Long-Term Preservation of Whole Blood Samples for Flow Cytometry Analysis in Normal and HIV-Infected Individuals from Africa

1990 ◽  
Vol 1 (1) ◽  
pp. 38-45 ◽  
Author(s):  
Renu B Lal ◽  
Subhash K Hira ◽  
Rita R Dhawan ◽  
Peter L Perine
Keyword(s):  
Flow Cytometry ◽  
Whole Blood ◽  
Phenotypic Analysis ◽  
Blood Samples ◽  
Remote Areas ◽  
Immunodeficiency Virus ◽  
Whole Blood Samples ◽  
The Stability ◽  
Proliferation Assays ◽  
Lymphocyte Phenotypes

A whole blood method requiring less than 4 ml of heparinized blood was developed to assess the practicality of preparing whole blood samples that could be easily stored, transported and readily used to determine the lymphocyte phenotypes and proliferation responses of individuals from remote areas who are infected with the human immunodeficiency virus. Minor modifications in standard whole blood procedure for lymphocyte phenotyping have significantly increased the stability of light scatter and fluorescence intensity of the cells for subsequent flow cytometry (FC) analysis. These changes include removal of lysis solution prior to fixation, fixation of monoclonal antibody-stained cells in 1% paraformaldehyde for 30 minutes and storage of fixed samples in medium containing 1% bovine serum albumin. Lymphocyte subsets and their functional subsets could reliably be determined on samples stored for up to 4 weeks. Further, blood samples could be kept at room temperature for up to 96 hours or at ambient temperature during transportation from Africa before staining for FC without affecting their quantitation. While samples could be processed for FC analysis under field-laboratory conditions, proliferation assays could only be performed on samples that were transported within 48 hours of their collection. The whole blood method saves time and expense and decreases the volumes of blood required to perform phenotypic analysis and functional assays on specimens collected in remote areas.

Effect of transport conditions on the stability of biochemical markers in blood.

1989 ◽  
Vol 35 (12) ◽  
pp. 2313-2316 ◽  
Author(s):  
S E Hankinson ◽  
S J London ◽  
C G Chute ◽  
R L Barbieri ◽  
L Jones ◽  
...  
Keyword(s):  
Whole Blood ◽  
Apolipoprotein B ◽  
Biochemical Markers ◽  
Room Temperature ◽  
Density Lipoprotein ◽  
Alpha Tocopherol ◽  
Endogenous Hormones ◽  
Blood Samples ◽  
Whole Blood Samples ◽  
The Stability

Abstract We examined the stability of lipids, carotenoids, alpha-tocopherol, and endogenous hormones in plasma prepared from whole blood that had been mailed to a central location for processing. Initially, to simulate transport conditions, whole-blood samples were stored in the laboratory, either at room temperature or cooled, for up to 72 h before processing. In the latter samples, lipid concentrations changed up to 1.4% per day, carotenoids up to -5.5%, and hormones up to 9.5%. In a second study, analyte concentrations in plasma from cooled whole blood mailed via overnight courier were compared with those from plasma that had been immediately separated, frozen, and mailed via overnight courier. Concentrations of cholesterol, high-density lipoprotein subfraction 3, apolipoprotein B, and retinol were stable. Overall, for each marker except estradiol, the between-person variation was at least twice the within-person variation. In a third study, at least 340 micrograms of DNA was recovered from 30 mL of cool-shipped whole blood. Our results indicate that shipping whole-blood samples by overnight courier is feasible for assay of several biochemical markers of interest in epidemiological research.

Flow Cytometry Detection of Platelet Procoagulant Activity and Microparticles in Patients with Unstable Angina Treated by Percutaneous Coronary Angioplasty and Stent Implantation

2001 ◽  
Vol 86 (09) ◽  
pp. 784-790 ◽  
Author(s):  
Catherine Vidal ◽  
Christian Spaulding ◽  
Françoise Picard ◽  
Frédéric Schaison ◽  
Josiane Melle ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Stent Implantation ◽  
Whole Blood ◽  
Coronary Angioplasty ◽  
Annexin V ◽  
Percutaneous Coronary Angioplasty ◽  
Blood Samples ◽  
Percutaneous Coronary ◽  
Whole Blood Samples

SummaryPlatelet activation is known to participate to the pathogenesis of acute coronary syndromes. Aminophospholipid exposure and micro-particles shedding are hallmarks of full platelet activation and may account for the dissemination of prothrombotic seats. Using flow cytometry analysis of annexin V binding to externalized aminophospholipids, we followed platelet procoagulant activity (PPA) and platelet microparticles (PMP) shedding in venous and coronary whole blood samples from 30 patients with unstable angina before and after percutaneous coronary angioplasty (PTCA) and stent implantation. Baseline values of PPA and PMP were significantly more elevated in patients than in control subjects (p <0.005). PMP percentage was significantly higher in coronary than in venous blood, and in coronary blood of patients with proximal instead of mid/distal lesions of coronary arteries. No enhancement of platelet reactivity to TRAP and collagen was induced by procedure. Whereas activated GpIIb-IIIa and P-selectin expression decreased 24 h and 48 h after procedure, PPA and PMP remained as elevated as before. Thus, flow cytometry is a reliable method for detection of fully activated platelets in whole blood samples. Annexin V binding analysis demonstrates the persistance of in vivo platelet activation, despite the use of antiaggregating agents.

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 Rapid Quantification of NETs In Vitro and in Whole Blood Samples by Imaging Flow Cytometry

2019 ◽  
Vol 95 (5) ◽  
pp. 565-578 ◽  
Author(s):  
Patrick M. Lelliott ◽  
Masatoshi Momota ◽  
Michelle S.J. Lee ◽  
Etsushi Kuroda ◽  
Norifumi Iijima ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Whole Blood ◽  
Blood Samples ◽  
Imaging Flow Cytometry ◽  
Whole Blood Samples ◽  
Rapid Quantification

Applicability of flow cytometry γH2AX assay in population studies: suitability of fresh and frozen whole blood samples

2021 ◽  
Author(s):  
Blanca Laffon ◽  
María Sánchez-Flores ◽  
Natalia Fernández-Bertólez ◽  
Eduardo Pásaro ◽  
Vanessa Valdiglesias
Keyword(s):  
Flow Cytometry ◽  
Whole Blood ◽  
Population Studies ◽  
Blood Samples ◽  
Whole Blood Samples

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.

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