Flow Cytometric Analysis of Peripheral Blood Stem Cells

Bender, James; Unverzagt, Kristen

doi:10.1089/scd.1.1993.2.421

Cited by 11 publications

(4 citation statements)

References 47 publications

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“…Sites reported viabilities of 57% or greater and less variable results. It is well documented that phenotyping cells from cryopreserved specimens introduces additional problems for analysis (11,12). Results of the remaining 19 specimens, from fresh collections with high viabilities, still demonstrated alarming variability.…”

Section: Resultsmentioning

confidence: 99%

North American Multicenter Study on Flow Cytometric Enumeration of CD34+ Hematopoietic Stem Cells

Brecher¹,

Sims²,

Schmitz³

et al. 1996

Journal of Hematotherapy

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Transplant centers often rely on CD34+ cell quantitation by flow cytometry to ensure adequacy of hematopoietic progenitor cell collection. Because of variation in interpretation, a lack of interlaboratory proficiency studies, and no generally accepted methodology, comparison of CD34 data from site to site is difficult. Twenty-one samples from marrow and peripheral blood stem cell collections were shipped to 10 participating North American laboratories for analysis. Duplicate samples were included to assess reproducibility. Participants were surveyed for methodology. Three centers had previously attempted to standardize their methodology among themselves. The variability observed in the CD34 values ranged from a max/min. reported value per sample of 2.9 to 749 (median 76). Exclusion of two outlying sites reduced the variability of results to 1.2 to 27 (median 3.1). Variation among the three standardized sites ranged from 1.2 to 4.4 (median 1.6). Overall reproducibility (excluding the outlying sites B and G) ranged from a minimum of 0-16.5 (percent mean difference) for site C to a maximum of 4.1-133 for site H. Strategies for gating were found to largely influence results. We observed an alarming variation among the CD34 cell counts reported from different laboratories. Standardization substantially reduced observed variation. The need for standardized methodology, reporting, quality control, and proficiency testing is underscored by these findings.

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Section: Resultsmentioning

confidence: 99%

North American Multicenter Study on Flow Cytometric Enumeration of CD34+ Hematopoietic Stem Cells

Brecher¹,

Sims²,

Schmitz³

et al. 1996

Journal of Hematotherapy

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“…5. Example of an incorrect ISHAGE Protocol Gating Strategy set up showing that the participant was actually using a ''Bender'' type gating strategy (9) and not the ISHAGE Protocol Gating Strategy as claimed. þ cell counts of 9 and 47 cells/ll, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…In addition to determining yield, the number of CD34 þ cells mobilized to the peripheral blood is also a predictor of the success of apheresis (5) and can be used to monitor, ''on-line,'' the yield of CD34 þ cells (6). Although simple flow methods for counting CD34 þ cells date back to the late 1980's (7)(8)(9)(10)(11)(12)(13), a standardized multi-parameter protocol that was based on the structural characteristics of the CD34 molecule and the epitopes detected by various CD34 monoclonal antibodies did not emerge until 1994 (14). The latter used the maximum information available of four parameters; forward and side light scatter and the intensity of CD34 and CD45 staining and subsequently formed the basis of a Clinical Guideline for CD34 þ cell quantitation in peripheral blood (PB) and PBSC for the International Society for Hematotherapy and Graft Engineering (ISHAGE) (15).…”

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confidence: 99%

ISHAGE protocol: Are we doing it correctly?

Whitby

Fletcher

et al. 2011

Cytometry Part B Clinical

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Background: Flow cytometric CD341 stem cell enumeration is routinely performed to optimize timing of peripheral blood stem cell collections and assess engraftment capability of the apheresis product. While a number of different flow methodologies have been described, the highly standardized ISHAGE protocol is currently the most widely employed, with 204/255 (81%) international participants in the UK NEQAS CD341 stem cell enumeration program indicating their use of this method. Recently, two laboratories were identified as persistent poor performers, a fact attributed to incorrect ISHAGE protocol usage/ setup. This prompted UK NEQAS to question whether other laboratories were making similar errors and, if so, how this might affect individual EQA performance.Methods and Results: In send out 0801, where two stabilized samples were issued, the EQA center surveyed 255 participants with flow analysis data and subsequent results collected. One hundred and ninety-six laboratories returned results with 103 returning dot plots. Eighty-three out of one hundred and three stated that they used the ISHAGE protocol gating strategy but 43% (36/83) were incorrectly setup. Analysis of the data showed those incorrectly using single platform ISHAGE gating strategy were twice as likely to fail an EQA exercise compared to those using the protocol correctly. This failure rate increased two fold when incorrect ISHAGE protocol was used in a dual platform setting.Conclusion: This study suggests a widespread fundamental lack of understanding of the ISHAGE protocol and the need to deploy it correctly, potentially having significant clinical implications and highlights the need to monitor participants rigorously in their deployment of the ISHAGE protocol. It is hoped that once these findings have been disseminated, performance can be improved. V C 2011 International Clinical

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“…The expression profile of CD45 permits the identification of hematopoietic progenitor cells because they express CD45 dimly. CD45 is rapidly lost during erythrocyte maturation and the CD45 density increases during the maturation of the monocyte, NK, B-, and T-lymphoid cell lineages (25)(26)(27)(28)(29). Figure 3 illustrates a peripheral blood sample of a patient treated with chemotherapy to which 50,000 beads were added, and was incubated with SY-III-8 and CD45 PE/CY5.…”

Section: Resultsmentioning

confidence: 99%

Automated Enumeration of CD34⁺Cells in Peripheral Blood and Bone Marrow

Chen¹,

Lin²,

Shye³

et al. 1994

Journal of Hematotherapy

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We have developed a rapid and accurate method to enumerate the number of CD34+ cells in peripheral blood, bone marrow, and leukopheresis samples. The method consists of a two-tube assay and a dedicated software program for data acquisition and analysis. The first reagent combination consists of (a) a nucleic acid dye to identify nucleated cells, (b) a CD45 monoclonal antibody labeled with PE/CY5 to discriminate progenitor cells from mature lymphoid, neutrophil, erythroid, and monocytic cells, (c) an IgG1 control antibody labeled with PE to establish the boundary between specific and nonspecific staining, and (d) a known number of fluorescent beads to determine an absolute count of cells. In the second reagent combination the IgG1 control antibody is replaced by a CD34 antibody labeled with PE that is used to identify the CD34+ cells in the location established by the control reagent combination. The software program uses the fluorescent beads to adjust the forward light scatter, orthogonal light scatter, and three fluorescence detectors of the flow cytometer. The expected location of the CD34+ cells is then established with the control reagent combination followed by the enumeration of the CD34+ cells per microliter of sample with the reagent combination containing the CD34 antibody. This method is sensitive enough to detect CD34+ cells in peripheral blood of normal donors and can reliably determine an increase in CD34+ cells in the peripheral blood of patients treated with chemotherapy and/or growth factors. The method alleviates some of the difficulties encountered when small numbers of CD34+ cells are enumerated. The system allows for more precise evaluations of the grafts used for bone marrow transplantation.

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Flow Cytometric Analysis of Peripheral Blood Stem Cells

Cited by 11 publications

References 47 publications

North American Multicenter Study on Flow Cytometric Enumeration of CD34+ Hematopoietic Stem Cells

North American Multicenter Study on Flow Cytometric Enumeration of CD34+ Hematopoietic Stem Cells

ISHAGE protocol: Are we doing it correctly?

Automated Enumeration of CD34⁺Cells in Peripheral Blood and Bone Marrow

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Flow Cytometric Analysis of Peripheral Blood Stem Cells

Cited by 11 publications

References 47 publications

North American Multicenter Study on Flow Cytometric Enumeration of CD34+ Hematopoietic Stem Cells

North American Multicenter Study on Flow Cytometric Enumeration of CD34+ Hematopoietic Stem Cells

ISHAGE protocol: Are we doing it correctly?

Automated Enumeration of CD34+Cells in Peripheral Blood and Bone Marrow

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Product

Resources

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Automated Enumeration of CD34⁺Cells in Peripheral Blood and Bone Marrow