The stability of ethanol in human whole blood controls: an interlaboratory evaluation.

Sterile whole human blood control materials were commercially prepared in batches containing anticoagulants and preservatives and approximately 90, 150, and 230 mg/dL ethanol with and without 0.3% (w/v) sodium azide. Aliquots in sealed vials were stored by the manufacturer at 2-8 degrees C until shipped monthly to three academic toxicology laboratories that analyzed them in duplicate by gas chromatographic headspace methods at monthly intervals for one year. The resulting data were pooled, and grand mean values were statistically analyzed to determine the respective alcohol stability in these azide-free and azide-containing blood samples. Azide-containing blood samples showed no alcohol losses during the 1-year period. Azide-free blood containing 1.0% (w/v) sodium fluoride and anticoagulants had small alcohol decreases over time, the total losses after one year being less than 5% of the original alcohol concentrations. The initial alcohol concentration of approximately 40 mg/dL also did not change during storage of additional samples of azide-free blood for one month at 4 degrees C. We concluded that addition of sodium azide to performance-test and control blood specimens for alcohol analysis is unnecessary and unwarranted and that alcohol losses in such blood samples can be minimized by simple appropriate treatments and conditions.

Regulation of brain m calpain Ca2+ sensitivity by mixtures of membrane lipids: activation at intracellular Ca2+ level.

Combinations of certain phospholipids and gangliosides increase the specific activity of m calpain and can activate m calpain at 1 to 10 microM Ca2+ concentration. However, this level of calcium is still greater than the normal intracellular calcium level. We have used combinations of lipids to demonstrate the m calpain activity at the physiological Ca2+ level. GD1a (100 microM) and cerebroside (Cerb; 750 microM; 1:7.5) mixture was the most effective. At 0.5 microM to 1.0 microM Ca2+ concentrations, 15-20% of the maximal activity was detected for the purified myelin and cytosolic m calpains. Other combinations were GD1a (100 microM), GM1 (100 microM), Cerb (750 microM), sulfatide (Sulf; 750 microM), and phosphatidylinositol (PI; 300 microM) at a ratio of 1:1: 7.5:7.5:3, respectively. These lipid mixtures stimulated calpain activity at three- to tenfold less calcium concentration than control. The other mixtures, including GD1a:Sulf (1:9) > GD1a:PI (1:4) > PI:Sulf (1:5) > Cerb:Sulf (1:5) and PI:Cerb (1:2.5), also stimulated calpain activity at 1.0 microM Ca2+ concentration. Triton X-100, oxidized glutathione (GSSG), and calpain activator did not affect the Ca2+ requirement. Liposomes containing GD1a, Cerb, and m calpain also showed recognizable calpain activity at a significantly reduced Ca2+ concentration (0.4 microM), confirming the glycolipid-mediated enzyme modulation. These studies indicate that specific lipid mixtures can stimulate m calpain activity at an intracellular level of Ca2+.

Analytical evaluation of methods for serum creatine kinase-MB. Electrophoresis, immunoinhibition and solid phase separation.

The purpose of this study was to evaluate immunoassay methods for the measurement of serum cardiac creatine kinase isoenzyme (CK-MB) with respect to sensitivity and specificity. The CK-MB electrophoretic assay (Helena Laboratories) was used as the reference. Two principles of immunoassay were included in the evaluation,--immunoinhibition and solid phase separation. The direct immunoinhibition techniques were from Beckman Instruments (CKMB reagent) and DuPont Medical Products (CKMB). Three solid phase separation techniques were from Abbott Laboratories (IMx CKMB), DuPont (acaPlus MCKMB), and Tosoh Medics Inc. (AIA-Pack CKMB). The electrophoretic method for separation of the CK isoenzymes has good specificity but lacks sensitivity for CK-MB in low concentrations. The immunoinhibition methods lack specificity and correlate poorly with the electrophoresis method and with the solid phase methods. The solid phase separation techniques are highly sensitive and show an excellent correlation with electrophoresis when based on specificity. The solid phase separation methods correlate well with each other.

High-resolution proton nuclear magnetic resonance spectroscopy in the detection of low molecular weight volatiles.

Proton nuclear magnetic resonance spectroscopy (1H MRS) has been used to identify ethanol in vivo and to detect other exogenous low molecular weight volatiles in human serum. 1H MRS was used to detect and quantitate 15 human sera containing various concentrations and combinations of ethanol, isopropanol, acetone, and methanol as previously quantitated by headspace gas chromatography. The 1H MRS method was linear for each alcohol. The lowest detectable alcohol concentration was 15 mg/L (peak height equal to three times the signal-to-noise ratio), and 30 mg/L (+/- 10% relative standard deviation) was the lowest level reproducibly quantitated. Within-run and day-to-day coefficients of variation (CV) ranged from 0.8 to 2.0% and 0.9 to 1.2%, respectively, for methanol; 0.5 to 1.9% and 0.6 to 1.3% for acetone; and 0.5 to 1.6% and 0.3 to 2.2% for isopropanol. In all cases, the lowest CVs for a particular compound were obtained for the highest measured concentration (1500 mg/L), and the highest CVs were observed for the lowest concentration (250 mg/L). The 1H MRS method for detection of these volatiles does not require sample pretreatment and is nondestructive, which allows for further analysis by other methods.

High-resolution proton nuclear magnetic resonance spectroscopy in the detection and quantitation of ethanol in human serum.

A precise, accurate, and nondestructive method for the detection and quantitation of serum ethanol in humans using proton (1H) nuclear magnetic resonance spectroscopy (MRS) was developed. The 1H MRS method was linear within the range of 30-1500 mg/L. The lowest detectable ethanol concentration was 15 mg/L, with 30 mg/L being the lowest level reproducibly quantitated. Within-run and day-to-day coefficients of variation (CV) ranged from 0.6 to 2.7% and 0.5 to 3.5%, respectively. The excellent day-to-day CVs indicate a negligible loss of ethanol due to volatilization during analysis. Fifteen human serum samples found to be negative for ethanol by headspace gas chromatography (HSGC) had no ethanol as detected by 1H MRS. Twenty-eight human serum samples with ethanol concentrations (determined by HSGC) ranging from 370 to 4440 mg/L were accurately reproduced by 1H MRS. The 1H MRS method required no pretreatment and was nondestructive, thereby allowing for further analysis by confirmatory methods.

Irradiation effect on aging red blood cells.

Irradiation of stored red blood cells (RBC) is increasingly utilized for patients who are immunosuppressed or on chemotherapeutic regimens. With the growing demand for irradiated cellular blood products, there has been an increasing need for transfusion services to store previously irradiated blood until needed for transfusion. The effect of irradiation on aging stored RBC has not been studied to date. Five units each of group A, RBC collected in CPD-Adsol (AS-1) with a prior shelf-life of 10, 20, 30, and 40 days, respectively, were divided equally utilizing a sterile docking device and stored at 1 to 6 degrees C. Baseline samples from each bag were obtained for the measurement of extracellular potassium (K+), plasma free hemoglobin (PFH), total lactate dehydrogenase (LD), and erythrocyte 2,3-DPG activity. One of each pair received 2,000 rads of gamma irradiation. Samples were obtained at 3 and 7 days post-irradiation, and multiples of 7 days until expiration. All irradiated units reached a state of K+ equilibrium at 60 to 70 mmol per L irrespective of the length of previous storage with an inverse relationship of RBC age at irradiation and the time required to reach the state of equilibrium. Increased K+ leakage from irradiated aging RBC suggests the need for including in vivo studies of cell survival to establish a post-irradiation storage life. Length of storage prior to irradiation had no effect on PFH, LD activity, and 2,3-diphosphoglycerate (2,3-DPG) activity compared to paired controls.

Serum alpha-fetoprotein: II. Clinical evaluation of two automated immunoassays for maternal serum screening.

A prospective clinical evaluation is reported for analysis of maternal serum alpha fetoprotein (s.AFP) testing by recently developed enzymeimmunoassay automated systems. The reference method for the study was a manual competitive binding radioimmunoassay which has been utilized by us in the Maternal Serum AFP Screening Program at the Medical University of South Carolina for the past five years. The patients were classified by weeks of gestation, 16 to 20. Mean concentrations of s.AFP increased with the increase in gestational week for each method; calculated multiples of median (MoM) values remained relatively constant regardless of the week of gestation. Mean and median concentrations of s.AFP measured by the radioimmunoassay and the coated tube enzymeimmunoassay methods showed close agreement, indicating the direct transferability of the methods for clinical purposes. The microparticle capture enzymeimmunoassay yielded higher results of s.AFP indicating the necessity for a correction if the method transfer is to be considered for sequential patient testing. (Multiples of median values for each method were uncorrected for maternal age, weight, or diabetes mellitus; lack of correction would not affect comparison of methods for clinical application.

Serum alpha-fetoprotein: I. Evaluation of quantitative assays adapted to automated immunoassay systems.

Serum alpha-fetoprotein (s.AFP) has been established as a useful tool in monitoring of high-risk pregnancies, as an indicator of fetal neural tube defects, and has been used as an adjunct tumor marker and for monitoring therapeutic efficacy in the treatment of certain tumors. To date, the methods for measuring s.AFP are based upon the immunologic principle and are manual methods. The purpose here is to relate the evaluation of two automated systems for the assay of s.AFP. The automated systems are based upon the following immunoassay methods: a microparticle capture enzyme separation and final quantitation by reflectance fluorescence, and a solid phase 'sandwich' separation coupled with enzyme activity measurement (EIA). The reference method is a competitive binding radioimmunoassay. It has been found by us that the automated methods directly transfer analytically with the manual assay. All methods are referenced to the same standard (WHO 1st Intl. Std. for AFP 72/225).

Effects of irradiation on red cells stored in CPDA-1 and CPD-ADSOL (AS-1).

Red blood cells (pRBC) collected in citrate, phosphate, dextrose, adenine-formula 1 (CPDA-1) and citrate, phosphate, dextrose-adenine, manitol saline solution (CPD-ADSOL [AS-1]) anticoagulants are increasingly being stored for variable periods in transfusion service inventories following irradiation. While anecdotal reports of increased K+ following irradiation and storage have recently appeared in the literature, concomitant in vitro biochemical changes resulting from differences in anticoagulants have not been reported. Utilizing two venipunctures, two units each of 225 mL of blood from five volunteers were collected in anticoagulant-adjusted CPDA-1 and AS-1 bags. Within two hours of collection, each unit was equally divided. One of each pair was irradiated (2000 rads). Samples were analyzed on Days 0, 1, 3, 7, and every seven days to expiration. Irradiation resulted in a 2.3 fold increase in K+ during the first seven days of storage for both anticoagulants, although significantly greater K+ levels were observed in the CPDA-1 pairs compared to the AS-1 pairs. Comparison of glucose utilization, plasma free hemoglobin, 2,3-diphosphoglycerate (2,3-DPG) and lactate dehydrogenase between control and irradiated CPDA-1 and AS-1 pairs and between anticoagulants were documented.

Reassessment of cross-reactivity of spironolactone metabolites with four digoxin immunoassays.

Spironolactone and one of its metabolites, canrenone, cross-react with some digoxin immunoassays to result in erroneous serum digoxin concentrations. Recently, additional compounds, 7-alpha-thiomethylspirolactone (7-a-TMS) and 6-beta-hydroxy-7-alpha-thiomethylspirolactone (6-B-OH-7-a-TMS), have been reported to be quantitatively important metabolites of spironolactone. This study was initiated to evaluate the cross-reactivity of these metabolites, canrenone, and spironolactone with four different digoxin immunoassays. Blank serum was spiked with each compound to yield concentrations reported to occur in vivo. Samples were analyzed in duplicate by each of the following immunoassays: fluorescence polarization immunoassay (FPIA); affinity-column-mediated immunoassay (ACMIA); radioimmunoassay (RIA); and enzyme immunoassay (EIA). The 7-a-TMS metabolite cross-reacted with both the RIA and ACMIA methods. Apparent digoxin concentrations were as great as 0.39 ng/ml for this metabolite at the highest concentration evaluated, 600 ng/ml. At the lowest concentrations evaluated with the 7-a-TMS metabolite, 50 ng/ml, apparent digoxin concentrations as high as 0.28 ng/ml were reported. The 6-B-OH-7-a-TMS metabolite did not cross-react to a significant extent with any of immunoassays studied. Canrenone cross-reacted with the ACMIA method at a concentration of 100 ng/ml. The EIA method exhibited no apparent cross-reactivity with any of the compounds, whereas the FPIA method exhibited minimal cross-reactivity. The results of this study indicate that the 7-a-TMS metabolite cross-reacts to a significant extent with some immunoassays; however, this is not true for the 6-B-OH-7-a-TMS metabolite.

Effect of data collection method on results of serum digoxin concentration audit.

The appropriateness of serum digoxin concentration (SDC) orders was evaluated with respect to indication for use, sampling time, and action taken by physicians when the reported SDC was out of the normal therapeutic range; the effect of the two data-collection methods used (retrospective and concurrent audits) on the results was studied. Criteria for the appropriate use of SDCs were approved by the medical staff through the pharmacy and therapeutics committee. Patients on adult medicine services were entered into the study as daily SDC determinations were reported by the clinical laboratory. Most of the SDCs were evaluated using approved criteria by primary pharmacist clinicians who were concurrently monitoring drug therapy and participating with the treatment team. A retrospective audit of the same patients was conducted, using only chart review. A total of 134 SDCs involving 78 patients were evaluated. Concurrent-audit results indicated that 18.7% of the SDCs were ordered without an appropriate indication, 16.4% were sampled incorrectly with respect to proper timing, and 8.2% did not result in dosage adjustments when indicated. With respect to appropriate sampling time and overall use of SDCs, significantly more SDCs met the standards under concurrent audit than under retrospective audit. The retrospective chart review method of auditing may not detect as much pertinent information as is desirable.

Definitive characterization of uric acid as an interferent in peroxidase indicator reactions and a proposed mechanism of action.

We have characterized and identified uric acid as an interferent to peroxidase catalyzed reactions where hydrogen peroxide is generated at relatively low concentrations. The implications of these findings are important for those utilizing peroxidase as an indicator reaction where low primary substrate concentrations require their preliminary extraction or chemical modification. We have shown that the elimination of uric acid as an interferent from biologic fluid obviates the necessity for such treatment. In amniotic fluid, our data suggests that uric acid represents the only interference to peroxidase-catalyzed reactions, especially when using p-substituted phenols as proton donors. The removal of uric acid has been shown to eliminate hydrogen peroxide reduction and should allow for an increase in sensitivity and specificity for measurements incorporating a peroxidase-coupled indicator reaction, hence, more effective use of these reaction sequences. To our knowledge, this is the first report of a mechanism to eliminate hydrogen peroxide reduction in amniotic fluid.

Lack of apparent effect of assay methodology on the pharmacokinetics of digoxin.

The purpose of this study was to determine if serum digoxin concentration data using three different automated immunoassay methods would produce similar pharmacokinetic values in normal volunteer subjects. Area under the curve (AUC), steady-state volume of distribution/bioavailability ratio (Vd/F), terminal elimination rate constant (beta), clearance/bioavailability ratio (CL/F), maximum digoxin concentration (Cmax), minimum digoxin concentration (Cmin), and time of peak (Tp) were evaluated. Ten healthy volunteers received digoxin capsules 0.2 mg daily for 10 days. On day 10, 16 serial blood samples were collected over a 24-h dosing interval and analyzed by radioimmunoassay (RIA) (Concept 4, Micromedic Systems), fluorescence polarization immunoassay (FPIA) (TDx, Abbott Laboratories), and affinity column-mediated immunoassay (ACMIA), (aca, duPont Instruments). When comparing RIA and FPIA, the mean of the percent differences for AUC, Vd/F, beta, and CL/F were 9, 4, 10, and 6%, respectively. The mean of the percent differences were 2, 3, 44, and 6%, respectively, when comparing RIA and ACMIA. However, none of these differences were statistically significant. Although a trend toward higher Cmax values by RIA was noted, there was no statistical difference in Cmax, Cmin, and Tp. Orthogonal regression of all serum digoxin concentrations showed that FPIA = 0.76 RIA + 0.19, r = 0.967 (p less than 0.001); and ACMIA = 0.92 RIA + 0.04, r = 0.943 (p less than 0.001). At serum digoxin concentrations less than 1 ng/ml, FPIA overestimated RIA results (p less than 0.005), while ACMIA was approximately equal to the RIA results.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin in human serum.

We describe a method for analyzing the choline-containing phospholipids, namely phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin that is highly sensitive, easy to perform, and very reliable. The method employs enzymic hydrolysis of choline from these phospholipids by phospholipase D with subsequent oxidation of choline by choline oxidase and generation of hydrogen peroxide. Hydrogen peroxide is then reduced by peroxidase with p-hydroxy-phenylacetic acid acting as an oxygen acceptor to form a fluorescent product which is stable for at least 24 h without loss of linearity. The analysis requires a 5 microL sample volume and demonstrates linearity over a wide range (133 mumol/L-6.65 mmol/L). The CVs are less than 3%.

Time-related changes in the plasma concentrations of prolactin, gonadotropins, sex hormone-binding globulin, and certain steroid hormones in female runners after a long-distance race.

Running is associated with an increase in plasma concentrations of certain anterior pituitary hormones and adrenal steroids. This study reports such increases after a marathon race. Six trained female runners, 26 to 42 years old, participated in a marathon race. Fasting (resting) blood samples were collected a few weeks before the race (baseline) and immediately (0 hour), 1 hour, and 4 hours after the run. The data were analyzed with the use of two-way analyses of variance (F-test), paired t-test, and Page's test. At 0 hour, compared with baseline, significant increases were observed in the plasma concentrations of testosterone (T), dehydroepiandrosterone sulfate (DHEA-S), cortisol (F), free T index (T/SHBG), and prolactin (PRL). At 1 hour, levels of these steroid hormones and PRL declined, some significantly. At 4 hours, levels of all hormones except DHEA-S returned to baseline. No significant changes were observed in concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and sex hormone-binding globulin (SHBG), as evaluated by F-test. Running-associated changes in plasma hormonal concentrations revert to baseline in four hours, although DHEA-S may take a little longer.

Interference of digoxin-like immunoreactive substances with three digoxin immunoassays in patients with various degrees of renal function.

The effect of renal function and digoxin use in adult patients on interference from digoxin-like immunoreactive substances (DLIS) with three digoxin immunoassays was studied. Hospital patients entered into the study were categorized into the following groups according to renal function: group I (serum creatinine less than 1.5 mg/dL), group II (serum creatinine 1.5-2.5 mg/dL), group III (serum creatinine greater than 2.5 mg/dL, not on hemodialysis), and group IV (serum creatinine greater than 2.5 mg/dL, on maintenance hemodialysis). Medical records were reviewed to determine whether or not patients were receiving digoxin. Excess sera for analysis of serum digoxin concentrations (SDCs) was collected from routine laboratory tests. Serum samples were assayed singly by fluorescence polarization immunoassay (FPIA, Digoxin I, Abbott), radioimmunoassay (RIA, Micromedic), and affinity-column-mediated immunoassay (ACMIA, aca, E.I. du Pont). Correlation of SDCs obtained by RIA and ACMIA with FPIA results was determined using linear-regression analysis. A total of 177 patients met the study criteria; 98 were receiving digoxin. In patients on digoxin, SDCs by RIA were significantly higher than those obtained by FPIA in group II and III patients. SDCs obtained by ACMIA correlated well with and were not significantly different from those obtained by FPIA in any of the patient groups. Maximum differences and mean absolute differences in SDCs obtained by RIA were greater than those for ACMIA when compared with FPIA values in all patient groups. Over 40% of patients with renal dysfunction not on digoxin had false-positive SDCs by RIA; the highest of these values was seen in groups II and III.(ABSTRACT TRUNCATED AT 250 WORDS)

Study of forensic and clinical source hemoglobin interference with the duPont aca ethanol method.

The enzymatic alcohol (ethanol, ALC) analytical pack for the duPont aca is intended primarily for the analysis of serum or plasma samples containing no visibly detectable free hemoglobin (non-hemolyzed samples). It has become apparent, through clinical and medicolegal consultation, that this methodology has been applied for assay of ethanol content in hemolyzed samples (sample sources: clinical and forensic cases). The influence of sample free hemoglobin on the ace ethanol method was investigated. With 'hemolyzed' clinical samples (containing primarily oxyhemoglobin) a significant decrease was found in known ethanol concentration at 0.5 g per dl and greater of hemoglobin with the influence being greatest at lower ethanol concentration. With forensic samples (containing varying amounts of oxy-, reduced, and methemoglobins), the same observation was made with measured decrease in ethanol concentration beginning at 0.1 g per dL hemoglobin and greater. In essence, the higher the sample hemoglobin concentration the greater would be the effect in decreasing the apparent ethanol concentration. Preparation of protein-free trichloroacetic acid supernatants and their assay on the aca and by head-space gas chromatography resulted in excellent recovery of the sample ethanol. It is concluded that the effect of sample free hemoglobin is spectral in nature, and its presence results in potential and real overblanking for the aca ethanol enzymatic reaction sequence.

A computerized system for rapid retrieval and compilation of critical or interesting patient data.

The timely availability and presentation of critical and interesting patient data is essential to ensure quality patient care as well as ease of recognition and, secondarily, to provide material for teaching and research. The ability to accomplish these goals expeditiously in a complex and busy clinical laboratory may be difficult to acquire. An on-line, computerized system has been developed which automatically searches each patient record for up to 600 different laboratory tests, compares each result against individually established limits, and then formats pertinent test results for maximum discrimination. The report format also includes patient and specimen collection data, diagnosis, attending physician's name, and other desired associated test results. An additional feature of the system is that selected critical or interesting results for each laboratory division may be displayed on video display terminals located throughout the laboratory and at nursing stations. The system was written using the FORTRAN programming language and runs on a Control Data Cyber 18 computer.

Vascular permeability in experimental spinal cord injury.

Following spinal cord injury in rats there was a time-dependent change of vascular permeability as reflected by extravasation of 125I-labelled serum albumin. The change of vascular permeability correlated with tissue calcium and water accumulation suggesting that cord exposure to plasma calcium as a consequence of vascular injury may contribute to the progressive post-traumatic cord necrosis.

Serum osmolality in acute intoxication: a prospective clinical study.

The authors prospectively performed simultaneous determinations of serum delta osmolality (delta-Osm), enzymic (alcohol dehydrogenase [ADH]) quantitation of serum ethanol (EtOH), and urine drug screens on 339 acutely intoxicated patients. In addition, the authors established reference ranges for measured and calculated serum osmolalities in a group of 55 healthy volunteers. The authors determined the clinical utility of the combined delta-Osm/ADH procedure for detecting the presence of EtOH or other low molecular weight (Mr) volatiles. In the reference population, the measured osmolality (M-Osm) and calculated osmolality (C-Osm) was 285.1 +/- 4.3 (SD) mOsm/kg and 287.4 +/- 5.1 (SD) mOsm/kg, respectively. The correlation between delta-OsM and serum EtOH was 0.968 in 151 patients in whom EtOH was detected. The presence of drugs in 67 (44%) patients or absence of drugs in 84 (55.6%) patients was shown to have no significant effect on the delta-Osm. The delta-Osm/ADH method failed to detect a volatile other than EtOH in only two cases (0.6%) or in addition to EtOH in three cases (0.9%). The concentrations of these volatiles were not clinically significant. The sensitivity for detecting EtOH by means of the delta-Osm calculation was 98.1% with a specificity of 98.2%. A disparity (delta-Osm greater than 10 mOsm/kg) between delta-Osm and the EtOH determination suggested a volatile other than EtOH in five cases (1.5%), which was confirmed by head-space gas chromatographic (GC-HS) analysis. A volatile in addition to EtOH in seven cases (2.1%) was suggested but not confirmed by GC-HS analysis. The delta-Osm/ADH procedure provides an efficient, rapid, and readily available method to evaluate the acutely intoxicated patient for the presence of EtOH and/or other low Mr volatiles.