One tube with eight antibodies for 14‐part bone marrow leukocyte differential using flow cytometry

Jacob, Marie‐Christine; Souvignet, Alice; Pont, Julie; Solly, Françoise; Mondet, Julie; Kesr, Sanae; Pernollet, Martine; Dumestre-Pérard, Chantal; Campos, Lydia; Cesbron, Jean‐Yves

doi:10.1002/cyto.b.21369

Cited by 10 publications

(16 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As several human blood disorders are associated with an unbalanced distribution of hematopoietic cells in both PB and BM, the generation of tools for the analysis of the hematopoietic system composition remains one crucial goal to understand the causes of hematological diseases, to design novel therapeutic approaches and to test their efficacy. Over the past three decades, flow cytometry has been widely applied for immune-phenotyping in clinical and research settings (1,2,29,30,47,(65)(66)(67)(68)(69)(70). A 17-multiparamentric assay has been developed in the past for analyzing in details PB mature cells (68).…”

Section: Discussionmentioning

confidence: 99%

“…Morphological examination is one of the routine laboratory tests performed for the diagnosis of hematopoietic/ immunological disorders, when alterations of the physiological differentiation of hematopoietic cells are suspected (1,29,47). Still, this test requires experienced investigators, it is time consuming, not accurate in quantification and the identification of rare blood cell types through this method could be difficult.…”

mentioning

confidence: 99%

“…Still, this test requires experienced investigators, it is time consuming, not accurate in quantification and the identification of rare blood cell types through this method could be difficult. Conversely, through the simultaneous measurement of multiple fluorescencent antibodies each binding to a specific surface or intracytoplasmatic molecule, flow cytometry provides a highly quantitative and reproducible technique (1,2,30,(47)(48)(49). To assess the relative frequencies of blood cellular components, cytometric analyses are commonly performed (1,2,(48)(49)(50)(51)(52) by splitting the available biological sample on different test tubes, each dedicated to the analysis of a restricted group of leukocyte subpopulations through a limited set of surface markers.…”

mentioning

confidence: 99%

“…To assess the relative frequencies of blood cellular components, cytometric analyses are commonly performed (1,2,(48)(49)(50)(51)(52) by splitting the available biological sample on different test tubes, each dedicated to the analysis of a restricted group of leukocyte subpopulations through a limited set of surface markers. As a result, the standard analyses do not allow identifying co-expression of >6-8 independent markers at a time and the relative frequency of all the different blood cell subtypes cannot be concomitantly assessed on the same sample (1,47,51,53). When applied to the study of human HSPC subtypes, cytometric evaluation is generally confined to the CD341 compartment or performed on CD341 cells purified from BM samples through magnetic beads or FACS-sorting (34)(35)(36)(37)54).…”

mentioning

confidence: 99%

See 3 more Smart Citations

Multiparametric Whole Blood Dissection: A one‐shot comprehensive picture of the human hematopoietic system

et al. 2017

View full text Add to dashboard Cite

Human hematopoiesis is a complex and dynamic system where morphologically and functionally diverse mature cell types are generated and maintained throughout life by bone marrow (BM) Hematopoietic Stem/Progenitor Cells (HSPC). Congenital and acquired hematopoietic disorders are often diagnosed through the detection of aberrant frequency or composition of hematopoietic cell populations. We here describe a novel protocol, called “Whole Blood Dissection” (WBD), capable of analyzing in a single test‐tube, hematopoietic progenitors and all major mature cell lineages composing either BM or peripheral blood (PB) through a multiparametric flow‐cytometry analysis. WBD allows unambiguously identifying in the same tube up to 23 different blood cell types including HSPC subtypes and all the major myeloid and lymphoid lineage compartments at different stages of maturation, through a combination of 17 surface and 1 viability cell markers. We assessed the efficacy of WBD by analyzing BM and PB samples from adult (n = 8) and pediatric (n = 9) healthy donors highlighting age‐related shift in cell composition. We also tested the capability of WBD on detecting aberrant hematopoietic cell composition in clinical samples of patients with primary immunodeficiency or leukemia unveiling expected and novel hematopoietic unbalances. Overall, WBD allows unambiguously identifying >99% of the cell subpopulations composing a blood sample in a reproducible, standardized, cost‐, and time‐efficient manner. This tool has a wide range of potential pre‐clinical and clinical applications going from the characterization of hematopoietic disorders to the monitoring of hematopoietic reconstitution in patients after transplant or gene therapy. © 2017 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC.

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Multiparametric Whole Blood Dissection: A one‐shot comprehensive picture of the human hematopoietic system

et al. 2017

View full text Add to dashboard Cite

show abstract

“…This rather subjective classification is also limited by the efficiency of the investigators and their medical knowledge, but also with respect to its discrimination power. Alternatively, the combination of leukocyte analysis by flow cytometry (22,23), in combination with automatic lobulation diagnosis by the rule of thirds and the circularity index could be a promising approach. This has been explained by the replacement of visual examination of blood smears by automated leucocyte counting which do not reveal these morphological changes adequately (16).…”

Section: Discussionmentioning

confidence: 99%

Image analysis of neutrophil nuclear morphology: Learning about phenotypic range and its reliable analysis from patients with pelger‐Huët‐anomaly and treated with colchicine

Schnipper

Stassen

Kallinich

et al. 2017

Cytometry Part B Clinical

View full text Add to dashboard Cite

The nuclear morphology of neutrophils depends on different endogenous and exogenous factors, which can lead to hypo- or hypersegmentation of the normally 2-4 segmented nucleus. Hyposegmentation can be due to mutations in the LBR-gene (Pelger-Huët-Anomaly) or can be induced, for example, by colchicine treatment. The range of this phenotypic variation is known as "norm of reaction," which can be of major relevance for clinical diagnosis and therapeutic intervention. In this project, we studied the norm of reaction in 26 subjects with 0-3 wild type LBR alleles. In addition, the phenotypic variation was analyzed in 3 patients with Familial Mediterranean Fever (FMF), before and after colchicine treatment. We measured the phenotype nuclear segmentation of neutrophils based on two conventional qualitative methods, the "rule of threads" and the "rule of thirds." In addition, we tested a morphometric quantitative approach, the "circularity index." The circularity index was superior in cases with hyposegmentation; the rule of thirds with respect to hypersegmentation. Approximately 65% of the observed phenotypic variance was explainable by the number of LBR wild type alleles. The gene-dosage effect followed a non-additive, hysteresis-like characteristic with lower and upper plateaus. Colchicine treatment had a clear, although minor phenotypic effect compared to the number of LBR wild type genes or the mutation type. Thus, the nuclear morphology of granulocytes and its norm of reaction can be regarded as an excellent model both for detailing the interplay between endogenous and exogenous factors and for clinical diagnostic purposes. © 2016 International Clinical Cytometry Society.

show abstract

Considerations for Use of Pharmacodynamic Biomarkers to Support Biosimilar Development – (II) A Randomized Trial with IL‐5 Antagonists

Gershuny

Sun

Schrieber

et al. 2022

Clin Pharma and Therapeutics

View full text Add to dashboard Cite

The US Food and Drug Administration (FDA) guidance describes how pharmacodynamic (PD) biomarkers can be used to address residual uncertainty and demonstrate no clinically meaningful differences between a proposed biosimilar and its reference product without relying on clinical efficacy end point(s). Pilot studies and modeling can inform dosing for such PD studies. To that end, we conducted a randomized, double-blinded, placebo-controlled, single-dose, parallel-arm clinical study in healthy participants to evaluate approaches to address information gaps, inform best practices for analysis of biomarker samples and study results, and apply emerging technologies in biomarker characterization. Seventy-two healthy participants (n = 8 per arm) received either placebo or 1 of 4 doses of the interleukin-5 inhibitors mepolizumab (3-24 mg) or reslizumab (0.1-0.8 mg/kg). A clinical study using doses lower than approved therapeutic doses was combined with modeling and simulation to evaluate the dose-response relationship of the biomarker eosinophils. There was no dose-response relationship for eosinophil counts due to variability, although the mepolizumab 24 mg and reslizumab 0.8 mg/kg doses showed clear effects. Published indirect-response models were used to explore eosinophil data across doses from this study and the unstudied therapeutic doses. Simulations were used to calculate typical PD metrics, such as baseline-adjusted area under the effect curve and maximum change from baseline. The simulation results demonstrate sensitivity of eosinophils as a PD biomarker and indicate doses lower than the approved doses would have PD responses overlapping with variability in the placebo arm. The simulation results further highlight the utility of model-based approaches in supporting use of PD biomarkers in biosimilar development.

show abstract

One tube with eight antibodies for 14‐part bone marrow leukocyte differential using flow cytometry

Abstract: A very simple, rapid, and reproducible flow cytometric approach, using a combination of eight antibodies allows determination of the cellular composition of bone marrow with high precision. © 2016 International Clinical Cytometry Society.

Cited by 10 publications

References 29 publications

Multiparametric Whole Blood Dissection: A one‐shot comprehensive picture of the human hematopoietic system

Multiparametric Whole Blood Dissection: A one‐shot comprehensive picture of the human hematopoietic system

Image analysis of neutrophil nuclear morphology: Learning about phenotypic range and its reliable analysis from patients with pelger‐Huët‐anomaly and treated with colchicine

Considerations for Use of Pharmacodynamic Biomarkers to Support Biosimilar Development – (II) A Randomized Trial with IL‐5 Antagonists

Contact Info

Product

Resources

About