Translational and clinical applications of salivary diagnostics.

There have been significant advances in techniques for the detection of biomarker signals in the oral cavity (e.g., ELISAs for proteins, PCR for RNA and DNA) as well as the engineering and development of microfluidic approaches to make oral-based point-of-care (POC) methods for the diagnosis for both local and systemic conditions a reality. In this section, we focus on three such approaches, namely, periodontal disease management, early markers for systemic diseases, and salivary markers useful for pharmacogenomic studies. Novel approaches using non-invasive, salivary samples and user-friendly devices offer results that are as sensitive and specific as laboratory-based analyses using blood or urine.

Infrared up-converting phosphors for bioassays.

The development of up-converting phosphor reporter particles has added a powerful tool to modern detection technologies. Carefully constructed phosphor reporters have core-shell structures with surface functional groups suitable for standard bio-conjugations. These reporters are chemically stable, possess the unique property of infrared up-conversion, and are readily detected. In contrast to conventional fluorescent reporters, up-converting phosphor particles do not bleach and allow permanent excitation with simultaneous signal integration. A large anti-Stokes shift (up to 500 nm) separates discrete emission peaks from the infrared excitation source. Along with the unmatched contrast in biological specimens due to the absence of autofluorescence upon infrared excitation, up-converting phosphor technology (UPT) has unique properties for highly-sensitive particle-based assays. The production and characteristics of UPT reporter particles as well as their application in various bioassays is reviewed.

Use of up-converting phosphor reporters in lateral-flow assays to detect specific nucleic acid sequences: a rapid, sensitive DNA test to identify human papillomavirus type 16 infection.

BACKGROUND: A lateral-flow (LF) device using the new reporter up-converting phosphor technology (UPT) was applied to DNA (hybridization) assays for the detection of specific nucleic acid sequences, thereby aiming to perform the test outside well-equipped laboratories. The methodology reported here is sensitive and provides a rapid alternative for more elaborate gel electrophoresis and Southern blotting. In a preliminary study, it was applied to screen for the presence of human papillomavirus type 16 (HPV16) in a defined series of cervical carcinomas. METHODS: A LF assay was used to capture haptenized DNA molecules and hybrids, which were immunolabeled (before LF) with 400-nm UPT particles. These particles emit visible light after excitation with infrared in a process called up-conversion. Because up-conversion occurs in only the phosphor lattice, autofluorescence of other assay components is virtually nonexistent. RESULTS: The use of the UPT reporter in LF-DNA tests, as compared with colloidal gold, improved the detection limit at least 100-fold. UPT LF-DNA tests were successfully applied to detect (in a blind test) the presence of HPV16 in DNA extracts obtained from cervical carcinomas. Test results matched 100% with previous characterization of these carcinomas. CONCLUSIONS: The use of UPT in LF assays to detect specific nucleic acids provides low attamole-range sensitivity. Hybridization and consecutive detection of PCR-amplified HPV16 sequences were successful in a background of 10 microg of fish-sperm DNA. The sensitivity of UPT detection in these complex mixtures indicates that detection of viral infections without PCR or other amplification technique is achievable.

Detection of marijuana use by oral fluid and urine analysis following single-dose administration of smoked and oral marijuana.

We compared oral fluid testing to urine testing in subjects who were administered single doses of marijuana by smoked and oral routes. Oral fluid specimens were collected with the Intercept DOA Oral Specimen Collection Device, screened for THC with the Cannabinoids Intercept MICRO-PLATE Enzyme Immunoassay (EIA) utilizing a 1.0-ng/mL cutoff concentration, and confirmed for THC by gas chromatography-tandem mass spectrometry (GC-MS-MS) with a 0.5-ng/mL cutoff concentration. Urine specimens were screened for 11-nor-carboxy-delta9-tetrahydrocannabinol (THCCOOH) by immunoassay utilizing a 50-ng/mL cutoff concentration and confirmed for THCCOOH by GC-MS with a 15-ng/mL cutoff concentration. Oral fluid specimens tested positive following smoked marijuana (N = 10) consecutively for average periods (+/-SEM; range) of 15 (+/-2; 1-24) and 13 h (+/-3; 1-24) by EIA and GC-MS-MS, respectively. The average THC detection times of the last oral fluid positive specimen following smoked marijuana by EIA and GC-MS-MS were 31 (+/-9; 1-72) and 34 h (+/-11; 1-72), respectively. In comparison to oral fluid, urine specimens generally tested negative for THCCOOH immediately after marijuana use. The average times to detection of the first urine specimen positive for THCCOOH by EIA and GC-MS were 6 (+/-2; 1-16) and 4 h (+/-1; 2-8), respectively. Urine specimens tested positive consecutively for average periods of 26 (+/-9; 2-72) and 33 h (+/-10; 4-72) for EIA and GC-MS, respectively. The average THCCOOH detection times of the last specimen by EIA and GC-MS were 42 (+/-10; 2-72) and 58 h (+/-6; 16-72), respectively. Considering the noninvasive nature of oral fluid collection and improved detection of recent marijuana use compared to urine testing, it was concluded that oral fluid testing for THC offers specific advantages over other means of marijuana testing when used in safety-sensitive testing programs.

Laboratory analysis of remotely collected oral fluid specimens for opiates by immunoassay.

The performance characteristics of a method for detecting opiates (morphine, codeine, heroin, and 6-acetylmorphine [6-AM]) in oral fluid specimens were examined and compared with methods for urine specimens. The oral fluid was easily obtained using a simple device that collects between 1 and 1.5 mL of fluid for laboratory analysis. Simultaneously collected specimens from 60 known opiate abusers from a drug-treatment center were first tested using an immunoassay cutoff of 10 ng/mL in oral fluids and 2,000 ng/mL in urine. Using a second aliquot, opiate confirmation in urine was performed by gas chromatography-mass spectrometry (GC-MS) and in oral fluids by GC-MS-MS. The combined immunoassay and GC-MS-MS procedures were completed with less than 250 pL of oral fluid. Opiates identified in oral fluid specimens from heroin users included morphine, codeine, heroin, and 6-AM. The immunoassay was tested for precision, stability, and the effects of potential cross-reactants. The results yielded 93.6% agreement between oral fluid and urine, suggesting that oral fluid may be a reliable matrix for opiate detection.

Detection of analytes by immunoassay using up-converting phosphor technology.

Up-Converting Phosphor Technology (UPT) is based on lanthanide-containing, submicrometer-sized, ceramic particles that can absorb infrared light and emit visible light. Biological matrices do not up-convert; hence, there is no contribution to test background from sample autofluorescence. Up-converting phosphors do not photobleach and are inert to common assay interferants such as hemoglobin. A reader called UPlink has been developed to interrogate lateral flow test strips that utilize UPT labels. The reader contains a miniaturized, 1-W, infrared laser with peak emission at 980 nm. Preliminary assays that use up-converting phosphor labels, including tests for a drugs of abuse panel and Escherichia coli O157:H7, have been developed. In a "sandwich" assay format, 10(3) org/mL E. coli O157:H7 organisms were detectable in a negative control background of 10(9) other organisms per milliliter of culture medium. Coefficients of variation in concentrations tested from 0 to 10(7) org/mL were all < or =10%. In a competitive inhibition assay format, a multiplexed test simultaneously detected amphetamine, methamphetamine, phencyclidine, and opiates in saliva. For all assays, the percent displacement at 10 ng/mL was > or =40% demonstrating performance comparable with lab-based, commercially available EIAs. All assays were complete in 10 min. The development of rapid tests using UPT creates new applications for on-site testing with sensitivity not available using other label technologies.

Up-converting phosphor reporters for nucleic acid microarrays.

An important application of robotically spotted DNA microarrays is the monitoring of RNA expression levels. A clear limitation of this technology is the relatively large amount of RNA that is required per hybridization as a result of low hybridization efficiency and limiting detection sensitivity provided by conventional fluorescent reporters. We have used a recently introduced luminescent reporter technology, called UPT (up-converting phosphor technology). Down-converting phosphors have been applied before to detect nucleic acids on filters using time-resolved fluorometry. The unique feature of the phosphor particles (size 0.4 microm) used here is that they emit visible light when illuminated with infrared (IR) light (980 nm) as a result of a phenomenon called up-conversion. Because neither support material of microarrays nor biomolecules possess up-conversion properties, an enhanced image contrast is expected when these nonfading phosphor particles are applied to detect nucleic acid hybrids on microarrays. Comparison of the UPT reporter to cyanin 5 (Cy5) in a low-complexity model system showed a two order of maginitude linear relationship between phosphor luminescence and target concentration and resulted in an excellent correlation between the two reporter systems for variable target concentrations (R2 = 0.95). However, UPT proved to be superior in sensitivity, even though a wide-field microscope equipped with a xenon lamp was used. This higher sensitivity was demonstrated by complementary DNA (cDNA) microarray hybridizations using cDNAs for housekeeping genes as probes and complex cDNA as target. These results suggest that a UPT reporter technology in combination with a dedicated IR laser array-scanner holds significant promise for various microarray applications.

Immunoassay for detection of cocaine/metabolites in oral fluids.

There is growing interest in the use of alternate biological fluids for drug testing. An advantage of oral fluids is that collection can be made from individuals under direct observation without undue embarrassment or invasion of privacy. This study evaluated the STC Cocaine Metabolite MICRO-PLATE EIA for use in detection of cocaine and metabolites in oral fluids. Intra- and interassay precision of the EIA was < 10%. The EIA was cross-reactive to benzoylecgonine (100%), cocaine (> 12.9%), and cocaethylene (13.8%), but did not demonstrate detectable cross-reactivity with other commonly encountered medicants. Evaluation of a series of potential adulterants of oral fluids indicated that common household chemicals and foodstuffs did not alter the outcome of EIA testing for cocaine metabolite. Analysis by EIA and by gas chromatography-mass spectrometry (GC-MS) of oral fluids and urine specimens collected from current drug users in treatment programs and subjects participating in research studies involving controlled dosing of cocaine provided assessment of the clinical sensitivity and specificity of the STC Cocaine Metabolite EIA. Analysis of the data by means of receiver operating characteristic (ROC) plot indicated that the optimal cutoff concentration for the oral fluids EIA was 10 ng/mL. In comparison to GC-MS (10-ng/mL combined cutoff concentration for cocaine and benzoyleogonine), the EIA (10-ng/mL cutoff concentration) demonstrated a sensitivity, specificity, and accuracy of 95%, 82%, and 88%, respectively. The oral fluids EIA was slightly less sensitive than the urine EIA (300-ng/mL cutoff concentration) for the detection of cocaine metabolite with a sensitivity, specificity, and accuracy of 73%, 85%, and 88%, respectively. Overall, testing of oral fluids for cocaine metabolite with the STC Cocaine Metabolite MICRO-PLATE EIA appears to offer a viable alternative to urine for detection of recent cocaine use.

Highly sensitive micro-plate enzyme immunoassay screening and NCI-GC-MS confirmation of flunitrazepam and its major metabolite 7-aminoflunitrazepam in hair.

Flunitrazepam (Rohypnol) is a benzodiazepine used in the treatment of insomnia as a sedative hypnotic and as preanesthetic medication in European countries and Mexico. Although it has no medicinal purpose in the United States, the occurrence of its abuse is increasing. Sexual abuse of both men and women while under the influence of so-called "date-rape" drugs has been the focus of many investigations. Reported date-rape drugs include flunitrazepam (FN), clonazepam, diazepam, oxazepam, gamma-hydroxybutyrate, and many others. FN has been banned in the United States because of its alleged use in such situations. Unfortunately, the detection of FN or its metabolites 7-aminoflunitrazepam (7-AFN) and desmethylflunitrazepam in a single specimen such as urine or blood is difficult in criminal situations because of the likelihood of single-dose ingestion and the length of time since the alleged incident. Hair provides a solution to the second of these problems in that drugs tend to incorporate into hair and remain there for longer periods of time than either urine or blood. There are various techniques for the detection of FN in plasma, blood, and urine, but little work has been done with hair. Hair collection is a virtually noninvasive procedure that can supply information on drug use for several months preceding collection. The objective of this paper was to determine if a commercially available micro-plate enzyme immunoassay system was sufficiently sensitive for the routine screening of 7-AFN in hair by the development of extraction procedures and optimization of the immunoassay kit. Further, this study used the same solid-phase extraction to isolate FN and its major metabolite, 7-AFN, and gas chromatography-mass spectrometry with negative ion chemical ionization for confirmation. Two seven-point standard curves were established ranging from 0.5 pg/mg to 100 pg/mg for 7-AFN and 2.5 pg/mg to 200 pg/mg for FN with respective deuterated internal standards. A replicate analysis of controls was performed to establish inter- and intraday variabilities. Two suicide cases along with one alleged date-rape case and one case of an emergency room patient whose blood screened positive for benzodiazepines were analyzed. All the hair specimens screened positive for benzodiazepines using micro-plate enzyme immunoassay. Two cases, including the date-rape case, were negative for FN and 7-AFN, and two postmortem hair samples were confirmed positive for FN and its metabolite.

The detection of cotinine in hydrolyzed meconium samples.

To date, the screening of meconium for the determination of tobacco exposure in newborns has proven difficult. It was hypothesized that cotinine forms reversible Schiff base bonds with free amino functions on proteins, therefore, hydrolysis of meconium would be necessary for the detection of 'free' cotinine. One-hundred-and-two (102) meconium samples received into our laboratory were extracted using a routine non-hydrolysis screening procedure for drugs of abuse. Separate aliquots of the specimens were hydrolyzed and re-extracted according to the same procedure. The results of the two methods were compared using a highly specific cotinine micro-plate enzyme immunoassay procedure (EIA). Of the non-hydrolyzed samples, 33% were positive for cotinine, while 79% of the hydrolyzed samples were cotinine-positive. Common drugs of abuse did not interfere with the analysis. Micro-plate EIA provides a rapid, simple and reliable screening method for the determination of cotinine in meconium following hydrolysis and extraction. In general, the meconium specimens received into our laboratory are from newborns considered to be at risk for post-natal problems due to suspected drug and/or alcohol abuse during pregnancy.

The detection of cocaine in hair specimens using micro-plate enzyme immunoassay.

The analysis of hair for drugs of abuse is becoming increasingly popular and is under consideration by the Division of Health and Human Services as a possible alternative or adjunct to urinalysis in workplace programs. The detection of cocaine in human hair using a commercially available micro-plate enzyme immunoassay is described for the first time. Sample size and incubation time were the major variables in the optimization of the method. In order to validate the procedure, the method was applied to 105 consecutive hair samples routinely received into our laboratory. The samples were simultaneously analyzed by the Micro-Plate immunoassay (EIA), as well as our current fluorescence polarization immunoassay (FPIA) procedure and gas chromatography-mass spectrometry (GC/MS). The sensitivity of the EIA and FPIA assays were 75% and 67.8% respectively; specificity 97.4% and 80.5% respectively; and efficiency 91.4 and 77.1% respectively. The Micro-Plate EIA was shown to be a valid alternative to other immunoassay screening methods for the detection of cocaine in hair by demonstrating increased sensitivity, specificity and efficiency over our current technique.

Immunization of rats reduces nicotine distribution to brain.

The effect of active immunization against nicotine on the initial distribution of nicotine to brain was studied in anesthetized rats. Animals received nicotine 0.03 mg/kg nicotine (equivalent to the nicotine dose absorbed by a human smoking two cigarettes) as a rapid injection in the jugular vein. In control animals, the arterial serum nicotine concentration initially exceeded the venous concentration 4.6-fold, similar to the initial arteriovenous difference produced by cigarette smoking in humans. Animals immunized with the nicotine analog CMUNic maintained this arteriovenous gradient, but with both arterial and venous nicotine concentrations several times higher than in controls. The arterial nicotine concentration was higher in immunized animals even at the first (7.5 s) sampling time. The brain nicotine concentration at 3 min was 36% lower in the immunized animals. The time course of nicotine distribution to brain was studied in a second group of animals. Brain nicotine concentration was reduced in rats immunized with CMUNic over the entire 6-min sampling period immediately following nicotine dosing (mean reduction 38%). A reduction was found at the earliest sampling time (30 s) and was maximal at 1 min (48%). Nicotine protein binding in serum was markedly increased in animals immunized with CMUNic compared to controls (91.2 versus 10.9%), and the unbound nicotine concentration in serum was lower (10.0 versus 13.4 ng/ml). The reduction in brain nicotine concentration correlated with antibody affinity for nicotine, and the percentage of nicotine bound in serum. These data demonstrate that nicotine-specific antibodies produced by active immunization rapidly bind nicotine in arterial blood, reduce the unbound nicotine concentration, and reduce the early distribution of nicotine to brain. Effects were observed using a clinically relevant nicotine dose and route of administration. These data suggest that the use of immunization to modify the behavioral effects of nicotine may be possible.

Detection of cell and tissue surface antigens using up-converting phosphors: a new reporter technology.

A novel luminescent reporter for the sensitive detection of antigens in tissue sections or on cell membranes is described. It consists of submicron-size phosphor crystals (0.2-0.4 microm), which are surface labeled with avidin or antibodies and capable of binding specifically to antigens on intact cells or in tissue sections. These phosphor reporters exhibit two-photon, anti-Stokes luminescence by up-converting infrared to visible light and are named Up-converting Phosphor Technology (UPT). They typically consist of yttriumoxysulfides doped with two different lanthanides exhibiting photostable, strong emission in the visible (blue, green, and red) upon excitation in the infrared. This report describes the conjugation of phosphor particles to NeutrAvidin with the subsequent use of this conjugate in a model system consisting of prostate-specific antigen in tissue sections and the CD4 membrane antigen on human lymphocytes. An epi-illumination fluorescence microscope was adapted to provide near-IR excitation using a xenon lamp for visualization of the visible emission. Advantages of UPT are (i) permanent, strong, anti-Stokes emission of discrete wavelengths; (ii) unmatched contrast in biological specimens due to the absence of autofluorescence upon excitation with IR light; (iii) simultaneous detection of multiple target analytes; and (iv) low-cost microscope modifications. The new methodology has not only high potential value in diagnostic pathology as described here, but may offer advantages for the detection of proteins or nucleic acids when applied to molecular biology, genomic research, virology, and microbiology.

Detection of methamphetamine in sweat by EIA and GC-MS.

Sweat was collected with the PharmChekTM sweat patch and drugs were eluted from the collection pad of the patch. A solid phase, enzyme immunoassay using microtiter plates was modified for analysis of methamphetamine in sweat. After methamphetamine administration, sweat contains primarily parent methamphetamine. The immunoassay was determined to have crossreactivity relative to 100% for the methamphetamine (MA) calibrators; to 144% for methylenedioxymethamphetamine (MDMA); to 30% for d-amphetamine; to 21% for methylenedioxyamphetamine (MDA); and to 8% for I-methamphetamine. The optimum cutoff concentration for this modified assay was determined by receiver operating characteristic analysis to be 10 ng/mL amphetamine equivalents. At this cutoff concentration the assay had a diagnostic sensitivity of 84.5% and a diagnostic specificity of 93.2% versus gas chromatography-mass spectrometry (GC-MS). The positive predictive value at a prevalence of 50% was 86%. The intra-assay precision at 10 ng/mL was 9.9% (coefficient of variation, CV) and the interassay CV was 13%. Analysis of spiked patches at plus or minus 25 and 50% around the cutoff gave a percent positive threshold of approximately 50% at a cutoff of 10 ng/mL and a 95% confidence level for a positive result by the EIA between 15 and 20 ng/mL. Of 18 potential adulterants that might be injected into or under the patch, two (tile cleaner and cough syrup) caused a false-positive response by immunoassay. All results were confirmed by GC-MS. The clinical sensitivity and specificity of the overall analysis system (sweat collection and analysis) were 85 and 100%, respectively, using known methamphetamine dosing of volunteers (10, 20, and 25 mg) as the reference standard.

Enzyme immunoassay validation for qualitative detection of cocaine in sweat.

A solid-phase enzyme immunoassay (EIA) involving microtiter plates was modified for analysis of cocaine in sweat. Sweat was collected with the PharmChek sweat patch and drugs were eluted from the collection pad of the patch. The sweat contained primarily parent cocaine. The assay was determined to have cross-reactivity for cocaine of 102% relative to 100% for the benzoylecgonine (BE) calibrators and for cocaethylene of 148%. The optimum cutoff concentration for this modified assay, determined by receiver-operating characteristic curve analysis, was 10 micrograms/L cocaine or BE equivalents. At this concentration the assay had 94.5% sensitivity and 99.1% specificity vs gas chromatography-mass spectrometry (GC-MS) as an acceptable indicator of the true clinical state. The positive predictive value at a prevalence of 50% was 99%. Threshold analysis for positives suggested that the 95% confidence interval for a positive result by the EIA was between 12.5 and 15 micrograms/L and that quality-control samples at 5 and 15 micrograms/L could be run with each batch to certify the precision around the cutoff. All positive samples must be confirmed by GC-MS. The sensitivity and specificity of the overall analysis system (immunoassay screen and GC-MS confirmation) was 86% and 97%, with known cocaine dosing of volunteers as the acceptable indicator of the true clinical state.

An enzymatic cycling procedure for beta-NADP+ generated by 3'-phosphodiesterase, 2':3'-cyclic nucleotide.

An enzymatic cycling procedure for beta-NADP+ generated by the enzyme 3'-phosphodiesterase, 2':3'-cyclic nucleotide (EC 3.1.4.37) from its substrate 2':3'-cyclic NADP+ is described. The enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and diaphorase (EC 1.8.1.4) are used to cycle the cofactor between its oxidized and reduced forms in the presence of glucose-6-phosphate and p-iodonitrotetrazolium violet (INT) with the concomitant production of colored INT-formazan, monitored at 492 nm. The amplification is about 400-fold per hour and is sensitive enough to detect 6 x 10(-13) mol of NADP(H). A simple procedure for the optimization of this cycling assay is also described. Conjugates to 3'-phosphodiesterase, 2':3'-cyclic nucleotide may be used in heterogeneous enzyme immunoassays for the detection of small quantities of haptens or proteins in biological fluids.

A new generation of oral hypoglycemics. Their use, measurement, and effects on insurance risk assessment.

The increased use of oral hypoglycemics among the more than 14 million diabetics in the United States and the development of new pharmaceutical formulations requires the continued surveillance of their use in the insurance applicant population. Routine monitoring of urine glucose or even blood glucose or fructosamine measurements often fail to detect the diabetic using oral hypoglycemics. Urine analysis for the use of first generation sulfonylureas has shown a declining prevalence of their use due to the increased number of prescriptions written for newer and more effective drugs. In order to monitor the use of these second-generation oral hypoglycemics, an immunoassay was developed to detect the use of glyburide, 1-[4-[2-(5 chloro-2-methoxybenzamido) ethyl]-phenyl] sulfonyl]-3-cyclohexylurea. After testing in a defined population of known users of this class of sulfonylureas, the assay was applied to a random population of 12,000 applicants applying for life insurance. Results demonstrate the predictive value of this assay in determining the prevalence of use of this drug in the insurance-buying population and its assistance in risk assessment.

A spectrophotometric assay for nanogram quantities of biotin and avidin.

Parameters and conditions of an enzyme based assay for biotin and avidin are presented. Biotinylated glucose-6-phosphate dehydrogenase when complexed with avidin becomes inactivated. Thus it was possible to construct a competitive assay system for biotin. The assay is sensitive between 100-500 ng/ml and could detect as little as 10 ng in 0.1 ml with a between run error of 2.4%. It requires a 60 min incubation at 21 degrees C and 5 min to assay. The avidin assay, based on the degree of inactivation of biotinylated-glucose-6-phosphate dehydrogenase in relation to the concentration of avidin, could detect as little as 0.25 ng in 0.1 ml or 2.5 ng/ml with an assay time of 10 min with a between run error of 3.9%. Both assays are rapid with significant improvements over other non-isotopic methods in sensitivity and comparable to radioisotopic methods in sensitivity with the added advantage of ease of method.

The predictive value of lipid studies in patients undergoing cardiac catheterization and open heart surgery.

All patients admitted for cardiac catheterization prior to possible coronary bypass surgery have a lipid profile ordered as part of their preadmission laboratory work. These studies, usually the first and only performed on the patient, include: high and low density lipoprotein-cholesterol, total cholesterol, triglyceride, and apolipoproteins A-I and B. This study was designed to determine the diagnostic significance and sensitivity of these tests based on a one-time determination as predictors of coronary artery disease. The three groups studied included those patients with surgically confirmed coronary artery disease (n = 247), aortic and/or mitral stenosis (n = 34), and normals, those free of disease (n = 30). The total population was 311 subjects, ranging in age from 20-85 years, which comprised 250 males and 61 postmenopausal females. The prevalence of the disease was 77% over the total population, with 238 of those with coronary artery disease going on to bypass surgery. Surgical results of cardiac catheterization were collected as percent occlusion. No correlations were found between lipoproteins and percent occlusion in the diseased group. Calculations of sensitivity and specificity for various lipid values suggested no clinical use for a one-time determination of these apo- and lipoproteins.