State-of-the-art model for derivation of analytical performance specifications: how to define the highest level of analytical performance technically achievable.

To be accurate and equivalent among assays, laboratory results should be traceable to higher-order references and their quality should fulfill maximum allowable measurement uncertainty (MU) as defined to fit the intended clinical use. Accordingly, laboratory professionals should estimate and validate MU of performed tests using appropriate analytical performance specifications (APS). Current consensus supports the derivation of APS by using one of the three models established by the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Strategic Conference held in Milan in 2014. It is recognized that some models are better suited for certain measurands than for others and the attention should be primarily directed towards their biological and clinical characteristics. Among others, model 3 should reflect the state of the art of the measurements that can be defined as the best analytical performance that is technically achievable. Taking serum C-reactive protein and ferritin as examples, here we describe the theoretical premises and the experimental protocol to be used to derive APS for MU when a measurand is allocated to this model. Although the model lacks a direct relationship with clinical outcomes, useful information about the in vitro diagnostic medical device performance and the average quality of provided results may be obtained.

Establishment of reference intervals of clinical chemistry analytes for the adult population in Egypt.

BACKGROUND: This is the first Egyptian nationwide study for derivation of reference intervals (RIs) for 34 major chemistry analytes. It was conducted as a part of the global initiative by the IFCC Committee on Reference Intervals and Decision Limits (C-RIDL) for establishing country-specific RIs based on a harmonized protocol. METHODS: 691 apparently healthy volunteers aged ≥18 years were recruited from multiple regions in Egypt. Serum specimens were analyzed in two centers. The harmonization and standardization of test results were achieved by measuring value-assigned serum panel provided by C-RIDL. The RIs were calculated by parametric method. Sources of variation of reference values (RVs) were evaluated by multiple regression analysis. The need for partitioning by sex, age, and region was judged primarily by standard deviation ratio (SDR). RESULTS: Gender-specific RIs were required for six analytes including total bilirubin (TBil), aspartate and alanine aminotransferase (AST, ALT). Seven analytes required age-partitioning including glucose and low-density lipoprotein cholesterol (LDL-C). Regional differences were observed between northern and southern Egypt for direct bilirubin, glucose, and high-density-lipoprotein cholesterol (HDL-C) with all their RVs lower in southern Egypt. Compared with other collaborating countries, the features of Egyptian RVs were lower HDL-C and TBil and higher TG and C-reactive protein. In addition, BMI showed weak association with most of nutritional markers. These features were shared with two other Middle Eastern countries: Saudi Arabia and Turkey. CONCLUSION: The standardized RIs established by this study can be used as common Egyptian RI, except for a few analytes that showed regional differences. Despite high prevalence of obesity among Egyptians, their RVs of nutritional markers are less sensitive to increased BMI, compared to other collaborating countries.

Serum amyloid A in healthy subjects: assessment of reference value using ELISA method.

Serum amyloid A (SAA) is a family of acute-phase reactants. The rise of SAA concentration in blood circulation during the acute-phase response is a clinical marker of active inflammation. Despite its practical and analytical advantages, SAA measurement by enzyme-linked immunosorbent assay (ELISA) has been used mainly as a research tool rather than for the routine laboratory testing. This may be partly explained by the lack of robust reference data in the literature for the different commercially available immunoassays. Using the recommended procedures for the production of reference intervals published by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), we developed the SAA reference interval for a well-defined Italian healthy population and investigated the correlation among SAA and C-reactive protein (CRP), the commonly used acute-phase marker. After data normalization, the reference cutoff was calculated as 225 ng/ml. A good correlation between SAA and CRP was found (P < .05). No statistically significant differences was found between males and females when the means of SAA values were compared, suggesting that not gender-partitioned reference range is recommended for this analyte. This study allowed to define a widely accepted reference cutoff for the SAA detected by ELISA, responding to an unmet need of laboratory medicine.

Predictive values of inflammatory back pain, positive HLA B27 antigen and acute and chronic magnetic resonance changes in early diagnosis of Spondyloarthritis. A study of 133 patients.

OBJECTIVES: To analyse the predictive values of inflammatory back pain (IBP), positive HLA B27 antigen, increased C-reactive protein (CRP), Spondyloarthritis (SpA) features, familial history (FH), magnetic resonance sacroiliac joints (MRI-SIJ) imaging and its weight in early SpA diagnosis. METHODS: 133 patients with back pain, aged <50, duration of the pain <2 years were included. Data such as IBP, HLA B27, increased CRP, SpA features, FH, SIJ´s radiography and MRI were collected for each patient. STIR sequences were classified as strongly positive bone morrow oedema (SPBME ≥2), clearly present and easily recognisable as positive according to the ASAS criterion, weakly positive (WPBME ≥2), suggestive, but not easily recognisable and, clearly negative none of those features. T1-weighted sequences were assessed as positive/negative for erosion, fat metaplasia, backfill and sclerosis, if ≥1, for each lesion was present. MRI images were read by three blinded readers. RESULTS: The average age was 38.9 years. 47 (35.3%) patients received SpA diagnosis according to the clinical opinion. IBP was highly specific, 0.81 and sensitive, 0.83. HLA B27 was positive in a half of the SpA patients. SPBME ≥2 provided a great specificity, 0.94 and an acceptable sensitivity, 0.79. Erosion was significantly more frequent in SpA patients (72% vs 7%), specificity 0.93. The addition of erosion ≥1 to the WPBME ≥2 noticeably improved specificity, 0.98, although slightly decreased sensitivity, 0.64. Fat metaplasia and backfill were highly specific, but poorly sensitive. Factors forecasting positive diagnosis were IBP, followed by SpA features and increased CRP. CONCLUSIONS: At the onset, IBP might be a good marker for selecting patients with suspicion of SpA. The addition of erosion to the ASAS criterion might be helpful for early diagnosis, especially in patients with doubtful STIR imaging where BME is present but it is hard to determinate whether the ASAS "highly suggestive" criterion is met.

Accuracy of C-reactive protein, procalcitonin, serum amyloid A and neopterin for low-dose CT-scan confirmed pneumonia in elderly patients: A prospective cohort study.

OBJECTIVE: The diagnosis of pneumonia based on semiology and chest X-rays is frequently inaccurate, particularly in elderly patients. Older (C-reactive protein (CRP); procalcitonin (PCT)) or newer (Serum amyloid A (SAA); neopterin (NP)) biomarkers may increase the accuracy of pneumonia diagnosis, but data are scarce and conflicting. We assessed the accuracy of CRP, PCT, SAA, NP and the ratios CRP/NP and SAA/NP in a prospective observational cohort of elderly patients with suspected pneumonia. METHODS: We included consecutive patients more than 65 years old, with at least one respiratory symptom and one symptom or laboratory finding suggestive of infection, and a working diagnosis of pneumonia. Low-dose CT scan and comprehensive microbiological testing were done in all patients. The index tests, CRP, PCT, SAA and NP, were obtained within 24 hours. The reference diagnosis was assessed a posteriori by a panel of experts considering all available data, including patients' outcome. We used area under the curve (AUROC) and Youden index to assess the accuracy and obtain optimal cut-off of the index tests. RESULTS: 200 patients (median age 84 years) were included; 133 (67%) had pneumonia. AUROCs for the diagnosis of pneumonia was 0.64 (95% CI: 0.56-0.72) for CRP; 0.59 (95% CI: 0.51-0.68) for PCT; 0.60 (95% CI: 0.52-0.69) for SAA; 0.41 (95% CI: 0.32-0.49) for NP; 0.63 (95% CI: 0.55-0.71) for CRP/NP; and 0.61 (95% CI: 0.53-0.70) for SAA/NP. No cut-off resulted in satisfactory sensitivity or specificity. CONCLUSIONS: Accuracy of traditional (CRP, PCT) and newly proposed biomarkers (SAA, NP) and ratios of CRP/NP and SAA/NP was too low to help diagnosing pneumonia in the elderly. CRP had the highest AUROC. CLINICAL TRIAL REGISTRATION: NCT02467092.

The Prognostic Role of Glasgow Prognostic Score and C-reactive Protein to Albumin Ratio for Sarcoma: A System Review and Meta-Analysis.

BACKGROUNDS: Both pretreatment serum CRP (C-reactive protein) level and ALB (albumin) level have been found to be predictive of survival for multiple malignancies including sarcoma. Since both of the GPS (Glasgow prognostic score) and CAR (C-reactive protein to albumin ratio) are based on the combination of CRP and ALB, we conducted a meta-analysis to evaluate the prognostic role of these two parameters for sarcoma patients. METHODS: A detailed literature search was conducted in MEDLINE, Embase, and Cochrane Library for relevant research publications written in English. Patients' clinical characteristics, outcomes of overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS) were extracted. Pooled hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) were combined to evaluate the prognostic role of GPS or CAR. RESULTS: Twelve articles containing 2695 patients were identified as eligible studies. The results showed that an elevated GPS was significantly correlated with poor OS (HR = 2.42; 95% CI: 1.98-2.94; p < 0.001; fixed-effects model), DSS (HR = 2.28; 95% CI: 1.75-2.97; p < 0.001; fixed-effects model), DSS (HR = 2.28; 95% CI: 1.75-2.97; p < 0.001; fixed-effects model), DSS (HR = 2.28; 95% CI: 1.75-2.97; p < 0.001; fixed-effects model), DSS (HR = 2.28; 95% CI: 1.75-2.97; p < 0.001; fixed-effects model), DSS (HR = 2.28; 95% CI: 1.75-2.97. CONCLUSION: An elevated GPS is predictive of poor survival in patients with sarcomas and is promising to be used as a factor for risk stratification. A higher CAR value is also predictive of poor survival; however, the optimal CAR cut-off value is still to be determined.

Evolution of the slopes of ST2 and galectin-3 during marathon and ultratrail running compared to a control group.

Background Previous studies have suggested that exercising may induce cardiac damage. Galectin-3 (Gal-3) and soluble suppression of tumorigenicity 2 (ST2) are very interesting biomarkers for heart failure and myocardial fibrosis. We aimed to compare the kinetics of emerging fibrosis cardiac biomarkers as Gal-3 and ST-2 in endurance runners, and recreational runners before and after a running event represented by a marathon and an ultratrail event. Methods Blood samples were taken from 19 healthy non-elite marathon runners (42 km), 27 ultratour runners (67 km), and 14 recreational runners who represented the control group (10 km) just before the run (T0), just after (T1) and 3 h after (T2), in order to analyze Gal-3, ST2, hsTnT, NT-proBNP, CKMB and hsCRP. We compared the percentage of evolution and the slopes obtained from T0 to T1 (pT0T1) and from T1 to T2 (pT1T2), between the different groups of runners participating in three different races. Results Plasma cardiac biomarker concentrations increased significantly from baseline to immediately post-exercise and most of the time decreased over the subsequent 3-h period. For pT0T1 and pT1T2, the markers Gal-3 and ST2 showed a significant difference between types of run (p < 0.05 and p < 0.0001, respectively). During the recovery time, Gal-3 returned to the baseline values but not ST2 which continued to increase. Conclusions Gal-3 and ST2 are considered as a reflection of cardiac fibrosis and remodeling. The evolution of both was different, particularly after the recovery time. ST2 values exceeding cutoff values at any time.

Age and Sex Distributions of Age-Related Biomarker Values in Healthy Older Adults from the Long Life Family Study.

OBJECTIVES: To determine reference values for laboratory tests in individuals aged 85 and older. DESIGN: Cross-sectional cohort study. SETTING: International. PARTICIPANTS: Long Life Family Study (LLFS) participants (N~5,000, age: range 25-110, median 67, 45% male). MEASUREMENTS: Serum biomarkers were selected based on association with aging-related diseases and included complete blood count, lipids (triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, total cholesterol), 25-hydroxyvitamin D2 and D3, vitamin D epi-isomer, diabetes mellitus-related biomarkers (adiponectin, insulin, insulin-like growth factor 1, glucose, glycosylated hemoglobin, soluble receptor for advanced glycation endproduct), kidney disease-related biomarkers (albumin, creatinine, cystatin), endocrine biomarkers (dehydroepiandrosterone, sex-hormone binding globulin, testosterone), markers of inflammation (interleukin 6, high-sensitivity C-reactive protein, N-terminal pro b-type natriuretic peptide), ferritin, and transferrin. RESULTS: Of 38 measured biomarkers, 34 were significantly correlated with age. Summary statistics were generated for all biomarkers according to sex and 5-year age increments from 50 and up after excluding participants with diseases and treatments that were associated with biomarkers. A biomarker data set was also generated that will be useful for other investigators seeking to compare biomarker levels between studies. CONCLUSION: Levels of several biomarkers change with older age in healthy individuals. The descriptive statistics identified herein will be useful in future studies and, if replicated in additional studies, might also become useful in clinical practice. The availability of the reference data set will facilitate appropriate calibration of biomarkers measured in different laboratories.

Impact of lipid markers and high-sensitivity C-reactive protein on the value of the 99th percentile upper reference limit for high-sensitivity cardiac troponin I.

OBJECTIVES: i) To assess the relationship between lipid markers and high-sensitivity C-reactive protein (hs-CRP), and high-sensitivity cardiac troponin I (hs-cTnI) in the reference population, and ii) to evaluate the impact of lipid markers and hs-CRP on the 99th percentile upper reference limit (URL) for hs-cTnI. METHODS: 531 questionnaire-identified presumably healthy individuals were enrolled in a single-center, cross-sectional study. Surrogate biomarkers for diabetes, myocardial and renal dysfunction were used to refine the healthy cohort (n=408). Lipid profile, total cholesterol:high-density lipoprotein cholesterol (HDL-C) ratio, non-HDL-C, apolipoprotein AI (apoAI), apolipoprotein B (apoB), apoB:apoAI ratio, lipoprotein(a), small dense low-density lipoprotein cholesterol (LDL-C) and hs-CRP were determined. RESULTS: Individuals with detectable vs. non-detectable hs-cTnI concentrations more often showed elevated LDL-C (60% vs. 46%; p=0.002), apoB (73% vs. 61%; p=0.008), apoB:apoAI ratio (53% vs. 40%; p=0.005) and lipoprotein(a) (15% vs. 7%; p=0.015). The apoB:apoAI ratio and to a lesser extent other lipid markers, but not hs-CRP, were positively associated with hs-cTnI concentration in univariate and multivariate analyses. Exclusion of individuals with elevated apoB:apoAI ratio or apoB, but not hs-CRP, lowered the 99th percentile URL in the healthy cohort respectively by 12.9% (6.2 vs. 5.4ng/L) and 14.5% (6.2 vs. 5.3ng/L). The corresponding reduction for both lipid biomarkers in the presumably healthy population was 24.0% (7.5 vs. 5.7ng/L). CONCLUSION: Our study demonstrates that atherogenic lipid markers, particularly apoB:apoAI ratio or apoB, influence the 99th percentile URL for hs-cTnI.

Development of C-reactive protein certified reference material NMIJ CRM 6201-b: optimization of a hydrolysis process to improve the accuracy of amino acid analysis.

To standardize C-reactive protein (CRP) assays, the National Metrology Institute of Japan (NMIJ) has developed a C-reactive protein solution certified reference material, CRM 6201-b, which is intended for use as a primary reference material to enable the SI-traceable measurement of CRP. This study describes the development process of CRM 6201-b. As a candidate material of the CRM, recombinant human CRP solution was selected because of its higher purity and homogeneity than the purified material from human serum. Gel filtration chromatography was used to examine the homogeneity and stability of the present CRM. The total protein concentration of CRP in the present CRM was determined by amino acid analysis coupled to isotope-dilution mass spectrometry (IDMS-AAA). To improve the accuracy of IDMS-AAA, we optimized the hydrolysis process by examining the effect of parameters such as the volume of protein samples taken for hydrolysis, the procedure of sample preparation prior to the hydrolysis, hydrolysis temperature, and hydrolysis time. Under optimized conditions, we conducted two independent approaches in which the following independent hydrolysis and liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) were combined: one was vapor-phase acid hydrolysis (130 °C, 24 h) and hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) method, and the other was microwave-assisted liquid-phase acid hydrolysis (150 °C, 3 h) and pre-column derivatization liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The quantitative values of the two different amino acid analyses were in agreement within their uncertainties. The certified value was the weighted mean of the results of the two methods. Uncertainties from the value-assignment method, between-method variance, homogeneity, long-term stability, and short-term stability were taken into account in evaluating the uncertainty for a certified value. The certified value and the expanded uncertainty (k = 2) of CRM 6201-b are (40.0 ± 1.6) µmol kg(-1).

Histopathologic and molecular evaluation of the Organ Procurement and Transplantation Network selection criteria for intestinal graft donation.

BACKGROUND: The Organ Procurement and Transplantation Network (OPTN) has formulated criteria for the selection of donors for intestinal transplantation. To date, however, no study has correlated histologic findings of intestinal injury with the OPTN criteria. We aimed to describe histopathologic and molecular features of allograft injury in relation to donor conditions defined by the OPTN criteria. MATERIALS AND METHODS: Graft histology (Park Score), Claudin-3 staining, systemic inflammatory markers (C-reactive protein/lipopolysaccharide-binding protein) and expression of heat shock protein 70, heme oxygenase 1, and interleukin 6 were evaluated in multiorgan deceased donors (donation after brain death [DBD] and donation after cardiac death [DCD]). RESULTS: Ninety-seven samples (52 jejunum/45 ileum) were recovered from 59 donors (46 DBD/13 DCD). The OPTN criterion cold ischemia time correlated with histologic injury (Park score) to which the jejunum appeared more susceptible than the ileum. Claudin-3 staining was higher, and heat shock protein 70 expression lower in donors meeting the OPTN criteria compared with donors not meeting the criteria and in DBD versus DCD. In DBD donors, interleukin 6 expression was higher compared with DCD donors and inversely related to C-reactive protein. CONCLUSIONS: Our multiparameter analysis suggests that the OPTN criteria can be discriminative concerning intestinal graft quality. Our data suggest that DCD intestinal allografts are qualitatively inferior and that the jejunum is more sensitive to ischemia than the ileum.

Evaluation of disease activity indices in Korean patients with rheumatoid arthritis.

The aim of this study is to examine the validity of the rheumatoid arthritis (RA), disease activity score (DAS), 28-C-reactive protein (CRP), the simplified disease activity index (SDAI), and the clinical disease activity index (CDAI) against the DAS28-erythrocyte sedimentation rate (ESR) and determine cut-off values for each tool in Korean patients with RA. A total of 223 RA patients were consecutively recruited from the Hanyang University Hospital for Rheumatic Diseases in Seoul, Korea. DAS28-CRP, SDAI, and CDAI were measured and compared with DAS28-ESR. The correlation coefficients of DAS28-ESR with DAS28-CRP, SDAI, and CDAI were 0.93, 0.85, and 0.84, demonstrating strong linear relationships. The cut-off values of DAS28-CRP classifying RA patients into four categories of disease activity were defined as 2.19, 2.60, and 4.07. SDAI cut-off values were defined as 3.75, 7.50, and 16.88. CDAI cut-off values were defined as 3.62, 7.38, and 16.50. DAS28-CRP, SDAI, and CDAI are valid and sensitive assessment indices of disease activity that are comparable to DAS28-ESR. The cut-off values of each tool derived in this study might be useful for routine monitoring and therapeutic decision-making in Korean RA patients.

Serum C-reactive protein concentrations in healthy Miniature Schnauzer dogs.

BACKGROUND: C-reactive protein (CRP) is a sensitive marker for inflammation in people and dogs. In people, an association between CRP concentration and atherosclerosis has been reported. Atherosclerosis is rare in dogs, but the Miniature Schnauzer breed may be at increased risk for developing this vascular disease. It is not known if CRP concentrations in Miniature Schnauzer dogs differ from those in other dog breeds. OBJECTIVES: Our objectives were to validate an automated human CRP assay for measuring CRP in dogs and compare CRP concentrations in healthy Miniature Schnauzer dogs with those in non-Miniature Schnauzer breeds. METHODS: Sera from 37 non-Miniature Schnauzer dogs with inflammatory disease were pooled and used to validate a human CRP immunoturbidimetric assay for measuring canine CRP. Blood was collected from 20 healthy Miniature Schnauzer dogs and 41 healthy dogs of other breeds. Median serum CRP concentration of healthy Miniature Schnauzer dogs was compared with that of healthy non-Miniature Schnauzer dogs. RESULTS: The human CRP assay measured CRP reliably with linearity between 0 and 20 mg/L. CRP concentration for healthy Miniature Schnauzer dogs (median 4.0 mg/L, minimum-maximum 0-18.2 mg/L) was significantly higher than for the healthy non-Miniature Schnauzer dogs (median 0.1 mg/L, minimum-maximum 0-10.7 mg/L); 17 of the 20 Miniature Schnauzer dogs had values that overlapped with those of the non-Miniature Schnauzer dogs. CONCLUSIONS: Median CRP concentration of Miniature Schnauzer dogs was slightly higher than that of other breeds of dogs. A relationship between higher CRP concentration in Miniature Schnauzer dogs and idiopathic hyperlipidemia, pancreatitis, and possible increased risk for atherosclerosis remains to be determined.

Cardiac troponin T measured by a highly sensitive assay predicts coronary heart disease, heart failure, and mortality in the Atherosclerosis Risk in Communities Study.

BACKGROUND: We evaluated whether cardiac troponin T (cTnT) measured with a new highly sensitive assay was associated with incident coronary heart disease (CHD), mortality, and hospitalization for heart failure (HF) in a general population of participants in the Atherosclerosis Risk in Communities (ARIC) Study. METHODS AND RESULTS: Associations between increasing cTnT levels and CHD, mortality, and HF hospitalization were evaluated with Cox proportional hazards models adjusted for traditional CHD risk factors, kidney function, high-sensitivity C-reactive protein, and N-terminal pro-B-type natriuretic peptide in 9698 participants aged 54 to 74 years who at baseline were free from CHD and stroke (and HF in the HF analysis). Measurable cTnT levels (≥0.003 µg/L) were detected in 66.5% of individuals. In fully adjusted models, compared with participants with undetectable levels, those with cTnT levels in the highest category (≥0.014 µg/L; 7.4% of the ARIC population) had significantly increased risk for CHD (hazard ratio=2.29; 95% confidence interval, 1.81 to 2.89), fatal CHD (hazard ratio=7.59; 95% confidence interval, 3.78 to 15.25), total mortality (hazard ratio=3.96; 95% confidence interval, 3.21 to 4.88), and HF (hazard ratio=5.95; 95% confidence interval, 4.47 to 7.92). Even minimally elevated cTnT (≥0.003 µg/L) was associated with increased risk for mortality and HF (P<0.05). Adding cTnT to traditional risk factors improved risk prediction parameters; the improvements were similar to those with N-terminal pro-B-type natriuretic peptide and better than those with the addition of high-sensitivity C-reactive protein. CONCLUSIONS: cTnT detectable with a highly sensitive assay was associated with incident CHD, mortality, and HF in individuals from a general population without known CHD/stroke.

[Significations of extremely elevated C-reactive protein: about 91 cases in a French hospital center]. / Significations d'une protéine C-réactive supérieure à 500mg/l: à propos de 91 prélèvements dans un centre hospitalier brestois.

This report is devoted to 91 extremely elevated protein C-reactive levels. Patients characteristics, etiologies of inflammation, comorbidities and microbiology are subjected to wide variations. Mortality is high (37 % and 51 %, 30 and 90 days later).

Analysis of the physicochemical state of C-reactive protein in different preparations including 2 certified reference materials.

BACKGROUND: Standardization of clinical measurements of C-reactive protein (CRP) is based on the availability of reference materials. The serum protein reference material ERM-DA470 was used as a calibrant for various commercially available immunoassays but has now been exhausted. The recently released ERM-DA470k/IFCC was intended to fully replace ERM-DA470. However, the new material was not suited for the certification of CRP because of a bias introduced by the lyophilization process that caused loss of about 20% of CRP measurable by routine immunoassays, compared with the nonlyophilized material that was stored in a liquid frozen state. METHODS: We investigated the physicochemical state of CRP in a set of 4 lyophilized and 2 nonlyophilized serum-based CRP-containing materials by semi-native gel electrophoresis, Western blotting, and gel filtration. RESULTS: We detected a monomeric form of CRP (mCRP) in lyophilized materials at a concentration significantly higher than seen in the materials not subjected to lyophilization. Different reconstitution protocols led to variations of the monomeric CRP fraction found in reconstituted, previously lyophilized material. CONCLUSIONS: Most of the 20% loss in measured CRP after lyophilization of the material can be accounted for by the dissociation of natively pentameric CRP into subunits. The observed dissociation results from lyophilization and subsequent reconstitution of the material at insufficient concentration levels of calcium ions. In the presence of various protein forms, differences in antibody specificity and reactivity between immunoassays and alterations of stoichiometry of antigen-antibody interactions can contribute to the divergence of the measured values.

Reproducibility of high-sensitivity C-reactive protein as an inflammatory component of metabolic syndrome in Japanese.

BACKGROUND: Cutoff points for high-sensitivity C-reactive protein (hs-CRP) as a component of metabolic syndrome (MetS) in Japanese have been proposed as 0.40-0.45 mg/L for men and 0.25-0.35 mg/L for women. However, there are some concerns about the reproducibility of hs-CRP. METHODS AND RESULTS: Reproducibility of hs-CRP as a component of MetS was examined using receiver-operating characteristic (ROC) curves for diagnosing MetS in 1,274 men and 673 women whose serum levels of hs-CRP were measured twice at annual health screening tests. The Spearman's correlation coefficient between baseline hs-CRP and hs-CRP at the next year's test was 0.68 in men and 0.71 in women. The area under the ROC curves of baseline hs-CRP, hs-CRP at the next year's test, and the mean of the 2 hs-CRP tests for diagnosing baseline MetS were 0.71, 0.71, and 0.72, respectively, in men and 0.75, 0.74, and 0.74, respectively, in women. Optimal cutoff points of baseline hs-CRP, hs-CRP at the next year's test, and the mean of 2 tests for diagnosing baseline MetS were all 0.40 mg/L in men and 0.35 mg/L in women. CONCLUSIONS: The serum level of hs-CRP was stable enough for use as a measure of the inflammatory component of MetS, and the optimal cutoff point of hs-CRP was 0.40 mg/L for men and 0.35 mg/L for women in a Japanese health-screening population.

Development and preparation of a new serum protein reference material: feasibility studies and processing.

BACKGROUND: The availability of matrix reference materials is essential for the standardisation of (immuno)assays used to measure proteins. The reference material ERM-DA470 (previously called CRM470) certified in 1993 has led to a large degree of harmonisation of these assays. A new serum protein reference material has now been produced (ERM-DA470k). It is intended to replace ERM-DA470, and will additionally be certified for beta(2)-microglobulin (B2M). METHODS: Serum from 390 healthy donors was pooled and processed so as to stabilise, delipidate and 'maturate' it. Purified C-reactive protein (CRP) and recombinant B2M were added. Pilot batches were produced to study the stability, homogeneity, and commutability of the material. On the basis of the results with the trial batches it was decided to proceed with the processing of the main batch of a candidate reference material. RESULTS: Two pilot batches were produced and the processed and spiked serum lyophilised after filling (1 mL). The B2M in the material was shown to be stable and commutable. For CRP, it was discovered that freeze-drying led to a decrease in measurable protein. The main batch of candidate reference material was produced and fulfilled the required criteria in terms of optical transparency, homogeneity and stability. CONCLUSIONS: A new serum protein reference material has been produced with the properties required for a serum protein reference material for 14 proteins. An apparent loss of CRP of approximately 20% was observed upon freeze-drying of the material.

[Report of the NEDO project "Research and development to promote the creation and utilization of an intellectual infrastructure: development of reference materials for laboratory medicine" "Development of pure substance-type certified reference materials"].

There is an increasing demand to establish a metrological traceability system for in vitro diagnostics and medical devices. Pure substance-type reference materials are playing key roles in metrological traceability, because they form the basis for many traceability chains in chemistry. The National Metrology Institute of Japan (NMIJ), in the National Institute of Advanced Industrial Science and Technology (AIST), has been developing purity-certified reference materials (CRMs) in this field, such as cholesterol, creatinine, and urea. In the New Energy and Industrial Technology Development Organization (NEDO) project, entitled: "Research and Development to Promote the Creation and Utilization of an Intellectual Infrastructure: Development of Reference Materials for Laboratory Medicine", several pure substance-type CRMs were developed. For a pure protein solution CRM, amino acid analysis and nitrogen determination were chosen as the certification methods. The development and certification processes for the C-reactive protein (CRP) solution CRM were completed, with the recombinant human CRP solution as a candidate material. This CRP solution CRM is now available as NMIJ CRM. For cortisol CRM, a purified candidate material and highly pure primary reference material were prepared. Each impure compound in the materials was identified and quantified. The pure cortisol CRM will be available in 2009. These two CRMs provide a traceability link between routine clinical methods and the SI unit.