Evaluación De Quimerismo En Pacientes Con Injerto De Células Progenitoras Hematopoyéticas En Panamá Del 2000­20 / Evaluation of Chimerism in Patients with Graft Hematopoietic Progenitor Cells in Panama from 2000­2018

Introducción: El Injerto de células progenitoras hematopoy éticas (ICPH) es actualmente un trata­miento para diferentes desórdenes hematológicos malignos y no malignos. El análisis del quimeris­mo post ICPH, y la cuantificación de cada población celular, deben ser monitoreados. El presente trabajo tiene como objetivo: el evaluar los res ultados de quimerismo completo y mixto en sangre periférica del receptor pos trasplante obtenidos por método cualitativo y cuantitativo del año 2000 al 2018. Material y método: El presente es un estudio descriptivo, observacional, transversal de dos mé­ todos de quimerismo efectuados a receptores y donantes de ICPH alogénico. Resultados: De los 79 pacientes estudiados por el método cualitativo: 65 (82.2%) resultaron con qui­ merismo completo y 14 (17.7%) con quimerismo mixto. No fue posible cuantificar por este método el % de células del donante y del receptor.Conclusión: El método cuantitativo es un método exacto, que determina el % de células del receptor y del donante presentes en la muestra. Con este método se evalúan un mayor número de marcadores genéticos que con el método cualitativo, y se obtienen un mayor número de loci informativos del quimerismo al compararlo con el método cualitativo.

Introduction: Hematopoietic progenitor cell grafting (ICPH) is currently a treatment for different ma­ lignant and non­malignant hematological disorders. The analy sis of post­ICPH chimerism, and the quantification of each cell population, should be monitored. The present work has as objective: to evaluate the results of complete and mixed chimerism in peripheral blood of the post­trans plant reci­pient obtained by qualitative and quantitative method from the year 2000 to 2018. Material and method: The present is a descriptive, observational, cross­sectional study of two met­ hods of chimerism performed on allogeneic ICPH recipients and donors . Results: Of the 79 patients studied by the qualitative method: 65 (82.2%) resulted with complete chi­ merism and 14 (17.7%) with mixed chimerism. It was not possible to quantify by this method the% of donor and recipient cells. Conclusion: The quantitative method is an exact method, which determi­nes the% of recipient and donor cells present in the sample. With this method, a greater number of genetic markers are evaluated than in the qualitative method, and a greater number of information loci of chimerism are obtained than with the qualitative method.

Late chimerical status after bone marrow transplantation in severe aplastic anemia according to two different preparatory regimens

ABSTRACT Background: This study investigated the influence of two conditioning regimens on the chimerical status of 104 patients with acquired severe aplastic anemia. Methods: Patients were monitored for at least 18 months after related bone marrow transplantation and reaching partial or complete hematologic recovery. Group I patients (n = 55) received 200 mg/kg cyclophosphamide alone and Group II (n = 49) received 120 mg/kg cyclophosphamide associated with 12 mg/kg busulfan. Patients were classified in three chimerism levels according to the percentage of donor cells in the peripheral blood. Results: Chimerism ≤50% occurred in 36.4% of Group I and none of Group II; chimerism 51-90% was found in 20.0% of Group I and 10.2% of Group II; and chimerism >90% was found in 43.6% of Group I versus 89.8% of Group II. A significant association (p-value < 0.001) was found between conditioning type and chimerism levels. A higher number of infused cells was associated with higher levels of chimerism only in Group I (p-value = 0.013). Multivariate analysis showed that chimerism >90% is associated with the cyclophosphamide plus busulfan conditioning (p-value < 0.001) and higher number of infused cells (p-value = 0.009), suggesting that these factors are predictive of graft outcome. Regarding hematological recovery, higher chimerism levels were associated with higher neutrophil (p-value = 0.003) and platelet counts (p-value < 0.001) in Group I only. These results show that myeloablative conditioning favors full donor chimerism and non-myeloablative conditioning predisposes to mixed chimerism or autologous recovery of hematopoiesis. Conclusion: These data show that autologous recovery depends on the intensity of immunosuppression and that the immunosuppressive function of cyclophosphamide alone can induce this type of hematopoietic recovery.

The role of fetal-maternal microchimerism as a natural-born healer in integrity improvement of maternal damaged kidney

ABSTRACT Purpose: To identify the fetal stem cell (FSC) response to maternal renal injury with emphasis on renal integrity improvement and Y chromosome detection in damaged maternal kidney. Materials and Methods: Eight non-green fluorescent protein (GFP) transgenic Sprague-Dawley rats were mated with GFP-positive transgenic male rats. Renal damage was induced on the right kidney at gestational day 11. The same procedure was performed in eight non-pregnant rats as control group. Three months after delivery, right ne- phrectomy was performed in order to evaluate the injured kidney. The fresh perfused kidneys were stained with anti-GFP antibody. Polymerase chain reaction (PCR) assay was also performed for the Y chromosome detection. Cell culture was performed to detect the GFP-positive cells. Technetium-99m-DMSA renal scan and single-photon emission computed tomography (SPECT) were performed after renal damage induction and 3 months later to evaluate the improvement of renal integrity. Results: The presence of FSCs was confirmed by immune histochemical staining as well as immunofluorescent imaging of the damaged part. Gradient PCR of female rat purified DNA demonstrated the presence of Y-chromosome in the damaged maternal kidney. Moreover, the culture of kidney cells showed GPF- positive cells by immuno- fluorescence microscopy. The acute renal scar was repaired and the integrity of dam- aged kidney reached to near normal levels in experimental group as shown in DMSA scan. However, no significant improvement was observed in control group. Conclusion: FSC seems to be the main mechanism in repairing of the maternal renal injury during pregnancy as indicated by Y chromosome and GFP-positive cells in the sub-cultured medium.

Cytogenetics and Molecular Genetic Analysis of Chimerism in Marmosets (Callithrix: Primates)

ABSTRACT The birth of fraternal twins is a characteristic frequently observed in callitrichids. Cytogenetic studies have demonstrated hematopoietic chimerism in marmosets with the occurrence of two cell lines 2n=46,XX/46,XY in females and males co-twins, without phenotypic changes. Amplification by PCR have also been used to verify the presence of the SRY gene in female chimaeras. Our aim was to verify the occurrence of chimerism in Callithrix sp. individuals considered as hybrids according to their intermediate phenotypes between C. jacchus and C. penicillata. Blood samples from 37 Callithrix sp. individuals were collected. Hematopoietic chimerism 2n=46,XX/46,XY was detected by cytogenetic analysis in five individuals, three males and two females. A fragment of approximately 200bp of the SRY gene was amplified in seven females with normal external genitalia. The percentage of 32% of chimeric individuals detected in the present study is similar to that observed for pure specimens of Callithrix. These data suggests that hybridization probably does not interfere with the occurrence of twin gestation, nor of chimerism. Although cytogenetics is the main tool to identify the two cell lineages present in cases of chimerism, the amplification of the SRY gene by PCR has proved to be more efficient to identify the Y chromosome in cases of chimeric female marmoset.

Implicaciones médico legales del quimerismo / Medicolegal implications of chimerstry

Los recientes avances en tecnologías biomédicas proporcionan herramientas a la Medicina Legal para el esclarecimiento de casos complejos y ejercer justicia basada en evidencia científica. ¿Pero qué ocurre cuando se imponen obstáculos como el quimerismo que podrían llevar a decisiones equivocadas? A pesar de que este fenómeno se creía casi inexistente, se han reportado interesantes casos, que han puesto a prueba la utilidad de las pruebas de ADN. El quimerismo se define como la presencia de líneas celulares con distinto material genético proveniente de diferentes orígenes en un único cuerpo. Esto tiene grandes implicaciones medico legales, principalmente en la investigación criminal. Por ejemplo en una escena de crimen, se pueden encontrar muestras de tejidos de un mismo individuo pero estas podrían tener ADN distinto si se trata de un individuo quimérico llevando a una mala interpretación de la información. También cabe resaltar las implicaciones del quimerismo en las pruebas de paternidad, ya que este fenómeno puede ocasionar falsos negativos en estas pruebas y por lo tanto un diagnóstico incierto de paternidad. El objetivo de esta revisión es describir las diferentes implicaciones médico legales del quimerismo y proponer posibles soluciones a los conflictos que podrían presentarse ante tales casos.

Recent advances in biomedical technologies are able to clarify today’s complex cases that are faced by Legal Medicine; allowing this branch of medicine to exercise justice based in scientific evidence. But what happens when an obstacle such as chimerism is present and may lead to false decisions? Although this phenomenon was thought to be inexistent, interesting cases have been reported. Chimerism is defined as the presence of different cell linings in a unique organism. This phenomenon has important implication in Legal Medicine, especially in criminal investigation. For example, in a crime scene the samples gathered may present different DNA and lead to misinterpretation of the information. On the other hand, paternity tests are also implicated in the presence of a chimera since it may originate a false negative result. The purpose of this review is to describe different Legal Medicine’s implications linked to chimerism and propose possible solutions to the conflicts that may arouse from a case of a chimera.

Quantification of mixed chimerism allows early therapeutic interventions

Hematopoietic stem cell transplantation is the curative option for patients with myelodysplastic syndrome; however, it requires a long post-transplantation follow-up. A 53-year-old woman with a diagnosis of myelodysplastic syndrome underwent related donor allogeneic hematopoietic stem cell transplantation in July 2006. Three months after transplantation, a comparative short tandem repeat analysis between donor and recipient revealed full chimerism, indicating complete, healthy bone marrow reconstitution. Three years and ten months after hematopoietic stem cell transplantation, the patient developed leukopenia and thrombocytopenia. Another short tandem repeat analysis was carried out which showed mixed chimerism (52.62%), indicating relapsed disease. A donor lymphocyte infusion was administered. The purpose of donor lymphocyte infusion is to induce a graft-versus-leukemia effect; in fact, this donor's lymphocyte infusion induced full chimerism. Successive short tandem repeat analyses were performed as part of post-transplantation follow-up, and in July 2010, one such analysis again showed mixed chimerism (64.25%). Based on this finding, a second donor lymphocyte infusion was administered, but failed to eradicate the disease. In September 2011, the patient presented with relapsed disease, and a second related donor allogeneic hematopoietic stem cell transplantation was performed. Subsequent short tandem repeat analyses revealed full chimerism, indicating complete bone marrow reconstitution. We conclude that quantitative detection of mixed chimerism is an important diagnostic tool that can guide early therapeutic intervention...

Microquimerismo en dermtología / Microchimerism in dermatology

Se denomina microquimerismo a la presencia en un organismo de una pequeña población de células o ADN que proviene de otro individuo genéticamente diferente. La causa más común de microquimerismo adquirido en forma natural es el tráfico bidireccional de células que se produce durante el embarazo entre la madre y el feto. Puede tener efectos benéficos o nocivospara el huésped, dependiendo de múltiples factores. Ha sido implicado en la patogenia de enfermedades autoinmunes como la esclerodermia, el lupus eritematoso sistémico, el lupusneonatal y la dermatomiositis, en la reparación de tejidos y el cáncer...

Etiopatogenia de la esclerosis sistémica progresiva / Etiopathogenesis of systemic sclerosis

La esclerosis sistémica progresiva (ESP) es una enfermedad autoinmune crónica del tejido conectivo. Se caracteriza por una alteración vascular inicial, una respuesta inmune alterada con producción de autoanticuerpos y un exceso de síntesis y depósito de fibras de colágeno en la piel y tejido conectivo. La activación y el daño endotelial son eventos tempranos en la patogenia de la enfermedad; sin embargo, el factor gatillante continúa siendo desconocido. Se piensa que el evento principal sería la interacción entre eventos autoinmunes y cambios vasculares tempranos, lo cual resulta en la generación de fibroblastos activados considerados como las células efectoras de la enfermedad. Se reconocen dos subgrupos clínicos de ESP: la variedad cutánea limitada y la variedad cutánea difusa, las cuales presentan distintos patrones de compromiso orgánico, autoanticuerpos, evolución y sobrevida.

Progressive systemic sclerosis is a chronic autoimmune disease of connective tissue. It is characterized by early vascular changes, altered immune response with production of auto-antibodies, and excessive synthesis and deposition of collagen fibers in the skin and connective tissue. Activation and endothelial cell damage are early events in the pathogenesis of the disease, but the precise triggering event(s) remain elusive. The main event would be the interaction between autoimmune events early vascular changes, resulting in the generation of activated fibroblasts, regarded as effector cells of the disease. There are two major subgroups of SSc, the limited cutaneous and the diffuse cutaneous variety, which have distinct patterns of organ involvement, self-auto-antibodies, evolution and survival.

Microquimerismo em glândulas salivares labiais de pacientes submetidas a transplante de células tronco hematopoiéticas / Microchimerism in labial salivary glands of hematopoietic stem cell transplanted patients

Objetivo: o Microquimerismo se refere a uma pequena quantidade de células ou DNA de um indivíduo presentes em outro indivíduo. O seu papel nas doenças autoimunes com semelhanças à doença do enxerto contra o hospedeiro tem sido muito estudado. Este trabalho teve como objetivo determinar se o microquimerismo do cromossomo-Y está presente em glândulas salivares labiais de mulheres submetidas a transplantes de células tronco hematopoiéticas com doadores do gênero masculino. Metodologia: onze amostras de glândulas salivares labiais de pacientes submetidos a transplante de células tronco hematopoiéticas alogênicos foram obtidas dos arquivos do Laboratório de Patologia Bucal da Faculdade de Odontologia da UFMG. Nenhum dos autores teve conhecimento prévio do graus de cGVHD das mesmas. Destas amostras, cinco eram de transplante entre gêneros feminino-feminino (grupo controle) e cinco entre masculino-feminino (grupo estudo), além de uma do gênero masculino (controle positivo)...

Quimerismo genético un nuevo paradigma para la medicina legal / Genetic chimerism a new paradigm for legal medicine

El quimerismo genético un concepto no bien esclarecido en la comunidad científica, aún confundido con mosaicismo. En la presente revisión pretendemos definirlo con un enfoque legal y ejemplificarlo con una serie de casos descritos en la literatura médica, los cuales nos orientan a la diferencia clara entre mosaicismo y quimerismo. Dentro de esta serie separamos los casos que se relacionan con hermafroditismo y los no relacionados. Además, hacemos referencia a un nuevo concepto que genera polémica en la investigación, el microquimerismo, relacionado con transfusiones sanguíneas, transplantes y la génesis de las enfermedades autoinmunes.

Genetic chimerism is a not well defined concept among the scientific community; it is still confused with mosaicism. In the following review we intend to define the concept with a legal point of view and exemplify it with a series of cases described in medical texts. The cases lead us to find the clear difference between mosaicism and chimerism. Contained in these series of cases we have separated the ones related with hemaphroditism from the ones that are not related. We also make reference to a new concept that generates controversy in the investigation; microchimerism, which is related with blood transfusions, transplants and genesis of autoimmune diseases..

Cariotipo del tití gris (Saguinus leucopus): similitudes con el cariotipo humano / Karyotype of gray marmoset (Saguinus leucopus): similarities with the human karyotype

El tití gris (Saguinus leucopus) es un primate endémico de Colombia cuyo cariotipo se describe en el presente estudio a partir de una pareja de individuos ubicados en el Centro de Rehabilitación de Fauna Silvestre del Oriente de Caldas, Colombia. Las muestras de sangre fueron recolectadas de la vena femoral y anticoaguladas con heparina de sodio. Los cromosomas se obtuvieron por el método clásico de cultivo de linfocitos y bandeamiento Q y G Los individuos presentan 46 cromosomas (2n = 46: 30Bi, 14A); cromosomas sexuales XX en la hembra y XY en el macho (quimerismo 46,XX/46,XY en este último). Se propone un ideograma para el cariotipo del Tití Gris. Se observan amplias semejanzas en los cromosomas X y 5 de S. leucopus con los cromosomas X y 19 humanos, respectivamente. Otras similitudes parciales se evidenciaron entre los cromosomas 1 de ambas especies, 2 y 14 de S. leucopus con el 7 humano. La comparación del tamaño de regiones exónicas de dos genes de S. leucopus y Homo sapiens no arrojó diferencia.

Avaliação do valor prognóstico da detecção do status quimérico de pacientes após o transplante alogênico de células progenitoras hematopoéticas / Evaluation of prognostic importance of the detection of chimerical status in post-allogeneic progenitor cell transplantation patients

Este trabalho teve por objetivo correlacionar o status quimérico de pacientes pós -TCPH alogênico com parâmetros clínicos, para avaliar o valor preditivo dos achados laboratorias de quimerismo. Amostras de sangue de 98 pacientes (67 em seguimento e 31 novos casos) foram submetidas à análise do status quimérico pós-TCPH. Os "loci"analisados por biologia molecular foram CS1PO, TPOX, F13A1, FESFPS, HUMTH01, VWA, SE33, HUMARA, HUMD21S11 e Amelogenina. Precocidade da evidência laboratorial de quimerismo misto (QM), em relação ao aparecimento dos sintomas clínicos de recaída, foi observada em 9 dos 12 pacientes nas LA, ou seja, nesses casos, a primeira manifestação de QM foi detectada pelo exame laboratorial antes de qualquer evidência citológica ou clínica de recaída. Em todos eles, houve uma mudança terapêutica relacionada com esse momento do aparecimento do QM. Em 100 por cento dos pacientes com QM na LMC, a detecção do quimerismo pelo exame laboratorial foi anterior a qualquer evidência citológica ou clínica de recaída. De uma maneira geral, o exame laboratorial da avaliação do status quimérico pós-TCPH alogênico pela análise dos "loci"hipervariáveis do genoma, mostrou ser um exame sensível, com detecção de até 1 por cento de QM e precoce, visto que, muitas vezes, foi a primeira manifestação de doença residual antes de qualquer evidência citológica ou clínica da mesma. A associação da existência de QM e a recaída clínica e/ou óbito fica mais evidente nos casos de LA do que nos casos de LMC e AAS.

This study aimed to correlate the chimerical status in post-allogeneic hematopoietic stem cell transplantation (HSCT) patients to clinical patterns in order to evaluate the predictive value of chimerism laboratorial findings. Blood samples from 98 patients (67 current and 31 new cases) were submitted to post-HSCT chimerical status analysis. The CS1PO, TPOX, F13A1, FESFPS, HUMTH01, VWA, SE33, HUMARA, HUMD21S11 and Amelogenian loci were analyzed. Precocity of Mixed Chimerism (MC) laboratorial evidence in relation to recurrent clinical symptom manifestations was observed in 9 out of 12 patients in AL, i.e., in these cases the first MC manifestation was detected in laboratory tests before any cytological or clinical evidence. In all cases, there was a therapeutic change due to MC onset. Chimerism detection through laboratorial examinations was prior to any cytological or clinical evidence in 100 percent of patients presenting MC in CML. Considering that it was the first manifestation of residual disease, before any cytological or clinical manifestation, laboratorial examination to evaluate chimerical status in post-allogeneic hematopoietic stem cell transplantation through analysis of genome hyper-variable loci, turned out to be a more sensitive examination and presented a detection rate of up to 1 percent for early MC. The association of MC to clinical recurrence and/death is more evident in AL cases than in CML and SAA.

Utilização da técnica de PCR para monitoramento molecular de quimerismo pós-transplantes / Using PRC for molecular monitoring of p´post transpalntation chimerism

O monitoramento pós-transplante de órgãos sólidos e medula óssea tornou-se uma ferramenta imprescindível para aumentar a sobrevida dos enxertos, sendo o termo quimerismo utilizado paradescrever a presença de células do doador nos pacientes receptores de transplantes. O presente artigo descreve as diversas aplicações e avanços nas técnicas que podem ser utilizadas para a detecção e monitoramento quantitativo do quimerismo póstransplantes,em especial a metodologia de PCR utilizando oligonucleotídeos fluorescentes e seqüenciadores automáticos para amplificação e detecção de marcadores conhecidos como microssatélites, ou STRs.

Estudio de quimerismo: aplicación en pacientes con trasplante de progenitores hematopoyéticos / Chimerism study: application in patients with allogeneic progenitor cell transplantation

El trasplante alogénico de progenitores hematopoyéticos (TAPH) es una técnica que ha cambiado el pronóstico de muchas enfermedades hematológicas malignas y no malignas. En determinados tiempos post trasplante coexisten células hematológicas de receptor y dador, por lo cual el individuo posee dos sistemas hematopoyéticos. El término Quimera se utiliza para indicar el origen dual de las células hematológicas. Este análisis es indispensable para saber si existe prendimiento o rechazo del trasplante. La determinación de quimerismo se realiza mediante la amplificación por PCR de secuencias cortas repetidas tandem (STRs del ingles short tandem repeat sequence). Este examen es fácil de realizar, reproducible, altamente sensible y específico. El resultado del quimerismo es de vital importancia a la hora de tomar decisiones clínicas, sobre todo si se utilizan técnicas no mieloablativas de acondicionamiento, infusión de linfocitos del donante, o modificaciones en los protocolos de inmunosupresión, donde existe una lata variabilidad en el prendimiento del injerto y el desarrollo de enfermedad de injerto contra huésped (EICH) e injerto contra tumor (ICT).

Allogeneic progenitor cell transplantation (ALoPCT) is a procedure that has changed the prognosis of many malignant and non-malignant hematologic diseases. For a period of time post-trasplant, cellular subtypes from the donor and the host coexist, giving the patient two hematopoietic systems. The term Chimera is used to indicate the dual origin of blood cells. This analysis is important to determine whether engrafment or rejection has occurred. The determination of chimerism is based on PCR pf Short Tandem Repeat sequences (STRs). The PCR technique is easy to perform, reproducible, highly sensitive and specific. Chimerism determination helps to improve the clinical approach to the patient, specifically when non-mieloablative conditioning, lymphocyte donor infusion or modification of the immunosuppressive protocols have employed. All of these manipulations produce a high variability in engraftment, development of graft versus host disease (GVHD) and the likelihood to eliminate tumor cells through graft versus tumor (GVT) effects.