RESUMEN
BACKGROUND: Immunofixation electrophoresis (IFE) is the standard method for confirming the presence of a monoclonal protein (M-protein) at multiple myeloma (MM) diagnosis. IFE is also essential at assessment of complete response (CR) and stringent CR during treatment. As the CR assessment is influenced by daratumumab and isatuximab, HYDRASHIFT assays were developed. METHODS: Samples from patients under treatment that included daratumumab or isatuximab were tested and monitored by IFE on the HYDRASYS system using HYDRASHIFT assays (HYDRASYS/HYDRASHIFT) and by IFE on the Epalyzer2 system (Epalyzer). RESULTS: The IFE using HYDRASYS/HYDRASHIFT avoided a false positive caused by drug-related IgG-κ and contributed to accurate assessment of CR. Furthermore, HYDRASYS/HYDRASHIFT detected small M-proteins at early relapse and detected free light chains (FLCs) in patients with renal impairment exhibiting high serum FLCs despite being often missed on Epalyzer. CONCLUSION: Sensitivity and specificity of M-protein detection vary greatly depending on the IFE system and reagents used.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Anticuerpos Monoclonales , Mieloma Múltiple , Proteínas de Mieloma , Humanos , Anticuerpos Monoclonales/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/sangre , Anticuerpos Monoclonales Humanizados/uso terapéutico , Proteínas de Mieloma/análisis , Femenino , Anciano , Masculino , Persona de Mediana Edad , Inmunoelectroforesis/métodos , Resultado del Tratamiento , Inmunoglobulina G/sangre , Cadenas kappa de Inmunoglobulina/sangre , Indicadores y ReactivosRESUMEN
Serum immunofixation electrophoresis (IFE) is often performed for screening monoclonal proteins (M proteins) in immunoglobulin light-chain amyloidosis (AL amyloidosis). However, the performance of serum IFE for detecting M protein in AL amyloidosis patients is often insufficient. In this study, we examined the detection rate of serum M protein in newly diagnosed AL amyloidosis patients and analyzed differences in M protein detection between IFE methods. Among 60 patients newly diagnosed with AL amyloidosis, 22 had undetectable serum M protein by IFE with the Epalyzer2 system. Samples with undetectable M protein had significantly lower involved serum-free light-chain (iFLC) and a smaller difference between involved and uninvolved serum-free light-chain (dFLC) values than samples with IFE-detectable monoclonal light chains. When samples that tested negative for M protein by the Epalyzer2 system were retested by IFE with the HYDRASYS 2 system, 50% had IFE-detectable monoclonal light chains. The IFE system and reagents used may affect serum monoclonal immunoglobulin light-chain detection in AL amyloidosis patients, especially those with low iFLC or low dFLC samples. More attention should be paid to the performance of IFE systems, since it may affect the diagnostic and therapeutic evaluation of AL amyloidosis patients.
Asunto(s)
Cadenas Ligeras de Inmunoglobulina , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas , Humanos , Cadenas Ligeras de Inmunoglobulina/sangre , Femenino , Masculino , Anciano , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/diagnóstico , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/sangre , Persona de Mediana Edad , Inmunoelectroforesis/métodos , Anciano de 80 o más AñosRESUMEN
BACKGROUND: Isatuximab, an IgG-kappa (IgGκ) anti-cluster of differentiation 38 (CD38) monoclonal antibody approved for use in patients with relapsed or refractory multiple myeloma (MM), can potentially interfere with the visualization of endogenous monoclonal protein (M-protein) on standard immunofixation electrophoresis (IFE) and lead to inaccurate classification of a patient's response to therapy. The Hydrashift 2/4 isatuximab IFE assay (Hydrashift isatuximab assay) removes isatuximab interference from IFE. Using samples from patients enrolled in clinical trials of isatuximab-based therapy for MM, we demonstrate how the Hydrashift isatuximab assay improves the ability to detect residual M-protein and offer recommendations for when the assay is most useful. METHODS: Samples from 141 patients with a variety of known M-protein isotypes were selected and analyzed by standard IFE and the Hydrashift isatuximab assay. A positive control containing isatuximab was run on every standard IFE and Hydrashift gel. RESULTS: The Hydrashift isatuximab assay reliably shifted the migration of isatuximab in patient samples. Standard IFE was adequate for determining 104 patients' M-protein status, and the Hydrashift isatuximab assay confirmed these results. In samples from 37 patients with a history of IgGκ MM and a single IgGκ band visible on standard IFE near the isatuximab migration site, the Hydrashift isatuximab assay was able to separate isatuximab from endogenous M-protein, identifying residual M-protein in 17 samples and preventing false-positive interpretations of standard IFE in 20 samples. CONCLUSIONS: The Hydrashift isatuximab assay is most useful in patients with known IgGκ MM when a single IgGκ band appears near the isatuximab migration site on standard IFE during isatuximab-based therapy. CLINICALTRIALS.GOV REGISTRATION NUMBERS: NCT03275285 and NCT03319667.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Inmunoelectroforesis , Mieloma Múltiple , Proteínas de Mieloma , Humanos , Mieloma Múltiple/sangre , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/inmunología , Mieloma Múltiple/diagnóstico , Inmunoelectroforesis/métodos , Proteínas de Mieloma/análisis , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales/uso terapéuticoRESUMEN
OBJECTIVES: Some therapeutic monoclonal antibodies, like daratumumab and elotuzumab, produce interfering monoclonal bands on serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE). Whether other common therapeutic antibodies also produce interference has not been systematically evaluated. DESIGN AND METHODS: SPEP/IFE from patients receiving isatuximab (48 patients), belantamab mafodotin (BM; 41), and denosumab (41) were retrospectively reviewed for therapeutic antibody interference. Cases exhibiting isatuximab interference were quantified and the maximum duration of isatuximab effect was evaluated. To characterize band position, neat human serum was spiked with BM or denosumab at supratherapeutic concentrations. Band migration patterns were compared on SPEP and IFE, with band position expressed relative to other constant protein fractions. RESULTS: Isatuximab-induced IFE interference was common (81.3 % of evaluated patients) with a maximum observed duration of 8 weeks. 10.4 % of isatuximab patients had IgG kappa monoclonal gammopathies that co-migrated with the drug; this subset could benefit from HYDRASHIFT 2/4 isatuximab testing. 8.3 % of IFE cases were negative for an isatuximab band but showed large, endogenous M-spikes migrating elsewhere. All patients in this group expired within 1 year of this finding. We hypothesize that an inability to detect isatuximab in this setting corresponds to a large residual myeloma burden that reduces isatuximab serum concentration. This observation may serve as a negative prognostic factor. Spiking studies demonstrated that BM and denosumab produce interference in vitro, but sustained interference was not observed in >40 treated patients. CONCLUSIONS: Therapeutic antibody interference in patients receiving isatuximab is common, and can persist for at least 8 weeks after administration. >10 % of patients receiving isatuximab may benefit from HYDRASHIFT testing post-therapy. In contrast, BM and denosumab fail to produce sustained interference in treated patients.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Denosumab , Mieloma Múltiple , Humanos , Denosumab/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Estudios Retrospectivos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/sangre , Electroforesis de las Proteínas Sanguíneas/métodos , Femenino , Masculino , Anciano , Persona de Mediana Edad , Anticuerpos Monoclonales , Inmunoelectroforesis/métodosRESUMEN
BACKGROUND: A substantial number of patients with multiple myeloma (MM) who have bone destruction are initially admitted into the orthopedic service at the hospital. However, routine laboratory testing usually fails to identify these patients, thus delaying optimal therapy. Therefore, there is a clear medical need for early diagnosis of MM in these patients. METHODS: Between 2019 and 2021, 42 patients receiving treatment for orthopedic conditions had normal hemoglobin (Hb), total protein (TP), albumin (ALB), creatinine (CREA), and blood calcium (Ca) levels before their surgical procedure(s) but were subsequently pathologically confirmed to have MM, based on their presenting orthopedic symptoms. During the same period, 52 patients with orthopedic conditions were pathologically excluded from the diagnosis of MM and were recruited into our control group. Serum free light chain (sFLC) testing was performed in 94 consecutive patients in the orthopedic service using Siemens N Latex FLC kits. The levels of Hb, TP, ALB, CREA, and Ca were also measured. All 42 patients with MM were divided into group A (n = 25: κ proliferation) and group B (n = 17: λ proliferation) by the pathology department. RESULTS: There were no significant differences in levels of Hb, TP, ALB, CREA, and Ca between group A and group B and the control group. However, the sFLC κ/λ ratio of group A and B was also significantly different from that of the control group (P < .001). The results of serum immunofixation electrophoresis (IFE) testing demonstrated negative results in 14 cases (58.3%) in group A and 4 cases (25.0%) in group B. CONCLUSIONS: Some patients with orthopedic conditions who do not have typical MM laboratory results, such as those with abnormal Hb, TP, ALB, CREA, and Ca levels before their operation(s), actually have MM. MM should be highly suspected in patients with unexplained bone lesions and with an abnormal sFLC κ/λ ratio. Further tissue or bone marrow biopsy is needed in these patients even if serum and urine IFE results are negative and light chain ratio is normal.
Asunto(s)
Calcio , Mieloma Múltiple , Humanos , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/sangre , Femenino , Masculino , Persona de Mediana Edad , Anciano , Calcio/sangre , Hemoglobinas/análisis , Creatinina/sangre , Cadenas Ligeras de Inmunoglobulina/sangre , Cadenas Ligeras de Inmunoglobulina/orina , Inmunoelectroforesis/métodos , Anciano de 80 o más Años , Adulto , Proteínas Sanguíneas/análisisRESUMEN
BACKGROUND: Matrix assisted laser desorption ionization time of flight mass spectrometry coupled to immune enrichment (MASS-FIX) as an alternative to serum immunofixation electrophoresis has demonstrated increased sensitivity in monoclonal protein (MP) detection with improved laboratory workflow. This study explored similar replacement of urine immunofixation electrophoresis (u-IFE) with urine MASS-FIX (u-MASS-FIX) by method comparison. METHODS: Residual urine (n = 1008) from Mayo Clinic patients with a known plasma cell disease were assayed neat by u-MASS-FIX analysis. Each sample was paired with the following: u-IFE, urine total protein, urine protein electrophoresis, serum κ/λ free light chain (LC) ratio (rFLC), and serum MASS-FIX (s-MASS-FIX). Analytical sensitivities were measured in pooled urine spiked with daratumumab. RESULTS: u-IFE and u-MASS-FIX had 91% agreement in determining the presence/absence of MPs (Cohen kappa = 0.8200). In discrepant cases, serum rFLC statistically aligned more closely with positive u-MASS-FIX cases than u-IFE. Patients positive by both s-MASS-FIX and u-MASS-FIX had matching MP masses (±20 daltons) in 94% of cases. The u-MASS-FIX spectra further identified κ/λ LC fragments and glycosylated LCs not appreciated on u-IFE. The unconcentrated u-MASS-FIX limit of detection of 0.156 mg/mL was determined equivalent to 100× concentrated u-IFE. CONCLUSION: u-MASS-FIX is a reliable alternative to u-IFE with the added benefits of LC glycosylation detection and MP mass tracking between serum and urine. Furthermore, u-MASS-FIX is performed using neat urine. Eliminating the need to concentrate urine for u-IFE has potential to increase productivity by decreasing labor minutes per test.
Asunto(s)
Paraproteinemias , Humanos , Cadenas kappa de Inmunoglobulina , Espectrometría de Masas , Inmunoelectroforesis/métodos , Anticuerpos Monoclonales , Cadenas Ligeras de InmunoglobulinaRESUMEN
BACKGROUND: Immunofixation electrophoresis (IFE) is important for diagnosis of plasma cell disorders (PCDs). Manual analysis of IFE images is time-consuming and potentially subjective. An artificial intelligence (AI) system for automatic and accurate IFE image recognition is desirable. METHODS: In total, 12 703 expert-annotated IFE images (9182 from a new IFE imaging system and 3521 from an old one) were used to develop and test an AI system that was an ensemble of 3 deep neural networks. The model takes an IFE image as input and predicts the presence of 8 basic patterns (IgA-, IgA-, IgG-, IgG-, IgM-, IgM-, light chain and ) and their combinations. Score-based class activation maps (Score-CAMs) were used for visual explanation of the models prediction. RESULTS: The AI model achieved an average accuracy, sensitivity, and specificity of 99.82, 93.17, and 99.93, respectively, for detection of the 8 basic patterns, which outperformed 4 junior experts with 1 years experience and was comparable to a senior expert with 5 years experience. The Score-CAMs gave a reasonable visual explanation of the prediction by highlighting the target aligned regions in the bands and indicating potentially unreliable predictions. When trained with only the new system images, the models performance was still higher than junior experts on both the new and old IFE systems, with average accuracy of 99.91 and 99.81, respectively. CONCLUSIONS: Our AI system achieved human-level performance in automatic recognition of IFE images, with high explainability and generalizability. It has the potential to improve the efficiency and reliability of diagnosis of PCDs.
Asunto(s)
Aprendizaje Profundo , Paraproteinemias , Humanos , Reproducibilidad de los Resultados , Inteligencia Artificial , Inmunoelectroforesis/métodos , Inmunoglobulina A , Inmunoglobulina G , Inmunoglobulina MRESUMEN
BACKGROUND: Multiple myeloma (MM) accounts for approximately 10% of hematological malignancies. The monoclonal immunoglobulin G kappa (IgG-κ) daratumumab can bind to CD38 on MM cells and be detected in serum immunofixation (IF), causing pitfalls in M-protein quantification. OBJECTIVES: To determine the efficacy of mitigating the interference of IgG MM treated with daratumumab. METHODS: Levels of Ig, free light chains (FLC) kappa (κ) and lambda (λ), serum protein electrophoresis (SPE)/IF, and Hydrashift 2/4 assays were assessed following manufacturer's instructions in three patients. RESULTS: Patient 1 was a 70-year-old male diagnosed with IgG-λ MM. The IF distinguished two monoclonal bands (IgG-κ and IgG-λ). With the Hydrashift assay, the daratumumab-anti-daratumumab immune complex shifted the IgG-κ to the α zone, suggesting that the monoclonal IgG-κ band corresponded to daratumumab. Patient 2 was a 63-year-old male with IgG-κ MM who was receiving daratumumab once every other week. SPE/IF assay revealed a faint monoclonal IgG-κ band in the ï§ zone. A stronger monoclonal band was observed after administration. The IgG-κ band disappeared on the Hydrashift assay, while the daratumumab-anti-daratumumab complex appeared as a broad smear in the α-region. Patient 3, a 63-year-old male diagnosed with IgG-λMM, was receiving daratumumab once every other month. The IF assay showed two distinct bands (IgG-κ and IgG-λ) post-daratumumab administration. The shift to the α zone of the IgG-κ bands on the Hydrashift assay confirmed that the additional band observed post-infusion was due to the daratumumab. CONCLUSIONS: The Hydrashift assay can help distinguish daratumumab from endogenous M-spike.
Asunto(s)
Cadenas Ligeras de Inmunoglobulina , Mieloma Múltiple , Masculino , Humanos , Anciano , Persona de Mediana Edad , Inmunoelectroforesis/métodos , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/tratamiento farmacológico , Inmunoglobulina G , ElectroforesisRESUMEN
BACKGROUND: We report the case of a monoclonal immunoglobulin of IgM Lambda isotype associated with monoclonal lambda-type free light chains not detected by capillary electrophoresis but identified by immunofixation. METHODS: Capillary electrophoresis showed hypoproteinemia and an inflammatory syndrome. The IF realized on Hydrasys 2 Scan Focusing Sebia® reveals an IgM monoclonal band and two monoclonal bands in the total lambda. A second IF is performed using anti IgM, anti IgD, anti IgE and anti-total and -free lambda light chains as antisera. It reveals the presence of a monoclonal protein isotype IgM Lambda with free light chains. In view of these discordant results, an immunosubtraction was performed on the same sample showing no abnormality. RESULTS: Our patient has a monoclonal IgM Lambda with lambda monoclonal free light chains all masked on capillary electrophoresis and therefore not detected. CONCLUSIONS: Capillary electrophoresis techniques are incrementally becoming the techniques of choice in medical laboratories as a replacement for gel electrophoresis, due to their automation and better sensitivity. However, in some cases, a monoclonal immunoglobulin may not be detected by capillary technique and may cause an inaccurate interpretation.
Asunto(s)
Cadenas Ligeras de Inmunoglobulina , Paraproteinemias , Anticuerpos Monoclonales , Electroforesis Capilar/métodos , Humanos , Inmunoelectroforesis/métodos , Cadenas kappa de Inmunoglobulina , Cadenas lambda de Inmunoglobulina , Paraproteinemias/diagnósticoRESUMEN
BACKGROUND: Plasma cell disorders (PCDs) are typically characterized by excessive production of a single immunoglobulin, defined as a monoclonal protein (M-protein). Some patients have more than one identifiable M-protein, termed biclonal. Traditional immunofixation electrophoresis (IFE) cannot distinguish if two bands of the same isotype represent biclonal proteins or M-proteins with some other feature. A novel assay using immunoenrichment coupled to matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (Mass-Fix) was applied to determine whether two bands of the same isotype represented (1) monomers and dimers of a single M-protein, (2) an M-protein plus a therapeutic monoclonal antibody (t-mAb), (3) an M-protein with light chain glycosylation, or (4) two distinct biclonal M-proteins. METHODS: Patient samples with two bands of the same isotype identified by IFE were enriched using nanobodies against IgG, IgA, IgM, or κ and λ light chains then analyzed by Mass-Fix. Light chain masses were used to differentiate IgGκ M-proteins from t-mAbs. Mass differences between peaks were calculated to identify N-glycosylation or matrix adducts. High-resolution mass spectrometry was used as a comparator method in a subset of samples. RESULTS: Eighty-one residual samples were collected. For IgA, 93% (n = 25) were identified as monoclonal. For IgG, 67% (n = 24) were monoclonal, and 33% (n = 12) were truly biclonal. Among the monoclonal IgGs, the second band represented a glycosylated form for 21% (n = 5), while 33% (n = 8) had masses consistent with a t-mAb. 44% (n = 8) of IgM samples were biclonal, and 56% (n = 10) were monoclonal, of which one was glycosylated. CONCLUSIONS: We demonstrate the utility of mass spectrometry in the characterization of multiple IFE bands of the same isotype. Improved reporting accuracy of M-proteins is useful for monitoring of patients with PCDs.
Asunto(s)
Anticuerpos Monoclonales/sangre , Inmunoelectroforesis/métodos , Proteínas de Mieloma/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/química , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/química , Masculino , Persona de Mediana Edad , Mieloma Múltiple/sangre , Proteínas de Mieloma/química , Multimerización de Proteína , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Accurate determination of complement component C1q is hampered by the fact that C1q is an immune complex binding protein. Consequently, immunochemical techniques which rely on immune complex formation in fluid phase such as nephelometry and turbidimetry tend to give results which differ from those obtained by, for example, ELISA and other solid phase-based assays. In this chapter, we discuss the pros and cons of different techniques for the quantification of C1q and present a comprehensive protocol for a newly developed magnetic bead-based sandwich immunoassay which has replaced nephelometry in our complement diagnostic laboratory at the University Hospital in Uppsala.
Asunto(s)
Complemento C1q/análisis , Proteínas del Sistema Complemento/análisis , Inmunoelectroforesis/métodos , Nefelometría y Turbidimetría/métodos , Electroforesis de las Proteínas Sanguíneas/métodos , Pruebas Diagnósticas de Rutina/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Separación Inmunomagnética/métodosRESUMEN
Detection of complement activation products can be carried out in a number of ways, and different methods are used in different laboratories. No international standard for measuring complement activation in the clinical setting has been agreed upon.Here we describe a modified assay for measuring C3dg. The assay is simple, inexpensive and stable. The estimation of C3dg directly reflects complement turnover independently of activation pathway.
Asunto(s)
Precipitación Química , Complemento C3b/análisis , Técnica del Anticuerpo Fluorescente/métodos , Fragmentos de Péptidos/análisis , Animales , Biomarcadores/análisis , Biomarcadores/sangre , Electroforesis de las Proteínas Sanguíneas , Activación de Complemento/fisiología , Complemento C3b/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/métodos , Fluoroinmunoensayo/métodos , Humanos , Inmunoelectroforesis/métodos , Inflamación/sangre , Inflamación/diagnóstico , Mediadores de Inflamación/análisis , Mediadores de Inflamación/sangre , Fragmentos de Péptidos/aislamiento & purificación , Polietilenglicoles/química , ConejosRESUMEN
BACKGROUND: The treatment of multiple myeloma (MM) has been revolutionized by the introduction of therapeutic monoclonal antibodies (tmAbs). Daratumumab, a human IgG1/κ tmAb against CD38 on plasma cells, has improved overall survival in refractory MM and was recently approved as a frontline therapy for MM. Work on tmAb interference with serum protein electrophoresis (SPE) during MM monitoring has failed to provide information for laboratories on incidence of interference and effective methods of managing the interference at a practicable level. We aimed to evaluate daratumumab and elotuzumab interference in a large academic hospital setting and implement immediate solutions. METHODS: We identified and chart reviewed all cases of possible daratumumab interference by electrophoretic pattern (120 of 1317 total cases over 3 months). We retrospectively reviewed SPE cases in our laboratory to assess clinical implications of tmAb interference before the laboratory was aware of tmAb treatment. We supplemented samples with daratumumab and elotuzumab to determine the limits of detection and run free light chain analysis. RESULTS: Approximately 9% (120 of 1317) of tested cases have an SPE and/or immunofixation electrophoresis (IFE) pattern consistent with daratumumab, but only approximately 47% (56) of these cases were associated with daratumumab therapy. Presence of daratumumab led to physician misinterpretation of SPE/IFE results. Limits of daratumumab detection varied with total serum gammaglobulin concentrations, but serum free light chain analysis was unaffected. CONCLUSIONS: Clinical laboratories currently rely on interference identification by electrophoretic pattern, which may be insufficient and is inefficient. Critical tools in preventing misinterpretation efficiently include physician education, pharmacy notifications, separate order codes, and interpretive comments.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Anticuerpos Monoclonales , Errores Diagnósticos/prevención & control , Cadenas Ligeras de Inmunoglobulina/análisis , Mieloma Múltiple , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados/análisis , Anticuerpos Monoclonales Humanizados/farmacocinética , Anticuerpos Monoclonales Humanizados/uso terapéutico , Electroforesis de las Proteínas Sanguíneas/métodos , Humanos , Inmunoelectroforesis/métodos , Factores Inmunológicos/análisis , Factores Inmunológicos/farmacocinética , Factores Inmunológicos/uso terapéutico , Límite de Detección , Mieloma Múltiple/sangre , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/tratamiento farmacológico , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: Measurement of monoclonal immunoglobulins is a reliable estimate of the plasma cell tumor mass. About 15% of plasma cell myelomas secrete light chains only. The concentration of serum free light chains is insufficient evidence of the monoclonal light chain burden. A sensitive quantitative estimate of serum free monoclonal light chains could be useful for monitoring patients with light chain myeloma. We describe such an assay that does not require mass-spectrometry equipment or expertise. METHODS: Serum specimens from patients with known light chain myelomas and controls were subjected to ultrafiltration through a membrane with pore size of 50 kDa. The filtrate was concentrated and tested by immunofixation electrophoresis. The relative area under the monoclonal peak, compared to that of the total involved light chain composition, was estimated by densitometric scanning of immunofixation gels. The proportion of the area occupied by the monoclonal peak in representative densitometric scans was used to arrive at the total serum concentration of the monoclonal serum free light chains. RESULTS: Using an ultracentrifugation and concentration process, monoclonal serum free light chains were detectable, along with polyclonal light chains, in all 10 patients with active light chain myelomas. Monoclonal light chains were identified in serum specimens that did not reveal monoclonal light chains by conventional immunofixation electrophoresis. The limit of detection by this method was 1.0 mg/L of monoclonal serum free light chains. CONCLUSION: The method described here is simple enough to be implemented in academic medical center clinical laboratories and does not require special reagents, equipment, or expertise. Even though urine examination is the preferred method for the diagnosis of light chain plasma cell myelomas, measurement of the concentration of serum free light chains provides a convenient, albeit inadequate, way to monitor the course of disease. The method described here allows effective electrophoretic differentiation of monoclonal serum free light chain from polyclonal serum free light chains and provides a quantitation of the monoclonal serum free light chains in monitoring light chain monoclonal gammopathies.
Asunto(s)
Anticuerpos Monoclonales/sangre , Inmunoelectroforesis , Cadenas Ligeras de Inmunoglobulina/sangre , Ultrafiltración , Biomarcadores , Humanos , Inmunoelectroforesis/métodos , Inmunoelectroforesis/normas , Cadenas kappa de Inmunoglobulina/sangre , Cadenas lambda de Inmunoglobulina/sangre , Mieloma Múltiple/sangre , Mieloma Múltiple/diagnóstico , Sensibilidad y Especificidad , Ultrafiltración/métodos , Ultrafiltración/normasRESUMEN
BACKGROUND: Serum protein electrophoresis (SPE) is a widely used laboratory technique to diagnose patients with multiple myeloma (MM) and other disorders related to serum protein. In patients with MM, abnormal monoclonal protein can be detected by SPE and further characterized using immunofixation electrophoresis (IFE). There are several semi-automated agarose gel-based systems available commercially for SPE and IFE. In this study, we sought to evaluate the analytical performance of fully automated EasyFix G26 (EFG26) and semi-automated HYDRASYS 2 SCAN (H2SCAN) for both SPE and IFE. METHODS: Both instruments were operated according to manufacturer's instructions. Samples used include a commercially available normal control serum (NCS) and patients' specimens. The following were evaluated: precision and comparison studies for SPE, and reproducibility and comparison studies for IFE. Statistical analyses were performed using Microsoft Excel. RESULTS: For SPE repeatability study, our results showed that EFG26 has higher coefficient of variation (%CV) compared with H2SCAN for both samples except for monoclonal component with %CV of 0.97% and 1.18%, respectively. Similar results were obtained for SPE reproducibility study except for alpha-1 (4.16%) and beta (3.13%) fractions for NCS, and beta fractions (5.36%) for monoclonal sample. Subsequently, reproducibility for IFE was 100% for both instruments. Values for correlation coefficients between both instruments ranged from 0.91 to 0.98 for the five classic bands. CONCLUSION: Both instruments demonstrated good analytical performance characterized by high precision, reproducibility and correlation.
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Electroforesis de las Proteínas Sanguíneas/instrumentación , Proteínas Sanguíneas/análisis , Inmunoelectroforesis/instrumentación , Automatización de Laboratorios , Electroforesis de las Proteínas Sanguíneas/métodos , Proteínas Sanguíneas/inmunología , Humanos , Inmunoelectroforesis/métodos , Proteínas de Mieloma/análisis , Reproducibilidad de los ResultadosRESUMEN
Drug-induced liver injury (DILI) to flucloxacillin is rare and is classified as idiosyncratic, as it is dependent on individual susceptibility, unpredictable, and dose-independent. The authors present the case of a 74 - year - old man with a history of monoclonal gammopathy under investigation and alcoholic habits of 24 g/day, with asthenia, anorexia, nausea, abdominal discomfort, and fever with three days of evolution. He was treated with two courses of antibiotic therapy with flucloxacillin to erysipelas previously (3 months and 2 weeks before admission). Lab tests showed serum AST levels of 349 U/L, ALT 646 U/L, alkaline phosphatase 302 U/L, GGT 652 U/L, total bilirubin 3.3 mg/dL and direct bilirubin 2.72 mg/dL. Infectious, autoimmune, and metabolic causes were ruled out. Magnetic resonance cholangiopancreatography showed normal results. Liver biopsy showed mild multifocal (predominantly microvesicular) steatosis; marked changes in the centrilobular areas (sinusoidal dilatation, marked congestion, hemorrhage, and multifocal hepatocyte collapse); expansion of the portal areas with the formation of bridges; proliferated bile ducts and inflammatory infiltrate of variable density, predominantly mononuclear type. The HLA-B*5701 screening test was positive. Hepatic biochemical tests remain abnormal with a significative increase in total bilirubin, which reached levels of 24.1 mg/dL, with the development of jaundice, pruritus, and choluria. DILI was assumed, and the patient was treated with ursodeoxycholic acid. There was favorable evolution, without evidence of blood coagulation dysfunction or encephalopathy. The analytic normalization was, however, slow, with evolution to chronicity. The authors present this case to remind the possibility of moderate/severe drug-induced liver injury to flucloxacillin, an antibiotic commonly used in clinical practice and association with the HLA-B * 5701 allele reported in the literature.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Floxacilina/efectos adversos , Antígenos HLA-B/efectos de los fármacos , Anciano , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Humanos , Inmunoelectroforesis/métodos , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Factores de RiesgoRESUMEN
BACKGROUND: Specific Pseudomonas aeruginosa (PA) precipitating immunoglobulin G antibodies in serum are correlated with PA biofilm infection and are used as diagnostic and prognostic markers in cystic fibrosis (CF). The aim of this study was to examine the change of PA antibody response in CF patients after bilateral sequential lung transplantation (LTx). METHODS: PA antibodies and airway bacteriology were retrospectively evaluated in 20 chronically infected CF patients, who underwent LTx between 2001 and 2016 at Rigshospitalet, Copenhagen. Yearly precipitin counts from one year before LTx and up to five years after LTx were compared. Monthly airway cultures were examined in the five-year period after LTx. In addition, crossed immunoelectrophoresis (CIE) were analysed for each patient for antigenic similarities from time of infection, pre-LTx and post-LTx. RESULTS: All patients experienced a significant drop in PA antibodies from one year pre-LTx to one year post-LTx (p < 0.0001). The PA antibody level did not differ between those, who became reinfected immediately after LTx, and those, who did not. No patients regained the high pre-LTx precipitin levels in the following five years. The antigenic specificities of the sera post-LTx were in each patient similar to the antigenic specificities at the beginning of infection indicating a decades long memory of their immune response like an "immunological fingerprint". CONCLUSIONS: After LTx a significant and continuous reduction in PA antibodies was observed. The reduction was independent of immediate reinfection after LTx. A novel three-factor explanatory model is presented.
Asunto(s)
Anticuerpos Antibacterianos , Formación de Anticuerpos/inmunología , Fibrosis Quística , Trasplante de Pulmón/métodos , Infecciones por Pseudomonas , Pseudomonas aeruginosa/inmunología , Adulto , Anticuerpos Antibacterianos/análisis , Anticuerpos Antibacterianos/sangre , Enfermedad Crónica , Fibrosis Quística/epidemiología , Fibrosis Quística/inmunología , Fibrosis Quística/microbiología , Fibrosis Quística/cirugía , Dinamarca/epidemiología , Femenino , Humanos , Inmunoelectroforesis/métodos , Memoria Inmunológica/inmunología , Masculino , Periodo Posoperatorio , Infecciones por Pseudomonas/sangre , Infecciones por Pseudomonas/fisiopatología , Estudios RetrospectivosRESUMEN
Introducción: La inmunoelectroforesis es una técnica de precipitación que permite la caracterización de muestras biológicas complejas. En el Departamento de Inmunología del Instituto de Ciencias Básicas y Preclínicas Victoria de Girón se cuenta con un antisuero hiperinmune obtenido por inmunizaciones de carneros contra proteínas totales séricas humanas y con otro antisuero anti IgA de calostro humano. Objetivo: Identificar IgG, IgM e IgA en suero humano y determinar respuesta anti IgM humana en el antisuero anti IgA de calostro humano obtenido en carnero. Material y Métodos: Se realizó un estudio observacional, descriptivo y transversal desde noviembre de 2017 hasta junio de 2018. Se desarrolló una inmunoelectroforesis de suero humano normal empleando el antisuero hiperinmune. Resultados: Se identificaron IgG, IgM e IgA además de albúmina y otras fracciones proteicas y se determinó respuesta anti IgM humana en el antisuero anti IgA de calostro humano obtenido en carnero. Conclusiones: Este trabajo permitió identificar y determinar la respuesta anticlases mayores de inmunoglobulinas en la muestra de estudio(AU)
Introduction: Immunoelectrophoresis is a precipitation technique that allows the characterization of complex biological samples. The Immunology Department of the Institute of Basic and Pre-Clinical Sciences Victoria de Girón has a hyperimmune antiserum obtained by immunization of sheep against human serum total proteins and it also has an anti-human IgA antiserum obtained from human colostrum. Objective: The aim of this study was to identify IgG, IgM and IgA in human serum and to determine response to anti-human IgM in human colostral IgA with antiserum obtained in sheep. Material and Methods: An observational descriptive cross-sectional study was conducted from November 2017 to June 2018. Immunoelectrophoresis of normal human serum was performed using hyperimmune antiserum. Results: These procedures allowed to identify IgG, IgM and IgA in addition to albumin and other protein fractions and to determine response to anti-human IgM in human colostral IgA with antiserum obtained in sheep. Conclusions: This work allowed us to identify and determine significant anti-class responses of immunoglobulins in the sample studied(AU)
Asunto(s)
Humanos , Animales , Inmunoelectroforesis/métodos , Sueros Inmunes/inmunología , Afinidad de Anticuerpos/genética , Epidemiología Descriptiva , Estudios TransversalesRESUMEN
SUMMARY Drug-induced liver injury (DILI) to flucloxacillin is rare and is classified as idiosyncratic, as it is dependent on individual susceptibility, unpredictable, and dose-independent. The authors present the case of a 74 - year - old man with a history of monoclonal gammopathy under investigation and alcoholic habits of 24 g/day, with asthenia, anorexia, nausea, abdominal discomfort, and fever with three days of evolution. He was treated with two courses of antibiotic therapy with flucloxacillin to erysipelas previously (3 months and 2 weeks before admission). Lab tests showed serum AST levels of 349 U/L, ALT 646 U/L, alkaline phosphatase 302 U/L, GGT 652 U/L, total bilirubin 3.3 mg/dL and direct bilirubin 2.72 mg/dL. Infectious, autoimmune, and metabolic causes were ruled out. Magnetic resonance cholangiopancreatography showed normal results. Liver biopsy showed mild multifocal (predominantly microvesicular) steatosis; marked changes in the centrilobular areas (sinusoidal dilatation, marked congestion, hemorrhage, and multifocal hepatocyte collapse); expansion of the portal areas with the formation of bridges; proliferated bile ducts and inflammatory infiltrate of variable density, predominantly mononuclear type. The HLA-B*5701 screening test was positive. Hepatic biochemical tests remain abnormal with a significative increase in total bilirubin, which reached levels of 24.1 mg/dL, with the development of jaundice, pruritus, and choluria. DILI was assumed, and the patient was treated with ursodeoxycholic acid. There was favorable evolution, without evidence of blood coagulation dysfunction or encephalopathy. The analytic normalization was, however, slow, with evolution to chronicity. The authors present this case to remind the possibility of moderate/severe drug-induced liver injury to flucloxacillin, an antibiotic commonly used in clinical practice and association with the HLA-B * 5701 allele reported in the literature.
RESUMO A hepatotoxicidade à flucloxacilina é rara e classifica-se como idiossincrática, uma vez que é dependente da suscetibilidade individual, não expectável e independente da dose. Apresentamos o caso de um homem, 74 anos, antecedentes de gamapatia monoclonal e hábitos alcoólicos de 24 g/dia, com quadro de astenia, anorexia, náuseas, desconforto abdominal e febrícula com três dias de evolução. Referência a dois ciclos de antibioterapia com flucloxacilina por erisipela (três meses e duas semanas antes da admissão). Analiticamente com AST 349 U/L, ALT 646 U/L, FA 302 U/L, GGT 652 U/L, bilirrubina total 3,3 mg/dL, bilirrubina direta 2,72 mg/dL. Excluídas etiologias infecciosa, autoimune, metabólica, bem como patologia das vias biliares por colangio-RM. Biópsia hepática mostrou esteatose multifocal ligeira (predominantemente microvesicular); alterações acentuadas nas áreas centrolobulares (dilatação sinusoidal, congestão acentuada, hemorragia e colapso multifocal de hepatócitos); expansão das áreas portais com constituição de pontes; ductos biliares proliferados e infiltrado inflamatório de densidade variável, predominantemente de tipo mononucleado. Tipagem de HLA-B*5701 positiva. Agravamento analítico atingindo bilirrubina total 24,1 mg/dL, com desenvolvimento de icterícia, prurido e colúria. Admitida a hepatotoxicidade, iniciou terapêutica com ácido ursodesoxicólico. Verificou-se evolução favorável, sem evidência de coagulopatia ou encefalopatia. A normalização analítica foi, no entanto, lenta, com evolução para cronicidade. Os autores apresentam este caso para alertar para a possibilidade de hepatotoxicidade moderada a grave à flucloxacilina, antibiótico de uso comum na prática clínica e associação com o alelo HLA-B*5701 relatada na literatura.