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Clonal hematopoiesis (CH) of indeterminate potential has been described in blood samples from large series of patients. Its prevalence and consequences are still not well understood because sequencing methods vary and because most studies were performed in cohorts comprising individuals with nonhematologic diseases. Here, we investigated the frequency of CH in 82 paired bone marrow and blood samples from carefully selected healthy adult volunteers. Forty-one genes known to be mutated in myeloid malignancies were sequenced with a 1% threshold of detection. In bone marrow samples, clones were found in almost 40% of healthy volunteers more than 50 years old. The most frequent mutations were found in DNMT3A and TET2, with 1 individual carrying 3 variants. Variant allele frequencies were highly concordant between blood and bone marrow samples. Blood parameters were normal except for those in 2 individuals: 1 had a mild macrocytosis and 1 had a mild thrombocytosis. Furthermore, no morphologic abnormalities or dysplasia were detected when bone marrow smears were carefully evaluated. Individuals with CH differed from others by age (62.8 vs 38.6 years; P < .0001) and platelet count (294 vs 241 ×109/L; P = .0208), the latter being no more significant when removing the 2 individuals who carried the JAK2 p.V617F mutation. These results confirm that CH is a very common condition in healthy adults over 50 years old. Consequently, the detection of driver myeloid mutations should be interpreted with caution in the absence of cytologic abnormalities in the blood and/or the bone marrow.

INTRODUCTION Cytokines are involved in many processes, including hematopoiesis and inflammation. Aging is associated with the onset of clonal hematopoiesis (CH) of indeterminate potential, putatively associated with a higher risk of progression to hematological malignancies such as myelodysplastic syndromes or acute myeloid leukemia. Moreover, CH may participate to create a pro-inflammatory environment contributing to the pathogenesis of age-related diseases, such as cardiovascular diseases. This is likely to be driven by or translated in changes in bone marrow (BM) and/or peripheral blood (PB) soluble factors for which reference values still remain unclear, because BM cytokines levels have never been determined in strictly selected healthy people. Indeed, control BM samples classically used in studies are from subjects undergoing surgeries for non-hematologic causes, such as total hip replacement or cardiac surgery, patients suffering from immune thrombocytopenic purpura, brain death patients or allogeneic BM donors. In this study, the BM and PB plasma concentrations of 49 hematopoietic and inflammatory cytokines were measured in a representative panel of 94 healthy adult volunteers and the results were analyzed considering their age and presence of CH. METHOD Ninety-four healthy donors aged from 18.6 to 80.1 years old (yo), including 58 women were recruited for this study (HEALTHOX protocol, CPP Tours, AFSSAPS identifier ID-RCB: 2016-A00571-50 and ClinicalTrials.gov # NCT02789839). The presence or absence of CH (>1% of variant allele frequency) in this cohort is already known (Guermouche H, Ravalet N et al, Blood Adv 2020;4(15):3550-3557). BM samples were obtained through sternal aspiration using a classical procedure in France, and PB sampling was performed at the same time by venipuncture. Samples were collected on sodium heparin or EDTA, centrifuged twice (1200 g, 10 min, 20 C°), aliquoted and stored at -80°C. A 48-plex human cytokine panel assay was used to quantify 48 human cytokines in PB and BM plasmas [beta-NGF, CCL2, CCL3, CCL4, CCL5, CCL7, CCL11, CCL27, CLEC11A, CXCL1, CXCL8, CXCL9, CXCL10, CXCL12, FGF-2, G-CSF, GM-CSF, HGF, IFN-alpha-2, IFN-gamma, IL-1 alpha, IL-1 beta, IL-1ra, IL-2, IL-2-RA, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-12B, IL-13, IL-15, IL-16, IL-17A, IL-18, KITLG, LIF, LT-alpha, M-CSF, MIF, PDGF subunit B, TNF, TNFSF10, VEGF-A]. MIF and FLT3L quantification were performed by ELISA. Regarding CH, the controls were subjects older than 50 yo without CH. Statistical analyses were performed with the R (3.6.3) and Rstudio version 1.2.5042 (www.rstudio.org) software. Comparisons were computed by Wilcoxon and Kruskal-Wallis tests. All pairwise multiple comparisons were performed using Dunn's-test for multiple comparisons of independent samples (PMCMR package). Correlations between cytokine levels in PB and BM were tested with Pearson and Spearman methods. Correlation matrices were plotted using the "corrplot" package. RESULTS CH was detected in 16 volunteers, mostly in individuals over 50 yo. BM and PB plasma samples were studied in 3 age-groups: 18-40, 40-60 and 60-80 yo. With aging, variations were observed for 18 BM cytokine levels, with 7 increasing (FLT3L, CXCL9, HGF, FGF-2, CCL27, IL-16, IL-18) and 8 decreasing (G-CSF, TNF, IL-2, IL-15, IL-17a, IL-4, LT-alpha, IL-1 alpha). In PB, 10 cytokines significantly increased with age (CXCL9, FLT3L, CCL27, CXCL10, HGF, CCL11, IL-16, IL-6, IL-1 beta, CCL2). CH was associated with significantly higher BM levels of MIF and IL-1 beta, lower BM levels of IL-9 and IL-5 and higher PB levels of IL-15, VEGF-A, IL-2, CXCL8, CXCL1 and G-CSF (Table). CONCLUSION In this study and for the first time, we concomitantly analyzed BM and PB concentrations of a panel of hematopoietic and inflammatory cytokines in a cohort of strictly selected healthy volunteers. In addition to the establishment of reference values, useful for various biological studies, and correlations between blood and BM levels, we identify variations in the BM of key cytokines according to age and CH. The differences in these cytokine concentrations, either as causes or as consequences, may shape both the BM microenvironment and hematopoietic processes, eventually leading to the beginning of age-related myeloid malignancies or inflammatory conditions. Figure 1 Figure 1. Disclosures Hirsch: Daiichi Sankyo Oncology: Consultancy; Novartis Pharma: Consultancy. Suner: Sanofi - Genzyme: Consultancy. Delhommeau: Celgene: Consultancy; BMS: Consultancy; Novartis: Consultancy.

Le microtitrage anti-D, technique d’hemagglutination en support gel sur hematies R0r (RH : 1,−2,−3,4,5) permet de quantifier les faibles taux d’anti-D grâce a une gamme de standard anti-D. Realisee depuis 1999 au CNRHP et automatisee sur Evo clinical Base Tecan en 2008 (dilutions et distribution), elle est adaptee a la prophylaxie Rh. L’objectif de l’etude etait d’evaluer la technique sur l’IH-500 de Bio-Rad® en comparaison avec celle du CNRHP. Sur IH-500, la reactivite des reactifs Bio-Rad® a ete comparee a celle des reactifs du CNRHP (R0r, standard anti-D). Les performances de la methode ont ete evaluees a l’aide de 3 CQI (2 CNRHP et 1 Bio-Rad®) sur R0r papainees PLC et natives NLC. Enfin une comparaison de methode a partir de serums de patientes variant de 1,5 a 48 ng/ml a ete faite. Les resultats des 3 CQI sont comparables entre les differents reactifs utilises. Il n’y a pas de difference significative entre les 2 types d’hematies sauf pour la limite de detection : 1,5 ng/ml en PLC – 6 ng/ml en NLC. Pour les 3 CQI, les tests de repetabilite, reproductibilite sur les rangs de dilution ont des CV entre 0 a 15 %, performances similaires a celles retrouvees avec l’Evo Clinical Base. La correlation avec la technique CNRHP realisee sur 50 echantillons en PLC et 44 echantillons en NLC a ete satisfaisante (Deming PLC : r2 = 0,80 Y = 0,89X + 0,78–NLC : r2 = 0,86 Y = 1,08X−0,25). Le microtitrage anti-D sur l’IH-500 offre des performances similaires a la methode realisee au CNRHP. L’IH-500 permet une lecture automatisee des gels. Cependant, il ne dispose pas d’algorithme de calcul ni d’interpretation permettant un rendu des concentrations en anti-D qui reste manuel necessitant un personnel forme.

Key Clinical Message Occurrence of two different hematological malignancies is very infrequent, and it is nevertheless important not to neglect basic examinations in patients follow‐up. A 65‐year‐old patient was referred to our institution for his chronic lymphocytic leukemia (CLL) checkup; we report the different steps leading to the diagnosis of a second hematological malignancy.

L’arrete du 15 mai 2018 relatif aux examens d’immuno-hematologie erythrocytaire ouvre la voie a l’utilisation de methodes de titrage d’anticorps en carte-gel, qui ont l’avantage de pouvoir etre automatisees jusqu’a l’etape de lecture. Dans ce contexte, nous avons evalue les performances de la methode de titrage en carte-gel Liss Coombs IgG sur l’automate IH-500 (Bio-Rad®). Une comparaison avec la methode de titrage en tube a egalement ete realisee, afin de definir des valeurs seuils pour le declenchement d’une surveillance echographique fœtale adaptee. Les tests de repetabilite et de reproductibilite, effectues a partir des scores du CQI anti-RH1 prepare au CNRHP montrent des CV proches de 2 % sur l’IH-500, comparativement aux valeurs de 6 % a 20 % etablies pour la methode manuelle de titrage en gel ou les methodes de titrage en tube. Les titres obtenus par les methodes tubes et gel ont ete compares pour 122 echantillons de specificites differentes : 105 echantillons avaient des valeurs superieures en technique gel, 15 des titres identiques et 2 des titres inferieurs. Les ecarts de titre les plus importants (en moyenne superieurs de 2 a 3 dilutions) concernaient les anticorps du systeme RH. Les specificites anti-KEL1 et anti-MNS1 comptaient le plus d’echantillons ayant des titres egaux, voire inferieurs d’une dilution, en technique gel. Si les seuils definis pour les titrages en tube semblent pouvoir etre extrapoles a la technique gel, il sera probablement necessaire, pour certaines specificites, de les revoir a la hausse, sous couvert d’etudes avec des donnees cliniques, afin d’eviter d’induire un suivi de grossesse lourd, anxiogene et inutile, pour un certain nombre de patientes.