Clinical Chemistry and Laboratory Medicine (CCLM)

Published by De Gruyter

Volume 39 Issue 11

Issue of Clinical Chemistry and Laboratory Medicine (CCLM)

Contents
Journal Overview

June 1, 2005

The Present State of Protein Analysis and Interpretation

John Whicher, Myron Johnson

Page range: 1017-1018

July 27, 2005

In Memoriam Carl-Bertil Laurell

Joyce Carlson, Jan-Olof Jeppsson

Page range: 1019-1019

June 1, 2005

From Paper Electrophoresis to Computer-supported Interpretation of Capillary Electrophoresis – Clinical Plasma Protein Analysis in Malmö, Sweden

Joyce Carlson

Page range: 1020-1024

Abstract

Protein analyses have been used in Malmö as a routine clinical diagnostic tool since 1953. Most serum samples are submitted for “protein profiles” including capillary zone electrophoresis and rate immune nephelometric quantification of nine proteins (five in urines), although analysis of single proteins may be requested. Standardization between laboratories in our region has been greatly improved by automation, CRM 470 calibration and external quality assurance. We are further extending standardization by developing computer supported interpretations using a program with improved user interface and graphical representation of electrophoretic curves superimposed upon a shaded reference interval. Programming is underway to provide complete automatic interpretation of these curves. Together, capillary electrophoresis (with access to mathematical analysis) and immunochemical quantifications allow a highly automated process accessible to further digital analysis and automated interpretation. Rapid, cost-effective and standardized analysis of serum protein profiles should improve the diagnostic evaluation of many categories of patients.

June 1, 2005

The Pavia Approach to Clinical Protein Analysis

Giampaolo Merlini, Sabrina Marciano, Chiara Gasparro, Irene Zorzoli, Tiziana Bosoni, Remigio Moratti

Page range: 1025-1028

Abstract

The protein chemistry laboratory of the Pavia University Hospital is specialized in the study of monoclonal components in body fluids. It is closely connected with the research laboratory devoted to the structural and functional study of pathogenic proteins and with the Clinical Chemistry Central Laboratory. The analyses are performed on specific medical request. The analytical approach is mainly based on analysis of patient's serum and urine by high-resolution agarose gel electrophoresis and immunofixation with the possible addition of the quantification of specific proteins. The interpretation of the protein pattern and the final reporting result from the integration of the laboratory data with the clinical information. This labor-intensive approach requires skills in the performance of protein analysis and in the interpretation/referral phase, as well as close communication with the attending physician.

June 1, 2005

How the Foundation for Blood Research (FBR) Has Managed Serum Protein Testing for New England Clinicians

Robert F. Ritchie

Page range: 1029-1034

Abstract

Serum protein analysis has been the primary focus of the laboratories at the Foundation for Blood Research (FBR) since 1965. Designed by clinicians to assist in the diagnosis and management of their patients, the Foundation's serum protein analyses became tools to answer questions that were difficult or impossible to answer at the bedside or by traditional chemistry tests. Research on the subject quickly led to services that required computer assistance. Measurement of individual proteins expanded as need dictated and finances allowed. Serum protein electrophoresis was added as a necessary test early in the process. Research, expansion of the number of test offered, and test volumes have demanded automation of both testing and interpretation. Testing now includes assays of 15 serum proteins, serum iron, and autoantibodies and is tailored to meet the needs of general practitioners, pediatricians, several internal medical specialties, and paramedical personnel. Samples rather than patients are sent to the laboratory and reports are returned by mail or electronic means. Physicians review all complex reports.

June 1, 2005

Evidence-Based Laboratory Interpretation System Built on a Large Collection of Case Records with Well-Defined Diagnoses

Kiyoshi Ichihara, Kazutaka Sato

Page range: 1035-1044

Abstract

A computer-based system for laboratory diagnosis was created to promote evidence-based practice of laboratory medicine. It runs on a database consisting of 1992 well-defined untreated cases from 38 common diagnostic categories. Clinical symptoms, signs, and severity were recorded together with laboratory test results both general and specific to the clinical diagnosis. The system has two modes in which to view the database: either single or multiple diagnostic categories at a time. In the single mode, it allows flexible filtering and quick subgrouping of cases within the diagnostic category by specifying parameters of interest. It is also capable of computing a similarity index of a case at hand to those in the database. The index is defined as a weighted sum of log-likelihood calculated dynamically from parameters chosen for the query. In the multi-mode, only the parameters commonly recorded in selected categories are retrieved from the database. The system offers a between-category comparison view of any parameter. The similarity indices can be also computed among categories to see how well a set of parameters differentiates a reference category from the others.

June 1, 2005

A Knowledge-Based System to Aid with the Clinical Interpretation of Complex Serum Protein Data

Robert F. Ritchie

Page range: 1045-1053

Abstract

In every area of science workers are finding increasing difficulty in managing the volume of available data. In medicine, the accelerating pace has the worrisome overtones of our failing to provide up-to-date care for our patients. In other information-intensive areas, we rely heavily on software that manages much of the complexity unseen. Patient care could benefit enormously from the incorporation of “knowledge-based” programs to aid with diagnosis and management of many disorders. This article describes such a system designed to organize complex data which can be viewed as a test cluster aimed at many disorders pertinent to serum proteins. This program performs complex tasks such as reference range adjustment, ICD-9 code assignment, and searching for diagnostic “signatures”, to generate clinically relevant text and simple graphics. The results have been remarkably accurate and produce repeatable results at the rate of ~10 cases per minute. The reluctance to embrace software assistance in laboratory medicine may have serious consequences in the short term and disastrous results within a decade. Expanding the limited algorithm described here to include more traditional chemistry testing could provide the very assistance that all in clinical care desire, a laboratory tool as powerful and adaptable as the traditional physical exam.

June 1, 2005

Markers of the Acute Phase Response in Cardiovascular Disease: An Update

John Whicher, Nader Rifai, Luigi M. Biasucci

Page range: 1054-1064

Abstract

There is now widespread agreement that inflammation is a key component in the progression of atheromatous lesions. Inflammatory cells are present at all stages in the development of the atheromatous plaque, and gene knockout experiments in mice show that atheroma is largely prevented in the absence of the normal inflammatory mediators. In humans reduction of inflammation accompanies successful treatment strategies for atheroma. An increasing number of studies suggest that the acute phase protein, C-reactive protein, provides increased prognostic information over and above existing markers of atheroma severity or progression in healthy individuals and in the acute coronary syndromes. Recent advances in our knowledge of the normal variability of C-reactive protein levels and in precise and sensitive measurements strengthen the arguments for adding this marker to the repertoire of the routine laboratory assessment of cardiovascular disease.

June 1, 2005

Protein Aggregation

Giampaolo Merlini, Vittorio Bellotti, Alessia Andreola, Giovanni Palladini, Laura Obici, Simona Casarini, Vittorio Perfetti

Page range: 1065-1075

Abstract

Protein aggregation occurs in vivo as a result of improper folding or misfolding. Diverse diseases arise from protein misfolding and are now grouped under the term “protein conformational diseases”, including most of the neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, the prion encephalopathies and Huntington's disease, as well as cystic fibrosis, sickle cell anemia and other less common conditions. The hallmark event in these diseases is a change in the secondary and/or tertiary structure of a normal, functional protein, leading to the formation of protein aggregates with various supramolecular organizations. In most cases the aggregates are organized in structurally well-defined fibrils forming amyloid deposits. The crucial feature of the amyloidogenic proteins is their structural instability induced either by mutations, post-translational modifications, or local conditions, such as pH, temperature, and co-solutes. The conformational change may promote the disease either by gain of a toxic activity or by the lack of biological function of the natively folded protein. As different molecular mechanisms are involved in the formation of the various forms of protein aggregates, the laboratory diagnostic approach remains frequently elusive.

June 1, 2005

Protein Standardization I: Protein Purification. Procedure for the Purification of Human Prealbumin, Orosomucoid and Transferrin as Primary Protein Preparations

Søren Blirup-Jensen

Page range: 1076-1089

Abstract

A procedure for the purification of human prealbumin, orosomucoid and transferrin as primary protein preparations has been developed. The procedure describes in detail the chemicals, the fractionation equipment and the purification of the three proteins from the same starting material (a 90-donor plasma pool). The fractionation steps involve methods like salting out, various anion and cation exchange chromatographies, preparative electrophoresis, and finally, size chromatography. Only mild and highly reproducible fractionation methods are used in order to obtain high recoveries. All of these provide the best guarantee that no serious subfractionation has taken place. At the same time, high recoveries are a necessity for the pure protein to be representative of the same protein in vivo . The following recoveries were obtained: prealbumin 55%, orosomucoid 70% and transferrin 85%. The pure proteins are produced as liquid calibrators (primary protein preparations) dissolved in one electrolyte (0.1 mol/l KCl) and stored in sealed glass ampoules at −80 °C. These three pure proteins were used as primary reference preparations in the certification of the international reference preparation CRM 470.

June 1, 2005

Protein Standardization II: Dry Mass Determination. Procedure for the Determination of the Dry Mass of a Pure Protein Preparation

Søren Blirup-Jensen

Page range: 1090-1097

Abstract

A procedure has been developed for the determination of the dry mass of a pure protein preparation dissolved in one electrolyte. The procedure not only renders the concentration (in g/l), but additionally gives the partial specific volume of the protein (in ml/g). The latter is an important parameter for the characterization of a specific protein. Furthermore, the importance of extensive dialysis against one electrolyte is discussed. For the drying of human serum proteins it is clearly shown that KCl is preferred to NaCl due to its stable temperature curve. By observing the parallel fluctuations in the weight of the empty vials during drying, an average correction factor is introduced, which greatly minimizes these changes. The assay principle is discussed from a mathematical as well as from a practical point of view. A detailed procedure is described and the final results for three primary pure proteins (prealbumin, orosomucoid and transferrin) are presented. Finally, important parameters such as the wavelength of maximum absorbance, the absorption coefficient and the specific refractive increment are discussed and values for the three proteins are presented. When these parameters have been established the future determination of concentration and the characterization of pure protein solutions are greatly facilitated. These procedures were important tools for ascribing mass values for prealbumin, orosomucoid and transferrin to the international reference preparation CRM 470.

June 1, 2005

Protein Standardization III: Method Optimization. Basic Principles for Quantitative Determination of Human Serum Proteins on Automated Instruments Based on Turbidimetry or Nephelometry

Søren Blirup-Jensen

Page range: 1098-1109

Abstract

Quantitative protein determinations in routine laboratories are today most often carried out using automated instruments. However, slight variations in the assay principle, in the programming of the instrument or in the reagents may lead to different results. This has led to the prerequisite of method optimization and standardization. The basic principles of turbidimetry and nephelometry are discussed. The different reading principles are illustrated and investigated. Various problems are identified and a suggestion is made for an integrated, fast and convenient test system for the determination of a number of different proteins on the same instrument. An optimized test system for turbidimetry and nephelometry should comprise high-quality antibodies, calibrators, controls, and buffers and a protocol with detailed parameter settings in order to program the instrument correctly. A good user program takes full advantage of the optimal reading principles for the different instruments. This implies - for all suitable instruments - sample preincubation followed by real sample blanking, which automatically corrects for initial turbidity in the sample. Likewise it is recommended to measure the reagent blank, which represents any turbidity caused by the antibody itself. By correcting all signals with these two blank values the best possible signal is obtained for the specific analyte. An optimized test system should preferably offer a wide measuring range combined with a wide security range, which for the user means few re-runs and maximum security against antigen excess. A non-linear calibration curve based on six standards is obtained using a suitable mathematical fitting model, which normally is part of the instrument software.

June 1, 2005

Protein Standardization IV: Value Transfer. Procedure for the Assignment of Serum Protein Values from a Reference Preparation to a Target Material

Søren Blirup-Jensen, A. Myron Johnson, Marianne Larsen

Page range: 1110-1122

Abstract

A new approach for the assignment of values to serum proteins in a target material using a reference preparation has been developed. The procedure describes the general as well as the practical principles involved in the value assignment (with examples). Two models have been developed: 1) The direct value transfer between serum matrices and 2) the indirect value transfer from a pure protein preparation to a serum protein material. The necessary mathematical equations are developed and explained. The data reduction and statistical evaluation are discussed. The practical procedure (the transfer protocol) is based on six dilutions of the reference preparation assayed together with six dilutions of the target material. In this way imprecision is reduced and the proportionality of the two materials ( i.e. the presence or absence of matrix effects) can be assessed directly by evaluating a single regression plot. If no matrix effects are found, the regression line will pass through zero with a slope equal to the ratio of the concentrations of the two materials. The transfer protocol is based on a multiple point value assignment obtained by several measurements a day repeated on several days, an important prerequisite being that all reconstitutions and dilutions are controlled by weighing.

June 1, 2005

Effect of Certified Reference Material 470 (CRM 470) on National Quality Assurance Programs for Serum Proteins in Europe

A. Myron Johnson, John T. Whicher

Page range: 1123-1128

Abstract

The introduction of the international reference material for serum proteins, CRM 470, has resulted in significant reduction of the among-laboratory variance for most proteins assayed in European national quality assurance programs. In general, the CVs have decreased by 5 to 65%. However, both among- and within-manufacturer variances in many cases remain unacceptably high. In addition, concentration-dependent differences in variance and bias are present for some proteins. Although some variance will persist, reducing variance and bias to levels required for the institution of universal reference ranges will necessitate more accurate transfer of values to calibrants and controls and improved calibration curve fitting by manufacturers, as well as better quality control within laboratories.

June 1, 2005

Commutability of Serum Protein Values: Persisting Bias among Manufacturers Using Values Assigned from the Certified Reference Material 470 (CRM 470) in the United States

Thomas B. Ledue, A. Myron Johnson

Page range: 1129-1133

Abstract

Significant among-manufacturer differences in values for serum proteins persist 7 years after the introduction of the international reference material (Certified Reference Material 470; Reference Preparation for Proteins in Human Serum). In some cases, such as transthyretin and C4, the biases actually continue to increase. Further efforts at standardization are needed in order to improve commutability of results among laboratories and are essential if universal reference intervals are to be developed.

June 1, 2005

The Existing Interim Consensus Reference Ranges and the Future Approach

Francesco Dati, A. Myron Johnson, John T. Whicher

Page range: 1134-1136

Abstract

The release of the reference material for serum proteins, CRM 470/RPPHS, in 1993, has given rise to a great improvement in the between-laboratory variability of serum protein measurements worldwide. However, conversion to the new reference material has resulted in significant changes in reference values for some proteins. The establishment of new reference ranges is currently in progress; in the interim, several professional societies and diagnostic companies have agreed to use consensus reference ranges based on studies that were already undertaken.

June 1, 2005

Using Multiples of the Median to Normalize Serum Protein Measurements

Glenn E. Palomaki, Louis M. Neveux

Page range: 1137-1145

Abstract

Quantification of serum proteins is useful in the diagnosis and clinical management of many disorders. With the introduction of automated analyzers and standardized reference materials, one of the last barriers to more widespread utilization of these measurements is the lack of availability of reliable and transferable age- and gender-specific reference ranges. One normalization method that deserves consideration is converting values to multiples of the median (MoM) for age and gender. When two analytic methods agree, or differ only by a proportional amount, conversion to MoM can be used to simplify the clinical interpretation of serum protein results. As a test of this method, assay results for IgG, transferrin, and albumin from three Swedish hospitals were normalized using published reference ranges from the United States. All assays were standardized to CRM 470. IgG results were in agreement in mass units, and transferrin measurements were proportionally different. However, there were important, non-proportional differences in albumin measurements. After converting IgG and transferrin measurements to MoM, published reference ranges were appropriate for the Swedish Hospitals.

June 1, 2005

Strategy for Determining Racial and Environmental Similarities and Differences for Plasma Proteins

Per Hyltoft Petersen, John T. Whicher, A. Myron Johnson, Yoshi Itoh, Anders B. Carlström

Page range: 1146-1153

Abstract

The aim of this protocol is to establish a common basis for the production of reference values and well-defined and documented reference intervals for plasma proteins, based on common standardization, using the IFCC/BCR/CAP Certified Reference Material CRM 470. The strategy is to search for racial and environmental/geographical similarities and sources of differences in order to describe the main causes for variability among smaller or larger groups in selected societies and to estimate the sizes of differences for the different proteins according to the investigated sources. For this purpose, groups of reference individuals are selected according to race and geographical/environmental location, e.g. African Americans and Caucasians from the US. The reference individuals are groups of approximately 160 healthy male blood donors, 20 to 60 years of age. Rule-out criteria are positivity for HIV, hepatitis B and C antibodies and blood hemoglobin below the lower reference limit. Exclusion in relation to different C-reactive protein (CRP) levels will be investigated. Coagulation, storage conditions, transport, and the procedure for thawing are specified. The laboratories undertaking the measurements must have adequate analytical performance, and calibration and quality of performance are defined and documented, together with recommended control materials and procedures. Statistical models for describing distributions and for comparing groups are described. It is recommended that the data be presented as reference limits with 90% confidence intervals of those limits.

June 1, 2005

Standardization of Immunoassay for CRM-Related Proteins in Japan: From Evaluating CRM 470 to Setting Reference Intervals

Yoshihisa Itoh, Kiyoshi Ichihara

Page range: 1154-1161

Abstract

The Japan National Institute of Health (JNIH), in close collaboration with academic societies, commercial companies, and the Japan Society of Medical Technologists, has led in the attempt to standardize plasma protein assays since the mid 1980s. Under a framework of global standardization, they used WHO primary reference materials to reduce discrepancies in values reported for proteins assayed using different systems, thus laying the foundations for a protein immunoassay standardization system in Japan. With the introduction of CRM 470 in 1993, the Japanese Committee for Clinical Laboratory Standards (JCCLS) has taken the initiative in promoting the use of the new material and bringing about the re-evaluation of all systems of quality assurance in clinical laboratories. This eventually led to the establishment of reference intervals in Japanese populations of children and adults after preparation of assigned calibrators from CRM 470 for each assay system. Here we review the history of a series of projects carried out in Japan and describe several remaining problems, through which we will attempt to evaluate the potential value of protein immunoassay standardization.

June 1, 2005

Soluble Transferrin Receptor (sTfR): Biological Variations and Reference Limits

Ghiath Raya, Joseph Henny, Josiane Steinmetz, Bernard Herbeth, Gérard Siest

Page range: 1162-1168

Abstract

The aim of this study was to establish soluble serum transferrin receptor (sTfR) reference limits. sTfR was measured in 885 healthy subjects from 3 to 91 years old (433 men, 409 women), without hematological abnormalities, using an immunonephelometric assay. The sTfR median concentrations in our population decreased gradually from the group aged 3–10 years to the group aged 21–40 years, then there were no changes in the older groups except for the females >60 years of age. The interindividual variability ranged from 12.6% to 30.3% among different age groups, and the analytical variability was 5%. Biological factors and other factors associated with sTfR concentration variation were examined and accounted for 35% of the sTfR variability in men aged 20 years or less, and 18% in those older than 20 years. Also, they accounted for 45% of the variability in women aged 20 years or less and 14% in those older than 20 years. The main factors statistically associated with sTfR concentration in males were ferritin, orosomucoid, hemoglobin, and tobacco in all age groups and only mean corpuscular volume (MCV) in males less than 20 years old. In the females the main factors were age, orosomucoid, and hemoglobin in all age groups, MCV and tobacco in females less than 20 years old, and ferritin and physical activity in females more than 20 years old. These factors were used to define the exclusion and partition criteria for obtaining the reference samples. Medians for reference values were: 1.60 mg/l in the 3–10-year old group (males and females); 1.42 mg/l in males between 11 and 20 years of age, and 1.33 mg/l in females of the same age. In the other age groups, the median of the reference values was 1.16 mg/l, except in females over 60 years old, for whom it was 1.26 mg/l.

June 1, 2005

High Sensitivity C-Reactive Protein (CRP) Reference Intervals in the Elderly

Bernard Herbeth, Gérard Siest, Joseph Henny

Page range: 1169-1170

Abstract

High sensitivity C-reactive protein is a useful marker in clinical practice; however, reference intervals are not available for all age groups. Therefore, the aim of this study was to determine reference intervals for elderly people. The non-parametric reference limits were calculated for the two genders, subdivided into two age classes (50–64 and 65–91 years). In our selected sample population, we did not observe significant gender differences.

June 1, 2005

High Sensitivity Immunoassays for C-Reactive Protein: Promises and Pitfalls

Thomas B. Ledue, Nader Rifai

Page range: 1171-1176

Abstract

C-reactive protein (CRP) has historically been measured in the clinical laboratory for the detection and monitoring of occult infection and inflammation, using immunoturbidimetric or immunonephelometric techniques. The recent commercial availability of automated high-sensitivity assays has enabled investigators to measure CRP at levels previously unattainable on a routine basis and to explore its clinical utility in apparently healthy individuals. CRP concentrations increased above the individuals' baselines but still within the normal reference intervals have been observed in association with increasing age, obesity, and smoking and in individuals with chronic infections such as Chlamydia pneumoniae and Helicobacter pylori . More importantly, however, data from prospective studies have shown CRP to be a strong and independent predictor of future coronary events in subjects with and without coronary heart disease. An algorithm for risk assessment of coronary risk employing both CRP and lipid concentrations has recently been proposed. However, in order for this approach to be incorporated into clinical practice, agreement among the various CRP methods must be achieved. Of critical importance to this process is a basic understanding of issues affecting assay performance. Factors such as assay precision, sensitivity, matrix effects, calibration, and standardization need to be addressed adequately by the in vitro diagnostic industry and the clinical laboratory.

July 27, 2005

Meetings and Awards

Page range: 1177-1180

About this journal

Objective
Clinical Chemistry and Laboratory Medicine (CCLM) publishes articles on novel teaching and training methods applicable to laboratory medicine. It is focused on basic and applied research and cutting-edge clinical laboratory medicine. CCLM is one of the leading journals in the field, with an impact factor over 8. All contributions submitted for publication in CCLM are single-blind peer reviewed by at least two experts in the field. CCLM is led by a multi-institutional editorial board. It is issued monthly, both in print and electronically. Letters to the Editor and Congress Abstracts are published online only.

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Topics

clinical biochemistry
clinical genomics and molecular biology
clinical haematology and coagulation
clinical immunology and autoimmunity
clinical microbiology
drug monitoring and analysis
evaluation of diagnostic biomarkers
disease-oriented topics (cardiovascular disease, cancer diagnostics, diabetes)
new reagents, instrumentation and technologies
new methodologies
reference materials and methods
reference values and decision limits
quality and safety in laboratory medicine
translational laboratory medicine
clinical metrology

Article formats
Research Articles, Reviews, Mini Reviews and Opinion Papers on all aspects of clinical chemistry and laboratory medicine, Guidelines and Recommendations, Point/Counterpoint Articles, Letters to the Editor, Editorials

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