Transfusion and Apheresis Science 50 (2014) 129–135
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Cord blood banking activity in Iran National Cord Blood Bank: A two years experience Mostafa Jamali a,b, Kamran Atarodi a,b, Mozhdeh Nakhlestani a,b, Hasan Abolghasemi c, Hosein Sadegh a, Mohammad Faranoosh a, Khadije Golzade a,b, Razieh Fadai a, Fereshte Niknam a,b, Mahin Nikougoftar Zarif a,b,⇑ a b c
Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran Iran National Cord Blood Bank, Iran Blood Transfusion Organization, Tehran, Iran Pediatric Congenital Hematologic Disorders Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 11 May 2013 Received in revised form 9 September 2013 Accepted 30 September 2013
Keywords: Cord blood bank Cord blood transplantation Hematopoietic stem cells
a b s t r a c t Today umbilical cord blood (UCB) has known as a commonly used source of hematopoietic stem cells for allogeneic transplantation and many cord blood banks have been established around the world for collection and cryopreservation of cord blood units. Herein, we describe our experience at Iran National Cord Blood Bank (INCBB) during 2 years of activity. From November 2010 to 2012, UCBs were collected from 5 hospitals in Tehran. All the collection, processing, testing, cryopreservation and storage procedures were done according to standard operation procedures. Total nucleated cells (TNC) count, viability test, CD34+ cell count, colony forming unit (CFU) assay, screening tests and HLA typing were done on all banked units. Within 3770 collected units, only 32.9% fulfilled banking criteria. The mean volume of units was 105.2 ml and after volume reduction the mean of TNC, viability, CD34+ cells and CFUs was 10.76 108, 95.2%, 2.99 106 and 7.1 105, respectively. One unit was transplanted at Dec 2012 to a 5-year old patient with five of six HLA compatibilities. In our country banking of UCB is new and high rate of hematopoietic stem cell transplants needs expanding CB banks capacity to find more matching units, optimization of methods and sharing experiences to improve biological characterization of units. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Since the first successful umbilical cord blood (UCB) transplantation in 1988, the use of UCB has been increased for treatment of a variety of hematopoietic malignancies, immunological and some hereditary disorders [1–4]. Several researches have focused on systematically evaluation of human UCB hematopoietic stem cells (HSC) potential and developing practical and efficient methods for the volume reduction, cryopreservation and storage of UCB units ⇑ Corresponding author at: Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran. Tel.: +98 21 88601575; fax: +98 21 88601576. E-mail address: [email protected] (M.N. Zarif). 1473-0502/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.transci.2013.09.012
[5–8]. In parallel, many cord blood banks have been established worldwide in order to bank UCB units for unrelated donor transplantation. For improving the quality of banked UCB samples, some international organizations were created. Netcord and Foundation for the Accreditation of Cellular Therapy (FACT), in collaboration with National Marrow Donor Programme (NMDP) established international standards whose major objects are to improve quality throughout all phases of UCB banking, including good practices in UCB storage, facilitate donor search, improve the quality of grafts, standardize excellence criteria on an international scale and importantly established procedures for bank accreditation, and cooperation of large experienced UCB banks [9–11]. Today, more than 600000 UCB have been banked and more than 30000 UCB units
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transplanted to children and adults [12]. The optimal number of cord blood units to cover maximum necessities is not really known, because of HLA diversity [13–15], but, based on some reports; it should approach 9 for 10000 inhabitants [16]. However, because of high prevalence of cancers and hematologic disorders in Iran [17] and consequently high necessity of transplant therapy, also, different HLA allele’s distribution among racial groups, it is justified having public CB banks in Iran. Over the past 2 years, Iran National Cord Blood Bank (INCBB) has been established under supervision of Ministry of Health in order to increase the pool of potential CB units for allogeneic transplantation. The purpose of this article is to describe experiences and current results of 1732 UCB units that were banked in INCBB during 2 years. All the procedures were done according to the last version of international standards published by Netcord/FACT organization and some local criteria.
2. Materials and methods 2.1. Donor selection Selection of donors is one of the most important steps in UCB banking. At first, a trained physician interviewed mothers and obtained extensive and accurate medical history of the family from informed consent donors. If some cases did not fulfill selection criteria, based on the local and the Netcord/FACT standards, they were excluded from collection. The selected donors signed consent form and were candidate for donation. 2.2. Cord blood collection To reduce the risk of microbial and maternal contamination closed collection system was used. Trained midwives collected UCB units at 5 maternity hospitals in Tehran. The samples were collected into cord blood collection bags (JMS, Singapore) contained 22 ml citrate phosphate dextrose adenine (CPDA) after normal vaginal delivery (NVD) or caesarean section (CS). 20 ml of mother venous blood were obtained in 2 tubes with anticoagulant reagent and 2 other tubes without anticoagulant (BD Pharmingen, CA, USA) within 4 h of delivery. All samples were labeled and transferred to the CB bank laboratory at 16–24 °C together with the documents. Medical exam of mother and newborn were done after 24 h of delivery to rule out any complications. 2.3. Cord blood pre volume reduction/processing criteria After checking documents, appearance and characteristic of units, CB volume was calculated. If the volume was more than 60 ml, some aliquots were removed for testing. Total nucleated cell (TNC) count and viability test were done and if the absolute nucleated cells were more than 8 108 per units and more than 85% of TNCs were viable, it was selected for the processing. The TNC cut off was
increased to 12 108 per units after 20 months, therefore; affected data presented in different groups. 2.4. Cord blood volume reduction/processing In order to perform cord blood volume reduction, the Sepax (Biosafe, Geneva, Switzerland) was used as automated cell separation method. For better Red Blood Cell (RBC) depletion and more TNC recovery, Hydroxyl Ethyl Starch (HES 200, 0.6%, Fresenius Kabi, Hamborg, Germany) in concentration of 2:5 (total volume of CB and CPDA) was added to the samples; after 30 min incubation at room temperature (RT) CB bag was attached to the Sepax processing kit (Sepax kit 530.1, Biosafe, Geneva, Switzerland) under sterile condition and run with the UCB-HES programme in cell separator. In this process, buffy coat was extracted under centrifugation and the bag of buffy coat was detached. The TNC, viability, CD34+ cell count and colony forming unit (CFU) assays were performed on the extracted buffy coat before cryopreservation. Where TNC count was more than 6 108, the buffy coat bag was placed in an automated mixing and cooling device (Coolmix-210, Biosafe, Geneva, Switzerland). Dimethyl sulfoxide (DMSO) and dextran 40 (CryoSure-Dex40, WAK-Chemie, Steinbach, Germany) was added to the buffy coat in 10 min cooling and mixing (concentration of 1:4 buffy coat volume). Three samples were prepared for the pre transplantation tests and sealed buffy coat bag was protected in an aluminum canister, transferred to a programmable control rate freezer (Planer, Middlesex, UK) in order to reduce the sample temperature to 100 °C. The freezing programme is illustrated in Table 1. Cord blood units were stored in vapor phase of nitrogen quarantine tank at 150 °C up to the second mother screening test. Sample safety and confirmed second mother screening test, after 6 to 9 months of delivery, allow storage of samples in liquid nitrogen tanks at 196 °C. 2.5. TNC count, viability test, CD34+ absolute count and CFU assay TNC count and viability test were done on the units, pre and post volume reduction; CD34+ cell count and CFU assay only on post volume reduction. Two Wright stained slides were prepared from each sample for nucleated RBC (nRBC) and differential count. The TNC was calculated based on the White Blood Cell (WBC) extracted from Sysmex X800i (Sysmex, Tokyo, Japan) and nRBC percentage. For detection of viable TNCs, Propidium Iodide (PI) (Sigma, St. Louis, USA) in concentration of 0.01% in PBS was used. As the dead cells absorb PI dye, any cells that emitted more red-fluorescent in comparison to control cells (without adding PI), was considered as dead cells. The absolute count of viable CD34+ cells were quantified using CD34 count kit (DAKO, Glostrup, Denmark) containing microbeads by flowcytometery (Partec, Munster, Germany). According to the manufacturer’s instruction, CD45 dim cells that expressed bright CD34 and did not absorb 7 Amino Actinomycin D. Colonogenic capacity of HSCs was determined using culture of 50000 TNC in 1 ml of commercially prepared
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M. Jamali et al. / Transfusion and Apheresis Science 50 (2014) 129–135 300
300
R=0.209
R=0.665 250
p=0.000
CB Volume (ml)
CB Volume (ml)
250 200 150 100
p=0.000
200 150 100 50
50
0
0 0
5
10
15
20
25
30
35
0
40
5
TNC (x10e8)
15
20
25
30
25
30
30
20
R=0.435
R=0.429 25
p=0.000
15
Total CFU (x10e5)
Total CD34 (x10e6)
10
Total CFU (x10e5)
10
5
p=0.000
20 15 10 5 0
0 0
5
10
15
20
25
0
30
5
10
15
20
TNC (x10e8)
TNC (x10e8) 60
20
R= -0.126
R=0.434 p=0.000
15
Nucleated RBC %
Total CD34 (x10e60)
50
10
5
p=0.000 40 30 20 10 0 -10
0
0
5
10
15
20
25
30
70
75
80
85
90
95
100
105
Viability %
Total CFU (x10e5) Fig. 1. Correlation between CB unit parameters.
Table 1 The freezing programme of control rate freezer for the sample temperature reduction from +4 °C to 100 °C. Temperature induction phase
Temperature alteration per minutes (°C)
First phase Second phase
Third phase
1 20 +10 1 10
Start (°C)
Stop (°C)
+4
+4
+4 4 60 30 75 100
4 60 30 75 100 100
methylcellulose medium containing cytokines (Methocult GF H4434, Stem cell technologies, Vancouver, Canada). This medium supports the growth of Burst Forming Unit
Erythroid (BFU-E), Colony Forming Unit Granulocyte Monocyte (CFU-GM) and Colony Forming Unit Granulocyte Erythroid Monocyte Megakaryocyte (CFU-GEMM). After 14 days incubation at 37 °C and 5% CO2, all the colonies were counted and differentiated.
2.6. Screening tests Microbial culture and all the screening tests [Cytomegalovirus antibody (IgG, IgM), Rapid Plasma Regain (RPR) for syphilis, Hepatitis virus B and C (HBs Ag, Anti HBs, Anti HBc and Anti HCV), Human Immunodeficiency Virus (HIV Ag– Ab) and Human T-Lymphotrophic Virus (Anti HTLV1&2)] were done on the cord blood plasma extracted after processing. In case of any indeterminate results, nucleic acid based test (NAT) was done for more evaluation.
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2.7. HLA typing HLA typing was performed on DNA obtained from the buffy coat samples. A PCR based method with sequence specific primers (SSP) and sequence specific oligonucleotide probes (SSOP) were carried out for the class I (A and B alleles) and class II (DRB1 alleles) detection. Further HLA typing on the other alleles and high resolution typing was done based on the transplant center request. Cellulose Acetate Electrophoresis at Alkaline pH was done to identify hemoglobinopathies and in case of more investigation, HPLC was used. ABO and Rh grouping were done on all samples. 2.8. Mother samples Buffy coat extracted from the EDTA samples for confirmation of one haplotype in case of release was stored at 80 °C. All screening tests, except microbial cultures that were done on the cord blood were done also on the mother serum, at the time of delivery and again after 6–9 months of delivery. 2.9. Sample release When a unit was selected for transplantation, before reserving, a group of tests including TNC count, viability test, and CD34+ absolute count and CFU assay were done on the segment. In case of transplantation, the unit was placed in the shipment Dewar in liquid nitrogen and together with all documents transferred to the transplant center. 2.10. Statistical analysis Statistical analysis was performed using SPSS software, version 16.5. Based on the distribution pattern of data, descriptive analysis was used to evaluate the biological characteristic of CB units. Multivariate linear regression analysis and Pearson’s correlation coefficient were used for detection of association between CB unit parameters. 3. Results 3.1. Collection activities From November 2010 to November 2012, a total of 6472 pregnant women at 5 hospitals in Tehran near the INCBB, were interviewed and about 81% of them agreed to CB donation. Within 5261 cases, 1491 (28.34%) were excluded at the collection sites, while, 2038 (38.74%) were rejected at the CB bank laboratory and only 1732 (32.92%) were successfully processed and stored, also; 30 units were selected for the quality control tests randomly. All major activities and the main reasons of exclusion at the collection sites and laboratory are summarized in Table 2. The comparison of banking rate between two pre processing TNC cut off groups revealed 33.52%, when the TNC cut off was 8 108, while it decreased to 29.23% when it was 1.2 108 (p value < 0.5).
The average of pregnancy age was 38.7 ± 0.9 weeks and 53.2% of infants were male and 46.8% were female. 3.2. The biologic characteristics of the units The mean ± SD of CB volume was 105.2 ± 24.9 ml and the buffy coat volume decreased to 22.11 ± 1.48 ml after volume reduction with Sepax cell separator. This criterion for the post volume reduction TNC, was 10.76 ± 3.03 108 and the TNC recovery rate was 79.1 ± 9.2% (range: 58.9– 99.7%). Other parameters are listed in Table 3. The amount of total CD34+ cells was 2.99 ± 2.08 106 (range: 0.51–16.37 106) after volume reduction. The total CFUs per units were 7.06 ± 3.66 105 (range: 7– 229 105), while the GM colonies were 3.97 ± 2.13 105 (range: 4–132 105). Comparative analysis of post volume reduction of TNC, viability, TNC recovery, CD34+ absolute count and CFU assay among different pre processing TNC cut off groups were done by ANOVA test and presented in Table 4. 3.3. Screening tests From 1778 successfully processed CB units, 38 (2.1%) had positive sterility testing. The rates of aerobic and anaerobic infectious were 32 (1.8%) and 6 (0.3%): all were excluded from banking. The positive rate of HBs Ag, Anti HBc, Anti HCv, Anti HIV1/2 and Anti HTLV I/II were 0.17% (3/1778), 0.11% (2/ 1778), 0.06% (1/1778), 0.06% (1/1778), 0.06% (1/1778) respectively. All the viral infection tests were confirmed by Nucleic Acid test (NAT) methods and compatibility of infant and mother results were notified. The average of CMV IgG Ab and IgM Ab were 2.58 ± 1.60 (range 0–34) and 0.20 ± 1.65 (range 0–57). Hb electrophoresis results showed presence of fast Hb in 0.17% (3/1778) of CB units; also in one case HBs were identified. These cases are under more investigations. 3.4. HLA typing For the HLA typing 19 HLA-A alleles, 34 HLA-B alleles and 13 DRB1 alleles were identified in Iran Blood Transfusion Organization HLA Laboratory. The most frequent A alleles were A02, A24, A03, A01, A11, A32 and A26 and among HLA-B alleles, B35, B51, B52, B18 and B38 were more frequent and finally DRB111, DRB104, DRB115, DRB113, DRB107, DRB103 were the most frequent alleles within DRB1 alleles. 3.5. Transplantation One CB unit was transplanted to a 5-year-old boy, affected by Chédiak–Higashi in Dec 2012. Five out of six alleles were matching according to the HLA high-resolution typing. The post volume reduction criterion for this unit was: TNC = 31.2 108 with more than 85% viable cells, total CD34+ cells = 14.6 106 and total CFU count per unit = 27.3 105. No adverse events reported during transplant infusion. The engraftment was investigated using PCR method and results showed presence of more than
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M. Jamali et al. / Transfusion and Apheresis Science 50 (2014) 129–135 Table 2 Summarized report of collected cord bloods and main reasons of rejection.
Rejection at the collection sites
Rejection at the cord blood bank
Banked
Variables
Number
Percent
Total n (%)
Medical history Delivery data and time CB collection and volume failure Mother sample failure Documentation failure Medical exam of mother and infant Clot Exceed time Low volume Low TNC count During 20 months 1.2 10 .
Table 3 Statistic regarding the volume, cellularity, Hb, Hct, nRBC and viability of UCBs pre and post volume reduction at INCBB. Variables
Volume (ml)
TNC/unit (108)
Hb (g/dl)
Hct (%)
nRBC (%)
Viability (%)
Pre volume reduction Mean ± SD (range)
105.2 ± 24.9 (60.0–248.1)
12.08 ± 3.74 (8.11–34.03)
11.9 ± 1.2 (7.0–16.6)
39.0 ± 4.04 (24.7–53.2)
4.3 ± 3.9 (0–50)
96.8 ± 3.0 (85.5–99.9)
Post volume reduction Mean ± SD (range)
22.11 ± 1.48 (17.6–27.7)
10.76 ± 3.03 (6.01–22.88)
14.7 ± 2.9 (3.4–51.8)
47.8 ± 7.8 (15.7–74.0)
3.6 ± 3.0 (0–36)
95.2 ± 4.3 (70.5–99.7)
95% of graft cells in recipient’s bone marrow after 25 days of transplantation, absolute neutrophil count reached to more than 500 after 22 days and platelet engraftment also occurs after 43 days. 3.6. Correlation of cord blood unit parameters A multiple comparison test demonstrated that TNC in CBs with higher volumes was significantly higher in comparison to those with lower volumes. Furthermore, CB units with more TNCs significantly created more CFUs and contained more CD34+ cells; but, there is no significant correlation between total CD34+ cells and CB volume. Presence of nRBCs, as well as more duration between CB collection and freezing caused a significant decrease of nucleated cells viability (Table 5 and Fig. 1). 4. Discussion Practical advantages of CB transplantation raised the necessity of available large number of high quality CB units. For this reason CB banks are being established in several countries [18,19]. Because of high prevalence of malignant and nonmalignant hematopoietic stem cells disorders, and consequently high rate of hematopoietic stem cells transplants in Iran, INCBB was established in Tehran. In order to offer high quality CB units, all the
procedures are done according to FACT standards. Donor selection issues are similar to those involved in Iran Blood Transfusion Organization. Trained staffs in CB collection sites and extensive donor selection caused less prevalence of infection diseases within the units in comparison to similar centers [20,21]. During 2-year experience 1732 units from 5261 cases were banked (32.9%) in INCBB. It means 866 units per year that is higher than some centers [20,22–24], it should be noticed that, the rate of banking depends directly on TNC cut off and in this experience increasing of TNC cut off from 8 108 to 12 108 caused 4.2% fall in banking rate. About 28% of our units were discarded at the collection sites, because of inadequate quality. Equipped collection sites with the cell counter and scale caused decrease in banking cost and just the CBs with acceptable criteria were carried to the bank. A total of 2038 (38.7%) units were excluded because of biological characteristics, mainly low volume and TNC. As previously reported, there is a meaningful correlation between CB volume and TNC in CB units; so, we decided to modify our standards in 2013 and we established new cut off for volume and TNC (70 ml and 15 108, respectively) [25]. For volume reduction, we used Sepax automated cell processor and TNC recovery was 79.1% in our laboratory; this result was similar to Valencia CB bank report, meanwhile some centers reported 83% and 87.0% TNC recovery after volume reduction with the same instrument [5,7,26,27]. However, according to Papassavas study, increasing the
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Table 4 Comparison of post volume reduction viability, TNC, TNC recovery, CD34 absolute count and CFU assay among different pre processing TNC cut off groups. Group 1: 8.00–10.00 108, group 2: 10.01–12.00 108, group 3: more than 12.01 108. Dependent variable
Mean ± SD
Pre processing TNC cut off groups
P value
Viability post (%)
93.88 ± 4.86 3.00 95.42 ± 3.82 3.00 95.39 ± 3.88 2.00
1.00
.000a
Final TNC/unit (108)
TNC Recovery (%)
CD34 absolute count/unit (106)
CFC/unit/unit (105)
a
2.00 3.00
831.00 ± 101.13 3.00 990.13 ± 111.96 3.00 1336.98 ± 282.04 2.00
1.00
78.03 ± 6.58 3.00 77.83 ± 7.09 3.00 76.20 ± 7.49 2.00
1.00
2.07 ± 1.31 3.00 2.96 ± 1.69 3.00 3.77 ± 2.42 2.00
1.00
5.62 ± 2.79 3.00 7.11 ± 3.75 3.00 9.52 ± 5.21 2.00
1.00
2.00 3.00
2.00 3.00
2.00 3.00
2.00 3.00
2.00 .000a 1.00 .910 1.00 .910
.000a .000a
2.00 .000a 1.00 .000a 1.00 .000a
.000a
2.00 .000a 1.00 .000a 1.00 .000a
.683
2.00 .000a 1.00 .000a 1.00 .000a
.000a
2.00 .000a 1.00 .000a 1.00 .000a
.000a
.000a .000a
.683 .000a
.000a .000a
.000a .000a
Meaningful difference.
Table 5 Multivariate analysis of CB unit parameters. Dependent variable
Independent variable
CB volume (ml) adjusted R2 = 0.467
Gestational age (weeks) TNC (108) Total CD34+ cells (106) CB volume (ml) Total CD34+ cells (106) CFU (105) CB volume (ml) TNC (108) CFU (105) Poly morpho nuclear cells% Nucleated RBC% Duration of collection to volume reduction (hours)
TNC (108) adjusted R2 = 0.517
Total CD34+ cells (106) adjusted R2 = 0.232
Viability (%) adjusted R2 = 0.090
a
Standardized coefficients, b 0.034 0.735 0.039 0.539 0.308 0.435 0.042 0.281 0.304 0.183 0.169 0.328
P value 0.161 0.000a 0.141 0.000a 0.000a 0.000a 0.148 0.000a 0.000a 0.000a 0.000a 0.000a
Significant correlation.
product volume caused more TNC recovery, but more RBCs too. In our volume reduced units, the mean of total CD34+ cells was 2.99 106, whereas this criterion was 6.3 106 in Solves study, 4 106 in North East Mexico CB bank, 1.8 106 in Omori study and 15.19 106 in a Chinese CB bank. About the CFU assay, we found the mean of 7.1 105 cells with colonogenic capacity in CB units (11.6 105 in Omori study). Low CD34+ cells and CFUs, in comparison to some centers, is the result of low TNC cut off in our center, so that; comparison of total CD34+ cells and CFU assay in different pre volume reduction
TNC groups showed meaningful difference, as well as, post volume reduction TNC. Low TNC recovery rate in the group with high TNC is noteworthy and could suggest that it is better to processed high TNC units in two parts. The results of CB screening tests revealed 2.2% positivity in sterility tests and 0.46% positivity in viral infections, which is slightly lower than the reports published by other centers [6,20,21,25]. HLA allele’s prevalence in INCBB is in compatible with previous reports from Iranian population, although those were done on smaller populations [28,29]. It should be notified that the cost of CB unit processing,
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freezing and storage is very high; therefore; many attempts were focused on investigation of the CB units’ biological parameters to minimize the processing and testing costs, also; to identify the actually needed tests. In this manner, correlation of CB volume, TNC, CD34+ cells and CFU assay were analyzed in some studies [25,30,31]. In our results, we found a significant correlation between CB volume, CFU assay and TNC; as previously reported, of course; this correlation is not close in some area, TNC and CD34 counting are mandatory, according to the FACT standards. About the CFU assay, some recent reports confirmed close correlation between the CFU assay and PMNs recovery after the CB transplantation; thus this test is considered a necessary quality control of CB units [32]. Most of the CB banks do not have any cut off for the viability test, before cryopreservation, because they reported that, decrease in viability is the result of PMNs death. In our experience, there was no correlation between PMN cells and viability, while, more nRBCs caused significantly lower viability. Finally, use of CB transplantation is relatively new in our country and there are limited numbers of BMT centers. Therefore, just one engrafted CB transplant was done from our units and 9 units are reserved for transplantation in new future. All together, the need of hematopoietic stem cell transplant is increasing and CB is an available and low risk source; thus, establishment of more CB banks and improvement of CB units quality for more efficient hematopoietic stem cell transplants as well as universal standardization of banking, is highly considered in developing countries. Acknowledgements The authors would like to express the deepest appreciation to Professor E. Gluckman, Professor P. Rebulla, Dr. R. Saccardi and S. Urbani for their valuable guidance during INCBB establishment. References [1] Gluckman E. History of cord blood transplantation. Bone Marrow Transpl 2009;44:621–6. [2] Rocha V, Kabbara N, Ionescu I, Ruggeri A, Purtill D, Gluckman E. Pediatric related and unrelated cord blood transplantation for malignant diseases. Bone Marrow Transpl 2009;44:653–9. [3] Rubinstein P. Cord blood banking for clinical transplantation. Bone Marrow Transpl 2009;44:635–42. [4] Rocha V, Garnier F, Ionescu I, Gluckman E. Eurocord and European. Blood and marrow transplant group. Hematopoietic stem-cell transplantation using umbilical-cord blood cells. Rev Invest Clin 2005;57:314–23. [5] Dazey B, Duchez P, Letellier C, et al. Cord blood processing by using a standard manual technique and automated closed system ‘‘Sepax’’ (Kit CS 530). Stem Cells Dev 2005;14:6–10. [6] Solves P, Mirabet V, Larrea L, et al. Cord blood volume reduction with AXP system (abstract). Biology and clinical applications of cord blood cells. Mandelieu–France, October 2008. [7] Solves P, Planelles D, Mirabet V, Blanquer A, Carbonell-Uberos F. Qualitative and quantitative cell recovery in umbilical cord blood processed by two automated devices in routine cord blood banking: a comparative study. Blood Transfus 2012;12:1–8. [8] Miura J, Minegishi M, Itoh T, Kitaura T, Fukawa N, Takahashi H, et al. Quality evaluation of umbilical cord blood progenitor cells cryopreserved with a small-scale automated liquid nitrogen system. Cryobiology 2008;57:178–81.
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[9] Rocha V, Gluckman E. Eurocord and European blood and marrow transplant group. Clinical use of umbilical cord blood hematopoietic stem cells. Biol Blood Marrow Transplant 2006;12:34–41. [10] Rocha V, Garnier F, Ionescu I, Gluckman E. Eurocord and European blood and marrow transplant group. Hematopoietic stem-cell transplantation using umbilical-cord blood cells. Rev Invest Clin 2005;57:314–23. [11] Foundation for the Accreditation of Cellular Therapy and Joint Accreditation Committee ISCT–EBMT. FACT-JACIE international standards for cellular product collection, processing, and transplantation. 4th ed. Omaha, NE: FACT; 2011. [cited 15.03.2012] [12] Gluckman E. Milestones in umbilical cord blood transplantation. Blood Rev 2011;25:255–9. [13] Farjadian S, Ghaderi A. HLA diversity in Iran. Iran J Immunol 2007;4:85–93. [14] Farjadian S, Safi S. Genetic connections among Turkic-speaking Iranian ethnic groups based on HLA class II gene diversity. Iran J Immunol 2006;3:106–13. [15] Khazaei H, Rezaei N, Aghamohammadi A, Amirzargar AA, Mollaei Ghasemi KH, Miri I, Nikbin B. Human leukocyte antigen profile of two ethnic groups in southeast of Iran. Iran J Allerg Asthma Immunol 2007;6:223–4. [16] Gluckman E, Rocha V. Cord blood transplantation: state of the art. Haematologica 2009;94:451–4. [17] Mousavi S, Gouya M, Ramazani R, Davanlou M, Hajsadeghi N, Seddighi Z. Cancer incidence and mortality in Iran. Ann Oncol 2009;20:556–63. [18] Rebulla P. Cord blood banking and transplantation in 2010. Transfus Apheresis Sci 2010;42(5):253–4. [19] Sozos J, Fasouliotis MD, Joseph G, Schenker MD. Human umbilical cord blood banking and transplantation: a state of the art. Eur J Obstet Gynecology Reprod Biol 2000;90:13–25. [20] Perez J, Robles R, Pedraza J, Guerra C, Romo L, Almaguer D. Cord blood banking activities at a university hospital in northeast Mexico: an 8-year experience. Transfusion 2012;52:2606–13. [21] Liu J, He J, Chen Sh, Qin F, Wang F, Xu G, et al. Cord blood banking and transplantation in China: a ten years experience of a single public bank. Transfus Med Hemotherapy 2012;39:23–7. [22] Davey S, Armitage S, Rocha V, Garnier F, Brown J, Brown CJ, et al. The London cord blood bank: analysis of banking and transplantation outcome. Br J Haematol 2004;125:358–65. [23] Tamburini A, Malerba C, Picardi A, Amadori S, Calugi A. Placental/ umbilical cord blood: experience of St. Eugenio hospital collection center. Transplant Proc 2005;37:2670–2. [24] Wu JY, Liao C, Xu ZP, Chen JS, Gu SL, Huang YN, et al. Banking and transplantation of umbilical cord blood in Guangzhou China. Cytotherapy 2006;8:488–97. [25] Omori A, Hirai M, Chiba T, Takahashi K, Yamaguchi S, Takahashi T, et al. Quality-assessments of characteristics of placental/umbilical cord blood associated with maternal age- and parity-related factor. Transfus Apheresis Sci 2012;46:7–13. [26] Lapierre V, Pellegrini N, Bardey I, et al. Cord blood volume reduction using an automated system (Sepax) vs. a semi-automated system (Optipress II) and a manual method (hydroxyethyl starch sedimentation) for routine cord blood banking: a comparative study. Cytotherapy 2007;9:165–9. [27] Papassavas AC, Gioka V, Chatzistamatiou T, et al. A strategy of splitting individual high volume cord blood units into two half subunits prior to processing increases the recovery of cells and facilitates ex vivo expansion of the infused haematopoietic progenitor cells in adults. Int J Lab Hem 2008;30:124–32. [28] Yari F, Bagheri N, Zaman Vaziri M, Sobhani M, Sabaghi F, Talebian A. HLA DRB1 polymorphism in the Iranian population. Sci J Iran Blood Transfus Org 2007;4:199–203. [29] Einollahi B, Rostami Z, Teimoori M. Human leukocyte antigen variation among Iranian renal transplant recipients. J Nephropathology 2012;3:164–9. [30] Al-Sweedan S, Musalam L, Obeidat B. Factors predicting the hematopoietic stem cells content of the umbilical cord blood. Transfus Aphresis Sci 2013 (Article in press). [31] Aroviita P, Teramo K, Westman P, Hiilesmaa V, Kekomaki R. Associations among nucleated cell, CD34+ cell and colony-forming cell contents in cord blood units obtained through a standardized banking process. Vox Sang 2003;84:219–27. [32] George TJ, Sugrue MW, George SN, Wingard JR. Factors associated with parameters of engraftment potential of umbilical cord blood. Transfusion 2006;46:1803–12.
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Transfusion and Apheresis Science journal homepage: www.elsevier.com/locate/transci
Cord blood banking activity in Iran National Cord Blood Bank: A two years experience Mostafa Jamali a,b, Kamran Atarodi a,b, Mozhdeh Nakhlestani a,b, Hasan Abolghasemi c, Hosein Sadegh a, Mohammad Faranoosh a, Khadije Golzade a,b, Razieh Fadai a, Fereshte Niknam a,b, Mahin Nikougoftar Zarif a,b,⇑ a b c
Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran Iran National Cord Blood Bank, Iran Blood Transfusion Organization, Tehran, Iran Pediatric Congenital Hematologic Disorders Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 11 May 2013 Received in revised form 9 September 2013 Accepted 30 September 2013
Keywords: Cord blood bank Cord blood transplantation Hematopoietic stem cells
a b s t r a c t Today umbilical cord blood (UCB) has known as a commonly used source of hematopoietic stem cells for allogeneic transplantation and many cord blood banks have been established around the world for collection and cryopreservation of cord blood units. Herein, we describe our experience at Iran National Cord Blood Bank (INCBB) during 2 years of activity. From November 2010 to 2012, UCBs were collected from 5 hospitals in Tehran. All the collection, processing, testing, cryopreservation and storage procedures were done according to standard operation procedures. Total nucleated cells (TNC) count, viability test, CD34+ cell count, colony forming unit (CFU) assay, screening tests and HLA typing were done on all banked units. Within 3770 collected units, only 32.9% fulfilled banking criteria. The mean volume of units was 105.2 ml and after volume reduction the mean of TNC, viability, CD34+ cells and CFUs was 10.76 108, 95.2%, 2.99 106 and 7.1 105, respectively. One unit was transplanted at Dec 2012 to a 5-year old patient with five of six HLA compatibilities. In our country banking of UCB is new and high rate of hematopoietic stem cell transplants needs expanding CB banks capacity to find more matching units, optimization of methods and sharing experiences to improve biological characterization of units. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Since the first successful umbilical cord blood (UCB) transplantation in 1988, the use of UCB has been increased for treatment of a variety of hematopoietic malignancies, immunological and some hereditary disorders [1–4]. Several researches have focused on systematically evaluation of human UCB hematopoietic stem cells (HSC) potential and developing practical and efficient methods for the volume reduction, cryopreservation and storage of UCB units ⇑ Corresponding author at: Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran. Tel.: +98 21 88601575; fax: +98 21 88601576. E-mail address: [email protected] (M.N. Zarif). 1473-0502/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.transci.2013.09.012
[5–8]. In parallel, many cord blood banks have been established worldwide in order to bank UCB units for unrelated donor transplantation. For improving the quality of banked UCB samples, some international organizations were created. Netcord and Foundation for the Accreditation of Cellular Therapy (FACT), in collaboration with National Marrow Donor Programme (NMDP) established international standards whose major objects are to improve quality throughout all phases of UCB banking, including good practices in UCB storage, facilitate donor search, improve the quality of grafts, standardize excellence criteria on an international scale and importantly established procedures for bank accreditation, and cooperation of large experienced UCB banks [9–11]. Today, more than 600000 UCB have been banked and more than 30000 UCB units
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transplanted to children and adults [12]. The optimal number of cord blood units to cover maximum necessities is not really known, because of HLA diversity [13–15], but, based on some reports; it should approach 9 for 10000 inhabitants [16]. However, because of high prevalence of cancers and hematologic disorders in Iran [17] and consequently high necessity of transplant therapy, also, different HLA allele’s distribution among racial groups, it is justified having public CB banks in Iran. Over the past 2 years, Iran National Cord Blood Bank (INCBB) has been established under supervision of Ministry of Health in order to increase the pool of potential CB units for allogeneic transplantation. The purpose of this article is to describe experiences and current results of 1732 UCB units that were banked in INCBB during 2 years. All the procedures were done according to the last version of international standards published by Netcord/FACT organization and some local criteria.
2. Materials and methods 2.1. Donor selection Selection of donors is one of the most important steps in UCB banking. At first, a trained physician interviewed mothers and obtained extensive and accurate medical history of the family from informed consent donors. If some cases did not fulfill selection criteria, based on the local and the Netcord/FACT standards, they were excluded from collection. The selected donors signed consent form and were candidate for donation. 2.2. Cord blood collection To reduce the risk of microbial and maternal contamination closed collection system was used. Trained midwives collected UCB units at 5 maternity hospitals in Tehran. The samples were collected into cord blood collection bags (JMS, Singapore) contained 22 ml citrate phosphate dextrose adenine (CPDA) after normal vaginal delivery (NVD) or caesarean section (CS). 20 ml of mother venous blood were obtained in 2 tubes with anticoagulant reagent and 2 other tubes without anticoagulant (BD Pharmingen, CA, USA) within 4 h of delivery. All samples were labeled and transferred to the CB bank laboratory at 16–24 °C together with the documents. Medical exam of mother and newborn were done after 24 h of delivery to rule out any complications. 2.3. Cord blood pre volume reduction/processing criteria After checking documents, appearance and characteristic of units, CB volume was calculated. If the volume was more than 60 ml, some aliquots were removed for testing. Total nucleated cell (TNC) count and viability test were done and if the absolute nucleated cells were more than 8 108 per units and more than 85% of TNCs were viable, it was selected for the processing. The TNC cut off was
increased to 12 108 per units after 20 months, therefore; affected data presented in different groups. 2.4. Cord blood volume reduction/processing In order to perform cord blood volume reduction, the Sepax (Biosafe, Geneva, Switzerland) was used as automated cell separation method. For better Red Blood Cell (RBC) depletion and more TNC recovery, Hydroxyl Ethyl Starch (HES 200, 0.6%, Fresenius Kabi, Hamborg, Germany) in concentration of 2:5 (total volume of CB and CPDA) was added to the samples; after 30 min incubation at room temperature (RT) CB bag was attached to the Sepax processing kit (Sepax kit 530.1, Biosafe, Geneva, Switzerland) under sterile condition and run with the UCB-HES programme in cell separator. In this process, buffy coat was extracted under centrifugation and the bag of buffy coat was detached. The TNC, viability, CD34+ cell count and colony forming unit (CFU) assays were performed on the extracted buffy coat before cryopreservation. Where TNC count was more than 6 108, the buffy coat bag was placed in an automated mixing and cooling device (Coolmix-210, Biosafe, Geneva, Switzerland). Dimethyl sulfoxide (DMSO) and dextran 40 (CryoSure-Dex40, WAK-Chemie, Steinbach, Germany) was added to the buffy coat in 10 min cooling and mixing (concentration of 1:4 buffy coat volume). Three samples were prepared for the pre transplantation tests and sealed buffy coat bag was protected in an aluminum canister, transferred to a programmable control rate freezer (Planer, Middlesex, UK) in order to reduce the sample temperature to 100 °C. The freezing programme is illustrated in Table 1. Cord blood units were stored in vapor phase of nitrogen quarantine tank at 150 °C up to the second mother screening test. Sample safety and confirmed second mother screening test, after 6 to 9 months of delivery, allow storage of samples in liquid nitrogen tanks at 196 °C. 2.5. TNC count, viability test, CD34+ absolute count and CFU assay TNC count and viability test were done on the units, pre and post volume reduction; CD34+ cell count and CFU assay only on post volume reduction. Two Wright stained slides were prepared from each sample for nucleated RBC (nRBC) and differential count. The TNC was calculated based on the White Blood Cell (WBC) extracted from Sysmex X800i (Sysmex, Tokyo, Japan) and nRBC percentage. For detection of viable TNCs, Propidium Iodide (PI) (Sigma, St. Louis, USA) in concentration of 0.01% in PBS was used. As the dead cells absorb PI dye, any cells that emitted more red-fluorescent in comparison to control cells (without adding PI), was considered as dead cells. The absolute count of viable CD34+ cells were quantified using CD34 count kit (DAKO, Glostrup, Denmark) containing microbeads by flowcytometery (Partec, Munster, Germany). According to the manufacturer’s instruction, CD45 dim cells that expressed bright CD34 and did not absorb 7 Amino Actinomycin D. Colonogenic capacity of HSCs was determined using culture of 50000 TNC in 1 ml of commercially prepared
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300
R=0.209
R=0.665 250
p=0.000
CB Volume (ml)
CB Volume (ml)
250 200 150 100
p=0.000
200 150 100 50
50
0
0 0
5
10
15
20
25
30
35
0
40
5
TNC (x10e8)
15
20
25
30
25
30
30
20
R=0.435
R=0.429 25
p=0.000
15
Total CFU (x10e5)
Total CD34 (x10e6)
10
Total CFU (x10e5)
10
5
p=0.000
20 15 10 5 0
0 0
5
10
15
20
25
0
30
5
10
15
20
TNC (x10e8)
TNC (x10e8) 60
20
R= -0.126
R=0.434 p=0.000
15
Nucleated RBC %
Total CD34 (x10e60)
50
10
5
p=0.000 40 30 20 10 0 -10
0
0
5
10
15
20
25
30
70
75
80
85
90
95
100
105
Viability %
Total CFU (x10e5) Fig. 1. Correlation between CB unit parameters.
Table 1 The freezing programme of control rate freezer for the sample temperature reduction from +4 °C to 100 °C. Temperature induction phase
Temperature alteration per minutes (°C)
First phase Second phase
Third phase
1 20 +10 1 10
Start (°C)
Stop (°C)
+4
+4
+4 4 60 30 75 100
4 60 30 75 100 100
methylcellulose medium containing cytokines (Methocult GF H4434, Stem cell technologies, Vancouver, Canada). This medium supports the growth of Burst Forming Unit
Erythroid (BFU-E), Colony Forming Unit Granulocyte Monocyte (CFU-GM) and Colony Forming Unit Granulocyte Erythroid Monocyte Megakaryocyte (CFU-GEMM). After 14 days incubation at 37 °C and 5% CO2, all the colonies were counted and differentiated.
2.6. Screening tests Microbial culture and all the screening tests [Cytomegalovirus antibody (IgG, IgM), Rapid Plasma Regain (RPR) for syphilis, Hepatitis virus B and C (HBs Ag, Anti HBs, Anti HBc and Anti HCV), Human Immunodeficiency Virus (HIV Ag– Ab) and Human T-Lymphotrophic Virus (Anti HTLV1&2)] were done on the cord blood plasma extracted after processing. In case of any indeterminate results, nucleic acid based test (NAT) was done for more evaluation.
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2.7. HLA typing HLA typing was performed on DNA obtained from the buffy coat samples. A PCR based method with sequence specific primers (SSP) and sequence specific oligonucleotide probes (SSOP) were carried out for the class I (A and B alleles) and class II (DRB1 alleles) detection. Further HLA typing on the other alleles and high resolution typing was done based on the transplant center request. Cellulose Acetate Electrophoresis at Alkaline pH was done to identify hemoglobinopathies and in case of more investigation, HPLC was used. ABO and Rh grouping were done on all samples. 2.8. Mother samples Buffy coat extracted from the EDTA samples for confirmation of one haplotype in case of release was stored at 80 °C. All screening tests, except microbial cultures that were done on the cord blood were done also on the mother serum, at the time of delivery and again after 6–9 months of delivery. 2.9. Sample release When a unit was selected for transplantation, before reserving, a group of tests including TNC count, viability test, and CD34+ absolute count and CFU assay were done on the segment. In case of transplantation, the unit was placed in the shipment Dewar in liquid nitrogen and together with all documents transferred to the transplant center. 2.10. Statistical analysis Statistical analysis was performed using SPSS software, version 16.5. Based on the distribution pattern of data, descriptive analysis was used to evaluate the biological characteristic of CB units. Multivariate linear regression analysis and Pearson’s correlation coefficient were used for detection of association between CB unit parameters. 3. Results 3.1. Collection activities From November 2010 to November 2012, a total of 6472 pregnant women at 5 hospitals in Tehran near the INCBB, were interviewed and about 81% of them agreed to CB donation. Within 5261 cases, 1491 (28.34%) were excluded at the collection sites, while, 2038 (38.74%) were rejected at the CB bank laboratory and only 1732 (32.92%) were successfully processed and stored, also; 30 units were selected for the quality control tests randomly. All major activities and the main reasons of exclusion at the collection sites and laboratory are summarized in Table 2. The comparison of banking rate between two pre processing TNC cut off groups revealed 33.52%, when the TNC cut off was 8 108, while it decreased to 29.23% when it was 1.2 108 (p value < 0.5).
The average of pregnancy age was 38.7 ± 0.9 weeks and 53.2% of infants were male and 46.8% were female. 3.2. The biologic characteristics of the units The mean ± SD of CB volume was 105.2 ± 24.9 ml and the buffy coat volume decreased to 22.11 ± 1.48 ml after volume reduction with Sepax cell separator. This criterion for the post volume reduction TNC, was 10.76 ± 3.03 108 and the TNC recovery rate was 79.1 ± 9.2% (range: 58.9– 99.7%). Other parameters are listed in Table 3. The amount of total CD34+ cells was 2.99 ± 2.08 106 (range: 0.51–16.37 106) after volume reduction. The total CFUs per units were 7.06 ± 3.66 105 (range: 7– 229 105), while the GM colonies were 3.97 ± 2.13 105 (range: 4–132 105). Comparative analysis of post volume reduction of TNC, viability, TNC recovery, CD34+ absolute count and CFU assay among different pre processing TNC cut off groups were done by ANOVA test and presented in Table 4. 3.3. Screening tests From 1778 successfully processed CB units, 38 (2.1%) had positive sterility testing. The rates of aerobic and anaerobic infectious were 32 (1.8%) and 6 (0.3%): all were excluded from banking. The positive rate of HBs Ag, Anti HBc, Anti HCv, Anti HIV1/2 and Anti HTLV I/II were 0.17% (3/1778), 0.11% (2/ 1778), 0.06% (1/1778), 0.06% (1/1778), 0.06% (1/1778) respectively. All the viral infection tests were confirmed by Nucleic Acid test (NAT) methods and compatibility of infant and mother results were notified. The average of CMV IgG Ab and IgM Ab were 2.58 ± 1.60 (range 0–34) and 0.20 ± 1.65 (range 0–57). Hb electrophoresis results showed presence of fast Hb in 0.17% (3/1778) of CB units; also in one case HBs were identified. These cases are under more investigations. 3.4. HLA typing For the HLA typing 19 HLA-A alleles, 34 HLA-B alleles and 13 DRB1 alleles were identified in Iran Blood Transfusion Organization HLA Laboratory. The most frequent A alleles were A02, A24, A03, A01, A11, A32 and A26 and among HLA-B alleles, B35, B51, B52, B18 and B38 were more frequent and finally DRB111, DRB104, DRB115, DRB113, DRB107, DRB103 were the most frequent alleles within DRB1 alleles. 3.5. Transplantation One CB unit was transplanted to a 5-year-old boy, affected by Chédiak–Higashi in Dec 2012. Five out of six alleles were matching according to the HLA high-resolution typing. The post volume reduction criterion for this unit was: TNC = 31.2 108 with more than 85% viable cells, total CD34+ cells = 14.6 106 and total CFU count per unit = 27.3 105. No adverse events reported during transplant infusion. The engraftment was investigated using PCR method and results showed presence of more than
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M. Jamali et al. / Transfusion and Apheresis Science 50 (2014) 129–135 Table 2 Summarized report of collected cord bloods and main reasons of rejection.
Rejection at the collection sites
Rejection at the cord blood bank
Banked
Variables
Number
Percent
Total n (%)
Medical history Delivery data and time CB collection and volume failure Mother sample failure Documentation failure Medical exam of mother and infant Clot Exceed time Low volume Low TNC count During 20 months 1.2 10 .
Table 3 Statistic regarding the volume, cellularity, Hb, Hct, nRBC and viability of UCBs pre and post volume reduction at INCBB. Variables
Volume (ml)
TNC/unit (108)
Hb (g/dl)
Hct (%)
nRBC (%)
Viability (%)
Pre volume reduction Mean ± SD (range)
105.2 ± 24.9 (60.0–248.1)
12.08 ± 3.74 (8.11–34.03)
11.9 ± 1.2 (7.0–16.6)
39.0 ± 4.04 (24.7–53.2)
4.3 ± 3.9 (0–50)
96.8 ± 3.0 (85.5–99.9)
Post volume reduction Mean ± SD (range)
22.11 ± 1.48 (17.6–27.7)
10.76 ± 3.03 (6.01–22.88)
14.7 ± 2.9 (3.4–51.8)
47.8 ± 7.8 (15.7–74.0)
3.6 ± 3.0 (0–36)
95.2 ± 4.3 (70.5–99.7)
95% of graft cells in recipient’s bone marrow after 25 days of transplantation, absolute neutrophil count reached to more than 500 after 22 days and platelet engraftment also occurs after 43 days. 3.6. Correlation of cord blood unit parameters A multiple comparison test demonstrated that TNC in CBs with higher volumes was significantly higher in comparison to those with lower volumes. Furthermore, CB units with more TNCs significantly created more CFUs and contained more CD34+ cells; but, there is no significant correlation between total CD34+ cells and CB volume. Presence of nRBCs, as well as more duration between CB collection and freezing caused a significant decrease of nucleated cells viability (Table 5 and Fig. 1). 4. Discussion Practical advantages of CB transplantation raised the necessity of available large number of high quality CB units. For this reason CB banks are being established in several countries [18,19]. Because of high prevalence of malignant and nonmalignant hematopoietic stem cells disorders, and consequently high rate of hematopoietic stem cells transplants in Iran, INCBB was established in Tehran. In order to offer high quality CB units, all the
procedures are done according to FACT standards. Donor selection issues are similar to those involved in Iran Blood Transfusion Organization. Trained staffs in CB collection sites and extensive donor selection caused less prevalence of infection diseases within the units in comparison to similar centers [20,21]. During 2-year experience 1732 units from 5261 cases were banked (32.9%) in INCBB. It means 866 units per year that is higher than some centers [20,22–24], it should be noticed that, the rate of banking depends directly on TNC cut off and in this experience increasing of TNC cut off from 8 108 to 12 108 caused 4.2% fall in banking rate. About 28% of our units were discarded at the collection sites, because of inadequate quality. Equipped collection sites with the cell counter and scale caused decrease in banking cost and just the CBs with acceptable criteria were carried to the bank. A total of 2038 (38.7%) units were excluded because of biological characteristics, mainly low volume and TNC. As previously reported, there is a meaningful correlation between CB volume and TNC in CB units; so, we decided to modify our standards in 2013 and we established new cut off for volume and TNC (70 ml and 15 108, respectively) [25]. For volume reduction, we used Sepax automated cell processor and TNC recovery was 79.1% in our laboratory; this result was similar to Valencia CB bank report, meanwhile some centers reported 83% and 87.0% TNC recovery after volume reduction with the same instrument [5,7,26,27]. However, according to Papassavas study, increasing the
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Table 4 Comparison of post volume reduction viability, TNC, TNC recovery, CD34 absolute count and CFU assay among different pre processing TNC cut off groups. Group 1: 8.00–10.00 108, group 2: 10.01–12.00 108, group 3: more than 12.01 108. Dependent variable
Mean ± SD
Pre processing TNC cut off groups
P value
Viability post (%)
93.88 ± 4.86 3.00 95.42 ± 3.82 3.00 95.39 ± 3.88 2.00
1.00
.000a
Final TNC/unit (108)
TNC Recovery (%)
CD34 absolute count/unit (106)
CFC/unit/unit (105)
a
2.00 3.00
831.00 ± 101.13 3.00 990.13 ± 111.96 3.00 1336.98 ± 282.04 2.00
1.00
78.03 ± 6.58 3.00 77.83 ± 7.09 3.00 76.20 ± 7.49 2.00
1.00
2.07 ± 1.31 3.00 2.96 ± 1.69 3.00 3.77 ± 2.42 2.00
1.00
5.62 ± 2.79 3.00 7.11 ± 3.75 3.00 9.52 ± 5.21 2.00
1.00
2.00 3.00
2.00 3.00
2.00 3.00
2.00 3.00
2.00 .000a 1.00 .910 1.00 .910
.000a .000a
2.00 .000a 1.00 .000a 1.00 .000a
.000a
2.00 .000a 1.00 .000a 1.00 .000a
.683
2.00 .000a 1.00 .000a 1.00 .000a
.000a
2.00 .000a 1.00 .000a 1.00 .000a
.000a
.000a .000a
.683 .000a
.000a .000a
.000a .000a
Meaningful difference.
Table 5 Multivariate analysis of CB unit parameters. Dependent variable
Independent variable
CB volume (ml) adjusted R2 = 0.467
Gestational age (weeks) TNC (108) Total CD34+ cells (106) CB volume (ml) Total CD34+ cells (106) CFU (105) CB volume (ml) TNC (108) CFU (105) Poly morpho nuclear cells% Nucleated RBC% Duration of collection to volume reduction (hours)
TNC (108) adjusted R2 = 0.517
Total CD34+ cells (106) adjusted R2 = 0.232
Viability (%) adjusted R2 = 0.090
a
Standardized coefficients, b 0.034 0.735 0.039 0.539 0.308 0.435 0.042 0.281 0.304 0.183 0.169 0.328
P value 0.161 0.000a 0.141 0.000a 0.000a 0.000a 0.148 0.000a 0.000a 0.000a 0.000a 0.000a
Significant correlation.
product volume caused more TNC recovery, but more RBCs too. In our volume reduced units, the mean of total CD34+ cells was 2.99 106, whereas this criterion was 6.3 106 in Solves study, 4 106 in North East Mexico CB bank, 1.8 106 in Omori study and 15.19 106 in a Chinese CB bank. About the CFU assay, we found the mean of 7.1 105 cells with colonogenic capacity in CB units (11.6 105 in Omori study). Low CD34+ cells and CFUs, in comparison to some centers, is the result of low TNC cut off in our center, so that; comparison of total CD34+ cells and CFU assay in different pre volume reduction
TNC groups showed meaningful difference, as well as, post volume reduction TNC. Low TNC recovery rate in the group with high TNC is noteworthy and could suggest that it is better to processed high TNC units in two parts. The results of CB screening tests revealed 2.2% positivity in sterility tests and 0.46% positivity in viral infections, which is slightly lower than the reports published by other centers [6,20,21,25]. HLA allele’s prevalence in INCBB is in compatible with previous reports from Iranian population, although those were done on smaller populations [28,29]. It should be notified that the cost of CB unit processing,
M. Jamali et al. / Transfusion and Apheresis Science 50 (2014) 129–135
freezing and storage is very high; therefore; many attempts were focused on investigation of the CB units’ biological parameters to minimize the processing and testing costs, also; to identify the actually needed tests. In this manner, correlation of CB volume, TNC, CD34+ cells and CFU assay were analyzed in some studies [25,30,31]. In our results, we found a significant correlation between CB volume, CFU assay and TNC; as previously reported, of course; this correlation is not close in some area, TNC and CD34 counting are mandatory, according to the FACT standards. About the CFU assay, some recent reports confirmed close correlation between the CFU assay and PMNs recovery after the CB transplantation; thus this test is considered a necessary quality control of CB units [32]. Most of the CB banks do not have any cut off for the viability test, before cryopreservation, because they reported that, decrease in viability is the result of PMNs death. In our experience, there was no correlation between PMN cells and viability, while, more nRBCs caused significantly lower viability. Finally, use of CB transplantation is relatively new in our country and there are limited numbers of BMT centers. Therefore, just one engrafted CB transplant was done from our units and 9 units are reserved for transplantation in new future. All together, the need of hematopoietic stem cell transplant is increasing and CB is an available and low risk source; thus, establishment of more CB banks and improvement of CB units quality for more efficient hematopoietic stem cell transplants as well as universal standardization of banking, is highly considered in developing countries. Acknowledgements The authors would like to express the deepest appreciation to Professor E. Gluckman, Professor P. Rebulla, Dr. R. Saccardi and S. Urbani for their valuable guidance during INCBB establishment. References [1] Gluckman E. History of cord blood transplantation. Bone Marrow Transpl 2009;44:621–6. [2] Rocha V, Kabbara N, Ionescu I, Ruggeri A, Purtill D, Gluckman E. Pediatric related and unrelated cord blood transplantation for malignant diseases. Bone Marrow Transpl 2009;44:653–9. [3] Rubinstein P. Cord blood banking for clinical transplantation. Bone Marrow Transpl 2009;44:635–42. [4] Rocha V, Garnier F, Ionescu I, Gluckman E. Eurocord and European. Blood and marrow transplant group. Hematopoietic stem-cell transplantation using umbilical-cord blood cells. Rev Invest Clin 2005;57:314–23. [5] Dazey B, Duchez P, Letellier C, et al. Cord blood processing by using a standard manual technique and automated closed system ‘‘Sepax’’ (Kit CS 530). Stem Cells Dev 2005;14:6–10. [6] Solves P, Mirabet V, Larrea L, et al. Cord blood volume reduction with AXP system (abstract). Biology and clinical applications of cord blood cells. Mandelieu–France, October 2008. [7] Solves P, Planelles D, Mirabet V, Blanquer A, Carbonell-Uberos F. Qualitative and quantitative cell recovery in umbilical cord blood processed by two automated devices in routine cord blood banking: a comparative study. Blood Transfus 2012;12:1–8. [8] Miura J, Minegishi M, Itoh T, Kitaura T, Fukawa N, Takahashi H, et al. Quality evaluation of umbilical cord blood progenitor cells cryopreserved with a small-scale automated liquid nitrogen system. Cryobiology 2008;57:178–81.
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