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1.
Biochem Med (Zagreb) ; 31(3): 030705, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34658646

RESUMO

Introduction: MicroRNAs are small, non-coding RNA molecules that are becoming popular biomarkers in several diseases. However, their low abundance in serum/plasma poses a challenge in exploiting their potential in clinics. Several commercial kits are available for rapid isolation of microRNA from plasma. However, reports guiding the selection of appropriate kits to study downstream assays are scarce. Hence, we compared four commercial kits to evaluate microRNA-extraction from plasma and provided a modified protocol that further improved the superior kit's performance. Materials and methods: We compared four kits (miRNeasy Serum/Plasma, miRNeasy Mini Kit from Qiagen; RNA-isolation, and Absolutely-RNA MicroRNA Kit from Agilent technologies) for quality and quantity of microRNA isolated, extraction efficiency, and cost-effectiveness. Bioanalyzer-based Agilent Small RNA kit was used to evaluate quality and quantity of microRNA. Extraction efficiency was evaluated by detection of four endogenous control microRNA using real-time-PCR. Further, we modified the manufacturer's protocol for miRNeasy Serum/Plasma kit to improve yield. Results: miRNeasy Serum/Plasma kit outperformed the other three kits in microRNA-quality (P < 0.005) and yielded maximum microRNA-quantity. Recovery of endogenous control microRNA i.e. hsa-miR-24-3p, hsa-miR-191-5p, hsa-miR-423-5p and hsa-miR-484 was higher as well. Modification with the inclusion of a double elution step enhanced yield of microRNA extracted with miRNeasy Serum/Plasma kit significantly (P < 0.001). Conclusion: We demonstrated that miRNeasy Serum/Plasma kit outperforms other kits and can be reliably used with a limited plasma quantity. We have provided a modified microRNA-extraction protocol with improved microRNA output for downstream analyses.


Assuntos
MicroRNAs , Biomarcadores , Humanos , MicroRNAs/genética , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real
2.
Br J Haematol ; 2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34476808

RESUMO

High-sensitivity multicolour flow cytometry (MFC)-based B-lymphoblastic leukaemia (B-ALL) measurable residual disease (BMRD) assay is increasingly being used in clinical practice. Herein, we describe six consistently present low-level populations immunophenotypically mimicking abnormal B-ALL blasts in 441 BMRD samples from 301 children. These included CD19+ CD123+ plasmacytoid dendritic cells differentiating from lymphoid precursors, CD10+ transitional B cells with CD10+ /CD38dim-to-negative/CD20bright/CD45bright phenotype, CD19+ natural killer (NK) cells, CD73bright/CD10+ mesenchymal stromal/stem cells, CD73bright/CD34+ endothelial cells, and a CD34+ CD38dim-to-negative/CD10- /CD20bright/CD45bright subset of mature B cells. We provide the proportions, comprehensive immunophenotype, and practical clues for proper identification of these low-level populations. Knowledge regarding the presence and immunophenotype of these mimics is essential for accurate interpretation in high-sensitivity MFC-BMRD analysis.

3.
Immunol Cell Biol ; 2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34582592

RESUMO

Recent studies have highlighted multiple immune perturbations related to severe acute respiratory syndrome coronavirus 2 infection-associated respiratory disease [coronavirus disease 2019 (COVID-19)]. Some of them were associated with immunopathogenesis of severe COVID-19. However, reports on immunological indicators of severe COVID-19 in the early phase of infection in patients with comorbidities such as cancer are scarce. We prospectively studied about 200 immune response parameters, including a comprehensive immune-cell profile, inflammatory cytokines and other parameters, in 95 patients with COVID-19 (37 cancer patients without active disease and intensive chemo/immunotherapy, 58 patients without cancer) and 21 healthy donors. Of 95 patients, 41 had severe disease, and the remaining 54 were categorized as having a nonsevere disease. We evaluated the association of immune response parameters with severe COVID-19. By principal component analysis, three immune signatures defining characteristic immune responses in COVID-19 patients were found. Immune cell perturbations, in particular, decreased levels of circulating dendritic cells (DCs) along with reduced levels of CD4 T-cell subsets such as regulatory T cells (Tregs ), type 1 T helper (Th1) and Th9; additionally, relative expansion of effector natural killer (NK) cells were significantly associated with severe COVID-19. Compared with patients without cancer, the levels of terminal effector CD4 T cells, Tregs , Th9, effector NK cells, B cells, intermediate-type monocytes and myeloid DCs were significantly lower in cancer patients with mild and severe COVID-19. We concluded that severely depleted circulating myeloid DCs and helper T subsets in the initial phase of infection were strongly associated with severe COVID-19 independent of age, type of comorbidity and other parameters. Thus, our study describes the early immune response associated with severe COVID-19 in cancer patients without intensive chemo/immunotherapy.

4.
Int J Lab Hematol ; 43(5): 990-999, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33432783

RESUMO

INTRODUCTION: Many new markers are being evaluated to increase the sensitivity and applicability of multicolor flow cytometry (MFC)-based measurable residual disease (MRD) monitoring. However, most of the studies are limited to childhood B-cell lymphoblastic leukemia/lymphoma (B-ALL), and reports in adult B-ALL are extremely scarce and limited to small cohorts. We studied the expression of CD304/neuropilin-1 in a large cohort of adult B-ALL patients and evaluated its practical utility in MFC-based MRD analysis. METHODS: CD304 was studied in blasts from adult B-ALL patients and normal precursor B cells (NPBC) from non-B-ALL bone marrow samples using MFC. CD304 expression intensity and pattern were studied with normalized-mean fluorescent intensity (nMFI) and coefficient of variation of immunofluorescence (CVIF), respectively. MFC-based MRD was performed at end of induction (EOI; day-35), end of consolidation (EOC; day 78-80), and subsequent follow-up (SFU) time points. RESULTS: CD304 was positive in 120/214(56.07%) and was significantly associated with BCR-ABL1 fusion (P = .001). EOI-MRD and EOC-MRD were positive in 129/214(60.3%) and 50/81(61.72%), respectively. CD304 was positive in a significant percentage of EOI (48%, 62/129) and EOC (52%, 26/50) MRD-positive B-ALL samples. Its expression was retained, lost, and gained in 73.7%, 26.3%, and 11.3% of EOI-MRD and 85.7%, 14.3%, and none of EOC-MRD samples, respectively. Low-level MRD (<0.01%) was detectable in 34 of all (EOI + EOC + SFU = 189) MRD-positive samples, and CD304 was found useful in 50% of these samples. CONCLUSION: CD304 is commonly expressed in adult B-ALL and clearly distinguish B-ALL blasts from normal precursor B cells. It is a stable MRD marker and distinctly useful in the detection of MFC-based MRD monitoring, especially in high-sensitivity MRD assay.


Assuntos
Neoplasia Residual/diagnóstico , Neuropilina-1/análise , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/análise , Medula Óssea/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasia Residual/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Adulto Jovem
5.
Cytometry B Clin Cytom ; 100(4): 434-445, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-32896101

RESUMO

Accurate knowledge of expression patterns/levels of commonly used MRD markers in regenerative normal-B-cell-precursors (BCP) is highly desirable to distinguish leukemic-blasts from regenerative-BCP for multicolor flow cytometry (MFC)-based measurable residual disease (MRD) assessment in B-lymphoblastic leukemia (B-ALL). However, the data highlighting therapy-related immunophenotypic-shift in regenerative-BCPs is scarce and limited to small cohort. Herein, we report the in-depth evaluation of immunophenotypic shift in regenerative-BCPs from a large cohort of BALL-MRD samples. Ten-color MFC-MRD analysis was performed in pediatric-BALL at the end-of-induction (EOI), end-of-consolidation (EOC), and subsequent-follow-up (SFU) time-points. We studied normalized-mean fluorescent intensity (nMFI) and coefficient-of-variation of immunofluorescence (CVIF) of CD10, CD19, CD20, CD34, CD38, and CD45 expression in regenerative-BCP (early, BCP1 and late, BCP2) from 200 BALL-MRD samples, and compared them with BCP from 15 regenerating control (RC) TALL-MRD samples and 20 treatment-naïve bone-marrow control (TNSC) samples. Regenerative-BCP1 showed downregulation in CD10 and CD34 expression with increased CVIF and reduced nMFI (p < 0.001), upregulation of CD20 with increased nMFI (p = 0.014) and heterogeneous CD45 expression with increased CVIF (p < 0.001). Immunophenotypic shift was less pronounced in the BCP2 compared to BCP1 compartment with increased CVIF in all but CD45 (p < 0.05) and reduced nMFI only in CD45 expression (p = 0.005). Downregulation of CD10/CD34 and upregulation of CD20 was higher at EOI than EOC and SFU time-points (p < 0.001). Regenerative-BCPs are characterized by the significant immunophenotypic shift in commonly used B-ALL-MRD markers, especially CD10 and CD34 expression, as compared to treatment-naïve BCPs. Therefore, the templates/database for BMRD analysis must be developed using regenerative-BCP.

6.
Cytometry B Clin Cytom ; 100(4): 421-433, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-32812702

RESUMO

Measurable/minimal residual disease (MRD) status has been suggested as a powerful indicator of clinical-outcome in T-cell lymphoblastic leukemia/lymphoma (T-ALL). Multicolor flow cytometric (MFC)-based T-ALL MRD reports are limited and traditionally based on the utilization of markers-of-immaturity like TdT and CD99. Moreover, studies demonstrating the multicolor flow cytometric (MFC) approach for the assessment of T-ALL MRD are sparse. Herein, we describe an 11-marker, 10-color MFC-based T-ALL MRD method using an "approach of exclusion." METHODS: The study included 269 childhood T-ALL patients treated with a modified-MCP841 protocol. An 11-marker, 10-color MFC-based MRD was performed in bone marrow (BM) samples at the end-of-induction (EOI) and end-of-consolidation (EOC) time-points using Kaluza-version-1.3 software. RESULTS: We studied EOI-MRD in 269 and EOC-MRD in 105 childhood T-ALL patients. EOI-MRD was detectable in 125 (46.5%) samples (median, 0.3%; range, 0.0007-66.3%), and EOC-MRD was detectable in 34/105 (32.4%) samples (median, 0.055%; range, 0.0008-27.6%). Leukemia-associated immunophenotypes (LAIPs) found useful for MRD assessment were dual-negative CD4/CD8 (40.9%), dual-positive CD4/CD8 (23.3%) and only CD4 or CD8 expression (35.8%); dim/subset/dim-negative surface-CD3 (39%), dim/subset/dim-negative/negative CD5 (28.3%), dim/dim-negative/negative/heterogeneous CD45 (44.7%) and co-expression of CD5/CD56 (7.5%). EOI-MRD-positive status was found to be the most-relevant independent factor in the prediction of inferior relapse-free and overall survival. CONCLUSION: We described an 11-marker 10-color MFC-based highly sensitive MRD assay in T-ALL using an approach of exclusion. The addition of CD4 and CD8 to the pan-T-cell markers in a 10-color assay is highly useful in T-ALL MRD assessment and extends its applicability to almost all T-ALL patients.

7.
Cytometry B Clin Cytom ; 100(3): 331-344, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-32738100

RESUMO

INTRODUCTION: Myeloid neoplasm with blasts showing mast cell (MC)-differentiation and MC-component less than 10% of all nucleated cells but not fulfilling the criteria for systemic mastocytosis with associated hematological neoplasm (SM-AHN) or myelomastocytic leukemia (MML) has not been described in the literature. Herein, we report a study of diverse myeloid malignancies with blasts showing MC-differentiation but not meeting the criteria for SM-AHN or MML. We also evaluated the utility of flow-cytometric immunophenotyping (FCI) in the characterization of immature-MCs (iMCs). METHODS: We identified nine patients of myeloid neoplasms and studied their morphological, FCI, immunohistochemistry, cytogenetic and molecular characteristics. We also compared the immunophenotypic features of MCs from patient samples with control samples. RESULTS: The study included patients with newly-diagnosed acute myeloid leukemia (n = 4), chronic myelomonocytic leukemia (n = 1), and chronic myeloid leukemia on follow-up (n = 4) showing MC differentiation in leukemic-blasts. These patients had mildly increased MCs (range, 0.5%-3%) in bone-marrow morphology, including immature-forms and did not meet the criteria for either SM-AHN or MML. On FCI, iMCs were positive for bright-CD117, heterogeneous-CD34, dim-to-negative-HLADR, and moderate-CD203c expression. Expression-levels of CD123 and CD38 were higher (p < 0.001) but CD33 and CD45 were lower in iMCs compared to mature-MC from control samples (p = 0.019 and p = 0.0037). CONCLUSION: We reported a rare finding of MC differentiation of leukemic blasts in diverse myeloid neoplasms and proposed it as a potential pre-myelomastocytic leukemia condition. We described the distinct immunophenotypic signature of immature-MCs using commonly used markers and highlighted the utility of FCI for the diagnosis of this entity.

8.
Cytometry B Clin Cytom ; 100(2): 206-217, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32865882

RESUMO

INTRODUCTION: In 2016, Children Oncology Group (COG) described a new high-risk subtype of acute myeloid leukemia (AML) with a distinct immunophenotypic-signature, RAM-phenotype (RAM-AML). Data on clinical and laboratory features of RAM-AML are still limited to COG report only. Herein, we report the clinicopathological characteristics and detailed immunophenotypic features of RAM-AML patients. In COG report, 38% of RAM-AML belonged to acute megakaryoblastic leukemia (AMKL)-subtype. Hence, we further compared the immunophenotypic features RAM-AML with non-RAM-AMKL diagnosed during the same study period. METHODS: We included RAM-AML and non-RAM AMKL patients diagnosed between January 2017 and December 2019. We studied their morphological, cytochemical, immunophenotyping, cytogenetic, and molecular characteristics. Mean fluorescent intensity (MFI) and expression-pattern of immunophenotypic markers of RAM-AML were compared with non-RAM AMKLs patients. RESULTS: We identified 11 RAM-AML (1%) and 21 non-RAM AMKL (1.9%) patients in 1102 pediatric-AML patients. Seven of 11 (63.64%) patients belonged to FAB-M7-subtype. CD56, CD117, and CD33 demonstrated overexpression, whereas CD45 and CD38 showed under-expression in RAM-AML patients. CD36 was consistently negative in RAM-AML, whereas moderate-bright positive in non-RAM AMKLs patients (p < 0.0001). On principle component analysis, addition of CD36 enhanced the visual-separation between RAM-AML and non-RAM AMKL clusters. Cytogenetic and molecular studies did not show any recurrent abnormality; however, RNA-sequencing study revealed CBFA2T3-GLIS2-fusion in three of seven (42.8%) RAM-AML patients. CONCLUSION: We report the clinicopathological characteristics and the detailed immunophenotypic profile in the world's second series of RAM-AML patients. We further report a novel finding of CD36-negative expression as an additional parameter to the multidimensional immunophenotypic signature of this entity.

9.
Front Oncol ; 10: 577, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32391267

RESUMO

Background: Measurable/minimal residual disease (MRD) status is suggested as a powerful indicator of clinical-outcome in T-cell lymphoblastic leukemia/lymphoma (T-ALL). Contrary to B-cell ALL, reports on T-ALL MRD are limited and mostly based on molecular methods, mainly from developed countries. Multicolor flow cytometry (MFC)-based T-ALL studies are very few. Clinically relevant cut-off levels and ideal time-point for MRD assessment are still inconclusive. In view of lack of T-ALL MRD data from the developing world, we evaluated the prognostic value of MFC-based post-induction (PI)-MRD assessment in T-ALL in the context of standard practice. Methods: We included 256 childhood-T-ALL patients (age < 15 years) treated with a modified-MCP841 protocol, which uses high-dose cytarabine during consolidation, as a part of standard hospital practice. MRD was studied using 10-color 11-antibody MFC with any level of detectable disease being considered positive. Post-induction (PI)-MRD was available in all patients, and post-consolidation (PC) MRD was available mostly in PI-MRD-positive patients (n = 88). Results: Three years cumulative-incidence-of-relapse (3years-CIR) in PI-MRD-positive patients was inferior to negative patients (46.3% vs. 18.4%). The median relapse-free-survival (RFS), event-free-survival (EFS) and overall-survival (OS) with hazard ratio (HR) of PI-MRD-positive patients were 21.4 months vs not reached (p < 0.0001, HR-4.7), 21.6 months vs. not-reached (p = 0.0003, HR-2.01) and 37.3 months vs. not reached (p = 0.026, HR-1.64) respectively. RFS, EFS and OS of patients with PI-MRD<0.01% (n = 17) were as inferior as PI-MRD ≥ 0.01% in comparison with MRD-negative patients with HR of 4.7 (p < 0.0001), 2.45 (p = 0.0003), and 2.5 (p = 0.029), respectively. Three-years-CIR of patients with hyperleukocytosis (≥100 × 109/L) was also higher (50.5 vs. 27.6%) with inferior RFS, EFS, and OS. Among PI-MRD-positive patients, 3years-CIR, RFS, EFS, and OS of PC-MRD-positive were also inferior to that of negative patients. On multivariate analysis any-level detectable PI-MRD and hyperleukocytosis remained independently associated with inferior RFS, EFS, and OS. A combination of PI-MRD-positive status and hyperleukocytosis identified the patients with the worst clinical outcomes. Conclusion: Detectable PI-MRD using MFC was found to be the strong predictive factor of inferior clinical outcome in T-ALL patients. The combination of PI-MRD status and hyperleukocytosis provides the most influential tool for the management of T-ALL in resource constrained settings from developing world.

10.
J Immunother Cancer ; 8(1)2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32439800

RESUMO

BACKGROUND: Recently, anti-CD38 monoclonal antibody (Mab) therapy has become a focus of attention as an additional/alternative option for many hematological neoplasms including T-cell acute lymphoblastic leukemia (T-ALL). It has been shown that antitumor efficacy of anti-CD38-Mab depends on the level of CD38 expression on tumor cells. Reports on CD38 expression in T-ALL are scarce, and data on the effect of cytotoxic chemotherapy on CD38 expression are limited to very few samples. Moreover, it lacks entirely in refractory disease and in adult T-ALL. We report the flow cytometric evaluation of CD38 expression in T-ALL blasts at diagnosis and the effect of cytotoxic chemotherapy on its expression in measurable residual disease (MRD), refractory disease (MRD≥5%), and relapsed disease in a large cohort of T-ALL. METHODS: The study included 347 samples (188 diagnostic, 100 MRD, 24 refractory and 35 relapse samples) from 196 (children: 85; adolescents/adults: 111) patients with T-ALL. CD38-positive blasts percentages (CD38-PBPs) and expression-intensity (mean fluorescent intensity, CD38-MFI) were studied using multicolor flow cytometry (MFC). MFC-based MRD was performed at the end-of-induction (EOI-MRD, day 30-35) and end-of-consolidation (EOC-MRD, day 78-85) subsequent follow-up (SFU-MRD) points. RESULTS: Patients were classified into early thymic precursor subtype of T-ALL (ETPALL, 54/188, 28.7%), and non-ETPALL (134/188, 71.3%). Of 188, EOI-MRD assessment was available in 152, EOC-MRD was available in 96 and SFU-MRD was available in 14 patients. CD38 was found positive in 97.9% (184/188) of diagnostic, 88.7% (110/124) MRD (including 24-refractory) and 82.9% (29/35) relapsed samples. Median (95% CI) of CD38-PBPs/MFI in diagnostic, MRD, refractory, and relapsed T-ALL samples were, respectively, 85.9% (82.10%-89.91%)/4.2 (3.88-4.47), 74.0% (58.87%-83.88%)/4.6 (3.67-6.81), 79.6% (65.25%-96.11%)/4.6 (3.33-8.47) and 85.2% (74.48%-93.01%)/5.6 (4.14-8.99). No significant difference was noted in CD38 expression between pediatric versus adult and patients with ETPALL versus non-ETPALL. No change was observed in CD38-MFI between diagnostic versus MRD and diagnostic versus relapsed paired samples. However, we noticed a mild drop in the CD38-PBPs in MRD samples compared with the diagnostic samples (p=0.016). CONCLUSION: We report an in-depth analysis of CD38 expression in a large cohort of T-ALL at diagnosis, during chemotherapy, and at relapse. Our data demonstrated that CD38 is robustly expressed in T-ALL blasts with a little effect of cytotoxic chemotherapy making it a potentially effective target for antiCD38-Mab therapy.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Glicoproteínas de Membrana/metabolismo , Recidiva Local de Neoplasia/metabolismo , Neoplasia Residual/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Seguimentos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Neoplasia Residual/tratamento farmacológico , Neoplasia Residual/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Prognóstico , Taxa de Sobrevida , Adulto Jovem
11.
Cytometry B Clin Cytom ; 98(4): 328-335, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31944572

RESUMO

BACKGROUND: Measurable residual disease (MRD) assessment using multicolor flow cytometry (MFC) has become the center point of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) risk stratification and therapeutic management. The addition of new markers can improve the accuracy and applicability of MFC-based MRD assay further. Herein, we evaluated the utility of a new marker, CD304/neuropilin-1, in the assessment of MFC-based MRD. METHODS: Expression patterns of CD304 were studied in leukemic blasts from BCP-ALL patients and in normal precursor B cells (NPBC) from uninvolved non-BCP-ALL bone marrow samples using 10-color MFC. MRD was monitored at end-of-induction (EOI; Days 35-40) and end-of-consolidation (Day 78-80) time points. RESULTS: We studied CD304 expression in 300 pediatric BCP-ALL patients and found it positive in BCP-ALL blasts in 41.7% of diagnostic samples. It was significantly associated with ETV6-RUNX1 (p < .001) as well as BCR-ABL1 (p = .019) and inversely associated with TCF3-PBX1 fusion gene (p = .0012). It was found clearly negative in NPBC. EOI-MRD was detectable in 152/300 (50.7%; ≥0.01% in 35.33% and <0.01% in 15.33%) samples, in which CD304 was positive in 72/152 (47.4%) diagnostic and 63/152 (41.4%) MRD samples. It was positive in 45.7% (21/46) of low-level (<0.01%) MRD samples. In comparison with diagnostic samples, its expression was retained in 68.06% (49/72), lost in 31.94% (23/72), and gained in 14/80 (17.5%) of EOI-MRD samples. CONCLUSIONS: CD304 is commonly expressed in leukemic blasts of BCP-ALL. It is very useful in distinguishing residual disease from hematogones and is a fairly dependable marker. Hence, it is a valuable addition for enhancing the sensitivity and applicability of MFC-based MRD assay in BCP-ALL.


Assuntos
Biomarcadores Tumorais/genética , Neoplasia Residual/genética , Neuropilina-1/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Adolescente , Linfócitos B/patologia , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Regulação Leucêmica da Expressão Gênica/genética , Humanos , Lactente , Masculino , Neoplasia Residual/diagnóstico , Neoplasia Residual/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/patologia
12.
Cytometry B Clin Cytom ; 98(1): 57-67, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31197916

RESUMO

BACKGROUND: Flow-cytometric minimal residual disease (FC-MRD) monitoring is a well-established risk-stratification factor in B-lymphoblastic leukemia/lymphoma (-B-ALL) and is being considered as a basis for deintensification or escalation in treatment protocols. However, currently practiced standard FC-MRD has limited sensitivity (up to 0.01%) and higher false MRD-negative rate. Hence, a highly sensitive, widely applicable, and easily reproducible FC-MRD assay is needed, which can provide a reliable basis for therapeutic modifications. METHODS: A 10-color high-event analysis FC-MRD assay was studied for the evaluation of MRD status at postinduction, (PI; day-35), postconsolidation, (PC; day-78), and subsequent follow-up time-points (SFU) in bone marrow samples from pediatric B-ALL. RESULTS: One-thousand MRD samples (PI-62.2%; PC-26.5%; and SFU-11.3%) from 622 childhood B-ALL patients were studied. High-event analysis was performed with median 4,452,000 events (range, 839,000 to 8,866,000 events) and >4 million events in 71% samples. MRD was measurable in 43.2% of PI-samples, in 29.4% PC-samples, and in 32.7% SFU-samples. To simulate comparison with standard FC-MRD, we reanalyzed MRD results gating only first 500,000 and first 1000,000 events in 122 PI-MRD positive samples with MRD levels <0.02%. Of these samples gated for 500,000 events and 1000,000 events, 32% and 21.3% were found to be falsely MRD-negative, respectively. CONCLUSIONS: We report an easily reproducible high-sensitivity 10-color FC-MRD assay with the sensitivity of 2-in-106 (0.0002%). It allowed the detection of low-level MRD in samples, which could have been reported negative using the standard FC-MRD with limited event analysis. Thus, this high-sensitivity MRD-methodology can provide a reliable basis for therapeutic modifications in B-ALL. © 2019 International Clinical Cytometry Society.


Assuntos
Citometria de Fluxo/métodos , Neoplasia Residual/diagnóstico , Neoplasia Residual/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Adolescente , Medula Óssea/patologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Sensibilidade e Especificidade
13.
Cytometry B Clin Cytom ; 94(3): 509-519, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29316178

RESUMO

BACKGROUND: Current flow-cytometric plasma cell (PC) gating is based on CD138, CD38, and CD45 expression. CD138 is known for variable expression and loss during storage and processing. Introduction of anti-CD38 and anti-CD138 monoclonal-antibody therapies has limited the use of these markers during follow-up. Hence, additional reliable PC-gating markers are required. Recently, CD229 has been claimed as an alternative PC-gating marker. However, these studies are limited to a small cohort of samples. We evaluated the utility of CD229 as a new PC-gating marker in routine laboratory practice. METHODS: Expression of CD229 was studied in 310 bone marrow (BM) samples (251 plasma-cell disorders and 59 controls) and compared with CD138 and CD38 expression. We also evaluated the effect of additional processing for cytoplasmic immunoglobulin-light-chains (CyIgL) staining on the quantitation of PC. RESULTS: Expression of CD229 was consistently stronger on PC than other hematopoietic-cells (p < 0.001). PC-percentages using CD229 in combination with CD38 or CD138 and CD45 revealed high correlation with a reference gating-strategy using CD138, CD38 and CD45 (r = 0.98, r = 0.99 r = 0.99 respectively) and r = 0.92 using CD229 and CD45 without CD38 or CD138. In contrast, CD138 expression showed significant variability (CV-MFI, 97.5) and loss from PC in 53% of samples. Quantitation of PC was found to be lower in 69.3% and higher in 30.7% samples processed for CyIgL-staining as compared to surface-staining. CONCLUSIONS: CD229 is a reliable new alternative PC-gating marker in routine laboratory practice. Quantitation of PC based on CD138 expression or from samples processed for CyIgL-staining should be used with caution. © 2018 International Clinical Cytometry Society.


Assuntos
Biomarcadores/metabolismo , Paraproteinemias/metabolismo , Plasmócitos/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/metabolismo , Estudos de Casos e Controles , Feminino , Citometria de Fluxo/métodos , Humanos , Cadeias Leves de Imunoglobulina/metabolismo , Imunofenotipagem/métodos , Masculino , Pessoa de Meia-Idade , Paraproteinemias/diagnóstico
14.
Cytometry B Clin Cytom ; 94(1): 100-111, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-27718302

RESUMO

BACKGROUND: Multiparametric flow cytometry (MFC) is a popular technique for minimal residual disease (MRD) analysis. However, its applicability is still limited to 90% of B-cell precursor acute lymphoblastic leukemia (BCPALL) due to two major issues, i.e. a proportion of cases do not express adequate leukemia associated immunophenotype (LAIPs) with currently used markers and drug-induced antigen modulation. Hence, the incorporation of additional reliable markers is required for the further improvement of MFC-based MRD evaluation. We studied the utility of new markers in improvising MFC-based MRD detection in BCPALL. METHODS: Expression-patterns of six new markers, i.e. CD24, CD44, CD72, CD73, CD86, and CD200 were studied in leukemic-blasts from ninety childhood BCPALL patients and in hematogones from 20 uninvolved staging bone marrow (BM) and ten postinduction non-BCPALL BM samples using eight-color MFC. The utility of these new markers in the day 35 postinduction MRD evaluation was determined. RESULTS: Frequencies of LAIPs of CD73, CD86, CD72, CD44, CD200, and CD24 in diagnostic samples were 76.7, 56.7, 55.6, 50, 28.9, and 20%, respectively. Differential expression of all new markers was highly significant (P < 0.01) between early (CD10+ CD19+ CD34+) hematogones, late (CD10+ CD19+ CD34-) hematogones and BCPALL blasts except between early hematogones and BCPALL blasts for CD200 (P = 0.1). In MRD-positive samples, CD73 showed the maximum (83%) frequency of LAIP and CD86 showed the highest (100%) stability of aberrant expression. Inclusion of CD73 and CD86 increased the applicability of MFC-MRD assay to 98.9% MRD samples. CONCLUSION: CD73 and CD86 are the most relevant markers to incorporate in the routine MRD evaluation of BCPALL. © 2016 International Clinical Cytometry Society.


Assuntos
5'-Nucleotidase/metabolismo , Antígeno B7-2/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasia Residual/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Adolescente , Adulto , Linfócitos B/metabolismo , Medula Óssea/metabolismo , Criança , Pré-Escolar , Feminino , Citometria de Fluxo/métodos , Proteínas Ligadas por GPI/metabolismo , Humanos , Imunofenotipagem/métodos , Lactente , Masculino , Adulto Jovem
15.
Curr Protoc Cytom ; 80: 6.38.1-6.38.15, 2017 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-28369766

RESUMO

The clinical use of flow cytometric DNA ploidy assay has been extended towards stratifying the risk of diseases, such as monoclonal gammopathies or B cell acute lymphoblastic leukemia, and to detect circulating tumor cells, both of which require detection of minute cell populations. This unit describes a protocol for determining DNA ploidy in fixed samples with simultaneous surface immunophenotyping. It is an easy method for simultaneous 6- to 8-color immunophenotyping and DNA content analysis using FxCycle Violet (FCV; DAPI) dye. This protocol is a one-step modification of routine multicolor immunophenotyping that includes surface staining followed by fixation and then DNA staining with FCV. It utilizes mature lymphocytes from the sample as an internal control for determination of DNA index. It is a sensitive method that allows DNA-ploidy determination and cell cycle analysis in a rare tumor population as low as 100 events, as well as DNA ploidy determination in various subsets of hematopoietic cells in the same sample based on their immunophenotype. © 2017 by John Wiley & Sons, Inc.


Assuntos
Corantes/química , DNA/análise , Imunofenotipagem/métodos , Ploidias , Contagem de Células , Linhagem Celular Tumoral , Humanos , Coloração e Rotulagem
16.
Cytometry B Clin Cytom ; 92(4): 315-318, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-27018867

RESUMO

BACKGROUND: CD19 is a B-cell specific marker, expressed on all stages of B-lymphocytes including plasma cells. It is widely used in the flow cytometric immunophenotyping (FCI) of B-cell and plasma cell malignancies. The analysis approach of FCI for the diagnosis and monitoring of B-cell acute lymphoblastic leukemia (B-ALL) is totally based on the CD19-based primary gating strategy and it would be challenging to study B-ALL without CD19 expression. Since CD19 negative B-ALL are extremely rare, we report three cases of B-ALL with negative expression of CD19 and discussed its implication in the diagnosis, residual disease monitoring and future targeted therapy. METHODS: Peripheral blood (PB) and bone marrow (BM) samples from three cases suspicious of acute leukemia were studied for morphology, cytochemistry, immunophenotyping and cytogenetics. FCI was performed using a comprehensive six to eight-color multiparametric assay. The cytogenetic studies for standard recurrent genetic translocations were performed by FISH & Karyotyping. RESULTS: The three cases studied were diagnosed as B-ALL based on the expression of CD10, CD20, CD22, CD34, and CD79a by leukemic blasts. CD19 was studied using two different clones (i.e. J3-119 & HIB-19) and found to be severely down regulated in all three cases. There were no significant differentiating features in morphology. Cytogenetic studies were negative for recurrent translocations. CONCLUSION: We report three cases of extremely rare CD19 negative B-ALL. Lack of awareness of negative CD19 expression in B-ALL can leads to incorrect immunophenotypic diagnosis and monitoring of B-ALL, especially in laboratories using limited markers. © 2016 International Clinical Cytometry Society.


Assuntos
Antígenos CD19/genética , Linfócitos B/patologia , Células da Medula Óssea/patologia , Imunofenotipagem/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Adulto , Anticorpos/química , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos CD19/imunologia , Linfócitos B/imunologia , Células da Medula Óssea/imunologia , Criança , Pré-Escolar , Regulação para Baixo , Feminino , Citometria de Fluxo , Expressão Gênica , Humanos , Cariotipagem , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia
17.
Cytometry A ; 89(3): 281-91, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26671309

RESUMO

Abnormal DNA ploidy is a valuable prognostic factor in many neoplasms, especially in hematological neoplasms like B-cell acute lymphoblastic leukemia (B-ALL) and multiple myeloma (MM). Current methods of flow-cytometric (FC) DNA-ploidy evaluation are either technically difficult or limited to three- to four-color immunophenotyping and hence, challenging to evaluate DNA-ploidy in minute tumor population with background rich of its normal counterpart cells and other hematopoietic cells. We standardized a novel sensitive and easy method of simultaneous evaluation of six- to seven-color immunophenotyping and DNA-ploidy using a dye-FxCycle Violet (FCV). Linearity, resolution, and coefficient of variation (CV) for FCV were studied using chicken erythrocyte nuclei. Ploidy results of FCV were compared with Propidium iodide (PI) in 20 samples and intra-assay variation for FCV was studied. Using this six-color immunophenotyping & FCV-protocol DNA-ploidy was determined in bone-marrow samples from 124 B-ALL & 50 MM patients. Dilution experiment was also conducted to determine the sensitivity in detection of aneuploidy in minute tumor population. FCV revealed high linearity and resolution in 450/50 channel. On comparison with PI, CV of Go/G1-peak with FCV (mean-CV 4.1%) was slightly higher than PI (mean-CV 2.9%) but had complete agreement in ploidy results. Dilution experiment showed that aneuploidy could be accurately detected up to the limit of 0.01% tumor cells. Intra-assay variation was very low with CV of 0.005%. In B-ALL, hypodiploidy was noted in 4%, hyperdiploidy in 24%, near-hyperdiploidy in 13% and remaining 59% were diploid. In MM, hypodiploidy was in 2%, hyperdiploidy in 58%, near-hyperdiploidy in 8% and remaining 30% were diploid. FCV-based DNA-ploidy method is a sensitive and easy method for simultaneous evaluation of six-color immunophenotyping and DNA analysis. It is useful in DNA-ploidy evaluation of minute tumor population in cases like minimal residual disease and MM precursor conditions.


Assuntos
DNA de Neoplasias/análise , Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Mieloma Múltiplo/diagnóstico , Ploidias , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Animais , Anticorpos/química , Antígenos CD/análise , Antígenos CD/genética , Antígenos CD/imunologia , Medula Óssea/imunologia , Medula Óssea/patologia , Núcleo Celular/ultraestrutura , Galinhas , DNA de Neoplasias/genética , DNA de Neoplasias/imunologia , Eritrócitos/ultraestrutura , Corantes Fluorescentes/química , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Sensibilidade e Especificidade
18.
Indian J Pathol Microbiol ; 55(1): 66-71, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22499304

RESUMO

BACKGROUND: Leukemic involvement in mantle cell lymphoma (MCL) is common, and can be secondary to nodal or extranodal disease or can be de-novo. There is paucity of literature that describes the morphological spectrum. AIM: This study was aimed at studying the morphological spectrum of leukemic MCL and to correlate the morphology with other features. MATERIALS AND METHODS: Twenty six such cases diagnosed over a period of four years were studied. Peripheral blood and bone marrow aspiration smears stained with Wrights stain were examined by three hematopathologists. Immunophenotyping was done using multicolor flow cytometry. Fluorescence in situ hybridization (FISH) done in 12 cases showed t(11;14)(q13:q32). RESULTS: Six cases had de-novo leukemic involvement; while 20 cases had secondary involvement. Morphologically, the cells were small (less than twice the size of red blood cell) or large. Small cell morphology in turn showed irregular nuclear border (n=13) or round nuclear contour (n=6). Large cells had blastic morphology (n=5) or had central prominent nucleoli resembling prolymhphocytes (n=2). Twenty cases showed characteristic immunophenotype of CD5+/CD19+/CD20+/FMC7+/CD10-/CD23- and light chain restrictions. Three cases expressed CD23 and two cases were negative for FMC7. Five out of 12 cases, where FISH was done, showed cytogenetic abnormalities in addition to t(11;14)(q13;q32). CONCLUSION: Morphological spectrum of leukemic MCL ranges from small cells resembling chronic lymphocytic leukemia (CLL) or follicular lymphoma (FL) to large cell mimicking prolymphocytic leukemia (PLL) or acute leukemia. Large cell morphology was associated with more frequent additional cytogenetic abnormality as well as a poorer outcome.


Assuntos
Células Sanguíneas/citologia , Medula Óssea/patologia , Linfoma de Célula do Manto/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Masculino , Microscopia , Pessoa de Meia-Idade
19.
Cytometry B Clin Cytom ; 78(4): 279-85, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20229505

RESUMO

BACKGROUND: We compared the international flow reference method (IRM) platelet counts with those obtained from CellDyn Sapphire (impedance and optical counts), LH750 (impedance counts), and the flowcytometry based ReaPanThrombo Immunoplatelet method (ReaMetrix). We further evaluated the degree of agreement of above methods with the IRM at the transfusion thresholds of 10 x 10(9) l(-1) and 20 x 10(9) l(-1). METHODS: A total of 104 thrombocytopenic blood samples with platelet count of <50 x 10(9) l(-1) were selected for the study. All samples were tested in parallel by various methods within 6 h of blood collection. RESULTS: For bias estimation, a Bland-Altman analysis was done by taking the IRM as the standard method. The bias for CDS-I counts was +6.505 x 10(9) l(-1) (95% LA -2.110 to +15.122), for CDS-O counts the bias was -3.779 x 10(9) l(-1) (95% LA -8.950 to +1.392), for LH750 the bias was +0.111 x 10(9) l(-1) (95% LA -5.862 to +6.084) and that for ReaMetrix was -1.602 x 10(9) l(-1) (95% LA -7.400 to +4.194). The LH750 had the least average bias and it overestimated platelet counts marginally. The ReaMetrix method showed the highest degree of agreement with the IRM, at both the threshold points with a K value of 0.960 (threshold < or = 10 x 10(9) l(-1)) and 0.923 (threshold < or = 20 x 10(9) l(-1)). CONCLUSIONS: Impedance platelet counts from LH750 were more accurate than optical methods in thrombocytopenic patients. ReaMetrix immunoplatelet counts show the maximum degree of agreement with the IRM at clinically relevant transfusion thresholds. We conclude that as current platelet transfusion thresholds are based on results of automated hematology analyzer methods, the true thresholds may be determined using the IRM and CD41/61 based single-platform immunoplatelet methods.


Assuntos
Citometria de Fluxo/métodos , Internacionalidade , Contagem de Plaquetas/métodos , Kit de Reagentes para Diagnóstico , Trombocitopenia/sangue , Eritrócitos/patologia , Humanos , Transfusão de Plaquetas , Padrões de Referência
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