Activation in stored platelet concentrates: correlation between membrane expression of P-selectin, glycoprotein IIb/IIIa, and beta-thromboglobulin release.

By using two distinct measurements of alpha-degranulation (surface P-selectin [alpha-granule membrane protein-140] expression and beta-thromboglobulin [beta-TG] release) and quantitation of glycoprotein (GP) IIb/IIIa surface density, stored platelet concentrates were evaluated to determine a) which method of measuring platelet alpha-granule release was more sensitive in detecting early platelet activation; b) whether Day 1 levels of activation predicted the extent of activation or cell lysis on Day 5 of storage; and c) whether changes in surface GPIIb/IIIa density were primarily dependent on platelet activation. By using samples from paired and unpaired units stored for 1, 3, and 5 days, four observations could be made. 1) A flow cytometric assay for the percentage of P-selectin-positive platelets was more sensitive for early detection of platelet activation than was measurement of beta-TG release. This finding was most likely due to enhanced sensitivity in detecting platelets that had undergone partial alpha-granule release. 2) Total P-selectin expression correlated with beta-TG release, which indicated that the extent of alpha-granule membrane fusion with the external platelet membrane was proportional to the amount of alpha-granule contents released into the supernatant. 3) All of the activation measurements on Day 1 predicted the activation values, but did not predict the degree of cell lysis (measured by lactate dehydrogenase discharge), on Day 5 of storage. 4) Surface GPIIb/IIIa density was increased on the subset of P-selectin-positive platelets as compared with the P-selectin-negative subset at all times during storage, but, within each subset, GPIIb/IIIa surface density did not significantly increase over the time of storage.

A macro creatine kinase isoenzyme in serum of apparently healthy individuals.

Activity of creatine kinase B subunit, as measured by an immunoinhibition assay, is persistently increased in certain healthy, asymptomatic adults, whose values for total CK activity are within normal limits. Serum samples from two such individuals were investigated by electrophoresis, heat inactivation, determination of activation energies, and immunosorption (on protein A). The results demonstrated the presence of a circulating complex of IgG and CK-BB, which has been described as macro CK, type I. To our knowledge, this is the first report of such macro CK isoenzymes in healthy individuals.

Acute effects of albumin infusion on blood volume and renal function in premature infants with respiratory distress syndrome.

The purpose of this study was to determine the acute effects of albumin infusion on blood volume and renal function in preterm infants with RDS and low total serum protein values. Ten infants (gestational age 28 to 36 weeks, body weight 0.88 to 2.46 kg) were given albumin 1 gm/kg (as 25% iv solution) over a ten-minute period. Within ten minutes after infusion was completed, total serum protein concentration, colloid osmotic pressure, and blood volume rose significantly while hematocrit fell from their preinfusion levels (P < 0.0005). Mean arterial blood pressure showed a smaller and less clear-cut increase (P < 0.05). Creatinine clearance rose significantly with infusion; even though preinfusion clearances correlated poorly with gestational age (r = 0.43), postinfusion clearances correlated well (r = 0.92). No significant rises in urinary flow rate Uosm/Posm, or free-water clearance were observed. These results indicate that albumin infusion acutely increases both blood volume and glomerular filtration in premature infants with RDS.

Automated radioimmunoassay of nicotine.

We have developed an automated nonequilibrium procedure for the radioimmunoassay of nicotine. The use of a unique iodinated nicotine derivative in this procedure gave a sensitivity of 10 micrograms/l for nicotine with a between-run precision of 7.4% and within-run precision of 6.0%. Nicotine levels of 60 to 67 micrograms/ml were found in subjects 15 min after smoking one standard cigarette. The technique herein reported is a very rapid, and sensitive radioimmunoassay for nicotine and facilitates the determination of nicotine in smoking subjects during the actual process of smoking.

Enzyme-linked immunosorbent choriomammotropin assay.

We have devloped an enzyme-linked immunosorbent assay for determining choriomammotropin (human placental lactogen) in serum. Unlabeled hormone competes with choriomammotropin-beta-galactosidase conjugate for antibody bound to polystyrene tubes. The entire assay can be performed in 2.5 h with good precision. The coefficient of variation for one sample with a mean concentration of 5.6 mg/L, assayed 10 times on the same day, was 5.7%. The coefficient of variation for nine samples (3.5 to 9.0 mg/L) assayed on five different days was 7.9%. Forty-eight clinical samples were assayed (y) and compared with results obtained by radial immunodiffusion (x). The resulting regression equation was: y = 1.05x + 0.78; r = 0.91.

An automated turbidimetric rate method for immunoglobulin assays.

A turbidimetric rate method for the determination of immunoglobulins IgG, IgA, and IgM has been adapted to an automatic kinetic rate analyzer. The procedure can be run on mildly lipemic sera without correction for sample light scatter. We report correlations with results by an immunodiffusion method and a manual laser nephelometric technique. The automated rate procedure described provides a rapid, accurate, precise and sensitive way to measure immunoglobulins.

Evaluation of EMIT methods for the determination of the five major antiepileptic drugs on an automated kinetic analyser.

We have evaluated the performance of enzyme-multipled immunoassay methods for the five major antiepileptic drugs on an automated system, the Perkin-Elmer Model KA-150 Kinetic Analyzer. The precision in the normal duplicate mode was found to be in the range of 6% to 10% for all five tests over a typical working day. All EMIT methods were compared to gas-liquid chromatographic procedures and, in addition, the phenytoin and phenobarbital assays were compared to a liquid-chromatographic method. The phenytoin assay was also compared to RIA and to a manual spectroscopic method. In general, most of the comparison studies resulted in acceptable correlation, although one gas chromatographic method did not correlate very well with the phenytoin and phenobarbital immunoassays.

A totally automated system for enzyme immunoassay of theophylline in serum.

We describe a procedure for enzyme immunoassay of theophylline (1,3-dimethylxanthine) in which all phases of the assay are totally automated in a Kinetic Analyzer (KA-150). This system permits assay of 75 10-mul samples per hour, with results available at 30-s intervals after initial sample preparation and preincubation. We compared results for 138 clinical samples by an ultraviolet method (x) and the present method (y). The slope of the comparison curve was 0.902, the y-intercept 0.402, and the correlation coefficient 0.984. The coefficient of variation for samples run in duplicate on the same day was 4.9%; it was 8.1% for samples run on different days. Specificity, sensitivity, simplicity, speed, and small reagent requirement all make this an attractive alternative to chromatographic procedures.

Comparative determination of phenytoin by spectrophotometry, gas chromatography, liquid chromatography, enzyme immunoassay, and radioimmunoassay.

Sera from patients being treated with phenytoin were analyzed for the drug by spectrophotometry, gas chromatography, radioimmunoassay, enzyme immunoassay, and liquid chromatography. The essay values obtained were intercompared statistically. Enzyme immunoassay and liquid chromatography appear to be attractive alternatives to the more traditional methods of spectrophotometry and gas chromatography. Our radioimmunoassay data correlated poorly with results by the four other methods.

A direct enzyme-linked immunosorbent assay (ELISA) for detection of antibodies for rubella virus in human sera.

A direct enzyme-linked immunoassay (ELISA), based on the "sandwich" principle on an antigen-coated plastic disc, was used for the rapid detection of rubella antibody. Results were obtained the same day, and the prior adsorption of sera to remove non-specific inhibitors was not necessary. The ELISA was compared to the hemagglutination-inhibition (HAI) test on 500 serum samples. There was general agreement between the two methods; most discrepancies occurred with low-titered HAI positive sera. There was excellent correlation between the tests with serum samples negative for rubella antibodies and those samples with HAI titers greater than or equal to 1:40.

A practical routine gas chromatographic determination of phenobarbital, primidone and diphenylhydantoin.

A gas chromatographic method for the routine determination of phenobarbital, primidone and diphenylhydantoin using alkaline extraction and on-column methylation has been developed. The procedure has been optimized for recovery of all three anticonvulsant drugs with particular attention to primidone. Correlation with photometric methods for phenobarbital and diphenylhydantoin show the technique offers significant advantages in both accuracy and precision over other methods.

Serum biochemistry values in normal pygmy goats.

Blood serum of pygmy goats (both sexes, and castrated males) was analyzed to establish biochemical reference values. Influence of age on reference values was also studied. Serum biochemical analyses were made for urea nitrogen, creatinin, bilirubin, lactate dehydrogenase, aspartate aminotransferase, alkaline phosphatase, glucose, uric acid, and total lipids. These serum values for pygmy goats were similar to those reported for man, except as follows: Aspartate aminotransferase activities were slightly higher than those reported for man. Glucose concentrations in pygmy goats were slightly lower than in human beings, and uric acid levels were significantly lower than the values for man. Female and castrated male goats had lower total lipid concentrations than did human beings, whereas intact males had higher concentrations. Thus, of the 9 measured variables for pygmy goats, 5 were comparable to human values. This, together with other attributes, including the small size which conduces to economics of maintenance and enhances the desirability of using pygmy goats in research.

Serum electrolytes in normal pygmy goats.

Serum from pygmy goats was analyzed to determine normal base lines for electrolytes. Animals of both sexes were used. Influence of age on these variables was also investigated. The electrolyte values were as follows: sodium, 147 +/- 6.0 mEq/L; potassium, 5.6 +/- 1.0 mEq/L; chloride, 106.0 +/- 4.2 mEq/L; calcium, 4.9 +/- 0.3 mEq/L; magnesium, 2.1 +/- 0.3 mEq/L; and inorganic phosphorus, 4.8 +/- 0.9 mEq/L. Most of the serum electrolyte values for pygmy goats were similar to reports for other ruminant species and human beings.

Radioimmunoassays of drugs of abuse in humans: a review.

Only recently has radioimmunoassay been used for the detection of drugs of abuse in body fluids. While conventional assay methods are time-consuming, relatively insensitive, and require a larger sample volume, the radioimmunoassay method is rapid, sensitive, specific, and can be performed with a minimum of sample. Performance of individual assays is identical, and requires about 1 hour to complete. Herein are reviewed the radioimmunoassays which have been developed for measurement of drugs of abuse in humans. These new techniques are of importance in screening and in further research into the effect of these drugs on the functions of the human body.

Automated turbidimetry of amylase activity by use of a discrete kinetic analyzer.

We adopted an automated turbidimetric rate method for determining amylase activity to the KA-150 Kinetic Analyzer. In the method, an insoluble amylopectin substrate is used with activity determined by the rate of decrease in turbidity. Run-to-run CV for 59 samples with activities up to 400 units (arbitrary amylase units per 100 ml of sample) was 2.8%. A comparison with a similar method, performed by nephelometry, for 104 sera, showed a correlation coefficient (r) of 0.992, with a slope of 1.02. In an additional comparison with an amyloclastic method, for 52 sera r was 0.997, with a slope of 1.01. Day-to-day precision for control sera with activities near the upper limit of normal (279 and 216 units) averaged 2.5% during two months. Measured and calculated activity were linearly related to well above the upper limit of normal (normal range, 60-200 arbitrary units), showing a deviation from linearity of about 10% at 450 units. Commercial reagents available for the Perkin-Elmer Model 91 Amylase Lipase Analyzer can be used with the KA-150.

Enzyme immunoassay of hepatitis associated antigen (HAA).

Due to its high sensitivity, radioimmunoassay has become of great importance in the detection and measurement of levels of proteins and steroids in body fluids. However, this method involves the use of expensive equipment and radioactive material. Herein is described an alternate method to radioimmunoassay, which uses an enzyme-labeled rather than a radioactively-labeled antibody. An enzyme immunoassay procedure, the Cordia HAA-enzyme Immunoassay, for the detection of hepatitis-associated antigen has been evaluated. With this technique a sandwich type immunoassay with an alkaline phosphatase tagged second antibody is used. The presence of antigen is detected by the p-nitrophenyl phosphotase activity of the bound enzyme. In 1083 clinical samples from patients of Jackson Memorial Hospital, only 19 discrepant results were found when tested by both the Cordia and the Ausria II methods. Eight had sufficient sera for retesting, yielding two positive Cordia, negative Austia; one negative Cordia, positive Ausria; one borderline positive; and four unconfirmed false positives by Cordia.

Automated radioimmunoassay of choriomammotropin (human placental lactogen).

We describe a totally automated procedure for radioimmunoassay of choriomammotropin, in which all phases of the assay are automated in a single system ("Centria"). This system permits the simultaneous incubation and separation of many samples in a nonequilibrium assay, and measurements are obtained in less than 30 min. Results for clinical samples by reference radioimmunossay methodology and with the Centria system compared uniformly well: y=0.91x - 0.87;r=0.94. The coefficient of variation for samples run in duplicate on the same day was 5.2%, 7.4% for samples run on different days. The specificity, sensitivity, simplicity, and speed of this system makes it a useful new tool for kinetic, nonequilibrium immunoassay.