Current status of CD34+ cell analysis by flow cytometry: The ISHAGE guidelines (original) (raw)
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Instrument- and protocol-dependent variation in the enumeration of CD34+ cells by flow cytometry
Transfusion, 2006
The information regarding the minimum number of CD34+ cells that are necessary to reconstitute hematopoiesis in patients undergoing peripheral blood progenitor cell transplantation is quite controversial. Some of the differences in these figures might be due to the selection of antibodies, staining protocols, and acquisition strategies for the flow cytometric enumeration of these cells. STUDY DESIGN AND METHODS: Twenty-seven human umbilical cord blood samples and 33 leukapheresis products were consecutively collected for this study. Cells were stained following two different protocols, both using monoclonal antibodies to CD45 and CD34, and analyzed by the same operator in two different flow cytometers to enumerate the percentage of CD34+ mononuclear cells. RESULTS: Relevant differences in the proportion of cells were encountered, and the correlation between the results yielded by both instruments and protocols, although statistically valid, was suboptimal. CONCLUSIONS: Both interinstrument and interprotocol variation can provide additional explanation for the redundantly reported discrepancies concerning the numbers of CD34 cells that suffice to secure hemopoietic grafting. These results point to the need for new and different standardization approaches in this clinically relevant field.
Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells
Cytometry, 1998
The need for a rapid and reliable marker for the engraftment potential of hematopoietic stem and progenitor cell (HPC) transplants has led to the development of flow cytometric assays to quantitate such cells on the basis of their expression of CD34. The variability associated with enumeration of low-frequency cells (i.e., as low as 0.1% or 5 cells/l) is exceedingly large, but recent developments have improved the accuracy and precision of the assay. Here, we review and compare the major techniques. Based on the current state of the art, we recommend 1) bright fluorochrome conjugates of class II or III monoclonal antibodies (mAbs) that detect all glycoforms of CD34, 2) use of a vital nucleic acid dye to exclude platelets, unlysed red cells, and debris or use of 7-amino actinomycin D to exclude dead cells during data acquisition, 3) counterstaining with CD45 mAb to be included in the definition of HPC, 4) during list mode data analysis, Boolean gating to resolve the CD34 ؉ HPCs from irrelevant cell populations on the basis of the low levels of CD45 expression and low sideward light-scatter signals of HPCs, 5) inclusion of CD34 dim and CD34 bright populations in the CD34 ؉ cell count, 6) omission of the negative control staining, and 7) for apheresis products, enumeration of at least 100 CD34 ؉ cells to ensure a 10% precision. Unresolved technical questions are 1) the replacement of conventional dual-platform by single-platform assay formats, i.e., derivation of absolute CD34 ؉ cell counts
Sao Paulo Medical Journal, 2009
CONTEXT AND OBJECTIVE: Counting and separating hematopoietic stem cells from different sources has importance for research and clinical assays. Our aims here were to characterize and quantify hematopoietic cell populations in marrow donors and to evaluate CD34 expression and relate this to engraftment. DESIGN AND SETTING: Cross-sectional study on hematopoietic stem cell assays, using flow cytometry on donor bone marrow samples, for allogenic transplantation patients at two hospitals in São Paulo. METHODS: Immunophenotyping of marrow cells was performed in accordance with positive findings of CD34FITC, CD117PE, CD38PE, CD7FITC, CD33PE, CD10FITC, CD19PE, CD14FITC, CD13PE, CD11cPE, CD15FITIC, CD22PE, CD61FITC and CD56PE monoclonal antibodies in CD45PerCP+ cells, searching for differentiation and maturation regions. CD34+ sorting cells were analyzed for CD38 and CD117. Rh-123 retention was done before and after sorting. Antigen expression and CD34+ cells were correlated with engraftment...
Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines
Cytometry, 1998
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 reproducibility of CD34 determinations due to the reduction in sample handling and calculation of results. Cytometry (Comm. Clin. Cytometry) 34:61-70, 1998.
Bone Marrow Transplantation, 1999
Three different methods for determination of CD34 ؉ cells in G-CSF-mobilized peripheral blood were compared. The methods were: the Milan/Mulhouse protocol, the ISHAGE guidelines for CD34 ؉ cells enumeration and our own protocol. The procedure we have adopted is essentially a Milan/Mulhouse protocolderived methodology combined with a multiparametric approach using the PAINT-A-GATE software analysis program. The samples were collected from 70 patients affected by acute leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, myeloma and breast cancer who were scheduled to receive autologous PBSC transplantation. PBSC collection was performed following mobilization with subcutaneous G-CSF at 5-10 g/kg/day. A minimum target of 2 × 10 6 /kg CD34 ؉ cells was considered an acceptable harvest to ensure a safe transplant. On average, three aphereses per patient were performed and a total of 204 apheresis samples were analyzed. Regression analysis of the percentage and absolute number of CD34 ؉ cells, as calculated with each method, achieved an excellent correlation in spite of methodological differences. In fact, both CD34 ؉dim and CD34 ؉ CD45 ؊ events were included in our gating strategy. In the setting of a triple staining associating CD34, CD38 and CD45, we identified a variable fraction of CD34 ؉ CD38 ؉ CD45 ؊ cells which would be otherwise undetected due to its CD45 negativity. To this end, we used a new technology referred to as laser-scanning cytometry (LSC) which allowed the isolation and morphological identification of CD34 ؉ CD45 ؊ cells. By comparing CD34 ؉ CD45 ؉ and CD34 ؉ CD45 ؊ cells, we found that they share a common morphology, thus confirming the hypothesis that the latter are to be considered for CD34 ؉ cell calculation. The median number of CD34 ؉ cells/kg, as calculated by the three methods, was: 4.79 × 10 6 /kg (range 1-570) for the Milan/Mulhouse protocol, 3.9 ؋ 10 6 /kg (range 0.8-498) for the ISHAGE one, and 5.17 ؋ 10 6 /kg (range 2-599) for our protocol. The median time to ANC and PLT engraftment was 11 (range 9-24) and 20 (range 10-70) days, respectively. Our protocol achieved the best correlation between CD34 ؉ cells/kg and time to ANC/PLT recovery according to the Spearman's rank test (r ؍ ؊40 and P Ͻ 0.015 for ANC, r ؍ ؊46 and P ؍ 0.005 for PLT). We conclude that (1) CD45 does not appear the ideal partner of HPCA-2 for determination of hematopoietic progenitors in mobilized peripheral blood; and (2) for clinical application, a single staining with 8G12 appears simple, reliable and feasible when rigorous procedures for sample preparation and acquisition are followed and an adequate software for multiparametric analysis is available.
Cytotherapy, 2012
Background aims. Stem cells are commonly enumerated with bead-based methods in blood and marrow progenitor cell transplantation centers. We compared the International Society of Hematotherapy and Graft Engineering (ISHAGE) bead-based method with a true volumetric one that obviates the use of fl uorescent beads for enumeration. Methods. From 31 samples, including 15 peripheral blood samples and 16 leukapheresis products, CD34 ϩ cells were enumerated with the single-platform bead-based ISHAGE method and a true volumetric method. After exclusion of two outliers, one from the peripheral blood group and the other from the leukapheresis group, the results were compared. Results. In the peripheral blood category, no signifi cant difference was observed. However, a proportional systematic error was seen in the leukapheresis group. The systematic error was corrected in the leukapheresis group using a regression line equation. The 95% confi dence interval of differences was [ -5.83, 2.18] for the peripheral blood and [ -38.40, 38.77] for the leukapheresis group after correction of the systematic error. Conclusions. The true volumetric method is a simple and reliable approach that can be used instead of the more popular bead-based procedures.
Hematopoietic stem cells in research and clinical applications: The “CD34 issue”
In this paper, experimental findings concerning the kinetics of hematopoietic reconstitution are compared to corresponding clinical data. Although not clearly apparent, the transplantation practice seems to confirm the basic proposals of experimental hematology concerning hematopoietic reconstitution resulting from successive waves of repopulation stemming from different subpopulations of progenitor and stem cells. One of the "first rate" parameters in clinical transplantations in hematology; i.e. the CD34+ positive cell dose, has been discussed with respect to the functional heterogeneity and variability of cell populations endowed by expression of CD34. This parameter is useful only if the relative proportion of stem and progenitor cells in the CD34+ cell population is more or less maintained in a series of patients or donors. This proportion could vary with respect to the source, pathology, treatment, processing procedure, the graft ex vivo treatment and so on. Therefore, a universal dose of CD34+ cells cannot be defined. In addition, to avoid further confusion, the CD34+ cells should not be named "stem cells" or "progenitor cells" since these denominations only concern functionally characterized cell entities.