Dataset Information

Validation of the single-platform ISHAGE protocol for enumeration of CD34+ hematopoietic stem cells in umbilical cord blood in a Brazilian center.

ABSTRACT:

Background

This study aims to validate the single-platform method for enumeration of CD34+ cells, by comparing the performance of two different commercial kits, as well as to evaluate the efficiency of the Accuri^TM C6 cytometer in providing direct counts of absolute cell numbers.

Method

We evaluated 20 samples from umbilical cord blood (UCB), comparing the two different methodologies for enumeration of CD34+ cells: single and dual-platform. For the assessment of the single-platform, Procount and SCE kits were used, both of which use fluorescent beads as a counting reference to obtain absolute CD34+ cells numbers. Moreover, after the acquisition of samples in flow cytometer Accuri^TM C6, following the protocol established for each kit, the number of CD34+ cells was recalculated, considering the cell count provided by the Accuri^TM C6.

Main results

In our analysis, the results showed a strong correlation between the number of CD34+ cells/μL (r²=0.77) when comparing the SCE kit and the current dual-platform method. On the other hand, the comparison between Procount kit and dual-platform results showed a moderate correlation for the number of CD34+/μL cells (r²=0.64).

Conclusion

Our results showed that the Accuri^TM C6 flow cytometer can be used safely, applying both the dual and single platform analysis strategy. Considering the ISHAGE protocol-based single-platform approach, as the most appropriate methodology for CD34+ cells enumeration, our results demonstrated that the SCE kit has great potential for national standardization of UCB samples analysis methodology.

SUBMITTER: Pedrosa de Lira de Morais CC

PROVIDER: S-EPMC8885393 | biostudies-literature | 2022 Jan-Mar

REPOSITORIES: biostudies-literature

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Publications

Validation of the single-platform ISHAGE protocol for enumeration of CD34+ hematopoietic stem cells in umbilical cord blood in a Brazilian center.

Pedrosa de Lira de Morais Carla Cristina CC Dias Alves Pinto Juliana J Wagner de Souza Karen K Izu Marina M Fernando da Silva Bouzas Luis L Henrique Paraguassú-Braga Flávio F

Hematology, transfusion and cell therapy 20201204 1

<h4>Background</h4>This study aims to validate the single-platform method for enumeration of CD34+ cells, by comparing the performance of two different commercial kits, as well as to evaluate the efficiency of the Accuri<sup>TM</sup> C6 cytometer in providing direct counts of absolute cell numbers.<h4>Method</h4>We evaluated 20 samples from umbilical cord blood (UCB), comparing the two different methodologies for enumeration of CD34+ cells: single and dual-platform. For the assessment of the sin ...[more]

PMID: 33358615

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Project description:BackgroundUmbilical cord blood (UCB) is a clinically relevant alternative source of hematopoietic stem/progenitor cells (HSPC). To overcome the low cell number per UCB unit, ex vivo expansion of UCB HSPC in co-culture with mesenchymal stromal cells (MSC) has been established. Bone marrow (BM)-derived MSC have been the standard choice, but the use of MSC from alternative sources, less invasive and discardable, could ease clinical translation of an expanded CD34+ cell product. Here, we compare the capacity of BM-, umbilical cord matrix (UCM)-, and adipose tissue (AT)-derived MSC, expanded with/without xenogeneic components, to expand/maintain UCB CD34+-enriched cells ex vivo.MethodsUCB CD34+-enriched cells were isolated from cryopreserved mononuclear cells and cultured for 7 days over an established feeder layer (FL) of BM-, UCM-, or AT-derived MSC, previously expanded using fetal bovine serum (FBS) or fibrinogen-depleted human platelet lysate (HPL) supplemented medium. UCB cells were cultured in serum-free medium supplemented with SCF/TPO/FLT3-L/bFGF. Fold increase in total nucleated cells (TNC) as well as immunophenotype and clonogenic potential (cobblestone area-forming cells and colony-forming unit assays) of the expanded hematopoietic cells were assessed.ResultsMSC from all sources effectively supported UCB HSPC expansion/maintenance ex vivo, with expansion factors (in TNC) superior to 50x, 70x, and 80x in UCM-, BM-, and AT-derived MSC co-cultures, respectively. Specifically, AT-derived MSC co-culture resulted in expanded cells with similar phenotypic profile compared to BM-derived MSC, but resulting in higher total cell numbers. Importantly, a subpopulation of more primitive cells (CD34+CD90+) was maintained in all co-cultures. In addition, the presence of a MSC FL was essential to maintain and expand a subpopulation of progenitor T cells (CD34+CD7+). The use of HPL to expand MSC prior to co-culture establishment did not influence the expansion potential of UCB cells.ConclusionsAT represents a promising alternative to BM as a source of MSC for co-culture protocols to expand/maintain HSPC ex vivo. On the other hand, UCM-derived MSC demonstrated inferior hematopoietic supportive capacity compared to MSC from adult tissues. Despite HPL being considered an alternative to FBS for clinical-scale manufacturing of MSC, further studies are needed to determine its impact on the hematopoietic supportive capacity of these cells.

Dataset Information

Validation of the single-platform ISHAGE protocol for enumeration of CD34+ hematopoietic stem cells in umbilical cord blood in a Brazilian center.

Background

Method

Main results

Conclusion

Publications

Validation of the single-platform ISHAGE protocol for enumeration of CD34+ hematopoietic stem cells in umbilical cord blood in a Brazilian center.

Similar Datasets

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