Commutability of serum protein values: persisting bias among manufacturers using values assigned from the certified reference material 470 (CRM 470) in the United States.

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.

High sensitivity immunoassays for C-reactive protein: promises and pitfalls.

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.

Effect of a new international reference preparation for proteins in human serum (certified reference material 470) on results of the College of American Pathologists Surveys for plasma proteins.

OBJECTIVE: To examine the impact of a new international reference preparation for serum proteins on the among-manufacturer variance in the College of American Pathologists Surveys. DATA SOURCE: The results of the Surveys for the years 1993-1998, supplied by the College of American Pathologists. DATA EXTRACTION AND SYNTHESIS: Mean values for manufacturers' assays were compared for each protein in the quality control samples. Results were separated by the reported reference material from which values for calibrators had been transferred. Standard statistical parameters (means, standard deviations, and coefficients of variation) were calculated from the reported means. CONCLUSIONS: Among-manufacturer coefficients of variation have dropped significantly for most serum proteins since the introduction of the new reference material. Possible reasons for continued differences are discussed.

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.

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 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.

Analytical evaluation of particle-enhanced immunonephelometric assays for C-reactive protein, serum amyloid A and mannose-binding protein in human serum.

Against a background of growing interest in more sensitive assays for quantifying various acute phase proteins, we evaluated the performance of recently developed tests for C-reactive protein (CRP), serum amyloid A (SAA) and mannose-binding protein (MBP) on the Behring nephelometer II (BNII). Sample results outside the calibration ranges of 3.5 to 220 mg/L for CRP, 3.3 to 215 mg/L for SAA and 0.09 to 5.6 mg/L for MBP were automatically re-measured at another dilution. The lower limits of detection were 0.01, 0.7 and 0.01 mg/L for CRP, SAA and MBP, respectively. The coefficients of variation (CV) for intra- (n > or = 20) and inter- (n > or = 15) assay precision were < 5.2% and < 8.5%, respectively, for the three proteins at concentrations representing low, normal and high. Linearity for each method was within 5% of the expected values throughout the calibration range. We observed no significant interference from bilirubin (up to 300 mg/L) or haemoglobin (up to 10 g/ L) for the three tests. Method comparison studies performed for CRP and SAA yielded the following results: y (CRP on BNII) = 0.75x (ELISA, Hemagen) -0.25 mg/L (r = 0.981, Sy/x = 2.1 mg/L; y (SAA on BNII) = 1.44x (ELISA, Hemagen) -9.9 mg/L (r = 0.972, Sy/x = 6.9 mg/L), where ELISA is enzyme-linked immunosorbent assay. Reference intervals established in 261 adult blood donors (aged 36.2 +/- 9.0 years) were found to be log-normal with 2.5th, 50th, and 97.5th centiles of < 0.17, 1.00 and 10.1 mg/L for CRP, < 0.84, 2.10 and 9.70 mg/L for SAA; and 0.30, 1.28 and 4.10 mg/L for MBP. We observed no relationship with CRP concentration and age; however, SAA levels increased with age while MBP levels decreased. The BNII provides a simple, rapid and sensitive system for measuring CRP, SAA and MBP in human serum.

Serum triglyceride: a possible risk factor for ruptured abdominal aortic aneurysm.

BACKGROUND: We aimed to determine the relationship between ruptured abdominal aortic aneurysm (AAA) and serum concentrations of lipids and apolipoproteins. METHODS: A cohort of 21 520 men, aged 35-64 years, was recruited from men attending the British United Provident Association (BUPA) clinic in London for a routine medical examination in 1975-1982. Smoking habits, weight, height and blood pressure were recorded at entry. Lipids and apolipoproteins were measured in stored serum samples from the 30 men who subsequently died of ruptured AAA and 150 matched controls. RESULTS: Triglyceride was strongly related to risk of ruptured AAA. In univariate analyses the risk in men on the 90th centile of the distribution relative to the risk in men on the 10th (RO10-90) was 12 (95% confidence interval [CI] : 3.8-37) for triglyceride, 5.5 (95% CI: 1.8-17) for apolipoprotein B (apoB) (the protein component of low density lipoprotein [LDL]), 0.15 (95% CI : 0.04-0.56) for apo A1 (the protein component of high density lipoprotein [HDL]), 3.7 (95% CI: 1.4-9.4) for body mass index and 3.0 (95% CI: 1.1-8.5) for systolic blood pressure. Lipoprotein (a) (Lp(a)) was not a significant risk factor (RO10-90 = 1.6, 95% CI: 0.6-3.0). In multivariate analysis triglyceride retained its strong association. CONCLUSION: Triglyceride appears to be a strong risk factor for ruptured AAA, although further studies are required to clarify this. If this and other associations are cause and effect, then changing the distribution of risk factors in the population (by many people stopping smoking and adopting a lower saturated fat diet and by lowering blood pressure) could achieve an important reduction in mortality from ruptured AAA.

New laboratory guidelines for serologic diagnosis of Lyme disease: evaluation of the two-test protocol.

Recent guidelines established by the Association of State and Territorial Public Health Laboratory Directors (ASTPHLD) and the U.S. Centers for Disease Control and Prevention (CDC) recommend the use of a two-test protocol for the serologic diagnosis of Lyme disease (LD). The two-test protocol relies on a sensitive screening test, which is followed by specific immunoglobulin M (IgM) and/or IgG immunoblotting (IB), depending on the date of disease onset, of all samples with equivocal and positive screening test results. We evaluated a commercially available IgM-IgG enzyme-linked immunosorbent assay (ELISA) and separate IB tests for IgM and IgG antibodies to Borrelia burgdorferi as candidate assays for the two-test protocol. Serum samples obtained from healthy controls (n = 29), from patients with diagnoses or laboratory findings associated with serologic cross-reactivity to LD (n = 24), and from patients with well-documented early- and late-stage LD provided by the CDC and the College of American Pathologists (n = 53) were examined to determine each assay's individual sensitivity and specificity. No false-positive results were detected among the healthy controls by either ELISA or IB, whereas four false-positive ELISA results were recorded within the cross-reactive group. None of these sera, however, were positive for either IgM or IgG reactivity according to IB band criteria. With regard to the patients with LD, we determined the sensitivity and specificity of the ELISA to be 96 and 100%, respectively, compared with the reference data provided for these specimens. When we compared our IB results with data from CDC, the assay sensitivity and specificity were 80 and 96.2%, respectively, for IgM and 81.8 and 95.8%, respectively, for IgG. Pursuant to this evaluation we assessed the suitability of the two-test protocol by performing a retrospective analysis using clinical history to define samples as positive or negative for LD. We determined clinical sensitivity and specificity for all study subjects (n = 112) to be 50 and 100%, respectively. A reduction in the clinical sensitivity of the two-test protocol was associated with a lack of antibody response or seroconversion in LD patients treated with antibiotics. We conclude that the CDC-ASTPHLD guidelines provide useful criteria for test performance and interpretation aimed at standardizing the serologic diagnosis of LD.

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.