Original Paper Vox Sang 1992;62: 141-145
I. C. Rittmeyer U. E. Nydegger Central Laboratory of Haematology, University of Berne, and Regional Red Cross Blood Transfusion Center, Berne. Switzerland
Influence of the Cryoprotective Agents Glycerol and Hydroxyethyl Starch on Red Blood Cell ATP and 2,3-Diphosphoglyceric Acid Levels ................................................................................................. Abstract
Because hydroxyethyl starch (HAES) is used for volume replacement therapy and as a cryoprotectant for frozen red blood cells (RBCs), this compound, in contrast to glycerol, does not require labor-intensive removal from thawed cells prior to transfusion. We here report the effect of both glycerol and HAES on the RBC organic phosphates ATP and 2,3-diphosphoglyceric acid (2,3DPG). The CPD-A,-stabilized RBCs of 20 healthy donors (3 females, 17males) were separately frozen in either 40% glycerol or 6% HAES, of molecular weight 200,000. ATP and 2,3-DPG concentrations were determined in CPD-A RBCs before addition of cryoprotectant and in cryopreserved thawed RBCs after 24 h storage at -80°C (glycerol) and -196°C (HAES). It appears that HAES, but not glycerol, significantly reduces ATP concentrations whereas both lead to a reduction of 2,3-DPG concentrations; this reduction was more pronounced with glycerol than with HAES. Experiments with the blood of 6 donors demonstrated that HAES affects autohemolysis by l6%, in contrast to glycerol, after which cryoprotectant autohemolysis was affected by 3.1% only. RBC recoveries were comparable using glycerol or HAES as cryoprotectants. A distinct pattern of reduction of 2,3-DPG levels by glycerol and less by HAES, and of ATP levels by HAES but not by glycerol, emerges. Our findings may be of importance if HAES is to be introduced as a convenient cryoprotectant .
.....................
Introduction
With transfusion of autologous blood components becoming standard practice and with improvements in freezer technology for storage of blood, a broader interest in cryopreservation of red blood cell (RBC) concentrates both for elective surgery and disaster medicine can be noted [ 1-41,There are two kinds of cryoprotective agents,
Received: July 23. 1WI Revised manubcript received: Octnher ?. I V J I Acceptcd: October 2. 1MI
those reducing intracellular crystal formation [e.g. glycerol, dimethylsulfoxide (DMSO)] and those acting on the extracellular environment [e. g. hydroxyethyl starch (HAES), polyvinylpyrrolidone and bovine serum albumin [5,6]. The routine prodecure for cryoconservation of RBCs involves glycerol with freezing at -80°C [6-81, but glycerol needs to be washed off upon thawing in order to reestablish normal osmolarity of the red cells. DMSO is
U.E. Nydegger. MD Central Laboratory of Haematology lnselspital CH-3010Berne (Switzerland)
CJ 19Y2 S. Karger AG. Bawl Ixwz-wY17/9?/1)62.1-(I1JI $2.75/(1
not used for freezing RBCs because of its toxicity [6]. More recently, HAES is being studied as a n alternative cryoprotectant because it is a well-tolerated volume substitute used in intensive care [9]. The levels of ATP and 2,3-diphosphoglyceric acid (2,3DPG) are a widely used criterion for the estimation of the quality of stored RBCs under blood banking conditions. Whereas some authors claim a significant correlation between postinfusion viability of RBCs and ATP levels [lo121, others found that RBC ATP maintenance does not by its own insure satisfactory survivability [13, 141; 2,3-DPG is an allosteric effector of hemoglobin that governs release and load of oxygen by hemoglobin [15]. While low 2 3 DPG and ATP levels in transfused RBCs are held to become regenerated [lo, 16-18] in part 3-8 h after transfusion, low levels of 2,3-DPG in transfused RBCs are hazardous in massive transfusions such as exchange transfusions of newborns or patients in need for massive transfusions [ 19-22]. The effects of glycerol and HAES on RBC ATP and 2,3-DPG have been studied previously [16,23]. On the basis of these studies, it was concluded that freezing of RBCs in the presence of HAES leads to an accelerated reduction of ATP levels. In addition to quantitating organic phosphates, the autohemolysis phenomenon is an alternative procedure to assess functional performance of the red-cell membrane and is used for diagnosis of a number of hemolytic anemias; accelerated autohemolysis may be a consequence of impaired phosphate transfer [24]. In view of the importance of organic phosphates and autohemolysis for estimation of red-cell quality, we have used their quantitation for the study of the efficacy and safety of glycerol and HAES for cryoprotection.
tocrit, ATP and 2,3-DPG. Thereafter. the tubes were centrifuged at 1,900g (3,500 rpm) for 20 min and the supernatants were discarded. Five milliliters of sedimented cells (hematocrit -0.9) were then divided into two portions. to one of which 1.0 nil glycerol was added in small portions during 3 min under continous agitation: the formula of glycerol was650 gllglycerol in0.006 MKC1.O.OO1 Mdinatriuni hydrogen phosphate and 0.16 M natrium acetate, pH 6.8. After an equilibration phase of 3 min, 5.0 ml of glycerol were added: the final concentration of glycerol was 40%. and the tubes (NUNC Cryotubes 60 x 12.5 mm, Roskilde. Denmark) were stored frozen at -80°C in an open Styropor support. The other tube was added 0.44nil 40% HAES. molecular weight 200.000, final concentration 6% (obtained as dry powder from Fresenius, Stans. Switzerland: diluted to 40% with0.9% NaCI), for subsequent storage in liquid nitrogen at -196°C. After 24 h, both samples were thawed in a water bath set at 37.0"C to thaw glycerolized RBCs and at 43.5"C to thaw HAES-protected RBCs. The glycerol-stored samples were added 2.5 ml NaCl 12% in distilled H 2 0 followed by 5 min equilibration, in turn followed by fractional addition of 10 ml1.6'70 NaCl stretched over 10 min. For the present down-scale of the automated washing procedure normally used with full-size RBC concentrates, the subsequent washing process for removal of glycerol was simulated by 3 washings with 1.6% NaCl by centrifugation. The final preparation was kept in 0.15 M NaCl and 0.2% glucose. To the tubes containing HAES-protected RBCs. 1 nil of 0.15 M NaCl was added to prepare the final solutions which were tested for hemoglobin concentration using a quantitative, colorimetric determination method provided by Sigma (Buchs, Switzerland) and for hematocrit. The 2.3-DPG and ATP levels were determined by kits also obtained from Sigma. using enzymatic determination methods within 2 h upon thawing. After all these tests were completed. the tubes were centrifuged again and hemolysis was measured by hemoglobin determinations in the supernatants. It should be mentioned that loss of hemoglobin was monitored during all washing steps by hemoglobinometry in the supernatants. Autohemolysis was tested according to Brown [27] and Bucher [28]: in brief. red cells were incubated for 48 h at 37.0"C and hemolysis was evaluated and expressed as percent of total hemoglobin content.
-
Results Donors and Methods Do1ror.r Twenty-four healthy blood donors (21 men. 3 women) between the ages of 23 and 64 years served for this study - 12 were blood group 0, I1 were of the A type and 1of the AB type. Fourteen ofthem were RhD-positive and the other 10 Rh-negative. The range of hemoglobin concentration was between 12.1and 17.4 g/dl as determined by Haemocue". a method described by Vanzetti [2S]. The mean ATP concentration was 4.4 k 0.42 pmollg Hb (X 1SD) and the mean 2.3DPG concentration was 13.9 k 1.6 pmol/g Hb. For cryopreservation with glycerol [6] or HAES [26]. published procedures were followed but down-scaled for the purpose of our study. Thirty milliliters of blood were drawn into plastic tubes on 4.7 nil sterile CPD-A I obtained form Biotrans (Dreieich. FRG). with the formula identical to the one used in blood bags. The tubes were left for 1 h at room temperature during which time samples were taken for the measurements of baseline hemoglobin concentrations. hema-
*
I32
RittmeyedNydegger
lnjluence of Glycerol on Recovery, Autohernolysis atid R B C Organic Phosphates In a first experiment, RBCs of 20 donations were frozen at -80°C under protection with glycerol. After thawing, they were assessed for RBC recoveries, and for residual ATP and 2,3-DPG concentrations (fig. 1,2; table la). The mean recovery was close to 90% and mean ATP concentrations after freezing were not statistically different from the values before freezing. In contrast, glycerol cryoprotection led to a significant drop of mean 2,3-DPG concentrations by 22%. In order to study a direct effect of glycerol on the osmotic fragility of RBCs, the degree of autohemolysis was compared between RBCs that were frozen in glycerol for
2.3-DPG and ATP in Red Cells after Cryoprotection
17 -r
1 4
After cryoconservation with GLYCEROL
I Before freezing
3 1
)I After cryoconservation with HAES
Fig.1. ATP concentrations per gram hemoglobin RBCs from 20 donations before and after freezing with glycerol (-80°C) or HAES (-196°C). Single data points connected by lines belong to the same donation. Note loss of ATPconccntrations aftercryoprotection with HAES but not with glycerol. Same data as used for table 1.
Table 1. Effect of glycerol. HAES and freezing temperatures on recovery. organic phosphates and autohemoly5is of RBCs
1-1
After cryoconservation with GLYCEROL
Before freezing
After cryoconservation with HAES
Fig.2. 2,3-DPG concentrations per gram hemoglobin RBCs from 20 donations before and after freezing with glycerol (-80°C) or HAES (-196°C). Single data points connected by lines belong to the same donation. Note more distinct loss of 2,3-DPG concentrations after cryoprotection with glycerol than with HAES: see also table 1.
Before freezing
After freezing +glycerol at -80°C
After freezing +HAES at - 196°C
X
X
X
SD
SD
significance"
SD
significance.'
n) Integrity of 20 donations as evaluated by measurement of hemolysis and organic phosphates
Hb. gldl Recovery. YO ATP, pmol/g Hb 2.3-DPG. Iimol/g Hb
15.8
f 1.1
4.4
f 0.4 f 1.6
13.Y
89.5 4.7 10.3
f3.2 fo.6 +2.7 ~~
NS, p>n.oi p>O.001
87.6 1.6 11.7
t2.4 t0.4 k 1.7
~
p
I. C. Rittmeyer U. E. Nydegger Central Laboratory of Haematology, University of Berne, and Regional Red Cross Blood Transfusion Center, Berne. Switzerland
Influence of the Cryoprotective Agents Glycerol and Hydroxyethyl Starch on Red Blood Cell ATP and 2,3-Diphosphoglyceric Acid Levels ................................................................................................. Abstract
Because hydroxyethyl starch (HAES) is used for volume replacement therapy and as a cryoprotectant for frozen red blood cells (RBCs), this compound, in contrast to glycerol, does not require labor-intensive removal from thawed cells prior to transfusion. We here report the effect of both glycerol and HAES on the RBC organic phosphates ATP and 2,3-diphosphoglyceric acid (2,3DPG). The CPD-A,-stabilized RBCs of 20 healthy donors (3 females, 17males) were separately frozen in either 40% glycerol or 6% HAES, of molecular weight 200,000. ATP and 2,3-DPG concentrations were determined in CPD-A RBCs before addition of cryoprotectant and in cryopreserved thawed RBCs after 24 h storage at -80°C (glycerol) and -196°C (HAES). It appears that HAES, but not glycerol, significantly reduces ATP concentrations whereas both lead to a reduction of 2,3-DPG concentrations; this reduction was more pronounced with glycerol than with HAES. Experiments with the blood of 6 donors demonstrated that HAES affects autohemolysis by l6%, in contrast to glycerol, after which cryoprotectant autohemolysis was affected by 3.1% only. RBC recoveries were comparable using glycerol or HAES as cryoprotectants. A distinct pattern of reduction of 2,3-DPG levels by glycerol and less by HAES, and of ATP levels by HAES but not by glycerol, emerges. Our findings may be of importance if HAES is to be introduced as a convenient cryoprotectant .
.....................
Introduction
With transfusion of autologous blood components becoming standard practice and with improvements in freezer technology for storage of blood, a broader interest in cryopreservation of red blood cell (RBC) concentrates both for elective surgery and disaster medicine can be noted [ 1-41,There are two kinds of cryoprotective agents,
Received: July 23. 1WI Revised manubcript received: Octnher ?. I V J I Acceptcd: October 2. 1MI
those reducing intracellular crystal formation [e.g. glycerol, dimethylsulfoxide (DMSO)] and those acting on the extracellular environment [e. g. hydroxyethyl starch (HAES), polyvinylpyrrolidone and bovine serum albumin [5,6]. The routine prodecure for cryoconservation of RBCs involves glycerol with freezing at -80°C [6-81, but glycerol needs to be washed off upon thawing in order to reestablish normal osmolarity of the red cells. DMSO is
U.E. Nydegger. MD Central Laboratory of Haematology lnselspital CH-3010Berne (Switzerland)
CJ 19Y2 S. Karger AG. Bawl Ixwz-wY17/9?/1)62.1-(I1JI $2.75/(1
not used for freezing RBCs because of its toxicity [6]. More recently, HAES is being studied as a n alternative cryoprotectant because it is a well-tolerated volume substitute used in intensive care [9]. The levels of ATP and 2,3-diphosphoglyceric acid (2,3DPG) are a widely used criterion for the estimation of the quality of stored RBCs under blood banking conditions. Whereas some authors claim a significant correlation between postinfusion viability of RBCs and ATP levels [lo121, others found that RBC ATP maintenance does not by its own insure satisfactory survivability [13, 141; 2,3-DPG is an allosteric effector of hemoglobin that governs release and load of oxygen by hemoglobin [15]. While low 2 3 DPG and ATP levels in transfused RBCs are held to become regenerated [lo, 16-18] in part 3-8 h after transfusion, low levels of 2,3-DPG in transfused RBCs are hazardous in massive transfusions such as exchange transfusions of newborns or patients in need for massive transfusions [ 19-22]. The effects of glycerol and HAES on RBC ATP and 2,3-DPG have been studied previously [16,23]. On the basis of these studies, it was concluded that freezing of RBCs in the presence of HAES leads to an accelerated reduction of ATP levels. In addition to quantitating organic phosphates, the autohemolysis phenomenon is an alternative procedure to assess functional performance of the red-cell membrane and is used for diagnosis of a number of hemolytic anemias; accelerated autohemolysis may be a consequence of impaired phosphate transfer [24]. In view of the importance of organic phosphates and autohemolysis for estimation of red-cell quality, we have used their quantitation for the study of the efficacy and safety of glycerol and HAES for cryoprotection.
tocrit, ATP and 2,3-DPG. Thereafter. the tubes were centrifuged at 1,900g (3,500 rpm) for 20 min and the supernatants were discarded. Five milliliters of sedimented cells (hematocrit -0.9) were then divided into two portions. to one of which 1.0 nil glycerol was added in small portions during 3 min under continous agitation: the formula of glycerol was650 gllglycerol in0.006 MKC1.O.OO1 Mdinatriuni hydrogen phosphate and 0.16 M natrium acetate, pH 6.8. After an equilibration phase of 3 min, 5.0 ml of glycerol were added: the final concentration of glycerol was 40%. and the tubes (NUNC Cryotubes 60 x 12.5 mm, Roskilde. Denmark) were stored frozen at -80°C in an open Styropor support. The other tube was added 0.44nil 40% HAES. molecular weight 200.000, final concentration 6% (obtained as dry powder from Fresenius, Stans. Switzerland: diluted to 40% with0.9% NaCI), for subsequent storage in liquid nitrogen at -196°C. After 24 h, both samples were thawed in a water bath set at 37.0"C to thaw glycerolized RBCs and at 43.5"C to thaw HAES-protected RBCs. The glycerol-stored samples were added 2.5 ml NaCl 12% in distilled H 2 0 followed by 5 min equilibration, in turn followed by fractional addition of 10 ml1.6'70 NaCl stretched over 10 min. For the present down-scale of the automated washing procedure normally used with full-size RBC concentrates, the subsequent washing process for removal of glycerol was simulated by 3 washings with 1.6% NaCl by centrifugation. The final preparation was kept in 0.15 M NaCl and 0.2% glucose. To the tubes containing HAES-protected RBCs. 1 nil of 0.15 M NaCl was added to prepare the final solutions which were tested for hemoglobin concentration using a quantitative, colorimetric determination method provided by Sigma (Buchs, Switzerland) and for hematocrit. The 2.3-DPG and ATP levels were determined by kits also obtained from Sigma. using enzymatic determination methods within 2 h upon thawing. After all these tests were completed. the tubes were centrifuged again and hemolysis was measured by hemoglobin determinations in the supernatants. It should be mentioned that loss of hemoglobin was monitored during all washing steps by hemoglobinometry in the supernatants. Autohemolysis was tested according to Brown [27] and Bucher [28]: in brief. red cells were incubated for 48 h at 37.0"C and hemolysis was evaluated and expressed as percent of total hemoglobin content.
-
Results Donors and Methods Do1ror.r Twenty-four healthy blood donors (21 men. 3 women) between the ages of 23 and 64 years served for this study - 12 were blood group 0, I1 were of the A type and 1of the AB type. Fourteen ofthem were RhD-positive and the other 10 Rh-negative. The range of hemoglobin concentration was between 12.1and 17.4 g/dl as determined by Haemocue". a method described by Vanzetti [2S]. The mean ATP concentration was 4.4 k 0.42 pmollg Hb (X 1SD) and the mean 2.3DPG concentration was 13.9 k 1.6 pmol/g Hb. For cryopreservation with glycerol [6] or HAES [26]. published procedures were followed but down-scaled for the purpose of our study. Thirty milliliters of blood were drawn into plastic tubes on 4.7 nil sterile CPD-A I obtained form Biotrans (Dreieich. FRG). with the formula identical to the one used in blood bags. The tubes were left for 1 h at room temperature during which time samples were taken for the measurements of baseline hemoglobin concentrations. hema-
*
I32
RittmeyedNydegger
lnjluence of Glycerol on Recovery, Autohernolysis atid R B C Organic Phosphates In a first experiment, RBCs of 20 donations were frozen at -80°C under protection with glycerol. After thawing, they were assessed for RBC recoveries, and for residual ATP and 2,3-DPG concentrations (fig. 1,2; table la). The mean recovery was close to 90% and mean ATP concentrations after freezing were not statistically different from the values before freezing. In contrast, glycerol cryoprotection led to a significant drop of mean 2,3-DPG concentrations by 22%. In order to study a direct effect of glycerol on the osmotic fragility of RBCs, the degree of autohemolysis was compared between RBCs that were frozen in glycerol for
2.3-DPG and ATP in Red Cells after Cryoprotection
17 -r
1 4
After cryoconservation with GLYCEROL
I Before freezing
3 1
)I After cryoconservation with HAES
Fig.1. ATP concentrations per gram hemoglobin RBCs from 20 donations before and after freezing with glycerol (-80°C) or HAES (-196°C). Single data points connected by lines belong to the same donation. Note loss of ATPconccntrations aftercryoprotection with HAES but not with glycerol. Same data as used for table 1.
Table 1. Effect of glycerol. HAES and freezing temperatures on recovery. organic phosphates and autohemoly5is of RBCs
1-1
After cryoconservation with GLYCEROL
Before freezing
After cryoconservation with HAES
Fig.2. 2,3-DPG concentrations per gram hemoglobin RBCs from 20 donations before and after freezing with glycerol (-80°C) or HAES (-196°C). Single data points connected by lines belong to the same donation. Note more distinct loss of 2,3-DPG concentrations after cryoprotection with glycerol than with HAES: see also table 1.
Before freezing
After freezing +glycerol at -80°C
After freezing +HAES at - 196°C
X
X
X
SD
SD
significance"
SD
significance.'
n) Integrity of 20 donations as evaluated by measurement of hemolysis and organic phosphates
Hb. gldl Recovery. YO ATP, pmol/g Hb 2.3-DPG. Iimol/g Hb
15.8
f 1.1
4.4
f 0.4 f 1.6
13.Y
89.5 4.7 10.3
f3.2 fo.6 +2.7 ~~
NS, p>n.oi p>O.001
87.6 1.6 11.7
t2.4 t0.4 k 1.7
~
p
