Prostate cancer and other xenografts from cells in peripheral blood of patients.

Good models for the investigation of human prostate cancer are few. Cells from approximately 9.2-21 ml of peripheral blood from patients with metastatic prostate cancer or metastatic colon cancer were injected s.c. into nude mice. Prostate cancer from 2 of 11 patients and colon cancer from 1 of 3 patients were found to be growing as metastases in the lungs of the nude mice. To our knowledge, this is the first report of the formation of xenografts from carcinoma cells taken directly from the peripheral blood of patients. Expanding circulating cancer cells with this approach may have important translational applications including: (a) development of models of human cancers; and (b) sampling of cancers from specific patients for novel molecular and therapeutic approaches.

A study of experimental lancets for blood collection to avoid bone infection of infants.

We describe a study made at two pediatric centers to test experimental lancets for blood collection by skin puncture of infants' heels or fingers. Our primary goal is to decrease the hazard of osseous injury while collecting adequate blood, by using three lancet widths at a constant length of 1.0 mm. The three widths used were 1.0, 1.25, and 1.5 mm. When success at skin puncture was defined rigidly on the basis of the blood volume obtained, the data show that success was related neither to the lancets' dimensions as tested nor to the age of the child, but rather to the phlebotomist's skill and experience, which improved with time.

A rapid toxicology screen for emergency and routine care of patients.

I have adapted, for use with a centrifugal analyzer, reagents from 10 different EMIT (Syva Co.) enzyme immunoassay urine drug-detection kits, reagents from another five serum kits, and reagents for determining ethanol. Assays for individual drugs in 100 microL of urine and in 100 microL of serum are performed simultaneously rather than sequentially in less than 15 min, with one-tenth the usual amount of reactants (sample + reagents). These assays, when combined with other easily performed tests, provide a rapid toxicology screen well-suited for evaluating acute emergency patients, and for performing a small number of daily routine screens. The total screen requires 500 microL of urine and 500 microL of serum. I summarize my experience with this procedure in its first year of use, and discuss differences between testing for the care of patients compared with searching for evidence of illegal drug use.

Turbidimetric determination of transthyretin (prealbumin) with a centrifugal analyzer.

We describe a simple, rapid turbidimetric method for measuring transthyretin in human serum by use of a centrifugal analyzer (Cobas-Bio) and commercially available antiserum. The immunoturbidimetric reaction is monitored at 340 nm for 180 s, and the changes in absorbance after the antiserum is added are used to prepare a stable standard curve. The range of measurement is 10 to 520 mg/L. The CV for between-day precision was less than 5.0% for normal and low controls. Bilirubin up to 310 mumol/L and hemoglobin up to 225 mumol/L did not interfere, but triglycerides greater than 2.3 mmol/L falsely depress the results. The normal reference interval for adults, derived from data on subjects 20 to 65 years old (both sexes), is 193 to 376 mg/L. Concentrations are lower in children (67-302 mg/L) and in adults 65 or more years old (113-292 mg/L).

Calculating unknown concentrations from nonlinear standard curves.

We describe an algorithm and program for calculating unknown concentration values from nonlinear standard data. The program was developed as an alternative to current data-handling techniques used for therapeutic drug enzyme immunoassay (EMIT) systems. The shape of the standard curve for a given analyte is required to be stable through the lifetime of a reagent lot. The relationship between the net change in milliabsorbance and the natural log of standard concentration is linearized once for each lot of reagents. For each subsequent time the assay is performed, one corrects the individual standard points by using the linearized relationship. Unknown concentrations are then calculated by using quadratic Lagrangian interpolation. The algorithm is faster and simpler than curve-fitting by nonlinear least squares, gives comparable results for unknowns, and rejects fewer assays for poor curve fit.

Adaptation of EMIT reagents to the Cobas Bio centrifugal analyzer.

Our adaptation of EMIT enzyme immunoassay reagents to the Cobas Bio centrifugal analyzer avoids the decrease in precision at the upper end of the therapeutic range reported in other adaptations. Long-term (12-month) CVs for lidocaine, valproic acid, and tobramycin were 5% or less; for quinidine, acetaminophen, and phenytoin, long-term CVs ranged from 7.2% to 8.6%.

Human collagen digestion: II. Theoretical model and application to data.

A kinetic mechanism for the digestion of collagen by bacterial collagenase has been proposed which is consistent with experimental data. Arguments are presented justifying the application of a homogeneous approach to the digestion of insoluble collagen. The mechanism takes account of increased kinetic blockages with aging and enzyme complexity. It is shown to be applicable to collagen from various tissues, including relatively impure preparations. However, the mechanism is not applicable to collagen from the immature period of the lifespan. Kinetic blockages are shown to increase three- to four-fold from age 20 to 70 years, within the main helical region of the collagen molecule. There is no increase of kinetic blockages in the N-terminal region of the molecule over this age range.

Human collagen digestion: I. Nature of the digestion kinetics as a function of age and structure.

The digestion of human collagen by bacterial collagenase was studied in order to relate collagen structure to the digestion kinetics. The digestion was shown to be first order in collagen and in collagenase on given material from run to run. There was no product inhibition and no enzyme deactivation. However, the rate changed with age, and also within a run as a function of conversion. It was shown that the normalized time (time X enzyme concentration), to reach a given amount of digestion can be plotted as a linear function of the sample age, from 20 to 70 years. The digestion broke down into two regions. The first was a rapid first order region which amounted to about 10% of the collagen. The second region could be graphed as a second order plot to obtain a straight line. This implied that the rate of digestion was functionally second order in collagen; first order in concentration and first order in conversion of collagen.

Apparent accelerated aging of human collagen in diabetes mellitus.

The chronologic ages of human subjects were determined experimentally by enzymatic digestion of tendon collagen samples. Determined age closely matched actual age for individuals dying with a variety of major diseases. Juvenile diabetics did not fit this pattern; their experimentally determined ages were significantly greater than their actual ages. This raises the possibility of relationships between diabetes mellitus, changes in connective tissue, and accelerated aging.