The increased use of Peripheral Blood Stem Cells (PBSC) to reconstitute hematopoiesis in autotransplant and, more recently, allotransplant settings has not been associated with a consensus means to quality control the PBSC product. Since the small population of cells that bear the CD34 antigen are thought to be responsible for multilineage engraftment, graft assessment by flow cytometric quantitation of CD34+ cells should provide a rapid, reliable, and reproducible assay. Unfortunately, although a number of flow cytometric assays for CD34 enumeration have been described, the lack of a standardized method has led to the generation of widely divergent data. Furthermore, none of these assays has been validated as to interlaboratory reproducibility and suitability for widespread clinical application. In early 1995, the International Society of Hematotherapy and Graft Engineering (ISHAGE) established a Stem Cell Enumeration Committee, the mandate of which was to validate a simple, rapid, and sensitive flow cytometric method to quantitate CD34+ cells in peripheral blood and apheresis products. We also sought to establish its utility on a variety of flow cytometers in clinical laboratories and its reproducibility between transplant centers. Here, we describe the four-parameter flow methodology adopted by ISHAGE for validation in a multicenter study in North America.

Background:Paroxysmal nocturnal hemoglobinuria (PNH) is a life‐threatening disorder caused by an inability to make glyco‐phosphatidyl‐inositol (GPI) anchors. While flow cytometry is the method of choice to detect the loss of GPI‐linked proteins, the development and validation of sensitive, standardized, methodologies have been hampered by the rarity of this disease and by technical difficulties in the accurate identification of PNH cells.Methods:Guidelines for the diagnosis and monitoring of PNH by flow cytometry were recently published by the International Clinical Cytometry Society (ICCS). However, specific reagent cocktails, and associated detailed analytic strategies were not directly addressed therein. In this supporting document based on the ICCS guidelines, we provide concise practical protocols for the high‐sensitivity detection of PNH RBCs and WBCs (both granulocytes and monocytes).Results:The CD235aFITC/CD59PE assay described was capable of detecting as few as 20 Type III PNH RBCs per million cells. Frequencies of Type III PNH cells in 10 normal samples were in the 0–6 per million RBCs. The high‐resolution granulocyte/neutrophil assays described in this study could detect PNH phenotypes consistently at a level of 0.01% sensitivity. Frequencies of PNH phenotypes in normal individuals were in the 0–10 per million granulocytes/neutrophils range.Conclusions:The careful screening and selection of specific antibody conjugates has allowed the development of reagent cocktails suitable for high‐sensitivity flow cytometric detection of PNH RBCs and PNH WBCs. The reagent cocktails described herein can be used on a variety of clinical flow cytometers equipped with four or more photo multiplier tubes. © 2012 International Clinical Cytometry Society

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.

No abstract

Summary:Reduced CD34 + cell viability due to cryopreservation has unknown effects on subsequent hematopoietic engraftment in autologous transplantation. Thirty-six consecutive autologous peripheral stem cell collections were analyzed for absolute viable CD34 + cell numbers at the time of stem cell collection and prior to reinfusion. Viable CD34 + cells were enumerated using single platform flow cytometry and the molecular exclusion

Solid cancers are a leading cause of death worldwide, primarily due to the failure of effective clinical detection and treatment of metastatic disease in distant sites. There is growing evidence that the presence of circulating tumor cells (CTCs) in the blood of cancer patients may be an important indicator of the potential for metastatic disease and poor prognosis. Technological advances have now facilitated the enumeration and characterization of CTCs using methods such as PCR, flow cytometry, image-based immunologic approaches, immunomagnetic techniques, and microchip technology. However, the rare nature of these cells requires that very sensitive and robust detection/enumeration methods be developed and validated in order to implement CTC analysis for widespread use in the clinic. This review will focus on the important technical and statistical considerations that must be taken into account when designing and implementing CTC assays, as well as the subsequent interpretation of these results for the purposes of clinical decision making.

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/μl) 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 CD34dim and CD34bright 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 from a single flow cytometric assessment instead of from combined flow cytometer (percent CD34+) and hematology analyzer (absolute leukocyte count) data, 2) the cross‐calibration of the available single‐platform assays, and 3) the optimal method for sample preparation. An important clinical question to be addressed is the definition of the precise phenotypes and required numbers of HPCs responsible for short‐ and long‐term recovery to optimize HPC transplant strategies. Cytometry (Comm. Clin. Cytometry) 34: 128–142, 1998. © 1998 Wiley‐Liss, Inc.

Flow cytometry is the method of choice to ‘diagnose’ paroxysmal nocturnal hemoglobinuria (PNH) and has led to improved patient management. Most laboratories have limited experience with PNH testing, and many different flow approaches are used. Careful selection and validation of antibody conjugates has allowed the development of reagent cocktails suitable for detection of PNH RBCs, CD71+ reticulocytes, and WBCs in clinical/sub‐clinical PNH samples. A CD235a‐FITC/CD59‐PE assay was developed capable of detecting Type III PNH RBCs at 0.01% sensitivity. A protocol targeting immature CD71+ RBCs can detect PNH reticulocytes at similar sensitivity. Four‐color FLAER‐based neutrophil and monocyte assays were developed to detect PNH phenotypes at a level of 0.01% and 0.04% sensitivity, respectively. For instrumentation with five or more PMTs, a single‐tube 5‐color FLAER/CD157‐based assay to simultaneously detect PNH neutrophils and monocytes is described. Using these standardized approaches, results have demonstrated good intra‐ and inter‐laboratory performance characteristics even in laboratories with little prior experience performing PNH testing. © 2015 by John Wiley & Sons, Inc.