[What to do in the event of a suspicion of analytical interference during an immunoassay?] / Conduite à tenir devant une suspicion d'interférence analytique lors d'un immunodosage.

Identifying analytical interference is a challenge for the medical biologist in providing advice to the prescriber. Indeed, these analytical interferences often have deleterious consequences on the care of patients. Understanding their mechanisms and mastering corrective procedures is essential to limit these management errors. Faced with the many questions from clinicians in current practice, we propose an algorithm for managing a sample when interference is suspected.

Heterophile antibody interference affecting multiple Roche immunoassays: A case study.

BACKGROUND: Analysis of many clinically important analytes is dependent on antibody-based assays. However, depending on the design, these assays are vulnerable to interference from endogenous molecules including circulating antibodies and free biotin. In this case report, we describe a patient whose laboratory findings from immunoassay based methodologies, are inconsistent with the clinical presentation. CASE PRESENTATION: A 14-year-old male was referred to Pediatric Endocrinology for suspected hyperthyroidism based on critically elevated free thyroxine (fT4) levels although clinical assessment was inconsistent with hyperthyroidism. Because repeat testing was discrepant, Endocrinology questioned the validity of the results prompting consultation with the laboratory to investigate the source of the inconsistent findings. Review of discordant results revealed that fT4 levels measured in laboratories utilizing Roche instrumentation were critically high, while results from laboratories using alternative platforms (i.e. Siemens Centaur) were within normal limits. CONCLUSION: After a comprehensive evaluation which included testing of paired specimens on multiple platforms, measurement of serially diluted specimens and a formal evaluation for the presence of heterophile antibodies, it was determined that a heterophile antibody interference was the most likely cause of the aberrant results in this patient.

Human chorionic gonadotropin suspected heterophile interference investigations in immunoassays: a recommended approach.

Background Heterophile antibody (HAb) interferences in immunoassays can cause falsely elevated hCG concentrations leading to incorrect diagnosis and treatments options. When results are not consistent with the clinical findings, hCG HAb interference investigation may be requested by the physician. A retrospective evaluation of the frequency of HAb interference was performed among cases of physician-requested investigations and the effectiveness of commercially available blocking reagents to detect HAb interference in two immunoassay systems was evaluated. Methods One hundred and thirteen physician requests for hCG HAb investigation from 2008 to 2017 were reviewed. The primary method used to measure hCG was the Beckman Coulter Access Total ßhCG (2008-2010) and the Roche Elecsys HCG+ß (2014-2017). HAb investigation included measurement by two immunoassays before and after treatment of samples with heterophile blocking reagents and serial dilution studies. Results Five cases of HAb and HAb-like interference were identified. The interference frequency was 6.7% for the Beckman assay and 2.9% for the Roche assay. The presence of HAb was detected using heterophile blocking reagents and an alternative method in three cases. The other two cases were detected due to discrepant results with an alternative method and non-linear serial dilutions (HAb-like). Conclusions HAb interference was observed in the Beckman and the Roche assays. The heterophile blocking reagents failed to detect 40% of interference cases. Blocking reagents should not solely be used for these investigations. Multiple strategies including the use of serial dilutions and using an alternative platform are critical when troubleshooting interferences in hCG immunoassays.

False-Positive Monospot in a Returning Traveler with Dengue Fever.

The heterophile antibody (Monospot), initial test of choice for Epstein-Barr virus (EBV)-associated infectious mononucleosis, is both sensitive (70-92%) and specific (96-100%). False positives have been demonstrated in cases of viral hepatitis, human immunodeficiency virus, leukemia, lymphoma, pancreatic cancer, systemic lupus erythematosus, and rubella. We present a case of a 46-yr-old male who developed fever, chills, headaches, myalgia, fatigue, and photophobia 1 d after returning from the Philippines. He demonstrated a mild transaminitis and significant thrombocytopenia (12,000 cells/µL). His initial evaluation revealed a positive heterophile antibody test. Without a classic EBV presentation, a fever in returning traveler evaluation was instituted resulting in a positive dengue test by direct fluorescence IgM (8.82 IU) and IgG (7.13 IU), respectively. Both his EBV DNA polymerase chain reaction and IgM by viral capsid antigen were negative. Dengue, an RNA flavivirus, and the dengue antibody have demonstrated cross-reactivity with other flaviviruses including Japanese encephalitis virus, yellow fever virus, West Nile virus, and St. Louis encephalitis. However, EBV is a double-helix DNA herpesvirus and structurally very different. To our knowledge, this is the first reported case of cross-reactivity between dengue and EBV that describes a potential false positive for the heterophile antibody test.

Heterophilic antibodies in sera from individuals without loxoscelism cross-react with phospholipase D from the venom of Loxosceles and Sicarius spiders

Background Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. Methods The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. Results We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. Conclusions People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.

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How to use the Monospot and other heterophile antibody tests.

Epstein-Barr virus (EBV) is a highly prevalent virus, transmitted via saliva, which often causes asymptomatic infection in children but frequently results in infectious mononucleosis in adolescents. Heterophile antibody tests, including the Monospot test, are red cell or latex agglutination assays, which detect antired cell antibodies produced as part of a polyclonal antibody response occurring during EBV infection. Heterophile antibody tests are rapid, cheap and specific tests that can be performed from the onset of symptoms of infectious mononucleosis. In adolescents, heterophile antibody tests have high specificity and sensitivity in the diagnosis of primary acute EBV infection. However, the tests have low sensitivity and low negative predictive value in young children and are not useful under the age of 4. Heterophile tests may be positive in other viral infections, autoimmune disease and haematological malignancies, but do not appear to be positive in primary bacterial infection. Virus-specific serology is required in children under the age of 4 or if an older child is heterophile negative. Virus-specific serology allows diagnosis and the pattern of positivity and negativity enables the clinician to stage the EBV infection. Virus-specific serology appears to have better sensitivity in young children, but there is cross-reaction with other herpesvirus infections, a longer turnaround time and it is more expensive to perform. Further research is needed to establish which children benefit from and hence require testing for heterophile antibodies, the cost-effectiveness of EBV investigations and whether heterophile titres have predictive value for the severity of infection and the likelihood of complications.

Physiological indices of stress in wild and captive garter snakes: correlations, repeatability, and ecological variation.

Glucocorticoids and leukocyte ratios have become the most widespread variables employed to test hypotheses regarding physiological stress in wild and captive vertebrates. Little is known, however, regarding how these two indices of stress covary in response to stressors, their repeatability within individuals, and differences in response time upon capture. Furthermore, few studies compare stress indices between captive and wild populations, to assess potential alteration of stress physiology in captivity. To address these issues, we examined corticosterone (CORT) and heterophil to lymphocyte (H:L) ratios in two ecotypes of the garter snake Thamnophis elegans. We found that CORT and H:L ratios were not correlated within individuals, and both variables showed little or no repeatability over a period of months. CORT levels, but not H:L ratios, were higher for individuals sampled after 10min from the time of capture. However, both variables showed similar patterns of ecotypic variation, and both increased over time in gravid females maintained in captivity for four months. We suggest that CORT and H:L ratios are both useful, but disparate indices of stress in this species, and may show complex relationships to each other and to ecological and anthropogenic variables.

Validity of a point-of-care based on heterophile antibody detection for the diagnosis of infectious mononucleosis in primary care.

OBJECTIVE: To evaluate the validity of a point-of-care test to diagnose infectious mononucleosis (IM) compared with Epstein-Barr virus (EBV) specific serology. METHODS: Patients over 14 years with sore throat and four Centor criteria--tonsillar exudate, fever, lymph glands tenderness and absence of cough--and negative pharyngeal testing for group A ß-haemolytic streptococcal antigen were consecutively recruited. All patients underwent pharyngotonsillar swab for microbiological culture, the rapid OSOM MonoTest for the diagnosis of IM in whole blood, the Paul-Bunnell test and complete blood analysis with serology for EBV and cytomegalovirus the day after the visit and at 15 days. Sensitivity and specificity were determined. RESULTS: We included 145 patients with a mean age of 24 ± 6.8 years. Of these, serology was determined in 129 subjects, with IM being diagnosed in 14 (10.9%). Both the MonoTest and the Paul-Bunnell test were positive in 13 patients with IM (92.9%) with no patient without disease being positive for either test--sensitivity of 92.9% (95% CI: 64.2-99.6%) and specificity of 100% (95% CI: 96-100%). The culture showed streptococcus A infection in 1 case (0.7%) and streptococcus C in 62 cases (42.8%). A total of 78 patients presented past infection by EBV (60.5%). CONCLUSIONS: Only one out of 10 patients with sore throat, four Centor criteria and negative rapid test for streptococcal infection presents IM. Despite the MonoTest presenting optimum sensitivity and specificity, it was found to have the same validity as the Paul-Bunnell test, with serological study continuing to be necessary for precise diagnosis of IM.

Effects of a premolt calcium and low-energy molt program on laying hen behavior and heterophil-to-lymphocyte ratios.

The objectives of this study were to compare the behaviors, postures, and heterophil-to-lymphocyte ratios (H:L) of laying hens housed in a cage system when offered a Ca premolt treatment and low-energy molt diets vs. a traditional feed withdrawal (FW) treatment during and after molt. A total of 144 Hy-Line W-36 hens (85 wk of age), housed 3 hens/cage (413 cm(2)/hen), were used. Hens were allotted to treatments according to a randomized complete block design, with the cage location and initial BW as the blocking criteria. Six treatments were compared in a 2 × 3 factorial arrangement with 2 Ca premolt treatments (fine or coarse) and 3 low-energy molt diets (FW, soybean hulls, or wheat middlings). The 2 Ca premolt treatments differed only in Ca particle size (fine was 0.14 mm and coarse was 2.27 mm mean diameter). Two postures and 5 behaviors were recorded and H:L was measured. Data were analyzed using the MIXED procedure of SAS, with P < 0.05 considered significant. There were no differences in behaviors, postures, or H:L during the premolt baseline period. The Ca premolt treatment had no carryover effects during or after molt for behaviors or postures. During molt, hens in the FW treatment were more active, and they ate and drank less compared with hens fed soybean hulls or wheat middlings, but there were no differences in aggression, nonnutritive pecking, or sitting. Drinking and aggression during and after molt were not different, but hens postmolt engaged in more sitting and feeding and less activity, nonnutritive pecking, and preening compared with during molt. There were no differences in H:L during or after molt. In conclusion, a Ca premolt treatment did not affect the behavior of the laying hen. The low-energy molt diets did not adversely affect behavior compared with FW and did not increase H:L; therefore, they could be useful alternatives for inducing molt in laying hens.

Lemierre's and Lemierre's-like syndromes in association with infectious mononucleosis.

OBJECTIVE: This study aimed to review cases of Lemierre's and Lemierre's-like syndromes in paediatric patients, to examine a possible association with Epstein-Barr virus as a predisposing factor, and to assess the impact of this virus on the severity of illness. METHODS: We performed a retrospective analysis of data from the in-patient database at Winthrop University Hospital, from January 2001 to October 2007. We reviewed clinical and laboratory findings as well as the outcome of infection in patients aged 21 years or less with a diagnosis of Lemierre's syndrome. An additional case of Lemierre's-like syndrome was also included. The illness severity and duration of in-patient management of those testing positive for heterophile antibody were then compared with the same parameters in patients who tested negative. RESULTS: Of the five patients diagnosed with Lemierre's syndrome, two had concomitant acute infection with Epstein-Barr virus. Additionally, a 19-year-old adolescent was admitted during this period with acute infectious mononucleosis, Fusobacterium necrophorum sepsis, sinusitis, frontal lobe abscess and ophthalmic vein thrombosis. The clinical presentation of all patients included fever, sore throat, and ear or neck pain. The duration of symptoms ranged from two days to three weeks prior to admission. The patients with acute Epstein-Barr virus infection had been diagnosed with infectious mononucleosis prior to admission, and tested positive for heterophile antibody. These patients subsequently underwent more extensive in-patient treatment, including intensive care management and ventilator support. The patients who tested negative for heterophile antibody experienced a milder course of illness, with a shorter duration of in-patient management. CONCLUSION: Two patients diagnosed with Lemierre's syndrome, and a third with Fusobacterium necrophorum sepsis, had coexisting acute Epstein-Barr virus infection. Patients who tested positive for heterophile antibody experienced a more severe course of illness. These observations suggest a possible association between Epstein-Barr virus infection and the severity of concomitant Lemierre's syndrome.

Gal knockout pig pericardium: new source of material for heart valve bioprostheses.

BACKGROUND: Although glutaraldehyde fixation is known to reduce immunogenicity and degeneration of heart valve bioprostheses, some degree of immunogenicity persists, which may trigger calcification. The aims of this study were to: (1) define the role of alpha-1,3-galactosyltransferase (alpha-Gal) antigen in valve calcification by comparing alpha-Gal-positive and alpha-Gal-deficient (GT-KO) pig pericardium; and (2) elucidate the role of human anti-Gal antibodies in the process of calcification and to determine the potential influence of different tissue-fixation techniques. METHODS: Glutaraldehyde-treated pericardium from alpha-Gal-positive and GT-KO pigs, with or without pre-labeling with human anti-Gal antibodies, were implanted in rats during 1 month. RESULTS: In glutaraldehyde-fixed pericardium, calcification levels were significantly lower in GT-KO pig pericardium (132.8 +/- 5.8 microg/mg) as compared with alpha-Gal-positive pig pericardium (155.7 +/- 7.1 microg/mg) (p < 0.015). In glutaraldehyde-fixed pig pericardium followed by a mix of formaldehyde, ethanol and Tween 80 (FET), the calcification levels were lower in GT-KO pig pericardium (0.35 +/- 0.1 microg/mg) as compared with alpha-Gal-positive pig pericardium (4.6 +/- 4.2 microg/mg). In glutaraldehyde-fixed pig pericardium + FET pre-incubated with human anti-Gal antibodies, calcification levels were significantly greater in alpha-Gal-positive pig pericardium (43.8 +/- 8.5 microg/mg) as compared with GT-KO pig pericardium (5.7 +/- 2.9 microg/mg) (p < 0.0001). CONCLUSIONS: This study demonstrates the role of alpha-Gal antigen and human alpha-Gal antibodies in the calcification process of valvular bioprostheses. It is suggested that GT-KO pig pericardium could be beneficial as a new source of material for heart valve bioprostheses.

The fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry.

Immunoassays have made it possible to measure dozens of individual proteins and other analytes in human samples for help in establishing the diagnosis and prognosis of disease. In too many cases the results of those measurements are misleading and can lead to unnecessary treatment or missed opportunities for therapeutic interventions. These cases stem from problems inherent to immunoassays performed with human samples, which include a lack of concordance across platforms, autoantibodies, anti-reagent antibodies, and the high-dose hook effect. Tandem mass spectrometry may represent a detection method capable of alleviating many of the flaws inherent to immunoassays. We review our understanding of the problems associated with immunoassays on human specimens and describe methodologies using tandem mass spectrometry that could solve some of those problems. We also provide a critical discussion of the potential pitfalls of novel mass spectrometric approaches in the clinical laboratory.

Evaluation of analytical performance of the Pathfast cardiac troponin I.

BACKGROUND: Cardiac troponins are considered the cornerstone for risk stratification and diagnosis of patients with acute coronary syndrome (ACS). Following Clinical Laboratory Standards Institute (CLSI) guidelines, we assessed the analytical performances of the Pathfast (Mitsubishi, Japan) cTnI method. METHODS: We evaluated different sample types. Control materials and lithium heparin plasma pools were used to determine: limit of blank (LoB), limit of detection (LoD), imprecision and linearity. The effects of potential endogenous interfering substances and the possibility of falsely increased cardiac troponin I (cTnI) concentrations attributable to the presence of heterophilic antibodies (HA), rheumatoid factor (RF) and human anti-mouse antibodies (HAMA) in high concentrations were evaluated. The 99th percentile limit of the cTnI value distribution was determined from 320 Caucasian reference individuals. RESULTS: No significant differences were found when cTnI concentrations of 40 lithium-heparin plasma samples were compared with the matched values of K(2)-EDTA plasma, whole blood and serum samples. The LoB and the LoD of the cTnI method were 0.0048 and 0.0066 microg/L, respectively. cTnI mean values from 0.66 to 6.0 microg/L showed a total CV% from 6.0 to 6.4. cTnI at a concentration of 0.02 microg/L was associated with a total CV of 9.6%. The method gave a linear response for cTnI concentrations within the measurement range. In six of 12 samples containing HA, a positive interference was demonstrated. The 99th percentile limit of the cTnI distribution in the reference population was 0.013 microg/L. CONCLUSIONS: The data indicate that the cTnI Pathfast method may be suitable for helping clinicians in the management of patients with ACS.

Algunos aspectos que el endocrinólogo debe conocer sobre los métodos de determinaciones hormonales / Hormone assays: some aspects that endocrinologists should know

Desde los trabajos pioneros de Yalow y Berson, que introdujeron el radioinmunoanálisis (RIA), los métodos de análisis de hormonas han evolucionado gradualmente con mejoras en todos los aspectos de su diseño, desde los análisis inmunorradiométricos a la automatización. Un ejemplo de esta evolución son los análisis de tirotropina y paratirina. A pesar de la gran precisión y la fiabilidad de los métodos hormonales utilizados en la actualidad, es importante revisar algunas limitaciones, como la interferencia por autoanticuerpos, anticuerpos heterofílicos o macroprolactina o el efecto gancho Objetivo: Conocer el proceso de adaptación a la diabetes mellitus tipo 1 (DM1) y analizar su correspondencia con las etapas del proceso de duelo descritas por Kübler-Ross. Sujetos y método: Estudio etnográfico mediante entrevistas en profundidad a 20 pacientes, 10 familiares y 12 profesionales (6 médicos y 6 enfermeras). Para el análisis se siguió el esquema de análisis de datos cualitativos de Miles y Huberman. Resultados: El paciente diagnosticado de DM1 y su familia afrontan la pérdida del estilo de vida y los objetos reales o imaginarios de su vida pasada. Enfermos y familiares experimentan reacciones emocionales que, en algún caso, pueden asemejarse a las etapas de duelo descritas por Kübler-Ross en una enfermedad terminal (negación, rebeldía, negociación, depresión y aceptación), pero hay diferencias que dependen de factores personales y psicosociales. Los profesionales tienden a relacionar la mala adherencia con la negación de la enfermedad, pero algunos pacientes se sienten amenazados por las exigencias de tratamiento y control y por sus consecuencias en su calidad de vida, y conscientemente optan por no seguir las recomendaciones. Es más realista hablar de adaptación a la enfermedad que de aceptación, puesto que los procesos de pérdida son constantes y el enfermo debe reconstruir nuevas identidades según su estado. El proceso de duelo afecta también a la familia y puede ser diferente que el del enfermo en tiempo, intensidad y valoración de los problemas. Conclusiones: La adaptación es un proceso complejo en el que intervienen muchas variables. Se observan diferencias en los mecanismos que utiliza cada sujeto en particular. Los profesionales sanitarios y, particularmente la enfermera, deben considerar las múltiples dimensiones psicosociales de la enfermedad crónica (AU)

Since the pioneering works of Yalow and Berson that introduced radioimmunoassays (RIA), hormone assays have been developed gradually, with improvements in all aspects of their design, from immunoradiometric assays to automatization. Examples of this evolution are the thyrotropin (TSH) and parathyroid (PTH) assays. Despite the strong accuracy and reliability of currently used hormone assays, some limitations should be reviewed, such as interference by autoantibodies, heterophile antibodies or macroprolactin and the hook effect (AU)