Method for quantifying free hemoglobin, distinct from the hemoglobin-haptoglobin complex, in human serum.

The measurement of free hemoglobin (free Hb) in blood is crucial for assessing the risk of organ damage in patients with hemolytic diseases. However, the colorimetric method, commonly used in clinical practice, does not distinguish between free Hb and the hemoglobin-haptoglobin complex (Hb-Hp) in the blood, instead reflecting the total Hb level. Although size-exclusion high-performance liquid chromatography (SEC-HPLC) can specifically measure free Hb, its clinical use is limited by long assay times. Here, we developed a novel assay method for the rapid quantification of free Hb in serum, distinguishing it from Hb-Hp, using a latex agglutination immunoturbidimetric assay (LATIA). This method could be used to measure free Hb in sera in the range of 1-100 µg/mL in approximately 15 min using an automatic biochemistry analyzer. Using Hb-spiked serum samples from healthy adults, there was a high correlation with Hb levels determined using the newly developed method and SEC-HPLC, indicating a high specificity for free Hb. This novel assay can be used to monitor levels of free Hb in patients with various hemolytic diseases and to design therapeutic strategies based on measured values. However, further studies are required to assess its clinical performance.

Nephelometry vs. Immunoturbidimetry assay: Analytical performance on IgG subclasses.

Interest in measuring immunoglobulin G Subclasses (IgG Subclasses) is increasing as more information is gathered and understanding regarding conditions associated with deficiencies of each IgG Subclass grows. Different methodologies are available for the measurement of IgG Subclasses, but their specificities vary. As a result, laboratories choose the methodology that better suits their routine, but which may not necessarily align with the needs of their population. In addition, the lack of standardization for the quantification of IgG Subclasses causes diagnostic gaps when comparing results provided by different methodologies. Thus, the purpose of our research is to compare the analytical performance of The Binding Site's (TBS) Optilite® human Immunoglobulin G (IgG) and IgG Subclasses Immunoturbidimetry assay, with the Nephelometry method routinely used in our clinical laboratory, Siemens BNII®. Our results show that the Immunoturbidimetry assay appears to be the most reliable to evaluate IgG Subclasses: the sum of IgG Subclasses and Total IgG correlate better than by Nephelometry. Although these methodologies share a similar principle, the comparison of results appears to be compromised. Therefore, prior to switching methodologies, further studies should be conducted to assess which methodology could be better applied to specific populations. It is also essential to standardise IgG Subclasses assays to reduce discrepancies that arise from comparing results.

A Case of False Elevation of D-Dimer and Elimination Methods.

BACKGROUND: There are many methods for the detection of D-dimer in clinical laboratories. Immunoturbidimetric assays are widely used because of its high sensitivity and specificity [1-3]. However, this method may be affected by the interference of rheumatoid factor (RF), heterophilic antibodies, and other unknown proteins, and its falsity will increase, thus affecting clinical diagnosis. METHODS: This paper reports the cause analysis of a case of spurious D-dimer increase and four corresponding elimination methods: double dilution of the original specimen, detection of fibrin degradation product (FDP) level, addition of heterophilic blocking reagent, and comparison between different instruments. RESULTS: It was confirmed that there were special antibodies in the patient's body by four methods, which had non-specific reactions with D-dimer reagents, resulting in false increases of results. CONCLUSIONS: When the coagulation function results of patients show isolated increases in D-dimer, or the results are inconsistent with clinical symptoms, laboratory personnel should consider the possibility of interference factors, and conduct effective treatment to obtain correct test results, and thus reduce the occurrence of medical adverse events caused by inaccurate test results.

Performance of an automated immunoturbidimetric assay for bovine serum amyloid A.

BACKGROUND: Acute phase proteins are a group of vital constituents of the innate immune system, which may also serve as circulatory biomarkers of inflammation. The major acute phase protein serum amyloid A (SAA) is a reliable and sensitive biomarker in cows, allowing for rapid detection of inflammatory disease. A multispecies automated immunoturbidimetric assay (VET-SAA, Eiken) has been validated for horses, dogs, and cats, and it has been used to measure SAA concentrations in bovine samples. OBJECTIVES: The aim of the present study was to perform an analytical validation of the VET-SAA immunoturbidometric assay based on monoclonal antihuman SAA antibodies for the measurement of SAA in clinical samples from cows. METHODS AND RESULTS: The validation included an assessment of imprecision, inaccuracy, and detection limit, as well as an evaluation of the overlap performance, using banked serum from healthy and sick cows with or without inflammatory disease. Intra- and interassay variation ranged from 0.91% to 2.9% and 2.5% to 5.8%, respectively. The assay was performed with acceptable accuracy within a clinically relevant range of SAA, although minor signs of inaccuracy were detected. Overlap performance was acceptable, with the VET-SAA assay able to differentiate between healthy cows and cows with inflammatory and noninflammatory conditions. The automated VET-SAA assay is considered acceptable for the measurement of SAA in cows.

Development of toluidine red particle agglutination-based turbidimetric immunoassay for anticardiolipin antibody detection in syphilis.

INTRODUCTION: Serological tests of non-treponemal and treponemal types are the most frequently used for syphilis diagnosis. Nontreponemal tests are used to monitor disease activity. Toluidine red unheated serum test (TRUST), as one of nontreponemal tests, is generally applicable to hospitals at different levels. However, accurate judgment of TRUST results is inseparable from an experienced and accurate operator. To reduce current shortcomings of manual TRUST method, we attempted to convert the manual TRUST test into automatic TRUST test, that is, to determine the degree of aggregation of toluidine red particles by detecting the absorbance value of serum after reaction with toluidine red particles. METHODS: 50 µL of serum sample and 80 µL toluidine red particles were added to 96-well plate. Then, the 96-well plate was placed on a microplate reader at medium grade for 8 min to mix. Then, plasma reagin reacted with toluidine red particles and promoted the aggregation of toluidine red particles to form a large clot, which eventually caused a decrease in the absorbance at 540 nm. RESULTS: The results showed that the specificity of the automatic TRUST test was 100%, the sensitivity was 87%. And this method showed 93.5% correlation with manual TRUST test. The developed method is simple and involves less subjectivity in reading results, opening new avenues for syphilis diagnostic testing. CONCLUSION: Turbidimetric immunoassay can avoid the shortcomings of subjective interpretation, time-consuming and manual operation of manual TRUST method, and is more suitable for large-scale screening in health examination.

Cistatina C: Biomarcador de riesgo cardiovascular en VIH / Cystatin C: Biomarker of cardiovascular risk in HIV

INTRODUCCIÓN: Los pacientes infectados por el virus de la inmunodeficiencia humana (VIH) tienen mayor riesgo cardiovascular (RCV). El desarrollo de enfermedad cardiovascular (ECV) en esta población involucra factores de RCV tradicionales y factores relacionados con la propia infección, como el estado proinflamatorio crónico, la disfunción inmune y el tratamiento antirretroviral recibido. La cistatina C (CC) ha demostrado utilidad para valorar la presencia de factores de RCV y ECV establecida en población general, ancianos y enfermos renales. Analizamos dicha asociación en una población VIH+. MATERIAL Y MÉTODOS: Estudio analítico, observacional, transversal. Se recogieron factores de RCV y presencia de ECV en pacientes VIH+, obteniendo determinación de CC. Se establecieron 2 grupos: grupo1=CC elevada (≥0,95mg/L) y grupo 2=CC normal (<0,95mg/L). RESULTADOS: Se incluyeron 95 pacientes, grupo1=27 (28,4%) y grupo2=68 (71,5%). Un valor de CC≥0,95mg/L se relacionó con la presencia de ECV (p = 0,01); con aumento de medias de circunferencia de cintura (p = 0,05), circunferencia de cuello (p = 0,04), presión arterial sistólica (p = 0,04), presión arterial diastólica (p = 0,01), puntaje al score de riesgo de Framingham (p = 0,03) y puntaje al score de riesgo de Framingham adaptado para VIH (p = 0,01). Después de realizarse análisis multivariado con incorporación de variables con asociación bivariada a ECV, solo CC≥0,95mg/L continuó relacionándose con ECV. CONCLUSIÓN: CC≥0,95mg/L se relacionó de forma independiente con la presencia de ECV. Este punto de corte también se vinculó a mayores niveles de presión arterial y mayor RCV a 10 años calculado por score de Framingham y score adaptado para población VIH

INTRODUCTION: Patients infected with the human immunodeficiency virus (HIV) have a higher cardiovascular risk (CVR). The development of cardiovascular disease (CVD) in this population involves traditional CVR factors and factors related to the infection itself, such as chronic inflammatory status, immune dysfunction, as well as the antiretroviral therapy received. Cystatin C (CC) has shown to be useful in assessing the presence of CVR factors and CVD established in the general population, the elderly population, and patients with chronic kidney disease. An analysis was performed on this association in an HIV positive population (HIV+). MATERIAL AND METHODS: Analytical, observational, cross-sectional study was conducted, and included collecting information about CVR factors and CVD in HIV+, as well as measuring CC. The patients were divided into 2 groups: Group1=high CC (≥0.95mg/L) and Group2=normal CC (<0.95mg/L). RESULTS: A total of 95 patients were included. Group1=27 (28.4%) and Group2=68 (71.5%). A value of CC≥0.95mg/L was related to the presence of CVD (P=.01). It was also related with and an increase in waist circumference (P=.05), neck circumference (P=.04), systolic blood pressure (P=.04), diastolic blood pressure (P=.01), Framingham score (P=.03), and Framingham score adapted for HIV (P=.01). After performing multivariate analysis with incorporation of variables associated with CVD in the bivariate analysis, only CC≥0.95mg/L continued to be related to CVD. CONCLUSION: CC≥0.95mg/L was independently associated with CVD. This cut-off point was also linked to higher levels of blood pressure, and higher CVR at 10 years using the Framingham Score and Framingham Score adapted for HIV population

Recomendaciones para la estandarización de la medida de lípidos y lipoproteínas. Recomendación (2018) / Recommendations for the standardisation of lipids and lipoproteins measurements. Recommendation (2018)

Este documento describe recomendaciones para la estandarización de la medida de las magnitudes lipídicas, puesto que resultan críticas para la toma de decisiones clínicas. Deben emplearse métodos recomendados validados frente a un método de referencia o definitivo, y materiales de control que cumplan con la Directiva Europea sobre Diagnóstico in vitro; deben cumplir también los objetivos recomendados por el National Cholesterol Education Program (NCEP) y la Sociedad Española de Medicina del Laboratorio (SEQCML). La determinación de colesterol de HDL por métodos homogéneos en equipos automatizados se considera aceptable para la práctica rutinaria, y la fórmula de Friedewald utilizable para estimar la concentración de colesterol de LDL siempre que las concentraciones de triglicéridos sean iguales o inferiores a 200mg/dL (2,3mmol/L); en otro caso, se recomienda utilizar la concentración de colesterol-no-HDL. La cuantificación rutinaria de apolipoproteínas A1 y B, y lipoproteína (a), puede efectuarse por métodos de inmunonefelometría e inmunoturbidimetría, con calibradores trazables a materiales de referencia

Some recommendations are presented for standardising the measurement of lipids and lipoproteins, as they are critical for clinical decisions making. Recommended methods validated against a reference or definitive method should be employed, as well as the use of control materials that comply with European Directives on in vitro diagnostics. Additionally, the chosen methods must comply with the objectives set forth by the National Cholesterol Education Program (NCEP) and by the Spanish Society of Laboratory Medicine (SEQCML). Determination of HDL cholesterol using automatic homogenous methods is considered acceptable for normal clinical practice, and the Friedewald Formula is considered to be usable to estimate LDL cholesterol concentration when triglyceride concentrations are below 200mg/dL (2.3mmol/L). If this should not be the case, the use of non-HDL cholesterol is recommended. Routine quantification of apolipoproteins A1 and B, and lipoprotein (a) can be measured using immunonephelometric or immunoturbidimetric methods, with calibrators that are traceable to reference materials

Evaluación económica de la implantación del autoanalizador Alfred 60/AST de detección de crecimiento bacteriano con sembrador automático / Economic evaluation of the Alfred 60/AST device implantation for bacterial growth detection with automatic sewing machine

Introducción: En los laboratorios de microbiología se impone cada vez más utilizar sistemas de cribado automatizados para descartar las orinas negativas. Nuestro objetivo fue estimar el umbral presupuestario a partir del cual el autoanalizador Alfred-60/AST sería rentable para nuestro hospital. Material y métodos: Estudio de minimización de costes mediante árboles de decisión, realizado en un Hospital General. Se comparó el coste del urocultivo tradicional con el procesamiento automático mediante Alfred-60/AST. El procesamiento tradicional supone el cultivo manual de todas las orinas recibidas en agar sangre y MacConkey e identificación de todos los microorganismos aislados con el sistema Vitek-2. El autoanalizador sembraría solo las orinas positivas en un medio cromogénico que identificaría directamente los aislamientos de Escherichia coli. Resultados: Las variables con mayor impacto económico en el modelo fueron la probabilidad de obtener un cultivo positivo, la prevalencia de E. coli en los urocultivos y el coste por muestra del sembrador. El análisis de sensibilidad multivariante mostró que el modelo es sólido. El análisis de sensibilidad bivariable mostró que el modelo es sensible a la modificación de los costes, principalmente del sembrador automático. A un valor umbral de 1,40 euros por determinación, el procesamiento automático reduciría los costes anuales en 2.879 euros. Conclusión: La introducción del autoanalizador Alfred-60/AST en nuestro laboratorio a un precio de 1,40 euros por determinación reduciría la carga de trabajo en el procesamiento de orinas, ahorrando tiempo y costes

Introduction: It is becoming increasingly necessary to automatize screening of urine samples to culture at Microbiology laboratories. Our objective was to estimate the budget threshold from which the Alfred 60/AST device would be profitable for our hospital. Material and methods: Cost minimization study by decision trees, carried out in a General Hospital. The cost of traditional urine culture and urine processing using Alfred-60/AST were compared. Traditional processing involves the culture of all urine specimens received onto blood and MacConkey agar, and identification of every microorganism isolated by Vitek-2 system. The autoanalyzer would only inoculate the positive urines onto a chromogenic media, directly identifying the Escherichia coli isolates. Results: The variables with the greatest economic impact in the model were the probability of obtaining a positive culture, the prevalence of E. coli in the urine cultures and the cost per sample using Alfred-60/AST. The multivariate sensitivity analysis showed that the model was solid. The bivariate sensitivity analysis showed that the model is suceptible to cost modification, mainly of the automatic device. At a threshold value of 1.40 euros/determination, the automatic processing would decrease the annual costs in 2,879 euros. Conclusion: The introduction of the Alfred-60/AST device in our laboratory at 1.40 euros/determination would reduce urine processing workload, saving time and costs

Association Between Increased Levels of Cystatin C and the Development of Cardiovascular Events or Mortality: A Systematic Review and Meta-Analysis / Associação entre Níveis Elevados de Cistatina C e o Desenvolvimento de Eventos Cardiovasculares ou Mortalidade: Uma Revisão Sistemática e Meta-Análise

Abstract Background: Cystatin C seems promising for evaluating the risk of cardiovascular events and mortality. Objective: To evaluate the association between high levels of cystatin C and the development of cardiovascular events or mortality. Methods: The articles were selected in the Medline/PubMed, Web of Science, and Scielo databases. The eligibility criteria were prospective cohort observational trials that assessed the association of high serum levels of cystatin C with the development of cardiovascular events or mortality in individuals with normal renal function. Only studies that evaluated the mortality outcome compared the fourth with the first quartile of cystatin C and performed multivariate Cox's proportional hazard regression analysis were included in the meta-analysis. A p value < 0,05 was considered significant. Results: Among the 647 articles found, 12 were included in the systematic review and two in the meta-analysis. The risk of development of adverse outcomes was assessed by eight studies using the hazard ratio. Among them, six studies found an increased risk of cardiovascular events or mortality. The multivariate regression analysis was performed by six studies, and the risk of developing adverse outcomes remained significant after the analysis in four of these studies. The result of the meta-analysis [HR = 2.28 (1.70-3.05), p < 0.001] indicated that there is a significant association between high levels of cystatin C and the risk of mortality in individuals with normal renal function. Conclusion: There is a significant association between high levels of cystatin C and the development of cardiovascular events or mortality in individuals with normal renal function.

Resumo Fundamento: A cistatina C tem-se mostrado promissora para avaliação do risco de eventos cardiovasculares e mortalidade. Objetivo: Avaliar a associação entre níveis elevados de cistatina C e o desenvolvimento de eventos cardiovasculares ou mortalidade. Métodos: A seleção dos artigos foi realizada por meio das bases de dados Medline/PubMed, Web of Science e Scielo. Os critérios de elegibilidade foram estudos observacionais de coorte prospectivos que avaliaram a associação entre níveis séricos elevados de cistatina C e o desenvolvimento de eventos cardiovasculares ou mortalidade em indivíduos com função renal normal. Apenas os estudos que avaliaram o desfecho mortalidade, que compararam o quarto com o primeiro quartil de cistatina C e que realizaram análise de regressão multivariada de riscos proporcionais de Cox foram incluídos na meta-análise. Foi considerado significativo o valor p < 0,05. Resultados: Dentre os 647 artigos encontrados, 12 foram incluídos na revisão sistemática e dois na meta-análise. O risco de desenvolvimento dos desfechos adversos foi avaliado por oito estudos por meio do cálculo do hazard ratio. Dentre estes, seis estudos encontraram um maior risco de eventos cardiovasculares ou mortalidade. A análise de regressão multivariada foi realizada por seis destes estudos, e o risco de desenvolvimento dos desfechos adversos permaneceu significativo após realização desta análise em quatro destes estudos. O resultado da meta-análise [HR = 2,28 (1,70-3,05), p < 0,001] indicou que há uma associação significativa entre níveis elevados de cistatina C e o risco de mortalidade nos indivíduos com função renal normal. Conclusão: Há uma associação significativa entre níveis elevados de cistatina C e o desenvolvimento de eventos cardiovasculares ou mortalidade em indivíduos com função renal normal.