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Reference intervals are an essential part of laboratory medicine, and accreditation standards require that every laboratory result is accompanied by an appropriate reference interval to provide guidance in the interpretation of the test. Furthermore, the Clinical Laboratory Improvement Act of 1988 (CLIA '88) requires laboratories to verify that the reference interval accompanying a laboratory result is appropriate for the patient population the laboratory serves. These 2 tasks are fundamental to providing quality laboratory services. In this review, we will consider the statistical methods that can be applied to establish and validate reference intervals. [ABSTRACT FROM AUTHOR]

Accurate measurement of the glomerular filtration rate (GFR) is essential for detecting renal insufficiency in living kidney donors. Iohexol is a "near-ideal" exogenous filtration marker for GFR measurement that has attracted increasing interest in clinical practice because it is non-toxic, non-radioactive, readily available, and easy to measure. In this chapter, we describe a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure iohexol in serum and to calculate GFR based on the rate of iohexol clearance. In this procedure, the contrast agent iohexol is administrated to the study subject in an outpatient setting, and three timed blood samples are collected. The serum proteins are precipitated, and the supernatant containing iohexol and the internal standard 2 H 5 -iohexol is diluted prior to LC-MS/MS analysis. The LC-MS/MS method utilizes a Thermo Vanquish UHPLC coupled with TSQ Endura triple quadruple mass spectrometer, with a total run time of 2.5 min. The LC-MS/MS method has demonstrated good analytical performances, and the workflow can be used to reliably measure GFR in apparently healthy individuals without impaired renal function, such as living kidney donors., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Vitamin D has received significant attention from clinical societies, researchers, and the general population in recent years. While 25-hydroxyvitamin D (25(OH)D) is the most commonly-used biomarker of vitamin D status, 1α,25-dihydroxyvitamin D (1,25(OH) 2 D), its bioactive form, plays a critical role in regulating calcium and phosphorus homeostasis and is also involved in the immune system and cellular differentiation. Consequently, accurate measurements of 1,25(OH) 2 D can aid in the differential diagnosis of calcium-related disorders such as hypocalcemia in vitamin D-dependent rickets and hypercalcemia due to inappropriate increase of serum 1,25(OH) 2 D in granulomatous diseases. However, due to its lipophilicity and very low circulating concentration, the measurement of 1,25(OH) 2 D is particularly challenging. Over the past several decades, numerous efforts have been made to develop sensitive, specific, and practical laboratory methods for measuring 1,25(OH) 2 D. Methods using radioreceptor assay, radioimmunoassay, enzyme immunoassay, enzyme-linked immunosorbent assay, automated chemiluminescent immunoassay, and liquid chromatography-tandem mass spectrometry have been described. Each of these methods has unique advantages and limitations, and some are no longer used. Despite the sophisticated methods in use today, substantial variations between methods still exist. A concerted effort toward standardization of 1,25(OH) 2 D measurement is needed to ensure accurate and reliable results across laboratories and methods.

Background: An accurate measurement of the glomerular filtration rate (GFR) is essential for detecting renal insufficiency in living kidney donors. Iohexol is a "near-ideal" exogenous filtration marker for GFR measurements that has attracted increasing interest in clinical practice because it is non-toxic, non-radioactive, readily available, and easy to measure. In this study, we aimed to set up a laboratory test to conveniently assess the plasma clearance of iohexol in living kidney donors., Methods: A workflow was established in the institution's infusion clinic to administer iohexol and to collect three timed blood samples from renal transplant donors. Iohexol was thereafter measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The serum proteins were precipitated and the supernatant containing iohexol was diluted prior to the LC-MS/MS analysis. The LC-MS/MS method was developed on a Thermo Vanquish UHPLC coupled with a TSQ Endura triple quadruple mass spectrometer with a total run time of 2.5 min. The analytical performance of the method was assessed., Results: The LC-MS/MS method demonstrated a good analytical performance. To calculate the iohexol clearance rate and the GFR, automated data integration and a result calculation were accomplished by using a custom Python script. Automated result reporting was achieved using a laboratory informatics system (LIS) vendor's direct media interface., Conclusions: We developed and implemented a laboratory test to assess the plasma clearance of iohexol. A workflow was established in the hospital to reliably measure the GFR in living kidney donors, with a potential to be further expanded into other areas where an accurate GFR measurement is needed.

Background: The circulating concentration of 1α,25-dihydroxyvitamin D [1α,25(OH)2D] is very low, and the presence of multiple isomers may lead to inaccurate quantitation if not separated prior to analysis. Antibody-based immunoextraction procedures are sometimes used to remove structurally related isomers of 1α,25(OH)2D prior to an LC-MS/MS analysis. However, immunoextraction increases sample preparation time and cost. In addition, some dihydroxyvitamin D metabolites are not completely removed by immunoextraction., Method: We developed an HPLC method using a phenyl-hexyl column to investigate interfering isomers of 1α,25(OH)2D., Result: Using this method, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) derivatization product of 1α,25(OH)2D was found to be present as 2 epimers, which were separated chromatographically with an area ratio of 2:1. PTAD derivatized metabolite of 25-hydroxyvitamin D3 [i.e., 4β,25-dihydroxyvitamin D3 (4β,25(OH)2D3)] eluted out between 6R and 6S epimers of derivatized 1α,25(OH)2D3. If not chromatographically resolved, 4β,25(OH)2D can affect 1α,25(OH)2D quantitation. In a method comparison study, it was found that the presence of 4β,25(OH)2D produced positive bias up to 127% on 1α,25(OH)2D3 quantitation., Conclusion: The LC-MS/MS method we developed without an immunoextraction procedure was able to resolve the major interference peak from 1α,25(OH)2D and achieved reliable quantitation of 1α,25(OH)2D., (© American Association for Clinical Chemistry 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Herein, we present 2 cases referred to the North Carolina Office of the Chief Medical Examiner (NC OCME) in which ethanol results reported by different hospital laboratories, using alcohol dehydrogenase (ADH)-based assays, were positive, whereas results of headspace gas chromatography testing performed in the NC OCME laboratory were negative. Literature reports suggest that false-positive ethanol measurements from ADH-based assays can occur when a combination of elevated lactate and lactate dehydrogenase (LD) are present in the specimen. The results were reported in perimortem specimens collected from 2 children with unrelated medical conditions. The cases and associated clinical parameters are considered based on the lactate/LD explanation for the false-positive results, to facilitate the recognition of circumstances that can produce erroneous serum ethanol results., (© American Society for Clinical Pathology 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Urine drug testing is an essential component in the evaluation and management of individuals with opioid use disorders, including those on buprenorphine or methadone maintenance therapies. Notwithstanding its centrality in adherence monitoring, studies have shown that clinicians have important knowledge deficiencies regarding the ordering and interpretation of drug tests. In this review, we discuss the scope and frequency of testing, the advantages and disadvantages of immunoassay- (presumptive) and liquid chromatograph-mass spectrometry- (definitive) based techniques, indications for definitive testing, medical necessity, and other considerations. Optimal use of urine drug testing depends on collaboration between clinicians and laboratory scientists., (© American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Urine drug testing (UDT) has become an essential component in the management of patients prescribed opioid analgesics for the treatment of chronic non-malignant pain. Several laboratory methods are available to monitor adherence with the pharmacological regimen and abstinence from illicit or unauthorized medications. Immunochemical screening methods are rapid and economical, but they have limitations, including lack of specificity, and confirmatory methods are often necessary to verify presumptive positive results. We analyzed the results of confirmatory assays in an outpatient setting to determine the predictive value of presumptive positive urine drug screen results using an automated immunoassay for eight common drugs or drug classes. Positive predictive values (PPVs), in descending order, were as follows: cannabinoids (100%), cocaine (100%), opiates (86.8%), benzodiazepines (74.6%), oxycodone (67.6%), methadone (44.1%) and amphetamines (9.3%). The number of positive barbiturate results was too small to be included in the statistical analysis., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Urine drug testing (UDT) is an emerging standard of care in the evaluation and treatment of chronic non-cancer pain patients with opioid analgesics. UDT may be used both to verify adherence with the opioid analgesic regimen and to monitor abstinence from non-prescribed or illicit controlled substances. In the former scenario, it is vital to determine whether the drug is present in the urine, even at low concentrations, because failure to detect the drug may lead to accusations of opioid abuse or diversion. Opiate immunoassays typically are developed to detect morphine and are most sensitive to morphine and codeine. Although many opiate immunoassays also detect hydrocodone (HC) and/or hydromorphone (HM), sensitivities for these analytes are often much lower, increasing the possibility of negative screening results when the drug is present in the urine. We selected 112 urine specimens from patients who had been prescribed HC or hydromorphone but were presumptive negative by the Roche Online DAT Opiate II™ urine drug screening assay, which is calibrated to 300 ng/mL morphine. Using a GC/MS confirmatory method with a detection limit of 50 ng/mL both for HC and for HM, one or both of these opiates were detected in 81 (72.3%) of the urine specimens. Examination of the raw data from these presumptive negative opiate screens revealed that, in many cases, the turbidity signal was greater than the signal obtained for the negative control, but less than the signal for the 300 ng/mL (morphine) threshold calibrator. A receiver operating characteristic curve generated for the reciprocal of the ratio of turbidity measurements in the patient specimens and negative (drug-free) controls, against the presence or absence of HC and/or HM by confirmatory analyses, produced an area under the curve of 0.910. We conclude that this opiate immunoassay has sufficient sensitivity to detect HC and/or HM in some urine specimens that screen presumptive negative for these commonly prescribed opiates at the established threshold., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Urine drug testing (UDT) services are provided by a variety of clinical, forensic, and reference/specialty laboratories. These UDT services differ based on the principal activity of the laboratory. Clinical laboratories provide testing primarily focused on medical care (eg, emergency care, inpatients, and outpatient clinics), whereas forensic laboratories perform toxicology tests related to postmortem and criminal investigations, and drug-free workplace programs. Some laboratories now provide UDT specifically designed for monitoring patients on chronic opioid therapy. Accreditation programs for clinical laboratories have existed for nearly half a century, and a federal certification program for drug-testing laboratories was established in the 1980s. Standards of practice for forensic toxicology services other than workplace drug testing have been established in recent years. However, no accreditation program currently exists for UDT in pain management, and this review considers several aspects of laboratory accreditation and certification relevant to toxicology services, with the intention to provide guidance to clinicians in their selection of the appropriate laboratory for UDT surveillance of their patients on opioid therapy.

Purpose: The aim of this study was to validate the formula derived by Purssell et al. that relates blood ethanol concentration to the osmolar gap and determine the best coefficient for use in the formula. The osmolar gap is often used to help diagnose toxic alcohol poisoning when direct measurements are not available., Methodology: Part I of the study consisted of a retrospective review of 603 emergency department patients who had a concurrent ethanol, basic metabolic panel and a serum osmolality results available. Estimated osmolarity (excluding ethanol) was calculated using a standard formula. The osmolar gap was determined by subtracting estimated osmolarity from the actual osmolality measured by freezing point depression. The relationship between the osmolar gap and the measured ethanol concentration was assessed by linear regression analysis. In Part II of this study, predetermined amounts of ethanol were added to aliquots of plasma and the estimated and calculated osmolarities were subjected to linear regression analysis., Results: In the cases of 603 patients included in Part I of the study, the median ethanol concentration in these patients was 166 mg/dL (Q1: 90, Q3: 254) and the range ethanol concentrations was 10-644 mg/dL. The mean serum osmolality was 338 mOsm/kg (SD: 30) and a range of 244-450 mOsm/kg. The mean osmolar gap was 47 (SD: 29) and a range of - 15 to 55. There was a significant proportional relationship between ethanol concentration and osmolar gap (r(2) = 0.9882). The slope of the linear regression line was 0.2498 (95% CI: 0.2472-0.2524). The slope of the linear regression line derived from the data in Part II of the study was 0.2445 (95% CI: 0.2410-0.2480)., Conclusions: The results of our study are in fairly close agreement with previous studies that used smaller samples and suggest that an accurate conversion factor for estimating the contribution of ethanol to the osmolar gap is [Ethanol (mg/dL)]/4.0.

The aim of this study was to investigate the accuracy of imprint cytology (IC) of breast core biopsy under ultrasound guidance and to assess the value of a rapid on-site preliminary diagnosis of breast lesions. A total of 437 breast core needle biopsies under ultrasound guidance with touch imprint cytology, histology, and final diagnosis were reviewed. These cases were collected from archived files at our institution. Of 437 core biopsies, IC classified 241 (55%) as benign; 22 (5%) as probably benign; 28 (6%) as probably malignant; 107 (25%) as malignant; and 39 (9%) as inadequate for IC diagnosis. Histological classifications for the 437 cases were: 285 (65%) benign; 132 (30%) malignant; 16 (4%) atypical hyperplasia; and 4 (1%) inadequate specimen. The overall sensitivity and specificity indices of IC were 95% and 96%, respectively, for benign and probably benign lesions vs malignant and probably malignant breast lesions. The overall positive and negative predictive values were 91% and 97%, respectively. The overall accuracy was 95% (379 of 398 cases, excluding specimens inadequate for IC diagnosis). IC of ultrasound-guided core needle biopsy provides a rapid and reliable preliminary diagnosis for breast lesions; it also serves as a means to verify the adequacy of biopsy specimens and to optimize the biopsy procedure. Use of IC may reduce anxiety in patients with benign lesions and expedite the diagnosis and assessment of treatment options in patients with breast cancer.

To determine the degree of ethanol absorption and the resultant formation and urinary excretion of its conjugated metabolites following intensive use of high ethanol content mouthwash, 10 subjects gargled with Listerine(®) antiseptic 4 times daily for 3¼ days. First morning void urine specimens were collected on each of the four study days and post-gargle specimens were collected at 2, 4, and 6 h after the final gargle of the study. Urine ethanol, ethyl glucuronide (EtG), ethyl sulfate (EtS), and creatinine were measured. Ethanol was below the positive threshold of 20 mg/dL in all of the urine specimens. EtG was undetectable in all pre-study urine specimens, but two pre-study specimens had detectable EtS (6 and 82 ng/mL; 16 and 83 μg/g creatinine). Only one specimen contained detectable EtG (173 ng/mL; 117 μg/g creatinine). EtS was detected in the urine of seven study subjects, but was not detected in the single specimen that had detectable EtG. The maximum EtS concentrations were 104 ng/mL and 112 μg/g creatinine (in different subjects). Three subjects produced a total of eight (non-baseline) urinary EtS concentrations above 50 ng/mL or 50 μg/g creatinine and three EtS concentrations exceeding 100 ng/mL or 100 μg/g creatinine. In patients being monitored for ethanol use by urinary EtG and EtS concentrations, currently accepted EtG and EtS cutoffs of 500 ng/mL are adequate to distinguish between ethanol consumption and four times daily use of high ethanol content mouthwash.

To assess the degree of ethanol absorption and subsequent formation of urinary ethyl glucuronide (EtG) and ethyl sulfate (EtS) following sustained application of hand sanitizer, 11 volunteers cleansed their hands with Purell(™) hand sanitizer (62% ethanol) every 5 min for 10 h on three consecutive days. Urine specimens were obtained at the beginning and end of each day of the study, and on the morning of the fourth day. Urinary creatinine, ethanol, EtG, and EtS concentrations were measured. EtG was undetectable in all pre-study urine specimens, but two pre-study specimens had detectable EtS (73 and 37 ng/mL). None of the pre-study specimens had detectable ethanol. The maximum EtG and EtS concentrations over the course of the study were 2001 and 84 ng/mL, respectively, and nearly all EtG- and EtS-positive urine specimens were collected at the conclusion of the individual study days. Only two specimens had detectable EtG at the beginning of any study day (96 and 139 ng/mL), and only one specimen had detectable EtS at the beginning of a study day (64 ng/mL), in addition to the two with detectable EtS prior to the study. Creatinine-adjusted maximum EtG and EtS concentrations were 1998 and 94 μg/g creatinine, respectively. In patients being monitored for ethanol use by urinary EtG concentrations, currently accepted EtG cutoffs do not distinguish between ethanol consumption and incidental exposures, particularly when urine specimens are obtained shortly after sustained use of ethanolcontaining hand sanitizer. Our data suggest that EtS may be an important complementary biomarker in distinguishing ethanol consumption from dermal exposure.

Tangier disease is a rare genetic disorder of lipid metabolism characterized by low concentrations of plasma high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol with normal or elevated levels of triglycerides. In this case report we describe a patient with diabetes who experienced an episode of urosepsis with a plasma lipid profile resembling Tangier disease. Experimental evidence in the literature suggests that similar lipid changes may occur due to cytokines released during sepsis. Clinicians should be aware of these changes to avoid misdiagnosis of lipid disorders.

The terms 'false-positive' and 'false-negative' are widely used in discussions of urine drug test (UDT) results. These terms are inadequate because they are used in different ways by physicians and laboratory professionals and they are too narrow to encompass the larger universe of potentially misleading, inappropriate and unexpected drug test results. This larger universe, while not solely comprised of technically 'true' or 'false' positive or negative test results, presents comparable interpretive challenges with corresponding clinical implications. In this review, we propose the terms 'potentially inappropriate' positive or negative test results in reference to UDT results that are ambiguous or unexpected and subject to misinterpretation. Causes of potentially inappropriate positive UDT results include in vivo metabolic conversions of a drug, exposure to nonillicit sources of a drug and laboratory error. Causes of potentially inappropriate negative UDT results include limited assay specificity, absence of drug in the urine, presence of drug in the urine, but below established assay cutoff, specimen manipulation and laboratory error. Clinical UDT interpretation is a complicated task requiring knowledge of recent prescription, over-the-counter and herbal drug administration, drug metabolism and analytical sensitivities and specificities.

Amoxicillin has been causally linked in the lay and medical literature to false-positive urine drug screens for cocaine metabolites. An exhaustive search of the peer-reviewed medical literature revealed no data to support this link. We hypothesized that amoxicillin does not cause false-positive urine drug screens for cocaine metabolites. To test this hypothesis, we examined the urine of 33 subjects administered a course of amoxicillin, subjecting the specimens to four common urine screening immunoassays. Thirty-one specimens were negative for the cocaine metabolite, benzoylecgonine (BE), by all four screening methods; two were positive for BE by all four screening methods. Both positive specimens were confirmed by gas chromatography-mass spectrometry (GC-MS) for the presence of BE at > 150 ng/mL. Three specimens that screened negative, but produced absorbance values that were intermediate between negative and positive controls, were submitted for GC-MS analysis; BE was detected in all three specimens at concentrations of 54, 94, and 119 ng/mL. Twenty-eight specimens produced screening results indistinguishable from negative controls. Within the limitations of the study design, we conclude that amoxicillin is unlikely to produce false-positive urine screens for cocaine metabolites.

Objective: To determine the proficiency in urine drug test interpretation among family medicine physicians who order these tests to monitor adherence in their patients on chronic opioid therapy., Methods: A seven-question instrument, consisting of six, five-option, single-best-answer multiple choice questions and one yes/no question was administered to 80 family medicine physicians attending a University of Kentucky Family Medicine Review Course. We calculated frequencies and performed chi2 analyses to examine bivariate associations between urine drug test utilization and interpretive knowledge., Results: The instrument was completed by 60/80 (75 percent) of eligible physicians (44 order urine drug testing; 16 do not). None of the physicians who order urine drug testing answered more than five of the seven questions correctly, and only 20 percent answered more than half correctly. Physicians who order urine drug testing performed better than physicians who do not order urine drug testing on only four of the seven questions, although there were no statistically significant differences between the groups on any question., Conclusions: Family medicine physicians who order urine drug testing to monitor their patients on chronic opioid therapy are not proficient in their interpretation. This study highlights the need for improved physician education in this area. It is imperative for physicians to work closely with certified laboratory professionals when ordering and interpreting urine drug tests.

Urine drug testing (UDT) has become an essential feature of pain management, as physicians seek to verify adherence to prescribed opioid regimens and to detect the use of illicit or unauthorized licit drugs. Results of urine drug tests have important consequences in regard to therapeutic decisions and the trust between physician and patient. However, reliance on UDT to confirm adherence can be problematic if the results are not interpreted correctly, and evidence suggests that many physicians lack an adequate understanding of the complexities of UDT and the factors that can affect test results. These factors include metabolic conversion between drugs, genetic variations in drug metabolism, the sensitivity and specificity of the analytical method for a particular drug or metabolite, and the effects of intentional and unintentional interferants. In this review, we focus on the technical features and limitations of analytical methods used for detecting drugs or their metabolites in urine, the statistical constructs that are pertinent to ordering UDT and interpreting test results, and the application of these concepts to the clinical monitoring of patients maintained on chronic opioid therapy.

Background: Acetaminophen was falsely detected in the plasma of a severely jaundiced patient, and a methodologic interference from bilirubin was suspected., Methods: Acetaminophen was measured by an enzymatic method (GDS Diagnostics). The putative bilirubin interference was investigated in 12 hyperbilirubinemic specimens and in bilirubin linearity calibrators. The analytical method was modified to correct for background absorbance at a second wavelength. Hyperbilirubinemic specimens were fortified with acetaminophen to assess the effect of the interference on acetaminophen measurements., Results: Acetaminophen was detected in 12 specimens from hyperbilirubinemic patients without a history of recent acetaminophen exposure. Dilution of hyperbilirubinemic specimens produced a nonproportional decrease in apparent acetaminophen concentrations, and no acetaminophen was detected when bilirubin was <50 mg/L. Background correction at a second wavelength failed to compensate for the interference. Although erroneous acetaminophen concentrations were detected in all specimens with high bilirubin, acetaminophen measurements in fortified specimens were accurate., Conclusion: The data are consistent with bilirubin interference in the enzymatic and/or chromogenic reactions involved in the acetaminophen method.

Discordant results were observed for serum CA-125 (carbohydrate antigen-125) assays in a patient who was monitored for recurrence of ovarian cancer. Serum CA-125 levels in this patient were normal when measured in one laboratory, but >5-times the upper limit of normal (35 U/mL) when measured in another laboratory. Both laboratories used dual antibody heterogeneous immunoassays, but from different manufacturers. Cross-linking heterophilic antibodies were suspected as a cause of the discrepancy, but the interference was not alleviated after 10-fold dilution. Assay of the patient's serum for human anti-mouse antibodies was positive, but only slightly above the reference range. Addition of blocking antibodies eliminated the interference, showing that human anti-mouse antibodies were the cause of the discrepant CA-125 results. These findings indicate that relatively low concentrations of human anti-mouse antibodies can cause significant interference in two-site immunoassays.

A method was developed for measuring cocaine and its metabolites, benzoylecgonine, ecgonine methyl ester, norcocaine, ecgonine ethyl ester, cocaethylene, and m-hydroxybenzoylecgonine, in breast milk by gas chromatography/mass spectrometry. Limits of detection for this method ranged from 2.5 to 10 ng/mL, and limits of quantitation ranged from 5 to 50 ng/mL. For each of the compounds measured by this method, linear response was demonstrated to 750 ng/mL. Breast milk was collected from 11 mothers who admitted to drug use during pregnancy and ten drug-free volunteers serving as control subjects. Cocaine was detected in six of the specimens obtained from drug-exposed subjects, and in none of the drug-free control subjects. In breast milk specimens where cocaine and one or more of its metabolites were detected, the concentration of parent compound was greater than any of the metabolites. The highest cocaine concentration found was over 12 microg/mL. Breast-fed infants of cocaine abusing mothers may be exposed to significant amounts of drug orally.

Respect for persons, beneficence, and justice are the cardinal principles that guide the ethical conduct of research on humans. Past abuses of human research subjects prompted medical organizations and governmental agencies to develop guidelines that ensure the protection of human research subjects. Human research funded by the U.S. government is strictly regulated, and Institutional Review Board approval of the experimental protocol is required prior to the award. Under limited circumstances, human research may be exempted from review, or review may be expedited. Research involving specimens submitted for pathological examination or diagnostic studies sometimes qualifies for these special categories of limited review. Academic pathologists and laboratorians should be aware of the regulations that apply to research on human subjects.

In the course of a clinical comparison involving 204 parallel total creatine kinase (CK), creatine kinase-MB isoenzyme (CK-MB), and cardiac troponin I (cTnI) measurements, 12 patients were identified in whom cTnI was elevated while total CK was normal, as well as 2 patients in whom CK-MB was elevated while cTnI was normal. CK-MB relative index was elevated in 6 of the twelve cTnI-positive patients with normal total CK; only 2 of these patients had a discharge diagnosis of acute myocardial infarction (AMI). All of the 12 patients in this group had medical conditions that are associated with greater risk for acute cardiac events. Both patients with normal cTnI but elevated total CK and CK-MB index had chronic renal insufficiency; one of these patients had a positive stress test and a diagnosis of AMI. The other cTnI-negative patient died 2 days after admission, and autopsy revealed evidence of ischemic changes, but not acute infarction. Significant differences were apparent between traditional CK-MB results and cTnI measurements. Using total CK elevation as a prerequisite for subsequent CK-MB measurement may limit the clinical sensitivity of this enzyme marker for detecting subacute ischemic damage to the myocardium. Elevated total CK and CK-MB isoenzyme without corresponding elevations in cTnI, on the other hand, may reflect changes in enzyme elimination kinetics due to renal failure, or cross-reactivity of the cTnI assay with non-cardiac antigens.

Testing for drugs of abuse in urine is commonplace in emergency departments and neonatal units. However, the clinical sensitivity of immunochemical screening methods is limited by the threshold concentrations used to distinguish between positive and negative specimens. Immunochemical screening methods for cocaine metabolite (benzoylecgonine), cannabinoids, and opiates in urine were recalibrated to detect drugs at lower threshold concentrations. The precision and linearity of the signals at the modified thresholds were verified by diluting drug-positive urine specimens to concentrations below the conventional cutoff concentration and measuring the rate signals in triplicate. To assess the clinical performance of the modified methods, specimens that tested negative using the unmodified assays were re-screened at the lower threshold, and specimens that re-screened positive were submitted for gas chromatographic/mass spectrometric (GC/MS) confirmation. Reproducibility of sub-threshold measurements was comparable to the unmodified assays, and rate separations between successive dilutions were sufficient to give semi-quantitative results. Using the lower thresholds, drugs were detected in 4-5% of the subjects that had screened negative at the conventional threshold concentration. GC/MS analysis confirmed the presence of cannabinoids and cocaine metabolite in 74% and 84%, respectively, of urine specimens that re-screened positive. Morphine, codeine, hydromorphone, or hydrocodone was detected by GC/MS analysis in 31% of opiate-positive re-screens.

Technical improvements in the application of molecular biology methods to detection and identification of specific nucleic acid sequences have resulted in more widespread incorporation of these techniques into clinical laboratory services. Gene amplification techniques have been refined and automated, making possible the rapid and economical detection of attomolar and smaller quantities of genetic material. Of particular benefit to clinical chemistry applications is the remarkable specificity of a DNA probe for its complementary sequence. Applications of molecular biology techniques in clinical chemistry include diagnosis of infectious, neoplastic, genetic, and hematological diseases. In addition, the use of oligonucleotides as molecular recognition probes may provide new analytical strategies for a wide range of diagnostic applications that currently rely on antibodies.

The increased use of cocaine by women of child-bearing age has left many health care scientists searching for improved methods of detecting prenatal cocaine exposure. To that end, a study of the determination of cocaine and its metabolites in amniotic fluid and umbilical cord tissue was undertaken. Amniotic fluid (n = 32) and umbilical cord tissue (n = 70) specimens were collected from pregnant subjects admitted to labor and delivery at Shands Hospital at the University of Florida (Gainesville, FL). Subjects were interviewed regarding drug use during each trimester. Subjects reporting cocaine use were designated as target subjects, and those denying use were control subjects. The specimens were subjected to solid-phase extraction and analyzed for cocaine and its metabolites by gas chromatography-mass spectrometry. Cocaine analytes (predominantly benzoylecgonine) were detected in 28.1 and 18.5% of the amniotic fluid and umbilical cord tissue specimens, respectively. Other cocaine analytes frequently detected included ecgonine methyl ester and m-hydroxy-benzoylecgonine in amniotic fluid specimens and ecgonine methyl ester, norcocaine, and m-hydroxybenzoylecgonine in umbilical cord tissue specimens. This study has shown that cocaine and its metabolites are readily detected in specimens of maternal and fetal origin.