Gas chromatographic determination of polychlorinated biphenyls (as Aroclor 1254) in serum: collaborative study.

A gas chromatographic-electron capture detection method for determining the concentration of polychlorinated biphenyls (PCBs) as Aroclor 1254 (AR 1254) in serum was evaluated through a 2-phase collaborative study. In Phase I, each collaborator's lot of Woelm silica gel (70-150 mesh) was evaluated for elution and recovery of AR 1254, which had been added in vitro at 25 ng/mL to a serum extract. In Phase II, each collaborator analyzed a series of bovine serum samples that contained the following: (1) in vitro-spiked AR 1254; (2) in vivo AR 1254 and 8 in vitro-spiked chlorinated hydrocarbons; (3) in vivo AR 1254 only; (4) 8 in vitro-spiked chlorinated hydrocarbons only; and (5) neither AR 1254 nor chlorinated hydrocarbons above the detection limit of the method. In Phase I, the average recovery of AR 1254 from silica gel for the 6 collaborators was 87.9 +/- 15.44% (mean +/- 1 SD; N = 18; range = 52.3-105.8%). In Phase II, the analysis of in vitro spikes of AR 1254 in serum at 8.58, 16.8, 41.8, and 84.3 ppb gave mean (means) interlaboratory recoveries of 89.0, 83.3, 79.4, and 76.9%, respectively, with within-laboratory (repeatability) relative standard deviations (RSDr) of 18.8, 20.5, 10.2, and 14.1%, respectively, and among-laboratory (reproducibility) relative standard deviations (RSDR) of 21.5, 21.1, 14.6, and 20.8%, respectively. The determination of in vivo AR 1254 in samples containing approximately 10, 25, 50, and 100 ng/mL of AR 1254 resulted in interlaboratory means of 10, 22, 39, and 79 ng/mL, respectively, with RSDr = 6.7, 9.7, 6.4, and 5.8%, respectively, and RSDR = 20.6, 16.0, 10.9, and 10.3%, respectively. The precision of the method for incurred AR 1254 showed a maximum RSDr of less than 10% and a maximum RSDR of less than 21% for a concentration range of 10-100 ng/mL. The accuracy of the method as demonstrated by the mean recovery of in vitro-spiked AR 1254 over a concentration range of 8.58-843 ng/mL was 82.2%. The method has been approved interim official first action.

Standardization of lipid and lipoprotein determinations for pediatric screening procedures.

Standardization of lipid and lipoprotein measurements is of concern to laboratories that serve pediatric as well as adult populations, since preventive measures taken during pediatric years can help control atherosclerotic cardiovascular problems that can arise during adult years. Multiple analytical and physiologic problems that exist inherently in the lipids and lipoproteins and in application of measurements to the pediatric population must be controlled to gain standardization and produce highly precise and accurate results. Reference values from well-described, observed pediatric populations have been published for use in interpreting analytical results. In collaboration with NHLBI, the CDC has offered lipid and lipoprotein standardization services for the United States since 1957 and, on an international scale, has cooperated with WHO since 1962. Standardization activities have been primarily concerned with total and HDL cholesterol and triglyceride determinations for cardiovascular epidemiologic and clinical trial investigations.

International Federation of Clinical Chemistry (IFCC). Scientific Committee, Analytical Section. IFCC/WHO principles and recommendations on evaluation of diagnostic reagent sets used in health laboratories with limited resources. Part 3. Selection and evaluation using reference materials. General considerations.

The purpose of this document is to provide general considerations for the selection and evaluation of clinical chemistry kits in laboratories with limited resources. Separate documents have been developed to provide guidance on experimental procedure, the statistical treatment and interpretation of the data and criteria for acceptable performance of diagnostic kits designed to measure specific analytes.

Determination of steroid hormones in a human-serum reference material by isotope dilution--mass spectrometry: a candidate definitive method for cortisol.

We report a method, based on isotope dilution--mass spectrometry, for determining cortisol in a pooled specimen of human serum. Isotopically labeled cortisol is added to 5.0 mL of serum so that the molar concentrations of labeled cortisol and unlabeled cortisol are approximately equal. The specimen and two calibration standards are extracted with dichloromethane, and the extracted cortisol is converted to the methoxime-trimethylsilyl ether derivative. Samples and standards are analyzed by gas chromatography--mass spectrometry by monitoring the peak areas for m/z 605 and 608. The cortisol concentration is calculated by linear interpolation between the two bracketing standards. Variances of data collected during six weeks showed that the overall coefficient of variation (CV) was 0.69% (n = 32); the within-vial CV, 0.63%; the among-vial CV, 0.22%; and the among-day CV, 0.15% (means = 3.973 nmol/vial). Method specificity was demonstrated by liquid chromatographic as well as C8 mini-column cleanup of samples before derivation, by alternative ion monitoring at m/z 636 and 639, and by negative-ion chemical ionization at m/z 459 and 462. Derivatives of all observed degradation products of cortisol under basic, neutral, and acidic conditions did not interfere.

Development of a reference material for alkaline phosphatase.

In developing a Reference Material for alkaline phosphatase, we studied the stability, kinetic properties, and commutability of separate preparations of the purified enzyme from human liver, intestine, bone, and placenta. The Michaelis constants (Km) for the preparations from liver, bone, and intestine agreed well with the Km values we obtained for five human serum specimens, whereas that for the placental isoenzyme differed significantly. The first three isoenzymes exhibited nearly identical response-surface patterns, which closely paralleled those observed for 12 human serum specimens (commutability), but not that of the placental isoenzyme. Thus, we believe that a reference material could equally well consist of either the bone, intestinal, or liver isoenzyme. All four isoenzymes were satisfactorily stable in temperature-accelerated degradation studies. We chose the liver isoenzyme as an appropriate reference material because liver tissue is easier to obtain than bone or intestine and the isoenzyme is abundant in liver, is easy to extract, and is the one most commonly increased in human serum. This material is stable at -20 degrees C, is free of interfering and degradative enzymes and, being of human origin, is commutable with the enzyme in human serum.

Design and demonstration of a quality assurance program for labor-intensive analytical systems.

An interlaboratory quality assurance program was designed and implemented for the analysis of serum for polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs). The two primary means of quality control were analysis of known and blind quality control samples and analysis of blind duplicate serum samples.

Evaluation of potential analytical approach for determination of polychlorinated biphenyls in serum: interlaboratory study.

Forty-four laboratories participated in evaluation of a method for determining polychlorinated biphenyls (PCBs) as AR 1254 in serum at the parts per billion level. The method involves deproteinating serum with methanol, extracting with hexane-ethyl ether, and eluting PCBs from deactivated silica gel for gas-liquid chromatographic determination with electron capture detection. Compounds are quantitated by using the Webb-McCall factors. Five serum pools, 4 containing in vivo-fortified PCBs (as AR 1254) or 8 in vitro-fortified chlorinated hydrocarbons (CHs), or both, were used. For PCB fortification levels of 9.89 (EP 2), 24.74 (EP 3), and 74.20 ppb (EP 4), interlaboratory coefficients of variation (CVs) for collaborators that adhered to protocol were 92.7, 67.6, and 25.8%, respectively. CVs on the same pools analyzed by the Centers for Disease Control (CDC) were 7.4, 7.8, and 4.6%, respectively. Average interlaboratory recoveries for pools EP 2, EP 3, and EP 4 were 138.1, 111.2, and 91.1%, respectively, and 99.8, 89.6, and 90.4%, respectively, for CDC on the same pools. There was a general decrease in the mean error for those laboratories that had participated in an earlier study in which they were allowed to use their own methods.

Chronological trend in blood lead levels between 1976 and 1980.

Analysis of a chronological trend in data from the second National Health and Nutrition Examination Survey indicated that average blood lead levels in the United States dropped approximately 37 per cent (5.4 micrograms per deciliter) from February 1976 through February 1980. There was no evidence that this trend was due to errors in laboratory measurement or to the design of the survey. The trend was present even after accounting for differences in race, sex, age, region of the country, season, income, and degree of urbanization. Changes in exposure to lead in paint or in the diet are unlikely explanations of the trend. However, the correlation of blood lead levels with the lead level in gasoline was highly significant (P less than 0.001) overall and in population subgroups defined by race, sex, and age. Although strong correlation does not prove cause and effect, the most likely explanation for the fall in blood lead levels is a reduction in the lead content of gasoline during this period.

Gas-liquid chromatographic determination of polychlorinated biphenyls and a selected number of chlorinated hydrocarbons in serum.

A method is proposed for concurrently determining the levels of multiple intact exogenous compounds in serum, particularly polychlorinated biphenyls (PCBs) as Aroclor (AR) 1254 and chlorinated hydrocarbons (CHs). Bovine serum pools containing in vivo-bound PCBs (as AR 1254) and in vitro-spiked CHs are used to evaluate the method, which encompasses serum denaturation with methanol, mixed solvent extraction, multiple solvent fractionation from activated silica gel, and determination by electron capture gas-liquid chromatography. Mean recoveries of the in vitro-spiked 9 CHs at levels of 2.0-29.1 ppb ranged from 52.8 to 98.4% from trial environmental pools; mean recoveries of the in vivo-bound PCBs (as AR 1254) were 114.1 and 92.6% at levels of 10 and 50 ppb, respectively.

Assessment of methods to determine PCB levels in blood serum: interlaboratory study.

Twenty-five laboratories participated in a study to determine the levels of polychlorinated biphenyls (PCBs) in 3 bovine serum pools, referred to as trial environmental pools (TEPs). TEPs 2 and 3 contained, respectively, low (9.97 ppb) and high (49.64 ppb) levels of PCBs (as in vivo-bound Aroclor 1254) and the same level of 9 commonly occurring chlorinated hydrocarbons. TEP 1 contained only naturally occurring levels of these analytes. Laboratories analyzed each sample in duplicate by the method used in their laboratory for measuring PCBs in blood serum. The coefficients of variation (CV) for the 12 laboratories reporting quantitative data and the required number of analyses for TEP 2 and TEP 3 were 37.0 and 30.7%, respectively. The mean recoveries for these 12 laboratories were 239.3 and 165.4% for TEP 2 and TEP 3, respectively. Three laboratories reported data with mean values for TEP 2 and TEP 3 within +/- 3 standard deviations of the CDC characterized mean. Their coefficients of variation were 12.4 and 18.8% for TEP 2 and TEP 3, respectively. The mean recoveries for these 3 laboratories were 150.7 and 98.4% for TEP 2 and TEP 3, respectively. Our most significant observations were the laboratories' failure to separate PCBs from DDTs and the excessive background of the reagent blanks. The widely discrepant results indicate a definite need to standardize methodology for this analysis.

Interlaboratory comparison of serum thyroxine analyses.

Approximately 300 clinical chemistry laboratories participated in a survey of measurements of the thyroxine (T4) concentration in 13 lyophilized serum specimens prepared by spiking a base serum pool with several different levels of T4. Of 35 commercially available kit methods, 4 kits were used by 30 or more laboratories, and 9 by 10 or more laboratories. Values obtained with the Abbott or Nuclear Medical laboratories, kits, which were used by one-third of all laboratories that named a specific kit, averaged 1 to 2 micrograms/dL greater than those obtained with the other methods. Within-run coefficients of variation were 10 to 20% for specimens containing less than 3 micrograms/dL of T4 and 3 to 6% for specimens with more elevated T4 levels.

A candidate reference method for determination of total protein in serum. II. Test for transferability.

The transferability of the candidate Reference Method for total serum protein was tested in eight laboratories in the United States and Europe. National Bureau of Standards SRM 927 (bovine serum albumin) was used in each analytical run as the calibration standard. The mean absorptivity value obtained for this material was 0.2983 L g-1 cm-1. Four serum pools prepared at the Centers for Disease Control were analyzed on each of 15 days. Within-run variation of the protein values (expressed as CV) in the eight laboratories ranged from 0.1 to 2.5% and day-to-day (total) variation in six of the laboratories ranged from 0.4 to 1%.

A candidate reference method for determination of total protein in serum. I. Development and validation.

We developed a candidate Reference Method for measuring total serum protein by use of the biuret reaction. The method involves a previously described biuret reagent (Clin. Chem. 21: 1159, 1975) and Standard Reference Material (SRM) 927 bovine albumin (National Bureau of Standards) as the standard. At 25 degrees C, color development for 30 or 60 min provides identical serum protein values. Glucose (up to 10 g/L) and bilirubin (up to 300 mg/L) do not interfere. Hemoglobin, at 3 g/L, increases apparent serum protein by 0.4 g/L. The presence of dextran in serum causes easily detected turbidity, but this interference can be eliminated by centrifuging the reaction mixture. Therapeutic concentrations of ampicillin, carbenicillin, penicillin, oxacillin, nafcillin, chloramphenicol, cephalothin, and methicillin in blood do not interfere, nor do triglycerides up to 10 g/L. Within-run and day-to-day standard deviations of the method are 0.1 and 0.4 g/L, respectively.

Characterization and intermethod relationships of materials containing purified human pancreatic and salivary amylase.

We describe the preparation and characterization of materials containing human pancreatic and salivary alpha-amylase (EC 3.2.1.1) and examine their relationship to endogenous amylase in human serum. Amylase was purified from human pancreas and saliva by solvent- and salt-fractionation and column chromatography to specific activities of 63 and 279 kU/g, respectively. Four liquid pools, differing only in activity, were prepared from each source of amylase, each in a matrix containing, per liter: 30 g of human albumin, 50 mmol of sodium chloride, 1 mmol of calcium chloride, and 50 mmol of Tris hydrochloride buffer, pH 7.4. Characterization of the pools showed that the amylase activity in the materials was stable for at least six months at 25 degrees C; among-vial variability of amylase activity was less than or equal to 0.5% (2 CV); and the pools were free from eight possible contaminating enzymes. Plots of salivary vs pancreatic amylase activity measure in our materials with eight commercially available methods showed least-squares slopes ranging from 0.51 to 1.0. The intermethod "commutability" of the materials (i.e., how closely they mimic endogenous serum amylase) was examined in relationship to approximately 100 human sera.

A coupled-enzyme equilibrium method for measuring urea in serum: optimization and evaluation of the AACC study group on urea candidate reference method.

We describe a coupled-enzyme equilibrium method for measuring urea in serum, which is performed on supernates prepared by treating each specimen with Ba(OH)2 and ZnSO4 (Somogyi reagent). Analytical recovery of [14C]urea added to a variety of matrices was essentially complete (mean, 100.6%) for the supernates after precipitation. Nine variables were univariately examined in arriving at the reaction conditions for the method: glutamate dehydrogenase, urease, 2-oxoglutarate, ADP, Tris . HCI, NADH, EDTA, pH, and temperature. The reagent is stable for at least 48 days at--20 degrees C and for 23 days at 4 degrees C. Mean analytical recovery of urea (14 mmol/L) added to seven different specimens (three different matrices) was 100.8%. The analytical linear range of the method extends to 30 mmol of urea per liter. Of 22 potential interferents, only bilirubin at 1 mmol/L (580 mg/L), hemoglobin at 10 g/L, and hydroxyurea at 6 mmol/L showed more than 2% interference. We discuss precision and effects of specimen dilution, and compare results for 100 human serum specimens with those measured for the same specimens with four other urea methods. We examined the effects of measuring a blank, consisting of sample and reagent without urease, with each specimen.