The Influence of Flow Cytometric Gating Strategy on the Standardization of CD34+ Cell Quantitation: An Australian Multicenter Study

Abstract

The Australian Multi-centre study on CD34+ cell quantitation by flow cytometry documented, first, the extent of variation of CD34+ cell enumeration and, second, the influence of flow cytometric gating on CD34+ cell measurement. A PBSC harvest analyzed by 20 participating centers showed results ranging from 0.64% to 2.80%, with a median of 1.54% CD34+ cells. Of 20 centers, 9 obtained results within +/-10% of the median (the criteria for reproducibility suggested by the ISHAGE Guidelines). The flow cytometric gating strategy was identified as one of the major variables among the methods used. In stage 2, list mode data from two samples were analyzed by 24 Australian and overseas centers, including the authors of three published guidelines. Significantly different CD34+ results were obtained when different gating strategies were used (p < 0.006). When all the centers used the same gating strategy, the measurement of CD34+ cells fell within a narrow range, with 0-7% of results outside +/-10% of the median. However, when different gating strategies were used, the results were more widely scattered, with 17% of centers outside +/-10% of the median. This study demonstrated the potential impact of flow cytometric gating strategy on the reproducibility of CD34+ cell enumeration.

Peripheral blood CD34+ cell count reliably predicts autograft yield

“…Importantly, we have previously verified our CD34 + cell enumeration methodology with the ISHAGE guidelines. 5,6 Consequently, our findings could potentially find wider applicability.…”

“…The mobilization strategy employed various chemotherapeutic agents supplemented with rhGCSF (10 g/kg), or (2)(3)(4)(5)(6)(7)(8)(9) utilized rhGCSF (10 g/kg) alone (Table 2). Following chemotherapy and cytokine mobilization, PBPC collection was initiated when the patient's PB WBC returned to Ͼ1.5 × 10 9 /l and/or the PB absolute neutrophil count (ANC) reached 1.0 × 10 9 /l following the nadir.…”

“…5,6 Briefly, 1 × 10 6 cells, either in whole blood or the apheresis collection product, were added to an aliquot of anti-CD34 PE and anti-CD45 PerCP which had previously been titrated to provide a saturating antibody concentration, and incubated for 20 min at room temperature. This was followed by addition of red cell lysing solution (FACS lysing solution, Becton Dickinson), followed by brief vortex mixing and incubation for 10 min at room temperature.…”

Enumeration of CD34+ hematopoietic progenitor cells for clinical transplantation: comparison of three different methods

“…In this regard, the sequential gating strategy of the ISHAGE protocol requires more manual work as compared to Milan/Mulhouse and our protocol, and still involves a certain degree of subjectivity. As the gating procedure was identified as one of the major variables affecting the reproducibility of the methods for CD34+ cell enumeration, 36 the demand for simple, even though standardized, strategies is obvious. In conclusion, for clinical purposes the use of PE-conjugated 8G12 in a single staining assay appears reliable and feasible when rigorous procedures for sample preparation and acquisition are followed and an adequate software for multiparametric analysis is available.…”

Flow cytometric CD34+ stem cell enumeration: Lessons from nine years' external quality assessment within the Benelux countries

“…Therefore, various national and international guidelines for CD34þ cell enumeration have been formulated (24,(26)(27)(28). Nevertheless, in various multicentre studies (29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) large variations between results of individual centers were observed. The outcome of CD34þ cell enumeration assays depends on several variables, such as sample source, sample processing, technical experience, and data analysis techniques.…”