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Monoclonal gammopathy (MG) is a spectrum of diseases ranging from the benign asymptomatic monoclonal gammopathy of undetermined significance to the malignant multiple myeloma. Clinical guidelines and laboratory recommendations have been developed to inform best practices in the diagnosis, monitoring, and management of MG. In this review, the pathophysiology, relevant laboratory testing recommended in clinical practice guidelines and laboratory recommendations related to MG testing and reporting are examined. The clinical guidelines recommend serum protein electrophoresis, serum immunofixation and serum free light chain measurement as initial screening. The laboratory recommendations omit serum immunofixation as it offers limited additional diagnostic value. The laboratory recommendations offer guidance on reporting findings beyond monoclonal protein, which was not required by the clinical guidelines. The clinical guidelines suggested monitoring total IgA concentration by turbidimetry or nephelometry method if the monoclonal protein migrates in the non-gamma region, whereas the laboratory recommendations make allowance for involved IgM and IgG. Additionally, several external quality assurance programs for MG protein electrophoresis and free light chain testing are also appraised. The external quality assurance programs show varied assessment criteria for protein electrophoresis reporting and unit of measurement. There is also significant disparity in reported monoclonal protein concentrations with wide inter-method analytical variation noted for both monoclonal protein quantification and serum free light chain measurement, however this variation appears smaller when the same method was used. Greater harmonization among laboratory recommendations and reporting format may improve clinical interpretation of MG testing.
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Gamopatia Monoclonal de Significância Indeterminada , Paraproteinemias , Humanos , Gamopatia Monoclonal de Significância Indeterminada/diagnóstico , Paraproteinemias/diagnóstico , Laboratórios , Cadeias Leves de ImunoglobulinaRESUMO
Objective: To assess the association of serum protein electrophoresis abnormalities with clinicopathological characteristics, and its impact on overall survival in chronic lymphocytic leukaemia patients. METHODS: The prospective study was conducted at Haematology and Immunology departments of the University of Health Sciences, Lahore, Pakistan, from 2019 to 2022, and comprised newly diagnosed chronic lymphocytic leukaemia patients. Lactate dehydrogenase and beta-2 microglobulin levels were measured by spectrophotometric principle, whereas serum protein electrophoresis was determined through commercially available capillary electrophoresis systems. Patients were followed up for 2 years post-diagnosis. Data was analysed using SPSS 21. RESULTS: Of the 50 patients, 40(80%) were males and 10(20%) were females. The overall mean age was 60±11 years. Serum protein electrophoresis was available for 40(80%) patients, and, among them, 12(30%) patients had abnormal levels, while 29(72.5%) required treatment. Overall response rate was 25(86.2%), and median two-year overall survival was 16.5 months (95% confidence interval: 10-20 months). Abnormal serum protein electrophoresis was significantly associated with Binet stage C, lower mean haemoglobin levels and higher median levels of lactate dehydrogenase and beta-2 microglobulin (p<0.05)). Regarding overall survival, the survival curves of chronic lymphocytic leukaemia patients with normal and abnormal serum protein electrophoresis status differed significantly (p=0.04). Conclusion: Abnormal serum protein electrophoresis could be considered a surrogate marker for advanced chronic lymphocytic leukaemia disease.
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Eletroforese das Proteínas Sanguíneas , L-Lactato Desidrogenase , Leucemia Linfocítica Crônica de Células B , Microglobulina beta-2 , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/mortalidade , Leucemia Linfocítica Crônica de Células B/diagnóstico , Feminino , Masculino , Pessoa de Meia-Idade , Prognóstico , Idoso , Estudos Prospectivos , Microglobulina beta-2/sangue , Eletroforese das Proteínas Sanguíneas/métodos , L-Lactato Desidrogenase/sangue , Paquistão/epidemiologia , Hemoglobinas/análise , Hemoglobinas/metabolismo , Taxa de Sobrevida , Estadiamento de Neoplasias , Proteínas Sanguíneas/análiseRESUMO
Serum protein electrophoresis (SPEP) is a valuable laboratory test that separates proteins from the blood based on their electrical charge and size. The test can detect and analyze various protein abnormalities, and the interpretation of graphic SPEP features plays a crucial role in the diagnosis and monitoring of conditions, such as myeloma. Furthermore, the advancement of artificial intelligence (AI) technology presents an opportunity to enhance the organization and optimization of analytical procedures by streamlining the process and reducing the potential for human error in SPEP analysis, thereby making the process more efficient and reliable. For instance, AI can assist in the identification of protein peaks, the calculation of their relative proportions, and the detection of abnormalities or inconsistencies. This review explores the characteristics and limitations of AI in SPEP, and the role of standardization in improving its clinical utility. It also offers guidance on the rational ordering and interpreting of SPEP results in conjunction with AI. Such integration can effectively reduce the time and resources required for manual analysis while improving the accuracy and consistency of the results.
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PURPOSE: This study aimed to investigate the correlation between calculated globulin (CG, total protein level minus albumin level) and the gamma globulin fraction (Gamma), obtained from serum protein electrophoresis with serum IgG levels in adults (≥ 18 years). METHODS: Using linear regression models, analyses of CG and Gamma levels correlation with IgG levels in adults were performed. Receiver-operator curves were created to determine cutoff values and the respective sensitivity and specificity measures. RESULTS: A total of 886 samples were analyzed. CG and Gamma were positively and statistically correlated with IgG levels (r2 = 0.4628 for CG, and = 0.7941 for Gamma, p < 0.0001 for both analyses). For the detection of hypogammaglobulinemia, i.e., IgG level below the reference value (6 g/L), a CG cutoff value of 24 g/L showed a sensitivity of 86.2% (95% CI 69.4-94.5) and a specificity of 92% (90.0-93.6). A Gamma cutoff value of 7.15 g/L yielded a sensitivity of 100% (88.3-100) and a specificity of 96.8 (95.3-97.8). CONCLUSION: Both CG and Gamma levels determined by protein electrophoresis analysis may be used to screen for antibody deficiencies in adults, enabling earlier diagnosis of antibody deficiencies in a routine clinical setting.
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Agamaglobulinemia , Doenças da Imunodeficiência Primária , Humanos , Adulto , Eletroforese , Globinas , Imunoglobulina GRESUMO
In order to accelerate Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), here we propose an optimized version of the technique enabled by experimental tuning reinforced by theoretical description. In the resulting system, the gel buffer was diluted twofold and supplemented with glycine at a low concentration, whereas a higher voltage was applied. This approach reduced runtime from 90 to 18 min. It is important to emphasize that, despite the high voltage applied to the gel, the resolution of the bands did not decrease compared to the original Laemmli method. The proposed acceleration approach can be used in other variants of SDS-PAGE.
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Glicina , Proteínas , Proteínas/análise , Dodecilsulfato de Sódio , Eletroforese em Gel de PoliacrilamidaRESUMO
OBJECTIVES: This study aims to investigate the effect of natural ultralipemic material (NULM) and intravenous lipid emulsion (IVLE) on capillary serum protein electrophoresis (SPEP). METHODS: NULM material was prepared from leftover patients' lipemic serum sample (triglyceride concentration >2,000 mg/dL) pool by a refrigerated high-speed centrifuge, and IVLE Omegaven lipid emulsion (30%) was used. Serum pools for interference study were prepared from patient samples for which serum protein electrophoresis was studied as Normal SPEP and M Peak SPEP. For both types of lipemia (DULM and IVLE), five pools with triglyceride concentrations of â¼4.52 mmol/L, â¼7.91 mmol/L, â¼14.69 mmol/L, â¼21.47 mmol/L, and â¼28.25 mmol/L were prepared. SPEP was studied in each pool with Sebia Capillarys Minicap. A repeated measure ANOVA test was used to determine the difference between the pools, and interferograms were used to evaluate the interference effect. RESULTS: Interference was not detected in IVLE added Normal SPEP and M Peak SPEP pools, either % or concentrations of fractions. In NULM-added Normal SPEP and M Peak SPEP pools, significant positive interference in albumin % (p=0.002 and p<0.001 respectively) and significant negative interference in gamma% (p<0.001 and p=0.005 respectively) and M protein peak (p=0.002) fractions were detected. However, significant positive interference was seen only for albumin concentration fractions (p<0.001 for both pools). CONCLUSIONS: It is vital to use NULM instead of IVLE solutions in lipemia interference studies for all laboratory tests, including CZE SPEP. The fractions concentration values calculated with the total protein concentration should be used for evaluating SPEP results.
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Emulsões Gordurosas Intravenosas , Hiperlipidemias , Humanos , Eletroforese , Triglicerídeos , Proteínas Sanguíneas/análise , AlbuminasRESUMO
OBJECTIVES: Alpha-1 antitrypsin (A1AT) deficiency was first identified in patients with emphysema by the absence of the α1 band on serum protein electrophoresis (SPE). Today, capillary zone electrophoresis is widely performed in laboratories. Here, we compared two SPE systems to detect decreased A1AT concentrations to optimize their use as a screening tool for A1AT deficiency. METHODS: Serum protein electrophoresis was performed on 200 samples on the Capillarys 2 and the V8 Nexus. The latter presents two α1 bands (α1 band 1 and 2) while the Capillarys 2 has only one (Capillarys 2 total α1). The measures of A1AT and α1 acid glycoprotein (AAG) were performed as well as the phenotyping of M, S and Z alleles. RESULTS: At a A1AT cutoff of 0.80 g/L, a cutoff of 1.21 g/L using the V8 Nexus α1 band 2 corresponded to a 100% sensitivity and a 92.4% specificity while a 1.69% cutoff corresponded to a 100% sensitivity and a 92.4% specificity. The performance of the α1 band 1 was suboptimal and rather corresponded to AAG. On the Capillarys 2, a cutoff of 2.0 g/L corresponded to a 75.0% sensitivity and a 86.6% specificity, while a 3.2% cutoff showed a 96.4% sensitivity and a 67.4% specificity. The V8 Nexus α1 band 2 was the method the most correlated with A1AT (r=0.90-0.94). CONCLUSIONS: The V8 Nexus α1 band 2 was the best predictor of A1AT deficiency, probably owing to a better resolution. The use of SPE was however unable to predict each phenotype. Phenotype or genotype studies are therefore still advisable in case of A1AT deficiency.
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Deficiência de alfa 1-Antitripsina , Humanos , Deficiência de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/genética , Genótipo , Fenótipo , Eletroforese CapilarRESUMO
Protein electrophoresis and immunoblotting are indispensable analytical tools for the characterization of proteins and posttranslational modifications in complex sample matrices. Owing to the lack of automation, commonly employed slab-gel systems suffer from high time demand, significant sample/antibody consumption, and limited reproducibility. To overcome these limitations, we developed a paper-based open microfluidic platform for electrophoretic protein separation and subsequent transfer to protein-binding membranes for immunoprobing. Electrophoresis microstructures were digitally printed into cellulose acetate membranes that provide mechanical stability while maintaining full accessibility of the microstructures for consecutive immunological analysis. As a proof-of-concept, we demonstrate separation of fluorescently labeled marker proteins in a wide molecular weight range (15-120 kDa) within only 15 min, reducing the time demand for the entire workflow (from sample preparation to immunoassay) to approximately one hour. Sample consumption was reduced 10- to 150-fold compared to slab-gel systems, owing to system miniaturization. Moreover, we successfully applied the paper-based approach to complex samples such as crude bacterial cell extracts. We envisage that this platform will find its use in protein analysis workflows for scarce and precious samples, providing a unique opportunity to extract profound immunological information from limited sample amounts in a fast fashion with minimal hands-on time.
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Técnicas Analíticas Microfluídicas , Microfluídica , Eletroforese , Immunoblotting , Microfluídica/métodos , Proteínas , Reprodutibilidade dos TestesRESUMO
C-reactive protein (CRP), fibrinogen, and d-dimer are determined in the human plasma of 2745 hospitalized patients with and without coronavirus disease 2019 (COVID-19) by automated-latex enhanced immunoassay and immuno-turbidimetric assay. SARS-COV-2 RNA qualitative test, real time polymerase chain reaction (RT-PCR) based, is performed in nasopharyngeal swabs to confirm those with SARS-COV-2 positivity. Furthermore, serum proteins are separated and quantified in all the patients by serum protein electrophoresis (SPE). A new SPE parameter, inflammatory protein ratio (IPR), is elaborated for the first time by a mathematical equation that considers the albumin, α1-globulin, and α2-globulin. IPR normal reference range (10.7%-28.3%) is calculated considering the normal reference range of albumin, α1-globulin, and α2-globulin obtained for controls. Analysis of variance (ANOVA), Pearson's, Kruskal-Wallis, and Spearman's tests application show that IPR significantly correlates with direct proportionality with d-dimer, CRP, and fibrinogen. Significant (p < 0.001) increase of these parameters, IPR included, is detected in COVID-19 patients only. Our results show that IPR is more specific for monitoring inflammatory status thanks to its correlation with the only three serum proteins involved in inflammation: albumin, α1-globulin, and α2-globulin. Furthermore, IPR can simplify the interpretation of SPE results about inflammatory status, being of unique value compared to the six-serum protein classes separately presented in the typical SPE clinical reports.
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COVID-19 , Albuminas , COVID-19/diagnóstico , Fibrinogênio , Humanos , Prognóstico , RNA Viral , SARS-CoV-2RESUMO
Annona squamosa is a medicinal plant that has been used in folk medicine since antiquity. The goal of this study is to see how effective Annona squamosa leaf extract (A.S.L.E) or its niosomal-entrapped preparation is at protecting skin from UVA irradiation. The prepared niosomal-entrapped A.S.L.E has been characterized via spectrophotometry and transmission electron microscopy imaging. Furthermore, the entrapment efficiency and in vitro release of A.S.L.E were determined. In this study, ex vivo and freshly prepared samples from the dorsal region of the rats' skin were used as biological samples, which were divided into five groups: control UVA-unexposed, unprotected UVA-exposed, A.S.L.E-protected UVA-exposed, and niosomal-entrapped A.S.L.E UVA-exposed. UVA irradiation was performed by exposing the skin samples to a UVA-producing lamp for 4 h. Samples from various groups were then examined using FTIR spectroscopy, histopathology, and protein electrophoresis methods. The results showed that A.S.L.E has a skin protective effect against UVA irradiation. The niosomal-entrapped A.S.L.E was more effective than the native plant leaf extract in protecting skin from the damaging effects of UVA. Therefore, the nanotechnologically formulated preparation, niosomal-entrapped A.S.L.E, can be used as an effective photoprotector (sunscreen) against the adverse effects of UVA radiation.
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Annona , Animais , Ratos , Extratos Vegetais/farmacologiaRESUMO
BACKGROUND: Common variable immunodeficiency (CVID) is a rare disease that affects children and adults and is often difficult to diagnose. Despite being one of the most frequent causes of immunodeficiency, involving gastrointestinal (GI), respiratory, and hematological systems, the disease onset can have heterogeneous and intermittent symptoms, frequently leading to diagnostic delay. GI symptoms are common and can include diarrhea, but the asymptomatic periods lead to overlooking the recurrent pattern. The same can occur with respiratory infections, thus delaying CVID suspicion. The starting point for CVID diagnosis is the decreased gamma globulin levels in serum protein electrophoresis (SPE), also observed through direct immunoglobulin's dosage. CASE PRESENTATION: The patient is a 38 years-old man who had intermittent diarrhea and recurrent airway infections for 19 years, but the CVID diagnosis was achieved only after SPE was carried out. At that time, he was already malnourished, and developed other complications related to CVID in a short period. CONCLUSIONS: SPE is readily available and inexpensive, but is not part of the laboratory approach in diarrhea. According to the case presented herein, it can be useful for patients with recurrent infections or other clues of the disease.
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Imunodeficiência de Variável Comum , Diagnóstico Tardio , Adulto , Proteínas Sanguíneas , Imunodeficiência de Variável Comum/complicações , Imunodeficiência de Variável Comum/diagnóstico , Diagnóstico Tardio/efeitos adversos , Diarreia/etiologia , Eletroforese , Humanos , MasculinoRESUMO
Neonates admitted to neonatal intensive care units are at a risk of developing healthcare-associated infections, leading to increased risk of mortality. This study aimed to identify organisms causing such late-onset infections in neonates and determine whether these isolates were genetically identical to those from the surrounding environmental surfaces and hands of healthcare workers (HCWs). A cross-sectional study was carried out over a period of 4 months in a university neonatal intensive care unit (NICU). Samples were collected from all neonates with symptoms of late-onset infections (n = 180). Fingerprint samples of 21 healthcare workers as well as 330 random environmental samples were also taken from the unit. Isolates from neonates, environment and fingerprints were subjected to protein electrophoresis followed by sequencing to detect genetic similarities. Almost half of neonatal samples were culture positive (91/180, 50.6%), out of which 72% of bacterial isolates (49/68) were multi-drug resistant. Klebsiella pneumoniae (32.6%) and Candida spp. (28.4%) were the commonest neonatal isolates. A cluster of two homologous Klebsiella pneumoniae strains was isolated from a neonate and an examining bed, while another homologous cluster was from a neonatal sample and a portal incubator. A third cluster was isolated from hands and three neonatal samples. This cluster (caused by Klebsiella pneumoniae strain NH54 chromosome) was found to perpetuate over the 4 months of the study. All three clusters were multi-drug-resistant Klebsiella pneumoniae. A homologous pair of each of Candida tropicalis and Candida glabrata was isolated from the blood of two neonates, and one neonatal and a crash cart sample, respectively. Overall, 8.8% (8/91) of neonatal samples were found to be homologous to other neonatal/environmental/hand isolates, denoting perpetuation of pathogens between neonates themselves and also other reservoirs of infections.Conclusion: The hands of HCWs, crash carts and incubators are reservoirs of pathogens and can lead to nosocomial infections. Clusters of multi-drug-resistant Klebsiella pneumoniae and Candida spp. were the predominant neonatal pathogens in this NICU. What is Known: ⢠The role of hands and the environment in transmission of infections to neonates is a subject of debate. ⢠Genetic sequencing provides solid evidence for detecting homologous strains. What is New: ⢠K. pneumoniae was the most frequently isolated pathogen, and concomitant isolation was found in two cases from the neonatal surroundings (bed/incubator) and hands. ⢠Candida spp. with homology were also found in different neonates and environmental samples suggesting risk of transmission.
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Infecção Hospitalar , Infecções por Klebsiella , Preparações Farmacêuticas , Infecção Hospitalar/epidemiologia , Estudos Transversais , Pessoal de Saúde , Humanos , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Infecções por Klebsiella/epidemiologia , Klebsiella pneumoniae/genética , Testes de Sensibilidade MicrobianaRESUMO
Capillary electrophoresis is a method with a long history of developments which enables monitoring of several pathological processes and has an irreplaceable role in screening for presence of M-protein. The aim of this study was to assess analytical performance of Sebia and Helena systems, as well as their screening efficiency for M-protein by identifying characteristic electrophoretic pattern abnormalities. The controls were analyzed in triplicate over a five-day period. Comparability testing was performed on 46 (Capillarys3Octa) and 49 (V8Nexus) serum samples with routinely used Capillarys2. Electropherograms (EPGs) were reviewed by two specialists independently to select samples for further processing by immunofixation. All precision test results met the eligibility criteria by Ricos et al. The correlation coefficients higher than 0.8 indicated excellent comparability although the results were slightly more comparable among the same manufacturer systems. There were no variations in observed abnormalities in EPGs when Capillarys systems were compared, but a disparity was detected in 11/49 EPGs on comparing Capillarys2 and V8Nexus. The cause of the detected difference could be in a different graphical presentation of the findings and in a lesser resolution of the applied buffer. The impression is that the V8Nexus system combined with the utilized buffer provides greater resolution in the alpha-1 and alpha-2 globulin fractions, but that it declines in the gamma globulin fraction. The precision and estimated accuracy criteria were met by both systems. Comparison results implied that capillary systems are not equally effective in M-protein screening, highlighting the necessity to include system screening efficiency in analytical evaluation.
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Paraproteinemias , Eletroforese em Gel de Ágar/métodos , Eletroforese Capilar/métodos , Humanos , Paraproteinemias/diagnósticoRESUMO
Three dogs under 12 months old were diagnosed with atypical multiple myeloma (MM), having an aggressive multifocal anaplastic round cell sarcoma in bone marrow, viscera, and/or peripheral blood, which were confirmed by cytology and immunohistochemistry to be of plasma cell origin. The intramedullary sarcomas caused myelophthisis, osteolysis, and hypercalcemia. Complete or free light chain monoclonal gammopathy in the serum and/or urine was demonstrated by protein electrophoresis and immunofixation. The polymerase chain reaction for antigen receptor rearrangement assay performed on 2 cases identified a clonally rearranged immunoglobulin gene. Neoplastic cells lacked expression of CD45, CD3, CD18, CD21, CD34, and MHCII by flow cytometry. Immunohistochemistry revealed MUM1 immunoreactivity of the neoplastic cells. Combining all data, the diagnosis was MM. An aggressive form of MM in young dogs should be a differential diagnosis for patients with an immunoglobulin-productive, B cell-clonal, CD45-negative, MUM1-positive discrete cell neoplasm arising from the bone marrow.
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Doenças do Cão , Mieloma Múltiplo , Animais , Linfócitos B , Medula Óssea , Doenças do Cão/diagnóstico , Cães , Citometria de Fluxo/veterinária , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/veterinária , PlasmócitosRESUMO
The erythrocyte sedimentation rate (ESR) is a widely used marker of inflammation, but the detailed mechanisms underlying the ESR remain unclear. We retrospectively collected laboratory data from our hospital's laboratory information system, and performed multiple linear regression analysis and correlation analysis to determine relationships between the ESR and other laboratory test parameters. The alpha-2, beta-2, and gamma fractions from serum protein electrophoresis, serum immunoglobulin (Ig) G, IgA, IgM, and complement C3 levels, plasma fibrinogen levels, and platelet count showed positive effects on the ESR; however, the serum albumin level showed negative effects. Since erythrocytes are negatively charged, an increase in positively charged proteins and a decrease in negatively charged albumin were suggested to increase the ESR. Notably, C-reactive protein (CRP) showed the third-strongest correlation with the ESR despite having no significant effect on the ESR. We also reviewed cases with discordant ESR and CRP levels to compare the disease profiles of high ESR/low CRP patients and low ESR/high CRP patients. The patients with high ESR/low CRP had a completely different disease profile from those with low ESR/high CRP. Since the ESR and CRP have different roles, they should be used as markers in a context-dependent manner.
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Proteína C-Reativa , Complemento C3 , Biomarcadores , Sedimentação Sanguínea , Proteína C-Reativa/análise , Complemento C3/análise , Fibrinogênio/análise , Humanos , Imunoglobulina A , Imunoglobulina M , Laboratórios Clínicos , Estudos Retrospectivos , Albumina Sérica/metabolismoRESUMO
BACKGROUND: Serum protein electrophoresis (SPE) is a common clinical laboratory test, mainly indicated for the diagnosis and follow-up of monoclonal gammopathies. A time-consuming and potentially subjective human expertise is required for SPE analysis to detect possible pitfalls and to provide a clinically relevant interpretation. METHODS: An expert-annotated SPE dataset of 159 969 entries was used to develop SPECTR (serum protein electrophoresis computer-assisted recognition), a deep learning-based artificial intelligence, which analyzes and interprets raw SPE curves produced by an analytical system into text comments that can be used by practitioners. It was designed following academic recommendations for SPE interpretation, using a transparent architecture avoiding the "black box" effect. SPECTR was validated on an external, independent cohort of 70 362 SPEs and challenged by a panel of 9 independent experts from other hospital centers. RESULTS: SPECTR was able to identify accurately both quantitative abnormalities (r ≥ 0.98 for fractions quantification) and qualitative abnormalities [receiver operating characteristic-area under curve (ROC-AUC) ≥ 0.90 for M-spikes, restricted heterogeneity of immunoglobulins, and beta-gamma bridging]. Furthermore, it showed highly accurate at both detecting (ROC-AUC ≥ 0.99) and quantifying (r = 0.99) M-spikes. It proved highly reproducible and resilient to minor variations and its agreement with human experts was higher (κ = 0.632) than experts between each other (κ = 0.624). CONCLUSIONS: SPECTR is an algorithm based on artificial intelligence suitable to high-throughput SPEs analyses and interpretation. It aims at improving SPE reproducibility and reliability. It is freely available in open access through an online tool providing fully editable validation assistance for SPE.
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Inteligência Artificial , Aprendizado Profundo , Proteínas Sanguíneas , Eletroforese , Humanos , Reprodutibilidade dos TestesRESUMO
OBJECTIVES: The value of the serum protein gap (PG, difference between total protein and albumin) in the detection of hyper- or hypogammaglobulinemia is not well established. We assessed the performance of PG for the detection of hyper- or hypogammaglobulinemia in a large sample of patients. METHODS: We reviewed all paired measurements of serum total protein, albumin, quantitative immunoglobulins, and serum protein electrophoresis tested between March 2014 and June 2017 at the Eastern Ontario Regional Laboratory Association. Sensitivity, specificity, positive predictive value, negative predictive value and likelihood ratios of PG at thresholds between 18 and 44 g/L for the detection of hyper- and hypogammaglobulinemia were assessed. RESULTS: There were 19,575 and 5,426 simultaneous paired data points to assess hyper- and hypogammaglobulinemia identified by serum protein electrophoresis (SPE) and nephelometry, respectively. The mean PG was 36.3 g/L (SD 8.6). The prevalence of hypergammaglobulinemia (>16 g/L by SPE) and hypogammaglobulinemia (IgG <7 g/L) was 21.9 and 5.5%, respectively. High PG (≥38 g/L) had sensitivity and specificity of 76.2 and 71.5% respectively for hypergammaglobulinemia. PG ≥38 g/L had a negative predictive value (NPV) of 93.1% for monoclonal, and 96.9% for polyclonal gammopathy. A PG threshold of ≤18 g/L had of sensitivity of 0.4%, specificity of 100%, PPV of 100% and NPV of 80.1% to detect hypogammaglobulinemia (IgG <7 g/L). CONCLUSIONS: High and low PG values were not sensitive in detecting hyper- or hypogammaglobulinemia, although negative predictive values were high for both. Performance of PG should be further evaluated prospectively in specific populations at risk of for abnormal IgG levels.
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Agamaglobulinemia , Hipergamaglobulinemia , Agamaglobulinemia/sangue , Agamaglobulinemia/diagnóstico , Albuminas , Eletroforese das Proteínas Sanguíneas , Humanos , Imunoglobulina GRESUMO
OBJECTIVES: Studies that investigate the accuracy and precision of M-protein quantification are scarce. These studies are prone to give a biased view, since they are exclusively performed by institutions with international top-expertise on M-protein diagnostics. To obtain a realistic impression of the accuracy and precision of M-protein quantification, we studied results of 73 laboratories participating in the Dutch External Quality Assessment (EQA) program for M-protein diagnostics. METHODS: To measure accuracy, healthy serum was spiked with respectively 1 and 5 g/L human IgG-kappa monoclonal antibody daratumumab. To measure precision, five sera were selected to be repeatedly send to all blinded EQA-participants. RESULTS: The reported concentrations for the EQA-sample spiked with 5 g/L daratumumab ranged from 2.6 to 8.0 g/L (mean 4.9 g/L, between-laboratory CV = 23%). 98% of the participants detected and correctly characterized the 1 g/L daratumumab band. Both the accuracy (mean 1.7 g/L) and precision (between-laboratory CV = 46%) of this 1 g/L M-protein was poor. In the five EQA-samples that were repeatedly send to the same 73 participating laboratories, between-laboratory precision (mean CV = 25%) was significantly different than the within-laboratory precision (mean CV = 12%). Relatively poor precision was observed in sera with small M-proteins. CONCLUSIONS: The EQA-data reveal a large variation in reported M-protein concentrations between different laboratories. In contrast, a satisfactory within-laboratory precision was observed when the same sample was repeatedly analyzed. The M-protein concentration is correlated with both accuracy and precision. These data indicate that M-protein quantification to monitor patients is appropriate, when subsequent testing is performed within the same laboratory.
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Confiabilidade dos Dados , Laboratórios , Coleta de Dados , HumanosRESUMO
OBJECTIVES: The therapeutic monoclonal antibody (t-mAb) daratumumab, used to treat multiple myeloma (MM) patients, interferes with routine, electrophoretic based M-protein diagnostics. Electrophoretic response assessment becomes increasingly difficult when multiple t-mAbs are combined for use in a single patient. This is the first study to address the analytical challenges of M-protein monitoring when multiple t-mAbs are combined. METHODS: In this proof-of-principle study we evaluate two different methods to monitor M-protein responses in three MM patients, who receive both daratumumab and nivolumab. The double hydrashift assay aims to resolve t-mAb interference on immunofixation. The MS-MRD (mass spectrometry minimal residual disease) assay measures clonotypic peptides to quantitate both M-protein and t-mAb concentrations. RESULTS: After exposure to daratumumab and nivolumab, both t-mAbs become visible on immunofixation electrophoresis (IFE) as two IgG-kappa bands that migrate close to each other at the cathodal end of the γ-region. In case the M-protein co-migrates with these t-mAbs, the observed interference was completely abolished with the double IFE hydrashift assay. In all three patients the MS-MRD assay was also able to distinguish the M-protein from the t-mAbs. Additional advantage of the MS-MRD assay is that this multiplex assay is more sensitive and allows quantitative M-protein-, daratumumab- and nivolumab-monitoring. CONCLUSIONS: Daratumumab and nivolumab interfere with electrophoretic M-protein diagnostics. However, the M-protein can be distinguished from both t-mAbs by use of a double hydrashift assay. The MS-MRD assay provides an alternative method that allows sensitive and simultaneous quantitative monitoring of both the M-protein and t-mAbs.
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Mieloma Múltiplo , Anticorpos Monoclonais/uso terapêutico , Humanos , Imunoeletroforese , Laboratórios , Espectrometria de Massas , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/tratamento farmacológicoRESUMO
Serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) are standard tools for multiple myeloma (MM) routine diagnostics. M-protein is a biomarker for MM that can be quantified with SPE and characterized with IFE. We have investigated combining SPE/IFE with targeted mass spectrometry (MS) to detect and quantify the M-protein. SPE-MS assay offers the possibility to detect M-protein with higher sensitivity than SPE/IFE, which could lead to better analysis of minimal residual disease in clinical laboratories. In addition, analysis of archived SPE gels could be used for retrospective MM studies. We have investigated two different approaches of measuring M-protein and therapeutic monoclonal antibodies (t-mAbs) from SPE/IFE gels. After extracting proteotypic peptides from the gel, they can be quantified using stable isotope labeled (SIL) peptides and measured by Orbitrap mass spectrometry. Alternatively, extracted peptides can be labeled with tandem mass tags (TMT). Both approaches are not hampered by the presence of t-mAbs. Using SIL peptides, limit of detection of the M-protein is approximately 100-fold better than with routine SPE/IFE. Using TMT labeling, M-protein can be compared in different samples from the same patient. We have successfully measured M-protein proteotypic peptides extracted from the SPE/IFE gels utilizing SIL peptides and TMT.