Faster clinical decisions in B-cell acute lymphoblastic leukaemia: A single flow cytometric 12-colour tube improves diagnosis and minimal residual disease follow-up.

Assessing minimal residual disease (MRD) in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) is essential for adjusting therapeutic strategies and predicting relapse. Quantitative polymerase chain reaction (qPCR) is the gold standard for MRD. Alternatively, flow cytometry is a quicker and cost-effective method that typically uses leukaemia-associated immunophenotype (LAIP) or different-from-normal (DFN) approaches for MRD assessment. This study describes an optimized 12-colour flow cytometry antibody panel designed for BCP-ALL diagnosis and MRD monitoring in a single tube. This method robustly differentiated hematogones and BCP-ALL cells using two specific markers: CD43 and CD81. These and other markers (e.g. CD73, CD66c and CD49f) enhanced the specificity of BCP-ALL cell detection. This innovative approach, based on a dual DFN/LAIP strategy with a principal component analysis method, can be used for all patients and enables MRD analysis even in the absence of a diagnostic sample. The robustness of our method for MRD monitoring was confirmed by the strong correlation (r = 0.87) with the qPCR results. Moreover, it simplifies and accelerates the preanalytical process through the use of a stain/lysis/wash method within a single tube (<2 h). Our flow cytometry-based methodology improves the BCP-ALL diagnosis efficiency and MRD management, offering a complementary method with considerable benefits for clinical laboratories.

Waldenstrom's macroglobulinemia highlighted by turbidity interference: a case report / Découverte d'une maladie de Waldenström via une interférence de turbidité : à propos d'un cas

A 65-year-old female was admitted to the Emergency Department for a fall. Upon admission, a blood sample was drawn for routine laboratory tests. Blood glucose measurement using the hexokinase method was made impossible due to a high lipemia index measured by the instrument despite a clear plasma specimen. It is well known that hemolysis, icterus, and lipemia interferences disrupt spectrophotometric quantifications. However, in this case, the absence of lipemia was confirmed by plasma triglycerides measurement in the normal reference range. Protein electrophoresis along with an immunofixation were carried out to characterize a monoclonal gammopathy since the presence of a monoclonal gammopathy in very high concentrations generates turbidity and interferes with spectrophotometric assays. A gamma peak evoking a monoclonal gammopathy was found on the serum electrophoresis and the immunofixation was positive for a monoclonal IgM kappa. Myelogram showed plasmocytes, lymphoplasmacytic cells and mast cells infiltration which led to the diagnosis of Waldenstrom macroglobulinemia. Based on this case, we propose a course of action to be taken in the case of a high lipemia index and clear plasma.

Une femme de 65 ans est admise aux urgences pour une chute. À l'admission, un échantillon de sang a été prélevé pour réaliser un bilan biologique de routine. Sur ce bilan, la mesure spectrophotométrique de la glycémie par la méthode de l'hexokinase était impossible en raison d'un indice de lactescence trop élevé et ce malgré un plasma d'aspect clair. Il est bien connu que l'hémolyse, l'ictère et la lactescence interfèrent dans le dosage spectrophotométrique. Cependant, dans ce cas, il n'y a pas de perturbation du bilan lipidique chez la patiente. Une prestation de conseil a donc été réalisée par le service de biochimie afin de recommander la réalisation d'une électrophorèse des protéines sériques car la présence d'une gammapathie monoclonale à des concentrations très élevées génère de la turbidité et interfère avec les dosages spectrophotométriques. Un pic dans la fraction des gammaglobulines évoquant une gammapathie monoclonale a été retrouvé sur l'électrophorèse sérique, l'immunofixation confirme la présence d'une IgM kappa monoclonale. Le myélogramme montre une infiltration de plasmocytes, de lymphoplasmocytes et de mastocytes ce qui a conduit au diagnostic de maladie de Waldenström. Sur la base de ce cas, nous proposons une conduite à tenir en cas d'indice de lactescence élevé avec un plasma d'aspect clair.

Applicability of the long-term uncertainty in measurement (LTUM) method for analytical performance assessment in clinical cytometry laboratories.

BACKGROUND: The estimation of uncertainty in measurement for quantitative analyses is an international obligation of the ISO15189 standard for laboratories. The most widespread method is the Internal Quality Control and External Quality Assessment (IQC + EQA). METHODS: We compared two methods to assess uncertainty in measurement for the quantification of the number of CD34+ stem cells and of the different lymphocyte subpopulations in blood samples: the IQC + EQA method and the Long-Term Uncertainty in Measurement (LTUM) method. RESULTS: We focused on the CD3+/CD4+ T lymphocyte subpopulation for a target value of 350 CD3+/CD4+/µl. The range in terms of uncertainty in the measurement of 350 CD3+/CD4+ cells/µl with the IQC + EQA method was [292.8; 407.2]. With the LTUM method, the uncertainty was 19.1% of the measured value. This represented a range of [283.2; 416.9]. CONCLUSIONS: The relative uncertainty calculated with the LTUM method can be adapted to any level of the measured parameter. IQC and EQA calculate the absolute uncertainty and need a clustering of values at different levels. This clustering can lead to some approximations in the uncertainty in measurement determination, particularly around the cut-off values. Unlike previous reports, uncertainty values were higher when calculated with the LTUM than with the IQC + EQA method. However, LTUM might be more representative of the daily routine practice with patient samples.

Efficiency of nilotinib to target chronic phase-chronic myeloid leukaemia primary mature CD34- and immature CD34+ cells.

Accumulation in target cells is an essential pharmacokinetic step of targeted therapies. Tyrosine Kinase Inhibitors (TKI) against the BCR-ABL fusion protein in Chronic Phase-Chronic Myeloid Leukaemia (CP-CML) cells constitute a unique model in terms of efficacy, specificity, and in vivo demonstration of response heterogeneity by target cells. The overall therapeutic response to nilotinib is heterogeneous with no satisfactory explanation. To better understand the patients' response heterogeneity, we quantified nilotinib uptake by primary CP-CML cells in standardized conditions using flow cytometry, which allowed also distinguishing mature (polymorphonuclear cells) from immature (CD34+) cells. Nilotinib was undetectable in 13.3% of PMN and 40% of CD34+ cells. Moreover, in CD34+ cells, intracellular nilotinib did not completely abolish BCR-ABL activity (monitored by CrkL phosphorylation inhibition), although nilotinib accumulated well in most CD34+ cell samples. Intracellular nilotinib concentration was inversely correlated with disease burden parameters, Sokal score, and early haematologic response at day 6 ± 1 only in PMN, suggesting an intrinsic ability to limit nilotinib entry in the forms with higher tumor cell burdenat diagnosis. These findings suggest that nilotinib accumulation in CP-CML cells is influenced by individual characteristics and intra-clonal heterogeneity, and might be used for pharmacokinetic studies and to assess the therapeutic response.

Impact of Epicatechin on the Procoagulant Activities of Microparticles.

Microparticles play a role in cardiovascular disease pathology. The flavanol-like epicatechin is increasingly considered due to its cardioprotective effects. The aim of this study was to investigate the impact of epicatechin on microparticle generation, phenotype and procoagulant properties. Plasma samples from 15 healthy subjects were incubated with increasing concentrations of epicatechin (1 to 100 µM). Then, the expression of glycoprotein IIb, phosphatidylserine (PS), glycoprotein Ib (GPIb) and P-selectin was assessed by flow cytometry analysis after (or not) platelet stimulation. Microparticle procoagulant activity was determined using ZymuphenTM MP and ZymuphenTM MP-TF for phospholipid and tissue factor content, and with thrombin generation (TG) assays for procoagulant function. Platelet microparticles that express GPIb (/µL) decreased from 20,743 ± 24,985 (vehicle) to 14,939 ± 14,333 (p = 0.6), 21,366 ± 16,949 (p = 0.9) and 15,425 ± 9953 (p < 0.05) in samples incubated with 1, 10 and 100 µM epicatechin, respectively. Microparticle concentration (nM PS) decreased from 5.6 ± 2.0 (vehicle) to 5.1 ± 2.2 (p = 0.5), 4.5 ± 1.5 (p < 0.05) and 4.7 ± 2.0 (p < 0.05) in samples incubated with 1, 10 and 100µM epicatechin, respectively. Epicatechin had no impact on tissue factor-positive microparticle concentration. Epicatechin decreased TG (endogenous thrombin potential, nM.min) from 586 ± 302 to 509 ± 226 (p = 0.3), 512 ± 270 (p = 0.3) and 445 ± 283 (p < 0.05). These findings indicate that epicatechin affects microparticle release, phenotype and procoagulant properties.