Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines

Keeney, Michael; Chin‐Yee, Ian; Weir, K; Popma, Jan; Nayar, Rakash; Sutherland, D. Robert

doi:10.1002/(sici)1097-0320(19980415)34:2<61::aid-cyto1>3.0.co;2-f

Cited by 431 publications

(219 citation statements)

References 22 publications

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“…cell population was used to identify the CD34? subpopulation according to the single-platform guidelines of the International Society of Hematotherapy and Graft Engineering (ISHAGE) [23]. Adding a known number of FlowCount fluorescent beads enabled the determination of an absolute CD34?…”

Section: Tnc Countmentioning

confidence: 99%

Quality of long-term cryopreserved umbilical cord blood units for hematopoietic cell transplantation

et al. 2011

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The aim of this study was to evaluate the quality of long-term cryopreserved umbilical cord blood (CB) units for hematopoietic cell transplantation (HCT). The recovery of the number of total nucleated cell (TNC), hematopoietic progenitor cells (HPCs; CD34+ cells, colony-forming units-granulocyte/macrophages [CFU-GMs]), and the percentage of viable cells, CD34+ CD38- cells, and CD34+ CXCR4+ cells of CB units cryopreserved for 10 years for HCT were examined. Eighteen CB units cryopreserved for 10 years (as the study group) and for 1 month (as the control group), respectively, were analyzed. The recovery rate of TNC, CD34+ cells and CFU-GMs were 88.72 ± 16.40, 68.39 ± 18.37 and 42.28 ± 38.16% for the study group and 80.17 ± 14.46, 72.67 ± 20.38 and 49.61 ± 36.39% for the control group (p = 0.106, p = 0.513 and p = 0.559, respectively). There were no significant differences in the recovery rate of TNC, CD34+ cells and CFU-GMs between the study group and the control group. The mean basal percentage of viable cells, CD34+ CD38- cells, and CD34+ CXCR4+ cells after thawing were 83.69 ± 9.45, 9.11 ± 4.13 and 81.65 ± 10.82% for the study group. These results indicate that long-term cryopreservation does not negatively affect the quality of CB units for HCT.

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Section: Tnc Countmentioning

confidence: 99%

Quality of long-term cryopreserved umbilical cord blood units for hematopoietic cell transplantation

et al. 2011

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“…In CD34ϩ HPC dual-platform analysis, the reference cells are the total leukocytes and no morphologically or phenotypically defined cell subset can be identified as the reference population. CD45 gating is also highly recommended when counting CD34ϩ HPC using the logical gate strategy developed by the International Society of Hematotherapy and Graft Engineering (ISHAGE; 119), and subsequently modified by others (47,51,53,120). The ISHAGE protocol and its modifications may be the best current examples of routinely applicable logical gating.…”

Section: Precise Identification Of Reference Populationsmentioning

confidence: 99%

“…FlowCount beads are larger than TruCount, and thus can be used with reduced FSC thresholding without a need for an associated fluorescent marker. However, an appropriate polygonal acquisition gate is necessary to eliminate the high amount of debris and electrical noise (53,120). The Beckman-Coulter method involves a fourcolor system using a single Argon-ion laser and the beads emit on all four channels (86,148).…”

Section: Count Standard and Spherotech-rainbow Beads But Theymentioning

confidence: 99%

Cytofluorometric methods for assessing absolute numbers of cell subsets in blood

et al. 2000

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The enumeration of absolute levels of cells and their subsets in clinical samples is of primary importance in human immunodeficiency virus (HIV)؉ individuals (CD4؉ T-lymphocyte enumeration), in patients who are candidates for autotransplantation (CD34؉ hematopoietic progenitor cells), and in evaluating leukoreduced blood products (residual white blood cells). These measurements share a number of technical options, namely, single-or multiple-color cell staining and logical gating strategies. These can be accomplished using single-or dual-platform counting technologies employing cytometric methods. Dual-platform counting technologies couple the percentage of positive cell subsets obtained by cytometry and the absolute cell count obtained by automated hematology analyzers to derive the absolute value of such subsets. Despite having many conceptual and technical limitations, this approach is traditionally considered as the reference method for absolute cell count enumeration. As a result, the development of single-platform technologies has recently attracted attention with several different technical approaches now being readily available. These single-platform approaches have less sources of variability. A number of reports clearly demonstrate that they provide better coefficients of variation (CVs) in multicenter studies and a lower chance to generate aberrant results. These methods are therefore candidates for the new gold standard for absolute cell assessments. The currently available technical options are discussed in this review together with the results of some cross-comparative studies. Each analytical system has its own specific requirements as far as the dispensing precision steps are concerned. The importance of precision reverse pipetting is emphasized. Issues still under development include the establishment of the critical error ranges, which are different in each test setting, and the applicability of simplified low-cost techniques to be used in countries with limited resources. Cytometry (Comm. Clin. Cytometry) 42:327-346, 2000.

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“…Still, these methods are intrinsically designed for fresh blood samples and disregard cell viability. Then again, stem cell enumeration according to ISHAGE guidelines includes a dead cell dye together with counting beads and cell definition through light scatter properties and antigen expression [14,15]. We have therefore adopted this protocol to measure the concentration of T cells.…”

Section: Introductionmentioning

confidence: 99%

ISHAGE-based single-platform flowcytometric analysis for measurement of absolute viable T cells in fresh or cryopreserved products: CD34/CD133 selected or CD3/CD19 depleted stem cells, DLI and purified CD56+CD3− NK cells

et al. 2007

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Considering the growing use of immunotherapeutic strategies in paediatric stem cell transplantation associated with risk of graft-versus-host disease, an accurate method for the enumeration of residual T cells/kg recipient's body weight is of paramount importance. Therefore, we propose a multi colour-flow cytometric strategy for correct absolute vital T cell enumeration in manipulated cell preparations for clinical use. The gating strategy is based on the ISHAGE single-platform stem cell enumeration method in combination with experiences from lymphocyte subtyping, using low scatter, high expression of CD3 and CD45 antigens and 7-AAD staining in a no-wash-preparation with counting beads. In spiking experiments, the detection limit was determined to be at 0.7 +/- 0.5 CD3(+) cells/microl with a minimum of 50 T cell events acquired. The cell preparations analysed contained a median absolute CD3(+) T cell number of 221 x 10(3) (0.09%, CD34 selected grafts, n = 187), 900 x 10(3) (0.004%, CD3/CD19 depleted grafts, n = 15) and 283 x 10(3) (0.012%, CD3 depleted/CD56 enriched NK-cells, n = 14), respectively. The results differed of those from conventional T cell measurement in cell products after extensive manipulation. Our method provides reliable residual T cell enumeration even at extremely low concentrations.

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Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines

Cited by 431 publications

References 22 publications

Quality of long-term cryopreserved umbilical cord blood units for hematopoietic cell transplantation

Quality of long-term cryopreserved umbilical cord blood units for hematopoietic cell transplantation

Cytofluorometric methods for assessing absolute numbers of cell subsets in blood

ISHAGE-based single-platform flowcytometric analysis for measurement of absolute viable T cells in fresh or cryopreserved products: CD34/CD133 selected or CD3/CD19 depleted stem cells, DLI and purified CD56+CD3− NK cells

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