DEFINITIVE GLOBAL REPORT

Transcription

1 EXPERTISE, SERVICE PROVISION AND CUSTOMER RELATIONS QUALITY OF MEDICAL LABORATORIES CLINICAL BIOLOGY COMMISSION COMMITTEE OF EXPERTS EXTERNAL QUALITY ASSESSMENT IN CLINICAL BIOLOGY DEFINITIVE GLOBAL REPORT FLOW CYTOMETRY: CD34+ STEM CELL ENUMERATION SURVEY 2014/2 IPH-2014/2/CD34/11 Expertise, service provision and customer relations Quality of medical laboratories J. Wytsmanstreet, Brussels Belgium

2 ISSN: COMMITTEE OF EXPERTS IPH (secretariat) TEL: 02/ FAX: 02/ Scheme coordinator: Dr. Van Blerk M. Alternate coordinator: Dr. Vernelen K. TEL: TEL: 02/ / Experts Dr. Bossuyt X. TEL: 016/ FAX: 016/ Dr. Chatelain B. TEL: 081/ FAX: 081/ Dr. Demanet C. TEL: 02/ FAX: 02/ Dr. De Schouwer P. TEL: 03/ FAX: 03/ Dr. Hougardy N. TEL: 063/ FAX: 063/ Dr. Kestens L. TEL: 03/ FAX: 03/ Dr. Pradier O. TEL: 02/ FAX: 02/ Dr. Van Bockstaele D. TEL: 015/ FAX: 015/ Committee of experts: 17/06/2014 Authorisation to release report: by Marjan Van Blerk (scheme coordinator) on 11/07/2014 All the reports are also available on our webpage: FORM 43/124/E v5. 2/18

3 TABLE OF CONTENTS Sample material 4 Participation 5 Methodology of the Belgian clinical laboratories 6 Results 7 Conclusion 15 Interpretation of the individual report 16 Next survey 18 FORM 43/124/E v5. 3/18

4 SAMPLE MATERIAL Sent out specimens The survey comprised 2 fresh umbilical cord blood samples (FC FC 12849) and The sample material was kindly provided by Dr. Pradier (Hôpital Erasme, Brussels) and distributed into aliquots at the Institute of Public Health (IPH, WIV-ISP). The sample material was collected into citrate-phosphate-dextrose. The samples were sent by Taxipost 24h and the laboratories were informed by of the send-out of the control material (day 0: 13 th of May 2014). The samples were tested negative for HIV 1 and 2, hepatitis B surface antigen, hepatitis C and syphilis. 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. We would like to thank Dr. Pradier for providing the control material. FORM 43/124/E v5. 4/18

5 PARTICIPATION Twenty-five Belgian clinical laboratories, 1 clinical trial laboratory, and 1 commercial cord blood bank participated in this survey. The laboratories were able to submit their results over the internet using the url: (toolkit). 92.6% of the participants returned their results this way. FORM 43/124/E v5. 5/18

6 METHODOLOGY OF THE BELGIAN CLINICAL LABORATORIES (n=25) Fifteen laboratories (60.0%) used a single platform approach for determining the absolute CD34+ cell count. Of these laboratories, 8 used Trucount technology (BD Biosciences), 5 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 13 Beckman-Coulter Cytomics FC Beckman-Coulter Navios 4 BD Biosciences FACSCanto 2 BD Biosciences FACSCalibur 1 Miltenyi Biotec MACSQuant analyzer 1 Sample preparation Fourteen participants used a sample volume of 50 µl and 9 participants a sample volume of 100 µl. Most of the laboratories (n=22, 88.0%) 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) 8 BD Biosciences Pharm Lyse 5 Beckman-Coulter VersaLyse 4 BD Biosciences FACS Lyse 3 BD Biosciences Ammonium chloride lysing solution 2 Cytognos Quicklysis 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=21, 84.0%) evaluated CD34+ cell viability using 7-AAD (7-Amino-actinomycin D, n=20) or TO-PRO-3 (n=1). Gating strategy With 3 exceptions (Beckman-Coulter Stem-Kit (1), Bender protocol (1), BD Biosciences Stem Cell Enumeration Kit (1)), all participants applied the ISHAGE (International Society of Hematotherapy and Graft Engineering) gating protocol. FORM 43/124/E v5. 6/18

7 RESULTS All participants received the blood samples on day % of the participants (n=25) performed the analyses on day 1 and 7.4 % on day 2 (n=2). Since the samples are fresh and not stabilised, it is extremely important to perform sample testing as soon as possible upon receipt. Statistics for the evaluation are solely based on the results of the Belgian clinical laboratories that performed the analyses on day 1 or 2 (n=25). The median % viable CD34+ cells within total WBC was 0.250% for sample FC and 0.260% for sample FC The overall CVs were 20.7 and 18.8%, respectively. The median absolute viable CD34+ cell count was 23.5 cells/µl for sample FC and 32.9 cells/µl for sample FC The overall CVs were 22.0 and 20.0%, respectively. FC Median CV % P25 P75 Range N % CD34+ cells within total WBC Absolute CD34+ cell count (cells/µl) FC Median CV % P25 P75 Range N % CD34+ cells within total WBC Absolute CD34+ cell count (cells/µl) FORM 43/124/E v5. 7/18

8 The following boxplots and histograms show these data graphically: FC12848 FC12849 Number of participants % CD34+ cells within total WBC % CD34+ cells within total WBC-FC12848 Number of participants % CD34+ cells within total WBC-FC12849 FORM 43/124/E v5. 8/18

9 FC12848 FC12849 Number of participants Absolute CD34+ cell count (cells/µl) Absolute CD34+ cell count (cells/µl)-fc12848 Number of participants Absolute CD34+ cell count (cells/µl)-fc12849 FORM 43/124/E v5. 9/18

10 In the next graph, the z-scores obtained for sample FC 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). % CD34+ cells within total WBC Z-scores for FC Z-scores for FC12848 FORM 43/124/E v5. 10/18

11 Absolute CD34+ cell count (cells/µl) Z-scores for FC Z-scores for FC12848 FORM 43/124/E v5. 11/18

12 The next tables and box-and-whisker plots compare the results from the double (n=10) and single (n=15) platform users: FC Median CV P25 P75 Range N cells/µl % cells/µl cells/µl cells/µl Double platform Single platform FC Median CV P25 P75 Range N cells/µl % cells/µl cells/µl cells/µl Double platform Single platform FC12848 n= 10 n= 15 FC12849 n= 10 n= 15 CD34+ cells/µl CD34+ cells/µl Double platform Single platform Double platform Single platform The median WBC count obtained by the laboratories using a double platform approach was /L for sample FC and /L for sample FC (n=10). The overall CVs were 3.3 and 4.4%, respectively. The next table shows the individual results (10 9 /L) per type of haematology analyser: Haematology analyser FC FC Abbott Cell-Dyn Sapphire Siemens Advia Sysmex XE 2100/XE Sysmex XN FORM 43/124/E v5. 12/18

13 The next tables compare the absolute CD34+ cell counts obtained by the laboratories using a lyse no wash procedure for sample preparation (n=22) and those employing a lyse-wash method (n=3): FC Median/Results CV P25 P75 Range N cells/µl % cells/µl cells/µl cells/µl Lyse/no wash Lyse/wash FC Median/Results CV P25 P75 Range N cells/µl % cells/µl cells/µl cells/µl Lyse/no wash Lyse/wash The next tables compare the absolute CD34+ cell counts obtained by the laboratories evaluating CD34+ cell viability (n=21) or not (n=4): Median/ Results cells/µl No determination of viability FC CV % P25 cells/µl P75 cells/µl Range cells/µl Determination of viability N 4 Median/ Results cells/µl No determination of viability FC CV % P25 cells/µl P75 cells/µl Range cells/µl Determination of viability N 4 FORM 43/124/E v5. 13/18

14 The median viability of CD45+ cells was found to be 90.7% in sample FC and 89.8% in sample FC The median viability of CD34+ cells was found to be 99.2% in sample FC and 99.6% in sample FC FC Median CV P25 P75 Range N % % % % % Viability of CD45+ cells Viability of CD34+ cells FC Median CV P25 P75 Range N % % % % % Viability of CD45+ cells Viability of CD34+ cells Viability of CD45+ cells (%) Viability of CD34+ cells (%) FC FC FC FC FORM 43/124/E v5. 14/18

15 CONCLUSION % CD34+ cells within total WBC (n=25) Twenty-two Belgian clinical laboratories (88.0%) reported results within the global median value +/- 2 SD. None of the Belgian clinical laboratories obtained an unacceptable result (z-score >3). Absolute CD34+ cell count (n=25) Twenty-four Belgian clinical laboratories (96.00%) reported results within the global median value +/- 2 SD. None of the Belgian clinical laboratory obtained an unacceptable result (z-score >3). FORM 43/124/E v5. 15/18

16 INTERPRETATION OF THE INDIVIDUAL REPORT Besides this global report, an individual report is at your disposal via internet (toolkit: Following information is provided: Your result (R) Your method Global median (M g ): central value of the results obtained by all laboratories (all methods confounded). Global standard deviation (SD g ): measure of the spread of the results obtained by all laboratories (all methods confounded). Global median of your method (M m ): central value of the results obtained by the laboratories using your method. Standard deviation of your method (SD m ): measure of the spread of the results obtained by the laboratories using your method. The coefficient of variation CV (expressed in %) for all laboratories and for the laboratories using your method: CV m = (SD m / M m ) * 100 (%) and CV g = (SD g / M g ) * 100 (%). Z score: difference between your result and the median of your method (expressed as a number of SD): Z m = (R - M m ) / SD m and Z g = (R - M g ) / SD g. U score: relative deviation of your result from the median of your method (expressed in %): U m = ((R - M m ) / M m ) * 100 (%) and U g = ((R - M g ) / M g ) * 100 (%). A graphical interpretation of the position of your result (R) towards the results of all the participants as well as the results of the participants using your method, based on the method of Tukey, for each parameter and for each analyzed sample. R : your result M m/g : median H m/g : percentiles 25 and 75 I m/g : internal limits (M ± 2.7 SD) O m/g : external limits (M ± 4.7 SD) FORM 43/124/E v5. 16/18

17 The global graph and the one of your method are presented on the same scale, which allows you to compare them. These graphs give you a rough estimation of the position of your result (R) towards the medians (M m/g ). More information can be found in 3 brochures available on our website (only in Dutch and French): (choose brochures in the menu) or directly on the following webpage (only in Dutch and French): Informatiebrochure over de externe kwaliteitsevaluatieprogramma's voor klinische laboratoria (Algemene informatiebrochure over de externe evaluatie)/ Brochure d information sur les programmes d évaluation externe de la qualité pour les laboratoires cliniques (Brochure d information générale sur l évaluation externe). 2. Statistische brochure (Algemene statistische berekeningsprocedure opgesteld door Professor Albert)/ Brochure statistique (Procédure générale de calcul statistique mis au point par le professeur Albert). 3. Verwerking van gecensureerde waarden (Statistische berekeningsprocedure toegepast op de gecensureerde waarden opgesteld door Professor Albert)/ Traitement des valeurs censurées (Procédure de calcul statistique appliquée aux valeurs censurées rédigée par le Professeur Albert). FORM 43/124/E v5. 17/18

18 NEXT SURVEY The next survey is scheduled for the 14 th of October END Scientific Institute of Public Health, Brussels This report may not be reproduced, published or distributed without the consent of the WIV-ISP. FORM 43/124/E v5. 18/18