Homogeneous enzyme immunoassay for tobramycin evaluated and compared with a radioimmunoassay.

We evaluated a commercially available homogeneous enzyme immunoassay (EMIT, Syva Co.) for tobramycin against a reference radioimmunoassay (RIA) method. Between-assay precision (CV) was 2.9% at 6.2 mg/L and 3.0% for values in the range of 1.0-7.6 mg/L. Accuracy based on a recovery experiment (1.0-13.0 mg/L) yielded an analytical recovery of 88-112%. A correlation study with 75 sera from patients on tobramycin therapy showed that EMIT = 0.984 RIA - 0.0808, r = 0.993. Neither the EMIT nor the RIA procedure was affected by the presence of gentamicin, amikacin, and vancomycin. Absorbance data from the EMIT system calculated with the conventional RIA logit-log algorithm correlate well with results generated by the Syva data-handling system (logit-log = 1.077 Syva - 0.318, r = 0.998). A reagent stability study indicated that the EMIT reagents, once reconstituted, remain stable for at least 17 days when stored at refrigerated temperatures, or 11 days if stored at room temperature, thus enabling frequent "stat" assays without the need to prepare a calibration curve each time.

Albumin and beta 2-microglobulin radioimmunoassays applied to monitoring of renal-allograft function and in differentiating glomerular and tubular diseases.

Blood and urine were samples daily from 11 renal-allograft recipients from one to six weeks after the transplant. Clearances of both albumin (Calb) and beta2-microglobulin (C beta 2 mu) were significantly increased in all 11 patients. Five patients (Group I) with acute allograft rejection showed markedly increased Calb and moderately increased C beta 2 mu, concurrent with decreased creatinine clearance (CCr). Five other patients (Group II) with no evidence of rejection demonstrated episodes of grossly increased C beta 2 mu with minimally increased but stable Calb and normal CCr. One patient had no evidence of rejection nor indications of glomerular or tubular proteinuria. While changes in serum beta 2-microglobulin concentration closely paralleled those of serum creatinine in the Group II patients, the results diverged in the Group I patients because the increase in serum beta 2-microglobulin exceeded that of serum creatinine and preceded the increased in creatinine by one to five days, suggesting that measurement of serum beta 2-microglobulin might afford earlier indication of the nature and extent of renal damage in the allograft recipients.

Gravimetric determination of urine volumes.

A procedure for estimating urine volumes from urine weights is presented. Regression analysis was used to assess correlation between weight-volume errors. The urine weight was divided by 1.016, the mean urine specific gravity in our patient population. It was found that the gravimetric method gave results lower than the volume method by one percent or less. We conclude that this difference is acceptable for clinical purposes. The gravimetric procedure has the advantages of rapidity, cost-effectiveness, and safety.

beta 2-Microglobulin clearance as measured by radioimmunoassay.

We describe a radioimmunoassay for beta 2-microglobulin (beta 2 mu) in serum and urine. We incubated aliquots of diluted samples at room temperature for 1 h with 125I-labeled beta 2 mu and a rabbit antiserum monospecific for human beta 2 mu, and separated the phases by the double-antibody technique. The logit-log transformed dose-response curve was linear in the range 2 to 64 ng, equivalent to 0.5 to 16 mg/L of serum and 0.5 to 320 mg/L of urine. Assay sensitivity was 2.4 ng of beta 2 mu. Validation studies included tests of precision, accuracy, antibody specificity, and parallelism of the dose-response curves for standard and unknown. In a study of 25 normal individuals, serum and urine beta 2 mu ranged from 1.1 to 2.3 mg/L and 40 to 360 micrograms/24 h; the clearance of beta 2 mu was 8 to 130 microL/min. In 21 renal allograft recipients tested one to five weeks after transplantation, serum and urine beta 2 mu ranged from 3.9 to 15.6 mg/L and 7.2 to 611 mg/24 h; beta 2 mu clearance was 0.60 to 33.3 mL/min. Values for both serum and urine correlated well with severity of allograft rejection.

Prednisolone clearance in renal transplantation.

Although prednisolone is the most common immunosuppressive agent used in renal transplantation, an accurate formulation of the optimal dose regimen remains to be established. A sensitive and precise radioimmunoassay was developed for this purpose. Fifty microliters of 1:20 dilutions of serum or 1:200 dilutions of urine were incubated at room temperature for two hours with 130 pg of 3H-prednisolone (150muCi/microgram) and a rabbit antiserum against prednisolone (1:1,600 dilution). Serial prednisolone clearances (Cp) for ten renal transplant patients representing variable allograft functions were compared with their corresponding creatine clearances (Cer). The data show that renal metabolism of prednisolone is well correlated with Ccr. Increasing (decreasing) Ccr is accompanied by increasing (decreasing) Cp. The availability of an assay for serum prednisolone, together with a knowledge of the ratio between these two clearances, may prove useful for regulating therapy in renal transplant patients.

Embolus radiolabelling in a new canine model.

Embolus radiolabelling with 131I fibrinogen was studied in a canine model of internal carotid artery embolization. The dog was chosen as the experimental animal because of its maxillocarotid artery which permits collateral flow round the occlusion and helps to prevent strokes. Clot was prepared by incubating blood at room temperature to inactivate plasminogen activators and then refrigerating it to promote clot retraction. Emboli persisting 48 hours were seen in 80% of animals. Major strokes were not seen when 0.25 to 0.30 cm3 were used. Autoradiography and well counting revealed uptake of isotope. The test, when refined, should provide a tool for the investigation of thromboemboli.

Radioimmunoassay for immunologlobulin G in serum and urine.

We describe a radioimmunoassay for immunoglobulin G (IgG) in serum and urine. Aliquots of diluted samples and 125I-labeled IgG were incubated in antibody-coated tubes at 37 degrees C for 24 h, the supernates were decanted, and the radioactivity in tubes containing the bound fraction was counted. The dose-response curve in the range of 0.4--500 mg/L of urine or 640--40 000 mg/L of serum was linear on logit-log transformation and iterative weighted regression. Assay sensitivity was 10 ng of IgG. Validation studies included testing for precision, accuracy, antibody specificity, and parallelism of the dose-response curves for standard and unknown. In a study of 14 apparently normal individuals, serum IgG = 4.0-10.9 gL, urine IgG = 1.1-4.8 mg/24 h, and IgG clearance = 0.2 X 10(-4) to 4.8 X 10(-4) mL/min. In 20 patients with renal allografts, serum IgG = 15.8-66 g/L, urine IgG - 9.6-626 mg/24 h, and IgG clearance = 9 X 10(-4) to 1.99 X 10(-1) mL/min. IgG values correlated well with severity of renal allograft rejection.

Radioimmunoassay for urinary albumin.

We describe a rapid, sensitive, and precise radioimmunoassay for urinary albumin (Ualb). Aliquots of diluted urine were incubated at room temperature for 1 h with 125I-labeled albumin and a rabbit antiserum monospecific for human albumin. Phase separation was effected by the double-antibody technique. The dose-response curve as linear in the range of 15.6-10000 ng, equivalent to 4 to 3000 mg/liter of urine. The limit of sensitivity was 16 ng of albumin. The coefficient of assay variation was 4.8%, both at 44 mg/liter and at 1304 mg/liter. A displacement curve obtained with a serially diluted urine sample of high albumin concentration was completely superimposable with the curve for which human albumin was used as a standard. In 26 normal individuals the range for Ualb was 2.2--12.6 mg/24h, and for albumin clearance (Calb, 1.8 x 10(-5)-19.6 x 10(-5) ml/min. After renal homografts in 25 patients, Ualb ranged from 16.9 to 9928 mg/24 h, and Calb from 2.7 x 10(-4) to 1.7 x 10(-1) ml/min. Both increased Ualb and Calb correlated well with the severity of renal homograft rejection.

Decreased angiotensin-converting enzyme in the adult respiratory distress syndrome.

Using hippuryl-L-histidyl-L-leucine as substrate, serum angiotensin-converting enzyme was measured in 13 patients who had adult respiratory distress syndrome, eight patients with respiratory failure without adult respiratory distress syndrome, and two groups of controls: 24 healthy blood donors and 24 hospitalized patients with a variety of conditions but without respiratory failure or adult respiratory distress syndrome. Serum angiotensin-converting enzyme expressed in units/ml was 14.60 +/- 5.60 for adult respiratory distress syndrome compared with 28.92 +/- 6.60 for the blood donors, 20.76 +/- 5.87 for the patients with respiratory failure without adult respiratory distress syndrome and 20.20 +/- 5.94 in the hospitalized patients without respiratory failure or adult respiratory distress syndrome. These differences were significant, P less than .001 when adult respiratory distress syndrome was tested against the blood donors and P less than .01 against the other two groups. The significance of these findings is not clear, but the possibility is raised that the decrease of angiotensin-converting enzyme in adult respiratory distress syndrome results from a loss of pulmonary endothelial cells, which are known both to produce angiotensin-converting enzyme and to be damaged in adult respiratory distress syndrome.

Spectrofluorometry of free and protein-bound plasma tryptophan.

We report a rapid, simple, and sensitive spectrofluorometric procedure for total serum or plasma tryptophan. Plasma deproteinization, tryptophan extraction, and pH optimization are all carried out with a buffered cellulose/ethanol reagent. The fluorescence is measured at an emission wavelength of 350 nm, on excitation of 290 nm. Tryptophan fluorescence relative to concentration is linear up to 50 mg/liter. The high precision, evidenced by a between-run coefficient of variation of 3.1%, negates the need for duplicate analysis of samples. Analytical recovery of tryptophan from plasma up to 50 mg/liter is 90-100%. We observed no significant difference between values for tryptophan in serum and plasma from the same individual. Assay of up to 32 samples requires 60 min. Commonly used anti-depressants have no detectable effect on tryptophan determination by this procedure.

A quality control program for the radioimmunoassay laboratory.

The major problems in applying quality control to radioimmunoassay measurements are (a) nonlinearity of the dose-response curve, and (b) nonuniformity of the residual variance. A logit-log transformation of the dose-response variables combined with an iterative weighted regression analysis appears to overcome most of the difficulties. This technic is particularly helpful when applied to substandard runs where extraneous standard points tend to distort assay results. The authors describe a quality control program that involves recording control values on calendar and histogram formats, monitoring assay variables by charting, and comparing the computer-calculated slope with the graphic plot to reveal "outliers." This program is useful in guiding technologists to locate possible causes for "out-of-limits" runs.

Determination of ionized calcium in serum that has been exposed to air.

We examined changes in ionized calcium concentration in serum after its exposure to air. Samples with total protein concentrations ranging from 50 to 90 g/liter were equilibrated with CO2 in nitrogen (5/95, by vol) or CO2 alone, to produce pH values of 7.0 to 8.0. Ionized calcium was then measured with an Orion flow-through electrode system. Curves relating pH and ionized calcium concentration had statistically identical slopes regardless of protein concentration. A factor was derived, based on pH change, for correcting values for ionized calcium in serum exposed to air, and its validity was confirmed by comparing corrected values for samples allowed to stand at ambient temperature (23 degrees C) without anaerobic precautions with values initially obtained on anaerobic aliquots of the same samples.

Improved method for euglobulin clot lysis time.

1. The use of Evans Blue dye to facilitate endpoint determination the elimination of 4 degrees, assay conditions are technical improvements in the euglobulin clot lysis test. 2. Plasma samples have limited stability at 30 degrees or 4 degrees, but are stable for prolonged periods at minus 20 degrees. Samples with accelerated clot lysis are much less stable than normal samples at 30 degrees. 3. The normal range is determined as greater than 70 minutes for citrated plasma and greater than 50 minutes for oxalated plasma. There is no sex difference in the normal range.

Estimation of norms from a controlled sample survey. II. Influence of body habitus, oral contraceptives, and other factors on values for the normal range derived from the SMA 12-60 screening group of tests.

PIP: The Westenberg-Mood 2-sample percentile test was used to determine if ranges of normal values for subgroups varied for variables measured on the SMA 12/60 ("AutoAnalyzer 12/60", analyzes serum). Data on 12 frequently determined serum constituents was collected from people age 20-49. Subjects were clinically normal men (423) and women (557). Individuals were subgrouped according to 1) sex, 2) geographical region of the U.S., 3) body habitus (weight), 4) season of the year when serum was drawn, 5) time of day, and 6) medications, if any, being used. Some of the more striking observations were 1) obese persons had higher concentrations of uric acid, 2) albumin and inorganic phosphate concentrations were decreased in women using oral contraceptives, and 3) lactate dehydrogenase activities were lower in the winter months. A laboratory test has increased usefulness when the normal range is narrowed; analysis of serum constituent levels in subgroups achieves this purpose.^ieng