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Analytical Methods

Overview of B3 Core Services

B3 Core services include the following:

  • Processing and storing of blood, urine, and DNA samples;
  • Providing accurate and timely measurements of biomarkers;
  • Providing field training to ensure quality control and efficiency of study personnel;
  • Answering specific research and technical questions; and
  • Designing laboratory protocols and supporting investigator applications.

B3 Core has its own extensive quality assurance and quality control measures that exceed certification requirements and achieve the high standards of a reference laboratory. B3 Core has been active in numerous federally and non-federally funded research protocols and provides comparability, standardization, and opportunities for intellectual collaboration with other studies.

Selected Summary of Assay Methods

Lipoproteins. Lipids are analyzed in serum on the Mindray BS-200 Chemistry Analyzer. For the quantitative determination of total cholesterol, an enzymatic assay is used. Cholesterol esters in the sample are hydrolyzed by cholesterol esterase to release free cholesterol. The free cholesterol is oxidized by cholesterol oxidase to release hydrogen peroxide. Phenol + 4-AAP + hydrogen peroxide, in the presence of peroxidase, produces a quinoneimine dye that is measured at 500nm. The absorbance at 500nm is proportional to the concentration of cholesterol in the sample. The CV for total cholesterol is 1.5-2.2%. For the quantitative determination of high-density lipoprotein cholesterol (HDL-C), a homogeneous method is used to directly measure serum HDL-C levels without the need for any off-line pretreatment or centrifugation steps. The method is in a two-reagent format. The first reagent contains α-cyclodextrin and dextran sulfate to stabilize LDL, very low-density lipoprotein, and chylomicrons. The second reagent contains PEG modified enzymes that selectively react with the cholesterol present in the HDL particles. Consequently, only the HDL-C is subject to cholesterol measurement. CV is 0.7-1.8% for HDL-C. For the quantitative determination of triglycerides, an enzymatic/glycerol phosphate oxidase (GPO) trinder assay is used. Serum triglycerides are hydrolyzed to glycerol and free fatty acids by lipase. In the presence of ATP and glycerol kinase, the glycerol is converted to glycerol-1-phosphate. The glycerol-1-phosphate is then oxidized by GPO to yield hydrogen peroxide. The condensation of hydrogen peroxide with 4-chlorophenol and 4-aminophenazone (4-AA) in the presence of peroxidase produces a red colored quinonimine dye which absorbs at or near 500nm. The intensity of the colored complex formed is directly proportional to the triglycerides concentration of the sample. CV is 1.9-2.3 for triglyceride. Low-density lipoprotein cholesterol (LDL-C) is calculated. However, when triglycerides are >400 mg/dL, the direct determination of LDL-C is performed. The CV for direct LDL-C averages 1.7% on the Mindray BS-200.

Glucose. Glucose is analyzed in serum or plasma on the Mindray BS-200 Chemistry Analyzer, using the hexokinase/G6PD method. In this method, glucose is phosphorylated by hexokinase in the presence of adenosine triphosphate (ATP) and magnesium to form glucose-6-phosphate (G-6-P) and adenosine diphosphate (ADP). G-6-P is then oxidized by glucose-6-phosphate dehydrogenase (G-6-PDH) in the presence of nicotinamide adenine dinucleotide (NAD), producing 6-phosphogluconate and NADH. The formation of NADH causes an increase in absorbance at 340 nm, which is directly proportional to the concentration of glucose in the sample. CV averages 1.2-1.3% for this assay.

Insulin. Insulin is measured in serum, using either automated assays on the Immulite or ELISA (Millipore). The Immulite 2000 method uses reagents from Diagnostic Products. It is a solid-phase, two-site chemiluminescent assay with a linearity of 0.2-400 IU/mL and a CV <10%. The Millipore ELISA is 2-hour assay and needs a sample <50ul. The assay range is 2–200 μU/mL and CV is 4.6-11.4%.

Hemoglobin A1c. The assay is performed on fresh or frozen whole blood samples on the Mindray BS-200 Chemistry Analyzer using an immunoturbidimetric method. This method utilizes the interaction of antigen and antibody to directly determine the HbA1c in whole blood. Total hemoglobin and HbA1c have the same unspecific absorption rate to latex particles. When mouse antihuman HbA1c monoclonal antibody is added (R2), latex-HbA1c-mouse anti human HbA1c antibody complex is formed. Agglutination is formed when goat anti-mouse IgG polyclonal antibody interacts with the monoclonal antibody. The amount of agglutination is proportional to the amount of HbA1c absorbed on to the surface of latex particles. The amount of agglutination is measured as absorbance. The HbA1c value is obtained from a calibration curve. CV is 1.0-1.5% for this assay.

Urinary Microalbumin. Urine microalbumin is measured on the Mindray BS-200 Chemistry Systems, using immunoturbidimetric assay. When a sample is mixed with anti-human goat antiserum, agglutination is caused by the antigen-antibody reaction. The turbidity is measured at 340nm and 700nm, and albumin in the sample is quantitatively determined. The assay range is 0.5-30 mg/dL and CV averages 2.2-5.2%.

Urinary Creatinine. Urine creatinine is measured on the Mindray BS-200 Chemistry Analyzer, using a modified Jaffe assay. In this assay, creatinine reacts with picric acid in alkaline conditions to form a color complex that absorbs at 510 nm. The rate of formation of color is proportional to the creatinine in the sample. CV averages 1.9-4.0% for this assay.

High sensitivity C-reactive protein (hsCRP). hsCRP in serum is measured on the Mindray BS-200 Chemistry Analyzer, using the latex particle enhanced immunoturbidimetric method. Latex particles coated with antibody specific to human CRP aggregate in the presence of CRP from the sample forming immune complexes. The immune complexes cause an increase in light scattering, which is proportional to the concentration of CRP in the serum. The light scattering is measured by reading turbidity (absorbance) at 570 nm. The CRP concentration is determined from a calibration curve developed from CRP standards of known concentration. The assay range is 0.10-160.0 mg/L, and the CV is 0.5-1.9%.

Chemistry Profile. A study-specific serum chemistry profile, measured on the Mindray BS-200 Chemistry Analyzer, routinely includes albumin (Alb); alanine transaminase (ALT); alkaline phosphatase (Alk); aspartate aminotransferase (AST); blood urea nitrogen (BUN); calcium; chloride; creatinine; GGT; LDH; potassium; sodium; phosphate; total protein; BUN/creatinine ratio; and uric acid. All reagents are tailored to the specific analyte to be measured. All have a CV <5% except for total bilirubin, which has a CV <10 %.Free Fatty Acid (FFA). FFAs are measured in serum using an enzymatic colorimetric assay run on the Mindray BS-200 ChemistryAanalyzer. This method relies on the acylation of coenzyme A (CoA) by fatty acids in serum in the presence of added acyl-CoA systhetase. The acyl-CoA produced is oxidized by added acyl-CoA oxidase with generation of hydrogen peroxide. Hydrogen peroxide, in the presence of peroxidase, permits the oxidative condensation of 3-methyl-N-ethyl-N-(hydroxyethyl)-aniline with 4-aminoantipyrine to form a purple-colored adduct, which can be measured colorimetrically at 550 nm. The amount of colored product formed is proportional to the amount of FFA in the serum that can be quantitatively determined from the measured optical density. The sensitivity of the assay is 0.01mEq/L, and the CV < 5%.

Anti-HBc and Anti-HCV IgG Antibodies. Both anti-HBc and anti-HCV IgG antibodies are measured on the ECi immunoanalyzer (Ortho Clinical Diagnostics). Assay precision is excellent, with high sensitivity (1.1 for anti-HBc and 0.9 for anti-HCV antibodies). Reportable ranges for both anti-HBc and anti-HCV are qualitative (negative, retest, reactive).

Custom Immunoassays. Many biomarker measurements using RIA, ELISA, or bead-based (xMAP) multiplexed assays on the Bioplex (BioRad) platform are available at B3 Core. These include cytokines, chemokines, adipokines, hormones, inflammatory factors, growth factors, antigens, and antibodies.

Sample Storage

The B3 Core has facilities for sample receipt and processing (hoods and refrigerated centrifuges), short- and long-term storage (refrigerators, -20° C and -80° C freezers with emergency power backup), supplemental air conditioning (AC), and remote alarm monitoring. Upon receipt of specimens, the B3 Core Specimen Processors open the package, check the sample label, and count the number of specimens (keeping them on dry ice). The specimens are logged, the due date is noted, and processing and assaying schedules based on the study needs are requested. Samples for storage are treated upon arrival as tests (i.e., assigned ID numbers and storage freezer and box numbers). The computerized storage of this information allows timely inventory of stored material and quick retrieval when needed.

Sample Storage and Tracking. Samples are stored in -80° C freezers at two separate sites redundantly monitored by B3 Core staff, in Washington, DC (on the MedStar Washington Hospital Center campus), and in Hyattsville, MD (at MHRI headquarters). Each specimen logged into a customized and study-specific Freezerworks database is assigned a unique, bar-coded inventory number, with the following information requested and entered: study name, coded participant ID, laboratory accession number, type of specimen, volume, description of storage tube, condition of specimen, storage box number, grid location within the storage box, and freezer number. Additionally, freeze/thaw cycles and disposition of specimens are electronically tracked when specimens are removed from the freezer for assay. The freezers are in locked, secured spaces, with supplemental AC and JCAHO-compliant gas generator backup power; they are continually and redundantly monitored for variations in freezer or ambient temperature and for power loss, and they also are connected to two robotic alarm systems that telephone, page, and email laboratory supervisors and technical personnel (in a redundant call tree) if a temperature or power deviation is sensed. The two biorepository sites are separated by 5.5 miles (12 minutes by car), facilitating easy access by B3 Core staff, yet providing an additional layer of security in the event of a natural disaster, terrorist attack, or other catastrophic event in the vicinity. All biorepository procedures and safeguards are in accord with International Society for Biological and Environmental Repositories and National Cancer Institute guidelines,

Quality Control

The B3 Core participates in extensive internal and external control programs to ensure stable, accurate, and precise measures. Quantitative measurements are performed according to strict written guidelines conforming to those of the College of American Pathologists (CAP). Good Laboratory Practice rules are used throughout the laboratory. Instrumentation is maintained according to manufacturer's standards, and performance is monitored according to CAP guidelines. Reagents are purchased from stable sources and purity is monitored according to CAP regulations. Assays are checked for linearity, sensitivity, parallelism, effect of freeze/thaw, recovery, and within-batch and between-batch coefficients of variation. All sample storage is at -80° C to minimize degradation. Controls at several levels are run with every batch and plotted on Levy-Jennings plots. All assay results are reviewed by a technical supervisor before final release into the data system.

Data are downloaded electronically from the autoanalyzer and randomly audited for accuracy. Our data system has multiple levels of electronic redundancy, culminating in an off-site daily copy. Access to the Laboratory Information System is double-password-secured, and samples are only identifiable by their study ID and laboratory sequence numbers. A clear trail of sample processing and handling is available to allow tracing of the pathway of any given sample through the laboratory. The data system is fully documented and maintained according to CAP regulations.

Key Equipment

Key equipment includes the following: Mindray BS-200 Chemistry Analyzer (MedTest) and ECiQ autoanalyzers (Ortho Clinical Diagnostics), Hitachi 717 autoanalyzer, Packard Cobra automated gamma counter, Synergy HT multi-model microplate reader, Bioplex multiplex HTF system (BioRad), NanoDrop Lite spectrophotometer, Scepter handheld automated cell counter, Beckman X-14R, 6R, Spinchron centrifuges, microcentrifuges, analytical balances, autoplate washer, water baths, sonicators, vacuum pumps, fume hoods, rockers, and vortexers. Forty five-80° C freezers are maintained at the main and satellite laboratories in rooms with backup diesel generator power and supplemental AC, redundant alarm monitoring systems, standby freezers, and 365-day x 24-hour on-call staff (with redundant coverage in a telephone, pager, and email call tree). Sample tracking is based on bar-coded cryolabels in a Freezerworks database.

Emergency power sources maintain the system in case of blackout, and the network is backed up every 24 hours. Weekly images of the database are kept off-site in fireproof safes. The laboratory is equipped with Intel core 2 computers in a Dell PowerEdge NT network, maintained by MHRI Information Technology staff, with daily backups kept in multiple offsite secure storage facilities.


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