Evaluation of hemoglobin interference in capillary heel-stick samples collected for determination of neonatal bilirubin

OBJECTIVE: In this study, we determined the assay performance criteria necessary to produce acceptable results for >or=98% of neonate bilirubin samples collected by capillary heel-stick. STUDY DESIGN AND METHODS: We determined serum free hemoglobin levels in 151 heel-stick serum samples to determine the hemolysis level. We then tested the effect of hemolysis on total bilirubin levels determined by four commercially available assays. RESULTS: The mean level of serum free hemoglobin was 1.62 g/L. Of the serum total bilirubin assays tested, the Total Bilirubin Special (Roche Diagnostics) and the TBILI (Roche Diagnostics) reagents did not show significant interference at the concentrations of free hemoglobin observed in >or=98% of heel-stick samples. The Vitros Bu/Bc slide (Ortho-Clinical Diagnostics) showed significant interference only at normal bilirubin concentrations; while the Bilirubin DPD reagent (Amresco Inc.) showed significant interference starting at hemoglobin concentrations of 1.0 g/L. CONCLUSIONS: Bilirubin assays that are not sensitive to approximately 6 g/L free hemoglobin should provide accurate results for most samples obtained via capillary heel-stick. Of the four assays tested, the Bilirubin DPD reagent (Amresco Inc.) was the most susceptible to the presence of free hemoglobin and will result in a higher rejection rate of neonate capillary heel-stick samples.     

Salicylate-induced increases in free triiodothyronine in human serum: Evidence of inhibition of triiodothyronine binding to thyroxine-binding globulin and thyroxine-binding prealbumin

Addition of sodium salicylate to human serum at concentrations often obtained during aspirin therapy causes 100-200% increases in free triiodothyronine (T(3)) and free thyroxine (T(4)) as estimated by ultrafiltration. The increase in free T(3) was unexpected since previous data had suggested that salicylate inhibits binding of T(4) only to thyroxine-binding prealbumin (TBPA) and that T(3) is not bound to this protein. Using ultrafiltration techniques, we demonstrated binding of T(3) to TBPA. The affinity constant for T(3)-TBPA binding appears to be slightly greater than that for albumin-T(3) binding. While salicylate inhibits the binding of T(3) (and T(4)) to TBPA, it can be predicted that little change will be observed in the free T(3) (or free T(4)) without inhibition of thyroid hormone binding to thyroxine-binding globulin (TBG). Using a competitive-binding protein displacement technique, it has been shown that sodium salicylate, like diphenylhydantoin (DPH), inhibits the binding of T(3) and T(4) to TBG. The magnitude of the increase in free T(3) and free T(4) induced by salicylates suggests that interference with TBG binding is its major effect.Aspirin was administered orally to two normal subjects in quantities sufficient to obtain serum salicylate levels of 20-25 mg/100 ml. This resulted in a decrease of 20-30% in total serum T(3) and T(4) levels. This decrease in T(4) levels is similar in magnitude to that previously observed in subjects receiving DPH. Unlike what has been observed with DPH treatment, therapeutic salicylate levels are associated with increases of 50-75% in the unbound fraction of both T(3) and T(4) which persist throughout an 8-10 day treatment period.     

Comparative multicentre study of a panel of thyroid tests using different automated immunoassay platforms and specimens at high risk of antibody interference.

The introduction of automation for immunoassays in recent years has brought about important and evident improvements in assay precision. Increasing standardization and comparability between platforms should enable the development of clinical guidelines and diagnostic algorithms for appropriate clinical decision making. A continuing source of variation between different automated immunoassay platforms is the sporadic effect of interfering antibodies or substances, thus causing aberrant results not supporting the patient's clinical status. The aim of this study was to describe current thyroid panel variation between automated immunoassay platforms including population specimens at risk of antibody interference. A multisite design with laboratories in three different countries using four different automated immunoassay platforms (Roche-Boehringer Mannheim Elecsys (Italy), Roche-Boehringer Mannheim ES300 (Wales), Bayer Immuno 1 and the Bayer ACS:180 evaluated the thyroid panel of thyrotropin (TSH), triiodothyromine (T3), free thyroxine (FT4) and free triiodothyronine (FT3). A common set of 158 randomly selected patient samples of non-thyroid and thyroid disorders, with and without treatment, was tested. Included were 62 patient samples at risk for endogenous antibody interference with high antimicrosomal antibody, anti-TSH receptor antibody and increased rheumatoid factor sub-populations. Across all controls and between platforms, precision measurements were comparable and varied between 0.7% and 12.8% for TSH, 2.8% and 13% for FT4, 1.8% and 10.5% for FT3 and 3.1% and 16% for T3 assay. Acceptable correlation and reproducibility were found between the three Bayer Immuno 1 platforms at each country's site with all four thyroid panel assays demonstrating r-values of 0.989 to 1.000 and slopes of 0.915 to 1.078. Comparisons between the different platforms showed acceptable correlation for all thyroid panel assays. Specimens containing rheumatoid factor were associated with a significantly increased variation between systems for the FT4 and FT3 assays (p < 0.01). This effect did not appear to be selective for a given platform. For specimens with raised autoimmune antibodies and therefore at risk of assay antibody interference, no variation could be observed between the platforms.     

Elevation of free thyroxine measurements in patients without thyrotoxicosis

The recent development of single step analogue assay techniques to measure free thyroxine in serum has meant that this estimation has become widely used as a first line test of thyroid function. However these assays are subject to in-vitro interference in two ways: where there is a variant serum albumin in familial dysalbuminaemic hyperthyroxinaemia, or where there are circulating thyroid hormone binding antibodies. In both these situations a spuriously high result for free thyroxine is obtained. This may have serious implications for patient management and we describe 10 patients in whom the finding of a misleadingly high free thyroxine concentration led to confusion in diagnosis and, in eight of the 10, to inappropriate antithyroid treatment. Clinicians should be aware of these technical problems and where a result for the free thyroxine concentration seems inappropriate to the patient's clinical state then measurement of thyrotrophin (TSH) by a sensitive immunometric method should be performed. If the results remain confusing the presence of a variant serum albumin or thyroid hormone binding antibodies should be sought.     

Interference by anti-immunoglobulin G antibodies in immunoradiometric assays of thyrotropin involving mouse monoclonal antibodies

"Sandwich"-type assays are subject to positive interference by the patient's "heterophile" antibodies. If present, these bind to the animal immunoglobulins in the assay reagents, forming artefactual sandwiches indistinguishable from those formed with the analyte itself. Immunoglobulins from non-immunized animals, added to the assay reagents, can diminish this effect by blocking the patient's antibodies. Elsewhere, we studied several patients with anti-mouse immunoglobulin activity, whose serum gave spuriously high results for thyrotropin (TSH) concentrations. Here we have studied this phenomenon by adding, to pooled zero-TSH serum, antibodies to mouse, goat, and horse immunoglobulins and then assaying TSH by several other sandwich-type assays involving mouse monoclonal antibodies. Assays not supplemented with blocking immunoglobulins from mice or other animals were more susceptible to this effect. When large amounts of antibody were added, the antibody excess diminished the interference. However, the presence of blocking immunoglobulins could reverse such antibody excess, actually enhancing, instead of diminishing, the positive interference. Users should be aware that blocking immunoglobulins may diminish but not necessarily eliminate this problem with such assays.     

Heterophilic antibody interference affecting multiple hormone assays: Is it due to rheumatoid factor?

Assay interference with heterophilic antibodies has been well described in literature. Rheumatoid factor is known to cause similar interference leading to falsely elevated hormone levels when measured by immunometric methods like enzyme-linked immunosorbent assay (ELISA) or multiplex immunoasays (MIA). We report a case of a 60-year-old male patient with a history of rheumatoid arthritis referred to our endocrine clinic for investigation of hypogonadism and was found to have high serum levels of LH, FSH, SHBG, Prolactin, HCG and TSH. We suspected assay interference and further tests were performed. We used Heteroblock tubes and PEG precipitation to eliminate the interference and the hormone levels post treatment were in the normal range. We believe the interference was caused by high serum levels of rheumatoid factor. Although he was treated with thyroxine for 3 years, we believe he may have been treated inappropriately as his Free T4 level was always normal despite high TSH due to assay interference. Our case illustrates the phenomenon of heterophilic antibody interference likely due to high levels of rheumatoid factor. It is essential for clinicians and endocrinologists in particular to be aware of this possibility when making treatment decisions in these groups of patients.     

Elevation of Free Thyroxine Measurements in Patients without Thyrotoxicosis

The recent development of single step analogue assay techniquesto measure free thyroxine in serum has meant that this estimationhas become widely used as a first line test of thyroid function.However these assays are subject to in-vitro interference intwo ways: where there is a variant serum albumin in familialdysalbuminaemic hyperthyroxinaemia, or where there is a Varientserum albumin in familial dysalbuminaemic hyperthyroxinaemia,or where there are circulating thyroid hormone binding antibodies.In both these situations a spuriously high result for free throxineis obtained. This may have serious implications for patientmanagement and we describe 10 patients in whom the finding ofa misleadingly high free thyroxine concentration led to confusionin diagnosis and, in eight of the 10, to inappropriate antithyroidtreatment. Clinicians should be aware of these technical problemsand where a result for the free thyroxine concentration seemsinappropriate to the patient's clinical state then measurementof thyro-trophin (TSH) by a sensitive immunometric method shouldbe performed. If the results remain confusing the presence ofa variant serum albumin or thyroid hormone binding antibodiesshould be sought.     

Interference of dextran in biuret-type assays of serum proteins.

Dextran interference in biuret-type assays of total serum proteins was investigated in a Belgian National External Quality Assurance Survey with 256 participants. In vitro supplementation of therapeutic (10% Gentran 70) dextran concentrations showed a broadly varying (from 0 to 20%) negative interference. The analytical interference was found to depend on both the sodium hydroxide and tartrate concentrations in the reagent formulation. The dry chemistry biuret method was not affected by the dextran interference. In a number of cases, the effects observed may be of clinical importance. Both clinicians and laboratory staff should be aware of the persistence of this analytical problem.     

Interference in thyroid-function tests in postpartum thyroiditis

Three women are described from a study of patients with postpartum thyroiditis whose sera gave spuriously increased concentrations of free thyroid hormone because of antibody binding of radiolabeled thyroxin (T4) and triiodothyronine (T3) analogs. All of the women showed increased serum concentrations of thyroid autoantibodies. The antibody binding of radiolabeled analogs and its effect on free T4 and free T3 assays disappeared by 48 weeks postpartum. Postpartum women who develop thyroid autoantibodies have approximately 2% prevalence of increased binding of radiolabeled analogs, which can result in an interference in thyroid hormone assays involving T4 and T3 analogs.     

Is free thyroxine accurately measurable at room temperature?

Two recently developed two-step, or "back-titration" assay kits for free thyroxine (FT4)--one based on a europium-labeled derivative of T4, the other on conventional radiolabeled T4--were compared with both symmetrical dialysis and an indirect FT4 radioimmunoassay. Discrepancies between the two-step and the other two methods were observed, particularly in samples that had very low to zero thyroxine-binding globulin (TBG) content. In those instances the two-step methods gave values almost twice as high as the other methods. This effect could be largely reversed in one of the two-step assays and completely reversed in the other by performing the first incubation step at 37 degrees C rather than at room temperature, as prescribed by the manufacturers. When symmetrical dialysis is performed at both temperatures, FT4 at room temperature is about 40% of the amount determined at 37 degrees C, except in zero-TBG samples, where it averages almost 80% of the value at 37 degrees C. Moreover, we demonstrated that the affinity of TBG for T4 is much more temperature-dependent than the affinity of transthyretin and albumin for T4, so that the net temperature effect on the FT4 in a sample depends on the relative contribution of TBG to total binding. We conclude that performing FT4 assays at room temperature is principally incorrect and leads to falsely increased values when samples with very low TBG concentrations are analyzed.     

Interference of anti-streptavidin antibodies: More common than we thought? In relation to six confirmed cases

Automated immunoassays are extensively used in routine laboratory diagnostics of endocrine disorders because of their advantages, such as high sensitivity, precision, and specificity. However, these methods are limited by the susceptibility of the immunochemical reaction to various interferences. They may present interferences related to the assay’s design, for example, the endogenous presence of anti-streptavidin antibodies (ASA) in platforms that use the biotin-streptavidin interaction. To date, there have been few reports in the literature of interference from endogenous ASA. However, such antibodies would potentially lead to falsely decreased or increased results of hormones that can lead to incorrect diagnoses.We report six patients with unusual thyroid function tests, incongruent to their clinical findings. They present elevated concentrations of total T3 and T4 and TSH values within the reference range when measured at Cobas 8000® e801 module (Roche Diagnostics®). Neither patient had been taking biotin; however, all demonstrated the presence of ASA causing falsely high results on competitive assays and also falsely low results on sandwich assays. The hormone panel was also analyzed in the same samples using a different platform available in our laboratory: Cobas 6000® e601 module (Roche Diagnostics®). Nine samples were sent to an external laboratory to be measured with the chemiluminescent method: ADVIA Centaur® (Siemens® Healthcare Diagnostics). The interference seems to affect e801 module and competitive assays the most without affecting results obtained by this chemiluminescent method. This interference could potentially affect other assays performed on the same platform, such as ATPO and estradiol.Finally, laboratories should suspect the presence of interference when there is no correlation between the hormone profile and the patient’s clinic. The biotin neutralization protocol demonstrated its effectiveness to eliminate ASA interference.     

The determination of thyroxine and thyroxine uptake with new homogeneous enzyme immunoassays using Boehringer Mannheim/Hitachi analysis systems.

New homogeneous enzyme immunoassays for the determination of thyroxine and thyroxine uptake have been developed. The CEDIA assays are based on the cloned enzyme donor immunoassay technology, which involves fragments of beta-galactosidase prepared by genetic engineering. The assays have been adapted for Boehringer Mannheim/Hitachi analysers. The CEDIA T4/T Uptake assays were evaluated in eleven clinical chemistry laboratories on various Boehringer Mannheim/Hitachi analysis systems, using a 2-point calibration. The analytical range of the T4 test was 10 to 258 nmol/l thyroxine. The T uptake test had a measuring range between 20-50%. Depending on the concentration of the analyte (samples from hypo-, eu- or hyperthyroid patients), mean coefficients of variation ranged from 1.8 to 4.8% within-run and from 4.1 to 6.5% between-run for the T4 assay. Even better coefficients of variation were obtained for the T uptake assay (1.4 to 2.3% within-run, 2.8 to 3.3% between run). The relative inaccuracy of the CEDIA assays with respect to values assigned by other tests was satisfactory in various control sera. The T4 assay was compared with one radioimmunoassay, one enzyme immunoassay and one fluorescence polarisation immunoassay. Slopes ranging from 0.9 to 1.1 and intercepts ranging from -10 to +10 nmol/l thyroxine were obtained with two exceptions. The results of the T uptake test correlated reasonably with those of other thyroxine-binding methods. No interference was observed with icteric and lipaemic sera. Haemoglobin up to 4 g/l had no significant influence. Results of the CEDIA T Uptake test are mainly used for calculation of the free thyroxine index, in which the thyroxine value is corrected for variations of thyroxine-binding protein concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemically blocked analog assays for free thyronines. II. Use of equilibrium dialysis to optimize the displacement by chemical blockers of T4 analog and T3 analog from albumin while avoiding displacement of T4 and T3 from thyroxin-binding globulin

Chemical blockers used to displace thyronine analog from albumin in analog kits for assay of free thyroxin (FT4) or free triiodothyronine (FT3) may also displace thyroxin (T4) or triiodothyronine (T3) from thyroxin-binding globulin (TBG), resulting in an apparent TBG dependence of results of free hormone estimates. We used equilibrium dialysis and antibody binding to assess the displacement of thyronine analogs and thyronines from albumin and TBG by use of chemical blockers. We chose a combination of two chemical blockers, which eliminated thyronine analog-albumin binding but minimized thyronine displacement from TBG for use in FT4 and FT3 assays. These blocked-analog free-hormone assays yielded accurate clinical results in euthyroid patients, hypo- and hyperthyroid patients, and in pregnant women. FT4 results were not entirely normalized in all nonthyroidally ill patients, indicating that decreased analog-albumin binding is not the only factor resulting in low FT4 results. In current Diagnostic Products Corp. (DPC) FT4 and FT3 blocked-analog kits, the blocker concentrations are the same as we used in these assays.     

Estimation of thyroxin, triiodothyronine, thyrotropin, free thyroxin, and triiodothyronine uptake by use of magnetic-particle solid phases

We evaluated solid-phase radioassays involving small, uniform magnetic particles (MAGIC, Corning Medical), obtaining data on total serum thyroxin (T4), triiodothyronine (T3), thyrotropin (TSH), free thyroxin (FT4), and T3 uptake for a total of 301 serum samples from euthyroid patients; patients receiving replacement thyroxin; patients receiving estrogen, or who were pregnant; hyperthyroid, hypothyroid, and nonthyroidally ill patients; and patients receiving salicylates, phenytoin, or heparin. We found each procedure to be technically simple and precise (between-assay CVs for T4 2.8-8.2%, T3 9-7.4%, TSH 2.9-4.5%, FT4 3.6-11%, T3 uptake 2.2-3.7%). The magnetic separations are rapid (2 to 5 min), reproducible, and complete. Each MAGIC assay produced clinically appropriate results. These assay systems offer the convenience associated with noncentrifugation assays and excellent analytical performance.     

A direct free thyroxine (T4) immunoassay with the characteristics of a total T4 immunoassay

BACKGROUND: Direct free thyroxine (T(4)) measurements have been linked to both T(4)-binding serum protein concentrations and protein-bound T(4) concentrations. Whether this is evidence of a relationship to total T(4) concentrations has not been reported. METHODS: We compared an analog-based direct free T(4) immunoassay and a total T(4) immunoassay. Each assay was applied to the fractions of serum T(4) obtained by ultrafiltration and equilibrium dialysis. Both were applied to serum-based solutions in which free T(4), T(4)-binding proteins, protein-bound T(4), and total T(4) were systematically varied, held constant, or excluded. RESULTS: Neither the free T(4) assay nor the total T(4) assay detected dialyzable or ultrafilterable serum T(4). Both assays detected and reported the T(4) retained with serum proteins. Both free and total T(4) results were related to the same total T(4) concentrations in the presence and absence of T(4)-binding proteins. Both results were similarly related to total T(4) concentrations when free T(4) was held constant while total T(4) was varied. Both were similarly related to a total T(4) concentration that was held constant while free T(4) progressively replaced protein-bound T(4). These free T(4) results, like total T(4) results, were unresponsive to a 500-fold variation in dialyzable T(4) concentrations. CONCLUSION: New experiments extend the characterization of a longstanding and incompletely characterized analog-based free T(4) immunoassay. These free T(4) measurements relate to total T(4) concentrations in the same way that total T(4) measurements do.     

Transient hyperthyroidism after total parathyroidectomy for tertiary hyperparathyroidism: A report of two cases

Surgical removal of enlarged parathyroid glands is the treatment of choice in most cases of tertiary renal hyperparathyroidism. Complications of this surgical procedure are rare. We report two cases of patients who developed acute hyperthyroidism after total parathyroidectomy with parathyroid autotransplantation for refractory tertiary hyperparathyroidism. The patients had no history or biochemical or radiologic evidence of thyroid disease. They were not taking drugs affecting thyroid function. Thyroid function (thyroid stimulating hormone, free T(3) and free T(4)) was measured preoperatively, immediately after surgery and again three months later. Total parathyroidectomy was successful in both patients. Circulating levels of parathyroid hormone were at the lower limit of normal values. Postoperative thyroid function tests demonstrated acute hyperthyroidism with a rapid increase in free T(3) and T(4) levels above normal and a drop in thyroid stimulating hormone below normal in both patients. The course of hyperthyroidism was short (normalization of fT(3) and fT(4) values within 14-21 days). Neither patient had symptoms of thyrotoxicosis. Transient hyperthyroidism may be an under-recognized complication of total parathyroidectomy for tertiary hyperparathyroidism. These patients should be monitored with thyroid function tests and assessed for clinical signs attributable to thyrotoxicosis.     

Performance of direct equilibrium dialysis and analogue-type free thyroid hormone assays, and an immunoradiometric TSH method in patients with thyroid dysfunction

Direct equilibrium dialysis and analogue-type radio-immunoassays for free triiodothyronine (FT3) and free thyroxine (FT4) in serum were compared in 168 subjects with various states of thyroid function. A good diagnostic efficacy for FT3 and FT4 by either type of assay was observed in hyperthyroidism. In hypothyroidism the free thyroid hormone assays, particularly the FT3 assays, performed diagnostically less well, partly because patients with mild disease were included in the study. No significant differences in the percentages of misclassifications of thyroid dysfunction patients by corresponding dialysis and analogue assays were found. We observed a good linear correlation between dialysis and analogue methods for FT3 (r = 0.98) and FT4 (r = 0.97) in this study comprising out-patients not suffering from severe non-thyroidal disease, known from earlier studies in this and other laboratories to interfere in these assays. It is concluded that analogue assays may be used on out-patients in whom severe systemic diseases are less frequent than in hospitalized patients. There are, however, other limitations to the use of analogue assays than systemic diseases. We observed two euthyroid patients with thyroxine auto-antibodies causing very high FT4 concentrations as determined by analogue assay; their dialysable FT4 concentrations were normal. We also tested a recently developed immunoradiometric serum TSH assay, which was found to perform well in primary hypo- and hyperthyroidism. Serum TSH was elevated in one patient hyperthyroid because of a TSH-producing pituitary adenoma, and within the reference limits in a patient with secondary hypothyroidism.     

Definition of T cell idiotypes using anti-idiotypic antisera produced by immunization with T cell clones

Alloreactive T cell clones with distinct specificities were used to raise anti-idiotypic antisera via an F1 anti-(parent anti-F1) protocol. Antisera were raised that could stimulate the proliferation of the appropriate T cell clone, but not other clones. The active fraction of the antisera for T cell proliferation was immunoglobulin. In addition to proliferation, an anti-idiotypic antiserum could induce the appropriate T cell clone to secrete substantial amounts of interleukin 2 (IL-2). Production of IL-2 appeared independent of the involvement of accessory cells. These accessory cells may be unnecessary for IL-2 production in our assay, or their effect may be produced by anti-idiotype. Thus, anti-idiotype may provide two or more specific T cell signals.     

Infusion of plasma expanders may lead to unexpected results in urinary protein assays.

Overt proteinuria was detected in the urine of a potential kidney donor, ultimately leading to the refusal of the kidneys for transplantation purposes. Histological examination of the kidneys did not reveal any abnormalities. Searching for substances that could have interfered with the urinary total protein assay, the role of infused, modified gelatin plasma expanders was investigated. We therefore measured the concentration of protein before and after the addition of various artificial plasma expanders to urine. Only when Biuret reagent or Pyrogallol Red dye were used did we find elevated concentrations of protein. Other methods, including the turbidimetric assays, did not detect additional amounts of protein in the spiked urine. We conclude that the infusion of modified gelatin solutions may cause apparent proteinuria. This effect is not observed with starch-based plasma expanders. Clinical chemists and clinicians should be aware of this phenomenon and possibly repeat the analysis with a different technique.     

Evaluation of four commercially available assays for free thyroxin

We assessed two two-step and two analog assays for measuring free thyroxin (FT4) in serum: Clinical Assays' "GammaCoat Free/Total T4" (CA), Vitek's "KinetiCount Phase II Free T4" (VTK), Diagnostic Products Corporation's "Coat-a-Count Free T4" (DPC) kit (June 1987 version), and Amersham's "Amerlex-M Free T4" (AMX). The VTK assay is automated except for the initial pipetting step. Interassay results correlated well except for samples with abnormal serum albumin concentrations. FT4 values for hypoalbuminemic samples showed a highly significant (P less than 0.0001) correlation with serum albumin concentration in the DPC and AMX assays. The relationships are described by the equations y = 0.382albumin (g/L) + 0.81 pmol/L and y = 0.450albumin (g/L) - 3.20 pmol/L, respectively. When we used an equation derived from the Law of Mass Action to adjust FT4 values to values expected at an ideal albumin concentration, the observed correlation of albumin and FT4 was abolished completely in the DPC assay, and partly so in the AMX assay. The precision of CA was comparable with that of the analog assays; the CV for the VTK assay was approximately twice that for the other three assays.