Portable simultaneous multiple analyte whole-blood analyzer for point-of-care testing.

We describe a portable clinical chemistry analyzer for point-of-care measurements of multiple analytes in less than 10 min from approximately 40 microL of whole blood (fingerstick or venous). Whole blood is applied directly to a 7.9-cm-diameter, single-use plastic rotor containing liquid diluent and greater than or equal to 4-12 tests in the form of 1- to 2-mm-diameter dry reagent beads. The reagent/rotor is immediately placed in a portable instrument along with a ticket/label results card. As the instrument spins the rotor, capillary and rotational forces process the blood into diluted plasma, distribute the patient's diluted sample to cuvettes containing the reagent beads, and mix the diluted sample with the reagents. The instrument monitors the chemical reactions optically at nine wavelengths; sample volume and temperature are also measured optically. The calibration data for each reagent are read from a bar code on the periphery of each rotor. The instrument processes all the measurements to calculate, store, print, and communicate the results. Each reagent/rotor contains an enzymatic control that must be within a defined range before the results from that analysis are reported.

Optimal conditions and comparison of lactate dehydrogenase catalysis of the lactate-to-pyruvate and pyruvate-to-lactate reactions in human serum at 25, 30, and 37 degrees C.

We report optimal conditions for assaying highly purified human lactate dehydrogenase isoenzymes with the lactate-to-pyruvate and pyruvate-to-lactate reactions, as they apply to human serum. Interconversion of results between reactions is not practicable. Measurements of lactate dehydrogenase in either reaction direction at 25, 30, or 37 degrees C can be equally reliable if the volume fraction and the resulting deltaA/min is small. However, for interinstrument and interlaboratory comparisons, results from the lactate-to-pyruvate reaction are more reliable.

Quantitative toxicology: interlaboratory and intermethod evaluation in New York State.

The New York State Department of Health has conducted a proficiency evaluation program in quantitative toxicology since 1974. Serum samples containing a barbiturate and phenytoin, together with either glutethimide, procainamide, or theophylline, are sent to participating laboratories quarterly. Within the first two years of the program the percentage of laboratories able to quantitate 75% of the test samples to within 25% of the gravimetric values increased from 25 (1974-1975) to 40% (1975-1976). This improvement was partly due to licensure requirements, improved technology for sample preparation and analysis, and the availability of better quality-control practice. An obstacle to obtaining uniform accuracy is the lack of adequate calibration or testing materials. To overcome these obstacles, pure drugs are weighed into a bovine serum matrix, and the weights are confirmed by reference laboratories and used as the target values in the testing program. Comparison of the methods used by participants in this program for barbiturate and phenytoin yielded equations different from those found in other method evaluations.

Optimal reaction conditions for assaying human lactate dehydrogenase pyruvate-to-lactate at 25, 30, and 37 degrees C.

Optimal reaction conditions for assaying human lactate dehydrogenase pyruvate-to-lactate were determined for isoenzymes 1 and 5 at 25, 30, and 37 degrees C. Three of the nine different buffers examined--imidazole, triethanolamine, and N-tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid--are satisfactory. Beta-NADH, pyruvate, and hydrogen ion concentrations were chosen to measure both isoenzymes with maximal-equal-sustainable efficiency at the lowest substrate concentrations. Approximately 95% of each isoenzyme is measured, for activities up to threefold the upper normal limit, if the measurements are made immediately after the reaction is initiated. The Arrhenius relationship for each isoenzyme is unique. Interconversion of results from one temperature to another is practical only with reservations. Results at 37 degrees C are not as reliable as those at 25 degrees C.

The effect of temperature on the kinetic constants of human lactate dehydrogenase 1 and 5.

The kinetic constants of human lactate dehydrogenase 1 and 5 (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27), assayed lactate-to-pyruvate increase with temperature. The reaction mechanism is ordered sequential as has been found with lactate dehydrogenase from other sources. The KM values for each substrate are larger for isoenzyme 5 than for 1. For lactate dehydrogenase 1 the KM(lactate) increases from 1.07 mM at 25 degrees C to 3.95 mM at 37 degrees C and for lactate dehydrogenase 5 it increases from 5.37 mM at 25 degrees C to 6.88 mM at 37 degrees C. The KM(NAD+) for lactate dehydrogenase 5 is 0.14 mM at 25 degrees C and 0.29 mM at 37 degrees C. The increase in the KM for each substrate with increasing temperature confirms that additional substrate is required for optimal reaction conditions at higher temperatures.

Optimal conditions for assaying human lactate dehydrogenase by the lactate-to-pyruvate reaction: Arrhenium relationships for lactate dehydrogenase isoenzymes 1 and 5.

Optimal reaction conditions to sassay human lactate dehydrogenase (lactate-to-pyruvate) were established for isoenzymes 1 and 5 at 25, 30, and 37 degrees C in diethanolamine and 2-amino-2-methyl-1,3-propanediol. Different substrate concentrations are required at each temperature. The conditions permit measurement of lactate dehydrogenase 1 and 5 with the lowest substrate concentrations that allow for the highest equal sustainable efficiency in measuring both isoenzymes. About 95% of each isoenzyme activity is measured if the assay is performed within the first minute after the reaction is initiated even for activities as high as triple the upper limit of normal. The Arrhenius relationship is different for each isoenzyme, but results obtained for each at one temperature can be compared with results at another temperature by use of simple conversion equations. Assays at 25 and 30 degrees C are more economical and less variable than assays at 37 degrees C.

Proficiency testing: purification of lactate dehydrogenase 1 and results of its use as a reference material in the New York State program.

Highly purified human lactate dehydrogenase 1 has been used in an interlaboratory evaluation and improvement progran in clinical chemistry in New York State since 1971. Although there are difficulties in determining and assigning the most nearly accurate values for test samples in the absence of a reference method and a reference material, we have minimized these difficulties by using human lactate dehydrogenase preparations purified as we describe here and suspended in the same matrix, and by utilizing "reference laboratories" that routinely are doing multiple assays to determine the most nearly accurate value. The lactate dehydrogenase used in the program is stable for longer than 1.5 years. Conversion factors were used to convert all results to U/liter at 30 degrees C. Review of the data for 1972-75 shows a marked improvement in the accuracy of virtually all methods used to determine this enzyme.

Interconverting measurements of human lactate dehydrogenase activities in three buffers.

The lactate-to-pyruvate reaction for serum lactate dehydrogenase (LD) is most frequently assayed in one of three buffers, pyrophosphate (PPi), tris(hydroxymethyl)amino-methane (Tris), or 2-amino-2-methyl-1-propanol (AMP). We described interconverting results for serum samples and for highly purified LD isoenzymes I (dissolved in one of these matrixes) assayed in these buffers under optimized reaction conditions. The equation for converting results obtained for sera in Tris (x) to those in PPi(y) (both at 30 degrees C) is y = 0.74x+10 (n = 98). Since AMP is used extensively in Technicon procedures, we determined the LD activity of sera with an SMA 12/60, at 37 degrees C. The equation for convering these AMP results to results obtained in PPi at 30 degrees C is y = 0.45x-16 (n = 90). Very different equations were obtained with highly purified LD isoenzyme I maintained in two different matrixes and with both isoenzymes assayed in the same matrix. The matrix in which LD is dissolved and the proportion of various LD isoenzymes affect the magnitude of difference in observed LD activity under various conditions. Therefore, in clinical laboratories that use more than one analytical method or when conversion equations are used in the comparison of interlaboratory results, it is important to define the LD source, isoenzyme content, and the matrix, as well as the reaction conditions, and to use many samples with a wide range of activities when determining the conversion equations. For any changes in reagent source, substrate concentration, or alteration in procedure, a new normal range and new conversion equations should be determined.

A search for the best buffer to use in assaying human lactate dehydrogenase with the lactate-to-pyruvate reaction.

Highly purified human lactate dehydrogenases I and V were assayed in 17 different buffers, at a variety of reaction pH's. Diethanolamine and 2-amino-2-methyl-1,3-propanediol provided the best measurements of the enzyme, assayed lactate-to-pyruvate. However, the commercial preparation of 2-amino-2-methyl-1,3-propanediol contained insoluble matter and was relatively expensive. All of the four buffers nowmost commonly used were found to present difficulties. Glycine and pyrophosphate were inhibotory tolactate dehydrogenase activity with increasing buffer concentration. 2-Amino-2-methyl-1-propanol had three major disadvantages: it is chemically unstable during reagent preparation; activity is dependent on buffer concentration; and the pH optima for isoenzymes I and V are vastly different. The pKa of tris(hydroxymethyl)aminomethane is 8.0 at 30 degrees C, whereas to measure total activity the reaction pH should be greater than 8.5; thus tris(hydroxymethyl)aminomethane has limited buffering capacity at the reaction pH.

Effect of reaction initiator on human lactate dehydrogenase assay.

Human lactate dehydrogenase isoenzymes I and V have decreased activities when the reaction is initiated with lactate. No loss in lactate dehydrogenase I activity was found when the reaction was initiated with enzyme or NAD+. For lactate dehydrogenase V an NAD+-initiated reaction, as compared to an enzyme-initiated reaction, yields lower activity in sodium pyrophosphate buffer but higher activity in tris(hydroxymethyl)aminomethane buffer. Both isoenzymes have higher lactate-to-pyruvate activity when assayed in the latter buffer than when assayed in the former. Human lactate dehydrogenase V (but not I) exhibited different activities when assayed with lactate from two different commercial sources. Human lactate dehydrogenase assayed by the pyruvate-to-lactate reaction is not affected by the choice of reaction initiator.

Effects of caffeine on postreplication repair in xeroderma pigmentosum cells.

Compared to its effects on unirradiated cells, caffeine inhibited DNA chain elongation and joining in UV-irradiated xeroderma pigmentosum (XP) cells but not in irradiated normal human cells. The drug also inhibited the ability of XP cells to recover and make DNA of normal size at long times after irradiation.

DNA repair in Potorous tridactylus.

The DNA synthesized shortly after ultraviolet (UV) irradiation of Potorous tridactylis (PtK) cells sediments more slowly in alkali than that made by nonirradiated cells. The size of the single-strand segments is approximately equal to the average distance between 1 or 2 cyclobutyl pyrimidine dimers in the parental DNA. These data support the notion that dimers are the photoproducts which interrupt normal DNA replication. Upon incubation of irradiated cells the small segments are enlarged to form high molecular weight DNA as in nonirradiated cells. DNA synthesized at long times ( approximately 24 h) after irradiation is made in segments approximately equal to those synthesized by nonirradiated cells, although only 10-15% of the dimers have been removed by excision repair. These data imply that dimers are not the lesions which initially interrupt normal DNA replication in irradiated cells. In an attempt to resolve these conflicting interpretations, PtK cells were exposed to photoreactivating light after irradiation and before pulse-labeling, since photoreactivation repair is specific for only one type of UV lesion. After 1 h of exposure approximately 35% of the pyrimidine dimers have been monomerized, and the reduction in the percentage of dimers correlates with an increased size for the DNA synthesized by irradiated cells. Therefore, we conclude that the dimers are the lesions which initially interrupt DNA replication in irradiated PtK cells. The monomerization of pyrimidine dimers correlates with a disappearance of repair endonuclease-sensitive sites, as measured in vivo immediately after 1 h of photoreactivation, indicating that some of the sites sensitive to the repair endonuclease (from Micrococcus luteus) are pyrimidine dimers. However, at 24 h after irradiation and 1 h of photoreactivation there are no endonuclease-sensitive sites, even though approximately 50% of the pyrimidine dimers remain in the DNA. These data indicate that not all pyrimidine dimers are accessible to the repair endonuclease. The observation that at long times after irradiation DNA is made in segments equal to those synthesized by nonirradiated cells although only a small percentage of the dimers have been removed suggests that an additional repair system alters dimers so that they no longer interrupt DNA replication.

Effect of caffeine on postreplication repair in human cells.

DNA synthesized shortly after ultraviolet (UV) irradiation of human cells is made in segments that are smaller than normal, but at long times after irradiation the segments made are normal in size. Upon incubation, both the shorter and the normal segments are elongated and joined by the insertion of exogenous nucleotides to form high molecular weight DNA as in nonirradiated cells. These processes occur in normal human cells, where UV-induced pyrimidine dimers are excised, as well as in xeroderma pigmentosum (XP) cells, where dimers are not excised. The effect of caffeine on these processes was determined for both normal human and XP cells. Caffeine, which binds to denatured regions of DNA, inhibited DNA chain elongation and joining in irradiated XP cells but not in irradiated normal human or nonirradiated cells. Caffeine also caused an alteration in the ability to recover synthesis of DNA of normal size at long times after irradiation in XP cells but not in normal cells.

Recovery of the ability to synthesize DNA in segments of normal size at long times after ultraviolet irradiation of human cells.

DNA synthesized in human cells within the first hour after ultraviolet (UV) irradiation is made in segments of lower molecular weight than in nonirradiated cells. The size of these segments approximates the average distance between pyrimidine dimers in the parental DNA. This suggests that the dimers interrupt normal DNA synthesis and result in gaps in the newly synthesized DNA. However, DNA synthesized in human cells at long times after irradiation is made in segments equal or nearly equal to those synthesized by nonirradiated cells. The recovery of the ability to synthesize DNA in segments of normal size occurs in normal human cells, where the dimers are excised, and also in cells of the human mutants xeroderma pigmentosum (XP), where the dimers remain in the DNA. This observation implies that the pyrimidine dimer may not be the lesion that causes DNA to be synthesized in smaller than normal segments.