Baseline Assessment of 25-Hydroxyvitamin D Assay Performance: A Vitamin D Standardization Program (VDSP) Interlaboratory Comparison Study.

The Vitamin D Standardization Program (VDSP) coordinated an interlaboratory study to assess the comparability of measurements of total 25-hydroxyvitamin D [25(OH)D] in human serum, which is the primary marker of vitamin D status. A set of 50 individual donor samples were analyzed by 15 different laboratories representing national nutrition surveys, assay manufacturers, and clinical and/or research laboratories to provide results for total 25(OH)D using both immunoassays (IAs) and LC tandem MS (MS/MS). The results were evaluated relative to bias compared with the target values assigned based on a combination of measurements at Ghent University (Belgium) and the U.S. National Institute of Standards and Technology using reference measurement procedures for the determination of 25(OH)D2 and 25(OH)D3. CV and mean bias for each laboratory and assay platform were assessed and compared with previously established VDSP performance criteria, namely CV ≤ 10% and mean bias ≤ 5%. Nearly all LC-MS/MS results achieved VDSP criteria, whereas only 50% of IAs met the criterion for a ≤10% CV and only three of eight IAs achieved the ≤5% bias. These results establish a benchmark for the evaluation of 25(OH)D assay performance and standardization activities in the future.

Baseline Assessment of 25-Hydroxyvitamin D Reference Material and Proficiency Testing/External Quality Assurance Material Commutability: A Vitamin D Standardization Program Study.

The Vitamin D Standardization Program (VDSP) coordinated a study in 2012 to assess the commutability of reference materials and proficiency testing/external quality assurance materials for total 25-hydroxyvitamin D [25(OH)D] in human serum, the primary indicator of vitamin D status. A set of 50 single-donor serum samples as well as 17 reference and proficiency testing/external quality assessment materials were analyzed by participating laboratories that used either immunoassay or LC-MS methods for total 25(OH)D. The commutability test materials included National Institute of Standards and Technology Standard Reference Material 972a Vitamin D Metabolites in Human Serum as well as materials from the College of American Pathologists and the Vitamin D External Quality Assessment Scheme. Study protocols and data analysis procedures were in accordance with Clinical and Laboratory Standards Institute guidelines. The majority of the test materials were found to be commutable with the methods used in this commutability study. These results provide guidance for laboratories needing to choose appropriate reference materials and select proficiency or external quality assessment programs and will serve as a foundation for additional VDSP studies.

Difference in bias approach for commutability assessment: application to frozen pools of human serum measured by 8 direct methods for HDL and LDL cholesterol.

BACKGROUND: We used a difference in bias approach to evaluate the commutability of 4 frozen serum pools for 8 direct methods for measurement of HDL and LDL cholesterol (HDLC and LDLC). METHODS: Freshly collected nonfrozen sera from 138 diseased and 37 nondiseased patients and 4 frozen pools from the CDC Lipid Standardization Program were measured by direct methods and by the beta-quantification reference measurement procedure of the CDC. We used an error components model to estimate the difference in the bias component of error plus its uncertainty for frozen pools vs patient samples between the direct method and the reference procedure. Frozen pools with bias differences less than a critical value determined by either medical requirements for bias or the random error components of the measurement procedures were considered commutable. RESULTS: On the basis of medical requirement criteria, 1 of the 4 frozen pools was commutable for most of the HDLC methods for both diseased and nondiseased patients, and none was commutable for LDLC methods. On the basis of random error criteria, all of the frozen pools were generally commutable for all of the HDLC methods for both diseased and nondiseased patients, and 1 of the 4 frozen pools was generally commutable for most of the LDLC methods for both diseased and nondiseased patients. CONCLUSIONS: Commutability was assessed as the closeness of agreement of the difference in bias between a reference material and a set of patient samples. Criteria for commutability could be based on fixed medical requirements for bias or on random error components.

Assessment of the relation between biomarkers for smoking and biomarkers for acrylamide exposure in humans.

Smoking is an important source of acrylamide exposure in the general population. We assessed the relationship between hemoglobin adducts of acrylamide (HbAA) and glycidamide (HbGA) as biomarkers of acrylamide exposure and plasma cotinine (PC) as biomarkers of tobacco smoke exposure in 94 men and 67 women. The median (5th-95th percentile) biomarker concentrations (pmol/g Hb) in the group of individuals with PC concentrations of 10 ng/mL [194 (87-403) and 107 (41-215) for HbAA and HbGA, respectively]. In individuals with PC concentrations of <1 ng/mL, HbAA and HbGA were similar to those observed in the group with PC values of 10 ng/mL. Although HbAA and HbGA could be categorized into distinguishable groups using PC concentration ranges commonly used to categorize presumed smokers and nonsmokers, no significant relationship was observed between these two biomarkers and PC within each group. The different exposure periods reflected by these biomarkers and the resulting different susceptibility to short-term variations in exposure patterns may in part explain these observations. The findings suggest that tobacco smoke exposure in individuals with PC values of <1 ng/mL has only a minimal effect on HbAA and HbGA.

Approaches for improving glucose monitor measurements for self-monitoring of blood glucose: from measurement harmonization to external quality assessment programs.

Self-monitoring of blood glucose (SMBG) is an important component in diabetes management, helping patients to achieve and maintain normal blood glucose levels. The benefit of SMBG depends on the quality of the measurement performed. Therefore, it is important to know the factors affecting the measurements and to assure that the quality of SMBG measurements is at the highest achievable level possible. To accomplish this, all aspects of the measurement procedure need to be taken into consideration. Sources of variability can be related to the monitor itself, its calibration and use, including blood collection. Improving the variability caused by each source requires specifically designed and targeted efforts. Variability related to the monitor can be assessed in studies that minimize other sources of variability. Variability related to monitor calibration can be assessed and minimized through harmonization or standardization programs, while variability related to the use of the monitors can be addressed through patient-oriented assessment and training. The latter may follow procedures similar to external quality assessment (EQA) programs used in clinical laboratory medicine. However, to obtain an optimal impact on patient care, such programs need to have a wide reach and the social and cultural competency to work efficiently with all patients. The EQA approach or approaches that would provide the most benefit to the patient remain to be determined.

Assessment of trueness of a glucose monitor using interstitial fluid and whole blood as specimen matrix.

BACKGROUND: Interstitial fluid (ISF) is a specimen of increasing interest for glucose measurements because it can be obtained in a minimally invasive manner. Our previous study showed that sufficient ISF can be obtained using microneedles to measure glucose with a conventional electrochemical glucose monitor. The aim of this study was to assess the trueness of this glucose monitor using split-sample comparison with whole blood. We used ISF as specimen and our gas chromatography/mass spectrometry (GC/MS) method as reference. METHODS: We obtained 50 ISF samples and 40 whole blood samples from hairless Sprague- Dawley rats and analyzed for glucose by both methods. RESULTS: For whole blood, a non-significant bias of 5.7% (+/-2 SD: -54.9% to 66.3%) was determined. ISF glucose measurements showed a significant constant bias of 29.5% (+/-2 SD: -85.0% to 144%), which seems to be caused in part by the lack of red blood cells in ISF. The correlation coefficients were 0.782 and 0.679 for whole blood and ISF, respectively. CONCLUSIONS: The assessed electrochemical glucose monitor shows a close agreement with our GC/MS reference method for whole blood, for which this monitor was optimized. When glucose measurements are performed with ISF as matrix, the observed bias needs to be taken into consideration. Further studies are necessary to elucidate the reasons for the wide dispersion of data for ISF.

Assessment of microwave-assisted enzymatic digestion by measuring glycated hemoglobin A1c by mass spectrometry.

Enzymatic digestion of proteins and analysis of the resulting peptides by mass spectrometry is an established approach in proteomics and in clinical and environmental chemistry. The long digestion times of several hours prevent the fast turnover of samples and results. Qualitative applications showed that microwave radiation profoundly shortens enzymatic digestion. However, its usefulness for quantitative applications had not been assessed. In this study, the microwave-assisted enzymatic digestion of hemoglobin at different temperatures, buffer concentrations, and digestion times was assessed and compared with conventional digestion for the proteolytic enzymes trypsin and Glu-C. A microwave-assisted enzymatic digestion method optimized for digestion time and temperature was applied for the analysis of glycated hemoglobin HbA1c and compared with a reference method. Using trypsin, complete digestion was obtained at 50 degrees C within 20 min. Under these conditions, the digestion efficiency was 20% higher than with conventional trypsin digestion. These effects were not observed with Glu-C as enzyme, probably because of the decreased stability of Glu-C at elevated temperatures in comparison with the trypsin used. The comparison of the optimized microwave-assisted digestion method using trypsin with the reference method for HbA1c using Glu-C gave a close correlation in the results (R2: 0.996). A significant bias of 0.33% HbA1c was observed, with higher values obtained with the microwave-assisted tryptic digest; this finding might have resulted from the use of a different enzyme. This study showed that microwave-assisted enzymatic digestion can substantially reduce digestion times to minutes and can be used in qualitative as well as quantitative applications.

Assessment of trueness of glucose measurement instruments with different specimen matrices.

BACKGROUND: The trueness of glucose monitors is commonly assessed using whole-blood samples and a clinical analyzer as a comparison method. In this study, the effect of specimen matrix on trueness of one clinical analyzer and one glucose monitor was compared with a gas chromatography-mass spectrometry (GC/MS) reference method, using split-sample comparison with capillary whole blood (CWB), venous whole blood (VWB), and plasma (PL). METHODS: CWB was analyzed by the glucose monitor and the GC/MS reference method. VWB was analyzed by the glucose monitor, clinical analyzer, and the GC/MS method. PL was analyzed by the clinical analyzer and the GC/MS reference method. RESULTS: For the glucose monitor, the bias was 0.4% and -18.2% for CWB and VWB, respectively. The clinical analyzer had a bias of -25.4% for VWB and -12.0% for PL and a proportional bias was detected in both specimens. Using the clinical analyzer as a comparison method, the glucose monitor had a proportional bias of -9.8%. CONCLUSION: The trueness of clinical analyzers can be affected by the specimen matrix that needs to be assessed before they are used as comparison method to assess trueness of glucose monitors.

Lipid and lipoprotein testing in resource-limited laboratories.

The role of total cholesterol (TC) and lipoproteins in the assessment of coronary heart disease (CHD) is firmly established from population and intervention studies. Total and low-density lipoprotein cholesterol (LDLC) levels are positively associated with CHD, and high-density lipoprotein cholesterol (HDLC) levels are negatively associated with CHD. Efforts to identify and treat people at increased risk based on cholesterol and lipoprotein levels have led to more lipid testing and the need for very reliable test results. Thus, quality laboratory services are an essential component of healthcare delivery and play a vital role in any strategy to reduce morbidity and mortality from CHD. In laboratories with limited resources, establishing laboratory capability to measure CHD risk markers may be a considerable challenge. Laboratories face problems in selecting proper techniques, difficulties in equipment availability and maintenance, and shortage of supplies, staffing, and supervision. The Centers for Disease Control and Prevention (CDC) has been providing technical assistance for more than 30 years to laboratories that measure lipids and lipoproteins and is willing to provide technical assistance as needed for other laboratories to develop this capability. CDC can provide technical assistance to establish lipid and lipoprotein testing capability to support a CHD public health program in areas with limited laboratory resources. This assistance includes: selecting a suitable testing instrument; providing training for laboratory technicians; establishing a simple quality control plan; and instructing staff on how to prepare frozen serum control materials suitable for assessing accuracy of lipid and lipoprotein testing.

The effects of errors in lipid measurement and assessment.

Accurate cholesterol and lipoprotein measurements have provided dependable and powerful basic risk factors for cardiovascular disease. A battery of total cholesterol (TC), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol, and triglycerides (TG) is recommended in the initial evaluation for classification of patients (based on lipids) into highly desirable, desirable, borderline, high, and very high lipid risk factor for cardiovascular disease. Treatment is based largely on the LDL cholesterol measurement result of the patient. The risk factor score of a patient greatly increases when other risk factors for cardiovascular disease exist, along with increased lipid risk factors. Attainment of the needed acceptable accurate lipid and lipoprotein measurements depends upon prevention or control of multiple sources of errors or variation that can exist in preanalytic, analytic, and postanalytic stages of determination of the reported result. Highly important is to control nonfasting, posture, diet, and alcohol intake in the preanalytic part, elimination of matrix effects and use of accurate calibrators in the analytic part, and check for transcription errors in preparation of reports in the postanalytic part of the measurement of lipids.

Assessment and recommendations on factors contributing to preanalytical variability of urinary pyridinoline and deoxypyridinoline.

BACKGROUND: Pyridinoline (PYD) and deoxypyridinoline (DPD) are two of the most extensively characterized biochemical bone markers, but the interpretation of results is hampered by biologic and other preanalytical variability. We reviewed factors contributing to preanalytical variation of pyridinium cross-links in urine. METHODS: We searched four databases for English-language reports on PYD and/or DPD in urine. Searches were restricted to humans, except for studies of stability, when the search was expanded to other species. The 599 identified articles were supplemented with references from those articles and with articles known to the authors. RESULTS: The mean reported within-day variability was 71% for PYD (range, 57-78%) and 67% for DPD (range, 53-75%). The mean interday variability was 16% for both DPD and PYD (range for PYD, 12-21%; range for DPD, 5-24%). The mean intersubject variabilities across studies were 26% for PYD (range, 12-63%) and 34% for DPD (range, 8-98%) for healthy premenopausal women and 36% (range, 22-61%) and 40%, (range, 27-54%) for postmenopausal women, respectively. Specimen instability and errors in creatinine measurements were additional sources of variability. CONCLUSIONS: Intra- and intersubject variability can be reduced by collecting specimens at a specific time of the day and by maintaining similar patient status at each specimen collection regarding factors such as medications and dietary supplements.