ISHAGE Guidelines

The requirements for the enumeration of CD34+ cells specified below are based on the 1996 ISHAGE guidelines,1 the modified 1998 “single platform ISHAGE protocol”,2 and a guidance document published in 2012 on behalf of UK NEQAS.6

ISHAGE Guidelines

AQUIOS STEM System

  • Universal precautions should be in effect, and appropriate safety procedures should be employed.1
  • AQUIOS CL accepts capped primary sample tubes, minimizing exposure to potentially biohazardous material such as open blood tubes.
  • Acid citrate dextrose formula A (ACD-A) is the anticoagulant used in apheresis procedures.1
  • EDTA is the anticoagulant of choice for peripheral blood samples.1
  • 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.
  • The validated anticoagulants include ACD-A for apheresis and EDTA for peripheral blood samples.
  • As a minimum, specimens should be labeled with the patient’s full name, patient identification number, date, and time the samples were collected.1
  • If specimen tubes are barcoded, and the LIS connection is enabled, the system automatically retrieves the test request from the LIS. For specimen tubes that are not barcoded, specimen ID and demographic data can be entered manually.
  • 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.
  • AQUIOS STEM Software allows users to enter additional data, such as Patient ID, Gender, Date of Birth, Collect Date, Analysis Date, Physician Name, Physician Code, Location, Harvest Volume, Body Weight, and specimen dilution factor.
  • Laboratory facilities must maintain instrument quality assurance records as well as acceptable quality control procedures. All reagents must be tested for acceptable reactivity.1
  • 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. Resulting QC data is monitored for each lot in the QC database.
  • AQUIOS STEM reagents use a unique barcode identity for tracking reagent type, lot number, container number, expiration dates, and remaining reagent levels. The reagent consumption is monitored by the system as the samples are processed.
  • Instrument PMT voltages should be reviewed and adjusted daily according to established laboratory practice.1
  • AQUIOS CL instrument voltages are monitored specifically for the AQUIOS STEM System by running Flow-Check Fluorospheres.
  • Instrument color compensation should be reviewed and adjusted according to established laboratory practice.1
  • 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.
  • Variability in the number of CD45+ cells between the duplicate runs (more than 3%) may be an indication of irregular instrument flow or incomplete red blood cell lysing.1
  • AQUIOS STEM Software displays the number of AQUIOS STEM Beads vs. the Time parameter.
  • AQUIOS STEM Software reports the number of both viable and total CD45+ cells (WBC) for each run.
  • List mode data should be stored on all specimens.1
  • The AQUIOS system stores test requests, results, and user information in an SQL server database. It is possible to generate backup versions of this database.
  • A hard copy printout of gating and analysis histograms must be stored.1
  • AQUIOS STEM Software allows the printing of reports for each run, including selected analysis histograms.
  • The identification of the personnel responsible for analysis must be recorded.1
  • AQUIOS STEM Software records the User logged in to the system when the test is performed. In addition, the name and ID of the physician performing the test can be entered manually. The printed report contains a signature field.
  • CD34+ cells are identified via light scatter properties and two-color immunofluorescence using CD45-FITC/CD34-PE.1, 2
  • AQUIOS STEM Kit contains a two-color fluorescent (FITC, PE) monoclonal antibody reagent and follows the gating and CD34+ identification pattern recommended by the ISHAGE guidelines.
  • CD34 clones: Class II or class III antibody, conjugated to a bright fluorochrome such as PE. Recommended clones: 581, 8G12, and QBend10.1, 2
  • AQUIOS STEM Kit uses CD34 clone 581 (class III) conjugated to PE.
  • CD45 clones: The antibody needs to detect all isoforms and all glycoforms. Conjugated to FITC. Preferred clone: J33. Potential alternatives: HLe-1 and KC56.1, 2
  • AQUIOS STEM Kit uses CD45 clone J33 conjugated to FITC.
  • Lysing reagent: A whole blood lysis technique is recommended for peripheral blood and apheresis products.1, 2
  • AQUIOS STEM Kit contains a ready-to-use red blood cell lysing reagent.
  • The lysing reagent used does not contain fixatives and obviates the requirement for washing/centrifugation steps. Ammonium chloride fulfills these requirements.2
  • The AQUIOS STEM Lysing Solution is a highly specific, ready-to-use and gentle lysing agent that does not contain fixatives. It lyses red blood cells in samples sufficiently to perform monoclonal antibody panel analysis without interference from red blood cells and allows analysis without damage to white blood cells.
  • The AQUIOS CL Flow Cytometer is a quantitative automated analyzer that performs the STEM diagnostic applications in a “no-wash” sample preparation process.
  • Viability of all samples should be checked routinely, either by adding a viability dye such as 7-AAD to the assay, or by trypan blue exclusion before samples are run.1
  • AQUIOS STEM Kit contains 7-AAD to analyze cell viability for each run.
  • Cell viability should be recorded for every specimen.1
  • AQUIOS STEM Software reports viability for CD45 and CD34 for every sample.
  • The method shall include a viability dye such as 7-AAD that discriminates between viable or nonviable CD34+ cells, and that allows for a direct assessment of the absolute number of CD34+ cells.2
  • AQUIOS STEM Kit contains 7-AAD to analyze cell viability.
  • AQUIOS STEM Software reports both the number of viable CD34+ cells AND the total number of CD34+ cells.
  • The inclusion of an extra viability plot that displays “all CD34+ events” is of use in samples potentially containing dead cells such as post-thawed samples, etc.6
  • AQUIOS STEM Software contains such a plot that displays viable CD34+ cells out of all CD34+ cells.
  • A sequential gating strategy is used to select the population of interest and simultaneously minimize interference from debris and mature cells to which antibodies can bind nonspecifically.1, 2
  • AQUIOS STEM Software follows the sequential gating strategy recommended by the ISHAGE guidelines.
  • The gating strategy shall reflect the following characteristics of true CD34+ cells:1, 2
    1. Express CD34 antigen
    2. Express CD45 antigen with staining intensity characteristics of blast cells (CD45 dim)
    3. Exhibit low side-angle and low to intermediate forward-angle light scatter characteristics of blast cells.
    4. The side and forward scatter of PBSC CD34+ cells is slightly higher than that of small lymphocytes
  • AQUIOS STEM Software follows the sequential gating strategy recommended by the ISHAGE guidelines.
  • Using the ISHAGE Boolean gating strategy, as originally described, allows the most accurate determination of CD34+ cell counts.6
  • AQUIOS STEM Software follows the Boolean gating strategy recommended by the ISHAGE guidelines.
  • In peripheral blood and apheresis products, it is recommended to use 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
  • 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.
  • The discriminator must be set below the forward scatter of small lymphocytes.1, 2
  • 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.
  • It is suggested that the enumeration of CD34+ cells is performed in duplicate.1, 2
  • AQUIOS STEM Software provides panels that perform CD34 enumeration in duplicate.
  • AQUIOS STEM Kit contains enough reagents to analyze 50 samples in duplicate plus negative control.
  • The assay shall be performed as follows:1, 2
    1. CD45-FITC/CD34-PE
    2. CD45-FITC/CD34-PE
    3. CD45-FITC/Isotype-control-PE
  • AQUIOS STEM System enables the analysis of all samples in duplicate plus negative control.
  • AQUIOS STEM Kit contains enough reagents to analyze 50 samples in duplicate plus negative control.
  • The number of CD34+ cells is derived from taking the average of two acceptable replicate CD34+ determinations, as assessed by CD45-FITC/CD34-PE staining, less the number of events representing nonspecific staining, as determined by the CD45-FITC/isotype-PE control.1, 2
  • AQUIOS STEM System enables the analysis of all samples in duplicate plus negative control.
  • AQUIOS STEM Software reports the average total number of CD34 cells, as well as the events associated to non-specific binding.
  • The number of CD34+ cells shall fall within 10% of the mean for the replicate samples.1
  • 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.
  • A PE-conjugated isotype control is combined with CD45-FITC to enumerate false positive signals in the PE fluorescence region.1, 2
  • AQUIOS STEM Kit contains a CD45-FITC / IsoClonic Control-PE reagent to check the non-specific binding of the CD34-PE monoclonal antibody.
  • AQUIOS STEM Kit contains enough reagents to analyze 50 samples in duplicate plus negative control.
  • The events that appear [in the region used for CD34 enumeration] are subtracted from the average total number of events identified with the CD45/CD34 combination.1, 2
  • AQUIOS STEM Software reports the average total number of CD34 cells, as well as the events associated to non-specific binding.
  • AQUIOS STEM Software notifies when the non-specific CD34 events are greater than 10% of the average CD34+ count when the CD34+ events are above 50 cells per microliter.
  • When the CD34+ events are below 50 cells per microliter, AQUIOS STEM Software notifies when the non-specific CD34 events are greater than 5 events.
  • The gating regions established for the duplicate CD34/CD45 samples remain unaltered for the analysis of the isotype/CD45 sample.1, 2
  • 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.
  • Given the selectivity of the sequential gating strategy, it is likely that the use of a negative control may be redundant.2
  • AQUIOS STEM Software provides IVD protocols that allow users to run samples with or without the use of a negative control.
  • A lyse/no wash methodology shall be used.2
  • AQUIOS STEM System is intended to be used as lyse/no wash methodology.
  • A single platform method shall be employed for absolute counting, with a known volume and assayed concentration of counting beads as internal standard.2
  • AQUIOS STEM System is a single platform methodology and uses AQUIOS STEM Count Beads for absolute counting.
  • Single platform ISHAGE approach is the best approach for enumerating CD34+ stem cells.6
  • AQUIOS STEM System is a single platform methodology.
  • It is recommended that a minimum of 100 CD34+ events and at least 75,000 CD45+ events are collected.1, 2
  • 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.
  • Especially for dual-platform methodologies, the absolute leukocyte count required for determination of absolute CD34+ cells in the PBSC sample must be obtained.1
  • AQUIOS STEM System is a single platform methodology. However, AQUIOS STEM Software reports the following parameters:
    • Number of viable CD45+ cells
    • Number of total CD45+ cells (WBC)
  • Ensure that the leukocyte count is no greater than 20,000 cells/µL.2
  • AQUIOS STEM System is validated for up to 30,000 cells/µL.
  • As the nucleated leukocyte count on some blood and apheresis samples is often higher than can be reliably counted by the instrument, samples may need to be prediluted, and the dilution factor must be taken account of in the final calculation.1, 2
  • AQUIOS STEM Software allows users to enter the dilution factor, which is then used to automatically calculate the corrected value.
  • The absolute number of CD34+ cells is multiplied by the absolute leukocyte count, and by the volume of the apheresis pack to derive the absolute CD34+ cell count per pack.1, 2
  • 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 absolute cell count per pack.
  • Precise pipetting of sample and fluorospheres is essential because the absolute count is dependent on an accurate measurement of CD34+ cells and fluorospheres.2
  • AQUIOS CL automatically pipettes all reagents, minimizing the need for human intervention.
  • Results should be reported as both percentage CD34+ events and absolute number x 106/L (peripheral blood) or the absolute number x 106 (apheresis products).1
  • AQUIOS STEM Software reports absolute counts for CD34 and CD45, as well as the percentage of CD34+ cells from viable CD45+ cells.
  • Add 100 µL of blood to each tube and add the volume of monoclonal antibody to the appropriately labeled tubes.1
  • AQUIOS STEM System uses 43 µL of sample material due to lower lysis reagent volumes.
  • After sample preparation, samples are stored on ice at 4°C in the dark and analyzed by flow cytometry within 1 hour.2
  • The reagents used in the AQUIOS STEM System allow for automated sample preparation and analysis at room temperature without a negative effect on cell viability within the time window required for automated sample preparation and analysis.

International guidelines and standards for CD34+ hematopoietic stem and progenitor cells analysis

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), 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/
  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: http://www.factwebsite.org/
  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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