CA 19-9: handle with care.

Since its inception in the mid-1980s of the 20th century testing for carbohydrate antigen 19-9 (CA 19-9) has raised expectation for an earlier diagnosis and accurate monitoring of several malignant diseases. After almost 30 years, the available evidences have confirmed the appropriateness and usefulness of determining CA 19-9 levels as a prognostic indicator and as a reliable tool for monitoring pancreatic and gastrointestinal cancer, but concerns have been raised about its applications in screening, which is actually not recommended, and in the diagnosis of malignancies, due to several interferences that limit the specificity and to the insufficient sensitivity of this marker. In this paper we aimed to review the basic concepts of CA 19-9 testing and its current applications, with a major focus on the most recent evidences dealing with assay interference, methods comparison and monitoring of malignant diseases. The prognostic value and monitoring recommendations for pancreatic, gastric and colorectal cancers are described in depth.

Comparisons of plasma aldosterone and renin data between an automated chemiluminescent immunoanalyzer and conventional radioimmunoassays in the screening and diagnosis of primary aldosteronism.

Determining values of plasma renin activity (PRA) or plasma active renin concentration (ARC), plasma aldosterone concentration (PAC), and aldosterone-to-renin ratio (ARR) is essential to diagnose primary aldosteronism (PA), but it takes several days with conventional radioimmunoassays (RIAs). Chemiluminescent enzyme immunoassays for PAC and ARC using the Accuraseed® immunoanalyzer facilitated the determination, but relations between Accuraseed® immunoanalyzer-based and RIA-based values in samples of PA confirmatory tests and adrenal venous sampling remained to be elucidated. We addressed this issue in the present study. This is a prospective, cross-sectional study. ARC and PAC values were measured by the Accuraseed® immunoanalyzer in samples, in which PRA and PAC values had been measured by the PRA-FR® RIA and SPAC®-S Aldosterone kits, respectively. The relations between Accuraseed® immunoanalyzer-based and RIA-based values were investigated with regression analyses. The optimal cutoff of Accuraseed® immunoanalyzer-based ARR for PA screening was determined by the receiver operating characteristic analysis. After log-log transformations, linear relations with high coefficients of determination were observed between Accuraseed® immunoanalyzer-based and RIA-based data of renin and aldosterone. Following the PA guidelines of Japan Endocrine Society, Accuraseed® immunoanalyzer-based cutoffs were calculated from the regression equations: the basal PAC for PA screening >12 ng/dL, PAC for the saline infusion test >8.2 ng/dL, ARC for the furosemide-upright test <15 pg/mL, and ARR for the captopril challenge test >3.09 ng/dL per pg/mL. The optimal cutoff of Accuraseed® immunoanalyzer-based ARR for PA screening was >2.43 ng/dL over pg/mL not to overlook bilateral PA patients. The present study provided conversion formulas between Accuraseed® immunoanalyzer-based and RIA-based values of renin, aldosterone, and ARR, not only in basal samples but also in samples of PA confirmatory tests and adrenal venous sampling. Although validation studies are awaited, the present study will become priming water of harmonization of renin and aldosterone immunoassays.

An optimised, highly sensitive radioimmunoassay for the simultaneous measurement of estrone, estradiol and estrone sulfate in the ultra-low range in human plasma samples.

Following the introduction of potent aromatase inhibitors for the treatment of breast cancer patients, highly sensitive methods have become mandatory to evaluate the influence of these drugs on plasma estrogen levels. Commercially available kits for estrogen measurements are not suitable for these kinds of evaluations due to their detection limits that are close to baseline estrogen levels in postmenopausal women. We describe here an optimised radioimmunoassay suitable for the simultaneous measurement of plasma estrone (E1), estradiol (E2) and estrone sulfate (E1S) levels in the ultra-low range. Following incubation with [3H]-labelled estrogens as internal standards, crude estrogen fractions were separated by ether extraction. The E1S fraction was hydrolysed with sulfatase followed by eluation on a Sephadex column. Free estrogens (E1, E2) were separated by chromatography (LH-20). Estrone and E1S (following hydrolysis) were converted into E2, and each estrogen fraction was measured by the same highly sensitive and specific radioimmunoassay using estradiol-6-(O-carboxymethyl)-oximino-2-(2-[125 I]-iodo-histamine) as ligand. Although several purification steps were involved, the internal recovery values for tritiated estrogens were found to be 88%, 90%, and 49% for E1, E2 and E1S, respectively. The intra-assay coefficient of variation was <5% for all recovery measurements. The detection limits were calculated following repeated blank measurements and found to be 1.14 pmol/L for E1, 0.67 pmol/L for E2, and 0.55 pmol/L for E1S, respectively. The intra-assay coefficient of variation (CV) was found to be 3.4% for E1, 5.1% for E2 and 6.1% for E1S, while the inter-assay CV was 13.6%, 7.6% and 7.5% for E1, E2, and E1S, respectively. Considering normal plasma levels for E2 (15 pmol/L), E1 (80 pmol/L) and E1S (400 pmol/L) in postmenopausal women, the method allows theoretically to detect suppression of plasma E2, E1 and E1S levels by 95.5%, 98.6% and 99.9% when starting from average, normal postmenopausal levels. Thus, the method presented here is to our knowledge the currently most sensitive assay available for plasma estrogen measurements in the ultra-low range and, as such, a reliable tool for a proper evaluation of potent aromatase inhibitors and other potential drugs influencing on plasma estrogen levels.

Thyroglobulin Antibody Screen Prior to Mass Spectrometry Provides Measurable Cost Savings and Optimal Laboratory Utilization.

OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods that allow accurate quantitation of thyroglobulin (Tg) in the presence of Tg antibodies (TgAbs) have recently become available. Due to cost differences between LC-MS/MS and immunoassay, some laboratories now offer a reflex test strategy that uses LC-MS/MS only for TgAb-positive samples. The goal of this study was to examine utilization of Tg testing strategies and cost savings. METHODS: Test ordering patterns were examined for over 150,000 orders for TgAb and Tg in our laboratory. The average list price was determined from three separate commercial laboratories offering this testing. RESULTS: Data showed that 89% of orders for Tg used the reflex test option, resulting in a savings of over $3 million compared with testing all samples by LC-MS/MS. Of the Tg by LC-MS/MS orders not using the reflex option, 1,663 also included a separate order for TgAb on the same patient sample, representing approximately $170,000 in potentially unnecessary costs from TgAb-negative samples. CONCLUSIONS: Identifying situations to use more expensive testing methods (eg, LC-MS/MS) only when necessary, such as for TgAb-positive patients, leads to considerable cost savings and a more economical use of valuable health care resources.

Radioimmunoassay of follistatin: application for in vitro fertilization procedures.

A sensitive radioimmunoassay (RIA) for follistatin was developed by using antisera raised in rabbits against purified porcine ovarian follistatin. The displacement curves generated with human follicular fluid and serum were parallel with the standard curve of porcine follistatin. Using this RIA, we have measured human serum follistatin immunoreactivity in six women undergoing ovarian hyperstimulation for in vitro fertilization (IVF). After treatment with a GnRH agonist and gonadotropin, serum estradiol and follistatin levels were increased, and there was a direct positive correlation between the elevated levels of estradiol and follistatin (r = 0.93, p less than 0.01). Thus, the level of circulating follistatin may likely reflect follicular maturation and be applicable in IVF procedures to determine the optimal timing for oocyte retrieval.

A homologous radioimmunoassay for rat insulin-like growth factor-I (IGF-I): implications for studies of human IGF-I physiology.

We report the development of a unique radioimmunoassay (RIA) for rat insulin-like growth factor-I (rIGF-I) which does not recognize human IGF-I (hIGF-I). The rIGF RIA uses a specific anti-rIGF-I antiserum with rIGF-I standards and radioligand. Rat sera were extracted by either an abbreviated acid/ethanol procedure or by acid-chromatography with virtually identical results. Assay sensitivity is congruent to 0.5 ng/tube and inter- and intra-assay coefficients of variation were 3.4-7.6% and 4.8-9.2%, respectively. Comparisons of the rIGF-I RIA with a typical heterologous RIA shows that the latter underestimates rIGF-I levels congruent to of 3-fold. Sera from rats treated with hIGF-I were also studied using the rIGF-I RIA and a hIGF-I- specific immunoradiometric assay (IRMA), and results indicate poor correlation between the actual total IGF-I levels (rIGF-I RIA + hIGF-IRMA) and the heterologous RIA estimates. The availability of both rIGF-I and hIGF-I specific immunoassays facilitates more precise studies of hIGF-I in the rat model.

A comparison of 13 different immunometric assay kits for gonadotropins: implications for clinical investigation.

To determine whether the new immunometric (sandwich) assays for gonadotropins achieve the alleged improvements over RIAs, we compared 13 commercial kits for LH and FSH with our established and validated polyclonal RIAs. Although the kits claimed detection limits as low as 0.05 IU/L, when sensitivity was tested with a uniform hormone standard (the Second International Reference Preparation of human menopausal gonadotropin urinary standard) and the requirement that the limit must be determined from a detectable standard point rather than the variance around zero, only 4 kits surpassed the detectability achieved by the existing LH and FSH RIAs. Seven of the 10 LH kits tested displayed greater than 10% cross-reactivity with either the alpha- or LH beta-subunit. The relationship between the kit standard and the Second International Reference Preparation of human menopausal gonadotropin uniform standard in each kit was nonlinear, so that attempts to compare results between assays with simple multiplication factors are inaccurate. Only 2 assays were designed to eliminate a hook effect. The following conclusions were reached. 1) Most available gonadotropin immunometric assays do not yet offer features not already available in existing validated polyclonal research assays. 2) Conversion of data from one assay to another is not straightforward. 3) Many of the manufacturers' claims do not appear justified. 4) Determination of the detection limit and other immunometric assay characteristics requires standardization of criteria. We propose the following minimum criteria for validating gonadotropin immunometric assays: 1) a uniform definition of the detection limit based on the lowest distinguishable standard concentration, 2) determination of the standard concentration at which a hook occurs, 3) determination of cross-reactivity to subunits as well as intact gonadotropins, and 4) establishment of an adequate normative data base.

Diurnal rhythm of 17 beta-estradiol secretion throughout pubertal development in healthy girls: evaluation by a sensitive radioimmunoassay.

Puberty is initiated by a nocturnal rise in gonadotropin secretion, which, in boys, results in an increased nocturnal secretion of testosterone. To characterize any similar diurnal rhythm of 17 beta-estradiol in healthy girls, we determined the secretion of 17 beta-estradiol before and during puberty. The study group consisted of 45 healthy girls whose height SD scores ranged from -3.7 to +4.9 compared with Swedish growth reference values. One to 6 profiles of 17 beta-estradiol (7 samples/24 h) were obtained from each girl during puberty and from 21 of the girls before clinical signs of puberty (a total of 76 serum profiles). Serum 17 beta-estradiol concentrations were determined using a modified RIA. The detection limit for the RIA was 1.8 fmol/tube, which corresponded to a serum level of 7.8 pmol/L in extracted serum. It was considered that levels above 50 pmol/L could be determined accurately without extraction. The serum levels of 17 beta-estradiol in prepubertal girls were, in most cases, below the detection limit, except in the morning, when in 17 of the 21 prepubertal girls, serum 17 beta-estradiol levels were just above the detection limit. All girls in early puberty (Tanner breast stage 2) had measurable serum levels of 17 beta-estradiol in the morning, whereas 10 of these 15 girls had levels below the detection limit around midnight. Later in puberty (Tanner breast stages 3 and 4), but before menarche, the diurnal rhythm was more obvious, with high levels of 17 beta-estradiol during the latter part of the night and in the morning. This diurnal rhythm was lost by 1 yr after menarche. There was a high degree of correlation between serum concentrations of 17 beta-estradiol and bone age, whereas there was much less, if any, correlation between 17 beta-estradiol and levels of sex hormone-binding globulin or dehydroepiandrosterone sulfate during puberty. We conclude that the nocturnal rise in gonadotropin secretion during puberty in girls is accompanied by an increased secretion of 17 beta-estradiol in the morning. This diurnal rhythm is lost 1 yr after menarche. Determination of 17 beta-estradiol levels in the morning could be useful in determining the initiation of puberty, whereas determinations in the late evening could provide information on the tempo of puberty.

Elevated 3,5-diiodothyronine concentrations in the sera of patients with nonthyroidal illnesses and brain tumors.

This study reports the development of a highly sensitive and reproducible RIA for the measurement of 3,5-diiodothyronine (3,5-T2) in human serum and tissue. The RIA employs 3-bromo-5-[125I]iodo-L-thyronine (3-Br-5-[125I]T1) as tracer, which was synthesized carrier free by an interhalogen exchange from 3,5-dibromo-L-thyronine (3,5-Br2T0). The detection limits were 1.0 fmol/g and 0.8 pmol/L in human brain tissue and serum, respectively. T3, diiodothyroacetic acid, and 3-monoiodothyronine cross-reacted with a 3,5-T2 antibody to the extent of 0.06%, 0.13%, and 0.65%, respectively. Serum concentrations of 3,5-T2 were measured in 62 healthy controls and 4 groups of patients with nonthyroidal illness, i.e. patients with sepsis (n = 24), liver diseases (n = 23), head and/or brain injury n = 15), and brain tumors (n = 21). The mean serum level of 3,5-T2 in the healthy subjects was 16.2 +/- 6.4 pmol/L. Concentrations of 3,5-T2 were significantly elevated in patients with sepsis (46.7 +/- 48.8 pmol/L; P < 0.01), liver diseases (24.8 +/- 14.9 pmol/L; P < 0.01), head and/or brain injury (24.1 +/- 11.3 pmol/L; P < 0.05), and brain tumors (21.6 +/- 4.8 pmol/L; P < 0.01). In all 4 patient groups, serum levels of T3 were significantly reduced, confirming the existence of a low T3 syndrome in these diseases. Serum concentrations of 3,5-T2 were significantly elevated in patients with hyperthyroidism (n = 9) and were reduced in patients with hypothyroidism (n = 8). The levels of T4, T3, and 3,5-T2 were measured in normal human tissue samples from the pituitary gland and various brain regions and in brain tumors. In normal brain tissue, the concentrations of 3,5-T2 ranged between 70-150 fmol/g, and the ratio of T3 to 3,5-T2 was approximately 20:1. In brain tumors, however, T3 levels were markedly lower, resulting in a ratio of T3 to 3,5-T2 of approximately 1:1. Recent findings suggest a physiological, thyromimetic role of 3,5-T2, possibly stimulating mitochondrial respiratory chain activity. Should this prove to be correct, then the increased availability of 3,5-T2 in nonthyroidal illness may be one factor involved in maintaining clinical euthyroidism in patients with reduced serum levels of T3 during nonthyroidal illness.

Dissociation between plasma bioactive and immunoactive ACTH concentrations in depressed patients.

Previous studies have reported dissociations between plasma cortisol and immunoactive adrenocorticotropic hormone (ACTH) concentrations in both normal controls and in patients with major depression. In order to investigate this issue further, placebo and dexamethasone (DEX) were administered to normal controls and depressed patients at 11 PM, and plasma cortisol and ACTH were measured the following morning at 7 AM. Plasma ACTH concentrations were quantitated by both immunoassay (I-ACTH) and by bioassay (B-ACTH). In 10 normal controls, DEX (0.25, 0.5, and 1.0 mg, PO, elixir) produced a dose-related suppression of cortisol, I-ACTH and B-ACTH, with all three hormones significantly suppressed by DEX (0.5 and 1.0 mg) (p < or = 0.01). In 20 depressed patients, 7 AM plasma ACTH and cortisol concentrations were assessed following a single dose of DEX (0.5 mg). Fifteen patients were classified as suppressors and five as escapers, as reflected by mean (+/- SEM) cortisol concentration of 19.9 +/- 3.0 ng/ml and 81.2 +/- 7.0 ng/ml, respectively. Mean I-ACTH concentrations were comparable in both the escapers (8.6 +/- 1.6 pg/ml) and in the suppressors (7.0 +/- 1.0 pg/ml). In contrast, the mean B-ACTH concentration was more than two-fold higher in the escapers (4.5 +/- 0.5 pg/ml) than in the suppressors (2.2 +/- 0.3 pg/ml) (p < or = 0.001). Eleven of the 20 patients received both placebo and DEX (0.5 mg) on two separate occasions. Although DEX significantly suppressed both cortisol (p < or = 0.0001) and B-ACTH (p < or = 0.01) concentrations, I-ACTH was not significantly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

The imprecision profile in immunoassay. A study using a resampling technique.

A resampling ('bootstrap') technique was applied to assess the reliability of the calculated imprecision profile (IP), as obtained from the dose/response curve and the response/error relationship (RER) using the cumulative data relative to two assays, i.e. a T4 radioimmunoassay (RIA) and a TSH immunofluorometric assay (IFMA), both run in duplicate. Mean values and the related uncertainty of the estimated dose errors were compared for different RER fitting conditions and different sizes of the duplicate response sets. The following observations were made: (a) compared to the maximum-likelihood procedure, the least-square fit proved to be unsuitable for estimating the parameters in the general RER equation variance(R) = aRb (where R indicates the response), (b) the simplifying assumption of a within-method constancy of the exponent in the RER equation, while acceptable for the T4 RIA, did not hold in the case of the TSH IFMA implying a much wider response range, (c) for both assays, response sets of ca. 100 duplicates were apparently compatible with an acceptable definition of the IP (+/- 10 to +/- 20% uncertainty).

A radioimmunoassay for the quantification of human ubiquitin in biological fluids: application to parasitic and allergic diseases.

Purified ubiquitin has been shown to share similar biological and physicochemical properties with a previously characterized lymphokine, platelet activity suppressive lymphokine (PASL). This lymphokine, which inhibits the cytotoxic function of activated platelets, is produced during schistosomiasis and Hymenoptera venom hypersensitivity (HVH). We have developed a radioimmunoassay specific for ubiquitin, in order to determine the ubiquitin levels in human sera and plasma from patients with these pathologies. The working range of the assay was between 60 and 500 ng/ml, and the sensitivity was 8-10 ng/ml. The reproducibility, specificity and accuracy were determined under standard condition (PBS-0.3% BSA) and using different biological fluids (human serum, plasma and T lymphocyte supernatant). Using this assay, we found that the ubiquitin concentrations were higher in both schistosomiasis and HVH (up to 150-300 ng/ml) compared with sera and plasma from healthy donors where the ubiquitin levels did not exceed 50 ng/ml.

Arginine vasopressin response to hypertonicity in hypertension studied by arginine vasopressin assay in unextracted plasma.

OBJECTIVE: To investigate whether plasma osmolality (Posm)-plasma arginine vasopressin (PAVP) response relationships with particular characteristics of sensitivity, gain or response pattern (linear/non-linear) occur more frequently in hypertensive than normotensive subjects. DESIGN: Analysis of Posm-PAVP curves observed in individual normotensive and hypertensive subjects rather than whole groups was considered appropriate for the described objective. METHODS: A sensitive and precise radioimmunoassay of PAVP in unextracted plasma was developed. Posm was raised in 11 normotensive and 19 hypertensive subjects by infusion of NaCl solution (0.86 mol/l, i.v.), and PAVP assayed at intervals. Best-fitting linear and non-linear equations for the Posm-PAVP relationship were established by computer. RESULTS: The mean rise in osmolality, blood pressure and blood volume was similar in the two groups. Hypertensive subjects showed a tendency towards higher rates of response (pmol AVP/l per mosm per kg) at Posm greater than 290 mosm/kg, leading to a non-linear response pattern compared with the normotensive subjects. CONCLUSIONS: The enhanced PAVP responses found in the hypertensive subjects at Posm greater than 290 mosm/kg may reinforce vascular and central nervous pathogenetic mechanisms.

Effect of high-performance liquid chromatography on plasma angiotensin II measurements in treated and untreated normotensive and hypertensive patients.

BACKGROUND: Angiotensin II (Ang II) levels are normally very low in human plasma, approximately 5 pg/ml. They are usually measured by radioimmunoassay after extraction and concentration. An additional high-performance liquid chromatography (HPLC) step is reportedly necessary for accurate measurement but it is laborious and time-consuming, severely limiting the number of samples that can be assayed. OBJECTIVE: To investigate whether the HPLC step was necessary for measuring Ang II in human plasma samples in our laboratory using our own Ang II antiserum. DESIGN: Human plasma Ang II levels, measured with and without the HPLC step, were compared in two different studies. Since the action of renin is the rate-limiting step in the production of Ang II in plasma, the relationships of plasma renin activity (PRA) to Ang II levels measured with and without HPLC were also evaluated. In the first study, 108 blood samples were collected from 29 hypertensive patients during placebo or treatment with the Ang II antagonist BMS-186295. In the second study blood samples were collected from 12 normal subjects before and during beta-adrenergic blockade. RESULTS: In samples collected during angiotensin II antagonism, which predictably increased plasma Ang II levels, a highly significant relationship between the Ang II measurements with and without HPLC was found (y = 0.99x + 1.7; r = 0.97, P < 0.001). The y intercept of 1.7 pg/ml suggested that the nonspecific immunoreactivity was close to 2 pg/ml in samples assayed without the HPLC step. During beta-adrenergic blockade, which predictably suppressed plasma renin levels, highly significantly linear relationships between HPLC and non-HPLC Ang II measurements (y = 1.3x + 1.6; r = 0.93. P < 0.001, n = 16) and between non HPLC Ang II and PRA (y = 1.9x + 1.7; r = 0.73, P < 0.001, n = 108) were again found. The relationship between PRA and HPLC Ang II was also highly significant (y = 1.4x + 0.04; r = 0.92, P < 0.001, n = 16), but the y intercept was significantly lower (P < 0.001), approaching zero, indicating the removal of nonspecific immunoreactivity during the HPLC step. CONCLUSIONS: These results demonstrate once more that, when using polyclonal antibody 182, the accuracy of the Ang II measurement in human plasma is improved by the inclusion of a HPLC step, especially for samples with Ang II levels in the normal-to-low range. They also show that plasma Ang II and PRA increase or decrease proportionally during treatment with Ang II antagonists or beta-adrenergic blockade, respectively.

Thyroglobulin measurement. Techniques, clinical benefits, and pitfalls.

The validity and reliability of serum Tg measurements is central to accurate diagnosis and cost-effective management of patients with differentiated thyroid carcinoma. Serum Tg is one of the most difficult biochemical tests for a laboratory to maintain at a high level of precision and reliability across the long clinical sampling interval typically employed when monitoring this condition. Physicians should be aware that the diagnostic value of serum Tg measurement is method-dependent, and that intermethod differences still approach 30% even after methods have been standardized on the new CBR reference preparation. This dictates that, ideally, serial serum Tg measurements in a patient should always be made in the same laboratory by the same method. Physicians need to consider a number of factors before selecting a laboratory to perform Tg measurements in patients with differentiated thyroid carcinoma. These factors include assay sensitivity, as judged by the discrimination between lower limit of the euthyroid range and the functional sensitivity limit, as well as assay specificity, especially with respect to serum TgAb interference. laboratories should use a sensitive TgAb immunoassay (not hemagglutination) to prescreen sera for TgAb and report the TgAb level if positive. Furthermore, it is the laboratory's responsibility to advise physicians fully about any change in Tg method as well as the direction of TgAb interference expected with that method. When TgAb is present, the serum Tg level should be a measure of the total (free and TgAb-bound) serum Tg level. Typically, IMA methods underestimate the serum total Tg level, especially when serum TSH is suppressed, whereas RIA methods tend to overestimate the serum total Tg level. The interpretation of a serum Tg level in a TgAb-positive patient should be made with caution and with consideration to any changes in TgAb levels. Serial TgAb monitoring of TgAb-positive patients may provide a physician with additional prognostic information on outcome. Precise, reproducible serial serum Tg measurements are critical, especially when patients are judged to have a high risk for recurrence, have tumors that are inefficient serum Tg secretors (as judged from the relationship between the preoperative serum Tg value and tumor mass or the serum Tg response to endogenous TSH stimulation), or have very high serum Tg values requiring dilution. In such patients, the banking of left-over sera (frozen) for concurrent intra-assay remeasurement with a more recent specimen significantly increases the clinical value of the test and facilitates earlier detection of recurrence or progression (see Fig. 2). Differentiated thyroid carcinoma is frequently diagnosed in young patients with decades of life expectancy. After their initial surgical treatment, these patients need life-long monitoring, because late recurrences and death from the disease can occur. The use of high-quality serum Tg measurements can significantly improve the cost-effective management of this disease by identifying low-risk patients in whom periodic radioiodine scans or therapy may be deferred in favor of serial serum Tg monitoring (on L-T4 suppression therapy). With this approach, expensive imaging procedures can be targeted to the minority of patients who are at high risk for recurrence.

Quantitation of platelet glycoprotein IV (CD36) in healthy subjects and in patients with essential thrombocythemia using an immunocapture assay.

Glycoprotein IV (GPIIb, CD36) is a major platelet membrane glycoprotein which is thought to participate in a number of adhesive reactions and to mediate signal transduction. In order to measure the total content of GPIV in human platelets, we have developed a simple and sensitive solid-phase radioimmunoassay based on the immunocapture of GPIV from Triton X-100-solubilized platelets. FA6-152, a monoclonal antibody to GPIV was coated on microtiter plates and bound antigen was quantified with a radiolabeled polyclonal antibody to GPIV. Using purified GPIV as a standard, the coefficients of variation of the assay were found to be less than 10% at concentrations of GPIV ranging from 0.15 to 0.75 micrograms/ml. The assay was validated by the parallelism obtained between purified GPIV dose-response curves and those obtained with platelet lysates, indicating a similar antigenic activity for GPIV in both samples. The level of GPIV in platelets from healthy donors was 0.23 +/- 0.05 (mean +/- SD, n = 15) micrograms per 100 micrograms of platelet proteins and a mean value of 27,440 +/- 6,200 (SD) molecules per platelet was calculated. The radioimmunoassay could be used to discriminate between the high level of platelet GPIV in patients with essential thrombocythemia (mean +/- SD = 81,850 +/- 27,780 molecules/platelet; n = 8) and the normal GPIV level in patients with secondary thrombocytosis (mean +/- SD = 26,810 +/- 4,030 molecules/platelet; n = 5), thereby demonstrating the clinical usefulness of the assay. The specific increase in platelet GPIV in patients with essential thrombocythemia was confirmed by immunoblot analysis whereas no increase in platelet GPIb or GPIIb-IIIa was observed by this technique.

Assessment of the relative contribution of different protease inhibitors to the inhibition of plasmin in vivo.

It has been shown that the most important inhibitor of plasmin is alpha 2-antiplasmin, however, other protease inhibitors are able to inhibit this proteolytic enzyme as well. The contribution of the various protease inhibitors to the inhibition of plasmin in vivo has never been quantitatively assessed. To assess the relative contribution of the different protease inhibitors on the inhibition of plasmin we developed a series of sensitive immunoassays for the detection of complexes between plasmin and the protease inhibitors alpha 2-antiplasmin, alpha 2-macroglobulin, antithrombin III, alpha 1-antitrypsin and C1-inhibitor, utilizing monoclonal antibodies that are specifically directed against complexed protease inhibitors and a monoclonal antibody against plasmin. It was confirmed that alpha 2-antiplasmin is the most important inhibitor of plasmin in vivo, however, complexes of plasmin with alpha 2-macroglobulin, antithrombin III, alpha 1-antitrypsin- and C1-inhibitor were also detected. Particularly during activation of fibrinolysis complexes between plasmin and inhibitors other than alpha 2-antiplasmin were detected. It was observed that during different situations the inhibition profile of plasmin was not constant e.g. in patients with diffuse intravascular coagulation plasma levels of plasmin-alpha 1-antitrypsin and plasmin-C1-inhibitor were increased whereas in plasma from patients who were treated with thrombolytic agents complexes of plasmin with alpha 2-macroglobulin and with antithrombin III were significantly elevated. In conclusion, we confirmed the important role of alpha 2-antiplasmin in the inhibition of plasmin, however, in situations in which fibrinolysis is activated other protease inhibitors also account for the inhibition of plasmin in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

A brief overview of nuclear medicine in China.

The year 1956 witnessed the birth of Nuclear Medicine in China, when the first course, Biomedical Applications of Isotopes, was offered in our country by the Peking Union Medical College (PUMC). This course was preceded by a training course in nuclear instruments in which students learned to construct the radiation detection devices required for performing experiments using radioisotopes. In 1958, several courses in clinical nuclear medicine brought up the first generation of nuclear medicine physicians in China. Historically, some of the chief events include: (1) operation of the first reactor, producing 33 radioactive isotopes in 1958; (2) first linear scanner built in 1960; (3) setting up an organization for the control of radiopharmaceuticals in 1961; (4) distribution of the first batch of cyclotron-produced isotopes in 1963; (5) development and use of the first radioimmunoassay (RIA) procedure in 1963; (6) production of tritium in 1964; (7) production of 99.8% enriched heavy water in 1965; (8) supply of 99mTc and 113mIn generators in 1972; (9) first gamma camera imported in 1972 and first homemade gamma camera installed in 1977; (10) founding of Chinese Society of Nuclear Medicine (CSNM) in 1980; (11) publication of the Chinese Journal of Nuclear Medicine beginning in 1981; (12) first single photon emission computed tomography (SPECT) imported in 1983. At present, there are 556 nuclear medicine departments in China with 4,000 staff.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of the substance immunologically cross-reactive with insulin in insulin RIA is an artifact caused by insulin tracer degradation: involvement of the insulin-degrading enzyme.

In previous literature, the existence of a new insulin-like substance found in tumor tissues, termed substance immunologically cross-reactive with insulin (SICRI), has been proposed. In these studies, insulin-specific radioimmunoassay (RIA) was the only detection method for SICRI. The mouse melanoma B16BL6 cell line was found to be a rich source of SICRI. In this paper, we show that SICRI is not expressed in B16BL6 cells. Previous RIA measurements were wrongly ascribed to SICRI. What was really measured was a positive artifact caused by insulin tracer degradation in RIA. Several lines of evidence indicate that protease responsible for insulin degradation in B16BL6 cells in insulin-degrading enzyme (IDE; EC 3.4.22.11). First, SICRI activity of B16BL6 cytosol measured by insulin RIA was inhibited by thiol protease inhibitor N-ethylmaleimide (NEM). Thiol active agents as well as metal chelators, both potent IDE blockers, inhibited also the insulin-degrading activity of the same sample. Second, cross-linking to 125I-labeled insulin of partially purified sample with highest insulin RIA activity specifically labeled only a single protein with molecular mass similar to IDE (110 kDa). Labeling was blocked by 'cold' insulin in excess. Third, kinetic studies of insulin degradation by RIA active chromatographic fractions revealed an apparent Kd of 90 nM which is very similar to the reported affinity of insulin for IDE (Kd = 100 nM). Additionally, in B16BL6 as well as in mouse myeloid leukemia cells, IDE gene is actively transcribed and this expression was found to be much stronger than in normal mouse tissues. In conclusion, our results strongly question the real existence of SICRI.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative genetic analysis of circulating levels of biochemical markers of bone formation.

Carboxyterminal propeptide of type 1 collagen (PICP) and bone Gla-protein-osteocalcin (BGP) are the most important components of the organic bone matrix and play a key role in bone formation. To investigate whether and to what extent variation of the plasma levels of these indices of bone turnover depends on genetic factors, we studied 355 adults belonging to nuclear pedigrees. Genetic analysis was carried out in 2 steps: 1) variance decomposition analysis was performed using the FISHER statistical package; and 2) complex segregation analysis implemented in the program package MAN. The effect of age and gender differences, gender hormones, as well as PTH and vitamin-D (calcidiol) plasma levels were evaluated simultaneously with the parameters of variance analysis. The results showed that about 50% of PICP variation is attributable to genetic factors. The effect of age was significant among men and postmenopausal women, whereas calcidiol influenced variation of PICP in premenopausal women. The results of variance analysis showed that some 40% of BGP, adjusted for confounding variables, can be explained in genetic factors. Age and PTH were important covariates for osteocalcin in men and premenopausal women. Exploration of the maximum likelihood estimates of the various hypotheses concerning the mode of intergenerational transmission of PICP and BGP demonstrated a good correspondence to the Mendelian mode of inheritance (i.e., major gene effect).