How the Foundation for Blood Research (FBR) has managed serum protein testing for New England clinicians.

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.

A knowledge-based system to aid with the clinical interpretation of complex serum protein data.

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 approximately 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.

Reference distributions for the positive acute phase proteins, alpha1-acid glycoprotein (orosomucoid), alpha1-antitrypsin, and haptoglobin: a comparison of a large cohort to the world's literature.

Limiting bedside use of positive acute phase protein measurements (alpha1-acid glycoprotein (orosomucoid), alpha1-antitrypsin, and haptoglobin) has been the lack of satisfactory methods for quantifying serum levels and a credible reference material. Great strides have been made in the last few years. The remaining barrier to more relevant and cost-effective use of serum protein data for diagnosis and prognosis is the availability of reliable reference intervals from birth to old age for both males and females. Sixty publications reporting reference intervals have been identified which meet the criteria used in our prior two studies, and these have been analyzed statistically. Previous small studies of these individual proteins agree on average, over their constrained age ranges, with our life-long reference ranges. This meta-analysis provides support for our reference ranges and places them in the perspective of previous publications.

Reference distributions for the positive acute phase serum proteins, alpha1-acid glycoprotein (orosomucoid), alpha1-antitrypsin, and haptoglobin: a practical, simple, and clinically relevant approach in a large cohort.

Most clinical conditions are accompanied by corresponding changes in serum levels of some, if not all, of the acute phase proteins. While conditions that affect the acute phase proteins are usually inflammatory in nature, non-inflammatory conditions also can cause changes (e.g., malnutrition, some malignancies without secondary inflammation, and genetic polymorphism). Only after the confounding effects of non-inflammatory conditions are taken into account can these measurements be used to detect and stage the inflammatory process and to evaluate the impact of treatment. In this third article in a series, reference ranges for serum levels for three of the acute phase proteins that increase during inflammation are examined: alpha1-acid glycoprotein (orosomucoid), alpha-antitrypsin, and haptoglobin. The study is based on a cohort of 55,199 Caucasian individuals from northern New England, tested in our laboratory between 1994 and 1999. Measurements were standardized against CRM 470 (RPPHS) and analyzed using a previously described statistical approach. Individuals with unequivocal laboratory evidence of inflammation (C-reactive protein of 10 mg/l or higher) were excluded. Levels of a,-acid glycoprotein changed little during life and between the sexes. Levels of alpha1-antitrypsin varied somewhat by age, rising slightly beyond age 55; males followed a pattern similar to that for females. For this protein, it was necessary to apply two equations to describe the lower levels associated with certain phenotypes. Haptoglobin levels fell significantly during the first decade of life for both males and females and climbed thereafter. Males and females displayed a similar pattern. When values were expressed as multiples of the age- and gender-specific median levels, the resulting distributions fitted a log-Gaussian distribution well over a broad range. When patient data are normalized in this manner, the distribution parameters can be used to assign a centile corresponding to an individual's measurement, thus simplifying interpretation.

Effect of low-density lipoprotein (LDL) antigen source on an enzyme-linked immunosorbent assay for autoantibodies against oxidized LDL.

We examined the effect of antigen source on an enzyme-linked immunosorbent assay (ELISA) for autoantibodies against oxidized low-density lipoprotein (LDL). Serum samples from 20 subjects with systemic lupus erythematosus (SLE) and from 20 controls were assayed for immunoglobulin G (IgG) and immunoglobulin M (IgM) autoantibodies against oxidized LDL, using either a pooled or individual (n = 3) LDL preparation as antigen. For IgG autoantibodies against oxidized LDL there was a relationship (r approximately 0.5, P < 0.01) between data obtained using individual versus pooled antigen preparations. Bias plots demonstrated consistent inverse, concentration-dependent relationships (r approximately -0.6, P < 0.001). The difference in IgG autoantibodies against oxidized LDL levels between SLE patients and controls was underestimated (39-58%) when assays used individual rather than pooled LDL antigen. For IgM autoantibodies against oxidized LDL the direct relationships were stronger (r approximately 0.8, P < 0.001) and the concentration-dependent relationships weaker (r approximately -0.3, significance variable) than for IgG autoantibodies against oxidized LDL. Variations between LDL preparations suggested that a pooled antigen would give a more stable assay. Thus, LDL antigen source is important in assays for both IgG and IgM autoantibodies against oxidized LDL.

The distribution of antinuclear antibody titers in "normal" children and adults.

OBJECTIVE: To characterize the antinuclear antibody (ANA) titer distributions and patterns in normal subjects, segregated by age and sex. METHODS: Sera were obtained from 183 blood donors (130 females, 53 males) aged 20-63 years, from 200 schoolchildren (100 females, 100 males) aged 10-19 years, and from 237 children (102 females, 135 males) aged 3 months to 9 years whose sera were received for unrelated clinical testing. ANA was assayed by indirect immunofluorescence using HEp-2 cells as substrate. RESULTS: In adults, ANA titers were slightly higher in females than in males (p=0.053); there was no sex effect in subjects aged <20 years. ANA titer increased significantly with age only among females (p<0.01). Homogeneous staining was associated with lower titers than speckled or nucleolar staining (p=0.058), at least in part because of antigen density in the test substrate itself. The frequency of cytoskeletal staining decreased (p<0.01) with age, while that of nucleolar staining increased (p<0.01). CONCLUSION: Reference ranges for ANA vary by age, sex, and immunofluorescence pattern. Therefore, all these variables must be considered in the interpretation of ANA results.

Reference distributions for the negative acute-phase serum proteins, albumin, transferrin and transthyretin: a practical, simple and clinically relevant approach in a large cohort.

Inflammation is associated with diverse clinical conditions accompanied by characteristic changes in serum levels of the acute-phase proteins that can be used to stage the inflammatory process and evaluate the impact of treatment. Some acute-phase proteins increase during inflammation, while others, such as albumin, transferrin, and transthyretin, decrease. The current study reports reference ranges for serum levels of albumin, transferrin, and transthyretin based on a cohort of over 124,000 Caucasian individuals from northern New England, tested in our laboratory between 1986 and 1998. Measurements were standardized against CRM 470 (RPPHS) and analyzed using a previously validated statistical approach. Individuals with laboratory evidence of inflammation (C-reactive protein of 10 mg/L or higher) were excluded. The levels of all three analytes varied by age, generally rising until the second or third decade of life and then decreasing thereafter. Albumin and transthyretin levels were higher during midlife among males as compared to females; the maximum being at 25 years for albumin (5%) and 35 years for transthyretin (16%). In contrast, above the age of 10 years, transferrin levels were increasingly higher among females (7% at 20 years). When values were expressed as multiples of the age- and gender-specific median levels, the resulting distributions fitted a log-Gaussian distribution. When patient data are normalized in this manner, the distribution parameters can be used to assign a corresponding centile to an individual's measurement simplifying interpretation. The ultimate interpretation of an individual's measurement relies upon the clinical setting.

Reference distributions for the negative acute-phase proteins, albumin, transferrin, and transthyretin: a comparison of a large cohort to the world's literature.

Clinical interpretation of the acute-phase proteins--albumin, transferrin, and transthyretin--has been hampered by the lack of accurate and precise methods for quantifying the levels and a stable and respected reference material. Now that these issues have been addressed, the community is faced with the need for credible age- and gender-specific reference values. The number of publications that address this issue, even for an analyte as familiar as albumin, is small and, in most cases, such publications lack the relevant data that would allow a combined experience to be created. We have identified 40 studies that meet our criteria: a description of the study participants' health status, of the statistical methodology, and of the laboratory technique and/or reference material used. Few of these studies reported values stratified by gender. A summary of the published median levels by age is presented for the three analytes, along with our own age- and gender-specific medians based on a large cohort. Ten of the studies presented a 95 percent reference range, in close agreement with ours where selection was based upon reported diagnosis rather than upon determination of individual health status. This meta-analysis provides support for the reliability of our recently published methodology and reference data for the clinical interpretation of individual albumin, transferrin, and transthyretin values. As with most laboratory measurements, clinical interpretation requires that other laboratory and clinical factors be considered.

Reference distributions for immunoglobulins A, G, and M: a practical, simple, and clinically relevant approach in a large cohort.

Serum immunoglobulins are measured millions of times each year, yet clinical interpretations remain hampered by inadequate age- and gender-specific reference limits. In order to provide more reliable and comprehensive reference distributions for IgA, IgG, and IgM measurements, we analyzed automated immunoassay values from 115,017 serum samples from northern New England patients (99% Caucasian) who were tested in our laboratory between 1986 and 1995. Measurements were standardized to reference material, CRM 470 (RPPHS). A simple, practical, and clinically relevant approach was used to determine reference distributions for the immunoglobulins over a wide range of ages for males and females. Levels of IgA and IgM varied considerably by age, and by gender for IgM. For each of the analytes, the observed 5th and 95th centiles were symmetric about the median and approximately constant over the entire age range. When immunoglobulin reference values are expressed as multiples of the age- and gender-specific regressed medians, the resulting distributions fit a log-Gaussian distribution well. This finding enables interpretation of serum immunoglobulin measurements using a common unit (multiples of the median) that is independent of age or gender. Insights gained from this study can help improve and simplify the interpretation of immunoglobulin measurements.

Reference distributions for immunoglobulins A, G, and M: a comparison of a large cohort to the world's literature.

Clinical interpretation of immunoglobulin measurements requires accurate and precise assays and widely accepted reference preparations, as well as reliable age- and gender-specific reference ranges. This last requirement, the topic of numerous publications, has not been addressed adequately. By a combination of computerized and hand searching of the literature from 1961 to 1997, we identified 109 publications presenting IgA, IgG, and/or IgM reference data in healthy individuals. After eliminating studies that lacked appropriate clinical, statistical, or reference material information, data from the 17 acceptable studies were converted to a common reference material, CRM 470/RPPHS. When median levels from our recently published large cohort study are superimposed on these published medians, they fall within the ranges of reported medians. The widths of published 95 percentile reference ranges (where each individual's health was verified) were also found to agree closely with the reference range widths found in our data (where inclusion was based on the reported diagnosis). The current combined study of narrowly applied reference ranges validates our recently published age- and gender-specific reference data for immunoglobulinsA, G, and M. Those new data can now be considered as a source of reliable reference ranges to be used by laboratories when interpreting immunoglobulin measurements.

Expert systems for the interpretation of serum proteins.

The ready availability of inexpensive desk-top computers with enormous disk storage has made the practicality of computer assisted medical interpretive software a reality. There seems little question that these programs could be of enormous help to physicians. However, there are daunting problems to their creation, including the lack of standards for clinical diagnostic precision or accuracy and paucity of helpful literature. As a result, the final products may be quite different. Little effort has been devoted in the laboratory to produce programs which could have great benefit in bridging the gap between laboratorians and clinicians. In a few circumscribed areas where the interpretation of laboratory measurements have been well studied in relation to patient demographics and to the final outcome, the impact has been enormous. The prime example is prenatal diagnosis of neural tube defects, and certain chromosomal and developmental abnormalities. Viewed as an obstacle by most people able and willing to attempt to create such programs is the omnipresence of necessary regulation. A brief overview of the general structure of a program to assist with the interpretation of serum proteins is given as a model in the perspective of current knowledge and state of the relevant literature.

Consensus of a group of professional societies and diagnostic companies on guidelines for interim reference ranges for 14 proteins in serum based on the standardization against the IFCC/BCR/CAP Reference Material (CRM 470). International Federation of Clinical Chemistry. Community Bureau of Reference of the Commission of the European Communities. College of American Pathologists.

The release in 1993 of a new reference material for serum proteins, CRM 470/RPPHS 5 has given rise to a great improvement in the between-laboratory variability of serum protein measurements worldwide. Conversion to the new reference material results in significant changes in reference values for some proteins. The establishment of new reference ranges will take a considerable time, and in the interim several professional societies and diagnostic companies have agreed to use consensus reference ranges based on studies already undertaken.

Anti-oxidized LDL antibodies and antiphospholipid antibodies in healthy subjects: relationship with lipoprotein- and oxidation-related analytes.

IgG autoantibodies against malondialdehyde-modified LDL (alpha oxLDL), antiphospholipid antibodies (APA) and oxidation- and lipoprotein-related analytes were assayed in sera from healthy subjects (51 males, 115 females, aged 22-63 years). alpha OxLDL levels were associated (P < 0.03) with IgG alpha cardiolipin (r = 0.18), IgM alpha cardiolipin (r = 0.17) and IgM alpha phosphatidyl-serine (r = 0.16) but not with age, cholesterol, triglyceride, apolipoproteins B and AI, lipoprotein(a), lipid peroxides, ceruloplasmin, copper, ferritin, transferrin or iron. APA levels were inversely associated with levels of both oxidation- and lipoprotein-related analytes. Ferritin (3.5%) and alpha oxLDL (1.4%) contributed independently to variation in IgG alpha cardiolipin levels, and apo B (2%) to variation in IgM alpha cardiolipin levels. These associations are small, indicating that there are no major biological associations between the measured variables. The lack of association between alpha oxLDL and lipoprotein- or oxidation-related analytes suggests that the relevant antigen is not in serum.

Oxidation-related analytes and lipid and lipoprotein concentrations in healthy subjects.

The relations between oxidation-related analytes and lipoprotein risk factors for coronary heart disease are poorly understood. To address this issue, ceruloplasmin, copper, iron, ferritin, cotinine, lipid peroxides, cholesterol, triglyceride, apoB, apoA-I, and lipoprotein(a) levels were measured in sera from apparently healthy subjects (51 men and 115 women). Pairwise comparisons revealed strong positive associations (P < .001) of copper and ceruloplasmin with lipid peroxides, total cholesterol, triglycerides and apoB, of transferrin with apoA-I and cholesterol, and of ferritin with triglycerides. Serum levels of oxidation-related analytes did not differ between smokers and nonsmokers. In multivariate analysis, serum copper was the major independent determinant of serum lipid peroxide level, accounting for 15% of the variability in concentration (ferritin accounted for 1.6%). Copper and ceruloplasmin accounted for 20.5% of the variation in triglyceride levels; triglycerides and apoB accounted for 12% of the variability in ferritin levels; apoB and apoA-I accounted for 9% of the variability in transferrin levels. The data suggest that serum copper contributes to lipid peroxidation in vivo. There are significant associations between lipoprotein and transition metal-related analytes, and further work is needed to elucidate the physiological basis for these relations.

ELISA of IgG antibody to oxidized low-density lipoprotein: effects of blocking buffer and method of data expression.

We describe an ELISA for serum IgG antibodies against malondialdehyde-modified low-density lipoprotein (mLDL). Optimal antigen concentration, serum dilution, and dilution of enzyme-conjugated second antibody were 25 mg/L, 1:250, and 1:5000, respectively, when 5 g/L human serum albumin was used for blocking. When data were expressed as mLDL/LDL (the ratio of IgG binding to mLDL vs LDL), within-run and between-run CVs were 7.0% and 8.9%, respectively. Antibody concentrations expressed as mLDL/LDL or as mLDL-LDL (the difference between IgG binding to mLDL and to LDL) were higher in women with systemic lupus erythematosus (n = 20) than in controls (n = 20) (P < 0.001). With bovine serum albumin or Superblock blocking buffers, only the mLDL-LDL data were significant. Thus, the choice of blocking agent and the method of data expression should be carefully considered when assaying IgG antibodies against mLDL.

New international reference preparation for proteins in human serum (RPPHS).

Quality-control surveys in recent years, in various parts of the world, have shown poor between-laboratory agreement for measurements of plasma proteins. Despite the existence of international reference materials distributed by the World Health Organization, standards produced by diagnostics manufacturers and professional organizations differ significantly in their ascribed values. The reasons for this are complex but include poor availability of the primary materials, confusion about their use, and the fact that their turbidity on reconstitution precludes their use in modern optical immunoassays. This unfortunate situation led to an important initiative to produce sufficient quantities of a widely available, optically clear secondary reference material for plasma proteins that could be used worldwide by manufacturers, professional organizations, and laboratories. Here we present an overview on how the laboratory community, including manufacturers, clinical laboratories, professional societies, and regulators, has reached what we consider is a successful conclusion to a difficult problem.