A 5-color flow cytometric method for extended 8-part leukocyte differential.

OBJECTIVES: Microscopic leukocyte differentials display many drawbacks. Several single 5 to 8-color tubes using multiparameter flow cytometry (MFC) are able to provide extended differentials with sequential gating-based analysis strategies. We investigated a new 5-color MFC method to perform an extended 8-part differential with a simplified gating strategy. METHODS: Whole blood was stained with a combination of antibodies including HLA-DR-FITC/CD19-PE/CD45-ECD/CD16-PC5 + CD71-PC5/CD5-PC7. RESULTS: An original approach was developed to exclude debris and straightforwardly gate the cells to identify sixteen populations. Strong correlations were obtained with the analyzer for neutrophils, lymphocytes, monocytes, and eosinophils (R2 >0.9). Abnormal cells, such as immature granulocytes and blast cells were identified with a good sensitivity and excellent correlation against cytomorphologic review (R2 =0.66 and 0.99, respectively). The choice of HLA-DR and CD5 improved specificity for the identification of activated T-lymphocytes and some lymphoid neoplastic cells, respectively. CONCLUSIONS: Here a new cytometric differential is proposed with a robust gating strategy which may be used even by unskilled cytometrists and can be easily automated. © 2017 International Clinical Cytometry Society.

Comparison of cross-platform flow cytometry minimal residual disease evaluation in multiple myeloma using a common antibody combination and analysis strategy.

BACKGROUND: A seven-color/eight-antibody approach has recently been proposed for the minimal residual disease (MRD) determination in multiple myeloma (MM), which was developed on FACSCantoII instruments. This strategy should be also applicable on different multiparameter flow cytometers (MFC), but this needs to be demonstrated before moving MRD assessment to local flow cytometry core facilities, nearer to patients, thereby reducing the risk of cell losses induced by sample transportation and delays in cell processing. METHODS: To evaluate the comparability of testing the same seven-color/eight-antibody single-tube on any instruments, MRD was evaluated concomitantly on two distinct MFC in a cohort of 80 bone marrow MM samples (i.e., 73 patients including seven with two MRD evaluations) in two French centers, Paris-Cochin and Dijon. RESULTS: No significant difference in the MM residual plasma-cells (MM-PCs) quantification was observed. Calculated on the basis of the whole amount of leukocytes assessed, the mean MRD percentage was, respectively, 0.1661% and 0.1458% using FACSCantoII or Navios instruments, with a very high correlation between instruments (r(2) = 0.9798) and a very minimal bias (-0.02). Moreover, there was no difference in MRD interpretation at 10(-4) threshold; whereas three MRD interpretation discordances were observed at 2.5 × 10(-5) threshold. CONCLUSION: This study demonstrates that this MRD-detection strategy is transposable between harmonized ≥ seven-color instruments. This shows that a homogeneous rapid MRD evaluation can be performed in most MFC platforms, in the near vicinity of clinical wards. However, the clinical validation of this approach needs to be strengthened, as well as its relevance compared to molecular approaches.

Comparison of cross-platform flow cytometry minimal residual disease evaluation in multiple myeloma using a common antibody combination and analysis strategy.

Background: A seven-color/eight-antibody approach has recently been proposed for the minimal residual disease (MRD) determination in multiple myeloma (MM), which was developed on FACSCantoII instruments. This strategy should be also applicable on different multiparameter flow cytometers (MFC), but this needs to be demonstrated before moving MRD assessment to local flow cytometry core facilities, nearer to patients, thereby reducing the risk of cell losses induced by sample transportation and delays in cell processing. Methods: To evaluate the comparability of testing the same 7-color/8-antibody single-tube on any instruments, MRD was evaluated concomitantly on two distinct MFC in a cohort of 80 bone marrow MM samples (ie. 73 patients including 7 with 2 MRD evaluations) in two French centers, Paris-Cochin and Dijon. Results: No significant difference in the MM residual plasma-cells (MM-PCs) quantification was observed. Calculated on the basis of the whole amount of leukocytes assessed, the mean MRD percentage was respectively 0.1661% and 0.1458% using FACSCantoII or Navios instruments, with a very high correlation between instruments (r2 = 0.9798) and a very minimal bias (-0.02). Moreover, there was no difference in MRD interpretation at 10-4 threshold; whereas 3 MRD interpretation discordances were observed at 2.5×10-5 threshold. Conclusion: This study demonstrates that this MRD-detection strategy is transposable between harmonized ≥7-color instruments. This shows that a homogeneous rapid MRD evaluation can be performed in most MFC platforms, in the near vicinity of clinical wards. However, the clinical validation of this approach needs to be strengthened, as well as its relevance compared to molecular approaches. © 2014 Clinical Cytometry Society.

Comparable flow cytometry data can be obtained with two types of instruments, Canto II, and Navios. A GEIL study.

Flow cytometry (FC) instruments settings classically rely on local establishment of photomultipliers (PMT) voltages adapted to the measurements expected to be performed. In the era of multiparameter FC (MFC), it appears more and more desirable that comparable patterns of fluorescence are obtained in different settings. This relies on a harmonization of settings between instruments. Although this has been shown to be feasible within a given brand of flow cytometers, little information is available about broader comparisons in a given center or in a multicenter fashion. Here, we report a two-phase series of experiments first performed between a Canto II (BD Biosciences) and a Navios (Beckman Coulter) instruments in the same center. PMT values adjusted on the reference instrument (RI) Canto II were used to establish target values for PMT settings on the paired Navios practice instrument (PI). This allowed to show the good correlation of all but peaks 1 and 2 of Rainbow(®) beads between RI and PI. Using 4- or 8-color stained leukocytes, the similitude of the settings was further confirmed. A complex set of matrices was then established between five centers all equipped with both instruments. Using Bland & Altman difference comparisons for median fluorescence values, it was shown that using either Rainbow beads or CD16 stained polymorphonuclears to set-up target values on the RI CantoII, highly superimposable results could be obtained on all 9 PI. The latter were obtained using Rainbow beads or Compbeads(®) for comparisons. In summary, this two-phase study demonstrates the feasibility of different methods allowing for a robust harmonization of settings for MFC.

Involvement of microtubules in the tolerance of cardiomyocytes to cold ischemia-reperfusion.

Before transplantation, the heart graft is preserved by the use of cold storage in order to limit ischemia-reperfusion stress. However, sustained exposure to low temperature may induce myocardial ultrastructural damage, particularly microtubules (MT) disruption. Previous data suggested that tubulin-binding agents are able to attenuate cold-induced cytoskeleton alterations. Thus, the aim of the present work was to study the influence of docetaxel (DX, a tubulin-binding taxane) on the effects of deep hypothermia (4 degrees C) and of simulated cold ischemia-reperfusion on the MT network and oxidative stress of cardiomyocyte (CM) in monolayer cultures prepared from newborn rat ventricles. The MT network was explored by immunocytochemistry and Western-blotting, the cell stress by tetrazolium dye assay (MTT) and lactate dehydrogenase (LDH) release, and the superoxide production by the dihydroethidium probe (DHE). The MT assembly remained stable after 4 and 8 h of hypothermia. Tubulin acetylation was promoted in CM subjected to 4-h hypothermia. Low temperature reduced the mitochondrial function and increased the basal LDH release. The cold ischemia during 4 and 8 h preserved MT network. Docetaxel promoted MT polymerization and tubulin acetylation in basal and in cold conditions. This drug decreased the release of LDH induced by cold ischemia. Moreover, hypothermia (4 h) significantly raised the anion superoxide production. Docetaxel decreased this oxidative stress in the control CM and in CM submitted to 4 h of hypothermia. These data demonstrated that stabilizing MT with DX exerted a protective effect on CM subjected to hypothermia and to cold ischemia-reperfusion. Tubulin-ligands should be thus considered to improve the tolerance of the heart graft toward stressing conservative conditions.