Relative Efficiency and Interchangeability of Huggins and American Red Cr.oss Red Cell Freezing Procedures M. HORNBLOWER A N D H.T. MERYMAN From the Americon Noiionol Red Cross Blood Reseorrh Laboroiory. Beihesdo. Morylond
Cells glycerolized in a low salt medium (Huggins) and deglycerolized by agglomeration were compared to cells glycerolized in a high salt medium (ARC) and deglycerolized by a centrifugal procedure. Both agglomeration and centrifugal deglycerolization produce entirely acceptable products. The average total recovery by agglomeration is somewhat less (86 per cent) than by the centrifugal method (93 per cent). The osmolalities and hemoglobin concentrations are comparable. Cells glycerolized in a high salt preparation are very stable while unfrozen and are relatively unaffected by failures of refrigeration and can be refrozen. Cells glycerolized in low salt media may increase in volume with time and are variable in their stability to unfrozen storage and to refreezing. Sickle trait cells do not agglomerate satisfactorily and cannot be deglycerolized by agglomeration but can be deglycerolized by a modification of the centrifugal method. Container surfaces can adversely influence the freeze-thaw hemolysis of high salt cells but appear to have no effect on the low salt preparation. Although cells glycerolized in high salt medium cannot be deglycerolized by agglomeration, the converse is possible. The standard 8.6M low salt glycerolizing solution did not permit optimal recovery by centrifugal wash. When cells were glycerolized in a Huggins low-salt formula modified to contain 6.2M glycerol in 400 ml of solution, the frozenthawed cells could be deglycerolized by either agglomeration or centrifugation with good recovery.
SMITH'^ REPORTED that a concentration of approximately 4M glycerol could protect human red blood cells against freezing injury. Subsequent experience showed that, although frozen, deglycerolized red blood cells were clinically effective, the problems of removing the high concentration of glycerol were a major obstacle to the practical application of frozen red blood cells to clinical use. I During t h e subsequent two decades, several d eg l y cerol izing procedures were
developed. T ~ l l i sused ' ~ the Cohn fractionator for both the glycerolizing and deglycerolizing of red blood cells. Although this device enabled him and his co-workers to process and transfuse several thousand units of frozen red blood cells,2 the procedure was too complex and expensive for other than experimental use. Pert et al. l o reported promising results with a method using approximately half the glycerol concentration, thereby simplifying deglycerolization, but requiring freezing and storage in liquid nitrogen. The method was subsequently developed further by Rowel' and Krijnen.' The first deglycerolizing procedure suitable for routine clinical use was th a t described by Huggin9 based on the observation that, at low pH and ionic strength, red blood cells agglomerate into large clumps and settle to the bottom of a container without centrifugation. This ingenious deglycerolization procedure, subsequently developed and licensed for clinical use, enabled many hospitals around the world to gain experience with frozen red blood cells. During the past five years, advances in the Cohn methodology, particularly the development of the Haemonetics disposable washing bowl* have led to substantial simplification and economy in the centrifugal washing method. Even more recently, an automated sequential batch washing device, the IBM Model 2991 cell washer,t has also proven effective and economical for deglycerolizing frozen red blood cells. As a result of the availability of these deglycerolizing
Received for publication June 1 I , 1976; accepted August I I , 1976. Contribution No. 353 from the American National Red Cross Blood Research Laboratory.
*Haemonetics Corporation, Natick, Massachusetts tIBM Corporation, Princeton, New Jersey
417 Transfusion scp1:oct. 1977
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418
HORNBLOWER A N D MERYMAN
procedures, the use of frozen red blood cells has expanded greatly and will undoubtedly continue to do so as their special virtues become more widely appreciated. Valeri and a ~ s o c i a t e s ' ~have . ' ~ reported studies comparing the Huggins agglomeration procedure w i t h t he centriffgal procedures. Three criticisms have been levelled against t he agglomeration procedure. First, it has been reported that roughly 25 per cent of the cell potassium is lost from the cells during the exposure to the low ionic medium.Ifi The cells regain their normal cation content within hours after transfusion. T h e second, more serious criticism of the agglomeration procedure has centered about cell recovery and t h e su pe r n at a n t hemoglobin content of t h e deglycerolized cell suspension. Valeri and Bond" have reported a mean loss of 25 f 6 per cent of the cells during deglycerolization with losses as high as 50 per cent in some units and as much as 5 grams of free hemoglobin in the cell suspension. Third, these same authorsI4 have reported that cells frozen by the Huggins method cannot be deglycerolized by agglomeration after more than two years of - 85 C storage without evidence of. poor post-thaw stability although deglycerolizing the same preparation by centrifugal methods using a saline wash resulted in fairly good wash stability. The reports of poor cell recovery and poor storage stability with the agglomeration method have been contested by hug gin^.^.^ We are unaware of any other comparative studies that might resolve this conflict. In accordance with a continuing policy of the Red Cross Blood Research Laboratory to evaluate and report the costs and relative effectiveness of the various methods and equipment used in red blood cell freezing, a study was undertaken to evaluate the Huggins agglomeration procedure in comparison to the procedure currently used by the Red The study had three general purposes. l ) To compare the Huggins agglomeration procedure and the Red Cross
Transfusion Sept.-Oct. 1977
procedure with respect to overall red blood cell recovery, the osmolality of the deglycerolized cell suspension, supernatant hemoglobin concentration, the cell stability when glycerolized but unfrozen, cost, technician time and general reliability. We wished, if possible, to reconcile the conflicting reports regarding cell recovery and to determine what are the assets and defects of the agglomeration method. 2) To confirm that cells glycerolized by the Huggins procedure could be deglycerolized by a centrifugal saline wash14 and to compare the recoveries with those of similar cells deglycerolized by agglomeration. We also hoped to determine what modifications of the Huggins glycerolizing procedure might be made which would give optimum recovery by either agglomeration or saline wash. The possibility was considered that a formulation permitting deglycerolization by either agglomeration or saline wash would serve to provide a greater flexibility in the choice of processing methods. 3) To determine what modifications of the Huggins agglomeration procedure might improve or simplify it. Based on our past experience3 we expected to find that the large area of polyvinylchloride surface to which the cells are exposed during freezing causes substantial post-thaw hemolysis which modifications in bag geometry and material might prevent. Our experience also suggested that the addition of glycerol directly to the packed cells with only a stirring bar to facilitate mixing could result in elevated post-thaw hemolysis.* We hoped to find it unnecessary, as in the Red Cross method, to prorate the quantity of glycerol solution depending on the weight of the packed cells.
Materials and Methods All blood was collected in full unit quantities in CPD anticoagulant by the standard Red Cross procedure. The plasma was removed from the blood units and the cells glycerolized within seven days of collection. All frozen units were stored at least overnight at -80 C in a mechanical freezer.
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RED CELL FREEZING
Standard Red Cross Procedure Glycerolizing.To one unit of packed cells 400 ml glycerol solution containing 6.2M glycerol, 0.14M sodium lactate, 5mM potassium chloride and 12.5mM sodium phosphate is added. The first 100 ml of glycerol solution is added while the collection bag is being vigorously shaken on a wrist action shaker. The remaining glycerol solution and the partially glycerolized cells are transferred to a 9 x I I inch freezing bag which is enclosed in an aluminum canister and allowed to freeze in a -80 C mechanical freezer. Thawing and Deglycerolizing. T h e frozen canister is put into a 37 C water bath for at least ten minutes to thaw. The freezing bag is then removed from the canister and connected to the Haemonetics washing apparatus. One hundred fifty ml of 12 per cent NaCl are added to the cells which are then pumped into the centrifugal washing bowl concurrently with a 1.6 per cent NaCl solution. Two liters of l .6 per cent NaCl followed by one liter of 0.8 per cent NaCI, 0.2 per cent glucose buffered with 12.5mM sodium phosphate to pH 7 completes the washing process. The cells are siphoned from the washing bowl into the final administration bag. Standard Huggins Procedure Glycerolizing. Packed cells are transferred to the blood freezing unit on the Cytoglomerator. An appropriate amount (depending on unit weight) of Huggins glycerol solution containing: 8.6M glycerol USP (79.2 per cent w/v), dextrose USP 6.0 gm/dl, fructose N F I .O gm/dl, disodium edetate USP 0.3 gm/dl and citric acid USP 0.006 gm/dl is added to the cells with agitation by a magnetic stirrer. The blood freezing unit is removed from the Cytoglomerator, folded, inserted in a cardboard fieezing container, and frozen in a -80 C mechanical freezer. Thawing and Deglycerolizing. The blood freezing unit is removed from the cardboard container and put into a 37 C water bath. After a few moments the unit is unfolded and thawed for seven minutes. The unit is then attached to the Cytoglomerator. An appropriate amount, depending on unit size, of 50 per cent dextrose is added with the magnetic stirrer on, followed immediately by two liters of 5 per cent fructose. The cells are allowed to agglomerate and settle and the supernate fluid is removed. The cells are then resuspended in two liters of 5 per cent fructose and allowed to agglomerate and settle a second time. The supernate fluid is removed and the cells are resuspended in.250 ml of normal saline in the final administration bag. This bag is then centrifuged and the supernate fluid is removed.
Modified Huggins" Solution The modified solution contained 400 ml of 6.2M glycerol USP (57.1% v/v), dextrose USP 6.0 gm/dl, fructose N F 1 .O gm/dl, disodium edetate USP 0.3 gm/dl and citric acid USP 0.006 gm/dl. This modified glycerol solution was used with the standard Red Cross glycerolizing and freezing procedure. Assays Hemoglobin concentrations were assayed using the I.L. Hemoglobinometer Model 231.1 Sodium and potassium concentrations were measured with the 1. L. Flame Photometer Model 143t after a preliminary packing of the cells in the Beckman Microfuge Model 152s and dilution in the Fisher Diluter Model 240.11 Recoveries were calculated by rinsing with saline the residual cells and hemoglobin from all of the tubing and containers used in deglycerolizing. This rinse was added to the waste from the deglycerolization procedure and the total hemoglobin content measured. The total hemoglobin of the washed cell suspension was also measured. The total process recovery was then determined by dividing the washed cell hemoglobin by the sum of washed cell plus waste hemoglobin. In addition, an aliquot of the waste was sedimented and the proportion of waste hemoglobin still present in intact cells was determined. This enabled a comparison of losses associated with hemolysis with losses resulting from cell washover and cells left in the apparatus.
Results In Table I data are presented showing days stored at -80 C, the supernatant hemoglobin content of the cell suspension before and after freezing and after deglycerolization, the final osmolality of the washed cell suspension, the proportion of waste hemoglobin that is in intact cells and the total process recovery. The mean recovery of cells is 93.4 per cent * 1.3 (S. D.) processed by the standard Red Cross method as compared to 86 per cent 3.4 (S. D.) for the standard Huggins method. T h e final supernatant hemoglobin concentrations and osmolalities are comparable for the two methods. The data for 12 units processed by the standard Red Cross method, using the modified Huggins glycerol solution, are essentially equivalent to that obtained with the Red Cross glycerol formula.
*
tlnstrumentation Lab., Inc. Watertown, Mass. §Beckman Instruments, Inc., Fullenton, Calif. IlFisher Scientific Co., Pittsburgh, Pa.
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HORNBLOWER A N D M E R Y M A N Table 1 .
Glycerolizing solution Deglycerolizingmethod
Sep1:Oct. 1971
Comparisonsbetween Standard and Modified Procedures
ARC ARC
Standard Huggins Agglom.
Modified Huggins ARC
Standard Huggins ARC
Modified Huggins Aaalom.
~ _ _ _ _ _
13 185 f 3
Number of units Days at - 80C Supt. Hgb. pre-freeze. mg % Supt. Hgb. post-thaw. mg % Deglycerolizedcells. Hct. Deglycerolizedcells. mOsm Deglycerolized cells. supt. hgb.. mg % %of waste hgb. in intact cells % recovery
11
12
10
8 f 5.3 88 f 49
4f3 10
2 1613 60120
1 54
95.6 f 51
263 f 107
102 & 72
127/98
328
53 f 3
81 k 9
52 f 6
49/49
70
356+ 16
349 f 12
366 f 32
3081326
353
160 f 90
92 f 44
148 f 109
1251109
128
5*2
50+ 1 1
5*3
212
46
93.4f 1.3
86&34
91 + 4
90185
91
1
NOTE: Data is presented as mean and standard deviation with the exception of column 4 in which the results of two units are both shown and column five where only one unit was studied.
-
E
.n C
120
c
0
E @ 80 I loot * c Q Q
CI
s
Q 0
CI
I-
0
20
40
60
80
Vol. Cell Suspension Iml) FIG. I. The total postthaw free hemoglobin present when portions of the same unit are frozen in differing volumes in identical plastic bags. Extrapolating the data to zero volume indicates the amount of lysis that must be attributed to interactions with the container.
Also in Table I data are presented for two units glycerolized and deglycerolized by the standard Red Cross procedure using the standard Huggins glycerol formula. The results were satisfactory but too few units were processed to enable a significant comparison with t h e two s t a n d a r d procedures. The modified Huggins glycerol solution avoids the need for prorating glycerol solutions of varying quantities, involves less introduction trauma to the cells and this is considerably more convenient than the standard Huggins solution. Data a r e also presented for one unit processed by the standard Huggins procedure using the modified Huggins glycerol formula, in an effort to simplify and improve the standard Huggins procedure. The recovery of cells is improved. Similar results have been reported by Huggins (per son al com m un ication ). Red blood cells glycerolized with the Red Cross glycerol lactate solution have shown an increase in freezing hemolysis with an increase in surface area of the freezing containerI5 particularly with polyvinylchloride (PVC) containers. Experiments were conducted to determine the effect on freezing hemolysis of the very large PVC freezing bag used in the Huggins procedure. One unit of blood was divided into two equal parts. One portion was glycerolized with half the usual amount of Huggins glycerol-glucose and the other portion with half the usual amount of Red Cross glycerollactate solution. The glycerolized cells were then divided into 20, 40, 60, and 80 ml aliquots in 300 ml PVC bags and frozen at -80 C. Figure I shows t h e mean total hemoglobin in t h e thawed supernatants for three different experiments. The projected intercept with the ordinate for the Hug-
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42 1
R E D CELL FREEZING
gins blood indicates that there is very little, if any, PVC surface effect while the hemolysis of the Red Cross blood confirms previous observations. Three units of sickle trait cells were glycerolized, frozen and deglycerolized by the standard Huggins procedure. Unit # I had not agglomerated two hours after the addition of the 5 per cent fructose. Unit #2 agglomerated slowly, the cells had settled to about six inches from the bottom of the bag 25 minutes after the fructose addition. Unit #3 agglomerated even more slowly. The cells had settled to about eight inches from the bottom of the bag IN hours after the fructose addition. One of the particular virtues of cells frozen in a high concentration of glycerol with isotonic electrolyte present is the great stability to fluctuations in temperature that these cells exhibit. Prolonged exposure for days a t refrigerator o r room temperature of cells glycerolized by the Red Cross procedure results in little or no evidence of serious changes. A 72-hour exposure to 22 C, for example, results in a reduction in mean cell potassium content of roughly 5 per cent and a negligible increase in free hemoglobin or of subsequent freeze-thaw lysis. Furthermore, cells glycerolized in this manner can be repeatedly frozen and thawed with no significant increase in per cent loss on subsequent freezes.* However, in a low ionic content, the red blood cell becomes relatively permeable to cations. We therefore investigated the response of cells glycerolized by the Huggins procedure to repeated freezing and thawing and to prolonged pre-freeze storage while glycerolized. A full unit of packed cells was divided into two equal parts, one of which was glycerolized with 200 ml of Huggins 6.2M glycerol glucose-fructose solution and the other with 200 ml of standard Red Cross 6.2M glycerol Na lactate solution. Each half was frozen and thawed, per cent hemolysis and intracellular K+ was measured, and each half was refrozen after approximately two hours. This procedure was repeated for a total of three freezings. As shown in Table 2 the Huggins and Red Cross glycerolized cells had the same recovery after the first freeze. The Huggins cells were somewhat low in intracellular K + . After the second freeze and thaw the Huggins cell survival was much worse than that of the Red Cross cells, and after the third freeze and thaw the Huggins cells were totally hemolyzed whereas only 4.7 per cent of the Red Cross cells were h emol yzed . Considering the possibility that the incubation at room temperature while glycerolized might be responsible for the lysis on refreezing we explored the effects of prolonged exposure to the glycerol solution before freezing. Two units of cells were glycerolized, one with the Huggins solution and
Table 2 .
Effects of Refreezing 1st freeze and thaw
2nd freeze and thaw
3rd freeze and thaw
Hugginsglycerol formula % hemolysis lntracellular K+
1.5 53
19
100
46
-
Red Cross glycerol formula % hemolysis lntracellular K+
1.5 75
2.7 71
4.7 75
~~
~~~
NOTE: Glycerolized cell suspensions were frozen at least
overnight, then thawed. incubated at room temperature for 2 hours. then refrozen. Progressive hemolysis and potassium loss were observed when the low ionic glycerolizing medium was used.
one with the Red Cross solution. Each unit was divided into 25 ml aliquots in Fenwal 300 ml transfer packs. Aliquots from each group were held at 4 or 22 C for various lengths of time before freezing. Figure 2 shows that prefreeze incubation of Huggins cells at 22 C results in 35 per cent postthaw hemolysis after 5 hours and 100 per cent after 24 hours. At 4 C, the increase in hemolysis
Duration of Pretreeze Incubation While Glycerolized I hrl
FIG.2. Cells incubated at room temperature following glycerolization with Huggins solution progressively increase in volume without lysis, approaching maximum volume after about 4 hours. A concomitant increase in postthaw lysis is seen if cells are frozen after such incubation.
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Transfusion
HORNBLOWER A N D M E R Y M A N Table 3.
Sept.-Oft. 1977
Estimated Costs ~~
~
~~~~
~
~
Solutions and disposables per unit processed
Capital Equipment
~
Haemonetics Shaker Model 17 Cell Washer Reuseable metal freezing Cannister Water bath
1 unit
360.00 4.285.00 3.95 277.00
Bowl Waste bag Washing set Transfer pack Freezing bag Solutions
$4.925.95
IBM Shaker
#2991 Cell Processor Reuseable Metal Freezing Cannister Water Bath
360.00 1 7,000.00 3.95 277.00
Freezing bag Processing set Transfer pack Solutions
4 units 3.75 .66 1.06 .90 3.50 5.48
$29.79
$1 5.35
3.50 7.50 .90 4.46
__
$16.36
$1 7.640.95 Huggins WS-1 Cytoglomerator Cytolight Cytobath
15.00 .66 4.25 .90 3.50 5.48
1.375.00 175.00 795.00
Processing set Solutions
18.3 1 12.00 $30.31
$2.345.00 NOTE: Costs quoted are based on small volume purchases as of May. 1975
was negligible after 24 hours. Cells in the high ionic Red Cross glycerol solution were unaffected at either temperature. Cell volume was measured periodically during incubation at 4 and 22 C. At 22 C, the cell volume in Huggins solution increased progressively until it approached lytic volume at about 4 hours. Longer incubation at 22 C resulted in no further volume increase and no hemolysis. At 4 C cell volume increased only from 70 to 90 u3 in 24 hours. Table 3 shows the comparable costs of the Haemonetics, IBM and the Huggins methods at the time the study was conducted. The cost of capital equipment for the Huggins method was less than the two Red Cross methods and the apparatus has the advantage of functioning in an emergency without electrical power. The cost per unit was higher than the IBM or the Haemonetics systems but in the latter case only when two or more units a r e washed through the same bowl.
Discussion The recovery of red blood cells processed by the Red Cross procedure averages approximately 7 per cent more than for the standard Huggins agglomeration procedure. Other recovery parameters such as os-
molality and supernatant hemoglobin are roughly equivalent. From the point of view of in vitro recovery, the Huggins agglomeration procedure is entirely statisfactory. Although we have confirmed the loss of cel I potassium following agg lom e r a t ion, there is no evidence that this is a significant shortcoming of the procedure. No evidence has been presented that the reduction in cell potassium has any adverse effect on cell physiology or clinical effectiveness. I t was not possible in this short term study to investigate the reports by ValeriiJ that, after more than two years frozen storage, there is a reduction of in vitro recovery, in vivo survival and postwash stability. TWO arguments can be used to minimize the importance of this criticism. First, in practice, very few units are stored in excess of two years. Of the roughly 20,000 units frozen in the Red Cross system two years or more before the study reported here, only 345 units, or less than two per cent were stored longer than two years. The mean storage
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RED CELL FREEZING
time for all units in the Red Cross system is less than two months. Second, Valeri's report indicates that if Huggins units stored more than 2 years are deglycerolized by saline wash, good recoveries and survival ~ e s u l t .Therefore, '~ should the report of deterioration after two years storage be confirmed, such units could be deglycerolized by the saline wash procedure rather than by agglorn eration. The tendency of cells glycerolized by the Huggins procedure to swell with incubation at room temperature and hernolyze with subsequent freezing is a problem not seen with cells frozen in t h e presence of a normal ionic content. The ability to refreeze cells that have been thawed is useful, particularly following freezer failure or temperature rise during shipping. However, it is unlikely that the federal regulations governing the manufacture of Red Cells, (Human) Frozen and Deglycerolized will include provisions for refreezing or permit the use of frozen cells that have been subjected to an accidental rise in storage temperature. The stability of cells glycerolized at a high ionic content may therefore not be a useable asset in practice. One shortcoming of the agglomeration procedure is the inability to deglycerolize sickle trait cells. Such cells also pack and hemolyze during deglycerolization by the standard Haemonetics or I B M method. However, a modified protocol has been devised for both of these devices which permits t h e deglycerolization of A-S Such cells glycerolized with the Huggins low ionic solution can also be deglycerolized with one of the modified high ionic centrifugal wash procedures. The capital costs for the agglomeration equipment are less than for either of the two centrifugal devices. The cost of solutions and disposables for the Huggins system is roughly twice that of the IBM system and equivalent to t h e Haemonetics system. However, in the Haemonetics system, more than one unit destined for the same recipient can be washed through the same disposables, thus reducing the cost per unit of disposables
by 30 per cent when two units are deglycerolized and by 50 per cent for four. It was the impression of our technicians that the agglomeration procedure was somewhat more demanding. During deglycerolization, the decision when to stop removing supernatant fluid after the cells have settled and the technicians's skill in rnanipulating the washing bag can influence the proportion of cells lost in the waste and, thereby, the in vitro recovery achieved. The modified Huggins solution permits satisfactory recovery of cells should they be deglycerolized by centrifugation rather than by agglomeration. This enhances the flexibility of a frozen red blood cell program and improves the yields in a blood center where both agglomeration and centrifugal deglycerolizing may be used, as well as making possible the exchange of frozen cells between centers using different methods of deglycerolizat ion. Using the Red Cross glycerolizing procedure, in which the first 100 ml of glycerol solution are added to the cells in the collection bag with shaking, increased the recovery of cells by only two to three per cent when the Huggins solution was used. Such mixing is absolutely mandatory for the high salt preparation. The increased postthaw hemolysis observed when cells in high ionic glycerol solutions are frozen in PVC bags was not observed when the cells were glycerolized in the non el ect rol y te solution. Contrary to our expectations, therefore, the very large area of PVC presented by the Huggins freezing is not a source of additional hemolysis. We feel that the modified Huggins glycerolizing solution offers the advantage over the standard Huggins procedure in that a single glycerolizing solution may be used rather than maintaining an inventory of and selecting from among several different concentrations of glycerol. References I.
Chaplin, H., and P. L. Mollison: Preservation of blood. In: Preservation and Transplantation of
424
HORNBLOWER AND MERYMAN
Normal Tissues. London, J . & A. Churchill, 1954,p.l21. 2. Haynes, L. L., W. C. Turville, M. T. Sproul, M. E. Henderson, J. W. Zemp, and J. L. Tullis: Long-term preservation-a reality. J. Mich. Med. SOC.61:1509, 1962. 3. Hornblower, M., and H. T. Meryman: Influence of the container material on the hemolysis of glycerolized red cells after freezing and thawing. Cryobiology 11:317, 1974. 4. Huggins, C. E.: Frozen blood. Eur. Surg. Res. 1:3, 1969.
preservation of blood by freezing. In: Red Cell Freezing, Technical Workshop, American Association of Blood Banks, 1973. Reversible agglomeration used to remove 6. -: dimethylsulfoxide from large volumes of frozen blood. Science 139504. 1963. 7. Krijnen, H. W., J. J . Fr. M. dewit. A. C. J. Kuivenhoven, and G. Reyden: Freezing of red cells with liquid nitrogen. I. Results with glycerol as an intracellular substance. Biblio. Haematol.
5. -:Practical
23:683, 1965. 8.
Meryman, H. T., and M. Hornblower: A method for freezing and washing red blood cells using a high glycerol concentration. Transfusion
9.
-,
12:145,1972.
and M. Hornblower: Freezing and deglycerolizing sickle-trait red blood cells. Transfusion 16:627, 1976.
Transfusion Sept.-Oct. 1977
10. Pert, J. H., P. K. Schork, and R. Moore: A new
method of low-temperature blood preservation using liquid nitrogen and a glycerol-sucrose additive. Clin. Res. 11:197, 1963. 1I .
Rowe, A. W., E. Eyster, and A. Kellner: Liquid nitrogen preservation of r e d blood cells for transfusion; a low glycerol-rapid freeze procedure. Cryobiology 5:119, 1968.
12.
Smith, A. U.: Prevention of haemolysis during freezing and thawing of red blood cells. Lancet 2:910, 1950.
13. Tubs, J. L.: Principles involved in glycerolization
and deglycerolization of red cells using Cohn fractionator. Cambridge, Massachusetts, Protein Foundation. Proceedings of 9th Conference on the Plasma Proteins and Cellular Elements of the Blood., p. 17. Valeri, C. R., and A. H. Runck: Long term frozen storage of human red blood cells: Studies in vivo and in vitro of autologous red blood cells preserved up to six years with high concentrations of glycerol. Transfusion 9 5 , 1969. 15. -, and J. C. Bond: Observations on the preservation of autologous human erythrocytes using glycerol, slow-freeze technic and agglomeration. Transfusion 6:254, 1966. 16. -, A. H. Runck, and C. E. Brodine: Current status of freeze-preservation of human red cells with glycerol. Biblio. Haematol. 38:249, 1971. 14.
Cells glycerolized in a low salt medium (Huggins) and deglycerolized by agglomeration were compared to cells glycerolized in a high salt medium (ARC) and deglycerolized by a centrifugal procedure. Both agglomeration and centrifugal deglycerolization produce entirely acceptable products. The average total recovery by agglomeration is somewhat less (86 per cent) than by the centrifugal method (93 per cent). The osmolalities and hemoglobin concentrations are comparable. Cells glycerolized in a high salt preparation are very stable while unfrozen and are relatively unaffected by failures of refrigeration and can be refrozen. Cells glycerolized in low salt media may increase in volume with time and are variable in their stability to unfrozen storage and to refreezing. Sickle trait cells do not agglomerate satisfactorily and cannot be deglycerolized by agglomeration but can be deglycerolized by a modification of the centrifugal method. Container surfaces can adversely influence the freeze-thaw hemolysis of high salt cells but appear to have no effect on the low salt preparation. Although cells glycerolized in high salt medium cannot be deglycerolized by agglomeration, the converse is possible. The standard 8.6M low salt glycerolizing solution did not permit optimal recovery by centrifugal wash. When cells were glycerolized in a Huggins low-salt formula modified to contain 6.2M glycerol in 400 ml of solution, the frozenthawed cells could be deglycerolized by either agglomeration or centrifugation with good recovery.
SMITH'^ REPORTED that a concentration of approximately 4M glycerol could protect human red blood cells against freezing injury. Subsequent experience showed that, although frozen, deglycerolized red blood cells were clinically effective, the problems of removing the high concentration of glycerol were a major obstacle to the practical application of frozen red blood cells to clinical use. I During t h e subsequent two decades, several d eg l y cerol izing procedures were
developed. T ~ l l i sused ' ~ the Cohn fractionator for both the glycerolizing and deglycerolizing of red blood cells. Although this device enabled him and his co-workers to process and transfuse several thousand units of frozen red blood cells,2 the procedure was too complex and expensive for other than experimental use. Pert et al. l o reported promising results with a method using approximately half the glycerol concentration, thereby simplifying deglycerolization, but requiring freezing and storage in liquid nitrogen. The method was subsequently developed further by Rowel' and Krijnen.' The first deglycerolizing procedure suitable for routine clinical use was th a t described by Huggin9 based on the observation that, at low pH and ionic strength, red blood cells agglomerate into large clumps and settle to the bottom of a container without centrifugation. This ingenious deglycerolization procedure, subsequently developed and licensed for clinical use, enabled many hospitals around the world to gain experience with frozen red blood cells. During the past five years, advances in the Cohn methodology, particularly the development of the Haemonetics disposable washing bowl* have led to substantial simplification and economy in the centrifugal washing method. Even more recently, an automated sequential batch washing device, the IBM Model 2991 cell washer,t has also proven effective and economical for deglycerolizing frozen red blood cells. As a result of the availability of these deglycerolizing
Received for publication June 1 I , 1976; accepted August I I , 1976. Contribution No. 353 from the American National Red Cross Blood Research Laboratory.
*Haemonetics Corporation, Natick, Massachusetts tIBM Corporation, Princeton, New Jersey
417 Transfusion scp1:oct. 1977
Volume 17
Number5
418
HORNBLOWER A N D MERYMAN
procedures, the use of frozen red blood cells has expanded greatly and will undoubtedly continue to do so as their special virtues become more widely appreciated. Valeri and a ~ s o c i a t e s ' ~have . ' ~ reported studies comparing the Huggins agglomeration procedure w i t h t he centriffgal procedures. Three criticisms have been levelled against t he agglomeration procedure. First, it has been reported that roughly 25 per cent of the cell potassium is lost from the cells during the exposure to the low ionic medium.Ifi The cells regain their normal cation content within hours after transfusion. T h e second, more serious criticism of the agglomeration procedure has centered about cell recovery and t h e su pe r n at a n t hemoglobin content of t h e deglycerolized cell suspension. Valeri and Bond" have reported a mean loss of 25 f 6 per cent of the cells during deglycerolization with losses as high as 50 per cent in some units and as much as 5 grams of free hemoglobin in the cell suspension. Third, these same authorsI4 have reported that cells frozen by the Huggins method cannot be deglycerolized by agglomeration after more than two years of - 85 C storage without evidence of. poor post-thaw stability although deglycerolizing the same preparation by centrifugal methods using a saline wash resulted in fairly good wash stability. The reports of poor cell recovery and poor storage stability with the agglomeration method have been contested by hug gin^.^.^ We are unaware of any other comparative studies that might resolve this conflict. In accordance with a continuing policy of the Red Cross Blood Research Laboratory to evaluate and report the costs and relative effectiveness of the various methods and equipment used in red blood cell freezing, a study was undertaken to evaluate the Huggins agglomeration procedure in comparison to the procedure currently used by the Red The study had three general purposes. l ) To compare the Huggins agglomeration procedure and the Red Cross
Transfusion Sept.-Oct. 1977
procedure with respect to overall red blood cell recovery, the osmolality of the deglycerolized cell suspension, supernatant hemoglobin concentration, the cell stability when glycerolized but unfrozen, cost, technician time and general reliability. We wished, if possible, to reconcile the conflicting reports regarding cell recovery and to determine what are the assets and defects of the agglomeration method. 2) To confirm that cells glycerolized by the Huggins procedure could be deglycerolized by a centrifugal saline wash14 and to compare the recoveries with those of similar cells deglycerolized by agglomeration. We also hoped to determine what modifications of the Huggins glycerolizing procedure might be made which would give optimum recovery by either agglomeration or saline wash. The possibility was considered that a formulation permitting deglycerolization by either agglomeration or saline wash would serve to provide a greater flexibility in the choice of processing methods. 3) To determine what modifications of the Huggins agglomeration procedure might improve or simplify it. Based on our past experience3 we expected to find that the large area of polyvinylchloride surface to which the cells are exposed during freezing causes substantial post-thaw hemolysis which modifications in bag geometry and material might prevent. Our experience also suggested that the addition of glycerol directly to the packed cells with only a stirring bar to facilitate mixing could result in elevated post-thaw hemolysis.* We hoped to find it unnecessary, as in the Red Cross method, to prorate the quantity of glycerol solution depending on the weight of the packed cells.
Materials and Methods All blood was collected in full unit quantities in CPD anticoagulant by the standard Red Cross procedure. The plasma was removed from the blood units and the cells glycerolized within seven days of collection. All frozen units were stored at least overnight at -80 C in a mechanical freezer.
Volume 17 Number 5
419
RED CELL FREEZING
Standard Red Cross Procedure Glycerolizing.To one unit of packed cells 400 ml glycerol solution containing 6.2M glycerol, 0.14M sodium lactate, 5mM potassium chloride and 12.5mM sodium phosphate is added. The first 100 ml of glycerol solution is added while the collection bag is being vigorously shaken on a wrist action shaker. The remaining glycerol solution and the partially glycerolized cells are transferred to a 9 x I I inch freezing bag which is enclosed in an aluminum canister and allowed to freeze in a -80 C mechanical freezer. Thawing and Deglycerolizing. T h e frozen canister is put into a 37 C water bath for at least ten minutes to thaw. The freezing bag is then removed from the canister and connected to the Haemonetics washing apparatus. One hundred fifty ml of 12 per cent NaCl are added to the cells which are then pumped into the centrifugal washing bowl concurrently with a 1.6 per cent NaCl solution. Two liters of l .6 per cent NaCl followed by one liter of 0.8 per cent NaCI, 0.2 per cent glucose buffered with 12.5mM sodium phosphate to pH 7 completes the washing process. The cells are siphoned from the washing bowl into the final administration bag. Standard Huggins Procedure Glycerolizing. Packed cells are transferred to the blood freezing unit on the Cytoglomerator. An appropriate amount (depending on unit weight) of Huggins glycerol solution containing: 8.6M glycerol USP (79.2 per cent w/v), dextrose USP 6.0 gm/dl, fructose N F I .O gm/dl, disodium edetate USP 0.3 gm/dl and citric acid USP 0.006 gm/dl is added to the cells with agitation by a magnetic stirrer. The blood freezing unit is removed from the Cytoglomerator, folded, inserted in a cardboard fieezing container, and frozen in a -80 C mechanical freezer. Thawing and Deglycerolizing. The blood freezing unit is removed from the cardboard container and put into a 37 C water bath. After a few moments the unit is unfolded and thawed for seven minutes. The unit is then attached to the Cytoglomerator. An appropriate amount, depending on unit size, of 50 per cent dextrose is added with the magnetic stirrer on, followed immediately by two liters of 5 per cent fructose. The cells are allowed to agglomerate and settle and the supernate fluid is removed. The cells are then resuspended in two liters of 5 per cent fructose and allowed to agglomerate and settle a second time. The supernate fluid is removed and the cells are resuspended in.250 ml of normal saline in the final administration bag. This bag is then centrifuged and the supernate fluid is removed.
Modified Huggins" Solution The modified solution contained 400 ml of 6.2M glycerol USP (57.1% v/v), dextrose USP 6.0 gm/dl, fructose N F 1 .O gm/dl, disodium edetate USP 0.3 gm/dl and citric acid USP 0.006 gm/dl. This modified glycerol solution was used with the standard Red Cross glycerolizing and freezing procedure. Assays Hemoglobin concentrations were assayed using the I.L. Hemoglobinometer Model 231.1 Sodium and potassium concentrations were measured with the 1. L. Flame Photometer Model 143t after a preliminary packing of the cells in the Beckman Microfuge Model 152s and dilution in the Fisher Diluter Model 240.11 Recoveries were calculated by rinsing with saline the residual cells and hemoglobin from all of the tubing and containers used in deglycerolizing. This rinse was added to the waste from the deglycerolization procedure and the total hemoglobin content measured. The total hemoglobin of the washed cell suspension was also measured. The total process recovery was then determined by dividing the washed cell hemoglobin by the sum of washed cell plus waste hemoglobin. In addition, an aliquot of the waste was sedimented and the proportion of waste hemoglobin still present in intact cells was determined. This enabled a comparison of losses associated with hemolysis with losses resulting from cell washover and cells left in the apparatus.
Results In Table I data are presented showing days stored at -80 C, the supernatant hemoglobin content of the cell suspension before and after freezing and after deglycerolization, the final osmolality of the washed cell suspension, the proportion of waste hemoglobin that is in intact cells and the total process recovery. The mean recovery of cells is 93.4 per cent * 1.3 (S. D.) processed by the standard Red Cross method as compared to 86 per cent 3.4 (S. D.) for the standard Huggins method. T h e final supernatant hemoglobin concentrations and osmolalities are comparable for the two methods. The data for 12 units processed by the standard Red Cross method, using the modified Huggins glycerol solution, are essentially equivalent to that obtained with the Red Cross glycerol formula.
*
tlnstrumentation Lab., Inc. Watertown, Mass. §Beckman Instruments, Inc., Fullenton, Calif. IlFisher Scientific Co., Pittsburgh, Pa.
420
Transiusion
HORNBLOWER A N D M E R Y M A N Table 1 .
Glycerolizing solution Deglycerolizingmethod
Sep1:Oct. 1971
Comparisonsbetween Standard and Modified Procedures
ARC ARC
Standard Huggins Agglom.
Modified Huggins ARC
Standard Huggins ARC
Modified Huggins Aaalom.
~ _ _ _ _ _
13 185 f 3
Number of units Days at - 80C Supt. Hgb. pre-freeze. mg % Supt. Hgb. post-thaw. mg % Deglycerolizedcells. Hct. Deglycerolizedcells. mOsm Deglycerolized cells. supt. hgb.. mg % %of waste hgb. in intact cells % recovery
11
12
10
8 f 5.3 88 f 49
4f3 10
2 1613 60120
1 54
95.6 f 51
263 f 107
102 & 72
127/98
328
53 f 3
81 k 9
52 f 6
49/49
70
356+ 16
349 f 12
366 f 32
3081326
353
160 f 90
92 f 44
148 f 109
1251109
128
5*2
50+ 1 1
5*3
212
46
93.4f 1.3
86&34
91 + 4
90185
91
1
NOTE: Data is presented as mean and standard deviation with the exception of column 4 in which the results of two units are both shown and column five where only one unit was studied.
-
E
.n C
120
c
0
E @ 80 I loot * c Q Q
CI
s
Q 0
CI
I-
0
20
40
60
80
Vol. Cell Suspension Iml) FIG. I. The total postthaw free hemoglobin present when portions of the same unit are frozen in differing volumes in identical plastic bags. Extrapolating the data to zero volume indicates the amount of lysis that must be attributed to interactions with the container.
Also in Table I data are presented for two units glycerolized and deglycerolized by the standard Red Cross procedure using the standard Huggins glycerol formula. The results were satisfactory but too few units were processed to enable a significant comparison with t h e two s t a n d a r d procedures. The modified Huggins glycerol solution avoids the need for prorating glycerol solutions of varying quantities, involves less introduction trauma to the cells and this is considerably more convenient than the standard Huggins solution. Data a r e also presented for one unit processed by the standard Huggins procedure using the modified Huggins glycerol formula, in an effort to simplify and improve the standard Huggins procedure. The recovery of cells is improved. Similar results have been reported by Huggins (per son al com m un ication ). Red blood cells glycerolized with the Red Cross glycerol lactate solution have shown an increase in freezing hemolysis with an increase in surface area of the freezing containerI5 particularly with polyvinylchloride (PVC) containers. Experiments were conducted to determine the effect on freezing hemolysis of the very large PVC freezing bag used in the Huggins procedure. One unit of blood was divided into two equal parts. One portion was glycerolized with half the usual amount of Huggins glycerol-glucose and the other portion with half the usual amount of Red Cross glycerollactate solution. The glycerolized cells were then divided into 20, 40, 60, and 80 ml aliquots in 300 ml PVC bags and frozen at -80 C. Figure I shows t h e mean total hemoglobin in t h e thawed supernatants for three different experiments. The projected intercept with the ordinate for the Hug-
Volume 17 Number 5
42 1
R E D CELL FREEZING
gins blood indicates that there is very little, if any, PVC surface effect while the hemolysis of the Red Cross blood confirms previous observations. Three units of sickle trait cells were glycerolized, frozen and deglycerolized by the standard Huggins procedure. Unit # I had not agglomerated two hours after the addition of the 5 per cent fructose. Unit #2 agglomerated slowly, the cells had settled to about six inches from the bottom of the bag 25 minutes after the fructose addition. Unit #3 agglomerated even more slowly. The cells had settled to about eight inches from the bottom of the bag IN hours after the fructose addition. One of the particular virtues of cells frozen in a high concentration of glycerol with isotonic electrolyte present is the great stability to fluctuations in temperature that these cells exhibit. Prolonged exposure for days a t refrigerator o r room temperature of cells glycerolized by the Red Cross procedure results in little or no evidence of serious changes. A 72-hour exposure to 22 C, for example, results in a reduction in mean cell potassium content of roughly 5 per cent and a negligible increase in free hemoglobin or of subsequent freeze-thaw lysis. Furthermore, cells glycerolized in this manner can be repeatedly frozen and thawed with no significant increase in per cent loss on subsequent freezes.* However, in a low ionic content, the red blood cell becomes relatively permeable to cations. We therefore investigated the response of cells glycerolized by the Huggins procedure to repeated freezing and thawing and to prolonged pre-freeze storage while glycerolized. A full unit of packed cells was divided into two equal parts, one of which was glycerolized with 200 ml of Huggins 6.2M glycerol glucose-fructose solution and the other with 200 ml of standard Red Cross 6.2M glycerol Na lactate solution. Each half was frozen and thawed, per cent hemolysis and intracellular K+ was measured, and each half was refrozen after approximately two hours. This procedure was repeated for a total of three freezings. As shown in Table 2 the Huggins and Red Cross glycerolized cells had the same recovery after the first freeze. The Huggins cells were somewhat low in intracellular K + . After the second freeze and thaw the Huggins cell survival was much worse than that of the Red Cross cells, and after the third freeze and thaw the Huggins cells were totally hemolyzed whereas only 4.7 per cent of the Red Cross cells were h emol yzed . Considering the possibility that the incubation at room temperature while glycerolized might be responsible for the lysis on refreezing we explored the effects of prolonged exposure to the glycerol solution before freezing. Two units of cells were glycerolized, one with the Huggins solution and
Table 2 .
Effects of Refreezing 1st freeze and thaw
2nd freeze and thaw
3rd freeze and thaw
Hugginsglycerol formula % hemolysis lntracellular K+
1.5 53
19
100
46
-
Red Cross glycerol formula % hemolysis lntracellular K+
1.5 75
2.7 71
4.7 75
~~
~~~
NOTE: Glycerolized cell suspensions were frozen at least
overnight, then thawed. incubated at room temperature for 2 hours. then refrozen. Progressive hemolysis and potassium loss were observed when the low ionic glycerolizing medium was used.
one with the Red Cross solution. Each unit was divided into 25 ml aliquots in Fenwal 300 ml transfer packs. Aliquots from each group were held at 4 or 22 C for various lengths of time before freezing. Figure 2 shows that prefreeze incubation of Huggins cells at 22 C results in 35 per cent postthaw hemolysis after 5 hours and 100 per cent after 24 hours. At 4 C, the increase in hemolysis
Duration of Pretreeze Incubation While Glycerolized I hrl
FIG.2. Cells incubated at room temperature following glycerolization with Huggins solution progressively increase in volume without lysis, approaching maximum volume after about 4 hours. A concomitant increase in postthaw lysis is seen if cells are frozen after such incubation.
422
Transfusion
HORNBLOWER A N D M E R Y M A N Table 3.
Sept.-Oft. 1977
Estimated Costs ~~
~
~~~~
~
~
Solutions and disposables per unit processed
Capital Equipment
~
Haemonetics Shaker Model 17 Cell Washer Reuseable metal freezing Cannister Water bath
1 unit
360.00 4.285.00 3.95 277.00
Bowl Waste bag Washing set Transfer pack Freezing bag Solutions
$4.925.95
IBM Shaker
#2991 Cell Processor Reuseable Metal Freezing Cannister Water Bath
360.00 1 7,000.00 3.95 277.00
Freezing bag Processing set Transfer pack Solutions
4 units 3.75 .66 1.06 .90 3.50 5.48
$29.79
$1 5.35
3.50 7.50 .90 4.46
__
$16.36
$1 7.640.95 Huggins WS-1 Cytoglomerator Cytolight Cytobath
15.00 .66 4.25 .90 3.50 5.48
1.375.00 175.00 795.00
Processing set Solutions
18.3 1 12.00 $30.31
$2.345.00 NOTE: Costs quoted are based on small volume purchases as of May. 1975
was negligible after 24 hours. Cells in the high ionic Red Cross glycerol solution were unaffected at either temperature. Cell volume was measured periodically during incubation at 4 and 22 C. At 22 C, the cell volume in Huggins solution increased progressively until it approached lytic volume at about 4 hours. Longer incubation at 22 C resulted in no further volume increase and no hemolysis. At 4 C cell volume increased only from 70 to 90 u3 in 24 hours. Table 3 shows the comparable costs of the Haemonetics, IBM and the Huggins methods at the time the study was conducted. The cost of capital equipment for the Huggins method was less than the two Red Cross methods and the apparatus has the advantage of functioning in an emergency without electrical power. The cost per unit was higher than the IBM or the Haemonetics systems but in the latter case only when two or more units a r e washed through the same bowl.
Discussion The recovery of red blood cells processed by the Red Cross procedure averages approximately 7 per cent more than for the standard Huggins agglomeration procedure. Other recovery parameters such as os-
molality and supernatant hemoglobin are roughly equivalent. From the point of view of in vitro recovery, the Huggins agglomeration procedure is entirely statisfactory. Although we have confirmed the loss of cel I potassium following agg lom e r a t ion, there is no evidence that this is a significant shortcoming of the procedure. No evidence has been presented that the reduction in cell potassium has any adverse effect on cell physiology or clinical effectiveness. I t was not possible in this short term study to investigate the reports by ValeriiJ that, after more than two years frozen storage, there is a reduction of in vitro recovery, in vivo survival and postwash stability. TWO arguments can be used to minimize the importance of this criticism. First, in practice, very few units are stored in excess of two years. Of the roughly 20,000 units frozen in the Red Cross system two years or more before the study reported here, only 345 units, or less than two per cent were stored longer than two years. The mean storage
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423
RED CELL FREEZING
time for all units in the Red Cross system is less than two months. Second, Valeri's report indicates that if Huggins units stored more than 2 years are deglycerolized by saline wash, good recoveries and survival ~ e s u l t .Therefore, '~ should the report of deterioration after two years storage be confirmed, such units could be deglycerolized by the saline wash procedure rather than by agglorn eration. The tendency of cells glycerolized by the Huggins procedure to swell with incubation at room temperature and hernolyze with subsequent freezing is a problem not seen with cells frozen in t h e presence of a normal ionic content. The ability to refreeze cells that have been thawed is useful, particularly following freezer failure or temperature rise during shipping. However, it is unlikely that the federal regulations governing the manufacture of Red Cells, (Human) Frozen and Deglycerolized will include provisions for refreezing or permit the use of frozen cells that have been subjected to an accidental rise in storage temperature. The stability of cells glycerolized at a high ionic content may therefore not be a useable asset in practice. One shortcoming of the agglomeration procedure is the inability to deglycerolize sickle trait cells. Such cells also pack and hemolyze during deglycerolization by the standard Haemonetics or I B M method. However, a modified protocol has been devised for both of these devices which permits t h e deglycerolization of A-S Such cells glycerolized with the Huggins low ionic solution can also be deglycerolized with one of the modified high ionic centrifugal wash procedures. The capital costs for the agglomeration equipment are less than for either of the two centrifugal devices. The cost of solutions and disposables for the Huggins system is roughly twice that of the IBM system and equivalent to t h e Haemonetics system. However, in the Haemonetics system, more than one unit destined for the same recipient can be washed through the same disposables, thus reducing the cost per unit of disposables
by 30 per cent when two units are deglycerolized and by 50 per cent for four. It was the impression of our technicians that the agglomeration procedure was somewhat more demanding. During deglycerolization, the decision when to stop removing supernatant fluid after the cells have settled and the technicians's skill in rnanipulating the washing bag can influence the proportion of cells lost in the waste and, thereby, the in vitro recovery achieved. The modified Huggins solution permits satisfactory recovery of cells should they be deglycerolized by centrifugation rather than by agglomeration. This enhances the flexibility of a frozen red blood cell program and improves the yields in a blood center where both agglomeration and centrifugal deglycerolizing may be used, as well as making possible the exchange of frozen cells between centers using different methods of deglycerolizat ion. Using the Red Cross glycerolizing procedure, in which the first 100 ml of glycerol solution are added to the cells in the collection bag with shaking, increased the recovery of cells by only two to three per cent when the Huggins solution was used. Such mixing is absolutely mandatory for the high salt preparation. The increased postthaw hemolysis observed when cells in high ionic glycerol solutions are frozen in PVC bags was not observed when the cells were glycerolized in the non el ect rol y te solution. Contrary to our expectations, therefore, the very large area of PVC presented by the Huggins freezing is not a source of additional hemolysis. We feel that the modified Huggins glycerolizing solution offers the advantage over the standard Huggins procedure in that a single glycerolizing solution may be used rather than maintaining an inventory of and selecting from among several different concentrations of glycerol. References I.
Chaplin, H., and P. L. Mollison: Preservation of blood. In: Preservation and Transplantation of
424
HORNBLOWER AND MERYMAN
Normal Tissues. London, J . & A. Churchill, 1954,p.l21. 2. Haynes, L. L., W. C. Turville, M. T. Sproul, M. E. Henderson, J. W. Zemp, and J. L. Tullis: Long-term preservation-a reality. J. Mich. Med. SOC.61:1509, 1962. 3. Hornblower, M., and H. T. Meryman: Influence of the container material on the hemolysis of glycerolized red cells after freezing and thawing. Cryobiology 11:317, 1974. 4. Huggins, C. E.: Frozen blood. Eur. Surg. Res. 1:3, 1969.
preservation of blood by freezing. In: Red Cell Freezing, Technical Workshop, American Association of Blood Banks, 1973. Reversible agglomeration used to remove 6. -: dimethylsulfoxide from large volumes of frozen blood. Science 139504. 1963. 7. Krijnen, H. W., J. J . Fr. M. dewit. A. C. J. Kuivenhoven, and G. Reyden: Freezing of red cells with liquid nitrogen. I. Results with glycerol as an intracellular substance. Biblio. Haematol.
5. -:Practical
23:683, 1965. 8.
Meryman, H. T., and M. Hornblower: A method for freezing and washing red blood cells using a high glycerol concentration. Transfusion
9.
-,
12:145,1972.
and M. Hornblower: Freezing and deglycerolizing sickle-trait red blood cells. Transfusion 16:627, 1976.
Transfusion Sept.-Oct. 1977
10. Pert, J. H., P. K. Schork, and R. Moore: A new
method of low-temperature blood preservation using liquid nitrogen and a glycerol-sucrose additive. Clin. Res. 11:197, 1963. 1I .
Rowe, A. W., E. Eyster, and A. Kellner: Liquid nitrogen preservation of r e d blood cells for transfusion; a low glycerol-rapid freeze procedure. Cryobiology 5:119, 1968.
12.
Smith, A. U.: Prevention of haemolysis during freezing and thawing of red blood cells. Lancet 2:910, 1950.
13. Tubs, J. L.: Principles involved in glycerolization
and deglycerolization of red cells using Cohn fractionator. Cambridge, Massachusetts, Protein Foundation. Proceedings of 9th Conference on the Plasma Proteins and Cellular Elements of the Blood., p. 17. Valeri, C. R., and A. H. Runck: Long term frozen storage of human red blood cells: Studies in vivo and in vitro of autologous red blood cells preserved up to six years with high concentrations of glycerol. Transfusion 9 5 , 1969. 15. -, and J. C. Bond: Observations on the preservation of autologous human erythrocytes using glycerol, slow-freeze technic and agglomeration. Transfusion 6:254, 1966. 16. -, A. H. Runck, and C. E. Brodine: Current status of freeze-preservation of human red cells with glycerol. Biblio. Haematol. 38:249, 1971. 14.
