Immunochemiluminometric and immunoradiometric determinations of intact and total immunoreactive parathyrin: performance in the differential diagnosis of hypercalcemia and hypoparathyroidism

Parathyrin (parathyroid hormone; PTH) was measured with three immunoassays: a two-site immunochemiluminometric (ICMA) and a two-site immunoradiometric (IRMA) method for intact PTH, and a sensitive radioimmunoassay for mid-region or "total" PTH, measuring both intact hormone and inactive fragments. Single specimens from normal subjects and from individuals with primary hyperparathyroidism, hypercalcemia associated with malignancy, and hypoparathyroidism were analyzed with all three methods. All individuals with primary hyperparathyroidism showed absolutely above-normal concentrations with the mid-region RIA, 28 of 29 did with the ICMA, and 21 of 29 did with the IRMA. PTH concentrations in primary hyperparathyroidism were most increased relative to normal subjects with the mid-region assay (10.4 times), less so with the intact assays (ICMA 5.5 times; IRMA 5.3 times). Concentrations of intact PTH were suppressed below normal in nearly all patients with hypercalcemia associated with malignancy, as measured with the ICMA (26 of 30) and the IRMA (28 of 30) assays. In marked contrast, results for mid-region PTH were normal or slightly above normal, consistent with studies suggesting that the parathyroids secrete both intact hormone and inactive fragments, the former being more sensitive to suppression by hypercalcemia. In hypoparathyroidism PTH concentrations were detectable but below normal in all patients by the intact assays and in all but one patient by the mid-region assay. These low concentrations are probably due to a nonspecific serum effect that could be resolved with selection of a more appropriate standard matrix. Although all three assays are useful in the differential diagnosis of hypercalcemia, two-site intact assays are more convenient and more specific in patients with compromised renal function.     

Lack of comparability of intact parathyroid hormone measurements among commercial assays for end-stage renal disease patients: implication for treatment decisions

BACKGROUND: Variability among assays used to measure intact parathyroid hormone (iPTH) is of particular concern because of the routine use of iPTH assay results to guide management of osteodystrophy and calcium metabolism in patients with end-stage renal disease (ESRD). The aim of this study was to determine the extent to which results from commercially available iPTH assays diverge from results obtained with the Nichols Allegro(R) Intact PTH immunoradiometric assay (IRMA), which was used as evidence in the development of the National Kidney Foundation's Kidney Disease Outcomes Quality Initiative Clinical Practice Guidelines. METHODS: We divided EDTA plasma from 46 dialysis patients with ESRD and measured iPTH values with the following commercially available iPTH assays: Nichols' Allegro iPTH IRMA, Nichols Advantage iPTH immunochemiluminescent assay (ICMA), Scantibodies' Total Intact PTH IRMA, DiaSorin's N-tact iPTH IRMA, DPC's Coat-A-Count iPTH IRMA, Roche's Elecsys iPTH ICMA, and DSL's Active iPTH IRMA. RESULTS: Method comparison showed considerable interassay differences in the measurement of iPTH in ESRD patients. IPTH values assessed by other methods ranged, on average, from 60% to 152% of the Nichols Allegro IRMA values. Of the 6 iPTH assays tested, only the Scantibodies Total Intact PT IRMA (P = 0.7554) and the Roche Elecsys iPTH ICMA (P = 0.1327) resulted in iPTH values not statistically different from those obtained with the Nichols Allegro iPTH IRMA. CONCLUSIONS: Noncomparability among iPTH assays remains a distinct problem for the management of ESRD patients. These results should be taken into consideration when determining the course of medical treatment based on measured iPTH concentrations.     

Early detection of myocardial infarction by an immunoradiometric procedure for creatine kinase MB

I investigated the diagnostic efficacy of the Embria-CK immunoradiometric assay (IRMA) in detecting myocardial infarction, using a single blood sample drawn at hospital admission 0 to 10 h after the onset of chest pain and serial testing over a 24- to 48-h interval after hospital admission. The sensitivity, specificity, and efficiency for hospital admission samples were 87.5, 83.3, and 85.0%, respectively, and for serial testing, 100, 79.2, and 87.5%, respectively. The diagnostic efficacy was comparable to that of electrophoresis and significantly better than the Du Pont CK-MB method.     

Validation of a new automated renin assay

BACKGROUND: Measurement of plasma renin is important for the treatment of patients with congenital adrenal hyperplasia (CAH) and in the evaluation of patients with suspected hyperaldosteronism. Immunologic assays for plasma renin offer easier implementation and standardization than enzyme-kinetic assays for plasma renin activity, but their sensitivity and specificity have been questioned. We studied a renin immunochemiluminescence assay on an automated platform. METHODS: Renin was measured by an enzymatic assay, by IRMA, and by the new Nichols Advantage Specialty System immunochemiluminometric assay (ICMA), in plasmas from unselected individuals from our outpatient departments and in samples from patients with selected diagnoses. RESULTS: The detection limit in the ICMA was 0.1 mU/L. The recovery was >90%, and the imprecision (CV) was generally <9%. Mean (SD) concentrations measured by ICMA were 32 (21)% lower than those measured by IRMA. Renin concentrations as measured by ICMA were identical in serum and EDTA-, heparin-, and citrate-anticoagulated plasmas. Prolonged incubation of whole blood at room temperature before centrifugation did not affect renin concentrations. The central 95% interval for 80 healthy adults was 6-85.5 mU/L. Plasma renin as assessed by ICMA in patients with primary hyperaldosteronism was <0.2 mU/L. CONCLUSIONS: The performance characteristics of the new renin ICMA allow its use for patients with CAH and for the diagnosis of mineralocorticoid hypertension. In view of the variability of renin concentrations, use for other forms of hypertension or physiologic research calls for the development of uniform sampling protocols.     

Simple immunochromometric assay for protein C activity

Protein C binds readily from human plasma to antibody-coated wells, where it may be quantitated with an iodine 125-labeled antibody to protein C. Treatment with thrombin results in a small reduction in the protein C antigen detectable by this immunoradiometric assay (IRMA). However, activated protein C resulting from thrombin treatment and retained by the antibody on a solid phase may be detected by an overnight incubation with chromogenic substrates S-2366 or CBS 34.47. The immunochromometric assay (ICMA, analogous to IRMA) described uses a heterologous antibody to protein C, activation with relatively low concentrations of bovine thrombin, and quantitation by hydrolysis of chromogenic substrate in a convenient 96-well microtiter plate system. The correlation between IRMA and ICMA protein C results was found to be good with normal persons and patients with liver disease. Patients taking oral anticoagulants had reduced protein C antigen (IRMA) but even lower protein C by ICMA, indicating that inactive forms were present.     

Evaluation of three current methods for the determination of creatine kinase-MB catalytic activity

Three current methods for the determination of the creatine kinase-MB activity were evaluated: electrophoresis (Beckman Inst.), ion-exchange MB-Zyme (J.T. Baker) and ion-exchange on aca (Du Pont). Eighty patients were selected out of three groups: acute myocardial infarction, heart and vascular surgery patients and a number of noncardiac cases. The precision of the ion-exchange methods was comparable; CV 5.7% (aca) and CV 5.2% (MB-Zyme) for a high creatine kinase-MB pool. Electrophoresis overestimated the creatine/kinase-MB/activity and was less precise (CV 9.6%) and less sensitive than the ion-exchange methods. The aca (Du Pont) method was less specific than the MB-Zyme. The main source of discrepancy was samples from certain patients in the surgery unit, which gave falsely high value by the aca method, and showed altered mobility in electrophoresis, suggesting an atypical creatine kinase. Three macro creatine kinase type I included in this study were erroneously determined as creatine kinase-MB by both the ion-exchange methods.     

Evaluation of a new chemiluminescence technique for human thyrotropin (BeriLux hTSH): diagnostic value of five immunometric assay methods.

A new commercially available human thyrotropin immunochemiluminometric assay (ICMA) kit was evaluated. The BeriLux assay (Hoechst Co., Germany) was compared with two other non-radioisotopic methods (AIA-1200 and IMx) and two other immunoradiometric assays (RIA-gnost TSH IRMA and EIKEN IRMA kits) in 32 normal subjects and 104 patients with Graves' disease, divided into seven groups: 1) untreated hyperthyroidism; 2) hyperthyroidism during treatment; 3) euthyroid with negative thyroliberin test (subclinical hyperthyroidism); 4) euthyroid with low thyroliberin test; 5) euthyroid with normal thyroliberin test; 6) euthyroid with high thyrotropin level (subclinical hypothyroidism); and 7) primary hypothyroidism. Patients in groups 2-6 were undergoing treatment with mercazole and propylthiouracil. The new immunoluminometric assay (ILMA) BeriLux kit was shown to have a remarkably improved analytical and clinical sensitivity. The minimal detectable level of thyrotropin in the assay was 0.006 mU/l. The precision was 2.8% and 6.1% at 0.093 +/- 0.003 mU/l and 0.028 +/- 0.002 mU/l, respectively, whereas the precision of the other methods was above 17.2% and 59.4% respectively. Seven patients from the untreated hyperthyroid group were given 500 micrograms thyroliberin i.v. (the thyroliberin test). The thyrotropin pattern before and after thyroliberin administration was always less than 0.006 mU/l with the BeriLux kit, whereas the other methods showed random fluctuations indicating their low accuracy at this concentration. Using the BeriLux kit, 7 of the 16 overt hyperthyroid patients undergoing treatment showed a measurable thyrotropin level below 0.01 mU/l but a negative thyroliberin test.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid and specific cyclosporine assay for the Du Pont aca discrete clinical analyzer performed directly on whole blood

A rapid, sensitive, and specific enzyme immunometric assay for cyclosporine in whole blood has been developed for the aca analyzer, using a monoclonal antibody from Sandoz, Ltd. Between-run CVs ranged from 6.5 to 7.6% for samples containing between 60 and 400 ng/mL cyclosporine. Sensitivity was better than 25 ng/mL in the assay, which has an effective upper range of greater than 600 ng/mL. Two correlation studies compared cyclosporine values from the Du Pont method to those determined by HPLC procedures in three hospital laboratories. The results from a total of 120 whole blood samples with CsA between 20 and 800 ng/mL showed excellent correlation between the methodologies. HPLC and Du Pont CsA values from 10 day serial studies also correlated well for samples from a kidney, kidney-pancreas, heart, and two liver transplant patients. We conclude that the Du Pont CSA assay provides accurate and reproducible results in a convenient format in less than 30 minutes.     

Comparison of results for 13 clinical laboratory determinations with three automated analytical systems.

We evaluated the Abbott Bichromatic Analyzer-100 (ABA-100) for use in the routine clinical chemistry laboratory by examining 13 different determinations that can be performed on the instrument. Results with the Du Pont "aca" and Technicon continuous-flow systems were compared to the ABA-100 in terms of upper limits of linearity, inter-run coefficient of variation, and results for samples from patients. The upper limits of linearity for the methods on the ABA-100 exceeded all of those for the continuous-flow systems, except for urea nitrogen. Precision of the ABA-100 was as good as or better than that of the aca for all determinations, except for glucose in a normal control serum and creatine kinase and creatinine in an above-normal control serum.     

Mass concentration and activity concentration of creatine kinase isoenzyme MB compared in serum after acute myocardial infarction

We compared three current methods (immunoinhibition, "Isomune-CK" immunoprecipitation, and the Tandem-E CKMB II immunoenzymometric assay) for determination of creatine kinase (CK; EC 2.7.3.2) isoenzyme MB in serum. Although results inter-correlated well, the immunoinhibition assay gave higher activity values. Atypical CK forms did not interfere with the immunoprecipitation and immunoenzymometric methods. In acute myocardial infarction the catalytic properties of CK decreased with the enzyme's age, as reflected by a steady increase in activation energy of the catalyzed reaction. In septicemia patients with very low CK and CK-MB catalytic activity, mean CK-MB mass concentration exceeded the upper reference limit, suggesting an increased rate of loss of activity concentration in these patients' sera. Because of the assay's lesser susceptibility to conformational changes at the active site of the enzyme, we suggest that measurement of CK-MB mass concentration is better suited for infarct sizing than measurement of catalytic activity.     

Determination of tricyclic antidepressants for ED analysis

Both the Biosite Triage (Biosite Diagnostics, San Diego, CA) method and the Du Pont aca (Du Pont Company, Wilmington, DE) method give qualitative tricyclic antidepressant (TCA) results to aid in the diagnosis of a TCA overdose. The Triage method uses urine samples and the aca uses serum samples. Although the cutoff values vary considerably between the two methods, the Triage results agreed well with the aca results. The Triage test has an advantage in instrument maintenance and time savings, allowing a reduction in turn-around time for our emergency department. Both urine and serum samples were obtained from 44 patients who were admitted to the emergency department with a diagnosis of “possible tricyclic overdose.” Discrepancies between the two methods were resolved by thin layer chromatography (Toxi-Lab, Ansys, Inc, Irvine, CA). Both methods were in agreement with the exception of five patients' samples. In this study, the Triage method allowed for detection of TCA using urine that is simple for the user and yielded higher sensitivity and specificity results compared with the Du Pont aca method.     

Evaluation and comparison of a radiative energy attenuation method for determining cholesterol in serum

Determination of cholesterol by a radiative energy attenuation (REA) technique was evaluated and compared with results obtained by the Boehringer Mannheim High Performance Cholesterol Assay and the Du Pont aca. Within-assay and between-assay CVs for the REA method, for two sets of controls, were both less than 5%. We observed no interference with lipemic samples. Analytical recovery averaged 102.8%. We used all three methods for parallel determinations of 217 patients' samples containing a wide range of cholesterol concentrations. Linear regression analysis of the REA results vs those of the comparison methods were as follows: REA = 1.03 Boehringer - 0.072 (r = 0.993) and REA = 1.02 aca - 0.048 (r = 0.995). We also discuss bilirubin interference with the REA method for cholesterol.     

Analytical and clinical evaluation of creatine kinase MB mass assay by IMx: comparison with MB isoenzyme activity and serum myoglobin for early diagnosis of myocardial infarction.

We analytically and clinically evaluated Abbott's IMx assay for creatine kinase (CK) isoenzyme MB (CK-MB) in serum. Over a 1-year period, the method was more specific but less precise than catalytic isoenzyme measurements by electrophoresis or immunoinhibition. Sera from different individuals without electrophoretic evidence of CK-MB but containing macro CK type 1 (n = 20), mitochondrial CK (n = 5), or CK-BB (n = 5) were scored as CK-MB negative by the IMx. Likewise, CK-MB-negative by the sera remained so after addition of purified human CK-MM (< or = 7600 U/L) or CK-BB (< or = 8100 U/L). For 39 patients admitted for suspicion of uncomplicated acute myocardial infarction (precordial pain for < or = 4 h), the diagnostic performance of the IMx CK-MB assay on admission and 4 h later was superior to that of total CK activity and compared well with that of CK-MB activity measured by electrophoresis or immunoinhibition. An admission, myoglobin showed a higher diagnostic sensitivity, specificity, and predictive value than did CK-MB and was the most informative test. Diagnostic performance on admission and 4 h later was further improved by considering positivity for myoglobin and for CK-MB by IMx and for the change in each over the first 4 h of hospitalization as criteria. Twelve hours after admission, diagnostic performance was further improved for all CK and CK-MB methods but began to decline for myoglobin.     

Estimation of reference intervals for total protein in cerebrospinal fluid

Protein in cerebrospinal fluid (CSF) was measured by a modified biuret procedure and in two automated instruments, the Du Pont aca and the Kodak Ektachem. The latter's dry-slide reagent was also evaluated for precision, linearity, and effect of potential interferents. In vitro, ampicillin and vancomycin increase the apparent value for CSF protein as measured with the Ektachem slides. We excluded patients with disorders of the central nervous system, and we estimated the central 95% percentile reference intervals for CSF protein for each of the three methods. We found no age or gender dependence of values. By the biuret procedure, the reference interval is 140 to 620 mg/L.     

An evaluation of immunoglobulin assays on the ACA III

We evaluated an immunoturbidimetric Du Pont ACA method for the determination of IgG, IgA and IgM in serum. Between-day reproducibilities of the ACA assays were comparable to those of a radial immunodiffusion, a Hyland nephelometric and a turbidimetric assay on Centrifichem 400. Correlations between results obtained by these methods were acceptable, but the results differed quantitatively. After simulated recalibration based on results of control sera or patient samples, the results for IgA and IgM became more interchangeable. IgM concentrations below 0.25 g/l, which are important for the assessment of intra-uterine infections, could not be quantified with the ACA. Moderate levels of haemoglobin, bilirubin or lipids do not interfere with the ACA assays. The evaluated assays are fast and easy to use.     

Laboratory evaluation of the Du Pont "Dimension Clinical Chemistry System"

We evaluated the analytical performance of the Du Pont Dimension Clinical Chemistry System to assess its suitability as a replacement for the Du Pont aca III, currently routinely used for analysis of pediatric, high-risk, and emergency specimens. Twenty-six analyses were studied. These generally fulfilled pre-set analytical goals for precision based on biological variation or "state of the art." Significant economies were achieved and we found the Dimension to be reliable, easy to operate, and simple to maintain. It also fulfilled safety requirements for analysis of specimens of high risk to laboratory staff.     

Two automated methods for plasma antithrombin III compared, and the clinical significance of the results

Antithrombin III (AT III) activity was determined with two different new chromogenic substances--Chromozym-TH (Tos-Gly-Arg-p-nitroanilide; Boehringer Mannheim) and alpha-N-carbobenzyl-oxy-L-lysine-thiobenzyl ester (Du Pont)--with both a discrete (aca) and a centrifugal analyzer (COBAS BIO). The correlation between the Chromozym-TH/centrifugal analyzer and Du Pont ester/aca methods was good (r = 0.9890). Precision within and between runs was similar to that for typical enzymic determinations. AT III in plasma of 226 healthy men and women ranged from 76.6 to 141.1% (100% = "normal"). We found no significant differences ascribable to oral contraceptives. AT III activity was decreased in 27% of patients with acute thromboembolic diseases (n = 62), in 48% of patients the first day after abdominal operations without complications (n = 78), and in 100% of patients with reversible or irreversible shock (n = 58). In patients receiving continuous therapy with heparin (1500 USP units/h) we saw no decrease in AT III within 96 h of beginning treatment. Plasma from 14 of 16 patients with disseminated intravascular coagulopathy showed a decrease in AT III of from 17 to 51% of normal before and during heparin therapy. We then treated all 16 patients with AT III concentrate. During such treatment, AT III in plasma must be monitored over short intervals to assure that sufficiently high proportions of AT III (greater than 70% of normal) are reached.     

Assays of serum lipase: analytical and clinical considerations

We evaluated three commercially available methods for determining lipase (EC 3.1.1.3) in serum--the Du Pont aca, Boehringer Mannheim Diagnostics (BMD), and Kodak Ektachem (EK) procedures--for their analytical properties and diagnostic efficiencies. Titrimetry was used as the comparative method. The BMD and EK methods showed better agreement with the titrimetric method, owing to the presence of the necessary cofactor, colipase, in their reagents. Colipase also increased the analytical sensitivity of the BMD and EK procedures as compared with the aca method. Determinations of serum lipase, by all methods, had a clinical sensitivity in excess of 80% for acute pancreatitis; the specificity of the lipase test was about 60%, or twice that of serum amylase. Serum lipase determinations with the current, simpler technology are superior to total amylase in the diagnosis of patients with acute pancreatitis. When a colipase-supplemented method is used, a serum lipase value greater than 10-fold the upper reference limit appears to be pathognomonic for acute pancreatitis or inflammation of organs close to the pancreas.     

Improved interassay correlation of digoxin results in patients with and without renal failure by elimination of digoxin-like immunoreactive factors

Use of immunoassays that do not detect endogenous digoxin-like immunoreactive factors (DLIF) in serum significantly improves the between-assay correlation of digoxin results for patients. We investigated five different immunoassay methods (Abbott, Clinical Assays, Corning, Du Pont, and Syva), measuring digoxin by all five assays in sera from 38 patients in renal failure and in 40 patients with normal renal function, all taking digoxin. The mean standard error of the estimate (Sy X x) of digoxin results (compared for all five assays) were significantly lower for patients with normal renal function than for patients in renal failure (0.148 vs 0.293 microgram/L, P less than 0.001). Assays previously shown (Clin Chem 1987;33:401) to be the least sensitive to DLIF (Syva and Corning) gave the lowest mean scatter about the regression (Sy X x = 0.192 microgram/L, renal failure; 0.114 microgram/L, normal renal function) for all 10 assay correlations. Evidently, discrepancies between digoxin values as measured by different immunoassay kits for patients with renal disease can be attributed to DLIF. Moreover, because inaccurate digoxin results attributed to DLIF may not be limited exclusively to groups of patients with known increased concentrations of DLIF, the possibility of "latent" DLIF interference may be a problem in many other human subjects.     

Clinical effectiveness of the Du Pont aca measurement of creatine kinase MB in serum from patients in a coronary-care unit

We evaluated the clinical effectiveness of measuring creatine kinase (CK; EC 2.7.3.2) isoenzyme MB and lactate dehydrogenase (LD; EC 1.1.1.27) isoenzymes in diagnosis of acute myocardial infarction. We used an agarose electrophoresis method to measure CK and LD isoenzymes and the Du Pont aca column method to measure CK-MB. Serial blood specimens were drawn from 100 patients consecutively admitted to our Coronary Care Unit. Because of the low diagnostic specificity for CK-MB measurements by both agarose electrophoresis and the discrete-analysis method, as compared with reported values, we re-evaluated our isoenzyme data by using Receiver Operating Characteristic curves. Such analysis of the data established optimal decision levels of greater than or equal to 25 U/L and greater than or equal to 18 U/L plus greater than or equal to 6% of total CK for serum CK-MB measured by the agarose electrophoresis and the aca methods, respectively, and an optimal decision level of greater than or equal to 0.92 for the ratio of LD 1/2 measured after agarose electrophoresis. At these decision levels we obtained a sensitivity of 100%, 100%, and 95% and a specificity of 94%, 92%, and 90% for CK-MB (agarose electrophoresis), CK-MB (aca), and the LD 1/2 ratio, respectively.