Improvement of storage of blood products
Project leader: Margriet Dijkstra-Tiekstra PhD
Stem cell studies
The Dept of Transfusion Monitoring (TraM) performs various research projects for the stem cell department, to optimize the freezing process for stem cells. Because hematopoietic progenitor cells (HPC) are not readily available for research purposes, a HPC simulant from buffy coats and plasma, with comparable cell counts and hematocrit but absence of CD34+ cells was developed by TraM for research purposes.
Various freezing bags have been tested and one of these has been validated for routine use, because the standard freezing bag will no longer have the requested CE mark.
Since the large molecular weight (200kDa) hydroxyethyl starch (HES) will no longer be available for sedimentation of bone marrow stem cells comparison experiments were done with lower molecular weight (130 kDa) HES and also sedimentation without HES was tested. For this the influence of volume and hematocrit was taken into account. The RBC depletion was highest for HES of 200 kDa and for no HES, while WBC recovery was acceptable in all tested conditions. The volume for sedimentation was optimal at 110-140 ml and the hematocrit influenced both WBC recovery and RBC depletion. While WBC recovery was best for a product with a low hematocrit (5 or 10%) the RBC depletion was best for a product with a hematocrit of 20-30%. Finally, the sedimentation process by either gravity, as is standard procedure, or by centrifugation was compared. It appeared that for RBC depletion there was no difference, but WBC recovery was much higher for sedimentation by gravity (about 80%) than by centrifugation (about 30%).
To optimize the freezing procedure for HPC experiments were performed to find the optimal volume for freezing stem cells. It appeared that 30 ml per freezing bag resulted in a better viability of WBC than 60 ml per freezing bag. However, a small volume per bag asks a large storage capacity. For this a balance between optimal and maximal need to be find.
Figure 1: Sedimentation of HPC-simulant with use of HAES, Voluven or no additive by centrifugation or gravity influences the WBC recovery. *, #, $ p<0.05 between sedimentation by centrifugation and gravity.
Dijkstra-Tiekstra MJ, Setroikromo AC, de Wildt-Eggen J. Freezing 'stem cells' in a bag and tube under various freezing conditions? Vox Sang 2012; 102(3):273.
Dijkstra-Tiekstra MJ, Setroikromo AC, Kraan M, Gkoumassi E, de Wildt-Eggen J. Optimization of the freezing process for hematopoietic progenitor cells: effect of precooling, initial dimethyl sulfoxide concentration, freezing program, and storage in vapor-phase or liquid nitrogen on in vitro white blood cell quality. Transfusion 2014 Aug 11 [Epub ahead of print].
Every year TraM organizes proficiency studies to compare the results of different instruments used within the different departments of the Blood Bank division. Samples are prepared centrally at TraM and distributed to the participants. Afterwards the results are collected and analyzed. Reports with conclusions are written and sent to the different departments. In 2013 again five national proficiency studies were executed.
1. Comparison of cell counters for counting platelets in platelet concentrates in plasma and storage solution
2. Comparison of flow cytometric instruments for counting leukocytes in plasma, red cell concentrates, platelet concentrates in plasma and platelet storage solution
3. Comparison of blood gas analyzers for measuring pH in platelet concentrates
4. Comparison of flowcytometric instruments for measuring platelet activation in platelet concentrates using CD62p expression and Annexin V binding
5. Comparison of flowcytometric instruments for counting platelets using CD41 expression.
For the proficiency study 1, 2, 3 and 5 trend analysis reports were also written, using data from 2006 to 2011, 2012 or 2013
Figure 2: Results of a trend analysis for low level leukocyte counting in platelet concentrate (TC) in plasma. The Z-score is used to measure the deviation from the ‘send-around’ mean and is expected to be zero. The Z-score is calculated as follows: (valuelab – averageoverall)/ SDoverall. The leukocyte levels (per µl) tested are shown in the legends of the figure.
Van der Meer PF, Karssing-van Leeuwen W, Kurtz J, Spengler HP, Blair A, Devine D, Harrison P, Lambrecht B, Vandenbroeke T, de Wildt-Eggen J, de Korte D; on behalf of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative. A flow cytometric method for platelet counting in platelet concentrates. Transfusion 2012; 52:173-80.
Gkoumassi E, Dijkstra-Tiekstra MJ, Hoentjen D, de Wildt-Eggen J. Hemolysis of red blood cells during processing and storage. Transfusion 2012; 52(3):489-92.
Gkoumassi E, Klein-Bosgoed C, Dijkstra-Tiekstra MJ, De Korte D, De Wildt-Eggen J. Non-invasive pH monitoring pH of platelet concentrates: a large field-test. Transfusion 2013; 53: 2287-92].