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Capillary zone electrophoresis (CZE) of serum proteins is a well-accepted method used in clinical chemistry laboratories to separate serum proteins and detect monoclonal (M) proteins. The detection is based on ultraviolet absorbance measurements at 200 nm (Capillarys, Sebia) or at 214 nm (Paragon 2000, Beckman Coulter), which correspond to the absorption of peptide bonds in proteins. Compared with gel electrophoresis, CZE offers the advantage that all proteins are quantified. Most interferences on CZE are caused by exogenous nonprotein substances that also absorb at 200/214 nm, such as radioopaque agents and antibiotics (1). Gelatin-based plasma expanders have also been reported to cause interference. Gay-Bellile and Gijbels reported an increase in the β/γ-region on CZE in samples from patients who had received infusions with …

Capillary zone electrophoresis (CZE) is documented to have high sensitivity and specificity for the detection of monoclonal (M) proteins. In two large (>1500 participants) prospective studies in which CZE was compared with “immunofixation/immunoelectrophoresis”, the sensitivity of CZE was 95% (1)(2). This is superior to the sensitivity of agarose gel electrophoresis (AGE), which Katzmann et al. (1) found to be 91% and Meunier (3) found to be 92.2% (evaluation of 2060 sera containing an M-protein). Especially in the β-globulin region, CZE with Paragon CZE 2000 (Beckman-Coulter) is superior to AGE for detection of low-concentration M-proteins (IgA and light chains) (3)(4). The specificity of CZE is reported to be high (98.6%) and comparable to that of AGE (1)(5). In a prospective study (2), we described three M-proteins with high pI values that migrated in the slow γ region on AGE but were not detected with Paragon. In addition, one high-concentration IgG|gl with a pI ≈7 that migrated in the mid-γ region on AGE was not separated by CZE. The Paragon has recently been upgraded [modified buffer, higher voltage (10.3 kV), more efficient cooling, adapted software (1.6.02)], and according … [↵][1]bAuthor for correspondence. [1]: #xref-corresp-2-1

The first case of the simultaneous presence of macroamylasemia and macrolipasemia in a patient with gluten enteropathy (celiac disease) is described. Both macroenzymes were formed by association of polyclonal IgA with amylase and lipase. Both macroenzymes had molecular masses > 300 kDa.

Background: Ischemic injury to the central nervous system causes cellular activation and disintegration, leading to release of cell-type–specific proteins into the cerebrospinal fluid (CSF). We investigated CSF concentrations of myelin basic protein (MBP), glial fibrillary astrocytic protein (GFAP), the calcium-binding protein S100B, and neuron-specific enolase (NSE) in acute ischemic stroke patients and their relation to initial stroke severity, stroke location, and long-term stroke outcome. Methods: CSF concentrations of MBP, GFAP, S100B, and NSE were assessed in 89 stroke patients on admission (mean 8.7 h after stroke onset) and in 35 controls. We evaluated the relation between CSF concentrations and (a) stroke severity (NIH Stroke Scale [NIHSS] score on admission, infarct volume), (b) stroke location, and (c) stroke outcome (modified Rankin Scale [mRS] score at month 3). Results: MBP concentration was significantly higher in subcortical than in cortical infarcts (median MBP, 1.18 vs 0.66 μg/L, P < 0.001). GFAP and S100B concentrations correlated with the NIHSS score on admission (GFAP, R = 0.35, P = 0.001; S100B, R = 0.29, P = 0.006), infarct volume (GFAP, R = 0.34, P = 0.001; S100B, R = 0.28, P = 0.008), and mRS score at month 3 (R = 0.42, P < 0.001 and R = 0.28, P = 0.007). Concentrations of NSE did not correlate with stroke characteristics. Conclusions: MBP, GFAP, S100B, and NSE display relevant differences in cellular and subcellular origins, which are reflected in their relation to stroke characteristics. MBP is a marker for infarct location. GFAP and S100B correlate with stroke severity and outcome.

To assess current practice in the detection, analysis, and reporting of cryoglobulins, a questionnaire was sent to 140 laboratories. Only 36% of laboratories used standard procedures (tube preheating, transport in container, and sedimentation and/or centrifugation at 37 °C) to ensure that the temperature did not drop below 37 °C until after serum separation. Time periods allowed for cryoprecipitation at 4 °C varied from 12 h to 9 days, with 30% of laboratories allowing precipitation for

Analysis of serum proteins by capillary zone electrophoresis (CZE) has become a routine method for the detection of dysproteinemias and monoclonal proteins. Detection is based on ultraviolet absorbance at 200 nm (Capillarys, Sebia) or 214 nm (Paragon 2000, Beckman Coulter), which corresponds to the absorption of peptide bonds in proteins. Compared with gel electrophoresis, CZE offers the advantage that proteins are more proportionately quantified compared with the variable dye-staining intensities exhibited by different serum proteins. A drawback of CZE, however, is the potential interference by exogenous nonprotein substances, such as radioopaque agents and antibiotics, which also absorb at these wavelengths (1). A 5-year old girl with protein-losing enteropathy underwent routine angiocardiography 4 years after a Fontan operation for …

Background: HEp-2 cells that overexpress the human 60-kDa SSA antigen have been used to improve sensitivity and specificity for the detection of anti-SSA antibodies by indirect immunofluorescence. We describe a survey on the detection of anti-SSA antibodies using a commercial substrate that overexpresses SSA.Methods: The evaluation was done on 18 371 consecutive samples submitted to the laboratory for detection of anti-nuclear antibodies, from which 188 anti-SSA antibody-containing and clinically documented samples were obtained. The presence of anti-SSA antibodies produced a distinct bright speckled pattern with nucleolar staining in 10–20% of interphase cells. The identity of all anti-SSA antibodies was confirmed by dot-blot analysis.Results:Samples containing anti-SSA antibodies were separated into three main groups: group I, distinctive SSA pattern and other nuclear staining (50%); group II, only the distinctive SSA pattern (29%); group III, nuclear staining but without the distinctive SSA pattern (21%). Anti-SSA antibodies with concurrent SSB antibodies were associated with group I, whereas anti-SSA antibodies with concurrent U1-RNP antibodies were associated with group III. Group I included mainly patients with Sjögren syndrome and systemic lupus erythematosus (SLE), whereas group III included patients with mixed connective tissue disease and SLE. Diseases not classically associated with the presence of anti-SSA antibodies were found in group II in >50% of the cases.Conclusions: SSA-positive individuals were identified in a population selected on the basis of HEp-2000 positivity. Our study highlights diseases associated with anti-SSA antibodies and associations between the presence of the distinctive SSA pattern on HEp-2000 and some clinical conditions.

Over the last 5 years, capillary zone electrophoresis (CZE) with the use of fused-silica capillaries has been increasingly introduced in clinical laboratories for routine serum-protein electrophoresis (1). The multichannel, automated Paragon CZE 2000 instrument (seven capillaries in parallel; Beckman Coulter) represented an especially attractive alternative to time-consuming manual techniques. CZE has been documented to perform reliably for the analysis of serum proteins and for the detection of monoclonal components (2)(3). We reported (4) that the sensitivity of the Paragon CZE 2000 system for the detection of monoclonal components (93%) was superior to the sensitivity of cellulose acetate gel electrophoresis (74%) and agarose gel electrophoresis (86%). In a prospective study, Katzman et al. (5) reported sensitivities of 95% and 91% for seven-capillary electrophoresis and agarose gel electrophoresis, respectively. When compared with agarose gel electrophoresis, CZE was able to detect more low-concentration IgA monoclonal components or light chains that were hidden in agarose gel electrophoresis because of comigration with transferrin or C3 (6). Paraproteins that are missed by CZE are typically very low-concentration monoclonal components that are also missed by agarose gel electrophoresis, but not by immunofixation. Problems with the detection of monoclonal components by CZE have been described (7)(8). Using a single-capillary Beckman P/ACE instrument with a capillary and buffer different from the Paragon CZE 2000 system, Jenkins and Guerin (7) performed a prospective study on 5500 specimens in which they compared CZE with agarose gel electrophoresis for the detection of monoclonal components. The authors identified six paraproteins that did not separate correctly on CZE. On agarose gel electrophoresis, all of these proteins migrated in the very slow γ region. The pI …

CASE DESCRIPTION A 67-year-old woman presented with dry eyes, dry mouth, intermittent polyarticular nonerosive arthropathy, bronchial and bronchiolar involvement with bronchiectasis, peripheral sensory neuropathy, cryoglobulinemic vasculitis (IgM |gk paraprotein), pronounced hypergammaglobulinemia (|gg-globulin fraction, 28.6 g/L; reference interval, 8.0–13.5 g/L), and rheumatoid factor positivity (rheumatoid arthritis latex test, 728 IU/mL; reference interval, ≤40 IU/mL). Both parotid glands were enlarged, especially on the left side (Fig. 1). Puncture of a parotid cyst revealed benign material suggestive of a sialocoele (salivary cyst). The results of …

Capillary zone electrophoresis (CZE) is an alternative method for separation of serum proteins (1). Two dedicated and automated multichannel instruments are available, the Paragon 2000 (Beckman-Coulter) and the Capillarys (Sebia). The sensitivity and specificity of Paragon 2000 for detection of monoclonal proteins have been reported to be 95%(2)(3)(4) and 78%(4), respectively. The low reported specificity reflects the frequent occurrence of slight abnormalities in the electropherogram at the anodal part of the γ-globulin fraction (fibrinogen region)(4). Only 2 studies (5)(6) evaluated the Capillarys for detection of monoclonal proteins and reported a high sensitivity, and prospective studies in a routine clinical setting are …

Luraschi et al. (1) recently published the first report describing the use of capillary zone electrophoresis (CZE) coupled with immunosubtraction to detect and characterize low concentrations of free γ heavy chains in serum. By contrast, we describe a case in which CZE failed to detect and characterize μ heavy chain disease. A 90-year-old woman who complained of weight loss presented with progressive enlargement of the left parotid gland, splenomegaly, and palpable inguinal lymph node enlargements. A combined positron emission tomography/computed tomography whole body scan confirmed left parotid involvement as well as supra- and infradiaphragmatic involvement of the lymph nodes. Laboratory investigation showed hemoglobin, leukocyte count and differentiation, and a platelet count within the appropriate reference intervals. Serum lactate dehydrogenase was increased. The IgG concentration was 5.46 g/L, …

The enhanced resolution of capillary zone electrophoresis (CZE) provides superior sensitivity for the detection of monoclonal (M) proteins in serum samples compared with agarose gel electrophoresis (AGE). This comes at the cost, however, of a lower specificity, calculated as 78% in our hands for the Paragon CZE 2000® with software version 1.602 (Beckman) (1). Abnormalities in the electropherogram that cause confusion are most frequently located in the anodal part of the γ-globulin fraction. Notably, fibrinogen, which remains in incompletely clotted serum samples, migrates at the junction between the β- and γ-fractions. Even when following the strict procedure of keeping serum samples for a minimum of 4 h at room temperature before electrophoresis, we found a suspected fibrinogen or possible M-protein peak in 39 (8.6%) of 454 consecutive routine samples in our laboratory. Recently, a simple method consisting of cold-ethanol precipitation was described for the removal of fibrinogen from serum specimens before agarose protein electrophoresis (2). We evaluated the use of this method in the routine analysis of serum samples by CZE in which a peak at the fibrinogen location could cause confusion with the detection of small M-proteins. Absolute ethanol was added to the specimens at a final concentration of 100 mL/L. …

Phosphate concentrations were determined in 52 cases of paraproteinemia. The unmodified acidic ammonium molybdate method produced 19% spuriously high results. The false increase of phosphate concentration was attributable to formation of precipitate in the reaction mixture. The precipitate was formed by interaction between immunoglobulins and the unmodified acidic ammonium molybdate reagent. The magnitude of interference bore no relation to the type, concentrations, or isoelectric point of the paraproteins or to the presence or absence of free light chains. Diluting the sample to approximately 40 g/L total protein reduced but did not always eliminate the interference. In some cases paraprotein concentration as low as 8 g/L falsely increased plasma phosphate results. Apparently, only IgG and IgM but not IgA paraproteins produced the interference. Deproteination by ultrafiltration or by treatment with trichloroacetic acid removed the interference. The Kodak slide method and the new modified Boehringer Mannheim phosphate test were found to be interference-free. However, in some cases the latter new formulation is sensitive to substantial changes in ionic concentration of the reaction mixture.

We wish to respond to the comments of K. Day and J. Zakowski concerning our letter in the August issue of Clinical Chemistry (1)(2) in which we discussed the capabilities of agarose gel electrophoresis/immunofixation and capillary zone electrophoresis for detecting rare monoclonal proteins. We first wish to respond to their comments that enhanced resolution often detects peaks that previously were not readily observable, that the gamma zone migrates more rapidly in CZE Version 1.6.02 and may be misinterpreted as a peak by …

Capillary zone electrophoresis (CZE) using fused-silica capillaries has become a well-accepted method for the separation of serum proteins and for the detection of monoclonal components in human serum (1)(2)(3)(4). In earlier methods, such as those that use agarose gels, quantification of the protein fractions was based on dye binding, whereas CZE uses ultraviolet detection at 214 nm for direct protein quantification via the peptide bonds. Any substance or drug that is present in serum and that absorbs at 214 nm potentially can interfere with CZE analysis. Few interfering substances have been reported. Radiocontrast media that absorb at 214 nm interfere with CZE and can simulate a monoclonal component (5)(6)(7), and the antibiotic piperacillin-tazobactam …

Capillary zone electrophoresis (CZE) is increasingly used in clinical laboratories for serum protein separation. The technique is a sensitive method for the detection of monoclonal (M) proteins. However, when compared with immunofixation, CZE fails to detect M proteins in 5% of samples (1)(2). These so-called “false-negative” results encompass low-concentration and “hidden” M proteins (e.g., in the transferrin peak). The goal of this study was to evaluate whether an assay for the detection of free light chains (FLCs) that has recently become available (Freelite®; The Binding Site) can detect the M proteins present in samples that produce false-negative results in CZE. The Freelite assay is a sensitive automated immunoassay for κ and λ FLCs in serum (3) and has been useful for the diagnosis and monitoring of patients with nonsecretory myeloma (4). Moreover, it has been reported that the quantification of FLCs in serum by nephelometry correlates with changes in urinary FLC excretion (5). Frozen sera from 54 patients that had previously been shown to contain M proteins by immunofixation (Sebia), but not …

Antibodies against extractable nuclear antigens (ENAs) are useful diagnostic markers for various autoimmune diseases. Anti-SSA and anti-SSB antibodies are found in Sjogren Syndrome (SS), anti-Sm antibodies are found in systemic lupus erythematosus (SLE), anti-RNP antibodies are found in mixed connective tissue disease (MCTD), and anti-Scl-70 antibodies are found in scleroderma. Antibodies against cytoplasmic Jo-1 and M2 are helpful markers for the diagnosis of, respectively, polymyositis and primary biliary cirrhosis. The antibodies can be identified by either counter immunoelectrophoresis (CIE) or immunoblotting. These techniques, however, are time-consuming, technically demanding, and do not provide for fast turnaround times. Over the last few years, alternative …