Potential problems in serum protein electrophoresis

Potential problems are described that one could encounter in carrying out an electrophoretic procedure including its ancillary phases of visualization (staining) and quantification (densitometry). Endpoint-like measurements for separated isoenzymes may provide artifactual kinetic values as well, because stain measurement is fixed at a single time whereas reagent blanking in the electrophoretic medium is substituted for the conventional serum initial absorbance readings of test-tube determinations. Truncation of separated electrophoretic zones or opacity of an electrophoretic anti-convection medium such as uncleared cellulose acetate may also interfere with absolute quantification procedures.     

A method of quantitative serum protein electrophoresis

A routine method of estimating quantitatively serum proteins by celluloseacetate electrophoresis is described suitable for most hospital laboratories.     

Automated serum protein electrophoresis by Capillarys.

Capillary zone electrophoresis (CZE) of serum proteins is increasingly gaining impact in clinical laboratories. In this report, we evaluate automated capillary zone electrophoresis by Capillarys (Sebia, France). Within-run and between-run imprecision for the five electrophoretic fractions was <2% and <6%, respectively. Data obtained with Capillarys correlated with results obtained with agarose gel electrophoresis and Paragon CZE 2000 (Beckman Coulter, USA). Analysis of serum obtained from patients with inflammation, nephrotic syndrome, bisalbuminemia, and alpha1-antitrypsin deficiency revealed that Capillarys was able to detect these abnormalities. Two hundred thirty eight samples were analyzed by agarose gel electrophoresis, Capillarys, capillary electrophoresis using Paragon CZE 2000 system, and immunofixation. Sample selection was based on the presence of a disturbed morphology (e.g., spike) of the protein profile or hypogammaglobulinemia on agarose gel electrophoresis and/or Capillarys. Immunofixation revealed the presence of a monoclonal protein, oligoclonal bands, polyclonal pattern, and a normal profile in, respectively, 89, 66, 19, and 64 samples. With Capillarys, Paragon, and agarose gel electrophoresis, a spike and/or disturbed morphology of the profile was found in 222, 182, and 180 samples, respectively. In these samples, immunofixation was negative in 73 (33%), 46 (25%), and 39 (22%) samples, respectively. These data indicate that Capillarys has a lower specificity than agarose gel electrophoresis and Paragon 2000. Of the 89 samples with a monoclonal protein, Capillarys, Paragon, and agarose gel electrophoresis failed to detect, respectively, three, three, and one monoclonal protein(s). Interferences by radio-opaque agents, complement degradation products, fibrinogen, and triglycerides are described. In conclusion, automated capillary zone electrophoresis with Capillarys provides for reproducible, rapid, and reliable serum electrophoresis.     

多发性骨髓瘤血清蛋白电泳与免疫学分型

目的对38例多发性骨髓瘤(MM)患者血清蛋白电泳、免疫电泳及免疫球蛋白进行定量分析,以探讨M蛋白、MM型别分布及其在MM中的临床诊断价值。方法血清蛋白和免疫电泳采用全自动琼脂糖凝胶电泳仪进行电泳和扫描;免疫球蛋白定量采用Beckman CS-免疫化学分析仪,以全自动方式进行速率散射比浊分析,测各样本的免疫球蛋白(IgG、IgA、Ig M)含量。结果38例MM全部检出M带,22例MM尿液检出本-周氏蛋白,检出阳性率为57.90%。血清免疫电泳分型IgGκ型14例,占36.84%;IgGλ型10例,占26.32%;IgAκ型5例,占13.16%;IgAλ型6例,占15.79%;Ig Mλ型3例,占7.89%,为最少。结论血清蛋白及免疫电泳能快速、准确地进行MM分型,对MM的诊断、病情判断、治疗及预后评估均具有重要的临床价值。     

Monoclonal protein reference change value as determined by gel-based serum protein electrophoresis

IntroductionThe International Myeloma Working Group recommendations for monitoring disease progression or response include quantitation of the involved monoclonal immunoglobulin. They have defined the minimum change criteria of ≧ 25% with an absolute change of no < 5 g/L for either minimal response or progression. Limited evidence is available to accurately determine the magnitude of change in a monoclonal protein to reflect a true change in clinical status. Here we determined the analytical and biological variability of monoclonal proteins in stable monoclonal gammopathy of undetermined significance (MGUS) patients.MethodAnalytical variability (CVa) of normal protein fractions and monoclonal proteins were assessed agarose gel-based serum protein electrophoresis. Sixteen clinically stable MGUS patients were identified from our clinical hematology database. Individual biological variability (CVi) was determined and used to calculate a monoclonal protein reference change value (RCV).ResultAnalytical variability of the normal protein fractions (albumin, alpha-1, alpha-2, beta, total gamma) ranged from 1.3% for albumin to 5.8% for the alpha-1 globulins. CVa of low (5.6 g/L) and high (32.2 g/L) concentration monoclonal proteins were 3.1% and 22.2%, respectively. Individual CVi of stable patients ranged from 3.5% to 24.5% with a CVi of 12.9%. The reference change value (RCV) at a 95% probability was determined to be 36.7% (low) 39.6% (high) using our CVa and CVi.ConclusionsSerial monitoring of monoclonal protein concentration is important for MGUS and multiple myeloma patients. Accurate criteria for interpreting a change in monoclonal protein concentration are required for appropriate decision making. We used QC results and real-world conditions to assess imprecision of serum protein fractions including low and high monoclonal protein fractions and clinically stable MGUS patients to determine CVi and RCV. The calculated RCVs of 36.7% (low) and 39.6% (high) in this study were greater that reported previously and greater than the established criteria for relapse. Response criteria may be reassessed to increase sensitivity and specificity for detection of response.     

Understanding and interpreting confidence and credible intervals around effect estimates

IntroductionReporting confidence intervals in scientific articles is important and relevant for evidence-based practice. Clinicians should understand confidence intervals in order to determine if they can realistically expect results similar to those presented in research studies when they implement the scientific evidence in clinical practice. The aims of this masterclass are: (1) to discuss confidence intervals around effect estimates; (2) to understand confidence intervals estimation (frequentist and Bayesian approaches); and (3) to interpret such uncertainty measures.ContentConfidence intervals are measures of uncertainty around effect estimates. Interpretation of the frequentist 95% confidence interval: we can be 95% confident that the true (unknown) estimate would lie within the lower and upper limits of the interval, based on hypothesized repeats of the experiment. Many researchers and health professionals oversimplify the interpretation of the frequentist 95% confidence interval by dichotomizing it in statistically significant or non-statistically significant, hampering a proper discussion on the values, the width (precision) and the practical implications of such interval. Interpretation of the Bayesian 95% confidence interval (which is known as credible interval): there is a 95% probability that the true (unknown) estimate would lie within the interval, given the evidence provided by the observed data.ConclusionsThe use and reporting of confidence intervals should be encouraged in all scientific articles. Clinicians should consider using the interpretation, relevance and applicability of confidence intervals in real-world decision-making. Training and education may enhance knowledge and skills related to estimating, understanding and interpreting uncertainty measures, reducing the barriers for their use under either frequentist or Bayesian approaches.     

五种疾病的血清蛋白电泳谱特点

目的分析了白/球蛋白(A/G)比值异常时血清蛋白电泳谱特点。方法(1)日立7060全自动生化分析仪双缩脲法检测血清总蛋白,溴甲酚绿法(BCG)检测白蛋白(ALB),计算A/G比值。(2)应用法国SEBIA全自动电泳仪琼脂糖凝胶电泳法测定血清蛋白谱。结果与健康对照组相比,113例A/G比值异常标本的血清蛋白电泳结果各组分结果差异均有统计学意义(P<0.05或P<0.01),主要表现为白蛋白降低,α1球蛋白、α2球蛋白、γ球蛋白增加。结论对A/G异常血清蛋白的进行电泳分析,有助于了解不同疾病中血清蛋白组分的变化情况,对疾病的临床诊断及疗效观测有较大价值。     

Precision and long-term stability of capillary electrophoresis measurement of serum protein zones

BACKGROUND: Analytical evaluations of an available system for capillary zone electrophoresis (CZE) of serum protein have been reported. However, data concerning long-term precision and stability of the system, operated under routine conditions, are lacking. We report data from an internal quality control (QC) scheme, obtained over a 1-year period. METHODS: Measurements were done with a pair of instruments (Beckman Paragon CZE 2000 system), each equipped with seven capillaries. After preliminary (1 month) assessment of possible inter-capillary and inter-instrument variations, the QC material (a home prepared serum pool stored in the frozen state) was assayed daily over a 1-year period. RESULTS: Maximum inter-capillary and inter-instrument differences were 3.1% and 2.4%. No significant trend was observed for daily values (205 measurements over 1 year); in the same period overall imprecision values (CV) were in the interval 1.2% (albumin) to 3.2-6.1% (globulin zones). Mean monthly imprecision (CV) values were in the interval 1.1% (albumin) to 5.2% (globulin zones). There was no significant trend of monthly means with time. The observed imprecision values were within the biological variation-derived goals for imprecision. CONCLUSIONS: It is concluded that the assessed analytical instruments, operated in routine conditions, show long-term stability and imprecision consistent with the clinical use of the results produced.     

Peaks and tails: Evaluation of irregularities in capillary serum protein electrophoresis

The increased analytical sensitivity of capillary electrophoresis detects additional irregularities that are suspicious for a monoclonal component. This is most noticeable in the beta-1-, beta-2- and gamma-globulin fractions. The causes of non-monoclonal irregularities are manifold, but are rarely reported back to the ordering physician. This article reviews the basic concepts to correctly identify irregularities, monoclonal and oligoclonal peaks by capillary electrophoresis. It then focuses on detecting and reporting typical non-monoclonal irregularities according to their electrophoresis fractions as well as their possible clinical implications.     

血清蛋白电泳分布规律对多种疾病的诊断价值

目的探讨血清蛋白电泳的分布特点及对多种不同疾病的临床诊断价值,并进一步分析其在不同肾病中的临床及实验室检查的特点。方法采用法国Sebia全自动毛细管电泳仪,对多种疾病和正常对照组血清进行电泳,分析其分布规律。结果各组疾病与对照组比较,除β1球蛋白差异无统计学意义(P>0.05)外,白蛋白、α1球蛋白、α2球蛋白、β2球蛋白、γ球蛋白以及M蛋白均有显著性差异(P<0.01),"山形峰"、"β-γ"桥、宽γ、"M"带显著;在肾病分型各组中肾病综合征中α2球蛋白、β2球蛋白显著高于其他组(P<0.05)。结论血清蛋白电泳分布规律分析对于各类疾病的临床诊断具有重要的参考价值。     

An expert system for the classification of serum protein electrophoresis patterns

BACKGROUND: With the improvement of capillary electrophoresis, much progress has been made in terms of sensitivity and automation, but the interpretation of the patterns, actually, depends totally on expert personnel. The aim of this work was to evaluate Neurosoft-Sebia, an expert system developed to discriminate between regular and anomalous serum protein electrophoresis patterns performed on Capillarys2. METHODS: Neurosoft-Sebia, based on six auto-associative neural networks, was trained to create the initial knowledge base. In the tuning phase, 3000 electrophoretic patterns were performed in three different laboratories, and the discordances between human experts and Neurosoft-Sebia classifications were added to the initial knowledge base. Finally, the performances of Neurosoft-Sebia were evaluated using a benchmark dataset. RESULTS: The initial knowledge base was created with 2685 fractions. In the tuning phase, 241 discordances were found: 56 as regular by Neurosoft-Sebia and anomalous by human experts, and 185 as anomalous by Neurosoft-Sebia and regular by human experts. Sensitivity values were evidenced as the ability of Neurosoft-Sebia in selecting anomalous fractions, with an increase from 66.67% using the initial knowledge base to 97.40% using the enriched knowledge base. CONCLUSIONS: This work demonstrated how the ability of Neurosoft-Sebia in selecting anomalous pattern was comparable to that of human experts, saving time and providing rapid and standardized interpretations.     

凝血机制障碍时血清蛋白电泳变化的探讨

目的 通过对凝血功能障碍患者血清蛋白电泳分析探讨血浆白蛋白、α、β、γ球蛋白蛋白变化。方法 选用健康体检者及凝血酶原时间测定(PT)、活化部分凝血活酶时间(APTT)测定、凝血酶时间(TT)测定延长的凝血机制障碍患者各20例,均分别作全自动电泳扫描分析。结果 凝血障碍组与健康组比较,其β-球蛋白、γ-球蛋白有极显著性差异(P<0.01),而α1及α2球蛋白均差异无显著意义。结论 凝血机制障碍病人血浆蛋白电泳异常。血清蛋白电泳分析可了解凝血功能异常时体内蛋白质变化概况。     

全自动毛细管血清蛋白电泳在肝癌中的临床价值

目的通过全自动毛细管血清蛋白电泳检测清蛋白、α1球蛋白、α2球蛋白、β1球蛋白、β2球蛋白、γ球蛋白,探讨以上指标在肝癌中的临床价值。方法收集2014年12月至2015年11月住院患者83例,其中原发性肝癌51例,继发性肝癌32例,运用全自动生化分析仪和全自动毛细管血清蛋白电泳检测患者血清的清蛋白(定量指标以g/L表示,定性指标以%表示)、总蛋白、α1球蛋白、α2球蛋白、β1球蛋白、β2球蛋白、γ球蛋白8项指标,并随机抽查100例健康体检者作为对照组,观察比较以上8项检测指标在各组的差异。结果原发性肝癌组治疗前与对照组比较,清蛋白(%)和γ球蛋白差异有统计学意义(P0.05);原发性肝癌组治疗后与对照组比较,清蛋白(g/L)、清蛋白(%)、β1球蛋白和γ球蛋白差异有统计学意义(P0.05);原发性肝癌组治疗前、后比较,清蛋白(g/L)和清蛋白(%)水平降低,差异有统计学意义(P0.05);γ球蛋白诊断原发性肝癌敏感度和特异度明显高于其余3项指标;同时对各组人群的血清γ球蛋白阳性率比较,原发性肝癌组患者γ球蛋白阳性率高于对照组和继发性肝癌组(P0.05)。结论通过全自动毛细管血清蛋白电泳检测发现,γ球蛋白在肝癌的诊治中具有一定的临床价值。