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 Table of Contents  
Year : 2020  |  Volume : 5  |  Issue : 2  |  Page : 182-186

Assessment of noncryopreserved stem cell viability (CD34+) at 4°C

1 Department of Transfusion Medicine, Rajiv Gandhi Cancer Institute and Research Center, Delhi, India
2 Department of Laboratory Medicine, Rajiv Gandhi Cancer Institute and Research Center, Delhi, India
3 Department of Laboratory Medicine, Sir Ganga Ram Hospital, Delhi, India
4 Department of TB, Jagjivan Ram Memorial Hospital, New Delhi, India

Date of Submission11-Aug-2020
Date of Decision25-Aug-2020
Date of Acceptance07-Oct-2020
Date of Web Publication13-Nov-2020

Correspondence Address:
Amardeep Pathak
Department of Transfusion Medicine, Rajiv Gandhi Cancer Institute and Research Center, Delhi
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/GJTM.GJTM_87_20

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Background and Objectives: Stem cell transplantation is one of the mainstay treatment modality for many benign and malignant disorders. Long- and short-term storage of stem cell is a crucial step. Long-term storage is using temperature-controlled preservation with liquid or vapor nitrogen at − 196°C. Short-term storage at 4°C ± 2°C is done for those products, which are intended to be infused within a few days after the collection. The current study was done to assess the viability of noncryopreserved stem cells at 4°C up to 9 days.
Methods: CD34 enumeration was done on day 0, 3, 5, 7, and 9 of the collection in 12 consecutive cases of stem cell harvest. Enumeration was done using ISHAGE protocol, and viability was measured using 7-AAD dye with “BD Stem kit” on single platform bead-based assay. Viability of the samples was compared at various time points and also with respect to their source (autologous versus allogeneic). The results were expressed as mean (range), and significance was calculated using Mann–Whitney test.
Results: A total of seven allogeneic and five autologous products were included. All the autologous products are stable till 5th day when stored at 4°C ± 2°C with none of the product showing >15% viability loss. However, the allogeneic products are stable up to day 3 only. On day 5, most 29% of the allogeneic products were showing >15% loss of viability.
Conclusions: Storage up to day 7 or 9 was associated with unacceptable loss of viability for both autologous and allogeneic products.

Keywords: ISHAGE, stem cell, viability

How to cite this article:
Pathak A, Tejwani N, Gudapati P, Sharma M, Tayal P, Mehta A. Assessment of noncryopreserved stem cell viability (CD34+) at 4°C. Glob J Transfus Med 2020;5:182-6

How to cite this URL:
Pathak A, Tejwani N, Gudapati P, Sharma M, Tayal P, Mehta A. Assessment of noncryopreserved stem cell viability (CD34+) at 4°C. Glob J Transfus Med [serial online] 2020 [cited 2021 Jul 26];5:182-6. Available from: https://www.gjtmonline.com/text.asp?2020/5/2/182/300642

  Introduction Top

Stem cell transplantation (autologous and allogeneic) is one of the mainstay treatment modality for many benign and malignant hematological disorders. Successful engraftment requires an infusion of a minimal stem cell dose per kilogram of the recipient weight. It has been observed that a minimal stem cell dose of 2 million cells/kg of the recipient is required for successful engraftment.[1] The dose at the time of transfusion is usually affected by three of the factors described ahead.

Donor factors

Autologous versus allogeneic

Previously conducted study at our center has shown that dose obtained in allogeneic transfusions is often satisfactory in comparison to autologous transplants. This is due to the fact that most of the patients receiving autologous transplants have often been exposed to multiple chemotherapy regimens which may have affected the stem cells pool of the marrow.


It has been noted that stem cell yield is often higher in younger individuals as compared to older individuals in allogeneic transplants.[2] This is probably explained by low marrow cellularity seen in old age individual. However, in very young donors, the absolute dose per harvest may be low as the amount of blood processed is often low.


Conventionally, granulocyte colony-stimulating factor (G-CSF) has been used at a dose of 10–15 mcg/kg/day for 5 continuous days as the preferred method for stem cell mobilization. This has been used for many years; however, in cases of autologous transplants, it has been seen that the use of plerixafor as an adjuvant to G-CSF has been associated with better results. This can be done as a preemptive technique in all cases, or its use can be guided by the use of preharvest CD34 counts on day 4.[3]

Pretransfusion storage

The harvested product often requires storage for long term or short term. The decision for the type of storage needs to be taken on an individual basis. Long-term storage is done by temperature-controlled aseptic environment cryopreservation. Short-term storage at 4°C ± 2°C is done for those products, which are intended to be infused within a couple of days after the collection.

There is a lack of standardized guidelines that describe the minimum viability after short-term storage at 4°C ± 2°C. As per the US Food and Drug Administration (FDA) and American Association of Blood Banks, a maximal permissible storage period is up to 48–72 h at 4 ± 2°C.[2] The Indian Council of Medical Research (ICMR) consensus document issued has advised maximal short-term storage up to 9 days.[4] However, there is no data describing the difference between the effect of storage on autologous and allogeneic product viability.

Keeping these factors in mind, the study was planned to study the viability of the stem cells on short-term storage and to look if there is a difference in the stability of autologous and allogeneic harvest products.

  Materials and Methods Top

This is a prospective study, which was taken as a pilot project for the duration of 9 months from September 2018 to June 2019 with 12 cases.


All the patients during this study period, who were taken up for peripheral blood stem cell (PBSC) transplantation, were enrolled after taking informed consent. The patients undergoing bone marrow harvests were excluded from the study.

The PBSC collection was done after the standard mobilization protocol using G-CSF ± Plerixafor.[3] On day 5 of mobilization, the harvest was done on SPECTRA OPTIA (Terumo Penpol) cell separator. After the collection of the desired volume, 2.5 ml representative harvest sample was separated from the final product collected. The final product remaining was processed as per the currently followed practice in our department of transfusion medicine (depending on the type of harvest, collected volume, and final stem cell dose in the bag). The 2.5 ml collected sample was aliquoted in five Eppendorf tubes after labeling (500 μl sample in each tube).

CD34 testing and viability was done using ISHAGE single platform bead-based assay with viability using 7-AAD [Figure 1]. The stem cells were counted as the events that are CD34 positive, CD45 dim positive and have a side scatter equal to scatter of the normal lymphocytes. The viability was measured using 7-AAD dye which binds to the nonviable cells.[5]
Figure 1: CD34 enumeration using single platform bead-based assay

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The first vial of these five Eppendorf tubes was analyzed for CD34 stem cell enumeration with viability testing using 7-AAD (day 0 viability). The remaining four Eppendorf tubes were kept at 4°C in the refrigerator. These tubes were analyzed for CD34 stem cell enumeration with viability testing on day 3, day 5, day 7, and day 9.

Statistical analysis

The results were expressed as mean (range), and significance was calculated using Mann–Whitney Test.

  Results Top

A total of 12 cases were included in this pilot project. Seven of them were allogeneic and five were autologous. The notable difference is significantly higher dose obtained in allogeneic collections than in autologous collections (17.5 versus 3.7, P ≤ 0.001). Patient characteristics are mentioned in [Table 1].
Table 1: Description of patient characteristic

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If we compare the viability data between two groups, than it appears that the autologous harvests were more stable as compared to allogeneic collections. However, the difference failed to attain statistical significance. However, the results may have been compromised due to the small sample size in two groups compared. The day-wise viability details of stem cell harvest of all the 12 cases are mentioned in [Table 2] and [Table 3].
Table 2: Viability of stem cell harvest product of 12 cases

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Table 3: Viability (mean±standard deviation) of all the 12 harvest products with P values

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The US FDA guidelines suggest that there should be >85% viability for stored stem cells harvest products before transfusion when stored at 4°C ± 2°C.[2],[6] We have compared the number of samples that show >15%, >30%, >40%, and >50% viability loss in autologous and allogeneic collections separately. It should be noted that all the autologous collections have >85% viability till day 5 collection. However, in allogeneic collection, it was seen that two products show <85% viability and one of these two also had <50% viability. The results on day 7 and 9 for both types of collections are not acceptable as per viability criteria with most of them showing <85% viability and many showing <50% viability. The loss of viability for autologous and allogeneic products is compared in [Table 4] and [Figure 2].
Table 4: CD 34+ viability fall (%) for stem cell harvest product of 12 cases

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Figure 2: Comparison of autologous and allogeneic peripheral blood stem cell harvest viability for CD 34+

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  Discussion Top

It is always the best practice that the harvested stem cells are cryopreserved immediately, but it cannot be possible all the time. Poor optimum storage condition can adversely affect the viability and recovery of the cells, especially in allogeneic bone marrow[7] and allogeneic PBSC transplantation.[8]

A total of 12 cases (donors/patients) were included in the study. Mean stem cell dose was higher in allogeneic harvests when compared to autologous collections (17.5 versus 3.7 million/kg, P ≤ 0.05). This is an expected finding as donors in most of the autologous collections have received multiple chemotherapy cycles which could have a deleterious effect on the stem cell pool. The same findings were also published in our previous study, where we have noted that there are higher rates of failure to achieve adequate stem cell dose in autologous collections as compared to allogeneic collections.[3]

There were two primary objectives of the current study. The first objective was to study the stability of the harvest products at 4°C ± 2°C. The first and the very important finding in the current study is that the product viability is severely compromised for both types of samples when stored beyond 5 days. The current findings are also supported by multiple other studies which have advised storage only up to 72 h at 1°C–6°C.[9],[10],[11],[12] The US FDA also approves short-term storage at 4°C ± 2°C for up to 3 days, and there should be at least 85% viability after thawing the product.[2],[6] This finding raises a conflict with the article by ICMR which mentions that storage up to 9 days at 4°C ± 2°C does not adversely affect the viability.[4] If the findings of this study and other published data are compared to ICMR consensus, than it should be observed that only 1 out of 12 harvest products has viability >85% at day 9 of storage.

The second objective of the study was to study the difference of stability between autologous and allogeneic harvests. Both the products were showing >85% viability up to 3 days of storage which goes as per the current US FDA recommendations of storage. If we study the overall pattern, than it appears that the autologous harvests are more stable than allogeneic harvests. This evident by the findings such as all the autologous products have >85% viability on day 5, and none had >40% viability loss up to day 7 of storage. It appears that autologous harvest stem cells are more resistant to death than allogeneic stem cells. This could be due to the fact that stem cells from autologous harvests have experienced many cycles of chemotherapy. This possibly had led to positive selection of most resistant stem cells. Thus, in autologous harvest, the stem cell dose is much less than allogeneic, but the stem cells collected are more resistant to adverse conditions. However, there was no significant difference found between two groups on any day of the storage.

There is an advantage associated with higher stability seen in autologous harvests at 4°C ± 2°C. Since there are higher failure rates in this group, it becomes possible to pool the consecutive day collection. This results in decreased costs and processing time without compromising neutrophil and platelet engraftment after infusion of progenitor cells.[13]

One of the major limiting factors for the comparison between two groups is the limited number of samples in each arm. The stem cell enumeration costs using flow cytometry are high due to which it was opted as a pilot project. In addition, the number of stem cell harvests done in a single institution is mostly limited. Thus, there is a need to further study the findings in a larger group as a multicentric trail for a longer duration.

  Conclusions Top

  • Autologous stem cell harvests are more resistant and can safely be stored at 4°C ± 2°C up to 5 days
  • The more resilient nature of these stem cells is possibly due to the positive selection of resistant stem cells due to chemotherapy given to donors as a part of disease management
  • Allogeneic harvests obtained from healthy donors can be safely stored at 4°C ± 2°C up to 3 days
  • Although there was no statistical significance of the study, which is owing to very small sample size, interesting and clinically relevant finding of our study urges the need of the same at a larger scale as a multicentric prospective study.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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. Clin Lab Haematol 1999;21:301-8.  Back to cited text no. 1
Roback JD, Combs MR, Grossman BJ, Hillyer CD. American Association of Blood Banks. Technical Manual. Bethesda, MD: American Association of Blood Banks; 2008.  Back to cited text no. 2
Agarwal P, Tejwani N, Pathak A, Kumar D, Agrawal N, Mehta A. Benefits of pre-harvest peripheral blood CD34 counts guided single dose therapy with PLERIXAFOR in autologous hematopoietic stem cell transplantation: A retrospective study at a tertiary care institute in India. Indian J Hematol Blood Transfus 2019;35:72-6.  Back to cited text no. 3
Bansal D, Totadri S, Chinnaswamy G, Agarwala S, Vora T, Arora B, et al. Management of neuroblastoma: ICMR consensus document. Indian J Pediatr 2017;84:446-55.  Back to cited text no. 4
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. J Hematother 1996;5:213-26.  Back to cited text no. 5
Applications for Minimally Manipulated, Unrelated Allogeneic Placental/Umbilical Cord Blood Intended for Hematopoietic and Immunologic Reconstitution in Patients with Disorders Affecting the Hematopoietic System. Available from: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/ind-applications-minimally-manipulated-unrelated-allogeneic-placentalumbilical-cord-blood-intended. [Last accessed on 2020 Feb 20].  Back to cited text no. 6
Lazarus HM, Kan F, Tarima S, Champlin RE, Confer DL, Frey N, et al. Rapid transport and infusion of hematopoietic cells is associated with improved outcome after myeloablative therapy and unrelated donor transplant. Biol Blood Marrow Transplant 2009;15:589-96.  Back to cited text no. 7
Jansen J, Hanks SG, Akard LP, Morgan JA, Nolan PL, Dugan MJ, et al. Slow platelet recovery after PBPC transplantation from unrelated donors. Bone Marrow Transplant 2009a; 43:499-505.  Back to cited text no. 8
Jestice HK, Scott MA, Ager S, Tolliday BH, Marcus RE. Liquid storage of peripheral blood progenitor cells for transplantation. Bone Marrow Transplant 1994;14:991-4.  Back to cited text no. 9
Petzer AL, Gunsilius E, Zech N, Clausen J, Hoflehner E, Nussbaumer W, et al. Evaluation of optimal survival of primitive progenitor cells (LTC-IC) from PBPC apheresis products after overnight storage. Bone Marrow Transplant 2000;25:197-200.  Back to cited text no. 10
Kao GS, Kim HT, Daley H, Ritz J, Burger SR, Kelley L, et al. Validation of short-term handling and storage conditions for marrow and peripheral blood stem cell products. Transfusion 2011;51:137-47.  Back to cited text no. 11
Moroff G, Seetharaman S, Kurtz JW, Greco NJ, Mullen MD, Lane TA, et al. Retention of cellular properties of PBPCs following liquid storage and cryopreservation. Transfusion 2004;44:245-52.  Back to cited text no. 12
Lazarus HM, Pecora AL, Shea TC, Koç ON, White JM, Gabriel DA, et al. CD34_ selection of hematopoietic blood cell collections and auto-transplantation in lymphoma: overnight storage of cells at 4°C does not affect outcome. Bone Marrow Transplant 2000;25:559-66.  Back to cited text no. 13


  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3], [Table 4]


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