FEDERAL PUBLIC SERVICE, HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT CLINICAL BIOLOGY COMMISSION COMMITTEE OF EXPERTS GLOBAL REPORT

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1 FEDERAL PUBLIC SERVICE, HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT CLINICAL BIOLOGY COMMISSION COMMITTEE OF EXPERTS GLOBAL REPORT EXTERNAL QUALITY ASSESSMENT IN CLINICAL BIOLOGY FLOW CYTOMETRY CD34+ STEM CELL ENUMERATION SURVEY 2011/1 IPH-11/1/CD34/2 Section Clinical Biology J. Wytsmanstreet, Brussels Belgium

2 ISSN: COMMITTEE OF EXPERTS Tel. Fax IPH (sec.) 02/ / Coordinator Dr. Van Blerk 02/ Dr. Bossuyt X. 016/ / Dr. Chatelain B. 081/ / Dr. Demanet C. 02/ / Dr. De Schouwer P. 03/ / Dr. Hougardy N. 063/ / Dr. Kestens L. 03/ / Dr. Pradier O. 02/ / Dr. Schaaf-Lafontaine N. 04/ / Dr. Van Bockstaele D. 015/ / All the reports are also available on our web page: Institut Scientifique de Santé Publique Wetenschappelijk Instituut Volksgezondheid, Brussels This report may not be reproduced, published or distributed without the consent of the WIV-ISP. CD34+ stem cell enumeration, global report 2011/1. Printing date: 05/08/2011 2/10

3 Sent out specimens The survey comprised 3 fresh umbilical cord blood samples (FC 11381, and ) collected on heparin. The samples were distributed by Taxipost 24h and the laboratories were informed by of the send-out of the control material (7 th of June: day 0). Requested analyses The participants were asked to perform flow cytometric CD34+ stem cell enumeration and to indicate the date of receipt, the date of sample analysis, and to provide details of the type of flow cytometer, the sample preparation technique, the source of antibodies, the gating strategy, and the data analysis software used. Since the samples were not stabilised, the laboratories were asked to perform sample testing as soon as possible upon receipt. Participation Twenty-three Belgian laboratories participated in this survey. Two laboratories were unable to participate in the survey since they received the samples only on day 7. With one exception, all other laboratories got the samples on day of the participants (21/23) performed the analyses on day 1. We would like to thank Dr. Pradier (Hôpital Erasme, Brussels) for providing the control material. CD34+ stem cell enumeration, global report 2011/1. Printing date: 05/08/2011 3/10

4 Methodology of the participants Eleven laboratories (47.8) used a single approach for determining the absolute CD34+ cell count. Of these laboratories, 6 used Trucount technology (BD Biosciences), 4 Flow-Count beads (Beckman-Coulter) and 1 Perfect-Count microspheres (Cytognos). The next table gives an overview of the flow cytometers used (n=22): Flow cytometer Number of laboratories BD Biosciences FACSCanto II 9 Beckman-Coulter Cytomics FC BD Biosciences FACSCalibur 3 BD Biosciences FACSCanto 2 Beckman-Coulter Epics XL 1 Sample preparation Eleven participants used a sample volume of 50 μl and 10 participants a sample volume of 100 μl. Most of the laboratories (n=16, 69.6) used a lyse no wash method. The 7 other participants employed a lyse-wash technique. The following table summarises the lysing reagents used (n=23): Lysing reagent Number of laboratories Ammonium chloride (NH 4 Cl) 10 BD Biosciences Pharm Lyse 4 BD Biosciences FACS Lyse 3 Beckman-Coulter VersaLyse 1 Beckman-Coulter OptiLyse C 1 Beckman-Coulter Immunoprep for Q-Prep 1 Cytognos Quicklysis 1 Dako Uti-Lyse 1 Qiagen EL-buffer 1 Monoclonal antibodies All but 3 laboratories (FITC) used a phycoerythrin (PE)-conjugated CD34 monoclonal antibody. The majority of the laboratories used a fluorescein isothiocyanate (FITC)- conjugated CD45 monoclonal antibody. Two participants used a PE-conjugated, 2 other participants a PerCP-conjugated, 1 laboratory an ECD-conjugated and another participant a PE-Cy7-conjugated CD45 monoclonal antibody. Viability The majority of the laboratories (n=15, 65.2) evaluated CD34+ cell viability using 7-AAD (7-Amino-actinomycin D, n=13) or TO-PRO-3 (n=2). Gating strategy With 4 exceptions (Milan + home made protocol, Bender protocol, Beckman-Coulter Stem-Kit, BD Biosciences Stem Cell Enumeration Kit), all participants applied the ISHAGE (International Society of Hematotherapy and Graft Engineering) gating protocol. CD34+ stem cell enumeration, global report 2011/1. Printing date: 05/08/2011 4/10

5 Results The median absolute CD34+ cell count was 19.0 for sample FC 11381, 21.0 for sample and 67.0 for sample. The overall s were 17.8, 27.5 and 22.9, respectively. FC Absolute CD34+ cell count Absolute CD34+ cell count Absolute CD34+ cell count The following histograms and box-and whisker plots show these data graphically. The boxes reflect the interquartile ranges (25 th -75 th percentile, -); the line in the middle represents the median. The whiskers extend to the lowest and highest value of the values laying within the range [-1.5(-);+1.5(-)] CD34+ stem cell enumeration, global report 2011/1. Printing date: 05/08/2011 5/10

6 Number of laboratories 4,5 4 3,5 3 2,5 2 1,5 1,5 0 FC Absolute viable CD34+ cells/ul Number of laboratories Absolute viable CD34+ cells/ul Number of laboratories Absolute viable CD34+ cells/ul CD34+ stem cell enumeration, global report 2011/1. Printing date: 05/08/2011 6/10

7 The next tables and box-and-whisker plots compare the results from the double (n=12) and single (n=11) users: FC Calculated absolute CD Direct absolute CD Calculated absolute CD Direct absolute CD Calculated absolute CD Direct absolute CD n= 12 n= n= 12 n= n= 12 n= 11 Double Single Double Single Double Single CD34+ stem cell enumeration, global report 2011/1. Printing date: 05/08/2011 7/10

8 The next tables and box-and-whisker plots compare the absolute CD34+ cell counts obtained by the laboratories evaluating CD34+ cell viability (n=15) or not (n=8): FC No determination of viability Determination of viability No determination of viability Determination of viability No determination of viability Determination of viability n= 8 n= n= 8 n= n= 8 n= 15 No viability Viability No viability Viability No viability Viability CD34+ stem cell enumeration, global report 2011/1. Printing date: 05/08/2011 8/10

9 The next tables and box-and-whisker plots compare the absolute CD34+ cell counts obtained by the laboratories using a lyse no wash procedure for sample preparation (n=16) and those employing a lyse-wash method (n=7): FC no wash wash no wash wash no wash wash n= 16 n= n= 16 n= n= 16 n= 7 no w ash wash no w ash wash no w ash wash CD34+ stem cell enumeration, global report 2011/1. Printing date: 05/08/2011 9/10

10 Conclusion Most of the participants (79.2) reported results within the global median value +/- 2 SD. One participant (1) obtained results that were systematically too high and three participants (2, 3, 4) results that were systematically too low. Finally, one participant (5) reported results that were extremely low. Participant FC CD34+ CD34+ CD CD34+ stem cell enumeration, global report 2011/1. Printing date: 05/08/ /10