A quality assurance program for human immunodeficiency virus seropositivity screening of dried-blood spot specimens.

Epidemiologic projects have been implemented for using dried-blood spot (DBS) specimens routinely collected on filter paper from neonates as a seroepidemiologic resource to monitor seroprevalence of human immunodeficiency virus (HIV) among childbearing women. To ensure the quality of the data base produced from the national and other epidemiologic surveys, a quality assurance program was developed to interact with all the neonatal screening laboratories. The focus of the Centers for Disease Control's quality assurance program for HIV seropositivity testing of neonatal blood specimens is to maintain a national program to produce, certify, and provide external quality control materials as DBSs on filter paper, to monitor the filter paper matrix, to operate an external performance surveillance program, and to provide other special and consultative services. The dried-blood control and surveillance materials are certified by rigorous testing for accuracy, stability, and homogeneity. Preliminary results from the first performance evaluation of screening laboratories indicated excellent performance.

Development and evaluation of a microtiter plate enzyme immunoassay for antibodies to 3,3'-dichlorobenzidine.

The authors obtained and evaluated antisera from rabbits injected with a derivative of a potent bladder carcinogen, dichlorobenzidine (DCB), conjugated to bovine serum albumin (BSA). A 14C-radioimmunoassay (RIA) was able to detect the presence of DCB antibodies, but its relative insensitivity led to the development of a more sensitive enzyme immunoassay (EIA). The EIA test was a "sandwich" method in which a second antibody, labeled with an enzyme (horseradish peroxidase), was used to measure antibody binding to transferrin (Tf)-conjugated DCB immobilized on a microtiter plate. Antibody titers measured by RIA were approximately 1:40; when measured by EIA, they were approximately 1:40,000. Antibody specificity was assessed by comparing the antibody binding activities of DCB, BSA, Tf, BSA-conjugated to DCB, and a number of N-substituted aromatic compounds that included benzidine (Bz). Among the compounds tested, the rabbit antiserum reacted only with DCB and the carrier protein, BSA. Moreover, antibody binding activity to Tf-conjugated DCB was significantly inhibited by unconjugated DCB concentrations between 30 and 500 ng/mL. The precision of antibody binding activities as a function of DCB concentration (expressed by the CV) ranged from 9% for low (30 ng/mL) DCB levels to 12% for higher (500 ng/mL) levels. This evaluation suggests that the antiserum obtained would be appropriate for detecting DCB levels at the ng/mL level.

Analysis of benzoylecgonine in dried blood spots by liquid chromatography--atmospheric pressure chemical ionization tandem mass spectrometry.

Residual samples from blood spots (i.e., whole blood spotted onto filter paper) are a useful source for epidemiological screening studies involving newborns. However, the small volume of blood available from residual blood spots complicates the assay. A method for analyzing benzoylecgonine (BZE; the primary metabolite of cocaine) in blood spots, in which the blood spot is eluted with aqueous ammonium acetate-methanol containing N-methyl trideuterated-BZE as an internal standard, followed by high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry using multiple reaction monitoring, has been developed. This approach provides a rapid, direct, sensitive (limit of detection, approximately 2 ng/mL, based on a 12-microL sample size), and highly specific means of determining BZE concentrations in blood spots. We have applied this method for confirmatory analyses in a large epidemiological study of the prevalence of cocaine use during late pregnancy.

Radioimmunoassay screening of dried blood spot materials for benzoylecgonine.

Residual samples of blood spots, which are routinely collected on almost all newborns in the United States, can be used to determine seroprevalence information on newborns and maternal exposures to various substances, including drugs of abuse. By modifying a commercial radioimmunoassay (RIA) kit for urinary samples, one can use blood spotted on filter paper as a matrix to quantitate the cocaine metabolite benzoylecgonine (BE). BE is stable for long periods of time in blood spots and we were able to quantitatively extract it with aqueous buffer. There were no matrix effects of the blood spot eluate on the RIA, and excess lipid in the blood did not alter measurement of BE. By using standards made up of BE in negative blood spot eluate and calibrators of blood that were spiked with BE and then spotted on filter paper to determine extraction efficiency, low levels of BE in blood could be measured. The limit of detection was 5 ng/mL, and the limit of quantitation was 10 ng/mL. Levels of BE in blood collected at autopsy in eluates of blood spots were measured, and they established excellent correlation (r2 = 0.93) with gas chromatography/mass spectrometry measurements. To test this technology, residual blood spots on 545 infants from three states were analyzed for BE.

An improved analysis for chlorinated pesticides and polychlorinated biphenyls (PCBs) in human and bovine sera using solid-phase extraction.

Chlorinated pesticides and polychlorinated biphenyls (PCBs) remain public health concerns because of their unresolved health impact and their persistence in humans. Current epidemiological studies of cancer, non-Hodgkins lymphoma, and endocrine disruption in National Center for Environmental Health (NCEH) laboratories require exposure assessment of many analytes in thousands of people. Previous methods of analyzing pesticides and PCBs in serum have proven inadequate for timely processing of the number of samples required for epidemiological studies. A new method that involves solid-phase extraction (SPE) and cleanup followed by dual-column gas chromatographic separation and electron capture detection has been developed. Nine surrogate compounds were added to the serum prior to sample workup to provide quality assurance for the SPE steps. These surrogates mimic the chemistry of the analytes in the extraction, cleanup, and gas chromatographic analysis steps. To increase selectivity, extracts were injected onto two gas chromatographs with different capillary columns, a DB-1701 and a DB-5. Recoveries of 17 pesticides, 28 PCB congeners, and one polybrominated biphenyl congener ranged from 40 to 80%. Recoveries from this procedure were found to be similar to those from the previously used liquid-liquid extraction method. Correlation of analyte and surrogate recoveries were compared to examine the ruggedness of the technique. The SPE method was found to provide improved sample throughput by a factor of 15.

Titration of a CD45-FITC conjugate to determine the linearity and dynamic range of fluorescence intensity measurements on lymphocytes.

To produce biologic calibrators for relative fluorescence intensity (RFI) measurements, we stained leukocytes with serial dilutions of CD45-FITC conjugate and processed them using our regular whole blood lysis procedure. Cells were stained with conjugate concentrations ranging from twice recommended to a million-fold lower. At the highest concentrations of conjugate, the RFI reached a plateau near the top of the third decade, indicating saturation of CD45 binding sites. As the concentration decreased, the RFI declined in a highly linear relationship between the dilution factor and the histogram channel number. For channel numbers corresponding to the lowest percentiles of the RFI distribution, linearity persisted down to the first half decade. The slope of this relationship revealed a true dynamic range of 4.5 decades, which was comparable to the value obtained with microbead standards calibrated in molecules of equivalent soluble fluorochrome (MESF). Our results suggest that the lower limit of linearity for fluorescence intensity from fluorescein isothiocyanate (FITC)-stained lymphocytes is below 500 MESF and that cellular autofluorescence is the major limiting factor in detecting and quantifying FITC-specific staining. This procedure provides an adroit way of characterizing the linearity and dynamic range of measurements for quantitative fluorescence cytometry using exactly the same matrix, stains, and preparation methods as those used for cellular analytes.

Impact of protein measurements on standardization of assays of apolipoproteins A-I and B1.

In 1989 the Committee on Apolipoproteins of the International Federation of Clinical Chemistry and the Centers for Disease Control conducted an international survey of total-protein measurements of isolated low-density lipoproteins (LDL) and delipidated high-density lipoproteins (HDL), and of their relationships to the National Institute of Standards and Technology (NIST) bovine serum albumin (BSA) Standard Reference Material (SRM). Most of the 93 apolipoprotein laboratories surveyed use the Lowry total-protein method. Results reported with the LDL preparations demonstrated a large bias and variation among methods; those with delipidated HDL were not as great, but were similar to those for BSA. Performance improved appreciably with use of the Lowry-sodium dodecyl sulfate method and the NIST BSA SRM for protein measurement. The total CVs, including among-laboratory and within-laboratory errors, averaged approximately 50% and 23% for LDL, 17% and 11% for HDL, and 18% and 11% for BSA solutions by all methods and by the Lowry methods, respectively. Regardless of the methods used, greater variability of the protein measurements was seen with the normally occurring LDL than with the nonlipoprotein BSA or delipidated HDL. The mean CV values for all samples among laboratories averaged between 10% and 15% with the modified Lowry methods; the biuret method gave the highest among-laboratory CV, 34%; the Kjeldahl had the lowest, 7.7%. Use of the same methodology and primary nonapolipoprotein standard is essential for comparability of protein results for apolipoprotein primary standard solutions. This is especially true for apolipoprotein B, because its inherent properties and lability make protein analysis difficult. This study supports the use of a standardized selected Lowry-sodium dodecyl sulfate method traceable to quantitative amino acid analysis as a point of reference for determining the protein concentration of primary calibration reference materials for apolipoproteins.

Method-dependent variations in the stability of apolipoprotein B in a stabilized liquid reference material.

Using accelerated Arrhenius-type short-term and long-term temporal studies, we evaluated the storage life of a stabilized, liquid-frozen reference material (SLRM) for human apolipoprotein B (apo B) developed by the International Federation of Clinical Chemistry. As measured by our candidate reference RIA, the concentrations of immunoreactive apo B in the SLRM showed pronounced degradation with exposure to increasing temperatures over time. The SLRM was stable for as long as 1 year when stored at - 70 degrees C, but its immunoreactive apo B declined by < 10% when stored at 4 degrees C for 10 months. Using radial immunodiffusion and an ELISA to assess the equivalency of measured mass for the accelerated thermal stability of the SLRM, we found a loss of immunoreactive apo B similar to that measured by RIA. Analyzing the same samples by liquid immunoprecipitation (nephelometry) resulted in the amount of apo B present being overestimated, especially in samples held for long periods. By using different immunological methods to evaluate this thermally aged SLRM, we demonstrated that its measured behavior varies depending on the method of quantitation.

Two-dimensional protein electrophoresis and multiple hypothesis testing to detect potential serum protein biomarkers in children with fetal alcohol syndrome.

Fetal alcohol syndrome (FAS) surveillance and intervention efforts are hampered by the lack of a specific biochemical test for diagnosis of the syndrome. Based on the hypothesis that abnormalities in growth and development (key features of FAS) involve altered protein metabolism, we analyzed serum proteins by two-dimensional gel electrophoresis and image analysis to search for potential protein biomarkers of FAS. Serum samples from 12 participants in whom FAS had been diagnosed and 8 sex- and age-matched participants whose mothers did not consume alcohol were analyzed in duplicate to determine whether the integrated intensities of matched proteins are significantly altered in children with FAS. Multiple hypothesis testing on 34 of the gels consisting of more than 1700 spots per gel revealed 21 proteins that we classified as potential protein biomarkers of FAS on the basis of significant t-test differences at p < 0.02. We classified 8 of the proteins as candidate biomarkers on the basis of significant concentration differences between case and control subjects at p < 0.01. One of the proteins is clearly an isoform of retinol binding protein; two appear in the area of the gel where alcohol dehydrogenase is expected to appear; one appears to be an isoform of alpha-1-antitrypsin; three appear to be isoforms of the beta-chain of haptoglobin; three may be forms of immunoglobulin light chains; and several others have not been associated with known proteins. No single protein differentiated all case subjects from control subjects, but stepwise canonical discriminant analyses revealed four groups of spots that distinguished between FAS case and control subjects with no misclassifications.(ABSTRACT TRUNCATED AT 250 WORDS)

An evaluation of the use of dried blood spots from newborn screening for monitoring the prevalence of cocaine use among childbearing women.

A collaborative March of Dimes study was designed to examine the utility of dried blood spot (DBS) materials routinely collected from newborns as a source for monitoring cocaine exposure and to assess the prevalence of cocaine use among childbearing women in Georgia. We used a modified urinary radioimmunoassay (RIA) to anonymously detect the cocaine metabolite benzoylecgonine (BE) in DBSs. Extensive efforts were undertaken to assure absolute nonlinkage of BE data to any individual. The positive results found by RIA were confirmed by a mass spectrometry (MS) method specifically developed to detect BE in DBSs. BE was measured in 23,141 DBSs collected during 2 months of routine newborn screening in Georgia. A good correlation was observed for RIA results versus MS results (r2 = 0.97). The estimated minimal statewide BE prevalence was 4.8 per 1000 childbearing women. We demonstrated that immunoassay testing for cocaine without confirmatory testing can yield falsely elevated prevalence rates. When proper confirmatory testing is done, DBSs are a valuable source for population-based monitoring of substance abuse among childbearing women.