Diffusion-based extraction of DMSO from a cell suspension in a three stream, vertical microchannel

Cells are routinely cryopreserved for investigative and therapeutic applications. The most common cryoprotective agent (CPA), dimethyl sulfoxide (DMSO), is toxic, and must be removed before cells can be used. This study uses a microfluidic device in which three streams flow vertically in parallel through a rectangular channel 500 μm in depth. Two wash streams flow on either side of a DMSO-laden cell stream, allowing DMSO to diffuse into the wash and be removed, and the washed sample to be collected. The ability of the device to extract DMSO from a cell stream was investigated for sample flow rates from 0.5 to 4.0 mL/min (Pe = 1,263-10,100). Recovery of cells from the device was investigated using Jurkat cells (lymphoblasts) in suspensions ranging from 0.5% to 15% cells by volume. Cell recovery was >95% for all conditions investigated, while DMSO removal comparable to a previously developed two-stream device was achieved in either one-quarter the device length, or at four times the flow rate. The high cell recovery is a ~25% improvement over standard cell washing techniques, and high flow rates achieved are uncommon among microfluidic devices, allowing for processing of clinically relevant cell populations.     

Cryopreservation and subsequent viability of human bone marrow in hematologic malignancies: Comparison of two different methods of cell reconstitution

Bone marrow cells collected from patients with hematologic malignancies were cryopreserved using DMSO as a cryoprotective agent. The growth kinetics of hemopoietic stem cells frozen to −196 °C was monitored immediately after thawing by the semisolid agar CFU-C assay and two different methods of cell reconstitution were compared. In the first procedure, thawed cells were plated after the removal of DMSO by washing the cell suspension; in the second, cell suspensions were cultured after a simple 1:1 dilution of DMSO with medium. The numbers of CFU-C per 2 × 10⁵ cells plated was higher by washing out the DMSO in all the groups studied. However, the absolute numbers of CFU-C contained in the whole ampoules after the freezing procedures was approximately the same using both methods. It is concluded that washing the cells only apparently yielded a better cloning efficiency, suggesting that such a procedure led to a higher mature nucleated cell loss with the consequence of a CFU-C concentration. This trend seems particularly evident in cells from the AML and CML patients.     

Study of a new device for washing and concentrating cryopreserved hematopoietic stem cells and mononuclear cells: a single center experience

Background aimsCryopreserved cellular products, as parts of hematopoietic progenitor cell (HPC) transplants, mononuclear cell reinjections for donor lymphocyte infusion or extracorporeal photopheresis, can be washed before being reinjected into the patient or infused directly, depending on local practices. The aim of washing is to reduce the incidence and severity of adverse reactions (ARs) due to the dimethyl sulfoxide (DMSO) used as a cryoprotective agent and other factors, such as dead cell debris. At the authors’ cell therapy laboratory (CTL) in Poitiers, France, as in 76% of Etablissement Français du Sang (EFS) CTLs, all cryopreserved products undergo thawing in a water bath followed by washing with the COBE 2991. As this device will soon cease to be available, an alternative process needs to be assessed.MethodsThe authors compared two closed systems: the authors’ semi-automatic system using the traditional centrifugation method (COBE 2991) and an automated device using spinning membrane filtration (Lovo). A total of 72 HPC bags available for research were used. The authors first performed a paired comparison, processing one or two HPC bags washed by each device. A second study was carried out to compare two different washing solutions generally used by EFS CTLs along with variable storage conditions. Finally, the authors studied the efficiency of the Lovo with three or four thawed bags. The main parameters studied were viable CD34+ cell recovery and viability, CD3+ cell recovery, stability up to 6 h after washing, DMSO elimination and center feasibility.ResultsThe Lovo device showed better CD34+ cell recovery compared with the COBE 2991 while maintaining CD34+ viability and stability over 6 h. Moreover, Lovo efficiency seemed to be independent of the number of thawed bags processed and washing solution used in the authors’ study. CD3+ cell recovery met the authors’ internal specifications (cell recovery >50%), with similar results seen when processing with either the COBE 2991 or Lovo. Additionally, on average, 97% of DMSO was removed after washing with Lovo, minimizing the risk of ARs. The storage conditions post-processing indicated preferred storage conditions of 7 ± 3°C. Finally, if processing time seemed shorter using COBE 2991 for one bag washed, the Lovo device required only one staff member regardless of the number of HPC bags processed.ConclusionsThe Lovo device seems to provide an opportunity to standardize HPC processing, ensuring patient safety, with, on average, 97% of DMSO removed, while improving recovery of cells of interest and maintaining viability over time in case of delayed transplant. The Lovo device consequently seems to be a serious alternative to the COBE 2991.     

Evidence for the involvement of oxygen free radicals in the ethanol-induced late cellular injury in mouse myeloma cells.

In this study, we analysed the ethanol-induced long term cell injury on a general cell model (Sp2/0-Ag14 cell line). Cells were incubated in 1, 5, 10, 15 and 20% of ethanol (EtOH) for 5 min. After washing cell viability was tested by the Trypan Blue exclusion test in 5, 60 min, 4 and 24 h after EtOH exposure. Free radicals were monitored every 30 min by electron spin resonance (ESR) with alpha-phenyl-N-tert-butylnitrone (PBN) spin trapping technique. Scavenger compounds such as glutathione (GSH), dimethyl sulfoxide (DMSO) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) were applied for 24 h incubation after EtOH exposure. EtOH concentration dependently decreased the cell viability immediately after 5 min exposure, but with 4 and 24 h, a secondary cell destruction was found. Using ESR-spin trapping technique, an increased free radical activity could be detected. DMPO, DMSO and GSH significantly, but in different period protected the cells against free-radical induced cellular damage. EtOH produces an early (immediately after EtOH exposure) and a late (in about 4 h) cellular damage on Sp2/0-Ag14 cells. The oxygen free radicals can be detected in a short time after EtOH exposure, its biological effect manifested as a secondary cell destruction at 4 and 24 h. This phenomenon can be prevented by scavenger compounds.     

Cryoprotectant removal temperature as a factor in the survival of frozen rice and sugarcane cells

Removal of cryoprotective additives through use of a room temperature (22 °C) washing step, instead of 0 °C, was found to improve the recovery of sugarcane suspension culture and rice callus tissues. Cultured cells were cryoprotected by gradual addition of a mixture of polyethylene glycol, glucose, and DMSO (PGD) to a final concentration of 10%-8%-10%, w/v, respectively, added at either 0 or 22 °C. After a programmed slow freezing of the cells, they were thawed rapidly and the cryoprotectants were gradually diluted and washed out using a 22 or 0 °C washing medium. Viability of suspension cultured sugarcane cells protected with PGD was greatly diminished when a cold washing solution was used, whether the cells had been frozen (?23 °C) or not. Two mutant lines of rice callus when frozen to ?196 °C in PGD and thawed showed less growth than unfrozen cells, but their growth was improved by washing the thawed cells with a 22 °C solution. With all cultures tested, the addition of PGD at 0 °C and post-thaw washing out at 22 °C gave improved survival. Particularly with the rice lines, optimizing the addition and washing procedures allowed culture survival of liquid nitrogen freezing not otherwise attained.     

Permeabilization of Cinchona ledgeriana cells by dimethylsulphoxide. effects on alkaloid release and long-term membrane integrity

Dimethyl sulphoxide (DMSO) has been used to permeabilize cells of Cinchona ledgeriana in suspension culture and promote the release of intracellular alkaloids. 5–6% v/v is required before any release is seen, and greater than 20% DMSO is required for full release. Even at these high levels of DMSO release is slow, taking in excess of seven hours to reach completion. Conditions which produce significant release of alkaloids have a deleterious effect on cells. Many of the membranes permeabilized did not recover their ability to selectively exclude compounds such as mannitol when the DMSO was removed. It is concluded that DMSO is not a suitable material for inducing alkaloid release in any biotechnological exploitation of alkaloid production by C. ledgeriana.Abbreviations DMSO Dimethyl sulphoxide - 2,4D 2,4-Dichlorophenoxyacetic acid     

Significance of the cell cycle in commitment of murine erythroleukemia cells to erythroid differentiation.

The relationship between differentiation of murine erythroleukemia cells (MEL) induced by DMSO and the cell division cycle has been analyzed. We demonstrate that incubation in the presence of DMSO increases the length of the G1 phase of the cell cycle. A method of synchronization of MEL cells by unit gravity sedimentation has been developed and characterized. Using this method, a series of synchronized cell populations covering the entire cell division cycle can be generated simultaneously. Cells synchronized by this technique were challenged with DMSO and analyzed for kinetics of commitment to the differentiation program. Our results indicate that populations of cells in G1 or G2 at the time of addition of inducer give rise to a greater proportion of committed cells than an unfractionated population, while cells in S phase result in a lower percentage of committed cells than the unfractionated population when cultured in DMSO.     

The possibility of the incorporation of macromolecules, including exogenous DNA, into the germ cells of male mice. The liposome method and Ca-P coprecipitation method

Liposomes loaded with FITC-labeled albumin in the presence of PEG-1,500 are actively sorbed on the membranes of mature spermatozoa and remain attached even after thorough washing. Immature sperm cells are able to incorporate alien DNA carried by liposomes. In contrast, the mature spermatozoa could not incorporate plasmid DNA loaded with positively charged liposomes. Chlortetracycline in Ca-P coprecipitate crystals is tightly fixed in the postacrosomal region of mature sperms. Intensity of staining of chlortetracycline is stimulated by DNA load in Ca-P coprecipitate as well as by DMSO or EDTA. The method of Ca-P coprecipitation could not provide for plasmid DNA incorporation into taure sperms. Foreign DNA incorporation in postacrosomal regions of sperm heads seems quite possible in experiments with dimethylsulphoxide (DMSO).     

Effect of DMSO and a freezing-thawing process on the 'ecto-ATPase' and AChE activities of human platelets

The activity of two membrane-bound enzymes of human platelets subjected to DMSO and a freezing-thawing process were analysed. Following treatment of fresh platelets with DMSO (5-20%) quantitative differences of AChE and 'ecto-ATPase' activities were seen. After cryopreservation of platelets in 5% DMSO the enzymatic activities of AChE and Mg2+-ATPase were no different from those obtained for fresh platelets. The lack of any changes in the activities of the enzymes of frozen platelets subjected to a washing procedure to remove DMSO, indicates that the mechanism of the DMSO-induced effect is reversible and that freezing-thawing process had no additional detrimental effects.     

Comparison of stem cell viability of bone marrow cryopreserved by two different methods

Two different cryogenic methods were used to study the preservation of murine bone marrow cells. Compared to the classical methods, in which separated mononuclear marrow cells in 10% dimethyl sulfoxide (DMSO) were cryopreserved in liquid nitrogen (-196 degrees C), a modified technique was carried out by cryopreservation of unfractionated marrow cells in a mixed protectant of 5% DMSO and 6% hydroxyethyl starch (HES) at -80 degrees C. Samples that were separately thawed after storage for 1, 4, 8, and 12 weeks were assayed for cell viability and recovery of CFU-GM and CFU-S. No macroscopic clumping of cells was noted either in fractionated or in unfractionated marrow cell cryopreservations. A mild damage, about 25% reduction of stem cells, was found at 1 week and did not deepen further. It seems that the greatest loss of stem cells occurred in the process of cryopreservation itself. Compared to prefreeze values, both a high number of cells that excluded trypan blue (87 +/- 3.4%) and a high recovery of CFU-GM (75 +/- 9.8%) and CFU-S (74 +/- 11.2) were observed in unfractionated marrow samples cryopreserved with the DMSO/HES mixture at -80 degrees C for 3 months and these results were very similar to those obtained from fractionated mononuclear marrow cells cryopreserved at -196 degrees C. The DMSO/HES protectant provides a simplified bone marrow cryopreservation technique that should be favorable to clinical application because of its high stem cell recovery and avoidance of cell-separation manipulation.     

Preliminary studies on the vitrification of red sea bream (Pagrus major) embryos

The objective was to identify an appropriate cryoprotectant and protocol for vitrification of red sea bream (Pagrus major) embryos. The toxicity of five single-agent cryoprotectants, dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycerol (GLY), and methyl alcohol (MeOH), as well as nine cryoprotectant mixtures, were investigated by comparing post-thaw hatching rates. Two vitrifying protocols, a straw method and a solid surface vitrification method (copper floating over liquid nitrogen), were evaluated on the basis of post-thaw embryo morphology. Exposure to single-agent cryoprotectants (10% concentration for 15 min) was not toxic to embryos, whereas for higher concentrations (20 and 30%) and a longer duration of exposure (30 min), DMSO and PG were better tolerated than the other cryoprotectants. Among nine cryoprotectant mixtures, the combination of 20% DMSO+10% PG+10% MeOH had the lowest toxicity after exposure for 10 min or 15 min. High percentages of morphologically intact embryos, 50.6+/-16.7% (mean+/-S.D.) and 77.8+/-15.5%, were achieved by the straw vitrifying method (20.5% DMSO+15.5% acetamide+10% PG, thawing at 43 degrees C and washing in 0.5M sucrose solution for 5 min) and by the solid surface vitrification method (40% GLY, thawing at 22 degrees C and washing in 0.5M sucrose solution for 5 min). After thawing, morphological changes in the degenerated embryos included shrunken yolks and ruptured chorions. Furthermore, thawed embryos that were morphologically intact did not consistently survive incubation.     

Effect of endocellular cryoprotectant upon polymorphonuclear neutrophil function during storage at low temperature.

The effect of cryoprotective agents dimethyl sulfoxide (DMSO), glycerol and ethylene glycol upon the function of polymorphonuclear neutrophils (PMNs) during storage between 0 ° and 4 °C was investigated.Increasing concentration of each cryoprotectant caused an increasing inhibition of chemotaxis with complete inhibition at 16.7%. At this concentration most PMNs were still able to exclude trypan blue dye. Chemotaxis was not inhibited if PMNs were exposed to 4.2 or 8.3% concentrations of cryoprotectants for 1 hr, and washed subsequently. However, the recovery of chemotaxis was not observed at 16.7% after 1 hr exposure to cryoprotectants. Moreover, a considerable number of PMNs could not exclude the dye. This would indicate that cells become fragile with cryoprotectants at a high concentration and the PMNs are easily damaged by washing. With 20 hr exposure PMNs, the inhibitory effect on chemotaxis was removed by washing when a 4.2% agent was used, but using an 8.3% agent, chemotaxis was not restored but PMNs exposed to DMSO displayed almost the same chemotaxis as a control. On the other hand, the ability of PMNs to ingest bacteria was not so markedly inhibited as the chemotaxis. With 1 hr exposure to cryoprotectants, the ingestive ability was hardly affected within 8.3%. As for 20 hr exposure, the same ingestive ability as that of a control was observed in all cases using a 4.2% concentration. However, using an 8.3% concentration, the DMSO-exposed PMNs retained a good ingestive ability.Judging from the above findings, DMSO would be suitable as a crypotrotective agent although the problem on toxicity remains to be resolved.     

Effect of polyethylene glycol and dimethyl sulfoxide on the fusion of bovine nuclear transfer using mammary gland epithelial cells

The effects of polyethylene glycol and dimethyl sulfoxide (PEG/DMSO) treatment of donor cells on the fusion and subsequent development of bovine nuclear transfer embryos using mammary gland epithelial (MGE) cells before electrofusion (fresh MGE cells) was studied. The same study was conducted on those cells that were frozen and stored in liquid nitrogen, and then thawed (frozen-thawed MGE cells). Experiment 1 showed that the exposure time and pH of PEG/DMSO solution affected the fusion of nuclear transfer, and that a higher fusion rate was obtained when fresh MGE cells were exposed to PEG/DMSO solution at pH 8.0 for 5 min. In Experiment 2, the proportion of fused oocytes with fresh PEG/DMSO-treated cells (70 +/- 6%) was significantly higher than that with non-treated cells (50 +/- 13%, p < 0.05). The same tendency was observed when frozen-thawed cells as donor nuclei were used (48 +/- 6% vs. 34 +/- 12%, p < 0.05). In addition, PEG/DMSO treatment has neither harmful nor beneficial effects on the cleavage and development of the blastocyst stage of reconstructed embryos (p > 0.05). The fusion and cleavage rates of frozen-thawed cells were significantly lower than those of fresh cells (p < 0.05). After 10 blastocysts, derived from fresh PEG/DMSO-treated cells, were transferred to five recipient heifers, one live female calf was obtained. Experiment 3 showed that PEG/DMSO treatment reduced the viability of both fresh and frozen-thawed MGE cells (p < 0.05). We conclude that the PEG/DMSO treatment of fresh MGE cells, as well as the frozen-thawed cells, before electrofusion has a positive effect on the fusion of nuclear transfer without decreasing the in vitro development of reconstructed embryos.     

Development of a novel microperfusion chamber for determination of cell membrane transport properties.

A novel microperfusion chamber was developed to measure kinetic cell volume changes under various extracellular conditions and to quantitatively determine cell membrane transport properties. This device eliminates modeling ambiguities and limitations inherent in the use of the microdiffusion chamber and the micropipette perfusion technique, both of which have been previously validated and are closely related optical technologies using light microscopy and image analysis. The resultant simplicity should prove to be especially valuable for study of the coupled transport of water and permeating solutes through cell membranes. Using the microperfusion chamber, water and dimethylsulfoxide (DMSO) permeability coefficients of mouse oocytes as well as the water permeability coefficient of golden hamster pancreatic islet cells were determined. In these experiments, the individual cells were held in the chamber and perfused at 22 degrees C with hyperosmotic media, with or without DMSO (1.5 M). The cell volume change was videotaped and quantified by image analysis. Based on the experimental data and irreversible thermodynamics theory for the coupled mass transfer across the cell membrane, the water permeability coefficient of the oocytes was determined to be 0.47 micron. min-1. atm-1 in the absence of DMSO and 0.65 microns. min-1. atm-1 in the presence of DMSO. The DMSO permeability coefficient of the oocyte membrane and associated membrane reflection coefficient to DMSO were determined to be 0.23 and 0.85 micron/s, respectively. These values are consistent with those determined using the micropipette perfusion and microdiffusion chamber techniques. The water permeability coefficient of the golden hamster pancreatic islet cells was determined to be 0.27 microns. min-1. atm-1, which agrees well with a value previously determined using an electronic sizing (Coulter counter) technique. The use of the microperfusion chamber has the following major advantages: 1) This method allows the extracellular condition(s) to be readily changed by perfusing a single cell or group of cells with a prepared medium (cells can be reperfused with a different medium to study the response of the same cell to different osmotic conditions). 2) The short mixing time of cells and perfusion medium allows for accurate control of the extracellular osmolality and ensures accuracy of the corresponding mathematical formulation (modeling). 3) This technique has wide applicability in studying the cell osmotic response and in determining cell membrane transport properties.     

Differing actions of penetrating and nonpenetrating cryoprotective agents.

A two-step freezing technique has been used to examine the role of cryoprotective agents during cooling. Chinese hamster fibroblasts were cooled to various subzero holding temperatures and subsequently thawed or cooled to ?196 °C before thawing. Cells were suspended in various concentrations of dimethylsulfoxide (DMSO) or hydroxyethyl starch (HES) before freezing. The results indicated differing protective actions of DMSO and HES. These differences were verified using glycerol as either a penetrating or a nonpenetrating agent.The results are consistent with the concepts that cryoprotection is based on the avoidance or minimization of intracellular freezing and the minimization of damage to the cell from the environment of concentrated solutes during cooling, and that the colligative action of both penetrating and nonpenetrating agents allows the cells to survive the conditions for a reduction of cell water content during cooling thereby reducing the amount of intracellular freezing. The results indicate that penetrating and nonpenetrating agents accomplish this in different ways. Penetrating agents create the environment for a reduction of cell water content at temperatures sufficiently low to reduce the damaging effect of the concentrated solutes on the cells. Nonpenetrating agents osmotically “squeeze” water from the cells primarily during the initial phases of freezing at temperatures between ?10 and ?20 °C when these additives become concentrated in the extracellular regions.     

CRYOPROTECTANT-INDUCED REDISTRIBUTION OF INTRAMEMBRANOUS PARTICLES IN MOUSE LYMPHOCYTES

This study demonstrates, by freeze fracture, clustering of intramembranous particles caused by cryoprotectant treatment of intact unfixed mouse lymphoid cells. Both T and B cells react in a similar fashion, while similar clustering of particles is not observed in some other cell types. The intramembranous particles can be aggregated by incubating unfixed cells in glycerol or dimethylsulfoxide (DMSO) before freezing. The aggregation phenomenon is dependent on the length of time of exposure and the concentration of the cryoprotectants. Further, the cells remain viable and the cryoprotectant-induced clustering is completely reversible. Prefixation of glycerol-treated cells with glutaraldehyde prevents the formation of these particle clusters, and unfixed nonglycerinated cells show no clusters. Thin sections of cells exposed to glycerol or DMSO without previous fixation exhibit bizarre membrane alterations and numerous other degenerative changes. These observations stress the importance of prefixation of lymphoid cells before exposure to glycerol or DMSO, as well as indicate that the membrane characteristics of certain cell types may be probed by glycerol treatment of unfixed cells.     

Analysis and cryopreservation of hematopoietic stem and progenitor cells from umbilical cord blood

BACKGROUND: Umbilical cord blood (UCB) is an important source of hematopoietic stem and progenitor cells (HSC/HPC) for the reconstitution of the hematopoietic system after clinical transplantation. Cryopreservation of these cells is critical for UCB banking and transplantation as well as for research applications by providing readily available specimens. The objective of this study was to optimize cryopreservation conditions for CD34+ HSC/HPC from UCB. METHODS: Cryopreservation of CD34+ HSC/HPC from UCB after mononuclear cell (MNC) preparation was tested in a research-scale setup. Experimental variations were concentration of the cryoprotectant, the protein additive and cell concentration. In addition, protocols involving slow, serial addition and removal of DMSO were compared with standard protocols (fast addition and removal of DMSO) in order to avoid osmotic stress for the cryopreserved cells. Viability and recoveries of MNC, CD34+ cells and total colony-forming units (CFU) were calculated as read-outs. In addition, sterility testing of the collected UCB units before further processing was performed. RESULTS: The optimal conditions for cryopreservation of CD34+ HPC in MNC preparations were 10% DMSO and 2% human albumin at high cell concentrations (5 x 10(7) MNC/mL) with fast addition and removal of DMSO. After cryopreservation using a computer-controlled freezer, high viabilities (89%) and recoveries for CD34+ cells (89%) as well as for CFU (88%) were observed. Microbial contamination of the collected UCB samples was reduced to a rate of 6.4%. DISCUSSION: Optimized cryopreservation conditions were developed for UCB MNC in respect of the composition of the cryosolution. In addition, our results showed that fast addition of DMSO is essential for improved cryopreservation and post-thaw quality assessment results, whereas the speed of DMSO removal after thawing has little influence on the recoveries of CD34+ cells and CFU.     

Effects of dimethylsulfoxide on membrane currents of neuroblastoma x glioma hybrid cell

Giga-ohm seal whole cell recording technique was used to examine ionic currents changes induced by dimethylsulfoxide (DMSO) in neuroblastoma X glioma hybrid NG 108-15 cells. DMSO (0.5-1%) reversible blocks sodium, potassium and calcium currents and shifts by about 6 mV the sodium inactivation curve towards more negative voltages.     

Storage of marine fish erythrocytes in liquid nitrogen

The preservation of erythrocytes from cod ( Gadus morhua ), saithe ( Pollachius virens ) and mackerel ( Scomber scombrus ) at −196° C was studied using dimethyl sulphoxide (DMSO) as a cryoprotectant. Erythrocyte recoveries of greater than 90% were obtained from all species and cod erythrocytes were stored for eighteen months with insignificant lysis. Larger quantities of blood were stored by removal of plasma from citrated blood prior to the addition of DMSO solution, and by storage of pelleted frozen blood in aluminium canisters in liquid nitrogen. Maximum recoveries of washed intact erythrocytes required thawing of pellets in 125% DMSO solution and washing with buffer containing decreasing concentrations of DMSO. Washed erythrocytes kept at 4° for at least two days showed little haemolysis, were morphologically similar to fresh erythrocytes and equally susceptible to the δ-haemolysin of Staphylococcus aureus .     

Fluorometric test of cell membrane integrity

A fluorometric test for assessing cell membrane integrity of bone marrow, based on the use of fluorescein diacetate (FDA), is described. It is demonstrated that the amount of fluorescein extracted from the labeled cells is directly proportional to the number of intact cells and that this relationship is not affected by the presence of the dead cells. The experimental conditions required for the accuracy of this test are as follows: (1) During the incubation with FDA the number of cells should not exceed ca. 3 × 10⁶ cells/ml. (2) With the fluorescein concentration of 2 μg/ml, the incubation temperature should be within the range of 20 to 30 °C for a period of 10 to 20 min. (3) To prevent the loss of fluorescein by labeled cells during washing, the cells must be maintained at a temperature near 0 °C. (4) If the cells are contaminated by hemoglobin, an appropriate correction of the fluorescein readings must be made. Provided that the controls are appropriately adjusted, the accuracy of the test should not be affected by the presence of serum and/or of DMSO in the reaction medium. Other potential sources of error may be the enhanced loss of fluorescein due to the presence of albumin or platelet-absorbed serum and the possible activation of esterases before the FDA treatment. It is concluded that the results of this test should be interpreted in terms of relative changes of the collective cell membrane integrity rather than in terms of cell viability, and that these results may be more meaningful than those based on the visual assessment of either the FDA-labeled cells or of the cells subjected to the dye exclusion test.