14 research outputs found
Spectroscopic evaluation of red blood cells of thalassemia patients with confocal microscopy: a pilot study
Hemoglobinopathies represent the most common single-gene defects in the world and pose a major public health problem, particularly in tropical countries, where they occur with high frequency. Diagnosing hemoglobinopathies can sometimes be difficult due to the coexistence of different causes of anemia, such as thalassemia and iron deficiency, and blood transfusions, among other factors, and requires expensive and complex molecular tests. This work explores the possibility of using spectral confocal microscopy as a diagnostic tool for thalassemia in pediatric patients, a disease caused by mutations in the globin genes that result in changes of the globin chains that form hemoglobin—in pediatric patients. Red blood cells (RBCs) from patients with different syndromes of alpha-thalassemia and iron deficiency (including anemia) as well as healthy (control) subjects were analyzed under a Leica TCS SP8 confocal microscope following different image acquisition protocols. We found that diseased RBCs exhibited autofluorescence when excited at 405 nm and their emission was collected in the spectral range from 425 nm to 790 nm. Three experimental descriptors calculated from the mean emission intensities at 502 nm, 579 nm, 628 nm, and 649 nm allowed us to discriminate between diseased and healthy cells. According to the results obtained, spectral confocal microscopy could serve as a tool in the diagnosis of thalassemia.This research was funded by Spanish Ministry of Economy and Competitiveness, grant number
DPI2017-89414-R. L.R.-B. thanks the Ministry of Science, Innovation and Universities for the PhD (FPI) grant she
has received.Peer ReviewedPostprint (published version
Hyperspectral imaging for skin cancer and blood disorders diagnosis
Hyperspectral imaging is a novel technology for acquiring an image at a large number of wavelengths, thus allowing the study of spectral and spatial details of a sample under analysis. This technology has emerged as a promising imaging modality to be used as a diagnostic tool in several medical applications where spectral information is relevant. In this paper, we outline our most recent achievements in this field. Firstly, hyperspectral imaging systems developed to improve non-invasive diagnosis of skin cancer, consisting of digital silicon and InGaAs cameras and light emitting diodes, are described. Secondly, we present our last advances using hyperspectral technology together with confocal microscopy to improve the diagnosis of blood diseases, in particular, hemoglobinopathies such as thalassemia and cell membrane diseases such as hereditary spherocytosis. Finally, new insights on these topics are discussed.This project has been funded by the Agencia Estatal de InvestigaciĂłn (AEI) (PID2020-112527RB-I00 / AEI / 10.13039/501100011033). L R-B thanks the Ministry of Science, Innovation and Universities for the PhD (FPI) grant she has received.Peer ReviewedPostprint (author's final draft
Membrane protein detection and morphological analysis of red blood cells in hereditary spherocytosis by confocal laser scanning microscopy
In hereditary spherocytosis (HS), genetic mutations in the cell membrane and cytoskeleton proteins cause structural defects in red blood cells (RBCs). As a result, cells are rigid and misshapen, usually with a characteristic spherical form (spherocytes), too stiff to circulate through microcirculation regions, so they are prone to undergo hemolysis and phagocytosis by splenic macrophages. Mild to severe anemia arises in HS, and other derived symptoms like splenomegaly, jaundice, and cholelithiasis. Although abnormally shaped RBCs can be identified under conventional light microscopy, HS diagnosis relies on several clinical factors and sometimes on the results of complex molecular testing. It is specially challenging when other causes of anemia coexist or after recent blood transfusions. We propose two different approaches to characterize RBCs in HS: (i) an immunofluorescence assay targeting protein band 3, which is affected in most HS cases and (ii) a three-dimensional morphology assay, with living cells, staining the membrane with fluorescent dyes. Confocal laser scanning microscopy (CLSM) was used to carry out both assays, and in order to complement the latter, a software was developed for the automated detection of spherocytes in blood samples. CLSM allowed the precise and unambiguous assessment of cell shape and protein expression.This publication is part of the project PID2020-112527RBI00, funded by CIN/AEI/10.13039/501100011033. Laura Rey-Barroso thanks the Ministry of Science, Innovation and Universities for the PhD (FPI) grant she has received (DPI2017-89414-R). The current study has been funded by
the Spanish National Agency of Investigation (AEI).Peer ReviewedPostprint (published version
EvaluaciĂłn de la microscopĂa confocal como herramienta de diagnĂłstico en enfermedades de los glĂłbulos rojos
La esferocitosis hereditaria (EH) provoca mutaciones en las proteĂnas de la membrana de los glĂłbulos rojos (GRs) que hacen que las cĂ©lulas se deformen y se vuelvan demasiado rĂgidas para poder viajar a travĂ©s de los vasos sanguĂneos. Estas cĂ©lulas anormales se destruyen masivamente en el bazo, lo que provoca anemia grave y esplenomegalia además de ictericia y cálculos biliares. El diagnĂłstico de la EH requiere la realizaciĂłn de complejas pruebas moleculares en la mayorĂa de casos. Para evitar la realizaciĂłn de dichas pruebas, la microscopĂa confocal espectral podrĂa utilizarse en el diagnĂłstico de estas y otras enfermedades. En este estudio, se tiñó la membrana de los GRs con tintes de color e inmunomarcadores, y, bajo un microscopio Leica TCS8, se analizaron los posibles defectos de membrana expresados como diferencias en color y forma en pacientes con EH
Mieloma mĂşltiple: canvis en el maneig clĂnic associats a la incorporaciĂł de nous fĂ rmacs
El mieloma mĂşltiple (MM) encara Ă©s una malaltia incurable. Tot i això, el seu maneig ha canviat considerablement en les dues darreres dècades per l’apariciĂł de quatre noves famĂlies de fĂ rmacs: els inhibidors del proteasoma (IP) com el bortezomib, els anomenats immunomoduladors (IMID) com la talidomida i la lenalidomida i darrerament els anticossos monoclonals i els limfòcits T amb receptors quimèrics d’antigen (CAR T). Aquests fĂ rmacs han demostrat eficĂ cia en combinaciĂł amb corticoides i amb alquilants, els agents utilitzats tradicionalment per al tractament del MM, o associats entre ells i actualment tenen indicaciĂł en el tractament de la recaiguda i de primera lĂnia en diferents combinacions. Bortezomib, talidomida i lenalidomida s’han incorporat plenament a la prĂ ctica assistencial sense que deixessin d’anar-se aprovant noves combinacions cada vegada d’eficĂ cia major.
En aquest context s’han dut a terme dos estudis retrospectius per avaluat l’impacte de la seqüenciació en el moment d’administrar els nous fà rmacs i els factors que podien preveure una resposta subòptima a esquemes amb alternatives més eficaces.
Per garantir l’absència de biaix de selecciĂł en les cohorts s’ha partit del registre de pacients amb MM del Servei d’Hematologia ClĂnica de l’Hospital Germans Trias i Pujol i de l’Institut CatalĂ d’Oncologia. S’han revisat i registrat totes les dades epidemiològiques, biològiques, de tractament i resposta de tots els pacients i s’han comparat mitjançant estadĂstics paramètrics i no paramètrics per garantir la comparabilitat entre cohorts. S’han utilitzat les definicions de consens de l’International Myeloma Working Group (IMWG) per definir les variables principals.
En el primer treball s’ha comparat la supervivència entre pacients tractats inicialment amb poliquimioterapia i a la recidiva amb IP o IMIDs (89 pacients), i pacients tractats amb IP o IMIDs incoporats a la primera lĂnia (65 pacients). En les nostres cohorts, la incorporaciĂł d’IMIDs i IP al tractament de primera lĂnia en pacients fins a 70 anys aporta una major supervivència que el tractament amb poliquimioterĂ pia seguit de tractament amb IMIDs i IP en el moment de la progressiĂł o recaiguda.
En el segon treball ens hem centrat en caracteritzar els pacients que podrien beneficiar-se més de la combinació de lenalidomida i dexametasona en la recaiguda, perquè tot i ser una combinació amb alternatives més eficaces el seu ús actual no és menyspreable. S’ha analitzat la cinètica de resposta al primer cicle i altres covariables de 227 pacients per determinar-ne l’impacte sobre la durada de la resposta i la supervivència global (SG). En la nostra cohort, l’assoliment d’una resposta parcial al primer cicle és la variable que s’associa a major supervivència lliure de progressió (SLP) i s’han identificat altres covariables amb impacte sobre la SLP, com l’antecedent d’una gammapatia monoclonal de significat incert (GMSI).Multiple myeloma is still an incurable disease. However, its treatment has changed considerably in the last two decades due to the emergence of four new drug families: proteasome inhibitors such as bortezomib, immunomodulators such as thalidomide and lenalidomide and lately monoclonal antibodies and CAR T-cells. These drugs have proven to be effective in combination with corticosteroids and alkylating agents, the agents traditionally used for the treatment of the MM, or associated with each other. Bortezomib, thalidomide and lenalidomide have been fully incorporated into everyday clinical practice, for MM first line treatment and treatment at relapse, whilst other new combinations have been approved due to their increased efficiency.
In this context, two retrospective studies have been carried out. The fist one, to evaluate the impact of treatment sequencing patterns regarding new drugs and the second one to study those factors that could predict a suboptimal response to schemes with more efficient alternatives.
All patients treated with MM in a consortium of three tertiary hospitals (Institut Català d’Oncologia) were registered at the time of starting therapy. To avoid selection bias, our studies were based in these registries. All epidemiological, biological, treatment and response data for all patients have been reviewed, recorded and compared using parametric and non-parametric statistics to ensure comparability between cohorts. The consensus definitions of the International Myeloma Working Group have been used to define the main variables.
In the first study, survival was compared between patients initially treated with conventional chemotherapy induction regimens and at relapse with IP or IMIDs (89 patients), and patients treated with IP or IMIDs upfront (65 patients). In our cohorts, the incorporation of IMIDs and IPs to first-line treatment in patients up to 70 years of age contributes to a greater survival than the treatment with conventional chemotherapy followed by treatment with IMIDs and IP at the time of progression or relapse.
Although some drug combinations have prove to be more efficient than lenalidomide and dexamethasone is still used. Our second study is focused on the characterization of patients who could benefit most from the combination of lenalidomide and dexamethasone at relapse. The kinetics of response to the first cycle and other covariates of 227 patients have been analyzed to determine the impact on response duration and overall survival. In our cohort, the achievement of a partial response after the first cycle is independently associated with a higher progression free survival. A prior history of monoclonal gammopathy of undetermined significance has also shown impact on progression free survival
Mieloma mĂşltiple : canvis en el maneig clĂnic associats a la incorporaciĂł de nous fĂ rmacs /
El mieloma mĂşltiple (07) encara Ă©s una malaltia incurable. Tot i això, el seu maneig ha canviat considerablement en les dues darreres dècades per l'apariciĂł de quatre noves famĂlies de fĂ rmacs: els inhibidors del proteasoma (IP) com el bortezomib, els anomenats immunomoduladors (IMID) com la talidomida i la lenalidomida i darrerament els anticossos monoclonals i els limfòcits T amb receptors quimèrics d'antigen (CAR T). Aquests fĂ rmacs han demostrat eficĂ cia en combinaciĂł amb corticoides i amb alquilants, els agents utilitzats tradicionalment per al tractament del 07, o associats entre ells i actualment tenen indicaciĂł en el tractament de la recaiguda i de primera lĂnia en diferents combinacions. Bortezomib, talidomida i lenalidomida s'han incorporat plenament a la prĂ ctica assistencial sense que deixessin d'anar-se aprovant noves combinacions cada vegada d'eficĂ cia major. En aquest context s'han dut a terme dos estudis retrospectius per avaluat l'impacte de la seqĂĽenciaciĂł en el moment d'administrar els nous fĂ rmacs i els factors que podien preveure una resposta subòptima a esquemes amb alternatives mĂ©s eficaces. Per garantir l'absència de biaix de selecciĂł en les cohorts s'ha partit del registre de pacients amb 07 del Servei d'Hematologia ClĂnica de l'Hospital Germans Trias i Pujol i de l'Institut CatalĂ d'Oncologia. S'han revisat i registrat totes les dades epidemiològiques, biològiques, de tractament i resposta de tots els pacients i s'han comparat mitjançant estadĂstics paramètrics i no paramètrics per garantir la comparabilitat entre cohorts. S'han utilitzat les definicions de consens de l'International Myeloma Working Group (IMWG) per definir les variables principals. En el primer treball s'ha comparat la supervivència entre pacients tractats inicialment amb poliquimioterapia i a la recidiva amb IP o IMIDs (89 pacients), i pacients tractats amb IP o IMIDs incoporats a la primera lĂnia (65 pacients). En les nostres cohorts, la incorporaciĂł d'IMIDs i IP al tractament de primera lĂnia en pacients fins a 70 anys aporta una major supervivència que el tractament amb poliquimioterĂ pia seguit de tractament amb IMIDs i IP en el moment de la progressiĂł o recaiguda. En el segon treball ens hem centrat en caracteritzar els pacients que podrien beneficiar-se mĂ©s de la combinaciĂł de lenalidomida i dexametasona en la recaiguda, perquè tot i ser una combinaciĂł amb alternatives mĂ©s eficaces el seu Ăşs actual no Ă©s menyspreable. S'ha analitzat la cinètica de resposta al primer cicle i altres covariables de 227 pacients per determinar-ne l'impacte sobre la durada de la resposta i la supervivència global (SG). En la nostra cohort, l'assoliment d'una resposta parcial al primer cicle Ă©s la variable que s'associa a major supervivència lliure de progressiĂł (SLP) i s'han identificat altres covariables amb impacte sobre la SLP, com l'antecedent d'una gammapatia monoclonal de significat incert (GMSI).El mieloma mĂşltiple (MM) encara Ă©s una malaltia incurable. Tot i això, el seu maneig ha canviat considerablement en les dues darreres dècades per l'apariciĂł de quatre noves famĂlies de fĂ rmacs: els inhibidors del proteasoma (IP) com el bortezomib, els anomenats immunomoduladors (IMID) com la talidomida i la lenalidomida i darrerament els anticossos monoclonals i els limfòcits T amb receptors quimèrics d'antigen (CAR T). Aquests fĂ rmacs han demostrat eficĂ cia en combinaciĂł amb corticoides i amb alquilants, els agents utilitzats tradicionalment per al tractament del MM, o associats entre ells i actualment tenen indicaciĂł en el tractament de la recaiguda i de primera lĂnia en diferents combinacions. Bortezomib, talidomida i lenalidomida s'han incorporat plenament a la prĂ ctica assistencial sense que deixessin d'anar-se aprovant noves combinacions cada vegada d'eficĂ cia major. En aquest context s'han dut a terme dos estudis retrospectius per avaluat l'impacte de la seqĂĽenciaciĂł en el moment d'administrar els nous fĂ rmacs i els factors que podien preveure una resposta subòptima a esquemes amb alternatives mĂ©s eficaces. Per garantir l'absència de biaix de selecciĂł en les cohorts s'ha partit del registre de pacients amb MM del Servei d'Hematologia ClĂnica de l'Hospital Germans Trias i Pujol i de l'Institut CatalĂ d'Oncologia. S'han revisat i registrat totes les dades epidemiològiques, biològiques, de tractament i resposta de tots els pacients i s'han comparat mitjançant estadĂstics paramètrics i no paramètrics per garantir la comparabilitat entre cohorts. S'han utilitzat les definicions de consens de l'International Myeloma Working Group (IMWG) per definir les variables principals. En el primer treball s'ha comparat la supervivència entre pacients tractats inicialment amb poliquimioterapia i a la recidiva amb IP o IMIDs (89 pacients), i pacients tractats amb IP o IMIDs incoporats a la primera lĂnia (65 pacients). En les nostres cohorts, la incorporaciĂł d'IMIDs i IP al tractament de primera lĂnia en pacients fins a 70 anys aporta una major supervivència que el tractament amb poliquimioterĂ pia seguit de tractament amb IMIDs i IP en el moment de la progressiĂł o recaiguda. En el segon treball ens hem centrat en caracteritzar els pacients que podrien beneficiar-se mĂ©s de la combinaciĂł de lenalidomida i dexametasona en la recaiguda, perquè tot i ser una combinaciĂł amb alternatives mĂ©s eficaces el seu Ăşs actual no Ă©s menyspreable. S'ha analitzat la cinètica de resposta al primer cicle i altres covariables de 227 pacients per determinar-ne l'impacte sobre la durada de la resposta i la supervivència global (SG). En la nostra cohort, l'assoliment d'una resposta parcial al primer cicle Ă©s la variable que s'associa a major supervivència lliure de progressiĂł (SLP) i s'han identificat altres covariables amb impacte sobre la SLP, com l'antecedent d'una gammapatia monoclonal de significat incert (GMSI).Multiple myeloma is still an incurable disease. However, its treatment has changed considerably in the last two decades due to the emergence of four new drug families: proteasome inhibitors such as bortezomib, immunomodulators such as thalidomide and lenalidomide and lately monoclonal antibodies and CAR T-cells. These drugs have proven to be effective in combination with corticosteroids and alkylating agents, the agents traditionally used for the treatment of the MM, or associated with each other. Bortezomib, thalidomide and lenalidomide have been fully incorporated into everyday clinical practice, for MM first line treatment and treatment at relapse, whilst other new combinations have been approved due to their increased efficiency. In this context, two retrospective studies have been carried out. The fist one, to evaluate the impact of treatment sequencing patterns regarding new drugs and the second one to study those factors that could predict a suboptimal response to schemes with more efficient alternatives. All patients treated with MM in a consortium of three tertiary hospitals (Institut CatalĂ d'Oncologia) were registered at the time of starting therapy. To avoid selection bias, our studies were based in these registries. All epidemiological, biological, treatment and response data for all patients have been reviewed, recorded and compared using parametric and non-parametric statistics to ensure comparability between cohorts. The consensus definitions of the International Myeloma Working Group have been used to define the main variables. In the first study, survival was compared between patients initially treated with conventional chemotherapy induction regimens and at relapse with IP or IMIDs (89 patients), and patients treated with IP or IMIDs upfront (65 patients). In our cohorts, the incorporation of IMIDs and IPs to first-line treatment in patients up to 70 years of age contributes to a greater survival than the treatment with conventional chemotherapy followed by treatment with IMIDs and IP at the time of progression or relapse. Although some drug combinations have prove to be more efficient than lenalidomide and dexamethasone is still used. Our second study is focused on the characterization of patients who could benefit most from the combination of lenalidomide and dexamethasone at relapse. The kinetics of response to the first cycle and other covariates of 227 patients have been analyzed to determine the impact on response duration and overall survival. In our cohort, the achievement of a partial response after the first cycle is independently associated with a higher progression free survival. A prior history of monoclonal gammopathy of undetermined significance has also shown impact on progression free survival
Multispectral imaging of healthy and diseased red blood cells using confocal microscopy
Red blood cell disorders represent the most common single-gene defects and pose a major public health problem, particularly in tropical countries, occurring with high frequency. Their diagnose can sometimes be difficult due to the coexistence of different causes of anemia, such as thalassemia and iron deficiency, and blood transfusions, among other factors, and requires expensive and complex molecular tests. In this work, blood samples from patients with different syndromes of alphathalassemia and iron deficiency (including anemia) as well as healthy (control) subjects were analyzed under a Leica TCS SP8 confocal microscope Samples exhibited autofluorescence when excited at 405 nm and three experimental descriptors calculated from the mean emission intensities at 502 nm, 579 nm, 628 nm, and 649 nm allowed us to discriminate between diseased and healthy cells. According to the results obtained, spectral confocal microscopy could serve as a tool in the diagnosis of thalassemia.Peer ReviewedPostprint (published version
Evaluation of confocal microscopy as a diagnosis tool on red blood cell diseases
In hereditary spherocytosis, mutations in red blood cell membrane proteins result in an overly rigid, misshapen cell whose deformability when traveling through the blood vessels is lost, causing severe anemia and splenomegaly, jaundice, and gallstones. In thalassemia, mutations in the globin genes can cause also severe anemia, skeletal and growth deficits and iron overload. Diagnosing these entities can be difficult due to the coexistence of other causes of anemia and blood transfusions, so complex molecular tests are required. In order to avoid these, we explored the possibility of using spectral confocal microscopy as a diagnostic tool for hereditary spherocytosis and thalassemia in pediatric patients. The red blood cell membrane was stained with different color dyes and immunolabels, to identify possible membrane defects expressed as differences in color and shape under a Leica TCS SP8. Staining the membrane and nuclei with lipophilic fluorescent dyes permitted the precise assessment of cell shape.Peer ReviewedPostprint (published version
Estudio espectroscĂłpico de glĂłbulos rojos mediante microscopĂa confocal en pacientes con talasemia: resultados preliminares
Peer ReviewedPostprint (published version
Estudio espectroscĂłpico de glĂłbulos rojos mediante microscopĂa confocal en pacientes con talasemia: resultados preliminares
Peer Reviewe