Volume 48, Issue 6

Rhabdomyolysis, a term used to describe the rapid breakdown of striated muscle, is characterized by rupture and necrosis of muscle fibers. This process results in the release of cell breakdown products into the bloodstream and extracellular space. Although direct muscle injury remains the most common cause of muscle injury, additional causes include hereditary enzyme disorders, drugs, toxins, endocrinopathies, malignant hyperthermia, neuroleptic malignant syndrome, heatstroke, hypothermia, electrolyte alterations, diabetic ketoacidosis and non-ketotic hyperosmolar coma, severe hypo- or hyperthyroidism and bacterial or viral infections. The classic triad of symptoms includes muscle pain, weakness and dark urine, although more than 50% of the patients do not complain of muscle pain or weakness. Additional systemic symptoms include fever, general malaise, tachycardia, nausea and vomiting. The laboratory diagnosis is based essentially on the measurement of creatine kinase in serum or plasma. Plasma and urine myoglobin measurement might be useful in the early stages of the syndrome and for identifying a subset of patients with minor skeletal muscle injury. Patient monitoring is pivotal (the mortality rate is as high as 8%), and should be focused on preventing the detrimental consequences, that often include renal disease and coagulopathy. In the pre-hospital setting, forced hydration with 1.5–2 L of sterile saline solution should be started immediately, followed by 1.5–2 L/h. Following hospital admission, continuous hydration should be ensured, alternating the saline solution with a 5% glucose solution. In the presence of myoglobinuria, urine should be alkalinized by use of sodium bicarbonate solution. Clin Chem Lab Med 2010;48:749–56.

Muscle tissue may be damaged following intense prolonged training as a consequence of both metabolic and mechanical factors. Serum levels of skeletal muscle enzymes or proteins are markers of the functional status of muscle tissue, and vary widely in both pathological and physiological conditions. Creatine kinase, lactate dehydrogenase, aldolase, myoglobin, troponin, aspartate aminotransferase, and carbonic anhydrase CAIII are the most useful serum markers of muscle injury, but apoptosis in muscle tissues subsequent to strenuous exercise may be also triggered by increased oxidative stress. Therefore, total antioxidant status can be used to evaluate the level of stress in muscle by other markers, such as thiobarbituric acid-reactive substances, malondialdehyde, sulfhydril groups, reduced glutathione, oxidized glutathione, superoxide dismutase, catalase and others. As the various markers provide a composite picture of muscle status, we recommend using more than one to provide a better estimation of muscle stress. Clin Chem Lab Med 2010;48:757–67.

The Kidney Disease: Improving Global Outcomes (KDIGO) have recently replaced the K/DOQI guidelines to help nephrologists in diagnosing, treating and monitoring chronic kidney disease mineral and bone disorders (CKD-MBD). Here, we comment on the KDIGO recommendations concerning the interpretation of parathyroid hormone (PTH) concentrations in dialysis patients: “In patients with CKD stage 5D, we suggest maintaining iPTH in the range of approximately two to nine times the upper normal limit of the assay. We suggest that marked changes in PTH concentrations in either direction within this range prompt an initiation or change in therapy to avoid progression to concentrations outside of this range”. Our comments concern the interpretation of PTH in dialysis patients, but also the more global problem of establishing normal values for PTH. Clin Chem Lab Med 2010;48:769–74.

Background: It is necessary to develop a highly specific and sensitive assay to quantify the exact amount of hepatitis C virus (HCV) RNA in blood of patients with hepatitis C. For this reason, a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for quantification of HCV RNA in human plasma was developed. Methods: A pair of primers as well as hybridization probes were selected. A real-time RT-PCR was set up and optimized. To establish the sensitivity of the assay, a serial dilution of HCV standards and reference sera, including the six major HCV genotypes, was used. The performance of the assay was evaluated with 191 known HCV-RNA positive and 100 negative samples. Results: The real-time assay had a sensitivity of 50 IU/mL, with a dynamic range of detection between 10 3 and 10 6 IU/mL. The coefficients of variation of threshold cycle values in intra- and inter-day-runs were <1.77% and 3.40%, respectively. Measurement of HCV-RNA positive samples yielded reproducible data with 100% specificity. Conclusions: The high sensitivity, simplicity, reproducibility, wide dynamic range, and low cost of this real-time HCV RNA quantification makes this method especially suitable for monitoring viral load during therapy and tailoring of treatment schedules accordingly. Clin Chem Lab Med 2010;48:777–84.

Background: Toll-like receptor 2 (TLR2) is essential for the immune response to tuberculosis (TB). The goal of the present study was to investigate whether the guanine-thymine (GT) repeat microsatellite polymorphism in intron 2 of the TLR2 gene might be correlated with susceptibility to TB in Han Chinese. Methods: The number of (GT)n repeats was determined by gene scanning from 244 patients with TB and 233 control subjects. The expression of TLR2 on CD14+ peripheral blood mononuclear cells was determined using flow cytometry. Results: No association in allelic polymorphism between control subjects and patients with TB was found. However, the S/M genotype of the microsatellite polymorphism was more frequent in TB patients than in healthy controls (p=0.01). The S/L genotype was more popular in controls than in patients with TB (p=0.007). TLR2 expression was higher in subjects with the S/L genotype than in those with the S/M genotype (p<0.05). Conclusions: Our data suggest that the S/M genotype of the microsatellite (GT)n polymorphisms in intron 2 of the TLR2 gene may increase susceptibility to TB in Chinese, and the S/L genotype may act as a negative risk factor. Clin Chem Lab Med 2010;48:785–9.

Background: Among markers of pregnancy complications, corticotropin-releasing hormone ( CRH ) mRNA, long pentraxin 3 (PTX3) protein and fetal and total DNA had been reported to be increased in the plasma of women with overt preeclampsia (PE). We developed an optimized protocol to evaluate whether concentrations of CRH mRNA, PTX3 mRNA and protein, fetal and/or total DNA are increased in fetal growth restriction (FGR), and whether they predict complications of pregnancy. Methods: The protocol included a preamplification step to enrich rare mRNA species. CRH and PTX3 mRNA, DNA and PTX3 protein were measured in the plasma of women with PE or FGR, in women at risk of developing these pathologies and in healthy women matched for gestational age. Results: CRH mRNA, fetal and/or total DNA and PTX3 protein were significantly increased in women with overt PE when compared to controls. Pregnant women who later developed PE or FGR during pregnancy showed total DNA levels that were significantly increased before the onset of both pathologies, while RNA markers were increased only in women who later developed PE. Conclusions: Our protocol for plasma RNA quantification may allow for the extension of a panel of predictive markers to be investigated in larger patient cohorts. Clin Chem Lab Med 2010;48:791–4.

Background: A new reference material for the liver enzyme aspartate transaminase (AST) (L-aspartate: 2-oxoglutarate-aminotransferase, EC 2.6.1.1), also called aspartate aminotransferase (ASAT), has been developed under the code ERM-AD457/IFCC. This certified reference material (CRM) for AST has been produced from a human type recombinant AST expressed in Escherichia coli and a buffer containing bovine serum albumin, and has been lyophilised. Methods: The homogeneity and the stability of the material have been tested and the catalytic activity concentration has been characterised by 12 laboratories using the reference procedure for AST at 37°C from the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). Results: The certified catalytic activity concentration and certified uncertainty of AST in the reconstituted material are (1.74±0.05) μkat/L or (104.6±2.7) U/L (with a coverage factor k =2; 95% confidence interval). Conclusions: Both the certified value and uncertainty are traceable to the International System of Units (SI). The material is aiming to control the IFCC reference procedure for AST at 37°C, which will then be used to assign values to calibrants and control materials. The present paper highlights the scientific challenges and innovations which were encountered during the development of this new CRM. Clin Chem Lab Med 2010;48:795–803.

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 β 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. Clin Chem Lab Med 2010;48:805–13.

Background: The usefulness of serum transferrin receptor (sTfR) as a marker of iron deficiency is limited by lack of standardization of commercial immunoassays for sTfR. An international collaborative study was performed to evaluate a lyophilized preparation of recombinant soluble transferrin receptor (rsTfR) for its suitability to serve as a World Health Organization (WHO) Reference Reagent to standardize immunoassays for sTfR. Methods: The concentration of pure rsTfR was determined from the A 280 nm using the adjusted theoretical extinction coefficient and molecular weight calculated from its sequence, before dilution and lyophilization in a sTfR-depleted serum matrix. Six manufacturers and a health protection laboratory assayed the candidate Reference Reagent, coded 07/202, along with three lyophilized serum samples, using commercial assays for sTfR. Results: Dose-response plots demonstrated acceptable overall parallelism between 07/202, manufacturers' in-house standards, and serum samples. However, there was poor agreement on the estimated (r)sTfR content of 07/202 and serum samples. Expressing the sTfR content of the serum samples relative to 07/202 markedly improved agreement between methods. Conclusions: Use of 07/202 would reduce inter-method variability. The preparation was established as the 1st WHO Reference Reagent for sTfR with assigned free rsTfR monomer values of 21.7 mg/L and 303 nmol/L (0.5 mL reconstitution). Clin Chem Lab Med 2010;48:815–20.

Background: Recent clinical and animal studies suggest that increased serum homocysteine (HCY) concentrations may be a risk factor for osteoporosis. In vitro studies showed that increasing HCY concentrations stimulate the activity of human osteoclasts. However, there is no data demonstrating that circulating HCY is related to structural and biomechanical properties of human bones. This study investigated the relationship between morphological as well as biomechanical bone properties and HCY serum concentrations in humans suffering from hip osteoarthritis (OA). Methods: Fasting blood samples and femoral heads were obtained from 94 males and females who underwent hip arthroplasty due to OA. Bones were assessed by dual energy X-ray absorptiometry (DXA), biomechanical testing (indentation method), and histomorphometry. Blood was collected for measurement of HCY, folate, vitamin B6, and vitamin B12. Subjects were classified as hyperhomocysteinemic (>12  μmol/L, n=47) and normohomocysteinemic (<12 μmol/L, n=47) according to their serum HCY concentrations. Results: Folate and vitamin B6, but not vitamin B12, were significantly lower in hyperhomocysteinemic subjects compared with controls. However, DXA, biomechanical testing, and histomorphometry did not reveal significant differences in bone quality between hyperhomocysteinemic subjects and controls. Conclusions: The results of the present study do not indicate a significant relationship between circulating HCY concentrations and morphological or biomechanical bone properties in humans with OA of the hip. Clin Chem Lab Med 2010;48:821–7.

Background: The goal of our study was to evaluate the diagnostic performance of the anti-cyclic citrullinated peptide 2 (anti-CCP2) assay in patients with autoimmune and inflammatory disorders. Methods: We tested the specificity and sensitivity of anti-CCP2 antibodies measured by ELISA in 787 patients with rheumatoid arthritis (RA), 1024 patients with other autoimmune/inflammatory rheumatic disease and 401 subjects without autoimmune rheumatic disease. The optimal cut-off value was defined as the value with the highest diagnostic accuracy (receiver operating characteristic curve analysis). Interval-specific likelihood ratios (LRs) were calculated for each range bounded by defined anti-CCP2 values. Results: To distinguish between patients with RA and controls, the cut-off value with the highest diagnostic accuracy for anti-CCP2 was 2.8 U/mL. Comparing the optimal cut-off value for anti-CCP2 to that recommended by the manufacturer (5.0 U/mL), an increase in prevalence between the proportions of test-positive patients was found for RA, undifferentiated connective tissue disease and undifferentiated arthritis. Evaluating interval-specific LRs for the selected ranges bound by two anti-CCP2 values, in RA and diseased controls, the LRs were 0.40 for values <5.0 U/mL, 6.66 for 5.0–15.0 U/mL, 27.01 for 15.1–30.0 U/mL and 28.89 for >30.0 U/mL. Conclusions: The cut-off value of 2.8 U/mL for anti-CCP2 has the highest diagnostic accuracy. A value of anti-CCP2 >15 U/mL is associated with an increase in the likelihood of RA disease. Clin Chem Lab Med 2010;48:829–34.

Background: A new interleukin-6 (IL-6) electrochemiluminescent immunoassay (ECLIA, Roche Diagnostics) was evaluated and compared to a previous semiquantitative immunoassay (Milenia Biotec). Reference ranges for cord blood plasma were also calculated. Methods: The new IL-6 ECLIA test was performed using a Cobas E601 analyzer (Roche Diagnostics). The comparison method (Milenia Biotec) was a semiquantitative lateral flow immunoassay, coupled to a digital image capture system (PicoScan). Healthy at term newborns were recruited to establish reference ranges for IL-6 in cord blood plasma. Results: Total imprecision ranged from 3.7% to 8.0% depending on the IL-6 concentrations. The calculated limit of detection for IL-6 measured by ECLIA was 2.63 pg/mL, almost twice as high as that claimed by the manufacturer (1.5 pg/mL). The linearity of the method was verified to 5000 pg/mL. The IL-6 reference limit, obtained from 148 cord blood samples, was 30 pg/mL (90% confidence interval: 19–48 pg/mL). Conclusions: The new quantitative ECLIA method showed good reproducibility, linearity and functional sensitivity. Additional clinically based studies are needed to elucidate the usefulness of the IL-6 reference limit, calculated here for the first time by ECLIA in cord blood plasma, to aid in the diagnosis of vertical transmission of sepsis. Clin Chem Lab Med 2010;48:835–8.

Background: Manual cell counting in cerebrospinal fluid (CSF) is technique-dependent, time-consuming, and thus costly and prone to inter-operator variability and low precision. Flow cytometry (FCM) with fast hematology analyzers (HAs) appears to improve accuracy and precision of CSF cell analysis; rapid CSF cell analysis is especially needed in emergency laboratories. Ten external trials of the German Society for Clinical Chemistry and Laboratory Medicine evaluated FCM with Coulter (LH750, 755), Abbott CD3200™, CD3500™, CD3700™, CD4000™, Sapphire™, ADVIA120 ® CSF assay, and Sysmex ® XE-2100 single platform analyzers. Methods: CSF controls were produced using native blood leukocytes and erythrocytes, resembling CSF and thus rendering the trials feasible and allowing comparison with native manual counting in a Fuchs-Rosenthal chamber and FACScan-CD45-CD14 dual platform analysis, which was used as the reference method. Statistical evaluation was performed using Passing/Bablok regression analysis. Results: Our evaluation revealed significant differences with respect to target values in leukocyte and erythrocyte counts, as well as leukocyte differentiation. These differences were attributed to inaccuracies produced by the HAs, due to blank correction in connection with impedance analysis, leukocyte loss, especially through monocyte injury due to the erythrocyte lysing agent, incomplete erythrocyte lysis, ADVIA cell sphering, cell differentiation using algorithms and peroxidase activity. Erythrocyte counting in the CSF controls was inaccurate with the Coulter and ADVIA analyzers. Conclusions: Evaluation of HAs by means of the CSF controls revealed inaccuracies in cell counting and leukocyte differentiation. Analyzer techniques, used for CSF cell assays, therefore need to be improved. Clin Chem Lab Med 2010;48:839–48.

Background : Circulating high-sensitivity C-reactive protein (CRP) is a good marker of chronic low-grade inflammation. The few studies that have addressed the relationship between coffee consumption and CRP concentrations report inconsistent findings. The authors of this study examined the relationship between coffee and green tea consumption and serum concentrations of CRP, and the interaction with alcohol consumption, smoking, and obesity in a large population of free-living Japanese men and women. Methods : Study subjects were 10,325 men and women, 49–76 years of age, living in Fukuoka City who participated in a baseline survey of a cohort study on lifestyle-related diseases. Coffee and green tea consumption and other lifestyle characteristics were assessed using a structured questionnaire. Anthropometric measurements and venous blood samples were also included. Results : CRP concentrations were progressively lower with increasing levels of coffee consumption, after adjustment for smoking and other covariates (p for trend=0.03) in men, but not in women. Stratified analysis indicated that this inverse association was primarily limited to men with a high consumption of alcohol (≥50 g/day). Green tea consumption showed no measurable relationship with CRP concentrations in either men or women. Conclusions : Coffee may be protective specifically against alcohol-induced hepatic inflammation. Further studies are warranted in different populations. Clin Chem Lab Med 2010;48:849–54.

Background: Currently, no satisfactory biomarkers are available to screen for esophageal squamous cell carcinoma (ESCC). The goal of this study was to find biomarkers and establish a serum protein fingerprint model for early diagnosis of ESCC using the ClinProt protocol of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Methods: Serum samples were collected from 62 patients with ESCC, nine patients with esophageal adenocarcinoma (EA) and 38 healthy individuals. Proteomic spectra of mass to charge ratio (m/z) were generated following the application of plasma to weak cationic-exchanger magnetic beads (WCX-MB). The spectral data were analyzed using a support vector machine, and potential biomarkers were chosen for system training and used to construct diagnostic models. Results: Three differential patterns were established using MALDI-TOF MS. Pattern 1, consisting of 11 protein peaks, separated ESCC patients from the healthy individuals with a sensitivity of 90.0% and a specificity of 88.4%. Pattern 2, consisting of eight protein peaks, separated ESCC in stage I and stage II from stage III and stage IV with a sensitivity of 92.9% and a specificity of 82.3%. Pattern 3, consisting of seven protein peaks, separated ESCC from EA with a sensitivity of 91.3% and a specificity of 80.0%. Conclusions: These results suggested that MALDI-TOF MS combined with MB separation yields significantly higher sensitivity and specificity for the detection of serum protein in patients with ESCC. Clin Chem Lab Med 2010;48:855–61.

Background: Empirical chemotherapy for patients with non-small cell lung cancer (NSCLC) is recommended, even without knowledge of the tumor's specific biological characteristics, and many patients may not benefit. The goal of this study was to identify potential serum biomarkers that influence resistance to chemotherapy, and to build a model that could be used to predict resistance to chemotherapy of patients with advanced NSCLC. Methods: A total of 97 NSCLC patients were classified as stage IIIB and stage IV. The chemotherapy regimen was cisplatin plus docetaxel. All patients received two cycles of chemotherapy. Serum protein profiles were detected using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and the spectra were analyzed with a support vector machine (SVM). Results: For the 93 eligible patients, 22 patients had a partial response (23.7%); 27 patients had stable disease (29.0%) and 44 (47.3%) had progressive disease. One hundred and twenty-eight mass peaks were detected from the chemotherapy sensitive group and the chemotherapy resistant group by receiver operator characteristic curve. The top 10 peaks with the highest area under the curve values were selected, randomly combined, and fed into the SVM. The SVM model with the highest accuracy was used as the diagnostic model. The model constructed using five protein peaks with mass/charge ratios of 3955 Da, 6207 Da, 7992 Da, 9214 Da, and 15,086 Da separated the chemotherapy resistant group from the chemotherapy sensitive group with a sensitivity of 83.3% and specificity of 85.7%. Conclusions: SELDI-TOF MS may provide a useful means in the search for serum biomarkers for predicting chemotherapy resistance in patients with advanced NSCLC. Clin Chem Lab Med 2010;48:863–7.

Background : Soluble mesothelin-related protein (SMRP) is increased in the sera of patients with malignant mesothelioma (MM), and has been suggested as a diagnostic tool for MM in an asbestos exposed population. However, factors affecting SMRP concentrations in normal subjects and those with other asbestos related disorders have not been investigated in any large population based study. Methods : Five hundred and thirty-eight subjects with a history of asbestos exposure were studied. Age, height, weight, body mass index (BMI), serum creatinine and glucose, estimated glomerular filtration rate (eGFR) and lung function were compared with SMRP concentrations. Results : The mean age [± standard deviation (SD)] of participants was 66.9 (±10.1) years, and mean (±SD) serum SMRP concentration was 0.91 (±0.67) nmol/L. SMRP values were inversely associated with weight (Pearson r=–0.1254, p=0.0036), BMI (Pearson r=–0.1594, p=0.0002), blood glucose (Pearson r=–0.1515, p=0.0004), single-breath carbon monoxide diffusing capacity (DLco) % predicted (Pearson r=–0.1847, p<0.0001), eGFR (Pearson r=–0.2835, p<0.0001) and single-breath carbon monoxide diffusing capacity per unit alveolar volume (DLco/VA)% predicted (Pearson r=–0.1872, p<0.0001) but were positively associated with age (Pearson r=0.2315, p<0.0001) and creatinine (Pearson r=0.3833, p<0.0001). Conclusions : This study has shown that demographic variables, physiological factors and lung function are associated with serum SMRP concentrations. Confounding factors should be considered when interpreting serum SMRP. Clin Chem Lab Med 2010;48:869–74.

Background: Whereas N-terminal pro-brain natriuretic peptide (NT-proBNP) is approved for risk stratification of patients with acute coronary syndromes (ACS), short-term temporal changes in NT-proBNP concentrations and the optimal time points for sampling are not clear. The purpose of this study was to better define the short-term changes in NT-proBNP in relation to clinical presentation, reperfusion and prognostic value in patients with ACS, as well as to identify the optimum time points for sampling. Methods: We studied daily plasma concentrations of NT-proBNP in 133 unselected patients with myocardial infarction (n=65), stable coronary artery disease (CAD, n=46) and no CAD (n=22) who underwent coronary angiography. Results: Patients with non-ST-elevation myocardial infarction (NSTEMI) presented with markedly higher NT-proBNP than patients with ST-elevation myocardial infarction (STEMI) [1305 (741–3208) ng/L vs. 170 (70–424) ng/L, p<0.001]. Also, time to presentation from onset of pain was much longer in NSTEMI as compared to STEMI (>48 h vs. <6 h, p<0.001). Patients with NSTEMI also presented with higher NT-proBNP as compared with CAD [224 (98–732) ng/L] and no CAD [47 (26–102) ng/L; p<0.001, NSTEMI vs. both]. Following successful percutaneous coronary intervention [thrombolysis in myocardial infarction (TIMI) 3-flow established], NT-proBNP increased markedly within 24 h in patients with STEMI [718 (379–1338) ng/L, p<0.01 vs. 0 h], whereas no change in NT-proBNP was noted in patients with NSTEMI [1190 (1010–2024) ng/L, p=0.88 vs. 0 h]. In both STEMI and NSTEMI, NT-proBNP decreased significantly 96 h after successful reperfusion [STEMI –52%, 372 (189–610) ng/L, p<0.05; NSTEMI –52%, 613 (365–724) ng/L, p<0.05]. Unsuccessful reperfusion (TIMI<3) was associated with unchanged or increased NT-proBNP. NT-proBNP at 96 h and peak NT-proBNP further displayed a strong correlation with cardiac troponin T (r=0.64 and r=0.54, p<0.001), a marker of infarct size, and NT-proBNP at 96 h was a strong predictor of long-term prognosis (hazard ratio 7.29, p=0.025). Conclusions: In patients with NSTEMI, NT-proBNP may be increased as high as concentrations usually associated with acute congestive heart failure despite the absence of clinical signs. In contrast, patients with STEMI and short time to presentation may present with completely normal NT-proBNP, but dramatic short-term increases following reperfusion. NT-proBNP reflects ischemic burden, reperfusion success and prognosis, and the current data support repetitive sampling in patients with ACS. Clin Chem Lab Med 2010;48:875–81.

Background: High-density lipoprotein (HDL) is believed to be protective against coronary heart disease (CHD). HDL is comprised of different subfractions. Of these, HDL 2b is believed to be the most important in preventing CHD. Current methods for HDL subfraction measurements are not standardized and often exhibit poor analytical performance, which can limit their usefulness in clinical practice. Methods: We have developed a microfluidic Lab-on-Chip electrophoresis system for serum HDL subfraction measurements. Linear polymers are used as a sieving buffer for HDL particle separation. Nine samples and two controls can be analyzed in 30 min. The percent HDL 2b that comprises total high-density lipoprotein cholesterol (HDL-C) is reported. Results: Samples with HDL-C of 0.259–2.072 mmol/L could be evaluated for percent HDL 2b with coefficient of variations (%CVs) below 8%. Total precision was typically below 1.5% and linearity was observed between 8% and 32% HDL 2b. Measurements were not affected by many therapeutic and biological compounds. Consistent results were obtained by three laboratories and showed r 2 values between 0.93 and 0.95. Conclusions: The assay allows for easy and reproducible measurement of percent HDL 2b. The experimental procedure and small size of the instrumentation needed for measurement make it viable in clinical settings. Clin Chem Lab Med 2010;48:883–9.