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

Abstract

In concert with the International Society of Hematotherapy and Graft Engineering (ISHAGE), we previously described a set of guidelines for detection of CD34 cells based on a four-parameter flow cytometry method (CD45 FITC/CD34 PE staining, side and forward angle light scatter). With this procedure, an absolute CD34 count is generated by incorporating the leukocyte count from an automated hematology analyser (two-platform method). In the present study, we modified the basic ISHAGE method with the addition of a known number of Flow-Count fluorospheres. To reduce errors inherent to sample washing/centrifugation, we implemented ammonium chloride lyse, no-wash no-fix sample processing. These modifications convert the basic protocol into a single-platform method to determine the absolute CD34 count directly from a flow cytometer and form the basis of the Stem-Kit from Coulter/Immunotech. A total of 72 samples of peripheral blood, apheresis packs, and cord blood were analysed and compared using the ISHAGE protocol with or without the addition of fluorescent microspheres. Comparison of methods showed a high correlation coefficient (r 0.99), with no statistically significant difference or bias between methods (P G 0.05). Linearity of the absolute counting method generated an R 2 value of 1.00 over the range of 0-250/l. Precision of the absolute counting method measured at three concentrations of CD34-stabilised KG1a cells (Stem-Trol, COULTER) generated a coefficient of variation (C.V.) ranging from 4% to 9.9%. In a further modification of the single-platform method, the viability dye 7-amino actinomycin D was included and demonstrated that both viable and nonviable CD34 cells could be identified and quantitated. Together, these modifications combine the accuracy and sensitivity of the original ISHAGE method with the ability to produce an absolute count of viable CD34 cells. It is the accurate determination of this value that is most clinically relevant in the transplant setting. These modifications may improve the interlaboratory reproducibil-ity of CD34 determinations due to the reduction in sample handling and calculation of results. Cytometry (Comm. Clin. Cytometry) 34:61-70, 1998.

Large volume leukapheresis: Efficacy and safety of processing patient’s total blood volume six times

“…CD34+ cell collection efficiency was lowest during the first TBV processing, but afterward remained stable. No significant interaction between the change in CD34+ cell Bojanic,11 collection efficiency and diagnosis (p=0.309) or the level of CD34+ cell mobilization were found (p=0.173).…”

“…CD34+ cells were analysed by flow cytometry using FACSCalibur (BD Biosciences, Heidelberg, Germany) following standard ISHAGE procedure for cell staining with anti-CD34-PE (clone 8G12) and anti-CD45-FITC (clone 2D1) monoclonal antibodies (BD Biosciences) [11]. CD34+ cell subsets were analyzed using monoclonal antibodies to CD38, HLA-DR, CD90, CD117, CD41 and CD33 (BD Biosciences).…”

Considerations for the control of background fluorescence in clinical flow cytometry

“…Instead, it can only be due to fluorochromeinduced binding. However, to the best of our knowledge, it has never been reported that this control resulted in significant numbers of ''positive'' events (8,9,38,43). We do not therefore, recommend the use of isoclonic controls quantitatively (i.e., subtract the isoclonic control-positive events from the sample-positive events that is under study) but instead solely as an indicator for whether antibody binding through fluorochrome binding in the sample of interest occurs.…”

“…An example of such a control (42) was incorporated into Coulter-Immunotech's Stemkit TM (43). An isoclonic control is specifically designed to determine undesirable antibody binding through fluorochrome-mediated binding.…”

A critical appraisal of factors affecting the accuracy of results obtained when using flow cytometry in stem cell investigations: Where do you put your gates?

“…However, stem cells have some unique characteristics and so some refinements are required to optimize the technique. Specific gating protocols exist for diagnostic and clinical purposes especially in relation to applications in haematology (11,12) but few standard protocols have been developed for other stem cell applications.…”