Relevance of flow cytometric enumeration of post‐thaw leucocytes: influence of temperature during cell staining on viable cell recovery

Fritsch, Gerhard; Frank, Nelli; Dmytrus, Jasmin; Frech, Christian; Pichler, Herbert; Witt, Volker; Geyeregger, René; Scharner, D.; Trbojevic, D.; Zipperer, Elke; Printz, Dieter; Worel, Nina

doi:10.1111/vox.12398

Cited by 8 publications

(9 citation statements)

References 22 publications

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“…Based on this work, we previously established a multicolor single‐platform flow‐cytometric assay to define at least six distinct CD34 progenitor subsets in different CD34 cell sources . According to this analysis and a second antibody panel defining lymphocyte subsets, we analyzed PBSC grafts obtained after either G‐CSF with or without chemotherapy without (n = 40) or with (n = 40) the addition of plerixafor, to investigate potential differences between these two mobilization regimens.…”

Section: Discussionmentioning

confidence: 99%

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Influence of plerixafor on the mobilization of CD34+ cell subpopulations and lymphocyte subtypes

Worel¹,

Frank

Frech

et al. 2017

Transfusion

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In contrast to the findings of other authors, our study shows a clear shift toward more committed CD34+ subsets after plerixafor in poor mobilizers and elucidates the importance of informative surface markers like CD45RA and CD133 in addition to CD38 to discriminate earlier from more committed CD34+ cells. Further studies are needed to analyze whether these findings have an impact on clinical outcome.

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Section: Discussionmentioning

confidence: 99%

“…The gating strategy for lymphocyte subsets was identical to the method described elsewhere. 20 Briefly, viable WBCs were first defined by their CD45 expression and their typical position in the FSC/SCC dot plot. In a next step, CD31 T cells, CD191 B cells, and CD561 NK cells were defined.…”

Section: Gating Strategymentioning

confidence: 99%

Influence of plerixafor on the mobilization of CD34+ cell subpopulations and lymphocyte subtypes

Worel¹,

Frank

Frech

et al. 2017

Transfusion

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“…Guidelines for postthaw evaluation by flow cytometry of these cryopreserved products are not standardized yet,[19] and it accounts for a careful assessment of cells considering the effect of freezing, thawing, processing,[20] and use of cryoprotectants (e.g., DMSO and HES, etc.). [2122] Our protocol included the assessment of the representative aliquot vial stored with each bag at −80°C; these aliquots were thawed at 37°C and evaluated immediately on flow cytometer based on the similar guidelines as the prefreezing samples were evaluated.…”

Section: Discussionmentioning

confidence: 99%

Outcome of 51 autologous peripheral blood stem cell transplants after uncontrolled-rate freezing (“dump freezing”) using −80°C mechanical freezer

Setia

Arora

Handoo

et al. 2018

Asian J Transfus Sci

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BACKGROUND AND OBJECTIVE:Controlled-rate freezing is a complicated, expensive, and time-consuming procedure. Therefore, there is a growing interest in uncontrolled-rate freezing (UCF) with −80°C mechanical freezers for cryopreservation of hematopoietic stem cells. This is a retrospective analysis of efficiency of UCF and outcome of autologous peripheral hematopoietic stem cell (PBSC) transplants at our center from December 2011 to June 2016.MATERIALS AND METHODS:Cryoprotectant solutions used included 5% dimethyl sulfoxide and 5% albumin with 2% hydroxyethyl starch and stored at −80°C mechanical freezer till transplant. Evaluation of cryopreservation was studied by analyzing the variation in cellularity, viability, and CD34+ stem cell dose recovery as well as clinical follow-up with engraftment.RESULTS:A total of 51 patients (23 females and 28 males) underwent autologous PBSC transplantations with a median age of 31 years (range: 3–60 years) for both hematological and nonhematological indications. Mean recovery post by UCF at −80°C mechanical was 92.9% ± 15.5% for nucleated cells, 86.6% ± 15.5% for viability, and 80% ± 21.5% in CD34+ dose. The median day to neutrophil engraftment was 10 (range 5–14 days) and platelets engraftment was 15 (range 8–45 days). The cryopreserved products were stored at −80°C for median 7 days (range 2-41 day) before transplant.DISCUSSION/CONCLUSION:Our analysis shows that PBSC can be successfully cryopreserved with mechanical uncontrolled rate freezing. This is a cheap and simple method to freeze the stem cells for a short period in resource-constrained setting.

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“…3. Standardized protocols need to be established regarding the handling of cryopreserved HSPCs in terms of the evaluation of CD34 + cells by flow cytometry, freezing/thawing conditions, and viability testing of CD34 + cells [41]. 4.…”

Section: Conclusion and Key Findingsmentioning

confidence: 99%

Flow Cytometric Characterization of Hematopoietic Stem and Progenitor Cell Subpopulations in Autologous Peripheral Blood Stem Cell Preparations after Cryopreservation

Heuer,

Löwhagen,

Uhlig

et al. 2023

Transfus Med Hemother

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Introduction: Autologous stem cell transplantation is a successful routine procedure with only a small number of non-engraftment cases, although the time to hematopoietic recovery may vary considerably across patients. While CD34 has been the decisive marker for enumerating hematopoietic stem and progenitor cells (HSPCs) for more than 30 years, the impact of CD34-positive cellular subpopulations in autologous HSPC grafts on hematopoietic reconstitution remains unclear. Methods: The two-color ISHAGE protocol represents the current gold standard for CD34+ cell enumeration but includes only the number of viable CD45+/CD34+ cells relative to the body weight of the recipient. We adapted a multicolor flow cytometry marker panel for advanced characterization of CD34 subpopulations in retained samples of autologous peripheral blood stem cell products (n = 49), which had been cryostored for a wide range from 4 to 15 years. The flow cytometric analysis included CD10, CD34, CD38, CD45, CD45RA, CD133, and viability staining with 7AAD. The findings were correlated with clinical engraftment data, including reconstitution of leukocytes, neutrophils, and platelets after transplantation (TPL). Results: We demonstrated that the identification of autologous HSPC subpopulations by flow cytometry after cryopreservation is feasible. Regarding the distribution of HSPC subpopulations, a markedly different pattern was observed in comparison to previously published data obtained using fresh autologous material. Our data revealed the largest ratio of lympho-myeloid progenitors (LMPPs) after freezing and thawing, followed by multipotent progenitors and erythroid-myeloid progenitors. A high ratio of LMPPs, representing an immature stage of differentiation, correlated significantly with early neutrophilic granulocyte and leukocyte engraftment (p = 0.025 and p = 0.003). Conversely, a large ratio of differentiated cells correlated with late engraftment of neutrophilic granulocytes (p = 0.024). Overall, successful engraftment was documented for all patients. Conclusion: We established an advanced flow cytometry panel to assess the differentiation ability of cryostored autologous peripheral blood stem cell grafts and correlated it with timely hematopoietic reconstitution. This approach represents a novel and comprehensive way to identify hematopoietic stem and progenitor subpopulations. It is a feasible way to indicate the engraftment capacity of stem cell products.

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Relevance of flow cytometric enumeration of post‐thaw leucocytes: influence of temperature during cell staining on viable cell recovery

Abstract: The post-thaw environment and, potentially, the cryoprotectant impact the outcome of cell enumeration, and results from the analysis tube may not be representative of the cells infused into a patient.

Cited by 8 publications

References 22 publications

Influence of plerixafor on the mobilization of CD34+ cell subpopulations and lymphocyte subtypes

Influence of plerixafor on the mobilization of CD34+ cell subpopulations and lymphocyte subtypes

Outcome of 51 autologous peripheral blood stem cell transplants after uncontrolled-rate freezing (“dump freezing”) using −80°C mechanical freezer

Flow Cytometric Characterization of Hematopoietic Stem and Progenitor Cell Subpopulations in Autologous Peripheral Blood Stem Cell Preparations after Cryopreservation

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