Quantification by Ultrafiltration and Immunofixation Electrophoresis Testing for Monoclonal Serum Free Light Chains.

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.

Moving towards harmonized reporting of serum and urine protein electrophoresis.

During the last decade, surveys by questionnaire in Canada, Australia and New Zealand revealed wide variation in reporting practices by laboratories and individual practitioners in the interpretation of serum and urine protein electrophoresis (PE). Such variation has potential to adversely impact patient outcomes if report structure is inconsistent or if the messaging is incorrectly perceived by the receiving physician. Concerted efforts have been initiated to promote harmonization in the use of interpretative comments. The primary goal is to add value through clear communication with requesting physicians in the interest of quality patient care. Resistance to a harmonized approach largely reflects longstanding personal reporting habits and preferences but change can be more readily embraced if the new system is intuitive, easy to use and saves time in reporting.

The Cutoff Level for Urine Protein in Urine Immunofixation Electrophoresis.

BACKGROUND: Immunofixation electrophoresis (IFE) maintains its importance in diagnosing monoclonal gammopathies. In particular, urine IFE detects free light chains (FLC) in urine samples even at low concentrations and offers higher sensitivity compared to serum electrophoresis and serum IFE. The aim of the present study was to determine the place and significance of quantitative urinary protein measurement before IFE in interpreting the results of subsequent IFE and to determine the most appropriate protein concentrations for the appearance of bands. METHODS: The records of a total of 600 patients, who underwent screening for Bence Jones proteinuria using IFE on 24-hour urine, were retrospectively reviewed. Urine IFE was performed using Helena SAS-I and SAS-I devices. The total protein concentration in the urine was quantitatively determined by the Pyrogallol red method, and the urine albumin level was determined using the immunoturbidimetric method. These analyses were measured on an Olympus/Beckmann AU5800. RESULTS: The evaluation of IFE results revealed that 311 patients had normal results, 108 patients had monoclonal bands, five patients had biclonal bands, 28 had polyclonal bands, and 148 patients had various degrees of proteinuria. ROC curves were created in order to determine the most appropriate urinary protein and albumin levels to observe bands in IFE. Accordingly, urine baseline protein level (mg/dL) showed the highest AUC value (cutoff value: 19.4 mg/dL, sensitivity: 92%, specificity: 98.2%, AUC: 0.972). CONCLUSIONS: The present study showed that quantitative protein measurement before IFE eliminated the disadvantages associated with the IFE method and its interpretation.

Validation of a lipoprotein(a) particle concentration assay by quantitative lipoprotein immunofixation electrophoresis.

BACKGROUND: Low-density lipoprotein (LDL) particle (P, or molar) concentration has been shown to be a more sensitive marker of cardiovascular disease (CVD) risk than LDL cholesterol. Although elevated circulating lipoprotein(a) [Lp(a)] cholesterol and mass have been associated with CV risk, no practicable method exists to measure Lp(a)-P. We have developed a method of determining Lp(a)-P suitable for routine clinical use. METHODS: Lipoprotein immunofixation electrophoresis (Lipo-IFE) involves rigidly controlled electrophoretic separation of serum lipoproteins, probing with polyclonal apolipoprotein B antibodies, then visualization after staining with a nonspecific protein stain (Acid Violet). Lipo-IFE was compared to the Lp(a) mass assay for 1086 randomly selected patient samples, and for 254 samples stratified by apo(a) isoform size. RESULTS: The Lipo-IFE method was shown to be precise (CV <10% above the 50 nmol/l limit of quantitation) and linear across a 16-fold range. Lipo-IFE compared well with the mass-based Lp(a) assay (r=0.95), but was not affected by variations in apo(a) isoform size. With a throughput of 100 samples in 90 min, the assay is suitable for use in the clinical laboratory. CONCLUSIONS: The Lipo-IFE method will allow Lp(a)-P to be readily tested as a CVD risk factor in large-scale clinical trials.

Immunoelectrophoresis: a method with many faces.

Immunoelectrophoresis (IEP) was the first practical method that combined electrophoresis and -immunoprecipitation for identifying and characterizing proteins within complex mixtures. Over the years, IEP has been extended to include a variety of techniques and, as a general name, has been applied to virtually any technique that involves electrophoresis and antigen-antibody precipitin reaction for proteins. Because of the diversity in technical details of different IEP versions, the method described here deals only with classic IEP. Although it requires some manual expertise, IEP is versatile, relatively easy to customize, and economical with no need for expensive instrumentation. Further, it can discern identity, partial identity, and nonidentity of the proteins. Any low-viscosity body fluid specimen or, possibly, culture fluid and tissue extract could be tested with IEP if proper antibodies are available. With these attributes, classic IEP remains a valuable tool for clinical diagnostic testing, purity checking of biochemical and pharmaceutical products, and research.

[Serum protein immunofixation in malignant hemopathies other than multiple myeloma and Waldenstrom's macroglobulinemia]. / Immunofixation des protéines sériques dans les hémopathies malignes autres que le myélome multiple et la maladie de Waldenström.

The interest of serum protein immunofixation in myeloma and Waldenstr m's macroglobulinemia is widely known. It is not so well defined in other malignant hemopathies. The purpose of this study was to determine immunofixation abnormalities in malignant hemopathies other than multiple myeloma and Waldenstr m's macroglobulinemia. We selected serum immunofixations of 61 patients affected by malignant hemopathies and 53 patients affected by other pathologies susceptible to give immunofixation's alterations. We showed that the frequency of immunofixation abnormalities was more important in patients affected by malignant hemopathies than in patients affected by other pathologies (70.5% vs 35.8%). A high frequency of monoclonal immunoglobulins was found in patients with lymphoma (53.3%) and oligoclonal immunoglobulins in other hemopathies (48.2%). No significant difference of the frequency of the monoclonal immunoglobulin isotypes was found. In summary, this retrospective study demonstrates a high frequency of immunofixation abnormalities in malignant hemopathies other than multiple myeloma and Waldenstr m's macroglobulinemia and different immunofixation characteristics between lymphomas and other hemopathies.

Padronizaçäo da técnica de imunofixaçäo na detecçäo e identificaçäo de paraproteínas séricas / Standartization of immunofixation technique for detection and identification of seric paraproteins

O diagnóstico e seguimento das paraproteinemias requer a identificaçäo e tipagem de paraproteínas (PP). A imunoeletroforese (IEF) é o método comumente usado embora demorado e pouco sensível. A técnica de imunofixaçäo (IF) é superior por ser mais sensível, rápida e de fácil interpretaçäo, particularmente no reconhecimento de PP presentes em baixa concentraçäo no soro e/ou urina. Consiste de fase eletroforética, seguida de fixaçäo, quando o anti-soro é colocado sobre o gel, precipitando a proteína. Objetivo. Este estudo objetiva padronizar a técnica de IF e compará-la à IEF. Métodos. Foram estudados os soros de 28 pacientes, sendo 25 portadores de mieloma múltiplo e 3 com hipergamaglobulinemia policlonal, comparados com 6 indivíduos normais. Todos foram submetidos à eletroforese (EF) em gel de agarose, à IEF e à IF. Resultados. O principal problema na padronizaçäo da IF foi a determinaçäo da diluiçäo que estabelecesse proporçäo ideal entre antígeno e anticorpo. A concentraçäo sérica ideal da PP, neste estudo, variou de 28 a 35 g/dL. A PP foi detectada e caracterizada por ambas as técnicas em 21 (84 por cento) dos indivíduos e näo detectada por nenhuma delas em 2 (8 por cento). Em outros 2, somente a IF conseguiu identificar a PP. Näo houve banda monoclonal à EF e à IEF que näo fosse identificada pela IF. Conclusäo. Nossos resultados permitem concluir que a IF é mais sensível que a IEF e deve ser incorporada à rotina de diagnóstico

[Standardization of immunofixation technique for the detection and identification of serum paraproteins]. / Padronização da técnica de imunofixação na detecção e identificação de paraproteínas séricas.

Diagnosis and follow up of paraproteinaemias require identification and typing of paraproteins. Immunoelectrophoresis is the most commonly used method, though a lengthy one and with low sensitivity. Immunofixation is more sensitive, faster and of easier interpretation, specially when monoclonal proteins are present in low concentration in the serum and/or urine. Immunofixation includes two steps. The first is electrophoresis; the second is immunofixation of the separated antigen by use of antiserum. The latter step is accomplished by layering the antiserum over the agarose gel immediately after electrophoretic separation of the proteins resulting in antigen/antibody precipitation. PURPOSE--The objective of this study is to standardize the technique of immunofixation and compare it to immunoelectrophoresis. METHOD--The serum of 28 patients (25 with multiple myeloma and 3 with polyclonal hypergammaglobulinaemia) was analysed and compared to 6 normal subjects. All were submitted to electrophoresis on agarose gel, immunoelectrophoresis and immunofixation. RESULTS--Dilution of the serum to produce a concentration suitable for immunofixation is critical. In our study the correct paraprotein concentration was 28 to 35 g/dl. Both methods detected and identified the paraprotein in 21 (84%) of the samples and in 2 (8%) it was not detected at all. In two of the samples, only immunofixation was able to detect and identify the paraprotein. There was not any monoclonal band observed either through the electrophoresis or immunoelectrophoresis that was not detected by the immunofixation. CONCLUSION--These results show that immunofixation is more sensitive than immunoelectrophoresis and therefore should be incorporated into diagnosis routine.

Evaluation of sperm specific lactate dehydrogenase isoenzyme C4 (LDH C4)--application to semen detection in stains.

The usefulness of LDH C (or LDH X) as a semen-specific marker has been tested. Many different methods have been applied for identification using various electrophoretic methods such as agarose gel electrophoresis, IEF, rocket-immunoelectrophoresis with subsequent enzymatical, immunological and immunochemical detection methods. Further methods applied were dot blot analysis for LDH C and PCR analysis for X/Y-chromosome differentiation. In blood/semen mixtures the LDH C-bands were detectable in dilutions up to 50-fold after staining for total LDH activity. An appropriate extraction procedure was developed. Immunological tests using a polyclonal antibody against LDH C were found to be highly specific and highly sensitive. The immunological test gave positive results with azoospermic stains up to a few weeks and in normal sperm stains positive reactions were obtained even after 8 months storage. A comparison of different detection methods for semen showed that immunological detection methods for LDH C are, at least after longer time periods of storage, superior to microscopical detection. Swabs showed positive LDH C-reactions up to 48 h after intercourse. This reaction was closely correlated to PCR for male cells and to microscopical searching.

Antigenic cross-reactivity among isolates of group JK corynebacteria.

Using rocket and rocket-line immunoelectrophoresis and immunoblotting it was demonstrated that a group of antibiotic-resistant bacteria, including several authenticated Corynebacterium jeikeium strains, shared many cross-reacting antigens. Only weak cross-reactivity was demonstrated with representatives of three other skin corynebacteria, C. bovis, C. hofmanii and C. minutissimum or with non-coryneforms. Differences within C. jeikeium are sufficient to permit the use of immunoblotting as an epidemiological tool.

Evaluation of co-agglutination (COA), counter immunoelectrophoresis (CIE), culture and direct microscopic (Dm) examination of cere-brospinal fluid (CSF) for detection of meningitis caused by common bacterial pathogens.

Cerebrospinal fluid from 260 children clinically diagnosed as meningitis were examined by Dm, culture, COA and CIE test. Dm revealed the presence of bacteria in 41 (15.8%) whereas culture showed growth of organism in 52 (20%) cases. COA and CIE test were done for the detection of antigen of H. influenzae, S. pneumoniae and N. meningitidis. Among the 3 methods viz. culture, COA and CIE test which were used for the detection of the above three organisms COA detected the maximum numbers (23.5%). COA test could detect antigen in both culture positive and culture negative CSF samples. COA test detected 100% of pneumococcal, 88.5% of H. influenzae and 66.7% of N. meningitidis antigens from CSF. Diagnosis by CIE in detecting H. influenzae and N. meningitidis antigens is inferior to culture and COA, whereas in detecting pneumococcal antigens CIE is superior to culture. So COA is a valuable, cheap, rapid and sensitive method for the diagnosis of meningitis caused by the above three organisms and when used along with culture 100% of cases can be diagnosed.

Evaluation of commercial imported fire ant extracts by crossed immunoelectrophoresis and radioallergosorbent test.

Imported fire ant whole-body extracts (IFAWBE) from three major U.S. commercial suppliers and a reference IFAWBE and imported fire ant venom (IFAV) preparation were compared by crossed immunoelectrophoresis and RAST-inhibition testing. Results of crossed immunoelectrophoretic studies showed major differences in antigen content between commercial preparations. IFAWBE and IFAV RAST-inhibition testing showed that no commercial extract had equivalent inhibitory activity to the reference IFAWBE. Reference IFAWBE reached 50% inhibition of IFAWBE and IFAV RAST at 52 and 492 micrograms/ml, respectively. IFAV gave 50% inhibition of IFAWBE and IFAV RAST at 5.9 and 58 micrograms/ml, respectively. Of the commercial extracts, only those from one supplier were able to reach 50% inhibition of the reference IFAWBE RAST, and no commercial extract from any supplier was able to reach 50% inhibition of IFAV RAST. Analysis of inhibition slopes and intercepts confirmed that there were both qualitative and quantitative differences among the commercial extracts and the reference IFAWBE and IFAV. Our findings suggest possible reasons why some individuals fail to respond to testing or immunotherapy with IFAWBE and show the urgent need for standardization of allergenic extracts for diagnosis and treatment of imported fire ant sensitivity.

Electroimmunoassay of albumin in human serum: accuracy and long-term precision.

An improved procedure for the Laurell "rocket" technique is described. Samples were electrophoresed in an agarose gel containing anti-human albumin. The gel plates were processed, the peaks stained, and peak heights used to calculate albumin concentrations. Factors affecting precision were (a) adequate heating of agarose gel before antibody is added, (b) accurate leveling of the gel surface during plate formation, (c) applied voltage during sample application, and (d) avoidance of the "edge" effect on sample placement in the gel. Multi-plate long-term precision (CV) for the method was 6.2% at a mean albumin concentration of 13 g/L and 3.0% at 37 g/L. Analytical recovery of 8 and 11 g of albumin per liter was 99 to 100%. There was negligible interference from hemoglobin and dextran as well as several common substances that bind to albumin--bilirubin and salicylate. Because of its high accuracy and good long-term precision, the method is a possible candidate reference method for serum albumin.

Immunochemical measurement of serum prostatic acid phosphatase (PAP). Clinical evaluation of radioimmunoassay and counter immunoelectrophoresis.

Two radioimmunoassay procedures (RIA-1 and RIA-2) were evaluated for the quantitation of prostatic acid phosphatase in serum and compared with the enzymatic method and counter immunoelectrophoresis method for their specificity and sensitivity. Sera from 168 patients were analyzed and these included: normals, 27; untreated prostatic cancer patients Stage A, 2; Stage C, 3; Stage D, 17; cancer of prostate treated with different modalities, 42; sarcoma of prostate, 1; prostatitis, 3; nonprostatic carcinoma, 17; and benign prostatic hyperplasia (BPH), 56. RIA-1 procedure appeared more sensitive (82% sensitivity) and specific (94.5% specificity) than the RIA-2 procedure (68% sensitivity and 91.8% specificity), but the differences were not statistically significant. The enzymatic method was found to be least sensitive (63.6% sensitivity) but also the most specific (100% specificity). Only 69 of the specimens were analyzed by counter immunoelectrophoresis, which showed sensitivity of 87% and specificity of 51.4%. False positives were observed more often in patients with nonprostatic cancer and BPH. The variations in diagnostic specificity of immunologic assays suggest the need of characterization of each antibody specificity.

Standardization of measures prior to cluster analysis.

A common problem in cluster analysis is the determination of a scale-free measure of distance between individuals. This paper presents a procedure for scaling measurements using a reference individual as a standard of comparison. The procedure is particularly useful in classification of the results of laboratory procedures, where a reference standard is routinely produced. An example is the clustering of patterns that result from crossed antigen-antibody electrophoresis for determining the phenotype of the serum protein alpha 1-antitrypsin. The procedure appears to remove extraneous variability while retaining the information necessary for classification.

Increasing the predictive value positive of the precipitin test for the diagnosis of deep-seated candidiasis.

Three hundred fifty human sera were tested by double immunodiffusion, crossed-line electrophoresis, and crossed immuno-affinoelectrophoresis with a concanavalin A intermediate gel for precipitating antibodies to antigens present in cytoplasmic extracts of Candida albicans. Sera from 48 of 287 hospitalized patients at risk of invasive candidiasis contained precipitating antibodies to Candida antigens. Of these 48 sera, 27 had precipitating antibodies only to cell-wall antigens present in the cytoplasmic extract, and 21 sera had precipitating antibodies to both cytoplasmic and cell-wall antigens. The latter sera came from patients who were 2.5 times as likely to have deep-seated candidiasis as those patients with precipitins exclusively to cell-wall antigens. Sera from seven of 22 patients with vaginal candidiasis and 10 of 41 patients with other fungal infections had precipitating antibodies to C. albicans cell-wall antigens; only two of these sera also contained precipitating antibodies to the cytoplasmic antigens. Crossed immunoaffinoelectrophoresis with concanavalin A reduced the number of false-positive results and increased the predictive value positive of the precipitin test for deep-seated candidiasis from 31% to 71%.