Quantitative method for determining serum alkaline phosphatase isoenzyme activity I. Guanidine hydrochloride: new reagent for selectively inhibiting major serum isoenzymes of alkaline phosphatase.

The potential use of the protein denaturant guanidine hydrochloride to inhibit selectively the enzyme activity of serum alkaline phosphatase isoenzymes from liver, bone, intestine, and placenta was investigated. Inhibition of each isoenzyme was shown to be dependent on time and concentration of inhibitor. In the presence of 0.3 mol/l (28.7 g/l) guanidine hydrochloride for 170 seconds 14%, 47%, and 90% of the total alkaline phosphatase activity remained in samples of bone, liver, and intestinal origins, respectively. In contrast, the activity of the placental isoenzyme increased by 24%. The degree of inhibition was shown to be independent of total alkaline phosphatase activity. Investigations were performed at 37 degrees C using the Cobas Bio centrifugal analyser. We conclude that this reagent has several practical advantages over urea as a selective inhibitor of alkaline phosphatase isoenzymes, including a faster and more reproducible inhibition at a much lower reagent concentration.

Quantitative method for determining serum alkaline phosphatase isoenzyme activity: estimation of intestinal component.

Intestinal alkaline phosphatase activity was measured using levamisole inhibition, and results were compared with a previously reported method using L-phenylalanine. Sixty two per cent intestinal, 39% placental, and 1.3% of either bone or liver alkaline phosphatase activity remained when alkaline phosphatase activity was inhibited in a 2-amino-2-methyl-1-propanol (AMP) buffer reagent system with 10 mmol/l levamisole (final assay concentration 8.1 mmol/l). The assay imprecision (SD) was 0.6 U/l compared with 3.9 U/l using L-phenylalanine for specimens with total alkaline phosphatase activity less than 250 U/l (reference range 30-120 U/l). In serum pools with raised total alkaline phosphatase activity errors in recovered intestinal activity were small (usually less than 3 U/l) when intestinal alkaline phosphatase was added. Much larger errors and many underestimated results were found using L-phenylalanine. For non-haemolysed specimens it is concluded that an assay based on levamisole inhibition provides a better measure of intestinal alkaline phosphatase activity than L-phenylalanine.

Quantitative method for determining serum alkaline phosphatase isoenzyme activity II. Development and clinical application of method for measuring four serum alkaline phosphatase isoenzymes.

A method for quantitating the liver, bone, intestinal and placental alkaline phosphatase activity of serum, using an algorithm for converting selective inactivation by guanidine hydrochloride, L-phenylalanine, and heat into equivalent isoenzyme activity is described. The method can individually quantify mixtures of isoenzymes to within a margin of 3%; it has acceptable reproducibility and has been used to develop both age and sex related reference ranges. Analysis time is about 30 minutes. The clinical reliability of this method has been shown in a study of 101 patients, in 79% of whom isoenzyme results were compatible with the final clinical diagnosis; in 10% a clinical diagnosis resulted from isoenzyme analysis, and in a further 11% the source of the increased alkaline phosphatase activity was identified and supported by electrophoresis, with a definite clinical diagnosis yet to be made.

Theoretical constraints in the measurement of serum bilirubin binding capacity.

The large majority of methods for measuring serum unoccupied bilirubin binding capacity involve adding an exogenous ligand to a diluted serum sample. If the added ligand binds specifically to bilirubin binding sites, the extrapolation is made that the amount of ligand which becomes bound represents the previously unoccupied bilirubin binding capacity of the original sample. This simple theory ignores the labile nature of the equilibrium reactions between bilirubin binding sites and the ligands with which they interact. The present theoretical and experimental study of these equilibrium reactions shows that (a) sample dilution alone results in changes in the proportional occupancy of bilirubin binding sites by bilirubin and other endogenous ligands, so that the extent of vacancy of those binding sites becomes exaggerated; (b) addition of an exogenous ligand to the diluted sample results is further displacement of native bilirubin and other endogenous ligands from bilirubin binding sites. The amount of added ligand which becomes bound is thus likely to represent a greater extent of vacancy of bilirubin binding sites than was present in the original sample. Results from such methods can therefore only overestimate the unoccupied bilirubin binding capacity of blood plasma in vivo. In order to avoid these analytical problems, current methods must be redesigned. It is suggested from this work that the ability of a serum to sequester safety additional bilirubin may best be assessed from measurement of its bilirubin buffering capacity. This parameter is different from the total or unoccupied binding capacity currently attempted, and its measurement is within the capability of published methods for measuring free (= unbound) bilirubin, modified to analyse minimally diluted samples.

Comparison of standardization techniques for manual colorimetric analyses.

The influence of both the mode of standardization and the type of standard on the precision of four manual colorimetric methods performed under optimal conditions variance is described. The terms variable calibration mode and constant calibration mode are proposed; these describe standardization by within-run standards and standardization by a predetermined calibration relationship between concentration and absorbance that remains constant over a fixed period of time. We show that calibration relationships and mode of standardization must be established for each and every individual method on objective evidence. Where within-run standards are used, they must be carefully selected for each method. The implications for method evaluation and quality control are discussed.

Assessment of the Beckman oxygen rate analyzer for the enzymatic assay of cholesterol.

We describe an evaluation of the Beckman oxygen rate analyzer for the determination of cholesterol. The precision and accuracy obtained did not fulfill the short-term criteria of the Center for Disease Control but the precision met the criteria of the College of American Pathologist for analysis of cholesterol in screening programs. Good overall correlation between the oxygen rate method and a continuous-flow method with prior extraction was found for patient samples. The results obtained by the oxygen rate method had net positive bias up to the limit of linearity of 8.6 mmol cholesterol/liter. The instrument is not recommended for laboratory or epidemiological studies but may prove useful in screening programs.

Comparison of standardization techniques for colorimetric analyses on a centrifugal analyzer.

The influences of the mode of standardization and the type of standard on the precision of four mechanized methods performed on a centrifugal analyzer are described. Experimental results show that the mode of standardization -- variable, using a standard in each analytical batch, and constant, using a direct relationship between concentration and absorbance -- must be objectively selected. If the variable mode is chosen, the type and level of standard must be carefully chosen and absorbance of assays of the standard must be carefully monitored for good quality control. It is recommended that the optimum standardization technique and standard, where applicable, should be assessed and subsequently documented in evaluations of methods, kits and instruments.

Evaluation of a kit for the colorimetric determination of inorganic phosphate.

An evaluation of a colorimetric kit method for the determination of inorganic phosphate (Pierce Phosphorus Auto/Stat Kit) is described. The within-batch and between-batch precisions were shown to fulfil current criteria, and recovery experiments, linearity studies, analyses of quality control materials, and studies of possible interfering substances evidenced good accuracy. Comparison of the results obtained on samples from patients with those obtained by the vanadate/molybdate continuous-flow method showed that the test method had a comparative positive bias. The use of a calibration reference serum as standard is recommended. The kit method is technically simple, requiring no protein precipitation, and analyses can be performed rapidly.

The effect of instrument variables on calculation factors for standardisation of colorimetric analyses.

A number of colorimetric methods, particularly enzyme activity assays, are usually standardised using calculation factors based on the molar absorptivity of a principle reactant or product. Such methods are subject to long-term variation. The relationship between long-term variation in results and instrument variables affecting calculation factors has not been quantitated. In this study, we have shown that, on a centrifugal analyser having a within-run coefficient of variation of less than 1%, instrument variables affecting calculation factor alone could result in changes in results of up to 8.5% over 75 days. We therefore advocate daily use of a solution of potassium dichromate to monitor instrument variables that can independently affect calculation factors and within-run imprecision. This procedure is useful for maintaining long-term performance and for differentiating problems of instrumental or chemical origin.

[Enzyme spectrophotometry determination of sodium, potassium and chloride ions in serum and urine]. / Enzymatische spektrophotometrische Bestimmung von Natrium-, Kalium- und Chloridionen im Serum und Urin.

The principles and main features of enzymatic methods for the measurement of sodium, potassium, and chloride are reviewed and their performance compared with current procedures. Each method makes use of a relatively specific enzyme, catalysing a reaction whose rate is sensitive to the ion to be determined. Where the (S)0.5 of the enzyme for the ion is much lower than the assay concentration, the ion concentration may be reduced by a binding agent. Alternatively, a competitive inhibitor may be used to raise the (S)0.5 of the enzyme. In the case of chloride determination with amylase the (S)0.5 of the enzyme is raised by limiting the concentration of free calcium. In the measurement of potassium, interfering ions such as sodium are removed by binding with Kryptofix 221 and improvement in performance is also achieved by use of a bacterial pyruvate kinase less sensitive to sodium. The enzymatic methods are applicable to measurement of sodium, potassium and chloride in blood or urine with good precision, accuracy, and specificity. They can be used on mechanized or manual instruments. There appears to be minimal interferences from compounds found in normal or pathological serum or urine.

Immunoturbidimetric measurement of transferrin.

This paper describes the development of an automated immunoturbidimetric assay for transferrin on a centrifugal analyser. Regression analysis of transferrin values measured immunoturbidimetrically demonstrates good agreement with data obtained by radial immunodiffusion (y = 0.997 + 0.024 g/l, r = 0.980, n = 50). The assay has a detection limit of 1.0 g/l and working range of approximately 1.0 to 6.0 g/l of transferrin. Day-to-day coefficient of variation is less than 3.5%. Immunoturbidimetric transferrin (g/l) and total iron binding capacity values (mumol/l) were compared using an established total iron binding capacity method (y = 0.050 X - 0.030 g/l, r = 0.967, n = 50). Minimal interference was found for lipaemic, haemolysed or icteric samples. Transferrin reference values with a mean of 3.05 g/l and 95% limits from 2.45 to 3.65 g/l were derived using serum from 300 apparently healthy subjects (150 males, 150 females). We conclude that the proposed transferrin method is more reliable and easier to perform than presently available total iron binding capacity methods.

Problems associated with clinical chemistry quality control materials.

Quality control methods and materials are widely used to monitor each and every facet of clinical chemistry laboratory performance. Quality control materials are also used in evaluation of methods and as secondary standards. A wide range of liquid and lyophilized materials are available from commercial sources and are prepared in individual laboratories. Many problems arise in the use of quality control materials. Problems discussed in this review include the use of nonhuman based materials and additives of animal origin, the physical and chemical characteristics of quality control materials that differentiate such samples from those from patients, attempts to generate quality control materials with elevated levels of particular analytes, the difficulties in handling and storage of quality control materials, the dangers of hepatitis, and the stability of quality control materials both during storage in the laboratory and after their reconstitution. The advantages and disadvantages of liquid and lyophilized quality control materials are discussed. The assignation of analyte values is of particular importance as the current trend is to consider inaccuracy of laboratory methods in addition to imprecision. This review assesses relevant publications in an area of fundamental importance to quality control in clinical chemistry.

Determination of inorganic sulfate in plasma with a centrifugal analyzer.

Turbidimetry of inorganic sulfate, after precipitation with barium sulfate, can be done simply in a Cobas Bio centrifugal analyzer. Polyethylene glycol is used as the precipitate-stabilizing agent. Reproducibility of precipitation is enhanced by the presence of BaSO4 particles, which function as seed nuclei. There is no interference by normal or above-normal concentrations of phosphate, heparin, bilirubin, hemoglobin, or erythrocyte contents, or by lipemia (triglyceride concentrations up to 6.5 mmol/L). Analytical recovery of added inorganic sulfate was found to be quantitative. Precision is similar to that for other methods for inorganic sulfate in plasma. This method is suitable for the rapid, routine analysis of plasma inorganic sulfate, and it is simple and less expensive to perform than alternative methods.

Manual and centrifugal analyzer determination of cholesterol and triglycerides with single-vial enzymatic reagents.

We have assessed Calbiochem single-vial enzymatic reagent systems for the analysis for cholesterol and triglycerides, using manual and centrifugal analyzer techniques. Analysis for cholesterol by both techniques had acceptable performance by the short-term criteria of the Center for Disease Control (CDC) during this study carried out under optimal conditions of variance. Analysis for triglycerides by the manual technique did not meet CDC criteria, and we do not recommend use of this method. Analysis for triglycerides by the centrifugal analyzer technique had acceptable short-term precision but had unacceptable accuracy at high concentrations of triglycerides; this method may be considered to have acceptable performance for routine laboratory use.

Laboratory evaluation of the Beckman Synchron CX3 clinical chemistry analyzer.

In this evaluation of the Beckman Synchron CX3, the multi-analyte clinical chemistry analyzer exhibited high precision, good linearity, and no carryover for each of the eight analytes measured. Results obtained correlated well with those produced by our routine instrumentation (Beckman Astra, Varian atomic absorption spectrophotometer). The instrument can process up to 75 samples per hour (600 tests per hour if all tests available are requested) and, after calibration, can provide urgent results for the complete panel of tests within 2 1/2 min. The performance characteristics of this instrument make it ideal as a routine or a "stat" analyzer for commonly requested tests in the clinical chemistry laboratory.

Effectiveness of an outpatient urine screening program.

We evaluated the effectiveness of a routine outpatient urinalysis screening program on a sample population of 2600 patients. The 189 abnormal urine results found in 182 patients were followed up by study of any new clinical and laboratory investigations or therapeutic modifications initiated on the basis of any abnormal test result. The urinalysis screening program appeared to have significant bearing on diagnosis or treatment in only 13 patients. Abnormalities found in 150 of the 182 patients were either not noted or no further positive action was taken. Thus we concluded that under the conditions of this study the urine screening program added to hospital costs without significant benefit to the patient.

Urinalysis by use of multi-test reagent strips: two dipsticks compared.

We compared Ames' "N-Multistix" with Boehringer's "Combur-8" ("Chemstrip-8") multi-test urine reagent strips by analysis of contrived urine specimens, testing accuracy, precision, specificity, and limits of detection of both products. Relative cost and ease of use were also examined. Each brand of urinary dipstick had specific advantages but it is unlikely that patient care would be adversely affected by preferential use of either product.