Overcoming Currently Unmet Needs in the Clinical Enumeration of CD34+ Hematopoietic Stem and Progenitor Cells

Dr. Andreas Böhmler, Director Strategic Marketing Clinical Solutions, Beckman Coulter Life Sciences, Krefeld, Germany

Clinical laboratories rely on commercially available IVD solutions for CD34+ enumeration in order to avoid time- and resource-consuming validation of user-defined tests. Most reagent kits and software packages were developed as a response to the 1996 and 1998 ISHAGE Guidelines but have not been updated to meet the evolving demands of diagnostic laboratories.

This Product Bulletin summarizes common unmet requirements and describes how the AQUIOS STEM System addresses these needs.

Common Unmet Needs

With the increased use of mobilized, hematopoietic stem and progenitor cells (HSPC) 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 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.

The hallmark of the ISHAGE Guidelines is a sequential gating strategy that derives the number of CD34+ cells from viable leukocytes. The Guidelines also require tests to be run in duplicate together with a negative control to correct for testing variability and unspecific cell and fluorochrome binding. Due to the selectivity of the sequential gating, the use of a negative control was made optional by the authors of the ISHAGE Guidelines in subsequent years, however the European Pharmacopoeia (Ph. Eur.) mandates its use3. 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.

Currently available software solutions that are part of an IVD system do not provide the flexibility to run panels for CD34+ enumeration either with or without a negative control while maintaining IVD status. In addition, not all CD34+ enumeration reagent kits contain a negative control reagent.

While the starting point of the sequential ISHAGE gating strategy, i.e., viable leukocytes, tremendously facilitates the correct quantification of rare CD34+ cell populations, it leads to the fact that as an end point all enumerated CD34+ cells are automatically viable, and it can be difficult to directly calculate the percentage of viable CD34+ cells out of all CD34+ cells. In addition, it may be challenging to retrieve the total number of CD45+ leukocytes from the applied gating strategy to assess overall specimen viability7.

For CD34+ cells derived from cord blood, the recently released 7th Edition of the NetCord - FACT International Standards for Cord Blood Collection, Banking, and Release for Administration requires the determination of both the total CD34+ count as well as the total viable CD34+ count for cord blood samples post-processing prior to cryopreservation, and the assessment of percent viability of CD34+ prior to release of a cord blood unit to the clinical program4.

In case CD34+ quantification is performed for QC purposes, laboratories seek the ability to not run the “full” ISHAGE panel consisting of duplicate tests plus negative control, but rather a single test, especially when only small specimen volumes are available for analysis. Current acquisition software does not provide the flexibility to adapt the panel accordingly as part of the IVD solution. Although for QC purposes an IVD system is not necessarily required, most labs performing these tests are highly regulated and therefore refrain from validating a separate user-defined test only for this purpose.

Laboratories performing CD34+ hematopoietic progenitor cell (HPC) enumeration are highly regulated in terms of data traceability and need to establish an extensive QC system, especially when undergoing accreditation6. Some key aspects of these control mechanisms are

  • The avoidance of sample misidentification throughout the process by adequate identification of all samples,
  • Adequate provisions for monitoring the reliability, accuracy, precision, and performance of test procedures and instruments,
  • Functional checks for instruments and reagents,
  • The use of appropriate reference material and the documentation of ongoing proficiency testing,
  • A process to prevent the use of expired reagents and supplies,
  • A mechanism that allows linking the lot number, expiration date, and manufacturer of supplies and reagents to each specimen.

The last two points can be especially challenging, as the documentation of reagents and specimens is often not interlinked and happens “offline”, i.e., not on the platform used for data acquisition and analysis.

In hemato-oncological laboratories, HPC samples often arrive as emergency (STAT) samples and require immediate attention, disrupting the routine workflow. Any issues with these samples duplicate efforts and thus increase the potential for human error. For these laboratories, it would be desirable to integrate HPC samples into the normal workflow, ideally in a way that minimizes the risk for sampling mistakes or other issues, as the analysis of CD34+ HPC is time critical. For laboratories that are specialized in CD34+ enumeration, such as cord blood facilities, a higher degree of automation would help to manage the increasing number of samples to be analyzed, while providing a high standard of traceability as outlined above.

Current IVD solutions for CD34+ enumeration by flow cytometry lack automation capability, mainly due to the use of red blood cell lysing reagents that negatively affect cell viability so that prepared samples need to be kept on ice prior to analysis. The ISHAGE Guidelines suggested the use of ammonium chloride because it basically was the only lysing reagent available at the time that was suited for a lyse/no wash approach without the need for additional fixatives, and without altering scatter properties of the cell population of interest. While the use of ammonium chloride is well established in basically all commercial CD34+ enumeration kits for flow cytometry, it prevents the implementation of CD34+ tests on automated sample preparation and flow cytometry platforms due to its effect on cell viability and because the working dilution needs to be prepared daily.

Today, ready-to-use red blood cell lysing reagents are available that specifically lyse red blood cells without pronounced impact on leukocyte viability during sample preparation, enabling performance of CD34+ enumeration on an automated flow cytometry system.

Evolution of the Gold Standard

A next generation CD34+ quantification kit for flow cytometry combines the benefits of established standards and protocols with enough flexibility to adapt reagent and software tools to today’s needs of a clinical laboratory. This includes acquisition and analysis panels for different lab needs without having to set up user-defined tests in parallel to IVD solutions, quality control mechanisms that meet the requirements of a highly regulated work environment, and a high degree of automation (Figure 1).

Figure 1. Characteristics of a solution for the identification and quantification of CD34+ hematopoietic progenitor cells that addresses the requirements of today’s clinical laboratories (based on quantitative market research).

AQUIOS STEM System

The AQUIOS STEM System was designed together with leading experts in the field of clinical CD34+ enumeration aiming to bring the Gold Standard to the next level. It is a modular approach for the automated analysis of CD34+ hematopoietic stem and progenitor cells on the AQUIOS CL Flow Cytometer (Table 1) and consists of

Panel flexibility

AQUIOS STEM Software provides a total of three different acquisition panels for clinical CD34+ enumeration (Figure 2). All protocols follow the sequential gating strategy of the ISHAGE Guidelines, and the panels provide the option to either run the “full” panel of three tests (duplicate plus negative control) as suggested by ISHAGE and mandated by the Ph. Eur., the optional ISHAGE panel without the use of a negative control, or a single test that can be used for QC purposes for rare specimen types. All panel combinations are part of the IVD solution without the need to create user-defined tests.

Figure 2. AQUIOS STEM System panel options for the clinical quantification of CD34+ HPC.

Antibody and reagent solutions

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. The kit contains enough reagent to analyze 50 samples in duplicate plus negative control. Reagents for CD34 and CD45 contain the clones recommended by the ISHAGE Guidelines.

Please refer to Table 2 for a list of all components of the AQUIOS STEM System, and to Table 3 for an overview of the parameters and specimen types that can be analyzed with the AQUIOS STEM System.

Automation concept

The AQUIOS CL Flow Cytometry System streamlines operations by incorporating automated loading, sample preparation, reagent management, and barcode scanning as well as data analysis and bidirectional laboratory information system (LIS) connectivity in one compact platform – this is what we call, Load & Go Flow Cytometry.

The AQUIOS STEM Software enables addition of CD34+ enumeration to the existing AQUIOS CL applications portfolio, so that most steps of the process can be automated. This is achieved using a red blood cell lysing reagent that fulfills the criteria of the ISHAGE Guidelines (no wash, no addition of fixatives, no alteration of scatter characteristics of the target population), but can be stored and handled at room temperature, is ready to use without the need for daily preparation of a working dilution from stock solutions and is gentle to the populations of interest.

The beads solution used for absolute counting has a carefully balanced buoyancy which does not require individual mixing before each pipetting step. Also, the bottle that contains the beads comes with a special cap that avoids evaporation.

Samples are loaded either using the cassette autoloader of the AQUIOS CL, or the Single Tube Loader for STAT samples that are then prioritized over other samples in the queue. This ensures a seamless workflow without the need to interrupt ongoing processes. Sample preparation takes place in 96 well deep-well plates, with individual probes for sample preparation and analysis, so that both steps happen in parallel, and samples are analyzed as soon as sample preparation is finished.

Figure 3. The autoloader accommodates a variety of tube sizes and can load up to 40 specimens at a time, with continuous random loading and unloading functionality. The system uses an internal barcode scanner for positive sample tracking of autoloader samples. The sample ID is matched to the request and the patient information is readily accessible throughout the sample process. A separate single-tube loader is available for STAT samples, open vial samples or irregular tube sizes.

AQUIOS CL system can be safely operated by non-flow cytometry experts, allowing the allocation of staff resources for maximum productivity.

Quality control mechanisms

Barcoded reagents. AQUIOS reagents use a unique barcode identity for tracking the expiration date, on board expiration, lot, and container numbers. The reagent consumption and plate usage are monitored by the system as the samples are processed. The barcode reader scans a reagent or plate upon first use, ensuring that the consumable information is error free. The AQUIOS Smart Track reagent tracking system provides real time consumable tracking to ensure that reagents with proper dating are used, and that there is sufficient reagent for each sample; this eliminates the risk of having to re-run samples because of insufficient reagents levels. The system will not run a test unless it finds the necessary antibody vials.

Specimen barcodes and positive specimen ID. The AQUIOS CL tube barcode reader can automatically read specimen barcodes without the use of a handheld barcode scanner. The AQUIOS CL flow cytometer matches the barcode on the tube to the LIS request. The specimen ID is recorded immediately before specimen aspiration to prevent misidentification, and the specimen ID is automatically tracked throughout the run.

Process controls. 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 two levels of CD34 with approx. 10 CD34+ cells/µL (Level 1) and approx. 30 CD34+ cells/µL (Level 2). Assay values are entered into the system by scanning the barcode of the Control Cell Assay Sheet.

The AQUIOS STEM Software QC module provides access to eIQAP, Beckman Coulter’s electronic Interlaboratory Quality Assurance Program, that allows users to obtain peer group comparison reports for their laboratory’s quality control data.

Automatically Pause when QC fails. The AQUIOS CL flow cytometer was designed as a Load and Go instrument, and the system has built in auto pause features that the user can enable to automatically proceed with patient samples once QC has passed. If the feature is enabled and QC fails, the instrument will not process patient samples and the user can be automatically alerted.

System checks. The system automatically verifies the laser power and that the vacuum is acceptable before each sample is run. The system also verifies internal communications, and that the hardware is functioning correctly at each step of the process. In addition, the system performs a fluidic check at the start of each run to ensure there is enough AQUIOS Sheath Solution available for the requested run.

Advanced reporting options

In order to assist with internal and external quality control standards and requirements, AQUIOS STEM Software reports several statistics parameters that go beyond traditional flow cytometry reports for CD34+ quantification (Table 1). Please refer to the Application Bulletin “How AQUIOS STEM System Addresses International Guidelines for the Clinical Enumeration of CD34+ Hematopoietic Stem and Progenitor Cells” for a complete list of features.

Table 1. Selected statistics and population parameters of AQUIOS STEM System that go beyond traditional flow cytometry reports for CD34+ quantification and support compliance with internal and external quality control standards and requirements.

Parameters Rationale

AQUIOS STEM Software reports the total number of CD34+ cells, the number of viable CD34+ cells, and percent CD34+ viability for each test.

AQUIOS STEM Software contains a plot that displays viable CD34+ cells out of all CD34+ cells.

Standards such as those from NetCord/FACT demand the determination of both the total number of CD34+ cells as well as the number of total viable CD34+ cells for CB samples post-processing and prior to cryopreservation.4

UK-NEQAS reported that “The inclusion of an extra viability plot...” that “...displays “all CD34+ events” and is of use in samples potentially containing dead cells such as post thawed samples, etc.”7

If a panel is selected where tests are run in duplicate, AQUIOS STEM Software directly reports the percent difference of the mean for viable CD34+ cells. The ISHAGE Guidelines require that “The number of CD34+ cells fall within 10% of the mean for the replicate samples.”1
AQUIOS STEM Software reports the total number of CD45+ cells, the number of viable CD45+ cells, and percent CD45+ viability for each test.

The ISHAGE Guidelines require that “The absolute leukocyte count required for determination of absolute CD34+ cells in the PBSC sample must be obtained.”1

While this requirement is mainly intended for dual platform tests, the overall CD45+ count facilitates the assessment of overall specimen viability and a better comparability of cell counts obtained from a hematology analyzer with the results obtained by flow cytometry.

The FACT/JACIE Standards demand that “For all cellular therapy products, a total nucleated cell count and viability measurement shall be performed.”5

AQUIOS STEM Software uses a total of 4 discriminators to ensure all CD34+ cells are captured: Forward Scatter, Side Scatter, Fluorescence 1 (CD45-FITC), and Fluorescence 2 (CD34-PE). An event is acquired if the value of only one parameter is above the defined discriminator.

The ISHAGE Guidelines require that “In peripheral blood and apheresis products, we recommend using the total number of CD45+ events above a noise discriminator (threshold) set on forward angle light scatter. This discriminator must not exclude any viable white blood cells (WBC)...” 1, 2

To ensure a true Load & Go solution, AQUIOS STEM System uses additional denominators to not lose CD34+ cells.

AQUIOS STEM System components and system parameters

The AQUIOS STEM System is a modular approach for the automated analysis of CD34+ hematopoietic stem and progenitor cells on the AQUIOS CL Flow Cytometer. The solution consists of the components specified in Table 2 and is intended to be used by laboratory professionals for the detection of the parameters in the specimen types shown in Table 3 on the automated AQUIOS CL Flow Cytometry System.

Table 2. Selected statistics and population parameters of AQUIOS STEM System that go beyond traditional flow cytometry reports for CD34+ quantification and support compliance with internal and external quality control standards and requirements.

Instrument Description
AQUIOS CL Flow Cytometry System AQUIOS CL Flow Cytometry System with Uninterruptible Power Manager
Software
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.
Reagents
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 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, CD45 and CD3 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 Beads, 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 3. 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

References

  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: https://pheur.edqm.eu/home
  4. 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: http://www.factwebsite.org/
  5. Foundation for the Accreditation of Cellular Therapy (FACT) - Joint Accreditation Committee (JACIE): FACT-JACIE International standards for hematopoietic cellular therapy product collection, processing, and administration. 8th Edition, 2021. Website: http://www.factwebsite.org/
  6. Dorn-Beineke A, Sack U: Quality control and validation in flow cytometry. J Lab Med 2016; 40 s1:1-13.
  7. 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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