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 2011/2 IPH-11/2/CD34/3 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 2011/2. Printing date: 14/12/2011 2/12

3 Sent out specimens The survey comprised 2 fresh umbilical cord blood samples ( and FC 11583) collected on heparin. The samples were distributed by Taxipost 24h and the laboratories were informed by of the send-out of the control material (11 th of October: 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-four Belgian laboratories participated in this survey. All laboratories got the samples on day % of the participants (18/24) 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 2011/2. Printing date: 14/12/2011 3/12

4 Methodology of the participants Thirteen laboratories (54.2%) used a single platform approach for determining the absolute CD34+ cell count. Of these laboratories, 8 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=24): Flow cytometer Number of laboratories BD Biosciences FACSCanto II 10 Beckman-Coulter Cytomics FC BD Biosciences FACSCalibur 3 BD Biosciences FACSCanto 2 Beckman-Coulter Navios 2 Monitoring of flow cytometer performance (n=23) All laboratories mentioned monitoring the performance of their flow cytometer, for which they all use commercial bead material. In addition, half of them also made use of commercial control material. The following 2 tables give an overview of the commercial bead and control material used: Commercial bead material N Frequency N BD Biosciences Cytometer Setup and Tracking (CST) beads 6 daily 2 weekly 2 2x/week 1 3x/week 1 Beckman Coulter Flow-Check Fluorospheres 4 daily 4 BD Biosciences 7-color setup beads + 3 weekly/ 1 Cytometer Setup and Tracking (CST) beads 2x/week weekly/ 1 weekly weekly/ 1 monthly BD Biosciences Rainbow Calibration Partikels (8 peaks) + 2 daily/ 2 Cytometer Setup and Tracking (CST) beads weekly BD Biosciences Calibrite 3 (+ APC beads) 1 weekly 1 BD Biosciences 7-color setup beads 1 weekly 1 DakoCytomation FluoroSpheres 6-peak 1 daily 1 BD Biosciences Rainbow Calibration Partikels (8 peaks) 1 weekly 1 Thermo Scientific Cyto-Cal Alignment Beads + 1 daily/ 1 Cyto-Cal Multifluor plus Violet Intensity Calibrator monthly Thermo Scientific Cyto-Cal Multifluor plus Violet Intensity 1 daily/ 1 Calibrator + BD Biosciences Calibrite 3 (+ APC beads) weekly Beckman Coulter Flow-Check Fluorospheres + 1 daily/ 1 Flow-set Fluorospheres weekly BD Biosciences Rainbow Calibration Partikels (8 peaks) + Beckman Coulter Flow-Check Pro Fluorospheres 1 per batch/ per batch 1 CD34+ stem cell enumeration, global report 2011/2. Printing date: 14/12/2011 4/12

5 Commercial control material N Frequency N BD Biosciences Stem Cell Control Kit 8 daily 2 weekly 2 2x/week 2 per batch 2 Beckman Coulter STEM-TROL Control Cells 3 daily 1 weekly 1 per batch 1 Streck Multicheck CD4 Low 1 daily 1 Sample preparation Ten participants used a sample volume of 50 μl and 9 participants a sample volume of 100 μl. Most of the laboratories (n=19, 79.2%) used a lyse no wash method. The 5 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) 8 BD Biosciences Pharm Lyse 4 BD Biosciences FACS Lyse 4 Beckman-Coulter VersaLyse 3 Beckman-Coulter OptiLyse C 1 Beckman-Coulter Immunoprep reagent system 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, 1 participant a PerCP-conjugated, 1 laboratory an ECD-conjugated, another laboratory a PE-Cy7-conjugated and still another laboratory a Krome Orangeconjugated CD45 monoclonal antibody. Viability Half of the laboratories (n=12, 50.0%) evaluated CD34+ cell viability using 7-AAD (7-Amino-actinomycin D, n=11) or TO-PRO-3 (n=1). Gating strategy With 4 exceptions (Bender protocol (n=2), Beckman-Coulter Stem-Kit (1), BD Biosciences Stem Cell Enumeration Kit (1)), all participants applied the ISHAGE (International Society of Hematotherapy and Graft Engineering) gating protocol. CD34+ stem cell enumeration, global report 2011/2. Printing date: 14/12/2011 5/12

6 Results The median absolute CD34+ cell count was 10.6 for sample, and 21.0 for sample FC The overall CVs were 21.9 and 14.4%, respectively. Median CV P25 P75 Range % Absolute CD34+ cell count FC Median CV P25 P75 Range % 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, P25-P75); the line in the middle represents the median. The whiskers extend to the lowest and highest value of the values laying within the range [P25-1.5(P75-P25);P75+1.5(P75-P25)]. CD34+ stem cell enumeration, global report 2011/2. Printing date: 14/12/2011 6/12

7 Number of participants Absolute viable CD34+ Number of participants FC Absolute viable CD34+ CD34+ stem cell enumeration, global report 2011/2. Printing date: 14/12/2011 7/12

8 In the next graph, the z-scores obtained for sample and FC are plotted against each other for each individual laboratory. The inner square of the plot represents the z-scores with absolute values <1, the next larger square represents the z-scores with absolute values <2, and the outer square represents z-scores with absolute values <3. Values situated outside of the outer square are considered unacceptable for at least one sample (z-score <-3 or >3). The values of one laboratory with strongly deviating results (: 89 CD34+, z-score: 33.8; FC 11583: 43 CD34+, z-score: 7.3) are not represented on the graph. CD34+ stem cell enumeration, global report 2011/2. Printing date: 14/12/2011 8/12

9 The next tables and box-and-whisker plots compare the results from the double (n=11) and single (n=13) platform users: Median CV % P25 P75 Range Calculated absolute CD Direct absolute CD FC Median CV % P25 P75 Range Calculated absolute CD Direct absolute CD The median WBC count obtained by the laboratories using a double platform approach (n=11) was /L for sample and /L for sample FC The overall CVs were 4.1 and 2.9%, respectively. The next table shows the individual results (10 9 /L) per haematology analyser: Haematology analyser FC Abbott Cell-Dyn Abbott Cell-Dyn Sapphire 8.4, , 8.1 Beckman Coulter LH Siemens Advia , , 7.4 Sysmex XE2 100/XE (2), 9.7, 9.8, , 7.3, 7.4 (2), 7.5 CD34+ stem cell enumeration, global report 2011/2. Printing date: 14/12/2011 9/12

10 The next tables and box-and-whisker plots compare the absolute CD34+ cell counts obtained by the laboratories evaluating CD34+ cell viability (n=12) or not (n=12): Median CV % P25 P75 Range No determination of viability Determination of viability FC Median CV % P25 P75 Range No determination of viability Determination of viability The median viability of CD45+ cells was found to be 97.0% (range: %, n=11) in sample and 95.8% (range: %, n=11) in sample FC The median viability of CD34+ cells was found to be 100.0% (range: %, n=8) in sample and 99.6% (range: %, n=8) in sample FC CD34+ stem cell enumeration, global report 2011/2. Printing date: 14/12/ /12

11 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=19) and those employing a lyse-wash method (n=5): Median CV % P25 P75 Range Lyse/no wash Lyse/wash FC Median CV % P25 P75 Range Lyse/no wash Lyse/wash CD34+ stem cell enumeration, global report 2011/2. Printing date: 14/12/ /12

12 Conclusion Nineteen laboratories (79.2%) reported results within the global median value +/- 2 SD. Four participants (16.7%) obtained an unacceptable result (z-score <-3 or >3, result depicted in bold) for at least one sample. Participant CD34+ z-score FC CD34+ z-score CD34+ stem cell enumeration, global report 2011/2. Printing date: 14/12/ /12