Cord blood banking - Nature

Bone Marrow Transplantation, (1999) 23, 505–509  1999 Stockton Press All rights reserved 0268–3369/99 $12.00 http://www.stockton-press.co.uk/bmt

Cord blood banking: volume reduction of cord blood units using a semi-automated closed system S Armitage1, D Fehily1, A Dickinson2, C Chapman3, C Navarrete1 and M Contreras1 1

National Blood Service – London and SE Zone, London Cord Blood Bank; 2University Department of Haematology, School of Clinical and Laboratory Sciences, Royal Victoria Infirmary, Newcastle upon Tyne; and 3National Blood Service – Newcastle Centre, Newcastle upon Tyne, UK

Summary: Clinical evidence indicates that placental/umbilical cord blood (CB) is an alternative source of haematopoietic stem cells for bone marrow reconstitution. To establish a CB bank large panels of frozen, HLA-typed CB units need to be stored. Cryopreserved, unprocessed CB units require vast storage space. This study describes a method, using the Optipress II Automated Blood Component Extractor (Opti II) from Baxter Healthcare Corporation, to reduce the volume of the CB collection, preserving the quantity and quality of the progenitor cells, in a closed system. The CB collection was transferred to a triple bag system, centrifuged to produce a buffy coat layer and processed using a standard Opti II protocol to separate the whole blood into three components: plasma, buffy coat and buffy coat-depleted red cell concentrate. The buffy coat volume was standardised to 25 ml; mean reduced volume of 24.5 ml (s.d. 1.5 ml) with 53% red cell depletion. Good recovery of cells was observed: 92%, 98%, 96% and 106% recovery of nucleated, mononuclear, CD34⫹ and total colonyforming cells, respectively. Using this method for processing CB units reduces storage requirement by two-thirds but preserves the quantity and quality of the progenitor cells. Keywords: cord blood; cord blood bank; processing; volume reduction; progenitor cells; transplantation

Cord blood (CB) is increasingly used as an alternative source of stem and progenitor cells for the reconstitution of BM and sustaining long-term haemopoietic recovery in both unrelated and related recipients.1–3 It offers several advantages over BM in that it is obtained without risk to either mother or infant, it has a decreased likelihood of transmitting infections – particularly pertinent for cytomegalovirus – and it can be stored fully tested and HLA typed, in the frozen state, available for immediate use. Recent clinical data1–3 indicate that CB transplants may result in reduced severity of acute GVHD, allowing the use of par-

Correspondence: S Armitage, London Cord Blood Bank, Deansbrook Road, Edgware, Middx HA8 9BG, UK Received 7 September 1998; accepted 24 September 1998

tially HLA-matched units and thus increasing the chance of finding donors for patients. To achieve a viable bank, providing a broad range of HLA-typed CB units, the storage of a large number of units is essential. The major logistical problem with such longterm banking is the required storage space, particularly when the CB units are stored unprocessed. Several different methods have been employed to reduce the volume of the CB unit, prior to cryopreservation of the cells, while minimising loss of either nucleated cells (NC) or progenitor cells: density gradient separation – standard4 or modified techniques5 – sedimentation of red cells by gelatin,6 rouleaux formation induced by hydroxyethyl starch and centrifugation to reduce both erythrocytes and plasma7,8 and differential centrifugation with manual9 or automated10 expression of RBC and plasma. The majority of these methods are not performed in a closed system and also require the addition of exogenous material. The aim of this study was to develop a method to reduce the volume of CB units, prior to the addition of cryoprotectant, using a closed system, compatible with large-scale processing and storage, without significant loss of NC or progenitor cells and without the risk of contamination. Using the Optipress II Automated Blood Component Extractor from Baxter Healthcare (Newbury, UK) partial removal of both plasma and RBC from the CB collection was achieved after one centrifugation step, with the standardisation of all CB units to 25 ml. To validate the volume reduction method fully, the units were cryopreserved, thawed and washed, as for transplantation. Viability, nucleated cell counts and clonogenic assays were used to demonstrate that the process of volume reducing the CB units caused no detrimental effect on the ultimate recovery of the cells.

Materials and methods CB collection CB was collected, by trained CBB staff, from the umbilical cord following delivery of the placenta, into a collection bag containing 21 ml CPD solution (Maco Pharma, Hampton upon Thames, UK). The units were stored at 22°C and all units with a volume of CB of 40 ml or above were processed within 24 h of collection.

Volume reduction of cord blood units S Armitage et al

506

Volume reduction

CD34⫹ cell enumeration

After thorough mixing, the CB collections were transferred to an Optipac triple system (Baxter Healthcare) from which the anticoagulant had been removed under sterile conditions. The Maco Pharma collection bag containing the CB was attached by sterile docking (Terumo, Haemonetics Corporation, MA, USA) to this triple system and the CB was transferred into the central collection bag. The CB in the triple bag system was centrifuged in oval buckets at 3300 g for 12 min at 22°C, ensuring that the bags were well supported to prevent creasing of the bag and disruption of the buffy coat layer. A standard protocol, programmed into the Optipress II, together with the standard backplate for buffy coat preparation, was used to process the CB units. The selected protocol No. 1 designed for the primary separation of whole blood into three components, automatically expressed the plasma and buffy coatdepleted RBC into the top transfer bag and the bottom bag, containing SAG-M, respectively, leaving the buffy coat component in the central collection bag. The programme was set with the following parameters: buffy coat volume of 25 ml, a buffy coat level of 6.5 and a force of 30. Samples for haematological and CD34⫹ cell counts, progenitor cell assays and both aerobic and anaerobic bacteriology cultures were removed from the CB unit pre- and post-separation.

Pre- and post-processing samples were dual labelled with a FITC-conjugated anti-CD45: (anti-HLe-1, Becton Dickinson (BD), Oxford, UK) and a PE-conjugated anti-CD34: (anti-HPCA-2, BD).11 Analysis of the CD34⫹ cell population was performed by flow cytometry on a BD FACSort using CellQuest software. The CD34 stained cells were determined as a percentage of the total NC and the results were expressed as absolute numbers. Viability of the samples was assessed by the addition of propridium iodide to the test.

Cryopreservation CB units were frozen in single 250 ml cryocyte bags (Baxter Healthcare) with the addition of an equal volume of 20% DMSO in 10% Dextran 40 (Baxter Healthcare). The units were double bagged and placed in aluminium cassettes for immediate freezing in a Kryo 10/16 controlled rate freezer (Planer Select, Sunbury, UK). On completion of the freezing programme when the units had been cooled to ⫺80°C, they were transferred to liquid nitrogen (LN2) vapour phase storage. Thawing and washing For experimental purposes the units were stored in the LN2 vapour for at least one week, then removed, thawed and washed according to the New York protocol.7 On removal from the LN2 vapour the bag was immersed in a 37°C waterbath and rapidly thawed. Immediately the CB was diluted with an equal volume of an isotonic salt solution containing 2.25% human albumin (BPL, Elstree, UK) and 5% Dextran 40, with continuous mixing. After centrifugation at 400 g for 15 min, the supernatant was removed and the sedimented cells were resuspended slowly in fresh albumin/dextran solution to the original 25 ml volume. Haematological cell counts A full blood count (FBC) and differential was performed on all samples using an automated haematology analyser, H3 Technicon (Bayer, Newbury, UK).

Progenitor cell assay For assessment of the colony-forming units (CFU) in the pre- and post-processing samples assays were performed using a commercially prepared complete methylcellulose medium, Methocult GF H4434 (StemCell Technologies; Metachem Diagnostics, Northampton, UK). Cells were plated unseparated, in triplicate at concentrations of 2.5 and 5.0 ⫻ 104 NC/ml. After 14 days incubation at 37°C in humidified air and 5% CO2, granulocyte–macrophage (CFU-GM), erythroid (BFU-E) and multipotential (CFUGEMM) colonies were scored by microscopic examination. Bacteriology To check for contamination, 0.5 ml samples from the preand post-processed CB units were incubated for aerobic and anaerobic micro-organisms, at 37°C for 14 days using an automated blood culture system (BacT/Alert; Organon Teknika, Cambridge, UK). Statistics Results are expressed as mean ⫾ s.d. The correlation between cell content of pre- and post-processed CB units was analysed by linear regression analysis. Results The results were analysed as two groups: the initial 30 CB units that were used during the development stage of this volume reduction method and the following 169 units handled in the routine processing laboratory. Volume reduction Table 1 shows the results of CB units which were volume reduced during the initial development stage (n = 30) and Table 1

Volume reduction and red cell depletion of CB units CB unit volume (ml)

Development (n = 30) Routine (n = 169)

Pre-process mean ± s.d. (range)

Post-process mean ± s.d. (range)

88.4 ⫾ 29.2 (46.6–174.1) 86.2 ⫾ 22.9 (52.3–185.1)

24.0 ⫾ 1.5 (19.5–25.9) 24.5 ⫾ 1.5 (18.5–27.5)

Red cell depletion (%) post-process mean ⫾ s.d. (range)

54.1 ⫾ 17.0 (24.6–83.1) 51.9 ⫾ 14.8 (12.6–83.8)


Table 2

Recovery of cells in buffy coat % Recovery

NCC MNC Lymphocytes CD34⫹ Total CFU

a

P value

Development mean ⫾ s.d. (range)

Routine mean ⫾ s.d. (range)

91.6 ⫾ 16.8 (65.9–116.4)a 97.7 ⫾ 14.6 (88.3–110.5)a 98.4 ⫾ 23.9 (71.6–120.0)a 96.2 ⫾ 13.9 (80.5–102.6)b 105.9 ⫾ 21.9 (90.6–121.1)b

83.3 ⫾ 9.7 (58.2–106.0)c 88.7 ⫾ 7.8 (70.7–114.8)c 92.4 ⫾ 7.3 (74.2–124.5)c 98.9 ⫾ 15.6 (81.0–108.8)d 102.9 ⫾ 15.6 (90.3–120.0)e

0.0647 NS NS NS NS

n = 30; bn = 20; cn = 169; dn = 49; en = 23.

after the process had been incorporated into the routine processing laboratory (n = 169). The pre-processing volume of the initial 30 CB units volume reduced using the Optipress II machine ranged from 46.6 to 176.1 ml, (mean ⫾ s.d. 88.4 ⫾ 29.2 ml); post-processing the reduced mean volume of the CB units was 24.0 ml (s.d. 1.5 ml), range 19.5– 25.9 ml. The results of the first 169 units processed under routine conditions, for inclusion into the unrelated bank, showed very similar results; a pre-processing mean volume of 86.2 ml (s.d. 22.9 ml) reduced to a mean volume of 24.5 ml (s.d. 1.5 ml) in the post-processed unit. A substantial red cell depletion was achieved in both groups, mean values of 54% and 52%, respectively. Cell recovery The volume reduction process was assessed by monitoring the NC, mononuclear cell (MNC), lymphocyte, CD34⫹ cell and total CFU content in both the pre- and post-process CB units. Cell recovery was reduced for all but the CD34⫹ cells when this method of volume reduction was introduced into the routine processing laboratory. The data for the cell recovery after processing are presented as a percentage in Table 2 and as absolute numbers in Table 3; Table 3 only Table 3

Cell counts for routine processing

Per unit

Pre-process mean ⫾ s.d. (range)

Post-process mean ⫾ s.d. (range)

r

NCC ⫻ 108

169 169

Lymphocytes ⫻ 108

169

8.5 ⫾ 3.7 (3.0–22.2) 4.6 ⫾ 2.1 (1.3–10.8) 4.1 ⫾ 1.9 (1.1–9.9) 2.9 ⫾ 2.5 (0.4–37.3) 1.1 ⫾ 1.0 (0.1–5.1)

0.95

MNC ⫻ 10

10.6 ⫾ 4.7 (3.7–25.1) 5.3 ⫾ 2.4 (1.6–11.3) 4.6 ⫾ 2.1 (1.3–10.2) 2.4 ⫾ 3.2 (0.1–33.9) 1.4 ⫾ 0.9 (0.2–7.7)

CD34⫹ ⫻ 106

49

CFU ⫻ 106

23

r = correlation coefficient.

Thawing and washing Twenty of the CB units were thawed, washed and assessed for their cell viability and percentage recovery of NC and CFU from the whole blood sample prior to processing. These results were compared with earlier quality assurance data obtained when the CB units were frozen as whole blood (Table 4). There was no significant difference of either NC (P = 0.4719) or CFU (P = 0.1187) recovery between units that had been frozen without any processing and those units processed in order to reduce their volume. The difference in viability of the thawed cells was also not significant (P = 0.3797). Bacteriology

n

8

shows results for the routine processing since the cell numbers for each group were similar. Pre-processing, the nucleated cell count (NCC) ranged from 3.7 to 25.1 ⫻ 108 per unit (mean 10.6 ⫾ 4.7 ⫻ 108). Post-processing the mean NCC was 8.5 ⫾ 3.7 ⫻ 108 (range 3.0–22.2 ⫻ 108) with a mean NC recovery of 83.3% when performed in the routine lab; this compared with 91.6% during the development stage. The mean initial MNC was 5.3 ⫾ 2.4 ⫻ 108 (range 1.6–11.3 ⫻ 108) and after processing was 4.6 ⫾ 2.1 ⫻ 108 (range 1.3–10.8 ⫻ 108), which gave a mean recovery of 88.7% and 97.7% in the development group. Lymphocyte recovery was 98.4% and 92.4% in the respective groups with a mean lymphocyte count of 4.6 ⫾ 2.1 ⫻ 108 (range 1.3–10.2 ⫻ 108) reduced after processing to 4.1 ⫾ 1.9 ⫻ 108 (range 1.1–9.9 ⫻ 108). CB units collected contained a mean of 2.4 ⫾ 3.2 ⫻ 106 CD34⫹ cells (range 0.1–33.9 ⫻ 106), post-processing this value was 2.9 ⫾ 2.5 ⫻ 106 (range 0.4–37.3 ⫻ 106) giving a recovery of 98.9%, 96.2% in the development group. The mean recovery of total CFU numbers was 100% in both groups. Linear regression analysis of the NC, MNC, lymphocyte, CD34 and total CFU counts pre- and post-processing gave correlation coefficients of 0.95, 0.97, 0.98, 0.97 and 0.98, respectively (Figure 1). Twenty of the CB units processed were assessed for cell viability and all showed more than 96% viable cells. The RC and the plasma fractions (n = 20), recovered after processing, were assessed for their NCC; a median of 4.1% and 1.0% NC were recovered in the RC and plasma components, respectively.

0.97 0.98 0.97 0.98

All CB units used in this study were negative for bacterial and fungal contamination both pre- and post-processing and post-thaw. Discussion Clinical results of CB transplants1–3 support the establishment of cord blood banks with large numbers of frozen, HLA-typed CB units available for immediate use. Cryopreservation of unmanipulated CB units in 10% DMSO solution for large-scale banking, although feasible, is not realistic. A number of papers indicate that processing of CB units to reduce their volume, prior to storage, is possible4–10

507


508

Nucleated cells

Mononuclear cells MNC post-processing (×108)

NCC post-processing (×108)

20

10

R2 = 0.9108 0

15

Lymph post-processing (×108)

15

30

10

5

R2 = 0.9506 0

0

10

20

30

NCC pre-processing (×108) Figure 1

Lymphocytes

0

5

10

MNC pre-processing (×108)

15

10

5

R2 = 0.9629 0 0

5

10

15

Lymph pre-processing (×108)

Relationship between total nucleated cell, mononuclear cell and lymphocyte counts in CB units pre- and post-processing (n = 169).

Table 4 Recovery cells after freezing and thawing, comparing units frozen as whole blood with units frozen after processing to reduce their volume

n % recovery NC % recovery CFU % viability

Whole blood mean ± s.d.

Volume reduced mean ⫾ s.d.

10 75.5 ⫾ 23.7 93.9 ⫾ 38.8 70.6 ⫾ 7.7

22 80.4 ⫾ 24.7 99.2 ⫾ 28.6 80.7 ⫾ 7.2

P

NS NS NS

and successful transplants with RBC-depleted CB units have been reported.1,7 However, the majority of these methods are performed in open systems and/or require the addition of exogenous material which is not compatible with large-scale processing. This paper describes a process, compatible with routine blood bank processing, for reducing the volume of the CB unit by partial removal of RBC and plasma in a closed system, by differential centrifugation followed by the automated expression of the RC and plasma using the Baxter Optipress II system. New processes often perform better during development, where one operator conducts the complete process on a small scale, under ideal conditions. Accuracy and consistency often decline when such processes are introduced into routine operation where a team of operators conduct the procedure in a busy, pressurised environment, on a large scale. Hence the decision to examine this process under these two conditions. Initial work using the Optipress II showed that to reduce the CB unit to less than 25 ml gave inconsistent results (data not shown). Having standardised the system to 25 ml, any unit that met all the criteria for banking, ie ⬎40 ml, collected within 24 h and stored at 22°C, was processed. Good separation of the CB unit during the centrifugation step was essential. This was achieved by using oval centrifuge buckets and providing good support for the triple bag system. The results show that the CB units (range 46.6–185.1 ml) could be processed to a standard volume of 25 ml, regardless of the initial starting volume, with 53% red cell

depletion. Reducing the CB unit to a standard volume consequently allows the addition of a standard volume of cryoprotectant to the cells and permits uniform freezing of the units and more rapid thawing. With the addition of an equal volume of 20% DMSO the units are frozen at a final volume of 50 ml which reduced our storage requirement by two-thirds. This saving will be further improved by the addition of 50% DMSO,7 allowing the units to be frozen at a volume of 31 ml with an 86% reduction in storage space. Good recovery of NC (92%/83%), MNC (98%/89%), lymphocytes (98%/92%), CD34⫹ cells (96%/99%) and total CFU (106%/103%) was demonstrated in both development and routine groups. Although recovery was generally higher in the development phase, as expected, the values were not significantly different other than for the nucleated cells (P = 0.0647). NC, MNC and lymphocyte recovery of the units processed in the routine lab, although slightly reduced, showed standard deviations of at least half those for the development phase reflecting the larger number processed and the reproducibility and reliability of the technique. Also, the freezing and thawing results demonstrated that processing of the units as described had no detrimental affect on the ultimate recovery of the cells. These results are comparable with those obtained using the alternative techniques of density gradient separation with either Percoll4 or Ficoll,5 3% gelatin sedimentation,6 hydroxyethyl starch sedimentation7,8 and differential sedimentation.9 The manipulations involved in these alternative methods, apart from those described by Rubinstein et al7 and Sousa et al,9 were performed in an open system which increases the risk of bacterial contamination. The two closed systems described7,9 involve two centrifugation steps, increasing the risk of damage to the bags and the processing time. In addition, the procedure described by Rubinstein et al7 requires the addition of hydroxyethyl starch for RC sedimentation. The use of the Optipress for volume reduction was first described by Ademokun et al10 to reduce CB units to 44 ml (n = 25). This report describes the optimisation of this process to reduce all CB units to 25 ml, which is the minimum volume possible with a standard Optipress II and standard backplate, in a routine processing laboratory.


Failure rate of the Optipress process was 1.53%; 0.94% of these failures were due to the presence of clots in the units, which block the bleedlines. These units should have been screened out as unsuitable for banking prior to processing. The remaining 0.59% were unexplained failures where either the whole collection was expressed into the top or bottom bag or the buffy coat ran over into the plasma in the top bag. In addition to space and cost savings, another advantage to reducing the volume of CB units is that samples for ABO/RhD typing and microbiology screening can be removed from the RC and plasma components, respectively, increasing the volume of transplantable material. In conclusion, the process described here demonstrates that CB units can be processed routinely, preserving the quantity and quality of the nucleated cells and progenitor cells in a closed system, without the addition of any exogenous material.

Acknowledgements We acknowledge the invaluable assistance of Yvonne Caffrey, Marcia Elderfield and Maureen Morgan.

References 1 Kurtzberg J, Laughlin M, Graham ML et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. New Engl J Med 1996; 335: 157–166.

2 Wagner JE, Rosenthal J, Sweetman R et al. Successful transplantation of HLA-matched and HLA-mismatched umbilical cord blood from unrelated donors: analysis of engraftment and acute graft-versus-host disease. Blood 1996; 88: 795–802. 3 Gluckman E, Rocha V, Boyer-Chammard A et al. Outcome of cord blood transplantation from related and unrelated donors. New Engl J Med 1997; 337: 373–381. 4 Charboro P, Newton I, Schaal JP, Herve P. The separation of human cord blood by density gradient does not induce a major loss of progenitor cells. Bone Marrow Transplant 1992; 9: 109–110. 5 Harris DT, Schumacher MJ, Rychlik S et al. Collection, separation and cryopreservation of umbilical cord blood for use in transplantation. Bone Marrow Transplant 1994; 13: 135–143. 6 Nagler A, Peacock M, Tantoco M et al. Separation of haematopoietic progenitor cells from human umbilical cord blood. J Hematother 1993; 2: 243–245. 7 Rubinstein P, Dobrilla L, Rosenfield RE et al. Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution. Proc Natl Acad Sci USA 1995; 92: 10199–10122. 8 Bertolini F, Battaglia M, Zibera C et al. A new method for placental/cord blood processing in the collection bag. I. Analysis of factors involved in red blood cell removal. Bone Marrow Transplant 1996; 18: 783–786. 9 Sousa T, de Sousa ME, Godinho MI et al. Umbilical cord blood processing: volume reduction and recovery of CD34⫹ cells. Bone Marrow Transplant 1997; 19: 311–313. 10 Ademokun JA, Chapman C, Dunn J et al. Umbilical cord blood collection and separation for haematopoietic progenitor cell banking. Bone Marrow Transplant 1997; 19: 1023–1028. 11 Sutherland DR, Keating A, Nayer R et al. The ISHAGE guidelines for CD34⫹ cell determination by flow cytometry. J Hematother 1996; 5: 213–226.

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