Bloodindex - Rejuvenation of Stored Red Cells

Stored red cells even at the end of their allowable shelf-life can be rejuvenated with solutions containing pyruvate, inosine, phosphate, glucose and adenine. Incubation with the rejuvenating solution at 37 oC for 3-4 hours greatly improves viability and restores 2, 3 DPG levels

Method

Add 50 ml of rejuvenating solution to the red cell concentrate.
Incubate at 37 oC for one hour prior to glycerolization and freezing

Freezing of Red Cells for Prolonged Storage

Red cells can be stored for longer period in frozen state. Frozen red cells improve cell survival and increase shelf life to 5 years or more. It has been seen that freezing and subsequent thawing of red cells produces cellular dehydration and mechanical trauma due to intracellular ice formation which ultimately causes haemolysis. A cryoprotective agent can prevent this freezing injury. The two agents commonly in clinical use are :

Glycerol
Dimethyl sulphoxide (DMSO)

Advantages

Long term storage of rare blood groups
Transfusion requirement for patients with antibodies against high frequency antigens
Storage of Autologous donations in patients with rare blood groups.
Fewer febrile and allergic transfusion reaction
Lower incidence of alloimmunization to HLA in transplant recipients
Ability to store rejuvenated outdated red cells
High level of 2,3 DPG in transfused blood.
Restoring blood stock and inventory.

Disadvantages

High cost
Short post-thawing shef-life (24 hrs)
Increased red cell loss in processing
Increased processing time in emergency

For freezing, red cells are used within 6 days of collection, however if a rejuvenating solution is used, the cells can be frozen even 2-3 days after expiry.

Two commonly used methods for freezing by glycerol employ:

Low glycerol solution (20%)
High glycerol solution (40%)

The rate of freezing and storage temperature determine the optimal glycerol concentration.

Storage of Blood and Blood Components

After the processing of blood into blood components safe and adequate storage and transportation.. are of immense value to preserve the viability and therapeutic efficacy of the components during their shelf-life or till the time it is transfused to the recipient.

Safe storage and adequate transportation of blood and blood component are vital for two main reasons.

to maintain therapeutic efficacy and life span of the constituent
to prevent bacterial over growth.

Whole blood

Whole blood and red cell concentrate must always be stored between 2°C and 8°C. A fall in temperature less than 2°C can cause freezing injury to the red cells leading to haemolysis. If haemolysed blood is transfused to a patient, it can lead to fatal consequences.

Temperatures more than 8°C can lead to overgrowth of bacteria which may have entered the blood unit during collections or component preparation.

As red cells consume glucose for their continued metabolism, storing a blood unit at 2-8°C will also decrease the rate of glycolysis. The shelf life of whole blood and red cell concentrate in commonly used anticoagulant, CPDA-1 is 35 days.

Plasma

Fresh frozen plasma (FFP)

FFP is usually transfused to restore or to maintain the clotting mechanism or as a volume replacement when crystalloids and colloids are not used. Fresh frozen plasma is prepared by removing plasma from a unit of blood within 6 hours of collection and snap freezing it to - 30^oC.

The speed of freezing is very important for maintaining the coagulation factors. FFP must always be stored at - 20°C or lower, otherwise the amount of clotting factors such as F VIII and F V will be greatly reduced. FFP should preferably be stored in cardboard boxes (containing full information about the ABO blood group, donation unit number and screening test results,) to prevent fixing up with the adjacent bags while freezing as it may lead to breakage of the bag.

Bag containing FFP meant for preparation of cryoprecipitate should be placed in between aluminium metal plates to achieve a uniform thickness of plasma which helps in rapid core snap freezing essential to maintain high yield of FVIII in the cryoprecipitate.

Liquid/frozen plasma

If plasma is removed from a unit of whole blood at any time upto 5 days after the expiry date and stored at 2-8°C, it is called as liquid plasma.

Liquid plasma can be stored frozen at - 20°C or lower as plasma which ‘has adequate amount of plasma proteins lacks the labile coagulation factors FV111 and FV. The shelf life of fresh frozen plasma and frozen plasma is one year if stored at - 20°C or lower.

Platelets Platelets are usually stored as concentrates. They are harvested from a single unit of blood either from PRP (platelet rich plasma) or from buffy coat and resuspended in 50 ml of autologous plasma.

Platelets should be stored at 22°C in plasma under conditions in which the pH is maintained at values above 6.8. Shelf life of platelet concentrate is no more than 5 days. The functional integrity of platelets depends on many factors.

Storage temperature : ideal temperature for platelet storage is 22°C. Temperatures below 200C during platelet preparation and storage cause a striking increase in the number of large aggregates in the platelet-rich plasma.

pH : A fail of pH of platelet concentrate due to lactate production from platelet glycolysis leads to loss of viability. A rise in pH above 7.3 also leads to loss of viability. The rate of fall of pH is affected by:

Number of platelets
Volume of plasma in which stored
Availability of 02

Plastics : Type of plastics used for storage of platelets is important. Availability of plastics with an increased permeability to 02 has made it possible to lengthen the period to which the platelets may successfully be stored. New second generation plastics such as polyolefin allow satisfactory storage upto S days. Other plastic bags in use are Teruflexa (Terumo) made of polyvinyl chloride (PVC) with reduced thickness and larger surface area, F-720 (Biotrans) made of PVC with a phthalate ester analogue as plasticizer, PL 2209 (Fenwel) made of PVC plasticized with BTHC.

Agitation Platelet concentrates must be gently and constantly agitated either on a flat bed agitator or a rotary agitator for better viability. Without agitation, there is a rapid fall in pH, due to collection of lactic acid metabolites.

Contamination with leucocytes : leucocyte contamination is associated with

Fall in pH.
Loss of glycoprotein lb from platelet surface leading to loss of platelet responsiveness to ristocetin and thrombin.

Storage of platelets in frozen state

Best results are found using 5% DMSO (dimethyl sulfoxide) as the cryopreservative agent. They can be stored satisfactorily in liquid nitrogen (-196^oC) or in 80^oC deep freeze.

Granulocytes

Granulocytes are stored generally in the same medium in which they are collected i.e. citrated donor plasma containing hydroxyethyl starch. Leucocytes tolerate storage poorly and lose their highly integrative function i.e. chemostaxis within 24 hrs.

Temperature : Storage is better at 22^oC rather than 4^oC.

Granulocyte concentrate should be transfused as soon as possible and should not be stored for more than 1 day.

Rejuvenation of Stored Red Cells

4. Rejuvenation of Stored Red Cells