Isolating Human Peripheral Blood Mononuclear Cells from Buffy Coats via High Throughput Immunomagnetic Bead Separation.

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in long-term storage, making them an ideal cell type for many downstream research purposes, including flow cytometry, immunoassays, and genome sequencing. Typically, PBMCs are isolated via density gradient centrifugation, however, it is a low-throughput workflow that is difficult and costly to scale. This article presents a high-throughput workflow using a magnetic bead-based PBMC isolation method that is quick to implement. Total cell concentration, viability, and population distribution with PBMCs obtained using density gradient isolation were compared, and cell viability and proportion of cell types were comparable for both techniques. Isolated PBMCs demonstrated over 70% viability up to 9 days after blood collection, although yield decreased by half after 5 days compared to PBMCs processed within 24 h of collection. In summary, this article describes a PBMC protocol that utilizes a bead-based approach to adapt to a high throughput workflow and demonstrates that both manual and automated bead-based methods can increase processing capacity and provide flexibility for various budgets.

Enriching Cardiomyocytes Derived from hiPSCs by Magnetic-Activated Cell Sorting (MACS).

Cardiomyocytes (CMs) derived from human-induced pluripotent stem cells (hiPSCs) are considered a promising platform for multiple applications, including disease modeling, regenerative medicine, screening of drug toxicity and investigation of cardiomyogenesis. Despite remarkable improvement in methodology enabling differentiation of hiPSCs into CMs, applied protocols generate heterogeneous cell populations composed of CMs along with differentiated non-cardiac cell-types and undifferentiated hiPSCs. Here we describea procedure of automated Magnetic-Activated Cell Sorting (autoMACS) for the purification of hiPSCs-derived CMs under sterile culture conditions. We illustrate that this approach led to a robust depletion of non-cardiac cells and enrichment of CMs, a result particularly crucial for hiPSC lines with poor cardiac differentiation efficiencies.

Peptide-Loaded HLA Class I Tetramer-Associated Magnetic Bead-Based Enrichment of HBV-Specific CD8+ T Cells.

HBV-specific CD8+ T cells are only present at the low frequency during chronic infection. Thus, they are often undetectable by conventional ex vivo staining methods using peptide-loaded HLA class I tetramers. Detection sensitivity can be increased by magnetic bead-based enrichment strategies following staining with peptide-loaded HLA class I tetramers. Additionally, some downstream applications like e.g., single cell RNA sequencing of virus-specific CD8+ T cells may also require a pre-enrichment step to increase the frequency of the cells of interest. For this, peptide-loaded HLA class I tetramers-associated magnetic bead-based enrichment is also a suitable method.

An integrated magnetic separation enzyme-linked colorimetric sensing platform for field detection of Escherichia coli O157: H7 in food.

An intelligent colorimetric sensing platform integrated with in situ immunomagnetic separation function was developed for ultrasensitive detection of Escherichia coli O157: H7 (E. coli O157: H7) in food. Captured antibody modified magnetic nanoparticles (cMNPs) and detection antibody/horseradish peroxidase (HRP) co-functionalized AuNPs (dHAuNPs) were firstly synthesized for targeted enrichment and colorimetric assay of E. coli O157: H7, in which remarkable signal amplification was realized by loading large amounts of HRP on the surface of AuNPs. Coupling with the optical collimation attachments and embedded magnetic separation module, a highly integrated optical device was constructed, by which in situ magnetic separation and high-quality imaging of 96-well microplates containing E. coli O157: H7 was achieved with a smartphone. The concentration of E. coli O157: H7 could be achieved in one-step by performing digital image colorimetric analysis of the obtained image with a custom-designed app. This biosensor possesses high sensitivity (1.63 CFU/mL), short detecting time (3 h), and good anti-interference performance even in real-sample testing. Overall, the developed method is expected to be a novel field detection platform for foodborne pathogens in water and food as well as for the diagnosis of infections due to its portability, ease of operation, and high feasibility.

Fluorescent identification of immunomagnetically captured CTCs using triplex-aptamer-targeted dendritic SiO2@Fe3O4 nanocomposite.

The enumeration of circulating tumor cells (CTCs) in peripheral blood plays a crucial role in the early diagnosis, recurrence monitoring, and prognosis assessment of cancer patients. There is a compelling need to develop an efficient technique for the capture and identification of these rare CTCs. However, the exclusive reliance on a single criterion, such as the epithelial cell adhesion molecule (EpCAM) antibody or aptamer, for the specific recognition of epithelial CTCs is not universally suitable for clinical applications, as it usually falls short in identifying EpCAM-negative CTCs. To address this limitation, we propose a straightforward and cost-effective method involving triplex fluorescently labelled aptamers (FAM-EpCAM, Cy5-PTK7, and Texas Red-CSV) to modify Fe3O4-loaded dendritic SiO2 nanocomposite (dmSiO2@Fe3O4/Apt). This multi-recognition-based strategy not only enhanced the efficiency in capturing heterogeneous CTCs, but also facilitated the rapid and accurate identification of CTCs. The capture efficiency of heterogenous CTCs reached up to 93.33%, with a detection limit as low as 5 cells/mL. Notably, the developed dmSiO2@Fe3O4/Apt nanoprobe enabled the swift identification of captured cells in just 30 min, relying solely on the fluorescently modified aptamers, which reduced the identification time by approximately 90% compared with the conventional immunocytochemistry (ICC) technique. Finally, these nanoprobe characteristics were validated using blood samples from patients with various types of cancers.

Microchip for Immunomagnetic Sorting of Circulating Tumor Cells (CTCs).

Circulating tumor cells (CTCs) isolated directly from whole blood opens new perspectives for cancer monitoring and the development of personalized treatments. However, due to their rarity among the multitude of blood cells, it remains a challenge to recover them alive with high level of purity, i.e., with few remaining white blood cells, and in a time frame compatible with the clinical context. Microfluidic chips have emerged as promising tools to address these challenges. We propose a two-step workflow including a pre-enrichment step, performed by a size-based pre-enrichment system, and a purification step, performed by an immunomagnetic chip. Here, we describe the protocol for the fabrication of the immunomagnetic microchip, the preparation of the sample, and the procedure for injection into the microchip allowing the sorting of the CTCs.

Integration of protein L-immobilized epoxy magnetic bead capture with LC-MS/MS for therapeutic monoclonal antibody quantification in serum.

In recent years, there has been a growing interest in the thriving monoclonal antibody (mAb) industry due to the wide utilization of mAbs in clinical therapies. Robust and accurate bioanalytical methods are required to enable fast quantification of mAbs in biological matrices, especially in the context of pharmacokinetics (PKs)/pharmacodynamics (PDs) and therapeutic drug monitoring (TDM) studies. In this investigation, we presented a novel immuno-magnetic capture coupled with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method designed for the quantification of immunoglobulin G-kappa-based mAbs in biological fluids. The immunoaffinity absorbent for mAb drug purification was meticulously crafted by immobilizing protein L onto monosize, magnetic poly(glycidyl methacrylate) (m-pGMA) beads, synthesized through dispersion polymerization. The microspheres were acquired with an average size of 1.6 µm, and the optimal binding of mAbs from the aqueous mAb solution was determined to be 45.82 mg g-1. The quantification of mAbs in 10 µL serum samples was achieved through affinity purification using m-pGMA@protein L beads (employing rituximab as an internal standard (IS)), on-bead reduction, and rapid tryptic digestion. Remarkably, the entire process, taking less than 2.5 hours, held significant potential for simplifying pretreatment procedures and minimizing analytical time. Furthermore, the developed method underwent validation in accordance with the European Medicines Agency (EMA) guidelines. The assay demonstrated commendable linearity within the 2-400 µg mL-1 range for both daratumumab and pembrolizumab. Intra- and inter-assay coefficients of variation fell within the range of 0.7% to 13.4%, meeting established acceptance criteria. Other validation parameters also conformed to regulatory standards. Ultimately, the efficacy of the method was substantiated in a pharmacokinetic study following a single-dose intravenous administration to mice, underscoring its applicability and reliability in real-world scenarios.

Procalcitonin Detection Using Immunomagnetic Beads-Mediated Surface-Enhanced Raman Spectroscopy.

The early detection of procalcitonin (PCT) is crucial for diagnosing bacterial infections due to its high sensitivity and specificity. While colloidal gold colorimetric and immune-chemiluminescence methods are commonly employed in clinical detection, the former lacks sensitivity, and the latter faces challenges with a brief luminescence process and an elevated background. Here, we introduce a novel approach for the quantitative analysis of PCT using surface-enhanced Raman spectroscopy (SERS), leveraging the enhanced properties of metal nanoparticles. Simultaneously, we employed a magnetic nanoparticle coating and surface biofunctionalization modification to immobilize PCT-trapping antibodies, creating the required immune substrates. The resulting magnetic nanoparticles and antibody complexes, acting as carriers and recognition units, exhibited superparamagnetism and the specific recognition of biomarkers. Then, this complex efficiently underwent magnetic separation with an applied magnetic field, streamlining the cumbersome steps of traditional ELISA and significantly reducing the detection time. In conclusion, the exploration of immunomagnetic bead detection technology based on surface-enhanced Raman spectroscopy holds crucial practical significance for the sensitive detection of PCT.

Analysis of maternal Fc gamma receptor IIIb isoantibodies using immunomagnetic negative selected neutrophils.

BACKGROUND AND OBJECTIVES: The isolation of neutrophils and subsequent detection of anti-human neutrophil antigens (HNA) antibodies are crucial in clinical medicine for the diagnosis of autoimmune neutropenia, neonatal alloimmune neutropenia (NAIN) and transfusion-related acute lung injury (TRALI). This study reports two cases of maternal anti-Fc-gamma-receptor-IIIb (FcγRIIIb) isoimmunization without NAIN symptoms and compares the efficiency of immunomagnetic negative selection (IMNS) with traditional dextran/Ficoll for neutrophil isolation in HNA serological assays. MATERIALS AND METHODS: Investigating two cases of maternal anti-FcγRIIIb isoimmunization, neutrophils from three donors were isolated from 8 mL of whole blood using IMNS and dextran/Ficoll. Serological assays included the granulocyte agglutination and immunofluorescence test, monoclonal antibody immobilization of granulocyte antigens and the LABScreen Multi (One Lambda). IMNS and dextran/Ficoll were compared in terms of cell yield, viability, time, cost and purity. RESULTS: Maternal anti-FcγRIIIb isoantibodies with FCGR3B gene deletion were detected in both cases. Newborns and fathers exhibited specific gene combinations: FCGR3B*02/FCGR3B*02 (Case 1) and FCGR3B*02/FCGR3B*03 (Case 2). IMNS outperformed dextran/Ficoll, yielding four times more neutrophils (average neutrophil counts: 18.5 × 103/µL vs. 4.5 × 103/µL), efficiently removing non-neutrophil cells and reducing processing time (30-40 min vs. 70-90 min), although it incurred a higher cost (2.7 times). CONCLUSION: Two cases of maternal anti-FcγRIIIb isoantibodies, unrelated to NAIN, were identified. Although neutropenia has not been described in these cases, we emphasize the importance of identifying asymptomatic cases with the potential for severe neutropenia. Additionally, IMNS is introduced as a rapid, high-yield, high-purity neutrophil isolation technique, beneficial for serological assays detecting anti-HNA antibodies.

A rapid immunomagnetic beads-based sELISA method for the detection of bovine αs1-casein based on specific epitopes.

Although αs1-casein poses significant health risks to individuals with milk allergies, the availability of quantification methods for this allergen remains limited. In this study, we developed an immunomagnetic beads-based immunoassay (IMBs-ELISA) for the precise quantitative detection of bovine αs1-CN, specifically targeting epitope AA173-194. No cross-reactivity was observed with the other 7 food allergens including milk allergen. The linear detection range of the established IMBs-ELISA method was 0.125 µg/mL-2.000 µg/mL, with a limit of detection of 0.099 µg/mL. The accuracy of this method was 1.048 %, and the intra-plate and inter-plate precision achieved 4.100 % and 6.777 %, respectively. Notably, the entire IMBs-ELISA process could be completed within 75 min, representing a substantial time-saving advantage over traditional ELISA methods. These results proved the reliability and rapidity of the IMBs-ELISA method for detecting αs1-CN in real food.

Isolation and Purification of Viable PGCs from Mouse Embryos.

In all organisms with sexual reproduction, sperm and oocytes derive from embryonic precursors termed primordial germ cells (PGCs) which pass on genetic information to subsequent generations. Studies aimed to unravel PGC development at molecular level in mammals can be traced at the early 1980s and were hampered by the difficulty in obtaining both sufficient quantities and purity of PGCs. For many laboratories, the isolation and purification methods of PGCs at different stages from embryos are the most shortcut and affordable tool to study many aspects of their development at cellular and molecular levels. In the present chapter, I focus on immunomagnetic cell sorting (MACS) and fluorescence-activated cell sorting (FACS) methods used in my laboratory for the purification of mouse PGCs from 10.5 to 12.5 dpc embryos before their differentiation in oogonia/oocytes in female and prospermatogonia in male.

Magnetic capture device for large volume sample analysis.

Immunomagnetic separation (IMS) techniques employing superparamagnetic particles can successfully isolate various components from mixtures. However, their utility can be limited for large-volume samples, viscous samples, or those containing a high density of particulate matter because of the need to generate high field gradients for particle recovery. Therefore, a new class of immunomagnetic particles was devised utilizing a single, macroscopic Pyrex spinbar conjugated with biorecognition elements to address these limitations. Advantages include an inherent capacity for effective mixing, an almost instantaneous recovery of the spinbar that can be performed without expensive equipment and with no loss of magnetic particles during processing, and reduced transfer of sample matrix. As a result, spinbars can provide an effective means for IMS with large-volume assays composed of complex matrices.

Development of a broad-spectrum one-step immunoassay for detection of glucocorticoids in milk using magnetosome-based immunomagnetic beads.

Immunomagnetic beads provide novel tools for high-throughput immunoassay techniques. In this study, protein G (PG) was immobilized onto bacterial magentic particles (BMPs) using an additional cysteine residue at the C-terminus. A broad-spectrum monoclonal antibody against glucocorticoids (GCs) was attached to BMPs through PG-Fc interaction, generating BMP-PG-mIgG immunomagentic beads. A sensitive one-step immunoassay was developed for GCs based on combination of BMP-PG-mIgG and dexamethasone-horseradish peroxidase tracer (DMS-HRP). The developed assay exhibited half inhibitory concentrations (IC50) for dexamethasone (DMS), betamethasone (BMS), prednisolone (PNS), hydrocortisone (HCS), beclomethasone (BCMS), cortisone (CS), 6-α-methylprednisone (6-α-MPNS), fludrocortisone acetate (HFCS) of 0.98, 1.49, 2.42, 9.29, 1.63, 6.13, 7.3, and 4.89 ng/mL, respectively. The method showed recoveries ranging rates from 86.5 % to 117 % with a coefficient of variation less than 12.3 % in milk sample, which showed a good correlation with LC-MS/MS. Thus, the proposed assay offers a rapid and broad-spectrum screening tool for simultaneous detection of GCs in milk.

A magnetic microneedle to isolate single immunomagnetically labeled cells.

Immunomagnetic enrichment of cell populations from bodily fluids followed by immunofluorescent labeling is an established sample preparation method often used for the detection and enumeration of rare cells such as circulating tumor cells. For a detailed analysis of the heterogeneous characteristics of these cells, the cells need to be retrieved individually. Although several technologies are available to obtain 100% pure cells either individually or in bulk, these are often expensive, have low specificity, or suffer from high cell losses, either inherent to the technology or caused by sample transfer into special chips. To solve this issue, we introduce the magnetic micro-needle approach, which allows for the isolation of immunomagnetically labeled target cells by the use of a magnetized microneedle directly from glass slides. The magnetic microneedle approach makes use of the already present magnetic labeling used for enrichment, while the glass-slide-based open sample container allows for easy and loss-free sample loading. Additionally, the system facilitates not only the isolation but also the precise placement of cells. As the used parts are low cost, the technology provides researchers with an affordable and efficient method to pick up and isolate, as well as specifically place magnetically labeled cells from enriched fractions, thereby enabling the researchers to isolate or analyze these rare cells in more detail.

Rapid detection of food-borne Salmonella contamination using IMBs-qPCR method based on pagC gene

Abstract Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1 mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 101 and 104 CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18 CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10 h, which is a promising rapid method to detect Salmonella in emergency.

Mycobacterium paratuberculosis detection in cow's milk in Argentina by immunomagnetic separation-PCR

Abstract The aim of this study was to standardize a diagnosis procedure to detect Mycobacterium avium subsp. paratuberculosis (Map) DNA in raw cow milk samples under field conditions. A procedure that combines both immunomagnetic separation and IS900 -PCR detection (IMS-IS1 PCR) was employed on milk samples from 265 lactating Holstein cows from Map infected and uninfected herds in Argentina. IMS-IS1 PCR results were analyzed and compared with those obtained from milk and fecal culture and serum ELISA. The extent of agreement between both tests was determined by the Kappa test. IMS-IS1 PCR showed a detection limit of 101 CFU of Map/mL of milk, when 50:50 mix of monoclonal and polyclonal antibodies were used to coat magnetic beads. All of the 118 samples from the Map uninfected herds were negative for the set of the tests. In Map infected herds, 80 out of 147 cows tested positive by milk IMS-IS1 PCR (55%), of which 2 (1.4%) were also positive by milk culture, 15 (10%) by fecal culture, and 20 (14%) by serum ELISA. Kappa statistics (95% CI) showed a slight agreement between the different tests (<0.20), and the proportions of agreement were ≤0.55. The IMS-IS1 PCR method detected Map in milk of the cows that were not positive in other techniques. This is the first report dealing with the application of IMS-IS1 PCR in the detection of Map in raw milk samples under field conditions in Argentina.

Association between respiratory and postural adaptations and self-perception of school-aged children with mouth breathing in relation to their quality of life

Objective: To investigate the respiratory and postural adaptations associated with mouth and nasal breathing and to evaluate the associations of such adaptations in mouth breathers' self-perceived quality of life. Method: Cross-sectional study with mouth breathers (initial n=116 and final n=48) and nasal breathers (initial n=131 and final n=24) from elementary school, aged between 7 and 14 years. Chest expansion, using cirtometry, the breathing pattern and the use of accessory muscles, by means of clinical evaluations and photogrammetry, and flexibility tests were evaluated in both groups. Subsequently, the mouth breathers were asked to complete the quality of life questionnaire. Statistical tests: Chi-square, odds ratio, Mann-Whitney, and binomial tests were first applied followed by logistic regressions. Results: Thoracic breathing (p=0.04), using of accessory muscles (p=0.03) and reductions in flexibility (p=0.001) increased the chances of an individual being a mouth breather when compared to nasal breathers. Subsequently, using of accessory muscles decreased the chances of snoring among mouth breathers (p=0.03); the presence of shoulder asymmetry reduced the chances of experiencing quiet sleep (p=0.05) and increased the chances of coughing or being tired when playing or running (p=0.008). Finally, forward head position reduced the chances of waking up at night (p=0.04) and experiencing shortness of breath (p=0.05). Conclusions: Respiratory and postural adaptations increased the chances of individuals persisting with mouth breathing. Additionally, these adaptations could be associated with mouth breathers' self-perceived quality of life. .

Detección inmunomagnética de células tumorales circulantes en cáncer de mama metastásico: nuevas tecnologías / Inmunomagnetic detection of circulating tumor cells in metastatic breast cancer: new technologies

Las metástasis hematógenas son la mayor causa de mortalidad en el cáncer de mama. Está documentado que una vez las células tumorales se diseminan el resultado es, generalmente, letal. Las células tumorales circulantes han sido consideradas por largo tiempo un reflejo de la agresividad de los tumores, y entre ellos uno de los más agresivos es el cáncer de mama metastásico. Los primeros resultados clínicos han permitido determinar una fuerte relación entre la detección y el número de las células tumorales circulantes, como un valor pronóstico y como marcador de la actividad antitumoral del tratamiento. El análisis inmunomagnético utilizando una nueva metodología permite determinar que un recuento de 5 células tumorales circulantes o más en 7,5 ml de sangre, en cualquier fase de la enfermedad, se asocia a un mal pronóstico, y es predictivo de una supervivencia global más corta.

Hematogenous metastasis is the major cause of mortality in breast cancer. Evidence indicates that tumor cells escape from the primary tumor mass into the blood stream and that these disseminated cells are the source of increased lethality. Circulating or metastatic tumour cells have been considered as useful indicators of the aggressiveness of breast cancer tumours. The first clinical results obtained with such assays strongly suggest that in metastatic breast cancer, circulating tumour cells detection and enumeration can be used to estimate prognosis and may serve as an early marker to assess anti-tumour activity of a treatment. Immunomagnetic analysis using a new methodology, determine that a circulating tumour cells count of 5 or more per 7,5 ml of blood, at any time during the course of the disease is associated with a poor prognosis and is predictive of shorter progression and overall survival.

Evaluation of an immunomagnetic capture method followed by PCR to detect Mycobacterium bovis in tissue samples from cattle / Evaluación de un método de captura inmunomagnética seguida de PCR para la detección de Mycobacterium bovis en tejidos de ganado

Tuberculosis is one of the most important infectious diseases worldwide. Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), an important animal pathogen with public health implications as it is a zoonosis. Currently, the diagnosis of BTB is based on the caudal fold test of the Tuberculin Skin Test (TST). Post-mortem bacterial culture is carried out to confirm the diagnosis, and then specific biochemical tests are performed for the characterization of the etiologic agent. Culture takes at least 4 to 8 weeks to develop. The diagnosis by molecular tests such as PCR can provide fast and reliable results, significantly decreasing the time of confirmation (from two months to two days), thus allowing the possibility of taking control actions to prevent the spread of the disease in herds. In this work the use of an immunomagnetic separation capture followed by PCR (IMS-PCR) based on the IS6110 element showed a detection threshold corresponding to 10 CFU in M. bovis-spiked PBS. In the case of infected bovine fresh tissues, after five replicates, the minimum value of detection was 1000 CFU in 100% of the trials (5/5). This paper attempts to provide a sensitive, rapid and specific technique for the diagnosis of bovine tuberculosis, and opens up the possibility of a direct application in the control and eradication of this cattle disease.

La tuberculosis es una de las enfermedades infecciosas más importantes. Mycobacterium bovis es el agente causal de la tuberculosis bovina (TBB), un patógeno animal y zoonótico. En la actualidad, el diagnóstico de TBB se basa en la prueba intradérmica de la tuberculina. El cultivo bacteriano post mortem se lleva a cabo para confirmar el diagnóstico y a continuación se realizan pruebas bioquímicas específicas para la caracterización del agente etiológico. El cultivo bacteriano toma por lo menos 4 a 8 semanas para su desarrollo. El diagnóstico mediante pruebas moleculares como PCR puede proporcionar resultados rápidos y robustos, con un considerable acortamiento hasta la confirmación del diagnóstico (de 2 meses a 2 días). En este trabajo, el uso de captura inmunomagnética seguida de PCR (IMS-PCR) dirigida al elemento IS6110 mostró un umbral de detección correspondiente a 10 UFC en M. bovis diluido en PBS. En el caso de tejidos bovinos inoculados experimentalmente después de 5 réplicas, el valor mínimo de detección fue de 1000 UFC en el 100% de los ensayos. Este artículo aspira a proporcionar una técnica sensible, rápida y específica para el diagnóstico de la tuberculosis bovina, con el fin de abrir la posibilidad de una aplicación directa en el control y la erradicación de esta enfermedad en el ganado.

Cuantificación inmunogenética de células tumorales circulantes en pacientes con cáncer de próstata: correlación clínica y patológica / Immunomagnetic quantification of circulating tumoral cells in patients with prostate cáncer: clinical and pathological correlation

OBJETIVOS: Detección y cuantificación de células tumorales prostáticas circulantes (CTC) en sangre periférica de pacientes con cáncer de próstata (CP) y estudiar la relación de las CTCs con los parámetrosclínico-patológicos.MÉTODOS: Estudio prospectivo con tres brazos: 26 pacientes (p) con CP localizado (CPL); 24p con CP metastático(CPM) y 30 controles voluntarios sanos. Se extrajouna única muestra de 7,5 mL de sangre periférica y se aislaron las CTC según un método inmunomagnéticobasado en el sistema CellSearch (Veridex). Las CTCs fueron identificadas como células nucleadas negativas para el CD45 (leucocitos) y positivas para las citoqueratinas(8, 18 y 19). Se estudiaron las relaciones del número de CTCs con los niveles de PSA, Gleason y clasificación TNM.RESULTADOS: Sólo el 10% de controles sanos tenían 1 CTC/7,5 mL, ninguno de los pacientes con CP localizadotuvo más de 3 CTC (88% ≤ 2 CTC) y aquellos con CPM presentaban niveles de CTCs significativamente más altos [m: 29 (1-178)] comparados con los otros dos grupos (P: 0.000). Se demostró una correlación positiva entre el número de CTC y cifras de PSA, con el tamaño del tumor y con la presencia o no de adenopatías.El grado Gleason fue el único parámetro que no mostró correlación con los niveles de CTC y aunque el número de CTC fue mayor en aquellos con metástasis viscerales [m: 297 (0-416)] comparado con los que tenían metástasis óseas [m: 68 (9,5-168)] estas diferenciasno fueron significativas.CONCLUSIONES: El análisis inmunomagnético nos permite cuantificar las CTC en sangre periférica y podríapresentar una posibilidad para lograr una estadificacióncorrecta y estimar un pronóstico adecuado de la enfermedad metastática(AU)

OBJECTIVES: To detect and enumerate circulating prostatic tumor cells (CTC) in the peripheral blood of patients with prostate cancer (PC) and study the relationship between CTCs and clinical-pathological parameters. METHODS: Prospective three-arm study: 26 patients (p) with localised PC (LPC); 24 P with metastatic PC (MPC) and 30 healthy volunteer controls. A single 7.5 ml sample of peripheral blood was retrieved; CTCs were isolated using an immunomagnetic method based on the CellSearch system (Veridex). CTCs were identified as nucleated cells negative for CD45 (leukocytes) and positive for cytokeratins. (8, 18 y 19) The relationship between CTC numbers and PSA levels, Gleason score and TNM classification was studied.RESULTS: Only 10% of the healthy controls had 1 CTC/7.5 mL, none of the patients with localised PC had more than 3 CTCs (88% ≤ 2 CTCs), and patients with MPC had significantly higher CTC levels [m: 29 (1-178)] compared with the other two groups (P: 0.000). A positive correlation was demonstrated between the CTC count and PSA levels, tumor size, and presen-ce or absence of enlarged lymph nodes. Gleason score was the only parameter that did not show any correlation with CTC levels, and although the number of CTCs was higher in patients with visceral metasta-ses [m: 297 (0-416)] compared with bone metastases patients [m: 68 (9.5-168)], these differences were not significant(AU)