Method of freezing red blood cells

Details Method of freezing red blood cells Method of freezing red blood cells

1991-08-07

1994-05-10

Capossela, Ronald C.

Refrigeration

Processes

Treating an article

62 78, 424529, F25D 2500

Patent

active

053097238

DESCRIPTION:

BRIEF SUMMARY
The present invention is concerned with the freezing of red blood cells (erythrocytes) for long term storage in such a way that they remain suitable for blood transfusion.
Storage of blood for use in transfusion services is well-known. Blood is usually stored in refrigerated conditions at 4.degree. C., where it has a maximum useful life of about 5 weeks. Maintenance of adequate supplies therefore requires the continuous co-operation of donors. Unfortunately there are periods, such as adjacent the Christmas period, where supplies can run short. There are also occasions when serious disasters leave a particular area short of supplies.
Also an increasing number of people desire to store their own blood, for example before elective surgery.
There is, therefore, a requirement for a method of long term storage of blood supplies.
Long life storage of blood is made possible by freezing of the blood to very low temperatures at which it is then stored. Unfortunately freezing blood as supplied has proved to be impossible. It is usual to store blood in standard units of about 450 ml, this being the volume donated by a donor at a single donation. Patients receiving transfusions are usually given an integral number of such units. Freezing such a volume of blood results in haemolysis of the red blood cells which renders the blood useless for future use in transfusions. Haemolysis results in the release of potassium, which is cardiotoxic, and of strome (cell debris) which can cause kidney failure.
It is generally considered that, to be acceptable for blood transfusion, red cells must not have substantially more than 1% haemolysis. In a method whereby blood can be stored by freezing, the blood is centrifuged to separate plasma and platelets from red blood cells, the red blood cells are mixed with a cryoprotective agent, and the mixture is frozen to a very low temperature, usually by using liquid nitrogen. The commonly used cryoprotective agent is glycerol. Unfortunately glycerol is toxic, and preparation of the red blood cells for use in transfusions requires several washings to remove the glycerol. This is a process which involves expensive equipment, requires a high standard of skill, experience and constant practice on the part of responsible personnel, and also requires an area of high sterility. The washing process takes about twenty minutes per unit, and results in the loss of between fifteen and twenty percent of cells.
A more attractive cryoprotective agent is hydroxyethyl starch (HES), or leavosan.
U.S. Pat. No. 3,758,382 describes how samples of 55 ml of whole blood containing an anti-coagulant (acidified citrate dextrose) were mixed with 40% w/v HES of average molecular weight 40,000 to 70,000 such that the final concentration was 12 to 14% w/v HES. The mixture was immersed in liquid nitrogen at -190.degree. C. and agitated at 200 cycles/minute until frozen. After storage at -140.degree. C. for up to a week the mixture was thawed by immersion for 60 seconds, with agitation at 160 cycles/minute, in a water bath at 47.degree. C. Recovery factors of up to 99% (average 97.4%) of red cells were achieved. The volumes used for freezing, namely 55 ml, are only fractions of standard units and would cause undesirable complications if used in conventional transfusion procedures.
In a development of this process, allowing for the freezing of standard units of blood, U.S. Pat. NO. 4,018,911 describes a method of freezing blood using HES of average molecular weight 150,000. The blood is centrifuged to separate plasma and platelets from the red blood cells, and some of the plasma is mixed with HES. The reason for the addition of plasma is that it was thought to be necessary for the protection of the red blood cells. The mixture of plasma and HES is then mixed with a roughly equal volume of cells giving a proportion of HES in the resultant mixture of the order of 14%. A standard unit donation of 450 ml has by this time been reduced to about 405 ml.
The mixture of plasma and HES is added to the red cells in a freezing bag and th

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