Establishment of a 21-color Panel for the Detection of Immune Cell Subsets in Human Non-small Cell Lung Cancer Tumor Tissues with Flow Cytometry / 中国肺癌杂志

BACKGROUND@#With the rise of multicolor flow cytometry, flow cytometry has become an important means to detect the immune microenvironment of lung cancer, but most of them are used to detect the proportion of cell subsets or the function of major cell subsets, and they cannot be detected at the same time. Therefore, a reliable 21-color flow cytometry protocol was established to detect the immune cell subsets in human non-small cell lung cancer (NSCLC) tumor tissues.@*METHODS@#Cell membrane surface antibodies cluster of differentiation (CD)45, CD3, CD19, CD4, CD8, programmed cell death 1 (PD-1), CD39, CD103, CD25, CD127, chemokine receptor 8 (CCR8), CD56, CD11c, human leukocyte antigen (HLA)-DR, CD38, CD27, CD69, CD62L, CD45RA, CCR7 and nucleic acid dye L/D were used to develop the protocol. Firstly, antibody titration experiments, voltage optimization, subtraction of one color staining and single color staining experiments were carried out for each antibody, and after the experimental conditions and detection schemes were determined, the feasibility of the scheme was verified by using peripheral blood mononuclear cells (PBMCs) specimens of six healthy adult volunteers. Tumor tissue samples from 6 NSCLC patients were tested and analyzed.@*RESULTS@#The established 21-color flow cytometry protocol was used to detect the tumor tissue samples of 6 NSCLC patients, and the proportion of each cell subset in lung cancer tissue, as well as the immunophenotype and differentiation of the main cell population, were analyzed.@*CONCLUSIONS@#The successfully established 21-color flow cytometry protocol is suitable for the detection of PBMCs and NSCLC tissue samples, which provides an effective new idea for monitoring the immune microenvironment status in lung cancer.

Estudio del líquido cefalorraquídeo en pacientes con neoplasias hematológicas malignas / Study of cerebrospinal fluid in patients with hematologic malignancies

Introducción: La infiltración del sistema nervioso central por células malignas constituye una complicación grave de algunas neoplasias hematológicas, principalmente leucemias agudas y linfomas agresivos. Objetivo: Resumir la base científica y la significación clínica de los métodos de estudio del líquido cefalorraquídeo para el diagnóstico y el seguimiento de la infiltración neuromeníngea en pacientes con neoplasias hematológicas. Métodos: Se buscó información durante abril de 2021 en las bases de datos PubMed, ScienceDirect y SciELO. Se seleccionaron las publicaciones en base a su tipología, actualidad, alcance y las limitaciones de los estudios. Conclusiones: El estudio citomorfológico del líquido cefalorraquídeo se considera el método estándar para el diagnóstico y el seguimiento de la infiltración neuromeníngea. La citometría de flujo resulta más sensible para la detección de infiltración oculta que la citología convencional; pero aún existen reservas sobre su significación clínica. Se investiga también la sensibilidad de otros estudios moleculares como el uso de la reacción en cadena de la polimerasa y la detección de biomarcadores(AU)

Introduction: Infiltration of the central nervous system by malignant cells constitutes a serious complication of some hematological malignancies, mainly acute leukemias and aggressive lymphomas. Objective: To summarize the scientific basis and clinical significance of cerebrospinal fluid study methods for the diagnosis and follow-up of neuromeningeal infiltration in patients with hematologic malignancies. Methods: Information was searched during April 2021 in PubMed, ScienceDirect and SciELO databases. Publications were selected based on their typology, timeliness, scope, and study limitations. Conclusions: The cytomorphological study of cerebrospinal fluid is considered the standard method for the diagnosis and follow-up of neuromeningeal infiltration. Flow cytometry is more sensitive for the detection of occult infiltration than conventional cytology, but there are still reservations about its clinical significance. The sensitivity of other molecular studies such as the use of PCR and biomarker detection is also investigated(AU)

Cobalt Chloride as a Hypoxia Mimicking Agent Induced HIF-1α and mTOR Expressions of Human Umbilical Cord Mesenchymal Stem Cells

ABSTRACT Objective: To assess the effects of cobalt chloride (CoCl2) as a hypoxia mimicking agent on human umbilical cord mesenchymal stem cells (hUCMSCs) expression of HIF-1α and mTOR for use in regenerative dentistry. Material and Methods: Human umbilical cord mesenchymal stem cells were isolated and then cultured. The characteristics of stemness were screened and confirmed by flow cytometry. The experiment was conducted on hypoxia (H) and normoxia (N) groups. Each group was divided and incubated into 24-, 48-, and 72-hours observations. Hypoxic treatment was performed using 100 µM CoCl2 on 5th passage cells in a conventional incubator (37°C; 5CO2). Then, immunofluorescence of HIF-1α and mTOR was done. Data was analyzed statistically using One-way ANOVA and Tukey's HSD. Results: Significant differences were found between normoxic and hypoxic groups on HIF-1α (p=0.015) and mTOR (p=0.000) expressions. The highest HIF-1α expression was found at 48 hours in the hypoxia group, while for mTOR at 24 hours in the hypoxia group. Conclusion: Hypoxia using cobalt chloride was able to increase human umbilical cord mesenchymal stem cells expression of HIF-1α and mTOR.

Effect of Survivin Down-Regulation by Egr1-Survivin shRNA Combined with Radiotherapy on Radiosensitivity of Esophageal Squamous Carcinoma / Efecto de la Regulación Negativa de Survivina por shRNA de Egr1-Survivina Combinado con Radioterapia sobre la Radiosensibilidad del Carcinoma Escamoso de Esófago

SUMMARY: This study is to investigate the effect of survivin down-regulation by Egr1-survivin shRNA combined with radiotherapy on the apoptosis and radiosensitivity of esophageal squamous cell carcinoma ECA109 and KYSE150 cells. ECA109 and KYSE150 cells were transfected with Egr1-survivin shRNA, and then treated with radiotherapy. After 24 h, the mRNA and protein levels of Egr1-survivin were detected by qPCR and Western-Blot. Cell cycle and apoptosis were detected by flow cytometry. Western blot also detected levels of cleavaged Caspase 3 and Caspase 9. YM155 was used as a positive control to inhibit survivin expression. The levels of survivin mRNA and protein in ECA109 and KYSE150 cells treated with Egr1-survivin shRNA combined with radiotherapy were significantly lower than those of the blank control group, the empty vector control group, and, the YM155 + radiotherapy group (P<0.05). Meanwhile, after survivin down-regulation, the ratio of G2 to S phase of ECA109 and KYSE150 cells increased significantly, leading to significant G2 and S phase arrest. Additionally, apoptosis of ECA109 and KYSE150 cells increased significantly (P <0.01). Further, protein levels of cleavaged Caspase 3 and Caspase 9 significantly increased in Egr1-survivin shRNA combined with radiotherapy group. Egr1-survivin shRNA combined with radiotherapy can down-regulate survivin expression, which further increases the apoptosis, and enhances the radiosensitivity of ECA109 and KYSE150 cells.

Este estudio tuvo como objetivo investigar el efecto de la regulación negativa de survivina por el shRNA de Egr1-survivina combinado con radioterapia sobre la apoptosis y la radiosensibilidad del carcinoma de células escamosas de esófago Células ECA109 y KYSE150. Las células ECA109 y KYSE150 se transfectaron con shRNA de survivina Egr1 y luego se trataron con radioterapia. Después de 24 h, los niveles de ARNm y proteína de Egr1-survivina se detectaron mediante qPCR y Western-Blot. El ciclo celular y la apoptosis se detectaron mediante citometría de flujo. La transferencia Western también detectó niveles de Caspasa 3 y Caspasa 9 escindidas. Se usó YM155 como control positivo para inhibir la expresión de survivina. Los niveles de ARNm y proteína de survivina en células ECA109 y KYSE150 tratadas con shRNA de survivina Egr1 combinado con radioterapia fueron significativamente más bajos que los del grupo control en blanco, el grupo control de vector vacío y el grupo de radioterapia YM155 + (P <0,05). Mientras tanto, después de la regulación negativa de survivina, la proporción entre las fases G2 y S de las células ECA109 y KYSE150 aumentó significativamente, lo que llevó a una detención significativa de las fases G2 y S. Además, la apoptosis de las células ECA109 y KYSE150 aumentó significativamente (P <0,01). Además, los niveles de proteína de Caspasa 3 y Caspasa 9 escindidas aumentaron significativamente en el shRNA de Egr1- survivina combinado con el grupo de radioterapia. El shRNA de survivina de Egr1 combinado con radioterapia puede regular negativamente la expresión de survivina, lo que aumenta aún más la apoptosis y mejora la radiosensibilidad de las células ECA109 y KYSE150.

Development, Histological, and Ultrastructural Evaluation of Sheep Hyperimmune Serum Therapy for COVID-19 / Desarrollo, Evaluación Histológica y Ultraestructural de la Terapia con Suero Hiperinmune de Oveja para COVID-19

SUMMARY: In response to the threat posed by new variants of SARS-CoV-2 and the urgent need for effective treatments in the absence of vaccines, the aim of this study was to develop a rapid and cost-effective hyperimmune serum (HS) derived from sheep and assess its efficacy. The utilization of a halal-certified, easily maintained in certain geographic regions, easy-to-handle animal such as sheep could provide a viable alternative to the expensive option of horses. Sheep were immunized with a whole inactivated SARS-CoV- 2 antigen to produce HS, which was evaluated for neutralizing potency using the PRNT50 assay. K18-hACE2 transgenic mice (n=35) were divided into three groups: control, SARS-CoV-2 exposure through inhalation, and SARS-CoV-2 exposed mice treated with HS. HS efficacy was assessed through serum proinflammatory cytokine levels, qRT-PCR analysis, histopathological examination of lungs and hearts, and transmission electron microscopy. Purified HS exhibited significant neutralizing activity (1/24,576). The SARS-CoV-2+HS group showed lower levels of TNF-α, IL-10, and IL-6 (P<0.01) and relatively lower levels of MCP-1 compared to the SARS-CoV-2 group. HS prevented death, reduced viral RNA levels in the lungs and hearts, protected against severe interstitial pneumonia, preserved lung tissue integrity, and prevented myocyte damage, while the SARS-CoV-2 group exhibited viral presence in the lungs. This study successfully developed a sheep-derived HS against the entire SARS-CoV-2 virus, resulting in a significant reduction in infection severity, inflammation, and systemic cytokine production. The findings hold promise for treating severe COVID-19 cases, including emerging viral variants, and immunocompromised patients.

En respuesta a la amenaza que suponen las nuevas variantes del SARS-CoV-2 y la urgente necesidad de tratamientos eficaces en ausencia de vacunas, el objetivo de este estudio fue desarrollar un suero hiperinmune (HS) rápido y rentable derivado de ovejas. y evaluar su eficacia. La utilización de un animal con certificación halal, de fácil mantenimiento en determinadas regiones geográficas y de fácil manejo, como las ovejas, podría proporcionar una alternativa viable a la costosa opción de los caballos. Las ovejas fueron inmunizadas con un antígeno de SARS-CoV-2 completamente inactivado para producir HS, cuya potencia neutralizante se evaluó mediante el ensayo PRNT50. Los ratones transgénicos K18-hACE2 (n = 35) se dividieron en tres grupos: control, exposición al SARS-CoV-2 mediante inhalación y ratones expuestos al SARS-CoV-2 tratados con HS. La eficacia de HS se evaluó mediante niveles de citoquinas proinflamatorias en suero, análisis qRT-PCR, examen histopatológico de pulmones y corazones y microscopía electrónica de transmisión. El HS purificado exhibió una actividad neutralizante significativa (1/24,576). El grupo SARS-CoV-2+HS mostró niveles más bajos de TNF-α, IL-10 e IL-6 (P<0,01) y niveles relativamente más bajos de MCP-1 en comparación con el grupo SARS-CoV-2. HS evitó la muerte, redujo los niveles de ARN viral en los pulmones y el corazón, protegió contra la neumonía intersticial grave, preservó la integridad del tejido pulmonar y evitó el daño de los miocitos, mientras que el grupo SARS-CoV-2 exhibió presencia viral en los pulmones. Este estudio desarrolló con éxito un HS derivado de ovejas contra todo el virus SARS-CoV-2, lo que resultó en una reducción significativa de la gravedad de la infección, la inflamación y la producción sistémica de citocinas. Los hallazgos son prometedores para el tratamiento de casos graves de COVID- 19, incluidas las variantes virales emergentes y los pacientes inmunocomprometidos.

Differential Effect of ITE on the Aryl Hydrocarbon Receptor Signaling in Breast Cancer Tissue: A Histopathological Study / Efecto Diferencial de ITE en la Señalización del Receptor de Hidrocarburo de Arilo en Tejido de Cáncer de Mama: Un Estudio Histopatológico

SUMMARY: Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is highly expressed in various types of cancers including breast cancer. However, the role of AhR with its endogenous ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) on the progression of breast cancer remains poorly understood. We aimed to investigate cell proliferation and migration states in breast cancer after activating AhR with the endogenous ligand ITE. Breast cancer tissue was evaluated by cell lines, immunohistochemistry, reverse transcription-polymerase chain reaction, cell proliferation, flow cytometry, migration assays and western blot techniques. We found that AhR was widely expressed in breast cancer tissues and metastasis lymph node tissues, but not in normal tissues. The expression AhR was independent between the age, grades and TNM classifications for breast cancer tissues. ITE treatment significantly induced the activation of AhR in a time-dependent manner in both MCF-7 and T47D breast cancer cell lines. Meanwhile, ITE did not affect the cell migration but significantly suppressed the cell proliferation in estrogen receptor positive (ER+) MCF-7 andT47D cells, which probably attribute to the induction of cell cycle arrest in G1 phase and shortened S phase. Further mechanism study showed that ERK1/2 and AKT signaling were required for the activation of AhR in MCF-7 cells. These data suggest that AhR is a potential new target for treating patients with breast cancer. ITE may be more potentially used for therapeutic intervention for breast cancer with the kind of ER(+).

El receptor de hidrocarburo de arilo (AhR) es un factor de transcripción activado por ligando que se expresa en gran medida en varios tipos de cáncer, incluido el cáncer de mama. Sin embargo, el papel de AhR con su ligando endógeno 2- (1'H-indol-3'-carbonil)-tiazol-4-ácido carboxílico metil éster (ITE) en la progresión del cáncer de mama sigue siendo poco conocido. Nuestro objetivo fue investigar la proliferación celular y los estados de migración en el cáncer de mama después de activar AhR con el ligando endógeno ITE. El tejido de cáncer de mama se evaluó mediante líneas celulares, inmunohistoquímica, reacción en cadena de la polimerasa con transcriptasa inversa, proliferación celular, citometría de flujo, ensayos de migración y técnicas de transferencia Western. Descubrimos que AhR se expresó ampliamente en tejidos de cáncer de mama y en linfonodos con metástasis, pero no en tejidos normales. La expresión AhR fue independiente entre la edad, grados y clasificaciones TNM para tejidos de cáncer de mama. El tratamiento con ITE indujo significativamente la activación de AhR de manera dependiente del tiempo en las líneas celulares de cancer de mama MCF-7 y T47D. Mientras tanto, ITE no afectó la migración celular, pero suprimió significativamente la proliferación celular en células MCF-7 y T47D con receptor de estrógeno positivo (ER+), lo que probablemente se atribuye a la inducción de la detención del ciclo celular en la fase G1 y la fase S acortada. Un estudio adicional del mecanismo mostró que las señales de ERK1/2 y AKT eran necesarias para la activación de AhR en las células MCF-7. Estos datos sugieren que AhR es un nuevo objetivo potencial para el tratamiento de pacientes con cáncer de mama. ITE puede ser utilizado más potencialmente en la intervención terapéutica para el cáncer de mama con el tipo de ER (+).

Evaluación comparativa de medición de linfocitos CD4+ por dos técnicas: estándar nacional versus test rápido de ejecución local / Comparative evaluation of CD4+ lymphocyte measurement by two techniques: national standard versus point-of-care rapid test

ANTECEDENTES: El recuento de linfocitos CD4+ (LTCD4) es una herramienta fundamental para la evaluación y seguimiento de los pacientes que viven con VIH. En Chile, la medición de LTCD4 estandarizada es por citometría de flujo. En el sistema público se realiza en forma centralizada en tres centros. Actualmente existen tecnologías de medición rápida de recuento de LTCD4 en el lugar de atención, permitiendo optimizar la atención de pacientes con infección por VIH. OBJETIVO: Comparar la precisión de un test rápido de ejecución local versus la técnica estándar. METODOLOGÍA: Realización de ambas técnicas en un grupo de 102 pacientes durante su control regular de salud. RESULTADOS: El rango de variación promedio de los resultados entre las dos técnicas fue de 10%, con una concordancia en los recuentos de LTCD4 de 97% para el rango de CD4 < 200 cél/uL, de 88% para los pacientes con recuento de LTCD4 entre 200 y 349 cél/uL y de 67% en los rangos superiores. CONCLUSIÓN: La técnica por test rápido es un sistema fácil de aplicar, de bajo costo, con alta concordancia con la técnica estándar, lo que debería considerarse en la atención de los pacientes que viven con VIH.

BACKGROUND: The CD4+ lymphocyte cell count is an instrumental tool for the assessment and follow-up in the therapeutic management of patients living with HIV. In Chile, the standardized CD4+ lymphocyte count technique is by flow cytometry. In the public health system, it is performed centralized in 3 sites. Currently, there are technologies that allow measuring the CD4 lymphocyte count at the point of care, allowing to optimize the care of HIV-infected patients. AIM: To compare the accuracy of a point of care rapid test versus the standard technique in patients under regular care at a single HIV center. RESULTS: The average variation of the results between the two techniques was 10%, with a 97% concordance in CD4 range values for patients with CD4 below 200 cells/uL, 88% for CD4 counts between 200 and 349 cells/uL. and 67% above that range. CONCLUSION: This point of care test is an easy-to-operate, low-cost system with high correlation with the standard technique and should be considered in the care of patients living with HIV.

Enfermedad mínima residual en leucemia mieloide aguda no promielocítica: Resultados preliminares de un estudio colaborativo internacional / Minimal residual disease in non-myelocytic acute myeloid leukemia: preliminary results of an international collaborative study

A pesar de los avances en los protocolos de tratamiento y en las medidas de soporte en pacientes con Leucemia Mieloide Aguda (LMA), 27% presentan recaídas de la enfermedad. Esto se debe, entre otras causas, a la persistencia de pequeñas cantidades de células malignas (blastos) resistentes a la terapia. Estas pequeñas cantidades de blastos remanentes se denominan Enfermedad Mínima Residual (EMR). La determinación de EMR requiere de técnicas no solo muy sensibles, sino también específicas, y permite evaluar la respuesta individual a la terapia. La introducción de la EMR como parámetro de respuesta y estratificación está bien definida en Leucemia Linfoblástica Aguda (LLA). Por el contrario, aunque existen publicaciones sobre el impacto pronóstico de la EMR en LMA, aún no se encuentra incluida en forma sistemática en los protocolos nacionales actuales, entre otros motivos, por lo laborioso de la determinación y por la necesidad de validación de la misma. Debe tenerse en cuenta que el inmunofenotipo de los blastos mieloides suele ser más heterogéneo que el de los blastos en LLA, presentando, en muchos casos, subpoblaciones diferentes entre sí, lo cual dificulta su detección certera y no hay consenso definido en cuanto a la metodología más eficaz. En este trabajo describimos una nueva estrategia de marcación y análisis estandarizada en un estudio multicéntrico internacional para LMA y la utilidad de la EMR como parámetro de respuesta y de estratificación. Asimismo, detallamos los resultados preliminares de nuestra cohorte de pacientes (AU)

Despite the improvement in treatment and supportive care of patients with Acute Myeloid Leukemia (AML), 27% of them relapse. This is due to the persistence of small amounts of malignant cells (blasts) resistant to therapy, among other causes. These small amounts of blasts are called Minimal Residual Disease (MRD). The determination of MRD requires not only techniques with high sensitivity but also with high specificity, and allows to evaluate the individual response to treatment. The introduction of MRD as a response parameter is well established in Acute Lymphoblastic Leukemia (ALL), and it is used in current stratification protocols. On the other hand, even though there are some reports regarding the prognostic impact of MRD in AML, it is still not included in the current national protocols due to the lack of validation of the determination, among other causes. This is due to the fact that the immunophenotype of myeloid blasts is more heterogeneous than in ALL, presenting different subpopulations, which difficults their accurate detection. Thus, there is still no consensus regarding the most effective approach. In this article, we describe a new staining and analysis strategy standardized by an international multicentric study, and the utility of EMR as a response and stratification parameter. Additionally, we show the preliminary results of our patient cohort. (AU)

99mTc-HYNIC/Cy7-Fab(Bevacizumab): su empleo como agente de imagen en mieloma múltiple / 99mTc-HYNIC/Cy7-Fab(Bevacizumab): its use as an imaging agent in Multiple Myeloma / 99mTc-HYNIC/Cy7-Fab (Bevacizumab): seu uso como agente de imagem em mieloma múltiplo

Introducción: el mieloma múltiple es un trastorno hematológico maligno y el segundo cáncer de la sangre más frecuente. El proceso de la angiogénesis tumoral es fundamental para el crecimiento y metástasis de muchos tipos de tumores, incluido en mieloma múltiple. Se sabe que la sobreexpresión del factor de crecimiento endothelial vascular se encuentra asociado a un mal pronóstico en esta patología, representando un blanco clave para la terapia anti-angiogénica en mieloma múltiple. El anticuerpo monoclonal Bevacizumab es capaz de unirse con gran afinidad al factor de crecimiento endothelial vascular bloqueando su acción. Objetivo: evaluar el Fab(Bevacizumab) marcado con 99mTc o Cy7 como potenciales agentes de imagen moleculares de la expresión de factor de crecimiento endothelial vascular en mieloma múltiple. Material y métodos: la expresión de factor de crecimiento endothelial vascular fue analizada mediante citometría de flujo en la línea celular huaman de mieloma múltiple, la MM1S. Fab(Bevacizumab) fue producido mediante digestión de Bevacizumab con papaína, conjugado a NHS-HYNIC-Tfa y radiomarcado con 99mTc. Se realizaron estudios de biodistribución y de tomografía computarizada por emisión del fotón simple. A su vez, Fab(Bevacizumab) fue marcado con Cy7 para obtener imágenes de fluorescencia in vivo hasta 96 horas. Resultados: el análisis por citometría de flujo en la línea celular MM1S reveló que la expresión de factor de crecimiento endothelial vascular es predominantemente intracelular. Los estudios de biodistribución y SPECT/CT del complejo 99mTc-HYNIC-Fab(Bevacizumab) mostraron una rápida eliminación sanguínea y una significativa captación a nivel renal y tumoral. Las imágenes por fluorescencia empleando Cy7-Fab(Bevacizumab) permitieron la visualización tumoral hasta 96 h p.i. Conclusiones: logramos visualizar la expresión de factor de crecimiento endothelial vascular in vivo en mieloma múltiple mediante el empleo del fragmento Fab del anticuerpo anti-VEGF (Bevacizumab) marcado con 99mTc y Cy7. Estos nuevos agentes de imagen molecular podrían ser empleados potencialmente en el ámbito clínico para la estadificación y el seguimiento de pacientes con mieloma múltiple, mediante la visualización radioactiva in vivo de la expresión de factor de crecimiento endothelial vascular en todo el cuerpo. La imagen óptica de estos trazadores mejoraría el muestreo tumoral y podría guiar la extirpación quirúrgica.

Introduction: Multiple myeloma is a hematologic malignancy and the second most common blood cancer. The process of tumor angiogenesis is central to the growth and metastasis of many types of tumors, including multiple myeloma. Overexpression of vascular endothelial growth factor is known to be associated with poor prognosis in this pathology, representing a key target for anti-angiogenic therapy in multiple myeloma. The monoclonal antibody Bevacizumab is able to bind with high affinity to vascular endothelial growth factor blocking its action. Objective: to evaluate 99mTc- or Cy7-labeled Fab(Bevacizumab) as potential molecular imaging agents of vascular endothelial growth factor expression in multiple myeloma. Methods: Vascular endothelial growth factor expression was analyzed by flow cytometry in the multiple myeloma huaman cell line, MM1S. Fab(Bevacizumab) was produced by digestion of Bevacizumab with papain, conjugated to NHS-HYNIC-Tfa and radiolabeled with 99mTc. Biodistribution and single photon emission computed tomography studies were performed. In turn, Fab(Bevacizumab) was labeled with Cy7 to obtain in vivo fluorescence images up to 96 hours. Results: Flow cytometry analysis in the MM1S cell line revealed that vascular endothelial growth factor expression is predominantly intracellular. Biodistribution and SPECT/CT studies of the 99mTc-HYNIC-Fab(Bevacizumab) complex showed rapid blood clearance and significant renal and tumor uptake. Fluorescence imaging using Cy7-Fab(Bevacizumab) allowed tumor visualization up to 96 h p.i. Conclusions: we were able to visualize vascular endothelial growth factor expression in vivo in multiple myeloma using the Fab fragment of the anti-VEGF antibody (Bevacizumab) labeled with 99mTc and Cy7. These new molecular imaging agents could potentially be employed in the clinical setting for staging and monitoring of patients with multiple myeloma by in vivo radioactive visualization of vascular endothelial growth factor expression throughout the body. Optical imaging of these tracers would improve tumor sampling and could guide surgical excision.

Introdução: O mieloma múltiplo é uma malignidade hematológica e o segundo câncer de sangue mais comum. O processo de angiogênese tumoral é fundamental para o crescimento e a metástase de muitos tipos de tumores, incluindo o mieloma múltiplo. Sabe-se que a superexpressão do fator de crescimento endotelial vascular está associada a um prognóstico ruim no mieloma múltiplo, representando um alvo importante para a terapia antiangiogênica no mieloma múltiplo. O anticorpo monoclonal Bevacizumab é capaz de se ligar com alta afinidade ao fator de crescimento endotelial vascular e bloquear sua ação. Objetivo: avaliar o Fab(Bevacizumab) marcado com 99mTc ou Cy7 como possíveis agentes de imagem molecular da expressão do fator de crescimento endotelial vascular no mieloma múltiplo. Métodos: A expressão do fator de crescimento endotelial vascular foi analisada por citometria de fluxo na linha celular de mieloma múltiplo MM1S. O Fab(Bevacizumab) foi produzido pela digestão do Bevacizumab com papaína, conjugado com NHS-HYNIC-Tfa e radiomarcado com 99mTc. Foram realizados estudos de biodistribuição e tomografia computadorizada por emissão de fóton único. Por sua vez, o Fab(Bevacizumab) foi marcado com Cy7 para geração de imagens de fluorescência in vivo por até 96 horas. Resultados: A análise de citometria de fluxo na linha celular MM1S revelou que a expressão do fator de crescimento endotelial vascular é predominantemente intracelular. Os estudos de biodistribuição e SPECT/CT do complexo 99mTc-HYNIC-Fab(Bevacizumab) mostraram uma rápida depuração sanguínea e uma captação renal e tumoral significativa. A imagem de fluorescência usando Cy7-Fab(Bevacizumab) permitiu a visualização do tumor até 96 horas p.i. Conclusões: Conseguimos visualizar a expressão do fator de crescimento endotelial vascular in vivo no mieloma múltiplo usando o fragmento Fab do anticorpo anti-VEGF (Bevacizumab) marcado com 99mTc e Cy7. Esses novos agentes de imagem molecular poderiam ser usados no cenário clínico para o estadiamento e o monitoramento de pacientes com mieloma múltiplo, visualizando radioativamente a expressão do fator de crescimento endotelial vascular in vivo em todo o corpo. A geração de imagens ópticas desses traçadores melhoraria a amostragem do tumor e poderia orientar a excisão cirúrgica.

Diacerein mitigates renal ischemia/reperfusion injury via inhibition of renal inflammation, dendritic cells maturation and apoptosis: the role of TLR4/NF-kB/NLRP3/IL-1beta signaling pathway / La diacereína mitiga la lesión por isquemia/reperfusión renal a través de la inhibición de la inflamación renal, la maduración de las células dendríticas y la apoptosis: el papel de la vía de señalización TLR4/NF-kB/NLRP3/IL-1beta

SUMMARY: One of the reasons for acute kidney damage is renal ischemia. Nevertheless, there are limited protective and therapeutic approaches for this problem. Diacerein is an anti-inflammatory drug characterized by numerous biological activities. We aimed to determine the ameliorative impact of diacerein on renal ischemia/reperfusion injury (I/R) condition, exploring the underlying mechanisms. Twenty-four male rats were allotted into four groups (n= 6): sham group; Diacerein (DIA) group; I/R group, in which a non-crushing clamp occluded the left renal pedicle for 45 min, and the right kidney was nephrectomized for 5 min before the reperfusion process; I/R + diacerein group, injected intraperitoneally with 50 mg diacerein/kg i.m 30 minutes prior to I/R operation. Ischemia/ reperfusion was found to affect renal function and induce histopathological alterations. The flow cytometry analysis demonstrated an elevated expression of innate and mature dendritic cells in I/R renal tissues. Moreover, upregulation in the expression of the inflammatory genes (TLR4, Myd88, and NLRP3), and overexpression of the pro-inflammatory cytokines (IL-1β), apoptotic (caspase-3) and pyroptotic (caspase-1) markers were observed in I/R-experienced animals. The aforementioned deteriorations were mitigated by pre-I/R diacerein treatment. Diacerein alleviated I/R-induced inflammation and apoptosis. Thus, it could be a promising protective agent against I/R.

La isquemia renal es una de los motivos del daño renal agudo. Sin embargo, los enfoques protectores y terapéuticos para este problema son limitados. La diacereína es un fármaco antiinflamatorio caracterizado por numerosas actividades biológicas. Nuestro objetivo fue determinar el impacto de mejora de la diacereína en la condición de lesión por isquemia/ reperfusión renal (I/R), explorando los mecanismos subyacentes. Veinticuatro ratas macho se distribuyeron en cuatro grupos (n= 6): grupo simulado; grupo de diacereína (DIA); grupo I/R, en el que una pinza no aplastante ocluyó el pedículo renal izquierdo durante 45 min, y el riñón derecho fue nefrectomizado durante 5 min antes del proceso de reperfusión; Grupo I/R + diacereína, inyectado por vía intraperitoneal con 50 mg de diacereína/kg i.m. 30 min antes de la operación I/R. Se encontró que la isquemia/ reperfusión afecta la función renal e induce alteraciones histopatológicas. El análisis de citometría de flujo demostró una expresión elevada de células dendríticas innatas y maduras en tejidos renales I/R. Además, se observó una regulación positiva en la expresión de los genes inflamatorios (TLR4, Myd88 y NLRP3) y una sobreexpresión de las citoquinas proinflamatorias (IL-1β), marcadores apoptóticos (caspasa-3) y piroptóticos (caspasa-1) en animales con experiencia en I/R. Los deterioros antes mencionados fueron mitigados por el tratamiento previo a la diacereína I/R. La diacereína alivió la inflamación y la apoptosis inducidas por I/R. Por lo tanto, podría ser un agente protector prometedor contra I/R.

Over-expression of gastrin inhibits apoptosis of gastric cancer cells via reactive oxygen species and annexin A2-mediated mitochondrial dysfunction / La sobreexpresión de gastrina inhibe la apoptosis de las células de cáncer gástrico a través de especies reactivas de oxígeno y disfunción mitocondrial mediada por anexina A2

SUMMARY: Gastrin plays a vital role in the development and progression of gastric cancer (GC). Its expression is up-regulated in GC tissues and several GC cell lines. Yet, the underlying mechanism remains to be investigated. Here, we aim to investigate the role and mechanism of gastrin in GC proliferation. Gastrin-overexpressing GC cell model was constructed using SGC7901 cells. Then the differentially expressed proteins were identified by iTRAQ analysis. Next, we use flow cytometry and immunofluorescence to study the effect of gastrin on the mitochondrial potential and mitochondria-derived ROS production. Finally, we studied the underlying mechanism of gastrin regulating mitochondrial function using Co-IP, mass spectrometry and immunofluorescence. Overexpression of gastrin promoted GC cell proliferation in vitro and in vivo. A total of 173 proteins were expressed differently between the controls and gastrin- overexpression cells and most of these proteins were involved in tumorigenesis and cell proliferation. Among them, Cox17, Cox5B and ATP5J that were all localized to the mitochondrial respiratory chain were down-regulated in gastrin-overexpression cells. Furthermore, gastrin overexpression led to mitochondrial potential decrease and mitochondria-derived ROS increase. Additionally, gastrin-induced ROS generation resulted in the inhibition of cell apoptosis via activating NF-kB, inhibiting Bax expression and promoting Bcl-2 expression. Finally, we found gastrin interacted with mitochondrial membrane protein Annexin A2 using Co-IP and mass spectrometry. Overexpr ession of gastrin inhibits GC cell apoptosis by inducing mitochondrial dysfunction through interacting with mitochondrial protein Annexin A2, then up-regulating ROS production to activate NF-kB and further leading to Bax/Bcl-2 ratio decrease.

La gastrina juega un papel vital en el desarrollo y progresión del cáncer gástrico (CG). Su expresión está regulada al alza en tejidos de CG y en varias líneas celulares de CG. Sin embargo, el mecanismo subyacente aun no se ha investigado. El objetivo de este estudio fue investigar el papel y el mecanismo de la gastrina en la proliferación de CG. El modelo de células CG que sobre expresan gastrina se construyó usando células SGC7901. Luego, las proteínas expresadas diferencialmente se identificaron mediante análisis iTRAQ. A continuación, utilizamos la citometría de flujo y la inmunofluorescencia para estudiar el efecto de la gastrina en el potencial mitocondrial y la producción de ROS derivada de las mitocondrias. Finalmente, estudiamos el mecanismo subyacente de la gastrina que regula la función mitocondrial utilizando Co-IP, espectrometría de masas e inmunofluorescencia. La sobreexpresión de gastrina promovió la proliferación de células CG in vitro e in vivo. Un total de 173 proteínas se expresaron de manera diferente entre los controles y las células con sobreexpresión de gastrina y la mayoría de estas proteínas estaban implicadas en la tumorigenesis y la proliferación celular. Entre estas, Cox17, Cox5B y ATP5J, todas localizadas en la cadena respiratoria mitocondrial, estaban reguladas a la baja en las células con sobreexpresión de gastrina. Además, la sobreexpresión de gastrina provocó una disminución del potencial mitocondrial y un aumento de las ROS derivadas de las mitocondrias. Por otra parte, la generación de ROS inducida por gastrina resultó en la inhibición de la apoptosis celular mediante la activación de NF-kB, inhibiendo la expresión de Bax y promoviendo la expresión de Bcl-2. Finalmente, encontramos que la gastrina interactuaba con la proteína de membrana mitocondrial Anexina A2 usando Co-IP y espectrometría de masas. La sobreexpresión de gastrina inhibe la apoptosis de las células CG al inducir la disfunción mitocondrial a través de la interacción con la proteína mitocondrial Anexina A2, luego regula el aumento de la producción de ROS para activar NF-kB y conduce aún más a la disminución de la relación Bax/Bcl-2.

BLOC1S1 promotes proliferation of goat spermatogonial stem cells / 生物工程学报

With the rapid development of gene editing technology, the study of spermatogonial stem cells (SSCs) holds great significance in understanding spermatogenesis and its regulatory mechanism, developing transgenic animals, gene therapy, infertility treatment and protecting rare species. Biogenesis of lysosome-related organelles complex 1 subunit 1 (BLOC1S1) is believed to have anti-brucella potential. Exploring the impack of BLOC1S1 on goat SSCs not only helps investigate the ability of BLOC1S1 to promote SSCs proliferation, but also provides a cytological basis for disease-resistant breeding research. In this study, a BLOC1S1 overexpression vector was constructed by homologous recombination. The BLOC1S1 overexpression cell line of goat spermatogonial stem cells was successfully constructed by lentivirus packaging, transfection and puromycin screening. The overexpression efficiency of BLOC1S1 was found to be 18 times higher using real time quantitative PCR (RT-qPCR). Furthermore, the results from cell growth curve analysis, flow cytometry for cell cycle detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining showed that BLOC1S1 significantly increased the proliferation activity of goat SSCs. The results of RT-qPCR, immunofluorescence staining and Western blotting analyses revealed up-regulation of proliferation-related genes (PCNA, CDK2, CCND1), and EIF2S3Y, a key gene regulating the proliferation of spermatogonial stem cells. These findings strongly suggest that the proliferative ability of goat SSCs can be enhanced through the EIF2S3Y/ERK pathway. In summary, this study successfully created a goat spermatogonial stem cell BLOC1S1 overexpression cell line, which exhibited improved proliferation ability. This research laid the groundwork for exploring the regulatory role of BLOC1S1 in goat spermatogonia and provided a cell platform for further study into the biological function of BLOC1S1. These findings also establish a foundation for breeding BLOC1S1 overexpressing goats.

Characterization and identification of primarily cultured astrocytes and microglia / 生物工程学报

The aim of this study was to investigate the growth characteristics of primarily cultured astrocytes and microglia of different generations and then optimize the method for obtaining primary astrocytes and microglia effectively. Primarily cultured microglia were isolated and purified from the cortices of neonatal mice. The proliferation curve of mixed glia cells was measured by Cell Counting Kit-8 (CCK-8) assay, the proportion of astrocytes and microglia was detected by flow cytometry, and the polarization of the two types of glia cells was identified by immunofluorescence staining. Cell growth results showed that the mixed glia cells of P0 and P1 generation had the best proliferative activity; 97.3% of the high purity microglia could be obtained by mechanical shaking at 170 r/min for 30 min, and there was no significant difference in the morphology of ionized calcium-binding adapter molecule 1 (Iba-1) positive microglia and the proportion of M1 and M2 phenotype among the P0, P1 and P2 generations of microglia isolated by the above methods. Moreover, 95.7 % of the high purity astrocytes could be obtained by astrocyte cell surface antigen-2 (ACSA-2) magnetic beads separation, and there was no significant difference in the morphology of glial fibrillary acidic protein (GFAP) positive astrocyte and the proportion of A1 and A2 phenotype among the P0, P1 and P2 generations of astrocyte isolated by the above methods. Taken together, this study observed the growth characteristics of primarily cultured microglia and astrocyte in vitro, and then proved the best generations for purifying microglia and astrocytes. Finally, we optimized the methods of obtaining microglia and astrocyte, and verified that continuous culture within 2 generations will not affect the functional phenotypes of glia cells. These results provide technical support for studying the molecular mechanism of inflammation-associated diseases in nervous system.

The consensus of Chinese experts on refined analysis of immune cell subsets in peripheral blood by multi-parameter flow cytometry / 中华预防医学杂志

The detection of immune cell subsets plays a very important role in the clinical diagnosis and treatment of various benign and malignant diseases and health management. In order to better carry out in-depth research on different functional immune cell subsets, establish reference intervals for clonality related indicators, establish special reference intervals for immune aging, individualized dynamic monitoring and treatment recovery, and discover the clinical significance of immune cells other than lymphocytes, it is urgent to analyze the peripheral blood immune cell subsets in a refined way. Multiparameter flow cytometry is an important technical method to detect immune cell subsets and evaluate immune function. In order to standardize the refined detection methods and protocols of peripheral blood immune cell subsets by flow cytometry, and further promote its application in clinical diagnosis and treatment of diseases and health management, Laboratory Medicine Committee of Chinese Association of Integrative Medicine (LMC-CAIM) organized experts to formulate this expert consensus.

Análise dos Critérios Imunofenotípicos por Citometria de Fluxo para Definição das Doenças Linfoproliferativas Crônicas de Células B / Analysis of Immunophenotypic Criteria by Flow Cytometry to Define B-Cell Chronic Lymphoproliferative Diseases / Análisis de Criterios Inmunofenotípicos por Citometría de Flujo para Definir Enfermedades Linfoproliferativas Crónicas de Células B

Introdução: A citometria de fluxo é uma metodologia importante para o diagnóstico das doenças linfoproliferativas crônicas de células B (DLPCB), contudo, por vezes, o citometrista não encontra subsídios suficientes para a definição exata da entidade patológica envolvida. Objetivo: Analisar os laudos emitidos a pacientes com doenças linfoproliferativas crônicas (DLPC) atendidos em um laboratório particular de Belém-PA, segundo os critérios de classificação estabelecidos pelos estudos de Matutes et al. e Craig e Foon. Método: Estudo retrospectivo com laudos de pacientes que realizaram imunofenotipagem por citometria de fluxo para diagnóstico de DLPCB no período entre setembro de 2015 a dezembro de 2019. Resultados: Depois de aplicados os critérios de Matutes et al. e Craig e Foon para os laudos analisados, observou-se concordância em: 45,24% casos de leucemia linfoide crônica de células B/linfoma linfocítico de pequenas células B; 14,29% casos de linfoma folicular; 4,76% casos de leucemia de células pilosas; e 21,43% de casos definidos como "outras DLPCB não classificáveis por citometria de fluxo". Entretanto, o teste de hipóteses de Hotelling (p=0,0409) mostrou haver diferença estatística para a definição das DLPCB segundo os critérios aplicados. Conclusão: Os resultados ressaltam que, mesmo sendo a citometria de fluxo importante para a caracterização das DLPCB, por vezes, o citometrista necessita incluir no laudo a categoria "outras doenças linfoproliferativas crônicas de células B não classificadas por citometria de fluxo" para induzir o prescritor a solicitar mais exames complementares.

Introduction: Flow cytometry is an important methodology for the diagnosis of chronic B-cell lymphoproliferative diseases (CBCLPD), however, sometimes the cytometrist does not find sufficient elements for the exact definition of the pathological entity involved. Objective: To analyze the reports issued to patients with chronic lymphoproliferative diseases (CLPD) tested at a private laboratory in Belém-PA, according to the classification criteria established by the studies by Matutes et al. and Craig and Foon. Method: Retrospective study with reports of patients who underwent immunophenotyping by flow cytometry for the diagnosis of CBCLPD from September 2015 to December 2019. Results: After applying the criteria by Matutes et al. and Craig and Foon to the reports analyzed, agreement was reached for 45.24% of the cases of chronic b-cell lymphocytic leukemia/small B-cell lymphocytic lymphoma, 14.29% of the cases of follicular lymphoma, 4.76% of the cases of hairy cell leukemia and 21.43% of the cases defined as "other CBCLPDs not classifiable by flow cytometry". However, Hotelling's hypothesis test (p=0.0409) showed a statistical difference for the definition of CBCLPD according to the criteria adopted. Conclusion: The results emphasize that even though flow cytometry is important for the characterization of CBCLPD, sometimes the cytometrist needs to include the category "other chronic B-cell lymphoproliferative diseases not classified by flow cytometry" in the report to induce the prescriber to request additional complementary exams.

Introducción. La citometría de flujo es una metodología importante para el diagnóstico de enfermedades linfoproliferativas crónicas de células B (ELPCB), sin embargo, en ocasiones el citometrista no encuentra suficientes subsidios para la definición exacta de la entidad patológica involucrada. Objetivo: Analizar los informes emitidos a pacientes con enfermedades linfoproliferativas crónicas (ELPC) tratados en un laboratorio privado en Belém-PA, de acuerdo con los criterios de clasificación establecidos por los estudios de Matutes et al. y Craig y Foon. Método: Retrospectivo con relatos de pacientes que se sometieron a inmunofenotipificación por citometría de flujo para el diagnóstico de ELPC de septiembre de 2015 a diciembre de 2019. Resultados: Tras aplicar los criterios de Matutes et al. y Craig y Foon a los informes analizados, se observó concordancia en: 45,24% de los casos de leucemia linfocítica crónica de células B/linfoma linfocítico de células B pequeñas; 14,29% casos de linfoma folicular; 4,76% casos de leucemia de células peludas; y 21,43% de los casos definidos como "otros ELPCB no clasificables por citometría de flujo". Sin embargo, la prueba de hipótesis de Hotelling (p=0,0409) mostró diferencia estadística para la definición de ELPCB según los criterios aplicados. Conclusión: Nuestros resultados enfatizan que si bien la citometría de flujo es importante para la caracterización de ELPCB, en ocasiones el citometrista necesita incluir en el informe la categoría "otras enfermedades linfoproliferativas crónicas de células B no clasificadas por citometría de flujo" para inducir al prescriptor a solicitar más exámenes complementarios.

The dynamics of genome size and GC contents evolution in genus Nicotiana / A dinâmica do tamanho do genoma e a evolução dos conteúdos de GC no gênero Nicotiana

Hybridization and Polyploidization are most common of the phenomenon observed in plants, especially in the genus Nicotiana leading to the duplication of genome. Although genomic changes associated with these events has been studied at various levels but the genome size and GC content variation is less understood because of absence of sufficient genomic data. In this study the flow cytometry technique was used to uncover the genome size and GC contents of 46 Nicotiana species and we compared the genomic changes associated with the hybridization events along evolutionary time scale. The genome size among Nicotiana species varied between 3.28 pg and 11.88 pg whereas GC contents varied between 37.22% and 51.25%. The tetraploid species in genus Nicotiana including section Polydiclae, Repandae, Nicotiana, Rustica and Sauveolentes revealed both up and downsizing in their genome sizes when compared to the sum of genomes of their ancestral species. The genome sizes of three homoploid hybrids were found near their ancestral species. Loss of large genome sequence was observed in the evolutionary more aged species (>10 Myr) as compared to the recently evolved one’s (<0.2 Myr). The GC contents were found homogenous with a mean difference of 2.46% among the Nicotiana species. It is concluded that genome size change appeared in either direction whereas the GC contents were found more homogenous in genus Nicotiana.

A hibridização e a poliploidização são os fenômenos mais comuns observados em plantas, principalmente no gênero Nicotiana, levando à duplicação do genoma. Embora as mudanças genômicas associadas a esses eventos tenham sido estudadas em vários níveis, o tamanho do genoma e a variação do conteúdo de GC são menos compreendidos devido à ausência de dados genômicos suficientes. Neste estudo, a técnica de citometria de fluxo foi usada para descobrir o tamanho do genoma e o conteúdo de GC de 46 espécies de Nicotiana, e comparamos as mudanças genômicas associadas aos eventos de hibridização ao longo da escala de tempo evolutiva. O tamanho do genoma entre as espécies de Nicotiana variou entre 3,28 pg e 11,88 pg, enquanto os conteúdos de GC variaramentre 37,22% e 51,25%. As espécies tetraploides do gênero Nicotiana, incluindo as seções Polydiclae, Repandae, Nicotiana, Rustica e Sauveolentes, revelaram aumento e redução do tamanho do genoma quando comparados à soma dos genomas de suas espécies ancestrais. Os tamanhos do genoma de três híbridos homoploides foram encontrados perto de suas espécies ancestrais. A perda da grande sequência do genoma foi observada nas espécies evolutivas mais velhas (> 10 Myr) em comparação com as que evoluíram recentemente (< 0,2 Myr). Os teores de GC foram homogêneos com diferença média de 2,46% entre as espécies de Nicotiana. Conclui-se que a mudança no tamanho do genoma apareceu em ambas as direções, enquanto os conteúdos de GC foram encontrados mais homogêneos no gênero Nicotiana.

Comparative analysis between cytomorphology and flow cytometry methods in central nervous system infiltration assessment in oncohematological patients

ABSTRACT Introduction Oncohematological patients require the evaluation for possible infiltration of the central nervous system (CNS) by neoplastic cells at diagnosis and/or during the monitoring of the chemotherapeutic treatment. Morphological analysis using conventional microscopy is considered the method of choice to evaluate the cerebrospinal fluid (CSF) samples, despite technical limitations. Objective This study aimed to compare the performance of the cytomorphology and flow cytometric immunophenotyping (FC) in the detection of CNS infiltration. Method We evaluated 520 CSF samples collected from 287 oncohematological patients for whom the detection of neoplastic cells was simultaneously requested by cytomorphology and FC. Results Laboratory analyses revealed 435/520 (83.7%) conclusive results by the two methods evaluated, among which 385 (88.5%) were concordant. Discordance between the methods was observed in 50/435 (11.5%) samples, 45 (90%) being positive by FC. Furthermore, the FC defined the results in 69/72 (95.8%) inconclusive samples by cytomorphology. The positivity of FC was particularly higher among hypocellular samples. Among 431 samples with a cell count of < 5/μL, the FC identified neoplastic cells in 75 (17.4%), while the cytomorphology reported positive results in 26 (6%). Among the samples that presented adequate cell recovery for evaluation by both methods (506/520), the comparative analysis between FC and cytomorphology revealed a Kappa coefficient of 0.45 (CI: 0.37-0.52), interpreted as a moderate agreement. Conclusion The data showed that the CSF analysis by FC helps in the definition of CNS infiltration by neoplastic cells, particularly in the cases with dubious morphological analysis or in the evaluation of samples with low cellularity.

A novel differential diagnosis algorithm for chronic lymphocytic leukemia using immunophenotyping with flow cytometry

Abstract Introduction The availability of a clinical decision algorithm for diagnosis of chronic lymphocytic leukemia (CLL) may greatly contribute to the diagnosis of CLL, particularly in cases with ambiguous immunophenotypes. Herein we propose a novel differential diagnosis algorithm for the CLL diagnosis using immunophenotyping with flow cytometry. Methods The hierarchical logistic regression model (Backward LR) was used to build a predictive algorithm for the diagnosis of CLL, differentiated from other lymphoproliferative disorders (LPDs). Results A total of 302 patients, of whom 220 (72.8%) had CLL and 82 (27.2%), B-cell lymphoproliferative disorders other than CLL, were included in the study. The Backward LR model comprised the variables CD5, CD43, CD81, ROR1, CD23, CD79b, FMC7, sIg and CD200 in the model development process. The weak expression of CD81 and increased intensity of expression in markers CD5, CD23 and CD200 increased the probability of CLL diagnosis, (p < 0.05). The odd ratio for CD5, C23, CD200 and CD81 was 1.088 (1.050 - 1.126), 1.044 (1.012 - 1.077), 1.039 (1.007 - 1.072) and 0.946 (0.921 - 0.970) [95% C.I.], respectively. Our model provided a novel diagnostic algorithm with 95.27% of sensitivity and 91.46% of specificity. The model prediction for 97.3% (214) of 220 patients diagnosed with CLL, was CLL and for 91.5% (75) of 82 patients diagnosed with an LPD other than CLL, was others. The cases were correctly classified as CLL and others with a 95.7% correctness rate. Conclusions Our model highlighting 4 markers (CD81, CD5, CD23 and CD200) provided high sensitivity and specificity in the CLL diagnosis and in distinguishing of CLL among other LPDs.