How the AQUIOS STEM System Addresses International Guidelines for the Clinical Enumeration of CD34+ Hematopoietic Stem and Progenitor Cells

Dr. Andreas Böhmler

The analysis of CD34+ hematopoietic stem and progenitor cells is one of the most regulated tests in clinical flow cytometry, as the enumeration of rare events such as stem cells can present a challenge to inter- and intralaboratory comparability and concordance.

This Product Bulletin summarizes the essential components and technical details for CD34+ enumeration specified in four commonly used international Guidelines and Standards and describes how the AQUIOS STEM System for CD34+ enumeration addresses these aspects, which are intended to support the laboratory with accreditation procedures.

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With the increased use of mobilized, peripheral stem and progenitor cells (PBSC) for transplantation purposes, Sutherland et al., in concert with the International Society for Hematotherapy and Graft Engineering (ISHAGE), described in 1996 a set of standards for CD34+ enumeration with the intent to provide a simple, sensitive method that allows for a high degree of accuracy and interlaboratory reproducibility. The resulting “ISHAGE Guidelines” soon became the gold standard for the enumeration of hematopoietic CD34+ progenitor cells by flow cytometry1.

In 1998, Keeney et al. published a modified version of the 1996 guidelines, by introducing beads for absolute counting, adding 7-AAD as viability dye to exclude dead cells, and a lysing reagent that doesn’t contain any fixatives2. These modifications converted the basic protocol into a single-platform method, and the resulting “Single platform with viability dye ISHAGE Guidelines” are still, more than 20 years later, mostly untouched. Many international, national, and regional governing bodies have adapted the essential components and technical details of the ISHAGE protocol for CD34+ enumeration, such as the European Pharmacopoeia (Ph. Eur.), a single reference work for the quality control of medicines in the European Union3. In contrast to the ISHAGE Guidelines, the Standards described in the Ph. Eur. are legally binding, as laid down in the Council of Europe Convention on the Elaboration of a European Pharmacopoeia and in EU and national pharmaceutical legislation.

Standards for the transplantation of hematopoietic progenitor cells are published by the Foundation for the Accreditation of Cellular Therapy (FACT) and JACIE, the Joint Accreditation Committee of ISCT and EBMT, the European Society for Blood and Marrow Transplantation4. In addition, the NetCord-FACT Standards provide guidelines for the use of hematopoietic progenitor cells from cord blood5.


The AQUIOS STEM System, comprised of AQUIOS STEM Software for the AQUIOS CL Flow Cytometry System, AQUIOS STEM-Kit Reagents, AQUIOS STEM CD34 Control Cells and Flow-Check Fluorospheres (Table 1), is an in vitro diagnostic medical device intended to be used by laboratory professionals for the detection of the parameters in the specimen types shown in Table 2, System Parameters on the automated AQUIOS CL Flow Cytometry System.

This is a quantitative assay used for the identification and enumeration of the cell populations specified in Table 2 in patients who are preparing for autologous or allogeneic hematopoietic progenitor cell transplantation, and in donors who undergo mobilization or collection schemes for autologous or allogeneic hematopoietic progenitor cell transplantations. Refer to Table 2 for details of the supported specimen types and anticoagulants.

Table 1. Components of the AQUIOS STEM System. All components are for In Vitro Diagnostic Use according to the regulations of the countries where the products are registered.

Instrument Description
AQUIOS CL Flow Cytometry System AQUIOS CL Flow Cytometry System with Uninterruptible Power Manager
AQUIOS STEM Software Software for CD34+ enumeration with automated gating algorithms for the AQUIOS CL. The AQUIOS STEM Software QC module provides access to eIQAP, Beckman Coulter’s electronic Interlaboratory Quality Assurance Program, which allows users to obtain peer group comparison reports for their laboratory’s quality control data.
AQUIOS STEM Kit, 50 tests AQUIOS STEM-Kit reagents consist of a CD45-FITC/CD34-PE murine monoclonal antibody reagent, a corresponding negative control (CD45-FITC/CD34-CTRL), an absolute count reagent (AQUIOS STEM-Count Fluorospheres), a cell viability reagent (7-AAD), and a ready-to-use lysing reagent (AQUIOS STEM Lysing Solution).
AQUIOS STEM CD34 Control Cells, 2 levels, 15 tests each AQUIOS STEM CD34 Control Cells are liquid preparations of stabilized human leukocytes for the verification of the parameters CD34 and CD45 as part of the AQUIOS STEM System. Each kit contains 2 levels of CD34 with approx. 10 CD34+ cells/µL (level 1) and approx. 30 CD34+ cells/µL (level 2).
Flow-Check Fluorospheres, 3x10 mL Flow-Check Fluorospheres are an assayed suspension of fluorospheres (fluorescent microspheres) used for daily verification of a flow cytometer's optical alignment and fluidics system.

Table 2. AQUIOS STEM System Parameters

Specimen Type Anticoagulant Kits/Parameters
K2EDTA K3EDTA Heparin Heparin ACD-A Mix ACD-A CPD AQUIOS Stem Kit
Whole Blood (WB) X X X       CD34 Percent
CD34 Absolute Count
CD45 Absolute Count
Mobilized whole blood (MB) X X X      
Fresh or thawed bone marrow (BM) X X X X    
Fresh or thawed Apheresis Product (AP)       X X  
Fresh or thawed Cord Blood (CB)         X X

This Product Bulletin summarizes the requirements for CD34+ enumeration specified in the above-mentioned guidance documents and describes how the AQUIOS STEM System addresses these aspects, intended to support laboratories with their accreditation procedures. The categories and requirements highlighted in this document are not exhaustive but focus on the technical aspects of the CD34+ enumeration procedure; other areas of the Guidelines and Standards that govern general laboratory, facility, and quality management requirements, are not considered.

International Guidelines and Standards for CD34+ Hematopoietic Stem and Progenitor Cells Analysis

To comply with all standards of care relevant to your organization, please consult the full texts of the documents referenced in this Product Bulletin, as well as the local, national, or international laws and regulations applicable in your geography.


  1. Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I: The ISHAGE guidelines for CD34+ cell determination by flow cytometry. International Society of Hematotherapy and Graft Engineering. J Hematother. 1996 Jun;5(3):213-26.
  2. Keeney M, Chin-Yee I, Weir K, Popma J, Nayar R, Sutherland DR: Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines. International Society of Hematotherapy and Graft Engineering. Cytometry. 1998 Apr 15;34(2):61-70.
  3. EDQM Council of Europe: European Pharmacopoeia (Ph. Eur.). 10th Edition 2019-2022. Website:
  4. Foundation for the Accreditation of Cellular Therapy (FACT), Joint Accreditation Committee – ISCT and EBMT (JACIE): International standards for hematopoietic cellular therapy product collection, processing, and administration. 8th Edition, Version 8.1, 2021. Website:
  5. Foundation for the Accreditation of Cellular Therapy (FACT): NetCord-FACT international standards for cord blood collection, banking, and release for administration. 7th Edition, 2019. Website:
  6. Whitby A, Whitby L, Fletcher M, Reilly JT, Sutherland DR, Keeney M, Barnett D: ISHAGE protocol: are we doing it correctly? Cytometry B Clin Cytom. 2012 Jan;82B:9-17.

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