Chromogranin-A and N-terminal pro-brain natriuretic peptide: an excellent pair of biomarkers for diagnostics in patients with neuroendocrine tumor.

PURPOSE: For the last decade chromogranin-A (CgA) has been a well-established marker for neuroendocrine tumor (NET), and N-terminal pro-brain natriuretic peptide (NT-proBNP) has been a useful marker for left ventricular dysfunction. This study examined the diagnostic value of CgA and NT-proBNP for carcinoid heart disease (CHD), and their prognostic value for overall survival in NET patients. PATIENTS AND METHODS: Serum samples were obtained and cardiac ultrasound studies performed in 102 NET patients. The criterion for mild and severe CHD was tricuspid regurgitation stage I/II and III/IV, respectively. Proportional odds and Cox proportional hazards models were constructed respectively to identify the association between CHD and overall survival with patient characteristics and the two markers. RESULTS: Severe CHD was found in 15 (15%) of 102 patients, 13 of whom had elevated NT-proBNP levels. In the univariate proportional odds model CHD was correlated with age (P = .007), CgA (P = .002), and NT-proBNP (P < .001), whereas in the multivariate model NT-proBNP and CgA were significantly associated with CHD (P < .001 and P = .01). In the univariate Cox models, age (P = .04), sex (P = .03), CgA (P = .003), and NT-proBNP (P = .04) were related to overall survival, and in the multivariate model CgA and NT-proBNP remained significantly related to overall survival (P = .002 and P = .04, respectively). CONCLUSION: NT-proBNP and CgA are very important markers in the diagnosis of CHD in patients with NET. Furthermore, patients with elevated NT-proBNP in addition to elevated CgA levels showed worse overall survival than patients with elevated CgA alone.

Influencia de la temperatura en las curvas de calibración en IRMA de enolasa neuronal específica y su interpretación fisicoquímica / Influence of temperature on the calibration curves in IRMA for neuron specific enolasa and its physicochemical interpretation

Objetivo: el análisis inmunorradiométrico (IRMA) es uno de los métodos principales utilizados en la determinación analítica de la enolasa neuronal específica (NSE). Se ha estudiado la influencia de la temperatura en las gráficas de calibración obtenidas por dicha técnica y se propone una justificación fisicoquímica basada en dos modelos teóricos. Material y métodos: usamos un kit comercial de IRMA para NSE y un contador gamma. Los resultados son analizados mediante el programa Statistica. Resultados y discusión: las actividades ligadas al anticuerpo aumentan con la temperatura, lo que da resultados que se ajustan a sendas modificaciones de las ecuaciones de los cuatro parámetros y Langmuir. Conclusiones: los dos modelos utilizados reproducen satisfactoriamente los resultados, pero es preferible el modelo de adsorción debido a su mayor simplicidad y significado físico más claro(AU)

Background: immunoradiometric assay (IRMA) is one of the principal methods used for the analytical determination of neuron specific enolase (NSE) concentration. We studied the influence of temperature on the calibration curves obtained by this method, and a physicochemical justification based on two theoretical models is proposed. Material and methods: we used a commercially available RIA kit for NSE and a gamma counter. Data was analysed using Statistical software. Results and discussion: activity bound to the antibody increases with temperature, producing results that are consistent with two modifications to the four parameter and Langmuir equations. Conclusions: the two models used successfully reproduce the results, with the adsorption model being preferable due to its greater simplicity and clearer physical significance(AU)

Immunoradiometric assay (IRMA) for human follicle stimulating hormone (FSH) and luteinizing hormone (LH) using common avidin solid phase.

This paper describes the use of avidin-biotin interaction as an affinity system, wherein avidin immobilized magnetizable particles (cellulose) are used as a common separation system in immunoradiometric assay (IRMA) for hormones of the human reproductive system, human follicle stimulating hormone (FSH), and luteinizing hormone (LH). Biotinylated probe was prepared by biotinylation of specific monoclonal antibody for respective antigen using the caproyl derivative of biotin N-hydroxysuccinimide. The detector antibody for the respective antigen was radiolabelled with 125I by a chloramine-T oxidation method and purified by gel filtration. In the IRMA procedure, standard/sample, respective biotinylated, and radiolabelled antibody as a single reagent, and avidin solid phase were added simultaneously to the assay tubes. After incubation for 3 h with shaking, the bound complex was quantitated for its radioactivity associated with the common avidin solid phase. Results showed that the developed assay protocol is applicable to IRMA of FSH and LH with good precision (intra and inter assay CV less than 8% and 11%, respectively), good assay range (0-200 mIU/mL) and analytical recovery (87-110%). The assay could detect 0.5 mIU/mL and 0.9 mIU/mL of FSH and LH, respectively, and showed good correlation with commercially available kits (FSH y = 0.98x + 0.21 and LH y = 0.99x + 0.18).

Immunoradiometric assay of chromogranin A in the diagnosis of small cell lung cancer: comparative evaluation with neuron-specific enolase.

The aims of our work were 1) to determine the diagnostic performance of an immunoradiometric assay of chromogranin A (CgA) in small cell lung cancer and 2) to compare its discriminatory power with that of neuron-specific enolase (NSE), the marker currently used for SCLC. We selected 166 cases of small cell (64) and non-small cell (102) lung cancer and 106 cases of non-malignant lung diseases as controls. Both CgA and NSE were assayed by immunoradiometric methods and cutoff values were established on the basis of a pre-fixed specificity of 95% in non-malignant lung diseases. The CgA assay showed better diagnostic sensitivity than NSE in SCLC (61% versus 57%), especially in limited disease, and a low positivity rate in NSCLC with respect to NSE (14% versus 22%). By contrast, NSE reflected disease extent more accurately than CgA (U test: CgA p<0.05, NSE p<0.001). Finally, we found that the CgA assay was not affected by hemolysis whereas NSE serum levels greatly increased in hemolyzed sera. In conclusion, CgA assaying by an IRMA method is a reliable procedure in the diagnosis of SCLC. NSE remains the marker of choice in staging and monitoring of the disease. Further studies are needed to evaluate the prognostic significance of the marker and its role in therapy monitoring and patient follow-up.

Serum insulin-like growth factor-1 (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) in healthy Thai children and adolescents: relation to height, weight, and body mass index.

The objective of this study was to evaluate the correlation between serum concentrations of insulin-like growth factor-1 (IGF-1), insulin-like growth factor binding protein-3 (IGFBP-3) and growth parameters (height, weight, and body mass index) in 260 healthy children and adolescents aged 5-20 years. The subjects were divided into 2 groups according to the age achieving final height. Group 1 included children with active growth consisting of girls aged under 14 years (N = 80) and boys aged under 16 years (n = 74). Group 2 included adolescents who achieved final height consisting of females aged at and over 14 years (n = 82), and males aged at and over 16 years (n = 24). In group 1, the serum concentrations of IGF-1 and IGFBP-3 were significantly positive correlated with all growth parameters. In group 2, although the correlation was insignificant, the concentrations of IGF-1 and IGFBP-3 seemed to be greater in individuals who were relatively taller and had lean body mass than those who were relatively short and over average body mass.

Comparison of two thyroglobulin immunoradiometric assays on the basis of comprehensive imaging in differentiated thyroid carcinoma.

The aim was to compare two thyroglobulin-immunoradiometric assays (Tg-IRMA) in the follow-up of patients with differentiated thyroid carcinoma (DTC) in order to set up interassay correlation, correlation to clinical background, and to determine whether a lower functional sensitivity (kit A: 0.5 ng/mL, kit B: 0.3 ng/mL) would allow an earlier detection of recurrences. Three hundred eight samples from 181 patients with DTC were investigated. The clinical interpretation of the Tg-IRMA results was based on comprehensive imaging and the clinical history before and during the study period. Groups were formed against this background and against the thyrotropin (TSH) levels of the samples (LT4- on and LT4-off). During a follow-up period that lasted until September 1998, the clinical situation was reevaluated in order to determine any changes in the patients' clinical status. The two assays presented a good interassay correlation of 0.838. Both assays had a high and comparably good sensitivity in the detection of recurrence of malignancy or distant metastases. Patients in remission had, in most cases, nonmeasurable or Tg values below 1 ng/mL. Kit B presented slightly measurable Tg results in a larger number of patients in remission; however, during the follow-up most of these slightly measurable Tg results were not reproducible, thus being most likely artifacts. Consequently, the functional sensitivity of 0.3 ng/mL of kit B showed no advantages in terms of an earlier tumor detection and seems to be unacceptably low. Negative consequences may be an increase in the number of investigations during the follow-up, which may be disconcerting for both the clinicians and the patients.

Analytical performance of the Tandem-R free PSA immunoassay measuring free prostate-specific antigen.

The analytical performance of the Tandem-R free PSA assay available from Hybritech Inc. was evaluated. Comparison of recoveries of purified free (unbound) prostate-specific antigen (PSA) diluted in female serum in the Tandem-R free PSA assay and the Tandem-R (total) PSA assay demonstrated a link in calibration between the assays and an accurate determination of percent free PSA. The cross-reactivity of the assay to purified PSA-alpha 1-antichymotrypsin was determined to be < 1%. The minimum-detectable concentration was < 0.05 microgram/L. The within-run and between-day CVs were < or = 5% for samples with > 0.3 microgram/L free PSA. Dilution and recovery showed no significant deviations from linearity across the assay range. The assay was insensitive to interference from blood components. The Tandem-R free PSA kit was shown to be an accurate, precise, and reliable assay for the measurement of free PSA.

Measurement of antibodies against glutamic acid decarboxylase 65 (GADA): two new 125I assays compared with [35S]GAD 65-ligand binding assay.

Recently, 65-kDa glutamic acid decarboxylase (GAD 65) antibodies (GADA) have been introduced as autoimmune markers in blood to confirm the diagnosis of insulin-dependent diabetes mellitus (IDDM). In this study, to evaluate two new assays that use 125I-labeled GAD 65, we assayed samples from 100 children with recent onset of diabetes and 100 control children, the results were compared with those of a [35S]GADA assay and with results for islet cell antibodies (ICA), the conventional autoimmune marker. Receiver operating characteristic (ROC) curve analysis showed one of the new assays (from RSR) to be more sensitive (P = 0.01) than the comparison ([35S]GADA) assay, whereas the second new assay (from Elias) was less sensitive (P < 0.001). The GADA frequency at 97.5% specificity was greatest in the comparison assay: 63 of 100 vs 41 of 100 (P < 0.01) and 53 of 100 (P = 0.16) in the RSR and Elias assays, respectively. Almost all GADA-positive patients had ICA, but one-third of the ICA-positive patients was GADA-negative. Accordingly, adding GADA analysis results to ICA testing increased the frequency of detection of autoimmune markers only slightly (from 81% to 85%). In conclusion, at 97.5% specificity the [35S]GADA assay seemed to be more efficient than the 125I assays, although the difference was significant only for the Elias 125I assay. Antigen-specific antibodies other than GADA may explain the difference in GADA and ICA frequencies.

LH isoform profiles during short-term pulsatile LHRH administration in elderly men.

LH isoform profiles were analyzed in sera resolved with isoelectrofocusing from 5 elderly men (age 70.6 +/- 2.95) and 5 young adult men (age 28.2 +/- 1.24), by using polyclonal antibodies (RIA), monoclonal antibodies directed against the beta-subunits (IRMA) and in vitro LH bioassay. Despite the fact that the elderly had lower testosterone levels than the young (293 +/- 38 vs 512 +/- 77 ng/dl, p < 0.05), no differences were noted in the isoforms detected by any of the assays, although each assay yielded a characteristic profile. Indeed, RIA showed most LH in the acidic range, while IRMA revealed LH profiles with a major peak in the basic range, thus resembling the profiles determined by means of the bioassay. In the elderly, the profiles were also analyzed on day 7 and day 14 of short-term pulsatile sc LHRH administration (150 ng/bw/120 min). Only the LH bioassay detected an LHRH-induced shift to more basic and bioactive forms; these changes accompanied an increased in testosterone levels on day 7 (396 +/- 83 ng/dl, p < 0.05 vs day 0) and on day 14 (320 +/- 58 ng/dl NS vs day 0). Our data suggest that: 1) the profiles obtained in young and elderly subjects are similar, irrespective of the antisera used; 2) as a result of treatment with LHRH in the elderly an increase in T levels occurs, possibly due to the observed changes in LH bioactivity; 3) the in vitro LH bioassay appears to be the most sensitive assay in detecting such changes, which consisted of an enrichment in more basic and bioactive glycoforms.

Growth hormone assays: early to latest test generations compared.

We compared the data from four growth hormone (GH) immunoassays for analyzing 24-h GH profiles in four apparently normal subjects and four obese subjects (508 serum samples). The detection limit was 0.02 microgram/L for one immunochemiluminometric assay (ICMA), 0.1 microgram/L for two IRMAs, and 0.4 microgram/L for one RIA. All GH pulses with a peak ICMA value > 1 microgram/L were detected by each of the other methods. Overall, the correlation coefficient between the values obtained with all four assays exceeded 0.90. However, for GH concentrations < or = 0.25 microgram/L, acceptable concordance (r2 > or = 0.80) was reached only between the ICMA and one IRMA; between the ICMA and the RIA, concordance was acceptable only for GH concentrations > or = 10 micrograms/L. In the normal subjects, the percentage of undetectable values was 0% with the ICMA but 29% with one of the IRMAs; in obese subjects, the corresponding values were 12% and 38%.

Analytical performance and clinical usefulness of a commercially available IRMA kit for measuring atrial natriuretic peptide in patients with heart failure.

We evaluated the analytical characteristics and clinical usefulness of a commercially available IRMA kit for measuring plasma concentrations of atrial natriuretic peptide (ANP) in healthy subjects and in patients with heart failure. The method uses two monoclonal antibodies prepared against sterically remote epitopes of the ANP molecule; the first antibody is coated on the solid-phase beads, and the second is radiolabeled with 125I. Fifty-nine healthy subjects and 77 patients with heart failure were studied. After subjects had rested 20 min in a recumbent position, blood samples were collected from a brachial vein into ice-chilled disposable polypropylene tubes containing aprotinin and EDTA. Plasma samples were immediately separated by centrifugation and stored at -20 degrees C until assay. The working range (CV <15%) was 10-2000 ng/L. The detection limit (2.13 +/- 0.91 ng/L) was similar to those reported for other IRMAs but was much better than those of RIAs. For healthy subjects, the results of this method (18.0 +/- 10.6 ng/L, range 4.7-63 ng/L, median 16.7 ng/L, n = 59) were similar to those generally reported for the most accurate methods, i.e., those using preliminary extraction and chromatographic purification of plasma samples. Measured plasma ANP was significantly associated with the severity of clinical symptoms, i.e., NYHA class (ANOVA, P <0.0001), and with the left ventricular ejection fraction (n = 62, r = 0.618, P <0.0001). Patients with severe heart failure showed greatly increased values (NYHA III-IV: 257.4 +/- 196.6 ng/L, n = 23).

Selective recognition of M235T angiotensinogen variants and their determination in human plasma by monoclonal antibody-based immunoanalysis.

The common M235T mutation of human angiotensinogen has been shown to be associated with a 10-20% increase in plasma angiotensinOgen level and increased frequency of essential and pregnancy-induced hypertension. The detection of such a common factor in the plasma of individuals at risk could be a useful tool for modern molecular-based medicine. The recognition of M235T variants was investigated using four monoclonal antibodies (mAbs) directed against human angiotensinogen; two immunometric assays were developed. The first assay (using mAbS 7B2 and 4G3) allowed the direct determination of angiotensinogen concentrations and did not show a significant difference with the enzymatic measurement of angiotensinogen. The second assay (using mAbs 1H8 and 1C11) showed a fine distinction between the T235 mutant and M235 wild-type forms of angiotensinogen, with a greater affinity for the latter, as confirmed by biosensor BIAcore experiments. This assay was extremely sensitive in measuring the proportions of the M235 and T235 forms present in the test samples, the first time such a distinction has been achieved in the serpin family. The simple immunoanalysis of the plasma allowed the direct determination of the M235T genotype of the individual tested. Furthermore, it was shown that the T174M mutation, described as being in complete linkage disequilibrium with the M235T mutation, had no influence on these results. Moreover, this assay suggested the presence of the M235 and T235 angiotensinogens in approximately equal amounts in heterozygous plasmas. In conclusion, the immunometric assay described in this study should provide original tools for investigating the relationship between M235T genotype, plasma angiotensinogen levels, and regulation of blood pressure.

Evaluation of a two-site immunoradiometric assay for measuring noncomplexed (free) prostate-specific antigen.

Serum prostate-specific antigen (PSA) in men is present as two different molecular forms separable by gel-filtration chromatography (GFC). We have evaluated a two-site IRMA that measures only the noncomplexed (free) form of PSA (F-PSA). Verification that the F-PSA assay measures solely F-PSA was obtained by assaying GFC-fractionated serum samples with both the F-PSA IRMA and a commercial PSA assay that measures total PSA (T-PSA: F-PSA plus alpha 1-antichymotrypsin-complexed PSA). The F-PSA assay detected only the 30-kDa peak corresponding to the free form of PSA, whereas the T-PSA assay detected two peaks: complexed PSA at approximately 90 kDa and F-PSA at approximately 30 kDa. The F-PSA assay had an analytical detection limit of 0.03 microgram/L and a measuring range up to 50 micrograms/L. The intraassay CV was 1.7-10% in the concentration range of 0.2-30 micrograms/L. The interassay CV was 3.4-12.5% in the same concentration range. Dilution and recovery studies showed no significant deviation from linearity across the assay range. The assay was insensitive to interference from hemoglobin, bilirubin, and total lipids up to concentrations of 5, 0.2, and 10 g/L, respectively. No significant loss of immunological activity (analyte stability) was seen day-to-day ( < or = 5) or after repeated freeze/thaw ( < or = 5) cycles. We conclude that the F-PSA IRMA is an accurate, precise, and reliable tool for measuring F-PSA in human serum.

Clinical validation of renin monoclonal antibody-based sandwich assays of renin and prorenin, and use of renin inhibitor to enhance prorenin immunoreactivity.

Newly developed IRMAs to measure the plasma concentrations of renin and prorenin were validated for clinical use and compared with a classical enzyme kinetic assay. The IRMAs involve two monoclonal antibodies, one that reacts equally well with renin and prorenin and one that recognizes renin well but prorenin only minimally. Prorenin reactivity with the second antibody was enhanced by adding the renin inhibitor, Remikiren, to plasma. The complex of prorenin with this active-site ligand undergoes a conformational change, whereby prorenin is converted into a form that cannot be differentiated from renin by the IRMA. The linear working range of the assay was 4.0-3000 mU/L. The concentration of prorenin was calculated by subtracting the assay result obtained without Remikiren (i.e., renin) from the result obtained with Remikiren (i.e., renin plus prorenin). No more than 2% of prorenin present in plasma was detected as renin. The interassay CVs for renin quantification were 18%, 13%, and 8% at low, medium, and high concentrations, respectively. The interassay CV for calculated prorenin was 8% at both low and high concentrations. The IRMA results were highly correlated with those of an enzyme kinetic assay in healthy subjects; in patients with such conditions as primary hyperaldosteronism, renovascular hypertension, and low-, medium-, and high-renin essential hypertension; and in women undergoing gonadotropin stimulation.

Influence of specimen carryover on sensitive thyrotropin (TSH) assays: is there a problem?

A relatively slow transition has occurred from so-called 1st-generation thyrotropin (TSH) assays (e.g., RIAs) through 2nd-generation assays (e.g., IRMAs) to 3rd-generation assays (e.g., immunochemiluminometric assays). Analysis of data from a modified internal quality-control design, followed up by a computer simulation, showed that specimen carryover has minimal effect on 2nd-generation TSH assays. However, extension of the simulation to a 3rd-generation assay showed the possibility of substantial effects in the subnormal region. Carryover of 1:1250 (0.08%), for example, may reduce the theoretical 10-fold precision improvement claimed for 3rd-generation assays to nearer fourfold. Simulation results suggest maximum allowable specimen carryover of approximately 1:10,000 (approximately 0.01%) for 3rd-generation TSH assays. We suggest that when automated specimen handling is used in a TSH assay, a well-designed carryover experiment should become a routine part of reports that claim 3rd-generation (or better) performance characteristics.

Applicability of short-lived radiometallic nuclide for high sensitivity two-site "sandwich" immunoradiometric assay: human growth hormone assay.

The sensitivity of the IRMA method is limited by the specific activity (SA) of the conventionally employed radioisotropic label and high sensitivity radioimmunoassay should theoretically be attained by the use of short-lived radiometallic nuclides. Our group have achieved radiolabeling of high SA IgG by using the radiometal, gallium-67 (67Ga) with a short half-life (T1/2 = 78 h) and deferoxamine (DF), a bifunctional chelating agent bound through a multispacer (dialdehyde starch, DAS) as the linker (J Nucl Med 32:825, 1991). In the present work, the application of the approach is attempted by employing a two-site IRMA for human growth hormone (hGH); the monoclonal antibody to hGH (MAB2) is bound to DF via DAS and the coupled DF-DAS-MAB2 is radiolabeled with 67Ga. The 67Ga-DF-DAS-MAB2 of high SA (4,884 MBq/mg versus 370-518 MBq/mg calculated for radioiodinated MAB2) was thus used for the two site 'sandwich' 67Ga-IRMA. Excellent correlation with the 125I-IRMA was registered, and higher detection capability obtained by using 67Ga over the 125I in the hGH IRMA offered a good basis for the exploitation of short-lived radio-nuclides in the IRMA system.