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Cryopreserved hematopoietic stem/progenitor cells stability program-development, current status and recommendations: A brief report from the AABB-ISCT joint working group cellular therapy product stability project team

Published:March 20, 2022DOI:https://doi.org/10.1016/j.jcyt.2022.03.001

      Abstract

      Background

      The AABB-ISCT Joint Working Group Stability Project Team (SPT) was assigned to roadmap a path toward standardization of cryopreserved hematopoietic stem/progenitor cell (HSPC) stability programs. HSPC stability encompasses a broad scope of conditions including non-frozen (“fresh”) and cryopreserved cell products, and varying methods for storage, thaw, and administration. This report assessed current practices and focused solely on cryopreserved HSPC cell therapy products to establish preliminary recommendations for a stability program roadmap.

      Methods

      A survey was prepared by the SPT and distributed to ISCT and AABB members. Survey results were summarized and recommendations were outlined based on the responses from the survey. This report highlights current practices for cryopreserved HSPC stability programs, including additional considerations and recommendations.

      Results and Discussion

      Eighty-two (82) centers worldwide participated in the survey. Survey results indicate variability across programs. HSPC stability depends on multiple factors within the processing facility (e.g., cryopreservation techniques, reagents used, and storage temperature) and independent variables (e.g., donor-related factors and starting material variability). While retention of hematopoietic engraftment potential is the primary goal for cryopreserved HSPC stability, engraftment results should not be used as the sole metric for stability programs. Based on the survey results, the SPT provides recommendations for consideration.

      Conclusions

      The SPT recommendations for best practices are not intended to replace existing standards. The survey results emphasize the need for the community to optimize best practices and consider initiating collaborative projects to improve the standardization of cryopreserved HSPC stability programs for cell therapy products.

      Keywords

      Abbreviations:

      AABB (association for the advancement of blood & biotherapies), ALDH (aldehyde dehydrogenase), CAP (college of american pathologists), CFU (colony forming unit), DMSO (dimethylsulfoxide), FACT (foundation for the accreditation of cellular therapy), FDA (food and drug administration), HAS (human serum albumin), HCT/Ps (human cells, tissues, and cellular and tissue-based products), HPC (hematopoietic progenitor cells), HSPC (hematopoietic stem/progenitor cells), ICH (the international council for harmonization), ISHAGE (international society of hematotherapy and graft engineering), LN2 (liquid nitrogen), NA (north america), PI (propidium iodide), QA (quality assessment), QC (quality control), SPT (stability project team), TB (trypan blue), 7-AAD (7-amino-actinomycin D)

      Introduction

      Hematopoietic stem/progenitor cells (HSPC) are multipotent cells widely used as potentially curative treatments for malignant and non-malignant hematologic disorders. HSPC is collected from either autologous or allogeneic donors from peripheral blood most commonly via apheresis, bone marrow via multiple aspirations, or from the placenta/cord blood after childbirth. HSPC cryopreservation and thawing, while beneficial in expanding patient access to transplantation, has a potential risk of loss of cell viability and potency. Multiple manufacturing factors may impact cell product quality and potency including storage conditions, shipping or transport conditions, manufacturing-specific processes, thawing procedure, and more. Within hours of removal from the body, HSPC viability starts to decline, with notable differences observed in as little as 20 hours and more dramatically after 48 hours [
      • Hahn S
      • Sireis W
      • Hourfar K
      • et al.
      Effects of storage temperature on hematopoietic stability and microbial safety of BM aspirates.
      ]. Most HSPCs - autologous, umbilical cord blood, and some allogeneic HSPC grafts - require cryopreservation for weeks to years prior to administration to patients, depending on the patient need and more recently for logistical reasons (i.e., COVID-19 pandemic). HSPC viability and potency are also influenced by factors independent of primary manufacturing and cryopreservation such as follows: cell source (e.g., bone marrow, peripheral blood, and umbilical cord blood), collection methodology, donor factors (e.g., age, past exposure to chemotherapy for autologous donors); cell concentration at the collection; granulocyte fraction within the graft; and length of time between collection to cryopreservation [
      • Hahn S
      • Sireis W
      • Hourfar K
      • et al.
      Effects of storage temperature on hematopoietic stability and microbial safety of BM aspirates.
      ,
      • Fry LJ
      • Giner SQ
      • Gomez SG
      • et al.
      Avoiding room temperature storage and delayed cryopreservation provide better postthaw potency in hematopoietic progenitor cell grafts.
      ,
      • Winter JM
      • Jacobson P
      • Bullough B
      • Christensen AP
      • Boyer M
      • Reems JA.
      Long-term effects of cryopreservation on clinically prepared hematopoietic progenitor cell products.
      ,
      • Uzoka C
      • Liu LC
      • Park Y
      • et al.
      Race/ethnicity and underlying disease influences hematopoietic stem/progenitor cell mobilization response: a single center experience.
      ]. Since each step from collection to infusion may impact the intended therapeutic function of HSPC, well-controlled and validated processes are recommended to maintain optimal target cell recovery and functional potency. Ultimately, stability programs should periodically evaluate key manufacturing steps (i.e., Critical Process Parameters or CPPs) via Critical Quality Attributes/Elements (CQAs, CQEs), with sufficient sample size, using a variety of standardized and validated assays, to mitigate the loss of viable cell recovery and function in the HSPC product.
      The United States Food and Drug Administration (FDA) provides a regulatory framework primarily under the Public Health Services Act Section 361, Title 21 CFR 1271 [
      Code Of Federal Regulation, Human Cells, Tissues, And Cellular And Tissue-Based Products
      Title 21 CFR Part 1271.
      ] (current Good Tissue Practices), 21 CFR 2106 and 21 CFR 2117(current Good Manufacturing Processes) and 21 CFR 6108(General Biological Products Standards) sections for the manufacturing of minimally manipulated human cells, tissues, and cellular and tissue-based products (HCT/Ps) intended for homologous use [
      Code of federal regulation
      Current good manufacturing practice in manufcturing, processing, packing, or holding of drugs.
      ,
      Code of federal regulation
      Current good manufacturing practice for finished pharmaceuticals.
      ,

      Code of federal regulation. General biological product standards. title 21 CFR Part 610. In. Washington, DC: US Government Printing Office; 2020 (revised annually).

      ].
      Products that are more than minimally manipulated, are not intended for homologous use, or come from an unrelated donor, are regulated under Section 351 of the PHS Act. Monitoring cell yield/recovery, viability, and potency (function) to support therapeutic efficacy are discussed but not specifically defined. Voluntary accreditation organizations including FACT (Foundation for the Accreditation of Cellular Therapy) and AABB (Association for the Advancement of Blood & Biotherapies) and international consensus-building organizations such as ICH (The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use) require or recommend establishing a written HSPC stability program for cryopreserved products but do not prescribe specific assays, use of standardized calculations, or definition of what constitutes acceptable results [
      FACT-JACIE international standards for cellular therapy product collection, processing. And administration.
      ,
      Standards for cellular therapy services.
      ]. Currently there is no single assay, which is accepted by consensus in the cell therapy field to accurately predict the engraftment potential of cryopreserved HSPC products. There are several challenges to establishing a stability program for cryopreserved HSPC, including the lack of 1) a consensus test or surrogate marker to predict HSPC hematopoietic engraftment efficacy post-transplant; 2) standardization for flow cytometry-based post-thaw viable target cell enumerations that can be paired with the International Society of Hematotherapy and Graft Engineering (ISHAGE) protocol; and 3) standardization regarding optimal sampling (number and frequency) of products to assess program effectiveness. The two-pronged goal of the AABB-ISCT Stability Project Team (SPT) is to understand current stability program practices among HSPC manufacturing facilities and to provide a roadmap toward standardization or best practices based on scientific evidence, using the general framework for stability testing found in 21 CFR 211.166.7 The standard for cell-based product stability covered under regulatory requirement 21CFR211.166 is required for cGMP finished pharmaceuticals, in combination with both AABB and FACT standards, can be used as a general guidance for HSPC cryopreserved product stability [
      Code of federal regulation
      Current good manufacturing practice for finished pharmaceuticals.
      ,
      FACT-JACIE international standards for cellular therapy product collection, processing. And administration.
      ,
      Standards for cellular therapy services.
      ]. Stability management of emerging novel and licensed commercial cell therapy products is also being considered but will be explored in depth later.

      Methods

      Survey preparation and distribution

      The Stability Program survey was developed by the AABB-ISCT SPT to identify gaps and opportunities for developing stability program guidance for cryopreserved HSPC products (Table 1). Questions were posed in a multiple-choice format to ensure that answers could be quantified. The survey was disseminated to the cellular therapy community via AABB and ISCT from April 2020 to May 2020. Results were curated by the AABB-ISCT Joint Working Group and made available to the SPT.
      Table 1HSPC stability program survey questions
      Survey questionSurvey response options
      Do you have a stability program in place?Yes, No, Other (Explain)
      Do you have constraints, which limit the implementation of your stability program? (Check all that apply)None, Knowledge, Staffing, Financial, Samples availability, Other (Explain)
      Which regulatory requirements are relevant to your stability program? (Check all that apply)FDA, FACT, JACIE, AABB, Other (Explain)
      What HPC products are included in your stability program? (check all that apply)HPC (A), HPC(M), HPC(CB)
      Is your stability program the same for all of your cryopreserved products?Yes, No
      Do you have an expiration time for cryopreserved HPC products?Yes, No
      What is your expiration time?< 5 years, 5 years, 10 years, > 10 years
      How did you establish your expiration time?In-house Validation, Published Literature, Arbitrary, Other (Explain)
      How long do you store your cryopreserved HPC products?1 year, < 5 years, 5 years, < 10 years, 10 years, > 10 years, Indefinitely, Other (explain)
      At which temperature do you store your cryopreserved product?< −150°C LN2, vapor, −80°C, < −150°C LN2, liquid, <−150 C for both liquid and vapor, 0ther (explain)
      What type of container do you use to package cryopreserved HPC products? (Check all that apply)Cryo-Bag, Cryo-Vial, Other (Explain)
      What type of retention samples do you keep for stability testing? (Check all that apply)Cryo-Bag, Cryo-Vial, Contiguous Segments, Other (Explain)l
      Are you able to compare pre-freeze and post-thaw results?Yes, No
      What type of stability testing do you perform? (Check all that apply)Total Nucleated Cell Count (TNC), CD34 Cell Count, Viability, Functional Potency Testing, Percent TNC Recovery, Percent CD34 Recovery, Other (Explain)
      What methodology do you currently use for viability? (Check all that apply)Trypan Blue Dye Exclusion, 7AAD - Viable Total Nucleated Cells, 7AAD - Viable CD34 Cells, Automated Fluorescence - Based Cell Counter, Other (Explain)
      What type of functional potency testing do you perform for stability? (Check all that apply)None, Colony Forming Unit (CFU), 7AAD-Viable CD34 Cells (Surrogate), ALDH, Other (Explain)
      What additional product attributes do you monitor in your stability program? (Check all that apply)None, Sterility, Cryo-Bag Integrity, Other (explain)
      How often do you test cryopreserved products to determine HPC stability? (Check all that apply)Annually, Every 2–3 years, Every 5 years, Other (explain)
      What is the nature of your organization (please select the best option)Community hospital, Academic medical center, Commercial entity Cord blood bank, public, Cord blood bank, private, Cord blood bank, hybrid, Government, other (explain)
      All survey results were merged into a single database. For each survey question, the total number of responders was counted. The answers to the survey questions were analyzed under 5 categories: survey participants, stability program, specimen type, stability testing, and storage and expiration time. For multiple-choice questions, the percentage of responders to each category was calculated out of the total participants that provided an answer. For the stability-testing category, the responders were separated into three groups based on their responses to the survey first question (Do you have a stability program?) (Table 2) and analyzed separately. When a responder chose the option “Other” in the multiple categories questions and provided a different description, each description was reviewed and assigned a standardized response.
      Table 2Availability of HSPC stability program and stability testing performed
      Survey question 1: Do you have a stability program?
      Stability testing performed (n)Yes (n = 67)No (n = 10)Other (n = 5)Total (n = 82)
      Total nucleated cell enumeration461148
      CD34 cell enumeration453250
      Viability testing523358
       Type of viability testing {n (%)
      Percentage indicated below are of those representing the type of viability testing.
      }:
       Trypan blue viability250126 (45%)
       7AAD CD34 viability402143 (74%)
       7AAD TNC viability300030 (52%)
       Automated fluorescence viability5117 (12%)
      Potency testing383243
       Type of potency testing {n (%)
      Percentage indicated below are of those representing the type of potency testing.
      }:
       Colony forming unit221124 (56%)
       7AAD-viable CD34 Cells193123 (53%)
       ALDH1012 (5%)
       Engraftment2002 (5%)
      Other attributes:
       Sterility testing310132
       Container integrity402143
       Label accuracy & integrity5005
       HLA typing2002
       CD3 enumeration & viability2002
      Compare pre & post507355
      No answer was provided for any of the stability testing questions147224
      a Percentage indicated below are of those representing the type of viability testing.
      b Percentage indicated below are of those representing the type of potency testing.
      The SPT used the survey results combined with available peer-reviewed literature and relevant regulatory, accreditation, and industry documents to outline current practices utilized to establish and monitor cryopreserved HSPC stability programs and to make recommendations or suggestions to improve standardization across programs. Throughout the manuscript, the term HSPC was used to describe cells procured from growth factor mobilized peripheral blood stem cells collected by apheresis, bone marrow harvest, or umbilical cord blood, as sources of the graft. In survey questions, the term HPC (hematopoietic progenitor cells) was used which should be read equivalent to the term HSPC.

      Results

      Stability program survey results

      The goal of the survey was to collect data from different types of organizations ranging from community hospitals, academic clinical centers, cord blood banks, and others engaged in HSPC processing to understand the current practices used by cell processing and storage facilities, as well as limitations and constraints. The survey included a broad array of questions addressing HSPC product types, storage conditions, expiration times, and methods used to assess stability (Table 1). Survey participants represent a wide range of organizational types and geographic locations. The survey results indicate most HSPC cell therapy programs (88%) currently have or support having a stability program. Although general regulatory frameworks and accreditation standards exist, some respondents identified the lack of specifically defined protocols as a primary constraint for the development of their HSPC stability program, reinforcing the potential value of this SPT. Survey results by topic are summarized below.

      Participating Centers

      Eighty-two (82) responders participated in the survey. Thirty-three respondents (40%) identified as an academic medical center, 7 (8.5%) identified as community or public hospital, and 2 (2.4%) identified as a cancer center. Thirteen (15.8%) responders identified as either a private, public, or hybrid cord blood bank. Three (3.6%) responders identified as either government or research institutes, and one center (1.2%) identified as affiliated with a not-for-profit donor registry. The rest of the participants (n = 23, 28%) did not provide information about their organization type.
      Forty-four out of 82 participants reported their organizations were in North America (NA) (53.6%), 12% (n = 10) in South America, 7.3% (n = 6) in Asia, 6% (n = 5) in Europe, and 4.8% (n = 4) in Australia. Thirteen (n = 13, 16%) centers did not identify their geographic location. The regulatory bodies and accreditation organizations overseeing stability programs included FACT (n = 55, 67%), AABB (n = 30, 36%), JACIE (n = 9, 11%), FDA (n = 41, 50% overall, but 41/44 of NA participants, and additional regional specific organizations (n = 10, 12%). Forty-nine (60%) participants identified as being regulated and/or accredited by at least two regulatory and accreditation agencies.

      HSPC Stability Programs

      Sixty-seven of 82 participants (82%) reported having an HSPC stability program in place and 10 participants (12%) reported lacking such a program. The remaining participants (n = 5, 6%) reported “Other”, including two developing stability programs at the time of the survey, two that monitor third party cell processing providers' stability programs, and one that does not have a stability program but tests all products for viability prior to distribution (Table 2).
      When asked about HSPC stability program constraints, less than half of the responders (n = 35, 43%) reported no constraints while the remaining (n = 47, 57%) listed at least one constraint including financial, knowledge, staffing, and test sample availability issues (n = 15, 18, 24, and 24, respectively). Twenty-Seven (33%) participants reported at least two constraints. Other indicated constraints were lack of appropriate guidance and published data (n = 2). Unavailability of properly validated tests and established stability criteria posed additional constraints to some stability programs (n = 3).

      Specimen Types

      The majority of responders (n = 62, 76%) included growth factor mobilized apheresis products as their source of HSPC graft in their stability program. Thirty-four responders (41.5%) included bone marrow and another 34 responders (41.5%) included cord blood, as graft sources in their HSPC stability programs. Twenty programs (24.4%) included aliquots both from apheresis products and bone marrow grafts, and 11 (13.4%) responders included all three graft sources in their stability programs. Most responders reported using cryobags as their primary storage container (n = 57, 70%). Of these, 23 (28%) programs indicated also using cryovials for cell storage. Two responders (2.4%) reported using only cryovials for product storage. For retention samples, 49 (60%) responders reported using cryovials, while 24 (29%) responders reported using cryobags. Fifteen (18%) responders reported using contiguous segments attached to the bag. Twenty-four (29%) responders used combinations of at least two of the three retention specimen containers. The majority (n = 56, 68%) indicated comparing pre-freeze and post-thaw results for their HSPC stability program.

      HSPC Testing

      Fifty-eight of the responders (71%) provided answers for the stability testing questions. All 58 (100%) responders stated that after thawing a cryopreserved HSPC graft, they test for cell viability (Table 2). Of these, 26 (45%) centers perform viability assessment by trypan blue (TB) dye exclusion and 30 (52%) centers perform 7-Amino-Actinomycin D (7-AAD) by flow cytometry to determine viable total nucleated cells (i.e., CD45 expression). Forty-three (74%) centers perform 7-AAD for viable CD34+ cell assessment, while 7 (12%) centers use automated fluorescence -based cell counter viability assays (Figure 1).
      FIGURE 1
      Figure 1Types of stability testing. (A) the majority of programs perform multiple tests to evaluate stability. Of 58 programs providing answers for type of stability testing questions, all perform viability testing (58/58), followed by CD34 enumeration 86% (50/58), Total nucleated cell (TNC) enumeration 83% (48/58), and potency 74% (43/58). The majority of programs, 93% (52/58), perform multiple stability tests. Stability testing only for viability is reported by 7% (4/58) programs. Regarding type of potency assay, 58 participants provided answers of these 15/58 (26%) responders report not testing for functional potency. B) Type of functional potency assay: 43 participants performed stability testing. There were total 51 potency tests from 43 responses having multiples types of potency tests being done for a single product. 24/43 (56%) perform colony forming unit (CFU) assay, while the remaining 23/43 (53%) responders use 7-AAD viable CD34+ cells as a surrogate for potency assay. Two centers reported using aldehyde dehydrogenase (ALDH) enzymatic function-based flow cytometry assay, and 2 centers reported using hematopoietic engraftment to determine potency of cryopreserved HSPC. (C) Types of viability testing performed: TNC viability by trypan blue 45% (26/58), TNC viability by 7-amino Actinomycin D (7AAD) 52% (30/58), CD34 viability by 7AAD 74% (43/58), and automated fluorescence 12% (7/58). The majority of programs, 64% (37/58), perform combination CD34 and TNC viability testing. Notably, 4/58 (7%) programs that reported viability testing as the only stability test perform CD34 7AAD [Color figure can be viewed at (Color version of figure is available online)].
      When asked about HSPC functional potency assay, 64 answered the question. Of these 15 (26%) responders reported not testing for functional potency. Regarding the type of potency assay, 43 participants perform some type of potency assays including multiple testing (51 tests) for some products (Figure 1). Of these 24 perform colony-forming unit (CFU) assay, while the remaining 23 responders use 7-AAD viable CD34+ cells as a surrogate for potency assay. Two centers reported using an aldehyde dehydrogenase (ALDH) enzymatic function-based flow cytometry assay, and two centers reported using hematopoietic engraftment to determine the potency of cryopreserved HSPC (Figure 1). Additional HSPC stability program attributes included product sterility (n = 32, 39%) and container integrity (n = 43, 52%).
      Most responders perform HSPC stability test verification on an annual basis (n = 54, 66%), with variability regarding the number of times per year and specimens tested. Taken together, HSPC stability testing identifies the lack of standardization or consensus in testing methods between laboratories highlighting the need for guidance toward best practice approaches.

      Storage and Product Expiration Time

      Fifty-nine (72%) participants reported their HSPC products' long-term storage temperature. Most of the responders (57/59, 97%) store cryopreserved HSPC in liquid nitrogen (LN2) using either vapor or liquid phase, or a combination of both. Two responders utilized a mechanical freezer (−80°C) for long-term storage. Fifty-seven out of 59 (97%) responders identified the storage duration. Most respondents store products indefinitely or until patients expire (n = 31, 54%); nine (16%) responders store 10–20 years, 12 (21%) responders store less than 10 years, and five (9%) responders store products 5 years or less.
      Thirty-two of 62 participants (51.6%) have a reported expiration time for cryopreserved HSPC product. Expiration time duration was recorded as 5 years or less (n = 9), 10 years (n = 12), or greater than 10 years (n = 10). Expiration time was determined by either in-house validation (n = 9, 15%), published literature (n = 15, 25%), in-house stability program (n = 2, 3%), or was arbitrarily determined (n = 7, 11%).

      Discussion

      The survey participants, who represent a wide range of organizational types and geographic locations, indicate that the majority of HSPC storage facilities have stated a willingness to establish a stability program. Interestingly, many respondents identified the lack of guidance as a constraint for the HSPC stability program development, which further emphasizes the need to develop guidance for the development and monitoring of an HSPC stability program.
      The FDA requires stability studies that include testing for drug attributes that are susceptible to change during storage conditions, which are likely to influence the quality, safety, and efficacy [
      Guidance for Industry
      Q1A(R2) Stability Testing of New Drug Substances and Products.
      ]. The stability program of cryopreserved HSPC products needs to be established based on a validation study with defined parameters that encompass viability, recovery of viable target cells, functional potency, and ultimately efficacy. Based on the validation data, the stability duration and expiration dates for cryopreserved HSPC products can be established. Literature continues to evolve regarding the age of products stored in LN2 and successful engraftment following transplantation of these older products years after storage [
      • Broxmeyer HE
      • Lee MR
      • Hangoc G
      • et al.
      Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood.
      ,
      • Broxmeyer HE
      • Cooper S.
      High-efficiency recovery of immature haematopoietic progenitor cells with extensive proliferative capacity from human cord blood cryopreserved for 10 years.
      ,
      • Berz D
      • McCormack EM
      • Winer ES
      • Colvin GA
      • Quesenberry PJ.
      Cryopreservation of hematopoietic stem cells.
      ].
      HSPC stability assays are important in the quality assessment (QA) of the manufacturing process and help ensure viability, potency, and dosing of the target cell product for the desired therapeutic efficacy.
      HSPC stability assays can be divided into three categories: (1) Quantification (i.e., cell number), (2) Viability, and (3) Function (Table 3). Enumeration of pre-cryopreservation viable CD34+ cells is routinely used to predict the suitability of the HSPC graft. CD34+ cell enumeration is precise, has a short turnaround time, and through the use of the ISHAGE protocol, has been largely standardized across laboratories [
      • Sutherland DR
      • Anderson L
      • Keeney M
      • Nayar R
      • Chin-Yee I.
      The ISHAGE guidelines for CD34+ cell determination by flow cytometry. International Society of Hematotherapy and Graft Engineering.
      ,
      • Keeney M
      • Chin-Yee I
      • Weir K
      • Popma J
      • Nayar R
      • Sutherland DR.
      Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines. International Society of Hematotherapy and Graft Engineering.
      ]. Yet viable CD34+ cell enumeration is not a functional potency assay and lacks a standardized protocol to be utilized for thawed cryopreserved samples. The CFU assay is a functional potency assay that reveals the clonogenic potential of the progenitor cells and their differentiation capacity in vitro. However, this assay has a long turnaround time, is not performed in many laboratories, and is not fully standardized across laboratories [
      • Kozlowska-Skrzypczak M
      • Gil L
      • Komarnicki M
      Factors affecting neutrophil recovery after autologous bone marrow-derived stem cell transplantation in patients with acute myeloid leukemia.
      ,
      • Coulombel L.
      Identification of hematopoietic stem/progenitor cells: strength and drawbacks of functional assays.
      ,
      • Pamphilon D
      • Selogie E
      • McKenna D
      • et al.
      Current practices and prospects for standardization of the hematopoietic colony-forming unit assay: a report by the cellular therapy team of the biomedical excellence for safer transfusion (BEST) collaborative.
      ,
      • Louis I
      • Wagner E
      • Dieng MM
      • Morin H
      • Champagne MA
      • Haddad E.
      Impact of storage temperature and processing delays on cord blood quality: discrepancy between functional in vitro and in vivo assays.
      ]. Alternative functional potency assays such as detection of the presence of aldehyde dehydrogenase (ALDH) enzyme activity, which is relatively higher in HSPCs compared to more mature lineage-committed progenitors, could be considered [
      • Moreb JS.
      Aldehyde dehydrogenase as a marker for stem cells.
      ]. Aldefluor™ is a fluorescent nontoxic substrate for ALDH which freely permeates into viable cells and is used in the flow cytometry platform. Unlike the CFU assay, however, the Aldefluor™ assay cannot serve as an indicator of HSPC differentiation potential. While each assay has a place in decision-making for HSPC stability programs, none are sufficiently reliable to be recommended as the sole assay used in all stability programs. Most of the assays listed in Table 3 can be affected by sample preparation variability due to the non-homogenous nature of HSPC, resulting in broad measurement bias [
      • Pegg DE.
      Viability assays for preserved cells, tissues, and organs.
      ,
      • Areman EM
      • Sacher RA
      • Deeg HJ.
      Processing and storage of human bone marrow: a survey of current practices in North America.
      ,
      • Areman EM
      • Sacher RA
      • Deeg HJ.
      Cryopreservation and storage of human bone marrow: a survey of current practices.
      ,
      • Katayama Y
      • Yano T
      • Bessho A
      • et al.
      The effects of a simplified method for cryopreservation and thawing procedures on peripheral blood stem cells.
      ].
      Table 3HSPC stability assays in common use
      PlatformQuantificationViabilityFunction
      Hematology analyzerTotal nucleated cell (TNC)NoNo
      Flow cytometryCD45+ and CD34+Yes; 7-aminoactinomycin D (7-AAD)Yes (not directly link to HSPC differentiation) aldehyde dehydrogenase (ALDH)
      Microscope/Image analysisTotal nucleated cellsTrypan blue, fluorescence based methodsNo
      Colony forming unit (CFU) assayCFUYes; CFUYes and specific to HSPC function; progenitor (CFU) viability, cellular differentiation
      The gold standard for HSPC functional potency assessment remains hematopoietic engraftment following transplantation. However, it lacks the sensitivity to uncover potential issues within the laboratory, is impacted by factors unrelated to manufacturing, and is determined too far downstream in manufacturing – potentially months later - to serve as the sole stability testing marker within a stability program. Often several-fold higher numbers of CD34+ cells than the historical minimum required dose [
      • Bai L
      • Xia W
      • Wong K
      • Reid C
      • Ward C
      • Greenwood M.
      Factors predicting haematopoietic recovery in patients undergoing autologous transplantation: 11-year experience from a single Centre.
      ] is infused to minimize the impact of testing variabilities or risk of potential loss of potency with cryopreservation/thawing on the kinetics of hematopoietic reconstitution.
      Although minimum acceptability limits for cell viability have not been established, some facilities use the FACT cord blood banking minimum acceptability limits of equal to or greater than 70% viability of CD34+ cells for post-thaw cord blood graft quality control (QC) samples [
      FACT-Netcord international standards for cord blood collection
      Banking, and release for Administartion.
      ].
      Notably, low percent recovery and/ or low viable target cell dose following cryopreservation, storage, and thaw, may indicate manufacturing, storage, and/or thawing, related protocol deviations and should be investigated to rule out suboptimal laboratory (manufacturing and analytical) processes.
      A cell viability assay determines the fraction of living cells within the total number of cells in the sample [
      • King MA.
      Detection of dead cells and measurement of cell killing by flow cytometry.
      ,
      • Kroemer G
      • Galluzzi L
      • Vandenabeele P
      • et al.
      Classification of cell death: recommendations of the nomenclature committee on cell death 2009.
      ]. “Living” does not preclude being ‘sick’ or on the path to dying. Viability assays, therefore, are best seen as a snapshot of what is considered ‘live’ at a given time as defined by the methodology in use. Viability testing alone is insufficient to characterize target cells as it fails to provide insight into potency.
      For cryopreserved HSPC products, recovery or yield indicates the total number of target cells retrieved post-thaw out of target cells measured pre-freeze. The target cells can be TNC, CD34+ cells or other cells present within the graft. TNC viability determination based on membrane permeability methods can underestimate cell death in comparison to apoptosis-based methods that are predominantly adopted by investigational laboratories and cord blood banks [
      • Moreb JS.
      Aldehyde dehydrogenase as a marker for stem cells.
      ,
      • Duggleby RC
      • Querol S
      • Davy RC
      • et al.
      Flow cytometry assessment of apoptotic CD34+ cells by annexin V labeling may improve prediction of cord blood potency for engraftment.
      ,
      • Radke TF
      • Barbosa D
      • Duggleby RC
      • Saccardi R
      • Querol S
      • Kogler G.
      The assessment of parameters affecting the quality of cord blood by the appliance of the Annexin V staining method and correlation with CFU assays.
      ]. It is also possible that apoptotic or dead cells have disappeared and are no longer detectable resulting in an overestimation of the viable fraction. It also does not represent sub-population, such as CD34+ HSPCs, which are relatively more resilient in comparison to committed mature blood cells or their precursors following the cryopreservation-thaw cycle [
      • Reich-Slotky R
      • Colovai AI
      • Semidei-Pomales M
      • et al.
      Determining post-thaw CD34+ cell dose of cryopreserved haematopoietic progenitor cells demonstrates high recovery and confirms their integrity.
      ].
      It is notable that most transplant centers utilize pre cryopreservation CD34+ cell number rather than post-thaw CD34+ cell number to determine the adequacy of a cryopreserved HSPC graft. Quantification of the post-thaw viable CD34+ cell number can demonstrate cell recovery, confirm cell integrity and serve as a valuable predictor of hematopoietic stem cell engraftment in transplantation [
      • Reich-Slotky R
      • Colovai AI
      • Semidei-Pomales M
      • et al.
      Determining post-thaw CD34+ cell dose of cryopreserved haematopoietic progenitor cells demonstrates high recovery and confirms their integrity.
      ,
      • Fournier D
      • Lewin A
      • Simard C
      • et al.
      Multi-laboratory assay for harmonization of enumeration of viable CD34+ and CD45+ cells in frozen cord blood units.
      ]. Declines in HSPC viability post-thaw are not uncommon and can be influenced by several factors including the concentration of cryoprotectant, unstable cell membrane immediately after thawing, and factors associated with the preparation of sample (i.e., thaw protocols, time-lapse following thaw to cell viability determination) among many others [
      • Yang H
      • Acker JP
      • Cabuhat M
      • McGann LE
      Effects of incubation temperature and time after thawing on viability assessment of peripheral hematopoietic progenitor cells cryopreserved for transplantation.
      ]. The variability can be minimized by adding thawed HSPC immediately to media containing human serum albumin (HSA) and allowing dilution of samples to ≤1% dimethylsulfoxide (DMSO) [
      • Fournier D
      • Lewin A
      • Simard C
      • et al.
      Multi-laboratory assay for harmonization of enumeration of viable CD34+ and CD45+ cells in frozen cord blood units.
      ,
      • Lee S
      • Kim S
      • Kim H
      • et al.
      Post-thaw viable CD34(+) cell count is a valuable predictor of haematopoietic stem cell engraftment in autologous peripheral blood stem cell transplantation.
      ,

      Mahmud N. Cryopreservation of stem cell product. In: Chandy M, Radhakrishnan VS, Sukumaran R. (eds), Contemporary Bone Marrow Transplantation. Organ and Tissue Transplantation. Springer, Cham. https://doi.org/10.1007/978-3-319-64938-2_16-1

      ,
      • Park Youngmin
      • Ganapathy GI.Amudha
      • Mahmud Dolores
      • Patel Pritesh
      • Rondelli Damiano
      Nadim Mahmud. Monitoring of stored hematopoietic stem/progenitor graft stability program in a single institute.
      ]. In addition, post-thaw storage of samples on ice or in a refrigerator as well as determining cell viability with minimal time-lapse after thawing can help as well.
      Despite the limitations described above, the current commonly used cryopreservation and storage methodologies, as well as the assays and calculations, are generally reliable on an intra-laboratory basis to detect changes in cell viability and potency during manufacturing and across cryopreservation and thawing.
      Based on the survey results as well as current practices, literature review, committee members' expertise, and relevant stability testing regulations and accreditation standards the SPT has put together the following points to consider and recommendations. The SPT considered current practice variability, resource limitations, lack of access to assays and variability among assay methodologies, and other limitations in drafting the recommendations. Recommendations from the project team are not intended to override organizational standards and should not be interpreted as organizational endorsements. The framework for a stability program is highlighted in Table 4. The following specific recommendations are proposed by the SPT to establish and maintain cryopreserved HSPC products.
      Table 4Framework for a HSPC stability program
      Defining product and population• Specify by HSPC product type (Apheresis, Marrow, Cord Blood) if cryopreservation technique is not identical

      • HSPC products & sample aliquot should have identical storage conditions (different containers require separate validation)
      Defining testing and other attributes• Specify tests for determining target cell product recovery, viability and potency (use validated techniques)

      • Additional considerations: Container and label integrity and HSPC product sterility
      Defining sample number and Frequency• Specify the minimal number of samples to include in the stability program

      • Specify the frequency of sample testing
      The committee recommends each center develop and implement a formal (written) HSPC Stability Program whose key components are validated and regularly verified. Written assessment of HSPC stability testing should address at a minimum container and label integrity, viability, recovery of viable target cells (i.e., CD34+ cells), potency, and sterility for the duration of storage or expiration. Stability assays may be used as QC parameters to predict cryopreserved HSPC product stability. HSPC product type needs to be specified if the cryopreservation technique is distinct for the product type (i.e., apheresis, bone marrow, or cord blood). If the cryopreservation technique is identical (autologous vs. allogeneic) separate metrics may not be warranted. The source material derived from autologous (sick patients) [
      • Uzoka C
      • Liu LC
      • Park Y
      • et al.
      Race/ethnicity and underlying disease influences hematopoietic stem/progenitor cell mobilization response: a single center experience.
      ] and allogeneic (healthy volunteers) donors may be different enough to warrant separating certain metrics, such as viable post-thaw CD34+ cells recovery.
      The committee recommends each center develop expiration times for each product based on internally validated data; however, primary data is not always available when establishing new programs or product lines. Adopting stability data from other validated programs or peer-reviewed literature (from programs with similar cell processing/cryopreservation techniques and storage conditions is acceptable only as an interim practice. Data gathered during initial validation and manufacturing should be actively monitored at frequent and regular intervals to ensure that the expected viability/potency of products is maintained.
      The established expiration date of cryopreserved products depends on the age of stored HSPC products utilized to validate the stability program. If cryopreserved HSPC products are stored in validated conditions and surrogate assays can demonstrate retention of acceptable viability and potency, the product may be considered beyond the expiration date with approval from a qualified designee [
      FACT-JACIE international standards for cellular therapy product collection, processing. And administration.
      ]. Data from these deviations can be used toward extending the expiration time, if adequate.
      The committee recommends regular verification of representative HSPC product aliquots and if feasible more frequent than annual verification. As a practical way to monitor the performance of personnel, equipment, and processes throughout the year, annual verification of established HSPC stability is necessary to ensure any impact of the process, personnel change or integrity of cryopreservation and storage system maintaining expected functionalities. The SPT suggests a higher frequency of monitoring than annual (quarterly or semi-annual) monitoring of HSPC stability to minimize the risk of the cumulative impact of unwanted findings during annual verifications of the HSPC stability program. The SPT recognizes that having a validated HSPC stability program will likely have ramifications on QC release criteria for cryopreserved HSPC products but release criteria can be independent of HSPC stability programs.
      The committee recommends a minimum of three sentinel samples (or data points such as one product per 5 products cryopreserved) annually per process or product type. The number of samples to be tested depends on the size, complexity of the program, and cell product type. At a minimum, there should be an adequate number of samples for statistical analysis to be performed for each attribute of the stability program to be tested such as CD34+ cell viability, viable CD34+ cell recovery, etc. Each unique product source and container (bag vs. vial/segment, etc.) as well as each separate manufacturing, cryopreservation, and storage condition should be separately validated and monitored. The samples can be included from routine processing and evaluation of products, or from sentinel QC samples, depending on the stability program.
      The committee does not recommend using engraftment data as the sole metric for stability. The committee discussed at length if post-transplant HSPC engraftment data serve as a marker of HSPC stability. The simple answer is No since the purpose of having the HSPC stability program is to provide predictability of stability and retention of the desired function prior to infusion, while engraftment is a prospective functional outcome indicator. Post-transplant hematopoietic engraftment data is important to correlate laboratory assessment of stability to transplant outcomes and is required to comply with FACT/AABB accreditation standards.
      The committee recommends facilities utilize the same assay(s) pre-freeze and post-thaw to determine the viability of the target cell product and allow for true comparison analysis within their stability program. Currently, several test methods exist to determine the cell viability of cryopreserved HSPC. The principles of viability testing vary between cell membrane integrity by dye exclusion (i.e., TB dye exclusion) or the ability of viable cells to prevent nucleic acid staining by membrane-impermeable fluorescent dye PI or 7-AAD.
      During discussions, the committee considered whether the TB dye exclusion test should be considered obsolete [
      • Takanashi M
      • Selogie E
      • Reems JA
      • et al.
      Current practices for viability testing of cryopreserved cord blood products: an international survey by the cellular therapy team of the biomedical excellence for safer transfusion (BEST) collaborative.
      ]. In the end, the committee agreed that while TB dye exclusion is not sufficient in most instances to identify rare target cells (i.e., CD34+) to serve as a sole testing method for HSPC stability, it may still be a useful part of a laboratory's program as long as its scope and limitations are considered.
      The committee suggests validating critical steps during post-thaw HSPC sample testing by flow cytometry (or other methodologies) to reduce intra- and inter-laboratory variation. The committee strongly encourages the community to work toward a standardized post-thaw viable CD34+ cell/target cell enumeration methodology similar to the ISHAGE protocol. The committee also suggests that proficiency testing, such as the College of American Pathologists (CAP) or other intra laboratories sample exchange, should work toward including cryopreserved HSPC samples. A previous multi-laboratory collaboration demonstrated ways to harmonize the enumeration of target cells after thawing of cryopreserved cord blood units [
      • Reich-Slotky R
      • Colovai AI
      • Semidei-Pomales M
      • et al.
      Determining post-thaw CD34+ cell dose of cryopreserved haematopoietic progenitor cells demonstrates high recovery and confirms their integrity.
      ,
      • Fournier D
      • Lewin A
      • Simard C
      • et al.
      Multi-laboratory assay for harmonization of enumeration of viable CD34+ and CD45+ cells in frozen cord blood units.
      ]. A similar approach can be adopted to harmonize target cell (i.e., CD34+) enumeration of other cryopreserved HSPC products Table 5. lists critical steps in post-thaw sample testing and key factors to consider reducing variability.
      Table 5Critical steps and key factors to consider to minimize post-thaw sample variability
      Critical stepsKey factors to consider
      Sample thawing and dilutionReduce intracellular ice nuclei formation and membrane destabilization by thawing rapidly and immediately diluting with isotonic solution containing protein source or colloids [
      • Bowman CA
      • Yu M
      • Cottler-Fox M.
      Evaluation of methods for preparing and thawing cryopreserved CD34+ and CD34- cell lines for use as reagents in flow cytometry of hematopoietic progenitor cells.
      ,
      • Woods EJ
      • Liu J
      • Derrow CW
      • Smith FO
      • Williams DA
      • Critser JK.
      Osmometric and permeability characteristics of human placental/umbilical cord blood CD34+ cells and their application to cryopreservation.
      ]. Adding a resting time post thaw to allow cells to recover their membrane structure was shown to be beneficial
      • Fournier D
      • Lewin A
      • Simard C
      • et al.
      Multi-laboratory assay for harmonization of enumeration of viable CD34+ and CD45+ cells in frozen cord blood units.
      .
      Post-thaw incubation time and storage temperatureOnce thawed and after initial membrane stabilization, cell viability reduces over time. The optimal sample storage temperature and storage time can be established by testing different storage temperatures and monitoring viability over time. Storage on ice can help reduce apoptosis by slowing down enzymatic activities. Cell viability can be maintained for at least 2 hours post thaw
      • Yang H
      • Acker JP
      • Cabuhat M
      • McGann LE
      Effects of incubation temperature and time after thawing on viability assessment of peripheral hematopoietic progenitor cells cryopreserved for transplantation.
      .
      Testing methodUse ISHAGE protocol. Establish optimal testing protocol that allows comparison of fresh and thawed samples. Use single platform methodology only to reduce the need for hematology analyzers that cannot discriminate between viable and non-viable cells
      • Allan DS
      • Keeney M
      • Howson-Jan K
      • et al.
      Number of viable CD34(+) cells reinfused predicts engraftment in autologous hematopoietic stem cell transplantation.
      . As long as the samples are diluted, the RBC lysis step can be skipped as it is not required for cryopreserved specimens
      • Fournier D
      • Lewin A
      • Simard C
      • et al.
      Multi-laboratory assay for harmonization of enumeration of viable CD34+ and CD45+ cells in frozen cord blood units.
      .
      Optimize and assess testing proficiencyTo improve the accuracy and reproducibility of sampling viscous specimens such as blood and bone marrow, use the reverse pipetting technique for all sampling
      • Barnett D
      • Janossy G
      • Lubenko A
      • Matutes E
      • Newland A
      • Reilly JT.
      Guideline for the flow cytometric enumeration of CD34+ haematopoietic stem cells. Prepared by the CD34+ haematopoietic stem cell working party. General Haematology task force of the British Committee for Standards in Haematology.
      and consider adding sampling reproducibility as part of competency assessment.
      The committee recommends facilities utilize a validated functional potency assay if available to determine the potency of the target cell product. It is understood that while no consensus test can be designated as the gold standard in assessing the stability of cryopreserved HSPC, determining flow cytometry-based viable CD34+ cell counts from post-thaw samples in combination with a functional potency assay, was currently considered best practice. Currently, there is no consensus regarding a functional potency test for HSPC; however, the CFU assay is well established and can provide information on both progenitor cell viability and hematopoietic differentiation capacity. Recently, a rapid flow cytometry assay was developed to measure post-thaw cord blood potency. The test is based on the detection of STAT5 phosphorylation after stimulation of CD34+ cells with interleukin-3 (IL-3) [
      • Simard C
      • Bonnaure G
      • Fournier D
      • Neron S.
      An objective flow cytometry method to rapidly determine cord blood potency in cryopreserved units.
      ] and is an indicator of stem cell activation and growth potential, while also correlating with the CFU assay results. The IL-3-pSTAT5 test has also been adapted to post-thaw HSPC (apheresis) and is currently being optimized. Aldefluor™ flow cytometry-based assay is an alternative functional potency assay. Use of surrogate potency assays (i.e., 7-AAD- viable CD34+ cells) is recommended to be minimized or phased out, as more specific assays become reasonably available and validated for clinical laboratories.
      The committee recommends container and label integrity and maintenance of microbial sterility be part of monitoring of HSPC Stability Program.
      The committee recommends the use of consensus terminology and associated calculations when available within a formal Stability Program to increase communication transparency and comparison of performance to clients and across programs.
      In summary, the AABB-ISCT SPT's recommendations for best practices are not intended to replace existing standards; but as the descriptor ‘best’ implies, to outline a unified approach to building a stability program, which seeks to maximize cryopreserved HSPC graft outcomes following thaw infusion and clarify what terms in a stability program means for a specific HSPC product instead of resting upon the minimal interpretation of current guidelines. From the survey results, it appears that the cell therapy community at large values a stability program that ensures the potency and safety of cryopreserved HSPC products within established expiration periods and beyond.

      Conflicts of interest

      This is a joint cooperation between the two organizations, ISCT and AABB, who have agreed to share the output of this work and to co-publish in both Transfusion (https://www.aabb.org/news-resources/news/transfusion-journal) and Cytotherapy (https://www.isct-cytotherapy.org/). This manuscript has not been published or is not under consideration by another journal. The authors of this manuscript have read and understood this journal's polices, and believe that the studies presented here do not violate any of these. The authors have disclosed no conflicts of interest.

      Acknowledgments

      Ms. Michele Sugrue, Dr. Christina Celluzzi, and Ms. Lizette Caballero are acknowledged for their guidance to the project team from the ISCT-AABB Joint Working Group. Ms. Rosemarie Bell is acknowledged as a member of the AABB-ISCT Stability Project Team.

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