A rapid one-step procedure for purification of mononuclear and polymorphonuclear leukocytes from human blood using a modification of the hypaque-ficoll technique

doi:10.1016/0022-1759(78)90143-6

Journal of Immunological Methods

Volume 24, Issues 3–4, December 1978, Pages 389-393

https://doi.org/10.1016/0022-1759(78)90143-6 Get rights and content

Abstract

A one-step procedure for purification of mononuclear and polymorphonuclear cells from human blood is described. It is a modification of the hypaque-Ficoll method with density of 1.095 g/ml. Centrifugation at 200 × g for 20–30 min resulted in the separation of mononuclear and polymorphonuclear cells into 2 distinct bands at the interface. The mononuclear cell fraction contained 83.9 ± 1.6% lymphocytes and 13.8 ± 2.3% monocytes, while the other consisted of highly purified neutrophils (96.4 ± 1.0%). Leukocyte recovery by this method was always greater than 80% and viability exceeded 98%. Both cell fractions retained their immunological functions.

References (10)

W.A. Skoog et al.
Blood
(1956)
Y.H. Thong et al.
J. Immunol. Methods
(1978)
R. Anderson et al.
Immunology
(1978)
A. Böyum
Scand. J. Clin. Lab. Invest.
(1968)
C. Dewer
J. Immunol. Methods
(1978)

There are more references available in the full text version of this article.

Cited by (288)

Acute hydrodynamic damage induced by SPLITT fractionation and centrifugation in red blood cells
2016, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Citation Excerpt :
Additionally, due to the low flow rates used, processing larger sample volumes may require larger SPLITT devices. Centrifugation in a Ficoll®-Paque density gradient is widely used to separate mononuclear and polymorphonuclear cells from whole blood, resulting in high purity fractions (83.9 ± 1.6% of lymphocytes; 13.8 ± 2.3% of monocytes; and 96.4 ± 1.0% neutrophils) with cell viability greater than 98% [35]. After the separation process, the remaining RBCs in the densest fractions show decreased cell size, suggestive of cellular dehydration [36].
Though blood bank processing traditionally employs centrifugation, new separation techniques may be appealing for large scale processes. Split-flow fractionation (SPLITT) is a family of techniques that separates in absence of labelling and uses very low flow rates and force fields, and is therefore expected to minimize cell damage. However, the hydrodynamic stress and possible consequent damaging effects of SPLITT fractionation have not been yet examined. The aim of this study was to investigate the hydrodynamic damage of SPLITT fractionation to human red blood cells, and to compare these effects with those induced by centrifugation. Peripheral whole blood samples were collected from healthy volunteers. Samples were diluted in a buffered saline solution, and were exposed to SPLITT fractionation (flow rates 1–10 ml/min) or centrifugation (100–1500 g) for 10 min. Cell viability, shape, diameter, mean corpuscular hemoglobin, and membrane potential were measured. Under the operating conditions employed, both SPLITT and centrifugation maintained cell viability above 98%, but resulted in significant sublethal damage, including echinocyte formation, decreased cell diameter, decreased mean corpuscular hemoglobin, and membrane hyperpolarization which was inhibited by EGTA. Wall shear stress and maximum energy dissipation rate showed significant correlation with lethal and sublethal damage. Our data do not support the assumption that SPLITT fractionation induces very low shear stress and is innocuous to cell function. Some changes in SPLITT channel design are suggested to minimize cell damage. Measurement of membrane potential and cell diameter could provide a new, reliable and convenient basis for evaluation of hydrodynamic effects on different cell models, allowing identification of optimal operating conditions on different scales.
Development of mini-Sep for nucleated cell purification and isolation
2012, Genomic Medicine, Biomarkers, and Health Sciences
Citation Excerpt :
Differential centrifugation initially separates cells and organelles by size, which often requires the additional use of density gradient centrifugation to isolate and purify the sample after centrifugation. Density gradient centrifugation uses gravity or centrifugal force to stratify and separate nucleated cells, and this process commonly involves the use of substances such as sucrose, glycerol, cesium chloride, rubidium chloride, or potassium tartrate.1–5 However, these isolation techniques require centrifugation or other purification devices to work, and they often require longer operating times.
Despite the availability of various methods that can isolate nucleated cells from blood or body fluids, these methods still require centrifugation and other purification devices to work. The main purpose of this study was to develop a simple cell purification and isolation device (mini-Sep) that does not require any centrifugation equipment to operate and can be used to isolate and concentrate nucleated cells from blood or body fluids for use in cell-related experiments. Mini-Sep works by using a syringe or pump to inject samples, thereby filtering out any impurities by removing residual nucleated cells and holding them in a containment space that was designed as part of the device. The results show that mini-Sep possesses the capability to isolate and concentrate nuclear cells, can be operated in simple and easy steps, and saves time typically spent on centrifugation. It can be easily adapted for use in various laboratories and places that lack laboratory equipment.
The effect of n-3 polyunsaturated fatty acids on leukotriene B4 and leukotriene B5 production from stimulated neutrophil granulocytes in patients with chronic kidney disease
2011, Prostaglandins Leukotrienes and Essential Fatty Acids
Citation Excerpt :
After a night fasting (at least 10 h) blood samples were drawn from the patients. The blood was anticoagulated with K-EDTA 1.6 mg/mL and underwent Hypaque-Ficoll (Nycomed, Oslo, Norway) discontinuous gradient centrifugation [28,29]. Neutrophil granulocytes were harvested into tissue culture medium (RPMI 1640, Sigma-Aldrich, Ayrshire, UK).
The proinflammatory leukotriene B₄ (LTB₄) may be of importance in the progression of chronic kidney disease (CKD). We investigated whether n-3 polyunsaturated fatty acids (PUFA) decrease LTB₄ and increase the formation of the less inflammatory leukotriene B₅ (LTB₅) in patients with CKD.
Fifty-six patients with CKD stage 2–5 were randomised to 2.4 g n-3 PUFA or olive oil for 8 weeks. Compared to controls, n-3 PUFA significantly decreased release of LTB₄ (p<0.001) and 5-hydroxyeicosatetraenoic acid (5-HETE) (p<0.01) and significantly increased release of LTB₅ (p<0.001) and 5-hydroxyeicosapentaenoic acid (5-HEPE) (p<0.001) from stimulated neutrophil granulocytes. Kidney function evaluated by creatinine clearance and proteinuria did not improve. In conclusion, n-3 PUFA supplementation for 8 weeks in patients with CKD stage 2–5 significantly decreased LTB₄ and 5-HETE and significantly increased LTB₅ and 5-HEPE. No effect was seen on kidney function.
Metabolism of 4-hydroxy-2-nonenal in human polymorphonuclear leukocytes
2010, Archives of Biochemistry and Biophysics
Citation Excerpt :
As scintillation solution Ultima Gold (high flash-point LSC-cocktail for aqueous and non-aqueous samples – Packard Instrument Company, Meriden, CT) was used. Human leukocyte preparations containing 90–98% of neutrophils and apparently free of contaminating erythrocytes were obtained by one-step procedure involving centrifugation of heparinized blood, freshly drawn from healthy donors and layered on Ficoll-Hypaque medium (Mono-Poly Resolving medium, ICN Pharm., Costa Mesa, CA) [34]. The cells were suspended in isotonic phosphate-buffered saline pH 7.4 with 5 mM glucose and stored on ice.
Intracellular metabolism of 4-hydroxy-2-nonenal (HNE), a major product and mediator of oxidative stress and inflammation, is analyzed in resting and fMLP-stimulated human polymorphonuclear leukocytes (PMNL), where this compound is generated during activation of the respiratory burst. HNE consumption rate in PMNL is very low, if compared to other cell types (rat hepatocytes, rabbit fibroblasts), where HNE metabolism is always an important part of secondary antioxidative defense mechanisms. More than 98% of HNE metabolites are identified. The pattern of HNE intermediates is quite similar in stimulated and resting PMNL – except for higher water formation in resting PMNL – while the initial velocity of HNE degradation is somewhat higher in resting cells, 0.44 instead of 0.28 nmol/(min × 10⁶ cells). The main products of HNE metabolism are 4-hydroxynonenoic acid (HNA), 1,4-dihydroxynonene (DHN) and the glutathione adducts with HNE, HNA, and DHN. Protein-bound HNE and water account for about 3–4% of the total HNE derivatives in stimulated cells, while in resting cells protein-bound HNE and water are 4% and 20%, respectively. Cysteinyl-glycine-HNE adduct and mercapturic acids contribute to about 5%.
Inhibition of the lipopolysaccharide-induced stimulation of the members of the MAPK family in human monocytes/macrophages by 4-hydroxynonenal, a product of oxidized omega-6 fatty acids
2008, American Journal of Pathology
Citation Excerpt :
Mononuclear leukocytes (MNLs) were prepared from the blood of healthy volunteers using an adaptation of the Hypaque/Ficoll method.21
The compound 4-hydroxynonenal (4-HNE) is the major aldehyde formed during lipid peroxidation of ω-6-polyunsaturated fatty acids and has been suggested to regulate inflammatory responses because it inhibits tumor necrosis factor (TNF) mRNA production in the human monocytic cell line THP-1. Here we demonstrate that 4-HNE inhibits TNF and interleukin-1β production in human monocytes in response to lipopolysaccharide. The main action of 4-HNE occurred at the pretranscriptional level; there was no effect on TNF mRNA production or stability when 4-HNE was added after stimulation. The mechanism of action of 4-HNE appears to be downstream of lipopolysaccharide-receptor binding. In the human monocytic MonoMac 6 cell line, 4-HNE caused selective inhibition of the activity of the mitogen-activated protein kinases p38 and ERK1/ERK2, but not JNK. However, in monocytes, the activities of all three kinases were inhibited, suggesting that the effects of 4-HNE were exerted at points upstream of ERK1/ERK2 and JNK as the levels of the phosphorylated kinases were reduced. In contrast, p38 phosphorylation was not inhibited, suggesting that 4-HNE affects kinase activity. 4-HNE also inhibited nuclear factor-κB activation in monocytes. In view of the roles of p38, ERK1/ERK2, JNK, and nuclear factor-κB in inflammation, the data suggest that 4-HNE, at nontoxic concentrations, has anti-inflammatory properties, most likely through an effect on these signaling molecules, and could lead to the development of novel treatments for inflammatory diseases.
Natural killer and CD8 T cells dominate the response by human peripheral blood mononuclear cells to inactivated Francisella tularensis live vaccine strain
2005, Human Immunology
Francisella tularensis is a category A biothreat agent, and as a result, it has recently generated much research interest. F. tularensis live vaccine strain (LVS) is an attenuated form of the virulent F. tularensis organism and has previously been used as a vaccine. However, because of safety concerns, it is no longer approved for this purpose. Thus, the use of inactivated organisms is preferable for vaccine purposes. Although many studies have been performed that examine human peripheral blood mononuclear cells (PBMC), and in particular CD4 T cells, responses to inactivated F. tularensis, there has been no study identifying the individual human cell populations within a mixed PBMC population that respond to this organism. We sought to address this deficit. Our results indicate that natural killer and CD8 T cells comprise the majority of cells responding to F. tularensis LVS. In addition, data suggest CD8 T cell responses are maximal when antibiotic-treated organisms are used and are minimal when formaldehyde-fixed organisms are used. Given the belief that CD8 T cells can play an important role in protection against F. tularensis infection, these studies have direct relevance to the development of F. tularensis vaccines that use inactivated organisms. In addition, important new knowledge is added to our understanding of the human immune response to F. tularensis LVS.

View all citing articles on Scopus

View full text

Short communicationA rapid one-step procedure for purification of mononuclear and polymorphonuclear leukocytes from human blood using a modification of the hypaque-ficoll technique

Abstract

Blood

J. Immunol. Methods

Immunology

Scand. J. Clin. Lab. Invest.

J. Immunol. Methods

Short communication
A rapid one-step procedure for purification of mononuclear and polymorphonuclear leukocytes from human blood using a modification of the hypaque-ficoll technique