The Effect of Temperature Variations on Cryoprecipitate

G. A. ROCK AND P. TITTLEY

From the Canadian Red Cross Blood Transfusion Service, Ottawa, Ontario, Canada

The effect of temperature on Factor VIII levels in blood and cryoprecipitate was assessed. Freshly col- lected units of blood required several hours to reach 4 C, but thls delay seemed of little importance since equal amounts of cryoprecipitated Factor VIII were recovered from blood stored either at 22 C or at 4 C. Freezing at -80, -60, or -40 C produced identical yields of Factor VIII, whereas freezing at -20 C resulted in si@fkantly lower recoveries. This might he expected if one considem the physkhemkal changes that occur during the *zing process.

MANY VARIABLES are known to affect the yield of Factor VIII by cryoprecipitation from human p l a ~ r n a . ~ * ~ * ~ . ~ Efforts to stand- ardize Factor VIII preparations have given rise to various recommendations including the storage of fresh whole blood 1 to 6 C prior to processing and free~ing,~ centrifuga- tion within six hours after collection8 and rapid freezing at -70 C.@

Freezing at -70 C involves the use of either dry ice or a cascade refrigerator sys- tem both of which are relatively expensive. In order to evaluate the necessity for low temperature freezing, we undertook an as- sessment of Factor VIII yields at various freezing temperatures. We also examined the effect of different blood storage tem- peratures on the subsequent Factor VIII level in the plasma and in the recovered cryoprecipitate.

Materials and Methods Blood Collection and Preparation

Blood was collected from randomly selected healthy men and women into double plastic bag

Received for publication November 28, 1977; ac- cepted January 29, 1978.

systems (Fenwal) using a citrate-phosphate-dex- trose (CPD) anticoagulant. To assess the effect of temperature on Factor VIII levels, ten units of whole blood were divided into two equal parts with one part being stored at 4 C and the other at 22 C. Cryoprecipitate was made from prepara- tions after six hours of storage.

To obtain a homogenous pool of plasma, in- dividual units of whole blood were centrifuged at 7000 x g at 4 C within one hour of collection. The supernatant plasmas were expelled into satellite bags, and were subsequently pooled with others of identical ABO group and Rh type in 2000 ml transfer packs. Two 5 ml aliquots were removed and immediately frozen at -80 C for later Factor VIII assay. The plasma pools were then divided into 200 ml lots in 300 ml Fenwal transfer packs.

Blood Temperature Measurement Assessment of the importance of immediate

refrigeration or cooling on the Factor VIlI re- covery requires determination of the time re- quired to "cool down" a unit of freshly col- lected whole blood. A temperature probe (YSI Model 4256 tele thermometer) was inserted directly into the blood bag immediately after collection and both the probe and bag were placed in a well-ventilated 4 C walk-in refrigera- tor. Continuous temperature recordings were ob- tained using a Fisher Recordall Series 5000.

Freezing Procedure All of the bags of pooled plasma were

placed in Harris PCF cassettes, four bags to a cassette, and frozen for 12 hours. The bags were compressed so that each plasma mass was held in a shape of relatively uniform thickness dur- ing freezing. All bags used in one experiment were processed simultaneously and all were frozen in the horizontal plane with the exception of those frozen at -80 C in the PCF (this machine holds the cassettes at a 35 degree angle). Plasmas were frozen at -80 C in a Harris

Transfusion J ~ u ~ r y - F e b r u ~ ~ y 1979

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86 Volume 19 Number 1

Volume 19 Number I TEMPERATURE AND FACTOR VIII 87

PCF, at -60 C in a Revco Chest freezer ULT-1234, at -40 C in a Revco Upright Freezer 1145-A-8, and at -20 C in a standard Sears freezer. The freezing coils of the Harris PCF came in contact with both the top and bottom of each cassette frozen at -80 C. In all other cases only the bottom of the cassette came in contact with the freezing coil. This was due to the structural design of the various freezers and could not be modified.

Thawing Procedure. Rapid thawing was accomplished with a Forma

2073B circulating waterbath at 4 C. This pro- cedure is described in detail in a previous publi- cation.' All of the supernatant plasmas and cryoprecipitates were refrozen in the storage compartment of a Harris PCF freezer at -80 C. They were stored for less than 24 hours prior to assay for factor VIII.

Factor VIII Assuy A modification of a one-stage assay:' was used.

Cryoprecipitates were thawed at 37 C, 10 ml of saline was added, and the samples were diluted 1: 10 in hemophiliac plasma. The factor VIII content of each sample was calculated using Hyland AHF PTC reference plasma as standard.

Results As shown in Figure 1 , there was a three hour

lag between collection of whole blood and its cooling to less than 10 C-even under ideal con- ditions. The presence of even a few other bags in the same cooling rack provided a significant insulating effect.

The effect of temperature on the Factor VIII

40- - 1 D U U O Y I - 1DAO SUIIROUNDID DV 110 CUL DAOKElO

DV fOUR fULL D U K E I S .- 1 MQ auaaouwmD

O ' ~ f i ~ ~ ~ ~ ~ ~ 1 0 2 4 0 8 10 18 14 10 1S 10

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FIG. I . Temperature of blood placed and maintained at ambient temperature of 4 C.

liar

1 I 1 I

0 2 4 0 D 1 0

wuaa FIG. 2. The effect of temperature on the factor VIII

level in whole blood. Solid line indicates 4 C; broken line indicates 22 C.

level in whole blood is shown in Figure 2. By six hours there was an approximately 15 per cent decrease in the total Factor VIII content of blood stored at 22 C. Levels were significantly higher (p > .05) when the blood was stored for six hours or 24 hours at 22 C rather than at 4 C. It can be seen from Table 1 that there was no significant difference (p > .05) between the levels of factor VIII activity in the cryoprecipitates made after 6 hours of storage at either temperature.

There was also no significant difference in the total Factor VIII activity recovered in the cryo- precipitate obtained by freezing at -40 C, -60 C or -80 C (Table 2). These values were however, significantly higher (p > .05) than those for cryoprecipitate made by freezing plasma at -20 C. While the total recovery of Factor VIII (cryoprecipitate plus supernatant activity) was the same for plasma samples frozen at -80 C, -60 C and -40 C, it was significantly reduced (to 62.2%) in the plasma frozen at -20 C.

Discussion

The very slow rate of cooling shown in Figure 1 indicates that low temperature storage is not achieved for a considerable period of time even under ideal conditions. In any case, it would appear that the factor VIII activity is actually higher in blood which is kept at 22 C rather than at 4 C. However this is not reflected in final yields since approximately equal amounts of factor VIII are cryoprecipitated at the two temperatures. This data is in agreement with

88 ROCK A N D TITTLEY Transfusion January-February 1979

Table 1. Factor Vll l Levels in Cryoprecipitate made after Six Hours of Storage at 4 C or 22 C

YO Factor Vlll Recovery'

Cryopre- Total cipitate Supernatant Recovery

4 C 41.96 2 10.4 15.93 f 1.75 57.89 5 10.94 22 C 41.08 2 12.2 15.36 2 33.2 56.44 2 12.85

* n = 10 in each case.

one previous report2 indicating no signifi- cant difference in factor VIII recovery after 4 C or 22 C storage. This suggests that the extra staff and manipulations involved in at- tempting to rapidly achieve 4 C storage are certainly not justified on the basis of factor VIII recovery.

It appears that insistance on very low temperature freezing (-70 C) is not neces- sary in order to obtain maximal factor VIII levels in the cryoprecipitate. Indeed, it is possible to freeze at -40 C (the tempera- ture available in many standard laboratory freezers) and still obtain good factor VIII yields. This fact is supported by the report of Kasper et who found no significant differences in mean recovery of factor VIII in aliquots of plasma frozen at -30 C in a freezer or between sheets of dry ice. Still, recommendations for -70 C freezing abounds . * s o

The relatively lower yield at -20 C is not surprising when one considers what hap- pens during the cryoprecipitation process. As the temperature is lowered, tiny ice crystals form and the solutes such as the

proteins and salts become more concen- trated in the intercrystalline spaces. If the temperature is lowered sufficiently the salt concentration reaches a level at which some of the protein components become insoluble and precipitate. Cryoprecipitation is there- fore essentially a salting out procedure at subzero temperatures. The major salt in the plasma is sodium chloride which has a eutectic point of -23 C. Therefore at temperatures above -23 C, tiny pockets of salt will remain in liquid solution at con- centrations which may not be sufficiently high to salt out concentrated proteins in the same pockets. It should also be noted that a significant portion of the liquid fraction of B lipoproteins is not extractable into ether when plasma is frozen at -20 C but is extractible if the temperature is -25 C or lower.6 Previous reports4 of association of factor VIII with the lipoprotein fraction of plasma suggest that there may be a casual link between the factors just cited and the necessity to freeze plasma at temperatures below -23 C in order to obtain maximal yields of factor VIII.

Acknowledgments We wish to acknowledge the cooperation of Miss

A. Baxter, Chief Technical Supervisor, and the tech- nical assistance of Mrs. Virginia Sherring of Quality Control section of the Ottawa Centre.

References

I . Brown, D. L., R. M. Hardistry, M. H. Kasoy, and C. Bracken: Antihemophilic globulin: Prepara-

Table 2. Factor Vl l l Recovered from Plasmas Frozen at Various Temperatures'

Cryoprecipitate Supernatant Total Ternpera-

ture Total Units YO Recovery Total Units Yo Recovery Units O/O Recovery

-80 c 75.3 2 14.2 53.1 2 9.7 29.8 f 3.3 21.4 2 4.1 105.1 f 14.8 74.9 2 12.8 -60 C 73.2 r 13 52.0 2 7.3 25.0 2 3.3 18.3 2 3.9 97.4 2 13.3 70.23 2 9.9 -40 C 71.1 f 9.9 52.4 2 8.3 24.8 2 3.1 18.9 f 3.5 96.5 11.0 73.0 2 14.3 -20 c 68.2 f 13.2 45.92 10.0 23.4 ? 2.2 16.2 2 4.5 91.6 5 13.1 62.4 2 13.8

n = 18 in each case.

Volume 19 Number 1 TEMPERATURE AND FACTOR VlIl 89

tion by an improved cryoprecipitation method and clinical use. Br. Med. J. 2:79, 1%7.

2. Burka, E. R., L. A. Harker, C. K. Kasper, S. V. Kevy, and P. M. Ness: A protocol for cryoprecipitate production. Transfusion 15307, 1975.

3. Hardisty, R. M., and J. C. Macpherson: A one- stage factor VIII (anti-hemophilic globulin) as- say and its use on venous and capillary plasma. Thromb. Diath. Haemorrah. 7:215, 1%2.

4. Hershgold, E. I., A. M. Davison, and M. E. Janszen: Human Factor VIIl (antihemophilic Factor): activation and inactivation by phospho- lipases. J. Lab. Clin. Med. 77:206, 1971.

5. Kasper, C. K., B. A. Myhre, J. D. McDonald, Y. Nakasoto, and D. I . Feinstein: Deter- minants of factor VIII recovery in cryopre- cipitate. Transfusion 1 5 3 12, 1975.

6. McFarlane, A. S.: Behaviour of lipoids in human serum. Nature 149:439, 1942.

7. Pool, J. G.: Cryoprecipitate quality and supply. Transfusion 15305, 1975.

8. Rock, G., and P. Tittley: Variations in cryoprecipi- tate production. Transfusion 1750, 1977.

9. Standards for Blood Banks and Transfusion Ser- vices, 8th ed. Washington, D.C., American As- sociation of Blood Banks. 1977.

G. A. Rock, Canadian Red Cross Blood Trans- fusion Service, 85 Plymouth Street, Ottawa KIS 3E2, Ontario, Canada.

P. Tittley, Canadian Red Cross Blood Transfusion Service.

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