[Novel criteria for assessing red blood cell viability in blood banks]. / Nouveaux critères d'évaluation de la viabilité des hématies destinées à la transfusion.

UNLABELLED: In light of recent results on the mechanism of programmed cell death of human red blood cells (RBC), the aim of the present study was to solve the enigma of the rapid clearance of transfused RBCs. MATERIALS AND METHODS: We describe new criteria of RBC viability founded on the use of flow cytometry. They were applied, in association with the classical ones: ATP and hemolysis measurements, to RBCs stored in SAGM medium for 42 days. RESULTS AND CONCLUSIONS: Application of an original method of flow cytometric quantitation of in vitro erythrophagocytosis showed that an important proportion of stored RBCs were phagocytized although the following classical signals for phagocytosis were absent, i.e.: desialylation, phosphatidylserine exposure in the outer leaflet of the RBC membrane, loss of CD47 receptor, an antiphagocytosis signal. In addition, ATP was still present and hemolysis was very low. This enigma was solved by the use of scanning electron microscopy, which showed the disappearance of discocytes and the presence of an important proportion of spheroechinocytes, which are the phagocytable forms of RBCs. The mechanism of this dramatic morphological transformation remains to be elucidated.

Programmed cell death in mature erythrocytes: a model for investigating death effector pathways operating in the absence of mitochondria.

Human mature erythrocytes have been considered as unable to undergo programmed cell death (PCD), due to their lack of mitochondria, nucleus and other organelles, and to the finding that they survive two conditions that induce PCD in vitro in all human nucleated cells, treatment with staurosporine and serum deprivation. Here we report that mature erythrocytes can undergo a rapid self-destruction process sharing several features with apoptosis, including cell shrinkage, plasma membrane microvesiculation, phosphatidylserine externalization, and leading to erythrocyte disintegration, or, in the presence of macrophages, to macrophage ingestion of dying erythrocytes. This regulated form of PCD was induced by Ca(2+) influx, and prevented by cysteine protease inhibitors that allowed erythrocyte survival in vitro and in vivo. The cysteine proteinases involved seem not to be caspases, since (i) proforms of caspase 3, while present in erythrocytes, were not activated during erythrocyte death; (ii) cytochrome c, a critical component of the apoptosome, was lacking; and (iii) cell-free assays did not detect activated effectors of nuclear apoptosis in dying erythrocytes. Our findings provide the first identification that a death program can operate in the absence of mitochondria. They indicate that mature erythrocytes share with all other mammalian cell types the capacity to self-destruct in response to environmental signals, and imply that erythrocyte survival may be modulated by therapeutic intervention.

Improved storage of erythrocytes by prior leukodepletion: flow cytometric evaluation of stored erythrocytes.

In vivo phagocytosis of senescent red blood cells (RBCs) by macrophages occurs 120 days after their release into the circulation. It depends on two sequential signals that trigger phagocytosis: (1) desialylation of membrane glycoconjugates with the exposure of the penultimate beta-galactosyl residues and (2) exposure of phosphatidylserine in the membrane outer leaflet. Leukodepleted and nonleukodepleted RBCs were compared using flow cytometric procedures to determine whether the in vitro deterioration of RBCs during storage might be attributable to an identical mechanism of desialylation induced by leukocyte neuraminidases, resulting in exposure of beta-galactosyl and subsequently phosphatidylserine residues - signals of senescent RBCs. Without prior leukodepletion, stored RBCs showed an increased population of senescent RBCs (using light scatter measurements), extensive desialylation with the exposure of beta-galactosyl residues (using specific fluorescein isothiocyanate [FITC]-lectins), significant exposure of phosphatidylserine in the outer leaflet of the RBC membrane (using FITC-annexin V), and extensive in vitro phagocytosis (using PKH-26-labeled RBCs). There were minimal changes observed with the leukodepleted RBCs. These results lead to the conclusion that leukocyte enzymes, including neuraminidases, are definitive contributers to the desialylation of RBCs during storage and to the exposure of phosphatidylserine residues. These deleterious effects resulting from highly active leukocyte enzymes are preventable by prior leukodepletion of the stored RBCs. Previously developed flow cytometric procedures to detect in vivo "RBC senescence" have been applied and proved to be reliable criteria to monitor the viability of stored RBCs.

Cellular and molecular mechanisms of senescent erythrocyte phagocytosis by macrophages. A review.

Human red blood cells (RBCs) have a life-span of 120 days in circulation, after which they are phagocytized by resident macrophages. Extensive studies have been undertaken by many investigators in order to elucidate the cellular and molecular mechanisms of the erythrophagocytosis. The critical questions addressed by physiologists, clinicians and biochemists are: 'which of the many traumatic blemishes that appear on the erythrocyte surface as it winds its way through the circulation is the primary signal for clearance of the effete RBC from the circulation?', or 'What is the critical signal that it, and it alone, will activate the resident macrophage to adhere to and engulf it?'. Numerous, and often conflicting, hypotheses have been proposed. Each investigator focusing on but one of the many modifications that afflict the cell surface of the ageing erythrocyte, viz changes in either or both the carbohydrate or peptidic moieties of glycoproteins; abolishment of the pre-existing asymmetry in the lipid bilayer with the exposure of phosphatidylserine residues; or alterations in spectrin, to mention but a few. Many of these investigators also have invoked an intermediary role for auto-immune antibodies that recognise the change(s) on the erythrocyte surface and thereby serve as opsonins as a prelude to the erythrophagocytosis. The objective of the present review is to evaluate the data in support of the various hypotheses, and to submit some of our own recent observations involving the use of flow cytometric procedures that: i) provide evidence that the cell surface sialic acid serves as a determinant of the life-span; ii) characterise the senescent erythrocyte population that is specifically captured and phagocytized by macrophages (utilising the rapid and sensitive procedure we developed for quantification of in vitro erythrophagocytosis); and finally iii) provide evidence for the existence of an alternative pathway that is independent of immunoglobulins.

Molecular mechanisms of erythrophagocytosis: flow cytometric quantitation of in vitro erythrocyte phagocytosis by macrophages.

A rapid, sensitive, and reproducible flow cytofluorimetric procedure is described for quantitation of erythrophagocytosis based on the use of red blood cells (RBCs) labeled with the fluorescent probe PKH-26. The procedure involves the following steps: i) incubation of PKH-26-labeled erythrocytes with macrophages, ii) removal of un-bound red blood cells, iii) lysis of membrane-bound RBCs, and iv) measurement of extent of phagocytosis by direct flow-cytometric analysis of intact macrophages. Each step was controlled by fluorescence microscopy. Use of fluorescent, instead of radio-labeled RBCs, makes the method more sensitive, rapid, and avoids radioactive hazards. Furthermore, this approach is multi-parametric and can distinguish different populations of macrophages with reference to their erythrophagocytic potential. This technology moreover, has broad applications from the initial step of contact (between effector and target cells to study the specific receptor mediated attachment) to the subsequent cascade of time-dependent changes resulting from that signal transduction.

Molecular mechanisms of erythrophagocytosis. Characterization of the senescent erythrocytes that are phagocytized by macrophages.

We have recently developed a flow cytometric assay for the quantitation of erythrophagocytosis, using PKH 26-labeled erythrocytes as the target cells. Using this assay we have shown that there is extensive phagocytosis of desialylated erythrocytes. Furthermore, we have demonstrated that it is the densest population of erythrocytes obtained on a self-forming gradient of Percoll that shows the greatest susceptibility to phagocytosis. We designate this population of erythrocytes as fraction X; it is even denser than the fraction 5 found previously. This population of erythrocytes corresponds to zone X previously seen in the dot-plot of the flow cytometric analyses of human erythrocytes. Further scrutiny of this fraction indicates that a) it shows the greatest reactivity with annexin V, which is specific for the detection of phosphatidylserine (PS) exposed on the outer leaflet of the erythrocyte membrane, b) it is the most susceptible to erythrophagocytosis by resident murine peritoneal macrophages, and c) this erythrophagocytosis of PKH 26-labeled erythrocytes can be inhibited by annexin V and by liposomes containing PS. Scanning electron microscopy of fraction X shows two populations of erythrocytes: (A) spheroechinocytes with filipodes and (B) echinocytes without filipods. After a 2-h period of phagocytosis, the cells remaining in fraction X show a decrease in population A, commensurate with a decrease in reactivity with FITC-labeled annexin V from 65.5 to 24%.

Flow cytofluorimetric analysis of young and senescent human erythrocytes probed with lectins. Evidence that sialic acids control their life span.

Comparing the properties of 'young' and senescent ('aged') O+ erythrocytes isolated by applying ultracentrifugation in a self-forming Percoll gradient, we demonstrate that the sialic acids of membrane glycoconjugates control the life span of erythrocytes and that the desialylation of glycans is responsible for the clearance of the aged erythrocytes. This capture is mediated by a beta-galactolectin present in the membrane of macrophages. The evidence supporting these conclusions is as follows: (1) Analysis by flow cytofluorimetry of the binding of fluorescein isothiocyanate labelled lectins specific for sialic acids shows that the aged erythrocytes bind less WGA, LPA, SNA and MAA than young erythrocytes. The binding of DSA and LCA is not modified. On the contrary, the number of binding sites of UEA-I specific for O antigen and of AAA decreases significantly. PNA and GNA do not bind to erythrocytes. (2) RCA120 as well as Erythrina cristagalli and Erythrina corallodendron lectins specific for terminal beta-galactose residues lead to unexpected and unexplained results with a decrease in the number of lectin binding sites associated with increasing desialylation. (3) The glycoconjugates from the old erythrocytes incorporate more sialic acid than the young cells. This observation results from the determination of the rate of transfer by alpha-2,6-sialyltransferase of fluorescent or radioactive N-acetylneuraminic acid, using as donors CMP-9-fluoresceinyl-NeuAc and CMP-[14C]-NeuAc, respectively. (4) Microscopy shows that the old erythrocytes are captured preferentially by the macrophages relative to the young ones. Fixation of erythrocytes by the macrophage membrane is inhibited by lactose, thus demonstrating the involvement of a terminal beta-galactose specific macrophage lectin. (5) Comparative study of the binding of WGA, LPA, SNA and MAA to the aged erythrocytes and to the in vitro enzymatically desialylated erythrocytes shows that the desialylation rate of aged cells is low but sufficient to lead to their capture by the macrophages.