Performance Validation of Revolution Centrifugal Force (RCF) for Plasma Depletion on Hematopoietic Progenitor Cell Apheresis Product Using Sorvall ST40R Centrifuge

Background: Original Performance Qualification on Sorvall ST40R Centrifuge for plasma depletion on Hematopoietic progenitor cell (HPC) product was set at 300g Revolution Centrifugal Force (RCF) 10 minutes at 20°C to achieve ≥ 80% Total Nucleated Cell (TNC) recovery. Cellular Therapy Lab (CTL) noticed the spun down product has unclear supernatant plasma, loosely packed cellular sedimentation with difficulty for separation. CTL has decided to re-validate the process to find an optimal RCF for a clear & defined separation between plasma and Buffy coat, maintaining ≥ 80% TNC recovery.

 Study Design and Methods: Research performed on practice recommendations and reference materials lead to select three variables of RCF: 700g, 1000g, and 1300g for this study. Ten samples were collected from different sources: single donor whole blood; pooled whole blood;  pre HPC,Apheresis peripheral  blood; HPC product and Donor Lymphocyte product. Sample size varied from 21ml – 120ml. Each sample was divided into three equal portions centrifuged for 10 minutes at 20°C at the selected RCF.  White blood cell (WBC) count was tested by the Sysmex cell counter. Viability was tested with Trypan Blue method.  The following data and test results were recorded for each portion pre and post centrifugation: sample volume,  WBC,  packed cell volume, plasma volume, Post plasma WBC, calculated TNC, Viability%, TNC % recovery, Clarity of spun sample, and Appearance of Buffy-coat. One way analysis of Variance was run to assess the P value for statistical significant difference. Optimal RCF was selected after this study. Validation of the selected RCF was performed on 5 HPC products from prior transplant recipients.  

Result: The study found no statistically significant difference among the 3 RCF variables with P=0.67. The difference between Pre & Post Viability was less than 3 % which indicates minimal cell damage during centrifugation. Observation found hazy plasma and loose Buffy Coat with 700g, clear and defined with 1000g and 1300g.  CTL selected RCF 1300g for plasma depletion. Validation study on 7 processes found post TNC plasma range of 0.01-0.10% of pre TNC, difference in Pre & Post Viability is 0-1%, five patients with successful absolute NC and platelet engraftment. Results of retro study compared to 7 random processes using prior RCF 300g found P= 0.352.

RCF selection Result: TNC % Recovery	RCF 700g	RCF 100g	RCF 1300g
Range	91-106	91-100	93-100
Median	95.5	98.5	97.0
Standard Deviation	4.22	2.62	2.41

Comparison Result:  TNC % Recovery	Prior  RCF 300g	Implemented RCF 1300g
Range	85-93	85-98
Median	88	90
Standard Deviation	3.13	3.99

Conclusion: The study found no significant difference in TNC % recovery using 700g, 1000g and1300g. There is defined cell pellet and clear plasma with higher RCF for ease of separation which help to harvest the final concentrated cellular product for cryo-preservation; especially using small volume bag.