Hepatosplenic T-cell lymphoma displays an original oyster-shell cytological pattern and a distinct genomic profile from that of gamma-delta T-cell large granular lymphocytic leukemia.

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Enteral citrulline supplementation versus placebo on SOFA score on day 7 in mechanically ventilated critically ill patients: the IMMUNOCITRE randomized clinical trial.

BACKGROUND: Restoring plasma arginine levels through enteral administration of L-citrulline in critically ill patients may improve outcomes. We aimed to evaluate whether enteral L-citrulline administration reduced organ dysfunction based on the Sequential Organ Failure Assessment (SOFA) score and affected selected immune parameters in mechanically ventilated medical intensive care unit (ICU) patients. METHODS: A randomized, double-blind, multicenter clinical trial of enteral administration of L-citrulline versus placebo for critically ill adult patients under invasive mechanical ventilation without sepsis or septic shock was conducted in four ICUs in France between September 2016 and February 2019. Patients were randomly assigned to receive enteral L-citrulline (5 g) every 12 h for 5 days or isonitrogenous, isocaloric placebo. The primary outcome was the SOFA score on day 7. Secondary outcomes included SOFA score improvement (defined as a decrease in total SOFA score by 2 points or more between day 1 and day 7), secondary infection acquisition, ICU length of stay, plasma amino acid levels, and immune biomarkers on day 3 and day 7 (HLA-DR expression on monocytes and interleukin-6). RESULTS: Of 120 randomized patients (mean age, 60 ± 17 years; 44 [36.7%] women; ICU stay 10 days [IQR, 7-16]; incidence of secondary infections 25 patients (20.8%)), 60 were allocated to L-citrulline and 60 were allocated to placebo. Overall, there was no significant difference in organ dysfunction as assessed by the SOFA score on day 7 after enrollment (4 [IQR, 2-6] in the L-citrulline group vs. 4 [IQR, 2-7] in the placebo group; Mann‒Whitney U test, p = 0.9). Plasma arginine was significantly increased on day 3 in the treatment group, while immune parameters remained unaffected. CONCLUSION: Among mechanically ventilated ICU patients without sepsis or septic shock, enteral L-citrulline administration did not result in a significant difference in SOFA score on day 7 compared to placebo. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT02864017 (date of registration: 11 August 2016).

Functional specialization of short-lived and long-lived macrophage subsets in human tonsils.

Macrophages play a central role in tissue homeostasis and host defense. However, the properties of human macrophages in non-diseased tissues remain poorly understood. Here, we characterized human tonsil macrophages and identified three subsets with distinct phenotype, transcriptome, life cycle, and function. CD36hi macrophages were related to monocytes, while CD36lo macrophages showed features of embryonic origin and CD36int macrophages had a mixed profile. scRNA-seq on non-human primate tonsils showed that monocyte recruitment did not pre-exist an immune challenge. Functionally, CD36hi macrophages were specialized for stimulating T follicular helper cells, by producing Activin A. Combining reconstruction of ligand-receptor interactions and functional assays, we identified stromal cell-derived TNF-α as an inducer of Activin A secretion. However, only CD36hi macrophages were primed for Activin A expression, via the activity of IRF1. Our results provide insight into the heterogeneity of human lymphoid organ macrophages and show that tonsil CD36hi macrophage specialization is the result of both intrinsic features and interaction with stromal cells.

The Negative Influence of Baseline Cell-free DNA on Long-term Survival in DLBCL Depends on Frontline Treatment Intensity.

PURPOSE: This study aims to investigate the relationship between the intensity of the initial treatment given to patients with de novo diffuse large B-cell lymphoma (DLBCL) and the impact of their baseline cell-free DNA (cfDNA) levels on their long-term survival. EXPERIMENTAL DESIGN: The GOELAMS 075 randomized clinical trial compared rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) with high-dose R-chemotherapy plus autologous stem cell transplantation (R-HDT) for patients aged ≤60. An interim PET assessment was used to refer patients for salvage therapy. With a median follow-up of more than 5.8 years, we analyzed the effects of the treatment arm, salvage therapy, and cfDNA level at diagnosis on overall survival (OS). RESULTS: In a representative group of 123 patients, a high cfDNA concentration (>55 ng/mL) at diagnosis was associated with poor clinical prognostic factors and constituted a prognostic marker, independently of the age-adjusted International Prognostic Index. A cfDNA level above a threshold value of 55 ng/mL at diagnosis was associated with significantly worse OS. In an intention-to-treat analysis, high-cfDNA R-CHOP patients (but not high-cfDNA R-HDT patients) had worse OS [HR (95% confidence interval), 3.99 (1.98-10.74); P = 0.006]. In patients with high cfDNA levels, salvage therapy and transplantation were associated with a significantly higher OS rate. Among 50 patients with complete response 6 months after the end of treatment, for 11 of 24 R-CHOP patients, the cfDNA did not fall back to normal values. CONCLUSIONS: In this randomized clinical trial, intensive regimens mitigated the negative influence of high cfDNA levels in de novo DLBCL, relative to R-CHOP.

Demultiplexing Ig repertoires by parallel mRNA/DNA sequencing shows major differential alterations in severe COVID-19.

To understand the fine differential elements that can lead to or prevent acute respiratory distress syndrome (ARDS) in COVID-19 patients, it is crucial to investigate the immune response architecture. We herein dissected the multiple layers of B cell responses by flow cytometry and Ig repertoire analysis from acute phase to recovery. Flow cytometry with FlowSOM analysis showed major changes associated with COVID-19 inflammation such as an increase of double-negative B-cells and ongoing plasma cell differentiation. This paralleled COVID-19-driven expansion of two disconnected B-cell repertoires. Demultiplexing successive DNA and RNA Ig repertoire patterns characterized an early expansion of IgG1 clonotypes with atypically long and uncharged CDR3, the abundance of this inflammatory repertoire being correlated with ARDS and likely pejorative. A superimposed convergent response included convergent anti-SARS-CoV-2 clonotypes. It featured progressively increasing somatic hypermutation together with normal-length or short CDR3 and it persisted until a quiescent memory B-cell stage after recovery.

Blood, Cellular, and Tissular Calcineurin Inhibitors Pharmacokinetic-Pharmacodynamic Relationship in Heart Transplant Recipients: The INTRACAR Study.

BACKGROUND: After heart transplantation, calcineurin inhibitors (CNI) (cyclosporin A and tacrolimus) are key immunosuppressive drugs to prevent graft rejection. Whole-blood concentration (C blood )-guided therapeutic drug monitoring (TDM) is systematically performed to improve graft outcomes. However, some patients will still experience graft rejection and/or adverse events despite CNI C blood within the therapeutic range. Other pharmacokinetic parameters, such as the intragraft, or intracellular concentration at the CNI site of action could refine their TDM. Nonetheless, these remain to be explored. The objective of the INTRACAR study was to describe the relationship between whole blood, intragraft, and intracellular CNI concentrations as well as their efficacy in heart transplant recipients (HTR). METHODS: In a cohort of HTR, protocol endomyocardial biopsies (EMB) were collected to assess rejection by anatomopathological analysis. Part of the EMB was used to measure the intragraft concentrations of CNI (C EMB ). C blood and the concentration inside peripheral blood mononuclear cells, (C PBMC ), a cellular fraction enriched with lymphocytes, were also monitored. Concentrations in the 3 matrices were compared between patients with and without biopsy-proven acute rejection (BPAR). RESULTS: Thirty-four HTR were included, representing nearly 100 pharmacokinetic (PK) samples for each CNI. C blood , C EMB , and C PBMC correlated for both CNI. BPAR was observed in 74 biopsies (39.6%) from 26 patients (76.5%), all except one was of low grade. None of the PK parameters (C blood , C EMB , C PBMC , C EMB/blood , and C PBMC/blood ) was associated with BPAR. CONCLUSIONS: In this cohort of well-immunosuppressed patients, no association was observed for any of the PK parameters, including C blood , with the occurrence of BPAR. However, a trend was noticed for the C EMB and C EMB/blood of cyclosporin A. Further studies in higher-risk patients may help optimize the use of C EMB and C PBMC for CNI TDM in HTR.

MONOCYTIC MYELOID-DERIVED SUPPRESSOR CELL EXPANSION AFTER CARDIAC SURGERY WITH CARDIOPULMONARY BYPASS INDUCES LYMPHOCYTE DYSFUNCTION.

ABSTRACT: Cardiac surgery with cardiopulmonary bypass (CPB) is associated with an immune paresis that predisposes to the development of postoperative infections and sepsis. Among factors responsible for CPB-induced immunosuppression, circulating myeloid-derived suppressor cells (MDSCs) have been found to induce early lymphocyte apoptosis and lymphocyte proliferation inhibition. However, the mechanisms involved are not fully understood. In this study, we found that the main lymphocyte subsets decreased significantly 24 h after cardiac surgery with CBP. As expected, cardiac surgery with CPB induced a monocytic MDSC expansion associated with an increased T-cell apoptosis and decreased proliferation capacity. Noteworthy, granulocytic MDSCs remain stable. Myeloid-derived suppressor cell depletion restored the ability of T-cell to proliferate ex vivo . After CPB, indoleamine 2,3-dioxygenase activity and IL-10 plasma level were increased such as programmed death-ligand 1 monocytic expression, whereas plasma level of arginine significantly decreased. Neither the inhibition of indoleamine 2,3-dioxygenase activity nor the use of anti-programmed death-ligand 1 or anti-IL-10 blocking antibody restored the ability of T-cell to proliferate ex vivo . Only arginine supplementation restored partially the ability of T-cell to proliferate.

Drug transporters are implicated in the diffusion of tacrolimus into the T lymphocyte in kidney and liver transplant recipients: Genetic, mRNA, protein expression, and functionality.

Because of a narrow therapeutic index and a wide inter- and intra-patient variability, therapeutic drug monitoring of the immunosuppressant drug tacrolimus (TAC) based on whole-blood concentrations (Cblood) is mandatory in solid organ transplant recipients. Using peripheral blood mononuclear cells concentrations (CPBMC) could improve patient outcomes. The poor correlation between Cblood and CPBMC makes hypothesize that drug transporters are implicated in the intracellular accumulation of TAC. The aim of this work was therefore to clinically study: i) the role of genetic variants and ii) the effect of mRNA and protein expression of 4 drug transporters on the TAC CPBMC/blood ratio. In addition, functional in vitro experiments were performed to mechanistically validate the clinical observations. Genetic variants of ABCB1/P-gp and SLC28A3/CNT3 did not influence TAC CPBMC in liver transplant recipients (LTR). ABCC2/MRP2 at the mRNA level; ABCB1/P-gp, SLC28A3/CNT3 and SLC29A1/ENT1 at the protein level; correlated with the CPBMC/blood in kidney and LTR. In vitro results suing transporter-expressing cells confirmed that TAC is substrate of P-gp but not MRP2, whereas experiments remained inconclusive for CNT3 and ENT1. In conclusion, the genetic-transcription-protein-functional approach presented in this work provides new insights in the understanding of TAC transport at the T lymphocyte plasma membrane.

Beneficial effects of citrulline enteral administration on sepsis-induced T cell mitochondrial dysfunction.

Severe sepsis induces a sustained immune dysfunction associated with poor clinical behavior. In particular, lymphopenia along with increased lymphocyte apoptosis and decreased lymphocyte proliferation, enhanced circulating regulatory T cells (Treg), and the emergence of myeloid-derived suppressor cells (MDSCs) have all been associated with persistent organ dysfunction, secondary infections, and late mortality. The mechanisms involved in MDSC-mediated T cell dysfunction during sepsis share some features with those described in malignancies such as arginine deprivation. We hypothesized that increasing arginine availability would restore T cell function and decrease sepsis-induced immunosuppression. Using a mouse model of sepsis based on cecal ligation and puncture and secondary pneumonia triggered by methicillin-resistant Staphylococcus aureus inoculation, we demonstrated that citrulline administration was more efficient than arginine in increasing arginine plasma levels and restoring T cell mitochondrial function and proliferation while reducing sepsis-induced Treg and MDSC expansion. Because there is no specific therapeutic strategy to restore immune function after sepsis, we believe that our study provides evidence for developing citrulline-based clinical studies in sepsis.

Comparative immune profiling of acute respiratory distress syndrome patients with or without SARS-CoV-2 infection.

Acute respiratory distress syndrome (ARDS) is the main complication of coronavirus disease 2019 (COVID-19), requiring admission to the intensive care unit (ICU). Despite extensive immune profiling of COVID-19 patients, to what extent COVID-19-associated ARDS differs from other causes of ARDS remains unknown. To address this question, here, we build 3 cohorts of patients categorized in COVID-19-ARDS+, COVID-19+ARDS+, and COVID-19+ARDS-, and compare, by high-dimensional mass cytometry, their immune landscape. A cell signature associating S100A9/calprotectin-producing CD169+ monocytes, plasmablasts, and Th1 cells is found in COVID-19+ARDS+, unlike COVID-19-ARDS+ patients. Moreover, this signature is essentially shared with COVID-19+ARDS- patients, suggesting that severe COVID-19 patients, whether or not they experience ARDS, display similar immune profiles. We show an increase in CD14+HLA-DRlow and CD14lowCD16+ monocytes correlating to the occurrence of adverse events during the ICU stay. We demonstrate that COVID-19-associated ARDS displays a specific immune profile and may benefit from personalized therapy in addition to standard ARDS management.

Combined Platelet and Erythrocyte Salvage: Evaluation of a New Filtration-based Autotransfusion Device.

BACKGROUND: The SAME device (i-SEP, France) is an innovative filtration-based autotransfusion device able to salvage and wash both red blood cells and platelets. This study evaluated the device performances using human whole blood with the hypothesis that the device will be able to salvage platelets while achieving a erythrocyte yield of 80% and removal ratios of 90% for heparin and 80% for major plasma proteins without inducing signification activation of salvaged cells. METHODS: Thirty healthy human whole blood units (median volume, 478 ml) were diluted, heparinized, and processed by the device in two consecutive treatment cycles. Samples from the collection reservoir and the concentrated blood were analyzed. Complete blood count was performed to measure blood cell recovery rates. Flow cytometry evaluated the activation state and function of platelets and leukocytes. Heparin and plasma proteins were measured to assess washing performance. RESULTS: The global erythrocyte yield was 88.1% (84.1 to 91.1%; median [25th to 75th]) with posttreatment hematocrits of 48.9% (44.8 to 51.4%) and 51.4% (48.4 to 53.2%) for the first and second cycles, respectively. Ektacytometry did not show evidence of erythrocyte alteration. Platelet recovery was 36.8% (26.3 to 43.4%), with posttreatment counts of 88 × 109/l (73 to 101 × 109/l) and 115 × 109/l (95 to 135 × 109/l) for the first and second cycles, respectively. Recovered platelets showed a low basal P-selectin expression at 10.8% (8.1 to 15.2%) and a strong response to thrombin-activating peptide. Leukocyte yield was 93.0% (90.1 to 95.7%) with no activation or cell death. Global removal ratios were 98.3% (97.8 to 98.9%), 98.2% (96.9 to 98.8%), and 88.3% (86.6 to 90.7%) for heparin, albumin, and fibrinogen, respectively. The processing times were 4.4 min (4.2 to 4.6 min) and 4.4 min (4.2 to 4.7 min) for the first and second cycles, respectively. CONCLUSIONS: This study demonstrated the performance of the SAME device. Platelets and red blood cells were salvaged without significant impact on cell integrity and function. In the meantime, leukocytes were not activated, and the washing quality of the device prevented reinfusion of high concentrations of heparin and plasma proteins.

Mass Cytometry Identifies Expansion of T-bet+ B Cells and CD206+ Monocytes in Early Multiple Sclerosis.

Multiple sclerosis (MS) is an immune-driven demyelinating disease of the central nervous system. Immune cell features are particularly promising as predictive biomarkers due to their central role in the pathogenesis but also as drug targets, even if nowadays, they have no impact in clinical practice. Recently, high-resolution approaches, such as mass cytometry (CyTOF), helped to better understand the diversity and functions of the immune system. In this study, we performed an exploratory analysis of blood immune response profiles in healthy controls and MS patients sampled at their first neurological relapse, using two large CyTOF panels including 62 markers exploring myeloid and lymphoid cells. An increased abundance of both a T-bet-expressing B cell subset and a CD206+ classical monocyte subset was detected in the blood of early MS patients. Moreover, T-bet-expressing B cells tended to be enriched in aggressive MS patients. This study provides new insights into understanding the pathophysiology of MS and the identification of immunological biomarkers. Further studies will be required to validate these results and to determine the exact role of the identified clusters in neuroinflammation.

Functional characterization of PD1+TIM3+ tumor-infiltrating T cells in DLBCL and effects of PD1 or TIM3 blockade.

In diffuse large B-cell lymphoma (DLBCL), tumor-infiltrating T lymphocytes (TILs) are involved in therapeutic responses. However, tumor-specific TILs can be dysfunctional, with impaired effector functions. Various mechanisms are involved in this exhaustion, and the increased expression of programmed cell death receptor 1 (PD1) and TIM3 on dysfunctional cells suggests their involvement. However, conflicting data have been published regarding their expression or coexpression in DLBCL. We evaluated the presence and phenotype of CD4+ and CD8+ TILs in freshly collected tumor tissues in DLBCL and compared the results with those in follicular lymphoma, classical Hodgkin lymphoma, and nonmalignant reactive lymphadenopathy. We found that TILs expressing both PD1 and TIM3 were expanded in DLBCL, particularly in the activated B cell-like subgroup. Isolated PD1+TIM3+ TILs exhibited a transcriptomic signature related to T-cell exhaustion associated with a reduction in cytokine production, both compromising the antitumor immune response. However, these cells expressed high levels of cytotoxic molecules. In line with this, stimulated PD1+TIM3+ TILs from DLBCL patients exhibited reduced proliferation and impaired secretion of interferon-γ, but these functions were restored by the blockade of PD1 or TIM3. In summary, the PD1+TIM3+ TIL population is expanded and exhausted in DLBCL but can be reinvigorated with appropriate therapies.

Venoarterial extracorporeal membrane oxygenation induces early immune alterations.

BACKGROUND: Venoarterial extracorporeal membrane oxygenation (VA-ECMO) provides heart mechanical support in critically ill patients with cardiogenic shock. Despite important progresses in the management of patients under VA-ECMO, acquired infections remain extremely frequent and increase mortality rate. Since immune dysfunctions have been described in both critically ill patients and after surgery with cardiopulmonary bypass, VA-ECMO initiation may be responsible for immune alterations that may expose patients to nosocomial infections (NI). Therefore, in this prospective study, we aimed to study immune alterations induced within the first days by VA-ECMO initiation. METHODS: We studied immune alterations induced by VA-ECMO initiation using cytometry analysis to characterize immune cell changes and enzyme-linked immunosorbent assay (ELISA) to explore plasma cytokine levels. To analyze specific changes induced by VA-ECMO initiation, nine patients under VA-ECMO (VA-ECMO patients) were compared to nine patients with cardiogenic shock (control patients). RESULTS: Baseline immune parameters were similar between the two groups. VA-ECMO was associated with a significant increase in circulating immature neutrophils with a significant decrease in C5a receptor expression. Furthermore, we found that VA-ECMO initiation was followed by lymphocyte dysfunction along with myeloid-derived suppressor cells (MDSC) expansion. ELISA analysis revealed that VA-ECMO initiation was followed by an increase in pro-inflammatory cytokines such as IL-6, IL-8 and TNF-α along with IL-10, a highly immunosuppressive cytokine. CONCLUSION: VA-ECMO is associated with early immune changes that may be responsible for innate and adaptive immune alterations that could confer an increased risk of infection.

SARS-CoV-2-Induced ARDS Associates with MDSC Expansion, Lymphocyte Dysfunction, and Arginine Shortage.

PURPOSE: The SARS-CoV-2 infection can lead to a severe acute respiratory distress syndrome (ARDS) with prolonged mechanical ventilation and high mortality rate. Interestingly, COVID-19-associated ARDS share biological and clinical features with sepsis-associated immunosuppression since lymphopenia and acquired infections associated with late mortality are frequently encountered. Mechanisms responsible for COVID-19-associated lymphopenia need to be explored since they could be responsible for delayed virus clearance and increased mortality rate among intensive care unit (ICU) patients. METHODS: A series of 26 clinically annotated COVID-19 patients were analyzed by thorough phenotypic and functional investigations at days 0, 4, and 7 after ICU admission. RESULTS: We revealed that, in the absence of any difference in demographic parameters nor medical history between the two groups, ARDS patients presented with an increased number of myeloid-derived suppressor cells (MDSC) and a decreased number of CD8pos effector memory cell compared to patients hospitalized for COVID-19 moderate pneumonia. Interestingly, COVID-19-related MDSC expansion was directly correlated to lymphopenia and enhanced arginase activity. Lastly, T cell proliferative capacity in vitro was significantly reduced among COVID-19 patients and could be restored through arginine supplementation. CONCLUSIONS: The present study reports a critical role for MDSC in COVID-19-associated ARDS. Our findings open the possibility of arginine supplementation as an adjuvant therapy for these ICU patients, aiming to reduce immunosuppression and help virus clearance, thereby decreasing the duration of mechanical ventilation, nosocomial infection acquisition, and mortality.

Linking the KIR phenotype with STAT3 and TET2 mutations to identify chronic lymphoproliferative disorders of NK cells.

Distinguishing chronic lymphoproliferative disorders of NK cells (CLPD-NK) from reactive NK-cell expansion is challenging. We assessed the value of killer immunoglobulin-like receptor(KIR) phenotyping and targeted high-throughput sequencing in a cohort of 114 consecutive patients with NK cell proliferation, retrospectively assigned to a CLPD-NK group (n = 46) and a reactive NK group (n = 68). We then developed an NK-cell clonality score combining flow cytometry and molecular profiling with a positive predictive value of 93%. STAT3 and TET2 mutations were respectively identified in 27% and 34% of the patients with CLPD-NK, constituting a new diagnostic hallmark for this disease. TET2-mutated CLPD-NK preferentially exhibited a CD16low phenotype, more frequently displayed a lower platelet count, and was associated with other hematologic malignancies such as myelodysplasia. To explore the mutational clonal hierarchy of CLPD-NK, we performed whole-exome sequencing of sorted, myeloid, T, and NK cells and found that TET2 mutations were shared by myeloid and NK cells in 3 of 4 cases. Thus, we hypothesized that TET2 alterations occur in early hematopoietic progenitors which could explain a potential link between CLPD-NK and myeloid malignancies. Finally, we analyzed the transcriptome by RNA sequencing of 7 CLPD-NK and evidenced 2 groups of patients. The first group displayed STAT3 mutations or SOCS3 methylation and overexpressed STAT3 target genes. The second group, including 2 TET2-mutated cases, significantly underexpressed genes known to be downregulated in angioimmunoblastic T-cell lymphoma. Our results provide new insights into the pathogenesis of NK-cell proliferative disorders and, potentially, new therapeutic opportunities.

Nonclassical Monocytes Are Prone to Migrate Into Tumor in Diffuse Large B-Cell Lymphoma.

Absolute count of circulating monocytes has been proposed as an independent prognostic factor in diffuse large B-cell lymphoma (DLBCL). However, monocyte nomenclature includes various subsets with pro-, anti-inflammatory, or suppressive functions, and their clinical relevance in DLBCL has been poorly explored. Herein, we broadly assessed circulating monocyte heterogeneity in 91 DLBCL patients. Classical- (cMO, CD14pos CD16neg) and intermediate- (iMO, CD14pos CD16pos) monocytes accumulated in DLBCL peripheral blood and exhibited an inflammatory phenotype. On the opposite, nonclassical monocytes (ncMOSlanpos, CD14low CD16pos Slanneg and ncMOSlanneg, CD14low CD16pos, Slanneg) were decreased in peripheral blood. Tumor-conditioned monocytes presented similarities with ncMO phenotype from DLBCL and were prone to migrate in response to CCL5 and CXCL12, and presented similarities with DLBCL-infiltrated myeloid cells, as defined by mass cytometry. Finally, we demonstrated the adverse value of an accumulation of nonclassical monocytes in 2 independent cohorts of DLBCL.

Multicentric MFI30 study: Standardization of flow cytometry analysis of CD30 expression in non-Hodgkin lymphoma.

CD30 transmembrane receptor, a member of the tumor necrosis factor receptor family, is expressed in different lymphomas. Brentuximab vedotin (BV), a CD30 monoclonal antibody (Ab)-drug conjugate, is effective in CD30-positive lymphomas. However, the response to BV is not always correlated to CD30 expression detected by immunohistochemistry (IHC). The objectives of this study were to standardize and evaluate CD30 intensity by flow cytometry (FCM) in non-Hodgkin's lymphomas. Twelve centers analyzed 161 cases on standardized cytometers using normalized median fluorescence intensity (nMFI30) of three different Abs, of which one clone can recognize the same epitope as BV. FCM distinguished four groups of cases: negative group (n = 110) which showed no expression with the three clones; high positive group (n = 13) which gave nMFI30 > 5% with all tested clones; dim positive group (n = 17) which showed nMFI30 > 1% with all tested clones and <5% for at least one; discordant group (n = 21) with positive and negative expression of the different clones. In consistency with the literature, CD30 was positive in all anaplastic large cell lymphomas, in some diffuse large B-cell lymphomas (DLBCL), and in other rare lymphomas. FCM results were concordant with those of IHC in 77% of cases. Discrepancies could be explained by clones-related differences, microenvironment, or intracytoplasmic staining. Interestingly, FCM was more sensitive than IHC in 11% of cases, especially in DLBCL. Multicenter standardized FCM of specific CD30 could improve case detection and extend the treatment of BV to various CD30-positive lymphomas.