A new method for isolation of reticulocytes: positive selection of human reticulocytes by immunomagnetic separation

Brun, Atle; Gaudernack, Gustav; Sandberg, S.

doi:10.1182/blood.v76.11.2397.2397

Cited by 38 publications

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“…Simple differential centrifugation (DC) provides a method to enrich reticulocytes from peripheral blood, but the enrichment is low (<3%) ; when used on hemochromatosis blood, DC can attain enrichments of 15% reticulocytes . Affinity‐based separation techniques and the direct growth of reticulocytes allows high purity (>90%) that is not matched by enrichments using AMPSs, but these methods are expensive and impractical for routine use . DC‐Percoll provides a means to enrich reticulocytes, but this method requires two separate centrifugation steps and two extraction steps; variation between steps compound to reduce reproducibility.…”

Section: Discussionmentioning

confidence: 99%

“…Current methods to obtain substantially enriched (>10% of erythrocytes) samples of reticulocytes are impractical, expensive, labor‐intensive, or not satisfactory for routine use for applications that require reproducible yields and readily available sources of blood. These methods include culture and development of progenitors , affinity‐based separation , differential centrifugation , and centrifugation over layered gradients . We describe these methods in detail in the Supporting Information.…”

Section: Introductionmentioning

confidence: 99%

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Enrichment of reticulocytes from whole blood using aqueous multiphase systems of polymers

et al. 2014

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This paper demonstrates the enrichment of reticulocytes by centrifuging whole blood through aqueous multiphase systems (AMPSs)-immiscible phases of solutions of polymers that form step-gradients in density. The interfaces of an AMPS concentrate cells; this concentration facilitates the extraction of blood enriched for reticulocytes. AMPS enrich reticulocytes from blood from both healthy and hemochromatosis donors. Varying the osmolality and density of the phases of AMPS provides different levels of enrichment and yield of reticulocytes. A maximum enrichment of reticulocytemia of 64 6 3% was obtained from donors with hemochromatosis. When used on peripheral blood from normal donors, AMPS can provide a higher yield of enriched reticulocytes and a higher proportion of reticulocytes expressing CD71 than differential centrifugation followed by centrifugation over Percoll. Blood enriched for reticulocytes by AMPS could be useful for research on malaria. Several species of malaria parasites show a preference to invade young erythrocytes and reticulocytes; this preference complicates in vitro cultivation of these species in human blood. Plasmodium knowlesi malaria parasites invade normal human blood enriched for reticulocytes by AMPSs at a rate 2.2 times greater (P < 0.01) than they invade unenriched blood. Parasite invasion in normal blood enriched by AMPS was 1.8 times greater (P < 0.05) than in blood enriched to a similar reticulocytemia by differential centrifugation followed by centrifugation over Percoll. The enrichment of reticulocytes that are invaded by malaria parasites demonstrates that AMPSs can provide a label-free method to enrich cells for biological research. IntroductionThe characterization of different cellular components of human whole blood plays an important role in medical diagnoses and research [1]. This task is often accomplished by isolating or enriching a specific cellular sub-population from the complex mixture of cells found in whole blood. Whole blood is composed of cells-leukocytes, platelets, and erythrocytes-suspended in protein-rich plasma; each type of cell is useful in the evaluation of the health of a patient. These cells can be further subdivided into important components. For example, the isolation of reticulocytes-immature erythrocytes-is important for research in malaria.Plasmodium (P.) vivax, P. ovale, and P. knowlesi-three causative agents of malaria in humans-preferentially invade human reticulocytes [2][3][4]. The study of these species of malaria suffers from the practical difficulty of in vitro cultures in whole human blood. Their cultivation in blood enriched for reticulocytes would provide concentrated host cells in which the parasites proliferate; continuous cultures of these parasites would make their study much more practical [4][5][6][7]. Enrichment is difficult because reticulocytes exist at a low concentration (0.5-2.5% of erythrocytes) in whole blood [8] and have a short half-life in culture ( 30 hr) [9]. Cryopreservation provides a method to store samples that hav...

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Enrichment of reticulocytes from whole blood using aqueous multiphase systems of polymers

et al. 2014

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“…The presence of the M 2 isozyme in PK‐deficient erythrocytes makes it difficult to diagnose PK deficiency in dogs using assays that measure total erythrocyte PK activity. Reticulocytes have considerably higher PK activity than do mature erythrocytes 34,35. Consequently, the finding of PK activity within or slightly above the reference interval in a dog with marked reticulocytosis is highly suggestive of PK deficiency.…”

Section: Pyruvate Kinase Deficiencymentioning

confidence: 99%

Pathogenesis, laboratory diagnosis, and clinical implications of erythrocyte enzyme deficiencies in dogs, cats, and horses

Harvey¹

2006

Veterinary Clinical Pathol

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Deficiencies of enzymes involved in erythrocyte metabolism can have significant effects on erythrocyte function and survival. Animals with pyruvate kinase (PK) or phosphofructokinase (PFK) deficiencies have shortened erythrocyte life spans and regenerative anemia. PK-deficient dogs (but not PK-deficient cats) develop progressive myelofibrosis and osteosclerosis of bone marrow and hemochromatosis and cirrhosis of the liver. PFK-deficient dogs have sporadic episodes of hyperventilation-induced intravascular hemolysis and hemoglobinuria. Cytochrome b5 reductase (Cb5R) deficiency in dogs and cats results in persistent methemoglobinemia and cyanotic mucous membranes. Severe deficiency of glucose-6-phosphate dehydrogenase, the rate-controlling enzyme in the pentose phosphate pathway, resulted in anemia with eccentrocytosis in an American saddlebred colt. Horses with erythrocyte flavin adenine dinucleotide (FAD) deficiency have both eccentrocytosis (attributable to severe deficiency in glutathione reductase activity) and methemoglobinemia (attributable to Cb5R deficiency); the dual enzyme deficiency occurs because FAD is a required cofactor for both enzymes. Erythrocyte enzyme deficiencies do not usually shorten life expectancy, except for PK-deficient dogs and potentially PFK-deficient dogs during a hemolytic crisis. Although enzyme deficiencies are rare causes of anemia and methemoglobinemia, the ability to diagnose deficient animals allows for the possibility of eliminating these undesirable traits in future breeding. DNA-based assays are available for PK and PFK deficiencies; whereas, biochemical tests of enzyme activity are required for other deficiencies. Continued research is needed to document additional enzyme deficiencies that likely occur and to develop additional DNA-based assays to detect heterozygous animals.

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“…It has been shown that transferrin receptors disappear during maturation, thereby suggesting that they are specific for immature red cells (Frazier et al, 1982). Bearing such difference in mind, a purification protocol using anti‐transferrin antibodies coupled to magnetic beads was developed (Brun et al, 1990); the results revealed 15–42% reticulocyte enrichment. However, the difficulty of eluting cells coupled to beads has been the main factor limiting this method.…”

Section: Reticulocyte Sourcesmentioning

confidence: 99%

Reticulocytes: Plasmodium vivax target cells

Moreno-Pérez

Ruíz

Patarroyo

2013

Biology of the Cell

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Reticulocytes represent the main invasion target for Plasmodium vivax, the second most prevalent parasite species around the world causing malaria in humans. In spite of these cells' importance in research into malaria, biological knowledge related to the nature of the host has been limited, given the technical difficulties present in working with them in the laboratory. Poor reticulocyte recovery from total blood, by different techniques, has hampered continuous in vitro P. vivax cultures being developed, thereby delaying basic investigation in this parasite species. Intense research during the last few years has led to advances being made in developing methodologies orientated towards obtaining enriched reticulocytes from differing sources, thereby providing invaluable information for developing new strategies aimed at preventing infection caused by malaria. This review describes the most recent studies related to obtaining reticulocytes and discusses approaches which could contribute towards knowledge regarding molecular interactions between target cell proteins and their main infective agent, P. vivax.

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A new method for isolation of reticulocytes: positive selection of human reticulocytes by immunomagnetic separation

Cited by 38 publications

References 0 publications

Enrichment of reticulocytes from whole blood using aqueous multiphase systems of polymers

Enrichment of reticulocytes from whole blood using aqueous multiphase systems of polymers

Pathogenesis, laboratory diagnosis, and clinical implications of erythrocyte enzyme deficiencies in dogs, cats, and horses

Reticulocytes: Plasmodium vivax target cells

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