RESUMEN
We describe a human lung disease caused by autosomal recessive, complete deficiency of the monocyte chemokine receptor C-C motif chemokine receptor 2 (CCR2). Nine children from five independent kindreds have pulmonary alveolar proteinosis (PAP), progressive polycystic lung disease, and recurrent infections, including bacillus Calmette Guérin (BCG) disease. The CCR2 variants are homozygous in six patients and compound heterozygous in three, and all are loss-of-expression and loss-of-function. They abolish CCR2-agonist chemokine C-C motif ligand 2 (CCL-2)-stimulated Ca2+ signaling in and migration of monocytic cells. All patients have high blood CCL-2 levels, providing a diagnostic test for screening children with unexplained lung or mycobacterial disease. Blood myeloid and lymphoid subsets and interferon (IFN)-γ- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated immunity are unaffected. CCR2-deficient monocytes and alveolar macrophage-like cells have normal gene expression profiles and functions. By contrast, alveolar macrophage counts are about half. Human complete CCR2 deficiency is a genetic etiology of PAP, polycystic lung disease, and recurrent infections caused by impaired CCL2-dependent monocyte migration to the lungs and infected tissues.
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Proteinosis Alveolar Pulmonar , Receptores CCR2 , Niño , Humanos , Pulmón/metabolismo , Macrófagos Alveolares/metabolismo , Proteinosis Alveolar Pulmonar/genética , Proteinosis Alveolar Pulmonar/diagnóstico , Receptores CCR2/deficiencia , Receptores CCR2/genética , Receptores CCR2/metabolismo , Reinfección/metabolismoRESUMEN
Adult T-cell leukemia (ATL) is a lymphoid neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1), which encodes the transcriptional activator Tax, which participates in the immortalization of infected T cells. ATL is classified into 4 subtypes: smoldering, chronic, acute, and lymphoma. We determined whether natural killer receptors (NKRs) were expressed in ATL. NKR expression (KIR2DL1/2DS1, KIR2DL2/2DL3/2DS2, KIR3DL2, NKG2A, NKG2C, and NKp46) was assessed in a discovery cohort of 21 ATL, and KIR3DL2 was then assessed in 71 patients with ATL. KIR3DL2 was the only NKR among those studied frequently expressed by acute-type vs lymphoma- and chronic/smoldering-type ATL (36 of 40, 4 of 16, and 1 of 15, respectively; P = .001), although acute- and lymphoma-type ATL had similar mutation profiles by targeted exome sequencing. The correlation of KIR3DL2 expression with promoter demethylation was determined by microarray-based DNA methylation profiling. To explore the role of HTLV-1, KIR3DL2 and TAX messenger RNA (mRNA) expression levels were assessed by PrimeFlow RNA in primary ATL and in CD4+ T cells infected with HTLV-1 in vitro. TAX mRNA and KIR3DL2 protein expressions were correlated on ATL cells. HTLV-1 infection triggered KIR3DL2 by CD4+ cells but Tax alone did not induce KIR3DL2 expression. Ex vivo, autologous, antibody-dependent cell cytotoxicity using lacutamab, a first-in-class anti-KIR3DL2 humanized antibody, selectively killed KIR3DL2+ primary ATL cells ex vivo. To conclude, KIR3DL2 expression is associated with acute-type ATL. Transcription of KIR3DL2 may be triggered by HTLV-1 infection and correlates with hypomethylation of the promoter. The benefit of targeting KIR3DL2 with lacutamab is being further explored in a randomized phase 2 study in peripheral T-cell lymphoma, including ATL (registered on https://clinicaltrials.gov as #NCT04984837).
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Infecciones por HTLV-I , Virus Linfotrópico T Tipo 1 Humano , Leucemia-Linfoma de Células T del Adulto , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Adulto , Productos del Gen tax/genética , Productos del Gen tax/metabolismo , Infecciones por HTLV-I/complicaciones , Infecciones por HTLV-I/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Leucemia-Linfoma de Células T del Adulto/patología , ARN , ARN Mensajero , Receptores KIR3DL2/genéticaRESUMEN
The gastrointestinal tract is the site of exciting immunological interactions between the epithelium and the mucosa-associated lymphoid tissue, leading to the immune response to food and microbial antigens in the digestive lumen. The objective of this review is to present the main dysimmune pathologies of the digestive tract leading to an enteropathy. As examples, we describe celiac and non-celiac enteropathies to clarify a florid diagnostic framework, by identifying a spectrum of elementary lesions, which must be confronted with the clinico biological context of the patient to orient the diagnosis. The microscopic lesions observed are most often non-specific and may be encountered in several diagnostic settings. Moreover, it is a set of elementary lesions in each clinical context that will orient the diagnostic framework. Celiac disease is the main etiology of enteropathy with villous atrophy, its diagnosis is multidisciplinary and there are many differential diagnoses. We will discuss celiac disease lymphomatous complications as enteropathy associated T-cell lymphoma including refractory sprue type 2. We will then present the non-celiac enteropathies. Among these, enteropathies of unknown etiology may be associated with a primary immune deficiency that may be reflected by florid lymphoid hyperplasia of the gastrointestinal tract and/or be associated with an infectious etiology that should also be constantly sought. Finally, we will discuss of induced enteropathy by new immunomodulatory treatments.
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Enfermedad Celíaca , Humanos , Enfermedad Celíaca/complicaciones , Enfermedad Celíaca/diagnóstico , Intestino Delgado/patología , Hiperplasia/patologíaRESUMEN
BACKGROUND: Donor availability and transplantation-related risks limit the broad use of allogeneic hematopoietic-cell transplantation in patients with transfusion-dependent ß-thalassemia. After previously establishing that lentiviral transfer of a marked ß-globin (ßA-T87Q) gene could substitute for long-term red-cell transfusions in a patient with ß-thalassemia, we wanted to evaluate the safety and efficacy of such gene therapy in patients with transfusion-dependent ß-thalassemia. METHODS: In two phase 1-2 studies, we obtained mobilized autologous CD34+ cells from 22 patients (12 to 35 years of age) with transfusion-dependent ß-thalassemia and transduced the cells ex vivo with LentiGlobin BB305 vector, which encodes adult hemoglobin (HbA) with a T87Q amino acid substitution (HbAT87Q). The cells were then reinfused after the patients had undergone myeloablative busulfan conditioning. We subsequently monitored adverse events, vector integration, and levels of replication-competent lentivirus. Efficacy assessments included levels of total hemoglobin and HbAT87Q, transfusion requirements, and average vector copy number. RESULTS: At a median of 26 months (range, 15 to 42) after infusion of the gene-modified cells, all but 1 of the 13 patients who had a non-ß0/ß0 genotype had stopped receiving red-cell transfusions; the levels of HbAT87Q ranged from 3.4 to 10.0 g per deciliter, and the levels of total hemoglobin ranged from 8.2 to 13.7 g per deciliter. Correction of biologic markers of dyserythropoiesis was achieved in evaluated patients with hemoglobin levels near normal ranges. In 9 patients with a ß0/ß0 genotype or two copies of the IVS1-110 mutation, the median annualized transfusion volume was decreased by 73%, and red-cell transfusions were discontinued in 3 patients. Treatment-related adverse events were typical of those associated with autologous stem-cell transplantation. No clonal dominance related to vector integration was observed. CONCLUSIONS: Gene therapy with autologous CD34+ cells transduced with the BB305 vector reduced or eliminated the need for long-term red-cell transfusions in 22 patients with severe ß-thalassemia without serious adverse events related to the drug product. (Funded by Bluebird Bio and others; HGB-204 and HGB-205 ClinicalTrials.gov numbers, NCT01745120 and NCT02151526 .).
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Terapia Genética , Globinas beta/genética , Talasemia beta/terapia , Adolescente , Adulto , Antígenos CD34 , Niño , Transfusión de Eritrocitos/estadística & datos numéricos , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos , Hemoglobinas/análisis , Hemoglobinas/genética , Humanos , Lentivirus/genética , Masculino , Mutación , Trasplante Autólogo , Adulto Joven , Talasemia beta/genéticaRESUMEN
Flow cytometry is broadly used for the identification, characterization, and monitoring of hematological malignancies. However, the use of clinical flow cytometry is restricted by its lack of reproducibility across multiple centers. Since 2006, the EuroFlow consortium has been developing a standardized procedure detailing the whole process from instrument settings to data analysis. The FranceFlow group was created in 2010 with the intention to educate participating centers in France about the standardized instrument setting protocol (SOP) developed by the EuroFlow consortium and to organise several rounds of quality controls (QCs) in order to evaluate the feasibility of its application and its results. Here, we report the 5 year experience of the FranceFlow group and the results of the seven QCs of 23 instruments, involving up to 19 centers, in France and in Belgium. The FranceFlow group demonstrates that both the distribution and applicability of the SOP have been successful. Intercenter reproducibility was evaluated using both normal and pathological blood samples. Coefficients of variation (CVs) across the centers were <7% for the percentages of cell subsets and <30% for the median fluorescence intensities (MFIs) of the markers tested. Intracenter reproducibility provided similar results with CVs of <3% for the percentages of the majority of cell subsets, and CVs of <20% for the MFI values for the majority of markers. Altogether, the FranceFlow group show that the 19 participating labs might be considered as one unique laboratory with 23 identical flow cytometers able to reproduce identical results. Therefore, SOP significantly improves reproducibility of clinical flow in hematology and opens new avenues by providing a robust companion diagnostic tool for clinical trials in hematology. © 2019 International Society for Advancement of Cytometry.
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Citometría de Flujo/métodos , Neoplasias Hematológicas/diagnóstico , Inmunofenotipificación/normas , Bélgica , Citometría de Flujo/instrumentación , Citometría de Flujo/normas , Fluorescencia , Francia , Neoplasias Hematológicas/sangre , Humanos , Inmunofenotipificación/métodos , Linfocitos/citología , Linfocitos/metabolismo , Monocitos/citología , Monocitos/metabolismo , Control de Calidad , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
ß-thalassemia, a hereditary blood disorder caused by defective synthesis of hemoglobin ß globin chains, leads to ineffective erythropoiesis and chronic anemia that may require blood transfusions. Sotatercept (ACE-011) acts as a ligand trap to inhibit negative regulators of late-stage erythropoiesis in the transforming growth factor ß superfamily, correcting ineffective erythropoiesis. In this phase II, open-label, dose-finding study, 16 patients with transfusion-dependent ß -thalassemia and 30 patients with non-transfusion-dependent ß-thalassemia were enrolled at seven centers in four countries between November 2012 and November 2014. Patients were treated with sotatercept at doses of 0.1, 0.3, 0.5, 0.75, or 1.0 mg/kg to determine a safe and effective dose. Doses were administered by subcutaneous injection every 3 weeks. Patients were treated for ≤22 months. Response was assessed as a ≥20% reduction in transfusion burden sustained for 24 weeks in transfusion-dependent ß-thalassemia patients, and an increase in hemoglobin level of ≥1.0 g/dL sustained for 12 weeks in non-transfusion-dependent ß-thalassemia patients. Sotatercept was well tolerated. After a median treatment duration of 14.4 months (range 0.6-35.9), no severe life-threatening adverse events were observed. Thirteen percent of patients reported serious but manageable adverse events. The active dose of sotatercept was ≥0.3 mg/kg for patients with non-transfusion-dependent ß-thalassemia and ≥0.5 mg/kg for those with transfusion-dependent ß-thalassemia. Of 30 non-transfusion-dependent ß-thalassemia patients treated with ≥0.1 mg/kg sotatercept, 18 (60%) achieved a mean hemoglobin increase ≥1.0 g/dL, and 11 (37%) an increase ≥1.5 g/dL, sustained for ≥12 weeks. Four (100%) transfusion-dependent ß-thalassemia patients treated with 1.0 mg/kg sotatercept achieved a transfusion-burden reduction of ≥20%. Sotatercept was effective and well tolerated in patients with ß-thalassemia. Most patients with non-transfusion-dependent ß-thalassemia treated with higher doses achieved sustained increases in hemoglobin level. Transfusion-dependent ß-thalassemia patients treated with higher doses of sotatercept achieved notable reductions in transfusion requirements. This trial was registered at ClinicalTrials.gov with the number NCT01571635.
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Anemia/tratamiento farmacológico , Anemia/etiología , Proteínas Recombinantes de Fusión/administración & dosificación , Talasemia beta/complicaciones , Adulto , Anemia/sangre , Anemia/diagnóstico , Biomarcadores , Transfusión Sanguínea , Terapia Combinada , Índices de Eritrocitos , Eritropoyesis/efectos de los fármacos , Femenino , Hemoglobinas , Humanos , Ligandos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes de Fusión/efectos adversos , Factor de Crecimiento Transformador beta/metabolismo , Resultado del Tratamiento , Talasemia beta/diagnóstico , Talasemia beta/tratamiento farmacológicoAsunto(s)
Leucemia Linfocítica Granular Grande , Receptores de Antígenos de Linfocitos T gamma-delta , Neoplasias del Bazo , Humanos , Leucemia Linfocítica Granular Grande/genética , Leucemia Linfocítica Granular Grande/patología , Leucemia Linfocítica Granular Grande/diagnóstico , Neoplasias del Bazo/genética , Neoplasias del Bazo/patología , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Linfoma de Células T/genética , Linfoma de Células T/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Femenino , Genómica/métodos , AncianoRESUMEN
In order to identify very early prognostic factors that can provide insights into subsequent clinical complications, we performed a comprehensive longitudinal multi-center cohort study on 57 infants with sickle cell anemia (55 SS; 2 Sß°) during the first 2 years of life (ClinicalTrials.gov: NCT01207037). Time to first occurrence of a severe clinical event-acute splenic sequestration (ASS), vaso-occlusive (VOC) event requiring hospitalization, transfusion requirement, conditional/ abnormal cerebral velocities, or death-was used as a composite endpoint. Infants were recruited at a mean age of 4.4 ±1 months. Median follow-up was 19.4 months. During the study period, 38.6% of infants experienced ≥1 severe event: 14% ASS, 22.8% ≥ 1 VOC (median age: 13.4 and 12.8 months, respectively) and 33.3% required transfusion. Of note, 77% of the cohort was hospitalized, with febrile illness being the leading cause for admission. Univariate analysis of various biomarkers measured at enrollment showed that fetal hemoglobin (HbF) was the strongest prognostic factor of subsequent severe outcome. Other biomarkers measured at enrolment including absolute neutrophil or reticulocyte counts, expression of erythroid adhesion markers, % of dense red cells, cellular deformability or Ï-globin genetic variants, failed to be associated with severe clinical outcome. Multivariate analysis demonstrated that higher Hb concentration and HbF level are two independent protective factors (adjusted HRs (95% CI) 0.27 (0.11-0.73) and 0.16 (0.06-0.43), respectively). These findings imply that early measurement of HbF and Hb levels can identify infants at high risk for subsequent severe complications, who might maximally benefit from early disease modifying treatments.
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Anemia de Células Falciformes/diagnóstico , Índice de Severidad de la Enfermedad , Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/terapia , Biomarcadores/análisis , Transfusión Sanguínea , Estudios de Cohortes , Femenino , Hemoglobina Fetal/análisis , Hemoglobinas/análisis , Hospitalización , Humanos , Lactante , Estudios Longitudinales , Masculino , PronósticoRESUMEN
BACKGROUND: We investigated 7 male patients (from 5 different families) presenting with profound lymphopenia, hypogammaglobulinemia, fluctuating monocytopenia and neutropenia, a poor immune response to vaccine antigens, and increased susceptibility to bacterial and varicella zoster virus infections. OBJECTIVE: We sought to characterize the genetic defect involved in a new form of X-linked immunodeficiency. METHODS: We performed genetic analyses and an exhaustive phenotypic and functional characterization of the lymphocyte compartment. RESULTS: We observed hemizygous mutations in the moesin (MSN) gene (located on the X chromosome and coding for MSN) in all 7 patients. Six of the latter had the same missense mutation, which led to an amino acid substitution (R171W) in the MSN four-point-one, ezrin, radixin, moesin domain. The seventh patient had a nonsense mutation leading to a premature stop codon mutation (R533X). The naive T-cell counts were particularly low for age, and most CD8+ T cells expressed the senescence marker CD57. This phenotype was associated with impaired T-cell proliferation, which was rescued by expression of wild-type MSN. MSN-deficient T cells also displayed poor chemokine receptor expression, increased adhesion molecule expression, and altered migration and adhesion capacities. CONCLUSION: Our observations establish a causal link between an ezrin-radixin-moesin protein mutation and a primary immunodeficiency that could be referred to as X-linked moesin-associated immunodeficiency.
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Linfocitos T CD8-positivos/inmunología , Cromosomas Humanos X/genética , Síndromes de Inmunodeficiencia/genética , Infecciones/genética , Proteínas de Microfilamentos/genética , Mutación/genética , Adolescente , Adulto , Anciano , Adhesión Celular , Movimiento Celular , Niño , Preescolar , Estudios de Asociación Genética , Humanos , Recuento de Linfocitos , Masculino , LinajeRESUMEN
Although purine analogues have significantly improved the outcome of hairy cell leukaemia (HCL) patients, 30-40% relapse, illustrating the need for minimal residual disease (MRD) markers that can aid personalized therapeutic management. Diagnostic samples from 34 HCL patients were used to design an 8-colour flow cytometry (8-FC) tube for blood MRD (B/RD) analysis (188 samples) which was compared to quantitative IGH polymerase chain reaction (Q-PCR) on 83 samples and to qualitative consensus IGH PCR clonality analysis on 165 samples. Despite heterogeneous HCL phenotypes at diagnosis, discrimination from normal B lymphocytes was possible in all cases using a single 8-FC tube, with a robust sensitivity of detection of 10(-4) , comparable to Q-PCR at this level, but preferable in terms of informativeness, simplicity and cost. B/RD assessment of 15 patients achieving haematological complete remission after purine analogues was predictive of a clinically significant relapse risk: with a median follow-up of 95 months; only one of the nine patients with reproducible 8-FC B/RD levels below 10(-4) (B/RD(neg) ) relapsed, compared to 5/6 in the B/RD(pos) group (P = 0.003). These data demonstrate the clinical interest of a robust 8-FC HCL B/RD strategy that could become a surrogate biomarker for therapeutic stratification and new drug assessment, which should be evaluated prospectively.
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Leucemia de Células Pilosas/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/sangre , Estudios de Casos y Controles , Femenino , Citometría de Flujo/métodos , Estudios de Seguimiento , Genes de las Cadenas Pesadas de las Inmunoglobulinas/genética , Humanos , Leucemia de Células Pilosas/tratamiento farmacológico , Leucemia de Células Pilosas/genética , Masculino , Persona de Mediana Edad , Neoplasia Residual/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Pronóstico , Recurrencia , Sensibilidad y EspecificidadAsunto(s)
Antineoplásicos/uso terapéutico , Mastocitosis Sistémica/tratamiento farmacológico , Estaurosporina/análogos & derivados , Edad de Inicio , Antineoplásicos/efectos adversos , Estudios de Seguimiento , Humanos , Análisis Multivariante , Estaurosporina/efectos adversos , Estaurosporina/uso terapéutico , Tasa de SupervivenciaRESUMEN
Extranodal NK/T-cell lymphoma (ENKTCL) is an Epstein-Barr virus (EBV)-related neoplasm with male dominance and a poor prognosis. A better understanding of the genetic alterations and their functional roles in ENKTCL could help improve patient stratification and treatments. In this study, we performed a comprehensive genetic analysis of 178 ENKTCL cases to delineate the landscape of mutations, copy number alterations (CNA), and structural variations, identifying 34 driver genes including six previously unappreciated ones, namely, HLA-B, HLA-C, ROBO1, CD58, POT1, and MAP2K1. Among them, CD274 (24%) was the most frequently altered, followed by TP53 (20%), CDKN2A (19%), ARID1A (15%), HLA-A (15%), BCOR (14%), and MSN (14%). Chromosome X losses were the most common arm-level CNAs in females (â¼40%), and alterations of four X-linked driver genes (MSN, BCOR, DDX3X, and KDM6A) were more frequent in males and females harboring chromosome X losses. Among X-linked drivers, MSN was the most recurrently altered, and its expression was lost in approximately one-third of cases using immunohistochemical analysis. Functional studies of human cell lines showed that MSN disruption promoted cell proliferation and NF-κB activation. Moreover, MSN inactivation increased sensitivity to NF-κB inhibition in vitro and in vivo. In addition, recurrent deletions were observed at the origin of replication in the EBV genome (6%). Finally, by integrating the 34 drivers and 19 significant arm-level CNAs, nonnegative matrix factorization and consensus clustering identified two molecular groups with different genetic features and prognoses irrespective of clinical prognostic factors. Together, these findings could help improve diagnostic and therapeutic strategies in ENKTCL. Significance: Integrative genetic analyses and functional studies in extranodal NK/T-cell lymphoma identify frequent disruptions of X-linked drivers, reveal prognostic molecular subgroups, and uncover recurrent MSN alterations that confer sensitivity to NF-κB inhibition.
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Cromosomas Humanos X , Linfoma Extranodal de Células NK-T , Humanos , Masculino , Femenino , Cromosomas Humanos X/genética , Linfoma Extranodal de Células NK-T/genética , Linfoma Extranodal de Células NK-T/virología , Linfoma Extranodal de Células NK-T/patología , Linfoma Extranodal de Células NK-T/metabolismo , Variaciones en el Número de Copia de ADN , Mutación , Persona de Mediana Edad , Animales , Adulto , Ratones , Pronóstico , Anciano , Perfilación de la Expresión Génica , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Adulto Joven , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/complicacionesRESUMEN
In order to standardize cellular hematology practices, the French-speaking Cellular Hematology Group (Groupe Francophone d'Hématologie Cellulaire, GFHC) focused on Perls' stain. A national survey was carried out, leading to the proposal of recommendations on insoluble iron detection and quantification in bone marrow. The criteria presented here met with a "strong professional agreement" and follow the suggestions of the World Health Organization's classification of hematological malignancies.
RESUMEN
Sickle cell disease (SCD) and transfusion-dependent ß-thalassemia (TDT) are the most prevalent monogenic disorders worldwide. Trial HGB-205 ( NCT02151526 ) aimed at evaluating gene therapy by autologous CD34+ cells transduced ex vivo with lentiviral vector BB305 that encodes the anti-sickling ßA-T87Q-globin expressed in the erythroid lineage. HGB-205 is a phase 1/2, open-label, single-arm, non-randomized interventional study of 2-year duration at a single center, followed by observation in long-term follow-up studies LTF-303 ( NCT02633943 ) and LTF-307 ( NCT04628585 ) for TDT and SCD, respectively. Inclusion and exclusion criteria were similar to those for allogeneic transplantation but restricted to patients lacking geno-identical, histocompatible donors. Four patients with TDT and three patients with SCD, ages 13-21 years, were treated after busulfan myeloablation 4.6-7.9 years ago, with a median follow-up of 4.5 years. Key primary endpoints included mortality, engraftment, replication-competent lentivirus and clonal dominance. No adverse events related to the drug product were observed. Clinical remission and remediation of biological hallmarks of the disease have been sustained in two of the three patients with SCD, and frequency of transfusions was reduced in the third. The patients with TDT are all transfusion free with improvement of dyserythropoiesis and iron overload.
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Anemia de Células Falciformes/terapia , Terapia Genética , Lentivirus/genética , Talasemia beta/terapia , Adolescente , Femenino , Terapia Genética/efectos adversos , Humanos , Masculino , Resultado del Tratamiento , Adulto JovenAsunto(s)
Leucemia Eritroblástica Aguda/complicaciones , Leucemia Eritroblástica Aguda/patología , Mieloma Múltiple/complicaciones , Mieloma Múltiple/patología , Humanos , Inmunofenotipificación , Leucemia Eritroblástica Aguda/terapia , Masculino , Persona de Mediana Edad , Mieloma Múltiple/terapia , PronósticoRESUMEN
BACKGROUND: As part of quality assurance in laboratories (labs) offering clinical cell analysis by flow cytometry (FCM), cell counting precision and robustness are evaluated but international desirable ranges are still missing. The aim of this study was to provide desirable interlaboratory variability reference values. METHODS: A prospective survey on monthly quality assessment was proposed to all French laboratories routinely performing lymphocyte subpopulation quantification, over one arbitrarily selected month (June 2017), regardless of instrument, counting system and quality controls used. Relative variabilities of the commercially available internal quality control (IQC) used locally were collected. Robust mean, standard deviation and CV were calculated on relative and absolute counts. RESULTS: Sixty-two labs participated, providing 91 sets of data on 82 instruments. All but three were enrolled in external quality assessment (EQA) and 46 in externalized IQA. The mean CV of five repeats ranged from 1.00 ± 0.33 for T cells to 4.78 ± 1.92 for NK cells and from 2.88 ± 1.46 to 5.87 ± 1.83 for relative and absolute counts, respectively. The precision correlated directly to the concentration of cells rather than the phenotype. Negligible differences were observed between IQC material: Multicheck™ (3.36 ± 1.30, n = 11); Immunotrol™ (3.62 ± 3.24, n = 21) and Statusflow™ (3.63 ± 1.87, n = 24) on CD4+ T cell, for example. Little difference was observed between counting systems such as Flowcount™ (4.55 ± 3.45, n = 19), Trucount™ (3.17 ± 1.40, n = 30) and the fully automated Aquios™ system (1.87 ± 0.75, n = 5) on single platforms, while dual platform CV was at 2.00 ± 0.58 (n = 2) on CD4+ T cell, as example. Robustness was measured on 21 ± 11 consecutive analyses of the same IQC material, providing CVs ranging from 4.45 ± 1.74 for T cells to 7.57 ± 2.19 for NK absolute counts. Average residuals calculated from the different low count IQC samples for CD4 T cells (median value below 200 cells/µl) were below 10 cells/µl, demonstrating their robustness for medical decisions. CONCLUSIONS: Real life variabilities in cell counting are directly related to the cell concentration and not to their phenotype. Desirable ranges within three SD are proposed according to different cell levels, based on 62 labs, different IQC material and systems. © 2018 International Clinical Cytometry Society.
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Citometría de Flujo , Linfocitos/citología , Servicios de Laboratorio Clínico/normas , Citometría de Flujo/normas , Humanos , Recuento de Linfocitos , Estudios Prospectivos , Control de CalidadRESUMEN
Performance of lymphocyte count by flow cytometry is difficult to evaluate because of the lack of desirable range for precision performance. The aim of this work was to propose recommendations for acceptable precision variability. Data of repeatability and reproducibility of two levels of IQC were collected from sixty-four laboratories. For each cell population, acceptable CV was fixed as the mean CV obtained with robust statistical methods plus 3 standard deviations. Performance limits for reproducibility varied from 2.5% to 9.1%, respectively for CD3+ and NK cells expressed as percentage, and from 9.7% to 14.2% respectively for absolute count of CD3+ and NK cells. Reproducibility data results were obtained from a mean of 21±11 data. Performance limits for reproducibility varied from 2.5% to 9.1% respectively for CD3+ and NK cells expressed as percentage, and from 9.7% to 14.2% respectively for absolute value of CD3+ and NK; 90% of the laboratories had CV inferior to the limit values. Both repeatability and reproducibility values were inversely related to the importance of cell population. These results highlight the accuracy of the method despite the variability of instruments, protocols, IQC and counting systems. It is thus possible to recommend a "state-of-the-art" maximum CV to evaluate and follow over time the performance of the method, and to extrapolate the results to rare cells counting.