James H. McBride

The elucidation of adrenal-cortical status by measuring two-hour urinary-free cortisol levels

Urinary-free cortisol (UFC) measured in 24-h samples has provided unambiguous data regarding adrenal-cortical status, but collection is cumbersome and compliance is a problem. We reasoned that measuring UFC in 2-h samples collected at strategic times would distinguish patients with adrenal-cortical disorders from normal subjects. Patients with Cushings syndrome demonstrated late P.M. (samples collected between 2000 and 2400 h) 2-h UFC levels that were greater than or equal to 12.0 micrograms/2 h (reference interval for this time period is 0.1-3.2 micrograms/2 h, n = 35). Several patients with various symptoms of Cushings syndrome displayed late P.M. levels within the reference interval. Those with adrenal insufficiency had morning (collected between 0500 and 0900 h) levels less than or equal to 0.2 microgram/2 h (reference interval 1.7-21.2 micrograms/2 h, n = 37). These data indicate that adrenal-cortical status is reflected by measuring UFC levels in 2-h samples.

The purification of human creatine kinase BB with high specific activity.

This paper describes the purification of human creatine kinase BB with high specific activity (1,122 U/mg). The procedure used resulted in a protein yield of 5.4 mg (21% recovery) from 150 g of brain tissue. Two-dimensional electrophoresis and PAGE studies indicated that purified CK-BB might exist as native isoenzyme along with structural aggregates since the multi-banded appearance was reduced to a single band with sodium dodecyl sulfate treatment but not with 2-mercaptoethanol. Investigators are cautioned not to store brain tissue for prolonged periods of time before isolation of the isoenzyme as this may lead to protein redistribution with additional bands becoming evident on PAGE.

Conversion of cardiac and liver transplant recipients from HPLC and FPIA (polyclonal) to an FPIA (monoclonal) technique for measurement of blood cyclosporin A

In an effort to replace HPLC and FPIA (polyclonal) for whole blood determination of Cyclosporin A (CsA), this study examined the application of FPIA (monoclonal) in patients post cardiac and liver transplantation. The assay had a minimum detectable dose of 15μg/ L, an overall recovery of 97% and was linear to 1200μg/ L, and gave inter‐assay precision values of < 5% (CV). On comparing FPIA (monoclonal) and HPLC for 59 cardiac transplant recipient blood samples, a correlation of FPIA (monoclonal) = 1.30 (HPLC) + 36.34, r = 0.96 was obtained. With liver transplant samples (n = 348), the correlation was FPIA (monoclonal) = 1.21 (HPLC) + 42.15, r = 0.98. Correlation on 131 cardiac transplant recipients gave FPIA (monoclonal) = 0.31 FPIA (polyclonal) + 43.97, r = 0.68. It is concluded that when converting from HPLC to FPIA (monoclonal) a positive bias of 21%–30% is observed, and in replacing FPIA (polyclonal) with FPIA (monoclonal), a negative bias of 50%–69% is seen with liver and cardiac patients respectively. These data indicate that therapeutic ranges should be reestablished or adjustments in CsA dosing would be necessary. J. Clin. Lab. Anal. 12:337–342, 1998.

Immunochemical extraction and automated measurement of plasma creatine kinase MB isoenzyme and creatine kinase MB2 isoform

Measurement of creatine kinase MB (CK‐MB) and its isoforms CK‐MB2 and CK‐MB1 are now applied in the diagnosis of acute myocardial infarction (AMI). The most common approach for analysis includes RIA, IRMA, and electrophoresis, all of which may be time‐consuming. This study examines determination of CK‐MB and CK‐MB2 by a rapid immunochemical extraction method followed by an automated measurement for both analytes. The automated method was sensitive to 2 U/L, linear to 180 U/L, and gave excellent interassay precision (<10% CV). Interference studies indicated that bilirubin, hemolysis, and lipemia caused analytical problems as did the presence of high activities of other CK isoenzymes, notably CK‐MM and CK‐BB, requiring dilution of samples prior to analysis. Application of immunochemical extraction gave a reference interval of CK‐MB (0–2.5 U/L) and CK‐MB2 (0.1–1.4 U/L) for blood donors (20–60 years), peak levels for ruled‐out AMI patients of CK‐MB (0.5–7.3 U/L) and CK‐MB2 (0.3–4.9), peak levels for ruled‐in AMI patients of CK‐MB (80–174 U/L) and CK‐MB2 (80–155 U/L). Coronary artery bypass patients (n = 24) and all trauma patients (n = 14) also demonstrated elevations in CK‐MB and CK‐MB2, whereas only five of the trauma patients demonstrated increased CK‐MB by IRMA. In patients (n = 7) having increased total CK and normal CK‐MB by IRMA, the extraction assay for CK‐MB and CK‐MB2 yielded increased values in all patients. This new approach to CK‐MB and CK‐MB2 analysis can be performed within 30 minutes of sample receipt. J. Clin. Lab. Anal. 11:163–168, 1997.

A Heterogeneous Fluorescence Immunoassay for Gentamich Using a Second Antibody Separation

A heterogeneous fluorescence immunoassay (FIA) for gentamicin was developed using a second antibody separation. The separation of bound from free fluorescence label, removes a number of endogeneous fluorescent interferences. Correlation with a standard radioimmunoassay (RIA) for gentamicin was acceptable (r = 0.91). We conclude that a heterogeneous FIA is a precise, accurate, and convenient alternative to monitoring antibiotic levels.

Evaluation of the Olympus Demand random access chemistry analyser.

We evaluated the Olympus Demand as a cost-saving measure, as a back up to a Technicon SMAC I, and to be the primary instrument for enzyme analysis. In general, it wasfound to be precise, reliable, easy to operate, but with only average turn-around time capabilities. Technically there were several limitations: (1) significant bilirubin interference in the analysis of serum bicarbonate and creatinine; (2) instability ofthe ion selective electrodes; (3) lengthy routine maintenance requiredfor the ion selective electrode module; (4) serum phosphorus and uric acid methods required blanking due to interferencefrom bilirubin, hemolysis, or lipemia; and (5) serum triglyceride analysis included measurement offree glycerol.