Cord blood banking: current status

Abstract

Objectives: Cord blood is increasingly used as a hematopoietic progenitor cell source for bone marrow transplantation.

Methods: Development of cord blood banks for altruistic use was essential to open the possibility of opting for cord blood therapy for patients lacking a conventional donor.

Results: Cord blood has major advantages as a resource for donor provision. First, it universalizes the access to the therapy since it allows transplantation of partially matched HLA grafts, benefiting ethnicities less represented in the adult volunteer list. Second, it is an off-the-shelf, ready-to-use source that avoids donor risk and attrition. Third, potentially it has better long term sustainability if a defined optimal size of the inventory is achieved; currently, more than 80% of patients, regardless ethnicity, can find a reliable cord blood donor with the current worldwide inventory.

Conclusion: In order that this approach may definitely solve the problem of an equitable access to the therapy, two aspects need to be improved: the quality of CB inventories (driven by a higher stringency of regulatory requirements), and clinical outcomes, particularly understanding the appropriate donor selection and focusing research on accelerating the speed of engraftment and immune reconstitution.

Keywords:

• Cord blood
• Banking
• Transplantation
• Unrelated donor

Cord Blood as a Haematopoietic Progenitor Cell Graft

Cord blood (CBU) contains naturally mobilized haematopoietic progenitor cells that have the capacity to engraft and reconstitute the haematopoietic system of an individual. This ability was first demonstrated when Gluckman et al. performed the first cord blood transplantation,1 with subsequent confirmation over the past 20 years with more than 20 000 procedures performed. A combination of large-scale banking and clinical transplantation development has made cord blood an alternative source for bone marrow transplantation.2

The use of CBUs as a source of stem cells for unrelated transplantation has several advantages over other sources of stem cells.3 These include an absent risk for the donor, a ready to use cellular product, and, clinically, low incidence of graft-versus-host disease which increase donor pool availability. However, the number of stem cells in a cord blood graft is relatively low for adult patients, delaying engraftment, and in principle there is no possibility to use donor lymphocytes infusion after transplantation. However, alternatives to overcome engraftment limitation have been developed like double cord blood transplantation with encouraging results. In this regard, therapy is extended to older patients using reduced toxicity conditioning and the double graft approach.

Cord Blood Banking

The Cord Blood Bank (CBB) is a multidisciplinary structure that is responsible for the recruitment and subsequent management of donors/mothers as well as the collection, processing, testing, cryopreservation, storage, listing, reservation, release, and distribution of CBUs. Public cord blood banks aim to store cord blood for allogeneic transplantation purposes. These banks are typically not for profit entities where units are banked at no cost to the donor. It is typically affiliated to a registry that offer CB units from multiple banks using defined search algorithm and sophisticated matching programs. Typically cord blood is not only searched by HLA match but cell dose and quality are important factors too. Nowadays, more than 500 000 units are registered in www.bmdw.org. The rapid growth in the global inventory resulted in a proportional decrease in the release percentage by each bank making public banking activities economically challenging. Recent publications are trying to address this issue, and it is still difficult to anticipate the long-term model of CB bank maintenance and sustainability.4

In addition to public banks, a large number of private banks have also been established. These banks aim to store cord blood for autologous or familiar use. In this setting, the cost of storage is covered by the donor, making them for-profit organizations with more solid structures. However, in contrast to allogeneic uses, the clinical benefit of autologous application remains to be proven.

Newly explored models of public–private partnership, the so called hybrid banks, are currently being proposed, notably driven by the need to improve the financial viability of cord blood banks. To be useful to families, these approaches will need to be more rigorously scrutinized by adoption of accreditation standards and improvement of the accuracy of their informed consent and other ethical aspects. These initiatives should share benefits to help research on cord blood cells. No question, sibling banking needs to be regularly offered when there is a clear indication for haemopoietic stem cell transplantation and be available to all patients in need.5

Cord blood processing and storage

Research and development of new processing tools and implementation of worldwide regulations will control and guarantee the quality, safety and potency of the future cord blood inventory.6 Despite this aspiration, many problems remain to be solved. Existing and new cord blood bank establishments need to design and develop quality management systems that ensure the product consistently attains its predefined quality at the end of the process.

There are several key steps where graft potency can be challenged. Cells are obtained after cord clamping. Collection techniques may influence the volume harvested but should not influence cell viability. In contrast, conditions during delivery influence the number of progenitors and cells in the graft, especially high in stressful labours. The majority of products range from 20 to 60 CD34+ cells per microlitre with some significant outliers. After collection, there is a preference to use cool environments for transit times longer than 24 hours. It is very important that each bank generates its own validation based on CD34+ viability or CFU assessment before selecting time and temperature parameters for non-frozen storage and transport. Transport containers need to minimize temperature changes and include temperature logs to monitor variations.

Many cord blood banks have threshold policies which remove units that are more than 48 hours old from collection and units below a defined volume or cell content. Nowadays, very high cellular thresholds are proposed, most of them largely higher than 1 billion total nucleated cells that make collection programmes inefficients. After volume reduction, cell can be at risk if transition times are not minimized. It is helpful to establish a minimum TNC yield for quality control (i.e. we use 60% as reference). Automation is beneficial to reduce processing failures especially in cord blood banks with high turnover. It is important to ensure that the processing of each single unit is done by a single technician in order to minimize risk of mislabelling. After freezing, units need to be sealed and packed within canisters. Particular attention needs to be paid to the type of bags used, the canister adaptability and the overwraps added to prevent cross contamination in case of bag damage. Cryovials should be avoided since sample handling especially thawing is complex and may generate unexpected variables. After freezing the unit needs are quarantined before being transferred to the long term storage inventory. Any event that results in an increase in temperature above −130°C after freezing is considered a significant warming event. Their accumulation during the storage can theoretically affect cell viability after thawing due to unstable glass transition. Duration of storage for cryopreserved CBU, including assigning an expiration date to CB units where appropriate, is still not defined. This time should be determined based on the medical literature and/or on the banks own experimental data.

We have demonstrated that quality tests of segment linked to the bag inside the overwrap correlates well with transplant bag and therefore can be used to perform batch quality control.7 Such tests may include assays for safety (infectious or genetic disease markers), identity (HLA, ABO, gender), and potency (CFU, viability, functional flow cytometry). In our bank, we use the ‘clonogenic efficiency (CLONE)’, a parameter based in the correlation between CFU and CD34+ cells, as a potency assay. It means that for a given amount of CD34+ cells, a defined number of CFUs are expected to growth. We hypothesize that any CLONE drop can predict function loses. In our experience, CB CD34+ cell expected CLONE is 35±12%. As a safety threshold, we arbitrarily chose 10% (equivalent to two standard deviations) as releasing criteria. Lastly, we are developing a new approach to shorten quality test before releasing. Application of ‘functional’ flow cytometry methodology assessing CD34 apoptosis resulted in encouraging prediction of CFU read-outs.8 This method is ideal to return immediate potency results. Finally, the most important quality check remains the transplant centre on data on cell yields and engraftment.

Cord Blood Provision

In order to make inventories available for transplantation, cord blood banks list their validated units using minimum essential data required for search procedures. Searches usually first sort potential candidates according to HLA match categories and then by cell dose. Other variables can be used to identify the best compatible donor between a few pre-selected candidates. Thus, data usually exported from the local processing databases to publicly accessible global databases are: (1) HLA typing of HLA–A, –B and –DRB1 loci using molecular techniques of low resolution for class I loci and high resolution for class II loci; (2) cell dose, with two main parameters, the amount of NC (including erythroblasts) contained in a CB unit, and CD34+ cell count as a surrogate indicator of graft potency, and (3) other qualitative data, including cord blood bank, year of production, ABO, other HLA types, and CFU.

Once a unit is listed, cord blood banks can be contacted for different request types: for instance, preliminary reports, additional testing (examples of most frequent activities related are extended typing, specific IDM to meet local regulations, and CFU post-thawing to confirm potency) and reservation to allocate a CBU for a defined patient and can include identity confirmation ideally using a segment attached to the transplant bag. When a decision is taken, cord blood bank receives a shipment request. It is recommended not to start patient conditioning before a unit has been safely received at the transplant centre. In spite cord blood grafts are off-the-shelf products, a number of administrative and technical procedures are required before transporting the unit using a door-to-door service. Factors including export/import issues and release tests sometimes prevent the immediate use of a unit.

Distribution of a frozen product is sometimes challenging. It is done using vapour-nitrogen cryoshippers approved for air shipment. All time sample need to be maintained below temperature of −150°C, and therefore temperature range during transportation shall be monitored.

After reception, inspection for bag integrity and labelling are important aspects to review before transiently storing the unit below −150°C. When all cord blood characteristics are reviewed and accepted, conditioning can be started. CB reconstitution for infusion is still controversial but usually a dilution/washing step using a dextran buffer is recommended to minimize infusion toxicity and maximize progenitor viability. Finally, every cord blood bank will require clinical information as part of their quality assurance plan. In this regard, Eurocord in Europe and CIBMTR in US are the main centres for retrospective studies on CB efficacy and provide important insights for future modification of standard practices.9

Towards More Rational Allogeneic Cord Blood Inventories

Use of unrelated donors as a source of stem cell for haematopoietic transplantation is steadily increasing and accounts for more than half of the allogeneic procedures currently performed. Combining adult and cord inventories allows transplant physicians the identification of suitable donors for most of their patients. This is due to an increase in both the efficiency of adult registers and the availability of high-quality cord blood units in accredited cord blood banks. Particularly, CB transplantation already contributed to 15% of the unrelated donor procedures performed within countries reporting to WMDA in 2010. Next challenge for CB banks is the ability for an immediate supply of reliable units defined by the four quality components: safety (sterility, IDM, haemoglobin), identity (HLA, blood group), purity (TNC and CD34) and potency (viability and CFU). Interestingly, purity and potency are becoming essential in CB transplantation. Assuring a good functional cell and progenitor dose after thawing is one of the most predictable factors of survival. Furthermore, for a particular CBB, availability of large numbers of accredited units with high cell numbers is crucial since those units are more and more selected in preference. Our experience at the Barcelona CBB at Programa Concordia Banc Sang i Teixits is a progressive increase on cells required for transplantation along time. Thus, last three years, only 5% of our almost 600 units shipped contained less than 12×10⁸ frozen TNCs.

We previously published, UK required 50 000 units to provide at least one donor to 80% of searches.10 There, we assumed five out of six donor-recipient matches (by A, B low resolution and DRB1 high resolution) will be the most common CB transplants in the future. We did a similar exercise for Spain and this number turned to be 35 000 (data unpublished). These big numbers are based only considering HLA distribution. However, as cell dose is becoming important, such size makes CBB economics difficult. For instance, to achieve 50 000 units of more than 15×10⁸ TNC at collection (resulting in 13×10⁸ frozen TNC after an average volume reduction lose of 15%), we will require the collection of 250 000 units that might be certainly unaffordable and expensive in a short period of time. Following these data, we now propose that a more cost-benefit inventory could be to plan an operational size of 15 000 units higher than 12×10⁸ frozen TNCs. Such bank would provide suitable units (five out of six) for approximately 2/3 of all searches. Further growth adds just marginal advantage but increasing enormously the cost (three times more investment will only provide 15% of additional compatibilities). Once achieved this size, aim would be maintaining numbers to reduce maintenance costs. Under this vision, and assuming similar or better clinical outcomes for CBT, CB could be the most sustainable stem cell source for the future.

Our recommendation is that CB banks should re-calculate size of their operational inventory defining their minimum frozen cell dose threshold (i.e. more than at least 12×10⁸ TNC and 4×10⁶ CD34). In addition, these operational units should be selected by standard quality criteria (i.e. Netcord-FACT) fulfilling strict eligibility questionnaires, appropriate batch record and validation, availability of reference samples for extended serology, and, critically, presence of an attached segment to perform identity/potency verification.

Conclusion

In summary, cord blood banking has developed extensively since the pioneering work by Pablo Rubinstein and colleagues in New York in 1993. In this presentation we will emphasize the need to re-enforce validation, in-process quality control and common international accreditation strategies such as those promoted by Netcord-FACT. Safety and identity issues remain the most critical parts of the cord blood bank responsibility. Extensive manipulation with significant variables such as transit time, storage temperature and cell composition result in unexpected viability problems. Finally, we encourage a revision of the available CBU inventories based on quality categories to help users to identify the most appropriate products. The priority of cord blood banks to improve outcome of CBT should now focus on building quality rather than quantity.