Proficiency test performance as a predictor of accuracy of routine patient testing for theophylline.

Proficiency testing (PT) is pivotal in assessing laboratory qualifications for certification and licensure. PT is expected to typify routine assay performance and determine whether the laboratory is producing clinically useful test results. Conventional schemes use mail-distributed test specimens and are often criticized as measuring the best possible laboratory performance, principally because of special practices associated with processing PT specimens. We used on-site proficiency tests and split samples to evaluate the ability of conventional PT schemes to accurately characterize routine laboratory performance. Using 412 assays of theophylline, performed routinely by 200 laboratories and subsequently in a reference laboratory, we found that the predictive value of PT performance in assessing quality of routine testing was high (100% for predicting substandard reliability of routine patient testing and 94% for excluding substandard reliability of patient testing). The imprecision of interlaboratory PT results was equivalent whether testing was observed (hand-carried specimens) or unobserved (mail-distributed specimens). Many methods used for determining theophylline concentration in serum were highly automated, closed, and precise analytical systems. The performance characteristics of these analytical systems are not easily manipulated by the analyst for purposes of improving PT outcome, and PT by use of mail-distributed test specimens is effective for assessing intralaboratory performance.

Evaluation of the rigor and appropriateness of CLIA '88 toxicology proficiency testing standards.

The proposed rule for the Clinical Laboratory Improvement Amendments of 1988 (CLIA '88) describes uniform standards for the design of proficiency testing (PT) challenges and for the evaluation of laboratory performance. Successful performance on PT is a condition for laboratory certification by the federal Department of Health and Human Services. We conducted a retrospective evaluation of PT data collected from laboratories participating in the New York State therapeutic substance monitoring/quantitative toxicology PT program to determine how well laboratories performed by proposed PT standards. We found that the unsuccessful performance rate is very low (less than 0.4%) and that laboratories providing limited services are more susceptible to program sanctions than are full-service laboratories. The performance criteria are empirically fixed limits that are not consistent with either the state of practice or the analytical goals based on clinical requirements for good patient care. We suggest that the performance standards, if adopted, would be a weak challenge to the capability of today's therapeutic drug monitoring service and will not provide the impetus to bring analytical performance characteristics into full compliance with analytical goals.

Measurement of alkaline phosphatase activity: characterization and identification of an inactivator in 2-amino-2-methyl-1-propanol.

An inactivator of alkaline phosphatase (EC 3.1.3.1) in 2-amino-2-methyl-1-propanol is demonstrated and characterized. This time-dependent inactivation results from chelation of enzyme-bound Zn2+; it is reversed by addition of Zn2+ and, to a lesser extent, other divalent metal ions. Cu2+ is an effective spectral indicator and can be used to determine the presence and quantity of inactivator. Data obtained from enzyme inactivation, Cu2+ absorbance spectra, "high-performance" liquid chromatography, thin-layer chromatography, Fourier-transform infrared spectroscopy, and mass spectroscopy indicate that the inactivator is 5-amino-3-aza-2,2,5-trimethylhexanol. This compound, even in trace amounts (less than 0.05% on a molar basis), shown to inactivate alkaline phosphatase.

Optimal conditions and comparison of lactate dehydrogenase catalysis of the lactate-to-pyruvate and pyruvate-to-lactate reactions in human serum at 25, 30, and 37 degrees C.

We report optimal conditions for assaying highly purified human lactate dehydrogenase isoenzymes with the lactate-to-pyruvate and pyruvate-to-lactate reactions, as they apply to human serum. Interconversion of results between reactions is not practicable. Measurements of lactate dehydrogenase in either reaction direction at 25, 30, or 37 degrees C can be equally reliable if the volume fraction and the resulting deltaA/min is small. However, for interinstrument and interlaboratory comparisons, results from the lactate-to-pyruvate reaction are more reliable.

Optimal reaction conditions for assaying human lactate dehydrogenase pyruvate-to-lactate at 25, 30, and 37 degrees C.

Optimal reaction conditions for assaying human lactate dehydrogenase pyruvate-to-lactate were determined for isoenzymes 1 and 5 at 25, 30, and 37 degrees C. Three of the nine different buffers examined--imidazole, triethanolamine, and N-tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid--are satisfactory. Beta-NADH, pyruvate, and hydrogen ion concentrations were chosen to measure both isoenzymes with maximal-equal-sustainable efficiency at the lowest substrate concentrations. Approximately 95% of each isoenzyme is measured, for activities up to threefold the upper normal limit, if the measurements are made immediately after the reaction is initiated. The Arrhenius relationship for each isoenzyme is unique. Interconversion of results from one temperature to another is practical only with reservations. Results at 37 degrees C are not as reliable as those at 25 degrees C.

The effect of temperature on the kinetic constants of human lactate dehydrogenase 1 and 5.

The kinetic constants of human lactate dehydrogenase 1 and 5 (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27), assayed lactate-to-pyruvate increase with temperature. The reaction mechanism is ordered sequential as has been found with lactate dehydrogenase from other sources. The KM values for each substrate are larger for isoenzyme 5 than for 1. For lactate dehydrogenase 1 the KM(lactate) increases from 1.07 mM at 25 degrees C to 3.95 mM at 37 degrees C and for lactate dehydrogenase 5 it increases from 5.37 mM at 25 degrees C to 6.88 mM at 37 degrees C. The KM(NAD+) for lactate dehydrogenase 5 is 0.14 mM at 25 degrees C and 0.29 mM at 37 degrees C. The increase in the KM for each substrate with increasing temperature confirms that additional substrate is required for optimal reaction conditions at higher temperatures.

Optimal conditions for assaying human lactate dehydrogenase by the lactate-to-pyruvate reaction: Arrhenium relationships for lactate dehydrogenase isoenzymes 1 and 5.

Optimal reaction conditions to sassay human lactate dehydrogenase (lactate-to-pyruvate) were established for isoenzymes 1 and 5 at 25, 30, and 37 degrees C in diethanolamine and 2-amino-2-methyl-1,3-propanediol. Different substrate concentrations are required at each temperature. The conditions permit measurement of lactate dehydrogenase 1 and 5 with the lowest substrate concentrations that allow for the highest equal sustainable efficiency in measuring both isoenzymes. About 95% of each isoenzyme activity is measured if the assay is performed within the first minute after the reaction is initiated even for activities as high as triple the upper limit of normal. The Arrhenius relationship is different for each isoenzyme, but results obtained for each at one temperature can be compared with results at another temperature by use of simple conversion equations. Assays at 25 and 30 degrees C are more economical and less variable than assays at 37 degrees C.

Interconverting measurements of human lactate dehydrogenase activities in three buffers.

The lactate-to-pyruvate reaction for serum lactate dehydrogenase (LD) is most frequently assayed in one of three buffers, pyrophosphate (PPi), tris(hydroxymethyl)amino-methane (Tris), or 2-amino-2-methyl-1-propanol (AMP). We described interconverting results for serum samples and for highly purified LD isoenzymes I (dissolved in one of these matrixes) assayed in these buffers under optimized reaction conditions. The equation for converting results obtained for sera in Tris (x) to those in PPi(y) (both at 30 degrees C) is y = 0.74x+10 (n = 98). Since AMP is used extensively in Technicon procedures, we determined the LD activity of sera with an SMA 12/60, at 37 degrees C. The equation for convering these AMP results to results obtained in PPi at 30 degrees C is y = 0.45x-16 (n = 90). Very different equations were obtained with highly purified LD isoenzyme I maintained in two different matrixes and with both isoenzymes assayed in the same matrix. The matrix in which LD is dissolved and the proportion of various LD isoenzymes affect the magnitude of difference in observed LD activity under various conditions. Therefore, in clinical laboratories that use more than one analytical method or when conversion equations are used in the comparison of interlaboratory results, it is important to define the LD source, isoenzyme content, and the matrix, as well as the reaction conditions, and to use many samples with a wide range of activities when determining the conversion equations. For any changes in reagent source, substrate concentration, or alteration in procedure, a new normal range and new conversion equations should be determined.

A search for the best buffer to use in assaying human lactate dehydrogenase with the lactate-to-pyruvate reaction.

Highly purified human lactate dehydrogenases I and V were assayed in 17 different buffers, at a variety of reaction pH's. Diethanolamine and 2-amino-2-methyl-1,3-propanediol provided the best measurements of the enzyme, assayed lactate-to-pyruvate. However, the commercial preparation of 2-amino-2-methyl-1,3-propanediol contained insoluble matter and was relatively expensive. All of the four buffers nowmost commonly used were found to present difficulties. Glycine and pyrophosphate were inhibotory tolactate dehydrogenase activity with increasing buffer concentration. 2-Amino-2-methyl-1-propanol had three major disadvantages: it is chemically unstable during reagent preparation; activity is dependent on buffer concentration; and the pH optima for isoenzymes I and V are vastly different. The pKa of tris(hydroxymethyl)aminomethane is 8.0 at 30 degrees C, whereas to measure total activity the reaction pH should be greater than 8.5; thus tris(hydroxymethyl)aminomethane has limited buffering capacity at the reaction pH.

Effect of reaction initiator on human lactate dehydrogenase assay.

Human lactate dehydrogenase isoenzymes I and V have decreased activities when the reaction is initiated with lactate. No loss in lactate dehydrogenase I activity was found when the reaction was initiated with enzyme or NAD+. For lactate dehydrogenase V an NAD+-initiated reaction, as compared to an enzyme-initiated reaction, yields lower activity in sodium pyrophosphate buffer but higher activity in tris(hydroxymethyl)aminomethane buffer. Both isoenzymes have higher lactate-to-pyruvate activity when assayed in the latter buffer than when assayed in the former. Human lactate dehydrogenase V (but not I) exhibited different activities when assayed with lactate from two different commercial sources. Human lactate dehydrogenase assayed by the pyruvate-to-lactate reaction is not affected by the choice of reaction initiator.