Ronan Foley & Pamela O'Hoski
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
Background
Historically obtained from the posterior pelvis of a donor under general anaesthesia, haemopoietic progenitor cells (HPCs) can now be obtained from peripheral blood as well as from the umbilical cord and placenta post-delivery. Recognition that haemopoietic growth factors (i.e. G-CSF) administered either alone or following chemotherapy results in significant mobilization of a blend of cell populations (including HPCs) into the peripheral blood has had a profound impact on stem cell collection both for autologous transplantation and for healthy stem cell donors. Identification of HPCs correlates with expression of the CD34+ antigen. Other markers such as the absence of CD38 or the presence of CD133, Flk2/Flt3 as well as aldehyde dehydrogenase activity may be more specific but remain investigational. Functional evaluation of colony-forming units (CFUs) of myeloid, erythroid, megakaryocytic and long-term culture initiating cells may be useful functional assays that can complement immunophenotypic analysis.
Terminology to describe HPC and other cell-based human products derived from a bone marrow harvest (HPC, Marrow), from mobilized apheresis peripheral blood (HPC, Apheresis), from umbilical cord (HPC, Cord Blood) or from steady-state apheresis for donor lymphocyte infusion (DLI) (Therapeutic Cells, T cells) (Table 40.1) have been suggested by the International Society for Blood Transfusion (ISBT 128 nomenclature). Each source exhibits different biological properties and graft compositions. If one compares a bone marrow graft (approximately 700–1500 ml) to mobilized blood progenitor cells (100–400 ml) collected by leucapheresis distinct differences are noted. A mobilized peripheral blood component typically contains a greater number of CD34+ progenitors but also a greater number of T-lymphocytes, which has raised concern for a greater incidence of chronic GVHD in patients undergoing allogeneic BMT [1]. Conversely, when a nonmyeloablative transplant is performed a higher dose of CD34+ cells may be of greater importance to ensure engraftment and stable chimerism, thus favouring an HPC, Apheresis product. A unit of cord blood (HPC, Cord Blood) may have less progenitor cells, but compensates with a higher proliferative potential and a lower risk of GVHD [2].
Table 40.1 Human haemopoietic progenitor cells (HPCs).
Transplant procedures
Autologous SCT
Expanding clinical indications support the use of autologous SCT in a variety of clinical settings, including multiple myeloma (single or tandem) and relapsed non-Hodgkin and Hodgkin lymphoma (B and T diffuse large cell, mantle cell lymphoma, Burkitts and follicular lymphoma). Autologous SCT is also a therapeutic option for patients with gonadal or retroperitoneal germ cell tumors refractory to cisplatin-based chemotherapy. At present autologous progenitor cells are almost exclusively obtained by leucapheresis. Use of HPC, Apheresis reduces the time to engraftment with shorter hospitalization, less transfusional support and the use of antimicrobials [3].
Allogeneic SCT
Allogeneic SCT involves replacement of a diseased bone marrow with haemopoietic elements from a healthy donor. It is now known that engraftment of both HPCs as well as donor immune T-lymphocytes are essential for long-term haemopoiesis and disease control. Donor lymphocytes contribute to both graft-versus-leukaemia (GVL) as well as graft-versus-host disease (GVHD). Shifting the balance of therapeutic efficacy solely from stem cell replacement to maintenance of a transplanted donor immune system has led to reduced intensity nonmyeloablative or ‘mini’ allogeneic transplants.
Allogeneic donors may be from a related sibling or obtained from a bone marrow registry. Matches are based on the human leucocyte antigen (HLA) system comprised of genes on chromosome 6 that encode cell-surface antigen presenting proteins linked to our immune system. The major histocompatibility complex (MHC) is made up of two basic classes involved in antigen presentation and subsequent immune activation. MHC class I includes HLA-A, HLA-B and HLA-C, whereas MHC class II includes HLA-DR, HLA-DQ and HLA DP. The proteins encoded by HLA define self and directly instruct the immune system to recognize self-versus-nonself. HLA typing previously employed simple serological testing (antibody-based) to provide low resolution typing. Although useful in the related setting, there have been concerns regarding sole use of low resolution typing use in unrelated donors. Many HLA laboratories now perform high resolution (HR) molecular typing to ensure that a potential unrelated donor/recipient pair is as highly matched as possible.
If an unrelated match cannot be found remaining options include related haploidentical transplantation or allogeneic transplant using stored cord blood. Haploidentical SCT involves a related donor/parent who has only a partial HLA match. Due to significant HLA barriers large-volume CD34+ product must be rigorously purified (T-depleted). This may result in lasting immune deficiency with a high risk of fulminant infection (viral, CMV, EBV) or relapse.
Donor lymphocyte infusions (DLI)
The cell products infused for DLI collected from the original HPC donor in an unstimulated state are called Therapeutic Cells-Apheresis and Therapeutic Cells-T Cells. DLI may be used to help convert mixed donor chimerism to full donor chimerism after allogeneic HPC transplant or as pre-emptive therapy for treatment of an early relapse by providing a direct GVL effect.
HPC products
Bone marrow
Use of bone marrow (HPC, Marrow) has decreased in recent years as other sources of progenitor cells have become available but in certain circumstances, like transplanting paediatric patients or patients with aplastic anaemia, a role for bone marrow still exists. Collecting bone marrow involves placing the donor under general anaesthesia and then aspirating 10–15 ml/kg recipient weight (maximum 20 ml/kg donor weight) from the posterior iliac crests and placing the product in a collection bag containing ACD/heparin anticoagulant. Collected product is passed through 500 and 200 μm filters to remove bone and other debris prior to infusion or further processing. The target nucleated cell dose (automated counter) is 2–4 × 108 /kg recipient weight. Use of marrow CD34+ enumeration suggests a CD34+ cell dose >3.0 × 106/kg correlates with improved recovery and 5-year survival while <1.2 × 106/kg correlates with inferior recovery [4,5].
Peripheral blood
Mobilization of CD34+ progenitor cells into blood with collection by leucapheresis (HPC, Apheresis) is the method of choice for patients undergoing autologous SCT. This procedure is based on obtaining sufficient CD34+ progenitor cells (defined as a minimum of 2.0 × 106 CD34+ cells/recipient weight and an optimal of 5.0 × 106 CD34+ cells/recipient weight). Ideally the mobilization strategy employed should result in an optimal product with predictable engraftment performance, minimal side effects, a reasonable DMSO volume and diminished risk of contamination with tumor cells. Lower doses of infused CD34+ cells can result in delayed or failed platelet engraftment, Case-by-case decisions are made to proceed with auto SCT when less than 2.0 × 106/kg CD34+ cells based on the clinical situation and stability of the underlying disease. For patients undergoing auto SCT two strategies can be employed: either growth factor(s) alone or growth factors that follow administration of chemotherapy. Use of growth factor(s) alone results in a more predictable schedule, less neutropenia-associated infections and reduced cost, but is associated with a higher chance of mobilization failure. A combined chemotherapy/growth factor approach is associated with greater toxicity and less predictability. The benefits of using chemotherapy include disease control during the procedure and theoretical benefit of reduced tumor cell contamination.
As an alternative to undergoing a marrow harvest, an allogeneic donor may be asked to provide mobilized peripheral blood progenitors collected by leucapheresis. Administration of G-CSF alone is the currently accepted strategy for mobilization of normal healthy donors. Use of HPC, Apheresis as opposed to HPC, Marrow appears to improve the time to haemopoietic recovery and offers a greater GVL effect [6], but carries a potentially higher risk of chronic extensive GVHD. Retrospective studies suggest children and adolescents with acute leukaemia or aplastic anaemia have inferior outcomes when an HPC, Apheresis graft is used [7].
Umbilical cord blood
Characteristics of banked cord products include highly functional HPCs, less CMV contamination and a lower risk of GVHD. It is generally accepted that the kinetics of haemopoietic recovery are significantly slower when using HPC, Cord Blood. This may relate to fewer and less mature HPCs. A minimum target of approximately 3.0 × 107 nucleated cells per recipient weight per unit of cord blood is required. A higher dose may be considered depending on HLA disparity. Measurement of CD34+ cells/recipient kg weight may be more informative. The mean collection volume for a cord sample is approximately 100 ml (50–200 ml) including anticoagulant [8]. Several techniques for cord blood collection may be performed either prior to or following delivery of the placenta. Closed system collection techniques have improved rates of bacterial contamination. Cells can be stored in a smaller volume by immediately removing plasma and red blood cells. Characterization of the cord unit includes: volume, weight, total nucleated count, CD34+ cell count, colony-forming analysis, ABO/Rh and HLA typing, full panel transmissible disease testing and haemoglobin electrophoresis [9]. Cord units should be processed quickly and immediately stored at 4°C with cryopreservation to occur as soon as possible. Once properly stored, it is currently not known how long HPC, Cord Blood components remain viable. In vitro analysis has suggested reasonable viability to as long as 15 years, perhaps longer [10]. In an effort to hasten time to reconstitution in larger adult recipients, double UCB are now being considered [11]. The strategy appears to improve significantly the time to engraftment. When evaluated 100 days posttransplant, typically only one cord unit, often the unit with a higher CD34+ count, seems to dominate reconstitution.
Donor lymphocytes
The collected product is known as TC, Apheresis and if the entire product is infused immediately it retains this name. In some instances there is concern that administration of a large single dose of donor T cells may precipitate fatal GVHD rather than the desired GVL. Graduated doses of donor T cells are often administered over time. Lymphocyte content is calculated using automated or manual differential as well as flow cytometry (CD3+). The laboratory will aliquot doses (defined by institution or protocol) based on CD3+ × 106/ kg of recipient weight, often giving the first dose fresh but cryopreserving other doses.
HPC product assessment and specialized procedures
CD34 enumeration
Flow cytometry on a fresh HPC product provides CD34+ enumeration in a timely manner (1 hour). Given the importance of accurate CD34+ enumeration the procedure should follow a standardized and validated methodology (i.e. ‘The ISHAGE guidelines for CD34+ cell determination by flow cytometry’) [12]. A CD34+ enumeration kit includes CD45-FITC/CD34-PE, isotype control PE, stem cell microbeads (known concentration/μL), lysing solution (ammonium chloride) and a viability dye 7-amino actinomycin D (7-AAD). HPC samples stored at 18--20°C should be processed within a few hours and samples kept overnight should be stored at 2--6°C. Total viable CD34, apoptotic and necrotic cells can be measured with calculations based on product volume.
CD34+ enumeration from peripheral blood on the morning of collection may be instructive and predictive. An absolute CD34+ cell number expressed per microlitre appears to be the most useful variable. In a recent analysis we assessed 258 donors undergoing autologous SCT. Greater than 20 CD34+/μL correlated with a successful collection (‘good mobilizers’), as opposed to donors with <10/μL (‘poor mobilizers’) (Table 40.2). Newer mobilization agents such as CXCR-4 binding inhibitors (AMD3100) and stem cell factor (hu-SCF) may be administered to auto SCT patients identified as high risk for mobilization failure.
Table 40.2 Predictive value of peripheral blood (PB) pre-CD34+ enumeration.
Viability assays
Trypan blue (TB) is a simple exclusion dye test indicating cell viability, which can be routinely performed in any laboratory. Cells that fail to exclude dye are considered nonviable and are readily identified. Use of fluorescent stains with dark-field microscopy may reduce background staining. 7-Aminoactinomycin (7-AAD) is a fluorescent chemical with affinity for GC-rich DNA. Nonviable cells lack membrane integrity and will take up 7-AAD, which can be measured by flow cytometry. If performing viability for cryopreserved HPC product it is important to keep in mind that small aliquots will have different cooling properties that may diminish viability. Thus viability results from a cryovial are simply an estimate of viability for an actual product contained in a bag.
In vitro HPC assays
Functional analysis of HPCs can be performed using cells grown in semi-solid methylcellulose (MC) to identify colony-forming units (CFU). Resultant CFU-erythroid, CFU-granulocyte, CFU-mixed (CFU-GEMM) and CFU-megakaryocyte colonies help to characterize the short-term multipotency of a given HPC product. These assays are time consuming (2 weeks) and do not provide real-time information for products that are administered shortly after collection. Facilities managing HPC, Cord products that are stored frozen over long periods may offer colony assay results along with CD34 content in order to characterize the available unit better. These assays do have utility in the evaluation of long-term storage and for validation of newer cryopreservation strategies. Longer cultures (2 months) on stromal cell layers followed by MC culture assess long-term culture initiating cells (LTC-IC) and may provide functional evidence for pluripotent HPCs.
Sterility testing
Sterility testing suitable to detect clinically significant bacteria and fungal contamination of an HPC product must be performed at a minimum postprocessing. It is our preference to collect cultures when the product arrives in the laboratory and after each step of processing. Most centres accomplish this by collection of aerobic and anaerobic blood cultures from the product. In some cases paediatric bottles can be used to minimize the volume of sample removed from the product. Cultures should be performed: (i) at the end of HPC product collection and the end of processing for cryopreservation and (ii) after each step of any reprocessing (washing cells, manipulation on cell processor, post CD34+ selection). Cultures may also be obtained from each bag at the time of re-infusion. If an investigative cell-based product is administered additional analysis for sterility, mycoplasma, endotoxin, identity, adventitial virus, purity and potency are required.
ABO incompatibility
HLA-matched HPC SCT can proceed even if the blood groups of donor and recipient do not match. There are two types of ABO mismatch, each with its own interventions, which may need to occur before the transplant product can be infused [13]. ABO major mismatch results when the recipient's plasma contains a potent ABO antibody directed against donor's red cells. HPC, Apheresis products have a haematocrit of 5–10%, whereas the red cell content of HPC, Marrow haematocrit is much higher, ranging from 25 to 30%. Significant intravascular lysis of red cells will cause a haemolytic transfusion reaction if the product is infused without an intervention to decrease the level of the ABO antibody. Recipient's with ABO antibody titre against donor's red cells greater than 1:16 should be prepared by performing several apheresis procedures to replace plasma with 5% albumin. The titre of antibody can be further reduced by infusing plasma containing soluble ABO substance that matches the problem antibody. These measures are generally sufficient to allow safe infusion of HPC, Apheresis, but significant residual antibody after other interventions may require processing of HPC, Marrow to remove most of the red cell content. Red cell depletion of marrow is most commonly performed using a blood processor such as the Cobe 2991 with or without the addition of a sedimenting agent like hydroxethyl starch (HES). Red cell content at completion of processing should be as low as possible while minimizing loss of progenitor cells.
An ABO minor mismatch results when the donor's plasma contains a potent antibody directed against recipient's red cells. The need for intervention is less common in this setting as the ratio of antibody to red cell antigen is much lower, but it is wise to assess the level of donor antibody against the intended recipient's red cells. The presence of an antibody with a titre above 1:256 may necessitate group O red cell exchange of the recipient if the product is HPC, Apheresis. HPC, Marrow should undergo plasma depletion to remove at least 80% of antibody either through manual centrifugation or in semi-closed mode using a Cobe 2991 or similar blood processor.
CD34+ enrichment
CD34+ enrichment of HPC products is performed for a variety of reasons: haploidentical transplant, reduction of potential tumour burden in autologous HPC products and providing a T-cell depleted product to a recipient at high risk of GVHD. Both HPC, Apheresis and HPC, Marrow can be CD34 enriched but the marrow product must first be processed to a buffy coat concentrate to reduce volume and red cell content. Commercially available monoclonal antibody-based CD34+ enrichment devices have proven highly effective. The CliniMacs™ instrument from Miltenyi Biotec produces an extremely pure product (average 98% T-cell depletion) while recovering 65–75% of initial CD34+ content. Briefly, the HPC product is labelled with antibody to CD34 antigen, which is attached to an iron dextran particle; then the product is run through a column that sits between the poles of a powerful electromagnet. Labelled cells are retained in the column until all the product is processed; then the magnet recedes and the CD34-enriched product is eluted into clinical grade PBS buffer containing 0.5% human serum albumin. The final product can be infused fresh or cryopreserved for use in the future.
Ex vivo expansion
Given the limitations of inadequate numbers of HPCs in some products (HPC, Cord Blood, poor mobilizers) the ability to ex vivo expand stem and progenitor cells has significant clinical potential. Numerous strategies to date have included cultures in combinations of cytokines including Flt-3 ligand, SCF, IL-3, IL-6, IL-11, G-CSF, GM-CSF and TPO grown with or without bone marrow stromal cells. In these studies it is important to determine if expansion is occurring in committed progenitor cells (short-term haemopoietic reconstitution) as opposed to early progenitor cell expansion (long-term reconstitution). Despite numerous efforts, attempts at clinical expansion have been limited to date and remain investigational. The ability to expand committed progenitors in an effort to improve short-term neutrophil and platelet recovery may be more feasible [14]. The ability to expand early progenitors has significant implications for investigational trials employing gene therapy.
T-cell depletion
Despite the use of potent immunosuppressive agents (i.e. Methotrexate, Cyclosporine) GVHD remains a common (up to 50%) complication for patients undergoing allogeneic SCT. GVHD is primarily mediated by T-lymphocytes, which can be successfully removed from the graft prior to administration. T-cell depletion can clearly reduce GVHD, but also may hinder engraftment, increase the incidence of leukaemic relapse and the risk of infections including posttransplant lymphoproliferative disorders. Ex vivo procedures include physical separation by density gradient (counterflow centrifugal elutriation), depletion with lectins, cytotoxic drugs and the use of anti-T-cell antibodies (examples are CD2, CD3, CD5, CD8, CD25 and CD52) alone or in combination (complement, conjugated to toxin). Despite an ability to significantly eliminate T cells to as low as <1 × 105 CD3+ cells/kg recipient weight and attenuate acute GVHD, no differences in chronic GVHD, transplant-related mortality and disease-free survival have been proven to date [15].
Storage of HPC products
In many instances the HPC product is stored for short periods of time (hours) in an unmanipulated liquid state. Reported temperatures suitable for short-term storage range from 4 to 37°C (see Table 40.3). Ambient temperature is often preferred for short-term storage of HPC, Marrow [16] but ‘ambient’ should be a specific temperature range, e.g. 18 to 22°C. HPC collected by apheresis can be held at room temperature for 1–2 hours if further processing is to occur imminently, but are most commonly stored at 4°C when longer storage is required. A lower temperature may minimize damage by nonspecific cytokine release from granulocytes and mononuclear cells. There is a progressive loss of progenitor cells during nonfrozen storage with the rate of loss influenced by cell concentration, quantity and type of other cells contained in the product, the storage bag and the storage temperature [17]. What is most important is that the definition of ideal conditions for storage be validated at the stem cell facility. Validation should include temperature, cell concentration (if the product is to be held overnight before processing the leucocyte count should be diluted with donor plasma to below a concentration below 2 × 108/mL), additives in the product, addition of extra plasma to dilute cell counts, viability and, if the technology is available, progenitor assays.
Table 40.3 Stem cell laboratory processing procedures.
|
Procedure |
Methods |
Indication |
|
Red cell depletion of HPC, Marrow |
Semi-automated – Cobe 2991 cell processor with or without HES |
Major ABO/other antigens |
|
Plasma depletion of HPC, Marrow |
Semi-automated – Cobe 2991 cell processor |
Minor ABO mismatch |
|
Buffy coat concentration |
Centrifugation |
Volume reduction |
|
Sterility |
Bacterial fungal detection |
HPC products precryopreservation and post – thaw |
|
CD34/CD3 enumeration |
Flow cytometry (i.e. ISHAGE) |
HPC, Apheresis |
|
CD34 enrichment |
Immunomagnetic bead-based separation |
Related haploidentical SCT |
|
T-cell depletion |
Antibody-based +/− toxin |
Investigational |
|
Cryopreservation |
DMSO, HES/DMSO controlled-rate freezing or freeze in –80°C |
Option for all HPC products |
The length of time the product can be stored should also be established by in-house viability measurements at the end of intended storage and an expiry date and time set for each type of product handled. All equipment used for storage must also be validated to maintain the established temperature range. Storage requirements should include designating a location dedicated only for HPC products and a separate, clearly labelled location for any product that must be quarantined. A mechanism for monitoring and documenting temperature that includes local and remote alarms must be in place. If the product is stored at ‘ambient temperature’, the temperature of the location of storage must be documented. There should be a posted contingency plan that deals with temperature or mechanical failure of the designated storage equipment.
Cryopreservation
The majority of allogeneic products are not cryopreserved; however, centres that perform large numbers of allogeneic transplants may cryopreserve collected product to allow increased flexibility in the timing of the transplant related to the donor collection. In other cases donor availability or a change in the recipient's condition may dictate that the collected product be cryopreserved. Virtually all products to be used for autologous SCT are cryopreserved in order to allow time to administer multiday conditioning regimens and ensure infusion of progenitor cells occurs once toxic chemotherapy drugs have been cleared from the circulation.
HPC products to be cryopreserved must be transported to the processing laboratory in a designated transport cooler that has been validated for transport time and temperature. On arrival the receiving staff document minimum/maximum and actual temperatures of transport and the product visual inspection for colour, leakage and correct labelling.
All materials to be used in the cryopreservation process should have lot number and expiration date recorded. Visual inspection of all equipment and reagents must also be performed and recorded. The product will be manipulated in a biohazard safety cabinet so appropriate cleaning, disinfection and checks of a magnehelic gauge to ensure proper airflow are done in advance so that all is ready when the product arrives. HPC, Marrow and HPC, Cord products require processing to reduce mature red cell content and volume reduction before cryopreservation processing can occur. In most instances the haematocrit of HPC, A is between 5 and 10%, which does not represent enough mature red cell contamination to cause a problem so these products can be cryopreserved without removal of mature red cells. Plasma collected from the donor (apheresis) or retained from red cell depletion (referred to as concurrent plasma) should always accompany the product to the processing laboratory in case there is a need to dilute the product.
Sterility testing must be performed on the product on arrival and after the addition of the cryoprotectant solution. It is also wise to collect a sterility sample from the prepared cryoprotectant solution to ensure reagents used are sterile. Once sterility samples are collected, samples are drawn for a nucleated cell count and CD34 assessment. Processing cannot begin until at least the nucleated cell count is known as products with a nucleated cell count higher than 4.5 × 108/mL require dilution by adding a calculated amount of concurrent donor plasma.
Techniques for cryopreservation are designed to interfere with mechanisms that cause cell damage or death during the freezing process [18]. HPC need to be protected from dehydration and ice crystal formation within the cell. Most often the cryoprotectant used to accomplish this protection is dimethyl sulfoxide (DMSO). Dimethyl sulfoxide is a ‘penetrating cryoprotectant’ that acts in two separate ways. First, when cooling is relatively slow ice crystals tend to form in the extra cellular space. Ice formation concentrates extracellular solutes resulting in increased osmolality. DMSO moderates the increasing concentration by slowing water absorption by the ice crystals. Second, rapid diffusion of DMSO through the cell membrane allows the intracellular concentration of DMSO to be equal to the extracellular concentration, which facilitates water to move from within to outside the cell without excessive osmotic stress and before ice crystal formation can occur.
At rapid rates of cooling intracellular ice can form even when cryoprotectant is used. The cooling rate should minimize ice formation potential and complement the cryoprotectant's adjustment of the solution's rate of cooling. The concentration of DMSO required in the solution is determined by the ‘colligative effect’. Colligative refers to the properties of a solution (i.e. rate of freezing) being dependent on the number of particles (solute) and not the composition of the particles. The optimum concentration of DMSO to achieve good penetration of cells and moderation of the freezing point of the extracellular water is 10% volume in volume. Reduced concentrations of DMSO (5%) can be used if DMSO is combined with a macromolecular cryoprotectant like HES [19]. This macromolecule does not penetrate the cells but protects by forming a viscous noncrystalline glassy shell that retards the movement of water, preventing progressive dehydration. Cryoprotection can be accomplished by using macromolecules alone. Addition of DMSO to the solution raises the temperature at which the glassy shell forms. This facilitates intracellular dehydration necessary to avoid ice crystal formation while extracellular solute concentration is stopped by the glassy formation. The ‘combined’ cryoprotectant seems to afford better cell recoveries than the use of macromolecule alone. This type of cryoprotectant solution is a complex blend of salts, sugars, DMSO and plasma proteins and requires careful attention to the recipe and process when being prepared. Plasma proteins also have cryoprotectant properties and the addition of serum proteins to a cryoprotectant solution appears to improve HPC survival. The range of protein concentration and the source of protein used in cryoprotectant solutions are variable, with some groups preferring donor plasma as their source while others use 5% human serum albumin.
Processing of products to remove mature red cells should be performed prior to cryopreservation for HPC, Marrow and HPC, Cord Blood, where infusion of large quantities of free haemoglobin from lysed red cells may cause renal toxicity. Large quantities of red cells can also cause clumping of the product during processing. The concentration of mature red cells in HPC, Apheresis is not usually high enough to cause problems during processing or at infusion. Bone marrow product must be processed before cryopreservation, not only to remove red blood cells but also to eliminate fat and the majority of plasma volume. Processing: (i) allows cryopreservation of a buffy coat suspended at a desirable cell concentration, (ii) minimizes the volume of cryoprotectant that is used and later infused to the recipient and (iii) reduces the amount of freezer storage space required. Processing of the bone marrow product to achieve these ends can be performed manually using caution to use centrifuge speeds that keep the ‘g’ between 800 and 1000 to minimize progenitor cell damage. Alternatively, the product can be processed in a semi-automatic manner using a Cobe 2991 cell processor.
High cell concentrations in a product to be cryopreserved can result in poor recovery of progenitor cells. Very high cell concentrations can lead to post-thaw clumping of cells possibly due to leucocyte agglutination. High cell concentrations may lead to increased neutrophil death precryopreservation, with consequent release of cytokines that may damage the progenitor cells. HPC products with very low cell concentrations can also result in poor post-thaw viability; these products should be volume reduced to increase cell concentration. Most laboratories freeze HPCs at concentrations between 1.0 and 5.0 × 108/mL but successful cryopreservation outcomes have been reported using concentrations as high as 8 × 108/mL and as low as 1 × 106/mL.
Cryopreserved HPCs have been stored at temperatures as warm as –80°C but there is risk of cell damage caused by recrystallization as water migrates from small to large crystals. Most centres avoid this risk by storing at or below –120°C in mechanical freezers or in the vapour or liquid phase of nitrogen. Products and temperature monitoring devices should be placed well below the rim of liquid nitrogen freezers to minimize the increase of temperature caused by opening the lid. Product exposed to frequent temperature change is at risk of progressive damage to stored cells. This gradient effect can be minimized by using aluminium storage canisters and frameworks inside freezers to allow better heat conductivity and moderation of temperature loss throughout the freezer. Storage of products immersed in liquid nitrogen provides a constant temperature of –196°C and insulation against temperature fluctuations. Measures must be taken to eliminate droplet or other contamination within the freezer as virus and bacteria can survive in liquid nitrogen and crosscontamination of products is a risk. Products immersed in liquid nitrogen should include some type of an overwrap on each bag before placing in the aluminium canister.
The duration of cryopreserved storage may be indefinite if temperatures are consistently maintained below –150°C. As previously noted, cord blood cells maintain functionality for 15 years or greater; a current report of autologous recipients receiving second transplants with product cryopreserved for up to 7 years describes engraftment kinetics identical to those seen at use for the first half of the product stored for only 1–2 months [20].
Thawing of cryopreserved haemopoietic progenitor cells
Thawing can occur at the patient bedside or in the laboratory depending on institutional policy. Recipient identification is verified and the product is retrieved from storage. Units should be transported to the thaw location in a liquid nitrogen dry shipper with continuous temperature monitoring. The temperature should be maintained below –120°C until immediately before the thaw.
In most instances thawing is performed in a 37°C waterbath. The water source can be freshly drawn tap or sterile water. If a waterbath maintained by thermostat is used there should be careful cleaning and disinfection between uses. Alternately, fresh water is placed into a sterile basin, using a new basin for each recipient. The monitored temperature range should remain between 35 and 39°C. If the cells are thawed too slowly there is risk of injury from ice recrystallization; if the temperature is too high there is loss of viability or clumping of protein material within the bag. To begin, the protective canister is opened and labels are confirmed by two technologists. If the cryopreserved product has been overwrapped at cryopreservation it can be placed into the prepared 37°C waterbath; otherwise the unit should be placed in a plastic bag prior to immersion to avoid water droplet contamination into exposed ports. The unit is gently massaged over 5 minutes to ensure all parts are liquid and there is no residual slush. Some centres collect a culture sample from each thawed bag immediately prior to infusion to document postprocessing sterility.
Toxicity of the cryoprotectant directly to HPCs has been described so exposure of thawed cells to DMSO should be limited both precryopreservation and at thaw. Cryoprotectant can cause toxicity to the recipient but if the dose of DMSO is carefully controlled it is not necessary to remove it prior to infusion. The DMSO dose should be limited to less than 1 gm/kg of recipient weight in a 24-hour period. If the total amount of DMSO exceeds this limit, infusion should occur over two days. In situations where DMSO is to be removed, serial dilutions of protein-based solution to avoid osmotic shock to cells can be employed. At completion cells are resuspended in a 5% protein-based solution.
Quality assurance
A quality programme defines the policies and environment necessary to attain acceptable outcomes and meet safety standards consistently. The components include SOPs that address all activities, standardized and controlled labelling, documentation/record keeping that ensures traceability, personnel qualifications and training, building, facilities and equipment validation, environmental monitoring, regular auditing and error and accident system/management. Regulatory authorities worldwide have placed major emphasis on the establishment of an effective quality programme along with strict compliance to best practices in clinical, collection and laboratory settings. At the processing laboratory the quality programme is the means by which good manufacturing practices are instituted and followed throughout product manufacturing and manipulation. Lot-to-lot variation is minimized and the safety, purity and potency of the product are guaranteed.
Key points
1. Clinical indications for both autologous and allogeneic stem cell transplants are increasing.
2. HPCs can be obtained from three sources: bone marrow, peripheral blood and umbilical cord blood. Characteristics of these products differ in terms of HPCs and other mature cells.
3. Validated methods for cryopreservation are a key requirement to ensure optimal graft performance following administration to a transplant recipient.
4. Quality assurance testing of products at multiple stages of processing is essential for safety, purity, identity, potency and stability.
5. Accurate nucleated cell counting, CD34+ enumeration, viability(/clonogenic) assays and sterility analysis before and after cryopreservation are essential.
6. An established quality assurance programme and formal accreditation are critical to establish high level standards in the field of HPC transplantation.
References
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3. Vellenga E, van Agthoven M, Coockewit AJ et al. Autologous peripheral blood stem cell transplantation in patients with relapsed lymphoma results in accelerated haemopoietic reconstitution, improved quality of life and cost reduction compared with bone marrow transplantation: the Hovon 22 study. Br J Haematol 2001; 114: 319–326.
4. Bittencourt H, Rocha V, Chevret S et al. Association of CD34 cell dose with haemopoietic recovery, infections, and other outcomes after HLA-identical sibling bone marrow transplantation. Blood 2002; 99: 2726–2733.
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Further reading
Martin-Henao GA, Torrico C, Azqueta C et al. Cryopreservation of HPC from apheresis at high cell concentrations does not impair the hematological recovery after transplantation. Transfusion 2005; 15: 1917–1924.
Thomas ED, Appelbaum FR, Blume KG, Forman SJ & Negrin RS Haemopoietic Cell Transplantation, 4th edn. Wiley-Blackwell; 2009.