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iv. PROCESSING, CRYOPRESERVATION AND INFUSION
- Does the volume reduction manipulation before cryopreservation influence cord blood cell recovery pretransplant? Screnci M, Salvatori S, Carmini D, Arcese W. Transfus Med. 2007;17:208-9
The authors point out that cell recovery pre-transplant could be crucial especially for large-sized patients or when cell content is not exceeding the minimum recommended. They cite two observations that have to be considered:
- a median cell loss of 25% after thawing with the removal of DMSO as reported by Laughlin et al (NEJM 2001;344:1815-1822)
- a better NC recovery when CBs are infused without manipulation (Hahn et al – Bone Marrow Transplant. 2003;32:145-150; and Laroche et al Transfusion 2005;45:1909-1916)
To investigate whether the volume reduction process including RBC reduction before freezing can influence progenitor cell recovery after thawing, the authors analyzed results obtained in 78 CB units, 54% of which were unmanipulated and 46% of which were manipulated. Volume reduction was carried out by several methods: HES sedimentation; by differential centrifugation with automated expression of erythrocytes and plasma; and centrifugation with manual expression of plasma. Before infusion, all 78 samples were thawed and washed according to the procedure described by Rubinstein (1995). There was no difference in total NC counts or time of storage between the two groups of CB units (unmanipulated vs. manipulated) before thawing.
After thawing and washing, NC recovery was better in the group of unmanipulated) CB than in the group of CB processed before storage (95.2 ± 14.7% recovery vs. 85 ± 15.4% recovery), and the difference was statistically significant (p=0.004). There was no difference in cell loss between the HES vs. non-HES volume reduction methods.
The authors conclude that their data indicate that cell recovery after thawing is significantly lower in CB units manipulated prior to storage. The authors state that in cases of CB units with a borderline cell dose, it could be reasonable to select the unmanipulated CB units. Alternatively as suggested by Hahn et al and Laroche et al, CB units could be infused immediately after thawing without additional manipulation.
- Analysis of hematopoietic cell transplants using plasma-depleted cord blood products that are not red blood cell reduced. Chow R, Nademanee A, Rosenthal J, Karanes C, Jaing TH, Graham ML, Tsukahara E, Wang B, Gjertson D, Tan P, Forman S, Petz LD. Biol Blood Marrow Transplant. 2007;13:1346-57.
The authors have developed and evaluated the use of CB products from which plasma but not RBC have been removed in order to minimize cell loss. CB units were processed by centrifugation in the original collection bag at 1680 X g for 10 minutes at room temperature followed by variable amounts of plasma removal into an attached empty bag using a plasma expressor to reach a final CB volume of 60-84 mL before adding cryoprotectant solution. The plasma depleted (PD) products were then pre-cooled at 4°C for at least 30 minutes prior to cryoprotectant addition. The products were subjected to controlled rate freezing and, after attaining -90°C, were immediately transferred from the controlled rate freezer to liquid nitrogen tanks. There was <0.1% total nucleated cell (TNC) loss incurred by processing as measured by recovery in the discarded plasma fraction (n=27); however, due to sampling for various tests and archival purposes and clumping after processing, an average of 98% TNC recovery was observed in the cryopreserved units in the inventory.
The effect of CB processing techniques on post-processing cell dose among the NMDP banks was studied. Using information derived from the NMDP Cord Blood Bank Performance data, the percentage of high cell dose units among the 2 types of inventories (PD vs. RBC-reduced) in the NMDP were compared. At all 3 TNC count levels (≥125, ≥150, and ≥200 X 107), the difference in proportions of inventories with high cell dose was significantly in favor of PD inventory (p<0.0001).
Analyses were carried on 118 infused products. Prior to infusion of the thawed CB, the product was washed in 67 instances whereas 50 products were infused without washing per the transplant center’s decision (information unavailable for 1 unit). Although CB products that were washed had a higher median and mean TNC dose than unwashed products prior to cryopreservation, the post-thaw TNC dose as reported by the transplant centers was lower for washed products than unwashed products. Unwashed products engrafted faster for ANC500 (20 days vs 27 days) (log rank test; p<.02) and platelets (47 days vs 54 days (log rank test; p=.0003). The incidence of grade III-IV aGVHD and extensive cGVHD among all patients were 13% ± 4% and 17% ± 6%, respectively. Relapse rate for malignancies was 25% +/- 6% and 100-day treatment-related mortality (TRM) was 16% +/- 3%. With a median follow-up of 557 days, the 1-year overall survival and relapse-free survival are 65% +/- 5% and 51% +/- 6%, respectively.
Analysis of infusion-associated adverse reactions was divided into patients who received post-thaw washed products (W) and those who received CB that were not washed after thaw (NW). Events occurring during or after infusion include hypertension (6NW, 4W), hives (1NW, 1W), nausea/vomiting (2NW, 4W), and dyspnea (1NW, 1W). Hemoglobinuria (9NW, 1W) is an expected occurrence for PD CB because most RBCs lyse after thawing. Most symptoms attributed to DMSO administration were self-limiting or easily managed. One patient developed seizure and encephalopathy, although the relationship to infusion was uncertain.
The authors point out that in centers that have opted not to wash the units after thawing, the additional 10%-30% cell loss due to wash is avoided. There were no clear benefits of performing post-thaw wash for PD CB.
The authors concluded that their results demonstrate that PD CBT is safe and effective, and that eliminating RBC reduction improves cell recovery during CB processing, resulting in a larger proportion of the inventory with high NC number.
- Assessment of cord blood unit characteristics on the day of transplant: comparison with data issued by cord blood banks. Wagner E, Duval M, Dalle JH, Morin H, Bizier S, Champagne J, Champagne MA. Transfusion. 2006;46:1190-8.
(Also see Citation #12)
The goal of this study was to compare the graft characterization results obtained upon thawing and washing to those provided by CBBs at selection. With tests that assess CB graft characteristics known to impact engraftment, CB units were analyzed after thaw and before infusion. The results were compared to data provided by CBBs to determine the impact on engraftment and assess how CBB-supplied information can affect future CB unit selection.
All CBBs provided information on unit volume and TNC content; 96% of CBBs provided CD34+ cell content; 78% provided data on CFC content; and 43% provided results of cell viability testing. Most of the CBBs (83%) provided such information on measurements before cyropreservation.
TNC content correlated rather well, both upon thawing (r2= 0.085) and after thawing and washing (r2=0.799). However, highly discrepant results were obtained for CD34+ cell enumeration, CFC content and cell viability.
The authors suggest that 7-aminoactinomycin D staining is preferable to trypan blue dye exclusion for testing cell viability and that the latter method is of little value in assessing CB units.
In six patients, important differences may have accounted for the lack of neutrophil engraftment. The discrepancies included finding only 8% of the reported CD34+ content, and no growth or very low growth (1% of reported CFU-GM content) of hematopoietic progenitor cells.
Recent studies suggest that CB samples from the attached segment of the freezing bag or a separate cryovial can accurately predict the graft content. If validated on a larger scale, the assays should ideally be performed on a thawed sample as precryopreservation testing may not predict the quality of a CB unit.
[Comment: How to evaluate the quality of an individual cord blood unit remains a problem in CB transplantation. Continued research on inter-laboratory standardization of methods is necessary. So far the results of such efforts have not been satisfactory. Results on a thawed sample would seem to be most representative of the infusion dose and may become the standard approach.]
- Multi-laboratory evaluation of procedures for reducing the volume of cord blood: influence on cell recoveries. Takahashi T, Rebulla P, Armitage S, van Beckhoven J, Eichler H, Kekomaki R, Letowska M, Wahab F, Moroff G, For The Biomedical Excellence For Safer Transfusion Collaborative. Cytotherapy. 2006;8:254-64.
Various procedures can be used to isolate stem and progenitor cells from cord blood. This study evaluated the hydroxyethyl starch sedimentation (HES) with two centrifugation steps, and the top and bottom (T&B) isolation of buffy coat following a single centrifugation, and two filter systems for processing cord blood, one developed by Asahi Kasei Medical (filter A) and the second by Terumo (filter B). Each of seven laboratories was randomly assigned the evaluation of either the HES or T&B method and one of the filter methods (n=8 cord blood units, per laboratory, for each method). The composite results obtained by the seven laboratories were summarized.
Results of cell recovery of cells with various methods were as follows:
| Method | Median TNC Recovery (%) | Median Mononuclear Cell Recovery (%) |
| HES | 79 | 72 |
| T&B | 86 | 96 |
| Filter A | 58 | 79 |
| Filter B | 61 | 70 |
| Group Traditional Methods | 82 | 87 |
| Filtration Methods | 58 | 75 |
The authors concluded that filters that capture stem and progenitor cells may be an appropriate methodology for processing cord blood collected for banking.
[Comment: Cell loss with each of the methods is significant. Cell dose has repeatedly been emphasized as critical to the success of cord blood transplantation. The cell losses reported in this study may significantly impact the appropriateness of a given unit for transplantation. Whether the value of removal of RBCs from the units compensates for the stem cell loss is being re-considered. This topic was discussed by Dr. Michael Creer at the Fourth Annual International Umbilical Cord Blood Transplantation Symposium. A CD with the audio and the slides of all speakers is available (See Home Page for details).]
- Multiple-laboratory comparison of in vitro assays utilized to characterize hematopoietic cells in cord blood. Moroff G, Eichler H, Brand A, Kekomaki R, Kurtz J, Letowska M, Pamphilon D, Read EJ, Lecchi LP, Reems JA, Sacher R, Seetharaman S, Takahashi TA; Biomedical Excellence for Safer Transfusion (BEST) Collaborative. Transfusion. 2006;46:507-15.
The authors conducted four exercises performed by multiple laboratories to assess assay variability on nucleated cell (NC), mononuclear cell (MNC) by hematology analyzers [HAs], and CD34+ cell (flow cytometry) measurements.
Intralaboratory reproducibility was highest for NC measurements and lowest for CD34+ cell measurements. Substantial variation was observed on measuring CD34+ cells
The authors concluded that it is important to recognize that the cell counts provided by processing laboratories to judge quality and suitability of a cord blood unit could be influenced by assay variability. Currently NC counts obtained with a HA is the preferred variable for judging quality and suitability. Although CD34+ cell counts may be a more important parameter for transplantation, the variation in assay results suggests that initially more standardized and interlaboratory validation of specific procedures is required. In addition, recent studies suggest that some of the CD34+ cells determined may be nonviable and that additional reagents may need to be utilized in the assays to exclude nonviable cells.
- Multiple-laboratory comparison of in vitro assays utilized to characterize hematopoietic cells in cord blood. Creer MH. Transfusion. 2006;46:498-500.
This is an editorial by the medical director of a large American cord blood bank. He reviewed the article by Moroff et al and pointed out that the results of the study are, in general, very consistent with the results of an NMDP cord blood external proficiency study. Collectively, these two studies demonstrate that significant improvements in accuracy and precision of measurements of WBC subsets and CD34+ cell concentration in cord blood will be required before we can expect to be able to demonstrate a consistent correlation between these measurements and cord blood transplant outcome.
- Preservation of immunological and colony-forming capacities of long-term (15 years) cryopreserved cord blood cells. Kobylka P, Ivanyi P, Breur-Vriesendorp BS. Transplantation. 1998;65:1275-8.
Cryopreserved cord blood may be stored for decades before being used for allogeneic stem cell transplantation. The authors examined the recovery, viability, clonogenic capacity, and T-cell reactivity to HLA alloantigens of cord blood samples cryopreserved up to 15 years.
Progenitor cell recoveries were studied by (colony-forming unit-granulocyte-macrophage) clonogenic assays from 18 cord blood samples short-term frozen for 2-8 weeks and from 8 samples cryopreserved for 15 years. Proliferative and cytotoxic responses against HLA antigens of thawed cord blood mononuclear cells after short-term or long-term cryopreservation were tested in standard mixed lymphocyte cultures and cell-mediated lympholysis assays.
After thawing, the mononuclear cell recovery from long-term frozen cord blood low-density fractions averaged 80% (range, 64% to 92%). The data show that long-term frozen cord blood cells keep their clonogenic potential. No damaging effect was seen on the proliferative and cytotoxic capacities of long-term frozen cord blood T cells.
The results support the possibility of long-term storage of progenitor cells from umbilical cord blood for future bone marrow reconstitution.
- Prospective flow cytometric evaluation of nucleated red blood cells in cord blood units and relationship with nucleated and CD34(+) cell quantification. Larghero J, Rea D, Brossard Y, Van Nifterik J, Delasse V, Robert I, Biscay N, Chantre E, Raffoux E, Socie G, Gluckman E, Benbunan M, Marolleau JP. Transfusion. 2006;46:403-6.
Nucleated red blood cells (NRBCs) are a physiological subset of CB population and high NRBC numbers can falsely elevate WBC counts requiring correction. The authors analyzed 826 CB units for total nucleated cells (TNCs), NRBCs, and CD34+ cells by flow cytometry. NRBCs were also counted conventionally by manual microscopy.
For 77.7% of the CB units, NRBC percentage was less than 10%. However, NRBC percentage was between 10 and 15% for 12% of the units and more than 15% for 10.3% of the units, with a maximum of 84%, thus demonstrating an important quantitative heterogeneity in CB.
The mean percentage of CD34+ cells was 0.27 percent (range, 0.01%-1.25%); when corrected for NRBC count, the mean percentage was 0.295% (p=0.0008 compared with the uncorrected percentage). The mean uncorrected TNC count was 16.26 x 108 and the corrected count was 14.8 x 108 (p<10-4 compared to the uncorrected count).
The authors indicate that their findings could have implications for CB QC given that CD34+ and TNC numbers predict engraftment and transplant outcomes. Also, a high NRBC percentage in a cord blood unit could lead the cord blood bank to further investigate is possible causes, such as sickle cell disease for which elevated numbers of circulating NRBC in the CB of neonates born to mothers with sickle cell trait has been described.
[Comment: This study emphasizes the obvious fact that a high percentage of NRBCs in a cord blood unit can result in misleading figures for WBC and CD34+ cell counts if corrections are not performed. Although one study has suggested that the presence of NRBCs in CB units does not reduce their engraftment potential, this result is counter-intuitive and has not been confirmed.]
