SCIENTIFIC INSTITUTE OF PUBLIC HEALTH QUALITY OF MEDICAL LABORATORIES CLINICAL BIOLOGY COMMISSION COMMITTEE OF EXPERTS GLOBAL REPORT

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1 SCIENTIFIC INSTITUTE OF PUBLIC HEALTH QUALITY OF MEDICAL LABORATORIES CLINICAL BIOLOGY COMMISSION COMMITTEE OF EXPERTS GLOBAL REPORT EXTERNAL QUALITY ASSESSMENT IN CLINICAL BIOLOGY FLOW CYTOMETRY CD34+ STEM CELL ENUMERATION SURVEY 2012/1 IPH-12/1/CD34/4 Section Quality of Medical Laboratories 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 2012/1. Printing date: 20/04/2012 2/12

3 Sent out specimens The survey comprised 2 fresh umbilical cord blood samples ( 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 (14 th of February: 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 received the samples on day % of the participants (21/24) performed the analyses on day 1. For the first time, the laboratories were able to submit their results over the internet using the url: (toolkit). Almost all the participants (87.5%) returned their results this way. We would like to thank Dr. Pradier (Hôpital Erasme, Brussels) for providing the control material. CD34+ stem cell enumeration, global report 2012/1. Printing date: 20/04/2012 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, 7 used Trucount technology (BD Biosciences), 5 Flow-Count or Stem-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 9 Beckman-Coulter Cytomics FC BD Biosciences FACSCalibur 2 BD Biosciences FACSCanto 2 Beckman-Coulter Navios 2 BD Biosciences Accuri C6 1 Monitoring of flow cytometer performance (n=24) 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 7 weekly 3 daily 2 2x/week 1 3x/week 1 Beckman Coulter Flow-Check Fluorospheres 5 daily 5 BD Biosciences 7-color setup beads + 2 weekly/ 1 Cytometer Setup and Tracking (CST) beads weekly weekly/ 1 monthly BD Biosciences Rainbow Calibration Partikels (8 peaks) + 2 daily/ 2 Cytometer Setup and Tracking (CST) beads weekly BD Biosciences Rainbow Calibration Partikels (8 peaks) + 2 per batch/ 1 Beckman Coulter Flow-Check Pro Fluorospheres per batch weekly/ 1 daily BD Biosciences Rainbow Calibration Partikels (8 peaks) + 1 weekly/ 1 BD Biosciences 7-color setup beads 2x/month DakoCytomation FluoroSpheres 6-peak 1 daily 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 2012/1. Printing date: 20/04/2012 4/12

5 Commercial control material N Frequency N BD Biosciences Stem Cell Control Kit 8 per batch 3 2x/week 2 daily 2 weekly 1 Beckman Coulter STEM-TROL Control Cells 3 daily 1 per batch 1 not mentioned 1 Sample preparation Twelve participants used a sample volume of 50 µl and 9 participants a sample volume of 100 µl. Most of the laboratories (n=18, 75.0%) used a lyse no wash method. The 6 other participants employed a lyse-wash technique. The following table summarises the lysing reagents used (n=24): Lysing reagent Number of laboratories Ammonium chloride (NH 4 Cl) 10 BD Biosciences FACS Lyse 5 Beckman-Coulter VersaLyse 2 BD Biosciences Pharm Lyse 2 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 (2), APC (1)) used a phycoerythrin (PE)-conjugated CD34 monoclonal antibody. All but 7 participants (PE (2), PerCP, ECD, APC-Cy7, APC-H7, Krome Orange) used a fluorescein isothiocyanate (FITC)-conjugated CD45 monoclonal antibody. Viability More than half of the laboratories (n=14, 58.3%) evaluated CD34+ cell viability using 7-AAD (7-Amino-actinomycin D, n=12) or TO-PRO-3 (n=2). 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 2012/1. Printing date: 20/04/2012 5/12

6 Results The median absolute CD34+ cell count was 12.8 for sample, and 39.8 for sample. The overall CVs were 22.4 and 20.5%, respectively. Median CV P25 P75 Range % Absolute CD34+ cell count 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 2012/1. Printing date: 20/04/2012 6/12

7 Number of participants Absolute viable CD34+ Number of participants Absolute viable CD34+ The following strongly deviating results were excluded from the subsequent tables and graphs: Participant CD34+ CD34+ z-score z-score * 18* *Probably sample switching error CD34+ stem cell enumeration, global report 2012/1. Printing date: 20/04/2012 7/12

8 In the next graph, the z-scores obtained for sample and 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). CD34+ stem cell enumeration, global report 2012/1. Printing date: 20/04/2012 8/12

9 The next tables and box-and-whisker plots compare the results from the double (n=10) and single (n=12) platform users: Median CV % P25 P75 Range Calculated absolute CD Direct absolute CD 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=10) was /L for sample and /L for sample. The overall CVs were 11.3 and 7.2%, respectively. The next table shows the individual results (10 9 /L) per haematology analyser: Haematology analyser Abbott Cell-Dyn Sapphire 7.2, , 16.8 Beckman Coulter LH Siemens Advia , 7.1, , 14.0, 14.3 Sysmex XE 2100/XE (2), 6.2, , 13.0, 13.1, 14.2 CD34+ stem cell enumeration, global report 2012/1. Printing date: 20/04/2012 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=14) or not (n=8): Median CV % P25 P75 Range No determination of viability Determination of viability Median CV % P25 P75 Range No determination of viability Determination of viability The median viability of CD45+ cells was found to be 91.3% (range: %, n=13) in sample and 95.6% (range: %, n=12) in sample. The median viability of CD34+ cells was found to be 95.6% (range: %, n=11) in sample and 98.3% (range: %, n=10) in sample. CD34+ stem cell enumeration, global report 2012/1. Printing date: 20/04/ /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=16) and those employing a lyse-wash method (n=6): Median CV % P25 P75 Range Lyse/no wash Lyse/wash Median CV % P25 P75 Range Lyse/no wash Lyse/wash CD34+ stem cell enumeration, global report 2012/1. Printing date: 20/04/ /12

12 Conclusion Twenty laboratories (83.3%) reported results within the global median value +/- 2 SD. Three participants (12.5%) obtained an unacceptable result (z-score <-3 or >3, result depicted in bold) for at least one sample. Participant z-score z-score CD34+ CD CD34+ stem cell enumeration, global report 2012/1. Printing date: 20/04/ /12