5.4 Therapeutic Protocol 
5.4.1 Isolation and enrichment of peripheral blood lymphocytes from 
donor. 
A phlebotomy or leukapheresis (performed by the NIH Blood Bank 
Apheresis Unit according to standard procedures) will be used to 
obtain lymphocytes from the donor. 
5.4.1. 1 Apheresis Technique: 
Leukapheresis will be performed either manually or with the use of an 
automated cell separator. If manual techniques are used, a unit of 
whole blood (volume not to exceed 10% estimated blood volume) will 
be withdrawn using a 1 :8 ratio of ACD-A to whole blood, and 
processed and separated into components by centrifugation. The 
leukocytes will be harvested and the remaining red cells and plasma 
returned to the donor. One to 5 units of whole blood may be 
sequentially processed in this manner. Only one venipuncture is 
necessary and the procedure requires 45 minutes per unit of whole 
blood processed. 
Apheresis may also be accomplished using automated cell separator 
devices. Whole blood is withdrawn from one venipuncture site at a 
rate of 40-60 ml/min and channeled into the cell separator, where 
cellular and plasma fractions are separated by centrifugation. The 
leukocytes are harvested into a component bag, and the red cells and 
plasma are reinfused into the patient via a second venipuncture site. 
Anticoagulation is achieved using ACD-A at a whole blood to 
anticoagulant ratio of 10:1. Maximum extracorporeal blood volume is 
300 ml (Fenwal, COBE models). One to two hours are required to 
process 1 to 5 liters of whole blood. 
Hazards and Precautions of Leukapheresis: Adverse reactions to 
apheresis procedures are rare and include vasovagal episodes 
related to needle insertions and transient volume loss, and cutaneous 
paresthesias related to citrate-induced hypocalcemia. The former 
reaction is handled by postural manipulation and fluid administration, 
the latter is usually relieved by slowing the rate of, or temporarily 
interrupting, the anticoagulant infusion. 
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