A Simple Quick Method of Draining Red Cells from Granulocyte Concentrates Ex Vivo without the Addition of Hetastarch after Granulocytapheresis
Authors: Aung Fleur M. , Lichtiger Benjamin 
Published: November 2020

Introduction
Granulocytapheresis from healthy volunteer donors are performed regularly at our institution to treat severely neutropenic leukemia/SCT patients with overwhelming bacterial/fungal infections. 500 to 750 mL of Hespan® (6% hetastarch in 0.9% sodium chloride injection) with citrate anticoagulant is administered by aseptic addition to the input line of the centrifugation apparatus at a ratio of 1:13 to venous whole blood.
The traditional method of gravity sedimentation of Granulocyte concentrates (GC) is the addition of 50 cc of 6% Hydroxyethyl starch in ACD anticoagulant which results in RBC volume reduction to &lt; 5ml (range acceptable for transfusion to ABO incompatible recipients according to AABB standards). The method is time consuming, tedious and adds additional HES to the product. We streamlined the process by allowing the sedimented red cells to drain via gravity upon completion of the apheresis procedure without the addition of Hetastarch.
Method:
GCs are obtained from prescreened eligible (meet AABB/FDA guidelines) family/friends of the patients. G-CSF is administered as a fixed dose of 480 mcg (donors &lt; 250 lbs.) or as 2 doses of 300/480 mcg (donors &gt; 250 lbs) plus 8 mg of dexamethasone (without history of cataracts) 12 hours prior to the collection. 30 ml of TriCitrasol Anticoagulant (46.7% Trisodium citrate) is added to 500 ml of 6% Hetastarch in 0.9% Sodium Chloride Injection for IV use only and not more than 1000 ml is used in a single GC apheresis procedure.
Our current practice is to test all O blood group donors for Anti-A Isoagglutinin titers. Cut off titers are set at &lt;128. GCs from group O donors with Anti-A titers &gt; 128 are drained of red cells if the recipient is non-O. For pediatric patients, our practice is to drain all GC products regardless of ABO compatibility between donor and recipient.
Process:
Upon completion of the Granulocytaheresis, the GC collection bag is left undisturbed hanging on the IV pole leaving all slips clamps attached. Red cells collected during the apheresis procedure settle at the base the collection bag. The collection line of the GC collection bag is clamped distal to the attachment of the sterile barrier filter inclusive of the sample bulb assembly. The clamp proximal to the sample bulb assembly needs to be left clamped. The collection bag is then sterile docked to a transfer bag (the plasma bag from the IDL set can be used) to create a closed sterile system. Two hemostats clamps are placed, one close to the inlet port of the collection bag and the other close to the sterile dock of the transfer bag. The clamps are opened slowly and the sedimented red cells are allowed to slowly drain into the transfer bag. Pressure using two fingers may be used to create a funnel to express the sedimented red cells into the port from the GC unit. When the desired amount of red cells are expressed into the transfer bag the process is stopped. The transfer bag is then removed by clamping and a relatively clean GC product is now available for transfusion. The entire process takes approximately 10-15 minutes. The GC bag is then sampled via the incorporated sample bulbs for the bag count, then labelled ready for release for transfusion.
Results:
We reviewed the results of six drained GCs by this method; the median Bag RBC was 0.8 x10e6/uL (range 0.06 - 0.23), Bag Hgb 0.8 g/dL (range 0.5-1.0) and Bag Hematocrit 0.7% (range 0.6-2.6). The median Bag WBC count was 10.6 x 10e10 (range 5.8 to 15.1), median bag volume 641 mL (range 462-774) and median volume processed 10526 ml (range 8800-12175 mL). We achieved our goal of reducing the red cells to &lt;2 ml, providing a GC with more than the minimum yield (1.0 x 10e10) required according to AABB standards and also the availability of a GC product within 2 hours of completion for transfusion.
Discussion:
The use of the newer Apheresis machine allows for efficient removal of the targeted component and monitors/adjusts the depth at which the cells are collected within the Buffy coat layer based on the desired hematocrit of the collected product. Our practice is to collect a relatively clean GC product where there is less than 2 mL of red cell contamination in the GC unit. However, this is not always achieved and the GC units do have to be drained. Approximately 10-25 mL of red cells are drained depending on the collection and the GC units drained in this manner are comparable to the GCs drained after the addition of 50 cc of Hetastarch to the GC product.

Disclosures
No relevant conflicts of interest to declare.