Biomedical subjects
Publications and source records attributed to D A Armbruster.
On-site workplace drug testing is fast becoming an attractive alternative to traditional methodologies. Among the reasons for its growing popularity is that these assays can be performed quickly and inexpensively by nonlaboratorians using simple equipment.
Large numbers of specimens (5000-18,000) were screened for amphetamines, barbiturates, cocaine, marijuana, opiates, and phencyclidine by RIA (Roche), Emit II (Syva), and a new immunoassay, CEDIA (cloned enzyme donor immunoassay, Microgenics). All immunoassays performed equivalently for cocaine, opiates, and phencyclidine. All immunoassays detected the same amphetamine/methamphetamine-positive specimens, but all also detected numerous specimens containing cross-reacting sympathomimetic amines. CEDIA detected 100%, Emit II 93%, and RIA 82% of the barbiturate-positive specimens. Emit II and CEDIA detected 86-88% of the specimens found by RIA to be marijuana positive. A subset of specimens was additionally screened by OnLine (Roche) and TDx (Abbott) for amphetamines, cocaine, and marijuana. OnLine and TDx also detected all of the amphetamine-positive specimens and numerous specimens containing cross-reacting sympathomimetic amines. All immunoassays performed equivalently for cocaine, and the four nonisotopic tests detected 86-89% of the marijuana positives found by RIA. Interfering sympathomimetic amine drug compounds can be eliminated by using an oxidizing agent, thus decreasing the number of unconfirmable amphetamine presumptive positives. The CEDIAs for all of the major drugs of abuse are reliable and effective for large-volume urine screening programs.
The limit of detection (LOD) for any analytical procedure, the point at which analysis is just feasible, may be determined by a statistical approach based on measuring replicate blank (negative) samples or by an empirical approach, consisting of measuring progressively more dilute concentrations of analyte. The limit of quantitation (LOQ), or concentration at which quantitative results can be reported with a high degree of confidence, may likewise be determined by either approach. We used both methods to determine LOD and LOQ for forensic gas chromatographic-mass spectrometric (GC-MS) analyses of abused drugs. The statistically determined LOD and LOQ values for these assays underestimated the LOD because of the large imprecision associated with blank measurements and the inability of blank samples to meet typical GC-MS acceptance criteria. The empirical method provided much more realistic LOD values, supported by reasonable experimental data, and are 0.5-0.03 the magnitude of the corresponding statistical LODs. The empirical LODs and LOQs are identical for these GC-MS assays. The observations made here about the LOD/LOQ for specific forensic GC-MS procedures are generally applicable to any type of analysis.
Prostate-specific antigen (PSA) is a glycoprotein produced exclusively by prostatic tissue. PSA's absolute tissue specificity makes it valuable as a forensic marker and, more important, as a tumor marker for prostatic cancer. Prostatic cancer is prevalent in the older male population and is a major cause of death in men. Previously, prostatic acid phosphatase (PAP) was used to help diagnose and monitor the efficacy of therapy for prostate cancer. PAP has now been displaced by PSA, which has greater clinical sensitivity even though it has less clinical specificity. PSA is useful for monitoring therapy, particularly surgical prostatectomy, because complete removal of the prostate gland should result in PSA being undetectable. Measurable PSA after radical prostatectomy indicates residual prostatic tissue or metastasis, and increasing PSA concentrations indicate recurrent disease. PSA is also useful for screening selected populations of patients with symptoms indicative of prostate cancer; its use for general screening is debatable because of its less-than-optimal specificity, the cost of unselected screening, and the lack of evidence that early detection of prostate cancer decreases morbidity and mortality. Distinguishing between patients with prostatic cancer and those with benign prostatic hypertrophy is particularly difficult because of the overlap in PSA values in the two groups. Determining the rate of change in PSA per year from serial measurements or calculating the ratio of PSA per volume of the prostate gland may allow these two groups to be more readily differentiated.
The newest formulation of the EMIT assay for drugs of abuse, EMIT II, and a new immunoassay, OnLine, using the kinetic interaction of microparticles in solution methodology, were evaluated for marijuana, cocaine, opiates, barbiturates, and phencyclidine. Both types of immunoassays were performed on an Hitachi 717 analyzer. Calibration curves, the degree of separation between negative and cutoff calibrators, precision, probability of carryover from positive to negative samples, and overall ease and speed of analysis were evaluated. EMIT II and OnLine were compared with RIA tests for the five drugs to determine each assay's ability to detect samples which confirm positive by GC/MS. The RIA and OnLine marijuana tests detected > 99% of confirmed positive samples while EMIT II detected about 90%. All three immunoassays performed equivalently for cocaine and opiates, each assay detecting at least 98% of positives. Barbiturates showed the greatest disparity with OnLine detecting 96%, EMIT II 85%, and RIA 79% of confirmed positive samples. Too few phencyclidine positive samples were detected for a method comparison study. The fully automated EMIT II and OnLine assays are preferable for a variety of reasons to our laboratory's current semi-automated RIA tests for large volume urine testing. The immunoassays offer comparable performance for some drugs but not for others.
The newest formulation of the Syva EMIT assay for drugs of abuse, EMIT II, and a new immunoassay, OnLine (Roche), utilizing the kinetic interaction of microparticles in solution (KIMS) methodology, RIA tests, and TDx fluorescence polarization immunoassay (FPIA) procedures were compared for marijuana, cocaine, opiates, and barbiturates. Both EMIT II and OnLine immunoassays were performed with a Hitachi 717 analyzer. Calibration curves, the degree of separation between negative and cutoff calibrators, precision, likelihood of carryover from positive to negative samples, and overall ease and speed of analysis were evaluated. RIA and OnLine detected 99% of gas chromatography/mass spectrometry (GC/MS)-confirmed marijuana samples; TDx, 95%; and EMIT II, 88%. All four immunoassays detected approximately 99% of confirmed cocaine-positive urines. RIA, OnLine, and TDx all detected 100% of opiate-confirmed samples; EMIT II, 97%. Barbiturate assays exhibited the greatest disparity, with OnLine and TDx detecting 100% of confirmed positives; EMIT II, 88%; and RIA, 78%. For a variety of reasons, we prefer the fully automated EMIT II and OnLine assays for high-volume urine testing, in comparison with our laboratory's semiautomated RIA tests and the limited-throughput TDx system. The four immunoassays investigated delivered comparable performance in terms of detection rates for GC/MS-confirmed positives for some drugs but not for others.
The accuracy and precision of the Packard Probe 1000, a computer-driven robotic sample processor, were determined using NCCLS Guideline 18-P, employing a rigorous gravimetric procedure. For volumes of 25, 100, and 200 muL, inaccuracy ranged from -0.5 to +0.6% (saline) and -0.8 to +0.7% (human serum), and imprecision from 0.1 to 0.6% (saline) and 0.02 to 0.8% (human serum), in the dip and sip mode of pipetting. For the same volumes of saline in the dispense-through pipetting mode, inaccuracy ranged from -0.1 to -0.9% and imprecision from 0.1 to 0.5%. To obtain this level of performance, the instrument's operating parameters were changed from the factory default settings. This automated liquid-handling system is capable of impressive accuracy and precision, but it must be calibrated in the same manner as any other volumetric device. Performance varies with the volume, type of liquid, and mode of pipetting. Inaccuracy of less than or equal to 1.0% and imprecision of less than =1.0% can be attained along with rapid, walkaway pipetting of calibrators, controls, and large numbers of patient samples.
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The Laboratory Services Branch (Epi Lab) of the Epidemiology Division, Brooks AFB, Texas, is designated by regulation to serve as the Air Force's central reference laboratory, providing clinical laboratory testing support to all Air Force medical treatment facilities (MTFs). Epi Lab recognized that it was not offering the MTFs a service comparable to civilian reference laboratories and that, as a result, the Air Force medical system was spending hundreds of thousands of dollars yearly for commercial laboratory support. An in-house laboratory upgrade program was proposed to and approved by the USAF Surgeon General, as a Congressional Efficiencies Add project, to launch a two-phase initiative consisting of a 1-year field trial of 30 MTFs, followed by expansion to another 60 MTFs. Major components of the program include overnight air courier service to deliver patient samples to Epi Lab, a mainframe computer laboratory information system and electronic reporting of results to the MTFs throughout the CONUS. Application of medical marketing concepts and the Total Quality Management (TQM) philosophy allowed Epi to provide dramatically enhanced reference service at a cost savings of about $1 million to the medical system. The Epi Lab upgrade program represents an innovative problem-solving approach, combining technical and managerial improvements, resulting in substantial patient care service and financial dividends. It serves as an example of successful application of TQM and marketing within the military medical system.
Recently developed fluorescent enzyme immunoassays (FEIs) for follitropin (FSH) and lutropin (LH) designed for the Stratus analyzer were compared with a typical dual FSH-LH radioimmunoassay (RIA) procedure. The FEIs use monoclonal antibodies in a sandwich reaction scheme similar to that of immunoradiometric procedures but with filter paper tabs rather than polystyrene beads and alkaline phosphatase as a tag instead of a radioisotope. Precision of the FEI was comparable with that of the RIA tests with CVs typically under 10%. The detection limit of the FSH test is 0.4 IU/L and that of the LH test is 0.6 IU/L. Measurable carryover exists but is clinically inconsequential. Neither assay exhibits total specificity, but the degree of cross-reactivity is unlikely to be problematic in most situations. Recovery with the FSH assay was 103%, with the LH assay, it averaged 108%. Moderate amounts of bilirubin and triglycerides and gross hemolysis caused no significant interferences. Method comparison studies yielded the following: FEI for FSH, 1.16 RIA + 3.1, r = 0.97, n = 114; FEI for LH, 0.77 RIA - 0.23, r = 0.96, n = 112. Difference analysis of the method comparison data revealed relatively poor agreement between the FEI tests and the dual RIA procedure. With the adoption of method-specific normal reference ranges, the FEI tests offer attractive alternatives to radioisotopic FSH and LH assays.
The Amerlite enhanced luminescence immunoenzymometric assays (LIA) for ferritin and free T4 (FT4) have been evaluated. The LIA assay system employs a two-site binding approach in which one antibody is coated on the interior of plastic microtiter wells and another antibody, or antigen, is tagged with horseradish peroxidase (HRPO). Signal, proportional to the amount of patient analyte, is produced when the HRPO catalyzes the oxidation of luminol resulting in light production. The light signal is increased in magnitude by the incorporation of an enhancing agent which also greatly prolongs the lifetime of the signal. Light production is monitored by an automated luminometer which uses a stored calibration curve to calculate patient results. FT4 within-run coefficients of variation (CVs) ranged from 2.7% to 6.6%; day-to-day CVs from 6% to 15.5% for three levels of control. Sensitivity is 0.03 ng/dl. T3 concentrations up to 8 ng/ml produced no cross-reactivity. Dilutional linearity was exhibited. Lipemia and icterus produced positive interferences. Linear regression analysis of Amerlite and radioimmunoassay (RIA) FT4 values yielded the following values: Amerlite = 0.63 RIA - 0.2, r = .87, n = 79. Ferritin within-run CVs ranged from 4.9% to 10.5%; day-to-day CVs from 5.8-12.5% for three levels of control. Sensitivity is less than 1 ng/ml. Recoveries ranged from 93% to 106% (av. = 99.4%). Dilutional linearity was exhibited. Lipemia caused a positive interference; hemoglobin caused a negative interference. Linear regression analysis of Amerlite and RIA ferritin values yielded the following values: Amerlite = 1.0 RIA + 11.1, r = .96, n = 55.
Assays for human choriogonadotropin (HCG) and thyroid stimulating hormone (TSH) have typically been performed using radioisotopic procedures. Two newer, nonradioisotopic approaches, enzyme immunoassay (EIA) and fluorometric enzyme immunoassay (FEI), were evaluated for the determination of HCG and TSH. Two TSH EIA assays (Abbott, Hybritech) and one FEI assay (Dade) were compared with each other and an immunoradiometric (IRMA) assay. The FEI assay performed better than the EIA assays with a sensitivity of 0.3 microIU/ml, within-run closing volumes (CVs) of less than 4.5%, and a carryover of 0.7%. All of the assays correlated well with each other and the IRMA assay. Three HCG EIA assays (Abbott, Hybritech, Roche) and one FEI assay (Dade) were evaluated. Again, the FEI assay performed better than the EIA assays with a sensitivity of 1.3 mIU/ml, within-run CVs of 4.0% or less, and a carryover of 1.7%. The new generation of nonradioisotopic assays offers performance comparable to radioisotopic assays coupled with various degrees of automation. A clinical laboratory may not only take advantage of these methods but can choose an assay system that is well suited to its particular requirements.
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The assay of cerebrospinal fluid (CSF) enzymes has been suggested for assessing the extent of damage and patient prognosis in cases of brain injury. A potential difficulty associated with using CSF enzyme levels as predictors of outcome is the possibility that enzyme concentrations may vary substantially from one brain region to another. We have determined the concentrations of seven enzymes in seven brain regions in the rat and cat. Acid phosphatase (ACP), aspartate aminotransferase (AST), isocitrate dehydrogenase (ICDH), lactate dehydrogenase (LD), and malate dehydrogenase (MDH) show little regional variability in the rat and cat while creatine kinase (CK) and glutamate dehydrogenase (GDH) both exhibit considerable regional variability in both animals. Lack of correlation between CSF enzyme levels and prognosis may possibly be explained by the observed regional variability. The enzymes demonstrating more homogeneous concentrations throughout the brain may be better candidates for predicting patient outcome by determination of the CSF enzyme level.
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Glucose molecules are joined to protein molecules to form stable ketoamines, or fructosamines, through glycation, a nonenzymatic mechanism involving a labile Schiff base intermediate and the Amadori rearrangement. The amount of fructosamine in serum is increased in diabetes mellitus owing to the abnormally high concentration of sugar in blood. The concentration of fructosamine in serum thus reflects the degree of glycemic control attained by the diabetic patient and is useful in monitoring the effectiveness of therapy in diabetes over a period of several weeks, in a manner analogous to the determination of glycated hemoglobin. Of the analytical approaches used to measure fructosamine, affinity chromatography with m-aminophenylboronic acid and the nitroblue tetrazolium reduction method appear to be the most practical means for clinical chemists to assay fructosamine quickly, economically, and accurately. Fructosamine values can readily distinguish normal individuals and diabetic patients in good glycemic control from diabetics in poor control. Unlike glycated hemoglobin, which reflects the average blood sugar concentration over the past six to eight weeks, fructosamine reflects the average blood sugar concentration over the past two to three weeks. Thus a clinical advantage is that fructosamine responds more quickly to changes in therapy, thereby allowing for improved glycemic control. Used in conjunction with determinations of blood sugar and (or) of glycated hemoglobin, or by itself, the fructosamine assay can provide clinically useful information for the detection and control of diabetes.
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