James L. Driscoll

Trypan blue dye uptake and lactate dehydrogenase in adult rat hepatocytes—Freshly isolated cells, cell suspensions, and primary monolayer cultures

SummaryLeakage of lactate dehydrogenase and staining by the vital dye trypan blue were investigated in adult rat hepatocytes at the time of isolation, in suspensions up to 3 h and in primary monolayer cultures up to 3 d. These two parameters of plasma membrane integrity were found to correlate closely in hepatocyte suspensions, but to a lesser degree in monolayer cultures. Functional activity was demonstrated in culture by glucose consumption and lactic acid production. There was a balance of total lactate dehydrogenase (LDH) activity over time for both hepatocyte suspensions and cultures. Loss of LDH activity in the cell fraction was accompanied by a corresponding increase in enzyme activity in the media fraction. Lactate dehydrogenase activity per dye-excluding hepatocyte was calculated to be 9.2±1.5×10−6 IU assayed at 37°C for 25 preparations of isolated hepatocytes.The results suggest that leakage of cytoplasmic enzyme and vital dye staining are of comparable sensitivity in evaluating hepatocyte preparations. Measurement of LDH leakage offers a less subjective alternative to cell counting procedures and is applicable to both attached and suspended cells.

Phenolsulfonphthalein (phenol red) metabolism in primary monolayer cultures of adult rat hepatocytes

SummaryThe sulfonic acid dye, phenolsulfonphthalein (PSP or phenol red), has been incorporated as a pH indicator in many tissue culture media formulations since the emergence of tissue culture methodologies. The present study was designed to examine the pathway, time course, and degree of metabolism of this anionic dye in monolayer cultures of adult rat hepatocytes. Thin layer chromatographic studies coupled with β-glucuronidase studies show that glucuronidation is the major metabolic pathway for PSP in vitro. About 20% of the dye is metabolized in the first 24 h, but this functional activity is decreased by approximately half at 48 h, and even further at 72 h of culture. This metabolic activity was not affected by continuous exposure to the dye. The effect of PSP concentration on its rate of metabolism by the adult rat hepatocyte in culture seemed to be biphasic, and at concentrations of less than 100µM there was indication of a saturable process. Although PSP seemed not to be toxic to hepatocyte cultures, it is partially metabolized by these cells (as opposed to no observed metabolism in human fibroblasts or HeLa cells). Therefore, its incorporation into tissue culture media formulations for use in hepatocyte cultures should be avoided, especially when studying the mechanism(s) of glucuronidation or metabolic pathways thought to be affected by this anionic dye.

Ponceau S: A sensitive method for protein determination in freshly isolated and cultured cells

A protein assay method is described that involves simultaneous dye binding and precipitation of protein. It has a sensitivity comparable to the widely used Lowry and Bradford methods without the chemical interferences sometimes encountered with these methods in tissue culture conditions. The assay uses readily available reagents and is applicable to a variety of cell preparations.

Economic Aspects of Instrument Evalution and Acquistion Part 1 Economic Aspects of Instrument Evalution

As the title implies, this two-part series will be devoted entirely to the economic aspects of instrument evaluation (Part 1) and acquisition (Part 2). The essential elements dealing with the economics of instrument testing are outlined in Table 1. A knowledge of comparative testing costs between the present technology utilized in the laboratory and that introduced via new instrumentation is as vital to management in its decision-making process as the instruments accuracy, precision, and observed effects on diagnostic testing. In situations where the technical data derived from instrument evalutions are essentially equivalent, the fine balance between making an acquisition or not wil undoubtedly be influenced strictly by economics, especially if savings in operational can be realized.

Instrument Optimization and Reliability

ABSTRACT As a continuation of past articles on instrument evaluation (1-4), two topics will be discussed: (i) optimization and (ii) reliability of instrumentation (or methodology). Although both have a great bearing and influence on much of the subject matter relating to the obtainment of valid technical data, they are of paramount importance today, specifically in areas of laboratory operations concerned with “cost-containment.”

Instrument Justification, Selection, and Technical Evaluation in Clinical Chemistry Part 2 Technical Aspects of Instrument Evaluation

The primary concern or objective of Part 2 is instrument evaluation based on a technical approach that is applicable to laboratory equipment of varying degrees of complexity [see item 1, Table 2, Part 1 (1)].

Instrument Justification, Selection, and Technical Evaluation in Clinical Chemistry Part 1 Instrument Justification And Selection

In a recent two-part series, the economic aspects of instrument evaluation and acquisition in clinical chemistry were discussed (1,2). In this present series, the various aspects of instrument justification and selection (Part 1) and technical evaluation (Part 2) in clinical chemistry will he covered.

Economic Aspects of Instrument Evaluation and Acquisition Part 2 Economic Aspects of Instrument Acquisition

As previously noted in Part 1 (1), three avenues of instrument acquisition are available to management if the decision is reached to obtain the instrument that has been evaluated in the clinical chemistry laboratory: namely, (i) purchase the instrument outright if funding is available, (ii) acquire the instrument via a financial loan at the most favorable interest rate over a given amortization period, or (iii) arrange for an operational lease. Beyond the cost of the instrument, the critical consideration of purchase as an acquisition option requires knowledge of several variables: depreciation, estimated life of the instrument, salvage value, time value of money (present value concepts), cash inflow/outflow, payback period, discounted cash flow rate, cost reimbursement by third-party payors, as well as the feasibility of equipment leases. How these are utilized in the financial decision-making process will be examined using the economic data generated in Part 1 of this two-part series using an excellent ...