Direct freezing of whole blood enables analysis of leucocyte markers by flow cytometry: a proof-of-concept study

2021 ◽  
Author(s):  
Pénélope Bourgoin ◽  
Inès Ait Belkacem ◽  
Isabelle Arnoux ◽  
Pierre-Emmanuel Morange ◽  
Fabrice Malergue
Keyword(s):  
Flow Cytometry ◽  
Whole Blood ◽  
Blood Cells ◽  
White Blood Cells ◽  
Proof Of Concept ◽  
Fresh Blood ◽  
Blood Samples ◽  
Step Flow ◽  
Freezing Method ◽  
One Step

Aim: A new one-step flow cytometry procedure has been recently demonstrated for identifying subjects with infections, but only for fresh whole blood samples. The goal of this study was to assess its applicability on frozen samples, by proposing a new method to perform the sample freezing directly and easily. Methods: Fresh blood was tested, then frozen either directly or with dimethylsulfoxide and serum. Common markers of white blood cells as well as infection-related biomarkers were tested. Results: All percentages of leucocyte subsets and levels of infection-related biomarkers were significantly correlated between frozen and fresh samples. Conclusion: The direct freezing method enables an accurate assessment of common cellular sub-populations and of levels of important infectious biomarkers via flow cytometry.

Sensitive Detection of Survival of Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones in Red Blood Cells, Granulocytes and Monocytes by High Resolution Multiparameter Flow Cytometry.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3436-3436
Author(s):  
Mayur K Movalia ◽  
Andrea Illingworth
Keyword(s):  
Flow Cytometry ◽  
High Resolution ◽  
Red Blood Cells ◽  
Whole Blood ◽  
Blood Cells ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Clone Size ◽  
Blood Samples ◽  
Whole Blood Samples ◽  
Pnh Clone

Abstract Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired clonal stem cell disorder characterized by intravascular hemolysis due to GPI-deficient red blood cells sensitive to complement-mediated lysis. Accurate and sensitive detection of PNH-type cells has become important not only to diagnose PNH but also because studies have shown PNH-type cells may indicate favorable response to therapy and favorable prognosis in patients with aplastic anemia and myelodysplastic syndrome. Previous studies have suggested optimal testing for PNH-type cells by flow cytometry should be limited to within 48 hours after collection of whole blood. Our laboratory has developed a very sensitive and specific high resolution flow cytometric method for detecting PNH-type cells based on testing over 3,000 patients with known PNH, aplastic anemia, myelodysplastic syndromes and other bone marrow failure syndromes. The aim for this study was to determine the longevity of PNH clones in whole blood samples, the day-to-day variability of these clones and the rate of deterioration of the PNH clones compared to normal blood cells. We analyzed 10 whole blood samples from patients known to have PNH-type cells on seven consecutive days utilizing a two-color assay with GPA-CD59 for the red blood cells, a 5-color assay with FLAER-CD24-CD14-CD15-CD45 for the granulocytes and a 5 color assay with FLAER-CD33-CD14-CD64-CD45 for the monocytes. The results are summarized in the table below. The initial PNH clone sizes ranged from 0.02% to 90.8%. The PNH cells showed an overall similar level of deterioration to the normal blood cells with even minor PNH clones of 0.02% able to be detected at day 7. The day-to-day variability of PNH clone sizes was generally less than 10%, with smaller clone sizes showing a higher degree of variation, up to 20%, due to their smaller absolute numbers. Interestingly, Type III PNH red blood cells showed slightly better overall survival than normal red blood cells and were detected in modestly increasing percentages throughout the study. Based on this data, we propose that accurate detection of PNH type cells can be achieved up to seven days after collection of whole blood when utilizing high resolution flow cytometry. PNH Clone Size on Sequential Days as Percentage of Original PNH Clone Size Original PNH Clone Sizes PNH Clone Sizes as Percentage of Original PNH Clone Size Cell Type Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Type III RBCs .02%–58.6% 102% 106% 107% 104% 108% 103% Granulocytes .29%–90.8% 100% 100% 93% 89% 79% 86% Monocytes .52%–89.9% 96% 96% 92% 94% 97% 85%

scholarly journals Negative enrichment of circulating tumor cells from unmanipulated whole blood with a 3D printed device

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chia-Heng Chu ◽  
Ruxiu Liu ◽  
Tevhide Ozkaya-Ahmadov ◽  
Brandi E. Swain ◽  
Mert Boya ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Whole Blood ◽  
Blood Cells ◽  
Point Of Care ◽  
White Blood Cells ◽  
Normal Blood ◽  
Blood Samples ◽  
Membrane Antigens ◽  
Tumor Types

AbstractReliable and routine isolation of circulating tumor cells (CTCs) from peripheral blood would allow effective monitoring of the disease and guide the development of personalized treatments. Negative enrichment of CTCs by depleting normal blood cells ensures against a biased selection of a subpopulation and allows the assay to be applied on different tumor types. Here, we report an additively manufactured microfluidic device that can negatively enrich viable CTCs from clinically-relevant volumes of unmanipulated whole blood samples. Our device depletes nucleated blood cells based on their surface antigens and the smaller anucleated cells based on their size. Enriched CTCs are made available off the device in suspension making our technique compatible with standard immunocytochemical, molecular and functional assays. Our device could achieve a ~ 2.34-log depletion by capturing > 99.5% of white blood cells from 10 mL of whole blood while recovering > 90% of spiked tumor cells. Furthermore, we demonstrated the capability of the device to isolate CTCs from blood samples collected from patients (n = 15) with prostate and pancreatic cancers in a pilot study. A universal CTC assay that can differentiate tumor cells from normal blood cells with the specificity of clinically established membrane antigens yet require no label has the potential to enable routine blood-based tumor biopsies at the point-of-care.

scholarly journals An easy and reliable whole blood freezing method for flow cytometry immunophenotyping and functional analyses

2020 ◽  
Author(s):  
Cecile Braudeau ◽  
Nina Salabert-Le Guen ◽  
Chevreuil Justine ◽  
Rimbert Marie ◽  
Jerome C. Martin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Whole Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Immune Monitoring ◽  
Fresh Blood ◽  
Peripheral Blood Mononuclear ◽  
Blood Samples ◽  
Whole Blood Samples

ABSTRACTBackgroundImmune profiling by flow cytometry is not always possible on fresh blood samples due to time and/or transport constraints. Besides, the cryopreservation of peripheral blood mononuclear cells (PBMC) requires on-site specialized lab facilities, thus severely restricting the extent by which blood immune monitoring can be applied to multicenter clinical studies. These major limitations can be addressed through the development of simplified whole blood freezing methods.MethodsIn this report, we describe an optimized easy protocol for rapid whole blood freezing with the CryoStor® CS10 solution. Using flow cytometry, we compared cellular viability and composition on cryopreserved whole blood samples to matched fresh blood, as well as fresh and frozen PBMC.ResultsThough partial loss of neutrophils was observed, leucocyte viability was routinely >75% and we verified the preservation of viable T cells, NK cells, monocytes, dendritic cells and eosinophils in frequencies similar to those observed in fresh samples. A moderate decrease in B cell frequencies was observed. Importantly, we validated the possibility to analyze major intracellular markers, such as FOXP3 and Helios in regulatory T cells. Finally, we demonstrated good functional preservation of CS10-cryopreserved cells through the analysis of intracellular cytokine production in ex vivo stimulated T cells (IFNg, IL-4, IL-17A,) and monocytes (IL-1b, IL-6, TNFa).ConclusionsIn conclusion, our protocol provides a robust method to apply reliable immune monitoring studies to cryopreserved whole blood samples, hence offering new important opportunities for the design of future multicenter clinical trials.

One-Step Microfluidic Purification of White Blood Cells from Whole Blood for Immunophenotyping

2019 ◽  
Vol 91 (20) ◽  
pp. 13230-13236 ◽  
Author(s):  
Byeongyeon Kim ◽  
Kyung Hwan Kim ◽  
Yunjung Chang ◽  
Suyeon Shin ◽  
Eui-Cheol Shin ◽  
...  
Keyword(s):  
Whole Blood ◽  
Blood Cells ◽  
White Blood Cells ◽  
One Step

scholarly journals An easy and reliable whole blood freezing method for flow cytometry immuno‐phenotyping and functional analyses

2021 ◽  
Author(s):  
Cecile Braudeau ◽  
Nina Salabert‐Le Guen ◽  
Justine Chevreuil ◽  
Marie Rimbert ◽  
Jerome C. Martin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Whole Blood ◽  
Freezing Method ◽  
Functional Analyses

EFFECTS OF BLOOD CELLS ON PLATELET AGGREGATION BY IMPEDANCE METHOD

1987 ◽  
Author(s):  
L Mannucci ◽  
R Redaelli ◽  
E Tremoll
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Blood Cells ◽  
White Blood Cells ◽  
Normal Subjects ◽  
Impedance Method ◽  
Induced Aggregation ◽  
Vitro Data ◽  
In Vitro Data

To evaluate the effects of blood cells on the response of platelets to aggregating agents using whole blood impedance aggregometer, studies were carried out on whole blood (WB) of normal subjects and of patients with: polycythemia vera (PV), iatrogenic anemia (IA), primary thrombocytosis (PT), idiopathic thrombotic purpura (ITP), myeloid chronic leukemia (MCL), iatrogenic leukopenia (IL). The in vitro effects of red blood cells (RBC) and of white blood cells (WBC) on platelet rich plasma (PRP) aggregation were also evaluated. WB, PRP, WBC and RBC were prepared by conventional methods. Aggregation was performed using the impedance aggregometer (mod. 540, Chrono Log Corp). In normal subjects the concentration of collagen giving 50 % aggregation (AC50 ) found in PRP did not differ from that of WB, indicating that hematocrit values within the normal range did not appreciably affect platelet aggregation. The results obtained in WB of patients are summarized in the table: In vitro data showed that aggregation in prp in wb of normal subjects was related to the number of platelets present in the sample. RBC added to PRP significant reduced aggregation only when the RBC number was greater than 4.101 cells. No effect of WBC on collagen induced aggregation of PRP was observed, whereas significant inhibition was detected after ADP. It is concluded that the aggregation evaluated in WB with impedance method is dependent on the platelet number. Also, in vitro data and studies in WB of patients indicate that aggregation is significantly affected by the presence of cells other than platelets only in conditions of changes of the ratio between platelets and leukocytes and/or red cells.

scholarly journals Analysis of the dynamics of Staphylococcus aureus binding to white blood cells using whole blood assay and geno-to-pheno mapping

2020 ◽  
Vol 310 (3) ◽  
pp. 151411
Author(s):  
Daria Gaidar ◽  
Alice Jonas ◽  
Ruslan Akulenko ◽  
Ulla Ruffing ◽  
Mathias Herrmann ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Whole Blood ◽  
Blood Cells ◽  
White Blood Cells ◽  
Whole Blood Assay ◽  
Blood Assay
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