► New data analysis method for WIMP dark matter searches with LXe dual-phase TPCs. ► Prompt scintillation light and ionization charge combined in Log (S1/C2) vs. C2. ► C2 is exhibits better nuclear ...recoil acceptance and superior energy resolution. ► Discrimination power of the TPC and its 3D event reconstruction fully maintained. ► New phase space is shown to be more appropriate to evaluate WIMP signal candidates.
A new data analysis method based on physical observables for WIMP dark matter searches with noble liquid Xe dual-phase TPCs is presented. Traditionally, the nuclear recoil energy from a scatter in the liquid target has been estimated by means of the initial prompt scintillation light (S1) produced at the interaction vertex. The ionization charge (C2), or its secondary scintillation (S2), is combined with the primary scintillation in log10(S2/S1) vs. S1 only as a discrimination parameter against electron recoil background. Arguments in favor of C2 as the more reliable nuclear recoil energy estimator than S1 are presented. The new phase space of log10(S1/C2) vs. C2 is introduced as more efficient for nuclear recoil acceptance and exhibiting superior energy resolution. This is achieved without compromising the discrimination power of the LXe TPC, nor its 3D event reconstruction and fiducialization capability, as is the case for analyses that exploit only the ionization channel. Finally, the concept of two independent energy estimators for background rejection is presented: E2 as the primary (based on C2) and E1 as the secondary (based on S1). log10(E1/E2) vs. E2 is shown to be the most appropriate phase space in which to evaluate WIMP signal candidates.
Introduction: Minimal residual disease (MRD) detected before and after hematopoietic stem cell transplantation (HSCT) is associated with poor outcomes in high-risk acute leukemia. This study ...prospectively evaluates the impact of MRD accessed by multiparameter flow cytometry (MFC) in a reference hospital in southern Brazil. Methods: Prospective analysis of 67 consecutive patients transplanted between 2019/Jun and 2022/Jun, with high-sensitive MFC analysis in the month before HSCT. Pre-transplant MRD was negative (MRD-) in 34 (50.7%) and positive (MRD+) in 33 (49.3%) patients (14 ALL and 18 AML). Posttransplant MRD evaluation showed MRD+ in five patients at d30 (5/26, 19.2%), two (2/23, 8.7%) at d60 and 5 (5/57, 8.8%) at d100. Results: There were no differences concerning pretransplant variables between positive and negative MRD pre-HSCT groups. Considering alive patients at d100: Pre-HSCT MRD negative versus posttransplant kinetic: Considering patients available at d100 (n = 57) in relation to pre-transplant MRD status, almost half (n = 30/57, 52.6%) had negative MRD both before and after transplantation. Two AML patients MRD negative became positive and subsequently relapsed, at d488 and d426. Both patients had AML with negative CD34, but this LAIP was not known prior to relapse. Pre-HSCT MRD positive versus posttransplant kinetic: In relation to the 27 patients with positive disease before transplantation who were alive at d100 (n = 27/35, 77.1%), the majority (n = 22/27, 81.5%) became MRD negative, suggesting minimal residual disease clearance in response to treatment. Four patients MRD+ relapsed within the first year after transplantation (d117, d136, d168 and d347) and two other two patients relapsed on d405 and d744. Cumulative incidence of relapse in total cohort was 11.4%, NRM was 22.4% and probabilities of EFS and OS were 64.2% and 70.4%, respectively. MRD positivity before transplantation was associated with decreased OS (88.2% versus 51.5%, p = 0.001) and EFS (82.4% versus 45.5%, p = 0.001), but relapse was not different (5.9% vs 18.2%, p = 0.036); non-relapse mortality was 8.8% vs 36.4% (p = 0.003). Extended follow-up showed a high risk of disease relapse in AML MRD+ subgroup (EFS 78.6% vs 36.8%, p < 0.001, and OS 92.9% vs 47.4%, p = 0.001), and this was associated to MRD positivity at d100. MRD analysis before transplantation had a high sensitivity (75%) in predicting survival, while MRD positivity at d100 was more specific in predicting subsequent relapse (90.5%) in this cohort. Conclusion: A highly sensitive assessment of MRD before HSCT in a real-life setting allowed the correct identification of patients with poor prognosis. Moreover, the persistence of MRD after transplantation identified patients at risk of relapse.
CD4 + lymphocyte counts are routinely ordered during the early phases of antiretroviral therapy and for prophylaxis of opportunistic infections in HIV-positive patients. Flow cytometry is the ...standard methodology for CD4 counts in Brazilian reference laboratories. However, these laboratories are located in large cities, frequently distant from patients, thus limiting patient access and delaying results. We compared a point-of-care test with flow cytometry determination of CD4(+) T lymphocyte counts in HIV patients. We analysed 107 consecutive samples by both methods. Overall, the point-of-care test performed well, with excellent agreement between it and the standard method. Test results were concordant for patients with CD4(+) T lymphocyte values above and below 200 cells/mm (3). The performance characteristics obtained were sensitivity 94% (95% CI 89.5-98.5%), specificity 93% (95% CI 88.2-97.8%), positive predictive value 86% (95% CI 79.4-92.6%), and negative predictive value 97% (95% CI 94-100%). The high sensitivity and specificity of the point-of-care test methodology suggest its utility as an alternative method for rapid measurement of CD4(+) T lymphocytes in patients with limited access to reference laboratories, enabling prompt therapeutic intervention for patients at risk of progression to AIDS.
Introdução: A Leucemia Aguda Indiferenciada (LAI) é caracterizada pela proliferação clonal de células hematopoiéticas primitivas, por definição, não possui marcadores específicos de linhagem linfoide ...ou mieloide sendo necessário excluir leucemias de linhagens incomuns como as de células dendríticas plasmo citoides, células NK, basófilos ou mesmo tumores não hematopoiéticos. Portanto o diagnóstico de LAI pode ser complexo e requerer outras análises além da imuno fenotipagem convencional. Dada sua raridade, pouco se sabe sobre sua incidência, sobrevida e manejo ideal. Os pacientes apresentam leucocitose, anemia, contagem variável de plaquetas e uma variedade de sintomas inespecíficos relacionados à hematopoese ineficaz (fadiga, sangramento e hematomas, infecções recorrentes, dor óssea) e/ou envolvimento de locais extramedulares (linfadenopatia, esplenomegalia, hepatomegalia). Relato de caso: Paciente masculino, 7 anos, astenia, 4 dias com febrícula, perda ponderal de 2 Kg. Hemograma externo: Hb: 7,4g/dL; VCM: 77,8fL (2% eritroblastos). Leuco: 4.000 uL; Neutro: 1.080 uL; Plaquetas: 13.000 uL. Exame Físico: Presença de linfonodos palpáveis em cadeia cervical anterior, posterior e supraclaviculares, móveis, fibroelásticos, < 1 cm, baço a 4 cm da borda costal esquerda; fígado a 6 cm da borda costal direita. Exames na admissão: Hb: 10,0 g/dL; VCM 80 fL; Leuco: 4.320 uL; Linf.: 1.666 mm3; Neutro: 648 uL; Blastos: 58,0%; Plt: 38.000 uL. Ferritina: 431 ng/mL; LDH: 269U/L; VHS: 50 mm. Medula óssea com celularidade diminuída, com presença de 55% de blastos de difícil diferenciação morfológica. Alguns blastos de tamanho pequeno e formato regular; núcleo ocupando quase todo o citoplasma, de cromatina frouxa e heterogênea sem nucléolo evidente; outros de tamanho mediano, formato regular; núcleo de formato irregular com cromatina frouxa e heterogênea por vezes com um nucléolo evidente; citoplasma escasso levemente basofílico e finamente granular. A Citometria de Fluxo mostrou no aspirado da MO 58,8% de blastos e na biópsia 67,0%. Positividade para: TdT-/+ nuclear (31,0%), CD15-/+ (58,0%), CD19+/++ heterogêneo, CD24+/++, (50,5%), CD33-/+ (26,6%), CD38++, CD45+/++, CD66c+/++, CD71-/+ (34,0%), CD123-/+ (40,0%), HLA-DR+/++ heterogêneo, NG2 (proteína 7,1)+/++ e negatividade para antígenos citoplasmáticos MPO, CD79, CD22, CD3, IgM e antígenos de membrana: CD3, CD7, CD10, CD11b, CD11c, CD13, CD16, CD22, CD34, CD35, CD36, CD41, CD42a, CD42b, CD56, CD61, CD64, CD105, CD117, IgM, IREM-2. Citogenética: 47, XY, +14/46, XY. Molecular: KMT2A-AFF1 por PCR Inconclusiva. ETV6-RUNX1: Negativo. FISH: KMT2A-AFF1 positivo, BCR:ABL negativo. As alterações cromossomo 10, 17,4 negativas. LCR sem infiltração. O paciente iniciou tratamento com protocolo GBTLI 2021, alto risco, e a Pesquisa de DRM D19 e D49 foram negativas. Técnica lise total Euroflow Positividade: CDD19, CD20, CD22, CD45, CD81, HLA-DR. Negatividade: CD10, CD15, CD34, CD38, CD66c/CD123, NG2 (proteína 7,1). LOD (limit of detection): 0,00086%. LLOQ (lower limit of quantification): 0,0021% e LOD: 0,0017% LLOQ: 0,0045% respectivamente. Conclusão: : Descrevemos um caso de leucemia aguda sem marcadores definidoras de linhagem. O prognóstico da AUL é geralmente ruim, mas tem melhorado ao longo do tempo. A imuno fenotipagem por Citometria de Fluxo Multiparamétrica (CFM) tornou-se uma ferramenta importante no manejo clínico das neoplasias hematológicas, tanto para fins de diagnóstico quanto de monitoramento (Doença Residual Mensurável ‒ DRM).
SARCOMA ERITROIDE Hoffmann, MFC; Beltrame, MP; Fava, F ...
Hematology, Transfusion and Cell Therapy,
10/2023, Volume:
45
Journal Article
Peer reviewed
Open access
Introdução: Sarcoma eritroide é uma variante extremamente rara do Sarcoma Mieloide (SM), anteriormente conhecido como sarcoma granulocítico ou cloroma, é uma neoplasia mieloide incomum que consiste ...em uma proliferação maligna derivada de blastos hematopoiéticos mieloides ocorrendo em um local anatômico diferente da Medula Óssea (MO). O SE pode preceder, seguir ou ocorrer na ausência de leucemia mieloide aguda sistêmica no contexto do diagnóstico ou recidiva em qualquer parte do corpo. O SEjá foi relatado em quase todos os locais anatômicos, mas é mais frequentemente encontrado na pele e tecidos moles, linfonodos e trato gastrointestinal. Relato de caso: Paciente masculino, 7 anos, 20 dias com dor em hipocôndrio, emagrecimento 5 Kg. RX do Tórax com DP ‒ Dreno Tórax e Tomo com massa torácica. Hb: 11 g/dL, VCM 68 fL, Leucócitos 7.310 uL, Linfócitos 7.240, Neutrófilos 4.094 uL, Plaquetas 469.000 uL, LDH 419 U/L, VHS 50 mm. Aspirado bilateral da medula óssea Mielograma e Citometria de Fluxo: Não evidenciaram blastos e/ou células não hematopoiéticas. Biópsia da MO negativa para neoplasia (AP e IH). Biópsia do linfonodo: Segmento de tecido adiposo contendo três linfonodos com histiocitose sinusal de aspecto reacional. AP da biópsia do mediastino: Neoplasia pouco diferenciada, de células redondas e azuis. Citometria de Fluxo do linfonodo: 31,7% de linfócitos B maduros não clonais e 65,2% de linfócitos T de tamanho e complexidade de baixos de fenótipo normal. Os linfócitos T CD3+TCRab+CD4-CD8- estão dentro do valor de referência (menor que 2,5%). Análise normal para pesquisa de clonalidade da TRBC-1 tanto para os linfócitos T CD4 quanto para os linfócitos T CD8. Não foram observadas células CD15/CD30/CD40/CD95 positivas. A Citometria de Fluxo do líquido pleural (660 leucócitos) mostrou 81,9% de células anormais. Positividade para CD36++, CD56-/+ (8,5%), CD71++, CD117++ e negatividade para antígenos citoplasmáticos MPO, CD79, CD3, CD61, antígeno nuclear Miogenina (nuclear) e antígenos de membrana CD3, CD4, CD5, CD7, CD8, CD9, CD10, CD11b, CD14, CD15, CD16, CD19, CD20, CD22, CD30, CD33, CD34, CD35, CD38, CD40, CD42a, CD44, CD45, CD61, CD64, CD73, CD81, CD95, CD99, CD105, CD123, CD271, GD2, Glicoforina A, HLA-DR, IREM-2, NG2 (proteína 7,1), cadeias leves Kappa e Lambda, GD2. Na amostra da biópsia do tumor com 110 leucócitos/uL mostrou 44,5% de células anormais com fenótipo semelhante ao líquido. Sarcoma eritroide? Tumor sólido? LCR sem infiltração. Na sequência a IH mostrou positividade para CD99, Ki-67 (60%) e TdT fraco, MPO positivo focal, E-caderina positivo difuso, Glicoforina A positivo focal. Após 3 meses recebemos amostra da medula óssea, sem infiltração.LOD (limit of detection): 0,00096% e LLOQ (lower limit of quantification): 0,0016%. Iniciado tratamento com protocolo GELMAI, entretanto, com pouca resposta tumoral. Optou-se por ressecção tumoral, laudo de AP com pequenas células redondas e azuis, aguardamos o resultado da imunohistoquímica. Conclusão: Descrevemos um caso de Sarcoma Eritroideque devido a raridade da doença foi possível diagnóstico rápido e preciso através da Citometria de Fluxo (CF) de 8 cores padronizada e com a utilização do painel Euroflow. Esta metodologia, é muito importante para investigar cuidadosamente a presença de celularidade eritroide atípica, mesmo em amostras de líquido e tecido de tumor. A CF fornece rapidamente resultados reprodutíveis e pode ser utilizada como uma ferramenta para diagnosticar de forma rápida e confiável esse tipo de malignidade.
Summary
Circulating immunoglobulin (Ig)G antibodies against M2 muscarinic acetylcholine receptors (M2 mAChR) have been implicated in Chagas' disease (ChD) pathophysiology. These antibodies bind to ...and activate their target receptor, displaying agonist‐like activity through an unclear mechanism. This study tested the ability of serum anti‐M2 mAChR antibodies from chronic ChD patients to modulate M2 muscarinic receptor–receptor interaction by bioluminescence resonance energy transfer (BRET). Human embryonic kidney (HEK) 293 cells co‐expressing fusion proteins M2 mAChR‐Renilla luciferase (RLuc) and M2 mAChR‐yellow fluorescent protein (YFP) were exposed to the serum IgG fraction from ChD patients, and BRET between RLuc and YFP was assessed by luminometry. Unlike serum IgG from healthy subjects and conventional muscarinic ligands, ChD IgG promoted a time‐ and concentration‐dependent increase in the BRET signal. This effect neither required cellular integrity nor occurred as a consequence of receptor activation. Enhancement of M2 receptor–receptor interaction by ChD IgG was receptor subtype‐specific and mediated by the recognition of the second extracellular loop of the M2 mAChR. The monovalent Fab fragment derived from ChD IgG was unable to reproduce the effect of the native immunoglobulin. However, addition of ChD Fab in the presence of anti‐human Fab IgG restored BRET‐enhancing activity. These data suggest that the modulatory effect of ChD IgG on M2 receptor–receptor interaction results from receptor cross‐linking by bivalent antibodies.
Previous studies showed that circulating autoantibodies against M2 muscarinic receptors (anti-M2R Ab) are associated with decreased cardiac parasympathetic modulation in patients with chronic Chagas ...disease (CD). Here we investigated whether the exposure of M2R to such antibodies could impair agonist-induced receptor activation, leading to the inhibition of associated signaling pathways. Preincubation of M2R-expressing HEK 293T cells with serum IgG fractions from chagasic patients with cardiovascular dysautonomia, followed by the addition of carbachol, resulted in the attenuation of agonist-induced Gi protein activation and arrestin-2 recruitment. These effects were not mimicked by the corresponding Fab fractions, suggesting that they occur through receptor crosslinking. IgG autoantibodies did not enhance M2R/arrestin interaction or promote M2R internalization, suggesting that their inhibitory effects are not likely a result of short-term receptor regulation. Rather, these immunoglobulins could function as negative allosteric modulators of acetylcholine-mediated responses, thereby contributing to the development of parasympathetic dysfunction in patients with CD.
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•The role of serum autoantibodies in Chagas disease-related dysautonomia was examined.•M2 muscarinic receptor antibodies inhibit agonist-induced Gi protein activation.•These antibodies also inhibit agonist-mediated arrestin-2 recruitment.•Anti-M2 receptor autoantibodies could function as negative allosteric modulators.•These autoantibodies could play a pathogenic role in chagasic dysautonomia.
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