TRANSFUSION MEDICINE UNIT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO.

QUANTITATION OF CD34 POSITIVE CELLS FOR STEM CELL TRANSPLANTATION HUSM/HEMA-UPT/STM-F2 VERSION NO. VERSION DATE. 1 24.03.2011

APPROVED BY: .. ASSOC. PROF. DR ROSLINE HASSAN HEAD OF HAEMATOLOGY DEPARTMENT

CONTROLLED COPY NO: 1 REGISTERED HOLDER TRANSFUSION MEDICINE UNIT

RECORD OF REVIEW/AMMENDMENT DATE VERSION NO. DETAIL OF AMMENDMENT BY

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TRANSFUSION MEDICINE UNIT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO. QUANTITATION OF CD34 POSITIVE CELLS FOR STEM CELL TRANSPLANTATION HUSM/HEMA-UPT/STM-F2 VERSION NO. VERSION DATE. 1 24.03.2011

PREPARED BY DESIGNATION CHECKED BY DESIGNATION AUTHORISED BY DESIGNATION

: MR LIM SENG HOCK : SCIENTIFIC OFFICER : ASSOC. PROF. DR RAPIAAH MUSTAFFA : HAEMATOLOGIST/LAB COORDINATOR : ASSOC. PROF. DR ROSLINE HASSAN : HAEMATOLOGIST/LAB DIRECTOR

1.

OBJECTIVE
To perform quantitation of CD34 positive cells.

2.

METHOD
Single platform ISHAGE gating strategy using flowcytometry.

3.

PRINCIPLE
The assay is performed by staining the blood sample with the reagent using individual BD TruCOUNTTM Tubes to obtain absolute counts. When a blood sample is added to the reagent, the fluorochrome-labeled antibodies in the reagent bind specifically to the cell surface. Additionally, the lyophilized pellet in the BD TruCOUNTTM Tube dissolves, releasing a known number of fluorescent beads. Ammonium chloride is added to lyse erythrocytes before the sample is acquired on a flow cytometer. During analysis, the absolute number of CD34-positive cells in the sample can be determined by dividing the number of CD34 cellular events by the number of fluorescent bead events, then multiplying by the bead concentration. Also a dye like 7-amino-actinomycin D (7-AAD) can be added to assess viability of the cells. Cells that are 7-AADpositive are not viable and can be gated out of further analysis. The gating strategy employed is based on the ISHAGE gating protocol.

4.
4.1

REQUIREMENTS
TYPE OF SAMPLES 4.1.1 Peripheral blood stem cell collect (PBSC) anticoagulated with ACDA 4.1.2 Peripheral blood stem cell in EDTA container (2ml / 3ml) 4.1.3 Bone marrow harvest (BM) anticoagulated with ACDA 4.1.4 Cord blood (CB) anticoagulated with CPD

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TRANSFUSION MEDICINE UNIT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO.
4.2 REAGENTS 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.3 Combined reagents of FITC-labelled anti-CD45 and PE-labelled antiCD34 (Cat No. 341071) BD Via-Probe Cell Viability Solution (7-AAD) (Material No. 555816) Ammonium chloride lysing solution, e.g. BD Pharm Lyse reagent (Cat No. 555899) BD FACSFlow (Cat No. 342003) BD Trucount tubes (Cat No. 340334)

QUANTITATION OF CD34 POSITIVE CELLS FOR STEM CELL TRANSPLANTATION HUSM/HEMA-UPT/STM-F2

VERSION NO. VERSION DATE.

1 24.03.2011

QC MATERIAL 4.3.1 4.3.2 R&D SYSTEMS CD34 Control (High) (Cat No. REF FC238) R&D SYSTEMS CD34 Control (Low) (Cat No. REF FC238)

4.4

EQUIPMENTS 4.4.1 4.4.2 Flowcytometer (FACS Calibur & FACsS Canto) Vortex mixer

4.4.3 Calibrated automatic pipettes

5.
NO 5.1

PROCEDURE
ACTIVITY RECEIVING SAMPLE Receive and register the sample (refer QP HUSM/MHT/PK1.06) Confirm that the information on the specimen label is identical to the request label. If there is a discrepancy, resolve it before proceeding and if there is any doubt about the identity of the patient, inform the ward concerned. RESPONSIBILITY MLT/SO

5.2

SAMPLE PREPARATION 5.2.1 Perform a white cell count (WCC) on all samples to be evaluated. Dilute the sample in sheath fluid (BD FACSFlow) to give a WCC of less than 50x10^3/L. Record the dilution factor for calculation of the final CD34 result.

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5.3

PROCEDURE: LYSE NO WASH Tube Set Up: 1)Tube 1: Dispense 20 L combined reagent of anti-CD45/CD34 onto the side of an appropriately labelled Trucount Tube just above
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TRANSFUSION MEDICINE UNIT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO. QUANTITATION OF CD34 POSITIVE CELLS FOR STEM CELL TRANSPLANTATION HUSM/HEMA-UPT/STM-F2 VERSION NO. VERSION DATE. 1 24.03.2011

the stainless-steel retainer. [Dispense 20 L 7-AAD reagent into the same tube if viability determination for stem cell harvest is required]. Do not touch the bead pellet. 2)Tube 1: Dispense accuratetly 50 L of well mixed sample by reverse pipetting into the appropriately labelled Trucount Tube. 3) Cap tube and vortex gently. Incubate 15 minutes @ 22C in the dark. 4) Add 1ml 1X BD PharM Lyse reagent. 5) Cap tube, vortex gently and incubate 3-5 minutes in the dark. The sample is now ready to be analysed on the flow cytometer. (Run samples within 2 hours of staining. If sample is not analyzed immediately, 5.4 vortex thoroughly just before acquisition) MLT/SO

GATING STRATEGY The gating strategy used is that recommended by the International Society of Hematotherapy and Graft Engineering (ISHAGE). It is based on the use of CD45 to identify the white cell population to be analysed and CD34 to identify the stem cell population. A number of plots are created to eventually gate the CD34 cells. During analysis, the absolute number of positive cells in the sample can be determined by comparing cellular events to bead events.

5.5

FLOW CYTOMETRY The flowcytometer have to be calibrated using calibrite prior to each run if calibration had not been done for the week. Refer BD Research Application Note: Absolute Stem Cell Counting Method (with Viability Option), page 4.

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5.6

ACQUISITION SET-UP Refer BD Research Application Note: Absolute Stem Cell Counting Method (with Viability Option), pages 4-6.

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5.7

ACQUISITION, ANALYSIS AND INTERPRETATION Refer BD Research Application Note: Absolute Stem Cell Counting Method (with Viability Option), pages 6-7. Interpretation criteria for a true stem cell based on ISHAGE guidelines are:
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TRANSFUSION MEDICINE UNIT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO.
-Positive CD34 expression -Dim CD45 expression -Low to intermediate FSC -Low SSC 5.8 ABSOLUTE CD34 COUNT CALCULATION = CD34 events x Bead count* x Sample dilution = cells/L Bead events Test volume
*Bead count value is printed on the Trucount package and varies from lot to lot.

QUANTITATION OF CD34 POSITIVE CELLS FOR STEM CELL TRANSPLANTATION HUSM/HEMA-UPT/STM-F2

VERSION NO. VERSION DATE.

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5.9

QUALITY CONTROL Run an appropriate control with assayed values for the percent and absolute values of CD34+ cells to conrm the staining and system integrity.

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6.0

REPORTING OF RESULTS Results obtained shall be immediately informed to the ward staff incharge by phone and followed by dispatch in the usual manner. Note down the time and name of staff receiving the results on the request form and in the record book for CD34 Assay.

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7.

LIMITATIONS 7.1 When determining absolute counts it is critical that the pipette used to add the sample to the BD TruCOUNT Tube be calibrated to deliver exactly 50 L. 7.2 Make sure you are using the bead count from the current lot of BD TruCOUNT Tubes when calculating the CD34 value. 7.3 Vortex samples immediately before running them on the ow cytometer to ensure thorough resuspension of cells and beads.

8.

REFERENCES 8.1 BD Research Application Note: Absolute Stem Cell Counting Method (with Viability Option) Flow cytometric enumeration and immunophenotyping of hematopoietic stem and progenitor cells. Gratama, J.W., Sutherland, D.R., Keeney, M. Semin. Hematol. (2001)
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8.2

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TRANSFUSION MEDICINE UNIT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO.
8.3 8.4 8.5 8.6 8.7

QUANTITATION OF CD34 POSITIVE CELLS FOR STEM CELL TRANSPLANTATION HUSM/HEMA-UPT/STM-F2

VERSION NO. VERSION DATE.

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Cell Quest TM Software Reference Manual (MHT/DL, 041) FacsCalibur TM System Users Guide (MHT/DL, 039) BD Cytometer Setup and Tracking Application Guide (MHT/DL 229) BD Stem Cell Control Insert. Catalog No. 340991 BD Cell Viability Solution Technical Data Sheet. Material Number: 555816

8.8 BD CD45/CD34 Monoclonal Antibodies Detecting Human Antigens Insert. Catalog No. 341071 8.9 BD CellQuest Pro Acquisition Notes. Flowcytometry Using Ishage Method. CD34 Enumeration by

Note: Safety precaution: When analyzing samples, always wear rubber gloves and adhere to standard safety procedures.

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