The ISHAGE Guidelines for CD34+ Cell Determination by Flow Cytometry

TLDR

Peripheral blood stem cell transplantation depends on CD34+ cells, yet no standardized flow cytometric assay exists, leading to inconsistent and unvalidated data across laboratories. The ISHAGE Stem Cell Enumeration Committee aimed to validate a simple, rapid, and sensitive flow cytometric method for quantifying CD34+ cells in peripheral blood and apheresis products. The committee adopted a four‑parameter flow cytometry protocol and tested it in a North American multicenter study to assess its applicability on various flow cytometers and reproducibility between transplant centers.

Abstract

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.