TRANSPLANTATION AND CELLULAR ENGINEERING Comparison of human umbilical cord blood processing with or without hydroxyethyl starch Milad Souri,1 Mahin Nikougoftar Zarif,1 Mahboobeh Rasouli,2 Khadijeh Golzadeh,1 Mozhdeh Nakhlestani Hagh,1 Nasim Ezzati,1 and Kamran Atarodi1
BACKGROUND: Umbilical cord blood (UCB) processing with hydroxyethyl starch (HES) is the most common protocol in the cord blood banks. The quality of UCB volume reduction was guaranteed by minimum manipulation of cord blood samples in the closed system. This study aimed to analyze and compare cell recovery and viability of UCB processed using the Sepax automated system in the presence and absence of HES. STUDY DESIGN AND METHODS: Thirty UCB bags with a total nucleated cell (TNC) count of more than 2.5 3 109 were divided in two bags with equal volume. HES solution was added to one bag and another was intact. Both bags were processed with the Sepax. To determine cell recovery, viability, and potential of colony-forming cells (CFCs), preprocessing, postprocessing, and thawing samples were analyzed. RESULTS: The mean TNC recovery after processing and after thaw was significantly better with the HES method (p < 0.01 for the postprocessing step and p < 0.05 for the postthaw step). There were no significant differences to mononucleated cells (MNCs) and CD341 cell recovery between the two methods after processing and after thaw. TNC and MNC viability was significantly higher without HES after processing and after thaw (p < 0.01). The results of the CFC assay were similar for both methods after processing and after thaw. CONCLUSION: These results showed that processing of UCB using the Sepax system with the without-HES protocol due to the lower manipulation of samples could be used as an eligible protocol to reduce the volume of UCB.
A
t present, umbilical cord blood (UCB) has been widely accepted as a valid source of hematopoietic stem cells (HSCs) for transplantation.1 After the first successful UCB transplantation which was performed by Gluckman and colleagues,2 the first unrelated cord blood bank was established in 1993 by Rubinstein and coworkers3 to provide an alternative source of HSCs when an adult marrow or peripheral stem cell donor cannot be found. In recent decades more than 50 UCB banks have been established worldwide with over 400,000 UCB units repertoire.4 Despite the presence of valuable markers such as CD341 cells, colony-forming cells (CFCs), and viability of cells to predict the outcome of transplantation, total nucleated cell (TNC) count due to ease of standardization is used as a processing criteria through all UCB banks.5 Efficient use of financial resources and inventory size of UCB banks are being guaranteed through processing and cryopreserving high quality UCB samples and UCB units, respectively.1,5
ABBREVIATIONS: BC 5 buffy coat; CFC(s) 5 colonyforming cell(s); HSC(s) 5 hematopoietic stem cell(s); UCB 5 umbilical cord blood. From the 1Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine; and the Department of Bio-Statistics, School of Public Health, Iran
2
University of Medical Sciences, Tehran, Iran. Address reprint requests to: Kamran Atarodi, IBTO Building, Hemmat Exp. Way, Next to the Milad Tower, PO Box 14665-1157, Tehran, Iran; e-mail:
[email protected]. This work was supported by the Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran (Grant 1394-0133-1828). Received for publication January 2, 2017; revision received March 27, 2017; and accepted June 16, 2017. doi:10.1111/trf.14290 C 2017 AABB V
TRANSFUSION 2017;57;2758–2766 2758 TRANSFUSION Volume 57, November 2017
CORD BLOOD PROCESSING WITHOUT HES
To optimize space and cost of cord blood storage, volume reduction as a suitable procedure that concentrates TNCs by reducing the plasma and red blood cells (RBCs) in the UCB units has been used.6 Numerous studies were performed to find the desirable method for UCB volume reduction. First Rubinstein and colleagues7 used hydroxyethyl starch (HES) for RBC sedimentation in volume reduction of UCB units, and most researchers followed this procedure as mentioned. UCB processing has undergone many changes. Various methods have been proposed from manual techniques such as multistage centrifugal, semiautomated methods such as Optipress II (Baxter Healthcare) to fully automated systems such as AutoXpress Platform “AXP” (Thermogenesis Corp.) and “Sepax cell separation system” (S-100, Biosafe S.A.).8-13 TNC recovery is the most challengeable task in these methods and none of them recover 100% of the TNC. Sepax system provides a functionally closed processing method along with high TNC recovery.11-13 The software of this system can reduce the volume of UCB with or without HES. However, the HES method is the most common protocol for UCB processing. The addition of dimethyl sulfoxide (DMSO) as cryopreservative during non-HES protocol can lead to produce the qualified UCB unit with low risk of contamination and decreased undesirable side effects. This study described the performance of non-HES protocol to compare with HES method to evaluate volume reduction of UCB with the Sepax system.
main Sepax unit and the single-use kits that provided sterile processing of samples. It has many different protocols for UCB and peripheral blood processing. Cord blood banks mainly use the HES protocol to reduce the volume of UCB. Thirty UCB bags with TNC count of at least 2.5 3 109 were chosen as suitable samples. Before processing, each UCB bag was connected to a 150-mL transfer bag (R4R2001, Baxter) with sterile connecting device (TSCD-II, Terumo). The first half of the cord blood bag volume was transferred to a sterile transfer bag with the balance. Second the volume of each bag was calculated separately and the absolute counts of TNCs, mononuclear cells (MNCs), and CD341 cells were determined in each bag. The HES containing solution (6% HES in 0.9 NaCl; Grifols) was added with 20% concentration of UCB volume directly to one of the bags under sterile conditions. Sepax processing special kit (CS-530.1) was connected to each bag and the HES containing bag was processed with the usual Sepax processing protocol (UCB-HES, V304) and the other was processed by the without-HES protocol (UCB, V218). All of the common variables such as buffy coat (BC) volume in two protocols were the same. During this process, whole blood was separated into three parts, containing BC (usually 23 mL), RBC, and plasma bags with monitoring of Sepax optical line sensor and pneumatic system. All processed units along with reference samples were cryopreserved in liquid nitrogen tank.
Laboratory analysis
MATERIALS AND METHODS UCB collection Before UCB collection mother donors were qualified based on FACT-NetCord standards. Written informed consent was obtained before delivery from the parents for UCB collection based on the Research Ethics Committee at High Institute for Research and Education in Transfusion Medicine (Tehran, Iran). UCB was collected by gravity by experienced midwives trained for cord blood collection. This procedure was performed using a cord blood collection bag (IBSP-NP-BSBCLP16GJ, JMS) containing 22 mL CPD-A1. The UCBs were stored at room temperature (16-248C) and transported to the bank at the same storage temperature. All UCB units were processed within 24 hours. The preprocessing sampling was performed on the UCB units with more than 80 mL cord blood volume (except CPD-A1) to determine TNC count, CD341 absolute count, and cell viability. In usual protocols only cord blood units containing TNC count of at least 1.2 3 109 were volume reduced and finally banked.
Volume reduction of CB with the Sepax automated device Sepax is a closed and fully automated device that is controlled with software. The Sepax system consists of the
After preprocessing sampling, TNC count and cell viability for choosing the suitable UCBs was determined and to calculate the recovery percentage of CD341 cells in comparison methods, the CD341 absolute count was performed in picked samples. Immediately after the volume reduction of UCB, RBC count, TNC count, cellular viability, CD341 absolute count, and CFCs were assessed from the BC samples. To determine TNC and MNC loss, the total counts of nucleated cells, MNCs, and polymorphonuclear leukocytes (PMNs) were evaluated on RBC and plasma bags.
Hematologic cell count Nucleated cell count and differential count were analyzed with autoanalyzer (Sysmex XS-800i, Toa Medical Electronic) and, the number of TNCs, MNCs, and RBCs were calculated.
Immunofluorescence analysis of CD341 cells The absolute count of CD341 cells was obtained with flow cytometer (Partec PASIII, Partec GmbH) using a progenitor cell enumeration kit (CD34 count kit, Dako) according to the manufacturer’s instruction. The cell analyzing and absolute viable CD341 cells were calculated Volume 57, November 2017 TRANSFUSION 2759
SOURI ET AL.
with the single-platform ISHAGE protocol14 and Flowmax software.
Viability assay The viability of TNCs, MNCs, and PMNs was performed with flow cytometer (Partec PASIII, Partec GmbH) using propidium iodide (Sigma-Aldrich) and the viable percentage of cells was evaluated using Flowmax software.
CFC assay The clonogenic assays were performed with commercially complete semisolid medium (Methocult GF H4434, Stem Cell Technologies). For both BC and thawed samples, 2.5 3 104 nucleated cells were plated in duplicate petri dishes (35 mm diameter) and then incubated for 2 weeks in a humidified atmosphere at 378C in 5% CO2. Colonies containing at least 50 cells were counted and extended to a whole BC bag volume based on TNC count.
Cryopreservation of BC Cryopreservation of UCB BC bags was performed with the standard method.15 To prepare the reference sample, approximately 200 lL of BC was transferred aseptically into a cryotube. After freezing with controlled-rate freezer (Planer Kryo 560-16, Planer PLC), BC bags along with reference samples were transferred to the liquid nitrogen (LN2) tank.
Reference sample thawing After 3 months, the reference samples were thawed and removed from liquid nitrogen and then embedded in overwrap placed in a water bath at 378C and transferred into a biosafety cabinet. After being thawed, the samples were diluted sixfold in a solution with a 5:1 ratio of 10% dextran 40 (molecular weight 40000; Hospira) and 20% human serum albumin (Biovitrum AB).
Statistical analysis Descriptive statistics were presented for UCB variables and results expressed as mean 6 SD. Statistical analysis was carried out using a statistical software program (SPSS, Version 23, SPSS, Inc.). To investigate the normality of data sets the Shapiro-Wilk test was performed. Paired t student test was used for comparing the mean of normal data sets and to analyze of two related samples the Wilcoxon’s test was applied. Finally the Pearson test was used for correlation check between different variables. p value of not more than 0.05 was considered significant. 2760 TRANSFUSION Volume 57, November 2017
RESULTS Cell recovery Overall, 30 UCB bags with a total TNC count of at least 2.5 3 109 were divided into two bags and reciprocally processed with or without HES using the Sepax system. White blood cell (WBC) recovery was measured using values of differential cell count before and after UCB units processing. In addition to determine whether nucleated cells fractionated in a different part of the processed unit, WBC differential counts were also determined on RBC and plasma bags. The absolute count of CD341 cells in UCB units processing before and after volume reduction were quantified with a flow cytometer using progenitor cell enumeration kit based on ISHAGE protocol. Cell recovery results for UCB units processing before and after volume reduction with the two methods are summarized in Table 1. To compare the effect of the two methods in freezing and thawing on BC units, reference samples were assessed in terms of all variables as processed UCB units. Table 1 also shows extended data to BC units for thawed reference samples. TNC recovery was significantly higher for UCB units processed by the HES method. However, the recovery rates of MNCs and CD341 cells were the same with the two methods (Fig. 1A). The analysis of extended data for reference samples has shown the same distribution of WBC population and WBC recovery profile for BC units processing with or without HES (Table 1 and Fig. 1B). A significant correlation between initial TNC count and postprocessing TNC count were observed in HES method (q 5 0.968, p < 0.001; Fig. 2A); however, there was no significant correlation between pre- and postprocessing TNC count by the without-HES method (q 5 0.335, p > 0.05; Fig. 2B). There are significant correlations between initial TNC count and postprocessing MNC count in both methods (p < 0.001; Figs. 2C and 2D). These results show that increasing recovery of nucleated cells in HES method refers to the more harvesting level of PMNs with the HES method compared with another method. The effective separation of plasma and BC was addressed by the low number of nucleated cells in the plasma bag in both methods. As well as the distribution profile of WBCs in the RBC bags through tracking, the loss of nucleated cells verified the results. The data for nucleated cell loss through RBC bags are summarized in Table 1. The count of TNCs and PMNs in RBC bags in the without-HES method (318.9 6 237.2 and 266.8 6 225.2, respectively) was significantly more than with the HES method (103.0 6 54.2 and 54.9 6 33.8, respectively; p < 0.001 for both variables). However, there was no significant difference in MNC count of RBC bags between the two methods. These results show that changes in methodology do not adversely affect the harvest of MNCs.
CORD BLOOD PROCESSING WITHOUT HES
TABLE 1. Data from UCB differential cells counts, cell recovery, and cellular distribution in all steps of UCB processing with two methods* Variable
With HES
Without HES
TNC count (3106) MNCs (%) PMNs (%) Total MNC count (3106) Total CD341 cell count (3105)
1551.0 6 336.1 45.3 6 9.1 54.7 6 9.1 727.6 6 232.0 68.3 6 42.0
TNC count (3106) TNC recovery after processing (%) MNCs (%) PMNs (%) Total MNC count (3106) MNC recovery (%) MNC loss after processing (%) Total CD341 cell count (3105) CD341 cell recovery (%) RBC depletion (%)
1340.3 6 277.6 86.6 6 4.7 45.6 6 7.3 54.4 6 7.3 603.8 6 140.9 85.0 6 10.0 15.0 6 10.0 60.0 6 33.29 86.93 6 12.39 81.0 6 7.2
Total TNC count TNC recovery (%) MNCs (%) PMNs (%) Total MNC count (3106) MNC recovery (%) Total CD341 cell count (3105) CD341 cell recovery (%)
1298 6 300.9 96.2 6 7.1 41.1 6 7.1 58.9 6 7.1 531.8 6 159.6 91.4 6 11.9 33.6 6 24.0 59.8 6 25.7
RBC bag TNC count (3106) RBC bag MNC count (3106) RBC bag PMN count (3106)
103.0 6 54.2 48.7 6 26.7 54.9 6 33.8
Before volume reduction 1556.0 6 346.8 45.3 6 9.1 54.7 6 9.1 729.0 6 235.2 68.7 6 43.9 After processing 1129.2 6 305.1 74.2 6 18.7 57.5 6 13.7 42.5 6 13.7 621.4 6 121.3 87.9 6 12.3 12.1 6 12.3 62.3 6 33.7 90.0 6 12.0 77.8 6 9.0 After thaw 1022 6 3 14 90.2 6 9.6 53.1 6 14.3 46.9 6 14.3 511.1 6 107.5 87.0 6 9.9 38.4 6 24.7 62.7 6 22.4 RBC bag cell distribution 318.9 6 237.3 52.1 6 26.6 266.8 6 225.2
p value 0.589
0.754 0.682 0.004 0.003
