Ultrasensitive Antibody Detection by Agglutination-PCR (ADAP).

Antibodies are widely used biomarkers for the diagnosis of many diseases. Assays based on solid-phase immobilization of antigens comprise the majority of clinical platforms for antibody detection, but can be undermined by antigen denaturation and epitope masking. These technological hurdles are especially troublesome in detecting antibodies that bind nonlinear or conformational epitopes, such as anti-insulin antibodies in type 1 diabetes patients and anti-thyroglobulin antibodies associated with thyroid cancers. Radioimmunoassay remains the gold standard for these challenging antibody biomarkers, but the limited multiplexability and reliance on hazardous radioactive reagents have prevented their use outside specialized testing facilities. Here we present an ultrasensitive solution-phase method for detecting antibodies, termed antibody detection by agglutination-PCR (ADAP). Antibodies bind to and agglutinate synthetic antigen-DNA conjugates, enabling ligation of the DNA strands and subsequent quantification by qPCR. ADAP detects zepto- to attomoles of antibodies in 2 µL of sample with a dynamic range spanning 5-6 orders of magnitude. Using ADAP, we detected anti-thyroglobulin autoantibodies from human patient plasma with a 1000-fold increased sensitivity over an FDA-approved radioimmunoassay. Finally, we demonstrate the multiplexability of ADAP by simultaneously detecting multiple antibodies in one experiment. ADAP's combination of simplicity, sensitivity, broad dynamic range, multiplexability, and use of standard PCR protocols creates new opportunities for the discovery and detection of antibody biomarkers.

Evaluation of risk-factor-based screening for thyroid peroxidase antibody positivity in pregnancy.

OBJECTIVE: To determine whether risk-factor-based screening for thyroid dysfunction in pregnancy performs well for detecting thyroid peroxidase antibodies (TPOAb), a marker for autoimmune thyroid disease. STUDY DESIGN: We prospectively evaluated pregnant women for thyroid dysfunction using The Endocrine Society's eleven screening questions. Serum was analysed for TPOAb. RESULT: We enrolled 546 women. TPOAb positivity was higher in women with a personal (odds ratio (OR) = 8·0; 95% confidence interval (CI) = 1·7-37·4; P = 0·02) or family history of thyroid disease (OR = 2·7; 95% CI = 1·3-5·7; P = 0·02). There was no association between the number of positive responses and TPOAb positivity (P = 0·41). Risk-factor-based screening missed 18 women (33%) with TPOAb. CONCLUSION: One-third of women with TPOAb were missed by the case-finding method. A personal or family history of thyroid disease was most strongly associated with TPOAb positivity.

Thyroglobulin (Tg) Testing Revisited: Tg Assays, TgAb Assays, and Correlation of Results With Clinical Outcomes.

CONTEXT: Measurement of thyroglobulin (Tg) by mass spectrometry (Tg-MS) is emerging as a tool for accurate Tg quantification in patients with anti-Tg autoantibodies (TgAbs). OBJECTIVE: The objective of the study was to perform analytical and clinical evaluations of two Tg-MS assays in comparison with immunometric Tg assays (Tg-IAs) and Tg RIAs (Tg-RIAs) in a cohort of thyroid cancer patients. METHODS: A total of 589 samples from 495 patients, 243 TgAb-/252 TgAb+, were tested by Beckman, Roche, Siemens-Immulite, and Thermo-Brahms Tg and TgAb assays, two Tg-RIAs, and two Tg-MS assays. RESULTS: The frequency of TgAb+ was 58%, 41%, 27%, and 39% for Roche, Beckman, Siemens-Immulite, and Thermo-Brahms, respectively. In TgAb- samples, clinical sensitivities and specificities of 100% and 74%-100%, respectively, were observed across all assays. In TgAb+ samples, all Tg-IAs demonstrated assay-dependent Tg underestimation, ranging from 41% to 86%. In TgAb+ samples, the use of a common cutoff (0.5 ng/mL) for the Tg-MS, three Tg-IAs, and the USC-RIA improved the sensitivity for the Tg-MSs and Tg-RIAs when compared with the Tg-IAs. In up to 20% of TgAb+ cases, Tg-IAs failed to detect Tg that was detectable by Tg-MS. In Tg-RIAs false-high biases were observed in TgAb+ samples containing low Tg concentrations. CONCLUSIONS: Tg-IAs remain the method of choice for Tg quantitation in TgAb- patients. In TgAb+ patients with undetectable Tg by immunometric assay, the Tg-MS will detect Tg in up to 20% additional cases. The Tg-RIA will detect Tg in approximately 35% cases, but a significant proportion of these will be clinical false-positive results. The undetectable Tg-MS seen in approximately 40% of TgAb+ cases in patients with disease need further evaluation.

In search of an unstimulated thyroglobulin baseline value in low-risk papillary thyroid carcinoma patients not receiving radioactive iodine ablation.

BACKGROUND: The clinical use of serum thyroglobulin (Tg) as a tumor marker in papillary thyroid cancer (PTC) patients following total thyroidectomy continues to evolve, due in part to the introduction of more sensitive (second generation) Tg immunometric assays (Tg(2G)IMA, functional sensitivity ≤ 0.10 ng/mL), and the implementation of new recommendations against radioactive iodine ablation (RAIA) for patients at the lowest risk of recurrence. As a result, there is a need to establish the optimal timing and interpretation of serum Tg values while on levothyroxine-induced suppression of thyrotropin (TSH) in thyroidectomized PTC patients with a thyroid remnant. This study examines the pattern of decline and eventual baseline value of unstimulated Tg (uTg) concentrations following total thyroidectomy in patients with low-risk PTC who did not receive RAIA. METHODS: The medical records of consecutive patients with thyroid cancer seen at the Los Angeles County + USC Medical Center were retrospectively reviewed. Serial uTg and TSH values from Tg-antibody negative low-risk PTC patients treated with total thyroidectomy and no RAIA were analyzed. Patients were stratified by degree of TSH suppression to assess the effect on uTg. Serial postoperative uTg values were evaluated for the temporal pattern of decline and ultimate baseline. Patients with medullary thyroid cancer (MTC) were studied as a surgical reference group. RESULTS: Records from 577 consecutive thyroid cancer patients were reviewed, of which 36 met all criteria for inclusion. By 6 months, uTg fell to <0.5 ng/mL in 61% of patients and all patients demonstrated uTg < 0.5 ng/mL 2 years after surgery. During a median follow up of 5.7 years, uTg values remained below this level. The median uTg values in patients with papillary microcarcinoma, PTC, and MTC were similar at 0.11, 0.12, and 0.09 ng/mL, respectively. Further decline in uTg was not observed once the TSH was <0.5 mIU/L. CONCLUSIONS: The uTg values during TSH suppression in Tg antibody-negative, low-risk PTC patients who did not receive RAIA were below 0.5 ng/mL by 6 months postoperatively in most cases and remained stable over the duration of patient follow-up.

Clinical review: Clinical utility of thyroglobulin antibody (TgAb) measurements for patients with differentiated thyroid cancers (DTC).

CONTEXT: Thyroglobulin autoantibodies (TgAb) are primarily measured in serum in conjunction with thyroglobulin (Tg)--the primary tumor marker used to monitor patients with differentiated thyroid cancers (DTC). Every specimen needs TgAb testing to authenticate that the Tg measurement is not compromised by TgAb interference. When present, TgAb concentrations per se can be monitored as a surrogate tumor marker. OBJECTIVES: The aims of the study were: 1) to review published reports concerning whether there are associations between DTC, thyroid autoimmunity (Hashimoto's thyroiditis), and the presence of TgAb; and 2) to evaluate the methodological factors that influence TgAb interference with serum Tg testing. DATA SOURCES: PubMed was used to identify studies published over the last 55 yr that focused on DTC relationships with thyroid autoimmunity and the presence of thyroid autoantibodies. RESULTS: Many studies have reported significant associations between papillary thyroid cancer and Hashimoto's thyroiditis that may have a favorable prognostic significance. TgAb is detected in approximately 20% of DTC patients and may be a more specific thyroid tumor marker than thyroid peroxidase antibodies. TgAb interferes with Tg immunometric assay (IMA) measurements, causing falsely low/undetectable Tg values, especially when TgAb concentrations are high and serum Tg concentrations (measured by RIA) are low. TgAb concentrations respond to changes in Tg-secreting thyroid tissue such that the TgAb trend can be used as a more reliable surrogate DTC tumor marker than Tg IMA. Current TgAb assays may not always detect interfering TgAb because of insensitivity and specificity differences. It is critical to retain the same method for long-term TgAb monitoring. CONCLUSIONS: Patients with Hashimoto's thyroiditis frequently have TgAb detected and may have a higher risk for papillary thyroid cancer. Although TgAb interferes with Tg IMA measurements, TgAb trends can be used as a surrogate DTC tumor marker in preference to Tg IMA, provided that the same method is used.

Mildly elevated TSH and cognition in middle-aged and older adults.

BACKGROUND: It is accepted that markedly elevated thyroid-stimulating hormone (TSH) levels are associated with impaired cognitive function. However, the findings regarding the association between mildly elevated TSH levels and cognition are equivocal. The objective of this study was to assess the relation between TSH levels in the normal to mildly elevated range (0.3-10.0 mIU/L) and several domains of cognitive function. METHODS: A healthy, community-based sample of 489 men and women (40-88 years old, mean = 60.5 years) enrolled in the B-Vitamin Atherosclerosis Intervention Trial were studied. A neuropsychological test battery was used to assess a broad array of cognitive functions. Four uncorrelated neuropsychological factors were extracted by principal component analysis. Using multivariable linear regression, performance on each factor was examined in relation to TSH levels, controlling for age, gender, race-ethnicity, education, homocysteine levels, low-density lipoprotein cholesterol levels, and smoking status. RESULTS: TSH levels were not associated with any of the four factor scores in the total sample or in younger (age < 60) or older (age >or= 60) subjects, although there was a trend for older subjects with higher levels of TSH to do more poorly on paragraph recall (p = 0.06). Gender-stratified analyses showed that TSH was positively associated with scores on word list learning for females only (p = 0.003). CONCLUSIONS: In this community-based sample of middle-aged to older individuals, increasing TSH levels were not associated with significantly reduced cognitive performance in any domain. Further exploration of the effects of gender on the association between TSH and cognition is warranted.

Measuring thyroglobulin and thyroglobulin autoantibody in patients with differentiated thyroid cancer.

Measurement of serum thyroglobulin is primarily used as a tumor marker in the postoperative management of patients with differentiated thyroid cancer. Unfortunately, the technical quality of current thyroglobulin assay methods varies and influences the clinical utility of this test. Two different methodologic approaches are used to measure serum thyroglobulin: the original competitive radioimmunoassay methodology and noncompetitive immunometric assay methods. Although the newer immunometric assays offer the technical benefits of eliminating the use of isotopes, using smaller specimen volumes, and having higher sensitivity potential, shorter turnaround times and the convenience of automation, immunometric assays also have a higher propensity for interference from both thyroglobulin autoantibodies and heterophilic antibodies, if present in the specimen. It is critical that physicians understand the technical limitations inherent in thyroglobulin measurement in order to effectively use this test for the postoperative management of patients with differentiated thyroid cancers.

Is screening for abdominal aortic aneurysm bad for your health and well-being?

BACKGROUND: The purpose of the present paper was to investigate whether screening for abdominal aortic aneurysm (AAA) causes health-related quality of life to change in men or their partners. METHODS: A cross-sectional case-control comparison was undertaken of men aged 65-83 years living in Perth, Western Australia, using questionnaires incorporating three validated instruments (Medical Outcomes Study Short Form-36, EuroQol EQ-5D and Hospital Anxiety and Depression Scale) as well as several independent questions about quality of life. The 2009 men who attended for ultrasound scans of the abdominal aorta completed a short prescreening questionnaire about their perception of their general health. Four hundred and ninety-eight men (157 with an AAA and 341 with a normal aorta) were sent two questionnaires for completion 12 months after screening, one for themselves and one for their partner, each being about the quality of life of the respondent. RESULTS: Men with an AAA were more limited in performing physical activities than those with a normal aorta (t-test of means P = 0.04). After screening, men with an AAA were significantly less likely to have current pain or discomfort than those with a normal aorta (multivariate odds ratio: 0.5; 95% confidence interval (CI): 0.3-0.9) and reported fewer visits to their doctor. The mean level of self-perceived general health increased for all men from before to after screening (from 63.4 to 65.4). CONCLUSIONS: Apart from physical functioning, screening was not associated with decreases in health and well-being. A high proportion of men rated their health over the year after screening as being either the same or improved, regardless of whether or not they were found to have an AAA.

Population based randomised controlled trial on impact of screening on mortality from abdominal aortic aneurysm.

OBJECTIVE: To assess whether screening for abdominal aortic aneurysms in men reduces mortality. DESIGN: Population based randomised controlled trial of ultrasound screening, with intention to treat analysis of age standardised mortality. SETTING: Community based screening programme in Western Australia. PARTICIPANTS: 41,000 men aged 65-83 years randomised to intervention and control groups. INTERVENTION: Invitation to ultrasound screening. MAIN OUTCOME MEASURE: Deaths from abdominal aortic aneurysm in the five years after the start of screening. RESULTS: The corrected response to invitation to screening was 70%. The crude prevalence was 7.2% for aortic diameter > or = 30 mm and 0.5% for diameter > or = 55 mm. Twice as many men in the intervention group than in the control group underwent elective surgery for abdominal aortic aneurysm (107 v 54, P = 0.002, chi2 test). Between scheduled screening and the end of follow up 18 men in the intervention group and 25 in the control group died from abdominal aortic aneurysm, yielding a mortality ratio of 0.61 (95% confidence interval 0.33 to 1.11). Any benefit was almost entirely in men aged between 65 and 75 years, where the ratio was reduced to 0.19 (0.04 to 0.89). CONCLUSIONS: At a whole population level screening for abdominal aortic aneurysms was not effective in men aged 65-83 years and did not reduce overall death rates. The success of screening depends on choice of target age group and the exclusion of ineligible men. It is also important to assess the current rate of elective surgery for abdominal aortic aneurysm as in some communities this may already approach a level that reduces the potential benefit of population based screening.

C-reactive protein levels and the expansion of screen-detected abdominal aortic aneurysms in men.

BACKGROUND: C-reactive protein (CRP) levels have been shown to predict a number of cardiovascular outcomes. CRP levels have also been found to be elevated in patients with abdominal aortic aneurysms (AAAs). The aim of this study was to assess the relation between CRP levels and rates of expansion of small AAAs. METHODS AND RESULTS: A cohort of men with small aneurysms was identified in a trial of screening with ultrasound scanning. After initial screening, men were rescanned at 6- to 12-month intervals. CRP levels were measured at the first follow-up visit. Rates of expansion and risk factors for expansion were assessed with the use of data from 545 men who attended for at least 1 scan after CRP levels were measured. These men were followed for a median of 48 (range, 5 to 69) months. The mean annual rate of expansion was 1.6 mm. The median CRP level was 2.6 mg/L in men with the smaller AAAs (30 to 39 mm, n=433) compared with 3.5 mg/L in men with larger AAAs (40 to 54 mm, n=112) (P=0.007). The multivariate age-adjusted logistic model confirmed initial aortic diameter to be the only factor associated with rapid expansion with an odds ratio of 7.2 (95% CI, 4.3,12.2) for an initial diameter of 40 to 54 mm relative to one of 30 to 39 mm. CONCLUSIONS: Most small aneurysms expand slowly. CRP levels are elevated in larger aneurysms but do not appear to be associated with rapid expansion. The most useful predictor of aneurysmal expansion in men is aortic diameter.