European Pharmacopoeia (Ph. Eur.)
The requirements specified below are based on the 10th edition 2019 of the Ph. Eur. and outlined in text number 01/2020:20723: Numeration of CD34/CD45+ cells in haematopoietic products (Reference 3).
Ph. Eur. Requirement
AQUIOS STEM System
- The determination is carried out by a single platform method using calibrated fluorospheres, after lysis of the sample red blood cells if necessary.3
- AQUIOS STEM System is a single platform methodology and uses AQUIOS STEM Count Beads for absolute counting.
- AQUIOS STEM Kit contains a ready-to-use red blood cell lysing reagent.
- This method applies to all types of preparations and whole blood. However, the characteristics of this method make it particularly suitable for preparations containing very low percentages of CD34/CD45+ cells.3
- AQUIOS STEM System is validated for the detection of the parameters in the specimen types shown in Table 1.1, System Parameters in Chapter 1 of the AQUIOS STEM System Guide.
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The technique used for enumeration of CD34/CD45+ cells must meet the following requirements:3
- high sensitivity, since haematopoietic stem cells are rare events;
- accuracy, to provide clinically relevant results;
- reproducibility, to provide clinically reliable results;
- speed, to provide real-time analysis
- The AQUIOS STEM System is a quantitative assay used for the identification and enumeration of the cell populations specified in Table 1.1 System Parameters (Chapter 1 of the AQUIOS STEM System Guide) 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 the AQUIOS STEM System Guide for details on performance characteristics.
- The flow cytometry assay uses commercially available, directly conjugated fluorochrome-labelled monoclonal antibodies, routine staining and whole blood lysing procedures, and a gating strategy using light scatter and immunofluorescence analysis using a pan-CD45/CD34 monoclonal antibody combination.3
- 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 Software follows the sequential Boolean gating strategy recommended by the ISHAGE guidelines.
- It is possible to determine CD34/CD45+ cell viability by appropriate nucleic acid staining with a stain that does not cross the intact cell membrane (for example, with 7-aminoactinomycin D).3
- AQUIOS STEM Kit contains 7-AAD to analyze cell viability for each run.
- Use class III CD34 antibodies that detect all glycosylation variants of the molecule (for example, clone 8G12 or 581). To detect rare events, use an antibody conjugated to the brightest fluorochrome excitable using an argon laser-based flow cytometer, for example phycoerythrin (PE).3
- AQUIOS STEM Kit uses CD34 clone 581 (class III) conjugated to PE.
- Pan-CD45 antibodies that detect all isoforms and all glycoforms of this structure are required. A CD45 antibody conjugated to fluorescein isothiocyanate (FITC) fluorochrome is generally used (for example, J33, HLe1, 2D1).3
- AQUIOS STEM Kit uses CD45 clone J33 conjugated to FITC.
- A negative control is analysed to detect any non-specific signal in the PE fluorescence region. If using an isotypic control, the PE-conjugated isotype is combined with CD45-FITC (or PerCP). If using an isoclonic control, the unconjugated (in excess) and PE-conjugated CD34 identical monoclonal antibody is combined with conjugated CD45. Alternative combinations may be used.3
- AQUIOS STEM Kit contains a CD45-FITC / IsoClonic Control-PE reagent to detect non-specific binding of the CD34-PE monoclonal antibody.
- AQUIOS STEM Kit contains enough reagents to analyze 50 samples in duplicate plus negative control.
- AQUIOS STEM Software provides panels that allow users to run tests in duplicate plus negative control as recommended by the ISHAGE guidelines.
- Depending on the technique used, the internal standard either consists of calibrated beads in suspension or is directly introduced into the associated tubes by the manufacturer.3
- AQUIOS STEM System is a single platform methodology and uses calibrated AQUIOS STEM Count Beads in suspension.
- The beads are automatically added by the system to the cell preparation immediately before analysis.
- Absolute count values are calculated by the AQUIOS STEM Software.
- The absolute number of CD34/CD45+ cells is calculated using the following expression: n x D x V
with
n = total number of CD34/CD45+ cells per microlitre;
D = dilution factor;
V = volume of the product to be tested, in microlitres.3
- AQUIOS STEM Software reports absolute counts for CD34 and CD45, as well as the percentage of CD34+ cells from viable CD45+ cells.
- AQUIOS STEM Software allows users to enter the dilution factor as well as the volume of the product to be tested, which are then used to automatically calculate the corrected value.
- Results are reported as both the percentage of CD34/CD45+ cells and the absolute number per microlitre. They may also be reported as the absolute number per kilogram of recipient body mass, where this is possible.3
- AQUIOS STEM Software reports absolute counts for CD34 and CD45, as well as the percentage of CD34+ cells from viable CD45+ cells.
- AQUIOS STEM Software allows users to enter the recipient body mass, which is then used to automatically calculate the number of CD34+ cells per kilogram body weight.
- The purpose of sequential gating is to select the population of interest and simultaneously minimise interference from debris and mature cells to which antibodies can bind non-specifically. If using a commercial kit, apply the gating recommended by the manufacturer.3
- AQUIOS STEM Software follows the sequential Boolean gating strategy recommended by the ISHAGE guidelines that fulfills these requirements.
- A sufficient number of events are analysed to maintain acceptable accuracy and precision, for example not fewer than 100 CD34+ events and not fewer than 60 000 CD45+ events; the total number of cells counted may be greater if the percentage of CD34 is 0.1 per cent or less.3
- AQUIOS STEM Software has stop criteria for 100 CD34+ cells AND 75,000 CD45+ cells OR 300 seconds.
- In the case where cell concentration prevents the collection of 100 CD34+ cells, a notification will inform the user that less than 100 CD34+ cells are acquired.
- AQUIOS STEM System is validated for the detection of the parameters in the specimen types shown in Table 1.1, System Parameters in Chapter 1 of the AQUIOS STEM System Guide.
- This includes ACD-A for apheresis and EDTA for peripheral blood samples.
- Fresh (less than 24 h old) apheresis products, whole blood samples, umbilical cord blood specimens or bone marrow samples can be processed. Old specimens (more than 24 h old) and specimens that have been frozen and thawed are stained with a viability dye.3
- AQUIOS STEM System is validated for the detection of the parameters in the specimen types shown in Table 1.1, System Parameters in Chapter 1 of the AQUIOS STEM System Guide.
- AQUIOS STEM Kit contains 7-AAD to analyze cell viability for each run.
- Ensure that the concentration of leucocytes is suitable prior to staining with monoclonal antibodies. If necessary, dilute the sample with medium that is compatible with the product to be tested and the lysing system. Record the dilution factor. It is recommended to perform the test with a negative control.3
- AQUIOS STEM System is validated for up to 30,000 cells/µL.
- AQUIOS STEM Software allows users to enter the dilution factor as well as the volume of the apheresis pack, which are then used to automatically calculate the corrected value.
- AQUIOS STEM System provides IVD reagents and protocols that allow users to run samples with or without the use of a negative control.
- Autostandardisation: For analysis of cells labelled with a commercially available kit, manufacturers have developed some quality tools for setting the flow cytometer. These settings are then automatically transferred on protocol analysis of samples. Specific fluorospheres are used to set the photomultiplier tube (PMT) on target values, compensation is set and the system is checked using a control preparation.3
- The AQUIOS CL Flow Cytometer is a quantitative automated analyzer that performs the STEM diagnostic applications in a “no-wash” sample preparation process. Since this system is intended to be an automated analyzer with hands-off processing of samples from specimen introduction to results reports, it is referred to as a Load & Go flow cytometer. The AQUIOS System Software and AQUIOS STEM Tests and Quality Control Reagents do not require user verification of standardization of light scatter, and fluorescence intensities. AQUIOS CL instrument settings are monitored specifically for the AQUIOS STEM System by running Flow-Check Fluorospheres.
- Discriminator/threshold: the forward angle light scatter threshold is set to exclude debris (low forward scatter) but not small lymphocytes from the light-scatter plot.3
- AQUIOS STEM Software uses a total of 4 discriminators in order 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 level.
- AQUIOS STEM Software follows the Boolean gating strategy recommended by the ISHAGE guidelines. This includes a Forward Scatter vs. Side Scatter plot that shows the scatter characteristics of lymphocytes vs. the scatter pattern of CD34+ cells.
- PMT high voltage settings: these must be consistent with cell-surface marker analysis and established within each laboratory so that negative and positive cell populations of moderate antigen density can be distinguished; PMT voltages are reviewed and adjusted periodically according to standardised laboratory procedures.3
- The AQUIOS System Software and AQUIOS STEM Tests and Quality Control Reagents do not require user verification of standardization of light scatter, and fluorescence intensities.
- AQUIOS CL instrument settings are monitored specifically for the AQUIOS STEM System by running Flow-Check Fluorospheres.
- Compensation: this must be acceptable for the colour spectra overlap (for example, FITC/PE) encountered in cell-surface marker analysis; colour compensation is analysed and adjusted according to standardised laboratory procedures.3
- Adjustment to compensation may be required in rare cases. Circumstances may include: At installation when compensation is performed the first time; after service is performed; if after reviewing a run, the operator determines that compensation needs to be adjusted.
- Flow rate: this must be consistent with routine cell-surface marker analysis.3
- AQUIOS STEM System uses a flow rate of 70 µL per minute.
- Gating regions: the gating regions established for the CD34/CD45 samples are maintained unaltered for the analysis of the negative region.3
- The AQUIOS STEM Software establishes the gating regions for the isotype/CD45 sample in an identical manner as the gating regions are established for the CD34/CD45 samples.
International guidelines and standards for CD34+ hematopoietic stem and progenitor cells analysis
- ISHAGE Guidelines
- European Pharmacopoeia (Ph. Eur.)
- FACT - JACIE International Standards for Hematopoietic Cellular Therapy Product Collection, Processing, and Administration
- NetCord - FACT International Standards for Cord Blood Collection, Banking, and Release for Administration
References
- 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.
- 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.
- EDQM Council of Europe: European Pharmacopoeia (Ph. Eur.). 10th Edition 2019-2022. Website: https://pheur.edqm.eu/home
- 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: http://www.factwebsite.org/
- 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/
- 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.
