DEFINITIVE ANNUAL REPORT FLOW CYTOMETRY: LYMPHOCYTE SUBSET ANALYSIS CD34+ STEM CELL ENUMERATION 2013

EXPERTISE, SERVICE PROVISION AND CUSTOMER RELATIONS QUALITY OF MEDICAL LABORATORIES CLINICAL BIOLOGY COMMISSION COMMITTEE OF EXPERTS EXTERNAL QUALITY ASSESSMENT IN CLINICAL BIOLOGY DEFINITIVE ANNUAL REPORT FLOW CYTOMETRY: LYMPHOCYTE SUBSET ANALYSIS CD34+ STEM CELL ENUMERATION 2013 IPH-2013/Flow cytometry/48 Expertise, service provision and customer relations Quality of medical laboratories J. Wytsmanstreet, 14 1050 Brussels Belgium www.wiv-isp.be

ISSN: 2294-3471 COMMITTEE OF EXPERTS IPH (secretariat) TEL: 02/642.55.22 FAX: 02/642.56.45 Scheme coordinator: Dr. Van Blerk M. Alternate coordinator: Dr. Vernelen K. TEL: e-mail: TEL: e-mail: 02/642.53.83 mvanblerk@wiv-isp.be 02/642.55.29 kvernelen@wiv-isp.be Experts Dr. Bossuyt X. TEL: 016/34.70.09 FAX: 016/34.70.42 e-mail: xavier.bossuyt@uz.kuleuven.ac.be Dr. Chatelain B. TEL: 081/42.32.43 FAX: 081/42.32.04 e-mail: bernard.chatelain@uclouvain.be Dr. Demanet C. TEL: 02/477.50.71 FAX: 02/477.50.63 e-mail: christian.demanet@uzbrussel.be Dr. De Schouwer P. TEL: 03/217.78.01 FAX: 03/217.78.12 e-mail: pieter.deschouwer@zna.be Dr. Hougardy N. TEL: 063/23.16.30 FAX: 063/23.17.61 e-mail: nicolas.hougardy@vivalia.be Dr. Kestens L. TEL: 03/247.62.29 FAX: 03/247.62.31 e-mail: lkestens@itg.be Dr. Pradier O. TEL: 02/555.36.51 FAX: 02/555.44.99 e-mail: olivier.pradier@erasme.ulb.ac.be Dr. Van Bockstaele D. TEL: 015/34.21.11 FAX: 015/34.21.47 e-mail: vanbocd@labcorp.com Committee of experts: 18/03/2014 Authorisation to release report: by Marjan Van Blerk (scheme coordinator) on 30/07/2014 All the reports are also available on our webpage: https://www.wiv-isp.be/clinbiol/bckb33/activities/external_quality/rapports/_nl/rapports_annee.htm http://www.wiv-isp.be/clinbiol/bckb33/activities/external_quality/rapports/_fr/rapports_annee.htm FORM 43/125/E v4. 2/34

TABLE OF CONTENTS 1. LYMPHOCYTE SUBSET ANALYSIS 4 1.1. Surveys 4 1.2. Methodology of the Belgian clinical laboratories 4 1.3. Results 7 1.4. Pz evaluation 27 2. CD34+ STEM CELL ENUMERATION 30 2.1. Surveys 30 2.2. Methodology of the Belgian clinical laboratories 30 2.3. Results 32 2.4. Pz evaluation 34 FORM 43/125/E v4. 3/34

1.1. Surveys 1. LYMPHOCYTE SUBSET ANALYSIS A triannual external quality assessment scheme for lymphocyte immunophenotyping is operational in Belgium since 2000. Each survey, participating laboratories are sent 3 fresh K 2 EDTA anticoagulated whole blood samples by overnight mail. The laboratories are surveyed for methodology and are asked to report white blood cell count (WBC), percentage of lymphocytes, percentages and absolute numbers of T (CD3+), B (CD19+) and NK cells, and of the CD4+ and CD8+ T cell subsets as well as the percentages of κ and λ chain expressing B cells and the κ/λ ratio. The samples are sent by Taxipost 24h and the laboratories are informed by e-mail of the send-out of the control material (day 0). In 2013, surveys were conducted in February (FC 12246, FC 12247, FC 12248), May (FC 12369, FC 12370, FC 12371) and October (FC 12547, FC 12548, FC 12549). 1 Canadian, 1 Latvian, 2 clinical trial laboratories and 46 Belgian clinical laboratories participated in these surveys. 2.2. Methodology of the Belgian clinical laboratories survey 2013/3 Eight laboratories (18.2%) used a single platform approach for determining the absolute lymphocyte subset counts. Of these laboratories, 5 used Trucount technology (BD Biosciences) and 3 Flow-Count beads (Beckman-Coulter). Following tables provide an overview of the haematology analysers and flow cytometers used: Haematology analyser Number of participants Sysmex XE 2100/XE 5000 15 Siemens Advia 2120 7 Abbott Sapphire 5 Beckman Coulter LH 750/780 4 Sysmex XN 3000/ XN 9000 4 Sysmex XT 2000i/XT 4000i 3 Beckman Coulter UniCel DxH 800 2 Sysmex XS 1000i 2 Abbott Cell-Dyn 3200 SL 1 Not mentioned 1 FORM 43/125/E v4. 4/34

Flow cytometer Number of participants BD Biosciences FACSCanto II 17 Beckman Coulter Cytomics FC 500 9 Beckman Coulter Navios 6 BD Biosciences FACSCanto 5 BD Biosciences FACSCalibur 4 Abbott Sapphire 1 BD Biosciences FACSCan 1 Beckman Coulter Epics XL 1 Monitoring of flow cytometer performance Except for one laboratory (Abbott Sapphire), all participants mentioned monitoring the performance of their flow cytometer, for which they all use commercial bead material. In addition, 75.6% also make use of commercial control material. CD3+, CD4+, CD8+, CD19+, and NK cells (n=44) All laboratories applied the whole blood lysis technique. More than half of the laboratories (58.1%) used a lyse no wash procedure. The following table summarises the lysing reagents used: Lysing reagent Number of laboratories BD Biosciences FACS Lyse 23 Ammonium chloride (NH 4 Cl) 7 Beckman-Coulter Optilyse C 4 Beckman-Coulter VersaLyse 4 BD Biosciences Pharm Lyse 2 Beckman-Coulter Immunoprep reagent system 2 Dako Uti-Lyse 1 Not mentioned 1 Most laboratories used 4-, 5- or 6-colour combinations. Number of participants CD3 + CD4 + CD8 + CD19 + NK 3 colours 2 2 2 2 4 4 colours 10 10 10 10 12 5 colours 5 5 5 5 2 6 colours 22 22 22 22 21 7 colours 1 1 1 1 1 8 colours 3 3 3 3 3 10 colours 1 1 1 1 1 FORM 43/125/E v4. 5/34

Belgian consensus recommendations have been issued for the performance of many flow cytometric-based tests (Acta Clinica Belgica 1999; 54:88-98). 95.5% of the laboratories used the recommended monoclonal antibody panels for performing CD3, CD4 and CD8 determinations (two colour systems: CD3/CD4 and CD3/CD8; three colour systems: CD3/CD4/CD45 and CD3/CD8/CD45; four colour systems: CD3/CD4/CD8/CD45). All but 9 participants (CD56 alone) used the combination of CD16 and CD56 to identify NK cells. All laboratories utilized CD45 as gating agent. Following table displays the sample quality control assessment procedures used by the participating laboratories: Sample quality control assessment Number 100% CD45 positive cells 1 + lymphosum 2 + CD3 consistency check 3 16 100% CD45 positive cells 1 + lymphosum 2 9 Lymphosum 2 9 Lymphosum 2 + CD3 consistency check 3 7 100% CD45 positive cells 1 + CD3 consistency check 3 1 100% CD45 positive cells 1 1 Not mentioned 1 1 CD45 Gating for routine flow cytometric analysis of human bone marrow specimens. Stelzer GT, Shults KE, Loken MR. Annals of the New York Academy of Sciences 1993; 677: 265 280 1 Use of CD45 fluorescence and side-scatter characteristics for gating lymphocytes when using the whole blood lysis procedure and flow cytometry. Nicholson JK, Hubbard M, Jones BM. Cytometry 1996;26:16-21 2 Sum of CD3 + % plus CD19 + % plus CD3 - CD16 + and/or CD56 + % (lymphosum) should equal the purity of lymphocytes in the gate ± 5%, with a maximum variability of 10% 3 Replicate results within a panel (e.g. CD3 + %) for the same sample should be within 5% of each other for FSC/SSC gating or within 3% for CD45/SSC gating κ and λ % B lymphocytes and κ/λ ratio (37 participants) One laboratory washed the samples only once. All other laboratories performed 2 (37.8%) or more (59.5%) washing steps. More than two thirds of the participants (75.7%) used polyclonal anti-κ/anti-λ reagents. All but 2 laboratories used anti-κ and anti-λ antibodies in combination with CD19 in one tube. All but 2 participants utilized CD19/SSC gating. All but 1 participant used the sum of the κ and λ chain expressing B cells for the technical validation of their analyses. FORM 43/125/E v4. 6/34

1.3. Results 97.8% (2013/2) to 100% (2013/1 and 3) of the Belgian clinical laboratories mentioning the day of receipt got the blood samples on day 1 and 0% (2013/1 and 3) to 2.2% (2013/2) received the blood samples on day 2 (day 0: send-out of blood samples). 77.8% (2013/2) to 90.9% (2013/3) of the Belgian clinical laboratories indicating the day of sample testing performed the analyses on day 1 and 9.1% (2013/3) to 17.8% (2013/2) on day 2 (day 0: send-out of blood samples). Statistics for the evaluation were solely based on the results of the Belgian clinical laboratories. Statistics for the evaluation of the WBC count, the percentage of lymphocytes by haematology analyser as well as the absolute counts for the different lymphocyte subsets were solely based on the results of the Belgian clinical laboratories who performed the analyses on day 1 or 2. The laboratories were able to submit their results over the internet using the url: https://qml.wiv-isp.be (toolkit). 85.7% (2013/1) to 91.7% (2013/3) of the participants returned their results this way. FORM 43/125/E v4. 7/34

The following tables show the medians and coefficients of variation obtained for the different parameters on the samples sent in 2013: WBC 10 9 /L FC 12246 4.80 4.7 43 FC 12247 4.80 3.8 43 FC 12248 4.41 3.4 43 FC 12369 5.09 4.1 41 FC 12370 5.05 2.1 41 FC 12371 5.10 3.3 41 FC 12547 7.27 5.5 44 FC 12548 7.18 5.0 44 FC 12549 6.53 4.4 44 Lymphocytes % Haematology analyser FC 12246 42.9 3.8 42 FC 12247 43.0 2.9 42 FC 12248 33.0 5.2 42 FC 12369 39.2 3.6 41 FC 12370 38.7 3.8 41 FC 12371 38.7 4.0 41 FC 12547 26.0 4.3 42 FC 12548 26.0 3.4 42 FC 12549 35.2 2.7 41 Lymphocytes % Flow cytometer FC 12246 42.3 10.5 34 FC 12247 42.6 8.7 34 FC 12248 32.8 10.4 34 FC 12369 37.5 7.4 34 FC 12370 38.4 7.6 34 FC 12371 38.3 7.5 34 FC 12547 25.2 7.3 36 FC 12548 25.0 7.1 36 FC 12549 34.8 5.5 36 FORM 43/125/E v4. 8/34

CD3 % FC 12246 75.8 2.0 45 FC 12247 75.7 1.7 45 FC 12248 76.8 2.1 45 FC 12369 75.1 1.9 45 FC 12370 74.9 2.2 45 FC 12371 74.3 1.7 45 FC 12547 80.6 2.0 44 FC 12548 80.6 2.3 44 FC 12549 80.6 2.0 44 CD3 10 9 /L FC 12246 1.549 9.1 45 FC 12247 1.571 8.8 45 FC 12248 1.117 8.0 45 FC 12369 1.519 7.3 42 FC 12370 1.453 8.7 42 FC 12371 1.469 8.6 42 FC 12547 1.544 9.6 44 FC 12548 1.509 9.5 44 FC 12549 1.690 7.4 44 CD4 % FC 12246 60.2 2.5 45 FC 12247 60.0 2.6 45 FC 12248 49.8 3.8 45 FC 12369 60.0 2.2 45 FC 12370 59.9 3.1 45 FC 12371 59.6 2.1 45 FC 12547 58.2 2.5 44 FC 12548 58.3 3.1 44 FC 12549 41.7 4.4 44 FORM 43/125/E v4. 9/34

CD4 10 9 /L FC 12246 1.224 9.1 45 FC 12247 1.230 9.9 45 FC 12248 0.720 9.7 45 FC 12369 1.199 6.9 42 FC 12370 1.164 8.7 42 FC 12371 1.169 7.5 42 FC 12547 1.115 10.0 44 FC 12548 1.074 7.1 44 FC 12549 0.910 9.8 44 CD8 % FC 12246 14.8 5.0 45 FC 12247 14.7 5.0 45 FC 12248 25.4 3.5 45 FC 12369 14.0 6.9 45 FC 12370 14.0 7.4 45 FC 12371 14.0 5.3 45 FC 12547 21.4 5.2 44 FC 12548 21.2 4.9 44 FC 12549 33.0 4.5 43 CD8 10 9 /L FC 12246 0.310 9.3 45 FC 12247 0.300 7.2 45 FC 12248 0.368 10.1 45 FC 12369 0.280 6.3 42 FC 12370 0.280 8.5 42 FC 12371 0.280 7.9 42 FC 12547 0.409 9.1 44 FC 12548 0.400 10.4 44 FC 12549 0.745 9.5 43 FORM 43/125/E v4. 10/34

CD19 % FC 12246 17.3 9.4 44 FC 12247 17.2 8.6 44 FC 12248 9.0 13.3 44 FC 12369 16.0 9.2 44 FC 12370 16.6 8.5 44 FC 12371 16.4 8.1 44 FC 12547 8.3 8.5 43 FC 12548 8.1 12.8 43 FC 12549 21.6 7.7 43 CD19 10 9 /L FC 12246 0.350 14.5 44 FC 12247 0.345 13.1 44 FC 12248 0.131 17.0 44 FC 12369 0.310 14.3 41 FC 12370 0.315 9.6 41 FC 12371 0.321 11.5 41 FC 12547 0.160 14.6 43 FC 12548 0.150 16.8 42 FC 12549 0.470 13.1 43 NK % FC 12246 6.0 16.0 44 FC 12247 6.0 20.4 44 FC 12248 12.3 12.8 44 FC 12369 7.9 9.6 44 FC 12370 8.0 9.7 44 FC 12371 7.9 9.4 44 FC 12547 9.6 11.0 43 FC 12548 9.7 12.2 43 FC 12549 2.6 22.8 43 FORM 43/125/E v4. 11/34

NK 10 9 /L FC 12246 0.128 22.1 44 FC 12247 0.120 24.7 44 FC 12248 0.174 20.8 44 FC 12369 0.158 13.6 41 FC 12370 0.155 14.3 41 FC 12371 0.158 15.9 41 FC 12547 0.180 18.3 43 FC 12548 0.184 18.1 43 FC 12549 0.060 22.2 42 κ % B lymphocytes FC 12246 59.8 2.7 37 FC 12247 59.7 3.0 37 FC 12248 54.8 7.8 36 FC 12369 60.1 3.0 39 FC 12370 60.0 4.3 39 FC 12371 60.0 2.5 39 FC 12547 57.7 4.5 37 FC 12548 57.0 4.4 37 FC 12549 59.5 5.5 36 λ % B lymphocytes FC 12246 39.0 7.2 37 FC 12247 38.3 4.1 37 FC 12248 42.9 9.7 36 FC 12369 38.4 5.4 39 FC 12370 38.0 5.1 39 FC 12371 38.7 5.4 39 FC 12547 41.0 7.9 37 FC 12548 41.6 7.6 37 FC 12549 40.0 6.3 36 FORM 43/125/E v4. 12/34

κ/λ ratio FC 12246 1.54 8.6 37 FC 12247 1.54 5.3 37 FC 12248 1.32 17.1 36 FC 12369 1.57 7.1 39 FC 12370 1.57 8.0 39 FC 12371 1.56 7.1 39 FC 12547 1.41 12.1 37 FC 12548 1.37 9.7 37 FC 12549 1.50 12.6 36 κ+λ % B lymphocytes FC 12246 99.2 2.2 37 FC 12247 99.2 1.3 37 FC 12248 98.5 2.2 36 FC 12369 99.4 1.5 39 FC 12370 99.1 1.9 39 FC 12371 99.4 2.2 39 FC 12547 99.2 1.6 37 FC 12548 99.3 1.4 37 FC 12549 99.6 1.9 36 Lymphosum % FC 12246 99.1 0.8 44 FC 12247 99.0 1.1 44 FC 12248 98.8 1.1 44 FC 12369 99.0 1.0 44 FC 12370 99.2 0.7 44 FC 12371 99.0 0.8 44 FC 12547 99.0 1.1 43 FC 12548 99.0 1.2 43 FC 12549 99.1 0.9 42 FORM 43/125/E v4. 13/34

The CVs for the WBC count ranged between 2.1 and 5.5%. The CVs for the % lymphocytes ranged from 2.7 to 5.2% for the % lymphocytes obtained with haematology analysers and ranged from 5.5 to 10.5% for the % lymphocytes obtained with flow cytometers. For the different lymphocyte subsets, the average between-laboratory variability was 2.0, 2.9, 5.3, 9.6, and 13.8% for the % of CD3+, CD4+, CD8+, CD19+, and NK cells, respectively. The average CVs of the absolute values were higher and amounted to 8.6, 8.7, 8.7, 13.8, and 18.9% for CD3+, CD4+, CD8+, CD19+, and NK cells, respectively. The overall CVs were larger for small subsets. For the percentages of κ and λ chain expressing B cells and the κ/λ ratio, the average CVs were 4.2, 6.5, and 9.7%, respectively. The following graphs show for the different parameters the evolution of the interlaboratory variability over the years. The black lines show the mean CV per survey. The red lines are a smoothed representation of the black lines and depict the evolution of the mean CV over time. Except for CD19+ cells and NK cells, interlaboratory variability has steadily decreased over the years. CD3 % CV (%) 2.0 2.5 3.0 3.5 4.0 4.5 5.0 2000 2002 2004 2006 2008 2010 2012 Year FORM 43/125/E v4. 14/34

CD3 CV (%) 6 8 10 12 14 2000 2002 2004 2006 2008 2010 2012 Year FORM 43/125/E v4. 15/34

CD4 % CV (%) 3 4 5 6 7 8 2000 2002 2004 2006 2008 2010 2012 Year FORM 43/125/E v4. 16/34

CD4 CV (%) 8 9 10 11 12 13 2000 2002 2004 2006 2008 2010 2012 Year FORM 43/125/E v4. 17/34

CD8 % CV (%) 4 6 8 10 12 14 16 18 2000 2002 2004 2006 2008 2010 2012 Year FORM 43/125/E v4. 18/34

CD8 CV (%) 8 10 12 14 16 18 20 2000 2002 2004 2006 2008 2010 2012 Year FORM 43/125/E v4. 19/34

CD19 % CV (%) 6 8 10 12 14 16 2000 2002 2004 2006 2008 2010 2012 Year FORM 43/125/E v4. 20/34

CD19 CV (%) 10 12 14 16 18 2000 2002 2004 2006 2008 2010 2012 Year FORM 43/125/E v4. 21/34

NK % CV (%) 10 15 20 25 2003 2005 2007 2009 2011 2013 Year FORM 43/125/E v4. 22/34

NK CV (%) 14 16 18 20 22 24 26 28 2003 2005 2007 2009 2011 2013 Year FORM 43/125/E v4. 23/34

kappa CV (%) 4 6 8 10 12 2005 2007 2008 2009 2010 2011 2012 2013 Year FORM 43/125/E v4. 24/34

lambda CV (%) 5 10 15 20 2005 2007 2008 2009 2010 2011 2012 2013 Year FORM 43/125/E v4. 25/34

kappa/lambda CV (%) 8 10 12 14 16 18 20 2005 2007 2008 2009 2010 2011 2012 2013 Year FORM 43/125/E v4. 26/34

1.4. P Z evaluation The performance of the laboratories was scored by means of the P Z evaluation. Methodology Each reported result is evaluated by means of a z-score: z = x M SD x: result M: median SD: standard deviation Z-scores reflect the performance of a laboratory with respect to its peer group. Z-scores <-3 or >3 (results falling beyond 3 SD from the median) are considered unacceptable. The performance of the laboratories is evaluated by means of the percentage of unacceptable z-scores (P Z, % of results falling beyond 3 SD from the median) obtained in the course of 1 year. P NZ = N Z 100 (%) N Z : number of results falling beyond 3 SD from the median N: number of reported results The next graph depicts the distribution of the P Z values of the participating laboratories for 2013: Pz (n=46) Cumulative proportion of laboratories (%) 100 90 80 70 60 50 40 30 20 10 0 5 10 15 20 25 30 35 Percentage of z-scores <-3 or >3 FORM 43/125/E v4. 27/34

The next table summarises for the different parameters the number of evaluated results and the percentage of results beyond 3 SD: Parameter Number of evaluated results % results >3 SD Leukocytes 10 9 /L 390 5.4 Lymphocytes % HA 381 5.0 Lymphocytes % FC 315 10.2 CD3 % 402 10.7 CD3 10 9 /L 399 1.3 CD4 % 402 8.5 CD4 10 9 /L 399 2.3 CD8 % 402 4.0 CD8 10 9 /L 399 3.3 CD19 % 393 5.6 CD19 10 9 /L 390 2.6 NK cells % 393 8.1 NK cells 10 9 /L 390 4.9 κ % B lymphocytes 337 5.3 λ % B lymphocytes 337 6.2 κ/λ ratio 337 5.9 κ+λ % B lymphocytes 337 9.5 Lymphosum 392 5.9 The following 3 tables show the percentage of results beyond 3 SD according to the methodology used (double vs single platform, lyse no wash vs lyse wash, use of polyclonal vs monoclonal antibodies for the determination of the κ and λ chain expressing B cells): Parameter Number of evaluated results % results >3 SD Double platform Single platform Double platform Single platform CD3 10 9 /L 324 75 1.5 0 CD4 10 9 /L 324 75 2.8 0 CD8 10 9 /L 324 75 3.7 1.3 CD19 10 9 /L 324 66 3.1 0 NK cells 10 9 /L 324 66 5.6 1.5 Parameter Number of evaluated results % results >3 SD Lyse and wash Lyse no wash Lyse and wash Lyse no wash CD3 % 168 234 11.9 9.8 CD3 10 9 /L 168 231 0.6 1.7 CD4 % 168 234 9.5 7.7 CD4 10 9 /L 168 231 1.2 3.0 CD8 % 168 234 5.4 3.0 CD8 10 9 /L 168 231 0.6 5.2 CD19 % 168 225 8.9 3.1 CD19 10 9 /L 168 222 4.2 1.4 NK cells % 168 225 14.9 3.1 NK cells 10 9 /L 168 222 10.1 0.9 Lymphosum 167 225 8.4 4.0 FORM 43/125/E v4. 28/34

For CD19 and NK, laboratories using a lyse no wash procedure obtained significantly less frequently results beyond 3 SD (Fisher s exact test). Parameter Number of evaluated results % results >3 SD Monoclonal Polyclonal Monoclonal anti-κ/anti-λ anti-κ/anti-λ anti-κ/anti-λ reagent reagent reagent Polyclonal anti-κ/anti-λ reagent κ % B lymphocytes 105 232 7.6 4.3 λ % B lymphocytes 105 232 9.5 4.7 κ/λ ratio 105 232 11.4 3.4 κ+λ % B lymphocytes 105 232 13.3 7.8 For the κ/λ ratio, laboratories using polyclonal anti-κ and anti-λ reagents obtained significantly less frequently results beyond 3 SD (Fisher s exact test). The next table shows the characteristics of the distribution of the Pz values in 2011, 2012 and 2013: number of evaluated participants (N), average (m) ± standard deviation (SD), percentiles, minimum and maximum: Year N m ± SD P 25 P 50 P 75 P 90 P 95 P 99 Min-max 2011 50 5.8 ± 8.8 1.2 3.0 5.6 15.6 22.8 39.2 0-45.8 2012 48 5.9 ± 7.7 0.8 2.6 10.0 14.4 17.6 32.7 0-40.3 2013 46 5.9 ± 6.9 0.8 4.0 9.0 13.9 17.3 29.1 0-32.5 The maximum of evaluated results per laboratory was 162. This table shows a.o. that Belgian laboratories reported an average of 5.9% results beyond 3 SD in 2013. Each participant is provided with an individual annual report summarising for each sample and parameter the result and z-score and mentioning the global P Z score. A result falling beyond 3 SD from the median (z-score <-3 or >3) is depicted in bold. Participants can compare their performance with that of other laboratories by means of the graph above. The Pz value is situated on the X-axis, the corresponding value on the Y-axis reflects the percentage of laboratories having an equal or better performance. Participants who obtained 10% of results with a z-score <-3 or >3 (P Z value 10) are considered as having unsatisfactory performance. If they are interested, participants who reported an outlying result for one or more parameters can contact the members of the expert committee to examine their data in order to find a possible explanation for the erroneous result. FORM 43/125/E v4. 29/34

2.1. Surveys 2. CD34+ STEM CELL ENUMERATION A triannual external quality assessment scheme for CD34+ stem cell enumeration is operational in Belgium since 2011. Each survey, participating laboratories are sent 2 fresh umbilical cord blood samples collected into heparin or citrate-phosphate-dextrose. The participants are 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. In 2013, surveys were conducted in February (FC 12181, FC 12182), May (FC 12372, FC 12373) and October (FC 12550, FC 12551). Twenty-seven Belgian clinical laboratories and 1 clinical trial laboratory participated in these surveys. The samples were sent by Taxipost 24h and the laboratories were informed by e-mail of the send-out of the control material (day 0). 2.2. Methodology of the Belgian clinical laboratories survey 2013/3 (n=26) Sixteen laboratories (61.5%) used a single platform approach for determining the absolute CD34+ cell count. Of these laboratories, 8 used Trucount technology (BD Biosciences), 6 Flow-Count or Stem-count beads (Beckman-Coulter) and 1 Perfect-Count microspheres (Cytognos). One participant used a volumetric single platform approach (MACSQuant analyzer (Miltenyi Biotec)). The next table gives an overview of the flow cytometers used: Flow cytometer Number of laboratories BD Biosciences FACSCanto II 12 Beckman-Coulter Cytomics FC 500 5 Beckman-Coulter Navios 4 BD Biosciences FACSCanto 3 BD Biosciences FACSCalibur 1 Miltenyi Biotec MACSQuant analyzer 1 Monitoring of flow cytometer performance Except for one laboratory, all participants mentioned monitoring the performance of their flow cytometer, for which they all use commercial bead material. In addition, 21 of them (80.8%) also make use of commercial control material. FORM 43/125/E v4. 30/34

Sample preparation Fourteen participants used a sample volume of 50 µl and 10 participants a sample volume of 100 µl. Most of the laboratories (n=23, 88.5%) used a lyse no wash method. The 3 other participants employed a lyse-wash technique. The following table summarises the lysing reagents used: Lysing reagent Number of laboratories Ammonium chloride (NH 4 Cl) 9 BD Biosciences Pharm Lyse 5 Beckman-Coulter VersaLyse 4 BD Biosciences FACS Lyse 3 BD Biosciences Ammonium chloride lysing solution 2 Cytognos Quicklysis 1 Dako Uti-Lyse 1 Qiagen EL-buffer 1 Monoclonal antibodies All but 3 laboratories (PC5.5/PE-Cy5.5, APC (n=2)) used a phycoerythrin (PE)- conjugated CD34 monoclonal antibody. All but 6 participants (APC-Cy7, APC-H7, Horizon V500 (n=2), Krome Orange, VioBlue) used a fluorescein isothiocyanate (FITC)- conjugated CD45 monoclonal antibody. Viability More than 80% of the laboratories (n=22, 84.6%) evaluated CD34+ cell viability using 7-AAD (7-Amino-actinomycin D, n=21) or TO-PRO-3 (n=1). Gating strategy With 5 exceptions (Beckman-Coulter Stem-Kit (2), Bender protocol (2), BD Biosciences Stem Cell Enumeration Kit (1)), all participants applied the ISHAGE (International Society of Hematotherapy and Graft Engineering) gating protocol. FORM 43/125/E v4. 31/34

2.3. Results 96.3% (2013/2) to 100% (2013/1 and 2013/3) of the participants mentioning the day of receipt got the blood samples on day 1 and 0% (2013/1 and 3) to 3.7% (2013/2) received the blood samples on day 2 (day 0: send-out of blood samples). 81.5% (2013/2) to 92.6% (2013/3) of the participants indicating the day of sample testing performed the analyses on day 1 of the survey and 7.4% (survey 2013/3) to 18.5% (survey 2013/2) on day 2 (day 0: send-out of blood samples). Statistics for the evaluation were solely based on the results of the Belgian clinical laboratories who performed the analyses on day 1 or 2. The laboratories were able to submit their results over the internet using the url: https://qml.wiv-isp.be (toolkit). 59.3% (2013/1) to 85.2% (2013/3) of the participants returned their results this way. The following table shows the median % viable CD34+ cells within total WBC and the median absolute CD34+ cell counts and coefficients of variation obtained for the samples sent in 2013: Sample Median CV N Median CV N % CD34+ cells within total WBC % CD34+ cells/µl % FC 12181 0.25 16.3 23 18.8 14.1 26 FC 12182 1.01 9.9 23 116.8 9.5 26 FC 12372 0.36 12.3 25 36.9 17.0 26 FC 12373 0.23 22.2 25 13.3 19.0 26 FC 12550 0.37 20.0 25 26.0 13.7 26 FC 12551 0.36 26.7 25 26.0 16.5 26 The median CD34+ cell count ranged between 13.3 and 116.8 CD34+ cells/µl. The overall CV varied from 9.5 to 19.0%. FORM 43/125/E v4. 32/34

The following graph shows the evolution of the interlaboratory variability over the years. The black lines show the mean CV per survey. The red lines are a smoothed representation of the black lines and depict the evolution of the mean CV over time. CD34 CV (%) 12 14 16 18 20 22 2011 2012 2013 Year FORM 43/125/E v4. 33/34

2.4. P Z evaluation The performance of the laboratories was also examined by means of the P Z evaluation. Given the very limited number of results available per year (2012: n=6, 2013: n=12), the P Z evaluation was based on the results obtained over 2 years. The next graph depicts the distribution of the P Z values of the participating laboratories for the period 2012-2013: Pz (n=27) Cumulative proportion of laboratories (%) 100 90 80 70 60 50 40 30 0 2 4 6 8 10 12 14 16 18 Percentage of z-scores <-3 or >3 The maximum of evaluated results per laboratory was 18. Eleven Belgian clinical laboratories (40.7%) reported no results beyond 3 SD in 2012 and 2013. Seven laboratories (25.9%) reported 1 result, 5 laboratories (18.5%) 2 results and 4 laboratories (14.8%) 3 results beyond 3 SD in 2012 and 2013. Each participant is provided with an individual annual report summarising for each sample and parameter the result and z-score and mentioning the global P Z score. A result falling beyond 3 SD from the median (z-score <-3 or >3) is depicted in bold. Participants can compare their performance with that of other laboratories by means of the graph above. The Pz value is situated on the X-axis, the corresponding value on the Y-axis reflects the percentage of laboratories having an equal or better performance. Given the limited number of results (n=18) available for the P Z evaluation for 2012 and 2013, unsatisfactory performance will be defined on the basis of results obtained over more than 2 years. END Scientific Institute of Public Health, Brussels 2014. This report may not be reproduced, published or distributed without the consent of the WIV-ISP. FORM 43/125/E v4. 34/34