Content: This review addresses the current state of knowledge regarding assays of HDL heterogeneity and function and their relationship to cardiovascular disease. HDL is highly heterogeneous, with subfractions that can be identified on the basis of density, size, charge, and protein composition, and the concept that certain subfractions of HDL may be better predictors of cardiovascular risk is attractive. In addition, HDL has been shown to have a variety of functions that may contribute to its cardiovascular protective effects, including promotion of macrophage cholesterol efflux and reverse cholesterol transport and antiinflammatory and nitric oxide–promoting effects.

Summary: Robust laboratory assays of HDL subfractions and functions and validation of the usefulness of these assays for predicting cardiovascular risk and assessing response to therapeutic interventions are critically important and of great interest to cardiovascular clinicians and investigators and clinical chemists.

Methods: We examined tHcy data in the prefortification NHANES III survey (phase II, 1991–1994) and in 3 postfortification survey periods (1999–2000, 2001–2002, and 2003–2004). We applied method adjustment equations to the survey data based on method comparison studies of separate samples. Persons with chronic kidney disease were excluded from the analyses.

Results: Mean plasma tHcy concentrations decreased by 8%, 9%, and 10% for adolescent, adult, and older men and by 6%, 3%, and 13% for women, respectively, from before to after fortification. Concentrations remained unchanged between the first and third postfortification survey periods. Prevalence estimates of increased plasma tHcy concentrations (>13 µmol/L) for older men and women decreased from prefortification (32% and 20%, respectively) to postfortification (14% and 5%, respectively) but remained unchanged thereafter (16% and 14%, respectively [males] and 5% and 9%, respectively [females]).

Conclusions: After adjusting for method changes, we quantified a prefortification to postfortification decrease in circulating tHcy concentrations of about 10% in a national sample of the US population. This change is similar to effects seen in intervention trials with folic acid and in smaller observational studies.

Methods: In this study, we investigated the relationship between CRP concentrations and TF and MCP-1 mRNA expression in unstimulated and lipopolysaccharide (LPS)-stimulated monocytes isolated from hypertensives with or without an increase in carotid intima-media thickness (IMT). We also investigated the expression of TF and MCP-1 mRNA and MCP-1 protein after in vitro addition of CRP to monocytes. We measured CRP (by immunonephelometry) and monocyte expression of TF and MCP-1 (by real-time PCR) in 80 untreated hypertensive patients without clinical cardiovascular disease (CVD) or additional risk factors for CVD compared with 41 controls. Based on IMT measured by carotid Doppler ultrasonography, patients were classified into the categories of normal (≤1 mm) or abnormal (>1 mm). TF and MCP-1 mRNA and MCP-1 protein (by Western blotting) were measured after in vitro addition of CRP to monocytes from 10 randomized controls as well as 10 hypertensives with IMT ≤1 mm and 10 with IMT >1 mm.

Results: CRP and TF and MCP-1 mRNA concentrations were significantly higher in IMT >1 mm hypertensives vs those with IMT ≤1 mm and controls. CRP had no effect on monocyte TF mRNA from either hypertensives or controls. CRP-stimulated monocytes from hypertensives, however, showed increased MCP-1 mRNA and protein expression compared with controls and LPS-stimulated cells.

Conclusions: Our findings suggest that the inflammatory response of blood monocytes plays an important role in the development of atherosclerosis and hypertension.

Methods: We used a universal mRNA–specific linear-amplification strategy in combination with Affymetrix Exon Arrays to amplify salivary RNA from 18 healthy individuals on the nanogram scale. Multiple selected candidates were preamplified in one multiplex reverse transcription PCR reaction, cleaned up enzymatically, and validated by qPCR.

Results: We defined a salivary exon core transcriptome (SECT) containing 851 transcripts of genes that have highly similar expression profiles in healthy individuals. A subset of the SECT transcripts was verified by qPCR analysis. Informatics analysis of the SECT revealed several functional clusters and sequence motifs. Sex-specific salivary exon biomarkers were identified and validated in tests with samples from healthy individuals.

Conclusions: It is feasible to use samples containing fragmented RNAs to conduct high-resolution expression profiling with coverage of the entire transcriptome and to validate multiple targets from limited amounts of sample.

Methods: We evaluated 2 previously characterized TNFSF4 gene variants (–921C>T and dbSNP rs3850641) with a) incident arterial events using a prospective case-cohort design with 344 incident CVD cases and 2254 control participants, all white, drawn from the Women’s Health Study cohort with 10 years of follow-up, and b) venous thromboembolism (VTE) risk using a nested, matched case-control design of 108 white male pairs (drawn from the Physicians’ Health Study cohort) and a case-cohort design of white female participants consisting of 125 cases and 2269 controls (drawn from the Women’s Health Study cohort), analyzed separately.

Results: Genotype distributions were in Hardy-Weinberg equilibrium. Results from a marker-by-marker regression analysis, adjusting for traditional risk factors, showed a significant association of –921C>T with an increased risk of VTE in women (additive: odds ratio 1.86; 95% CI 1.17–2.92, P = 0.008) in women. Furthermore, using a haplotype-based regression analysis, haplotype C-G was associated with a reduced risk of VTE relative to the referent haplotype, C-A (odds ratio 0.50; 95% CI 0.27–0.92; P = 0.02). In contrast, we found little evidence for an association of the variants/haplotypes with risk of VTE in men or CVD risk in women (as previously reported).

Conclusions: Our present findings, if corroborated in other prospective investigations, suggest that the TNFSF4 variants tested may be useful indicators for assessing the risk of venous thromboembolism.

Methods: We sequenced a region 6-kb upstream of the IL6 [interleukin 6 (interferon, beta 2)] transcription start site in a search for functional variants and detected 3 common variants: –6331T>C, –6101A>T, and –5617/–5616C/A>T/G. IL6 –6331T>C (C allele frequency, 0.20; 95% confidence interval, 0.16–0.24) showed strong negative linkage disequilibrium with –174G>C (D' = –0.97) and was studied further in 309 individuals who underwent coronary artery bypass grafting.

Results: Patients with the TT genotype had higher IL-6 concentrations 6 h after surgery than those with the CC genotype (mean, 199.4 ng/L vs 114.9 ng/L; P = 0.02). A similar association was seen in a cohort of 173 patients who underwent intensive periodontal therapy: Individuals with the CC genotype had significantly lower IL-6 concentrations 24 h after therapy than TT patients (mean, 0.78 ng/L vs 5.00 ng/L; P < 0.0001). A similar trend was observed in 203 healthy individuals from northern Europe (1.29 ng/L for the TT genotype vs 0.89 ng/L for the CC genotype; P = 0.07). Reporter assays that used a sequence flanking the –6331 single-nucleotide polymorphism spliced upstream to the IL-6 minimal promoter driving luciferase gene expression demonstrated a 1.3-fold increase in promoter activity (P < 0.01) for constructs containing –6331T. Electrophoretic mobility shift assays revealed enhanced binding of transcription factor Oct-1 to the T allele.

Conclusions: IL6 –6331T is associated with increased IL-6 concentrations in an acute inflammatory state via a mechanism involving binding of the Oct-1 transcription factor. This finding may help resolve conflicting studies based on the IL6 –174G>C variant.

Methods: We included 255 consecutive male patients presenting with intermittent claudication in the investigation and matched the patients by age and diabetes mellitus with 255 control individuals.

Results: Plasma bilirubin concentrations were significantly lower in patients than in controls [mean (SD), 12.5 (5.3) µmol/L vs 15.4 (7.9) µmol/L; P < 0.001]. We found a clear association between the number of TA repeats and plasma bilirubin concentration. Considering the 6/6 TA-repeat genotype as the wild type, we observed a slight increase in bilirubin concentration individuals with the heterozygous 6/7 genotype and pronounced increases for those with the homozygous 7/7 genotype. This association occurred in both controls and patients; however, patients and controls were not significantly different with respect to UGT1A1 TA-repeat genotype frequencies.

Conclusions: Our study of a well-phenotyped group of patients with intermittent claudication and control individuals revealed a clear association between low bilirubin concentrations and peripheral arterial disease but no association between the UGT1A1 polymorphism and the disease.

Methods: For affinity chromatography, we coupled Fab' fragments of polyclonal sheep antibodies specific for N-terminal proBNP (NT-proBNP) epitope 1–21 to silica beads. We connected a column (10 mm x 0.8 mm inner diameter) packed with these beads to a trypsin reactor and used a preconcentrator in combination with a fritless nanospray column to perform MS analyses of proBNP forms in preextracted and non-preextracted samples of plasma from patients with severe heart failure (HF). We used Western blotting in deglycosylation experiments to confirm the shifts in proBNP and NT-proBNP masses.

Results: Tandem MS experiments demonstrated the presence of both NT-proBNP and circulating proBNP in preextracted samples of plasma from patients with severe HF, and Western blotting analyses revealed 2 bands of approximately 23 kDa and 13 kDa that shifted after deglycosylation to positions that corresponded to the locations of recombinant proBNP and synthetic NT-proBNP.

Conclusions: We obtained clear evidence for circulating proBNP in patients with severe HF and provided the first demonstration of O-glycosylation of NT-proBNP. The higher molecular masses for NT-proBNP and proBNP observed in the Western blotting analyses than those expected from calculations can be explained by O-glycosylation of these peptides in vivo.

Methods: We analyzed endogenous NT-proBNP by several immunochemical methods using a broad panel of monoclonal antibodies specific to different epitopes of the NT-proBNP molecule.

Results: Treatment of endogenous NT-proBNP by a mixture of glycosidases resulted in significant improvement of the interaction between deglycosylated NT-proBNP and monoclonal antibodies (MAbs) specific to the mid-fragment of the molecule. MAbs specific to the N- and C-terminal parts of NT-proBNP (epitopes 13–24 and 63–76) were able to recognize glycosylated and deglycosylated protein with similar efficiency.

Conclusions: The central part of endogenous NT-proBNP is glycosylated, making it almost "invisible" for the antibodies specific to the mid-fragment of the molecule. Thus sandwich assays using even one antibody (poly- or monoclonal) specific to the central part of the molecule could underestimate the real concentration of endogenous NT-proBNP. MAbs specific to the N- and C-terminal parts of NT-proBNP (epitopes 13–24 and 63–76) are the best candidates to be used in an assay for optimal NT-proBNP immunodetection.

Methods: We developed a multiplexed, quantitative methylation-specific polymerase chain reaction (MSP) assay consisting of 3 methylation markers, GSTP1, RARB, and APC, and an endogenous control, ACTB, in a closed-tube, homogeneous assay format. We tested this format with urine samples collected after digital rectal examination from 234 patients with prostate-specific antigen (PSA) concentrations ≥2.5 µg/L in 2 independent patient cohorts from 9 clinical sites.

Results: In the first cohort of 121 patients, we demonstrated 55% sensitivity and 80% specificity, with area under the curve (AUC) 0.69. In the second independent cohort of 113 patients, we found a comparable sensitivity of 53% and specificity of 76% (AUC 0.65). In the first cohort, as well as in a combined cohort, the MSP assay in conjunction with total PSA, digital rectal examination status, and age improved the AUC without MSP, although the difference was not statistically significant. Importantly, the GSTP1 cycle threshold value demonstrated a good correlation (R = 0.84) with the number of cores found to contain prostate cancer or premalignant lesions on biopsy. Moreover, samples that exhibited methylation for either GSTP1 or RARB typically contained higher tumor volumes at prostatectomy than those samples that did not exhibit methylation.

Conclusions: These data confirm and extend previously reported studies and demonstrate the performance of a clinical prototype assay that should aid urologists in identifying men who should undergo biopsy.

Methods: The nanodot array luminometric immunoassay (NALIA) system consists of conventional 96-well membrane-bottomed plates in which antigens or antibodies are adsorbed onto the underside of the membrane. Current arrays use a 5 x 5 format (25 dots/well), which allows 10 analytes to be measured in duplicate: double-stranded DNA (dsDNA), centromere protein B (CENP-B), PCNA, Sm, Sm ribonucleoprotein (Sm-RNP), U1-snRNP, Scl70, SSA/Ro, SSB/La, Jo-1, and controls. The test fluid, control sera, and subsequent reagents are drawn through the membrane. The captured analytes are quantified by monitoring chemiluminescence with a charge-coupled device (CCD) and analyzed with commercial array software.

Results: The assay can detect <20 x 10³ IU/L of anti-dsDNA. The interwell CV was 10%–14%. There was an 83% concordance ( = 0.56) between the NALIA results obtained for anti-dsDNA assayed by β-testing in a routine immunology diagnostic laboratory and the results obtained with a conventional ELISA reagent set. The concordance values for Ro, La, Sm, and RNP were 98% (, 0.92), 93% (, 0.41), 97% (, 0.62), and 97% (, 0.73), respectively.

Conclusion: The NALIA approach promises to provide a highly economical platform for a wide range of applications that require assays of multiple analytes. The degree of concordance of our results with a conventional reagent set was no less than that occurring between different commercial products. A sample of serum from a finger stick provides a volume sufficient to perform the array assay.

Methods: We processed whole blood from hospitalized patients after burn injury and severe blunt trauma according to the microfluidic and standard macroscale leukocyte isolation protocol. Side-by-side comparison of RNA quantity, quality, and genome-wide expression patterns was used to clinically validate the microfluidic technique.

Results: When the microfluidic protocol was used for processing, sufficient amounts of total RNA were obtained for genome-wide expression analysis from 0.5 mL whole blood. We found that the leukocyte expression patterns from samples processed using the 2 protocols were concordant, and there was less variability introduced as a result of harvesting method than there existed between individuals.

Conclusions: The novel microfluidic approach achieves leukocyte isolation in <25 min, and the quality of nucleic acids and genome expression analysis is equivalent to or surpasses that obtained from macroscale approaches. Microfluidics can significantly improve the isolation of blood leukocytes for genomic analyses in the clinical setting.

Method: We used TCA to precipitate proteins in whole blood. Thiamine and its phosphate esters were derivatized using potassium ferricyanide to thiochromes, which were separated by gradient elution on a reversed-phase HPLC column and detected by fluorescence. The method was validated for linearity, limit of quantification, imprecision, accuracy, and interference. Results obtained with this method were compared with those produced by the method currently used in our clinical laboratory. Reference values of thiamine and its phosphate esters were determined in samples obtained from self-reported healthy adults who were not taking vitamin supplements. To shorten analysis time, our method used whole blood rather than washed erythrocytes, did not require lengthy enzymatic dephosphorylation, and had a simple mobile phase.

Results: The method was linear to 4000 nmol/L. The lower limit of quantification was 3 nmol/L. The within-run CV was <3.5% and total CV was <9.4%. This method correlated with our current method (r = 0.97). Approximately 90% of the total thiamine content in whole blood was present as thiamine diphosphate (TDP). The means (ranges) for an apparently healthy population were 114 (70–179) nmol/L for TDP and 125 (75–194) nmol/L for total thiamine. Results for separation and measurement of free thiamine and thiamine phosphate esters in whole blood were obtained within 5.5 min.

Conclusion: We developed an HPLC method that allows separation and measurement of free thiamine and thiamine phosphate esters in whole blood and provides more rapid results than other methods.

Methods: We retrospectively analyzed 64 895 measurements of triglycerides, total apo B, and LDL–apo B. Samples were ultracentrifuged, and 3 commercially available immunoassays that use different antibodies were used to measure LDL–apo B in the 1.006 infranate fraction.

Results: After adjusting for triglyceride concentration, we found total apo B and LDL–apo B measurements to be strongly correlated. We derived a simple linear equation for calculating LDL–apo B concentration (in milligrams per deciliter) from measurements of total apo B and triglycerides: LDL–apo B = apo B – 10 mg/dL – triglycerides/32. This equation accurately predicts LDL–apo B values within ±12% of the measured value in 75% of cases.

Conclusions: Our equation provides a convenient means of estimating LDL–apo B from commonly available measurements of total apo B and triglycerides without the need for ultracentrifugation. LDL–apo B measurements were also independent of the different apo B antibodies in the 3 assays used in this study. An equation that predicts LDL–apo B particle number may be useful, regardless of the apo B assay used.

Methods: We generated enhanced green fluorescent protein (EGFP)-expressing Eschericha coli by transforming E. coli with the plasmid vector pEGFP. We used these bacteria in a flow cytometric assay to measure both engulfment and degradation of living bacteria by monocytes and granulocytes in human whole blood from fresh, heparinized venous blood samples. To determine whether the test detected differences between healthy individuals and patients with secondary immunodeficiencies, we compared the phagocytosis of monocytes and granulocytes measured in blood samples from immunosuppressed kidney transplantation patients and from patients with postoperative sepsis in immunoparalysis with phagocytosis measured in samples from age-matched healthy individuals.

Results: In samples from healthy individuals, we found that in both monocytes and granulocytes bacterial degradation was negatively correlated with the age of the sample donor. Furthermore, we detected decreased bacterial engulfment in granulocytes from septic patients and decreased bacterial degradation in monocytes from immunosuppressed kidney transplantation patients.

Conclusions: This flow cytometric assay measures the engulfment and degradation of live bacteria by human blood monocytes and granulocytes. By means of this assay we detected significant differences between healthy controls and patients with secondary immunodeficiencies that may contribute to the increased incidence of infection complications seen in these patients.

Methods: A novel amperometric RSNO sensor, based on an immobilized organoselenium catalyst at the distal tip of an electrochemical nitric oxide detector, was used to determine RSNO species in diluted whole blood without centrifugation or pretreatment. Porcine blood was collected into aluminum foil–wrapped syringes via a 12-inch butterfly needle tube assembly. Two blood samples were collected from the same animal—one with the butterfly needle tubing wrapped in aluminum foil and one with the tubing exposed to ambient room light. The RSNO concentrations in these sequential blood samples were determined by a standard addition procedure.

Results: Eight sets of measurements were made in 6 animals. Samples exposed to light yielded RSNO concentrations only 23.6% (7.2%) [mean (SD)] of the RSNO concentrations determined in samples that were shielded from light and obtained from the same animals.

Conclusions: These results suggest significant photoinstablity of RSNOs in whole blood and indicate the critical importance of proper light protection during sampling and processing of blood samples for the accurate determinations of endogenous RSNO concentrations.

Methods: Mice were immunized with different conjugated proGRP peptides, including residues 31–98, 1–98, and preproGRP(-23–125). Pair combinations of the resulting monoclonal antibodies (mAb) were tested. The improved TR-IFMA was compared with the only other available proGRP assay, the proGRP ELISA (IBL).

Results: A panel of 12 high-affinity mAbs was produced. The best assay combination was between our original E146 mAb as solid-phase antibody and the new mAb M16 as tracer. The new TR-IFMA had a linear dose-response curve, a wide dynamic range (13–13 500 ng/L), and a limit of detection of 2.8 ng/L. Total CV was <5.6% over the whole measuring range. Bland-Altman difference analysis indicated a significant positive bias between the IFMA and the ELISA.

Conclusions: We describe a sensitive and robust mAb-based TR-IFMA for proGRP. The assay is fully automated and displays high quality performance.