key: cord-0962410-vwyze72e
authors: Chang, Annabella; Ragg, Scott J.; Ma, David D.
title: Meeting the COVID challenge: Optimizing vCD34(+) in cryopreserved HPC samples for implementation of an external QA Program
date: 2022-01-25
journal: Cytotherapy
DOI: 10.1016/j.jcyt.2021.10.009
sha: 3d82413a8793bd13870953475c5a8564d0dd3120
doc_id: 962410
cord_uid: vwyze72e

BACKGROUND: The COVID-19 pandemic has forced a fundamental change in the global procurement of allogeneic hematopoietic progenitor cells (HPCs) for transplantation. To better meet the emergent challenges of transporting cryopreserved allogeneic HPC during pandemics, there is an urgent need for External Quality Assurance (EQA) programs to evaluate reproducibility and harmonization of viable CD34(+) cell (vCD34(+)) HPC enumeration, as the current EQA programs are unsuitable for analysis of vCD34(+). The cost-effective distribution of HPC cryopreserved reference samples (CRSs) with acceptable reproducibility and specificity is key to the success of a vCD34(+) EQA program. METHODS: Cryopreserved HPC samples (n = 11) were either stored on dry ice for 1 to 4 days or for 1 day followed by liquid nitrogen (LN) storage for 1 to 3 days to assess optimal conditions for vCD34(+) EQA. Flow cytometric enumeration of vCD34(+) HPCs was performed using a single platform assay combined with 7-AAD viability dye exclusion. The optimum transportation condition was validated in pilot and multicenter national studies (n = 12). RESULTS: A combination of 1 day on dry ice followed by LN storage stabilized viability compared with continuous storage on dry ice. This study demonstrates that dispatch of CRSs on dry ice to recipient centers across a distance of ≤4000 km within 26 h, followed by LN storage, resulted in reproducible intercenter vCD34(+) enumeration. The estimated cost of safer and more convenient dry ice delivery is >20-fold lower than that of LN. CONCLUSION: This approach can form the basis for economically and scientifically acceptable distribution of CRSs for external vCD34(+) EQA.

The COVID-19 pandemic has fundamentally changed the global procurement of voluntary unrelated and related allogeneic hematopoietic progenitor cells (HPCs) for transplantation. Pre-pandemic, allogeneic donor HPCs were transported as fresh material using personal couriers on passenger flights. The global passenger aviation slowdown necessitated a rapid change in practice to the cryopreservation of allogeneic HPCs at the collection facility, with subsequent dry shipper transport on freight aircraft to the transplant center. Reference vials of the cryopreserved HPC product that accompany the package serve as a source of quality control material for assessment of engraftment potential at the transplant facility. A recent report [1] found highly variable vCD34 + HPC recoveries in cryopreserved allogeneic HPCs procured by national and international centers, with implications for patient safety after the rapid switch from couriertransported fresh HPCs to cryopreserved product because of COVID-19 travel restrictions. This finding illustrates the urgent need to establish External Quality Assurance (EQA) programs for vCD34 + HPC enumeration, and a major barrier is the expense and safety of goldstandard transport of CRSs using a liquid nitrogen (LN) shipper.

Rapid enumeration of CD34 + HPCs by flow cytometry is the accepted biomarker of engraftment potential [2] and also facilitates monitoring and optimal harvest timing of allogeneic and autologous blood HPCs by apheresis. Since the establishment of a widely accepted exclusion gating protocol [3] , considerable standardization of methodology has occurred, enabling interlaboratory comparisons [4] . Inclusion of a viability dye such as 7-aminoactinomycin D (7-AAD) allows enumeration of viable CD34 + cells (vCD34 + ), which is particularly useful for cryopreserved samples [5] .

EQA programs use a common reference sample to identify underperforming laboratories and enable targeted interventions and training to gain conformant outcomes in previously nonconforming facilities. National and international EQAs use fresh or stabilized cells for enumeration of total CD34 + to ensure that results in fresh HPC harvests are accurate, reproducible between centers and valid for clinical application [6À8] . Global participation in these external EQAs was enabled by the relatively low distribution costs of stable reference material at ambient temperature across a geographic expanse. However, the use of stabilized cells in an EQA precludes enumeration of vCD34 + HPCs.

The FACT-JACIE International Standards for Hematopoietic Cellular Therapy Product Collection, Processing and Administration (Eighth Edition) [9] requires that, " for HPC products intended for restoration of hematopoiesis, an assay measuring viable CD34 shall be performed." Furthermore, there is a requirement for facilities to have a program, such as vCD34 + HPC cell assessment, that regularly evaluates the viability and potency of cryopreserved cellular therapy products.

There are no acceptable commercial quality control materials or interlaboratory standardization programs for vCD34 + HPC enumeration on fresh or cryopreserved samples. Fresh samples must be stabilized before distribution, which makes these samples unsuitable for viability assessment using 7-AAD. Cryopreserved reference samples (CRSs) are generally not used due to the logistics and cost implications of transport to participating facilities. Although comparison studies between laboratories within close proximity may use a bulky LN dry shipper to transport CRSs, this method is expensive and impractical for interstate or international participants. An alternative low-temperature method is dry ice transport to allow the establishment of lower-cost external EQAs to better meet the needs of autologous HPC programs and the emergent challenges of transporting cryopreserved allogeneic HPCs during the COVID-19 pandemic.

This study evaluated the feasibility of distribution of CRSs on dry ice for vCD34 + enumeration. After initial simulation and pilot studies, a multicenter study was conducted in 12 laboratories across Australia, covering distances of 4000 km (»2500 miles) and transit times of 26 h. Shipping on dry ice, with return to storage in vapor-phase LN on receipt, facilitated nationwide delivery to testing laboratories of analytically comparable CRSs, with a >20-fold reduction in cost compared with transportation in an LN dry shipper. This approach may facilitate the establishment of an urgently needed, practical and costeffective EQA program for vCD34 + HPC enumeration.

Approval was obtained from the St Vincent's Hospital Human Research Ethics Committee (10/070) to collect G-CSFÀmobilized HPCs from autologous and allogeneic donors after receiving informed consent according to the Declaration of Helsinki. When the harvested yield of HPCs was greater than that required for transplantation, a 15-mL sample was obtained from each of the 10 HPC harvests (denoted A to J), diluted in autologous plasma and 10% DMSO at a cell count <250 £ 10 9 /L, aliquoted (300 to 500 mL per 1.8-mL cryovial) and immediately cryopreserved in a Planer Kryo 360-3.3 controlledrate freezer at À1°C/min to À50°C, then À5°C/min to À180°C. Vials were transferred to the vapor phase of LN for storage.

To simulate dry ice and LN transport and storage conditions, cryopreserved vials of harvested HPCs were stored under different conditions (Table 1) at the central laboratory, after which vCD34 + enumeration was performed.

Vials were thawed in a 37°C water bath with gentle mixing and 50 ml was immediately diluted 1/10 in DAS (5% Dextran 40, 2.5% Human Serum Albumen, 0.9% saline). 100 ml of diluted sample (»1 À 2.5 £ 10 6 cells), was transferred to duplicate Trucount tubes (Becton Dickinson) containing 20 ml CD45-FITC/CD34-PE (Becton Dickinson) and 20 ml 7-AAD viability dye (Becton Dickinson). Tubes were gently mixed and incubated in the dark at room temperature for 10 minutes.

Each tube was diluted with 500 ml DAS and the sample acquired with a FACSCanto II (8 or 10 color) flow cytometer (Becton Dickinson). Data files were analyzed using FACSDiva software (Becton Dickinson). The single platform exclusion gating strategy developed by Sutherland and Keeney [3, 5] was used to enumerate the vCD34 + HPC population.

To determine the effect of different storage conditions on vCD34 HPC, the reference point used was the mean vCD34/ml of the liquid nitrogen control (LNC) tested on Day-1 to Day-4 of each HPC harvest. The percent change in vCD34 of each storage condition from the LNC for the same HPC was calculated using the formula ðvCD34 þ per mL of storage condition À vCD34 þ per mL of the LNC meanÞ =vCD34 þ per mL of the LNC mean  100:

To extend the findings of the evaluation study, a pilot study was initiated with 2 HPC harvests (HPC-E and HPC-G). Two vials from each harvest were packed in »1.5 kg dry ice (day 0) and transported by overnight air freight from the central laboratory (Lab A) to 2 interstate laboratories (Labs B and C, »1600 and »900 km, respectively). One vial was tested on the day of arrival, i.e., 1 day on dry ice (D1). Table 1 Experimental design and nomenclature for the single-center study evaluating transport and storage conditions for cryopreserved HPC on dry ice with or without subsequent storage in LN. The second vial was transferred to LN upon arrival, then thawed and tested on day 4 (D1 + L3). The central laboratory also performed the same experiments, as well as LN-stored control samples. All results were collated by the central laboratory. Evaluation of reproducibility was assessed on the criteria of results within §10% of the median being deemed comparable, based on the coefficient of variation of the single platform method [5] .

After completion of the pilot study, a multicenter study was initiated by inviting Australian centers enrolled in the Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) CD34 + Program to take part. Twelve laboratories from five of the six Australian states participated.

A single cryopreserved vial from product HPC-D was delivered to each of these laboratories on dry ice as described in the pilot study. Participants were instructed to perform the vCD34 + assay on day 1 or 3 (after transferring the vial to LN storage) if testing on the day of arrival was not possible. Control samples stored in LN were also tested on day 1 and 3 by the central laboratory. All results were collated by the central laboratory. Evaluation of reproducibility was assessed on the criteria of results within §10% of the median being deemed comparable.

Analysis was performed with SPSS, version 24 (IBM, Chicago, IL). The t test for significantly different from null (0) [10] was applied to the absolute counts for each group and also used to assess differences between pairs of means and SD in each day and storage condition. P < 0.05 was considered to be significant. Bonferroni's correction [11] was applied for multiple comparisons between the same data. Correlation analysis of multicenter results was performed using R version 3.6.3 (R Core Team). Z scores for comparison of multicenter study results were calculated and interpreted as per ISO13528:2015 [12] .

For original data, please contact d.ma@amr.org.au.

Evaluation of dry ice and LN storage on vCD34 + in a single center Dry ice and LN transport and storage conditions ( Table 1) were simulated at a central laboratory, and the vCD34 + were enumerated ( Figure 1 ). No significant change in vCD34 + counts over time were observed during continuous storage in LN. Storage on dry ice for 1 day resulted in a significant decrease (P = 0.011) ( Figure 2 and Table 2 ) in vCD34 + content relative to the LNC mean. After this initial decrease, a stabilization of vCD34 + content was observed upon subsequent storage in LN (D1 versus D1 + L1; D1 versus D1 + L2, and D1 versus D1 + L3; P = NS). Samples stored in dry ice for 3 and 4 days showed a trend to decreased viable CD34 content ( Figure 1) ; however, it did not reach statistical significance. Note that three D4 samples (HSC-B, -F and -G) were unable to be analyzed owing to a technical fault, reducing the power of the D4 subset and requiring interpretative caution. These combined results suggested that storage and shipping of CRSs on dry ice for 1 day with subsequent storage in LN for up to 3 additional days warranted investigation as an alternative to LN storage and transport.

A pilot study was performed to evaluate the effect on vCD34 + HPC of shipping two CRSs from central Lab A to each of peripheral Lab B and Lab C on dry ice with subsequent storage in LN. Both Lab B and Lab C reported that the freighted CRSs arrived frozen with excess dry ice at unpacking (transport distances of »1600 and »900 km, respectively). For both samples HPC-E and HPC-G (Figure 3a and 3b) , Labs A and B provided vCD34 + /mL enumerations that were within acceptable limits of the D1 consensus value. Lab C, however, provided vCD34 + results outside the acceptable limits of both the consensus values and the LN control. Lab C's results were internally consistent for HPC-E but inconsistent for HPC-G, suggesting analytical issues rather than problems with sample stability. These findings suggested that overnight shipping of CRSs on dry ice might provide acceptably reproducible vCD34 + results when the assay was performed on arrival day or after further LN storage.

Findings from the pilot study were further explored in a multicenter study. A single vial of cryopreserved HPC was shipped in dry ice and delivered by next day transport to each of the 12 laboratories (Table 3) with excess dry ice still present at unpacking. The Data were allocated into groups A and B based on storage conditions to enable comparison between the different treatments. The difference between pairs of means and SD in each day and each storage condition was tested. The use of P < 0.05 to determine statistical significance was changed to P < 0.017 under Bonferroni's correction for multiple comparisons using the same data. * Statistical significance by t test. Black outlined rectangle shows the median §10% for samples exposed to dry ice for 1 day with or without further storage in LN.

transportation distances ranged from 0.5 to 4,000 km (median 513 km), with transit times ranging from 1 to 26 h (median 22.5 h). The 12 centers submitted vCD34 + counts, with six results obtained on day 1 (D1); one result was obtained on day 2 (D1 + L1) and five results were obtained on day 3 (D1 + L2). Overall, 8 of the 12 centers (67%) returned results that were within §10% of the median and were deemed comparable (Figure 4 ). There was no significant difference between samples tested on D1 or D1 + L1/L2 (P = 0.41). Interestingly, all results fell within the z score acceptability limits of |z| < 2, with z scores ranging from + 1.36 to À1.91. Correlation analysis showed no significant relationship between comparability of vCD34 + counts and (i) sample transit time (R = 0.67, P = 0.07) or (ii) distance traveled (R = 0.19, P = 0.55), demonstrating that laboratory outcome was unlikely to be related to sample transport.

Flow cytometric enumeration of vCD34 + HPCs is widely used as a quality indicator for assessing the engraftment potential of cryopreserved HPC harvests [13, 14] . Such assays on thawed reference vials representative of the cryopreserved HPC products are critical process and quality control points required under both international (e.g., FACT/JACIE [9] ) and national (e.g., NPAAC in Australia [15] ) clinical facility accreditation standards. However, the data on interlaboratory comparison of vCD34 + HPC enumeration of cryopreserved cells is limited. A recent Australian study [1] found highly variable vCD34 + HPC recoveries in cryopreserved allogeneic HPC procured by national and international centers before the pandemic and highlighted implications for international allogeneic HPC procurement after the sudden pivot from couriering HPCs as a fresh product to transporting as a cryopreserved product owing to COVID-19 travel restrictions. The lack of an EQA for vCD34 + HPC enumeration meant that interlaboratory variability in measurement could not be excluded as a cause. This report illustrates the urgent need to establish EQA programs for vCD34 + HPC enumeration in CRSs, but a major barrier to this is the expense of gold-standard transport of CRS using a LN shipper.

Multicenter studies of vCD34 + HPC enumeration using dry shipper transport have been restricted to the setting of cord blood units [16, 17] . Further investigation into this important area for harmonization and standardization has been hampered by the expense of LN dry shipper transport that arises from their limited availability, dangerous goods classification and weight. The estimated cost of dispatch and return of a 30-kg LN shipper by air freight between Australian states is approximately AUD$1500 to $2000 per unit, compared with AUD$70 for a 1.5-kg dry iceÀfilled insulated container across the same distance one way (4000 km). Thus, there is a >20-fold higher cost of LN delivery compared with the dry ice delivery. The capital outlay for LN dry shippers is also considerably more expensive than a reusable expanded foam container. Transport of clinical diagnostic material on dry ice (À78°C) is used widely and is cheaper and easier to handle in many practical senses than LN (À196°) [18] .

The present study evaluated the potential for dry ice to be used as a practical and economical transport medium for CRSs in a national multicenter vCD34 + HPC enumeration EQA program. For such a program to be useful and meaningful, it is essential that the reference sample delivered to each participating center has the same analytical properties, such that any resulting variances are attributable to testing methodology rather than the sample. Initial experiments demonstrated that cryopreserved HPC reference vials transferred from cryogenic LN vapor storage to dry ice underwent a significant decrease in vCD34 + HPC content in the first 24 h compared with controls stored continuously in LN. However, when the CRS was returned to LN storage, the vCD34 + HPC content did not show further significant deterioration. In the context of providing a stable and consistent reference EQA material, we hypothesized that instructing participants to transfer the cryopreserved EQA reference vial to LN after delivery on dry ice would extend the shelf life of the reference material without compromising its analytic comparability with other facilities. This would enable facilities that were unable to test the EQA material on the day of delivery to preserve the sample without invalidating their participation. The pilot and multicenter studies confirmed that CRS transportation on dry ice provided a consistent reference material to evaluate interlaboratory enumeration of vCD34 + HPCs in an EQA setting. In both cases, the majority of laboratories reported a consensus result that was within §10% of the median. Of note, in both studies, the laboratories with the longest sample transit times (26 h) and distance travelled (»4000 km) reported vCD34 + HPC counts that were within the limits of consensus. Importantly, and consistent with our singlecenter evaluation study, re-storage in LN returned a similar proportion of consensus results as samples tested immediately after dry ice delivery, enabling delayed testing of EQA material by recipient centers if required. The multicenter study demonstrated there is some variation in reported vCD34 + HPC enumeration within Australian bone marrow transplant facilities and, consistent with other studies [16, 17] , an EQA using cryopreserved HPCs is required to drive interfacility harmonization and standardization.

The aim of this study was to develop an analytically consistent CRSs for use in an EQA, and as such several limitations arise in interpretation of the pilot and multicenter studies. Instruments, reagents and protocols were not controlled or standardized, leading to potential sources of bias and analytical variability. Despite this, a high level of analytical consistency was achieved, thereby demonstrating the study aim. It is interesting to note that all multicenter study participants recorded a satisfactory z score of |z|< 2, which under ISO13528:2015 designates acceptable performance in proficiency testing by interlaboratory comparison [12] . This reinforces the suitability of the proposed CRS dry ice distribution for a vCD34 + HPC enumeration EQA. Further multicenter studies using this methodology will be undertaken to identify critical sources of variability and reduce the observed inconsistency between Australian centers performing vCD34 + HPC enumeration.

Effects comparable to those seen in our study were also noted on the post-thaw function of encapsulated liver spheroids that were similarly cryopreserved to À196°C and transferred to À80°C for 24 h to mimic a storage and distribution scenario [18] . The somewhat surprisingly reduced post-thaw function after short storage at À80°C after LN storage was postulated to be related to the fluidizing of the freeze concentrated matrix between ice crystals on rewarming to À80°C [19] . These findings support the present study that retransferring samples to LN after 24-h storage on dry ice halted the decline in vCD34 + HPC numbers. According to the above hypothesis, this is presumably due to extracellular re-vitrification removing the warminginduced damaging physical changes, and thereby preventing further vCD34 + HPC loss.

Given the critical reliance on vCD34 + HPC content as an indicator of graft quality and patient safety in HPCs derived from cord blood [20] and autologous HPC(A) [14] , it is imperative that risks associated with interlaboratory errors and variation in vCD34 + HPC enumeration are minimized through harmonization and standardization underpinned by an EQA. This has heightened relevance in light of the rapid transition to cryopreservation of nationally and internationally procured allogeneic HPCs resulting from COVID-19 disruption to passenger air travel and the identified discordant vCD34 + HPC counts reported by collection and transplant centers. Our study has demonstrated that dry ice transport of cryopreserved HPC for 26 h results in the provision of a stable CRS that can enable the commencement of EQA programs for vCD34 + HPC enumeration. This transit time will enable sample distribution across continental distances, while the shelf life can be extended by re-storage in LN at the recipient facility, providing analytic flexibility and convenience.

The authors declare no competing financial interests.

Variable CD34+ recovery of cryopreserved allogeneic HPC products: transplant implications during the COVID-19 pandemic

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We acknowledge the helpful discussion and technical assistance by Sandy Smith, Steven Li and Lisa Flores from SydPath, and Helen Tao, Haematology Department, St Vincent's Hospital. Emmanuel Dale and Louise Wienholt from RCPAQAP provided administrative support for the pilot and multicenter studies. We also thank the consented donors and all the participants in the pilot and multicenter studies; these centers cannot be identified due to the confidentiality provisions of the RCPAQAP. We also express our gratitude to David Bishop and Annette Trickett for reviewing the manuscript. This project is funded by research funds of the Department of Haematology, St Vincent's Hospital Sydney. RCPAQAP Haematology funded the courier costs for sending cryopreserved samples to participants.

DM and AC designed and performed the research, analyzed the data, and wrote the manuscript; SR analyzed the data and wrote the manuscript.