The document describes the process of red blood cell reduction for a bone marrow product received in multiple bags totaling 800-2000ml. The process involves taking initial samples, filtering the marrow, washing and concentrating it on a cell processor to extract the buffy coat layer containing stem cells. A sedimentation media is added to separate red blood cells from the buffy coat, which is then expressed into a new bag. A second sedimentation further reduces red cells, leaving a concentrated buffy coat of 2-5ml with minimal red cells. Albumin is added for stability and a final sample tests cell counts and sterility before infusion.
2. Weigh the Product
Marrow products are often received in multiple bags with a typical
cumulative volume between 800-2000 ml (pediatric products may be
significantly smaller). Red cell content can sometimes account for nearly
half of the total cell composition.
3. Take Initial Samples
A single bone marrow collection often arrives in multiple bags. In order
to assess the initial quality of the product, each bag is sampled for
ABO/RH, Cell Counts, Viability, and Sterility.
4. Filter the Marrow
The marrow product is filtered to remove contaminants (e.g. clots, fat
and bone fragments), in order to ensure efficient centrifugation,
concentration, and total cell recovery.
5. Wash and Concentrate the Marrow on the COBE 2991 Cell Processor
A cell washer is used to remove plasma, additives, and
excess red cell volume.
6. Centrifugation on the COBE Cell Processor creates a
cellular interface. The buffy coat layer at the inner
ring is collected (approximately 200ml) for further
processing.
Extract Stem Cells from Excess Supernatant and Red Cells
7. Remove Post Wash Sample
A post wash sample is removed for counts and sterility.
8. Prepare Sedimentation Media
A hydroxyethyl starch solution is added to the product creating a density
gradient. Mature red cells settle on the bottom, while the buffy coat
layer remains in the supernatant suspension with a small amount of red
cells. A culture media is also added to preserve PH and osmotic balance.
9. Express Supernatant After the First Sedimentation
After one hour, a distinct layer will be visible marking the supernatant
and red cell layers. The supernatant layer containing the buffy coat is
expressed into a new bag for further processing.
10. Weigh the Expressed Supernatant Buffy Coat Layer
The volume of the expressed product is determined after the first
sedimentation in order to calculate the correct ratio of media
required for an effective red cell reduction.
11. Express Supernatant After the Second Sedimentation
A second sedimentation is performed. After 45-60 minutes the
supernatant layer containing the buffy coat is collected into the
product bag .
12. Centrifuge the Expressed Product
The product is centrifuged, separating the buffy coat layer from the media.
13. Express the Media from the Product
The media is expressed from the product leaving a
concentrated buffy coat with minimal red cells
(typically 2-5 ml).
15. Add 5% Human Albumin to the Product.
5% human albumin is added to the product to stabilize
the cells for infusion.
16. Remove Pre-Infusion Sample
A final sample is removed and sent for cell counts, viability, and sterility. CD34
enumeration is performed for cell dose calculations, revealing how many
hematopoietic stem cells are present in the product. Representative QC
samples are also cryopreserved if potency testing is requested at a later date.
17. Final Marrow Product: Post Red Blood Cell Reduction
The final product volume is typically between 100-
150 ml, with an RBC content of 2-5 ml.
