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Although manufacturers are compelled by the European IVD Directive, 98/79/EC, to have traceability of the values assigned to their calibrators if suitable higher order reference materials and/or procedures are available, there is still no equivalence of results for many measurands determined in clinical laboratories. The adoption of assays with metrological traceable results will have a significant impact on laboratory medicine in that results will be equivalent across different laboratories and different analytical platforms. The IFCC WG on Allowable Errors for Traceable Results has been formed to define acceptable limits for metrological traceability chains for specific measurands in order to promote the equivalence of patient results. These limits are being developed based on biological variation for the specific measurands. Preliminary investigations have shown that for some measurands, it is possible for manufacturers to assign values to assay calibrators with a measurement uncertainty that allows the laboratory enough combined uncertainty for their routine measurements. However, for other measurands, e.g., plasma sodium, current assays are too imprecise to fulfil limits based on biological variation. Although an alternative approach based on probability theory is being investigated, the most desirable approach would be for industry to improve measurement methods so that they meet clinical requirements.

The use of multiple biomarkers representing various etiologic pathways of atherosclerosis may improve the prediction of interindividual variation in the ankle-brachial index (ABI). To this end, we investigated associations of 47 candidate biomarkers with the ABI and presence of peripheral arterial disease (PAD) in African-Americans (AAs) and non-Hispanic whites (NHWs).Study participants included 1,291 AAs (71.1% women, mean age, 63.4±9.3 years) and 1,152 NHWs (57.5% women, mean age 58.5±10.1 years) belonging to hypertensive sibships. Peripheral arterial disease was defined as an ABI ≤ 0.90. Circulating levels of 47 candidate biomarkers were log-transformed before analysis because of skewed distribution. Multivariate regression analyses were used to identify biomarkers associated with ABI or PAD independently of age, sex, conventional risk factors, and medication use.After adjustment for covariates, higher levels of nine biomarkers were associated with a lower ABI in AAs (all P ≤ 0.005); these biomarkers were C-reactive protein (CRP), interleukin-6, tumor necrosis factor receptor-II (TNF-R II), lipoprotein(a), N-terminal pro-brain natriuretic peptide (NT-proBNP), pro-atrial natriuretic peptide, C-terminal pro-arginine vasopressin, osteoprotegerin, and fibrinogen. Three biomarkers - myeloperoxidase, NT-proBNP, and D-dimer - were associated with ABI in NHWs (all P ≤ 0.01). C-reactive protein, interleukin-6, TNF-R II, lipoprotein(a), NT-proBNP, pro-atrial natriuretic peptide, D-dimer, and fibrinogen were associated with PAD (all P ≤ 0.005) in AAs after adjustment for covariates. None of the biomarkers were independently associated with PAD in NHWs.A multimarker approach improved the prediction of interindividual variation in the ABI in AAs and NHWs, and improved prediction of the presence of PAD in AAs.

Tryptophan (TRP) depletion was used to study serotonin because the ratio of TRP to large neutral amino acids (TRP/ LNAA) determines the quantity of TRP that enters the brain. Because TRP is not universally available, a modified technique of TRP depletion was developed where a 1/4 strength preparation of an amino acid mixture (AAM) replaces TRP as the placebo. Seven healthy subjects could not differentiate between the preparations in this double-blind, placebo-controlled study. Urinary 6-hydroxymelatonin sulfate (6-MS) was monitored as a biochemical marker of serotonin. The TRP/LNAA ratio (GG = 0.0001) and 6-MS secretion (GG = 0.024) were decreased, but placebo TRP levels (GG = 0.062) were not altered significantly. This modified technique facilitates the sue of TRP depletion in clinical research.

Objectives Nearly half of men with clinically localized prostate cancer are understaged. We evaluated whether knowledge of preoperative free prostate-specific antigen (f-PSA), complexed (c-PSA) and total (t-PSA) concentrations or the ratios thereof (f-PSA/t-PSA, c-PSA/t-PSA and f-PSA/c-PSA) could improve upon the staging of prostate cancer when compared with standard PSA testing (t-PSA). In addition, we examined their associations with tumor grade and deoxyribonucleic acid (DNA) ploidy. Methods Two hundred ninety patients with prostate cancer, 178 (61 %) of whom were treated with radical prostatectomy, formed the study group. Results Although there were significant differences in the f-PSA concentrations with respect to clinical stage, considerable overlap in PSA levels among the clinical substages was observed. Statistically significant differences but weak correlations were observed between the individual f-PSA, c-PSA and t-PSA concentrations with regard to pathologic stage (organ-confined versus extraprostatic) and grade. No significant relationship, however, was observed with the three ratios. Higher PSA values were not always associated with a pathologic stage of pT3 or greater and lower levels did not ensure that a tumor was organ-confined. Only a slight association was observed between c-PSA and t-PSA levels and DNA ploidy. No significant relationship was observed between the f-PSA levels as well as the three ratios with regard to DNA ploidy. A statistically significant improvement in predicting pathologic stage was observed when combining knowledge of preoperative t-PSA concentration with the c-PSA/t-PSA ratio. However, the area under the receiver operator characteristic curves was only slightly increased; as such this combination was of limited clinical utility. Conclusions Statistically significant but weak correlations were observed between the molecular forms of PSA and stage, grade and DNA ploidy. The significant overlap in f-PSA and c-PSA values among all stages, grades and ploidy values precluded any useful predictive information for the individual patient. As such, preoperative knowledge of f-PSA and c-PSA values and the three ratios provided no additional diagnostic information over standard PSA (t-PSA) values alone.

The major clinical role for total triiodothyronine (TT3) and (or) free T3 (FT3) is the assessment of hyperthyroidism in patients with suppressed sensitive thyrotropin (sTSH) concentrations. The assays are particularly important in hyperthyroid patients with normal free thyroxine (FT4) concentrations to assess potential T3 thyrotoxicosis. Other specialized uses for T3 and FT3 measurements are monitoring thyroid hormone replacement therapy, the evaluation of amiodarone-induced thyrotoxicosis, and predicting outcome of antithyroid drug therapy in patients with Graves hyperthyroidism. The roles of these tests in assessing heart function in cardiopulmonary bypass surgery, evaluation of patients with neuropsychiatric disorders, and monitoring of patients on anticonvulsant therapy are not well defined. These assays are not recommended for diagnosis of hypothyroidism. Analytic recommendations include CV < 5.2% for T3 and < 3.8% for FT3; < 0.2% cross-reactivity with L-T4; and < 1.0% cross-reactivity with D-T4, D-T3, and reverse T3.

BACKGROUND. Although prostate-specific antigen (PSA) has aided significantly in the diagnosis of prostate cancer, more sensitive and accurate assays are needed. Presently, we are developing a sensitive immunoassay for hK2 protein for the detection of prostate cancer. METHODS. Polyclonal and monoclonal antibodies specific for hK2 were produced and used for Western blot analysis and immunohistochemistry for detection of hK2 protein in serum and human tissues. The reverse-transcriptase polymerase chain reaction (RT-PCR) was utilized to detect hK2 mRNA from patient blood samples. RESULTS. Western blot analysis demonstrated that the antibodies used are monospecific for hK2. Immunohistochemistry showed that hK2 is expressed only in prostatic epithelia. An RT-PCR study showed that hK2 mRNA would be a useful candidate for early detection of prostatic micrometastasis. CONCLUSIONS. Monospecific antibodies for hK2 have been developed for detecting hK2 protein. Our studies indicate that hK2 may be a useful marker for prostate cancer.

This article proposes analytic performance goals for five quality indicators: precision, trueness, linearity, detection limits, and consistency across instruments and time. We defined our goals using methods linked to clinical practice data. Goals for desirable precision and trueness are based on biological variation. Linearity goals are related to total error recommendations. Detection limit goals are derived from 0.1 percentile of patient values. Goals for consistency are derived from the variability of distributions of patient test values. Data were collected and evaluated for each of these quality indicators for 46 chemistry tests measured on the Roche cobas 8000 analyzer.

Background: In a planned International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) worldwide study on reference intervals (RIs), a common panel of serum samples is to be measured by laboratories from different countries, and test results are to be compared through conversion using linear regression analysis. This report presents a validation study that was conducted in collaboration with four laboratories. Methods: A panel composed of 80 sera was prepared from healthy individuals, and 45 commonly tested analytes (general chemistry, tumor markers, and hormones) were measured on two occasions 1 week apart in each laboratory. Reduced major-axis linear regression was used to convert reference limits (LL and UL). Precision was expressed as a ratio of the standard error of converted LL or UL to the standard deviation (SD) comprising RI (approx. 1/4 of the RI width corresponding to between-individual SD). The allowable and optimal levels of error for the SD ratio (SDR) were set as

Objective To report the clinical responses and mediator-release profiles of an aspirin-sensitive man with systemic mast cell disease during aspirin desensitization. Material and Methods We quantified the release of six mediators during aspirin desensitization. Results Although aspirin was administered cautiously with an initial dose of 20 mg, successful aspirin desensitization necessitated complete monitoring and resuscitation capabilities of a medical intensive-care unit for 4.5 days because of frequent, severe anaphylactoid responses. To our knowledge, this is the first report of a pronounced increase in plasma levels of the vasodilator peptide calcitonin gene-related peptide during episodes of aspirin-induced hypotension. Increases in plasma levels of calcitonin and serum levels of tryptase paralleled those of calcitonin gene-related peptide, but plasma levels of calcitonin remained increased for up to 18 hours. Urinary excretion ofhistamine and I-methyl-4-imidazoleacetic acid also showed precipitous, although delayed, increases. Excretion of the prostaglandin D 2 metabolite 11β-prostaglandin F 2α : followed a bimodal pattern during aspirin desensitization; after severe hypotensive responses, the maximal value was more than 490,000 pg/mL, but the level decreased to less than 100 pg/mL after therapeutic serum levels of salicylate were attained. Conclusion These data suggest that the hypotensive responses to aspirin in some patients with systemic mast cell disease may result from the combined effects of several mediators.

The genomic and the cDNA clones of human glandular kallikrein (hK2), a member of the kallikrein family, have been isolated; however, the hK2 protein has not yet been identified and characterized. The deduced sequence of hK2 is highly homologous to prostate specific antigen (PSA), a widely accepted prognostic indicator of prostate carcinoma. Also, hK2 mRNA, like PSA mRNA, is exclusively expressed in prostatic epithelia. These two properties make hK2 a potentially useful marker for studying prostate cancer. In this paper, we describe for the first time the overexpression of the entire hK2 protein (pre-pro hK2:pphK2) in the E. coli system. Our system yields high levels of authentic pphK2 (as determined by partial amino acid sequence analysis) comprising about 40% of total cellular protein. pphK2 was purified to near homogeneity by preparative SDS/PAGE and used to generate anti-pphK2 antibodies in rabbits. The antibodies recognize the recombinant hK2 protein and a major band of approximately 34 kDa in seminal fluid.

Purpose: Reported prostate specific antigen values may differ substantially among assays using Hybritech (R) or WHO standardization. The Beckman Coulter (R) Prostate Health Index and [-2]proPSA are newly approved serum markers associated with prostate cancer risk and aggressiveness. We studied the influence of assay standardization on these markers. Materials and Methods: Prostate specific antigen, percent free prostate specific antigen and [-2]proPSA were measured using Hybritech calibration in 892 men from a prospective, multicenter study undergoing prostate biopsy. We calculated the Prostate Health Index using the equation, ([-2]proPSA/free prostate specific antigen) x root PSA. Index performance characteristics for prostate cancer detection were then determined using recalculated WHO calibration prostate specific antigen values. Results: The median Prostate Health Index was significantly higher in men with prostate cancer than in those with negative biopsies using WHO values (47.4 vs 39.8, p

The reference intervals (RIs) given in laboratory reports have an important role in aiding clinicians in interpreting test results in reference to values of healthy populations. In this report, we present a proposed protocol and standard operating procedures (SOPs) for common use in conducting multicenter RI studies on a national or international scale. The protocols and consensus on their contents were refined through discussions in recent C-RIDL meetings. The protocol describes in detail (1) the scheme and organization of the study, (2) the target population, inclusion/exclusion criteria, ethnicity, and sample size, (3) health status questionnaire, (4) target analytes, (5) blood collection, (6) sample processing and storage, (7) assays, (8) cross-check testing, (9) ethics, (10) data analyses, and (11) reporting of results. In addition, the protocol proposes the common measurement of a panel of sera when no standard materials exist for harmonization of test results. It also describes the requirements of the central laboratory, including the method of cross-check testing between the central laboratory of each country and local laboratories. This protocol and the SOPs remain largely exploratory and may require a reevaluation from the practical point of view after their implementation in the ongoing worldwide study. The paper is mainly intended to be a basis for discussion in the scientific community. Japan Society for the Promotion of Science (24256003)

Purpose: The aim of this study was to investigate the promoter hypermethylation as diagnostic markers to detect malignant prostate cells and as prognostic markers to predict the clinical recurrence of prostate cancer. Experimental Design: DNA was isolated from prostate cancer and normal adjacent tissues. After bisulfite conversion, methylation of 14,495 genes was evaluated using the Methylation27 microarrays in 238 prostate tissues. We analyzed methylation profiles in four different groups: (i) tumor (n = 198) versus matched normal tissues (n = 40), (ii) recurrence (n = 123) versus nonrecurrence (n = 75), (iii) clinical recurrence (n = 80) versus biochemical recurrence (n = 43), and (iv) systemic recurrence (n = 36) versus local recurrence (n = 44). Group 1, 2, 3, and 4 genes signifying biomarkers for diagnosis, prediction of recurrence, clinical recurrence, and systemic progression were determined. Univariate and multivariate analyses were conducted to predict risk of recurrence. We validated the methylation of genes in 20 independent tissues representing each group by pyrosequencing. Results: Microarray analysis revealed significant methylation of genes in four different groups of prostate cancer tissues. The sensitivity and specificity of methylation for 25 genes from 1, 2, and 4 groups and 7 from group 3 were shown. Validation of genes by pyrosequencing from group 1 (GSTP1, HIF3A, HAAO, and RARβ), group 2 (CRIP1, FLNC, RASGRF2, RUNX3, and HS3ST2), group 3 (PHLDA3, RASGRF2, and TNFRSF10D), and group 4 (BCL11B, POU3F3, and RASGRF2) confirmed the microarray results. Conclusions: Our study provides a global assessment of DNA methylation in prostate cancer and identifies the significance of genes as diagnostic and progression biomarkers of prostate cancer. Clin Cancer Res; 18(10); 2882–95. ©2012 AACR.

Background: Total PSA assays measure both complexed and non-complexed forms of PSA while free PSA assays only measure non-complexed forms. Free PSA is a mixture of isoforms including immature PSA (proPSA) with retained portions of the leader sequence (e.g. [- 7], [- 4], and [- 2]proPSA) and nicked forms (BPSA). ProPSA isoforms in male sera have been associated with prostate cancer. This study characterized the analytical performance of a chemiluminescent immunoassay for [- 2]proPSA. Methods: The Access Hybritech p2PSA assay is a sandwich immunoassay using an anti-[- 2]proPSA monoclonal antibody attached to paramagnetic beads and an anti-PSA monoclonal antibody conjugated to alkaline phosphatase calibrated with recombinant [- 2]proPSA. Analytical studies including sensitivity (CLSI EP17-A) and imprecision (CLSI EP5-A2) were performed. Results: The Access Hybritech p2PSA assay for [- 2]proPSA had a dynamic range of 0.5 to 5000 pg/ml. The total CV of the assay was

BACKGROUND Improved tests are needed for detection and management of prostate cancer. We hypothesized that differential gene expression in prostate tissue could help identify candidate blood biomarkers for prostate cancer and that blood from men with advanced prostate disease could be used to verify the biomarkers presence in circulation. METHODS We identified candidate markers using mRNA expression patterns from laser-capture microdissected prostate tissue and confirmed tissue expression using immunohistochemistry (IHC) for the subset of candidates having commercial antisera. We analyzed tissue extracts with tandem mass spectrometry (MS/MS) and measured blood concentrations using immunoassays and MS/MS of trypsin-digested, immunoextracted peptides. RESULTS We selected 35 novel candidate prostate adenocarcinoma biomarkers. For all 13 markers having commercial antisera for IHC, tissue expression was confirmed; 6 showed statistical discrimination between nondiseased and malignant tissue, and only 5 were detected in tissue extracts by MS/MS. Sixteen of the 35 candidate markers were successfully assayed in blood. Four of 8 biomarkers measured by ELISA and 3 of 10 measured by targeted MS showed statistically significant increases in blood concentrations of advanced prostate cancer cases, compared with controls. CONCLUSIONS Seven novel biomarkers identified by gene expression profiles in prostate tissue were shown to have statistically significant increased concentrations in blood from men with advanced prostate adenocarcinoma compared with controls: apolipoprotein C1, asporin, cartilage oligomeric matrix protein, chemokine (C-X-C motif) ligand 11 (CXCL11), CXCL9, coagulation factor V, and proprotein convertase subtilisin/kexin 6.

Objectives. To assess the comparability of the Tandem-R and IMx serum prostate-specific antigen (PSA) assays across levels of the ratio of free-to-total serum PSA found in a community-based population of healthy men. Methods. Banked serum samples from the baseline component of the Olmsted County Study of Urinary Symptoms and Health Status Among Men were thawed and analyzed using the Tandem-R and IMx PSA assays. Serum levels also were determined for the free, noncomplexed form of PSA, PSA complexed to alpha-1 antichymotrypsin, and total PSA with a research-based immunofluorometric assay. Results. The results of the Tandem-R and IMx assays were strongly correlated at all levels of the ratio of free-to-total serum PSA. The Spearman correlation coefficients ranged from 0.87 to 0.98 (all p < 0.001). The relationship between the Tandem-R and IMx assays, however, differed at low levels of free-to-total serum PSA compared with high levels. In the lowest 10th percentile of the ratio of free-to-total serum PSA (0.04 to 0.18}, the IMx assay read lower than the Tandem-R (slope _+ standard error = 0.92 +0.04, in- tercept _+ standard error = 0.21 _+0.14); whereas in the upper 10th percentile of free-to ~, total ratio (0.46 to 0.65) the IMx assay yielded values higher than the Tandem-R assay (slope = 1.21 +0.07, intercept = 0.14 +0.05). In the middle 90%, the slope did not sta- tistically differ from 1.0. Conclusions. For the majority of men, results of the Tandem-R and IMx PSA assays were virtually identical. The small differences found would not be of clinical significance for most men but should be considered when comparing results of different assays in sequential determinations for a specific man.

In tumor-induced osteomalacia, a rare syndrome characterized by hypophosphatemia, hyperphosphaturia, low plasma 1,25-dihydroxyvitamin D concentrations, and osteomalacia,1–5 all biochemical and pathological abnormalities disappear when the tumor is removed. Tumors associated with this syndrome are thought to secrete a substance that inhibits the renal tubular reabsorption of phosphate,1–5 but whether this factor interacts directly with renal tubular cells is not known. We investigated the ability of medium in which sclerosing hemangioma cells from a patient with oncogenic osteomalacia were cultured to alter sodium-dependent phosphate transport in opossum-kidney epithelial cells. We found that the medium inhibited phosphate transport, without increasing . . .

Background Serum prostate-specific antigen (PSA), when used in combination with existing detection methods, improves the clinician's ability to detect early and potentially curable prostate cancer. Findings This report describes clinically important issues about use of the serum PSA concentration for detecting early prostate cancer. Other PSA-related factors—PSA density, PSA velocity, and age-specific reference ranges—seem to enhance the ability of clinicians to distinguish benign prostatic conditions from early prostate cancer. Because digital rectal examination only minimally affects the serum PSA concentration, delaying a determination after this examination is unnecessary. Finasteride therapy for benign prostatic hyperplasia should be initiated only after the prostate has been evaluated for cancer because this 5α-reductase inhibitor lowers the serum PSA value by approximately 50%; however, reassessment of the prostate for cancer is necessary if the PSA level fails to decrease as expected or increases to more than 2 ng/mL during finasteride treatment. Conclusion Currently, PSA is the most important, accurate, and clinically useful tumor marker for prostate cancer.

Objective To determine the longitudinal changes of benign prostate-specific antigen (BPSA) and [−2]proPSA and how these changes relate to the outcomes. These markers have been shown to be predictive of prostate cancer (CaP) and benign prostatic hyperplasia treatment; however, little is known about longitudinal changes in these markers. Methods In 1990, a 25% subsample from a cohort of white men aged 40-79 years, who were randomly selected from Olmsted County, Minnesota residents, completed a detailed clinical examination. BPSA and [−2]proPSA were measured from frozen sera. The men were evaluated biennially (median follow-up 7 years; range 0-8.8). Mixed-effects regression models were used to estimate the longitudinal changes in the BPSA and [−2]proPSA levels overall and by outcomes. Spearman correlations were used to compare these changes with the baseline levels and the annualized changes in urologic measures. Results The median and 25th and 75th percentiles annualized percent change for [−2]proPSA and BPSA was 3.7%, 2.5% and 5.2% and 7.3%, 6.8%, and 7.7%, respectively. The annualized percent change for both markers correlated with the baseline and annualized changes in PSA and prostate volume. The annualized percent change increased with increasing age decade for [−2]proPSA but not for BPSA. The rate of increase in [−2]proPSA was significantly greater for men who developed enlarged prostates (median 3.5%, 25th and 75th percentile 2.6% and 4.4%, respectively) or CaP (median 8.1%, 25th and 75th percentile 6.6% and 9.8%, respectively) compared with those who did not develop enlarged prostates (median 1.9%, 25th and 75th percentile 0.9% and 3.0%, respectively) or CaP (median 3.5%, 25th and 75th percentile 2.3% and 4.8%, respectively). Conclusion BPSA and [−2]proPSA levels increase over time. The annualized percent change in [−2]proPSA increases with age and might be a useful predictor of CaP development.

Background: Accrediting organizations require laboratories to establish analytic performance criteria that ensure their tests provide results of the high quality required for patient care. However, the procedures for instituting performance criteria that are directly linked to the needs of medical practice are not well established, and therefore alternative strategies often are used to create and implement surrogate performance standards.Content: We reviewed 6 approaches for establishing outcome-related analytic performance goals: (a) limits defined by regulations and external assessment programs, (b) limits based on biologic variation, (c) limits based on surveys of clinicians about their needs, (d) limits based on effects on guideline driven medical decisions, (e) limits based on analysis of patterns for ordering follow-up clinical tests, and (f) limits based on formal medical decision models. Performance criteria were tabulated for 12 common chemistry analytes and 4 routine hematology tests.Conclusions: There is no consensus currently about the preferred methods for establishing medically necessary analytic performance limits. The various methods reviewed give considerably different performance limits. The analytic performance limits claimed by a laboratory should correspond to those limits that can be reliably maintained based on validated QC monitoring systems. These limits generally are larger than the observed CVs and bias parameters collected for assay validation. There is a major need for increased communication among laboratorians and clinicians on this topic, especially when the analytic performance limits that can be consistently maintained by a laboratory are inconsistent with the expectations of health care providers.

Some men have rapid increases in benign prostatic enlargement and lower urinary tract symptoms (LUTS), and it is not clear how sex steroid hormones contribute to the rates of change in these urologic outcomes. Therefore, the authors conducted a population-based cohort study of 648 men residing in Olmsted County, Minnesota, from 1990 to 2007, to examine associations between baseline sex steroid hormones, the rate of change in these hormones, and the rates of change in LUTS, maximum urinary flow rate, and prostate volume. Annual changes in hormone levels and urologic outcomes were calculated using mixed-effects regression models. Associations between hormone variables and rates of change in urologic outcomes were assessed with linear regression models. Higher baseline estradiol levels and rapid declines in estradiol over time were associated with rapid increases in LUTS and rapid decreases in maximum flow rate. Lower baseline bioavailable testosterone levels and more rapid declines in bioavailable testosterone were associated with more rapid increases in prostate volume. These results suggest that both absolute sex steroid hormone levels and the rates at which the levels change may be important in the development of urologic conditions in aging men.

This article provides an overview of the classification of thyroid carcinoma into well-differentiated forms (papillary and follicular) as well as the rare Hurthle cell, medullary, and anaplastic forms. The characteristics, relative frequency, and mortality rates of each form of thyroid cancer are summarized. The roles of history, physical examination, blood tests, such as thyroid function test, thyroglobulin (htg), calcitonin and CEA, radionuclide and ultrasound imaging, and fine-needle aspirates are outlined for the diagnosis and management of thyroid cancer. An algorithmic diagram of the monitoring procedures for long-term management of various forms of thyroid cancer is presented.

This article reviews the role of thyrotropin assays in clinical medicine and illustrates the advantages and limitations of assays that can measure subnormal concentrations. Analytic considerations that can significantly alter the performance of these assays are outlined. The concept of assay generations is defined, and the potential roles of third and fourth generation assays are postulated. In addition, a diagnostic strategy is presented for using TSH and other procedures in the diagnosis and management of thyroid disease.

Diagnosis and management of thyroid disease benefit substantially from close interactions between clinicians and laboratorians. Clinicians desire reliable tests for diagnosis and management of both hypothyroidism and thyrotoxicosis. Traditionally, multitiered testing has been used, beginning with an index of free thyroxine concentration, which, if abnormal, has been followed by basal thyrotropin (TSH) measurements (for hypothyroidism) or thyrotropin-releasing hormone-stimulated TSH measurements (for thyrotoxicosis). Improvements in analytical methods for TSH have made it practical to use basal TSH measurements in serum as the first-line test for thyroid disease. However, performance guidelines are needed, because not all TSH assays work well in this role. The performance guidelines developed by the American Thyroid Association are reviewed to illustrate how they help to assure consistent analytical testing. Further improvements in TSH assays (third- and fourth-generation assays), which support measurements down to 0.01 mlU/L and 0.001 mIU/L, may provide additional advantages for classifying thyrotoxic patients and monitoring thyroxine-suppressive therapy in patients with thyroid cancer. The potential advantages of these newer assays are illustrated with case examples. Additional analytical performance monitors are proposed to help ensure that these next-generation TSH assays meet the expanded clinical needs.

I propose a method for defining tolerance limits for assay bias and assay imprecision, based on the effects of these tolerance limits on the clinical specificity of the assay. An analytical "error budget" is defined as the squared sums of the imprecision and bias errors. The maximum limit for this error budget is set at a value corresponding to a 50% increase in the false-positive rate for classifying healthy subjects. For gaussian distributions with +/- 2 SD used as decision limits, this error budget equates to 0.45 SD of combined within-person and between-person biological variation (SDBiol). To provide reasonable power for bias detection in an assay, I recommend that the SD of the assay be kept at less than half the bias limit. Then, for the gaussian distribution, the maximum bias limit should be < 0.36 SDBiol and the SD of the assay should be < 0.18 SDBiol. Procedures are provided for using the same principles to define tolerance limits for decision limits other than +/- 2 SD and for nongaussian distributions.

Currently there is no simple test to detect pancreatic cancer (PC). Fasting plasma islet amyloid polypeptide (IAPP) was reported to be increased in patients with PC (1), and a soluble factor from PC cells was reported to stimulate IAPP secretion (2), suggesting the possibility of an increase of IAPP in the plasma of patients with PC. In a recent clinical study, however, IAPP was not a satisfactory marker for detecting PC (3). Human IAPP is easily degraded by proteases present in serum or plasma and also degraded during the clotting process. A possible cause for the loss of utility of IAPP for PC detection could be degradation of the polypeptide before analysis. Therefore, we designed a study to reevaluate this marker using plasma samples carefully collected …

Background Cardiovascular disease (CVD) susceptibility differs between men and women and varies with ethnicity. This variability is not entirely explained by conventional CVD risk factors. We examined differences in circulating levels of 47 novel protein markers of CVD in 2561 men and women of African-American (AA) and non-Hispanic White (NHW) ethnicity, enrolled at geographically distinct sites. Methodology/Principal Findings Participants (1,324 AAs, mean age 63.5 y, 71% women; 1,237 NHWs, mean age 58.9 y, 57% women) belonged to sibships ascertained on the basis of hypertension. Solid-phase immunoassays and immunoturbidometric, clot-based, chromogenic, and electrophoretic assays were used to measure the 47 protein markers in plasma or serum. Marker levels were log transformed and outliers were adjusted to within 4 SD. To identify markers independently associated with sex or ethnicity, we employed multivariable regression analyses that adjusted for conventional risk factors, prior history of CVD, medication use and lifestyle factors (physical activity, alcohol consumption and education). Generalized estimating equations were used to correct for intrafamilial correlations. After adjustment for the above covariates, female sex was associated with higher levels of 29 markers and lower levels of 6 markers. Female sex was independently associated with higher levels of several inflammatory markers as well as lipoproteins, adipokines, natriuretic peptides, vasoconstrictor peptides and markers of calcification and thrombosis. AA ethnicity was associated with higher levels of 19 markers and lower levels of 6 markers, including higher levels of several inflammatory makers, higher leptin and lower adiponectin levels, lower levels of vasodilator-natriuretic peptides, higher levels of vasoconstrictor-antidiuretic peptides and markers of calcification and thrombosis. Conclusions/Significance Plasma levels of several novel protein markers of CVD differ significantly in the context of sex and ethnicity. These results have implications for individualized CVD risk assessment.

Three immunometric assays of parathyroid hormone (PTH)--a commercial immunoradiometric assay, an in-house immunoradiometric assay, and an immunochemiluminometric assay--were evaluated in 50 patients with surgically proven primary hyperparathyroidism. Of these patients, 43 had increased values with the commercial assay (sensitivity, 86%), whereas 45 patients had increased concentrations with both the in-house immunoradiometric and the in-house immunochemiluminometric assays (sensitivities, 90%). Because of the results of this comparison study, we confidently chose the immunochemiluminometric assay as our routine assay; this assay was evaluated retrospectively in 361 patients with surgically proven primary hyperparathyroidism. In 45 patients, PTH values were below the upper limit of normal (sensitivity, 88%). The results indicate that the sensitivities of current immunometric assays are approximately 90%. Twenty patients who had hypercalcemia associated with malignant involvement were assessed with the immunochemiluminometric assay. Of these 20 patients, 19 had subnormal PTH values, and 1 had a value within the normal range. In contrast, in the past, PTH values determined with radioimmunoassays have often been in the normal range for such patients. Thus, an immunometric PTH assay is superior to a radioimmunoassay in the differential diagnosis of hypercalcemia associated with malignant disease.

The immunochemiluminometric assay described uses immobilized anti-human parathyrin (parathyroid hormone, hPTH)(1-44) and anti-hPTH(44-68) antisera and acridinium ester-labeled anti-hPTH(1-34) to simultaneously measure both intact hPTH and its amino-terminal fragments. Results by the assay correlate well with those by a cAMP-based bioassay and the Nichols Allegro immunoradiometric assay. The minimal detection limit is 0.08 pmol/L. The normal range is 1.0-5.0 pmol/L, and values are higher in older women. About 90% of study patients with surgically proven parathyroid adenomas had above-normal preoperative PTH concentrations, whereas patients with hypercalcemia of malignancy had normal or suppressed values. This assay was designed to detect both intact PTH and amino-terminal PTH fragments; however, chromatographic fractionation of pools of primary and secondary hyperparathyroid plasma showed virtually no amino-terminal fragment activity. Nonetheless, the design is important because the absence of carboxyl-terminal binding sites prevents interference by carboxyl-terminal fragments and because bioactive amino-terminal fragments will react in the assay if they are present in the patients' sera or if they are produced by in vitro proteolysis of intact PTH.

Background: The measurement of multiple protein biomarkers may refine risk stratification in clinical settings. This concept has stimulated development of multiplexed immunoassay platforms that provide multiple, parallel protein measurements on the same specimen.Content: We provide an overview of antibody-based multiplexed immunoassay platforms and discuss technical and operational challenges. Multiplexed immunoassays use traditional immunoassay principles in which high-affinity capture ligands are immobilized in parallel arrays in either planar format or on microspheres in suspension. Development of multiplexed immunoassays requires rigorous validation of assay configuration and analytical performance to minimize assay imprecision and inaccuracy. Challenges associated with multiplex configuration include selection and immobilization of capture ligands, calibration, interference between antibodies and proteins and assay diluents, and compatibility of assay limits of quantification. We discuss potential solutions to these challenges. Criteria for assessing analytical multiplex assay performance include the range of linearity, analytical specificity, recovery, and comparison to a quality reference method. Quality control materials are not well developed for multiplexed protein immunoassays, and algorithms for interpreting multiplex quality control data are needed.Summary: Technical and operational challenges have hindered implementation of multiplexed assays in clinical settings. Formal procedures that guide multiplex assay configuration, analytical validation, and quality control are needed before broad application of multiplexed arrays can occur in the in vitro diagnostic market.

Background: Multiplex arrays are increasingly used for measuring protein biomarkers. Advantages of this approach include specimen conservation, limited sample handling, and decreased time and cost, but the challenges of optimizing assay format for each protein, selecting common dilution factors, and establishing robust quality control algorithms are substantial. Here, we use measurements of 15 protein biomarkers from a large study to illustrate processing, analytic, and quality control issues with multiplexed immunoassays. Methods: We contracted with ThermoScientific for duplicate measurements of 15 proteins in 2322 participants from a community-based cohort, a plasma control, and recombinant protein controls using 2 custom planar microarrays with 6 (panel A) or 9 (panel B) capture antibodies printed in each well. We selected constituent analytes in each panel based on endogenous concentrations and assay availability. Protocols were standardized for sample processing, storage, and freeze-thaw exposures. We analyzed data for effects of deviations from processing protocols, precision, and bias. Results: Measurements were within reportable ranges for each of the assays; however, concentrations for 7 of the 15 proteins were not centered on the dose–response curves. An additional freeze-thaw cycle and erroneous sample dilution for a subset of samples produced significantly different results. Measurements with large differences between duplicates were seen to cluster by analyte, plate, and participant. Conventional univariate quality control algorithms rejected many plates. Plate-specific medians of cohort and plasma control data significantly covaried, an observation important for development of alternative quality control algorithms. Conclusions: Multiplex measurements present difficult challenges that require further analytical and statistical developments.

Quantitation of circulating tumor cells (CTCs) can provide information on the stage of a malignancy, onset of disease progression and response to therapy. In an effort to more accurately quantitate CTCs, we have synthesized fluorescent conjugates of 2 high-affinity tumor-specific ligands (folate-AlexaFluor 488 and DUPA-FITC) that bind tumor cells >20-fold more efficiently than fluorescent antibodies. Here we determine whether these tumor-specific dyes can be exploited for quantitation of CTCs in peripheral blood samples from cancer patients. A CTC-enriched fraction was isolated from the peripheral blood of ovarian and prostate cancer patients by an optimized density gradient centrifugation protocol and labeled with the aforementioned fluorescent ligands. CTCs were then quantitated by flow cytometry. CTCs were detected in 18 of 20 ovarian cancer patients (mean 222 CTCs/ml; median 15 CTCs/ml; maximum 3,118 CTCs/ml), whereas CTC numbers in 16 gender-matched normal volunteers were negligible (mean 0.4 CTCs/ml; median 0.3 CTCs/ml; maximum 1.5 CTCs/ml; p < 0.001, chi(2)). CTCs were also detected in 10 of 13 prostate cancer patients (mean 26 CTCs/ml, median 14 CTCs/ml, maximum 94 CTCs/ml) but not in 18 gender-matched healthy donors (mean 0.8 CTCs/ml, median 1, maximum 3 CTC/ml; p < 0.0026, chi(2)). Tumor-specific fluorescent antibodies were much less efficient in quantitating CTCs because of their lower CTC labeling efficiency. Use of tumor-specific fluorescent ligands to label CTCs in peripheral blood can provide a simple, accurate and sensitive method for determining the number of cancer cells circulating in the bloodstream.

BACKGROUND:Many men develop a rising PSA after initial therapy for prostate cancer. While some of these men will develop a local or metastatic recurrence that warrants further therapy, others will have no evidence of disease progression. We hypothesized that an expression biomarker panel can predict which men with a rising PSA would benefit from further therapy. METHODOLOGY/PRINCIPAL FINDINGS:A case-control design was used to test the association of gene expression with outcome. Systemic (SYS) progression cases were men post-prostatectomy who developed systemic progression within 5 years after PSA recurrence. PSA progression controls were matched men post-prostatectomy with PSA recurrence but no evidence of clinical progression within 5 years. Using expression arrays optimized for paraffin-embedded tissue RNA, 1021 cancer-related genes were evaluated-including 570 genes implicated in prostate cancer progression. Genes from 8 previously reported marker panels were included. A systemic progression model containing 17 genes was developed. This model generated an AUC of 0.88 (95% CI: 0.84-0.92). Similar AUCs were generated using 3 previously reported panels. In secondary analyses, the model predicted the endpoints of prostate cancer death (in SYS cases) and systemic progression beyond 5 years (in PSA controls) with hazard ratios 2.5 and 4.7, respectively (log-rank p-values of 0.0007 and 0.0005). Genes mapped to 8q24 were significantly enriched in the model. CONCLUSIONS/SIGNIFICANCE:Specific gene expression patterns are significantly associated with systemic progression after PSA recurrence. The measurement of gene expression pattern may be useful for determining which men may benefit from additional therapy after PSA recurrence.

Yasuharu et al1 found an inverse association between plasma resistin and estimated glomerular filtration rate in a community-based cohort of Japanese individuals, findings that are consistent with our report.2 Furthermore, they observed plasma resistin to be a significant determinant …

We developed and validated a radioimmunoassay for circulating human parathyroid hormone-related peptide (PTHrP), based on a commercial antiserum to the synthetic 1-34 fragment of PTHrP, 125I-Tyr degrees-PTHrP(1-34) as radioligand, and prior extraction of the native peptide from plasma with C-2 cartridges. We determined immunoreactive PTHrP concentrations in plasma samples from 48 healthy persons (mean +/- SD, 3.1 +/- 1.0 pmol/liter; range, less than 2 to 5 pmol/liter), 8 patients with primary hyperparathyroidism, 36 patients with hypercalcemia and a concurrent malignant lesion, and 9 normocalcemic patients with cancer and increased serum levels of carcinoembryonic antigen or prostate-specific antigen. PTHrP was normal in samples from patients with primary hyperparathyroidism (3.2 +/- 1.1 pmol/liter), secondary hyperparathyroidism (2.5 +/- 1.3 pmol/liter), and cancer without hypercalcemia (2.4 +/- 1.0 pmol/liter). In contrast, plasma immunoreactive PTHrP levels were increased (6.0 to 85.0 pmol/liter) in 47% of patients with hypercalcemia and cancer of various types, with or without bone metastatic lesions. Large amounts of PTHrP were also found in conditioned medium from cultured human prostatic carcinoma cells. Thus, PTHrP may be a causative factor for hypercalcemia associated with a malignant lesion in at least half of the cases. Measurement of circulating PTHrP may be of differential diagnostic help in hypercalcemic states.

To define the role of parathyroid gland function in the pathophysiology of bone loss in type I (postmenopausal) osteoporosis, we measured serum intact parathyroid hormone (PTH) concentration by immunoradiometric assay (IRMA) and by multisite immunochemiluminometric assay (ICMA) in 63 postmenopausal osteoporotic women (PMOp) with vertebral compression fractures and in 75 age-comparable postmenopausal normal women (PMNl). Also, tetracycline-based histomorphometric indices in cancellous bone were assessed in iliac biopsy samples from 61 PMOp and 28 PMNl women. Serum PTH concentrations by IRMA were similar in PMOp and PMNl (medians, 3.92 and 3.77 pmol/l; NS) but were significantly lower in PMOp by the more sensitive ICMA (medians, 2.82 and 3.14 pmol/l; P less than 0.01). By multiple linear regression analysis, serum PTH was directly related (P less than 0.001) to activation frequency, bone resorption rate, bone formation rate, and the calculated rate of bone loss. For each unit (pmol/l) increase in serum PTH by ICMA, activation frequency increased by 1.3%/year more (P = 0.01), bone resorption rate increased by 3.9%/year more (P = 0.003), and the rate of cancellous bone loss was 2.8% greater (P = 0.0003) in the PMOp women compared with the PMNl women. Concentrations of serum estradiol, but not serum estrone, had a weak opposing effect to PTH, especially for bone formation rate. These data suggest that in PMOp the bone has increased sensitivity to the biologic effects of PTH. This may represent one of the fundamental pathophysiologic defects in PMOp and, in the setting of estrogen deficiency, may explain, in part, their greater rate of bone loss.

The isolation and purification of prostate-specific antigen (PSA) and the development of a radioimmunoassay for this antigen represent major advancements for the detection of adenocarcinoma of the prostate and the monitoring of response to therapy in patients with this disease. Both monoclonal and polyclonal assays for PSA are available. In attempts to correlate pathologic tumor stage and PSA levels, tumors of higher stage (pathologic stages C1, C2, D1, and D2) have been associated with elevated PSA levels. Increased PSA levels have also been found in patients with benign prostatic diseases (benign prostatic hypertrophy and prostatitis). PSA has been shown to be an excellent marker after radical prostatectomy and for monitoring of radiation therapy. Patients with a persistently elevated PSA level for more than 6 months postoperatively should be assessed for residual or recurrent local or systemic disease. Thus far, routine use of PSA testing as a mass screening modality for prostatic cancer has not been considered cost-effective.

In late 1986, we initiated a hospital reflectance meter glucose program with the Phlebotomy Service performing the determinations. In 1988, it became necessary to replace the original meters, and an evaluation of the Lifescan One Touch Meter was undertaken. Twelve One Touch meters were used during the course of the evaluation. The One Touch reagent strips incorporate the well-established glucose oxidase/peroxidase chemistry. Precision studies on a single meter (within-day and day-to-day) were performed using manufacturer’s controls and heparinized whole blood when appropriate. Precision was acceptable, with the coefficient of variation ranging from 1.1% to 4.7% (within-run) and 2.8% to 4.6% (day-to-day). Recovery studies using a single meter demonstrated acceptable recoveries (94% to 106%) within the range of 72 to 397 mg/dL (4.0 to 21.8 mmoll L) of glucose. Correlation studies of the One Touch meters (x) with the Beckman Astra-8 (y) gave correlation coefficients of 0.979 (n=267) and 0.966 (n=198) in two separate studies (six meters and twelve meters). Linear- regression equations were, respectively, y=0.947×+2.7 mg/dL (n=267) andy=0.88×+14.4 mg/dL (n=198). In both split-sample comparison studies (n=267) and blind-sample comparison studies (n=279) with the clinical laboratory, 7.5% and 7.2% (respectively) of data pairs greater than 100 mg/dL (5.5 mmol/L) showed differences greater than 15% between the One Touch meters and laboratory method. In the majority of the cases, the One Touch meter underestimated rather than overestimated the glucose value.

• To assess the contribution of fecal blood testing to cancer detection in a clinical practice setting, we studied records from 160 patients with both a new tissue diagnosis of colorectal adenocarcinoma and a preceding stool blood test (HemoQuant, Mayo Medical Laboratories, Rochester, Minn) determination. In this group, 71% had suggestive colorectal symptoms (particularly stool changes, overt bleeding, and abdominal pain) or anemia at presentation, and 29% were asymptomatic. Fecal blood levels remained normal in more than 40% of both symptomatic and asymptomatic patients. In only 26 patients (16% overall) was an abnormal fecal blood level the sole heralding feature, but this subset of patients had a more favorable stage. Fecal blood levels were higher with advanced, larger, and more proximal tumors and with stools collected before purgation. We conclude that, in the practice setting, fecal blood level elevation alone is an uncommon but important manner of colorectal cancer presentation, most cancers present with symptoms, and fecal blood levels are often normal in both symptomatic and asymptomatic patients. ( Arch Intern Med . 1990;150:1041-1045)

Eight surgical and one autopsy specimen of pituitary adenomas (six cases of Cushing's disease, two of Nelson's syndrome. and one of hypopituitarism) were studied by histochemical, immunohistocytological, and ultrastructural methods. Eight tumors showed the characteristic histochemical profile of corticotroph adenoma—amphophilic to basophilic, and periodic acid-Schiff-positive to some extent. In all tumors, immunohistochemical studies revealed adrenocorticotropic hormone (ACTH) and à-subunit in the cytoplasm of some adenoma cells. By electron microscopy, seven tumors were found to be monomorphous; six were typical corticotroph adenomas and one was a subtype II silent corticotroph adenoma. One unique lesion was bimorphous—i.e., composed of corticotrophs as well as cells resembling glycoprotein cells. Immunoelectron microscopy by the double-labeling immunogold technique, performed on one corticotroph adenoma, demonstrated the presence of ACTH and à-subunit not only within the same adenoma cells but also within the same secretory granules. The cytogenesis of ACTH à-subunit tumors, a rare form of plurihormonal adenoma. remains to be elucidated. The duration of disease associated with these tumors exceeded the duration in patients with ordinary corticotroph adenomas. Given the low frequency with which increases in serum à-subunit are detectable in patients with such tumors—13% in this series—hormone production is not recognized at preoperative evaluation.