can prevent recurrent severe AKI in myeloma relapse. Survival data reflect the critically ill patient population with high tumor burden and high-risk of early death, but also show encouraging long-term myeloma and renal remissions. The benefit of the additional HCO dialysis in addition to conventional chemotherapy is currently investigated in a randomised multicenter trial (EuLITE study). Disclosures:Weisel:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.