Hemolysis of human red blood cells by freezing and thawing in solutions containing sucrose: Relationship with posthypertonic hemolysis and solute movements

Woolgar, A.E.

doi:10.1016/0011-2240(74)90037-6

Cited by 15 publications

(9 citation statements)

References 11 publications

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“…Notably, 15 minutes of direct contact with ice may be sufficient indeed to generate a significant degree of haemolysis in blood tubes or in blood gas syringes. As earlier demonstrated by Woolgar, maintaining a blood tube in a cooling bath (- 9 °C) for 10 minutes is sufficient to increase haemolysis up to 76% ( 15 ).…”

Section: Discussionmentioning

confidence: 67%

A paradigmatic case of haemolysis and pseudohyperkalemia in blood gas analysis

Salvagno

Demonte

Lippi

2018

Biochem. med. (Online)

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A 51-year old male patient was admitted to the hospital with acute dyspnea and history of chronic asthma. Venous blood was drawn into a 3.0 mL heparinized syringe and delivered to the laboratory for blood gas analysis (GEM Premier 4000, Instrumentation Laboratory), which revealed high potassium value (5.2 mmol/L; reference range on whole blood, 3.5-4.5 mmol/L). This result was unexpected, so that a second venous blood sample was immediately drawn by direct venipuncture into a 3.5 mL lithium-heparin blood tube, and delivered to the laboratory for repeating potassium testing on Cobas 8000 (Roche Diagnostics). The analysis revealed normal plasma potassium (4.6 mmol/L; reference range in plasma, 3.5-5.0 mmol/L) and haemolysis index (5; 0.05 g/L). Due to suspicion of spurious haemolysis, heparinized blood was transferred from syringe into a plastic tube and centrifuged. Potassium and haemolysis index were then measured in this heparinized plasma, confirming high haemolysis index (50; 0.5 g/L) and pseudohyperkalemia (5.5 mmol/L). Investigation of this case revealed that spurious haemolysis was attributable to syringe delivery in direct ice contact for ~15 min. This case emphasizes the importance of avoiding sample transportation in ice and the need of developing point of care analysers equipped with interference indices assessment.

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Section: Discussionmentioning

confidence: 67%

A paradigmatic case of haemolysis and pseudohyperkalemia in blood gas analysis

Salvagno

Demonte

Lippi

2018

Biochem. med. (Online)

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“…This solvent is in fact a solution containing all penetrating solutes, including sodium, penetrating cryoprotectants and, if the membrane should be sufficiently damaged, normally nonpenetrating cryoprotectants like sucrose. Farrant and Woolgar (1972b) and Woolgar (1972Woolgar ( , 1974 have reported that sucrose does not enter red cells during hypertonic exposure, but it does during freezing and thawing (Daw et al, 1973) which is the result one would expect if loading were to occur during dilution. Daw's result indicates that the membranes are able to reseal with an excess of internal solute and therefore stabilize at a greater volume than normal; up to 170% of normal should be possible because only 50% of human red cells lyse at this volume at room temperature (Pegg, 1984).…”

Section: Role Of Unfrozen Fraction Inmentioning

confidence: 98%

“…The cells that were initially in a hypertonic solution were still surrounded by a hypertonic medium at the end of the experiment, and the glycerol was not removed. Interestingly, Woolgar has shown that red cells frozen and thawed in 0.75 M sodium chloride solution are less damaged than cells frozen to the same temperature in 0.15 M sodium chloride (Woolgar, 1972 and1974). Clearly this result cannot be due to differing salt concentrations during freezing because they will be identical, but in the same papers, Woolgar also showed that posthypertonic hemolysis (which we discuss below) is reduced as the osmolality of the sodium chloride solutions to which the cells are returned, is increased.…”

Section: Role Of Unfrozen Fraction Inmentioning

confidence: 99%

“…The difference may be due to the fact that the posthypertonic hemolysis experiments involved resuspension in isotonic saline, whereas thawing of cells that had been frozen in sucrose/sodium chloride caused them to be resuspended in sucrose/sodium chloride. As Woolgar (1972, 1974 has shown, if sucrose is present during the posthypertonic phase, hemolysis is reduced, presumably because even in the leaking cell the reflection coefficient for sucrose is higher than that for sodium chloride.…”

Section: Role Of Unfrozen Fraction Inmentioning

confidence: 99%

See 1 more Smart Citation

On the mechanism of injury to slowly frozen erythrocytes

Pegg¹,

Diaper²

1988

Biophysical Journal

125

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When cells are frozen slowly in aqueous suspensions, the solutes in the suspending solution concentrate as the amount of ice increases; the cells undergo osmotic dehydration and are sequestered in ever-narrowing liquid-filled channels. Cryoprotective solutes, such as glycerol, reduce the amount of ice that forms at any specified subzero temperature, thereby controlling the buildup in concentration of those other solutes present, as well as increasing the volume of the channels that remain to accommodate the cells. It has generally been thought that freezing injury is mediated by the increase in electrolyte concentration in the milieu surrounding the cells, rather than reduction of temperature or any direct action of ice. In this study we have frozen human erythrocytes in isotonic solutions of sodium chloride and glycerol and have demonstrated a correlation between the extent of damage at specific subzero temperatures, and that caused by the action at 0 degrees C of solutions having the same composition as those produced by freezing. The cell lysis observed increased directly with glycerol concentration, both in the freezing experiments and when the cells were exposed to corresponding solutions at 0 degrees C, showing that the concentration of sodium chloride alone is not sufficient to account quantitatively for the damage observed. We then studied the effect of freezing in anisotonic solutions to break the fixed relationship between solute concentration and the volume of the unfrozen fraction, as described by Mazur, P., W. F. Rall, and N. Rigopoulos (1981. Biophys. J. 653-675). We confirmed their experimental findings, but we explain them differently. We ascribe the apparently dominant effect of the unfrozen fraction to the fact that the cells were frozen in, and returned to, anisotonic solutions in which their volume was either less than, or greater than, their physiological volume. When similar cell suspensions were subjected to a similar cycle of increase and then decrease in solution strength, but in the absence of ice (at 20 degrees C), a similar pattern of hemolysis was observed. We conclude that freezing injury to human erythrocytes is due solely to changes that occur in the composition of their surrounding milieu, and is most probably mediated by a temporary leak in the plasma membrane that occurs during the thawing (reexpansion) phase.

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“…In other instances, RBCs with rare antigens are identified and cryopreserved as reference cells for research, screening, and future diagnostic purposes. Current RBC storage techniques involve the use of glycerol 1,2 or sucrose 3‐5 as antifreeze agents to maintain cell membrane integrity in the frozen state and provide membrane stability upon thawing 2,5‐7 . Without a doubt, cryopreservation of RBCs offers a great way of providing a means for long‐term preservation; however, the cryopreservation technique has disadvantages.…”

mentioning

confidence: 99%