G N Bowers's research while affiliated with Massachusetts General Hospital and other places

We evaluated N-methyl-D-glucamine (MEG) as a buffer for assay of alkaline phosphatase (ALP; EC 3.1.3.1) and compared the MEG-based assay with the current International Federation of Clinical Chemistry Reference Method for ALP (IFCC/RM/ALP), in which 2-amino-2-methyl-1-propanol (AMP) is the pH buffer. The ALP assay in MEG at 30 and 37 degrees C shows excellent correlation with the IFCC/RM/ALP at 30 degrees C, but yields proportionately higher ALP activities (8.2% at 30 degrees C and 57% at 37 degrees C). ALP is unstable in both MEG and AMP at 37 degrees C. Serum incubated in MEG undergoes a pH-dependent biphasic loss of ALP activity: an initial rapid 5% loss after 1 min of incubation and a 10% loss per hour thereafter. A similar pattern was seen for incubation with AMP. The use of a serum-initiated reaction (no preincubation of enzyme with buffer) eliminated the early loss in activity. The addition of the metal ion buffer N-hydroxyethylethylenediaminetriacetic acid, along with low concentrations of Zn and Mg, as used in the IFCC/RM/ALP, reduced the slow loss in activity over time, as did decreasing the reaction temperature to 30 degrees C, but had no effect on the early rapid decay in activity seen in the first minute. Moderate transphosphorylation (45%) and nonenzymatic hydrolysis (3.3 U/L) were observed with MEG under the conditions of the assay (37 degrees C). A comparison of different lots of MEG from two manufacturers showed no significant difference in ALP activities.

Current quality assurance approaches will not be adequate to satisfy the needs for quality in the next decade. Quality management science (QMS), as evolving in industry today, provides the dynamic framework necessary to provide continuous improvement of quality. QMS emphasizes the importance of defining quality goals based on the needs and expectations (implied needs) of customers. The laboratory can develop customer-friendly goals and measures of quality by recognizing that customers' experiences are represented by a totality of results. Quality goals and measures are best communicated as "total performance" by specifying a limit and percentile of the distribution, rather than a mean and standard deviation. Application of quality goals within the laboratory will usually require partitioning the total performance goal into components and translating those components into specifications to guide the operation and management of production processes. QMS also extends beyond technical processes to people processes and provides guidance for improving the quality of worklife and caring for the laboratory's most essential resource--our people.

The manual Reference Method of the Centers for Disease Control for serum iron (CDC/RM/Fe) and a semiautomated adaptation of it were used to evaluate two working methods: one, a detergent solubilization procedure for the Roche Cobas-Bio analyzer, the other, the Kodak Ektachem 700 procedure, based on dry-film technology. The CDC/RM/Fe and its semiautomated version gave essentially the same results for 40 sera from hospital patients. This semiautomated version was in turn compared with the two working procedures in a study involving 200 patients. Each of the working methods correlated well with the semiautomated CDC/RM/Fe method. Separate recovery and interference studies indicated satisfactory analytical recovery of iron in all cases, but the detergent solubilization method was found to be susceptible to interference by hemoglobin, lipemia, and bilirubin.

Ion-selective electrode analyzers for measuring lithium (Li/ISE) in serum became available in early 1987. We compared results for patients' samples from three of them vs results from flame atomic emission spectrometry (FAES). Within-run and day-to-day imprecision ranged from 0.01 to 0.03 mmol/L and 0.01 to 0.04 mmol/L, respectively. Comparing Li/ISE results (y) with the FAES results (x) gave the following equations: y = 1.063x - 0.035 for AMDEV's Lytening 2, y = 1.020x + 0.038 for NOVA's Model 11, and y = 1.030x - 0.027 for AVL's Model 985. Unexplained positive errors greater than 0.2 mmol/L were observed for two of the 90 patients' samples, but only a few additional excessively high values were seen in 3000 patients' samples run subsequently (Lytening 2). Causes of error in clinical Li/ISE measurements are still unclear; simply characterizing them as "matrix effects" does not correct the underlying analytical problem. An increase in pH from loss of CO2 gave low results on two of the three Li/ISE analyzers but did not change FAES results. Trimethylammonium bicarbonate used in a reconstitution solution caused extremely high Li/ISE results but did not change FAES results. Performance specifications to help reduce and correct these errors are recommended.

The purity, spectral characteristics, and rate of nonenzymatic hydrolysis of 2,6-dichloro-4-nitrophenyl phosphate (DCNPP) were determined. Rates of DCNPP hydrolysis by prostatic acid phosphatase (PAP) and erythrocytic acid phosphatase (EAP) (both EC 3.1.3.2) were measured in the absence and in the presence of various alcohols. 1.5-Pentanediol was the most effective transphosphorylation agent for specifically enhancing the activity of PAP. 1,4-Butanediol also enhanced PAP activity but markedly inhibited EAP activity. Bovine and human serum albumin preparations also accelerated the hydrolysis of DCNPP. DCNPP can be used for the continuous or multipoint-rate assay of PAP.

During an evaluation of the IFCC reference method for alanine aminotransferase (ALT, EC 2.6.1.2), we noted that the specimen blank activity reaction was markedly increased. Experience with five different lots of D-alanine from four commercial sources indicated that substantial and varying negative bias (up to -10%) could be introduced into the blank-corrected ALT activity, depending on the lot of D-alanine used. Although the IFCC procedure for ALT mentions the possibility of this L-alanine contamination, we believe that the degree of contamination in commercial reagents is underestimated. Analyzing the five lots of D-alanine for L-alanine, we found the magnitude of negative bias to be correlated directly with L-alanine contamination. Here, we describe a quick, sensitive assay based on coupled reactions of L-amino acid oxidase/peroxidase for quantifying L-alanine in the concentration range of 0-15 mmol/L without a sample-dilution step. Results by this alternative L-alanine assay agreed well with those recommended in the IFCC ALT procedure. Further examination suggested an even simpler solution to the L-alanine contamination problem, because we found no difference in the blank-corrected ALT activity determined in Tris HCl buffer, with or without D-alanine (free of L-alanine). We therefore propose that D-alanine be omitted from the IFCC reference ALT procedure.

We describe two cases, hospitalized patients, in whom the activity of creatine kinase (EC 2.7.3.2) isoenzyme-MB was above normal. Both are particularly noteworthy in that creatine kinase-BB and a macro creatine kinase form thought to be type II of mitochondrial origin were also present. The macro creatine kinase component in both cases co-migrated electrophoretically with creatine kinase-MM but was easily identified after the latter was removed by precipitation with M-subunit-specific antibodies. In the first case, the patient had a readily diagnosable acute myocardial infarction while under observation in the cardiac intensive care unit: electrocardiographic changes and the rapid increase and decrease in total creatine kinase were as would be expected. In marked contrast, in the second case, we saw no abrupt changes in either of these characteristics. The latter patient's primary disease was a rectal carcinoma with massive metastases to the liver; however, the presence of abnormally high creatine kinase-MB activity raised the question of possible myocardial infarction.

We examined 17 lots of 2-oxoglutarate (seven acid forms, three K salt forms, and seven Na salt forms), obtained from eight commercial suppliers, for suitability for measuring aspartate aminotransferase (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2) in human serum. Measurements of the catalytic activity concentrations of these two aminotransferases with each of these 17 preparations were not sufficiently sensitive to distinguish good from poor-quality material. Thus, we ranked these lots for purity, by specific analysis with glutamate dehydrogenase and by liquid chromatography, and determined the water content, acid content, and spectral characteristics of each. On the basis of a 2-oxoglutarate assay value by glutamate dehydrogenase of 98% or greater, we considered seven of the preparations acceptable and 10 unacceptable. The molar absorptivities (L X mol-1 X cm-1, mean +/- SD) of the seven acceptable lots in 1 mol/L HCl were: epsilon 325 nm = 9.12 +/- 0.02 (CV = 0.2%), epsilon 279 nm = 2.63 +/- 0.23 (CV = 9.9%), and epsilon 245 nm = 37.9 +/- 4.1 (CV = 10.9%). Use of these spectrophotometric limits alone unambiguously distinguished the inferior lots of 2-oxoglutarate. We urge the inclusion of detailed spectrophotometric specifications for 2-oxoglutarate in Reference Methods for aminotransferase measurements.