Computer-supported detection of M-components and evaluation of immunoglobulins after capillary electrophoresis

BACKGROUND: Electrophoresis of serum samples allows detection of monoclonal gammopathies indicative of multiple myeloma, Waldenstr?m macroglobulinemia, monoclonal gammopathy of undetermined significance, and amyloidosis. Present methods of high-resolution agarose gel electrophoresis (HRAGE) and immunofixation electrophoresis (IFE) are manual and labor-intensive. Capillary zone electrophoresis (CZE) allows rapid automated protein separation and produces digital absorbance data, appropriate as input for a computerized decision support system. METHODS: Using the Beckman Paragon CZE 2000 instrument, we analyzed 711 routine clinical samples, including 95 monoclonal components (MCs) and 9 cases of Bence Jones myeloma, in both the CZE and HRAGE systems. Mathematical algorithms developed for the detection of monoclonal immunoglobulins (MCs) in the gamma- and ss-regions of the electropherogram were tested on the entire material. Additional algorithms evaluating oligoclonality and polyclonal concentrations of immunoglobulins were also tested. RESULTS: CZE electropherograms corresponded well with HRAGE. Only one IgG MC of 1 g/L, visible on HRAGE, was not visible after CZE. Algorithms detected 94 of 95 MCs (98.9%) and 100% of those visible after CZE. Of 607 samples lacking an MC on HRAGE, only 3 were identified by the algorithms (specificity, 99%). Algorithms evaluating total gammaglobulinemia and oligoclonality also identified several cases of Bence Jones myeloma. CONCLUSIONS: The use of capillary electrophoresis provides a modern, rapid, and cost-effective method of analyzing serum proteins. The additional option of computerized decision support, which provides rapid and standardized interpretations, should increase the clinical availability and usefulness of protein analyses in the future.     

Analysis of inflammatory response in human plasma samples by an automated multicapillary electrophoresis system.

A new automated multicapillary zone electrophoresis instrument with a new high-resolution (HR) buffer (Capillarys with HR buffer) for analysis of human plasma proteins was evaluated. Albumin, alpha(1)-antitrypsin, alpha(1)-acid glycoprotein, haptoglobin, fibrinogen, immunoglobulin (Ig)A, IgG and IgM were determined nephelometrically in 200 patient plasma samples. The same samples were then analyzed on the Capillarys system (Sebia, Paris, France). The albumin concentration from the nephelometric determination was used for quantification of the individual peaks in the capillary electrophoresis (CE) electropherogram. There was strong linear correlation between the nephelometric and electrophoretic determination of alpha(1)-antitrypsin (R(2) = 0.906), alpha(1)-acid glycoprotein (R(2) =0.894) and haptoglobin (R(2) = 0.913). There was also good correlation between the two determinations of gamma-globulins (R(2) = 0.883), while the correlation was weaker for fibrinogen (R(2) = 0.377). The Capillarys instrument is a reliable system for plasma protein analysis, combining the advantages of full automation, good analytical performance and high throughput. The HR buffer in combination with albumin quantification allows the simultaneous quantification of inflammatory markers in plasma samples without the need for nephelometric determination of these proteins.     

Identification of a specific marker for hepatitis C virus infection using capillary zone electrophoresis

BACKGROUND: Hepatitis C virus (HCV) infection is now becoming a common health problem in both developed and developing countries. The limitation of the available diagnostic approaches enhances the efforts to have a rapid, sensitive, and specific diagnostic testing for HCV infection. Capillary zone electrophoresis (CZE) is a fully automated analytical technique whose popularity is quickly increasing in the clinical chemistry laboratory. CZE can analyze nanoliters or less of samples with detection sensitivity at the attomole level (10(-18) mol) or less. METHODS: CZE was optimized for the identification of a specific marker of HCV infection. The performance characteristics of the CZE for the detection of HCV RNA peak were evaluated in comparison with standard nested PCR. RESULTS: A characteristic peak at 2.72 min was identified only in the CZE electropherogram of urine samples from HCV-infected individuals. The nature of the characteristic peak was investigated and confirmed to be HCV RNA using PCR and other biochemical treatments including RNase, DNase, and trypsin enzymes. CZE showed high degrees of sensitivity (94%) and specificity (96%) in comparison with the nested PCR. CONCLUSION: CZE provides a rapid, inexpensive, sensitive, and specific analytical method for diagnosis and mass screening of a large number of HCV-infected individuals.     

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.     

Leucocyte-associated plasma proteins. Association of prealbumin, albumin, orosomucoid, alpha 1-antitrypsin, transferrin and haptoglobin with human lymphocytes, monocytes, granulocytes and a promyelocytic leukaemic cell line (HL-60)

The distribution of prealbumin, albumin, orosomucoid, alpha 1-antitrypsin, haptoglobin and transferrin, including their electrophoretic heterogeneous variants, was studied in isolated lymphocytes, monocytes and granulocytes and in a human promyelocytic cell line (HL-60) by crossed immunoelectrophoresis. Prealbumin, albumin and transferrin were present in lymphocytes, monocytes and granulocytes, whereas the cellular variants of orosomucoid and haptoglobin were present only in granulocytes. alpha 1-Antitrypsin was present in four electrophoretic variants which were differently distributed among the various cell types. Synthesis of alpha 1-antitrypsin by monocytes, granulocytes and HL-60 cells was demonstrated by 14C-leucine incorporation. The six plasma proteins could not be removed from intact cells by incubation with the respective antibodies at 0 degrees C, or iodinated by lactoperoxidase catalysed iodination at 23 degrees C. They were, however, readily solubilized by freeze-lysis of the cells, suggesting an intracellular localization. Compared to their plasma counterparts none of the proteins differed in their hydrophobic properties but the carbohydrate residues of orosomucoid, alpha 1-antitrypsin and haptoglobin were different. The pattern of disappearance of the proteins from the cells during incubation suggested that the localization of albumin and transferrin in relation to the cells differed from that of the other proteins.     

Biochemical modifications in a case of analbuminemia (author's transl)]

A new case of analbuminemia is described for a six month old child of Algerian origin. The serum albumin concentration was 64 mg/l and its immunochemical action was identical to that of normal albumin. The system reacted by an increase of the synthesis of globulins. For the subject, the alpha1-antitrypsin, ceruloplasmin, haptoglobin, alpha2-macroglobulin, transferrin and immunoglobulins M contents were three times higher than the standard figures. However, it was possible to show that the presence of free bilirubin independent from proteins could be detected at a concentration of 17 mumol/l.     

Variability of plasma proteins according to molecular size. Long-term and short-term intra-individual variation

The intra-individual variations in serum concentrations of alpha 1-antitrypsin, albumin and alpha 2-macroglobulin were determined using high precision analytical methods. The long-term (3 months) variations were 8.2% for alpha 1-antitrypsin and 2.9% for alpha 2-macroglobulin in five males and five females. The coefficients of variation for albumin were 1.5 and 3.4% for males and females, respectively. In males the long-term variations of albumin and alpha 2-macroglobulin were highly correlated. The short-term (2 days) intra-individual variations in six males were 2.5, 3.8 and 3.4% for alpha 1-antitrypsin, albumin and alpha 2-macroglobulin respectively (coefficients of variation). A diurnal variation was found for albumin with maximal concentrations at 18.00 hours. At 6.00 and 10.00 hours the fractional concentrations of alpha 1-antitrypsin and albumin were lower than for alpha 2-macroglobulin. The variations of the three proteins were positively correlated.     

Genetic typing of alpha1-antitrypsin by immunofixation electrophoresis, identification of subtypes of Pi M.

Prolonged electrophoresis in alkaline agarose gels, followed by immunofixation, is a valuable addition to acid starch gel electrophoresis and isoelectric focusing for the genetic phenotyping of alpha1-antitrypsin. This technique is helpful in clarifying certain variants and in ascertaining types in serum and amniotic fluid samples with secondary changes. In addition, heterogeneity may be detected within the variants found at pH 4.95, analogous to hemoglobin polymorphism. Two "new" variants, PiMLamb and PiMBaldwin, have been detected by a combination of immunofixation electrophoresis and acid starch gel electrophoresis.     

3. The laboratory investigation of monoclonal gammopathies.

The monoclonal gammopathies are a group of disorders characterized by proliferation of a single clone of plasma cells that produce a homogeneous, monoclonal (M) protein. The structure of immunoglobulins, relationship of normal (polyclonal) immunoglobulins to myeloma and macroglobulin (monoclonal) immunoglobulins, pattern of monoclonal immunoglobulin overproduction, and laboratory methods for the recognition and study of monoclonal proteins in the serum and urine are reviewed. Interpretation of these laboratory tests is emphasized. The demonstration of a monoclonal protein in the serum or urine of a patient suggests multiple myeloma or one of its variants (solitary plasmacytoma of bone, extramedullary plasmacytoma, or plasma cell leukemia); Waldenstr?m's macroglobulinemia or, occasionally, lymphoma; heavy-chain diseases (gamma, alpha, and mu); primary amyloidosis; and monoclonal gammopathies of undertermined significance. The electrophoretic and immunoelectrophoretic patterns found in the monoclonal gammopathies are discussed. Periodic electrophoresis of the serum and urine is essential in the follow-up and management of patients with a monoclonal gammopathy.     

Immunological and electrophoretical approaches to macroamylase analysis.

The characteristics of abnormally large-size amylase (macroamylase) of the three patients were studied. Amylase isoenzyme patterns of the three macroamylase samples of agar gel electrophoresis were different from the normal one: a post-beta-globulin or middle-ganna-globulin zone was found in macroamylase, while a fast-gamma, and a pre-gamma-globulin or both zones is found in normal amylase. With gel chromatography at pH 3.4, macroamylase dissociated to normalsized amylase. The electrophoretic patterns of the normal-sized amylase were identical with those of normal amylase.The binding proteins of the macroamylase were identified as immunoglobulin IgG in onecase and immunoglobulin IgA in the other two cases by the immunochemical criteria and immunoenzyme electropherogram. These immunoglobulins bound with serum amylase having normal molecular weight and electophoretic mobility to form macroamylase.     

Nasal lavage as a tool for the assessment of upper-airway inflammation in adults and children

The prevalence of respiratory allergies has increased over the last 20 years, highlighting the need for a simple and noninvasive tool to investigate, in a clinical and epidemiological context, airway-inflammation mechanisms encountered in allergic and inflammatory processes. The nose, as the first region of the respiratory tract to come in contact with airborne pollutants, is easily explored with the use of nasal lavage (NL). We evaluated an NL method for adults and children, along with its reproducibility and capacity to separate different subgroups. NL reproducibility, assessed in 10 healthy, nonsmoking adults on three different occasions, was determined with the use of the intraclass coefficient of correlation for such inflammatory markers as total cell count, albumin, urea, neutrophil elastase, alpha(1)-antitrypsin, interleukin-6, and interleukin-8. Using this NL method, we analyzed nasal markers of 50 healthy adults (smokers and nonsmokers) and 12 healthy children. Our NL method demonstrated high reproducibility with regard to total cell count, albumin, urea, and alpha(1)-antitrypsin (intraclass correlation coefficient > 0.75). Compared with NL results in nonsmokers, NL in heavy smokers revealed significant increased concentrations of total cell counts and interleukin-8 and significant decreased concentrations of interleukin-6. These findings suggest that NL can be used as a tool in the assessment of inflammation because it has the correct reproducibility and can discriminate between heavy smokers and nonsmokers. Moreover, the use of this standardized method in children is feasible.     

Cellulose acetate membrane electrophoresis in the analysis of urinary proteins in patients with tubulointerstitial nephritis

Urinary proteins from 14 patients with tubulointerstitial nephritis were analyzed by cellulose acetate membrane electrophoresis. Urinary total protein concentrations were measured, and urinary 15 proteins (prealbumin, albumin, alpha(1)-microglobulin, alpha(1)-antitrypsin, alpha(2)-macroglobulin, haptoglobin, retinol binding protein, transferrin, beta(2)-microglobulin, IgA, IgG, kappa- and lambda-light chains, cystatin C, and lysozyme) were identified by the use of a rapid and highly sensitive colloidal silver staining reagent suited for use with cellulose acetate membranes, as reported previously by Matsuda et al. (J Clin Lab Anal 15:171-174, 2001; Clin Chem47:763-766, 2001) and Hiratsuka et al. (J Clin Lab Anal 10:403-406, 1996). We also analyzed urinary total protein concentration and urinary protein fractions according to the presence of acute or nonacute interstitial nephritis. In addition, the relationship between urinary protein fraction and complications of interstitial nephritis was analyzed. The goal of this work was to find a useful index for the diagnosis of tubulointerstitial nephritis.     

Identification and quantification of serum proteins secreted into the normal human jejunum

The in vivo transfer of serum proteins to the human intestinal lumen was characterized by crossed immunoelectrophoretic analyses of intestinal perfusates from four healthy volunteers. Serum proteins with molecular masses below 100 kDa and the immunoglobulins were found in human jejunal perfusates. Larger serum proteins were either absent (alpha and beta lipoproteins) or present in small amounts (alpha 2-macroglobulin, haptoglobulin and ceruloplasmin). These results demonstrate the existence of a selective transfer of serum proteins to the intestinal lumen under physiological conditions. The intestinal clearance rate was 0.1 ml serum per hour per 10 cm jejunum for albumin, prealbumin, alpha 1-antitrypsin, orosomucoid, transferrin and haemopexin. The rate of secretion of total protein to the jejunal lumen was 100 mg protein per hour per 10 cm jejunum. About 45% was due to immunoglobulins and further 10-15% due to the remaining serum proteins. It is suggested that the serum proteins pass through the epithelium by a transcellular mechanism.     

Improved resolution of serum bisalbumin on electrophoresis and investigation of bisalbumin in urine

OBJECTIVES:: The aims of this study were to improve the resolution of albumin variants (bisalbumins, especially albumin Naskapi) from albumin in commercial serum protein electrophoresis systems; to compare the separation of bisalbumin from albumin on these improved methods against the recognized gold standard of capillary electrophoresis; and to investigate the presence of albumin variants in urine. DESIGN AND METHODS:: Electrophoresis was performed using the Sebia Hydrasys 15/30 beta1beta2 system, as well as modified methods on the Sebia Hydrasys HR and the Beckman Paragon systems. The interpretation of electrophoretic gels was performed by manual visualization and densitometric scanning. Serum samples were also analyzed using capillary electrophoresis at Sebia Electrophoresé located in Paris, France. Urine samples were concentrated using Vivaproducts concentration tubes and were electrophoresed using the Sebia Hydrasys HR system. RESULTS:: Representative gels and scans of serum samples demonstrate the improved resolution of modified electrophoresis methods compared to routine methods. The raw data from the gels and scans were compiled to calculate concordance and discordance for each method. DISCUSSION:: The various commercial serum protein electrophoresis systems showed improved resolution using the modified methods. In comparison with capillary electrophoresis, the modified Sebia Hydrasys HR and Beckman Paragon methods using visualization demonstrated 100% concordance and thus performed equally as well as the gold standard. Urine studies found that variant albumins are also excreted in the urine in the same 50-50 ratio as that found in the serum.     

Application of isoelectric focusing in alpha1-antitrypsin phenotyping.

alpha1-Antitrypsin, the major protease inhibitor in human serum, occurs in a considerable number of variant forms, some of which are associated with lung and liver diseases. The identification of these genetic variants, generally called Pi-types, by means of isoelectric focusing is described, as well as investigations concerning the practical application of isoelectric focusing as a routine procedure for typing the variants of alpha1-antitrypsin. Finally, isoelectric focusing is compared with the most widely used Pi-phenotyping technique, namely acid starch-gel electrophoresis followed by immunoelectrophoresis in antibody-containing agarose gel.     

Genetic variants of alpha 1-antitrypsin and hemoglobin typed by isoelectric focusing in preselected narrow pH gradients with PhastSystem.

In this method for automatically running and staining isoelectric focusing (IEF) gels, pre-made dehydrated polyacrylamide gels were rehydrated before assays run with the PhastSystem (Pharmacia LKB Biotechnology). The typing of genetic variants of hemoglobin and alpha 1-antitrypsin in narrow pH gradients (pH 6.7-7.7 and 4.2-4.9, respectively) was simple, convenient, and reproducible. The clinically important variants of alpha 1-antitrypsin (ZZ and SZ) were identified from serum or dried blood on filter paper. The fast screening of abnormal hemoglobin samples (HbS) for cases in clinical medicine was easily performed. The total analysis time for the phenotyping with conventional protein staining was approximately 60 min.     

Distribution of alpha 1-antitrypsin in normal, granuloma, and tumor tissues in rats.

Purified rat alpha 1-antitrypsin and albumin were radiolabeled, and their distribution and catabolism in various tissues were studied in rats with inflammatory granuloma or transplanted sarcoma. Intravenously administered labeled alpha 1-antitrypsin accumulated remarkably in extravascular spaces of the granuloma or sarcoma tissues. Among the normal organs examined, the lung preferentially incorporated alpha 1-antitrypsin. Furthermore, most of the alpha 1-antitrypsin accumulated in these three tissues remained in a TCA-precipitable form throughout the observation period. Since alpha 1-antitrypsin is incorporated in large amounts into inflammatory or tumor tissues, it could play a role in regulation of inflammatory processes or in controlling the proliferation of a tumor. The studies on TCA fractionation also suggest that liver and kidney provide the main sites for degradation of this protein. Although the accumulation of labeled albumin in granuloma and sarcoma was less marked, it showed essentially the same distribution and degradation pattern as alpha 1-antitrypsin in both morbid states.     

A comparative evaluation of the use of immunoturbidimetry in clinical practice]

A variant of immunoturbidimetry available for any clinical diagnostic laboratory has been developed. The data of measurements of immunoglobulins, albumin, alpha 2-macroglobulin and some other proteins of pregnancy in the blood serum and other biologic fluids, carried out by immunoturbidimetry, coincided with the findings of radial immunodiffusion and rocket immunoelectrophoresis, but immunoturbidimetry is more rapid and simple.     

Replacement therapy of alpha 1-antitrypsin deficiency. Reversal of protease-antiprotease imbalance within the alveolar structures of PiZ subjects.

The emphysema associated with the inherited serum deficiency of alpha 1-antitrypsin appears to result from an imbalance between neutrophil elastase and its major inhibitor within the alveolar structures. In the present study we assessed the feasibility of reversing this biochemical defect within the lung via parenteral replacement therapy with an alpha 1-antitrypsin concentrate of normal plasma. A 20--40% polyethylene glycol precipitate of pooled human donor plasma was used to obtain an enriched alpha 1-antitrypsin concentrate devoid of hepatitis B antigen and immunoglobulins. Using this material, five individuals with severe serum alpha 1-antitrypsin deficiency (PiZ phenotype) and advanced emphysema received 4 g of alpha 1-antitrypsin intravenously at weekly intervals for four doses. During this period of weekly replacement therapy alpha 1-antitrypsin serum levels were maintained at greater than or equal to 70 mg/dl, the level likely required for effective antielastase protection of the lung. In addition, assessment of lower respiratory tract antielastase activity by bronchoalveolar lavage demonstrated that parenteral replacement of alpha 1-antitrypsin resulted in establishment of effective antielastase activity within the alveolar structures. There were no untoward side effects consequent to this approach to the replacement therapy of alpha 1-antitrypsin. These results demonstrate that the parenteral replacement of alpha 1-antitrypsin provides a means of obtaining elastase-antielastase balance within the lung of individuals with this serum protease inhibitor deficiency.